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34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1

34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC... Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 https://doi.org/10.1186/s40425-019-0763-1 MEETING ABSTRACTS Open Access 34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1 National Harbor, MD, USA. 6-10 November 2019 Published: 6 November 2019 About this supplement These abstracts have been published as part of Journal for ImmunoTherapy of Cancer Volume 7 Supplement 1, 2019. The full contents of the supplement are available online at https://jitc.biomedcentral.com/articles/supplements/volume-7-supplement-1. Please note that this is part 1 of 2. Results Poster Presentations Preliminary cohort of 10 patients includes 4 with recurrence at a median Biomarkers, Immune Monitoring, and Novel of 2.4 years and 6 with no recurrence at a median of 12 years post-LT. Technologies We found that patients with recurrence post-LT have significantly higher densities of MPO+ PMNs compared to those with no recurrence. This dif- P1 ference is primarily driven by PMNs located within the peritumoral Peritumoral neutrophil infiltration predicts recurrence of stroma (Median [interquartile range [IQR] 2.46 [1.99 - 2.92] vs 1.23 [0.723 hepatocellular carcinoma following liver transplantation -1.78], p=0.019). Intratumoral PMN infiltration was not associated with re- 1 1 1 1 Marc Najjar, MD , Michael Ross , Ayush Srivastava , Robyn Gartrell, MD , currence (Median [IQR] 0.91 [0.59 - 1.20] vs 1.33 [0.56 – 1.90], p=0.308). 1 1 1 Emanuelle Rizk, BA , Olivia Perez , Evan Lieberman , Charles Drake, MD, Moreover, density of CD3, both intratumoral and peritumoral, did not cor- 1 1 1 1 PhD , Ladan Fazlollahi , Helen Remotti , Elizabeth Verna , Karim relate with recurrence, nor did the tissue-derived NLR. Further, we found 2 1 1 Halazun , Jean Emond , Yvonne Saenger, MD that the tissue-derived NLR did not correlate with NLR in blood. 1 2 Columbia University Medical Center, New York, NY, United States; Weill Conclusions Cornell Medicine, New York, NY, United States Higher densities of peritumoral PMNs are associated with post-LT Correspondence: Marc Najjar (mn2594@cumc.columbia.edu) HCC recurrence. Evaluation of TME using qmIF can be used to predict Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P1 recurrence in post-LT HCC. Further, tissue based analysis of PMNs does not correlate with serum NLR allowing potential for composite Background biomarkers. As this is preliminary, further analysis is underway and Hepatocellular carcinoma (HCC) is the most common liver malig- will be validated on the larger cohort of patients. nancy and the 5th cause of cancer-related mortality worldwide. Though previous studies have found that serum neutrophil-to- Reference lymphocyte ratio (NLR) is predictive of survival post liver transplant 1. Gartrell RD, Marks DK, Hart TD, et al. Quantitative Analysis of Immune (LT), peritumoral neutrophil (PMN) infiltration in the tumor micro- Infiltrates in Primary Melanoma. Cancer Immunol Res 2018;6:481-93. environment (TME) of HCC has not been thoroughly investigated yet. In this study we sought to evaluate tissue based PMN infiltration in HCC post LT using quantitative multiplex immunofluorescence (qmIF), previously used to study the TME of several other tumor types[1]. Methods A database of 634 patients was created at Columbia University Irving Medical Center (CUIMC) including adult patients with available clin- ical follow up who underwent liver transplantation (LT) for HCC be- tween 1998 and 2018. We evaluated a preliminary cohort of 10 patients using qmIF, excluding patients with viral hepatitis. FFPE tumor sections were pre-selected by a GI pathologist. Slides were stained using qmIF for MPO (PMNs), CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), HLA-DR (immune activation), and Hep- Par1 (hepatocytes/tumor). Multiplex images were visualized using Vectra (Akoya) and processed using inForm (Akoya). Data was ana- lyzed using R Studio for concatenation, density, nearest neighbor Fig. 1 (abstract P1). Quantitative multiplex immunofluorescence and statistical analysis. Serum NLR was calculated using complete images of HCC blood counts collected prior to LT(Figure 1). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 2 of 272 P2 determined via flow cytometry. Analyte secretion was determined Single-cell RNAseq analysis of the effects of cryopreservation on from supernatant using Milliplex MAP Human CD8+ T-cell Panel. primary tumor tissue Results Shawn Fahl (shawn.fahl@dls.com) We detected pembrolizumab binding to T-cells in a dose dependent Discovery Life Sciences, Huntsville, AL, United States manner and an increase in the activation marker CD69 on T-cells fol- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P2 lowing tumor cell and pembrolizumab treatment in three of four pa- tients tested. We devised an initial E:T optimization screen to identify Background a patient-specific ratio which renders our subsequent therapy re- The tumor microenvironment is a complex mixture of multiple cell sponse profiling highly personalized. CD3+CD8+ T-cell mediated types, and numerous therapeutic interventions have been developed tumor cell death and enhanced killing was detected in the presence targeting distinct aspects of this environment. Tumor tissue samples of pembrolizumab. Immune cell infiltration as well as therapy related are an integral part of identifying and understanding potential thera- cell death was observed in our 3D microtumors. Altered patient spe- peutic targets within the tumor microenvironment of multiple cancer cific cytokine secretion was measured when the cultures were indications. As early biomarker discovery is often hindered by the lo- treated with pembrolizumab and significantly correlated with pem- gistical demands of sourcing fresh human tumor tissue, cryopre- brolizumab induced reduction of microtumor growth rates. served dissociated tumor cell suspensions provide a viable Conclusions alternative for accessing multiple, highly-annotated tumor samples The data generated from these two complex 3D in vitro models al- for complex studies. Previous evaluations of cryopreservation on vi- lows us to better understand immune responses to autologous able tumor tissue have relied on flow or mass cytometry which, while tumor cells and checkpoint blockade. Our models are therefore ideal powerful, are limited in the number of targets that can be analyzed. and complimentary for preclinical testing of new I/O agents as well Single cell gene expression can analyze the expression of signifi- as patient response predictions to I/O based therapies. cantly more targets and provide a clearer picture on the effects of Ethics Approval cryopreservation on the cellular composition of the tumor. Tissue was acquired with approval from Prisma Health's Institutional Methods Review Board, PRO# 00069834. Multiple unique primary tumor samples were dissociated to the single-cell level and profiled by flow cytometry. These single cell sus- P4 pensions were subsequently subjected to single cell RNASeq using Novel immune competent murine glioblastoma models derived the 10X Genomics platform prior to, and immediately following, cryo- T2 L/L L/L L/L from Nestin-CreER ; Quaking ; P53 ; PTEN mice preservation. Data was subsequently analyzed to determine how 1 1 2 Chao-Hsien Chen, MD , Renee Chin, MS , Genevieve Hartley, PhD , cryopreservation impacted the cellular composition of the tumor 1 2 2 Cheng-En Hsieh, MD , Rishika Prasad, MS , Takashi Shingu, PhD , David microenvironment. 2 2 2 Hong, MD , Jian Hu, PhD , Michael Curran, PhD The University of Texas MD Anderson Cancer Center UTHealth P3 Graduate School of Biomedical Sciences, Houston, TX, United States; Predicting patient response to checkpoint blockade therapy using The University of Texas MD Anderson Cancer Center, Houston, TX, in vitro 3D cultures United States Kathryn Appleton, PhD, Ashley Elrod, Qi Jin Guo, Dennis Ruder, Tessa Correspondence: Michael Curran (MCurran@mdanderson.org) DesRochers, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P4 KIYATEC, Inc., Greenville, SC, United States Correspondence: Tessa DesRochers (tessa.desrochers@kiyatec.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P3 The widely used glioblastoma multiforme (GBM) model GL261 is highly immunogenic and readily cured by checkpoint blockade limit- Background ing its use for pre-clinical modeling of immunotherapy for human Knowledge of immune responses that correlate with clinical outcome GBM [1,2]. We developed four novel murine immunocompetent glio- T2 is essential for the development of strategies to harness a patient’s blastoma stem cell (QPP) lines derived from Nestin-CreER Quaking L/L L/L L/L immune system to eradicate cancer. Pre-clinical platforms that recap- (QKI) ; P53 ; PTEN mice, reflecting a common set of alterations itulate the immune response in the context of cancer are necessary in patients [3-5]. The four QPP cell lines are syngeneic to C57BL/6J for adequate understanding and detection of clinical efficacy, how- mice and exhibit distinct responses to T-cell checkpoint blockade. ever, the technology to accurately test immuno-oncology (I/O) ther- Methods apy response is lacking. Despite the value animal models provide in The differential responsiveness of each QPP line was assessed a pre-clinical setting, they lack matched patient tumor and immune through analysis of tumor growth in the brain versus the flank in un- cell interactions. To address this shortcoming, we developed in vitro treated, αPD-1, or αCTLA-4 treated mice. The impact of tumor gen- 3D tissue models that maintain autologous patient tumor cells and omic landscape on responsiveness at each site was measured immune cells for the testing and prediction of immune cell re- through whole exome sequencing. To understand cellular factors sponses. We hypothesize that these 3D tissue models will recapitu- modulating responsiveness of these GBM lines to checkpoint block- late the patient tumor microenvironment and detect response to I/O ade, the immune microenvironments of sensitive (QPP7) versus re- agents. sistant (QPP8) lines were compared in the brain using high Methods parameter flow cytometry. Drivers of flank sensitivity versus brain re- Tumor cells and T-cells were obtained from seven melanoma patient sistance were also measured for QPP8. biopsies and screened for PD-L1 and lymphocyte populations prior Results to incorporation into 3D culture. Effector cell to Tumor cell (E:T) QPP GBM lines demonstrate a range of sensitivities to CTLA-4 and optimization assays were conducted with expanded T-cells at differ- PD-1 blockade when implanted on the flank ranging from complete ent densities and co-cultured at different time points with tumor sensitivity (QPP7) to complete resistance (QPP4). In the brain, QPP7 cells. Viability was measured using CellTiter-Glo® 3D. T-cell response remains sensitive to both antibodies, but QPP4 and QPP8 fail to re- was determined using flow cytometry following 24-hour co-culture spond to blockade of either checkpoint (Figure 1). Analysis of the with tumor cells. Microtumors were established using a biologically QPP8 immune infiltrate in skin reveals enhanced ratios of CD8s to inert scaffold and extracellular matrix components. Microtumor via- Treg and myeloid suppressors in response to checkpoint blockade; bility was determined using PrestoBlue and T-cell infiltration was however, none of these benefits manifest in the brain QPP8 except a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 3 of 272 very specific increase in CD8s relative to granulocytic suppressors (Figure 2). Brain-implanted QPP8 reacts adaptively to checkpoint blockade by upregulating PD-L1 expression across its myeloid stroma. In contrast, immune-responsive QPP7 does not induce PD-L1 and shows markers of enhanced CD8 T cell fitness. Consistent with these observations, genomic analysis reveals a higher mutation dens- ity in QPP7 versus the other QPP lines. Using checkpoint-insensitive QPP4/8, we have now identified agonists of the Stimulator of Inter- feron Genes (STING) pathway as highly promising therapeutics for treating these tumors in the brain. Conclusions We have developed novel syngeneic models of GBM with relevant genetics and immune sensitivities relative to human disease. Through comparing T cell checkpoint blockade sensitive versus in- sensitive variants of these QPP lines, and through comparing variant sensitivity dictated by site of implantation, we have begun to identify the genetic and cellular components that govern immunotherapeutic sensitivity of GBM. References 1. Reardon DA, Omuro A, Brandes AA, Rieger J, Wick A, Sepulveda J, Phuphanich S, de Souza P, Ahluwalia MS, Lim M, Vlahovic G, Sampson J (2017) OS10.3 Randomized Phase 3 Study Evaluating the Efficacy and Safety of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: CheckMate 143. Neuro-Oncology 19: iii21-iii21. 2. Reardon DA, Gokhale PC, Klein SR, et al. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model. Cancer Immunol Res. 2016;4(2):124-135. 3. Hu J, Ho AL, Yuan L, et al. From the Cover: Neutralization of terminal differentiation in gliomagenesis. Proc Natl Acad Sci U S A. 2013;110(36):14520-14527. Fig. 2 (abstract P4). Immune landscape of QPP8 TME in 4. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic different niches landscape of glioblastoma. Cell. 2013;155(2):462-477. 5. Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating P5 endolysosomal degradation. Nat Genet. 2017;49(1):75-86. Laminar Wash™ AUTO system: a reliable walk-away sample Ethics Approval preparation solution for better TIL recovery without centrifugation All experiments were conducted according to protocols approved by the 1 1 2 2 Ira Kim , Melvin Lye , Roberta Zappasodi, PhD , Isabell Schulze , University of Texas MD Anderson Cancer Center Institutional Animal Care 3 1 1 Christoph Eberle, PhD , Chyan Ying Ke , Kong Leong Cheng , Ih Chin and Use Committee. 1 1 2 1 Kon , Royce Pek , Taha Merghoub, PhD , Namyong Kim, PhD 1 2 Curiox Biosystems, Boston, MA, United States; MSKCC, New York, NY, United States; Charles River Laboratories, Worcester, MA, United States Correspondence: Namyong Kim (namyong@curiox.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P5 Background The naturally occurring tumor infiltrating lymphocytes (TILs) exist in a complex microenvironment containing the extracellular matrix, blood vessels, and stromal and endothelial components in addition to vari- ous immune cells. While a growing number of preclinical mouse models is under development, the heterogeneity of the cell compos- ition in a solid tumor poses considerable technical challenges in iso- lating and characterizing the TILs for the downstream analysis. One common problem with TILs preparation occurs during solid tumor dissociation, whereby the TILs are left in a mixture with tissue debris and dead cells in suspension. Consequently, a preparation of autolo- gous TILs often requires further costly and laborious processing such as density gradient centrifugation, immune cell sorting and enrich- ment, and dead cell and debris removal in combination with multiple centrifugation steps. We introduce a novel Laminar Wash™ technol- ogy, which can help overcome these technical challenges. Methods We performed pilot studies on syngeneic (MC38, CT26, Cloud- manS91, 4T1) and humanized mouse tumor models as well as with human PBMCs and tumor biopsies using the Laminar Wash™ technol- ogy. Briefly, we evaluated various functional parameters of TILs such as polyfunctional CD8+ T cell responses and glucose update effi- ciency (2-NBDG) as well as conducting a side-by-side comparison of the TIL recovery rate and immunophenotypic characteristics of lymphoid and myeloid subsets on the Laminar Wash™ and the Fig. 1 (abstract P4). Orthotopic QPP survival and immune sensitivity centrifugation-based systems. In addition, the cell retention rate, cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 4 of 272 viability, debris removal, epitope preservation and the overall pro- detectable in the periphery starting at 2 weeks with stable levels (up cessing time were assessed and compared. Furthermore, we intro- to 40% of live cells to 8 weeks) with increasing T effector memory duce a complete walk-away approach to sample preparation that cells over the course of study. All tumors evaluated had high levels eliminates operator-based variability while significantly enhancing re- of TIL as measured by flow cytometry and immunohistochemistry. producibility and consistency of downstream analysis. Costimulatory and inhibitory molecules evaluated on CD4 and CD8 Results included 4-1BB, TIM-3, LAG-3, OX-40, as well as PD-1, which was Our data demonstrate that the Laminar Wash™ method resulted in expressed on both peripheral blood cells and in TIL. Tumor growth higher cell retention and viability, more clearly defined immune sub- kinetics was unaltered by PBMC humanization through a 5-week sets, a lowered background signal, and an enhanced yield of the TILs study window. from freshly dissociated tumor samples compared to the Conclusions centrifugation-based counterparts. The Laminar Wash™ system can PBMC-humanized NSG-B2M mice may represent a model for evaluat- effectively remove the floating debris in suspension while keeping ing of IO therapeutics with a long study window due to the lack of the live cells unperturbed, allowing the cell surface architecture and xGVHD. While PBMC engraftment kinetics are donor dependent, simi- epitopes better retained for improved downstream analysis with flow lar phenotypes are observed and T cell subsets expressing several cytometer. Additionally, the Laminar Wash™ AUTO system offers a relevant therapeutic targets, including PD-1 are present. This model completely automated sample processing solution for dissociated may permit a rapid in vivo method to study checkpoint blockade tumor samples, simplifying and expediting cell preparation with en- and other T-cell-directed IO therapeutics. hanced consistency and reproducibility. Ethics Approval Conclusions The study was approved by Champions Oncology's Institutional Ani- Laminar Wash™ results in healthy, viable, and well defined popula- mal Care and Use Committee (IACUC). tion of TILs, while improving the overall quality of data. The AUTO station provides an automated, centrifuge-free, and walk-away work- P7 flow for dissociated tumor samples for cytometry-based assays. Development of a natural killer (NK) ImmunoGraft platform for the evaluation of the pharmacodynamics of immuno-oncology Acknowledgements therapeutics Laminar Wash™ results in healthy, viable, and well defined population of TILs, 1 1 2 Bhavana Verma, PhD , Bruce Ruggeri , Jon Weidanz, PhD , Amy Wesa, while improving the overall quality of data. The AUTO station provides an PhD automated, centrifuge-free, and walk-away workflow for dissociated tumor 1 2 Champions Oncology, Rockville, MD, United States; Abexxa Biologics, samples for cytometry-based assays. Arlington, TX, United States Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); P6 Amy Wesa (awesa@championsoncology.com) Development of a peripheral blood mononuclear cells (PBMC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P7 ImmunoGraft platform to evaluate the pharmacodynamics of Immuno-oncology therapeutics Background Bhavana Verma, PhD, Bruce Ruggeri, Amy Wesa, PhD Harnessing NK cell anti-cancer cytotoxicity has gained interest as a Champions Oncology, Rockville, MD, United States therapeutic strategy, and consequently improved preclinical models Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); supporting the translation of NK cell–mediated therapies to the clinic Amy Wesa (awesa@championsoncology.com) are desired. Reproducible models with human NK engraftment into Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P6 immunodeficient mice co-engrafted with cell line-derived xenograft or patient-derived xenograft tumor models have been lacking due to Background an inability to support NK cell engraftment and persistence. Here we Humanized immune system (HIS) mouse models enable in vivo stud- evaluated IL-15-NOG mice for the engraftment and sustained survival ies in the context of the human immune cells with a human tumor of both ex vivo expanded and primary human NK cell isolates for es- and are critical for the development of next generation immune- tablishing models that engraft effectively with both human NK cells oncology (IO) agents. Humanization of immunodeficient mice and a PDX or CDX tumor. through the adoptive transfer of normal adult PBMC leads to rapid Methods engraftment of human T cells to study immune-modulatory agents NK cells from normal adult peripheral blood mononuclear cells in the context of human tumor xenografts, but is limited by the de- (PBMC) donors (N=3) were expanded using two different commer- velopment of xenogeneic graft-versus host disease (xGVHD). In this cially available kits and evaluated for NK phenotype, expansion rates study, we evaluated the engraftment of PBMC in β2microglobulin and yields. Titrated doses of ex vivo expanded NK cells were adop- null super-immunodeficient mice NSG-B2M mice, that lack MHC Class tively transferred into IL-15-NOG mice for human chimerism, and the I on host tissues. A cell line-derived xenograft model (CDX) co- persistence and survival of NK cells and their immunophenotype engrafted with PBMC (PBMC-ImmunoGraft) was characterized for were assessed. In separate studies, naïve NK cells enriched from humanization, tumor infiltrating leukocytes (TIL) phenotype and PBMC were also evaluated for NK cell persistence and expansion tumor response to checkpoint inhibitors. in vivo. To establish an NK ImmunoGraft, NK cells were engrafted in Methods xenograft tumor bearing mice and tumor growth kinetics were PBMC from healthy donors (N=7) were implanted and engraftment characterized. in peripheral blood was assessed by flow cytometry. T cell memory Results phenotypes were assessed over time in a small cohort, and costimu- Donor dependent NK expansion was observed ex vivo, with 28 to latory and inhibitory T cell subsets were evaluated at the terminal 50-fold expansion by two weeks. NK cells expanded ex vivo were time point in blood and secondary lymphoid organs. Next, NSG-B2M CD3-CD16+CD56± and varied based on the expansion kit utilized. mice were co-implanted with MDA-MB-231 breast cancer cell line s.c, Nearly all CD45+ cells in circulation were NK cells, and these peaked humanized with PBMC and tumor growth kinetics were monitored. by week 2, and were maintained for up to 10 weeks in IL-15-NOG Efficacy studies evaluating check point inhibitors are currently mice. Primary NK cells engrafted with slower kinetics, with peak ongoing. abundance at 3-4 weeks. NK cells expressed granzyme B, and further Results functional studies are in progress. For all NK cell populations, cell Successful PBMC engraftment without xGVHD was observed in NSG- density-dependent engraftment was observed with a largely stable B2M mice up to 8 weeks, in contrast with MHC Class I expressing im- NK phenotype observed across the study. In the absence of any munodeficient mice that developed xGVHD within 4-5 weeks. Dose therapeutic treatment, NK cell persistence and expansion in vivo did and donor- dependent chimerism was observed. T cells were not inhibit tumor xenograft growth kinetics in IL-15-NOG mice Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 5 of 272 Conclusions sensitivity and as such could be utilized to assess efficacy of the IL-15-NOG mice support the survival and persistence of human NK monoclonal antibody administered. There are also potential applica- cells from both ex vivo expanded and naïve NK cells, suggesting the tions related to rapid drug screening using the patient-derived xeno- universality of this platform for human NK engraftment. Our prelimin- graft model. Our future plans are focused on adapting these ary studies support IL-15 NOG mouse model as a suitable system for solutions to the characterization of immune checkpoint inhibitor evaluation of NK cellular therapies or NK cell-modulating therapies in therapeutics in standard-of-care FFPE tissues obtained from patients the context of patient-derived or cell-line derived xenograft (PDX or undergoing immunotherapy. CDX) mouse models Ethics Approval P9 The study was approved by Champions Oncology's Institutional Ani- Immune checkpoint biomarkers in hepatocellular carcinoma (HCC): mal Care and Use Committee (IACUC). Assessment of PD-L1 and tumour mutation burden in tumour samples from clinical patients 1 1 1 1 P8 Hisani Horne, PhD, MPH , Young Lee , Todd Creasy , Rebecca Fish , 1 1 2 1 Monoclonal antibody detection from formalin-fixed paraffin- Jonathan Cairns , Paul Scorer , Janine Feng , Marietta Scott, PhD , Mark 1 1 1 embedded tumor tissues using Fab-selective proteolysis nSMOL Gustavson , Aleksandra Dudek-Madej , Craig Barker , Nicholas 1 1 2 2 coupled with liquid chromatography and triple quadrupole mass Holoweckyi , Rebecca Halpin , Peiyi Wang , Quinea Lassiter , Xiaoling 2 2 1 1 spectrometry Xia , Mohammed Abdelwahab , Weimin Li , Alejandra Negro , Jill 1 1 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Noriko Iwamoto, PhD , Walker 2 2 2 1 2 Yoshinobu Koguchi, MD, PhD , John Cha , Brian Piening, PhD , Eric Tran, AstraZeneca, Gaithersburg, MD, United States; Roche Tissue 2 2 2 2 PhD , Hong-Ming Hu, PhD , Bernard Fox, PhD , William Redmond, PhD Diagnostics, Oro Valley, United States 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Correspondence: Hisani Horne (hisani.madison@astrazeneca.com) Cancer Center, Portland, OR, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P9 Correspondence: Takashi Shimada (tashimada@shimadzu.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P8 Background Programmed cell death ligand-1 (PD-L1) expression and tumour Background mutation burden (TMB) have been shown to be predictive of re- With the development of immune checkpoint inhibitors, the focus of sponse to anti-PD-1/PD-L1 immunotherapies in various cancers. cancer therapy is shifting to immunotherapy. Our purpose is to de- The prevalence and distribution of PD-L1 expression and/or velop the drug efficacy index by rapid analysis of antibodies accumu- tumour mutations in HCC and correlation with clinical characteris- lating in cancer tissue using liquid chromatography and mass tics are poorly understood. A better understanding of these bio- spectrometry (LC-MS/MS) and by characterization of the antibody markers may help inform appropriate patient selection strategies distribution in the tumor microenvironment. Using a novel proteoly- in HCC. sis method in which antibody molecules are collected on a 100 nm Methods resin pore and trypsin is immobilized on a 200 nm nanoparticle sur- PD-L1 expression was evaluated on tumour cells (TC), immune cells face, we have developed a method for physicochemically limiting (IC), or combined TC and IC using the VENTANA PD-L1 (SP263) Assay trypsin access to antibody and identifying the structural specificity of in three independent HCC sample sets: 2 from commercial tissue complementarity-determining regions while minimizing extra pep- banks (n = 500 and n = 2417) and 1 from patients enrolled in tides and protease without depending on the type of antibody. NCT02519348, encompassing a wide range of stage and grade of dis- Using this method to detect antibodies from formalin-fixed paraffin- ease. NCT02519348 is a phase 2 study evaluating safety, efficacy out- embedded (FFPE) tumor tissues, we aim to develop novel diagnostics comes of durvalumab with or without tremelimumab in advanced that can aid in therapeutic dosing and predicting responses to HCC. TMB was assessed in tissue by whole exome sequencing in a antibody-based therapies. subset of 70 patients from NCT02519348. Methods Results To demonstrate the feasibility of these approaches, the human At a cut-off date of Feb 28, 2019, 282/335 (84.2%) patients en- breast and epidermoid carcinoma cell lines SKBR3 and A431 were in- rolled in NCT02519348 were successfully evaluated for PD-L1 cubated with either trastuzumab and cetuximab, which bind to (Table 1). Significant expression was seen in ICs relative to TCs. erbB2 and EGFR, respectively. FFPE cell blocks were then prepared Patients in NCT02519348 showed higher PD-L1 expression in TCs and proteins were extracted from 8 μm sections after deparaffiniza- than commercial cohorts. In a univariate analysis using 1% cut tion and decrosslinking. The extracted proteins were subjected to offs, higher TC (but not IC or combined TC and IC) PD-L1 expres- the Fab-selective proteolysis nSMOL, and the signature peptides of sion was associated with patients with HCV infection (p=0.003). each antibody, IYPTNGYTR for trastuzumab and SQVFFK for cetuxi- Of 70 study patients tested, 55 were evaluable for TMB (median mab, were detected via triple-quadrupole LC-MS/MS. SCID mice were 2.59, range 0.46 - 5.61 Mut/Mb). Among patients with available subcutaneously implanted with BT474 cells and 5 days later were in- TMB data there was no observed correlation between TMB and fused with 10 mg/kg or 20 mg/kg trastuzumab. 24 h after administra- PD-L1 expression. tion, tumor and other tissues were harvested and FFPE block were Conclusions prepared for trastuzumab quantitation in FFPE tissues. PD-L1 expression was observed in both TC and ICs in HCC, with the Results latter being more prevalent. Viral status and disease stage may im- As a result of the pretreatment protocol using the cell block, the con- pact PD-L1 expression in this setting, but further work is needed to ditions of deparaffinization, decrosslinking, and protein extraction confirm this. TMB and PD-L1 appear to identify distinct patient sub- were optimized. Mass spectra of the signature peptides from trastu- sets in HCC. zumab and cetuximab could be detected using 20,000 cells. This Trial Registration condition was also applied to xenograft tissue and the degree of NCT02519348 trastuzumab accumulation was detected in FFPE tumor tissue in a Ethics Approval dose-dependent manner. The study (NCT02519348) is performed in accordance with eth- Conclusions ical principles that have their origin in the Declaration of We show that these approaches can be utilized to quantify antibody Helsinki and are consistent with ICH/GCP, and applicable regula- concentrations in typically-challenging FFPE specimens with good tory requirements. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 6 of 272 Table 1 (abstract P9). See text for description different subsets, compared to DGM, potentially allowing more as- says to be executed from the same blood sample. Overall, this tech- nology has the potential to transform cell separation by automating a variable and labor-intensive processes, and therefore has utility in applications that require consistent cell quality and functionality. Table 1 (abstract P10). See text for description P10 Table 2 (abstract P10). See text for description Inertial microfluidics enables highly consistent separation and concentration of leukocytes from human peripheral blood for downstream B-cell and T-cell functional assays Sarah Mickool, Eric Smith, Aleksander Jonca, Gustavo Arnal, Mary Vincent Larcom, Melanie Scully, Peng Megn Kou, PhD, Nitin Kulkarni, Kyle Smith MicroMedicine, Inc., Waltham, MA, United States Correspondence: Kyle Smith (kyle@micromedicine.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P10 Background Cell separation plays a vital role in research and clinical settings for the development and monitoring of cutting-edge therapies. Despite its labor-intensiveness and variability, density gradient P11 centrifugation-based method (DGM) has remained the primary Early detection of breast cancer (BCa) through MDSC and method of upstream cell isolation for decades due to a lack of viable lymphocyte immunophenotyping: from manual gating to pattern alternatives. This is problematic as DGM is a non-scalable, manual recognition neural networks 1 1 1 process. To address this lack of innovation, we have developed an George Dominguez, PhD , John Roop , Alexander Polo, BS , Anthony 1 2 1 automated Microfluidic System based on inertial focusing that en- Campisi, BS , Dmitry Gabrilovich, MD/PhD , Amit Kumar, PhD 1 2 ables label-free white blood cell (WBC) separation and concentration Anixa Biosciences, San Jose, CA, United States; The Wistar Institute, from 3-75mL of whole blood in short timescale with high Philadelphia, PA, United States consistency, providing reliable sample preparation for downstream Correspondence: George Dominguez (george@anixa.com) functional assays. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P11 Methods WBCs were isolated from 15% ACD-A anticoagulated peripheral hu- Background man blood using the Microfluidic System or DGM. Cell number, via- Myeloid-derived suppressor cells (MDSCs) are contributors in sup- bility, and immune phenotype were evaluated by hematology porting tumor progression and escape [1,2]. Studies have quantified analyzer and flow cytometry. To assess B-cell function, cells were MDSCs to detect tumor development, monitor progression, and/or cryopreserved post separation, thawed, and stimulated with IL-2 and predict therapeutic responses [3, 4]. Here, we compared several ma- R848, followed by Human IgG and IgM ELISPOT. To assess T-cell func- chine learning (ML) approaches to analyze flow cytometry data to tion, thawed cells underwent bead-based granulocyte depletion and detect breast cancer (stage I/II) through manual gating and hyper- stimulation with CEF peptide pool, followed by Human IFNγ ELISPOT. voxelation of cell events. Results Methods The prototype Microfluidic System consistently processed 40mL of We used standard multiparametric flow cytometry techniques to anticoagulated blood in approximately 20 minutes with minimal measure myeloid-derived suppressor cell (MDSC), myeloid, and hands-on time as opposed to 60–90 minutes for DGM with signifi- lymphocyte cell populations found in the peripheral blood of 99 cant hands-on time. While DGM collects only peripheral mononuclear biopsy-confirmed early stage BCa patients and 88 healthy donor fe- cells (PBMCs), the System isolates the total WBC population and may male (HDF) controls. Manual gating was performed to generate be beneficial for immunophenotyping. As shown in Table 1, the gated values, and raw flow cytometry data were transformed using Microfluidic System consistently provided improved WBC or PBMC HyperVOX to generate hypervoxelated cytometry event counts. The recovery, viability, purity, RBC depletion, and platelet depletion as ML algorithms used were: support vector machine (SVM), Bayes SVM, compared to DGM. Immune phenotyping shows that the Microfluidic Ensemble SVM, k-nearest neighbor (kNN), and pattern recognition System also consistently resulted in improved recovery of lympho- neural network (PRNN). All algorithms were trained using data from cyte subsets, including CD19+, CD3+, CD4+, and CD8+ cells (Table 2). 64 BCa patients and 69 HDF controls. Predictions were evaluated B-cell and T-cell functionality were found to be equivalent between using the performance of each trained ML algorithm on 35 early the two cell isolation methods based on IgM/IgG and IFNγ secretion, stage BCa patients and 19 HDF that were not used for training (hold- respectively. With the improved cell recovery using the Microfluidic out test set). System, more target cells from the same blood sample may be col- Results lected for downstream assays. Using manually gated counts, the resulting accuracies were: SVM = Conclusions 75.4%, Bayes SVM = 71.3%, Ensemble SVM = 65.6%, and kNN = The Microfluidic System offers a faster, more reliable method than 69.7%. Using hypervoxelated event counts, the resulting accuracies DGM for upstream cell separation from whole blood. The System were: SVM = 78.7%, Bayes SVM = 77.1%, Ensemble SVM = 57.4%, consistently recovers more cells, including functional lymphocytes of kNN = 67.2%, and PRNN = 92.6%. Hypervoxelated data analyzed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 7 of 272 using PRNN resulted in the highest accuracy with a sensitivity of regulated; q-value > 0.05 and log2 fold change > 0.58). In addition to the 91.4% and a specificity of 94.7%; the resulting AUC = 0.9098 (95%CI acute phase proteins (e.g. CRP and SAA1) which were previously verified = 0.8031 to 1.000). Additionally, we tested 26 samples collected from to be elevated in subjects with NSCLC, partial least squares discriminant patients with confirmed ductal carcinoma in situ (DCIS) using hyper- analysis helped identify additional proteins that are differentially voxelated counts with a PRNN. Even though they are clinically expressed between the sample groups. Most relevant to immune func- deemed as pre-cancerous (stage 0), 18 out of 26 (AUC = 0.8421; tion was CLC (Galectin-10), which was elevated in NSCLC samples and 95%CI = 0.7163 to 0.9679) were classified as BCa suggesting utility has been identified as key component supporting the suppressive func- for detecting the existence of even a non-invasive cancerous lesion. tion of Tregs.[1] Furthermore, F13A1 was suppressed in the NSCLC sam- Conclusions ples which is known to be associated with macrophage activation. Although further study is needed, we believe that using PRNN with Conclusions MDSC immunophenotyping, in conjunction with other known clinical 162 proteins were identified as candidate biomarkers and reflect the risk factors, would allow for clinicians to make a more informed diag- host immune response via acute phase response signaling, innate im- nosis and treatment recommendation when screening and for mune response, and other proinflammatory stimuli. Several of these recommending subsequent interventions for early stage breast markers have been linked to patient outcomes and poor prognosis. cancer. Reference References 1. Kubach, J., et. al.; Blood 2007 110:1550-1558 1. Kumar V, Patel S, Tcyganov E, Gabrilovich D. The nature of myeloid- derived suppressor cells in the tumor microenvironment. Trends Immu- P13 nol. 2016; 37:208-220. Immunomodulatory effects of Interleukin 2 in the circulation of 2. Marvel D, Gabrilovich D. Myeloid-derived suppressor cells in the tumor melanoma patients and the added impact of VEGF inhibition with microenvironment: expect the unexpected. J Clin Invest. 2015; 125:3356- Ziv-aflibercept 1 2 3 Arjun Khunger, MD , Ghanashyam Sarikonda , Paul Frankel, PhD , Jenn 3. Elliott L, Doherty G, Sheahan K, Ryan E. Human tumor-infiltrating myeloid 2 2 2 2 Tsau, PhD , Zeni Alfonso, PhD , Jane Gao, MS , Anil Pahuja, BSc , cells: phenotypic and functional diversity. Front Immunol. 2017; 8:86. 2 2 2 Christine Vaupel, PhD , Naveen Dakappagari , Shabnam Tangri, PhD , 4. Okla K, Wertel I, Wawruszak A, Bobinski M, Kotarski J. Blood-based ana- Ahmad Tarhini, MD, PhD lyses of cancer: circulating myeloid-derived suppressor cells – is a new 1 2 Memorial Hospital West, Pembroke Pines, FL, United States; Navigate era coming? Crit Rev Clin Lab Sci. 2018. BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA, United Ethics Approval 3 4 States; City of Hope, Duarte, CA, United States; Emory University and The study was approved by the Virtua Oncology (#20161), University of Winship Comprehensive Cancer center, Atlanta, GA, United States Pennsylvania (#826544), and Cooper Health (#17-174) IRBs. Correspondence: Ahmad Tarhini (tarhiniaa@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P13 P12 Deep characterization of the depleted plasma proteome in Background subjects with NSCLC using data independent acquisition mass Interleukin 2 (IL-2) plays a key role in antitumor immunity by en- spectrometry reveal host immune response mechanisms hancing survival of antitumor cytotoxic T lymphocytes and nat- Nicholas Dupuis, PhD, Linda Sensbach, Sebastian Müller, Lukas Reiter ural killer (NK) cells and promoting proinflammatory cytokines, Biognosys AG, Schlieren, Switzerland that can lead to durable responses in patients with melanoma. Correspondence: Nicholas Dupuis (nicholas.dupuis@biognosys.com) High levels of vascular endothelial growth factor (VEGF) are asso- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P12 ciated with non-response to IL-2 and combination biotherapy with Ziv-aflibercept (inhibitor of the VEGF pathway) and high- Background dose IL-2 may lead to improved antitumor efficacy. Mechanistic Measurement of circulating biomarkers in cancer has proven utility studies utilizing peripheral blood of melanoma patients treated for early detection, differential diagnosis, and predicting pre- with this biotherapy may illuminate the underlying mechanisms treatment response to therapy. More recently, circulating proteomic of immune susceptibility and resistance [1]. biomarkers for pre-treatment prediction of therapeutic response Methods have received additional attention due to the heterogeneous re- Patients with stage III or stage IV inoperable melanoma were sponses to immunotherapies. To develop a greater understanding of treated with high-dose IL-2 alone or in combination with Ziv- the circulating plasma proteome in subjects with cancer we have op- aflibercept in a phase 2 clinical trial [1] (NCI8628; Tarhini et al. timized a depleted plasma proteomic workflow, based on label-free Cancer. 2018). Peripheral blood mononuclear cells (PBMC) from data independent acquisition (DIA) mass spectrometry, and applied it treated patients (N=89) on this trial were tested at baseline (be- to plasma from subjects with late stage NSCLC. This approach pro- fore initiating systemic immunotherapy), and 6-weeks (following vides a deep and unbiased description of the plasma proteome and immunotherapy initiation). High complexity (14-color) flow cytom- the dysregulated biological pathways associated with lung cancer. etry designed to detect key immunological biomarkers such as Methods myeloid-derived suppressor cells (MDSCs), regulatory T cells Plasma samples from subjects with Stage III-IV non-small cell lung (Tregs), proliferating T-cells, PD-1 and TIM3 expression on T-cells, cancer (NSCLC, n = 15) and age matched healthy donors (n = 15) and differentiation of T-cells into Th1, Th2 or Th17 phenotype were depleted of 14 high abundance proteins using MARS Hu-14 were used to evaluate the correlation between immunological spin columns (Agilent). All samples were prepared for mass spectro- biomarker expression and efficacy. Statistical significance was de- metric acquisition using two-hour gradients on a C18 column termined using ANOVA or paired student’st-test. coupled online to a Thermo Scientific Q Exactive HF-X operated in Results DIA mode. Targeted data extraction was performed using Spectro- Treatment with high dose IL-2 resulted in significant immune activa- naut (Biognosys) with a hybrid library approach. Statistical analysis tion as detected by significant increases in both proliferating CD4+ was conducted to identify disease associated biomarker candidates (p<0.0001) and CD8+ (p<0.0001) T-cells at 6-weeks post-treatment in and pathway analysis highlights dysregulated biological functions. both treatment arms in addition to increase in Tregs (CD4+ CD25+ Results Foxp3+ T-cells; p<0.0001). Addition of VEGF inhibition showed a gen- A comprehensive protein spectral library was created containing 1,827 eral trend towards decrease in classical monocytes (CD14+ CD16-; p= unique proteins. In DIA acquisition, in total 1,304 proteins were quantified 0.0769) as well as Th17 cells (defined as CD45RA- CCR6+ CXCR3- across all samples (1,105 average per sample). Univariate statistical testing CCR4+; p=0.0597). In patients receiving combination therapy, a identified 162 dysregulated proteins (125 up-regulated and 37 down- higher proportion of subjects experienced CBR (Clinically Beneficial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 8 of 272 Response = CR+PR+SD) compared to monotherapy and this CBR cor- P15 related with a decrease in CD4+ ICOS+ (p=0.0219), classical mono- Microsatellite instability detection with cell-free DNA next- cytes (CD14+ CD16-; p=0.0141), Th17 cells (CD45RA- CCR6+ CXCR3- generation sequencing CCR4+; p=0.0445) as well activated CD4+ T-cells (CD4+ CD38+ HLA- Ariane Lozac’hmeur, MS, Jason Perera, PhD, Denise Lau, PhD, Aly Khan, DR+; p=0.0285). PhD, Ariane Lozac’hmeur, MS Conclusions Tempus Labs, Chicago, IL, United States VEGF inhibition with Ziv-aflibercept adds significant immunomod- Correspondence: Ariane Lozac’hmeur ulatory effects when combined with IL-2. Further correlative ana- (ariane.lozachmeur@tempus.com) lyses determining the effect of combination therapy on Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P15 progression-free survival and identifying predictive biomarkers of therapeutic efficacy are ongoing and will be presented at the Background meeting. Microsatellite instability is a clinically actionable genomic indication for cancer immunotherapy. In microsatellite instability-high (MSI-H) tumors, Acknowledgements defects in DNA mismatch repair (MMR) can cause a hypermutated This United States (U.S.) National Cancer Institute (NCI)-sponsored study was phenotype where alterations accumulate in the repetitive microsatellite initiated by the California Cancer Consortium under N01 contract NO1-CM- regions of DNA. MSI detection is typically performed by subjecting 2011-00038. Laboratory correlatives were supported by Navigate BioPharma. tumor tissue (“solid biopsy”) to clinical next-generation sequencing or Trial Registration specific assays, such as MMR IHC or MSI PCR. Circulating cell-free tumor https://clinicaltrials.gov/ct2/show/NCT01258855 DNA (cfDNA) testing (“liquid biopsy”) is rapidly emerging as a less inva- sive method for cancer detection and monitoring disease progression. Reference Here, we explore the possibility of detecting MSI in cfDNA and develop 1. Tarhini AA, Frankel P, Ruel C, Ernstoff MS, Kuzel TM, Logan TF, et al. NCI a novel cfDNA MSI detection assay with high specificity. 8628: A randomized phase 2 study of ziv‐aflibercept and high‐dose Methods interleukin 2 or high‐dose interleukin 2 alone for inoperable stage III or The Tempus cfDNA targeted panel contains 39 highly informative IV melanoma. Cancer. 2018;124(22):4332-41. microsatellite loci previously used by the clinically validated Tempus Ethics Approval xT 595-gene panel. For each microsatellite locus, we identified all se- The study was initiated after approval by the ethics committee at the quencing reads that mapped to the corresponding microsatellite re- participating sites and was conducted in accordance with the gion and quantified the number of repeat units contained within the Declaration of Helsinki. sequencing read. Next, three distinct summary statistics were calcu- lated to characterize the distribution of the number of repeat units for each locus. Finally, using 54 labeled patient samples (17 MSI-H, P14 37 microsatellite stable) sequenced with the Tempus cfDNA panel, a Validation of dendritic cell and natural killer cell signatures for k-Nearest Neighbor (k-NN) classifier was trained to classify each locus clinical biomarker development for a new sample. Patient samples with more than 50% unstable loci Bolan Linghu, PHD, Pei Zhang, PhD, Marylens Hernandez, Mingchao Xie, were classified as MSI-H. PhD, Christine Barbon, Srimathi Srinivasan, Deanna Russell, MS, Anna Results Coenen-Stass, Deanna Mele, PhD, Patricia McCoon, PhD, Jonathan Dry, We validated the ability of our model to detect MSI on a new inde- Ben Sidders, Kris Sachsenmeier, PhD pendent validation dataset. MSI-H status was detected in 6 patient AstraZeneca, Waltham, MA, United States samples. In 3 of these patients (2 colorectal, 1 skin cancer), abnormal Correspondence: Ben Sidders (benjamin.sidders@astrazeneca.com); Kris MMR IHC confirmed the detected MSI-H status. In the other 3 pa- Sachsenmeier (kris.sachsenmeier@astrazeneca.com) tients (1 colorectal, 1 non-small cell lung cancer, and 1 endometrial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P14 cancer), MSI-H status was confirmed by our clinically validated solid tumor MSI assay. Furthermore, the reliability of the model was vali- Background dated in 10 technical replicates from 2 MSI-H patients in our training Quantification of immune cell abundance using gene signatures from dataset. The results were 100% concordant with all 10 replicates clas- mRNA profiling has the potential to inform clinical studies of cancer sified as MSI-H. immunotherapy. However, few of the signatures reported in previous Conclusions studies have been validated therefore the concordance of signature These results demonstrate the ability of our assay to detect MSI in scores with corresponding immune cell abundance is unknown. cfDNA with high specificity, providing a transformative opportunity Methods to report a clinically actionable insight alongside other somatic To tackle this challenge we designed a two-stage validation strategy. changes detected from cfDNA. Firstly we validate signatures computationally using previously pub- lished datasets. Secondly we generate expression profiling data from an immune cell spike-in experiment with human PBMCs. As a proof P16 of concept experiment, we implemented the method to validate two Circulating immunological biomarkers for predicting response to gene signatures for CD141+ dendritic cells (DC) and CD56+ natural neoadjuvant chemotherapy in TNBC patients killer (NK) cells. Charlotte Milton, PhD, Thanussuyah Alaguthurai, Atousa khiabany, Mres, Results Sheeba Irshad, MD PhD We demonstrate gene signatures for both CD56+ NK and CD141+ Kings College London, London, United Kingdom DC cell types show high and significant agreement to the corre- Correspondence: Sheeba Irshad (sheeba.irshad@kcl.ac.uk) sponding immune cell abundance. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P16 Conclusions This work establishes a starting point for validating gene signatures Background through an approach that is tractable yet recapitulates real-world Triple negative breast cancer (TNBC) accounts for 10-20% of breast variability we might expect in clinical use. cancer and is associated with particularly poor prognosis. Patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 9 of 272 are commonly treated with neoadjuvant chemotherapy (NAC) and decisions. In this work, we perform whole plasma and plasma- response to treatment is a strong predictor of overall survival. Re- derived exosome proteomic profiling to construct a predictive model cently, the ability of chemotherapeutics to stimulate an anti-tumour of immunotherapy response and toxicity, and to glean further bio- immune response has been appreciated as an important mechanism logic insight into the mechanisms underlying resistance to ICB. of action; possibly contributing to the elimination of distant micro- Methods metastatic disease by resetting of the attenuated functional immun- Whole plasma was analyzed in a cohort of 55metastatic melanoma ity. In TNBCs, higher levels of tumour-infiltrating lymphocytes correl- patients receiving anti-PD1 antibodies (MGH IRB #11-181) at baseline, ate with response to NAC and high intra-tumoral levels of immune- and on-treatment at 6 week and 6 month time-points. Exosomes related genes, including those associated with type I interferon re- were analyzed in 15 of these patients for all time-points. Proteomic sponses, and the presence of CD8+ cytotoxic T lymphocytes, correl- analysis was performed using an innovative multiplex proximity ex- ate with improved disease outcome. tension assay that enabled detection of more than 1000 proteins Methods simultaneously. A linear mixed model with maximum likelihood esti- The underlying hypothesis of this study is that phenotypic profiling mation for model parameters was used to analyze differences be- of peripheral blood cells have the potential to inform clinical deci- tween patient groups, and significant differences were determined sions and help predict therapeutic response, with lower costs and after Benjamini and Hochberg multiple hypothesis correction. higher compliance than serial tumour biopsies, due to their minimal Results invasiveness. Whilst significant research efforts have been made to Between plasma baseline and on-treatment time-points, 67 differen- assess circulating markers such as circulating tumour cells and circu- tially expressed proteins were identified including markers of inflam- lating tumour DNA as potential biomarkers; understanding the evolv- mation such as PD1, CXCL9, CXCL10, CXCL11, IL10, CCL3 and TNFR2. ing peripheral “immunological status” of TNBC patients on NAC is Exosome samples had a distinct protein signature over the treatment warranted. period compared to plasma, including differential expression of We therefore set out to analyse serial blood samples from TNBC pa- CXCL16, CCL18, CCL20, and IL6, among others. 41 proteins were dif- tients receiving NAC to monitor the changes in the peripheral im- ferentially expressed in plasma between ICB responders and non- mune response through deep analysis of functional and phenotypic responders including several inflammatory proteins such as CD28, immune markers. We investigated (1) whether chemotherapy affects TNFb, MCSFRa and IL8, and others implicated in melanoma resist- the immune phenotype; and (2) whether a defined peripheral blood ance, such as MIA and ERBB2. Similarly, exosome revealed a distinct immune phenotypic profile relates to treatment response. protein signature between responders and non-responders com- Results pared to plasma consisting of CXCL9, CXCL13, CXCL16, CCL19, CD8a, Here we present preliminary results from 10 TNBC patients receiving GZMA and CD5 expression. Whereas plasma proteins reflected a NAC. Analysis of 39 PBMC populations using mass cytometry by myeloid signature, exosome proteins reflected a lymphoid signature, time-of-flight (CyTOF), highlighted phenotypic changes in B cell pop- suggesting that the two compartments may capture elements of dif- ulations in response to treatment, in particular a dramatic increase in ferent immune processes. Integrating data from both plasma and circulating regulatory B cells (CD19+CD24+CD38+) post- exosome proteomics, we applied machine learning tools to build a chemotherapy (5.4% and 46.2% of B cells pre- and post- predictor of response. Further analysis to look for predictors of tox- chemotherapy, respectively, p=0.0004). We also detected an increase icity is currently underway. in expression of exhaustion markers (CD38+CD39+) on CD8+ T cells Conclusions which was associated with poor response to chemotherapy (0.8 and Overall, our work suggests that plasma and exosome protein signa- 2.7 fold increase from baseline in exhausted CD8+ T cells in patients tures are distinct and may reflect unique immunological processes. with pathological complete response and residual disease, respect- Proteomic analysis of these compartments may be an effective way ively, p=0.008). for non-invasive liquid biopsy to predict ICB response. Conclusions We now plan to integrate these data with Luminex profiling of 36 P18 serum cytokines, mass spectrometry analysis of circulating exosomes Liquid biopsy protein biomarkers to predict responses and and clinicopathological and standard of care blood monitoring. elucidate resistance to cancer immunotherapy Taken together, this study aims to provide a comprehensive analysis 1 2 3 Arnav Mehta, MD PhD , Marijana Rucevic, PhD , Gyulnara Kasumova , of the utility of immune monitoring to understand TNBC patient re- 2 2 3 Emmett Sprecher , Lina Hultin Rosenberg , Dennie Frederick , Ryan sponse to NAC. 3 3 3 3 Sullivan, MD , Nir Hacohen , Keith Flaherty , Genevieve Boland Ethics Approval 1 2 MGH and Broad Institute, Boston, MA, United States; Olink Proteomics, The study was approved by NRES Committee London - Chelsea, ap- Watertown, MA, United States; MGH, Hanover, MA, United States proval number 13/LO/1248. Correspondence: Marijana Rucevic (m.rucevic@olink.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P18 P17 Role of plasma-derived exosome in monitoring immunotherapy Background response and toxicity The response of metastatic melanoma to anti-PD1 is heterogeneous. 1 2 3 Arnav Mehta, MD PhD , Gyulnara Kasumova , Alvin Shi , Lina Hultin We performed proteomic profiling of patient plasma samples to 4 4 2 2 Rosenberg , Emmett Sprecher , Dennie Frederick , Ryan Sullivan, MD , build a predictor of immunotherapy response and uncover biological 2 1 2 Keith Flaherty , Nir Hacohen , Genevieve Boland , Marijana Rucevic, insights underlying primary resistance. PhD Methods 1 2 MGH and Broad Institute, Boston, MA, United States; MGH, Hanover, An initial cohort comprised 55 metastatic melanoma patients re- 3 4 MA, United States; MIT, Cambridge, MA, United States; Olink ceiving anti-PD1 (Pembrolizumab or Nivolumab) at Massachu- Proteomics, Uppsala, MA, United States setts General Hospital (MGH), and 116 additional patients Correspondence: Arnav Mehta (nawi214@gmail.com) comprised a validation cohort. Plasma samples were collected Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P17 baseline and on-treatment, at 6 weeks and 6 months’ time- points, and profiled for 1000 proteins by a multiplex Proximity Background Extension Assay (PEA, by Olink Proteomics). A subset of patients Immune checkpoint blockade (ICB) has revolutionized the treatment had single-cell RNA-seq (Smart-Seq2 protocol) performed on of many solid tumors, including metastatic melanoma. Despite recent tumor tissue. Group differences and treatment effects were eval- successes, many patients fail to respond or are overcome by severe uated using linear mixed models with maximum likelihood esti- toxicities that limit further treatment. To date, there are no non- mation for model parameters, and Benjamini and Hochberg invasive predictors of response and toxicity that can guide treatment multiple hypothesis correction. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 10 of 272 Results immunohistochemical staining of Sema4D of the tumor associated At the baseline, 6 differentially expressed proteins were identified inflammatory cells (TAIs). between responders (R) and non-responders (NR) whereas im- Results mune suppression marker ST2 and IL-6 were found significantly sSema4D levels in plasma of HNSCC and the autoimmune individuals higher among NR. Kaplan-Meier survival curves stratified by the (p=0.18, independent-samples Mann-Whitney test), were not statisti- baseline differentially expressed proteins were highly predictive cally significantly different, but sSema4D levels were significantly of overall survival (OS) and progression-free survival (PFS). At 6- higher in the HNSCC and the autoimmune groups compared to weeks on-treatment time point, 80 proteins were found differen- healthy donors (p<0.001 for both comparisons). Three histological tially expressed between R and NR including several proteins im- patterns of tumor inflammation were defined according to the extent plicated in primary or acquired resistance (IL8, MIA, TNFR1 of stromal inflammation and TAIs infiltrate into the tumor islands. among others). Several 6-weeks differentially expressed proteins First; the inflamed type (TAIs infiltrated the tumor cells), second the were highly predictive of survival (ICOSL, IL8, MIA). Furthermore, TAIs excluded type (inflamed stroma but TAIs did not infiltrate the 160 significantly differentially expressed (DE) proteins were identi- tumor islands and/or were excluded by a thin peri-tumoral fibromyx- fied across the treatment period majority of which are reflective oid zone) and third as deserted ( minimal to no TAIs in the peri- of immune activation under the pressure of the immunotherapy. tumroal stroma or the tumor islands). The paired tumor tissue and Analysis of single-cell RNA-seq data of tumor tissue from a subset blood samples collected at the same time point, showed that high of these patients revealed that gene expression of most proteins levels of sSema4D in plasma, correlated directly with TAIs excluded predictive of response were enriched among tumor myeloid cells, histological pattern of tumor inflammation (p= 0.04). with the remainder of proteins being reflective of exhausted T Conclusions cell states. Our data presents a novel role of Sema4D as a soluble immune bio- Conclusions marker that can read in real time the histological pattern of tumor in- These results unveil a putative role of myeloid cells within the flammation. This opens new avenues for personalized immunotherapy tumor microenvironment in anti-PD1 response or primary resist- and HNSCC patient stratification. ance. Whole plasma proteomic profiling of anti-PD1 treated pa- tients revealed DE proteins between R and NR that may enable a References liquid biopsy to predict anti-PD1 response. Importantly, we dem- 1. Derakhshandeh R, Sanadhya S, Lee Han K, Chen H, Goloubeva O, Webb onstrate the relationship of serum biomarkers to OS and PFS and TJ, Younis RH. Semaphorin 4D in human head and neck cancer tissue are currently attempting to build machine learning classifiers as and peripheral blood: A dense fibrotic peri-tumoral stromal phenotype. predictors of response to checkpoint therapy leveraging early and Oncotarget. 2018; 9:11126-11144. late on-treatment time points. 2. Younis RH, Han KL, Webb TJ. Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid- Derived Suppressor Cells. J Immunol. 2016; 196:1419-29. P20 3. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith E, Zauderer M, Evans Semaphorin 4D in peripheral blood of head and neck squamous EE, Allen CT. Semaphorin4D Inhibition Improves Response to Immune- cell carcinoma reads the histological pattern of tumor Checkpoint Blockade via Attenuation of MDSC Recruitment and Function. inflammation in real time Cancer Immunol Res. 2019; 7:282-291. Ioana Ghita, Manar Elnaggar, Risa Chaisuparat, John Papadimitriou, 4. Ayers M, et al. IFN-gamma-related mRNA profile predicts clinical response Joshua Lubek, Rania Younis, BDS, MDS, PhD, Soren Bentzen, PhD to PD-1 blockade. J Clin Invest. 2017; 127:2930–40 University of Maryland, Baltimore, MD, United States Ethics Approval Correspondence: Rania Younis (ryounis@umaryland.edu) The study was approved by University of Maryland institutional review board, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P20 Institutution‘s Ethics Board, approval number (HCR-HP-00073603) Background There is an urgent need for immune biomarkers that can monitor P21 the status of inflammation of cancer patients. Soluble biomarkers Activation Profiling of tumor infiltrating CD8+ T cells reveals CTLA- represent a convenient prognostic and diagnostic method. Sema- 4 mean fluorescence intensity correlates with response in phorin 4D (Sema4D) is a glycoprotein that can function as a trans- treatment naïve melanoma membrane protein or a cleaved soluble form (sSema4D), that we Lauren Levine, MD, Katy Tsai, MD, James Lee, MD, Clinton Wu, BS, Kelly previously detected in peripheral blood [1]. The role of Sema4D as Mahuron, MD, Alain Algazi, MD, Michael Rosenblum, MD PhD, Adil an inflammatory mediator in several pathological aspects and its role Daud, MD in tumor immune suppression [2,3], highlights its significance as a University of California, San Francisco, San Francisco, CA, United States molecule to be further investigated for translational potential. The Correspondence: Adil Daud (daudai@gmail.com) objective of this work was to investigate the level of sSema4D in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P21 plasma in relation to the histological pattern of tumor inflammation of head and neck squamous cell carcinoma (HNSCC) patients in real Background time. Background: Activation markers such as PD-1 and PDL-1 as well as Methods tumor mutation burden and IFN-gamma gene expression profiling Under University of Maryland institutional review board approval and have been explored as markers for response in melanoma and in upon patient consent, we obtained paired peripheral blood and other cancers. PD-1 inhibition activates checkpoint positive cytotoxic tumor tissue of thirty-nine HNSCC patients, collected at the same T lymphocytes (cpCTLs) inducing tumor regression. We have previ- time point to allow for real time correlative analysis. Thirty eight pa- ously demonstrated that baseline peCTL frequency predicts response tients of classic autoimmune conditions, thirteen allergy patients, to anti–PD-1 monotherapy and combination CTLA4/PD-1 blockade in seven osteoarthritis patients and thirty-one healthy donors were in- metastatic melanoma. We evaluated the frequency of this CD8+ T cell cluded as controls. The level of Sema4D in plasma was detected subset at baseline and after immunotherapy treatment and evaluated using tailored direct ELISA assay. The histological pattern of tumor in- the utility of the intensity of expression activation marker expression flammation [4] was analyzed by three pathologists using the as a surrogate for tumor response as assessed by flow cytometry. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 11 of 272 Methods proportional hazard modeling to associate gene expression and We identified 490 patients with melanoma biopsied pre and post signature scores with the clinical annotations. PD-1 therapy and available for analysis. Of these 148 patients Results had unresectable stage III or stage IV melanoma and were treat- In the PanCancer IO 360 analysis, genes and signatures are compared ment naïve and started PD-1 therapy following biopsy. An add- to clinical annotations through heat maps, volcano plots, forest plots, itional 61 patients were identified with PD-1 resistant melanoma. box plots, waterfall plots, swim lane plots, Kaplan Myer plots, scatter Approximately 2 × 106 cells were stained with anti-hCD3, anti- plots, and the IO 360 wheel plot. The report is delivered in an HTML hCD8, anti-hCD45, anti-CD4 , anti-Foxp3, anti–hCTLA-4 (14D3), format that provides interactive visualizations, quality control, and anti–PD-1 , anti–HLA-DR, anti–PD-L1, and LIVE/DEAD Fix- able downloadable results. Data are analyzed individually and as part of Aqua Dead Cell Stain (Life Technologies). Data were acquired by larger treatment groups. an LSRFortessa (BD Biosciences) and analyzed using FlowJo soft- Conclusions ware (Tree Star, Inc.). Objective Responses were evaluated by The PanCancer IO 360 assay is a tool for characterizing transcriptional RECIST 1.1, CR/PR were classified as “responders” and SD/PD as patterns associated with tumor-immune interactions that can be ap- “non-responders.” plied across a wide range of cancer types. Gene signatures enable ro- Results bust characterization of immune activity from small sample cohorts, : cpCTL percentage correlated with response. The mean cpCTL was and the report simplifies the interpretation of results. This combin- 27.1% for treatment naïve responders (R), 16.52% for treatment naïve ation enables researchers to have insight into clinically relevant biol- non-responders (NR) and 8.59% for PD-1 resistant patients post treat- ogy that will ultimately lead to help drive the immune-oncology ment (ANOVA p=0.0003 for R/NR, 801 (ANOVA p=0.0002). field. Conclusions PD-1 progressive patients are significantly depleted in cpCTL even References compared to treatment naïve non-responders, suggesting that add- 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-γ-related mRNA profile pre- itional T cell influx may be needed for effective checkpoint blockade dicts clinical response to PD-1 blockade. J Clin Invest. 2017;127(8):2930- in these patients. In treatment naïve melanoma, CD8+ activation as shown by CTLA-4 MFI has an optimal range along the activation- 2. Danaher P, Warren S, Dennis L, et al. Gene expression markers of Tumor dysfunction spectrum, and strongly correlates with response to PD-1 Infiltrating Leukocytes. J Immunother Cancer. 2017;5:18. checkpoint therapy. 3. Danaher P, Warren S, Lu R, et al. Pan-cancer adaptive immune resist- ance as defined by the Tumor Inflammation Signature (TIS): results Acknowledgements from The Cancer Genome Atlas (TCGA). J Immunother Cancer. We gratefully acknowledge the patients who participated in this study 2018;6(1):63. Ethics Approval The study was approved by UCSF's Ethics Board approval number 138510 P23 High dimensional immune monitoring of peripheral blood samples P22 from breast cancer patients using mass cytometry (CyTOF) 1 1 2 Transcriptomic characterization of immune response within Jose Villasboas, MD , Kaitlyn McGrath, MS , El-ad David Amir, PhD , 1 1 1 diverse tumor environments using the NanoString® nCounter® Roberto Leon-Ferre, MD , Matthew Goetz, MD , Judy Boughey, MD , 1 1 1 PanCancer IO 360™ assay Jody Carter, MD, PhD , Krishna Kalari, PhD , Liewei Wang, MD, PhD , 1 1 Jessica Perez, PhD, Lei Yang, David Henderson, PhD, Heather Brauer, Vera Suman, PhD , Richard Weinshilboum, MD , Stephen Ansell, MD, PhD, Sarah Warren, PhD PhD 1 2 NanoString, Seattle, WA, United States Mayo Clinic, Rochester, MN, United States; Astrolabe Diagnostics, Fort Correspondence: Sarah Warren (swarren@nanostring.com) Lee, NJ, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P22 Correspondence: Jose Villasboas (Villasboas@mayo.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P23 Background The efficacy of immune response in solid tumor settings is driven Background by many factors including the biology of the tumor, the immune CyTOF produces high dimensional single cell data allowing simultan- system, and the microenvironment. The Tumor Inflammation Sig- eous monitoring of multiple immune cell subsets. This enables nature (TIS) is an 18-gene Research Use Only (RUO) signature that characterization of the immune system in normal and disease states. measures the presence of a preexisting immune response on the We developed a standardized pipeline to study human peripheral nCounter platform and enriches for response to pembrolizumab blood mononuclear cells (PBMCs) of cancer patients. Here we detail [1]. We have incorporated TIS into the PanCancer IO 360 panel, a our process and present early findings on a cohort of 40 patients 770-gene RUO expression assay containing 48 additional signa- with early-stage triple-negative breast cancer (TNBC) treated with tures of tumor-immune biology. To accompany this panel, we neoadjuvant chemotherapy. have created analysis software that associates the gene expres- Methods sion and signature scores with annotations of the samples to Thirty commercially-available metal-tagged antibodies were opti- characterize the immune system, tumor, and stroma within the mized to identify major cell subsets using a 4-point titration scheme. tumor microenvironment to give insight into underlying biology Replicates of cryopreserved PBMCs from a pool of 4 healthy donors of response to treatment, disease progression, survival, and other were created for panel titration and used as longitudinal references. sample characteristics. We studied 40 cryopreserved PBMCs from patients with TNBC. We Methods stained samples individually using standard protocol, barcoded over- The PanCancer IO 360 assay relies on gene signatures to describe night during DNA intercalation, and pooled for acquisition. Debar- biological processes, measure the presence of 14 different im- coded output data was normalized on a per-batch basis to the mune cell populations, or report the expression of key thera- median intensity of EQbeads. We uploaded files to an automated peutic targets. Data from The Cancer Genome Atlas (TCGA) was platform for unbiased processing. Patient-level meta-data was added used for signature training and development. Signatures are ei- to experiment matrix to determine differential abundance of immune ther single genes, weighted linear sums of multiple genes with subsets across clinical and pathological groups. coregulated expression, or algorithms to determine under- Results expression of genes in a coregulated pathway [2,3]. The analysis We required 7 rounds of titration to optimize antibody concentra- software leverages differential expression analysis and Cox tions. Data was collected on over 23 million live single-cell events Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 12 of 272 (Table 1) assigned to 31 canonical populations (Figure 1A). The me- Table 1 (abstract P23). See text for description dian frequencies of main populations were: B cells (11.9%), T-CD4+ cells (34.3%), T-CD8+ cells (11.7%), NK cells (8.6%), and monocytes (11.3%). At the profiling level, 76 subsets were agnostically identified, with B, T, NK, and monocytes broken down into 10, 32, 8, and 13 subsets respectively. Activated (CD38+CD161+) CD16+NK cells (Fig- ure 1B) were more prevalent in TNBC samples (median 5.2%, range 0.5%-11.9%) compared to normal blood (median 0.76%, range 0.1%- 2.4%). A population with phenotype suggestive of myeloid derived suppressor cells (LineagenegHLA-DRLowCD66b+CD24+CD16+; Figure 1C) was also more prevalent in TNBC samples (median 1.2%, range P24 0.1%-17.3%) compared to normal blood (median 0.6%, range 0.2%- Molecularly guided digital spatial profiling for highly multiplexed 1.0%). These populations demonstrated opposite association trends analysis of gene expression with spatial and single cell resolution when patients were stratified by clinical outcomes. Activated NK cells 1 2 2 Anushka Dikshit, PhD , Chris Merritt, PhD , Jamie Rose Kuhar , Karen were more frequent in patients achieving pathological complete re- 2 2 1 1 Nyugen , Kristina Sorg , Bingqing Zhang , Courtney Anderson, PhD , sponse while MDSC-like cells were more frequent in those with re- Xiao-Jun Ma sidual disease (Figures 1D-1E). 1 2 Advanced Cell Diagnostics, Newark, CA, United States; NanoString Conclusions Technologies, Seattle, WA, United States We demonstrated the feasibility of a complete pipeline for deep phe- Correspondence: Xiao-Jun Ma (xiao-jun.ma@bio-techne.com) notyping of cryopreserved PBMCs in cancer patients. Our approach Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P24 identified rare cell subsets using an unbiased analysis tool, linking specific populations to opposite clinical outcomes. High dimensional Background immune monitoring is feasible and should be applied to study the The tumor microenvironment (TME) is a network of complex inter- immune system of cancer patients at large. actions between the tumor and surrounding immune cells. Im- munotherapies including immune checkpoint blockade have Acknowledgements demonstrated therapeutic efficacy and durable responses for sev- This work was part of the Mayo Clinic Cancer Immunome Project which is eral tumor types, however most patients are nonresponsive or de- supported by the Wohlers Family Foundation. Samples were obtained in velop resistance to such immunotherapies. To identify new collaboration with investigators from the Mayo Clinic Breast Cancer Genome predictive biomarkers to better stratify patients, it is essential to Guided Therapy (BEAUTY) study. The BEAUTY study is funded in part by the comprehensively characterize the immune cells within the TME at Mayo Clinic Center for Individualized Medicine; Nadia’s Gift Foundation; John the molecular level. Traditional methods to assess gene expression P. Guider; the Eveleigh Family; George M. Eisenberg Foundation for Charities; in tissues lack either spatial information or sensitivity/specificity. To generous support from Afaf Al-Bahar; and the Pharmacogenomics Research address this, we have developed a novel workflow combining the Network (PGRN). Other contributing groups include the Mayo Clinic Cancer single molecule and single cell visualization capabilities of the RNA- Center and the Mayo Clinic Breast Specialized Program of Research scope in situ hybridization (ISH) assay with the highly multiplexed Excellence (SPORE). spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler Ethics Approval (DSP) RNA assays (Research Use Only). The study was reviewed approved by the Mayo Clinic Institutional review Methods board (IRB). The fully automated RNAscope Multiplex Fluorescent assay was used to visually identify CD3E (T-cell)-enriched regions and CD19 and CD20 (B-cell)-enriched regions within FFPE human lung cancer tis- sues. Using the GeoMx DSP, 10 CD3E-enriched regions of interest (ROI) and 10 CD19-enriched ROI were spatially profiled for 78 genes related to immune-oncology research. The RNAscope Multiplex Fluor- escence assay was used again to visually confirm the differentially expressed genes between the T and B-cell-enriched regions with sin- gle cell resolution. Results To show a workflow combining RNAscope molecularly guided visualization and GeoMx DSP profiling is feasible, we confirmed that both assay protocols are compatible. We then examined con- cordance between GeoMx DSP and RNAscope ISH data, demon- strating that RNAscope and GeoMx DSP data can be obtained on the same section. To test the full automated workflow, we com- pared the differentially expressed genes within the T cell and B cell-enriched ROI. The RNAscope assay confirmed that, while the expression of the immunoregulatory molecules CTLA4, PD-L1, PD- 1, and ICOSLG were detected in both ROI, the CD3E (T-cell)- enriched ROI demonstrated significantly higher expression of these checkpoint markers. Compared to the CD19-enriched ROI, the CD3-enriched ROI also showed increased inflammatory signa- ture, demonstrated by elevated levels of cytokines and chemo- kines such as CCL5, CXCL9 and IFNG. Conclusions We present a robust workflow that overcomes the historical limita- tions of ISH and IHC by combining high resolution imaging with high plex profiling. With this workflow, the RNAscope ISH technology can molecularly guide the GeoMx DSP to precisely profile ROI while retaining the morphological context of heterogenous tumors. Fur- Fig. 1 (abstract P23). High dimensional immune monitoring of thermore, RNAscope assays can be used to confirm GeoMx DSP- breast cancer PBMCs identified gene expression signatures at single cell resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 13 of 272 P25 4. Borbulevych OY, Santhanagopolan SM, Hossain M, Baker BM. TCRs used A conserved MART-1 T cell receptor motif is predictive of in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens responses to checkpoint blockade via distinct mechanisms. J Immunol. 2011;187(5):2453-2463. 1 2 2 Ariel Isser, BS , Tatsuya Yoshida , Junya Ichikawa , Jeffrey Weber, MD, Ethics Approval 2 3 PhD , Jonathan Schneck, MD, PhD The protocol was approved by the NYU Institutional Review Board, i16-01975 1 2 Johns Hopkins University, Baltimore, MD, United States; New York School of Medicine, New York, NY, United States; Johns Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Jeffrey Weber (jeffrey.weber@nyulangone.org); Jonathan Schneck (jschnec1@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P25 Background Since the introduction of checkpoint blockade inhibitors for cancer immunotherapy, numerous studies have sought to identify bio- markers predictive of patient response [1]. However, the relevance of antigen-driven responses to the tumor has yet to be investigated. To address this question, we examined T cell responses to MART-1, an antigen overexpressed in melanoma cells and a target for melanoma clinical trials that have had variable degrees of success. We hypothe- sized that features of patients’ MART-1 CD8+ T cell repertoires could predict their response to checkpoint blockade. Methods To understand the MART-1 T cell repertoire, MART-1 CD8+ T cells were expanded from HLA-A2+ melanoma patients and healthy do- nors using artificial antigen presenting cells (aAPC) or peptide-pulsed dendritic cells. Tetramer positive cells were sorted after 14-22 days and CDR3β sequenced. Motif analysis based on sequence homology was performed using the Immunomap algorithm by clustering 11,252 unique MART-1 CDR3β sequences from 33 samples and 20 donors, including five nivolumab responders and five non- responders [2]. Fig. 1 (abstract P25). See text for description Results No significant difference in the frequency of MART-1 expanded T cells was seen between healthy donors and melanoma patients with or P26 without checkpoint therapy. There was no immunodominant Vβ gene Murine T cell phenotype and function in a single-well format: a usage and limited clonotype overlap between donors. However, se- novel, multiplexed and high-throughput assay workflow using the quence homology showed extensive overlap between donors, driven iQue platform by two clusters present in 60% and 80% of samples at average frequen- Veronica Bruce, PhD, Caroline Weldon, John O'Rourke, Veronica Bruce, cies of 10% and 14%, respectively. These clusters were homologous to PhD each other as well as the DMF4 T cell receptor (TCR), one of the first Sartorius, Albuquerque, NM, United States clinically used genetically engineered T cells, with a known crystal struc- Correspondence: John O'Rourke (John.ORourke@Sartorius.com) ture [3,4]. The core region of these clusters contained a conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P26 amino acid motif that was identical to contact residues between the DMF4 TCR and MART-1 peptide bound to HLA-A2. The motif identified Background from the core region of these clusters was highly conserved across Immunotherapy is an actively growing arena in oncotherapeutics re- samples, present almost exclusively in the junctional region between search and development. In this context, whether testing CAR-T cells, the D and J genes of the CDR3β, and encoded by a diverse range of checkpoint inhibitors, or novel bispecific antibodies, the ultimate nucleotides, all evidence of selective pressure. Despite its conservation, goal is to modulate the immune system to harness its tumor killing the frequency of this motif was nearly six times lower in pre-therapy power. T cells play a critical role in immune-regulated clearance of samples expanded from non-responders compared to responders (40% both liquid and solid tumors. Upon antigenic stimulation and activa- vs. 7%, p=0.0045, Figure 1). tion, T cells rapidly expand, secrete cytokines, and differentiate to Conclusions various functional subsets (e.g. effector T cells, memory T cells). On Since the frequency of the identified MART-1 TCR motif is significantly the other hand, suppression of T cells (i.e. exhaustion) leads to im- lower in non-responders compared to responders, it could potentially mune escape and the spread of tumor cells. Mouse models remain be used as a biomarker to predict response of HLA-A2+ melanoma pa- the most commonly used animal system for in vivo and in vitro can- tients to checkpoint blockade prior to the onset of therapy. cer biology research and drug discovery. As researchers move for- ward to either better understand the role of T cells in cancer biology References or to develop novel immunotherapies, there is a need for improved 1. Zappasodi R, Wolchok JD, Merghoub T. Strategies for Predicting methods to quickly gather comprehensive data on T cell biology in Response to Checkpoint Inhibitors. Curr Hematol Malig Rep. this model. To address this, we demonstrate a multiplexed, high- 2018;13(5):383-395. throughput, robust assay workflow capable of measuring multiple 2. Sidhom J-W, Bessell CA, Havel JJ, Kosmides A, Chan TA, Schneck JP. murine T cell biology endpoints quickly and reproducibly in a single- ImmunoMap: A Bioinformatics Tool for T-Cell Repertoire Analysis. Cancer well format. Immunol Res. January 2017; 6(2):151-162. Methods 3. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of Tumor-Infiltrating In our workflow, stimulated mouse T cells were assayed in a 96-well Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with plate using fluorescent antibodies against CD3, CD4, CD8, CD69, Metastatic Melanoma. N Engl J Med. 1988;319(25):1676-1680. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 14 of 272 CD44, CD62L, and PD-1, QBeads for cytokine detection, and markers the tumor tissue upon drug treatment. The impact of different for cell viability and proliferation. Data were acquired on the iQue3 immuno-oncology drug treatments ex vivo on TME will be discussed. technology (VBR configuration) and analyzed on a plate-based level Application of this platform in the clinical studies may also allow de- using the integrated ForeCyt software. termining the most effective combinatorial therapeutic strategies for Results individual patients. Our assay workflow enabled simultaneous evaluation of viability, in- terrogation of helper and cytotoxic T cells for markers of activation P28 and exhaustion, and identification of key memory subsets. Prolifera- Mass spectroscopy-based highly multiplexed super-resolution tion and secreted cytokines (IFN-gamma and IL-2) were also quanti- imaging method for fine details of tumor microenvironment fied. Data analysis and visualization of multiple endpoints was monitoring and tumor-immune cell interactions streamlined and performed in real time using the ForeCyt software. 1 1 1 2 Yunhao Bai, BS , Bokai Zhu , Michael Angelo, MD, PhD , Yongxin Zhao , Conclusions 1 1 1 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Garry Nolan, PhD The assay was completed in four hours, including data analysis. This 1 2 Stanford University, Stanford, CA, United States; Carnegie Mellon workflow saves the end user’s time and resources by combining mul- University, Pittsburgh, PA, United States tiple experiments into a single, multiplexed workflow, and helps Correspondence: Sizun Jiang (sizunj@stanford.edu); Xavier Rovira Clave minimize subject-to-subject variability. Altogether, our workflow al- (xrovira@stanford.edu); Garry Nolan (gnolan@stanford.edu) lows for easy phenotype and functional profiling of murine T cells in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P28 a single-well format while generating actionable results in a matter of hours. Background In tumor microenvironment, tumor-immune interactions are indi- P27 cated by cell surface proteins such as T cell receptor (TCR) and PD- Functional 3D-plEX quantitative multiplex immunofluorescence L1. The key workhorse for studying these cellular interactions is via platform to assess IO drug impact on tumor microenvironment in imaging; conventional imaging methods are limited by the number ex vivo treated intact 3D-tumor organoids of fresh patient tumor of channels and the spatial resolving capabilities. tissue A new modality of imaging, Multiplexed Ion Beam Imaging (MIBI) Jenny Kreahling, PhD, Vijayendra Agrawal, PhD, Melba Page, PhD, Mibel [1,2], can resolve >40 parameters simultaneously in biological sam- Pabon, PhD, Soner Altiok, MD, PhD ples. MIBI can current attain single cell resolutions but has difficulties Nilogen Oncosystems, Tampa, FL, United States in resolving fine subcellular features. Here, we present Expansion Correspondence: Soner Altiok (soner@nilogen.com) MIBI (ExMIBI), which combines a physical expansion of a biological Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P27 sample with the MIBI imaging method. ExMIBI will be critical for the scientific community to obtain previously inaccessible insights into Background the fine details of tumor microenvironment and cancer-immune cell The tumor stroma consists of various components of the tumor interactions, and promises to unravel fundamental insights in patient microenvironment including tumor cells, fibroblasts, immune cells immunotherapy responses. and the extracellular matrix. Spatial organization and dynamic inter- Methods play of the complex cell-to-cell interactions play an important role Expansion microscopy (ExM) [3,4] is a technique that can physical ex- in cellular phenotypes that can result in permanent alterations in pansion of biological specimens 4 to 10 folds through polymer cellular functions and response to oncology as well as immuno- chemistry, three-color fluorescent imaging of cellular features with oncology drug treatments. While informative, conventional 2D an apparent lateral resolution of 70 nm in diffraction-limited confocal tumor dissociated models do not maintain the stromal- microscopes has been achieved. However, the expanded gel is fragile stoichiometry of the tumor microenvironment, lacking vital support and contains up to 99.9% water, which limits its usage in imaging mechanisms necessary to accurately assess ex-vivo tumor cell via- method that requires high vacuum condition. We explored a way to bility and immune-cell activation after drug treatment. Here, we de- collapse the tissue-containing gel on a complementary charged sub- scribe a functional quantitative multiplex immunofluorescence strate to achieve a vacuum-compatible gel that can be imaged by platform, 3D-plEX, to quantify drug-mediated changes in tumor im- the MIBIscope, with lateral resolution <100 nm. Various methods for mune microenvironment and tumor cell viability in intact 3D tumor sample charging removal are systematically tested for imaging a organoids of patient tumor samples. non-conductive gel in MIBI. Methods Results All patient tumor samples were obtained with patient consent We have established a robust method, ExMIBI, that allows ExM and relevant IRB approval. Unpropagated live 3D tumoroids hydrogels to be compatible with the high vacuum imaging condi- measuring 100-150 micron in size were prepared from fresh pa- tions of the MIBI. This method can achieve 40 parameters. A vali- tient tumors using a proprietary technology, pooled together to dated panel of MIBI compatible antibodies, focusing on the immune represent tumor heterogeneity and equally distributed to differ- system, is being tested and established for ExMIBI in FFPE tissues ent treatment groups including nivolumab, ipilimumab, atezolizu- (Figure 1). mab and urelumab singly or in different combinations. Cell Conclusions media was collected for multiplex cytokine release assay. Tumor- The combination of ExM and MIBI, termed ExMIBI, permits highly oids were fixed and embedded for multiplex immunofluorescence multiplexed super resolution imaging of tissue samples. We will now studies. In addition to tumor cell killing, treatment-mediated be able to map previously inaccessible, finer details of the tumor changes in TME was analyzed in each treatment group side-by- microenvironment. The application of ExMIBI to dissect cellular im- side using multiplex immunofluorescence markers including CD4, mune interactions, in their spatial biological context, will allow a bet- CD8, FoxP3, CD68, Pan-CK, PD-L1 and Ki67. ter understanding into the basic principles of our immune system in Results healthy and disease states. Our results demonstrated that 3D-plEX platform using clinically rele- vant intact, uniformly sized tumoroids of fresh patient tumor tissue is Acknowledgements highly versatile and reliable approach to quantify drug-mediated We thank Matt Newgren for tireless technical support on the MIBI changes in cellular composition and spatial organization of the tumor instrument. This research has received advice and help from Prof. Michael immune microenvironment. Angelo, Prof. Sean Bendall and their research group. B.Z. is supported by the Conclusions Stanford Graduate Student fellowship. S.J is supported by a Stanford Dean’s Combination of this approach with multiplex cytokine release assay Fellowship and the Leukemia & Lymphoma Society Career Development allows a comprehensive understanding of dynamic changes within Program. X.R.-C. is supported by a long-term EMBO fellowship. This work was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 15 of 272 supported by grants from the FDA, NIH, Parker Institute for Cancer separate tumor and stromal compartments, detect tumor/stroma Immunotherapy, the Bill and Melinda Gates Foundation, as well as the margins, and identify leukocytes in immunostained tissues were im- Rachford and Carlota A. Harris Endowed Professorship to G.P.N. plemented in each tissue analyzed. Resulting image markups of cell detection and biomarker expression measured by image analysis References were reviewed by an MD pathologist for acceptance. Tissues not 1. Angelo M, Bendall SC, Finck R, et al. Multiplexed ion beam imaging of meeting acceptance criteria were re-analyzed until acceptable to the human breast tumors. Nature Medicine. 2014;20(4):436–42. reviewing pathologist. 2. Keren L, Bosse M, Marquez D, et al. A Structured Tumor-Immune Micro- Results environment in Triple Negative Breast Cancer Revealed by Multiplexed We demonstrate the synergistic value of layered image analysis algo- Ion Beam Imaging. Cell. 2018;174(6):1373-87.E19. rithms which provide context to biomarker expression in NSCLC tis- 3. Chen F, Tillberg PW, Boyden ES. Expansion Microscopy, Science. sues. Samples were grouped in to immune desert, excluded, and 2015;347(6221):543-8. inflamed phenotypes based on total leukocyte and CD8 expression 4. Tillberg PW, Chen F, Piatkevich KD, et al. Protein-retention expansion mi- patterns in the tumor, stroma, and margin. PD-L1 expression was croscopy of cells and tissues labeled using standard fluorescent proteins scored based on percentages of tumor and stromal expression, as and antibodies. Nature Biotechnology. 2016;34(9):987–92. well as digital representations of common PD-L1 scoring paradigms. Additionally, samples were stratified by PD-L1 patterns of constitu- tive, induced, immune, or ignorant expression. Conclusions Digital image analysis of IHC stained tissues creates comprehensive tissue biomarker profiles that are useful in assessment of tumor and immune interactions in IO drug development and patient stratifica- tion. Complex algorithms that utilize AI and machine learning can be overseen by MD pathologists to create clinically acceptable digital analysis solutions. References 1. Rimm DL, Han G, Taube JM, et al. A Prospective, Multi-institutional, Pathologist-Based Assessment of 4 Immunohistochemistry Assays for PD- L1 Expression in Non–Small Cell Lung Cancer. JAMA Oncol. 2017;3(8):1051–1058. 2. Hendry S, Salgado R, Gevaert T, et al. Assessing Tumor-infiltrating Lym- phocytes in Solid Tumors: A Practical Review for Pathologists and Pro- Fig. 1 (abstract P28). The workflow and sample images of ExM-MIBI posal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcin- oma In Situ, Metastatic Tumor Deposits and Areas for Further Research. P29 Adv Anat Pathol. 2017;24(5):235–251. Comprehensive image analysis of immunostained NSCLC tissues 3. Taube JM, Galon J, Sholl LM, et al. Implications of the tumor immune provides necessary context for immune oncology biomarker microenvironment for staging and therapeutics. Mod Pathol. profiling 2018;31(2):214–234. Charles Caldwell, PhD, Jenifer Caldara, BS, Will Paces, BS, Kelsey Weigel, 4. Silva MA, Ryall KA, Wilm C, Caldara J, Grote HJ, et al. PD-L1 immunostain- PhD, Roberto Gianani, MD ing scoring for non-small cell lung cancer based on immunosurveillance Flagship Biosciences, Westminster, CO, United States parameters. PLOS ONE 2018; 13(6): e0196464 Correspondence: Charles Caldwell (ccaldwell@flagshipbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P29 P30 Background Deep spatial profiling of the immune landscape of MSI and MSS Manual pathology assessments of Immunohistochemistry (IHC) colorectal tumors markers in immune oncology (IO) is often challenging and results Sarah Church, PhD, Jason Reeves, Daniel Zollinger, Jill McKay-Fleisch, can be highly variable[1,2]. Measuring biomarker presence in IO must Andrew White, BSc, Michael Bailey, Arya Bahrami, PhD, Chris Merritt, take in to account both immune and tumor environments and pro- PhD, Margaret Hoang, Sarah Warren, PhD, Joseph Beechem, PhD vide contextual information on the interaction between tumor and NanoString Technologies, Everett, WA, United States immune biomarker landscapes [3]. Due to the complex nature sur- Correspondence: Sarah Church (schurch@nanostring.com) rounding tissue biomarker interpretation in IO, digital image analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P30 (IA) solutions have been developed that layer complex artificial intelligence (AI) and machine learning algorithms to obtain full tissue Background biomarker profiles necessary for drug development and patient In colorectal cancer (CRC) there have been many recent advances in stratification[4]. immune-related biomarkers that are both prognostic and predictive Here, a comprehensive tissue analysis solution is presented in mono- of response to immunotherapy. Microsatellite instability (MSI)/mis- plex PD-L1 and CD8 stained slides that includes precise digital bio- match repair deficiency (dMMR) is present in ~15-20% of CRCs and marker scoring in tumor and stromal compartments, recapitulation of corelates with increased immunogenic mutations that often augment common scoring paradigms, analysis of biomarker expression at the lymphocyte infiltration into the tumor microenvironment (TME). Add- tumor/stroma interface (margin), and quantification, scoring, and itionally, location of tumor infiltrating T-cells in two areas of the TME, spatial localization of leukocytes in the tumor and stroma. Aggrega- the tumor center (CT) and invasive margin (IM) has also been shown tion of all cellular and biomarker data generates tissue phenotypes to be prognostic and predictive of response to immunotherapy. Here that characterize the IO landscape of each tissue. we use multiplexed protein and RNA digital spatial profiling to elicit Methods the immune landscape of MSI-MSS characterized CRC tumors. Serial sections of 20 NSCLC samples were IHC stained for PD-L1 and Methods CD8 expression. Stained slides were scanned at 20x magnification Forty-eight CRC tumors were analyzed for gene expression (GX) and analyzed using Flagship Biosciences’ image analysis solutions. using the NanoString® nCounter® PanCancer IO 360™ Research Use Image analysis algorithms which quantify biomarker expression, Only (RUO) Gene Expression Panel and assessed for 48 cell typing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 16 of 272 and biological signatures, including MMR loss/MSI predictor and the Polaris® with pre-defined acquisition parameters. Scans were un- Tumor Inflammation Signature (TIS). A subset of 18 CRC tumors (6 mixed and analyzed with inForm® software using a pre-configured al- MSI-TIS-hi, 6 MSS-TIS-hi, 6 MSS-TIS-lo) was selected for analysis with gorithm tailored to the PD1/PD-L1 Lung Cancer Panel Kit. Spatial the RUO GeoMx™ Digital Spatial Profiler (DSP) using 40 antibodies, analyses and visualizations were performed using the phenoptr and 84 or 1,600+ in situ probes. Selection of regions of interest (ROIs) in phenoptrReports R-based packages and custom scripts. two locations, CT and IM were guided by staining with fluorescent Results markers (CD45, CD3, pan-CK, DNA). 300-600 μM diameter circle ROIs The pre-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer Panel were selected, and in some cases segmented by pan-CK+/pan-CK-. Kit was able to visualize the panel targets (PD-L1, PD-1, CD8, CD68, Results FoxP3, and Cytokeratin) across the variety of lung cancer samples in Using whole tissue GX, we first confirmed MSI/dMMR characterization the TMA. Cell phenotyping and spatial analyses revealed core-to-core and TIS status of 48 CRC tumors using PanCancer IO 360 signatures. variations in cell densities and proximities among different markers. We selected 18 tumors within this cohort based on TIS status to fur- Measurement of the dynamic range of PD-L1 expression across dif- ther dissect the location-dependent immune contexture of the TME, ferent cores also revealed the improved sensitivity in PD-L1 detection with a particular emphasis on differentiating MSI-TIS-hi and MSS-TIS- provided by unmixing. hi CRCs. DSP confirmed loss of dMMR markers (MSH2/MLH1) and Conclusions identified an increased amount of potentially suppressive macro- The end-to-end Phenoptics staining, imaging, unmixing, and spatial phages (CD163+PD-L1+) in MSI-TIS-hi versus MSS-TIS-hi tumors. Seg- analysis workflow described here provides a robust and sensitive mentation of ROIs based on tumor versus stroma (pan-CK+/-) platform for exploring the immune landscape within the tumor identified samples with high proportions of tumor-invading TILs. microenvironment. These samples were then further profiled using probes against 1600+ mRNA targets revealing distinct pathways related immune cell Reference orientation within the TME. 1. Lu S, Stein JE, Rimm DL, et al. Comparison of Biomarker Modalities for Conclusions Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Here we show the use of novel high-plex spatial profiling to profile Review and Meta-analysis. JAMA Oncol. Published online July 18, 2019. location and pathways in the TME of MSI and MSS CRC tumors. doi:10.1001/jamaoncol.2019.1549 These findings elicit unique biology related to the location and sig- naling of immune cells, which have the potential to unveil targets for P32 therapeutic combinations. Differential immune contexture of human colorectal carcinomas with mismatch repair deficiency (MSI-H) and increased DNA P31 damage responses (DDR) 2 2 2 Applying multispectral unmixing and spatial analyses to explore Shruti Desai, PhD , Venkata Nagineni , Micaela Morgada , Aravind 2 2 1 2 tumor heterogeneity with a pre-optimized 7-color immuno- Kalathil , Ila Datar , Charles Fuchs, MD, MPH , Patricia LoRusso, DO , 2 2 oncology workflow Ranjit Bindra , Kurt Schalper, MD, PhD 1 2 Carla Coltharp, PhD, Bethany Remeniuk, PhD, Chichung Wang, Rachel Yale University, New Haven, CT, United States; Yale University, School Schaefer, Linying Liu, Glenn Milton, Victoria Duckworth, Michael McLane, of Medicine, New Haven, CT, United States Peter Miller, Yi Zheng, Carla Coltharp, PhD Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Akoya Biosciences, Hopkinton, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P32 Correspondence: Yi Zheng (YZheng@akoyabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P31 Background Tumor cells accumulate deleterious genomic alterations through sus- Background tained mutagenic exposure and defective DNA repair. Approximately The tumor microenvironment hosts a myriad of cellular interactions 15% of human colorectal carcinomas (CRCs) display mismatch repair that influence tumor biology and patient outcomes. Multiplex im- deficiency (MSI-H) associated with increased somatic mutations and munofluorescence (mIF) provides the ability to investigate a large sensitivity to immune checkpoint blockers. Advanced tumors can number of these interactions in a single tissue section, and has been harbor additional DNA-repair alterations with functional/therapeutic shown to outperform other testing modalities for predicting re- implications. Increased double strand DNA breaks have been re- sponse to immunotherapies [1]. ported across solid tumors and can be detected by changes in Multispectral imaging (MSI) improves the capabilities of mIF by pro- Serine139-phosphorylated histone H2AX (γH2AX). We studied the im- viding the ability to spectrally unmix fluorescence signals. This in- mune composition of human CRCs with MSI-H and elevated DDR. creases the number of markers that can be probed in the same scan Methods and allows for separation of true immunofluorescence signals from Using multiplexed quantitative immunofluorescence (QIF), we stud- tissue autofluorescence background. ied the level of major adaptive and innate immune markers in a Here, we apply MSI to explore spatial interactions observed in lung retrospective collection of 265 stage I-IV CRCs from Yale represented cancer samples using an end-to-end translational workflow based on in tissuemicroarrays. We used previously validated QIF panels includ- the PhenopticsTM platform. The workflow includes a pre-optimized ing the markers DAPI, cytokeratin, γH2AX, CD3, CD4, CD8, CD20, PD- 7-color staining panel kit along with a pre-configured analysis algo- L1, CD15, myeloperoxidase (MPO), IL-8, Ki-67, granzyme-B (GZB), rithm for cell phenotyping. Beta-2 microglobulin (B2M), HLA-class I and HLA-class II. The MSI sta- Using tissue microarrays (TMA), we demonstrate the heterogeneity of tus was determined using clinical-grade immunohistochemistry de- spatial interactions observed among different lung cancer samples tection of MLH1, MSH2, MSH6 and PMS2. We analyzed the and the improved sensitivity of detection afforded by unmixing association between localized measurement of markers and with multispectral scans. major clinicopathologic variables/survival. Methods Results A lung cancer TMA was created using the 3DHistech TMA Master II From 252 evaluable cases, 12.1% were classified as MSI-H. Relative to from five formalin-fixed paraffin-embedded lung cancer tissue blocks. MSS tumors, MSI-H CRCs showed significantly higher levels of PD-L1, The TMAs were stained using the Opal Polaris 7-Color PD1/PD-L1 lower CD20 and non-significant increases in CD3, CD4, CD8, T-cell Ki- Lung Cancer Panel Kit on the Leica BOND RXTM automated stainer 67 and T-cell GZB. MSI-H cases displayed lower tumor-cell B2M and using the associated preloaded Opal 7-Color Panel Kit protocol. increased stromal HLA-class II expression. MSI-H tumors also showed Whole slide MOTiFTM multispectral scans were acquired on Vectra significantly higher levels of IL-8 and MPO+ cells than MSS Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 17 of 272 counterpart. The level of γH2AX was comparable between MSI-H and clinicopathologic associations were found. γH2Ax was not prognostic MSS malignancies. Cases with increased tumor-cell γH2AX (> cohort as single marker. However, elevated simultaneous expression of median) showed significantly higher levels of PD-L1 and all studied γH2Ax and CD3 was associated with longer 5-year overall survival in lymphocyte markers than cases with lower γH2AX. In addition, these the immunotherapy-naïve cohorts. In patients treated with PD-1 axis tumors displayed significantly higher T-cell proliferation, mild in- blockers, elevated baseline γH2Ax/CD3 was associated with a clear creases in T-cell GZB and higher levels of HLA-class I/class II proteins. trend toward longer survival but did not reach statistical significance. The levels of IL-8 and MPO+ cells were comparable across the γH2AX Conclusions groups. The DNA repair and immune markers were variably associ- Active DDR as measured by tumor-cell γH2Ax expression occurs in a ated with 5-year overall survival in the cohort. high proportion of human NSCLCs and is associated with T-cell in- Conclusions flamed tumors. Despite their association with smoking, lung adeno- DNA repair deficiency defines human CRCs with distinct innate and carcinomas harboring activating mutations in KRAS display lower adaptive immune contexture. While mismatch repair deficiency is as- DDR markers than EGFR mutant or EGFR/KRAS wild type malignan- sociated with mild/moderate intratumor T-cell responses and prom- cies. Collectively, our results support the use of combination therapy inent myeloid cell features; elevated DDR display prominent adaptive targeting DDR and immunostimulatory therapies in a fraction of immunity and unaltered myeloid-cell changes. Our data indicate that NSCLC. MSI-H and DDR phenotypes are independent features in human CRC Ethics Approval and this could be used to design optimal therapeutic strategies. All tissues were used after approval from the Yale Human Investiga- Ethics Approval tion committee protocol #9505008219 which approved the patient All tissues were used after approval from the Yale Human Investiga- consent forms or waiver of consent. tion committee protocol #9505008219 which approved the patient consent forms or waiver of consent. P34 A fully optimized end-to-end solution for I/O multiplex P33 immunofluorescence staining using Opal Polaris 7-Color PD1/PD- DNA damage response (DDR) is associated with increased adaptive L1 Panel Kits for lung cancer and melanoma anti-tumor responses and PD-L1 expression in human non-small Yi Zheng, Rachel Schaefer, Linying Liu, Glenn Milton, Carla Coltharp, cell lung cancer 2 2 1 PhD, Victoria Duckworth, MS, Michael McLane, Peter Miller, MS Shruti Desai, PhD , Aravind Kalathil , Roy Herbst, MD, PhD , Ranjit 2 1 2 Akoya Biosciences, Hopkinton, MA, United States Bindra , Patricia LoRusso, DO , Kurt Schalper, MD, PhD 1 2 Correspondence: Peter Miller (pmiller@akoyabio.com) Yale University, New Haven, CT, United States; Yale University, School Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P34 of Medicine, New Haven, CT, United States Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P33 Understanding cellular heterogeneity and spatial relationships be- tween biomarkers within the tumor microenvironment (TME) is a key Background component to translational research in immuno-oncology. Multiplex Tumor cells accumulate genomic alterations as a consequence of immunofluorescence (mIF) on formalin-fixed, paraffin-embedded sustained mutagenic events and defective DNA repair mechanisms, (FFPE) tissue is the multiparameter assay most frequently chosen collectively called DNA damage response (DDR). Targeting DDR path- across all current I/O clinical trials, as it allows for quantitative assess- ways can induce synthetic lethality and prominent anti-tumor re- ment of these relationships in situ. Running medium to large scale sponses in neoplasms with DNA repair deficiency. In addition, translational studies on FFPE tissue demands an assay that is repro- increased DNA damage could favor anti-tumor immune responses by ducible, quantitative, easy-to-use, and standardized, yet still allows increasing the neo-antigenic load and T-cell recognition. Despite its for flexibility when detecting differentially expressing biomarkers therapeutic implications, the frequency and significance of DDR alter- across samples. In this study, we demonstrate a fully developed, flex- ations in human non-small cell lung cancer (NSCLC) remains poorly ible, end-to-end workflow solution for tissue biomarker discovery by understood. applying miF in lung cancer and melanoma. This newly developed Methods Phenoptics™ solution provides an integrated MOTiF™ workflow in- Using irradiated cell line preparations and expression controls, we cluding primary antibodies and image analysis algorithms enabling a standardized a multiplexed quantitative immunofluorescence more comprehensive and specific TME analysis with minimal user (mQIF) panel for simultaneous and localized measurement of optimization. DAPI (all cells), cytokeratin for tumor epithelial cells (AE1/AE3, Methods DAKO), γH2AX to map active DNA damage/repair responses FFPE samples from human lung cancer and melanoma were stained (JBW301, Millipore), CD3 for T-lymphocytes (Rabbit polyclonal, using Opal Polaris 7-Color PD1/PD-L1 Lung Cancer and Melanoma DAKO) and PD-L1 (E1L3N, CST) in formalin-fixed paraffin- Panel Kits. Staining was performed on the Leica BOND RX™ auto- embedded (FFPE) tissue samples. We used this panel to interro- mated stainer with the pre-loaded MOTiF protocol. Multispectral gate 4 retrospective NSCLC cohorts from Yale represented in tis- scans were acquired on Vectra Polaris® with pre-optimized acquisi- sue microarray format including immunotherapy-naïve cases tion parameters and analyzed with a pre-configured phenotyping al- (Cohort#1: n=297 and #2:n=175); lung adenocarcinomas tested gorithm in inForm®. Spatial analyses and visualizations were for major oncogenic mutations (Cohort #3, n=139); and baseline performed in R using phenoptr and phenoptrReports. NSCLC samples from patients treated with immune checkpoint Results blockers (Cohort #4, n=84). We analyzed the levels of the markers This simplified end-to-end solution results in better quantification of in different tumor tissue compartments and their association with cancer-immune interactions by providing: major clinicopathological variables. Results Detectable nuclear tumor-cell γH2Ax was recognized in 37-58% of  Well-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer NSCLCs. Elevated tumor-cell γH2Ax expression was consistently asso- and Melanoma Panel Kits. Along with the pre-loaded Leica ciated with smoking history, increased intratumor CD3+ T-cells and BOND RX automation protocol, we provide a staining workflow PD-L1 protein expression across the cohorts. The level of γH2Ax was with pre-defined primary antibody concentration, fixed staining significantly lower in KRAS mutant lung adenocarcinomas than in order, and Opal™ dye-antibody pairs, leaving Opal concentra- EGFR mutant or EGFR/KRAS wild type tumors. No additional tions as a flexible dial. Recommended image acquisition Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 18 of 272 parameters on the Vectra Polaris® that significantly simplify lymphoid aggregates. Micro-regions were picked and sequenced. visualization of multiple markers via multispectral isolation. Pre- Hierarchical clustering and differential expression analysis differ- configured image analysis algorithms that make quantitative entiated the three micro-region types and revealed tumor- and T analysis at a per-cell and per-slide level streamlined and cell-specific expression signatures. CIBERSORT demonstrated the standardized. presence of T cell-associated transcriptomic profiles in lymphoid aggregates and in TIL-containing micro-regions that were propor- tional to the number of T cells retrieved. Aligned RNA-seq reads Conclusions were further analyzed via TraCeR to identify TCR α and β chain The 7-Color PD1/PD-L1 panel kits utilizing MOTiF whole slide scan- sequences from retrieved TILs. ning enable visualization of multiple biomarkers at the whole slide Conclusions level, revealing distribution patterns and their spatial context across These data establish the potential of combining multi-parameter IF the entire tissue section. With these new assays, we have demon- microscopy with highly focused RNA sequencing as a powerful tool strated an easy-to-use yet comprehensive end-to-end Phenoptics re- for investigation and biomarker discovery for immuno-oncology. search workflow. We have radically simplified the Opal method and facilitated the development and optimization of translational multi- plex fluorescent assays by providing pre-defined staining conditions P36 while still giving researchers the flexibility to balance signals based The complexity of myeloid-derived suppressor cells in non-small on their tissue samples. Complementary pre-configured phenotyping cell lung cancer: A combinatorial multiplex IHC and flow cytometry provides researchers faster access to quantitative data across study approach samples. 1 2 1 1 Amanda Finan, PhD , Muriel Smet , Maroua Tliba , Manon Motte , Jean- 1 1 1 Philippe Coton , Domenico Lazzaro , Renaud Burrer 1 2 Histalim, Montpellier, France; Barc Lab, Ghent, Belgium P35 Correspondence: Renaud Burrer (rburrer@histalim.com) Pick-Seq®: a spatial analysis tool for immuno-oncology biomarker Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P36 discovery utilizing multi-parameter imaging and RNA sequencing of tissue micro-regions Background 1 1 1 Nolan Ericson , Rebecca Podyminogin , Jennifer Chow, PhD , Yu-An Lung cancer is the most common cause of cancer-related deaths 2 2 2 2 1 Chen , Jia-Ren Lin , Zoltan Maliga , Peter Sorger , Kyla Teplitz , Melinda worldwide with non-small cell lung cancer (NSCLC) representing the 1 1 1 Duplessis, PhD , Eric Kaldjian, MD , Tad George, PhD gross majority of the cases. The immune microenvironment of NSCLC 1 2 RareCyte, Seattle, WA, United States; Harvard Medical School, Boston, is diverse with many players that can impact tumor development MA, United States and clinical outcomes. In particular, myeloid-derived suppressor cells Correspondence: Tad George (tgeorge@rarecyte.com) (MDSC) are important components of the immunosuppressive net- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P35 work that can hinder the activity of T cells, natural killer cells, and dendritic cells. MDSC in the blood may represent prognostic markers Background for NSCLC patients and for monitoring a patient’s response to im- Pick-Seq is a novel workflow uniquely enabled by the RareCyte Cyte- munotherapies. There is a gap in the relevance of MDSC within the Finder® Instrument that combines visualization of multiple protein tissue context due to limitations with conventional immunohisto- markers with investigation of gene expression from selected micro- chemistry. Multiplex immunofluorescence offers a technical advan- regions on tissue slides, providing spatial and contextual investiga- tage by allowing the detection of co-expression and spatial tion of tumors and their microenvironment. organization of multiple targets within a preserved tissue architecture Methods on a single slide. Frozen breast carcinoma and formalin-fixed, paraffin-embedded ton- Methods sil sections were stained by multi-parameter immunofluorescence (IF) We have developed the multiplex immunofluorescence for markers of T cells, B cells, and cytokeratin. Slides were imaged Histoprofile-MDSC panel to identify monocytic MDSC (M-MDSC) with the CyteFinder® Instrument and 40 μm micro-regions were re- and polymorphonuclear MDSC (PMN-MDSC) in situ. Five human trieved with the integrated CytePicker® Retrieval Module. RNA was NSCLC tissue samples were investigated by multiplex immunofluor- isolated and whole transcriptome amplified (SMART-seq v4), followed escence and H&E staining. After multispectral acquisition, the MDSC by Nextera XT library preparation, sequencing on Illumina MiSeq, and populations were evaluated with the imaging software HALO. gene expression analysis. Differentially expressed genes were se- Paired peripheral blood was analyzed for circulating M-MDSC by lected to create a Pick-Seq-informed IF staining panel to confirm flow cytometry. RNA expression results. Cell compositions of each micro-region were Results deconvolved with CIBERSORT. The development and verification of the multiplex panel are pre- Results sented. The NSCLC subtype of the samples was determined by a Tonsil micro-regions from one T cell zone and two adjacent follicles pathologist from the H&E sections. Monocytes, neutrophils, M-MDSC, were retrieved for RNA sequencing. Transcriptomic analysis con- and PMN-MDSC were evaluated in the five tissue samples. The neu- firmed increased expression of B cell markers in follicles and T cell trophils, monocytes, and M-MDSC in the peripheral blood could be markers in the T cell zone. CIBERSORT analysis revealed distinct cellu- assessed by flow cytometry. A varying distribution of the cell popula- lar compositions between T cell zones and the B cell follicles. tions in the lung tissue and the peripheral blood of the different Principle component analysis of gene expression found that micro- NSCLC subtypes can be appreciated. The two approaches are regions retrieved from the two follicles clustered independently from compared. each other, and from the T cell zone micro-regions. Differential ex- Conclusions pression analysis between the adjacent follicles revealed distinct pat- We present an in-depth combined approach for MDSC investigation terns of CD21 expression, a marker which was not present in the in lung tissue and the peripheral blood of NSCLC patients. The ap- original IF staining panel. Subsequent staining confirmed differential proaches presented here demonstrate the power of multiplex immu- protein expression of CD21, indicating that only one follicle con- nohistochemistry and flow cytometry in the identification and tained a germinal center. In breast carcinoma, ROI were identified for quantification of multiple immune cell populations with a limited micro-region retrieval that included tumor cells, tumor cells with quantity of patient sample and the potential application of this interspersed tumor infiltrating lymphocytes (TIL), or adjacent method in both preclinical and clinical studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 19 of 272 P37 8. Otvos B, Silver DJ, Mulkearns-Hubert EE, et al. Cancer Stem Cell-Secreted ImmunoPET imaging of glioma-infiltrating myeloid cells using Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Sup- Zirconium-89-labeled anti-CD11b antibody pressor Cell Function and Facilitates Glioblastoma Immune Evasion. Stem Alexandra Foster, BS, Rajeev Kumar, Shubhanchi Nigam, Lauren McCarl, Cells. 2016; 34:2026–2039. Robert Edinger, Ian Pollack, Carolyn Anderson, Wilson Edwards, Gary 9. Meyer C, Cagnon L, Costa-Nunes CM, et al. Frequencies of circulating Kohanbash, Alexandra Foster, BS MDSC correlate with clinical outcome of melanoma patients treated with University of Pittsburgh, Pittsburgh, PA, United States ipilimumab. Cancer Immunol Immunother. 2014; 63:247–257. Correspondence: Gary Kohanbash (gary.kohanbash2@chp.edu) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P37 The study was approved by University of Pittsburgh's Institutional Animal Care and Use Committee (IACUC). Background Gliomas are the most common primary central nervous system tumor, P38 with malignant gliomas causing significant morbidity and mortality. Sensitive methodologies for tracking T cell immunotherapy by MRI Thirty percent of a glioma’s cellular mass may be attributed to immuno- 1 2 1 2 Brooke Helfer, PhD , Deanne Lister , Charles O'Hanlon III , Eric Ahrens , suppressive and pro-tumoral tumor-associated myeloid cells (TAMCs), Brooke Helfer, PhD primarily myeloid-derived suppressor cells (MDSCs) and tumor- 1 2 Celsense, Inc, Pittsburgh, PA, United States; UCSD, La Jolla, CA, United associated macrophages (TAMs) [1-4]. Multiple preclinical studies and States clinical trials have attempted to target these cells; however, monitoring Correspondence: Brooke Helfer (brooke@celsense.com) responses to these therapies remains a challenge. Quantifying TAMCs Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P38 within gliomas using an antibody-based tracer for non-invasive posi- tron emission tomography (immunoPET) may allow for better patient Background stratification, monitoring of treatment efficacy, and ultimately improve Cancer immunotherapies have made a great progress and hold much survival rates [5-9]. Integrin CD11b is a cellular marker expressed on the promise in the treatment of cancer. Specifically, in the case of B-cell surface of TAMCs frequently used to identify macrophages and micro- malignancies (such as Acute Lymphoblastic Leukemia, or ALL), CAR glia. We therefore hypothesized that radiolabeled anti-CD11b antibody (chimeric antigen receptor) and TCR (T-cell receptor) therapies have (Ab) could be used for immunoPET imaging of TAMCs in a preclinical demonstrated encouraging clinical results. As we begin to target solid orthotopic syngeneic glioma model. tumors with TCR and CAR T-cells, the hurdle of being able to select a Methods suitable target and achieve successful cellular delivery/homing to the The human/mouse cross-reactive anti-CD11b Ab (clone M1/70) was site of disease remains. With this in mind, being able to visualize a rap- conjugated with p-NCS-Bz-DFO chelator and radiolabeled with 89Zr idly dividing cellular population is another obstacle to consider. for PET imaging with specific activity of 2 μCi/μg. PET/CT imaging, Methods with or without a blocking dose of anti-CD11b Ab, was performed in Here we demonstrate the application of two clinically applicable per- mice bearing established orthotopic syngeneic GL261 gliomas. Flow fluorocarbon (PFC) tracers, one commercially available and a next- cytometry and histology in tissues collected from post-imaging bio- generation magnetic resonance imaging (MRI) probe called FETRIS. distribution validated targeting of CD11b+ TAMCs. Both of these agents enable the migration and persistence of cellular Results therapies to be noninvasively imaged by 19F MRI, while the FETRIS Standard uptake values (SUV) indicated significant 89Zr-anti-CD11b reagent adds additional detection sensitivity. Ab uptake in the tumor ipsilateral right brain (SUVmean = 2.6 ± 0.24) Results compared to contralateral left brain (SUVmean = 0.6 ± 0.11). Blocking Using a general T-cell expansion protocol, we show that adding a cellular with 10-fold lower specific activity 89Zr-anti-CD11b Ab reduced the label does not alter the viability or release characteristics of T cells. By SUV in right brain with (SUVmean = 0.11 ± 0.06). Spleen and lymph pairing the PFC signal with conventional proton MRI from the same im- nodes also showed high uptake, while bone and muscle showed low aging session, the images are able to be overlaid, allowing cells to be uptake. Biodistribution analysis confirmed these results. Additionally, traced to their anatomical location. With nominal exogenous fluorine nat- no uptake was observed in the brain of non-tumor bearing mice that urally present in tissue, labeled cells appear with little background. received 89Zr-ant- CD11b. Flow cytometry with QuantiBRITE Fluores- Conclusions cence Quantitation Kit demonstrated that the majority of tumor- Images of both reagents show the detection and sensitivity of the infiltrating immune cells expressed CD11b at an average of 54,076 method and how they can be applied to monitor the distribution of CD11b molecules per cell in GL261. cells over time. Conclusions Imaging TAMCs with 89Zr-labeled anti-CD11b Ab may be feasibility for preclinical studies, patient stratification, and monitoring of immunotherapy. P39 References Looking beyond the assay: Comparison of multiplex chromogenic 1. Gabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate and fluorescent immunohistochemistry for standardized immune immune cells resemble M0 macrophage phenotype. JCI insight. 2016; oncology profiling in non-small cell lung carcinoma patients 1 2 2 1(2). Ana Hidalgo Sastre, PhD , Lorenz Rognoni, PhD , Monika Baehner , 2 2 2 2 2. Kennedy BC, Showers CR, Anderson DE, et al. Tumor-associated macro- Marco Testori , Jessica Chan , Andreas Spitzmüller , Nicolas Brieu, PhD , 3 3 3 phages in glioma: friend or foe? J oncol; 2013. 2013. Bonnie Phillips, PhD , Katir Patel, PhD , Sean Downing, PhD , Alex 4 4 2 3. Kohanbash G, Okada H. Myeloid-derived suppressor cells (MDSCs) in gli- Haragan , John Field , Florian Leiss, PhD 1 2 3 omas and glioma-development. Immunolo invest. 2012; 41:658–679. Definiens, Munich, Germany; Definiens AG, Munich, Germany; Ultivue, 4. Lapa C, Linsenmann T, Lückerath K, et al. Tumor-associated macrophages Cambridge, MA, United States; Liverpool University Hospital, Liverpool, in glioblastoma multiforme-a suitable target for somatostatin receptor- United Kingdom based imaging and therapy? PloS one. 2015; 10. Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) 5. Kohanbash G, McKaveney K, Sakaki M, et al. GM-CSF Promotes the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P39 Immunosuppressive Activity of Glioma-Infiltrating Myeloid Cells through Interleukin-4 Receptor-α. Cancer Res. 2013; 73:6413–6423. Background 6. Raychaudhuri B, Rayman P, Huang P, et al. Myeloid derived suppressor cell Given the heterogeneity of tumors and the variety of potential bio- infiltration of murine and human gliomas is associated with reduction of markers in immune oncology, there is a need for quantitative standard- tumor infiltrating lymphocytes. J Neurooncol. 2015; 122:293–301. ized assays to reliably assess the immune status of a patient’stumor to 7. Okada H, Kohanbash G, Zhu X, et al. Immunotherapeutic approaches for be able to extract the true biological information across cohorts. Here, glioma. Crit rev immunol. 2009; 29:1–42. two different tissue-based approaches have been compared: multiplex Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 20 of 272 immunofluorescence (mIF) and multiplex chromogenic immunohisto- P40 chemistry (mIHC). Independently of the technique used, assay reproduci- Tumour immunity signatures to expand current diagnostic bility and standardized quantification of staining intensity are a approaches in mismatch repair deficient cancers in the context of prerequisite for obtaining consistent results. Using a cohort of non-small Lynch Syndrome through InSituPlex technology and Tissue cell lung carcinoma (NSCLC) patients, we identified patterns of immune Phenomics integration 1 2 3 cell infiltration that were comparable, independent of the assay applied. Ryan Hutchinson, Fellow , Armin Meier, PhD , Bonnie Philips ,Katir 3 3 3 1 Methods Patel, PhD , Sean Downing, PhD , Karan Sharma , Julia Como ,Simin 1 2 4 1 Formalin-fixed paraffin-embedded (FFPE) true consecutive slides from 7 Daneshvar , Gillian Livock , Ingrid Winship , Christophe Rosty ,Mark 5 2 1 NSCLC resections were stained with a multiplex chromogenic panel (in- Jenkins ,GunterSchmidt , Daniel Buchanan ,RyanHutchinson, cluding CD3, PD-L1, CD68, CD8, PD-1) at Mosaic Laboratories [1] and with Fellow the UltiMapper kits (I/O PD-L1 and I/O PD-1) from Ultivue. mIHC scans Victorian Comprehensive Cancer Centre, Melbourne, Australia; 2 3 were acquired with an Aperio AT Turbo scanner (Leica), while mIF scans Definiens AG, Munich, Germany; Ultivue, Cambridge, MA, USA, Boston, 4 5 were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss) both as whole MA, United States; Royal Melbourne Hospital, Melbourne, Australia; The slide images. mIHC and mIF images were co-registered, and Definiens University of Melbourne, Melbourne, Australia custom algorithms for digital image analysis were applied [2,3]. Correspondence: Daniel Buchanan (daniel.buchanan@unimelb.edu.au) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P40 Densities of immune cell populations and their locations in different com- partments (invasive margin vs tumor center and tumor epithelium vs Background tumor stroma) were measured (Figure 1). For instance, CD3 cell density Deficiency in the mismatch repair (dMMR) can result from had a Pearson correlation of 0,91 and a Spearman correlation of 0,89 be- inherited mechanisms (Lynch Syndrome (LS)) or from somatic tween both assays (mIHC vs mIF). Differentiation between tumor epithe- inactivation caused by hypermethylation of the MLH1 gene lium and tumor stroma was based on a histology-driven deep learning promoter (MLH1 methylated). A third subtype of dMMR colo- approach for mIHC and on pan Cytokeratin for mIF (Figure 1). rectal cancer (CRC) and endometrial cancer (EC) have neither Conclusions LS nor MLH1 promoter methylation and are referred to as sus- By applying mIHC and mIF in true consecutive tissue slides we re- pected Lynch syndrome (SLS). There remains a knowledge gap trieved the information of tumor immune cell infiltrates that was as to whether the tumour microenvironment (TiME) is different consistent across the different assays and distinguished it from infor- between LS, MLH1-methylated and SLS dMMR CRC and EC. The mation that is specific to either of the assays. We believe that being aimofthisstudy wastocharacterise and identify immune pat- able to relate across staining techniques could help pathologists and terns within the TiME that may enhance the current clinical research centers draw conclusions across cohorts that were stained triaging of LS, SLS and MLH1-methylated subtypes of dMMR with the same markers but with different assays. CRC and EC. Methods References Ten FFPE samples from seven individuals were studied: CRC (N=5; 1. Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of 1xLS, 2xMLH1 methylated, 1xSLS and 1x proficient MMR (pMMR)) inflammatory cell patterns and densities using multiplex and EC (N=2; 1xLS and 1xpMMR) and where available adjacent nor- immunohistochemistry immuno-oncology assays [abstract]. In: Proceed- mal tissue (N=5: 3xcolon and 2xendometrium) were characterized ings of the AACR-NCI-EORTC International Conference: Molecular Targets using the Ultivue UltiMapper I/O portfolio (InSituPlex) on a Leica and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia Bond autostainer and digitally acquired using the Zeiss Axio Scan Z1. (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl): Abstract nr B069. We evaluated CD3, CD8, CD11c, CD20, CD45RO, CD68, CD163, Gran- 2. Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated zyme B, Ki-67, MHC II, PD-1, PD-L1, and pan-cytokeratin. Tissue phe- quantification of whole-slide multispectral immunofluorescence images to nomic approaches were developed to spatially characterize, quantify identify spatial expression patterns in the lung cancer microenvironment. immune cell patterns and visualize heterogeneity within the TiME SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. (Figure 1). 3. Brieu, Nicolas & Meier, Armin & Kapil, Ansh & Schönmeyer, Ralf & Gavriel, Results Christos & Caie, Peter & Schmidt, Günter. (2019). Domain Adaptation- InSituPlex technology enabled the visualization of the based Augmentation for Weakly Supervised Nuclei Detection. heterogenous infiltration and co-localization patterns across LS Ethics Approval dMMR CRC (Figures 2&3). Tissue phenomic approaches demon- Ethical approval was granted by the Liverpool Research Ethics Committee, strated the following; within the SLS category the colon cancer reference number 97/141. had higher mean areas of intraepithelial (IE) PD-L1 (6% vs. 2%), CD8 (18% vs. 8%) and CD68 (28% vs. 12%) compared to the pMMR EC. The MLH1 methylated tumour with a high tumour mu- tation burden (33.84 mutations/MB) had a higher mean area of IE PD-L1 (8% vs. 2%) and CD8 (30% vs. 5%) while the tumour with low TMB had a higher mean area of IE CD68 (15% vs. 8%). Within LS, the EC had a higher mean area of IE PD-L1 compared to the CRC (14% vs. 0%), in contrast the CRC had a higher IE CD8 area (27% vs. 10%) (Figures 4&5). Conclusions This study evaluated the immune contexture within inherited and sporadic subtypes of dMMR CRCs and ECs, highlighting dif- fering immune infiltration patterns and phenomic densities. In- tegration of multiplex technologies and Tissue Phenomics can enhance the understanding of the dMMR TiME and with poten- tial utility in clinical triaging and to inform immune-oncology clinical trials. Acknowledgements We thank the investigators and participants of the ACCFR and ANGELS Fig. 1 (abstract P39). Consecutive slides from NSCLC resection studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 21 of 272 Fig. 2 (abstract P40). TiME regions of immune infiltration Fig. 1 (abstract P40). Phenotypes Fig. 3 (abstract P40). TiME regions of immune infiltration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 22 of 272 P41 Multiplexed Imaging for the simultaneous detection of nucleic acids and proteins to dissect the tissue immune landscape and microenvironment of viral diseases 1 1 2 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Chi Ngai Chan, PhD , Bokai 1 1 1 1 Zhu , Yunhao Bai, BS , Marc Bosse, PhD , David McIlwain, PhD , Sean 1 1 2 Bendall, PhD , Michael Angelo, MD, PhD , Jacob Estes, PhD , Garry Nolan, PhD 1 2 Stanford University, Stanford, CA, United States; Oregon Health and Sciences University, Stanford, CA, United States Correspondence: Jacob Estes (estesja@ohsu.edu); Garry Nolan (gnolan@stanford.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P41 Background Multiplexed Ion Beam Imaging (MIBI) is a novel imaging modal- ity capable of resolving >40 parameters simultaneously in bio- logical samples. Here, we developed viralMIBI, a highly sensitive method capable of detecting down to single copies of nucleic acids, in addition to protein epitopes. ViralMIBI en- ables the functional dissection of the immune landscape in viral driven diseases, such as that of tumor viruses (HBV, EBV, LCV) and others (HIV, SIV, Zika, Ebola). The combination of vir- alMIBI and cutting-edge cell neighborhood analytical methods will be paramount to better understand the immunological host-pathogen interactions for viral diseases, revealing insights into virus-induced immunodeficiency as well as virus-driven cancers. Methods To allow for the sensitive detection of nucleic acids, we took advantage of a customized branched DNA amplification method that can be easily adapted to a variety of multiplexed Fig. 4 (abstract P40). TiME regions of immune infiltration imaging platforms. Formalin-Fixed and Paraffin-Embedded (FFPE) tissue samples from Rhesus macaque animal models for a number of viral diseases were processed for viralMIBI nucleic acid and protein marker detection. Imaging was performed with the MIBIscope, a secondary ion mass spectrometry based device. Results We have established a robust method for highly multiplexed nucleic acid and protein epitope detection in FFPE tissue samples. As a proof of concept, we were able to detect down to single integrated copies of SIV. The establishment and validation of a Rhesus macaque spe- cific antibody panel allowed for the in-depth characterization of cel- lular identities at the single-cell level, while maintaining their tissue geopositions. Conclusions ViralMIBI enables the MIBI to achieve highly sensitive nucleic acid detection, in addition to its multiplexed protein capabil- ities. Here, we leveage this method for the detection of various viral pathogens. ViralMIBI is also applicable to other targets, such as genomic amplifications frequently seen in cancers, or gene expression studies. The ability to image >40 parameters in tissue samples will vital for a better understanding of im- mune regulation of diseases, such as the establishment of viral related cancers as well as latent tissue reservoirs of pathogens. These discoveries can then be translated to better immuno- therapy treatments against viral driven diseases. Acknowledgements We thank Matt Newgren for tireless technical support on the MIBI instrument, Rachel Finck, Xiao-Jun Ma and Bingqing Zhang for helpful discussions. S.J was supported by a Stanford Dean’s Fellowship and the Leukemia & Lymphoma Society Career Development Program. X.R.-C. was supported by a long-term EMBO fellowship. This work was supported by grants from the FDA, NIH, Parker Institute for Cancer Immunotherapy, the Bill and Melinda Gates Foundation, as well as Fig. 5 (abstract P40). TiME regions of immune infiltration the Rachford and Carlota A. Harris Endowed Professorship to G.P.N. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 23 of 272 P42 An integrated multiplexing approach for the immunoprofiling of the tumor microenvironment of ovarian granulosa cell tumors 1 2 1 1 Juncker-Jensen, PhD , Tyvette Hilliard , Nicholas Stavrou , Erinn Parnell , 1 1 1 1 Judy Kuo , Eric Leones , Flora Sahafi , Josette William, PhD, MD , Sharon 2 1 Stack , Anna Juncker-Jensen 1 2 NeoGenomics, Aliso Viejo, CA, United States; University of Notre Dame, South Bend, IN, United States Correspondence: Anna Juncker-Jensen (anna.juncker- jensen@neogenomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P42 Background Ovarian granulosa cell tumors (GCTs) are rare tumor accounting for 2-5% of all ovarian cancers. The main current treatment for GCT is surgery, however a subset require chemotherapy for residual and re- current disease. GCT malignancies are often low-grade, however a clinical characteristic of these tumors is a tendency for late recur- rence which is the most critical factor for GCT death. As the onset of recurrence is unpredictable, future research should focus on identify- ing both biomarkers for prognosis prediction, as well as targets that could help guide clinical trials in the development of targeted ther- apies for this rare indication. As GCTs are rare tumors making tissue Fig. 1 (abstract P41). Validation of viralMIBI: Detection of SIV in availability very limited, we used a dual multiplexing approach in infected tissue order to maximize the data output from a total of 14 FFPE tumor samples (6 primary tumors, and 8 recurrent tumors). Methods For protein multiplexing we have used MultiOmyx™, an immunofluores- cence (IF) multiplexing assay utilizing a pair of directly conjugated Cya- nine dye-labeled (Cy3, Cy5) antibodies per round of staining (Figure 1). Each round of staining is imaged and followed by dye inactivation en- abling repeated rounds of staining and deactivation, while deep learn- ing based cell classification algorithms identify positive cells for each biomarker. We generated a 15-marker panel consisting of CD3, CD4, CD8, FoxP3, CD68, CD163, HLA-DR, CD34, CTLA-4, PD-1, PD-L1, Ki67, vimentin, S100, and Pan Cytokeratin. For the gene expression analysis RNA was extracted from the adjacent 10 μm section and then analyzed using the Nanostring nCounter assay, specifically the 770 gene PanCan- cer Immune Panel. Hybridization, purification and immobilization and counts were based on manufacturer’sprotocol. Results On protein level we confirmed previous findings that ovarian GCTs are so-called “cold” tumors, with a very low density of T cell infiltra- tion. When we analyzed the presence of macrophages in the tumor microenvironment however, we found a 113% increase in TAM dens- ity in recurrent tumors compared to primary tumors. When searching for markers differentially expressed between primary and recurrent tumors we detected 4 genes in our PanCancer immune panel that were either significantly down-regulated (CCND3 or TOLLIP), or up- regulated (MAP3K and TNFSF4) in recurrent tumors. TNFSF4 encodes the protein OX40L, and interestingly a high expression of its receptor OX40 has previously been shown to be indicative for response to chemotherapy in recurrent ovarian cancer [1]. Conclusions We have used a dual multiplexing approach on both gene and pro- tein level in order to immunoprofile the tumor microenvironment of ovarian rare granulosa tumors. Reference 1. Ramser M, Eichelberger S, Däster S, Weixler B, Kraljević M, Mechera R, Tampakis A, Delko T, Güth U, Stadlmann S, Terracciano L, Droeser RA, Singer G. High OX40 expression in recurrent ovarian carcinoma is Fig. 2 (abstract P41). Detection of single integration events of SIV indicative for response to repeated chemotherapy. BMC Cancer. with viralMIBI 2018;18:425-433. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 24 of 272 custom panel of more than 25 markers on the PBMC samples and acquired the images using a Keyence benchtop microscope. Results The CODEX system was used to generate highly multiplexed im- mune profiles of human PBMC samples using an optimized cus- tom panel of CODEX antibodies. The images were processed using the CODEX Software Suite and cell phenotypes were clus- tered and annotated using the Multiplexed Analysis Viewer (MAV). Antibody specificity and panel performance were evalu- ated by assessing co-expression and mutually exclusive expres- sion of relevant immune markers with the CODEX analysis pipeline. Conclusions Simultaneous analysis of tens of markers in blood or plasma sam- ples can have several applications in the discovery of cellular bio- markers, immune monitoring and drug discovery and development. This preliminary study shows the compatibility of the CODEX system with cell suspensions for highly multiplexed, single-cell analysis and offers a more cost-effective method for immune profiling of blood samples. P44 Solar-IHC: Cell-to-cell distances in the tumour immune microenvironment of Hepatocellular Carcinoma has the potential Fig. 1 (abstract P42). Immunofluorescent overlay image of GCT to prognosticate survival 1 2 2 recurrent tumor Matthew Leong, NA , Toh Han Chong , Choo Su Pin , Kiat Hon Lim 3 2 2 4 Tony , Joycelyn Lee , David Wai , Poh Sheng Joe Yeong , Jin Miao Chen, PhD Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, Singapore; National Cancer Centre Singapore, Singapore, Singapore; Singapore General Hospital, Singapore, P43 Singapore, Singapore; Department of Anatomical Pathology, Singapore A novel platform for highly multiplexed, single-cell imaging of cell General hospital, Singapore, Singapore, Singapore; Singapore suspensions Immunology Network, Agency of Science, Technology and Research, 1 2 1 1 Anum Khan , Won-Mean Lee , Jon Mulholland , Dhananjay Wagh , John Singapore, Singapore, Singapore 1 2 Coller , Gabriel Mercado Correspondence: Jin Miao Chen (Chen_Jinmiao@immunol.a- 1 2 Stanford University, Palo Alto, CA, United States; Akoya Biosciences, star.edu.sg) Menlo Park, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P44 Correspondence: Won-Mean Lee (wmlee@akoyabio.com); Gabriel Mercado (gmercado@akoyabio.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P43 Hepatocellular carcinoma (HCC) is a lethal cancer, being the fourth leading cause of cancer-associated mortality worldwide Background due to its low five-year survival and high reoccurrence rates [1], Analyzing populations at the single cell level has become increas- and identifying indicators of prognosis is key in developing novel ingly important in the study of cancer and autoimmune disorders treatments and improving survival of HCC patients. With the ad- due to high levels of population heterogeneity and rare cell phe- vent of digital pathology, the immune-architecture of solid tu- notypes that can drive disease pathogenesis and progression. mours has become a central interest of cancer research and has Until recently, characterizing protein markers on single cells was been studied for the development of predictive and diagnostic limited to a handful of markers due to the technical and logis- applications. Here, we have assessed if intercellular Euclidean dis- tical challenges of flow cytometry platforms. New advancements tances in the tumour immune microenvironment can possibly be in single cell analysis technologies have enabled researchers to used to predict patient prognosis. study more than 30 parameters per cell. But these platforms are Methods expensive and require significant panel design, thereby limiting In this study, biopsies were taken from 110 HCC patients who access and usability. Here we demonstrate the use of the recently underwent surgical resection. The solar-IHC pipeline involves ar- launched CODEX System to generate an in-depth immune profile ranging the liver biopsies into tissue arrays and subsequently of human PBMC samples. studying them using automated multiplex immunohistochemistry/ Methods immunofluorescence (mIHC/IF) protocol developed in Singapore The CODEX® System is an affordable, benchtop instrument that General Hospital, with biomarkers Ecadherin, CD3, CD8, CD103 integrates with existing fluorescence microscopes and enables and PD1 [2], followed by image analysis software inForm version highly multiplexed imaging of over 40 markers in fresh frozen 2.4.2. and FFPE tissue samples. The CODEX technology uses a DNA- Results based barcode library to label antibodies and iterative cycles of Ecadherin was adopted as the tumour cell marker, and 26 im- adding and removing cognate dye-labeled oligonucleotides to mune cell phenotypes are defined by variable levels of immune reveal the staining of three markers per cycle. Data acquisition markers CD8, CD103, and PD-1 (as shown in Table 1) Dimension- is fully automated by the CODEX instrument. We tested a ality reduction and unsupervised clustering of the distances Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 25 of 272 between tumour and immune cell phenotypes showed distinct P45 clusters of patients with significant differences in clinical out- Multiplex immunofluorescence staining, whole slide imaging, and comes. Long cell-cell distances between immune cell phenotypes spatial phenotyping of T-cell exhaustion, regulatory T cells, and and tumor cells was associated with an improved overall survival myeloid-derived suppressor cells in tumor FFPE samples 2 3 1 1 (p-value = 0.02) and disease-free survival (p-value = 0.01), while Kyla Teplitz , Katir Patel, PhD , Michael Tomac, MS , Kate Lillard, PhD , 3 1 the opposite was true for short cell-cell distances between im- Mael Manesse, PhD , Anne Hellebust, PhD 1 2 mune cell phenotypes and tumor cells. This was observed in the Indica Labs, Inc., Alcester, United Kingdom; Rarecyte, Inc, Seattle, WA, analysis of all CD8+, CD8-, CD103+, CD103-, PD1+ and PD1- cells, United States; Ultivue, Inc, Cambridge, MA, United States possibly due to the suppressive immunomodulatory effect Ecad- Correspondence: Kate Lillard (kate@indicalab.com) herin+ tumour cells has on neighbouring immune cells [3]. Fur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P45 thermore, machine learning enabled the prediction of clusters with considerable accuracy, with a K-fold cross-validation of 91%. Background This indicates a strong association of cell-cell distances with pa- The immune cell milieu that comprises the tumor microenviron- tient survival, and a robust reproducibility of distance pattern- ment (TME) is highly heterogeneous and complex. Depending based predictors. on biological interactions and functional state, immune cell Conclusions populations can either promote or suppress tumor progression. In this study, our data suggests that the analysis of intercellular CD8+ T cells, for example, are the primary mediators of anti- distances has the potential to be used as a prognostic indicator tumor immunity; however, they are often ineffective either be- in HCC. Coupled with next generation machine learning tech- cause they are unable to infiltrate the tumor or because they niques, this novel approach to cell-to-cell distance analysis has become functionally exhausted [1,2], Pathologically activated the potential to be an easily implementable algorithm to predict myeloid-derived suppressor cells (MDSCs) which infiltrate the patient prognosis in HCC. This bioinformatics approach can also tumor are also associated with tumor progression [3,4]. Multiple be utilized in the analysis of other biomarkers and cancer types, biomarkers are required to accurately identify these individual and this brings exciting prospects for the future of cancer immune cell types and their functional states. In this work, we research. employ advanced multiplexing techniques to observe biologic- ally and functionally distinct T cell and MDSC populations and References to quantify their density and distribution within the TME of sev- 1. McGlynn KA, Petrick JL, London WT. Global epidemiology of eral tumor types. hepatocellular carcinoma: an emphasis on demographic and regional Methods variability. Clinics in liver disease. 2015;19(2):223-38. UltiMapper assays were used to perform multiplex immunofluor- 2. LimJCT,Yeong JPS, LimCJ, OngCCH,WongSC, Chew VSP, escence on multiple tumor FFPE samples (lung, colorectal, breast). Ahmed SS, Tan PH, Iqbal J: An automated staining protocol for Three multilpex panels were run in this study: UltiMapper PD-1 seven-colour immunofluorescence of human tissue sections [CD3, CD45RO, PD-1, CK/Sox1], UltiMapper T-reg[CD4, CD8, FoxP3, for diagnostic and prognostic use. Pathology. 2018 CK, Sox10], and UltiMapper MDSC[CD11b, CD14, CD15, HLA- Apr;50(3):333-341 DR].FFPE slides were stained using the BOND RX autostainer from 3. Nagl S, Haas M, Lahmer G, Büttner-Herold M, Grabenbauer GG, Fietkau R, Leica Biosystems and scanned on the CyteFinder® II HT Instru- et al. Cell-to-cell distances between tumor-infiltrating inflammatory cells ment from RareCyte, Inc. This instrument performs high-speed, have the potential to distinguish functionally active from suppressed in- whole-slide scanning in the 5 channels used in the UltiMapper flammatory cells. Oncoimmunology. 2016;5(5):e1127494. Kits and outputs an open source, stitched, pyramidal TIFF. Image Ethics Approval analysis was conducted using HALO 3.0 software to perform cell This study was approved by the Institutional Review Board (IRB), approval phenotyping, proximity analysis, image registration, and density number 2014/590/B. mapping. Results Cell counts for relevant phenotypes were obtained for each panel to identify exhausted T cells, T-regs, cytotoxic T cells, M-MDSCs, and Table 1 (abstract P44). See text for description PMN-MDSCs. Spatial analysis was employed to map the degree of T- cell infiltration and exhaustion correlating to T-reg and MDSC expres- sion in the tumor microenvironment. Conclusions Here we present a workflow for tackling the complexity of the tumor immune microenvironment by leveraging high-quality multiplex panels, high-speed whole-slide imaging, and quanti- tative spatial analysis. UltiMapper assays used in this study (PD-1, T-reg, and MDSC) were able to identify single-cell phe- notypes through co-localization and negative selection of markers. Using HALO image analysis, cell populations were enumerated and quantified to measure the level of T-cell ex- haustion caused by T-cell regulation and myeloid-derived im- mune cell suppression. References 1. Fridman WH, Zitvogel L, Sautes-Fridman C, Kroemer G. The immune con- texture in cancer prognosis and treatment. Nat. Rev. Clin. Oncol. 2017; 14:717–734. 2. Thommen DS, Schumacher TN. T Cell Dysfunction in Cancer. Cancer Cell. 2018; 33:547-562. 3. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 2012; 12:253–268. 4. Kumar V, Patel S, Tcyganov E, Gabrilovich DI. The Nature of Myeloid- Derived Suppressor Cells in the Tumor Microenvironment. Trends Immu- nol. 2016; 37:208–220. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 26 of 272 P46 medicine but without the complex workflow, safety, and half-life limi- Same-slide multiplex immunofluorescence and brightfield tations. In this study, we compared the behavior of monocytes histological staining as a new research tool for fast and loaded with nanoparticles in vitro and nanoparticles injected intra- comprehensive pathology assessment of the tumor venously and subsequently taken up by phagocytic cells (in situ load- microenvironment ing) and imaged using MPI the differences in biodistribution and Mael Manesse, PhD, Douglas Wood, PhD, Heike Boisvert, PhD, Sean migration of monocytes in in naïve and tumor-bearing and naïve Downing, PhD, Mael Manesse, PhD (control) mice. Ultivue, Cambridge, MA, United States Methods Correspondence: Mael Manesse (mael.manesse@ultivue.com) Twenty mice were implanted with 300,000 4T1 tumour cells in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P46 4th mammary fat pad. CD11b+ mouse monocytes were harvested using EasySep® Mouse CD11b positive selection kit II (StemCell Tech- Background nologies). The isolated monocytes were prelabeled with Vivotrax® Innovative and efficient translational research tools enabling a better (100 μg/mL). On day 7 post-implantation, either 5 million prelabeled understanding of the tumor and its microenvironment are a keystone cells or free Vivotrax (6 mg/kg) were intravenously injected into nor- of the development of digital pathology. Current immunohistochem- mal or tumor-bearing mice for in vitro or in situ targeting experi- istry (IHC) methods limit the depth of information from a single tis- ments (N=5 mice for all four groups). 3D MPI images using a sue sample to a single target in the case of chromogenic staining, or MOMENTUM MPI system (Magnetic Insight) were acquired 1, 4, 7 to sample morphology and general cell identification in the case of and 10 days after injection. MicroCT images (CT120, Trifoil Imaging) hematoxylin and eosin staining (H&E). True phenotyping requires the were acquired and co-registered using VivoQuant (Invicro). Tumors, use of a single section, as serial sections may not contain the same liver, spleen and draining lymph nodes were then harvested, imaged, cells, especially small immune cells such as T-cells. Multiplex im- fixed, and stained with Prussian blue and analyzed for iron contents. munofluorescence (mIF) methods have been established to provide Results insights into a wide number of markers of interest and their spatial Tumor-bearing mice showed a significant accumulation of nanoparti- context in a single sample. Here, we demonstrate a new research ap- cles for both the in situ and in vitro targeting methods, although the proach combining multiplexed detection of protein markers with time and amount of accumulation was different. For both experi- standard H&E pathology review in tumor samples, in a streamlined, ments, nanoparticles were predominately detected in the expanding single-day sample-to-answer workflow. margins of the tumor. For the in vitro labeled monocytes, accumula- Methods tion was rapid, with the maximum accumulation being at 24 hours InSituPlex technology was used to perform multiplex immunofluores- post-injection, while for the in situ labeled cells, accumulation was cence staining of formalin-fixed, paraffin-embedded (FFPE) samples slower. from human tonsil and primary tumor biopsies on the Leica Biosys- Conclusions tems BOND RX autostainer. The tissues were then imaged in five dis- By combining the sensitivity, specificity as well as accurate quantita- tinct fluorescent channels (DAPI, FITC, TRITC, Cy5, Cy7) before being tion potentials of MPI, information can be obtained on labeled stained using standard H&E protocols and imaged again. Fluorescent monocytes and their biodistribution in tumour models. Other cells and brightfield whole-slide images were acquired on a ZEISS AxioS- can also be labeled (dendritic cells, MDSCs, NKs, and T cells) and this can.Z1 slide scanner. Images of the same tissue section were co- information can be utilized to better understand the factors influen- registered and fused into a single image for analysis using Indica cing immune cell migration in and around tumors. Labs HALO software. Results P48 The InSituPlex technology enables deep phenotyping of immune Turning ‘cold’ tumours ‘hot’: Guided magnetic hyperthermia for cells through colocalization and co-expression of multiple protein tumour immune stimulation markers in tumor samples. Phenotypic information was then overlaid 1 1 2 1 Patrick Goodwill , Daniel Hensley , Zhi Wei Tay , Elaine Yu , James with the H&E image of the same section to facilitate identification 1 1 1 1 Mansfield, Msc , Blayne Kettlewell , Ryan Orendorff , Kyle Fields , Steve and immuno-profiling of specific cells in the tumor and its environ- Conolly ment. The fused images were also analyzed to provide cell counts, 1 2 Magnetic Insight, Alameda, CA, United States; University of California distance mapping, and expression levels of each of the markers. Berkeley, Berkeley, CA, United States Conclusions Correspondence: James Mansfield (jim@jmansfield.com) In this work, we present a new modality for pathology research with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P48 a convenient workflow that enables fast tissue review and deep immuno-profiling and phenotyping of the tumor via fusion of H&E Background and mIHC staining of the same tissue section. Cancer immunotherapy is now the “fifth pillar” of cancer thera- peutics [1]. Although hugely successful, there are limitations. In P47 many studies, less than half the patients are responsive to ther- Imaging cancer immunology: Systemic tracking of immune cells apy. One hypothesis is that refractory tumours are immunologic- in vivo with magnetic particle imaging ally ‘cold’– i.e., there are insufficient immune cells in the tumour 1 1 2 James Mansfield, Msc, Gang Ren , Jeff Gaudet , Yanrong Zhang , Sara for the therapy to be efficacious [2]. Thus, methods to stimulate 2 2 1 Ghobadi , Max Wintermark , Patrick Goodwill an immunogenic response in solid tumours to improve immuno- 1 2 Magnetic Insight, Alameda, CA, United States; Stanford University, Palo therapy efficacy are desirable. Alto, CA, United States Hyperthermia is known to induce a local immunogenic response, Correspondence: James Mansfield (jim@jmansfield.com) making it a potential adjunct to radiation and immune therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P47 One hyperthermia method is Magnetic Fluid Hyperthermia (MFH), which is based on electromagnetic heating of magnetic nanoparti- Background cles (MNPs) [3,4]. , However, poor control of heating localization and The rapid growth of research into immuno-oncology research has magnitude have prevented MFH’s widespread clinical adoption. fueled a need to track be able to determine the location of a variety Magnetic Particle Imaging (MPI) is an emerging tracer imaging tech- of immune cells systemically and in solid tumors. However, existing nique that directly detects and quantitates superparamagnetic iron- methods for cell tracking that have generally been insufficient. Mag- oxide nanoparticles with exceptional contrast and high sensitivity at netic Particle Imaging (MPI) is a novel tomographic molecular im- millimeter-scale resolutions [5]. MPI’s contrast is similar to nuclear aging technique that can be used to non-invasively track iron-oxide medicine, but without the complex workflow, safety, and half-life lim- tagged immune cells in 3D in vivo, with contrast similar to nuclear itations of a radioactive tracer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 27 of 272 Methods conventional Treg and CTL populations in GIT tissue sections Here we describe how MPI and MFH can be combined to pro- from IBD patients versus normal individuals by multiplex duce spatially localized heating and accurate control of heating immunofluorescence. magnitude. Spatial localization is achieved using a unique Methods mechanism, magnetic localization. Localization is effected by Conventional Treg are typically defined as lymphocytes with a CD3+/ using a strong magnetic field gradient to produce a “field-free CD4+/CD25+/FoxP3+ immuno-phenotype. This complex antigenic region” (FFR) where nanoparticles are heated, while nanoparti- signature has made it difficult to definitively label Treg populations cles outside the FFR are quenched and do not heat. The use of in tissue sections by immunohistochemistry. We combined a 5-plex an FFR thus enables millimeter-scale control over which MNPs (CD3, CD4, CD8α, CD25, FoxP3) immunofluorescence assay using Ulti- are heated [6-8]. vue InSituPlex® multiplex technology with image analysis using Results Indica Labs HaloTM software to identify, localize and enumerate: 1) MPI is first used to quantitate the MNPs prior to heating, to enable total CD3+ T cells, 2) CD8α+ cytotoxic T lymphocytes (CTL) and 3) treatment planning and prediction of the heating dose. MFH can CD3+/CD4+/CD25+/FoxP3+ conventional Treg in formalin-fixed then be induced in target regions of interest located anywhere in paraffin-embedded (FFPE) sections of GIT from patients with UC and the body while avoiding regions containing MNPs that should not be CD versus controls. Using this approach, we were able to definitively heated, such as the liver or lymph nodes. identify and enumerate these immune cell populations on single Conclusions FFPE tissue sections from each specimen. Combined MPI-MFH enables new treatment workflows that ex- Results ploit spatially localized MFH and accurate control of heating We found greater Treg and CTL cell densities (cells/mm2) in colon magnitude. These workflows may resemble image-guided radi- from CD and UC patients versus controls and higher densities of Treg ation therapy or image-guided high-intensity focused ultra- and lower densities of CTL in small intestine from patients with CD sound. Combined MPI-MFH also prevents damage to nearby versus controls. healthy tissue while enabling new applications such as targeted Conclusions immunogenic stimulation. MPI-MFH also enables new heat- The Ultivue InSituPlex© assay was capable of discretely localizing actuation applications involving systemic injection of MNPs conventional Tregs and CTL in human tissues. This multiplex platform followed by local targeting such as local release of a drug [9] could be used to simultaneously localize Tregs and CTL in FFPE surgi- (break thermally labile bonds/nanocarriers) without requiring ac- cal resections and biopsies of neoplastic tissue as well. tive chemical targeting. While currently only available for small animal use, its underlying physics does not prevent its transla- References tion to human sizes 1. Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, Sung JJY, Kaplan GG. References Worldwide incidence and prevalence of inflammatory bowel disease in 1. Zaidi, N; Jaffee, E. J Clin Invest (2018). the 21st century: a systematic review of population-based studies. Lancet. 2. Sharma, P. et al. Curr Opinion Immunol (2016). 2018; 390:2769-78. 3. Jordan, A. et al. Journal of Magnetism and Magnetic materials, (1999). 2. Corridoni D, Arseneau KO, Cominelli F. Inflammatory bowel disease. 4. Latorre, M. Puerto Rico health sciences journal, 28(3) (2009). Immunol Lett. 2014; 161:231-5. 5. Gleich, B. & Weizenecker, J. Nature 435,1214–1217 (2005). 3. van Herk EH, Te Velde AA. Treg subsets in inflammatory bowel disease 6. Murase, K. Physica Medica, 29(6), 624-630 (2013). and colorectal carcinoma: Characteristics, role, and therapeutic targets. J 7. Hensley, D. Physics in Medicine & Biology, 62(9), 3483 (2017). Gastroenterol Hepatol. 2016; 31:1393-404. 8. Tay, Z. ACS nano, 12(4), 3699-3713 (2018). 4. Yamada A, Arakaki R, Saito M, Tsunematsu T, Kudo Y, Ishimaru N. Role of 9. Liu, J. F., Small, 14(44), 1802563 (2018). regulatory T cell in the pathogenesis of inflammatory bowel disease. World J Gastroenterol. 2016; 22:2195-205. Ethics Approval P49 Human tissues were obtained from the National Disease Research Use of Ultivue InSituPlex® multiplex immunofluorescence to Interchange (NDRI) with support from NIH grant U42OD11158. Tissues localize and quantify regulatory T lymphocytes in formalin-fixed were collected for research purposes under IRB-approved informed paraffin-embedded human tissue sections consent and collection procedures and provided to Pfizer in accordance 1 1 2 Shawn O'Neil, DVM, PhD , Renee Huynh , Courtney Hebert , Jamie with applicable government regulations and guidelines. 2 2 1 1 Buell , Sean Downing, PhD , John Jakubczak, PhD , Yutian Zhan, MS 1 2 Pfizer, Cambridge, MA, United States; Ultivue, Inc., Cambridge, MA, United States P50 Correspondence: Shawn O’Neil (llospo@gmail.com) Rapid high-plex staining and simultaneous imaging for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P49 immunophenotyping of tissue sections 1 2 1 Benjamin Pelz, PhD , Daniel Migliozzi , Diego Dupouy , Anne-Laure 3 3 2 Background Leblond , Alex Soltermann , Martin Gijs 1 2 The inflammatory bowel diseases ulcerative colitis (UC) and Lunaphore Technologies, Lausanne, Switzerland; École Polytechnique Crohn’s disease (CD) are chronic, relapsing inflammatory disorders Fédérale de Lausanne, Lausanne, Switzerland; Universitätsspital Zürich, of the gastrointestinal tract (GIT) that affect millions of individuals Zurich, Switzerland worldwide [1]. The pathogenesis of these disorders is thought to Correspondence: Benjamin Pelz (benjamin.pelz@lunaphore.com) involve dysregulation of mucosal immune homeostasis in the GIT Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P50 in response to environmental factors in genetically susceptible in- dividuals [2]. Regulatory T cells (Treg) are CD4+ T lymphocytes Background that play a central role in peripheral immune tolerance, actively The tumor microenvironment plays a vital role in cancer development. inhibiting inflammation upon antigenic stimulation. There are two Multiplex immunostainings allow studying the interaction of different cell major populations of Treg: conventional Treg and TR1 cells [3]. types in the tumor microenvironment using a single tissue slide. Though Conventional Treg arise from the thymus (tTreg) or can be in- several techniques are available to perform high-plex stainings, they re- duced in the periphery (pTreg); both tTreg and pTreg constitu- quire intensive manual handling, are highly time consuming or not com- tively express FoxP3 and CD25 (IL-2Rα). An imbalance in patible with tissue sections on standard microscope slides. Here we conventional Treg and effector T cells in the GIT microenviron- present a fully automated microscope integrated method for rapid high- ment is thought to play a part in the pathogenesis of inflamma- plex sequential fluorescent immunostaining and imaging of tissue tory bowel disease (IBD) [4]. Thus, we sought to quantify sections. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 28 of 272 Methods P51 Formalin-fixed, paraffin-embedded tissue sections underwent manual dewax- Phenotypic and spatial analysis of inter- and intra-tumor ing and antigen retrieval step. All subsequent steps of staining, antibody elu- heterogeneity using multiplexed ion beam imaging tion and imaging were automated on the microscope integrated (MIBI) 1 2 2 microfluidic device. A single tissue section was stained sequentially for 24 dif- Jason Ptacek, PhD , Robert Johnson, PHD , Joann Palma, PHD , Jay 1 1 1 1 1 ferent immunophenotyping and tissue structural markers. Each staining cycle Tarolli , Rachel Finck , Murat Aksoy , Yi Zhang , Jessica Finn , Jason consisted of incubation of the tissue section with a pair of mouse and rabbit Ptacek, PhD 1 2 primary antibodies, followed by the corresponding fluorescently labelled sec- Ionpath, Inc, Menlo Park, CA, United States; AbbVie, North Chicago, IL, ondary antibodies and DAPI. The section was imaged after each staining United States cycle and subsequently eluted before staining the next pair of markers. Correspondence: Jessica Finn (jessica.finn@ionpath.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P51 Our microscope integrated microfluidic system allowed automated 24-plex staining with conventional primary and fluorescently labelled secondary Background antibodies in less than five hours, including image acquisition steps. The Elucidating both the cell types present in the tumor microenvir- microfluidic tissue processor enabled fast fluidic exchange and thereby re- onment and the spatial relationship between immune and sulted in reduced staining time down to 10-12min per marker. Integration cancerous cells is at the forefront of immunotherapy research. of a window into the microfluidic chip allowed direct tissue imaging under To address this, MIBI has been developed to image up to 40 the microscope avoiding the removal and mounting of the slide. Protocol markers at single cell resolution. optimization resulted in a high signal to background noise ratio for each Methods marker and complete elution of antibodies from the previous staining step. Staining of 10 NSCLC formalin-fixed paraffin embedded (FFPE) A comparison of a 10-plex staining with standard chromogenic stainings tissue sections was performed similarly to traditional IHC ex- on sequential sections showed high concordance for the stained area on cept that a panel of 20 metal labeled antibodies were stained tonsil as well as lung cancer tissue sections (Figure 1). simultaneously. The tissue was imaged at subcellular resolution Conclusions using an ion beam and time-of-flight secondary ion mass spec- With the microscope integrated microfluidic system, it is possible to trometry (ToF-SIMS). The masses of detected species were then perform fast multiplex stainings including image acquisition without assigned to target biomolecules given the unique label of each the need to handle the tissue slide. Moreover, due to the sequential na- antibody and multi-step processing and segmentation were ture of the system it would be easily possible to further increase the performed to create images of the TME and enable quantitative number of markers in the multiplex staining. We foresee this technique metrics of different cell subsets. to greatly facilitates the execution of high-plex stainings and thereby Results the discovery of novel tumor-microenvironment interactions. Control samples imaged at study start and end showed con- sistent marker quantification (inter-run R2>0.99), indicating MIBI staining and acquisition is reproducible and robust. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. Highly expressed nuclear, membrane, and cytoplasmic markers were utilized in conjunc- tion to accurately determine cell boundaries in tissue images. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relation- ships between cells of different types. At the highest level, cells were classified as positive for markers that are indicative of immune and tumor cells based on measured intensities of marker expression, such as CD45+ and keratin+ cells in epi- thelial cancers, respectively. Co-expression of markers were used to classify immune cells into subsets, including T cells and macrophages (Figure 1). Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. Conclusions MIBI offers high-parameter capability, at sensitivity and reso- lution uniquely suited to understanding the complex tumor im- mune landscape, including the spatial relationship of immune and tumor cells and the expression of immunoregulatory proteins. Fig. 1 (abstract P50). Automated microfluidics-assisted multiplexing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 29 of 272 Conclusions Anti-ILT3 mAb treatment induced a conversion from intra- tumoral immune suppression to activation in a SK-MEL-5 hu- NSG tumor model. CyTOF in anti-ILT3 drug discovery holds promise to effect a paradigm-shift in our ability to under- stand MOA and evaluate the impact of therapeutic interven- tions that can accelerate biomarker discovery and drug development. The anti-ILT3 mAb activity in human immune cells in vitro and tumor efficacy in vivo is presented in a companion poster. Ethics Approval The study was approved by Merck Institutional Animal Care and Use Committee, approval number 2022-200518-FEB. P53 Pixelwise H-score: a novel digital image analysis-based metric to quantify membrane biomarker expression from IHC images Amy-Jackson Fisher, Pamela Whalen, Cory Painter, Pamela Vizcarra, Eric Powell, MD, Sripad Ram, PhD Pfizer, Inc., San Diego, CA, United States Correspondence: Sripad Ram (sripad.ram@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P53 Fig. 1 (abstract P51). Cell segmentation and classification Background Immunohistochemistry (IHC) assays play a central role in evaluat- ing biomarker expression in tissue sections for diagnostic and re- P52 search applications. Manual scoring of IHC images, which is the CyTOF in anti-ILT3 mAb drug discovery - humanized tumor model current standard of practice, is based on qualitative criteria and selection and in vivo PD/biomarker exploration are known to have several shortcomings in terms of reproduci- Yujie Qu, MD, Alan Byford, Caniga Michael, Ying Huo, Barbara Joyce- bility and scalability to large scale studies. While digital image Shaikh, BS, Laurence Fayadat-Dilman, Veronica Juan, Carl Mieczkowski, analysis (DIA) based approaches hold significant promise to over- Laura Bald, Jeanne Baker, Michael Meehl, Scott Pruitt, MD, PhD, Stephen come these limitations, current DIA methods pose several chal- Alves, Lily Moy, Philip Brandish, PhD, Jie Zhang-Hoover, Jie Zhang- lenges that have limited their widespread use in analyzing Hoover clinical samples. Merck, Boston, MA, United States Methods Correspondence: Jie Zhang-Hoover (jie.zhang-hoover@merck.com) We introduce a novel DIA metric, the pixelwise H-score (pix H- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P52 score), that quantifies biomarker expression from whole-slide scanned IHC images. Pix H-score is unique in that it does not rely Background on the detection of individual cells or the delineation of subcellu- ILT3 on human monocytic myeloid cells is linked to immune tol- lar compartments (e.g. nucleus and cell membrane) which are ne- erance in transplantation and immune suppression in cancer. cessary for traditional scoring algorithms such as the H-score. All Anti-ILT3 mAb is being developed as a cancer immunotherapy to DIA metrics are calculated using either commercially available reverse the suppression and increase T cell activation. To evaluate (HALO, Visiopharm) or open-source (QuPath) digital pathology the effect of an anti-ILT3 mAb in vivo, we sought to select an ap- software packages. propriate humanized tumor model and identify immune activa- Results tion signatures in the model that are associated with the We compute the pix H-score, the ATM score [1] and the trad- treatment efficacy. itional H-score [2] from IHC images for several biomarkers includ- Methods ing PD-L1. Our results show that the pix H-score exhibit tight Humanized tumor models were generated by subcutaneously concordance to multiple orthogonal measurements such as mRNA implanting Panc 08.13 or SK-MEL-5 tumor cells in NSG mice levels and pathologist score, and provide consistently better per- engrafted with human cord-blood CD34+ hematopoietic stem cells formance over other DIA metrics. (hu-NSG). Tumor-bearing mice were treated with a human-mouse Conclusions chimeric anti-ILT3 mAb. Single cell mass cytometry (CyTOF) that sim- We anticipate that the new metric introduced here will be ultaneously quantifies over 40 cell surface and intracellular markers broadly applicable to quantify biomarker expression from a was used to phenotype tumor infiltrating cells (TILs) in these tumor wide variety of IHC images. Although not shown here, the new models. metric can also be applied to immunofluorescence images. Results Moreover, these results underscore the benefit of digital image The CyTOF phenotyping of untreated mice showed an overall im- analysis-based approaches which offer an objective, reprodu- mune suppressive environment in the tumor in both Panc 08.13 and cible and highly scalable strategy to quantitatively analyze IHC SK-MEL-5 hu-NSG models. ILT3 expression was detected in both images. models. However, the levels of ILT3 expression on CD14+ myeloid cells and percentage of CD14+ myeloid cells among TILs were higher in SK-MEL-5 compared to Panc 08.13 tumors, which led to the selec- References tion of the SK-MEL-5 model for further exploration. Anti-ILT3 mAb 1. Choudhury KR, Yagle KJ, Swanson PE, Krohn KA, Rajendran JG, A Robust treatment in the SK-MEL-5 hu-NSG model increased activation of Automated Measure of Average Antibody Staining in CD14+ myeloid sub-populations in TILs by viSNE CyTOF clustering Immunohistochemistry Images. J Histochem Cytochem. 2010; 58: 96-107. analysis. Furthermore, the treatment increased levels of CD69 and 2. Hatanaka Y, Hashizume K, Nitta K, Kato T, Itoh I, Tani Y, Cytometrical HLA-DR expression on CD4+ T cells, while reducing the percentage image analysis for immunohistochemical hormone receptor status in of naïve CD4+ T suppressor cells in CD45+ TILs. breast carcinomas. Pathol Int. 2003; 53: 693-699. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 30 of 272 P54 P55 Development of a 9-color immunofluorescence assay using Combining the best of two worlds: Transfer of multiplex tyramide signal amplification and multispectral imaging for immunofluorescence images from non-small cell lung carcinoma patients high-throughput studies on FFPE tissue sections into pseudo multiplex chromogenic immunohistochemistry images 1 2 1 1 Bethany Remeniuk, PhD, Carla Coltharp, PhD, Kristin Roman, MS, Lorenz Rognoni, PhD ,Ana HidalgoSastre, PhD ,Linda Brützel , Philipp Wortmann , 1 1 1 3 Chichung Wang, Clifford Hoyt, MS Monika Baehner ,Marco Testori ,Jessica Chan , Bonnie Phillips, PhD , Katir Patel, 3 3 4 4 1 Akoya Biosciences, Hopkinton, MA, United States PhD , Sean Downing, PhD , Alex Haragan ,JohnField , Florian Leiss, PhD 1 2 3 Correspondence: Clifford Hoyt (choyt@akoyabio.com) Definiens AG, Munich, Germany; Definiens, Munich, Germany; Ultivue, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P54 Cambridge, MA, United States; Liverpool University Hospital, Liverpool, United Kingdom Background Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) In cancer research, advancing our understanding of the under- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P55 lying mechanisms driving disease progression is key to devel- oping new therapeutic regimens and improving patient Background outcomes. Over the past several years, multiplex immunofluor- One of the biggest challenges in multiplex chromogenic IHC (mIHC) is to escence (mIF) has played a vital role in elucidating novel accurately identify and quantify double positive cells. Multiplex immuno- immune-tumor interactions and identifying targets of interest fluorescence (mIF) instead, allows for visualization of plenty of biomarkers for drug discovery and development. at once with true co-localization. However, visualizing tissue morphology Emerging studies utilizing mIF have revealed complex cell-to- in mIF images can be challenging and the vast color combinations over- cell interactions within the tumor microenvironment (TME), whelming. Pathologists are key to retrieve biological information from however, greater interrogation of the biology comprising multiplex assays and provide annotations for assay validation. To support these interactions, including cellular composition and pathologist analysis, resections of non-smallcelllungcarcinoma (NSCLC) functional status, require higher levels of multiplexing. With patients were stained with mIF and displayed as pseudo mIHC images. the rapidly increasing number of available multiplexing ap- Additionally, consecutive slides were stained with a mIHC panel. PD-L1 proaches, there is an inherent tradeoff between capability positive macrophages from the pseudo mIHC images were quantified and throughput. and compared to the readouts identified in the real chromogenic IHC. In this study, we demonstrate a streamlined workflow to Methods develop and optimize a 9-color assay on the Leica BOND RX™ 7 formalin-fixed paraffin-embedded (FFPE) resections from NSCLC patients autostainer. This methodology offers an optimal balance be- were stained using Ultivue’s UltiMapper I/O PD-L1 kit and I/O PD-1 kit and tween multiplexing and sample throughput to facilitate research whole image scans were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss). and support translational studies on whole formalin-fixed Consecutive slides were stained with a multiplex chromogenic panel (in- paraffin-embedded (FFPE) tissue. cluding CD68, CD8, PD-1) at Mosaic Laboratories (1) and scanned with an Aperio AT Turbo scanner (Leica). Images were analyzed using an automated Methods workflow for quantitative multiplex image analysis developed at Definiens Forthe9-colorassay,Opal™ fluorophores were used on serial (2). Afterwards, mIF images were converted into pseudo mIHC images. Pa- sections of lung cancer FFPE tissue. The panel was designed on thologists annotated double positive macrophages for CD68 and PD-L1 on Akoya’s Mantra 2 semi-automated multispectral microscope, both images. Results were compared with automatically detected double which allows for rapid analysis of staining performance. Once positive cells and across assays. In addition, pathologists qualitatively optimized, multispectral images were acquired on both the assessed visual similarity of real and artificial chromogenic images. Mantra 2 and Vectra Polaris of the same tissue regions and an- Results alyzed to show equivalence between the platforms. Cell counts, Pathologists annotated double positive macrophages for CD68 and densities, and spatial parameters were generated using Akoya’s PD-L1 markers on both images (mIF and pseudo mIHC). Results were inForm imageanalysissoftwareand theRscript package compared with those obtained using artificial intelligence to auto- phenoptrReports, which produces quick, summarized outputs of matically detect double positive cells and across assays. In addition, the image analysis data. These same analyses were also used pathologists qualitatively assessed visual similarity of real and artifi- to evaluate reproducibility of all markers when run in a high- cial chromogenic images (Figure 1). throughput process. Conclusions Results Transferring mIF into pseudo mIHC images helps to combine the advan- Dynamic range of measured per cell signals for all markers had tages from both approaches: true colocalization of biomarkers whilst main- a median of 200:1. Agreement between the Mantra 2 and taining tissue morphology, facilitating visual evaluation of digital images Vectra Polaris-based measurements was generally >95% when by pathologists. This technology could be used to complement research, comparing cellular expression signals and cell counts based on clinical routine diagnostic, drug development and biomarker discovery. cell phenotyping classifiers. Cross talk was undetectable after spectral unmixing despite significant spectral overlap inherent References in a 9-color assay. Reproducibility across three batches of five (1) Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of serial sections of lung cancer tissue was generally <10% coeffi- inflammatory cell patterns and densities using multiplex immunohistochemistry cient of variation for all markers in the assay, supporting a immuno-oncology assays [abstract]. In: Proceedings of the AACR-NCI-EORTC high-through process of approximately 20 resection samples per International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct day. 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Conclusions Suppl): Abstract nr B069. We have successfully established a standardized process for 9- (2) Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated color multiplexing that offers a balance between elucidating the quantification of whole-slide multispectral immunofluorescence images to intricate cellular biology driving disease progression and thera- identify spatial expression patterns in the lung cancer microenvironment. peutic responsiveness within the TME while simultaneously SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. providing a practical and reliable assay that can be imple- Ethics Approval mented to support translational, high-throughput studies in clin- Ethical approval was granted by the Liverpool Research Ethics Committee, ical research. reference number 97/141. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 31 of 272 using matched 3,3′-Diaminobenzidine chromogenic and single bio- marker fluorescent controls tonsil tissue, which were validated using tumor samples from patients with high grade glioma. We characterized the spatial arrangement of myeloid subpopulations, ex vivo and corre- lated the changes in spatial orientation, quantity and localization of cells to the tumor. We determined that dynamic re-arrangement of myeloid cells can be observed under pressure of immunotherapy within the tumor, and confirm both a time-dependent and dose- dependent effect of oHSV-1 on this immune cell modulation. Conclusions These data suggest a unique, multiplexed approach to study spatial ar- rangement of myeloid and T-cell populations and their spatial distribu- tion within tumors under basal growth conditions or in the presence of anticancer immunotherapies, which may implicate the activity of mye- loid cells with treatment responses. These findings could impact per- sonalized cancer immunotherapy for patients receiving care. Ethics Approval The samples were collected under IRB approval. Consent The samples were collected under written patient consent for publi- cation of this abstract. P57 An ex-vivo human system elucidates a role for natural killer cells in the anticancer effect of drug combinations in triple negative breast cancer Fig. 1 (abstract P55). mIF and pseudo mIHC 1 2 2 Aaron Goldman , Douglas Best , Saravanan Thiyagarajan , Misti Jain, 2 2 1 2 PhD , Basavaraja Shanthappa , Munisha Smalley, PhD , Hans Gertje, BS , Aaron Goldman P56 1 2 Brigham and Women's Hospital; Mitra Biotech, Woburn, MA, United States Interrogating the effect of oncolytic Herpes simplex virus-1 on Correspondence: Aaron Goldman (goldman1@mit.edu) spatial arrangement of myeloid cells in glioblastoma multiforme Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P57 using an ex vivo human system and multiplex immunohistochemistry 1 2 2 Background Munisha Smalley, PhD , Misti Jain, PhD , Saravanan Thyiagarajan , Emily 3 3 4 2 Response and resistance to cancer therapy relies on the presence of ac- Alonzo , Katherine Crosby , Douglas Best , Hans Gertje, BS , Basavaraja 2 5 5 1 tive immune cells in the tumor microenvironment, which recalibrate Shanthappa , Ralph Pulchalski , Charles Cobbs , E. Antonio Chiocca , 1 1 1 the body’s own defense largely by modulating exhaustion of cytotoxic Sean Lawler, PhD , Aaron Goldman , Munisha Smalley 1 2 lymphocytes including T cells and natural killer (NK) cells. However, Brigham and Women's Hospital, Woburn, MA, United States; Mitra there is a critical gap in our understanding for the role of immune cells Biotech RxDx, Bangalore, India; Cell Signaling Technologies, Danvers, to drive response or resistance to drugs and immunotherapies at the MA, United States; University of Birmingham, Birmingham, United individual patient level. This is primarily due to limitations in complex Kingdom; Swedish Neuroscience Institute, Seattle, WA, United States tumor-immune interfaces that exist in many current tumor models. Correspondence: Aaron Goldman (goldman1@mit.edu) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P56 Here, we deployed an ex-vivo human system that uses an explant of native, patient-derived solid tumors including autologous immune cells. Background Utilizing biopsied tumor tissue from patients diagnosed with triple- The role of myeloid cell populations within a tumor and their contribu- negative (ER- PR- HER2-) breast cancers (TNBC, N=7), we studied drug- tion to effective cancer immunotherapy is emerging with considerable induced cell death (cleaved caspase-3) and spatial heterogeneity of NK interest. However, assessing the role of intratumoral myeloid cells cells (CD3-CD56+PanCK-) using multiplex immunohistochemistry under therapy pressure has it’s challenges. Here, we implemented a (mIHC). Spatial orientation of cells in the microenvironment, including multiplex immunohistochemistry (mIHC) panel and a human ex vivo proximity of NK to tumor and NK cell density within regions of the system to interrogate key myeloid subsets as they affiliate with infiltra- tumor vs. stroma were performed using HALO-based quantitative ana- tion and activation of an emerging immunotherapy for glioblastoma lyses. Finally, we deployed in-vitro co-culture studies using 3-D TNBC multiforme (GBM) – oncolytic Herpes simplex virus-1 (oHSV-1). mIHC organoids and human-derived NK cells (NK-92MI). combined with advancements in digital pathology and machine learn- Results ing algorithms have enabled identification, quantification and spatial First, we report the ability of the ex-vivo human system to retain the orientation of multiple cell types in a single field of view (FOV). spatial orientation and total population of natural killer cells and T- Methods cells over the course of a 72h explant culture. Next, using Spearman Cell Signaling Technology antibodies (CD3e,D7A6E™, ID: 85061), (CD68, correlation analyses and principal component analysis (PCA), we de- D4B9C, ID: 76437), (CD11c, D3V1E, 49420), (MHC Class II (HLA-DRB) LGII- termined that drug response to both immunotherapy and conven- 612.14), (Pan-Keratin, C11, 4545) were optimized for mIHC staining, tional cancer drugs, indicated by high incidence of cleaved caspase-3 using a tyramide signal amplification approach to pin-point, in a single after drug pressure, is directly associated with changes to the tumor- FOV: intratumoral T-cells, defined by CD3e+; macrophages, defined by NK cell proximity and density of NK cells within the tumor bed vs. CD68 and conventional dendritic cells defined by CD11c+MHCII +, in the stroma. Finally, using the 3-D tumor organoid cultures with NK relation to the surrounding tissue architecture defined by pan cytokera- cells, we determined that activity and tumor cytolysis by NK cells is tin. These biomarkers were integrated with incidences of oHSV-1 infil- hampered through cancer cell-activated cytokines, which diminish tration and replication (via expression of green fluorescent protein). expression of activating biomarkers including NKG2D/C. Results Conclusions First, we confirmed an optimized protocol for treating GBM ex vivo Taken together, these results provide a method to study the spatial with oHSV-1 such that tissue viability, infiltration and replication of arrangement of immune cells in an entirely human system, which the virus are optimal. The staining of the mIHC panel was optimized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 32 of 272 can be perturbed with anticancer drugs to reliably influence the ex- P59 pression and growth patterns of immune cells. We further demon- Highly consistent automated multiplex immunofluorescence for strate that this strategy can help to guide in-vitro studies to further immunoprofiling of solid tumors in clinical trials: assay validation elucidate mechanisms of action of drugs, which influence response study using multispectral imaging and digital analysis 1 2 2 vs. resistance via immune cell activity. Michael Surace, PhD , Lorenz Rognoni, PhD , Farzad Sekhavati , Andrew 2 2 2 1 Ethics Approval Fisher, PhD , Andreas Spitzmueller , Sara Batelli, PhD , Karma Dacosta , 2 3 4 Anonymous breast cancer tissue samples were collected under IRB Vinay Pawar , Clifford Hoyt , Edwin Parra, MD, PhD , Jaime Rodriguez- approval with due written consent from each patient. Canales, MD 1 2 AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, 3 4 Germany; Akoya Biosciences, Hopkinton, MA, United States; UT - MD P58 Anderson Cancer Center, Houston, TX, United States Brain MRI performed within 4 weeks of PD-1 inhibitors as a Correspondence: Jaime Rodriguez-Canales potential prognostic marker for non-small cell lung cancer (rodriguezcanalesj@medimmune.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P59 Ammar Sukari, MD, Misako Nagasaka, MD, Seongho Kim, PhD, Tahmida Chowdhury, Natasha Robinette, MD Background Karmanos Cancer Institute, Detroit, MI, United States Novel multiplex immunofluorescent (mIF) platforms have been devel- Correspondence: Ammar Sukari (sukaria@karmanos.org) oped for immunoprofiling of solid tumors to understand the tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P58 microenvironment and to identify biomarkers for immunotherapy. One of these methods employ IF and tyramide signal amplification (TSA) to Background generate between 4 to 9-marker multiplex panels analyzed with multi- PD-1 inhibitors aim to re-instate the natural anti-cancer immune- spectral imaging. Although this method can provide reliable data, they mediated cytotoxicity. Although PD-1 inhibitors are now consid- can show variability in consistency depending on the markers [1], ered part of standard of care treatment in advanced metastatic which affects the reliability of mIF for use in clinical trials. The goal of NSCLC [1], little is known about the effects of PD-1 inhibitors on this study was to develop and validate a highly consistent mIF method asymptomatic central nervous system (CNS) metastases. We hy- for its use in clinical trials in the pharma and academic environments. pothesized that early MRI brain imaging due to the development Methods of neurological signs and symptoms following the initiation of A mIF panel for the analysis of carcinomas was optimized using an PD-1 inhibitor may help delineate a subset of NSCLC patients automated stainer (Leica) and automated multispectral scanner (Po- with asymptomatic and undiagnosed CNS metastases prior to ini- laris, Akoya Biosciences). The markers included keratins (AE1/AE3), tiation of therapy and may predict for worse outcomes. CD68, PD-L1, PD1, CD8 and Ki67. Each primary antibody was first per- Methods formed on standard chromogenic IHC according to previously vali- Data from NSCLC patients who received at least one dose of PD- dated protocols. The mIF panel was developed with a secondary 1 inhibitors between September 2013 through the data cut-off of antibody detection and TSA, using specific fluorophores for each May 2017 were captured from our institution’s pharmacy data- marker. Once optimized, the miF panel was tested on serial sections base. The primary objective was to describe the characteristics of of formalin-fixed human tonsil controls and six non-small cell lung patients with MRI brain being performed within 4 weeks of the carcinomas, including replicates, together with standard IHC staining first dose of PD-1 inhibitors and the secondary objectives were of each individual marker for comparison. A set of three repeats on estimation of progression free survival (PFS) and overall survival different days for each mIF with all cases was performed to test (OS) for the same population. consistency and reproducibility of the mIF method. All slides were Results digitally scanned and analyzed using Automated Definiens Insights 140 NSCLC patients received at least one dose of PD-1 inhibitors Platform with custom algorithms (Definiens AG, Munich, Germany), prior to data cut-off. Median age was 64 (range: 24-86). 83 (59%) comparing the cell populations in the serial section slides between were male. 64 (46%) were treated on a clinical trial. There were standard IHC and mIF, and between mIF repeated rounds. The data 92 (66%) adenocarcinoma, 41 (29%) squamous cell carcinoma was statistically analyzed using Pearson’s correlations. (SCC) and 7 (5%) poorly differentiated NSCLC. 84 (40%) had a Results pre-PD1 inhibitor MRI brain performed and 25 (18%) had been Using an automated workflow, the mIF data compared with standard diagnosed with baseline CNS metastases. 128 (91%; Group 1) did IHC showed correlations between 0.83 to 0.99. The data from the not have an MRI brain performed within 4 weeks of starting PD-1 three rounds of mIF performed on different days showed correlations inhibitors, while 12 (9%; Group 2) patients did. 9 out of 12 pa- between 0.89 and 0.99. The marker that showed the lowest correl- tients had new or worsening CNS metastases. Of the 9, 1 had ation was CD68 (r=0.83), the possible cause was the difficulties on WBRT, 1 had gamma knife, 1 went onto hospice while a decision cell segmentation due to morphologic irregularity shown by macro- was made to monitor imaging and symptoms in 6 patients. The phages. Overall our present data showed a much higher consistency median PFS was 5.28 months (95% CI, 3.90 to 8.03) and 1.75 than our previously published results using a non-automated mIF months (95% CI, 1.08 to NE) for Group 1 and Group 2, respect- protocol, which correlations ranged from 0.17 to 0.87[1]. ively. The median OS was not reached (95% CI, 15.38 to NE) and Conclusions 5.77 months (95% CI, 2.85 to NE) for Group 1 and Group 2, Our data demonstrates that using an automated workflow including respectively. automated staining, scanning and analysis, and with properly vali- Conclusions dated IHC markers, mIF becomes a highly consistent methodology In this retrospective analysis, patients who had MRI brain within and it is compatible for its use with clinical trial tissue specimens. 4 weeks of starting PD-1 inhibitors had worse outcomes. Trial Registration Not applicable Reference 1. NCCN Clinical Practice Guidelines in Oncology. Non-Small Cell Lung Can- Reference cer. Version 5. 2019- June 7, 2019. https://www.nccn.org/professionals/ 1. Parra ER, Uraoka N, Jiang M, Cook P, Gibbons D, Forget MA, Bernatchez physician_gls/default.aspx#site, last accessed 7/30/2019. C, Haymaker C, Wistuba II, Rodriguez-Canales J. Validation of multiplex Ethics Approval immunofluorescence panels using multispectral microscopy for immune- The study was approved by the Wayne State University Institution's Ethics profiling of formalin-fixed and paraffin-embedded human tumor tissues. Board, approval number 062616M1E. Sci Rep. 2017; 7:13380-13391. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 33 of 272 P60 P61 Combining transcriptomic immune population inference with Segmentation and classification of single cells using multiplexed automated digital masking of H&E images finds immune effectors ion beam imaging preferentially distribute within stroma regions Jay Tarolli, Rachel Finck, Murat Aksoy, Yari Sigal, Noah Newgren, Jessica 1 2 2 Christopher Szeto, PhD , Mustafa Jaber, PhD , Liudmila Beziaeva , Kevin Finn, Jason Ptacek, PhD 1 1 1 Kazmierczak , Steve Benz , Shahrooz Rabizadeh IONpath, Menlo Park, CA, United States 1 2 ImmunityBio, Santa Cruz, CA, United States; NantOmics, Culver City, Correspondence: Jay Tarolli (jay@ionpath.com) CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P61 Correspondence: Steve Benz (Steve.Benz@nantomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P60 Background When studying the tumor microenvironment, knowing not only Background the types of immune cells present but also the spatial distribu- Multiple methods to characterize immune-cell populations in tion and relationship of these immune cells to other immune and tumor microenvironment (TME) are being assessed as potential tumor cells provides crucial information. In the past, techniques biomarkers of immunotherapy response. These include manual used to analyze these spatial relationships have been limited by pathological assessment of lymphocyte infiltration, immunohisto- the number of biomarkers that could be simultaneously mea- chemical (IHC) staining for specific adaptive response markers sured. Recently, with the development of multiplexed ion beam such as CD8, and more recently transcriptomic-based deconvolu- imaging (MIBI), 40+ biomarkers can be simultaneously measured tions of immune populations such as xCell and TIMER. Here we in a single scan [1]. By probing with an ion beam, tissue sections combined digital masking using deep-neural nets with transcrip- can be imaged at a spatial resolution on the same order of mag- tomic deconvolution to infer where immune-subpopulations may nitude as light based techniques, providing subcellular resolution. reside in the TME. This combination of multiplexed biomarker measurements and Methods subcellular spatial resolution enables segmentation of the image An unselected set of 187 clinical samples from the ImmunityBio data- into individual cells, making possible subsequent cell type classifi- base were analyzed. Each had H&E stained diagnostic slides with cation and quantification. pathologist-annotated tumor regions, as well as deep whole- Methods transcriptomic sequencing (>200M reads). Deep neural networks pre- Samples of placenta, lung, tonsil, lymph node, thymus, and liver viously trained on TCGA slide images were used to generate digital were imaged with MIBI. Segmentation of these images was per- spatial masks for 3 characteristics: tumor-content, lymphocytes, and formed in two steps. First, a MaskRCNN [2] model was trained stroma. Patients were scored based on the presence of intratumoral to utilize multiplexed MIBI data to predict the location of cell lymphocytes (iTIL) and stromal lymphocytes (sTILs). Immune subpop- instances in a MIBI image for a single class of objects by learn- ulations were inferred from RNAseq expression of published ing features from a set of nuclear, cytoplasmic, and membrane immune-cell-specific genesets [1,2], as was Wnt-signaling level [3]. markers. The centroids of each predicted cell instance were Significant associations between immune subpopulations and level used as seed points and, after manual refinement of these seed of infiltration were analyzed. points, watershed segmentation was performed to determine Results boundaries between instances. Both the summed intensity of a Manually annotated positive tumor regions were accurately digitally marker as well as a weighted cell score which accounts for the masked as >83% tumor or lymphocyte. Wnt signaling was strongly spatial distribution of a marker’s expression throughout a cell associated with overall stromal content (Rho=0.47, p<0.0001). Strong instance were calculated and were used for cell type anti-correlation was observed between levels of sTILs and iTILs classification. (Rho=-0.42, p<0.0001), and remained significant when including Results overall stroma area as a covariate. Digital lymphocyte masks some- Cell population and densities were calculated for a number of what correlated with RNAseq-based deconvolution of lymphocyte different cell types, including T cells, B cells, and macrophages classes (Rho=0.30, p=0.0001) in line with reports from others [4], based on a combination of one or more coexpressed biomarkers however this decreased when comparing lymphocyte count within present within segmented cells. Figure 1 shows an example FOV annotated tumor regions only (Rho=0.17, p=0.03), despite high con- with several cell types classified in a single image. Expression of cordance of lymphocyte counts within and outside of annotated re- immunoregulatory proteins including PD-1 and PD-L1 were quan- gions overall (Rho=0.82, p<0.0001). RNAseq-based lymphocyte levels tified and assigned to specific cell types. Finally, nearest neighbor were more associated with sTILs than iTILs (Rho=0.19 vs. -0.28, p< distances between various cell types were determined to 0.01 respectively). characterize the spatial organization of cell populations within Conclusions each tissue image. Adaptive response effectors such as NK and T-cells are found Conclusions more resident in surrounding stromal tissue than infiltrating The ability to characterize the many different cell types within tumor tissue. Increased Wnt/B-catenin signaling in stromal re- the tumor microenvironment is made possible by the highly mul- gions, reported by others as immunosuppressive, may sequester tiplexed nature of MIBI data, the subcellular spatial resolution of immune effectors and aid in immune escape. the image data, and downstream analysis tools, including com- puter vision approaches, which enable cell segmentation, classifi- References cation, and spatial analysis. 1. Bindea G, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 2013; 39:782-795. References 2. Danaher P, et al. Gene expression markers of tumor infiltrating 1. Keren L, Bosse M, Marquez D, Angoshtari R, Jain S, Varma S, Yang S, leukocytes. J Immunother Cancer. 2017; 5:18. Kurian A, Van Valen D, West R, Bendall S, Angelo M. A Structured 3. Slattery ML,et al. Expression of Wnt-signaling pathway genes and Tumor-Immune Microenvironment in Triple Negative Breast Cancer their associations with miRNAs in colorectal cancer. Oncotarget. 2018; Revealed by Multiplexed Ion Beam Imaging. Cell. 2018; 174:1373- 9:6075. 1387. 4. Pai SG, et al. Wnt/beta-catenin pathway: modulating anticancer immune 2. He K, Gkioxari G, Dollár P, Girshick R. Mask R-CNN. IEEE International Con- response. J Hematol Oncol. 2017; 10:101. ference on Computer Vision (ICCV). 2017; 2961-2969. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 34 of 272 combinations for a seven-color panel. Using this method, the number of slides was reduced to three per target (18) plus con- firmation seven-color slides resulting in a panel containing CD3, CD8, CD68, Cytokeratin, FOXP3 and PD-L1 (Figure 2). Conclusions Multiplex IF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interaction/co-localization of multiple targets within a single tissue specimen. MIF panels can take eight or more weeks to optimize, however, researchers can save time and resources using validated antibodies and this antibody order guide. Fig. 1 (abstract P62). See text for description Fig. 1 (abstract P61). See text for description Table 1 (abstract P62). See text for description P62 Bringing the tumor microenvironment into focus: Simplified development of seven-color multiplex immunohistochemistry- immunofluorescence (mIF) panels Melissa Whiteman, PhD, Eric McIntush, PhD, Mike Spencer Bethyl Laboratories, Montgomery, TX, United States Correspondence: Mike Spencer (mspencer@bethyl.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P62 Background For the advancement of immunotherapeutics, the need to under- stand the tumor microenvironment has never been more press- ing. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers. This is critical in instances where sample is limited, such as a tumor bi- opsy or other clinical specimen. The applications of mIF are nu- merous, and span clinical, translational, and basic research applications. A seven-color mIF can take eight weeks, or more, to develop. Herein, we describe a simplified, faster approach. Methods FFPE human tissue was stained with PathPlex™ Panel 4 IHC vali- dated primary antibodies (Bethyl Laboratories [A810-004]), mouse or rabbit HRP-conjugated secondary antibodies (Bethyl Laborator- ies [A90-116P, A120-501P]) and detected using Opal™ Polaris 7- color IHC kit fluorophores (Perkin Elmer [NEL861001KT]). Primary antibody order was optimized utilizing tissue microarray serial sections, and three slides per target by staining after the first, third, or sixth heat-induced epitope retrieval (HIER). All three slides were imaged using the same exposure time and analyzed Fig. 2 (abstract P62). See text for description for target/nucleus counts, signal intensity, and background. Fi- nally, the order was tested in the seven-color mIF and compared to single stain for confirmation. Whole slide scans were gener- ated using the Vectra Polaris® and analyzed using InForm® image analysis package. P63 Results Clinical assay development and validation of multiplex Development time of a seven-color mIF was reduced using IHC immunofluorescent (mIHC) marker panel for evaluation of tumor validated antibodies and the optimized dilution. Antibody order infiltration myeloid cells in FFPE tissue sections 1 1 2 was guided by results of three slides stained after first, third or Lan Yi, PhD , Jonathan Juco, MD , Ashhad Mahmood, MD , Omar 1 1 1 sixth HEIR. The ratio of target staining/DAPI nuclear counts, aver- Laterza , Charo Garrido , Lan Yi, PhD 1 2 age intensity and overall background predicts the optimal order Merck & Co., Inc, Hillsborough, NJ, United States; Diagnostic Pathology of staining. Some targets reveal larger average area staining, Services, LLC, Skillman, NJ, United States higher intensity and lower background when stained last, for ex- Correspondence: Omar Laterza (omar_laterza@merck.com); Charo ample FOXP3 (Table 1, Figure 1), while the inverse may be true, Garrido (charo.garrido@merck.com) or no effect for other targets. There are 720 possible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P63 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 35 of 272 Background staining pattern of more than 20 parameters. The high-parameter anti- Myeloid-derived suppressor cells (MDSCs) are a group of leuco- body panel used was optimized with human FFPE tonsil tissues. Human cytes with myeloid origin and immune-suppressive function. lung cancer FFPE tissues were then analyzed with this same panel. Cell Ample recent evidence supports key contributions of MDSC to clustering using the MAV software identified tens of cell types within tumor progression through immune-mediated mechanisms. the tumor tissues. Immune cell sub-types were mapped onto the ori- MDSCs include two major subsets based on their phenotypic and ginal image data to assess infiltration and spatial associations. morphological features: polymorphonuclear (PMN)-MDSC and Conclusions monocytic (M)-MDSC. However, these cells remain less studied Unlike other cyclic IF approaches involving multiple antibody staining than T lymphocytes as their phenotypical, morphological and and stripping steps, the CODEX platform involves a single initial functional heterogeneity generate confusion in investigation and staining step and subsequent gentle and relatively fast manipulation analysis of their role. of the tissue thereafter. This provides a superior workflow and pre- Methods vents tissue degradation. CODEX data from various normal and can- With the progresses on multiplex IHC assay technology, we are now cer human FFPE tissue types is shown here with corresponding able to develop multiplex immunofluorescent marker panels to single-cell analysis of key tissue features. Overall, the CODEX platform evaluate the expression and localization of the main subpopulations is an accessible and versatile technology for high parameter, spatial of myeloid cells in the tumor microenvironment. profiling of tissue specimens. Results We have developed and validated CD14, CD66b, CD163 and P65 MHCII (HLA-DR) IHC multiplex marker panel to evaluate the main Mutation-targeted T cell responses in blood from patients with populations of myeloid cells, along with their activation status. solid tumors prior to treatment and which evolve with clinical After completing the validation for individual markers in a single benefit from anti-PD-1 therapies chromogenic IHC platform, we optimized the incorporation of 1 2 1 1 Benjamin Yuen, PhD , Fangfang Yin, PhD , Duo An, PhD , Boi Quach , each marker into the multiplex platform. In parallel, a multiplex 1 2 1 Linlin Guo, PhD , Joanne Tan, PhD , Songming Peng, PhD , Zheng Pan, image analysis algorithm (APP) was generated and validated to 1 1 1 1 PhD , Olivier Dalmas, PhD , Robert Bao, PhD , Kyle Jacoby, PhD , Barbara quantify each subpopulation of myeloid cells in the tumor area. 1 1 2 Sennino, PhD , Stefanie Mandl, PhD , Matt Walters, PhD , Juan Jaen, Last, fit for purpose analytical validation, including sensitivity, spe- 2 1 1 PhD , Alex Franzusoff, PhD , Benjamin Yuen, PhD cificity and precision was successfully carried out. 1 2 PACT Pharma, South San Francisco, CA, United States; Arcus Conclusions Biosciences, Hayward, CA, United States This validated assay is currently being used to support multiple on- Correspondence: Songming Peng (speng@pactpharma.com) going and future clinical trials. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P65 P64 Background Highly multiplexed single-cell spatial analysis of FFPE tumor T cells targeting tumor-exclusive neoepitopes (neoE) have been pos- tissues using CODEX® tulated to represent the primary mediators of clinical benefit for pa- Jessica Yuan, PhD, Gajalakshmi Dakshinamoorthy, Joseph Kim, Sejal tients with solid tumors treated with immunotherapies. Identifying Mistry, Nadya Nikulina, Roya Bashier, Cassandra Hempel, Maria Elena and tracking these T cells in patients can help to understand the Gallina, Julia Kennedy-Darling, Jessica Yuan, PhD mechanism for immune checkpoint inhibitor therapies, as well as Akoya Biosciences, Menlo Park, CA, United States provide new therapeutic candidates for personalized adoptive cell Correspondence: Julia Kennedy-Darling (j.kennedy@akoyabio.com) therapies. However, this has been hampered by the low frequency of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P64 neoE-specific T cells in peripheral blood. To this end, we demonstrate the use of the imPACT Isolation Technology®, an ultra-sensitive high- Background throughput technology, to capture neoE-specific CD8 T (neoE-T) cells Characterizing the complexities of the tumor microenvironment is elem- from peripheral blood. In addition, this technology can be utilized to ental to understanding disease mechanisms. The spatial relationships be- quantify and monitor neoE-T cells longitudinally during therapy. We tween infiltrating immune cells and the remodeling of the cellular matrix show here preliminary data applying the imPACT technology to clin- is widely recognized as a key component to defining tumor heterogen- ical trial samples for the characterization of mutation-targeted T cell eity. Current methodologies for analyzing the spatial dimension in tissues, responses from patients associated with clinical benefit. like traditional immunofluorescence (IF) and immunohistochemistry (IHC), Methods are limited to a few parameters at a time, restricting the scope of identifi- Peripheral blood mononuclear cells (PBMC) from patients with non- able cells. Conversely, single-cell technologies like mass cytometry and small cell lung cancer (NSCLC) and treated with combinations con- NGS-based tools provide multiplexing capabilities, but at the expense of taining an anti-PD-1 antibody were analyzed. Briefly, tumor-exclusive the associated spatial information. Here, we present the analysis of hu- neoE-HLA target candidates were predicted and barcoded snare li- man lung cancer FFPE tissues with CODEX using a panel of more than 20 braries comprising personalized neoE-HLA reagents were produced markers targeting the tumor microenvironment. for capture of neoE-specific CD8+ T cells from PBMCs. Longitudinal Methods analysis of neoE-T cells responses throughout the duration of treat- The CODEX technology, developed by Akoya, is comprised of a fluidics ment was performed to obtain valuable information on neoTCR se- instrument that interfaces with existing microscope hardware, as well as quences and neoE-T cell quantification & phenotype. a suite of reagents and associated control and analysis software. The Results CODEX technology involves labeling antibodies with oligonucleotide- A baseline neoE-specific CD8 T cell profile was identified in all sub- based Barcodes followed by a single staining step. Around 40 parameters jects prior to treatment. Among NSCLC subjects exhibiting objective can be measured within a single tissue through fully-automated, iterative responses to therapy, some neoE-T cell clones identified at baseline cycles of adding and removing corresponding dye-conjugated Reporters. persist in the blood and/or diversify in clonality over the course of Here, we apply this technology using a panel of antibodies targeting im- treatment. In some circumstances, new neoE-T cell clones have mune, cancer and other architectural features to measure cell subsets in emerged on treatment with anti-PD-1. Furthermore, phenotype ana- cancer FFPE tissues. Image data is processed using the CODEX analysis lysis suggested the neoE-T cells captured from blood have been acti- pipeline, including clustering, annotation and mapping of cell types to vated, indicating previous encounter with their respective neoE-HLA the original image data with the Multiplexed Analysis Viewer (MAV). targets. Results Conclusions The CODEX technology was used to ascertain complex cellular niches The imPACT technology was used to assess the phenotype & quan- and spatial associations between multiple cell types based on the tity of neoE-specific T cells in blood of trial participants over time. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 36 of 272 This approach revealed the evolution of mutation-targeted T cell re- Conclusions sponses in participants with clinical benefit and may prove to be a Future plans include analyzing the remainder of patients enrolled in powerful tool to provide mechanistic understanding of immune re- the trial and the utilization of CD8+ and MDSC-specific CyTOF panels sponses associated with clinical benefit. These data support further to further classify the aforementioned subpopulations to further elu- testing of the neoE-T cell capture technology, with the potential to cidate this relationship. This methodology offers insight into the pro- uncover the identity of neoE-specific T cells pre-existing in the blood gression of vaccine-induced patient immune responses and exhibits of patients and the evolution of immune attack to cancer. promise as a platform that may be extrapolated to other Ethics Approval immunotherapies. The study was approved by the institutional review boards or ethics committees of the participating sites in Arcus Biosciences’ clinical References studies. 1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW. The 2016 World Health Organization Classification of Tumors of the Cen- P66 tral Nervous System: A summary. Acta Neuropathol. 2016; 131:803– A novel mass cytometry-based immunomonitoring platform for characterizing the peptide vaccine-induced immune response of 2. Chheda Z, Kohanbash G, Okada K, Jahan N, Sidney J, Pecoraro M, Yang X, HLA-A*0201+ patients with K27M+ diffuse midline gliomas Carrera D, Downey K, Shrivastav S, Liu S, Lin Y, Lagisetti C, Chuntova P, 1 1 1 Jared Taitt, BA , Payal Watchmaker, PhD , Takahide Nejo, MD, PhD , Neil Watchmaker P, Mueller S, Pollack I, Rajalingam R, Carcaboso A, Mann M, 2 1 1 Almeida , Kaori Okada , Sabine Mueller, MD PhD , Hideho Okada, MD, Sette A, Garcia C, Hou Y, Okada H. Novel and shared neoantigen derived 1 1 PhD , Jared Taitt, BA from histone 3 variant H3.3K27M mutation for glioma T cell therapy. J University Of California, San Francisco, San Francisco, CA, United States; Exp Med. 2017; 215:141-157. George Washington University, Washington DC, United States Ethics Approval Correspondence: Sabine Mueller (sabine.mueller@ucsf.edu); Hideho The study was approved by UCSF IRB #: 16-20574 Okada (hideho.okada@ucsf.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P66 P67 Background Use of a regional integrated health record data network to identify Diffuse midline glioma, including diffuse intrinsic pontine glioma patients who received checkpoint therapy following cancer (DIPG) constitutes up to 20% of pediatric brain cancer and has a me- diagnosis as a foundation for exploring immunotoxic events dian survival of 9-10 months. Given the proximity of DIPG to paren- Theresa Walunas, PhD, Carlos Galvez, Saya Jacob, Jeffrey Sosman, MD, chymal regions that play vital homeostatic functions, surgical Abel Kho resections are often restricted in size and scope, leaving irradiation Northwestern University, Chicago, IL, United States and chemotherapy as the primary management options. The on- Correspondence: Theresa Walunas (t-walunas@northwestern.edu) going development of immunotherapy has shown significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P67 promise in many fields, including that of gliomas. Genetic studies revealed that greater than 70% of DIPG cases harbor an amino Background acid substitution from lysine (K) to methionine (M) at position 27 Immune related adverse events (irAE) occur in >80% of patients of histone 3 variant 3 (H3.3). We previously identified a novel receiving immune checkpoint inhibitors (ICI). Currently, most HLA-A*02:01-restricted neoantigen epitope encompassing the data about the incidence of irAE comes from clinical trials with H3.3K27M mutation. Accordingly, we have implemented a pilot restrictive eligibility requirements. With the wide use of ICI ther- vaccine through the Pacific Pediatric Neuro-Oncology Consortium apy as standard of care for many cancers, it is important to as- (PNOC). sess incidence of irAE in a general patient population. The Methods Chicago Area Patient Reported Outcomes Research Network Twenty-nine newly diagnosed DIPG patients who are HLA-A2+ and (CAPriCORN) is a clinical data research network containing med- H3.3K27M+ underwent radiation therapy, and then received the ical records for >9.5M patients who receive care in 11 institu- H3.3K27M peptide vaccine and tetanus toxoid (TT) peptide emulsified in tions spanning diverse patient populations and healthcare Montanide in combination with poly-ICLC every 3 weeks for a total of 24 settings [1]. Using CAPriCORN, we wanted to determine whether weeks. Our objective is to characterize vaccine-induced H3.3K27M-spe- we could identify a large, diverse cohort of patients who re- cific CD8+ T-cell and myeloid-derived immunosuppressive subpopula- ceived ICIs as a foundation for exploring the incidence of irAE tions in peripheral blood mononuclear cells utilizing a novel H3.3K27M- in a real-world data source. specific dextramer-based mass cytometry (CyTOF) method [1,2]. Methods Results We identified all patients within CAPriCORN who were 19-88 Through this approach, the temporal expansion of vaccine-reactive years old, had a diagnosis for an ICI-approved cancer, and re- CD8+ T-cells was observed in all patients who completed a minimum ceived an ICI from 1/1/2011 through 12/31/2018. Clinical experts of 24 weeks on the study (n = 4). Simultaneously, this expansion was identified the International Classification of Disease 9 and 10 not observed in 4 of 5 patients who withdrew from the regimen due codes used to document cancer diagnosis and the RxNorm [2] to progression. These T-cells were clustered on a tSNE plot using ca- codes for each ICI documented as a medication ordered in the nonical CD8+ T-cell activation markers and further classified by their medical record (Table 1). The query was developed against the expression profiles, revealing distinct effector memory, central mem- PCORnet Common Data Model version 4.1 [3], validated on the ory and transitional effector subpopulations. Chronological monitor- Northwestern University site node in CAPriCORN and distributed ing of these groups indicates the time course-dependent to all CAPriCORN sites. Six of 9 sites returned counts. Data was development and persistence of vaccine-reactive exhausted and ef- centrally aggregated and stratified by age, race, sex and therapy. fector memory CD8+ T-cells in 3 of the 4 initial patients analyzed. All data are aggregated counts. Furthermore, an analogous clustering and phenotyping approach Results was used for myeloid cells, allowing for the identification of myeloid- As shown in Table 2, we identified 6,541 patients within CAPri- derived suppressor cell (MDSC) subpopulations. A comparative ana- CORN who received ICI therapy for cancer. 45% are female, 75% lysis revealed a positive correlation between two monocytic myeloid- identify as white, 13% African American, 2% Asian and 1% Native derived suppressor cell (M-MDSC) subpopulations and progression- American, and 86% are 51-83 years of age. The most well repre- free survival. sented cancers were Non-small Cell Lung Carcinoma (50%) and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 37 of 272 Metastatic Melanoma (18%). Overall, 67% received anti-PD1 ther- P68 apies, followed by combination ICI therapies, anti-PDL1 and anti- Innate inflammatory pathways are associated with TIL growth and CTLA4, though usage varied within cancer types. response to adoptive immunotherapy 1 1 1 Conclusions Suhendan Ekmekcioglu, PhD , Dai Ogata, MD, PhD , Caitlin Creasy, MS , 1 1 1 Our results demonstrate that a large cohort of cancer patients who Marie Forget, PhD , Sun-Hee Kim, PhD , Jason Roszik, PhD , Mike 3 1 1 have received ICI therapy can be identified in an integrated medical Spencer , Patrick Hwu, MD , Elizabeth Grimm, PhD , Chantale 1 1 record data environment that spans 11 institutions in a major urban Bernatchez, PhD , Suhendan Ekmekcioglu, PhD center. This population is racially diverse, represents both sexes, a The Univeristy of Texax MD Anderson Cancer Center, Houston, TX, wide range of ages and includes all cancer types approved for ICI United States; Bethyl Laboratories, Inc, Montgomery, TX, United States therapy as of 2018. This real-world cohort will be an effective founda- Correspondence: Suhendan Ekmekcioglu tion on which to explore the incidence of irAE, particularly rare irAE (sekmekcioglu@mdanderson.org); Chantale Bernatchez that require large sample sizes to investigate. (cbarnatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P68 Acknowledgements The investigators would like to acknowledge the CAPriCORN network for Background query support. This work was supported by Patient Centered Outcomes Immune infiltration of T cells (TIL) into the melanoma microenviron- Research Institute (PCORI) CDRN-1306-04737. ment has been associated with improved survival for some patients, and also has been exploited to grow TIL in vitro for adoptive therapy. References However, prognostic significance of immune infiltrating cells in melan- 1. Kho AN, Hynes DM, Goel S, et al. CAPriCORN: Chicago Area Patient- oma and other tumors remains a relatively new concept, and markers Centered Outcomes Research Network. J. Am Med Inform Assoc. 2014; related to suppressive versus active functional TIL remain unclear. We 21:607-611. previously reported that in Stage III melanoma patients’ tumors, posi- 2. https://pcornet.org/download/pcornet-common-data-model-v4-1- tive expression of CD74 together with low or absent Macrophage Mi- specification-5-may-2018/?wpdmdl=1919&refresh= gration Inhibitory Factor (MIF) associates with favorable prognosis [1]. 5d42596fab3e11564629359 Methods 3. https://www.nlm.nih.gov/research/umls/rxnorm/ From an ongoing clinical trial using TIL intended for adoptive immuno- Ethics Approval therapy, we have studied the melanoma patient tumors specimens The study was approved under the CAPriCORN IRB, CHAIRb: Research Protocol (FFPE) from 20 patients whose autologous TIL lines grew to sufficient #14120201 “CAPriCORN Clinical Data Research Network Master Protocol”. number for possible use clinically. We also examined another 20 sets of melanoma tumor from which the TIL did not grow or not grow well. We analyzed the differences in the two groups of tumors (40 total FFPE) for Table 1 (abstract P67). See text for description CD74 regulated pathway features and inflammatory marker expression. Results CD74 regulated markers included CD44, MIF, and downstream in- flammatory targets including inducible Nitric Oxide Synthase (iNOS) and Nitrotyrosine (NT). Our findings confirm our previous report in that tumor CD74 expression significantly associates with favorable OS and PFS (both, p=0.0038) and provides new data that in this set of patients the CD74 also correlates with best irRC of TIL treated pa- tients. New findings include that the NT expression in tumor cells as- sociated with poor TIL growth (p=0.014), as well as lack of clinical response to TIL treatment (p=0.02). We have also found that tumor cell-derived MIF and iNOS expression correlate with unfavorable prognosis for both OS and PFS (p=0.016 and 0.018, respectively). Conclusions We have identified the protein expression of CD74, MIF and of iNOS as providing survival information, and proposed that CD74+/MIF-/iNOS- to- gether be considered to form a "signature" of good prognosis in general melanoma outcomes as well as TIL growth and favorable responses for these patients. Use of this signature for selecting patients for entry into TIL and possibly other immunotherapy trials, as well as research on the differential pathways of IFN-γ signaling in melanoma appear as important areas for future mechanistic research to improve patient outcome. Table 2 (abstract P67). See text for description Reference 1. Ekmekcioglu S, Davies MA, Tanese K, Roszik J, Shin-Sim M, Bassett RL Jr, Milton DR, Woodman SE, Prieto VG, Gershenwald JE, Morton DL, Hoon DS, Grimm EA.Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and Its Expression Associates with Favorable Sur- vival for Stage III Melanoma. Clin Cancer Res. 2016 Jun 15;22(12):3016-24. P69 Highly multiplexed single cell spatial analysis of the tumor microenvironment in lymphoma 1 1 2 Monirath Hav, MD, PhD , Anthony Colombo , Erik Gerdtsson , Mohan 2 2 2 2 Singh , Denaly Chen , Imran Siddiqi, MD PhD , James Hicks , Peter Kuhn, 2 1 1 PhD , Akil Merchant, MD , Akil Merchant, MD 1 2 Cedars Sinai Medical Center, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States Correspondence: Akil Merchant (akil.merchant@cshs.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P69 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 38 of 272 Background Diffuse large B cell lymphoma (DLBCL) being the most subtype of non-Hodgkin lymphoma. Despite evidence of expression of PDL-1 on lymphoma cells, less than 10% of DLBCL patients re- spond to PD1 therapy [1]. We hypothesize that a better characterization of spatial architecture of the tumour microenvir- onment (TME) in lymphoma will help explain differences in re- sponses to PD1/PDL-1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cy- tometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [2]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for rele- vant immunophenotypes, the spatial architecture of those pheno- types and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype (Figure 1A). Immune cell represented 33% of the cells represented by CD4 (36%), CD8 (30%), macro- phages (26%) and TREG (8%) (Figure 1B). Immune cell infiltra- tion in individual tumor samples ranged from 7% to 75% with marked heterogeneity. (Figure 1C-D. Analysis of immune Fig. 1 (abstract P69). See text for description marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure 1E) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macro- phages, and tumor cells (Figure 2A). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure 2B). Meta-clusters 2, 6, 8 and 11 were the 4 most dominant patterns of CD8 spatial interaction in the TME. Each CD8 spatial interaction pattern is dis- tinctive with case to case heterogeneity (Figures 2C-D). Risk as- sessment analyses of spatial clusters 1, 2 and 4 (“hazardous”)had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 (“protective”) (Figure 2E). In the “protective” spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREG pheno- types, with opposite in “hazardous” areas (Figures 3A-B). TIM-3 expression was high both on T cells and tumor cells in the “haz- ardous” neighborhoods. Conclusions Our novel approach to spatial analysis of the immune architecture re- veals clinically relevant insights into the TME. References 1. Ansell, S. M. et al. Nivolumab for Relapsed/Refractory Diffuse Large B-Cell Lymphoma in Patients Ineligible for or Having Failed Autologous Trans- plantation: A Single-Arm, Phase II Study. J. Clin. Oncol. 37, 481–489 (2019). 2. Giesen, C. et al. highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. methods | 11, 417 (2014). Ethics Approval Fig. 2 (abstract P69). See text for description The study was approved by USC IRB, approval number HS10-260 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 39 of 272 of PD-L1+ immunoreactivity in epithelial/tumor cells, increased im- munoreactivity for PD-L1 in CD68+ cells, and a closer relationship be- tween intra-epithelial and stromal CD8+ cells with PD-L1+ cells. In addition, areas in CRC and CD heavily infiltrated by immune cells or with tertiary lymphoid structures contained clusters of PD-L1+ cells that were negative for both CD68 and pan-Cytokeratin. Most of the CD3+ cells in non-lesional CD were PD-1 negative, except around ter- tiary lymphoid structures. In contrast, a greater percentage of CD3+ cells were also PD-1+ in CRC, and more so in lesional CD tissue. Conclusions The Ultivue UltiMapper multiplex fluorescence immunohisto- chemistry platform was effective in characterizing the PD-L1/PD- 1 axis and T cell exhaustion environment in FFPE tissue, in part due to the ability to clearly identify more complex immune cell phenotypes than traditional multiplex IHC. The application of the UltiMapper assays demonstrated many similarities between marker and cell type distribution between lesional colonic CD and CRC. P71 Refining tumor mutation burden values using variant expression Shannon Bailey, PhD, Muhammad Ekram, PhD, Jim Lund, Jeffrey Gulcher WuXi NextCODE Genomics, Arlington, MA, United States Correspondence: Jeffrey Gulcher (jgulcher@wuxinetcode.com) Fig. 3 (abstract P69). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P71 Background P70 Tumor mutation burden (TMB) is used as a surrogate marker for the Tissue-based characterization of T cell exhaustion in inflammatory neoantigen load of a tumor, and many studies have shown that TMB bowel disease and colorectal cancer using multiplex IHC predicts the success of immune-oncology (IO) treatments for cancers, 1 1 1 Marina Bleck, PhD, Diane Mierz , Ania Mikucki , Marie Marcher , Sidharth such as anti-PD-1 or anti-CTLA4 therapy. While IO treatment of pa- 1 1 2 Kerkar, MD , Gerald Nabozny, PhD , Sean Downing, PhD , Alexander tients with high TMB has led to success and excitement in the field, 1 1 Klimowicz, PhD , Alexander Klimowicz, PhD not all high TMB patients respond to IO treatment. TMB can be de- 1 2 Boehringer Ingelheim, Ridgefield, CT, United States; Ultivue, termined using next-generation sequencing, and both panel and Cambridge, MA, United States whole-exome DNA sequencing have been used to measure the mu- Correspondence: Alexander Klimowicz tation load of a tumor. (alexander.klimowicz@boehringer-ingelheim.com) Because all genes are not expressed in every cell, inclusion of the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P70 mutations found in unexpressed genes may confound the utility of TMB to predict neoantigen load. In this study, we explore improving Background TMB by taking into account both DNA mutation and RNA expression T cell exhaustion and the PD-L1/PD-1 checkpoint axis has been ex- with the hypothesis that using both criteria will lead to a TMB bio- tensively characterized in peripheral blood mononuclear cells and in marker that correlates better with neoantigen load. human tumor tissues. This has provided a better understanding of Methods the role this pathway plays in tumor immunology and of its clinical We examined DNA and RNA sequencing data for different cancer types in utility in predicting responsiveness to checkpoint inhibitor therapies. The Cancer Genome Atlas (TCGA) to determine refined TMB values. The T cell exhaustion is not only associated with tumor progression, but data were assessed to identify mutations with and without expression. has recently been associated with better prognosis and milder course Results of disease for a number of autoimmune and autoinflammatory disor- We found that a significant faction mutations included in a standard ders. We set out to characterize and contrast the T-cell exhaustion TMB calculations reside in genes that are not expressed, and this frac- environment between colonic Crohn’s disease (CD) and colorectal tion varies significantly among samples. A corrected TMB that incorpo- cancer (CRC). We applied the Ultivue UltiMapper multiplex fluores- rates gene expression is likely to be a better predictor of neoantigen cence IHC platform to capture complex immune cell phenotypes and load. In addition, our group has previously identified TCGA samples provide a more in depth characterization than traditional IHC. with allele specific expression in which up to 25% of the tumor muta- Methods tions are not expressed despite significant expression of the gene. The Commercially sourced FFPE surgical resections from n=5 colonic CD pa- fraction of unexpressed mutations is much higher in some cancer tients (matched lesional and non-lesional tissue) were compared to n= types. Adding this correction to the TMB calculation, including only var- 5 CRC tumor resections (3 hot and 2 cold tumors) using the Ultivue Ulti- iants that are expressed in the tumor in the TMB calculation, changes Mapper multiplex fluorescence immunohistochemistry platform. Two the average TMB values found among different cancer types, and cor- UltiMapper kits were used to evaluate the T cell environment in these rects the TMB in individual samples. This refined TMB value provides a tissues: UltiMapper I/O PD-L1 panel included the markers CD8, CD68, biomarker that reclassifies samples scored as high TMB to low TMB and PD-L1, and pan-Cytokeratin/Sox10; UltiMapper I/O PD-1 panel included is likely to better predict response to IO treatment. the markers CD3, CD45RO, PD-1, and pan-Cytokeratin/Sox10. All assays Conclusions were stained on Leica BOND RX autostainers. Whole-slide images were As our results suggest, adding RNA sequencing can be used to im- acquired on a ZEISS Axio Scan.Z1 slide scanner. Image analysis was per- prove the TMB biomarker to better separate treatment groups. The formed using Indica Labs HALO software. initial analysis was performed with TCGA exome data and is now be- Results ing extended to refine TMB values generated from gene panels in- Contrasted to non-lesional CD tissues, several similarities were ob- cluding TSO 500. We are also evaluating differences in tumor- served between CD lesional tissue and CRC, including the presence infiltrating lymphocytes in based on the refined TMB value. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 40 of 272 P72 attractive targets for immunotherapy. Identifying the full comple- Comprehensive and accurate prediction of presented neoantigens ment of peptides derived from a protein presented on a major class-I using ImmunoID NeXT and advanced machine learning algorithms HLA restriction provides a vital step toward increasing the speed and Dattatreya Mellacheruvu, PhD, Rachel Pyke, Charles Abbott, PhD, Nick viability of many immunotherapeutic strategies. Advances in next- Phillips, Rena McClory, John West, MBA, Richard Chen, Sean Boyle, PhD, generation sequencing (NGS) and single-cell technologies have en- Dattatreya Mellacheruvu, PhD abled the accurate capture of somatic mutations accumulated by a Personalis Inc., Menlo Park, CA, United States tumour, yet a significant hurdle remains how this information can be Correspondence: Sean Boyle (sean.boyle@personalis.com) utilized for immunotherapeutic benefit. Identifying which somatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P72 mutations produce neoantigens is crucial in providing the link be- tween genetic change and immunological impact. Background Methods Comprehensive detection of potential neoantigens and accurate pre- Directly identifying potential neoantigens using mass spectrometry diction of their MHC presentation are critical prerequisites for selecting offers a significant improvement over traditional approaches based neoepitopes that can be used for creating personalized cancer vac- on prediction. However, the relatively high sample requirement of cines. However, prediction models developed using in-vitro MHC- this approach inherently limits the depth of analysis that can be per- peptide binding assays cannot model upstream presentation machin- formed, with a significant risk that low abundance neoantigens are ery, such as proteasome cleavage and peptide loading. Advances in not detected. immuno-affinity purification followed by mass spectrometry (IP-MS) By integrating multiomics data from over 1000 experiments in 200 have enabled direct detection of MHC-bound peptides and can there- immortalised cell lines, we have generated a database of over two mil- fore be used for modelling native MHC-peptide presentation. Further, lion unique HLA-peptide sequences that offers near total coverage of genetically engineered cell lines that express a single HLA allele enable the protein-coding genome. Our comprehensive HLA class-I peptide unambiguous HLA-peptide assignment. Here, we present an overview atlas has been used as a reference tool to aid direct identification of of our MHC presentation prediction framework based on a large collec- neoantigens by targeted mass spectrometry, to probe indirectly for the tion of such mono-allelic cell lines and discuss its utility in conjunction presence of neoantigens, and to explore how many common driver with ImmunoID NeXT, our commercially available exome scale DNA mutations associated with cancer interact with the immunopeptidome. and RNA sequencing and analytics platform specifically designed to en- Results able the development of immuno-therapies. We have identified hundreds of neoantigens directly by mass spec- Methods trometry and found that mutated proteins follow the same pattern Mono-allelic cell lines were generated from K-562 null-HLA parental of antigen processing and presentation as their unmutated equiva- cells by transfecting each of the selected alleles. Cells were grown, lents. As a result, our HLA peptide atlas offers significant value in pre- screened for surface expression, lysed and immuno-affinity purified dicting the likelihood of a somatic mutation creating a neoantigen. using a column coated with HLA class I (W6/32) antibody. Peptides Comparing predicted neoantigens with those directly identified by were gently eluted and analyzed using LC-MS/MS. Peptide-to-spectrum mass spectrometry, we show effective prioritization of mutations by assignment was performed and filtered at 1% false discovery rate. accurately predicting the presence and relative abundance of neoan- Results tigens. Applying this process toward the five most commonly mu- The training data for our MHC presentation prediction framework were tated genes in cancer reveals a marked bias toward mutations that generated using a large collection of genetically engineered mono- either act negatively or are in ‘quiet’ areas of the immune landscape. allelic cell lines, encompassing approximately 60 HLA Class I alleles that As all mutated peptides contain novel amino acid sequence, and are are frequently present across various populations. The resulting hence able to elicit an immune response, this ability to convert ‘po- immuno-peptidomics data were comprehensive and of high quality - tential’ into ‘actual’ is crucial in establishing a mechanism for identify- the peptide yields were high (median of approx. 1600 unique peptides ing false positive results observed in cell-based assays. per allele) and the dominant motifs were in agreement with published Conclusions motifs. Our prediction framework is based on multiple modelling algo- An integrative multiomics approach to neoantigen identification rithms, including a multi-layer neural network, and uses proprietary and has delivered a powerful reference for developing novel standard features such as peptide sequence, peptide length, binding immunotherapies pocket sequence and abundance (measured by transcripts per million). We created allele-specific and pan-allele models and evaluated them P74 on an independent hold-out dataset. Both our allele-specific and pan- Integrating CD8 and CD4 effector neo-epitope content with allele models had superior performance compared to other public regulatory T cell epitope exclusion is a superior prognostic tools, with a higher precision across a range of recall (sensitivity) values. biomarker for bladder cancer patient compared to their tumor Conclusions mutation burden Our integrated pipeline for neoepitope discovery, which includes the 1 2 1 Guilhem Richard, PhD , Randy Sweis, MD , Matthew Ardito, BA , comprehensive profiling of putative neoantigens using ImmunoID 2 1 3 Tzintzuni Garcia , Leonard Moise, PhD , Michael Princiotta, MS, PhD , NeXT and accurate and sensitive prediction of MHC presentation of 3 1 3 Dominique Bridon , William Martin, BA MD , Gad Berdugo, MSc, MBA , such neoantigens across all HLA Class I alleles (using our pan-allelic 4 4 1 Arjun Balar , Gary Steinberg , Anne de Groot, MD models) enables the effective generation of neoepitopes that are crit- 1 2 EpiVax, Inc., Providence, RI, United States; University of Chicago, ical for developing personalized cancer vaccines. Chicago, IL, United States; EpiVax Oncology, New York, NY, United States; NYU Langone Health, New York, NY, United States P73 Correspondence: Gad Berdugo (gberdugo@epivaxonco.com) Large scale multiomics reveals a marked bias in driver mutations Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P74 toward areas not reliably presented to the immune system Alex Powlesland, PhD, Michael Cundell, PhD, Floriana Capuano, PhD, Background Brandon Higgs, David Lowne, BS, Ricardo Carreira, PhD We hypothesized that neo-epitope-based prediction using an ad- Immunocore Ltd, Abingdon, United Kingdom vanced in silico T cell epitope screening system (Ancer™) may better Correspondence: Alex Powlesland (alex.powlesland@gmail.com) identify patients with improved prognosis than tumor mutation bur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P73 den. Analysis of genomic data derived from the muscle-invasive bladder cancer (BLCA) cohort of The Cancer Genome Atlas (TCGA) Background database for CD4, CD8, and Treg neo-epitopes was performed to de- The repertoire of HLA-peptides presented to the immune system termine whether Ancer™ would improve prognostic stratification which derive from cancer-associated, viral, and mutated proteins are compared to tumor mutational burden (TMB). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 41 of 272 Methods BLCA patient mutanomes (n=412) were retrieved from the TCGA and evaluated with Ancer™, an innovative and automated neo-epitope screening platform that combines proprietary machine learning- based HLA I and HLA II neo-epitope identification tools with removal of inhibitory regulatory T cell epitopes for neo-epitope ranking and personalized cancer vaccine design. BLCA patients were separated based on median TMB or neo-epitope burdens. We investigated the effect of integrating both CD8 and CD4 neo-epitope burdens as most mutanome pipelines exclusively focus on the identification of Class I neo-epitopes. Overall survival was analyzed using the Kaplan-Meier method and differences analyzed by log-rank testing. Results Compared to low TMB, high TMB was significantly associated with improved survival (p = 0.0001, difference of 38.5 months in median survival, Figure 1). Improved differentiation of median survival times was obtained when separating patients based on their Class I neo- epitope content, as estimated by Ancer™ (p < 0.0001, difference of 59.8 months in median survival). Adding Class II neo-epitope burden further increased separation of OS times, showcased by a 69.6-month increase in median survival for BLCA patients with both high CD8 and high CD4 neo-epitope contents compared to other patients (p = 0.0001). Since we discovered that Class II neo-epitopes can induce in- hibitory responses, we further evaluated whether the screening of these detrimental sequences could improve our analysis. Upon iden- tifying Class II neo-epitopes likely to induce T effector (Teff) re- Fig. 2 (abstract P74). See text for description sponses, we found that the median survival of patients with high CD8 and high CD4 Teff contents was extended by nearly 4 months to 73.4 months compared, to the remainder of the cohort (p < 0.0001, Figure 2). P75 Conclusions targetSCAPE and ultraSCAPE: Simultaneous identification and deep Our analysis suggests that optimal host-immune recognition of profiling of human antigen-specific T cells and other immune cell CD8+, CD4+, and Treg epitopes plays a key role in cancer sur- subsets by mass cytometry 1 1 1 vival. While defining CD8 neo-epitope burden enhanced associa- David Roumanes, PhD , Faris Kairi , Alessandra Nardin, DVM , Evan 2 1 tions with OS, the inclusion of CD4 Teff neo-epitope burden Newell, PHD , Michael Fehlings 1 2 substantially helped identify long-term survivors. These results immunoSCAPE, Cambridge, MA, United States; Fred Hutchinson suggest that defining the number of true neo-epitopes using Cancer Research Center, Singapore, Singapore Ancer™ may represent a novel prognostic or predictive Correspondence: Alessandra Nardin biomarker. (alessandra.nardin@immunoscape.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P75 Background During clinical trial immune monitoring, especially in the field of im- munotherapy, it is critical to collect in-depth phenotypic information from multiple immune cell populations in order to assess the biological activity of the immunotherapy, to identify biomarkers of response or progression, and/or to identify new drug targets. However, patient sam- ples, for example peripheral blood mononuclear cells (PBMC) or tissues, are often only available in small amounts and current methods face limitations in either depth of analysis and/or cell throughput. Methods In order to identify therapy-relevant antigens and to facilitate a concur- rent in-depth characterization of cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling cap- abilities of cytometry by time of flight (CyTOF) and a unique method- ology allowing the identification and characterization of rare antigen- specific T-cell subsets (targetSCAPE). By implementing a new technol- ogy (ultraSCAPE) that combines flow and mass cytometry together with a combinatorial live cell barcoding strategy, we further increased the high-dimensional phenotyping capacities to over 100 different marker molecules through simultaneous in-depth profiling of up to three add- itional immune cell subsets from the same sample. Results We isolated 4 different immune cell populations from a single sample and combined 3 different phenotypic panels consisting of 35 makers each together with a combinatorial tetramer multiplex and phenotyp- ing panel for deep profiling of myeloid cells, NK cells, B cells and T cells. We demonstrate the potential of this novel immuno-phenotyping method, by tracking virus-specific T cells while simultaneously charac- Fig. 1 (abstract P74). See text for description terizing 4 immune cell subsets with over 100 distinct phenotypic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 42 of 272 markers from a single sample, which is currently impossible employing P77 modern flow cytometers or classical mass cytometry methods. Comprehensive profiling of tumor-immune interaction in anti-PD-1 Conclusions treated melanoma patients reveals subject-specific tumor escape With its ability to provide an unprecedented picture of the immune mechanisms 1 1 1 1 status within a single sample, including T cell specificity information Charles Abbott, PhD , Eric Levy, PhD , Rachel Pyke , Rena McClory , 2 1 1 and in depth profiling of relevant immune cell subsets, ultraSCAPE in Sekwon Jang, MD , Richard Chen, PhD , Sean Boyle, PhD 1 2 combination with targetSCAPE can provide detailed insights on the Personalis, Menlo Park, CA, United States; Inova, Fairfax, VA, United effects of immunotherapy on the immune cell population. Informa- States tion learned from in-depth immune phenotyping of several immune Correspondence: Sean Boyle (sean.boyle@personalis.com) cell subsets such as T, NK and myeloid cell subsets can be leveraged Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P77 for the development of novel diagnostics, for biomarker discovery and for monitoring therapeutic strategies in immunotherapy. Background Checkpoint inhibitor therapy has demonstrated meaningful antitu- mor activity for many patients, though the majority fail to achieve P76 complete response. Thus, it is of particular interest to identify bio- Development of immunopeptidomic platform for human leucocyte markers and mechanisms that promote positive response to im- antigens class I using microflow liquid chromatography and munotherapy. In the present study, we apply our comprehensive quadrupole time-of-flight mass spectrometry tumor immunogenomics platform (ImmunoID NeXT), integrating data 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Yoshinobu Koguchi, MD, from the tumor, tumor microenvironment and immune system to 2 2 2 2 PhD , John Cha , Brian Piening, PhD , Eric Tran, PhD , Hong-Ming Hu, create a comprehensive biological signature of patient response to 2 2 2 PhD , Bernard Fox, PhD , William Redmond, PhD therapy. 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Methods Cancer Center, Portland, OR, United States We characterized the immunogenomics of 52 unresectable, stage III/ Correspondence: Takashi Shimada (tashimada@shimadzu.com) IV melanoma patients who underwent anti-PD-1 therapy to assess Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P76 factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 12 months. Background For each patient, a single paired FFPE tumor and normal blood sam- The highly complex population of peptides associated with human ple was collected and profiled using Personalis’ ImmunoID NeXT plat- leucocyte antigens (HLA) is the human immunopeptidome. Compre- form; an augmented exome/transcriptome platform and analysis hensive characterization of the immunopeptidome is key in predict- pipeline, which produces comprehensive tumor mutation informa- ing immunotherapeutic responses by evaluating targets of T cell tion, gene expression quantification, neoantigen characterization, interaction and in developing the next generation of cancer im- HLA typing and LOH, TCR repertoire profiling and tumor microenvir- munotherapies. Mass spectrometry (MS) is a technology that holds onment profiling. Tumor molecular information was then analyzed significant promise for untargeted and complete identification of the together with clinical outcome. immunopeptidome. MS acquisition is mainly used an electrospray Results ionization (ESI) combined with nanoflow liquid chromatography (LC). Comprehensive profiling demonstrated that elevated pretreatment However, the analysis time and retention reproducibility could be is- neoantigen burden was predictive of response to PD-1 blockade, and sues. Therefore, we tried to develop an MS platform that can ensure significantly associated with progression-free survival. Additionally, the coverage while increasing throughput using a microflow LC. we observed increased response to anti-PD-1 therapy in patients Methods with elevated pretreatment TCR clonality. Patients with high neoanti- HLA class I complexes were purified from A431 cell lysate by W6/32 im- gen burden and TCR clonality that failed to achieve complete re- munoaffinity. Purified HLA peptides were eluted with 5% formic acid. sponse revealed potential resistance mechanisms to anti-PD-1 The peptides were fractionated by 10 kDa ultrafiltration. HLA peptides therapy. Specifically, we identified two patients with high expression were separated with L-column2 ODS (0.3x150 mm) using a trap-elute of IDO1 or CTLA4, which may facilitate PD-1-independent immune protocol of microflow LC (Nexera-Mikros) and quadrupole time-of-flight escape. Additionally, we found two patients with antigen presenta- MS (LCMS-9030). Flow rate was set at 5 μl/min with a gradient of aceto- tion machinery (APM) mutations. The first patient had independent nitrile in 0.1% formic acid for 18 min. MS/MS spectra were acquired HLA-A and HLA-B mutations, likely leading to loss of surface expres- using a data-dependent manner of top-10 precursor intensities. The sion of the proteins. In the second APM mutation patient we ob- precursor scan was first set from 400 to 600 Da. The charge states of served a high frequency (80% AF) frameshift variant in B2M, which precursors were set between 1 to 4, and MS/MS scan was from 200 to potentially prevents proper HLA class I folding and antigen presenta- 1200 Da. The data were analyzed by Mascot proteome server and tion. These APM mutations suggest reduced neoantigen presentation PEAKS sequencing software on SwissProt database. The mass toler- in these patients, which are probable mechanisms for tumor escape. ances of precursors and fragments were set at 0.05 Da and 0.3 Da. Min- By integrating neoantigen burden, HLA-LOH and APM mutational imal peptide length was set to 8 amino acids. data into a corrected neoantigen burden, we were able to increase Results the predictive strength of this biomarker. An initial round of optimizations was performed to establish optimal Conclusions parameters for immunopeptidome identifications. Using tryptic pep- In summary, our comprehensive cancer immunogenomic analyses tides from A431 lysate, we optimized the 50-100 msec repeat of MS/ demonstrate that genomic and immune profiling of pretreatment pa- MS scanning, top-10 of data-dependent acquisitions per scan, 50 Da tient samples can identify biomarkers and resistance mechanisms to scan range, 35V±10V spread of collision electrode voltage, and 3.0 kV immune checkpoint blockade, suggesting the potential efficacy of of electrospray voltage. These parameters were then applied for these as an integrated biomarker to optimize anti-PD-1 therapy pa- identification of HLA-associated peptides from A431 cells. From this, tient selection. we identified 4,217 MS/MS and 801 sequences from 34,042 spectra. Conclusions From these data, we demonstrate that similar sensitivity can be suffi- P78 ciently achieved with microflow platform as has been demonstrated Optimization of tumor nutation burden measurement in FFPE DNA preciously for nanoflow LC-MS. This has significant advantages in Janice Au-Young, PhD, Iris Casuga, PhD, Vinay Mittal, Dinesh Cyanam, terms of throughput, instrument maintenance, and widespread ap- MS, Elaine Wong-Ho, Fiona Hyland, Seth Sadis, Warren Tom, PhD plicability. Our future directions are to determine whether cancer Thermo Fisher Scientific, South San Francisco, CA, United States neoepitopes identified by these approaches may be recognized and Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) therapeutically targeted by patient T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P78 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 43 of 272 Background demonstrated [1]. Solid tumors systemically reprogram the lung Tumor mutation burden (TMB) measures the number of somatic mutations unique immune environment, dominated by intravascular neutro- and is a positive predictive factor for response to immune-checkpoint in- phil functions [2], to colonize this site. The concept of ‘oligopro- hibitors in multiple cancer types. While whole exome sequencing (WES) is gression’ has recently received mounting attention, due to its thegoldstandardfor TMBmeasurement,itisnot practicalfor routineuse. relevance and because it represents an interesting in vivo model TMB values measured using targeted sequencing have been shown to to study TIME, although the specific mechanisms of oligometa- have good correlation with WES. However, during FFPE preservation, DNA static process are relatively underinvestigated [3, 4]. may undergo cytosine deamination, resulting in false C>T substitutions Methods and elevated TMB values. We have assessed the effect of DNA damage RNA sequencing was performed on a retrospective collection of tis- andrepaironTMB values usingthe Oncomine TumorMutationLoad sue samples from primary renal cell carcinoma, melanoma, and Assay (OTMLA), a targeted next generation sequencing assay. NSCLC and paired lung oligometastases of untreated patients (Figure Methods 1). Enrichment of tumor-related pathways and transcripts that reflect We measured TMB from 37 FFPE colon, lung, endometrial and gastric tu- the enrichment of immune cell subsets was assessed by single sam- mors usingthe OTMLApanel on IonGeneStudiowith20ngofinput DNA ple gene set enrichment analysis. Differentially expressed genes be- from tumor only samples. The informatics workflow utilizes a custom vari- tween primary tumors versus the corresponding lung metastases ant calling and germline variant filtering algorithm to accurately estimate were used for pathway analysis. Neutrophils extracellular traps (NETs) somatic variants in tumor tissue. In parallel, TMB was measured by Whole were revealed by immunofluorescence, assessing extracellular DNA Exome Sequencing (WES) targeting 50Mb using 100ng of tumor and and citrullinated H4 histone co-localization and/or myeloperoxidase matched normal DNA on a HiSeq X instrument. We examined factors [5]. Autophagy was assessed the CYTO-ID® kit [5]. that affect OTMLA measurements: Deamination signature, degree of de- Results amination and allele ratio identify DNA samples with high levels of dam- While tumor-related pathway enrichment differed mostly according age due to FFPE preservation. A Uracil-DNA glycosylase (UDG) repair step to the primary tumor histology, perturbations of immune-regulatory was introduced to eliminate damaged targets and improve usable TMB pathways was observed during oligoprogression in the lung. Decon- values of DNA from FFPE tumor tissue. At the variant level, samples with volution of immune cell subpopulations identified increased imma- high deamination scores were analyzed dynamically as a function of al- ture dendritic cells and reduced T cell abundance in oligometastatic lele frequency to study TMB values for correlation with WES. lesions. Strikingly, a large proportion of differentially modulated path- Results ways were “immune” rather than “cancer-cell”-related. Core analysis OTMLA TMB values showed good correlation with WES-derived TMB; confirmed that the main transcriptomic network that is affected dur- however ~10% of tumor DNA samples had high TMB and deamination ing disease progression is immune-based, centered on a cross-link values outside the expected range. These samples were included as a between innate and adaptive immunity. Specifically, it was associ- subset of samples tested with and without the UDG repair step. UDG ated with decreased HLA, iCOS, IL-9, and IL-17 pathway activity and treatment decreased TMB and deamination scores, resulting in higher downregulation of interferon signaling. During progression, we ob- correlation with WES TMB values. Some samples with very high de- served coherent modulation of transcripts associated with NET gen- amination scores were unable to be rescued; however, TMB values in eration, related to upregulation of key autophagic genes, to samples with low deamination and minimal damage were not affected. competition of the HMGB1 molecule with CXCL12 and CXCR4 and Conclusions RAGE receptor (AGER) activation. Accordingly, NET expression was We show that deaminated cytosine bases can be enzymatically re- strikingly more abundant in lung metastases than in primary tumors. moved by treatment with UDG. In a subset of FFPE samples tested, Conclusions UDG treatment was demonstrated to reduce the OTMLA estimated Our results identify evident molecular mechanisms associated with SNP proportion consistent with deamination. This results in consist- suppression of the immune milieu during disease oligoprogression in ent and effective reduction of C>T artifacts without affecting true the lung across different tumors. They include innate-adaptive im- variants and can provide TMB values in a biologically relevant range. mune dysfunction HLA-mediated, and interferon dysregulation asso- ciated with neutrophil-mediated immune suppression. Since these tumors are targeted by immune checkpoint blockade (ICB) our data P79 highlights the relevance of characterizing the TIME composition in Molecular comparison of tumor microenvironment in primary paired primary and oligometastatic lesions during ICB treatment to lung, melanoma, and kidney tumors versus paired lung metastases optimize treatment approaches. reveals shared perturbations of the immune milieu during oligoprogression Acknowledgements 1 1 Davide Bedognetti, MD, PhD , Jessica Roelands, Master , Angelo Paola Nistico' and Gennaro Ciliberto are co-last authors. 2 2 3 3 Manfredi , Norma Maugeri , Francesca De Nicola , Ludovica Ciuffreda , 3 3 3 Matteo Pallocca , Maurizio Fanciulli , Francesca Di Modugno, PhD , References 3 3 3 Paolo Visca , Barbara Antoniani , Gabriele Alessandrini , Darawan Rinchai, 1. Angelova M, Mlecnik B, Vasaturo A, Bindea G, Fredriksen T, Lafontaine L, 1 1 3 PhD , Wouter Hendrickx, PhD , Paola Nistico', MD , Gennaro Ciliberto, et al. Evolution of Metastases in Space and Time under Immune MD Selection. Cell. 2018 Oct;175(3):751-765.e16. 1 2 Sidra Medicine, Doha, Qatar; IRCCS Ospedale San Raffaele, Milano, Italy; 2. Granton E, Kim JH, Podstawka J, Yipp BG. The Lung Microvasculature Is a Istituto Nazionale Tumori Regina Elena, Rome, Italy Functional Immune Niche. Trends Immunol. 2018 Nov;39(11):890-899. Correspondence: Gennaro Ciliberto (gennaro.ciliberto@ifo.gov.it) 3. Weichselbaum RR. The 46th David A. Karnofsky Memorial Award Lecture: Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P79 Oligometastasis-From Conception to Treatment. J Clin Oncol. 2018 Sep27:JCO1800847. Background 4. Stephens SJ, Moravan MJ, Salama JK. Managing Patients With The importance of tumor-host interactions during cancerogenesis Oligometastatic Non-Small-Cell Lung Cancer. J Oncol Pract. 2018 and metastatic progression has been now widely appreciated. Jan;14(1):23-31. More recently, the impact of the tumor immune microenviron- 5. Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli ment (TIME) to mold tumor evolution was convincingly C, Maiuri L, Maseri A, D'Angelo A, Bianchi ME, Rovere-Querini P, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 44 of 272 Manfredi AA.Activated platelets present high mobility group box 1 to Results neutrophils, inducing autophagy and promoting the extrusion of Overall survival (N=141, median 6.05 months) was not associated neutrophil extracellular traps. J Thromb Haemost. 2014 with PD-L1 positive (>=1% tumor or tumor+immune cells)/nega- Dec;12(12):2074-88. doi: 10.1111/jth.P689. tive, diffuse/non-diffuse, gastric/gastroesophageal, prior therapy Ethics Approval (median=2), tumor IFNgamma signature or CD8+ tumor infiltrate. The study was approved by IFO Institution’s Ethics Board, approval number Differential gene expression analysis identified GRB7, a down- 561/03. stream mediator of HER2 signaling and part of the HER2/ERBB2 amplicon in breast cancer [4], as one of two genes associated with survival >1 year (FDR=0.027). HER2-positivity (medical record) was associated with a 3.5-fold higher median expression of GRB7. Prolonged survival was associated with both HER2-positivity (n= 43/132; HR=0.58, p=0.01) and the top quartile of GRB7 expression (n=25/94; HR=0.48, p=0.007). The median survival for HER2- positive patients was 10.1 months versus 5.95 months for HER2- negative. HER2 status was not associated with PD-L1 status or CD8+ infiltrate. Nearly all HER2-positive (n=40/43) and 2 HER2- negative patients received trastuzumab (median 62 days post- trastuzumab). Prior or best response was not related to 1 year survival and 2 of 3 HER2-positive patients that did not receive trastuzumab had >1 year survival. TMB was also evaluated and significantly associated with HER2-positivity (N=61, p=0.041). In the subset of 61 patients with TMB data, patients with high TMB plus HER2-positivity had the longest median survival of 15.4 months compared to all other patients at 6.7 months (N=14 vs 47; HR=0.47; p=0.04). Conclusions HER2 was associated with improved survival with checkpoint blockade in advanced GEA patients, regardless of response to Fig. 1 (abstract P79). See text for description prior trastuzumab. This study was limited by the lack of pre- treatment biopsies, but consistent with a recent report on Asian GEA patients [5]. The combination of HER2-positivity and relatively higher TMB in a limited dataset led to the greatest observed me- dian survival time, suggesting an interaction between HER2 and P80 TMB that warrants exploration in future GEA studies involving HER2 is associated with prolonged survival in advanced checkpoint inhibition. gastroesophageal adenocarcinoma patients treated with checkpoint blockade Acknowledgements 1 1 2 Carrie Brachmann, PhD , Emon Elboudwarej , Manish Shah, MD , David The authors gratefully acknowledge the patients and their families who 3 4 5 Cunningham , Jean-Philippe Metges , Eric Van Cutsem, MD, PhD , Zev participated in this study. 6 1 1 1 Wainberg, MD , Jingzhu Zhou , Dung Thai , Pankaj Bhargava , Daniel Trial Registration Catenacci, MD Clinicaltrials.gov NCT02862535 1 2 Gilead Sciences, Foster City, CA, United States; Weill Cornell Medicine, NY Presbyterian, New York, NY, United States; Sutton and London References Hospital, London, United Kingdom; Brest University Hospital, Brest, 1. Shah M, Metges J-M, et al. A phase 2, open-label, randomized study to France; University Hospitals Leuven & KU Leuven, Leuven, Belgium; evaluate the efficacy and safety of andecaliximab combined with nivolu- 6 7 UCLA School of Medicine, Santa Monica, CA, United States; University mab versus nivolumab alone in subjects with unresectable or recurrent of Chicago Medical Center, Chicago, IL, United States gastric or gastroesophageal junction adenocarcinoma. ASCO Gastrointes- Correspondence: Carrie Brachmann (carrie.brachmann@gilead.com) tinal Cancers Symposium. 2019. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P80 2. Metges J-M, Elboudwarej E, et al. Exploratory evaluation of baseline tumor biomarkers and their association with response and survival in pa- Background tients with previously treated advanced gastric cancer treated with ande- The benefit of checkpoint blockade in advanced gastric cancer caliximab combined with nivolumab versus nivolumab. ASCO is limited and patient selection biomarkers are needed. In a ran- Gastrointestinal Cancers Symposium. 2019. domizedphase 2study in >=2ndlineadvancedgastroesopha- 3. Brachmann C, Zhang Y, et al. Evaluation of intratumoral T cells in geal adenocarcinoma (GEA) cancer in Europe, US and Australia, biopsies from advanced gastric patients treated with andecaliximab and there was no clinical benefit for the addition of andecaliximab nivolumab. ASCO Gastrointestinal Cancers Symposium. 2019. to nivolumab in the total population or evaluated subgroups 4. Ferrari A, Vincent-Saloman A, et al. A whole-genome sequence and tran- (including PD-L1) [1,2]. Pharmacodynamic analyses demon- scriptome perspective on HER2-positive breast cancers. Nature Commu- strated little to no impact of andecaliximab [3]. This exploratory nications. 2016. biomarker analysis included all patients as a nivolumab-treated 5. Satoh T, Kang Y-K, et al. Exploratory subgroup analysis of patients with prior population. trastuzumab use in the ATTRACTION-2 trial: a randomized phase III clinical Methods trial investigating the efficacy and safety of nivolumab in patients with ad- Evaluation of archival tumor tissue was described [2,3]. Tumor muta- vanced gastric/gastroesophageal junction cancer. Gastric Cancer. 2019. tion burden (TMB) was evaluated by whole exome sequencing with Ethics Approval matched normal. Survival analyses (cox proportional hazards) were This study was approved by the institutional review board or independent adjusted for age and sex. ethics committee appropriate for each site. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 45 of 272 P81 Table 1 (abstract P81). Associations between TMB and Baseline Association of tumor mutational burden with clinical, genomic, Characteristics and treatment characteristics in advanced non-small cell lung cancer 1 1 2 Connor Willis, PharmD , Hillevi Bauer, PharmD , Trang Au, PharmD ,Sudhir 1 1 3 Unni, PhD, MBA , Wallace Akerley, MD , Ashley Sekhon, MD , Firas Badin, 4 5 6 7 MD , John Villano, MD, PhD , Matthew Schabath, PhD , Bing Xia, MD , 8 9 9 Beth Gustafson, PharmD , Komal Gupte-Singh, PhD , Beata Korytowsky , 9 9 John-Michael Thomas, PharmD , Gabriel Krigsfield, PhD , Solomon 9 1 10 Lubinga, PhD , Diana Brixner, PhD , David Stenehjem, PharmD 1 2 University of Utah, Salt Lake City, UT, United States; Long Island University, Brooklyn, NY, United States; MetroHealth Medical Center, Cleveland, OH, United States; Baptist Health, Lexington, KY, United 5 6 States; Markey Cancer Center, Lexington, KY, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; University of Southern California, Los Angeles, CA, United States; Saint Luke's Cancer Institute, Kansas City, MO, United States; Bristol-Myers Squibb, Princeton, NJ, United States; University of Minnesota, Duluth, MN, United States Correspondence: David Stenehjem (stene032@d.umn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P81 Background Tumor mutational burden (TMB) is emerging as a potential predictor of response to immunotherapy in various tumor types. However, the association of TMB data with clinical, demographic, genomic, and treatment characteristics warrants further investigation. Methods Nine U.S. Comprehensive Cancer Centers participated in this observa- tional, cohort study; five centers are members of the Oncology Re- search Information Exchange Network (ORIEN). Adult patients with stage IV non-small cell lung cancer (NSCLC) with tissue-based TMB data from any testing platform were included and their treatment in- formation was abstracted using a standardized case report form. TMB reporting ranged from September 2014 through March 2019. TMB-High and TMB-Low were defined as >10 mutations/megabase (mut/Mb) and <10 mut/Mb, respectively. Clinical, demographic, gen- omic, and treatment characteristics were compared by TMB level. Results There were 426 patients enrolled in the study across seven of the Table 2 (abstract P81). Associations between TMB and Select nine sites. TMB results from comprehensive genomic profiling (CGP) Oncogene Mutations were available for 354 patients. CGP vendors included Foundation Medicine (79.9%), Caris Life Sciences (17.0%), Tempus (2.8%), and NantHealth (0.3%). The median time from diagnosis to CGP testing was 45 days. A comparison of clinical and demographic characteris- tics by TMB is presented in Table 1. TMB-High status was associated with male gender (p<0.01), and positive smoking history (p<0.01). No correlation was found between TMB and PD-L1 (Table 2). TMB-High was positively associated with multiple oncogenes including STK11, LRP1B, TP53, and KDM5C (Table 2). In addition, there were significant negative associations between TMB-High and individual occurrences of altered ALK (p=0.03), EGFR (p<0.01), and ROS1 (p=0.03). The pro- portion of patients receiving first-line immunotherapy increased yearly from 8.5% in 2015, 19% in 2016, 40% in 2017, and 46% in Conclusions These interim results demonstrate the feasibility of conducting multi- site observational electronic health record-based studies with CGP and TMB across a national cohort of comprehensive cancer centers. P82 Immunotherapy utilization has been increasing in the first-line set- High-throughput pairing of single T-cell α and β chains along with ting. Associations between TMB status and driver mutations are indi- phenotypic expression profiling 1 2 3 cative of cancer etiology and informative for treatment decision- Brittany Brown, BS , Miranda Byrne-Steele, PhD , Wenjing Pan, PhD , 2 2 2 4 making. Updated results will be presented with an expanded cohort Song Li , Mary Eisenhower, BS , Daniel Weber , Mollye Depinet, MS , 2 2 2 of patients and future publications with the final cohort (n~1000) will Xiahong Hou, PhD, MD , Alex Moore , Jian Han, MD PhD 1 2 explore treatment, survival and response data. iRepertoire, Inc., Huntsville, AL, United States; iRepertoire, Huntsville, AL, United States; HudsonAlpha Institute for Biotechnology, Huntsville, AL, Acknowledgements United States; iRepertoire.com, Huntsville, AL, United States This study was sponsored by Bristol-Myers Squibb. Recruitment efforts were Correspondence: Jian Han (jhan@irepertoire.com) supported by Mikaela Larson (Huntsman Cancer Institute) and M2Gen® Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P82 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 46 of 272 Background panel was compared to whole-exome sequencing (WES) derived The T-cell receptor (TCR) is responsible for recognizing antigens as TMB. LOH-MHC status reported from PGDx elio tissue complete was peptides bound to a major histocompatibility complex. TCRs typically compared to MHC status determined by WES for accuracy. Addition- contain both an alpha (α) and beta (β) chain that contribute to anti- ally, TMB and MHC status were analyzed together in FFPE samples gen specificity; however, we have seen multiple cases of a single cell from ICI treated patients and correlated to clinical outcomes. containing dual α or dual β chains as well. When analyzing bulk rep- Results ertoires, information about endogenous pairing of α and/or β chains 115 pan-cancer samples were analyzed for microsatellite status and is lost after bulk lysis of T-cell populations. Pairing α and β chains demonstrated an overall agreement of 100.0% with a PCR-based from a single cell while also analyzing the phenotypic expression al- method. TMB was determined in 118 pan-cancer samples and dis- lows us to track TCR specificity and T cell function. This information played a high level of concordance with WES-derived TMB (Pearson can provide direct calculations of clonal frequency in various cell sub- correlation, p=0.903) across a range of TMB scores (0.2-89.7 muts/ sets, allow tracking of specific lymphocytes with treatment, and reveal Mbp). FFPE tissue samples from 98 cancer patients previously treated paired information for both chains of the receptor for downstream Car- with ICIs were then tested for LOH-MHC and demonstrated 88% ac- T development. curacy of detection when compared to WES. Furthermore, analysis of Methods TMB and MHC status in tandem found that patients with both high Here, we developed a method for high-throughput pairing of TCR α TMB and normal MHC status were found to have a significantly and β chains along with expression profiling. We examined, on aver- higher PFS, suggesting greater efficacy of ICI therapy. age, around 15,000 CD4+ cells loaded onto the BD Rhapsody Express Conclusions system. The receptor information is amplified from the same cDNA These data demonstrate the feasibility of measuring MSI, TMB, and using iRepertoire’s proprietary method that incorporates a multiplex evaluating LOH-MHC with high accuracy in a single >500 gene NGS mix of primers associated with both the TCR α and β loci; phenotyp- assay. Additionally, the results presented herein suggest that measur- ing of the cell is obtained using the BD Rhapsody RNA-seq kit. Along- ing TMB and MHC status concurrently can provide added utility in side the high throughput data, we also performed FACS-based single predicting patient response to ICI therapy. cell sequencing on the same individual’s samples through our iPair method (presented previously) and examined the overlapping recep- P84 tor sequences between both methods. Panel-derived tumor mutational burden (TMB) correlates with Results immune checkpoint inhibitors (ICIs) response in gastrointestinal With this mid throughput method, we are able to accurately assess cancers the frequency of single cells containing dual alpha or dual beta TCRs, 1 2 1 San-chi Chen, MD , Kien-Thiam Tan, PHD , Ming-Huang Chen , Yi-Ping which can help to evaluate the high throughput data. 1 2 2 1 Hung, MD , Yi-Lin Hsieh , Yi-Hua Jan , Yee chao Conclusions Taipei Veterans General Hospital, Taipei, Taiwan, Province of China; The described high throughput application should be applicable to ACT Genomics Co., Ltd., Taipei, Taiwan, Province of China any oligo-dT based single cell strategy. Correspondence: Yee chao (ychao@vghtpe.gov.tw) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P84 This study was approved by New England IRB, IRB number 120160202 Background P83 Tumor mutational burden (TMB) has been emerging as a relatively Pan-cancer assessment of composite genomic biomarkers in new biomarker that is independent of PDL1 for the prediction of re- immuno-oncology to predict responses and resistances to immune sponse to the immune checkpoint inhibitor (ICI) treatment. A recent checkpoint inhibitor therapy study has shown that whole exome sequencing (WES)-derived TMB Gustavo Cerqueira, Laurel Keefer, Kelly Gerding, PhD, Kenneth correlates well panel-derived TMB that is estimated using targeted Valkenburg, Christina Oliveras, James White, Leila Ettehadieh, Christopher sequencing. Here, we evaluate the correlation between panel- Gault, James Hernandez, Eric Kong, Isabell Loftin, Samuel Angiuoli, derived TMB with response to ICI treatment in gastrointestinal Abigail McElhinny, John Simmons, PhD cancers. Personal Genome Diagnostics, Baltimore, MD, United States Methods Correspondence: Eric Kong (ekong@pgdx.com) FFPE tumor and normal tissues from 18 patients with gastrointestinal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P83 cancers who had previously received ICI therapy at Taipei Veterans Gen- eral Hospital were retrospectively underwent targeted next-generation Background sequencing (ACTOncoTM) for the identification of somatic variants across Therapeutic response to immune checkpoint inhibitors (ICIs) requires 440 genes and the calculation of TMB. NetMHC and IEDB were used to a prior, suppressed immune response that is released via the inter- predict neopeptide bound to patient-specific HLA class one genotype. action of the checkpoint receptors with their cognate ligands. Micro- RECIST criteria were used to categorize tumor response. satellite instability (MSI) and tumor mutation burden (TMB) have Results emerged as composite genomic metrics that may better predict pa- Patients were grouped into responder (PR or CR, n=10) and non- tient response to ICI treatment, compared to conventional PD-L1 ex- responder (PD or SD, n=7). Among all patients, responders had sig- pression. However, testing for MSI and TMB separately is labor nificantly higher TMB than non-responders (mean 7.37 muts/Mb vs. intensive, increases turnaround time, and consumes valuable tumor 1.24 muts/Mb, p=0.0007). The number of predicted neopeptides tissue samples. Furthermore, recent studies have demonstrated that were significantly higher in responders than in non-responders antigen presentation mechanisms may also play a role in predicting (mean 10.8 vs. 3.6, p=0.0226). Notably, a non-responder harboring outcomes, wherein loss of heterozygosity in MHC class I genes (LOH- EGFR gain-of-function mutation did not respond to the ICI treatment MHC) suggests low likelihood of ICI benefit. despite high TMB. Furthermore, patients harboring MUC16 mutation Methods demonstrated higher TMB than patients without MUC16 mutation Here, we propose that these varied immune-oncology metrics can be (mean 17.38 muts/Mb vs. 3.9 muts/Mb, p=0.0005) (Figure 1). measured together in a single assay, utilizing next-generation se- Conclusions quencing (NGS) technologies. To test this hypothesis, we analyzed Although the cohort size is small, our study showed that panel-based >200 pan-cancer FFPE tumor tissue samples using the PGDx elio™ tis- TMB is predictive of response to ICI. As in lung cancers, EGFR muta- sue complete assay (currently in development; >500 genes panel) to tion is associated with decreased efficacy of ICI in the GI cancers. measure MSI, determine TMB (across 1.3 Mb), and assess MHC status Ethics Approval in a single assay. Detection of MSI was assessed for accuracy against The study was approved by TPEVGH intuition’s Ethics Board, approval a validated PCR approach and TMB determination from our targeted number 2015-07-002BC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 47 of 272 of TMB using publicly available whole-exome cancer sequencing data resulted in high correlation (R2 = 0.902, 0-40 mutations/Mb) and was parallel to the performance of our existing Oncomine Tumor Muta- tion Load Assay (R2 = 0.901, 0-40 mutations/Mb). Empirical analysis and performance of the assay on a common set of cell lines based on a universal reference standard also resulted in a positive correlation. Conclusions A larger tumor only NGS assay was developed to support compre- hensive genomic profiling and routine clinical research in oncology. The assay design and informatics workflow support characterization of mutational signatures and provide normalized TMB estimates. Min- imal input material requirement and rapid sample to report time will have a high impact on clinical research. More detailed information on the assay and an update on performance will be presented. P86 Predictive Immune Modeling enables biomarker discovery in NSCLC patients treated with second line immunotherapy 1 1 2 Natalie LaFranzo, PhD , Steve Daniel, PhD , Walt Carney, PhD , Milan 3 1 Bhagat , Natalie LaFranzo, PhD 1 2 Cofactor Genomics, St. Louis, MO, United States; Walt Carney Biomarkers Consulting, LLC, Boston, MA, United States; TriStar Technology Group, LLC, Washington, DC, United States Fig. 1 (abstract P84). See text for description Correspondence: Natalie LaFranzo (natalie_lafranzo@cofactorgenomics.com) P85 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P86 Development of a pan-cancer NGS assay for detection of tumor mutational burden and targeted biomarkers from FFPE samples Background Dinesh Cyanam, MS, Vinay Mittal, Nickolay Khazanov, Paul Williams, While cancer checkpoint inhibitors have garnered much attention Janice Au-Young, Gary Bee, Sameh El-Difrawy, Aren Ewing, Jennifer due to their ability to generate durable responses and improved sur- Kilzer, Anelia kraltcheva, PhD, Scott Myrand, MS, Yu-Ting Tseng, Cristina vival, the actual number of patients who are eligible for, receive Van Loy, Elaine Wong-Ho, Chenchen Yang, Dinesh Cyanam, MS, treatment with, and respond to these therapies remains modest [1]. Santhoshi Bandla, Warren Tom, PhD, Seth Sadis This is driven by a dependency on legacy diagnostics, built on Thermo Fisher Scientific, Ann Arbor, MI, United States single-analyte biomarkers such as PD-L1, which have failed to cap- Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) ture the complexity of disease [2]. Even in the case of non-small cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P85 lung cancer (NSCLC), an indication where the benefit of IO therapies is considered significant, there is much to be learned about the biol- Background ogy of the patients who respond, or do not respond to these Next-generation sequencing (NGS) is being applied to support routine therapies. clinical research in oncology with a primary focus on evaluating known Methods oncogenic variants. However, the advent of cancer immunotherapies Multidimensional RNA models have emerged to move beyond requires that clinical research solutions must also address biomarkers these legacy methods to reveal the full scope of disease com- such as Tumor Mutational Burden (TMB) and Microsatellite Instability plexity, resulting in increased predictive accuracy. Leveraging a (MSI) for immune checkpoint inhibitors. Therefore, we developed a re- database of gene expression models built using Predictive Im- search use NGS solution for FFPE tissues that expanded upon our mune Modeling, immune context of the tumor microenvironment current Oncomine Tumor Mutation Load Assay by measuring bio- is quantified. In this study, a cohort of NSCLC patients who re- markers for both targeted and immune checkpoint therapies. ceived second-line immunotherapies (checkpoint inhibitors) were Methods evaluated retrospectively. Pre-treatment solid tumor FFPE tissue Gene content was prioritized based on the relevance and variant samples were processed using the ImmunoPrism immune profil- prevalence of biomarkers in solid tumors. Additional genomic regions ing assay to generate comprehensive, individual immune profiles. were added to supplement the coding sequence footprint to support Pathological, demographic, and survival data (including overall TMB. The assay used Ion AmpliSeq™ technology with automated survival and progression-free survival, indicative of therapy re- templating on the Ion Chef™ system and sequencing on the Ion Gen- sponse), was used to group patients for predictive biomarker eStudio™ S5 sequencing platform. An automated tumor-only work- discovery. flow for variant calling, TMB and MSI estimation and sample quality Results reporting was provided within Ion Reporter Software. Decision sup- Individual immune profiles of the patients are compared, both within port tools were used for variant interpretation and evaluation of po- and between relevant cohorts, and statistically-significant biological tential variant relevance. signals are reported. Machine-learning derived multidimensional Results biomarkers were also generated, which are defined by the optimal Over 500 genes with known DNA and RNA alterations were included. combination of all analytes measured in the assay, enabling improve- The panel has broad capability for variant calling, fusion detection, ments in predictive accuracy. This study represents the first data MSI status, in addition to TMB. Specifically, for TMB, DNA repair path- generated using the ImmunoPrism assay with patients receiving ways were comprehensively represented as alterations in these checkpoint inhibitor therapies. genes may lead to high mutation burden. A coding sequence foot- Conclusions print to support TMB was generated. In development studies, the Predictive Immune Modeling enables us to build multidimensional assay displayed high uniformity and consistent read depth to sup- models of disease. When combined with well-curated patient co- port robust variant calling. The automated workflow required min- horts, such as the NSCLC patients described here, predictive bio- imal input of FFPE tumor only DNA and RNA material. Sample to markers may developed which capture more facets of the complex report turnaround time was less than five days. In-silico assessment immune contexture than previously possible. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 48 of 272 References P88 1. Haslam A, Prasad V. Estimation of the Percentage of US Patients Evaluation of a tumor-only pan-cancer targeted semi-conductor With Cancer Who Are Eligible for and Respond to Checkpoint based next-generation sequencing (NGS) test for microsatellite Inhibitor Immunotherapy Drugs. JAMA Netw Open. 2019 May instability in FFPE samples 1 2 2 3;2(5):e192535. Sameh El-Difrawy, Ph D , Anelia kraltcheva, PhD , Vinay Mittal , Elaine 2 2 2 2 2. Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune- Wong-Ho , Dinesh Cyanam, MS , Seth Sadis , Jennifer Kilzer , Cristina 2 2 2 2 checkpoint blockade: response evaluation and biomarker development. Van Loy , Janice Au-Young, PhD , Aren Ewing , Sameh El-Difrawy Nat Rev Clin Oncol. 2017 Nov;14(11):655-668. ThermoFisher Scientific, South San Francisco, CA, United States; Ethics Approval Thermo Fisher Scientific, Carlsbad, CA, United States The human tissue samples utilized for this study were provided by Correspondence: Sameh El-Difrawy (Sameh.El- TriStar Technology Group and have written, informed donor consent Difrawy@thermofisher.com) permitting academic and commercial research for publication, as Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P88 well as approval from a competent ethical committee. Background Comprehensive genomic profiling using next-generation sequencing (NGS) has become an essential tool to support routine clinical research P87 in oncology. Advent of cancer immunotherapies also requires assess- All-in-One, quantitative immune repertoire profiling of PBMC and ment of immune checkpoint inhibitor biomarkers such as microsatellite FFPE for renal cancer treatment evaluation instability (MSI) and tumor mutational burden (TMB). 1 1 2 Mollye Depinet, MS , Wenjing Pan, PhD , Sang-gin Wu, MD, PhD , MSI arises from defects in the mismatch repair (MMR) system and is as- 1 1 1 Xiaohong Hou, MD PhD , Brittany Brown, BS , Mary Eisenhower, BS , sociated with hypermutability of short DNA sequence repeats, micro- 1 1 3 Daniel Weber , Miranda Byrne-Steele, PhD , Michael Lotze , Jian Han, satellite locations, throughout the genome. Such defects are commonly MD PhD observed in colorectal, gastric and endometrial cancers and have been 1 2 iRepertoire, Huntsville, AL, United States; National Taiwan University, shown to be predictive of response to immunotherapy treatment. Trad- Taipei City, Taiwan, Province of China; UPMC Hillman Cancer Center, itionally MSI testing has been done using single biomarker tests such Pittsburgh, PA, United States as PCR/fragment analysis or immunohistochemistry (IHC) that require Correspondence: Jian Han (jhan@irepertoire.com) high sample input and are time consuming. Therefore, we developed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P87 an RUO NGS solution appropriate for FFPE tissues that addresses bio- markers for targeted and immune checkpoint therapies. Background Methods Next generation sequencing of the immune repertoire is a com- The performance of our RUO NGS based MSI approach was tested in prehensive immune profiling methodology that allows detailed, the context of a large Ion AmpliSeq™ panel composed of more than sequence-specific insight into the adaptive immune response. 13,000 amplicons covering 500+ genes. The content includes a di- While immune repertoire analysis of bulk RNA typically focuses verse set of microsatellite markers targeting MSI locations comprised on a single receptor chain, understanding of the variable rear- of mono- and di-nucleotide repeats that range from 7 to 34 bp. Se- rangements of the immune repertoire as a whole provides a quencing was carried out on the Ion 550™ chip and the Ion GeneStu- broader view of the immune landscape with potential prognostic dio™ S5 system. In-sample standards were designed and value. This is accomplished through the study of all seven TCR incorporated as internal references utilized by the analysis pipeline and BCR chains together (i.e., TCR-alpha, TCR-beta, TCR-delta, and a novel algorithm was developed that leverages the unique sig- TCR-gamma, and BCR-IgK and -IgL). One of the key challenges nal processing properties inherent in semi-conductor sequencing. during immune receptor amplification is the formation of dimers, The test provides results for individual microsatellites and generates which can compete with the immune amplicons of interest dur- an MSI score and status for the sample of interest. ing library preparation. Results Methods The performance of the MSI solution was tested using a set of over We therefore developed a novel PCR technique, dimer avoided multi- 400 FFPE and cell-line samples from different tissue types and plex PCR (dam-PCR), that effectively avoids dimer formation during showed excellent concordance with orthogonal tests. We report on PCR and incorporates unique molecular identifiers for direct RNA the sensitivity and specificity of our tumor only approach and quantification and error removal. With one sample, dam-PCR allows propose ideas to utilize generated MSI score in combination with for the amplification of all seven TCR and BCR loci in a single, quanti- other bio markers. tative multiplex reaction. Here, we apply this method to the amplifi- Conclusions cation of both PBMC and FFPE RNA from renal cancer patients An NGS assay was developed to support comprehensive genomic undergoing treatment. profiling and routine clinical research in oncology. The assay design Results and unique informatics workflow support precise characterization of We found that both TCR-alpha and -beta diversity prior to treatment mutational signatures and provides normalized MSI and TMB esti- along with the expression ratio between B cells and T cells are good mates. The performance of the assay was verified over a large cohort predictors of treatment efficacy. of colorectal, gastric and endometrial cancer samples with MSI status Conclusions independently assigned by orthogonal tests. [For Research use Only. Our study suggests that examining multi-chain immune reper- Not for use in diagnostic procedures] toire composition can be valuable for predicting treatment re- sponse and evaluating treatment protocols. Additionally, this method shows promise for future applications in both clinical P89 settings and basic research, as it allows for a cost effective, all- Obesity related changes in AXL-driven inflammatory signaling inclusive, and quantitative immune-profiling analysis of immune impact survival in melanoma repertoires from a range of sample types, including FFPE, where Alicia Gingrich, MD, Kylie Abeson, BS, Alexander Merleev, PhD, Robert sample RNA may be both limited in quantity and degraded in Canter, MD, MAS, FACS, Emanual Maverakis, Amanda Kirane, MD, Alicia quality. Gingrich, MD Ethics Approval University of California, Davis, Sacramento, CA, United States This study was approved by the University of University of Pittsbur- Correspondence: Alicia Gingrich (agingrich@ucdavis.edu) gh's Ethics Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P89 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 49 of 272 Background The TYRO3, AXL and MERTK (TAM) receptor tyrosine kinase (RTK) family have been associated with a number of human cancers, including melanoma.[1-3] Effects attributed to oncogen- esis and metastasis (epithelial-to-mesenchymal transition) of the TAM receptors have been described.[2] Recent evidence correl- ating obesity with a paradoxical improved response to immuno- therapy in melanoma suggests both tumor microenvironment and clinical phenotype play a role in response.[4] Therefore, we sought to build a predictive model of response to therapy from biomarkers, using TAM receptors and conventional markers of checkpoint inhibition such as PD-1. This model was tested in the normal weight, overweight and obese populations. Methods TCGA-SKCM melanoma tumor mRNA expression and clinical data for metastatic melanoma patients were downloaded from the GDC legacy archive (https://portal.gdc.cancer.gov/legacy-archive) (n = 471).[5] Bio- markers were defined as “high” or “low” expression in each patient. Dif- ferences in Kaplan-Meier survival curves based on level of expression were tested using G-rho family tests. Strength of relationships between biomarkers were measured using Pearson’s correlation. All statistical analysis were performed using R package “survival”. Results Normal weight, overweight and obese patients had markedly dif- ferent biomarker profiles associated with survival (Figure 1). In Fig. 1 (abstract P89). KM curves by clinical phenotype the normal weight population, high CD8 (p=0.0093), PD1 (p= and biomarker 0.0093) and CD84 (p=0.022) were associated with improved sur- vival. In the overweight population, high CD8 (p=0.0098), PD1 (p=0.0004) and CD84 (p=0.0081) were associated with improved P90 survival, while high Gas6 (p=0.029) and MERTK (p=0.043) were as- Unique tumor immune microenvironments of potentially PD-L1/ sociated with decreased survival. And in the obese population, TGF-β trap responsive tumors high AXL expression was associated with improved survival (p= Sean Glenn, PhD, Sarabjot Pabla, MSc, PhD, BS, Erik Van Roey, Jonathan 0.004), while CD8 (p=0.91) and PD1 (p=0.89) demonstrated no as- Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, sociation. In correlation analysis, AXL expression was most closely Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, associated with macrophage markers CD163 (r=0.52), CD84(r= Yirong Wang, MS, Carl Morrison, MD, DVM 0.56) and MS4A4A(r=0.53) in the obese but not the normal OmniSeq, Inc., Buffalo, NY, United States weight population. Correspondence: Sean Glenn (sean.glenn@omniseq.com) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P90 Taken together, these data suggest that immunologic response in metastatic melanoma patients is driven by separate immune Background profiles for obese and non-obese populations. AXL appears to Tumors often do not respond to PD-1/PD-L1 axis inhibitors due to im- mediateresponsein theobesepopulation by a macrophage- mune escape mechanisms present in the tumor microenvironment. Bi- driven mechanism as opposed to T cell mediation. Collectively, functional antibody-based immunotherapies that simultaneously target the significant differences in the transcriptomic profiles between immune checkpoints and immunosuppressive cells are being devel- obese and non-obese patients suggest potential clinical implica- oped to slow tumor growth. Anti-PD-L1/ TGF-β trap fusion proteins are tions regarding targets for treatment and application to patients one approach being tested to counter the traditional immune check- based on clinical phenotype. point inhibition via PD-1/PD-L1 axes and simultaneously inhibit the pro-tumor/anti-inflammatory effects of TGF-β.Inthis study, we not only References describe the tumor immune microenvironment of tumors expressing 1. Dransfield I, Farnworth S. Axl and Mer receptor tyrosine kinases: distinct PD-L1 and TGF-β, but also describe potential patient selection strat- and nonoverlapping roles in inflammation and cancer? Adv Exp Med egies based on gene expression measurements of these tumor im- Biol. 2016;930:113-32. mune microenvironments from clinical samples. 2. Verma A, Warner SL, Vankayalapati H, et al. Targeting Axl and Mer kinases Methods in cancer. Mol Cancer Ther. 2011;10:1763-73. RNA-seq was performed for 395 immune transcripts on 1323 FFPE tu- 3. Wu X, Liu X, Koul S, et al. AXL kinase as a novel target for cancer therapy. mors of diverse histologies. To find true TGF-β high expressing tu- Oncotarget. 2014;5:9546-9563. mors, TGF-β gene expression was normalized by a tumor 4. McQuade JL, Daniel CR, Hess KR, et al. Association of body-mass index inflammatory score (average expression rank of 161 inflammation and outcomes in patients with metastatic melanoma treated wtih tar- genes derived from co-expression signature of 1323 tumors spanning geted therapy, immunotherapy, or chemotherapy: a retrospective, multi- 35 tumor histologies). Proportion of PD-L1 IHC positive, tumor muta- cohort analysis. Lancet Oncol. 2018;19:310-322. tional burden (TMB) high and cell proliferation categories was esti- 5. Guan J, Gupta R, Filipp FV. Cancer systems biology of TCGA SKCM: mated for TGF-β high expressing tumors. Inclusion and exclusion efficient detection of genomic drivers in melanoma. Sci Rep. criteria were developed based on PD-L1 and normalized TGF-β 2015;5:7857. expression. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 50 of 272 Results Results Gene expression revealed varying degrees of TGF-β high tumors RNA-seq libraries from control RNAs at both 10 and 100 ng input in all tumor types studied. Sarcoma, pancreatic cancer and breast have less than 10% rRNA reads, replicate correlations greater cancer had the highest proportion of TGF-β high tumors. Within than 99%, and gene-level and transcript-level detection rates that these TGF-β high tumors, 41% were PD-L1 IHC+ (TPS≥1%), and are highly concordant with a conventional library preparation kit. 28% were TMB-high. 11% (n=147/1323) tumors were both TGF-β Additionally, the data confirms comparable dynamic range and high and PD-L1 high making these tumor microenvironments linearity of response of the ERCC spike-in controls. Libraries pre- ideal for a potential PD-L1/TGF-β trap treatment. Interestingly, pared from FACS-sorted T cells show differential expression pro- 47% (n=69/147) of these tumors presented with strong/moderate files consistent with the expected patterns. inflammation, with 53% (n=78/147) being non-inflamed tumors. Conclusions Conversely, there were 11.7% (n=155/1323) tumors that were The Advanta RNA-Seq NGS Library Prep workflow simplifies the high- TGF-β low and PD-L1 low presenting suboptimal tumor micro- throughput generation of RNA-seq libraries, significantly minimizing environment for a potential treatment. Notably, only 26% (n=40/ hands-on time and costly reagent consumption, which will facilitate 155) of these tumors presented with strong/moderate inflamma- the incorporation of RNA sequencing into the immune-oncology tion with clear majority (74%; n=115/155) being strongly or mod- research toolkit. erately inflamed tumors. For Research Use Only. Not for use in diagnostic procedures. Conclusions This large clinically tested tumor cohort suggests an immune phenotype of potentially PD-L1/TGF-β trap responsive tumors ex- ists across multiple histologies. PD-L1/TGF-β high tumors have distinct immune profiles compared to PD-L1/TGF-β low tumors. A P92 clinical immune gene expression assay described in this study Survival benefits of comprehensive genomic profiling and could not only improve patient selection for anti-PD-L1/TGF-β treatment in metastatic non-small cell lung cancer 1 2 trap treatment, but for other bi-specific fusion protein based Alison Sexton Ward, PhD , Jennifer Johnson, MD ,Komal Gupte- 3 3 immunotherapies. Singh, PhD , Mohammad Ashraf Chaudhary, PhD ,Devender 3 1 1 Ethics Approval Dhanda, PhD , Oliver Diaz, PhD , Katherine Batt, MD, MSc , John De-identified specimens and data were analyzed by OmniSeq under Fox 1 2 IRB approved protocol BDR 080316 (Roswell Park Comprehensive Precision Health Economics, Oakland, CA, United States; Jefferson Cancer Center, Buffalo, NY). University Hospital, Philadelphia, PA, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Priority Health, Grand Rapids, MI, United States Correspondence: Komal Gupte-Singh (Komal.Singh@bms.com) P91 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P92 Expression profiling of T cells using nanoscale automation with a full-length RNA sequencing library preparation kit on a Background microfluidic circuit platform 1 1 1 Metastatic non-small cell lung cancer (mNSCLC) patients who receive Thomas Goralski, PhD , Sangpen Chamnongpol , Michael Phelan , 2 1 1 comprehensive genomic profiling (CGP) at diagnosis may be more Jennifer Snyder-Cappione , Julie Alipaz , Joel Brockman ,Brian 1 1 1 1 likely to receive optimal first line (1L) therapies than patients who re- Fowler , Jennifer A. Geis ,Christopher Kubu ,Raphael Kung , 1 1 1 ceive panel testing (PT) with the enhanced ability to identify bio- Benjamin Lacar , Naveen Ramalingam, PhD ,Mandi Wong ,Charles 1 1 markers with associated therapies. The incremental survival benefits Park ,David King 1 2 of receiving optimal treatments following CGP testing at diagnosis Fluidigm Corp, South San Francisco, CA, United States; Boston have yet to be estimated. University School of Medicine, Boston, MA, United States Methods Correspondence: Christopher Kubu (chris.kubu@fluidigm.com) A Markov simulation model of biomarker testing and treatment Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P91 assignment for mNSCLC was built to estimate the survival out- comes associated with CGP versus PT. Biomarkers identified with Background PT were EGFR, ALK, ROS1, BRAF, and PD-L1 (≥50%). All biomarker RNA sequencing (RNA-seq) provides hypothesis-free profiling of tests were tested simultaneously using single gene testing or transcript levels and isoforms. This profiling captures a compre- assay. CGP, which employed Next-Generation Sequencing, identi- hensive view of the peripheral immune system or the tumor fied all the above biomarker changes and estimated tumor muta- microenvironment. The resulting profiles can be used to tional burden (TMB). The model assumed that PD-L1 testing was characterize differential gene expression patterns that can further conducted together with CGP. Biomarker identification, except for the understanding of the immune system. To further enable TMB and PD-L1, was assumed to be mutually exclusive and to these types of studies we have developed a highly cost-effective, occur at published prevalence rates. Incremental false-negative nanoliter-volume microfluidics-based workflow and chemistry rates of each genetic test in PT relative to CGP were applied. compatible with Illumina® sequencing instruments to simultan- Treatment pathways followed NCCN guidelines and current pub- eously generate RNA-seq libraries from up to 48 samples. This lished clinical trial results. Key inputs and assumptions were method fully automates solid-phase capture of polyadenylated tested in sensitivity analyses. RNA, reverse transcription, and index PCR within a compact nano- Results scale integrated fluidic circuit (IFC) on our Juno™ system. The Patient overall survival for each biomarker test within each test- workflow includes reagents necessary to generate full-length, ing strategy are shown in Table 1. Patients receiving CGP had random-primed RNA-seq libraries from as little as 10 ng of total 8.5% (1.4 months) longer survival on average than those who re- RNA, while preserving strandedness information. ceived PT. Patients receiving CGP testing at presentation spent Methods more time on 1L therapies (40% vs. 33%), thereby less time on Multiple replicates of 10 ng and 100 ng of total RNA from con- 2L therapies (23% vs. 26%) compared to patients receiving PT at trol samples spiked with ERCC RNA Spike-In Mixes were used to presentation. prepare RNA-seq library using the Advanta™ RNA-Seq NGS Library Conclusions Prep Kit. The performance was compared to a conventional li- CGP testing among mNSCLC patients at the time of diagnosis re- brary preparation kit. We also used our platform to profile total sulted in survival gains in comparison to PT due to higher proportion RNA purified from FACS-sorted CD3+, CD8+, CD28–,and CD25+/ of patients receiving optimal 1L treatment. hi T suppressor cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 51 of 272 Table 1 (abstract P92). Overall survival (months) by testing strategy & analyzed using Adaptive immunoSEQ in 125 samples. Tumor im- biomarker mune phenotype classification was done using unsupervised consen- sus clustering based on the expression of ICR genes. Results We have built one of the most extensive high-quality datasets for immunogenomic alterations available so far in colon cancer. Our pre- liminary data supports a positive impact of ICR gene expression in colon cancer cohort: patients with a Th-1 polarized microenviron- ment display better survival. Integrative analysis encompassing som- atic mutation, copy number variations, and transcriptome is ongoing and will be presented at the conference (Figure 1). Conclusions This newly generated immune centric NGS dataset, generated in Qatar, will contribute dramatically to elucidating the genetic determi- nants of immune responsiveness in cancer. Acknowledgements This work was supported by Qatar National Research Fund (QNRF) with grant JSREP07-012-3-005 References 1. Wang, E., Worschech, A. & Marincola, F. M. The immunologic constant of rejection. Trends Immunol. 29, 256–262 (2008). 2. Spivey, T. L. et al. Gene expression profiling in acute allograft rejection: challenging the immunologic constant of rejection hypothesis. J. Transl. Med. 9, 174 (2011). 3. Bertucci, F. et al. The immunologic constant of rejection classification P93 refines the prognostic value of conventional prognostic signatures in The advanced immune-centric NGS cohort for colon cancer breast cancer. Br. J. Cancer (2018). doi:10.1038/s41416-018-0309-1 1 1 2 Wouter Hendrickx, PhD , Jessica Roelands, Master , Peter Kuppen , 4. Galon, J., Angell, H. K., Bedognetti, D. & Marincola, F. M. The Continuum 3 1 1 Francesco Marincola, MD , Najeeb Syed , Davide Bedognetti, MD, PhD of Cancer Immunosurveillance: Prognostic, Predictive, and Mechanistic 1 2 Sidra Medicine, Doha, Qatar; Leiden University Medical Center, Leiden, Signatures. Immunity 39, 11–26 (2013). Zuid-Holland, Netherlands; Refuge Biotechnologies, Half Moon Bay, CA, 5. Roelands, J. et al. Genomic landscape of tumor-host interactions with dif- United States ferential prognostic and predictive connotations. bioRxiv 546069 (2019). Correspondence: Davide Bedognetti (dbedognetti@sidra.org) doi:10.1101/546069 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P93 6. Pagès, F. et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. The Background Lancet 391, 2128–2139 (2018). The immune system has a substantial effect on the progression of 7. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with colon cancer. Typically, an immune response defined by a polarized low memory requirements. Nat. Methods 12, 357–360 (2015). Th1 phenotype, characterized by expression of chemokine-receptor 8. Risso, D., Schwartz, K., Sherlock, G. & Dudoit, S. GC-Content Normalization ligands, activation of interferon-stimulated genes, production of cyto- for RNA-Seq Data. BMC Bioinformatics 12, 480 (2011). toxic molecules by effector immune cells, and upregulation of im- Ethics Approval mune regulatory genes, has been associated with immune-mediated Sidra Medicine IRB approval : #1602002725 tumor rejection. We have previously introduced a gene signature, called Immunology Constant of Rejection (ICR), that reflects these im- mune components.[1–4] This signature was able to differentiate quite well the patients with an active immune environment and improved survival vs those who did not[5]. Virtually, all correlative analyses integrating exome and transcrip- tomic data in colon cancer based on publicly available date use the TCGA cohort. Although it is broadly accepted that T-cell infiltration influences prognosis in colon cancer[6], the association between transcriptomic immune signature and patient survival could not be observed in the TCGA colon cancer cohort. This is likely due to the per protocol exclusion of samples with low tumor purity (i.e., higher stromal/immune infiltration), as at that time TCGA consortium fo- cused on defining cancer genetic makeup. To gain more insight into the underlying mechanism of cancer tissue rejection by the immune system in colon cancer, we build an extensive data repository from high quality snap frozen colon cancer samples unbiased for tumor purity. Methods RNA and DNA were isolated from a cohort of 366 colon cancer pa- tients collected over the last decade at the University of Leiden Med- ical Center (LUMC), Netherlands. Tissue sections flanking the corresponding samples were hematoxylin- and eosin-stained. RNA- seq (HiSeq4000) data was obtained using HISAT2 alignment[7] and quantile normalized after GC-correction of the raw counts.[8] Whole Fig. 1 (abstract P93). The advanced immune-centric NGS cohort for Exome Sequencing (WES) (>100X) was performed for normal and colon cancer cancer tissue (366 RNA-seq and 608 WES). T-cell repertoire was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 52 of 272 P94 A potential mechanism of anti-cancer immune response activated by immune-related adverse events (irAEs) in urological cancer patients 1 1 1 Taigo Kato, MD, PhD , Motohide Uemura , Koji Hatano , Atsunari 1 1 1 2 Kawashima , Takeshi Ujike , Kazutoshi Fujita , Kazuma Kioytani , Norio Nonomura 1 2 Osaka University, Osaka, Japan; Japanese Foundation for Cancer Research, Tokyo, Japan Correspondence: Taigo Kato (kato@uro.med.osaka-u.ac.jp) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P94 Background With the spread of usage of Immune checkpoint inhibitors (ICIs), a certain number of patients face discontinuation of ICIs due to severe immune- related adverse events (irAEs). Recently, some reports have shown en- couraging efficacy among patients who discontinued ICIs, leading to the hypothesis that irAEs-experienced patients have strong and long-lasting anti-cancer immune responses. So far, the molecular mechanisms of the immune response, particularly for T cells that play pivotal roles in attack- ing cancer cells, still remain unclear. Thus, characterization of T cell reper- Fig. 2 (abstract P94). Expanded T cells in metastatic site are toire and immune signatures in peripheral blood mononuclear cells detected in systemic (PBMCs) and tumors before and after ICIs treatment should contribute to better understanding of irAEs-related anti-cancer immune responses. Methods P95 In this study, we collected PBMCs from 4 urological cancer patients, Single-cell RNA-sequencing from clinically relevant core needle before ICIs treatment and at the onset of severe irAEs. For 1 kidney biopsies for evaluation of tumor-immune cell interactions in the cancer patient who had long durable response after discontinuation tumor microenvironment of ICIs, we also collected metastatic tissue sample and applied a next Namit Kumar, PhD, Mohan Bolisetty, Peter Szabo, PhD, Xuan Li, Becky generation sequencing approach to characterize T cell receptor (TCR) Penhallow, Ryan Golhar, Alice Walsh, Saumya Pant repertoires using RNAs isolated from tumors and PBMCs. We also Bristol-Myers Squibb, Princeton, NJ, United States measured mRNA expression levels of immune-related genes in the Correspondence: Namit Kumar (Namit.Kumar@bms.com) PBMCs of pre- and post-ICIs treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P95 Results We found that elevated transcriptional levels of CD3, CD4, CD8, GZMA, Background PRF1, andFOXP3 alongwithhighGZMA/CD3and PRF1/CD3 ratiointhe Elucidating biomarkers associated with immunotherapy response peripheral blood at the onset of irAEs. TCR repertoire analysis revealed and resistance will allow for better informed patient selection and drastic expansion of certain T cell clones in metastatic tissue after irAEs treatment decisions as well as enhanced drug development strategy. (Figure 1). Interestingly, some of these abundant TCR clonotypes were Current biomarker strategies are based on cellular markers (eg, im- also increased in peripheral blood at the onset of irAEs (Figure 2). munohistochemistry) or bulk molecular averages (eg, whole-exome Conclusions sequencing). However, there is limited ability to integrate cellular Our findings revealed that a certain number of expanded- and irAEs- and molecular data. Single-cell RNAseq (scRNAseq) is a promising related T cell clones in cancer tissue may also circulate systemically technology allowing for an unbiased analysis of the tumor microen- and then attack tumor cells in distant regions, leading to durable re- viroment (TME) at cellular resolution. Despite the immense potential, sponse in the patients with irAEs. implementation of this technology in clinical trials has been limited due to lack of methodologies applicable to clinically relevant speci- mens such as core-needle biopsies (CNB). Here, we describe the de- velopment of clinically applicable scRNAseq technology and analysis. Methods Treatment-naïve commercially sourced tumor resections were used to gen- erate ex-vivo CNB for scRNAseq analysis with 10X genomics. Post-clustering, unsupervised cell-type identification was performed (SingleR), and down- stream analyses were carried out (Seurat v2, custom R). Cells from multiple patients/tumor types (endometrial, TNBC, NSCLC, ccRCC, gastrointestinal; n= 8), and healthy donors (peripheral blood mononuclear cells; n=3) were com- bined, batch-corrected and aligned using canonical correlation analysis (CCA); and differential gene expression was performed (MAST algorithm). Results CNB scRNAseq was optimized across 5 tumor types, and the resulting data from ~43,000 cells allowed for the unbiased identification of TME cellular components (stromal, epithelial, immune-cell subtypes). The cellular resolution of this dataset allowed us to identify cell pop- ulations with distinct gene signatures. For example, we identified 2 macrophage subclusters—a lung tumor-specific cluster and a tumor- independent cluster. Lung-specific macrophages showed upregula- tion of genes including SPP1, G0S2, RGCC, PHLDA1, and TREM. Differ- ential gene expression analysis evaluated similarities and differences between TME vs healthy PB cells and allowed for surrogate pharma- codynamics marker assessment. In our analysis, 1197 genes were dif- Fig. 1 (abstract P94). Clonal T cell expansion in ferentially expressed; the most enriched genes in tumor-derived pancreatic metastasis monocytes included HSPA1A, IL8, APOE, and SPP1 whereas PB Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 53 of 272 monocytes were enriched for genes including LGALS2, S100A12, P97 S100A9, AHNAK, and CSTA. TCRB repertoire convergence and clonal expansion define the Conclusions NSCLC tumor microenvironment of responders to anti-PD-1 We have demonstrated the feasibility of scRNAseq from single CNB monotherapy 1 2 2 through the development of protocols to enable identification of bio- Timothy Looney, PhD , Katharina Leonards , Ilaria Alborelli , Luca 1 2 markers related to pharmacodynamics, therapeutic response, or disease Quagliatta , Philip Jermann 1 2 progression. Further, we have optimized the bioinformatics workflow to Thermo Fisher Scientific, Austin, TX, United States; University of Basel, derive meaningful biological insights from these scRNAseq datasets, Basel, Switzerland such as mechanisms involved in immune response or resistance that Correspondence: Philip Jermann (philipmartin.jermann@usb.ch) are tumor extrinsic or intrinsic. Our pilot study sets the groundwork to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P97 explore including scRNAseq in future prospective clinical studies. Background Acknowledgements There is an outstanding need to identify predictive biomarkers for re- Bristol-Myers Squibb. sponse to anti-PD-1 monotherapy for NSCLC. Here we investigated TCRB clonal expansion and TCR convergence within the pretreatment tumor microenvironment as predictors of response in a cohort of 37 P96 FFPE-preserved biopsies. For context, we compared the predictive T-cell receptor alpha and beta repertoire profiling using an value of these features with TMB values from the same tumors. augmented transcriptome Methods Eric Levy, PhD, Pamela Milani, Sean Boyle, PhD, Gabor Bartha, Charles Total RNA from FFPE-preserved pretreatment NSCLC biopsies (11 re- Abbott, PhD, Robert Power, Rena McClory, Robin Li, John West, MBA, sponders, 14 non-responders) was extracted for TCRB repertoire se- Richard Chen quencing via the Oncomine TCRB-SR assay (15-265ng RNA input; Personalis, Inc., Menlo Park, CA, United States average 164ng) and the Ion Torrent Gene Studio S5. TMB values Correspondence: Richard Chen (richard.chen@personalis.com) were obtained from FFPE-preserved gDNA from the same biopsies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P96 using the Oncomine Tumor Mutation Burden Assay. TCR conver- gence and clonal expansion were evaluated independently or in a Background combined model as predictors of response. The promise of immunotherapy has revealed the need for compre- Results hensive profiling of the tumor and its immune microenvironment, in- TCRB sequencing revealed increased TCR convergence (p = .02, Wil- cluding analysis of the T-cell receptor (TCR) repertoire. To address coxon) and clonal expansion (p = .06, Wilcoxon) in those who bene- this challenge, we developed ImmunoID NeXT to provide a more fited from anti-PD-1 therapy. A logistic regression classifier comprehensive view of the tumor and tumor microenvironment combining both features was able to discriminate responders from (TME) from limited FFPE tumor biopsies. This includes profiling both non-responders with a sensitivity of .91 and specificity of .71 at the the TCR alpha and beta chains. We show that ImmunoID NeXT accur- optimal cutoff, per the Youden’s J method. The TCR-based classifier ately and reproducibly profiles abundant clones and provides infor- was able to identify responders who otherwise had low to intermedi- mation on the diversity of T-cells in tumor samples. ate (<10muts per Mb) TMB. Methods Conclusions We first analyze the reproducibility of ImmunoID NeXT using repli- TCRB clonal expansion and convergence warrant further evaluation cates of PBMCs. Then, we compare the concordance of clones from as potential predictive biomarkers of response. Importantly, TCRB se- ImmunoID NeXT to the top clones from a standalone TCR sequen- quencing may allow for identification of responders who are other- cing approach. We also analyze the reproducibility of clones in wise missed by TMB-based stratification. patient-derived FFPE samples, and compare to IHC quantification of CD3+ cells to highlight the intra-sample heterogeneity of T-cell abun- dance and diversity. We then analyze the clonal diversity of pre- P98 treatment tumor samples in a cohort of melanoma patients who Automated rarefaction analysis for precision human and mouse B underwent PD-1 blockade. Finally, we use ImmunoID NeXT to profile and T cell receptor repertoire profiling from peripheral blood and the clonal diversity across over 100 solid tumor samples. FFPE-preserved specimens Results Timothy Looney, PhD, Geoffrey Lowman, PhD, Michelle Toro, Jayde Abundances of clones shared between replicates of PBMC samples Chang, Denise Topacio-Hall, BS, MA, Loni Pickle, PhD, Fiona Hyland, have a very high concordance (R2>0.99 with both TRA and TRB). Timothy Looney, PhD Compared to the standalone TCR approach, we identify over 96% of Thermo Fisher Scientific, Austin, TX, United States the top 1000 TRA clones, and over 99% of the top 1000 TRB clones, Correspondence: Timothy Looney (timothy.looney@thermofisher.com) both with highly concordant abundances (R2>0.95 and R2>0.94 in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P98 TRA and TRB, respectively). Subsequent curls of a tumor FFPE sample also have a high concord- Background ance of clonal abundances (R2>0.89 and R2>0.91 in TRA and TRB, re- Identifying the optimal input amount and sequencing depth for B spectively). TCR sequencing also provides a view of the clonal diversity and T cell receptor repertoire profiling is challenging owing to vari- of T-cells in a sample, which is not available with quantification via IHC. ation in material quality and lymphocyte diversity in blood and FFPE Finally, in a melanoma cohort, clonality based on either TRA or TRB is preserved specimens. Rarefaction analysis has emerged as a potential significantly different in responders to checkpoint inhibition. approach for assessing whether immune repertoire libraries have Conclusions been sequenced to saturation. Here we present a novel automated The ImmunoID NeXT platform can provide insight into the diversity method for saturation analysis of IGH and TCRB chain libraries de- of the immune repertoire, highlighting the platform’s ability to pro- rived from sequencing of peripheral blood leukocytes (PBL) and vide comprehensive analysis of both the tumor and tumor micro- FFPE-preserved RNA and DNA. environment. We demonstrate that ImmunoID NeXT is reproducible, Methods sensitive, and accurate at profiling high-abundance TRA and TRB Human TCRB and IGH repertoire libraries were generated using the clones, as well as feasible with FFPE samples. We also highlight how Oncomine TCRB-SR and BCR IGH-SR assays from: (1) 25ng PBL total immune repertoire results from ImmunoID NeXT can be used to gain RNA (2) 500ng PBL gDNA (3) 150ng RNA from FFPE preserved NSCLC understanding about the immunological composition of the TME. Fi- and (4) 200ng gDNA from FFPE preserved brain tissue. Mouse TCRB nally, we show how ImmunoID NeXT can profile the diversity of the and IGH libraries were generated using the Ion Ampliseq TCRB-SR TCR repertoire in tumor samples. and BCR IGH-SR assays and 25ng RNA or 500 ngDNA derived from Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 54 of 272 spleen or lymph node. Libraries were sequenced on the Ion Torrent P100 Gene Studio S5 then analyzed with Ion Reporter to identify clono- Impact of obesity on immunity in gastroesophageal types, quantify clonal expansion and diversity, and for IGH chain li- adenocarcinoma [GEAC] 1 2 1 braries, identify B cell clonal lineages and assess isotype usage. We Sarbajit Mukherjee, MD, MS , Sami Ibrahimi , Yali Zhang , Jianmin 1 1 then repeated clonotyping and analysis of secondary repertoire fea- Wang , Pawel Kalinski, MD, PhD tures using data that had been downsampled to fixed read depths. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Results University of Oklahoma, Oklahoma City, United States We observed an asymptotic relationship between the sequencing depth Correspondence: Sarbajit Mukherjee and the number of B and T cell clones detected, clone Shannon diversity, (sarbajit.mukherjee@roswellpark.org) and B cell clonal lineage richness and diversity, indicating that libraries Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P100 had been sequenced to saturation. By contrast, T and B cell normalized Shannon entropy appeared robust to sequencing depth. Background Conclusions Obesity is associated with an elevated risk of GEAC [1], but the Automated downsampling analysis may serve as a convenient tool molecular mechanism remains unknown. Paradoxically, however, for optimizing sequencing depth and input amount for B and T cell obesity is associated with a superior response to anti-PD-1 treat- repertoire sequencing studies. We expect this approach to become a ment [2,3]. This may be explained by our recent observations routine component of immune repertoire analysis. that obesity enhances PD-1 mediated T-cell dysfunction in a mechanism involving leptin signaling. Prompted by this data, we aimed to identify obesity/leptin-regulated molecular biomarkers in P99 GEAC. TMBler: a bioinformatic tool for measuring and optimizing Tumor Methods Mutational Burden quantification from targeted sequencing panels Based on the body-mass index (BMI), we categorized patients Laura Fancello, Luca Mazzarella, MD PhD, Alessandro Guida, Arnaud into normal (BMI 18-24.9), overweight (BMI 25-29.9) and obese Ceol, Piergiuseppe Pelicci, Luca Mazzarella, MD PhD (BMI ≥30). We then retrospectively analyzed the clinical report of IEO Istituto Europeo di Oncologia IRCCS, Milano, Italy PD-L1 staining by IHC_22C3/Keytruda from metastatic GEAC pa- Correspondence: Luca Mazzarella (luca.mazzarella@ieo.it) tients treated at our institution between 2014-2019. Chi-squared Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P99 test was used to determine the association between categorical variables. Next, we performed RNA-seq analysis of 13 gastric can- Background cer FFPE specimens (8 obese and 5 normal weight) to identify Tumor mutational burden (TMB) is increasingly proposed as a predict- differential gene expression between these two groups. Gene ex- ive biomarker for immunotherapy response in cancer patients. pression was quantified by log-fold changes. Differentially TMB assessed by Whole Exome Sequencing (WES) is considered the gold expressed genes were identified by using DESeq2. Then we standard but remains confined to research settings. Targeted enrichment looked at the association between the expression of leptin and panels of various genomic sizes are emerging as a more sustainable method- immune-related genes from those specimens, using generalized ology for assessing TMB in the clinical setting. However, panel-based TMB linear model implemented in DESeq2. TCGA gastric cancer data- quantification has not been adequately standardized to date, leading to base (TCGA -STAD) was used to validate these associations (using major heterogeneities in TMB measurement and a lack of uniformly accepted Pearson test) independently. A p-value of <0.05. cutoff values, thus limiting the possibility to transfer results across settings. In Results particular, the choice of variants to include in TMB calculation (synonymous, Our analysis of the clinical report of 77 patients with metastatic cancer driver genes or low-allelic frequency mutations, or other features) may GEAC revealed that patients with a BMI =>25 were more likely to strongly affect results and in particular TMB predictive value [1] express PD-L1 than normal-weight individuals (p = 0.03)(Table 1). Methods Our RNA-seq analysis identified the following genes to be up- We developed "TMBler", an R package to calculate TMB from targeted regulated in the obese group: NOS2, FOXP3, IDO1, EOMES, sequencing panels. TMBler allows to select multiple filters on mutation CD160, and CXCR5 (p<0.05). Expression of these genes was posi- counts for TMB quantification. It also includes a set of functions to tively correlated with leptin in our database; however, these asso- simulate custom panels on WES and calculate predictive value based ciations did not reach statistical significance; possibly due to our on available data on immunotherapy response matched with sequen- small sample size. The same analysis within the TCGA-STAD data- cing data [2,3]. Finally, it allows to measure panel-based TMB concord- base identified a strong positive correlation between the expres- ance with WES-based TMB and its predictive value using Receiver sion of all six genes and leptin (p <0.05)(Figure 1). GSEA Operating Characteristic (ROC) curves. identified several up-regulated immune-related pathways (Adap- Results tive Immune System, Antigen Processing Cross Presentation etc.) By simulating custom and commercially available panels, we show that the in the obese group. application of specific filter combinations can significantly influence TMB cal- Conclusions culation and its predictive value, and we identify instances where risk of erro- Our preliminary data suggest that obesity, and specifically lep- neous assignment of patients to responder/nonresponder groups is highest tin, is associated with several immune markers in GEAC. Our Conclusions mechanistic studies will explore how obesity/ leptin regulates TMBler is a useful tool for quantifying TMB from targeted panels. It the immune system and promotes cancer. These studies may can analyze performance of existing panels, optimize analytical pipe- allow us to identify new leptin regulated pathways as thera- line and design novel custom panels through simulations. peutic targets. References References 1. Fancello L, Gandini S, Pelicci PG, Mazzarella L. Tumor mutational burden 1. Garai J, Uddo RB, Mohler MC, Pelligrino N, Scribner R, Sothern MS et al. quantification from targeted gene panels: major advancements and At the crossroad between obesity and gastric cancer. Methods Mol challenges. J Immunother Cancer. 2019 Jul 15;7(1):183 Biol.2015; 1238:689-707. 2. Hellmann MD, Nathanson T, Rizvi , et al. Genomic Features of Response 2. Ibrahimi S, Mukherjee S, Roman D, King C, Machiorlatti M, Aljumaily R. to Combination Immunotherapy in Patients with Advanced Non-Small- Effect of Body Mass Index and Albumin level on Outcomes of Patients Cell Lung Cancer. Cancer Cell. 2018 Receiving Anti PD-1/PD-L1 Therapy. J Clin Oncol 36, 2018 (suppl 5S; abstr May 14;33(5):843-852 213). 3. Samstein RM, Lee CH, Shoushtari AN. Tumor mutational load predicts 3. Wang Z, Aguilar EG, Luna JI, Dunai C, Khuat LT, Le CT et al. Paradoxical survival after immunotherapy across multiple cancer types. Nat Genet. effects of obesity on T cell function during tumor progression and PD-1 2019 Feb;51(2):202-206 checkpoint blockade. Nat Med. 2019 Jan; 25(1):141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 55 of 272 Ethics Approval P101 The study was approved by the Institutional Review Board at Roswell Park Immune-based classification of pleural malignant mesothelioma by Comprehensive Cancer Center, approval number STUDY00000894 / BDR using integrative transcriptome analysis 1 2 1 2 109419. Ernest Nadal, MD, PhD , Ania Alay , David Cordero , Elisabeth Aliagas , 1 1 3 3 José Ruffinelli , Ramón Palmero , Ricard Ramos , Ivan Macía , Anna 3 3 1 Ureña , Fran Rivas , Xavier Solé 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; Bellvitge Biomedical Research Institute, L'Hospitalet, Spain; Bellvitge University Hospital, Table 1 (abstract P100). See text for description L'Hospitalet, Spain Correspondence: Xavier Solé (x.sole@iconcologia.net) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P101 Background Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia. Immune checkpoint inhibitors in MPM demonstrated modest efficacy, partly due to lack of predictive biomarkers of clinical benefit from immunotherapy. The aims of this work were: to identify immune fractions associated with clinical outcome; to stratify MPM patients based on their immune contexture and to characterize the immune-based groups at the genomic and transcriptomic levels. Methods Seven gene-expression datasets of MPM were used to assess theimmunemicroenvironmentof516 samples. The abundance of 20 immune fractions in each sample was inferred using Gene Set Variation Analysis. Identification of clinically-relevant frac- tions was performed with Cox Proportional-Hazards Models ad- justed for age, stage, sex, and tumor histology. Results T-Helper 2 (Th2, HR=2.14, p=1.5x10-4) and cytotoxic T cells (CTC; HR=0.57, p=9.1x10-3) were found to be consistently asso- ciated with overall survival in multiple datasets. Three immune clusters (IG) were subsequently defined based on Th2 and CTC immune infiltration levels: IG1 (54.5% of samples) had high Th2/ low CTC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) had low Th2/high CTC levels. Immune clusters were associated with overall survival independently of tumor histology, with an improving survival from IG1 to IG3 (HR IG2=0.52, 95% CI 0.39–0.69; HR IG3=0.32, 95% CI 0.19–0.53; p= 8.4x10-8; Figure 1). IG3 was significantly enriched in epithelioid tumors (90% IG3 vs. 62% IG1, p=0.001) and patients were youn- ger compared to the other groups(60 yearsIG3 vs. 66years IG1, p=0.021). These groups showed differential molecular pro- files, being IG1 enriched for CDKN2A and IFN-related genes de- letions. No statistically significant differences in the tumor mutational burden was observed, howerver IG3 tumours had fewer mutations than IG1 and IG2 groups. At the transcriptional level, IG1 samples showed upregulation of cell proliferation and DNA repair-related gene-sets, while IG3 samples presented up- regulation of immune checkpoint inhibitors (Figure 2) and inflammation-related pathways. Finally, integration of gene ex- pression with functional signatures of in vitro drug response showed that IG3 patients are more likely to respond to immune checkpoint inhibitors, while IG1 patients might be more sensi- tive to PARP inhibitors. Conclusions Analysis of publicly available gene-expression data of MPM reveals three major immune-based groups, based on Th2 and CTC composition. These clusters are associated with distinct genomic profiles and clinical outcome. Further validation of this classification is warranted in an independent cohort of Fig. 1 (abstract P100). See text for description MPM. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 56 of 272 P102 CT antigens are frequently expressed non-inflamed tumors Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS , Erik Van Roey, Sean Glenn, PhD, Jonathan Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, Yirong Wang, MS, Carl Morrison, MD, DVM OmniSeq, Inc., Buffalo, NY, United States Correspondence: Sarabjot Pabla (sarabjot.pabla@omniseq.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P102 Background Cancer testis (CT) antigens are tumor antigens that have a highly tis- sue restricted expression in germ cells but are often expressed in di- verse malignancies. With their highly immunogenic expression limited to tumor cells, CT antigens have become a prime target for cancer vaccinations and T-cell based therapy with chimeric T-cell re- ceptors. In this study, we investigated the association of two CT anti- gens (NY-ESO-1 and LAGE-1a) with the immune microenvironment of real-world clinical tumors spanning multiple histologies. Furthermore, we describe the association of CT antigens with traditional bio- markers of immunotherapy such as PD-L1 immunohistochemistry (IHC) and tumor mutational burden (TMB), with inflammatory status and cell proliferation status with confirmatory studies performed on a large TCGA pan-cancer cohort of 11,001 tumors. Methods Unsupervised clustering was performed on gene-expression data of 395 immune transcripts of 1323 FFPE tumors to reveal three inflam- matory patient clusters and three distinct gene groups; CT-antigen, inflammatory and neoplastic clusters. Test for proportions was per- formed using Pearson’s chi-squared test to describe association of NY-ESO-1 and LAGE-1a with PD-L1 IHC, TMB, inflammatory cluster and cell-proliferation. A retrospective cohort (n=242) of checkpoint Fig. 1 (abstract P101). Overall survival analysis according to the inhibition (CPI) treated tumors was utilized to perform overall survival immune groups (Kaplan-Meier curves) and response to CPI therapy for CT antigen+ tumors. Survival analysis was confirmed against the Pan-Cancer TCGA cohort (n=11,001). Results Unsupervised clustering showed clear co-expression sub-clustering of CTA genes differentiated from “immune” and from “neoplastic expres- sion”. PD-L1 IHC status was not associated with NY-ESO-1 (p=0.71) or LAGE-1a (p=0.52) status. Interestingly, LAGE-1a positive cases were over-represented in TMB high cases (p=0.016), whereas, NY-ESO-1 sta- tus was not associated with TMB. NY-ESO-1 positive cases were highly over-represented in non-inflamed cluster (p=0.006), whereas, LAGE-1a status was not associated with inflammation status. Both NY-ESO-1 (p= 0.031) and LAGE-1a (p=0.008) were significantly associated with cell- proliferation status. NY-ESO-1 positive tumors have significantly (p= 0.014) higher response rate in retrospective cohort but this was not ob- served for LAGE-1a status. NY-ESO-1 and LAGE-1a status showed trend toward better (p=0.09 and p=0.06 respectively) survival in the retro- spective and TCGA pan-cancer cohort. Conclusions This study presents an in-depth analysis of the immune landscape of CT antigen positive tumors across multiple histologies. CT antigen bearing tumors not only have unique immune profiles but also have significant associations with biologically relevant emerging bio- markers such as inflammatory signature, TMB and cell proliferation. CT antigens are a viable target for non-inflamed tumors for check- point inhibition therapy. Ethics Approval De-identified specimens and data were analyzed by OmniSeq under IRB approved protocol BDR 080316 (Roswell Park Comprehensive Fig. 2 (abstract P101). Expression of immune checkpoint markers Cancer Center, Buffalo, NY). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 57 of 272 P103 Methods Detection of human leukocyte antigen class I loss of Stage I-III subjects (n=24) receiving curative-intent chemo (doxo- heterozygosity in solid tumor types by next-generation DNA rubicin, cyclophosphamide, paclitaxel) were monitored longitudin- sequencing ally (mixed effects linear model) with serial peripheral blood Jason Perera, PhD , Brandon Mapes, PhD, Denise Lau, PhD, Ameen mononuclear cell flow cytometry and quantitative immunose- Salahudeen, Aly Khan, PhD quencing of the T-cell receptor β locus (TCRseq) using the immu- Labs, Chicago, IL, United States noSEQ® assay (Adaptive Biotechnologies, Seattle, WA). To evaluate Correspondence: Jason Perera (jason.perera@tempus.com) for long-term chemo effects, these analyses were repeated in a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P103 cohort of recurrent breast cancer patients who received chemo >12 months preceding analysis (n=9). Wilcoxon rank sum and Background tests of slope were employed to screen for associations with Human leukocyte antigen (HLA) class I proteins are expressed on the chemo response, defined as complete pathologic response (pCR) surface of all nucleated cells and are vital for immune surveillance. at surgical resection. When tumor-specific mutations (neoantigens) are presented on HLA Results molecules to CD8+ T cells, this recognition can drive immune re- By TCRseq, chemo resulted in an acute decline in T-cell fraction (0-8 sponses against the tumor and lead to tumor destruction. One mech- weeks, p12 months following chemo. anism of immune escape for tumors is loss of heterozygosity in HLA Conclusions genes (HLA-LOH), which reduces the total number of neoantigens avail- Curative-intent chemo is associated with T-cell death followed able for presentation to T cells. Due to the highly polymorphic nature by reconstitution, with the resulting T-cell repertoire being more of HLA, the copy number status of HLA genes is extremely challenging clonal and less abundant in naïve T cells. These findings persist to assess by standard bioinformatics approaches. To investigate the at the time of metastatic recurrence, and therefore may contrib- prevalence of HLA-LOH, we developed a specialized pipeline to detect ute to immunotherapy non-response in metastatic disease. Con- HLA-LOH by DNA next-generation sequencing (NGS). versely, we identified T-cell reconstitution as a potential Methods biologic modifier of chemo response. T-cell reconstitution can A cohort of colorectal and non-small cell lung cancer samples be therapeutically targeted with inhibitors of androgen receptor underwent DNA sequencing on the Tempus xT panel using signaling, which in experimental models enables thymic matur- paired, formalin-fixed, paraffin-embedded tumor and normal ation of naïve T-cell clones and an increase in peripheral T-cell (blood or saliva) samples. To detect HLA-LOH from NGS data, we count. This hypothesis is beingevaluatedinanongoingphase used NGS-based HLA typing to resolve the patient’smostprob- II clinical trial of bicalutamide (androgen receptor antagonist) able HLA haplotype. Based on this haplotype, we adaptively rea- plus ipilimumab and nivolumab in metastatic breast cancer ligned reads, extracted a number of features describing the (NCT03650894). relative allele coverage in the tumor and normal samples, and Ethics Approval used these features to make a confident determination of allelic The study was reviewed and approved by Providence Heath and Ser- loss in the patient’s tumor sample. vices Internal review board, approval number 15-162. Results Evidence of HLA-LOH was detected in 16% of non-small cell lung tumor samples and 17% of colorectal tumor samples. We did not ob- P105 serve a significant association between LOH status and tumor muta- PD-L1 isoform as a potential biomarker to predict response for tional burden or neoantigen load. In the colorectal cancer cohort, anti-PD-(L)1 treatment HLA-LOH was observed in tumor samples classified as microsatellite Kunbin Qu (kunbin.qu@beigene.com) instability-high (MSI-H); however, the association between HLA-LOH BeiGene, USA, San Mateo, CA, United States status and MSI status was not statistically significant. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P105 Conclusions We developed a novel method of determining HLA-LOH by DNA Background NGS and demonstrated that HLA-LOH is a readily detectable feature anti-PD-1/anti-PD-L1 (anti-PD-(L)1) therapies have shown clinical ac- in human tumors. These results highlight the complexity of antigen tivity across different cancers. However, predicting patient response presentation, the potential importance of HLA-LOH as a biomarker of remains challenging. Here we explore PD-L1 splicing isoforms as a immunotherapy response and resistance, and lays the groundwork potential predictive biomarker for anti-PD-(L)1 therapy response. Four for future investigations. PD-L1 splicing isoforms exist, including one dominant wildtype tran- script and an alternative isoform which skips the second exon (del- taExon2_PD-L1). P104 Methods Impact of chemotherapy (chemo) on peripheral T-cell diversity and TCGA normalized mRNA transcript counts were downloaded implications for subsequent immunotherapy response in breast from Genomic Data Commons. anti-PD-1 treated melanoma cancer 1 2 3 RNA-Seq data was from Hugo et. al. [1]. Bioinformatics analyses Joanna Pucilowska, PhD , Paul Fields, PhD , Valerie Conrad, BS , David 3 3 3 were performed in statistical package R. Human wildtype and Page, MD , Alison Conlin, MD , Joanna Pucilowska, PhD , Catherine 2 3 3 3 deltaExon2_PD-L1 isoforms were stably transfected into the Sanders, PhD , Raina Tamakawa, MS , Brie Chun, MD , Isaac Kim, MD , mouse cell-line BW5147. A chimeric PD-1 receptor, P3Z, which Mark Schmidt 1 2 fuses the extracellular and transmembrane domains of human Providence Cancer Center, Portland, OR, United States; Adaptive PD-1 to the cytoplasmic domain of human CD3ζ, was stably Biotechnologies, Seattle, WA, United States; EACRI Providence Cancer transfectedintoHuT78 cellsasa reporter assay for PD-1 signal- Center, Portland, OR, United States ing and IL-2 production [2]. Correspondence: David Page (david.page2@providence.org) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P104 By examining protein crystal structures from Protein Data Bank, we found exon2 occupies the physical interface between PD-1 and PD- Background L1. It is also the interface between anti-PD-L1 therapeutics and PD- Immune checkpoint blockade is only modestly effective in metastatic L1. Therefore, anti-PD-L1 molecules may not effectively target PD-L1 breast cancer. One potential contributing factor is chronic lymphode- in patients harboring the deltaExon2_PD-L1 isoform and may lack pletion associated with preceding curative-intent chemo. Here, we clinical activity. The prevalence of the deltaExon2_PD-L1 isoform evaluate the short and long-term effects of chemo on peripheral T- across TCGA tumors is shown in Figure 1. There are 8 cancers where cell counts and clonal diversity in a cohort of breast cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 58 of 272 the isoform is present above 5%, including liver and endometrial cancers. The deltaExon2_PD-L1 isoform was successfully transfected into BW5147 as demonstrated by mRNA expression. In co-cultures of HuT78/P3Z with BW5147/PD-L1 (both the wildtype and deltaEex- on2_PD-L1), IL-2 was secreted from the wildtype but not from the deltaExon2_PD-L1. Incubation with anti-PD-1 reduced IL-2 in a dose- dependent manner with the wildtype only (Figure 2), indicating del- taExon2_PD-L1 does not support PD-1 signaling. Patients expressing only deltaExon2_PD-L1 or a higher ratio of deltaExon2_PD-L1/wildtype may not have optimal PD-(L)1 axis signaling and be less responsive to anti-PD-1 intervention. To test this hypothesis, a ratio metric between deltaExon2_PD-L1 and wildtype was applied to an anti-PD-1 treated melanoma cohort GSE78220. This biomarker ratio stratified responders from non- responders with a p-value of 0.027 (non-responders with no del- taExon2_PD-L1 isoform were excluded, Figure 3), whereas PD-L1 expression did not. Conclusions Patients with deltaExon2_PD-L1 isoform lack the interface be- tween PD-L1 and PD-1, the same interface necessary for anti-PD- L1 therapeutic binding. This may lead to non-optimal signaling through the PD-(L)1 axis. Suboptimal signaling and inability to bind anti-PD-L1 potentially could reduce response to both anti- PD-1 and anti-PD-L1 treatments. This hypothesis needs to be fur- ther validated in additional anti-PD-L1 and anti-PD-1 treated Fig. 2 (abstract P105). See text for description cohorts. Acknowledgements The authors would like to thank Vanitha Ramakrishnan and Jessica Li for scientific discussions. References 1. Hugo W. et. al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell 2016; 165:35- 2. Zhang T. et. al. The binding of an anti-PD-1 antibody to FcγRΙ has a pro- found impact on its biological functions. Cancer Immunol Immunother. 2018; 67:1079-1090. Fig. 3 (abstract P105). See text for description P106 Tumor mutational burden profile (TMB) of oncogenic driver mutations in non small cell lung cancer Paul Walker, MD, Nitika Sharma East Carolina University, Greenville, NC, United States Correspondence: Nitika Sharma (sharman@ecu.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P106 Background Tumor mutational burden has emerged as a potential biomarker pre- dictive of response to Immune checkpoint blockade (ICB) in lung cancer. The utility of this biomarker in oncogenic driver mutations, that account for nearly 20-50% of NSCLC, is still unknown. KRAS mu- tation in lung cancer is a prognostic biomarker whereas EGFR and Fig. 1 (abstract P105). See text for description BRAF pathogenic mutations are predictive of response to tyrosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 59 of 272 kinase inhibitors (TKI). ICB with bevacizumab has demonstrated clin- TMB was higher in current and former smokers (median TMB 10.7 v ical benefit in EGFR mutated lung cancers per IMpower150 clinical 6.4 mutations/megabase(Mb); p < 0.0001), patients without an identi- trial [1]. TMB analysis between actionable/pathogenic EGFR muta- fiable oncogenic driver mutation (median TMB 14.5 v 8.5 mutations/ tions (i.e. exon 19 del, exon 21 L858R, T790M) and EGFR uncommon/ Mb; p = 0.004), and patients with locoregional disease at the time of variants mutations may provide therapeutic implications [2]. To ex- diagnosis (median TMB 10.8 v 8.0 mutations/Mb, p = 0.02). TMB cor- plore the immunological basis for these findings, we evaluated the related closely across all matched tumor pairs (Pearson’s r = 0.85, Fig- immune biomarker profile of NSCLC patients using Caris next- ure 1). Significant increases or decreases in TMB were uncommon in generation sequencing (NGS) platform. paired samples, and we observed no significant change in median Methods TMB with increasing time between specimen collection or with inter- We studied tissue samples on 446 patients with NSCLC from 2016- vening chemotherapy, immunotherapy, radiation therapy, or tar- 18. TMB was measured by counting all non-synonymous somatic mu- geted therapy. tations per megabase of the genome coding area using targeted Conclusions NGS (592 genes). High TMB was defined as ≥ 10 mut/Mb. The ana- In NSCLC, TMB correlated closely across tumor pairs, and increasing lysis was conducted using SAS 9.4. Variables were tested using a Wil- time between sample collections and intervening treatments were coxon signed-rank test. not correlated with significant changes in TMB. Results Ethics Approval KRAS mutations were found in 85 pts (19%), BRAF in 9 pts (2%), EGFR This study was conducted under Dana-Farber/Harvard Cancer Center mutation in 36 pts (8%), EGFR pathogenic mutation in 22 pts (5%), Protocol 02-180. EGFR variants in 14pts (3%). The median TMB of KRAS mutant vs KRAS wt (wild type) was 10 vs 7 mut/Mb (range 0-31, p<0.01). Conclusions This study highlights the unique immune profile of certain oncogenic driver mutations in NSCLC. Our results show that KRAS and BRAF mu- tant subsets have a significantly higher TMB than KRAS and BRAF wild type. In addition, EGFR variants have a higher TMB as compared to actionable pathogenic EGFR mutations. These findings could have therapeutic implications in guiding patient selection for ICB and merit a prospective investigation. References 1. Socinski M, Jotte R,Cappuzzo F. Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC. N Engl J Med. 2018; 378:2288-2301. 2. Offin M, Rizvi H,Tenet M.Tumor Mutation Burden and Efficacy of EGFR- Tyrosine Kinase Inhibitors in Patients with EGFR-Mutant Lung Cancers. Clin Cancer Res. 2018;1102. Ethics Approval The study was approved by ECU Institutional Review Board, approval number UMCIRB 15-001311. P107 Changes in tumor mutational burden in serially biopsied non-small cell lung cancer 1 1 1 James Smithy, MD, MHS , James Smithy, MD, MHS , David Hwang, MD , 2 2 3 1 Yvonne Li , Liam Spurr , Andrew Cherniack, PhD , Lynette Sholl , Mark Awad, MD PhD 1 2 Brigham and Women's Hospital, Boston, MA, United States; Dana- Farber Cancer Institute, Boston, MA, United States; Broad Institute of MIT and Harvard, Boston, MA, United States Fig. 1 (abstract P107). See text for description Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P107 P108 Background Working towards precision medicine of the tumor High tumor mutational burden (TMB) has been associated with re- microenvironment 2 2 2 2 sponse to checkpoint blockade in non-small cell lung cancer (NSCLC) Kyung Kim, PhD , Jeeyun Kim , Seung-Tae Kim , Jung-Yong Hong , 1 1 and other malignancies. However, the degree to which TMB changes Laura Benjamin , Kristen Strand-Tibbitts, PhD 1 2 over time, across anatomical sites, and with intervening treatment re- Oncologie Inc, Waltham, MA, United States; Samsung Medical Center, mains unknown. To evaluate TMB changes across time points, we Seoul, Republic of Korea compared TMB in tissue specimens from patients with serially- Correspondence: Kristen Strand-Tibbitts biopsied NSCLC. (kristen@oncologie.international) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P108 Clinicopathologic characteristics and changes in TMB were analyzed from patients with NSCLC and more than one tissue specimen that Background had undergone targeted next generation sequencing (NGS, OncoPa- Immune therapies for cancer have generated an enhanced focus on nel) at the Dana-Farber Cancer Institute. Those representing distinct controlling cancer through modulation of biologies associated with primary tumors by histologic or genomic analysis were excluded. the tumor microenvironment, rather than the traditional approach of Results targeting cancer cell biology. As more and more targeted therapies 193 NSCLC patients with more than one interpretable NGS result are designed to modulate the tumor microenvironment, we need a were identified; 30 were excluded due to separate primary tumors. better understanding of microenvironmental heterogeneity in human Of the 163 remaining patients included in the analysis, the median cancer. Similar to what has been done to describe patient subsets time between samples was 14 months (range: 0 to 114 months). based on their cancer biology using DNA and RNA signatures, we are Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 60 of 272 working to describe patient subsets based on their microenviron- nivolumab treatment. The CD8 signature may be used alone and/or in ment biology. The ultimate goal is to find effective means of identify- combination with other relevant and potentially independent bio- ing patients for novel therapeutic treatments that target biological markers such as PD-L1 expression or TMB to identify patients likely to pathways that regulate the non-neoplastic cells and drive cancer benefit from anti–PD-1 therapies. progression. Methods Acknowledgements RNA from publicly available sources including microarray and RNA- Bristol-Myers Squibb. Professional medical writing and editorial assistance Seq were analyzed with respect to gene signatures that describe four were provided by Katerina Pipili, PhD, and Jay Rathi, MA, of Spark Medica Inc, different microenvironmental phenotypes. funded by Bristol-Myers Squibb. Results Trial Registration These four microenvironmental subtypes are prognostic, but also NCT02387996 show evidence of being predictive to existing modalities of cancer drugs when analyzed in retrospective analysis. We examined the im- References pact of cancer stage on the distribution of these subtypes and find 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene little variation. signature with CD8 expression by immunohistochemistry (IHC) in Conclusions multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. Future clinical trials to prospectively test these four unique signatures 2. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- as predictive biomarkers for therapy need to be designed. lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. 3. Galsky M, Saci A, Szabo P, et al. Impact of tumor mutation burden on P109 nivolumab efficacy in second-line urothelial carcinoma patients: explora- Predictive performance of a CD8-derived signature by gene tory analysis of the phase II CheckMate 275 study. Ann Oncol. 2017; expression profiling in patients with urothelial carcinoma from 28(Suppl 5): Abstract 848PD. CheckMate 275 Ethics Approval 1 2 1 Peter Szabo, PhD , Padmanee Sharma, MD, PhD , George Lee, PhD , The protocol was approved by site institutional review boards or 1 1 1 1 Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , Robin independent ethics committees and conducted according to Good 1 1 1 1 Edwards, PhD , Saumya Pant , Abdel Saci , Neeraj Adya , Matthew Clinical Practice guidelines, per the International Conference on Galsky, MD Harmonisation. Patients provided written informed consent based on 1 2 Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD Declaration of Helsinki principles. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Institute, New York, NY, United States Correspondence: Peter Szabo (Peter.Szabo@bms.com) P110 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P109 Tumor CD8+ T-cell infiltration assessed by gene expression profiling alone or by immunohistochemistry plus epithelial- Background mesenchymal transition gene expression in urothelial carcinoma in Gene expression profiling (GEP) has been used to identify biomarkers CheckMate 275 1 1 2 of response to immunotherapy. Using a GEP-based inflammation Peter Szabo, PhD , Abdel Saci , Padmanee Sharma, MD, PhD , George 1 1 1 1 1 assay, we derived and analytically validated a CD8 signature to assess Lee, PhD , Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , 1 1 1 T-cell infiltration in the tumor microenvironment (TME) [1]. Here, we Scott Chasalow , Michael Montalto , Robin Edwards, PhD , Saumya 1 1 1 3 retrospectively explore the association of the CD8 signature, alone Pant , Neeraj Adya , Bruce Fischer, MD , Matthew Galsky, MD 1 2 and in relation to established biomarkers PD-L1 and tumor muta- Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD tional burden (TMB), with clinical response to nivolumab treatment. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Methods Institute, New York, NY, United States In the phase 2 CheckMate 275 trial, 270 patients with platinum- Correspondence: Peter Szabo (Peter.Szabo@bms.com) resistant metastatic urothelial carcinoma (UC) and evaluable tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P110 PD-L1 expression received nivolumab treatment. Responses were de- termined by blinded, independent review committee assessments Background [2]. Minimal follow-up time for the current analysis was ~3 years. T- Close proximity of CD8+ T cells to cancer cells has been associated with cell infiltration in the TME was assessed using the CD8 signature and improved outcome with immunotherapy. Using a gene expression pro- by immunohistochemistry (IHC) using an automated commercial pro- filing (GEP)-based inflammation assay, we previously derived gene sig- prietary assay (Dako mouse clone C8/144B; Agilent Technologies Co) natures that defined CD8+ T-cell infiltration (CD8 signature) and [1]. PD-L1 expression on tumor cells was independently assessed by localization to tumor parenchymal and stromal compartments (CD8- IHC using the PD-L1 IHC 28-8 pharmDx assay (Dako). TMB was mea- topology signatures) in multiple tumor types [1,2]. In patients with sured by whole exome sequencing [3]. Cox proportional-hazards re- urothelial carcinoma (UC), high stromal/epithelial-mesenchymal transi- gression assessed the dependence of progression-free survival (PFS) tion (EMT) gene expression has been associated with T-cell exclusion or overall survival (OS), and logistic regression assessed the depend- and poor response to immunotherapy [3]. Here, we assess three CD8- ence of objective response (OR) on biomarker values. The linear ef- derived signatures and compare them with a CD8 immunohistochemis- fects of biomarkers and their multiplicative interaction were included try (IHC)-derived score combined with EMT gene expression (CD8.IH- when multiple biomarkers were evaluated. Likelihood-ratio tests (2- C_EMT) to evaluate associations between these biomarkers and with sided) were used to assess biomarker interaction effects. Associations response to nivolumab in patients with UC in CheckMate 275 [4]. with PFS and OS were investigated using Kaplan–Meier analyses with Methods biomarker scores categorized by tertile. 270 patients with platinum-resistant metastatic UC received nivolumab, Results with response assessed by blinded central review [4]. CD8+ T-cell infil- GEP was evaluable in 205 (76%) and GEP+TMB in 113 (42%) of 270 tration in the TME (assessed using the CD8 signature [1], CD8-topology treated patients. Baseline characteristics, OR, PFS, and OS were similar signatures (parenchymal, stromal) [2], and by IHC using a proprietary between all treated patients and the GEP-evaluable cohort. CD8 signa- commercial assay [Dako mouse clone C8/144B antibody; Agilent Tech- ture scores showed a positive association with OR (P=0.005), PFS (P= nologies Co]) and PD-L1 expression on tumor cells (Dako PD-L1 IHC 28- 0.005), and OS (P 8 pharmDx) were assessed on baseline tumor samples. Predictive per- Conclusions formance of the CD8 signature and CD8-topology signatures individu- These results suggest that the GEP-based CD8 signature may have util- ally, the combined CD8-derived signatures (triple CD8), and ity as a potential biomarker for predicting clinical response to CD8.IHC_EMT, was evaluated using Cox proportional-hazards regression Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 61 of 272 for overall and progression-free survival (OS, PFS) and with logistic re- (NGS). Although the 0.4 cutoff identifies MSI-high status, there is in- gression for objective response (OR). Odds ratios were scaled to reflect sufficient data for this score's repercussion for MSI-stable patients [1]. the difference between the 75th and 25th biomarker percentiles. Two- MSI-high status rarely occurs in lung cancer patients representing sided likelihood-ratio tests were used to assess biomarker and inter- less than 1% of the cases. Therefore, we aim to identify how MANTIS action effects. Associations with PFS and OS were also investigated score correlates with immune profile and clinical outcomes in MSI- using Kaplan–Meier analyses with biomarker scores categorized by stable lung cancer. tertile. Methods Results MANTIS score was calculated for two TCGA (The Cancer Genome GEP was evaluable in 205/270 (76%) patients. Baseline characteristics Atlas) cohorts: squamous cell carcinoma (SqCC, n= 501) and adeno- and clinical outcomes were similar in the overall population and the carcinoma (ADC, n=517). After excluding MSI-high patients (n=3 and GEP-evaluable cohort. Response and survival predictions from the 1, respectively) we stratified each cohort into quartiles. The highest triple CD8 and CD8.IHC_EMT overlapped, and both biomarkers pre- quartile was named MANTIS-high (M-H) and the lowest quartile dicted benefit from nivolumab independent of PD-L1 expression. MANTIS-low (M-L). Immune profile (immune cell infiltration and PD- Odds ratios for OR were 2.59 (95% CI, 1.59–4.21) for triple CD8, 2.12 L1 expression), tumor mutational burden (TMB), neoantigen burden (1.47–3.07) for CD8.IHC_EMT, 2.51 (1.42–4.43) for the CD8 signature, and survival outcomes were compared between M-H and M-L. Tumor and 1.74 (1.22–2.49) for the parenchymal CD8-topology signature. immune landscape was identified using signatures from immune Conclusions metagenes predicting infiltration for 31 immune cells. Combined CD8 and CD8-topology gene signatures (triple CD8) Results showed similar performance to CD8.IHC_EMT for predicting response M-H was associated with higher activated CD4, gamma delta and and survival in nivolumab-treated patients with UC. These data sug- Th17 T cell infiltration when compared with M-L in lung SqCC (all gest potential utility of testing biomarker combinations and support p <0.05). No statistically significant difference in tumor T cell infil- further evaluation of gene signatures associated with parenchymal vs tration was found in ADC (Figure 1,2). M-H patients had a higher stromal CD8+ T-cell localization for predicting response to immuno- TMB when compared with M-L patients in ADC (p<0.05) and the therapy in patients with cancer. same tendency was observed for SqCC (p=0.10) (Figure 3). Add- itionally, M-H correlated with lower PD-L1 (CD274) expression in Acknowledgements both SqCC and ADC (each p<0.05) when compared with M-L. No Bristol-Myers Squibb. Professional medical writing and editorial assistance significant differences in neoantigen burden were demonstrated. were provided by Bernard Kerr, PGDipSci, and Jay Rathi, MA, of Spark Medica M-H patients showed a trend towards lower median overall sur- Inc, funded by Bristol-Myers Squibb. vival in SqCC and ADC (75 vs 63 months p=0.21; 53 vs 50 Trial Registration months, p=0.14, Figure 3C,3D). NCT02387996 Conclusions This is the first report that illustrates the implications of a microsatel- References lite instability score on immune landscape, PD-L1 expression, TMB 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene and clinical outcome from a pool of more than a thousand MSS non- signature with CD8 expression by immunohistochemistry (IHC) in small cell lung cancer patients. multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. 2. Szabo PM, Lee G, Ely S, et al. CD8+ T cells in tumor parenchyma and Reference stroma by image analysis and gene expression profiling: potential 1. Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam biomarkers for immuno-oncology therapy. J Clin Oncol. 2019; 37(Suppl): AM, Waldner MJ, Bindea G, Mlecnik B, Galon J, Trajanoski Z. Abstract 2594. Characterization of the immunophenotypes and antigenomes of 3. Wang L, Saci A, Szabo PM, et al. EMT- and stroma-related gene expres- colorectal cancers reveals distinct tumor escape mechanisms and novel sion and resistance to PD-1 blockade in urothelial cancer. Nat Commun. targets for immunotherapy. Genome Biol. 2015; 16:64. 2018; 9:3503. 4. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. Ethics Approval The protocol was approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation. Patients provided written informed consent based on Declaration of Helsinki principles. P111 Clinical and immunologic implications of a microsatellite instability score in lung cancer 1 1 1 Pedro Viveiros, MD , Misuk Lee , Bhoomika Sukhadia, MD , Kyunghoon 1 2 2 1 Rhee , Victor Wang , Jeffrey Chuang , Young Kwang Chae, MD 1 2 Northwestern University, Chicago, IL, United States; Jackson Laboratory For Genomic Medicine, Farmington, CT, United States Correspondence: Misuk Lee (misuklee55@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P111 Background Microsatellite instability status is currently used to predict susceptibil- Fig. 1 (abstract P111). Immune landscape in squamous ity to immunotherapy. MANTIS score was originally developed to cell carcinoma identify microsatellite instability through next-generation sequencing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 62 of 272 Conclusions This rich and large dataset illustrates the power and scalability of the 10x Genomics Chromium Single Cell Immune Profiling Solution with Feature Barcoding technology and presents an exciting opportunity for researchers to explore and draw further conclusions about the mechanisms of TCR–pMHC interaction. Furthermore, this experiment serves as the next step on the path toward the even larger-scale ex- periments that will be necessary to fully comprehend the rules of antigen recognition in the adaptive immune system in response to cancer and infectious diseases and will be key in the development of successful immunotherapies. Acknowledgements This study was performed in collaboration with our 10x Genomics partners Fig. 2 (abstract P111). Immune landscape in adenocarcinoma Immudex and Biolegend. P112 P113 A new way of immunity exploration by linking highly Dynamic analysis and visualization of the immune infiltration in multiplexed antigen recognition to immune repertoire and human cancer by integrating TCGA data phenotype Mingchao Xie, PhD, Bolan Linghu, PhD, Zhongwu Lai, PhD, Jonathan 1 1 1 Dagmar Walter, PhD , Stephane Boutet , Michael Stubbington, PhD , Dry, Ben Sidders 1 2 1 1 Katherine Pfeiffer , Josephine Lee , Luz Montesclaros , Julia Lau , Daniel AstraZeneca, Waltham, MA, United States 1 1 3 Riordan , Alvaro Martinez Barrio , Liselotte Brix, PhD , Kivin Jacobsen, Correspondence: Jonathan Dry (Jonathan.Dry@astrazeneca.com); Ben 3 4 4 1 PhD , Bertrand Yeung , Xinfang Zhao , Tarjei Mikkelsen Sidders (benjamin.sidders@astrazeneca.com) 1 2 10x Genomics, Pleasanton, CA, United States; 10x Genomics.com, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P113 Pleasanton, CA, United States; Immudex, Copenhagen, Denmark; Biolegend, San Diego,CA, United States Background Correspondence: Tarjei Mikkelsen (tarjei@10xgenomics.com) Understanding of the complex interplay between tumors and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P112 their immunologic microenvironment is critical for immune- oncology (IO) studies, which can facilitate the discovery of Background novel prognostic biomarkers, identification of new drug targets, Recent progress in cancer immunotherapy emphasizes the and determination of drug resistance mechanisms. However, importance of understanding immune-regulatory pathways in due to a lack of proper analysis tools and datasets, systematic- cancer. It has been shown that immune cells play a crucial role ally exploring the tumor–immune interaction is still a big in the tumor microenvironment and can be used for targeted challenge. therapeutics. Therefore, it is important to understand and Methods characterize T cells and their antigen binding specificity and di- Here, we deconvoluted the immune cell compositions and per- versity in order to develop effective targeted immunotherapies. formed IO-related pathway/signature enrichment analysis for 9,721 Recent technological advancements have enabled the integra- primary tumor samples from 33 TCGA cancer types using transcrip- tion of simultaneous cell-surface protein, transcriptome, immune tomic data, and developed a web-based application, IO Browser. repertoire and antigen specificity measurements at single cell Results resolution, providing comprehensive, scalable, high-throughput The browser allows the user to visualize the immune infiltrations of a characterization of immune cells. sample or cohort, and to define disease segments or “immuno-types” Methods based on the presence of single/multiple immune cell types or IO- Using the 10x Genomics Single Cell Immune Profiling Solution related pathway/signatures. Users can then perform survival compari- with Feature Barcoding technology in conjunction with Biolegend sons, explore gene expression of key cancer and IO genes as well as oligo-conjugated antibodies and Immudex DNA barcoded generate oncoprints in the different segments. The browser also pro- peptide-MHC Dextramer® (pMHC), we performed multi-omic vides statistical analysis to identify the gene or mutations enriched in characterization of CD8+ T cell recognition of various virus and the immuno-typed disease segment, and correlate gene expression common cancer antigens in normal patients. Next generation se- or mutations with specific immune cell types in tumor microenviron- quencing libraries were made following the 10x Genomics work- ment (TME). flow, where gene expression and immune repertoire libraries are Conclusions generated alongside libraries from DNA barcodes conjugated to In summary IO Browser enables comprehensive analysis and antibodies or pMHC, allowing quantification of cell surface pro- visualization of the dynamic interactions between tumor and im- teins and identification of T cell receptor (TCR) specificities. Ana- mune landscape, and can aid our understanding of the interplay be- lysis was performed using the latest version of Cell Ranger (v3.0). tween tumor genomics and immune biology to facilitate line of sight The TCR-dist algorithm was used to identify clusters of related and disease segmentation. TCR sequences and enriched CDR3 motifs. Results P114 We performed multi-omic characterization of ~100,000 CD8+ T cells Tissutal immune profile and pathological complete response in from four MHC-matched donors. The multi-omic combination of triple negative breast cancer gene expression, paired alpha/beta T cell receptor (TCR) repertoire, 1 1 1 Andrea Botticelli, MD , Bruna Cerbelli , Simone Scagnoli , Maria Ida cell surface proteins and pMHC binding specificity allowed the identi- 1 1 2 2 Amabile , Alessandro De Luca , Lucio Fortunato , Leopoldo Costarelli , fication of CD8+ T cell subpopulations with specificity for pMHCs 1 1 1 Marianna Nuti, PhD , Giulia D'Amati , Paolo Marchetti within our panel. Within our data, we observed TCRs with cognate 1 2 Sapienza University of Rome, Rome, Italy; San Giovanni Addolorata antigens that had been reported previously, while also identifying Hospital, Rome, Italy entirely new TCR–pMHC interactions. In addition, we observed spe- Correspondence: Bruna Cerbelli (bruna.cerbelli@uniroma1.it) cific expanded non-naïve T cell clones along with more diverse bind- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P114 ing in the naïve compartment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 63 of 272 Background spectrometry to deeply characterize global tumor proteomes to In the neoadjuvant setting, pathological complete response (pCR) is identify proteins and pathways that are associated with pre- more frequently achieved by triple negative breast cancer subtype treatment response to anti-PD-1 immunotherapy. and patients who attain this status show improved survival; However, Methods standard neoadjuvant therapy results in pCR rates slightly over 30% Unbiased, data-independent acquisition (DIA) mass spectrometry of cases. The mechanism underlying the resistance to chemotherapy was used to analyze formalin fixed paraffin imbedded (FFPE) is still unclear and could be related both to the molecular heterogen- tumor tissue samples from subjects with Stage III-IV melanoma eity of cancer cells and to the activation of the treatment-related im- which were resected prior to initiation of first-line anti-PD-1 ICI mune response. For this reason, the search for immune biomarkers therapy. The selected samples represent two distinct clinical sub- able to predict the response to chemotherapy represents a new groups; those who received clinical benefit, with a partial re- promising frontier. Recent reports underscore the role of TILs, PDL-1 sponse or better (PR, SD and CR, n = 13), and those with no and CD73. The aim of our work is to define a novel tissutal immune clinical benefit (PD, n = 9) and no observable response to ther- profile (TIP) able to predict pCR [1,2]. apy. Samples were prepared for mass spectrometry using stand- Methods ard procedures. All samples were analyzed using 2-hour gradients We enrolled 61 pts who received NAC (EC for 4 cycles followed by on a LC-MS/MS setup operated in DIA mode. Data was extracted Paclitaxel q7 for 12 cycles or q21 for 4 cycles) between Jan 2011 and using Spectronaut (Biognosys) with a sample specific spectral li- June 2017 at Policlinico Umberto I and San Giovanni Addolorata Hos- brary which was combined with a large human tissue resource li- pital of Rome. We performed, in basal paraffin-embedded biopsies, brary. Statistical analysis was conducted to identify proteins that stromal TILS evaluation and immunohistochemistry for PD-L1 (Ven- are either up- or down-regulated with respect to benefit group. tana SP142 clone) evaluated both on tumor cells (TC) and tumor- Pathway analysis was also conducted to highlight dysregulated infiltrating immune cells (IC) and CD-73 assessed on TC. We defined biological functions and pathways. “positive tissutal immune profile” (TIP+) the pts with “high TILS” Results (>50%), “PD-L1 positive” ( >1% both on TC and IC ) and “low CD73” 7,590 proteins were quantified across all samples, with 6,627 (<40%), and the others as “negative tissutal immune profile” (TIP- quantified on average per sample. Univariate statistical testing ).Statistical analysis was performed with T di Student test and χ2 test. between groups identified 254 proteins that are dysregulated Results (120 up-regulated and 134 down-regulated) in subjects who re- We enrolled 61 females (median age: 50 y; range 28-75) affected by ceived clinical benefit. Through partial least squares discriminant TNBC. The clinical stage before NAC was as follow: 3 pts cT3 (5%), 3 analysis (PLS-DA) a set of 25 proteins was identified that describe pts cT4 (5%) and 28 pts were cN+ (38%). Twenty-three patients the variance between the two sample groups. When annotated (38%) showed pCR. No significant associations were found between to their sub-cellular location, all up-regulated species are identi- pR and cT, cN, age, and KI-67. Seven patients (11%) were TIP+ and fied as mitochondrial proteins, indicating an enhanced metabolic achieved pCR in 100% of cases; 54 patients were TIP- and pCR were environment, and the down-regulated species are cytosolic, lyso- showed in 16/54 of cases (30%) (p< 0,001). somal or membrane associated. This observation was also Conclusions reflected in pathway analysis which identified up-regulation of ar- TIP+ seems to be associated with higher pCR rate in TNBC patients ginine and citrulline metabolism and down-regulation of adhesion .These preliminary results suggest the possibility of using novel pro- related processes driven by MHC-II and integrins. files combining multiple immune- biomarkers. Conclusions Global profiling of the tumor proteome provides a unique References characterization of melanoma tumor biology. A pathway level ana- 1. Matsumoto H, Koo SL, Dent R, Tan PH, Iqbal J. Role of inflammatory lysis shows increased metabolic processes combined with decreases infiltrates in triple negative breast cancer. J Clin Pathol. 2015;68:506–510. in adhesion related proteins may underly the differences in benefit doi: 10.1136/jclinpath-2015-202944 139. related to ICI therapy. 2. Jiang T, Xu X, Qiao M et al. Comprehensive evaluation of NT5E/CD73 Ethics Approval expression and its prognostic significance in distinct types of cancers. The study was approved by the Istituto Nazionale Tumori IRCCS Fon- BMC Cancer. 2018 Mar 7;18(1):267. doi: 10.1186/s12885-018-4073-7. dazione “G. Pascale” of Napoli Institutution‘s Ethics Board, approval PubMed PMID: 29514610; PubMed Central PMCID: PMC5842577. number 33/17. Ethics Approval Consent CE 4181 Sapienza University of Rome Written informed consent was obtained from the patient for pub- lication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this P115 journal. Deep proteomic characterization of FFPE tumor samples from late- stage melanoma subjects treated with anti-PD-1 immunotherapy 1 1 Nicholas Dupuis, PhD , Jakob Vowinckel, PhD , Domenico Mallardo, P116 2 2 2 2 MD , Mariaelena Capone, MD , Madonna Gabriele , Antonio Sorrentino , Centrifuge-free red blood cell lysis and immunostaining of whole 2 1 2 Vito Vanella , Daniel Heinzmann , Paolo Antonio Ascierto, MD blood for flow cytometry using Laminar Wash™ system 1 2 Biognosys AG, Schlieren, Switzerland; Istituto Nazionale Tumori IRCCS, Ira Kim, Melvin Lye, Chyan Ying Ke, Nadiezda Fernandez Oropeza, Naples, Italy Sigeeta Rajaram, Kong Leong Cheng, Ih Chin Kon, Royce Pek, Namyong Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) Kim, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P115 Curiox Biosystems, Boston, MA, United States Correspondnce: Namyong Kim (namyong@curiox.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P116 Immune checkpoint inhibitors (ICI) have improved the treatment options for patients with advanced stage melanoma, with im- Background proved clinical responses and overall survival compared to stand- Blood cells are prime indicators of immuno-surveillance, and the ease ard systemic therapies. However, a large percentage of melanoma of blood sampling makes blood analysis a key interest for clinical patients do not respond to ICIs, highlighting the need for a and research applications. While current flow cytometry methods are greater understanding of the tumor environment and host im- high-throughput and provide fine resolution in the segregation of mune response. Here, we apply unbiased discovery proteomics, white blood cell (WBC) populations, WBC enrichment involving red based on label-free data independent acquisition (DIA) mass blood cell (RBC) lysis are laborious and typically performed manually, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 64 of 272 contributing to experimental variability especially as blood cells are Clonality assessments ; dualplex and multiplex immunohistochemis- sensitive to physical and chemical stress. try coupled to digital pathology analyses to assess Immune Cells Infil- Methods tration and PD-L1 mediated inhibition (Immunoscore® IC), Immune We describe RBC lysis and leukocyte immunostaining on a Suppression through Regulatory T cells and Myeloid-derives suppres- centrifuge-less platform Laminar Wash™, using a novel wall-less plate sor cells quantification, T-Cell Exhaustion status ; standardized and laminar flow washer. The Laminar Wash™ 24-well plate consists methods for assessment of endothelial activation markers ; flow cy- of an array of hydrophilic spots surrounded by hydrophobic surface, tometry for circulating immune cell subtypes quantification; ICI which functions as a virtual wall that separates each spot. Each well plasma exposure levels. A multimodal integrative Immunogram pres- is capable of staining and lysing 100uL of whole blood. During lysis, entation is proposed for each patients. WBCs settle to the surface of the spot, allowing the spent lysis buffer Conclusions to be removed by a gentle and continuous laminar-flow washing This preliminary study shows that multimodal immune profiling is process on the Laminar Wash™ system, eliminating centrifugation feasible and could be a new tool to understand the biology and and resuspension that may stress cells and disrupt antibody binding. pharmacology of lung cancer resistance to anti-PD1/L1 ICIs and po- Results tentially guide patient management décisions. We observed improved retention of CD45+ lymphocytes while lysing on Laminar Wash™ plates compared to conventional centrifuge Acknowledgements tubes. In studies comparing mouse whole blood lysis and antibody This work is supported by the French National Cancer Agency, Agence staining by conventional tube centrifuge and Laminar Wash, Laminar Nationale du Cancer, through the PIONeeR project financing. Wash achieved dramatically higher staining index and improved Trial Registration resolution of cell cluster by flow cytometry. ClinicalTrials.gov Identifier: NCT03493581 Conclusions Ethics Approval In summary, Laminar Wash system provides gentle, fast and convenient The study was approved by the French Ethic Comitee CPP Ouest II Angers, blood lysis, while improving data quality with superior antibody staining. approval number 2018/08. P117 P118 Immunogram to decipher PD1/L1 ICI resistance: a proof of concept HYDRA platform development to investigate Siglec-engaging in advanced Non-small cell lung cancer patients of the PIONeeR tumor immunosuppressive glyco-codes Project Li Peng, PhD, Adam Petrone, Adam Shoemaker, Jillian Prendergast, PhD, 1 2 3 Florence Monville, PhD , Frederic Vely , Joseph Ciccolini , Florence Zakir Siddiquee, Jenny Che, Lihui Xu, BS, Karl Normington, PhD, MBA, 2 2 1 4 Sabatier , Stephane Garcia , Vanina Leca , Marion Fabre , Christelle James Broderick, Li Peng, PhD 4 1 4 1 Piperoglou , Pernelle Outters , Laurent Arnaud , Laurent Vanhille, PhD , Palleon Pharmaceuticals, Waltham, MA, United States 1 1 1 Caroline Lauge, BA , Anna Martirosyan, Dr , Aurelie Collignon , Marie Correspondence: Li Peng (lpeng@palleonpharma.com) 5 6 7 2 Roumieux , Julien Mazieres , Maurice Perol , Françoise Dignat-George , Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P118 2 2 1 Eric Vivier , Fabrice Barlesi, MD, PhD , Jacques Fieschi, PhD 1 2 3 HalioDx, Marseille, France; AMU, APHM, Marseille, France; AMU, APHM, Background 4 5 IPC, Marseille, France; APHM, Marseille, France; AMU, Marseille, France; The glyco-immune checkpoint (Siglec/sialoglycan axis) has emerged 6 7 Toulouse Universitary Hospital, Toulouse, France; Centre Leon Berard, as a new mechanism of cancer immune escape and offers new thera- Lyon, France peutic interventions to overcome resistance to current immunother- Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) apies. Siglecs (sialic acid-recognizing Ig-superfamily lectins) are type I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P117 transmembrane sialoglycan binding proteins expressed on various immune cells (innate and adaptive). Humans express at least four- Background teen unique Siglecs which have distinct preferred sialoglycan ligands. In the management of advanced Non-Small Cell Lung Carcinoma Tumors upregulate certain sialoglycan patterns to facilitate immune (NSCLC), PD1/L1 immune checkpoint inhibitors (ICIs) have increased cell evasion by engaging these inhibitory Siglec receptors. This tumor overall survival (OS) over standard second-line chemotherapy. While inhibitory “glyco-code” consists of a heterogenous mixture of numer- this long-term increase in OS is driven by about 20% of patients, others ous sialoglycans, binding to Siglecs through low affinity and high display disease progression during the first weeks. PIONeeR workpack- avidity interactions. age 2 aims to understand and eventually predict response and/or re- Methods sistance to those ICIs in stage IV or recurrent NSCLC patients. For that Deciphering the hypersialylation glyco-code of tumors is key to iden- purpose, an Immunogram was designed that integrates a comprehen- tifying cancer patients for glyco-immune checkpoint blockade ther- sive set of biomarkers measured in the tumor microenvironment. apies. However, the heterogeneity and complexity of sialoglycans Methods make characterization of the tumor surface sialoglycome difficult The immune contexture from the PIONeeR trial’s patients is being with current technologies. To overcome this challenge, we developed characterized in a prospective manner and will be confronted to clin- a proprietary sialoglycan-probing reagent, HYDRA, to functionally de- ical data at the end of the study. This multi-modal approach, encom- tect inhibitory tumor sialoglycans engaging Siglecs. HYDRA mimics passing a range of immune scoring assays, is applied to blood and this natural avidity driven Siglec-sialoglycan interaction, consisting of tumor biopsy from each patient, both sampled before and through- multimeric fusions of a Siglec N-terminal extracellular domain con- out anti-PD1/L1 ICI treatment. This work aims at describing pre- taining the carbohydrate recognition domain (CRD), a trimerization treatment samples profiling. motif, and a Fc dimerization domain. Results Results We assessed the feasibility of such a profiling and provide descriptive We have generated several HYDRA constructs with robust expression multi-modal immune profiles for the 10 first PIONeeR-included pa- using a mammalian HEK293 system. Size-exclusion chromatography tients. These profiles combine raw results from more than ten tests, profiles of HYDRA demonstrate high purity and confirmed multimeric corresponding to the following technologies and biomarkers: Gen- assembly. HYDRAs have greater than fifteen-fold increase in binding omic Next Generation Sequencing for Tumor Mutational Burden affinity compared to Siglec-Fc dimers as measured using bio-layer (TMB), DNA mismatch-repair deficiency (MSI/MSS status) and T Cell interferometry Octet. HYDRA also demonstrates sialoglycan-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 65 of 272 binding, as its binding was eliminated when cells were treated with P120 sialidase (which removes terminal sialic-acids of sialoglycan) or using Evaluation of CD8 score by automated quantitative image analysis cells lacking sialoglycans from knocking out UDP-GlcNAc 2- in metastatic melanoma treated with PD1 blockade: preliminary Epimerase. Glycan array binding of HYDRA confirmed similar sialogly- results can preferences of its Siglec counterpart as described in the litera- Anjali Rohatgi, MD PhD, Douglas Hartman, Arivarasan Karunamurthy, ture, suggesting engineering did not alter glyco-recognition Julie Burkette, Yana Najjar, MD, John Kirkwood, MD, Hassane Zarour, MD, properties. These high-affinity and sialoglycan-specific HYDRAs en- Liron Pantanowitz, Diwakar Davar, MD abled us to develop a robust immunohistochemistry (IHC) assay to UPMC, Pittsburgh, PA, United States analyze cancer patient samples. A cohort of tissues (>2,500 patients) Correspondence: Diwakar Davar (davard@upmc.edu) from various indications were analyzed to enable indication Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P120 prioritization for glyco-immune checkpoint therapies. HYDRA IHC on healthy and cancerous human tissues demonstrate unique binding Background patterns with concordance between duplicate primary tumor cores PD1 blockade produces responses in 30-40% of metastatic melan- and primary tumor versus metastatic cores from the same patient in oma (MEL) with durable relapse-free benefit [1,2]. Pre-existing tumor- non-small cell lung, kidney and colon cancer samples. infiltrating CD8+T cell infiltrates (TIL), neoantigen burden and IFN-γ Conclusions gene expression signature (GES) correlate with clinical anti-tumor re- In summary, the HYDRA technology distills the structural hetero- sponse [3-5] to PD1 blockade. However, neoantigen burden and IFN- geneity of tumor surface sialoglycans to a straightforward func- γ GES are cost-prohibitive and time-consuming assays that are not tional readout of immunosuppressive glyco-codes engaging available for clinical use; while CD8 T cell analysis by immunohisto- inhibitory Siglecs, which may allow patient stratification based on chemistry (IHC) is cost-effective and operator-independent. The aim deciphering a tumor-specific surface glycan pattern. of this study is to develop and validate an image analysis algorithm to automatically quantify CD8+ T cells (CD8 score) in patients with metastatic MEL treated with PD1 blockade. P119 Methods Analytical validation of run-to-run and site-to-site performance of Included patients had advanced metastatic MEL treated with PD1 a human immune profiling assay and automated data analysis blockade. Radiographic response assessed using RECIST v1.1. For the solution for CyTOF mass cytometry technology purposes of this analysis, patients were defined as responders (R; Clare Rogers (clare.rogers@fluidigm.com) complete, partial response, stable disease) or non-responders (NR; pro- Fluidigm, South San Francisco, CA, United States gressive disease). Pre-treatment tumor biopsies from 58 patients were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P119 utilized. Brightfield image analysis results were cross-validated with fluorescence-based quantification (AQUA™). A nuclear image algorithm Background designed to run on whole slide images was optimized to manual count. Immune profiling is an essential method for quantifying changes The algorithm was locked down and used on a cohort of whole tissue in immune population numbers and states over time in health sections from MEL patients. All images were reviewed by independent and disease. A cornerstone in translational and clinical research, pathologist blinded to clinical outcomes. Response and outcomes were it is frequently used to investigate chronic inflammation, infec- statistically correlated with image analysis results. tious disease, autoimmune diseases, and cancer. The diversity of Results immune populations demands a high parameter approach to There were 40 R patients and 18 NR patients. Median CD8 score was more fully and efficiently quantify these changes. Mass cytometry, 101 cells/mm3 in R and 48.7 cells/mm3 in NR (p=0.098). Median PFS which utilizes CyTOF® technology, is a single-cell analysis platform were greater in R compared to NR (18 months vs. 2 months, p100 that hasusedasmanyas50 metal-taggedantibodies[1] to re- cells/mm3 (64%). solve discrete cell populations, all in a single tube of sample. It is Conclusions an ideal solution for routine enumeration of immune cell We report the successful technical development and clinical valid- populations. ation of an image algorithm to automate CD8 score for metastatic Methods MEL treated with PD1 blockade. Preliminary results demonstrate We have developed a sample-to-answer solution for human im- CD8 score was directly associated with response and improved mune profiling using mass cytometry: the Maxpar® Direct™ Im- PFS. CD8 score is an assay that could be carried out using exist- mune Profiling System. It includes an optimized 30-marker ing technology in pathology departments. Further analysis will immune profiling panel provided in a dried single-tube format, focus on validating these results in a larger cohort to permit clin- validated SOPs for human whole blood and PBMC staining, an in- ical use. strument data acquisition template, instructions for data acquisi- tion on a Helios™ system, and automated Maxpar Pathsetter™ References software for data analysis. 1. Ribas A, Hamid O, Daud A, Hodi FS, Wolchok JD, Kefford R, Joshua AM, Results Patnaik A, Hwu WJ, Weber JS, Gangadhar TC, Hersey P, Dronca R, Joseph Here we present assay analytical validation data on repeatability, re- RW, Zarour H, Chmielowski B, Lawrence DP, Algazi A, Rizvi NA, Hoffner B, producibility, software precision, software accuracy, and site-to-site Mateus C, Gergich K, Lindia JA, Giannotti M, Li XN, Ebbinghaus S, Kang reproducibility. The repeatability of eight identical donor samples ac- SP, Robert C. Association of Pembrolizumab With Tumor Response and quired on a single Helios instrument resulted in CVs 5% in fre- Survival Among Patients With Advanced Melanoma. JAMA. 2016; quency). Reproducibility of three identical samples acquired on three 315:1600-9. different Helios instruments resulted in CVs 2. Larkin J, Lao CD, Urba WJ, McDermott DF, Horak C, Jiang J, Wolchok JD. Conclusions Efficacy and Safety of Nivolumab in Patients With BRAF V600 Mutant and We conclude that this assay provides a robust solution for broad im- BRAF Wild-Type Advanced Melanoma: A Pooled Analysis of 4 Clinical Tri- mune profiling using mass cytometry, reducing sources of variability als. JAMA Oncol. 2015;4:433-40. and subjectivity in sample preparation and data analysis. 3. Tumeh P, Harview C, Yearley J, Shintaku I, Taylor E, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West A, Carmona M, Kivork C, Seja E, Reference Cherry G, Gutierrez A, Grogan T, Mateus C, Tomasic G, Glaspy J, Emerson 1. Simoni Y, Becht E, Fehlings M et al. Bystander CD8+ T cells are abundant R, Robins H, Pierce R, Elashoff D, Robert C, Ribas A. PD-1 blockade in- and phenotypically distinct in human tumour infiltrates. Nature. 2019; duces responses by inhibiting adaptive immune resistance. Nature. 557:575–579. 2014;515:568-71 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 66 of 272 4. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu to identify patient-specific patterns which might improve the predic- XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber tion of the response to therapy. AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Conclusions Tomassini JE, Loboda A, Kaufman D. Pan-tumor genomic biomarkers for We believe that the Cancer Immunogram has the potential to facili- PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362:197 tate drug development by providing a 360° vision of the tumour im- 5. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee mune contexture and may also help clinicians to personalize W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, advanced cancer patient care. Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN, Chan TA. Mutational References landscape determines sensitivity to PD-1 blockade in non-small cell lung 1. Galon J, Bruni D. Approaches to treat immune hot, altered and cold cancer. Science. 2015;348:124-8. tumours with combination immunotherapies. Nat Rev Drug Discov. Ethics Approval 2019;18:197-218. The study was approved by University of Pittsburgh‘s Institutional Review 2. Pagès F et al. International validation of the consensus Immunoscore for Board, approval number PRO18080253. the classification of colon cancer: a prognostic and accuracy study. Consent Lancet. 2018; 391:2128-2139. Written informed consent was obtained from the patient for publication of 3. Mlecnik B et al. Integrative Analyses of Colorectal Cancer Show this abstract and any accompanying images. A copy of the written Immunoscore Is a Stronger Predictor of Patient Survival Than consent is available for review by the Editor of this journal. Microsatellite Instability. Immunity. 2016; 44:698-711. 4. Blank CU et al. The "cancer immunogram". Science. 2016;; 352:658-60. 5. Sbarrato T et al, Combining multimodal biomarkers as an immunogram P121 to guide immunotherapy use: A proof of concept. Proceedings: AACR Cancer Immunogram: combining multi-parameter approach and Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. machine learning to capture the complexity of tumor immune contexture 1 1 1 Thomas Sbarrato, PhD , Laurent Vanhille, PhD , Mounia Filahi , Anna P122 1 1 1 Martirosyan, PhD , Véronique Frayssinet , Caroline Davin, BA , Caroline Microfluidic-based cell separation method improves workflow for 1 1 1 1 Laugé , Assil Benchaaben , Alboukadel Kassambara , Felipe Guimaraes , evaluation of rare lymphocytes from cancer patient samples 1 2 1 1 1 1 1 1 Régis Perbost , Jérôme Galon , Hélène Girardi , Jacques Fieschi, PhD Jodi Stone, BS , Megan Nichols , Amy Austin , Kala Bradshaw , Jessica E. 1 2 1 1 2 HalioDx, Marseille, France, Centre de Recherche des Cordeliers, Paris, Norris, BS, MT , Jennifer Montague, PhD , Peng Meng Kou , Nitin 2 2 2 2 France Kulkarni , Nirav Sheth , Anya Manning, MBA , Sarah M. Mickool , Kyle 2 2 1 Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) Smith , Ravi Kapur , John Powderly, MD, CPI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P121 Carolina BioOncology Institute, Huntersville, NC, United States; MicroMedicine, Waltham, MA, United States Background Correspondence: John Powderly (jpowderly@carolinabiooncology.org) To tailor clinical care and personalized treatment of cancer patients, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P122 the scientific community together with the practitioners have fo- cused into refining our understanding of cancer biology and resist- Background ance to treatments. In that perspective, the concept of Immunoscore Isolation of rare lymphocyte populations from peripheral blood prod- proposed by Galon et al [1, 2, 3] has highlighted the crucial role of ucts of cancer patients can be challenging due to technician variabil- immune response to the tumor. In parallel, immunotherapies by im- ity and substantial cell loss through standard cell separation mune checkpoint inhibitors (ICI) anti-PD-1/PD-L1 were approved in methods such as Mononuclear Cell Preparation Tubes™ (CPTs). An several cancer indications, such as Non-Small Cell Lung Cancer or automated microfluidic approach was evaluated to determine melanoma, even if only a minority of these patients respond posi- lymphocyte recovery, processing time, and ease of use. Furthermore, tively to the treatment. In addition, ICI are far less effective for other increased yields of rare cells from cancer patients’ peripheral blood high-incidence indications like colorectal cancer (CRC), thus suggest- could potentially substitute for leukapheresis when leukapheresis is ing that multiple factors may be critical for capturing the exact na- not a viable option. ture of the tumour microenvironment (TME). In this context, the Methods comprehensive identification and assessment of these factors could White blood cells (WBCs) or peripheral blood mononuclear cells be key to stratify patients and allow the selection of the optimal (PBMCs) were isolated from human peripheral blood using either treatment. MicroMedicine’s Microfluidic System (MS) or CPTs. Cell viability and Methods lymphocyte recovery were compared using a hematology analyzer In order to support clinical researchers and biopharmaceutical com- and flow cytometry. Further, a rare lymphocyte population was posi- panies in the evaluation of the efficacy of candidate drugs, HalioDx tively immunomagnetically selected from healthy volunteers and has developed the Cancer Immunogram, a solution based on Blank cancer patients. Immunophenotyping was performed pre- and post- CU et al. [4]. Our multi-parameter approach encompassing a unique cell selection, followed by in vitro expansion of the rare lymphocytes. range of immune scoring assays is based on the analysis and the un- Results derstanding of the immune contexture of tumors and offers a per- Using cells collected from healthy volunteers, the automated MS sonalized and dynamic “fingerprint” of tumor-immune system prototype consistently recovered 83.5 ± 10.1% lymphocytes in a total interaction. To address this, the Cancer Immunogram combines dif- of 31 ± 5.8 minutes, including hands-on time, compared to the ferent technologies and biomarkers to assess 1) the tumor character- standard CPT process, which recovered 43.2 ± 7.6% lymphocytes in istics (Tumor foreignness, MSI, PD-L1 expression, common mutation 72.2 ± 4.1 minutes, from 32 – 34 mL blood samples (n = 5). The via- drivers), 2) the immune infiltration (Immunoscore®, CD8/PD-L1 prox- bility was comparable at 97.9 ± 1.6% (MS) and 96.7 ± 3.0% (CPT). In imity, TCR clonality, immune expression signature), 3) the immune further studies with both healthy donors and cancer patients, a rare checkpoint status (T Cell Exhaustion BrightPlex panel) and 4) the im- lymphocyte population was successfully selected from WBCs isolated mune suppression status (Treg, MDSC and M1/M2 macrophage with the MS, enabling immunophenotyping of the rare cell popula- BrightPlex panels). tion and subsequent in vitro expansion. Expansion of this lymphocyte Results population from a colon adenocarcinoma patient was found to be Here, we consolidate our Proof of Concept for the Cancer Immuno- suppressed post-immunotherapy compared to pre-treatment. Cells gram in the context of CRC [5] by leveraging this meta-analysis on a isolated from patients with other malignancies were successfully ex- 20-patients cohort. Using machine learning algorithms to extract the panded. Finally, peripheral blood collected from cancer patients most relevant features, we show that the Cancer Immunogram allows yielded a greater number of rare lymphocytes using the MS for the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 67 of 272 cell expansion study in comparison to the CPT isolation method, NK cells, in mediating antitumor response after immune check- which correlates to having a higher lymphocyte recovery. point inhibition. Conclusions NanoString nCounter is Intended for Research Use Only. Not for Use The MS consistently recovered approximately twice the number in Diagnostic Procedures. of lymphocytes in half the time compared to the traditional CPT Ethics Approval method. The automated cell separation process improves the The study was approved by Yale University Human Investigation consistency of cell isolation while freeing up technician time. Rare Committee, approval number 9505008219. lymphocyte populations could be reliably recovered from periph- eral blood with significantly higher yield compared to the CPT P124 method. While leukapheresis enriches for MNCs and MS isolates A novel cell-mediated immunotherapy for treatment of lung and all WBCs, these results suggest that peripheral blood collection- breast cancer based MS has the potential to complement leukapheresis, espe- Indu Venugopal, PhD, Kathlynn Brown, Michael McGuire, Claire Gormley cially for small- to medium-scale studies. SRI International, Harrisonburg, VA, United States Correspondence: Kathlynn Brown (kathlynn.brown@sri.com) P123 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P124 Identification of mRNA signatures that predict response to immunotherapy in melanoma patients Background 1 2 2 Ioannis Vathiotis, MD , Amy Sullivan , Sarah Warren, PhD , Nicole Cancer Immunotherapies designed to generate a cell-mediated 1 1 1 Gianino , Sandra Martinez-Morilla, PhD , Pok Fai Wong, MD, MPhil , immune response against tumors are emerging as frontline 1 3 1 Harriet Kluger, MD , Konstantinos Syrigos , David Rimm, MD, PhD treatment options for cancer; however, concerns regarding effi- 1 2 Yale University, New Haven, CT, United States; NanoString cacy, safety and cost efficacy have limited the use of these Technologies, Seattle, WA, United States; University of Athens, Athens, treatments. Greece Methods Correspondence: David Rimm (david.rimm@yale.edu) To address these weaknesses, we developed a novel immunother- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P123 apy capable of delivering previously encountered antigenic pep- tides specifically to cancer cells and facilitating their presentation Background through the MHC class I pathway. It utilizes a synthetic nanoparti- Currently, there is no diagnostic test that can accurately predict cle delivery system comprised of three components: a neutral response in melanoma patients treated with immunotherapy. stealth liposome, encapsulated synthetic immunogenic HLA class I NanoString® nCounter® PanCancer IO 360™ panel (Research Use restricted peptides derived from measles virus (MV), and a tumor- Only) measures mRNA from 770 genes related to the tumor and targeting peptide on the external surface of the liposome. The host immune response. Here, we used this panel to assess the targeting peptide results in accumulation of liposomes specifically predictive value of individual genes and weighted gene signa- inside cancer cells, and facilitates presentation of MV-derived im- tures in a cohort of immunotherapy (ITx) treated melanoma munogenic peptides in HLA class I molecules (Figure 1). We refer patients. to this system as TALL (Targeted Antigen Loaded Liposomes). Methods Therefore, TALL can generate a secondary immune response spe- We used pretreatment, formalin-fixed paraffin-embedded (FFPE) cifically against the targeted tumor cells in a patient who has whole tissue sections from 59 melanoma patients that received been previously vaccinated against or infected by MV. In short, single agent or combination immunotherapy (pembrolizumab, we are attempting to trick the immune system into responding nivolumab, or nivolumab plus ipilimumab). Two slides from each as though the cancer cell is infected with MV without the use of patient were macrodissected and RNA was extracted. The mRNA viral particles. transcripts were hybridized and tagged by unique probes for the Results 770-plex PanCancer IO 360 panel and then measured on the We synthesized liposomes encapsulating H250, an immunogenic nCounter platform. RNA counts were correlated with best overall HLA class I restricted peptide identified from measles response (BOR), clinical benefit (CB), progression free survival hemagglutinin protein. These liposomes were targeted to breast (PFS) and overall survival (OS). and lung cancer cells via our targeting peptide, which was identi- Results fied using phage-display methodology. Treatment of lung cancer Indoleamine 2,3-dioxygenase 1 (IDO1) was the best single gene cells with TALL results in functional presentation of H250 in both predictor of BOR (Area under the curve (AUC) = 0.73) and CB MHC and HLA class I molecules. Our in-vitro and in-vivo studies (AUC =0.70).Among othergenes,IDO1 mRNAwas also foundto indicate that presentation of H250 is dependent on the cancer be significantly associated with longer PFS (P < 0.01, False dis- targeting peptide; liposomes that lack the targeting peptide did covery rate (FDR) = 0.18) and OS (P < 0.01, FDR = 0.052). The not accumulate in the cancer cells and presentation of H250 was previously described 18-gene tumor inflammation score (Ayers abrogated. Treatment with TALL substantially reduced growth of TIS) validated for the prediction of BOR (AUC = 0.68), PFS (P < LLC1 and 4T1 tumors in vaccinated C57BL/6 and Balb/c mice 0.05, FDR = 0.18) and OS (P < 0.001, FDR = 0.025). TIS also pre- respectively. dicted CB (AUC = 0.67). Its predictive value remained the same ir- Conclusions respective to immunotherapy agent administered. Nevertheless, it The outcome of our therapy is a robust cytotoxic T lymphocyte re- decreased for patients harboring the BRAF and NRAS mutations sponse directed specifically against the tumor. It's advantages in- (AUC = 0.76 versus 0.51 and 0.44 for patients with BRAF and clude: 1) Bypassing the need to identify tumor-associated antigens or NRAS mutations respectively). The best signatures for this cohort educate the immune system through a primary immune response; 2) were for Cytotoxicity, Immunoproteasome and CD56dim Cells It is anticipated to be effective against tumors with a low mutational which were predictive for BOR (AUC = 0.72, 0.71 and 0.70 re- load, making it efficacious on early-stage and metastatic cancer; 3) It spectively), CB (AUC = 0.69, 0.68 and 0.70 respectively), and OS does not use a live virus or biologically-derived material, allowing for (all FDRs < 0.05). Further work is underway to compare these complete synthetic manufacturing. It also does not require isolation Yale melanoma results with other cohorts. or ex-vivo manipulation of patient’s cells, reducing production time Conclusions and costs. Pretreatment mRNA counts of single genes or weighted signature scores are related to immunotherapy outcomes in melanoma pa- Acknowledgements tients. This work validated the Ayers TIS signature and Research reported in this work was supported by DOD under grant number highlighted the role of the immune microenvironment, especially W81XWH-16-1-0262 and SRI internal research funds Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 68 of 272 receptors. In contrast, ciforadenant inhibited induction of the AdenoSig and AMPSig in all experimental settings at the tran- script and protein level. Conclusions A2AR agonists and AMP induce specific GEPs dominated by im- munosuppressive mediators of MDSC and monocyte/macro- phage biology. These GEPs may be used as biomarkers for patient selection. CD73 antagonists alone may be limited by the induction of compensatory immunosuppressive pathways medi- ated by AMP accumulation. Combination ciforadenant and CPI- 006 treatment may synergize to activate anti-tumor immunity by 1) blocking adenosine production and signaling, 2) directly activating immune cells, and 3) blocking a compensatory induc- tion of AMPSig. This combination strategy is being evaluated in an ongoing Ph1/1b clinical trial in patients with advanced solid tumors. Trial Registration NCT02655822 and NCT03454451 Fig. 1 (abstract P124). TALL mechanism of action P125 Adenosine and AMP gene expression profiles predict response to adenosine pathway therapies and indicate a need for dual blockade of CD73 and A2AR with CD73 inhibitors Stephen Willingham, PhD, Drew Hotson, PhD, Jessica Hsieh, Brian Munneke, Long Kwei, PhD, Joseph Buggy, Richard Miller, MD Corvus Pharmaceuticals, Burlingame, CA, United States Correspondence: Stephen Willingham (swillingham@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P125 Background Extracellular adenosine in the tumor microenvironment gener- ates an immunosuppressive niche that promotes tumor growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. Various agents targeting the adenosine path- way are now in clinical trials as cancer therapies. Ciforadenant is a selective A2AR antagonist and CPI-006 is an anti-CD73 anti- body (Fc-mutant IgG1) that blocksthe enzymaticconversion of AMP to adenosine and directly stimulates immunity. Both agents are now being studied in clinical trials (NCT02655822 and NCT03454451). In this report, we evaluate the role of ad- Fig. 1 (abstract P125). See text for description enosine and AMP-related gene expression profiles (GEPs) that may predict the response of patients receiving adenosine path- way therapies. Ex vivo studies reveal a requirement for dual P126 blockade of CD73 and A2AR for optimal neutralization of AMP Discovery of biomarkers associated with benefit from PD-1 mediated immunosuppression. checkpoint blockade in non-small-cell lung cancer (NSCLC) using Methods high-plex digital spatial profiling Normal human PBMCs were stimulated ex vivo with NECA (stable ad- 1 1 2 Jon Zugazagoitia, MD, PhD , Swati Gupta, PhD , Kit Fuhrman, MS PhD , enosine analog) or AMP. RNA from tumor biopsies and PBMC was an- 1 1 1 Scott Gettinger, MD , Roy Herbst, MD, PhD , Kurt Schalper, MD, PhD , alyzed using NanoString. Renal cell cancer (RCC) tumor biopsies David Rimm, MD, PhD collected from patients treated with ciforadenant (100 mg BID) either Yale University School of Medicine, New Haven, United States; as a single agent (n=18) or in combination with atezolizumab (n=14). Nanostring, Inc., Seattle, WA, United States Results Correspondence: David Rimm (david.rimm@yale.edu) Ex vivo A2AR agonism resulted in dose-dependent increases in CXCR2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P126 ligands (CXCL1,2,3,5,8) and key mediators of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory media- Background tors such as IL-1beta and CCL2,3,7,8, 20 were also observed, as were in- Only a minority of patients with advanced NSCLC truly benefit from creases in SERPINB2, S100A8, PTGS2, THBS1. Preliminary biomarker single-agent PD-1 checkpoint blockade, and more robust predictive analysis suggests ciforadenant anti-tumor activity in RCC was associ- biomarkers are needed to optimally deliver these therapies. The ated with increased expression of select analytes (AdenoSig) in pre- GeoMx Digital Spatial Profiler (DSP) (NanoString, Inc.) allows high- treatment biopsies (Figure 1). plex protein expression analysis in a quantitative and spatially- Ex vivo AMP or AMPalphaS (a non-hydrolyzable AMP analog) resolved manner from single formalin-fixed paraffin embedded tissue stimulation induced a similar GEP (AMPSig), but included specific sections. Here we use this technology as a discovery tool to find pro- decreases in OAS3, BIRC5, CDK1, MX1, IFI27, and IFIT1. CD73 anti- tein markers associated with benefit from single-agent PD-1 check- body and small molecule antagonists amplified the AMPSig by point blockade in NSCLC. preserving AMP, which itself directly stimulates adenosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 69 of 272 Methods biopsies on the Leica Biosystems BOND RX autostainer. The tissues We used the GeoMx DSP in a cohort of 63 immunotherapy-treated were then imaged in five distinct fluorescent channels (DAPI, FITC, NSCLC cases represented in a tissue microarray, 52 of whom had TRITC, Cy5, Cy7) in 3 rounds of image acquisition on the ZEISS Axio pre-treatment samples and received single-agent PD-1 checkpoint Scan.Z1. HALO analysis software was used to identify cell phenotypes blockade. A panel of 40 photocleavable oligonucleotide-labeled pri- and spatial interactions across the whole slide images. UMAP and mary antibodies (NanoString Human IO panel) was used for protein PSDM were also used to characterize cellular phenotypes and similar- detection. Proteins were measured in 4 independent molecularly- ities amongst samples in the cohorts. Downstream H&E staining was defined tissue compartments by fluorescence co-localization (tumor performed on the same slides with a fourth imaging round to pro- [panCK+], leucocytes [CD45+], macrophages [CD68+], and non- duce a fused 12-plex fluorescent and brightfield image. immune stromal cells [CK-/CD45-CD68-/DNA+]). The photocleaved Results oligos were hybridized and digitally counted with the nCounter plat- The multiplex panel was able identify all 12 markers in FFPE samples form. Two cut-points (median and top tertile) were explored for each and immunophenotype single cells through co-expression of several maker. All statistical testing was performed using a two-sided signifi- biomarkers. Of the 4,096 possible phenotypes, the relevant pheno- cance level of α=0.05 without correction for multiple hypothesis types mapped included, but were not limited to: T cells, T-regs, Cyto- testing. toxic T-cells, Exhausted T-cells, B cells, NK cells, M1 and M2 Results macrophages, tumor cells, and expression along the PD-L1 and PD-1 160 protein variables were generated per case (normalized counts immune checkpoint axis. Distance mapping and infiltration indexes within molecularly defined compartments). In univariate analyses were measured in tumor regions, stroma compartments, and along using pre-specified cut-points, 10 markers were associated with clin- invasive margins. ical benefit (CB) or non-CB, 6 markers with PFS, and 13 markers with Conclusions OS. Of these, CD56 (top tertile) and CD4 (median) measured in the In this work, we introduce a tumor and immune cell phenotyping CD45 compartment were the only markers that significantly pre- multiplex immunofluorescence panel for the comprehensive dicted either CB (OR 6.7, p = 0.014 and OR 8.5, p = 0.014, respect- characterization of the tumor microenvironment and its applicability ively) longer PFS (HR 0.38, p = 0.011 and HR 0.33, p = 0.002, across a range of carcinoma and melanoma FFPE tissue samples for respectively) and longer OS (HR 0.44, p = 0.044 and HR 0.31, p = support of deep pathology assessment in drug discovery research. 0.002, respectively). After adjusting for 3 baseline clinical prognostic The ability to colocalize markers in the same compartment for the factors (performance status, liver metastasis, dNLR) in a multivariate identification of thousands of phenotypes when combined with Cox proportional hazard model, both CD56 and CD4 remained pre- brightfield pathological assessment within a single sample has the dictive for PFS (HR 0.39, p = 0.020 and HR 0.37, p = 0.017, respect- potential to accelerate immunotherapy research. ively), while only CD4 was predictive for OS (HR 0.28, p = 0.006). Conclusions P128 This pilot scale, discovery study shows the potential of the DSP tech- Pooled analysis of Programmed Death Factor Ligand 1 (PD-L1) nology in the identification of spatially-informed biomarkers of re- expression as a predictive biomarker using individual data on sponse to PD-1 checkpoint blockade in NSCLC. This works highlights 7,918 randomized study patients a previously undescribed role for CD56+ immune cells and CD4+ T- Andrea Arfe, Geoffrey Fell, Brian Alexander, MD MPH, Mark Awad, MD cells as potential predictors of immunotherapy outcomes in NSCLC. PhD, Scott Rodig, MD, PhD, Lorenzo Trippa, Jonathan Schoenfeld, MD, Ethics Approval MPH All tissue samples were collected and used with specific consent or Dana-Farber Cancer Institute, Boston, MA, United States waiver of consent under the approval from the Yale Human Investi- Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) gation Committee protocol #9505008219. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P128 P127 Background Development of a 12-marker immunofluorescence multiplex panel PD-L1 expression is one of the most studied biomarkers to predict for the in-depth investigation of the tumor immune landscape the efficacy of immune checkpoint inhibitors (ICIs), but its clinical sig- analyzing 4,096 phenotypes nificance is controversial. Several factors have limited the study of Courtney Hauck, Aditi Sharma, Monique Johnson, Wenya Yang, Bonnie PD-L1 expression. Most trials use of hazard ratios (HRs) to measure Phillips, PhD, Mark Burton, HTL ASCP, Douglas Wood, PhD, Stephanie treatment effects on survival outcomes, a questionable practice for Hennek, PhD, Mael Manesse, PhD, J Kent Moore, PhD, Katir Patel, PhD, immunotherapy studies. Additionally, trials use different cut-off Jamie Buell, Sean Downing, PhD values to dichotomize PD-L1 scores, complicating meta-analyses. Ultivue, Cambridge, MA, United States Therefore, we performed a pooled analysis to: i) estimate the distri- Correspondence: Sean Downing (sean.downing@ultivue.com) bution of PD-L1 expression scores in clinical populations, and ii) as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P127 sess the relationship between PD-L1 levels and ICIs’ effects on overall survival (OS). Instead of HRs, we used a more robust metric, i.e. differ- Background ences in restricted mean survival times (ΔRMSTs). Current IHC methods limit the depth of information from a single tis- Methods sue sample to a single target in the case of chromogenic staining, or Following PRISMA guidelines, we analyzed individual-level data re- to sample morphology and general cell identification in the case of constructed from the publications of 14 randomized clinical trials of H&E. Multiplex immunofluorescence (mIF) methods provide insights ICIs. We used an imputation-based approach to estimate i) the distri- into a wide number of markers of interest and their spatial context in bution of PD-L1 scores, ii) the survival distribution in different PD-L1 a single sample but limit the level of marker co-localization detection classes, and iii) pooled ΔRMST estimates. We show the advantage possible because of multiple antigen retrieval or photobleaching provided by meta-analytic estimates such as ours for the design of steps. Here, we demonstrate the utility of a new 12-plex mIF panel future studies in a simulation study. We simulated 10,000 NSCLC tri- using InSituPlex technology that can identify thousands of pheno- als (1:1 randomization; sample size: 500 patients) that compared ICIs types and spatial behavior through the co-localization of markers with standard chemotherapy. Simulated trials followed either i) a de- that was once limited to the domain of flow cytometry. sign that does not use prior information on the distribution of PD-L1 Methods levels and their association with ICIs’ effects, or ii) a design tailored The 12-Plex marker panel was developed including: CD3, CD4, CD8, to our meta-analytic estimates. CD20, Granzyme B, CD56, CD68, CD163, FoxP3, PD-1, PD-L1, and pan- Results Cytokeratin/Sox10 used the InSituPlex and DNA-Exchange technol- We reconstructed data on 7,918 individual patients, 3,496 with ogy to perform mIF staining of FFPE samples from tonsil and tumor NSCLC, 4,529 with other tumors. The estimated distribution of PD-L1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 70 of 272 expression is U-shaped, with most patients presenting a low or high Methods expression: only about 7% had an expression in the 5%-50% range. Serial peripheral blood mononuclear cells were obtained from pa- ΔRMST estimates suggest that i) ICIs provide an OS benefit to all pa- tients with RCC undergoing immunotherapy. Samples were obtained tients, and ii) the magnitude of OS benefits increases along with PD- at baseline (cycle 1) and initiation of each subsequent cycle (up to L1 score, although changes in ΔRMSTs were greater in NSCLC (Figure cycle 6). Flow cytometry identified longitudinal changes in T cell sub- 1). In the simulations, the power to detect a positive treatment effect sets. Additionally, recently activated CD8 T cells, identified by surface increased from 80% to 93% using a design tailored to meta-analytic expression of CD38 and HLA-DR, were sorted at baseline, post-cycle information. 1, and post-cycle 2, and analyzed by RNA seq. Clinical responses Conclusions were determined at the first restaging scans using RECIST v1.1 cri- By highlighting that higher PD-L1 scores predict increasing OS bene- teria, to define those with clinical benefit (complete response, partial fits, our findings extend those of recent meta-analyses that evaluated response, or stable disease) or no clinical benefit (progressive PD-L1 expression scores as predictors of ICIs’ efficacy. They also illus- disease). trate how meta-analytic estimates like ours can improve the power Results of future trials to detect ICIs’ benefits. Our findings also suggest that Of 27 patients analyzed, 10 received nivolumab, 7 nivolumab + the practice of dichotomizing the range of PD-L1 expression scores is NKTR-214, and 10 nivolumab + ipilimumab. Median age was 58 years inadequate for patient stratification. (range 33-78) with a male (70%), Caucasian (89%), and solely clear cell histology (83%) predominance. A burst in circulating activated CD8 T cells as defined by a ≥1.8 fold increase in CD38+HLA-DR+ CD8 T cells from baseline to post-cycle 1 (Figure 1A) was observed in 8/12 patients who had clinical benefit and 6/15 patients with no clinical benefit (Figure 1B). Transcriptional analysis revealed that in patients with the aforementioned immunological response, T-cells had upreg- ulated TCR signaling, CD28 signaling, enhanced glycolysis and iron uptake, and reduced TGF-beta signaling compared to patients with- out an immunologic response. Conclusions Peripheral blood immune monitoring of RCC patients while on im- munotherapy may provide an early predictor of response. One im- portant limitation identified is the treatment specific cycle length defined sample collection timing and therefore may miss transient early immunologic changes. This study advances knowledge regard- ing the newly generated effector CD8 T cells that contain important information about the immunobiology underlying response to immunotherapy. Acknowledgements This work was supported by funding from the NCI grant 1-R00-CA197891 and Nektar Therapeutics. We would like to acknowledge The Yerkes NHP Genomics Core which is supported in part by NIH P51 OD011132, the Emory Flow Cytometry Core (EFCC) supported by the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378, and NIH/NCI under award number, 2P30CA138292-04. Fig. 1 (abstract P128). See text for description References 1. Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, Castellano D, Choueiri TK, P129 Gurney H, Donskov F, Bono P, Wagstaff J, Gauler TC, Ueda T, Tomita Y, Correlation of circulating CD8 T cell activation with response to Schutz FA, Kollmannsberger C, Larkin J, Ravaud A, Simon JS, Xu LA, immunotherapy in advanced renal cell carcinoma Waxman IM, Sharma P, CheckMate I. Nivolumab versus Everolimus in Jennifer Carlisle, MD, Caroline Jansen, BS, Adriana Reyes, Nataliya Advanced Renal-Cell Carcinoma. N Engl J Med. 2015;373(19):1803-1813. Prokhnevska, BS, Deborah Baumgarten, MD, Viraj Master, MD, PhD, R. 2. Motzer RJ, Tannir NM, McDermott DF, Aren Frontera O, Melichar B, Donald Harvey, PharmD, Bradley Carthon, MD, PhD, Omer Kucuk, MD, Choueiri TK, Plimack ER, Barthelemy P, Porta C, George S, Powles T, Mehmet Bilen, Haydn Kissick Donskov F, Neiman V, Kollmannsberger CK, Salman P, Gurney H, Hawkins Emory University, Atlanta, GA, United States R, Ravaud A, Grimm MO, Bracarda S, Barrios CH, Tomita Y, Castellano D, Correspondence: Jennifer Carlisle (jennifer.w.carlisle@emory.edu) Rini BI, Chen AC, Mekan S, McHenry MB, Wind-Rotolo M, Doan J, Sharma Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P129 P, Hammers HJ, Escudier B, CheckMate I. Nivolumab plus Ipilimumab ver- sus Sunitinib in Advanced Renal-Cell Carcinoma. N Engl J Med. Background 2018;378(14):1277-1290. Although immunotherapy with PD-1 or dual PD-1/CTLA-4 blockade 3. Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, Sica GL, can be a successful treatment for patients with advanced renal cell Yu K, Koenig L, Patel NT, Behera M, Wu H, McCausland M, Chen Z, Zhang carcinoma (RCC), the majority do not respond [1, 2]. Prior peripheral C, Khuri FR, Owonikoko TK, Ahmed R, Ramalingam SS. Proliferation of PD- blood immune profiling studies have shown that a transient rise in 1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung activated CD8 T cells correlates with clinical response to PD1 block- cancer patients. Proc Natl Acad Sci U S A. 2017;114(19):4993-4998. ade in lung cancer [3, 4] and melanoma [5]. The effect of checkpoint 4. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, blockade on circulating CD8 T cells in RCC is unknown, as is the T cell Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, biology underlying clinical response. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 71 of 272 Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, approval of the NYU Institutional Review Board at the NYU Perlmut- Araki K, Ahmed R. Rescue of exhausted CD8 T cells by PD-1-targeted ter Cancer Center with informed consent [11]. therapies is CD28-dependent. Science. 2017;355(6332):1423-1427. Serum samples were run on HuProt Human Proteome Microarrays 5. Huang AC, Postow MA, Orlowski RJ, Mick R, Bengsch B, Manne S, Xu W, containing >19,000 human proteins by CDI Laboratories. Raw serum Harmon S, Giles JR, Wenz B, Adamow M, Kuk D, Panageas KS, Carrera C, IgG signal intensities were processed across staining cohorts via Wong P, Quagliarello F, Wubbenhorst B, D'Andrea K, Pauken KE, Herati interquartile range normalization. RS, Staupe RP, Schenkel JM, McGettigan S, Kothari S, George SM, Pre-existing antibody responses were defined as patient-specific IgG Vonderheide RH, Amaravadi RK, Karakousis GC, Schuchter LM, Xu X, signals >3.5 median absolute deviations above cohort median IgG Nathanson KL, Wolchok JD, Gangadhar TC, Wherry EJ. T-cell invigoration background (modified Z-score). Group statistics were computed to tumour burden ratio associated with anti-PD-1 response. Nature. (GraphPad Prism), and gene ontology enrichment analysis was per- 2017;545(7652):60-65. formed (Enrichr) [12]. Ethics Approval Results Samples are collected under an approved IRB protocol (The Urological Several pre-existing antigen-specific IgG autoantibody targets were Satellite Specimen Bank at Emory University, IRB00055316), and all observed to have associations with good outcomes (SD/PR) or ob- patients provided informed consent. jective clinical responses (PR/CR) versus patients with progressive dis- ease (POD). While final determination of the most predictive subsets is ongoing, many targets represent genes in an axis surrounding im- mune signaling pathways, hereditary neurodegenerative disease, and the ubiquitin proteasome pathway (ie, UBQLN1, UBQLN2). An exemplary example was observed in the autoantibody responses shared by >10% of all patients regardless of clinical outcome. Gene ontology enrichment analysis of these shared melanoma-patient autoantibodies versus KEGG 2019 [12] demonstrates this set of pro- teins is strongly enriched for neurotrophin signaling-associated pro- teins after multi-sample correction (P=0.004) (Table 1). Several other associations were observed cohort-wide for ontologies with tissue- specific enrichment in the brain, neurons, and neuronal processes. Conclusions In this pilot study, we found strong associations across the cohort for autoantibodies against nerve-growth-inducing neurotrophins and genes like UBQLN1 and UBQLN2 which have strong associations with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson’s, and Alzheimer’s – neurodegenerative diseases that are known to have incidences which correlate with melanoma [14–16]; this hints at a potential immunologic connection between the conditions, per- haps related to an antitumor / autoimmune axis involving the targets reported here. Acknowledgements We thank the patients and their families who consented to participate in this study. Funding support for the study was provided by the NYU Cancer Center and NIH/NCI Cancer Center Support Grant P30CA016087, the Marc Jacobs campaign to support melanoma research, Goldberg Charitable Trust, Fig. 1 (abstract P129). See text for description Wings for Things Foundation and Clayman Family Foundation to I. Osman; the American Medical Association foundation, the Melanoma Research Foundation and the American Skin Association grants to M. Gowen. Trial Registration P130 Patient samples included in this study were not part of a randomized Melanoma patients harbor pre-existing IgG autoantibodies controlled clinical trial. targeting neuronal proteins that associate with differential clinical outcomes following checkpoint blockade 1 2 2 2 References Tyler Hulett, PhD , Keith Giles , Michael Gowen, MD , Danny Simpson , 2 2 2 1. Nagele EP, Han M, Acharya NK, DeMarshall C, Kosciuk MC, Nagele RG. Jeremy Tchack , Una Moran , Zarmeena Dawood , Anna Pavlick, MD, 2 1 2 2 Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human MBA , Shaohui Hu , Hua Zhong , Michelle Krogsgaard , Tomas Kirchhoff, 2 2 Sera, and Their Number Is Influenced By Age, Gender, and Disease. PhD , Iman Osman 1 2 Tsokos GC, editor. PLoS ONE. 2013;8:e60726. CDI Laboratories, Portland, OR, United States; New York University 2. Larman HB, Zhao Z, Laserson U, Li MZ, Ciccia A, Gakidis MAM, et al. School of Medicine, New York, NY, United States Autoantigen discovery with a synthetic human peptidome. Nature Correspondence: Tyler Hulett (tyler.hulett@cdi-lab.com) Biotechnology. 2011;29:535–41. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P130 3. Meyer S, Woodward M, Hertel C, Vlaicu P, Haque Y, Kärner J, et al. AIRE- Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Background Autoantibodies. Cell. 2016;166:582–95. Autoantibody landscapes are very specific to the individual, can remain 4. Graff JN, Puri S, Bifulco CB, Fox BA, Beer TM. Sustained Complete stable for many years, and contain unique features reported in associ- Response to CTLA-4 Blockade in a Patient with Metastatic, Castration- ation with cancer, autoimmunity, infection, neurologic conditions, Resistant Prostate Cancer. Cancer Immunology Research. 2014;2:399–403. CD8+ T cell behavior, and checkpoint blockade adverse events [1–11]. 5. Gnjatic S, Ritter E, Büchler MW, Giese NA, Brors B, Frei C, et al. Seromic The goal of this work was to determine whether pre-existing antigen- profiling of ovarian and pancreatic cancer. Proceedings of the National specific features in melanoma patient autoantibody landscapes would Academy of Sciences. 2010;107:5088–5093. associate with clinical outcomes following checkpoint blockade. 6. Wongkulab P, Wipasa J, Chaiwarith R, Supparatpinyo K. Autoantibody to Methods Interferon-gamma Associated with Adult-Onset Immunodeficiency in Pre-treatment serum samples were collected from 117 melanoma pa- Non-HIV Individuals in Northern Thailand. Rottenberg ME, editor. PLoS tients prior to checkpoint blockade with anti-CTLA4 (N=60), anti-PD1 ONE. 2013;8:e76371. (N=38), or both in combination (N=16). All data was collected with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 72 of 272 7. Anderson KS, Sibani S, Wallstrom G, Qiu J, Mendoza EA, Raphael J, et al. P131 Protein Microarray Signature of Autoantibody Biomarkers for the Early Single cellular interrogation of tumor microenvironment enables Detection of Breast Cancer. Journal of Proteome Research. 2011;10:85–96. diagnosis and prognostication of malignancies 1 1 2 8. Miersch S, Bian X, Wallstrom G, Sibani S, Logvinenko T, Wasserfall CH, Wei Jian Tan , Jian Hang Lam , Mona Meng Wang , Paola Ricciardi- 1 2 3 et al. Serological autoantibody profiling of type 1 diabetes by protein Castagnoli , Anita Sook Yee Chan , Tony Kiat Hon Lim , Joe Poh Sheng 3 1 arrays. Journal of Proteomics. 2013;94:486–96. Yeong , Tong Seng Lim, PhD 1 2 9. Srivastava RM, Lee SC, Andrade Filho PA, Lord CA, Jie H-B, Davidson HC, Menarini Biomarkers Singapore, Singapore, Singapore; Singapopre Eye et al. Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate Research Institute, Singapore, Singapore; Singapore General Hospital, to Trigger Tumor Antigen-Specific T-cell Immunity in Head and Neck Singapore, Singapore Cancer Patients. Clinical Cancer Research. 2013;19:1858–72. Correspondence: Tong Seng Lim (tongseng.lim@mbiomarkers.com) 10. Hulett TW. Coordinated responses to individual tumor antigens by IgG Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P131 antibody and CD8+ T cells following cancer vaccination. 2018;14. 11. Gowen MF, Giles KM, Simpson D, Tchack J, Zhou H, Moran U, et al. Background Baseline antibody profiles predict toxicity in melanoma patients treated Tumor microenvironment contains a diverse array of cell types with immune checkpoint inhibitors. Journal of Translational Medicine with heterogeneous genomic and molecular profiles. Averaging [Internet]. 2018 [cited 2018 Nov 4];16. Available from: https://translational- the characteristics of all cells in a cancerous tissue no doubt ob- medicine.biomedcentral.com/articles/10.1186/s12967-018-1452-4 scures important variations in biomarkers among minority, but 12. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, critical, pathogenic cell populations. High resolution, single-cell Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list analyses are thus needed in the clinic to precisely delineate the enrichment analysis tool. BMC Bioinformatics. 2013;128(14). inherent heterogeneity of tumor microenvironment underlying 13. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, oncogenesis in each patient. Biopsies derived from tumor micro- Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, McDermott MG, environment are routinely used for medical diagnosis or prognos- Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene tications. The number of cells typically available from a biopsy is set enrichment analysis web server 2016 update. Nucleic Acids Research. limited and heterogeneous. The ability to distinguish, select, and 2016; gkw377 . sort rare malignant or pathogenic immune cells from either tissue 14. Olsen JH, Friis S, Frederiksen K. Malignant Melanoma and Other Types of or liquid biopsies poses a unique challenge for single-cell based Cancer Preceding Parkinson Disease: Epidemiology. 2006;17:582–7. diagnosis and prognostication. Heterogeneous cell populations 15. Freedman DM, Curtis RE, Daugherty SE, Goedert JJ, Kuncl RW, Tucker MA. with non-target immunoreactive cells in pausicellular biopsies The association between cancer and amyotrophic lateral sclerosis. Cancer complicate conventional bulk-cell analysis, and could lead to dis- Causes & Control. 2013;24:55–60. ease misdiagnosis or prognostication. 16. Roe CM, Fitzpatrick AL, Xiong C, Sieh W, Kuller L, Miller JP, et al. Cancer Methods linked to Alzheimer disease but not vascular dementia. Neurology. We adopted a state-of-the-art multi-modal strategy including the 2010;74:106–12. real-time imaging-based DEPArray with downstream molecular and Ethics Approval genomic assays [1], and quantitative multiplex immunofluorescent All data collected for this study was collected with approval of the NYU technique [2] in order to predict clinical outcome or direct therapy, Institutional Review Board at the NYU Perlmutter Cancer Center with based on single-cell based diagnostic or prognostic biomarkers de- informed consent. rived from tumor microenvironment. Consent Results No sensitive or patient identifiable information is included in the data We provided proof-of concepts that DEPArray technology enabled presented. automated isolation and recovery of rare malignant or pathogenic immune cells from liquid or tissue biopsies in several malignan- cies including vitreoretinal lymphoma (VRL), hepatocellular carcin- oma (HCC) and colorectal carcinoma (CRC). Rare target B lymphoma cells were distinguished and sorted from pausicellular ocular vitreous biopsies with high resolution and purity required Table 1 (abstract P130). Enrichment of anti-neuronal growth for sensitive single-cell based MYD88 mutational profiling to aid autoantibodies VRL diagnosis [1]. Single cellular imaging revealed the presence of large (>10μm), irregular shaped of a novel population of HCC- infiltrating macrophages in association with improved prognosis after surgery. A unique signature regulatory T-cells (Tregs) popu- lation was identified in both blood circulation and cancerous tis- sues of CRC. These signature Tregs expressed phenotypically distinct surface markers in association with better disease-free and overall survival of CRC patients. Conclusions Using real-time imaging-based, digital sorting DEPArray, we could distinguish, select and sort different types of malignant or target immune cells including B-cells, T-cells and macrophages from het- erogeneous tumor microenvironment or liquid biopsies with low cellularity. Comprehensive genomic and molecular characteriza- tions at single cell resolution revealed crucial biomarkers associ- ated with clinicopathological features that impact clinical outcome of patients. The single cell interrogation using DEPArray technology provides a novel precision medicine tool for diagnos- tics and prognostications of malignancies in future. Acknowledgements This study was supported by research funding from the research collaboration between A. Menarini Biomarkers Singapore Pte Ltd, Singapore Eye Research Institute and Singapore General Hospital. Some data presented here are part of patent filed on 21 August 2018 (#10201807097T). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 73 of 272 References P133 1. Tan, W.J., et al., Single-cell-MYD88 sequencing of isolated B cells High tumor expression of DKK1 is associated with improved from vitreous biopsies aids vitreoretinal lymphoma diagnosis. Blood, clinical benefit and longer progression free survival across 2019. multiple solid tumors when treated with a targeted anti-DKK1 2. Lim, J.C.T., et al., An automated staining protocol for seven-colour im- antibody (DKN-01) munofluorescence of human tissue sections for diagnostic and prognos- Michael Kagey, PhD, Girish Naik, MD, Michael Haas, PhD, Heidi tic use. Pathology, 2018. 50(3): p. 333-341. Heath, Franziska Schurpf-Huber, Walter Newman, PhD, Cynthia Sirard, Ethics Approval MD This study was approved by the SingHealth Institutional Review Board in Leap Therapeutics, Cambridge, MA, United States accordance with the Singapore Guidelines for Good Clinical Practice and Correspondence: Cynthia Sirard (csirard@leaptx.com) the Declaration of Helsinki, approval number #2009/907/B, 2012/104/F Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P133 and #2017/2494 Background Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, con- P132 tributes to an immune suppressive tumor microenvironment Harmony: Integrative tool to analyse and visualise multiplex- and promotes tumor growth, angiogenesis and metastasis. immunofluorescence single-cell data DKN-01, a DKK1 neutralizing antibody, has demonstrated clin- 1 2 3 4 Duoduo Wu , Joe Yeong, MBBS, PhD , Grace Tan , Marion Chevrier , ical activity across multiple solid tumors as both a monother- 5 5 4 Josh Loh , Tony Lim , Jinmiao Chen apy and in combination with checkpoint inhibitors and 1 2 National University of Singapore, Singapore, Singapore; Department of chemotherapies. Nonclinical studies indicate that DKN-01 effi- Anatomical Pathology, Sing, Singapore, Singapore; Nanyang cacy depends on a functioning immune system, notably nat- Technological University, Singapore, Singapore; Agency of Science, ural killer (NK) cells. High tumor expression of DKK1 correlates Technology and Resear, Singapore, Singapore; Singapore General with a worse clinical prognosis in many solid tumors. As such, Hospital, Singapore, Singapore, Singapore we evaluated tumor levels of DKK1 and association with clin- Correspondence: Tony Lim (Lim.Kiat.Hon@singhealth.com.sg); Jinmiao ical outcomes for DKN-01 based therapies. Chen (Chen_Jinmiao@immunol.a-star.edu.sg) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P132 DKK1 mRNA expression in patient tumor biopsies was evalu- ated with a RNAscope in situ hybridization assay. Expression Background levels were semi-quantified with QuPath or manually scored. In the advent of immuno-technology, newer single-cell flow cytome- Data was pooled from three separate clinical trials, DKN-01 as try techniques have greatly increased the capacity for the maximum monotherapy or in combination with paclitaxel or pembrolizu- number of immunological parameters measured. Notably, multiplex- mab in esophagogastric cancer (EGC) (NCT02013154), DKN-01 immunofluorescence (mIF) can perform measurements for 7 markers, as monotherapy or in combination with paclitaxel in epithelial flow cytometry can handle 20, and imaging mass cytometry can endometrial cancer (EEC) or epithelial ovarian cancer (EOC) process up to 37 biomarkers simultaneously. Hence, dimensionality (NCT03395080) and DKN-01 in combination with gemcitabine/ reduction techniques such as t-SNE and UMAP are becoming increas- cisplatin in biliary tract cancer (BTC) (NCT02375880). Survival ingly important for tumour single-cell data analysis. Using human he- analysis was performed by the Kaplan-Meier method and multi- patocellular carcinoma (HCC) tissue samples, we aim to compare and variable Cox proportional-hazards and logistic regression evaluate the use of a new technique, UMAP, as an alternative to t- models were used to study the association of DKK1 H-score SNE in mIF derived single-cell data. cutoffs (tertiles and quartiles) with survival and clinical benefit Methods (CR, PR or SD per RECIST v1.1) outcomes. We adopted an unsupervised clustering approach using FlowSOM to Results identify 8 major cell types present in human HCC tissues by staining Atotal of 120 patients (59EGC,28EEC,20EOC and13BTC) them with 7 markers, including immune-checkpoint molecules and had DKK1 tumor expression with response and survival out- one nuclear counterstain. Following that, UMAP and t-SNE were ran comes. Patients who had an H-score ≥ upper-quartile (≥50) of independently on the dataset to qualitatively compare the distribu- DKK1 expression versus < upper-quartile had a higher-odds of tion of clustered cell types in both dimensionality reduction tools. having clinical benefit/response with an adjusted OR of 4.46 Results (95% CI: 1.78, 11.71) and a longer PFS with an adjusted HR of The key advantage of UMAP is its superior runtime – it takes approxi- 0.49 (95% CI: 0.29, 0.81). Patients who had an H-score ≥ upper- mately one-fifth the time required to run t-SNE. Both techniques pro- tertile (≥35) versus < upper-tertile had a higher-odds of having vide similar arrangements of cell clusters, with the key difference clinical benefit/response with an adjusted OR of 2.82 (95% CI: being UMAP’s extensive characteristic branching. Also, increasing 1.21, 6.67) and a longer PFS with an adjusted HR of 0.53 (95% perplexity values in t-SNE results in a t-SNE visualisation with certain CI: 0.33, 0.85). degrees of branching like that of UMAP’s, albeit limited. When pa- Conclusions rameters such as standard deviation, minimum and maximum inten- Elevated DKK1 tumor expression was associated with a higher sity from the mIF image cytometry data were included, a t-SNE plot clinical benefit/response rate and longer PFS for DKN-01 based with virtually the same morphology as the resulting UMAP plot can treatments across multiple solid cancers. Tumor expression of be visualised. Most interestingly, UMAP’s branching highlighted bio- DKK1 may represent an important patient selection criterion logical lineages, especially in identifying potential hybrid tumour cells for further development of DKN-01 based therapies in solid (HTC). Survival analysis shows patients with higher proportion of HTC cancers. The contribution of high levels of tumor DKK1 expres- have a worse prognosis (p-value = 0.019). sion to an immune suppressive tumor microenvironment and Conclusions the role of NK cells is currently under investigation. We conclude that both techniques are similar in their visualisation capabilities, but UMAP has a clear advantage over t-SNE in runtime, Ethics Approval making it highly plausible to employ UMAP as an alternative to t-SNE Studies were approved by the Institutional Review Boards of each in single-cell data analysis. participating institution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 74 of 272 P134 Background Prognostic value of tumor microenvironment based on PD-L1 Loss of Y chromosome (LOY) is a well know established expression and CD8+ TILs density in locally advanced NSCLC phenomenon associated with cancers and ageing. Recently, LOY treated with concurrent chemoradiotherapy in peripheral blood cells was suggested as a possible biomarker 2 1 1 1 Lukas Käsmann , Kathrin Gennen , Julian Taugner , Chukwuka Eze , for different cancers in males. On the basis of previous findings, 1 1 1 Monika Karin , Olarn Roengvoraphoj , Jens Neumann , Amanda the present case-control study was conducted to evaluate the 3 1 1 1 Tufman , Michael Orth , Simone Reu , Claus Belka association of LOY in peripheral blood cells in prostate (PC) and 1 2 University Hospital, Munich, Germany; University of Munich, Munich, colorectal cancers (CRC) in males [1-4]. Germany; Thoracic Oncology Centre Munich, Munich, Germany Methods Correspondence: Lukas Käsmann (lkaesmann@gmail.com) 30 CRC patients (mean age = 44.03±10.8), 36 PC patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P134 (mean age = 60.8 ± 15.8 yrs) and 36 healthy control male cases (mean age = 54.6± 15.1 years) were recruited. DNA was Background extracted by using a standard phenol-chloroform method. The prognostic role of the tumor immunity microenvironment Multiplex quantitative fluorescent (QF) PCR was used to co- (TIME) in multimodal treatment for locally advanced non-small amplify the homologous sequences present on the Y chromo- cell lung cancer (LA-NSCLC) is unclear. Increasing evidence sug- some and other chromosome followed by their analysis on the gests treatment benefit depending on tumor cell PD-L1 expres- genetic analyzer (ABI 3500) and finally the Y/X ratio was calcu- sion. The purpose of this retrospective single-center study was to lated on the basis of the peak height obtained from the investigate the prognostic value of PD-L1 expression on tumor electropherogram. cells in combination with CD8+ tumor stroma-infiltrating lympho- Results cytes (TILs) density in inoperable LA-NSCLC treated with concur- The mean Y/X ratio was significantly lower in the whole group rent chemoradiotherapy (CRT). of cancer patients (0.709±0.02; p <0.0001) when compared to Methods the controls (0.92±0.044). Also, the Y/X ratio when calculated We collected retrospectively clinical characteristics and initial separately was found to be lower in CRC (0.701±0.078; p < tumor biopsy samples of 31 inoperable LA-NSCLC patients treated 0.0001) and PC (0.717±0.044; p <0.0001) cases, when compared with concurrent CRT. PD-L1 expression on tumor cells (0% versus to controls (0.92±0.044). Multivariate logistic regression was ≥1%), CD8+ TILs density (0-40% vs. 41-100%) and TIME according performed by matching cancer and control subjects with age to classification by Zhang et al. were evaluated for potential and the results suggest that LOY is not influenced by their prognostic value in terms of local control, progression-free (PFS) age. and overall survival (OS) as well as correlations with clinic- Conclusions pathological features investigated. The results support the significant association of LOY in peripheral Results blood cells carcinogenesis in males. LOY can also serve as a non- Median OS was 14 months (range: 3-167 months). The OS rates invasive cancer biomarker to improve the early diagnosis and man- at 1- and 2 years were 68% and 20%. Local control rates for the agement of cancer patients in males. entire cohort at 1 and 2 years were 74% and 61%, respectively. Median PFS and PFS at 1 and 2 years were 13±1.4 months, 58% Acknowledgements and 19%. PD-L1 expression <1% on tumor cells was associated We are highly grateful to Sanjay Gandhi Post Graduate Institute of with improved OS, PFS and local control in patients treated with Medical Sciences, Lucknow, Uttar Pradesh, India for providing the concurrent CRT. Univariate analysis showed a trend for improved infrastructure and lab facilities for research work. The authors also thank OS and local control in patients with low CD8+ TILs density. all the consultant and residents of SGPGIMS, who helped in carrying out Evaluation of TIME appears to be an independent prognostic fac- the study. Dr Ambreen Asim is the first author, who collected data, tor for local control, PFS and OS. The longest and shortest OS carried out all the practical work and drafted this abstract. Prof. Sarita were achieved in patients with type I (PD-L1neg/CD8low) and Agarwal is the corresponding and second author, who helped in type IV (PD-L1pos/CD8low) tumors (median OS: 57±37 vs. 10±5 finalizing, correcting and critical review of the work. Prof.Rakesh Kapoor months, p=0.05), respectively. and Prof. Neeraj Rastogi are the oncologist consultant who has provided Conclusions prostate and colorectal cancer patients blood samples after taking Assessment of the tumor immunity microenvironment (TIME) by informed consent for this study. PD-L1 expression on tumor cells and CD8+ TILs density is a pre- dictive biomarker in patients treated with concurrent CRT for in- References operable LA-NSCLC. 1. Noveski P, Madjunkova S, Stefanovska E. Loss of Y Chromosome in Peripheral Blood of Colorectal and Prostate Cancer Patients. Plos One. Acknowledgements 2016;11(1). The study was funded by the German Center for Lung Research (DZL). 2. Chang Y M, Perumal R, Keat P Y, Rita Y.Y. Yong, Daniel L.C. Kuehn, Ethics Approval Leigh Burgoyne. A distinct Y-STR haplotype for Amelogenin nega- The study was approved by the University Ethics Board, approval number tive males characterized by a large Yp11.2 (DYS458-MSY1-AMEL-Y) 493-16. deletion. Forensic Sci Int. 2007; 166(2-3):115-20. Consent 3. Donaghue C, Mann K, Docherty Z and Ogilvie C M. Detection of Written informed consent was obtained from the patient for publication of mosaicism for primary trisomies in prenatal samples by QF-PCR and this abstract and any accompanying images. A copy of the written consent karyotype analysis.Prenat Diagn .2005; 25: 65–72. is available for review by the Editor of this journal. 4. Plaseski T, Noveski P, Trivodalieva S, Georgi D. Quantitative Fluorescent- PCR Detection of Sex Chromosome Aneuploidies and AZF Deletions /Du- plications. Genetic Teasting, 2008 : 12: 4. P135 Ethics Approval Studying Loss of Y chromosome in colorectal and prostate cancers This study was approved by Sanjay Gandhi Post Graduate Institute of in males for non-invasive cancer biomarkers Medical Sciences Ethics Board; approval number IEC CODE – 2018- 53- Ambreen Asim, PhD, Sarita Agarwal, Rakesh Kapoor, Neeraj Rastogi IMP-103 dated 18th June 2018.” Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Consent India Written informed consent was obtained from the patient for publication of Correspondence: Sarita Agarwal (saritasgpgi@gmail.com) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P135 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 75 of 272 P136 Background Quantitative/spatial analysis of Tregs reveal a prominent PD-L1 protein expression by immunohistochemistry (IHC) meas- biomarker role in human non-small cell lung cancer (NSCLC) urement is the only FDA-approved protein diagnostic biomarker 1 1 1 Richa Gupta , Nicolas Rodriguez-Arriagada , Shruti Desai, PhD , for PD1/PD-L1 immunotherapies [1]. However, the tumor-immune 2 1 Konstantinos Syrigos , Roy Herbst, MD, PhD , Vamsidhar Velcheti, MD interaction is complex: PD-L1 expression alone is not predictive 3 1 1 FACP , David Rimm, MD, PhD , Sarah Goldberg, MD, MPH , Kurt of patient response [2]. This has led to the investigation of other Schalper, MD, PhD PD-1 ligands such as PD-L2 [2]. PD-L2 even in the absence of 1 2 Yale University, Burlington, CT, United States; Athens University, PD-L1 has been associated with clinical response to PD-1 block- Athens, Greece; NYU-Langone Medical Center, Pepper Pike, OH, United ade in multiple tumor types [2]. PD-L2 status has also been inves- States tigated where immunotherapy based on PD-L1 has been less Correspondence: Kurt Schalper (kurt.schalper@yale.edu) successful, such as prostate cancer, where PD-L1 expression is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P136 typically low [3]. Here, significantly higher levels of PD-L2 were associated with multiple survival and response measures [3]. Due Background to the diagnostic and therapeutic potential indicated by the pres- Regulatory T cells (Tregs) mediate potent tolerogenic signals, are involved ence of this key immune checkpoint ligand in patients irrespect- in adaptive anti-tumor immune responses and T-cell reinvigoration using ive of PD-L1 expression [2-4], dependable detection tools for immune checkpoint blockers. Despite their prominent immune suppres- investigating the presence and role of PD-L2 are crucial. To ad- sive role, the tissue distribution and contribution of Tregs to clinical out- dress this need, Abcam have developed and extensively charac- comes in human lung cancer is not well understood. terized and validated a recombinant rabbit monoclonal antibody Methods specific to PD-L2 (CAL28). For checkpoint inhibitors, Abcam The levels and tissue distribution of Tregs and major tumor infiltrating already has research use only versions of three anti-PD-L1 Rab- lymphocyte (TIL) subsets were measured using simultaneous detection MAb® antibodies employed in the clinical setting (73-10, 28-8 and of FOXP3, CD4, CD8, pancytokeratin and DAPI by multiplexed quantita- SP142), co-developed with pharmaceutical and diagnostic tive immunofluorescence in 619 formalin-fixed paraffin embedded companies. (FFPE) NSCLCs from 4 independent cohorts represented in tissue micro- Methods arrays (cohort #1 [Yale, n=210], cohort #2 [Greece, n=192]; cohort #3] A recombinant rabbit monoclonal antibody was generated using [80 immunotherapy-treated NSCLCs]; cohort #4 [Yale, n=137, adenocar- a direct B cell cloning process and characterized for IHC. The cinomas with mutation testing). Markers were measured in different tis- clone was tested using PD-L2-transfected and non-transfected sue compartments and cell phenotypes were used for individual cell HEK293 cells fixed in formaldehyde and processed into paraffin counts and machine-learning-based spatial analysis. We studied the as- wax (FFPE) and further validated alongside In Situ Hybridization sociation between T-cell populations, tissue distribution, clinicopatho- (ISH) for PD-L2 mRNA in FFPE commercial cell lines. Once speci- logic/molecular characteristics and outcomes. ficity was determined, it was tested in positive and negative tis- Results sues and TMAs of Head & Neck Squamous Cell Carcinoma Tregs (DAPI+/CD4+/FOXP3+ cells) were predominantly located in the (HNSCC), Prostate Carcinoma (PC) and Renal Cell Carcinoma stromal compartment and represented 3-10% of the total T-cell popula- (RCC). tion. The level of Tregs was positively associated with higher CD8+ T- Results cell infiltration across the cohorts. There was no consistent association CAL28 demonstrated positive IHC staining on PD-L2- between Treg levels and patient age, gender, smoking status, clinical overexpressed HEK293 cells processed in FFPE with a lack of stage or tumor histology. However, Tregs were significantly higher in staining in the parental line. Additionally, CAL28 demonstrated KRAS mutated lung adenocarcinomas than in EGFR mutant or KRAS/ IHC staining in FFPE cell lines where PD-L2 expression was con- EGFR wild-type cases. As a single marker, the level of Tregs was not sig- firmed with ISH for PD-L2 mRNA. Expression in tumor tissue in nificantly associated with survival. However, the Treg to CD8 signal ratio TMAs from HNSCC, PC and RCC was evaluated with no non- was associated with shorter 5-year overall survival across the cohorts. specific background staining. Reduced survival was also seen in cases with a higher 5-nearest neigh- Conclusions bor (5NN) mean distance between CD4+/Tregs and CD8+/CD4+ cells. We have demonstrated sensitivity, specificity and reproducibility Notably, the survival effect of the Treg-associated metrics was numeric- of a recombinant rabbit monoclonal antibody to PD-L2 in IHC ally higher in patients treated with immune checkpoint blockers. (CAL28). The global, commercial availability of this recombinant Conclusions clone to researchers, pathologists, clinicians and the biopharma- Tregs are prominently less abundant than other TIL subsets in NSCLC ceutical industry will enable further progress to be made in un- microenvironments and they are increased in T-cell inflamed tumors. derstanding the clinical relevance and predictive value that PD-L2 Their positive association with CD8+ cytotoxic TILs suggests their up- promises for cancer immunotherapy. regulation upon adaptive anti-tumor immune pressure and could ex- plain the inconsistent reported relationship between Tregs and References prognosis. Elevated Treg to CD8 signal ratio and reduced spatial clus- 1. Tsao MT, Kerr K, Yatabe Y, et al. PL 03.03 Blueprint 2: PD-L1 Immunohisto- tering between CD4-Tregs and CD8-CD4 are indicative of poor out- chemistry Comparability Study in Real-Life, Clinical Samples. J Thor Oncol. come preferentially in NSCLC patients treated with checkpoint 2017;12(Suppl 2):S1606 blockade suggesting a biomarker role. 2. Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Ethics Approval Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T. All tissues were used after approval from the Yale Human Investiga- Clin Cancer Res. 2017;23:3158-3167 tion committee protocol #9505008219 which approved patient con- 3. Zhao SG, Lehrer J, Chang SL, Das R, Erho N, Liu Y, Sjöström M, Den RB, sent forms or waivers of consent. Freedland SJ, Klein EA, Karnes RJ, Schaeffer EM, Xu M, Speers C, Nguyen PL, Ross AE, Chan JM, Cooperberg MR, Carroll PR, Davicioni E, Fong L, Spratt DE, Feng FY.The Immune Landscape of Prostate Cancer and P137 Nomination of PD-L2 as a Potential Therapeutic Target. J Natl Cancer Inst. Development and high specification validation of two recombinant 2019;111:301-310 rabbit monoclonal antibodies to accurately detect human PD-L2 4. Takamori S, Takada K, Toyokawa G, Azuma K, Shimokawa M, Jogo T, expression in FFPE tissue sections by immunohistochemistry Yamada Y, Hirai F, Tagawa T, Kawahara A, Akiba J, Okamoto I, Nakanishi Simon Renshaw, Will Howat, PhD, Subham Basu, PhD Y, Oda Y, Hoshino T, Maehara Y. PD-L2 Expression as a Potential Predict- Abcam, Cambridge, United Kingdom ive Biomarker for the Response to Anti-PD-1 Drugs in Patients with Non- Correspondence: Subham Basu (Subham.Basu@abcam.com) small Cell Lung Cancer. Anticancer Res. 2018;38:5897-5901 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P137 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 76 of 272 P138 3. Oaknin A, Duska LR, Sullivan RJ, et al. Preliminary safety, efficacy, and Review of evidence for predictive value of microsatellite pharmacokinetic/pharmacodynamic characterization from GARNET, a instability/mismatch repair status in response to non-anti-PD-(L)1 phase I/II clinical trial of the anti–PD-1 monoclonal antibody, TSR-042, in therapies in patients with advanced or recurrent endometrial patients with recurrent or advanced MSI-H and MSS endometrial cancer. cancer Gynecol Oncol. 2019; 154:17. 1 2 2 2 Cara Mathews, MD , Ellie Im , Liliana Alfaya , Karin Travers , Craig 4. Antill YC, Kok PS, Robledo K, et al. Activity of durvalumab in advanced Gibson endometrial cancer (AEC) according to mismatch repair (MMR) status: 1 2 Women and Infants Hospital, Providence, RI, United States; TESARO: A The phase II PHAEDRA trial (ANZGOG1601). J Clin Oncol 37, 2019 (suppl; GSK Company, Waltham, MA, United States abstr 5501). Correspondence: Cara Mathews(cmathews@wihri.org) 5. Djordjevic B, Bruegl A, Fellman B, et al. The prognostic effect of MLH1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P138 loss in endometrial endometrioid adenocarcinoma. Lab Invest. 2014; 94:280A–281A. Background 6. Cohen JG, Goodman MT, Karlan BY, Walsh C. Genomic characterization of Multiple immunotherapies have been evaluated in patients with ad- grade 3 endometrial carcinoma. Gynecol Oncol. 2014; 133:134–135. vanced or recurrent endometrial cancer (EC) using molecular bio- 7. Cosgrove CM, Cohn DE, Hampel H, et al. Epigenetic silencing of MLH1 in markers, including microsatellite instability-high (MSI-H) and stable endometrial cancers is associated with larger tumor volume, increased (MSS) status. Clinical outcomes appear to be different in patients with rate of lymph node positivity and reduced recurrence-free survival. Gyne- MSI-H/mismatch repair (MMR)-deficient status versus MSS/MMR-profi- col Oncol. 2017; 146(3):588–595. doi:10.1016/j.ygyno.2017.07.003. cient status when receiving anti-programmed cell death (ligand) 1 (PD- 8. Kim SR, Pina A, Albert A, et al. Does MMR status in endometrial cancer [L]1) therapies [1,2]. It is unclear if these differences are due to the ther- influence response to adjuvant therapy? Gynecol Oncol. 2018; 151:76–81. apies themselves or to differences inherent to the patient populations. 9. Aghajanian C, Filiaci V, Dizon DS, et al. A phase II study of frontline We sought to evaluate the association between MSI-H/deficient MMR paclitaxel/carboplatin/bevacizumab, paclitaxel/carboplatin/temsirolimus, (dMMR) status and response among patients with advanced or recur- or ixabepilone/carboplatin/bevacizumab in advanced/recurrent rent EC. endometrial cancer. Gynecol Oncol. 2018; 150:274–281. Methods We conducted a systematic review of the Embase, MEDLINE, and P139 Cochrane Central Register of Controlled Trials databases from 2000 Expression of GITR and GITR-L by head and neck squamous cell to present to identify publications (manuscripts and conference pro- cancer ceedings) on studies using chemotherapy, surgery, radiotherapy, hor- 1 2 Rachna Moudgil, MS , Christopher Paustian, PhD , Carmen Ballesteros- monal therapy, or biological therapy (or any combination thereof) in 1 1 1 Merino, PhD , Shawn Jensen, PhD , Hong-Ming Hu, PhD , Walter Urba, adult patients (≥18 years) with stage III or IV advanced or recurrent 1 1 1 MD, PhD , Carlo Bifulco, MD , Marcus Couey, MD, DDS , Traci Hilton, EC, and where MMR or MSI status was identified (by any means). To 2 1 1 1 PhD , Bernard Fox, PhD , Rom Leidner, MD , R. Bryan Bell, DDS, MD better understand the prognostic value of MSI-H/MSS status, we ex- 1 2 Earle A. Chiles Research Institute, Portland, OR, United States; UbiVac, cluded anti-PD-(L)1 therapies from the analysis, as recent evidence Portland, OR, United States suggests that there is a positive predictive value for these agents in Correspondence: Bernard Fox(foxb@foxlab.org) patients with MSI-H/dMMR status [2-4]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P139 Results Our systematic review of MSI/MMR status and recurrence-free survival Background (RFS), progression-free survival (PFS), and overall survival (OS) identified Head and neck squamous cell cancer (HNSCC) ranks as the 6th most a total of 5 studies. One study reported dMMR status was associated common cancer afflicting humans and remains a significant unmet med- with a reduction in RFS (hazard ratio, 2.02) [5], while another study ical need. While interfering with the PD-1/PD-L1 axis improves outcomes, found no significant effect [6]. A third study reported a trend towards a the majority of patients progress and die of their disease. To address this higher rate of recurrence among patients with advanced-stage EC with lack of efficacy our group has explored the immune makeup of HNSCC, dMMR than among patients with MMR proficiency (P value not re- hypothesizing that a better characterization of responders and non- ported) [7]. Two studies reported no significant association between responders will result in improved predictive biomarkers and insights into PFS and dMMR status [8,9]. Three studies found no statistically signifi- strategies to improve outcomes for the majority of patients. cant association between OS and dMMR status [5,6,8]. Methods Conclusions Over the past 7 years we have collected and processed more than This review could not identify a consistent association between 350 HNSCC specimens. When sufficient tumor material was available, dMMR or MSI-H status and recurrence, RFS, PFS, or OS among pa- tumor-infiltrating lymphocytes (TIL) and primary tumor cultures were tients with advanced or recurrent EC receiving therapy other than initiated and characterized for autologous tumor reactivity. Once anti-PD-(L)1. For RFS, where differences were present, they trended established, tumor cell lines were characterized for phenotypic towards worse outcomes for patients with MSI-H/dMMR status. Con- markers by flow cytometry. Flow cytometric analysis and RNASeq has sequently, we have identified no evidence of a prognostic or predict- been performed on some established cell lines and on FFPE tumor ive value of MSI-H or dMMR biomarker status for efficacy outcomes specimens. in patients with advanced or recurrent EC receiving non–anti-PD-(L)1 Results therapy. Further investigation into the prognostic or predictive value Consistent with previous reports increased expression of CD8 T cells of MSI-H/dMMR status is warranted. was associated with improved outcome. In preliminary studies in- creased expression of GITR was also associated with improved out- Acknowledgements come. Initial speculation was that GITR expression was coming from Clinical Trial Registration: N/A immune infiltrates. Subsequently a report suggested that GITR could be expressed by HNSCC. Using flow cytometry we detected low level References GITR expression on 3 HNSCC cell lines and low to high level expres- 1. Segal NH, Wainberg ZA, Overman MJ, et al. Safety and clinical activity of sion of GITR-L on a 8 HNSCC cell lines. Studies are continuing to ex- durvalumab monotherapy in patients with microsatellite instability–high pand on these preliminary observations. (MSI-H) tumors [abstract]. J Clin Oncol. 2019; 37(Suppl 4):670. Conclusions 2. Konstantinopoulos PA, Liu JF, Luo W, et al. Phase 2, two-group, two- Anti-GITR and GITR-L both have the potential to provide positive sig- stage study of avelumab in patients (pts) with microsatellite stable (MSS), nals to immune cells. In addition to APC’s, GITR-L expression by some microsatellite instable (MSI), and polymerase epsilon (POLE) mutated re- HNSCC cells may contribute to the make-up of the immune cells infil- current/persistent endometrial cancer (EC) [abstract]. J Clin Oncol. 2019; trating these cancers. 37(Suppl 15):5502. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 77 of 272 Acknowledgements P141 Funding Support: The Harder Family, Robert and Elsie Franz, Wes and One-year progression-free survival in lung cancer patients treated Nancy Lematta, Lynn and Jack Loacker, the Providence Portland Medical with immune checkpoint inhibitors is significantly associated with Foundation and the Oral and Maxillofacial Surgery Foundation, The Mur- a novel immunomodulatory signature but not PD-L1 staining 1 1 1 2 dock Trust. Harsha Ranganath, MD , Amit Jain , Justin Smith , Julie Ryder , Amina 1 2 2 2 3 Ethics Approval Chaudry , Emily Miller , Felicia Hare , Poojitha Valasareddy , Rob Seitz , 3 3 3 2 The study was approved by the institutional review board of the Providence David Hout , Brock Schweitzer , Tyler Nielsen , Janice Mullins , Gregory Portland Medical Center (12-075A). Vidal University of Tennessee Health Sciences, Indianapolis, IN, United States; 2 3 P140 West Clinic Cancer Center, Memphis, TN, United States; Insight Myeloid cell contexture and IL-8 expression as a candidate Genetics, Nashville, TN, United States immunotherapy target in non-small cell lung cancer (NSCLC) Correspondence: Gregory Vidal (gvidal@westclinic.com) 1 1 Venkata Vamsi Nagineni, MD , Kurt Schalper, MD, PhD , Shruti Desai, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P141 1 2 2 1 PhD , Ignacio Melero, MD , Miguel Sanmamed, MD, PhD , Richa Gupta , 1 1 Roy Herbst, MD, PhD , Venkata Vamsi Nagineni, MD Background 1 2 Yale University, Waukegan, IL, United States; University of Navarra, Immune checkpoint inhibitors (PD-(L)1 inhibitors) have shown Pamplona, Spain promising therapeutic outcomes and have been approved for Correspondence: Kurt Schalper (kurt.schalper@yale.edu) multiple indications. However, widespread use of PD-(L)1 inhibi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P140 tors has been limited by a low response rate and immune- related adverse events. Therefore, an improved method for Background predicting response to the immune checkpoint blockade would Interleukin-8 (IL-8) is a chemokine expressed in multiple cancer types, better identify patients misclassified by conventional testing. We including NSCLC. It exerts various functions in shaping cancer have evaluated a proprietary algorithm which utilizes gene vascularization, cell dedifferentiation and inflammation/immunity. IL-8 expression in solid tumors to assess the presence of an immu- was described as a chemotactic factor for neutrophils and it has been nomodulatory (IM) signature intended to predict immunother- proposed to mediate recruitment of tolerogenic myeloid cells favoring apy response. The purpose of this study was to evaluate the a pro-tumorigenic microenvironment. Although clinical trials targeting performance of the IM signature against progression-free IL-8 are ongoing, its expression and role in NSCLC is unclear. survival (PFS) of patients treated with immune checkpoint Methods inhibitors. We developed a multiplexed quantitative immunofluorescence (QIF) Methods panel for simultaneous and localized measurement of IL-8, myeloperox- In this retrospective study, archival tumor tissue from metastatic idase (MPO), CD15, cytokeratin (CK) and DAPI. We analyzed the expres- lung cancer patients treated with one of three PD-(L)1 inhibitors sion of these markers and their association with PD-L1, CD4 and CD8- (pembrolizumab, nivolumab, and atezolizumab) either as a single positive cells in 3 retrospective NSCLC immunotherapy-naive cohorts agent or in conjunction with standard chemotherapy, from whom represented in tissue microarrays (cohort #1, n=262; #2, n=145; and #3, response data was available, was tested for the IM signature. n=132); 1 cohort of NSCLC patients treated with immune checkpoint Patients were stratified into two groups based on IM signature blockers (#4, n=59) and 1 collection of lung adenocarcinomas (LAC) an- classification as positive or negative, which was compared to im- alyzed for activating mutations in EGFR and KRAS (#5, n=121). We stud- munohistochemistry PD-(L)1 testing with a primary endpoint of ied the level of the targets, their distribution and association with one-year progression-free survival. Additionally, the IM signature immune features, clinicopathological variables and survival. classification was compared with objective response by Spear- Results man’s correlation as a continuous variable. IL-8 protein signal was detected in ~85% of cases with cytoplasmic Results staining pattern and was higher in tumor than in stromal cells. Elevated A total of 71 metastatic lung cancer patients were included in the tumor IL-8 was consistently associated with higher MPO+ neutrophils study with a median follow-up of 29 months. The one-year PFS haz- and CD15+ tumor-associated myeloid cells across the cohorts, but not ard ratio for the IM positive group was 0.31 (95% CI 0.14 to 0.68; with CD4+ and CD8+ T-cells. Increased IL-8 expression was not associ- p=.004 - Figure 1). A total of 62 out of the 71 metastatic lung cancer ated with major clinicopathologic variables. Elevated MPO+ and CD15+ patients had previous PD-L1 staining. Head-to-head analysis of PD-L1 cells was significantly higher in KRAS mutated than in EGFR mutated and IM signature on these patients found the one-year PFS hazard LACs. High MPO and CD15 signal was associated with shorter 5-year ratio for the IM group to be 0.30 (95% CI 0.13 to 0.71; p=0.006) and overall survival in all NSCLC cohorts. The negative prognostic effect of the one-year PFS hazard ratio for PD-L1 positive staining to be 0.76 MPO and CD15 was comparable in both immunotherapy-naïve and (95% CI 0.31 to 1.82; p=0.533). The mean IM correlation value with immunotherapy-treated NSCLC collections. objective response for PD = -0.06; SD = -0.04; PR = 0.14; CR = 0.33; p Conclusions Conclusions IL-8 protein is frequently expressed in NSCLCs associated with increased The IM signature was significantly associated with prolonged one- tumor-associated myeloid cells but independent from intratumor T-cell year progression-free survival among patients treated with PD-L1 in- responses. KRAS mutated LACs have prominent MPO+/CD15+ expres- hibitors while PD-L1 staining failed to be significantly associated. Pa- sion, supporting an immune suppressive role of myeloid cells in these tients classified positive by the IM signature demonstrated a three- malignancies. CD15 and MPO are prognostic markers in NSCLC and IL-8 fold improved hazard ratio compared to those who were negative. blockade could mediate favorable immunomodulatory effects. Funding was provided by Insight Genetics working in cooperation Ethics Approval with West Cancer Center and Research Institute. All tissues were used after approval from Yale Human Investigation Ethics Approval committee protocol #9505008219 which approved the patient con- This study was approved by the West Cancer Clinic Institutional Re- sent forms or waivers of consent view Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 78 of 272 above. The classifier delivered stratifications for response vs nonre- sponse with 84% accuracy, 79% sensitivity, 92% specificity, 75% posi- tive predictive value (PPV) and 95% negative predictive value (NPV). The associations of EpiSwitch™ response calls with overall survival (OS) and progressive free survival (PFS) in the independent cohort were significant (OS and PFS: log-rank p Conclusions The established EpiSwitchTM classifier contains strong binary markers of epigenetic deregulation with features normally attributed to gen- etic markers; the binary status of these classifying markers is statisti- cally significant for survival. Altogether, these findings highlight the potential of the EpiSwitchTM approach for identifying responders and non-responders to immuno-oncology therapies. Acknowledgements The authors would like to thank patients enrolled in the EMR000070-001 JAVELIN Solid Tumor trial for agreeing for their samples to be used for research purposes. This work is funded by Merck KGaA, Darmstadt, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. References 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- cer. Front Genet. 2016; 7:194. Fig. 1 (abstract P141). See text for description 2. Carini C, Hunter E; Scottish Early Rheumatoid Arthritis Inception Cohort investigators, et al. Chromosome conformation signatures define P142 predictive markers of inadequate response to methotrexate in early Development and validation of baseline predictive biomarkers for rheumatoid arthritis. J Transl Med. 2018; 16:18. response to avelumab in second-line (2L) non-small cell lung 3. Jakub JW, Grotz TE, Jordan P, et al. A pilot study of chromosomal cancer (NSCLC) using EpiSwitchTM epigenetic profiling aberrations and epigenetic changes in peripheral blood samples to 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , identify patients with melanoma. Melanoma Res. 2015; 25:406-11. 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan 4. Bastonini E, Jeznach M, Field M, et al. Chromatin barcodes as biomarkers 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem for melanoma. Pigment Cell Melanoma Res. 2014; 27:788-800. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir 5. Yan H, Hunter E, Akoulitchev A, et al. Epigenetic chromatin conformation 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins changes in peripheral blood can detect thyroid cancer. Surgery. 2019; 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, 165:44-49. Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; Ethics Approval Mayo Clinic, Rochester, MN, United States The protocol was approved by the institutional review board or independent Correspondence: Parantu Shah (parantu.shah@emdserono.com); ethics committee at each center. Matthew Salter (matthew.salter@oxfordbiodynamics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P142 P143 Development and validation of baseline predictive biomarkers for Background response to immuno-checkpoint treatments in the context of Development of baseline predictive classifiers for response to treatment multi-line and multi-therapy cohorts using EpiSwitchTM epigenetic can provide advantages for programs of targeted immunotherapies, profiling development of successful combination therapies, and identification of 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , responder populations to active therapies. Chromosome conformations 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan represent strong systemic cellular network deregulations associated 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem with differences in clinical phenotypes and outcomes [1]. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir Methods 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, business of Merck KGaA, Darmstadt, Germany/Pfizer alliance," has ap- Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; plied its proprietary technology EpiSwitchTM to monitor systemic Mayo Clinic, Rochester, MN, United States epigenetic biomarkers for chromosome conformation signatures in Correspondence: Parantu Shah (parantu.shah@emdserono.com) baseline blood samples of patients with multiline anti–PD-L1 (avelu- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P143 mab) treatment of NSCLC. This application was based on the pub- lished methodology for validated predictive biomarkers for response Background to treatment [2], systemic blood-based monitoring of oncological Development of baseline predictive classifiers for response to treatment conditions [3-5], and proprietary programs in collaboration with the can provide advantages for programs of targeted immunotherapies, Mayo Clinic for predictive and response biomarkers in melanoma pa- development of successful combination therapies, and identification of tients treated with anti–PD-1 therapy (pembrolizumab). responder populations to active therapies. Changes in chromosome Results conformations represent strong systemic cellular network deregulations A 14-marker classifier was generated with 12 avelumab-treated pa- associated with differences in clinical phenotypes and outcomes [1]. tients in each response group; in this cohort, responders were de- However, questions remain about the applicability of classifiers across fined as patients with complete or partial response, and non- treatment lines, indications, and drug combinations. responders were defined as patients with progressive disease. Valid- Methods ation of the developed predictive markers was performed on an in- Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a dependent cohort of 75 patients treated with avelumab as either business of Merck KGaA, Darmstadt, Germany/Pfizer alliance, has ap- first-line (1L) or 2L therapy. In the validation cohort, patients with plied its proprietary technology EpiSwitchTM to monitor systemic stable disease were also considered as responders in addition to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 79 of 272 epigenetic biomarkers for chromosome conformation signatures at Methods baseline in patients with multiline anti–PD-L1 (avelumab) treatment Tyramide signal amplification detection was used to inform on the of non-small cell lung cancer (NSCLC). Additionally, epigenetic bio- tumor/stroma/immune contexture (CD8, PanCK, FAP, MHC-I, CD31) and markers to predict outcome and response in patients with melanoma to characterize T-cell functions (PD1, CD3, PanCK, GZMB, and PD-L1). treated with anti–PD-1 (pembrolizumab) and its combination with Whole slide digital pathology scoring algorithms were developed to another agent were identified in collaboration with the Mayo Clinic. identify all phenotypes represented by the markers and their specificity Results and sensitivity was verified against the results by expert observers. Three NSCLC classifiers predicting response to avelumab in first-line Results (1L), second-line (2L), and combined 1L + 2L cohorts were built and ap- Assay performance including accuracy, precision, and sequential markers plied to test sets. Average accuracy, positive predictive value (PPV), and detection was validated on > 200 unique cases of Gastric, Pancreatic, negative predictive value (NPV) for 10-fold cross-validation on data Breast, Lung, Urothelial and Colorectal carcinomas. For an early proof-of- splits were reported. An NSCLC patient set treated with 2L pembrolizu- concept, we analyzed paired pre- vs. post treatment tumor biopsies from mab served as an independent test set. The 2L NSCLC classifier three pancreatic cancer patients treated with a combination of atezolizu- achieved high (defined hereafter as > 0.7) predictive power (PPV, NPV, mab and chemotherapy. Digital pathology algorithms identified biologic- and accuracy) in the 2L test set but not in the 1L test set. A reduced ally and clinically relevant features: MHC-I is highly expressed in tumor version of this classifier achieved a PPV of 0.71 in the 2L pembrolizu- cells of the primary lesions and very rare tumor cells express MHC-I in the mab population. The 1L classifier was not applicable in patients who re- liver met samples. A patient with partial response (PR) showed a signifi- ceived 2L treatment for NSCLC. The 1L + 2L composite classifier had cantly increased tumor MHC-I upon treatment, while two patients with high predictive power in both 1L and 2L cohorts and a high PPV for stable disease (SD) did not show significant changes. The PR patient also identifying responders in the 2L pembrolizumab population. A fourth showed an increased density of CD3+, CD8+, and GZMB+ T cells within classifier starting with preselected NSCLC markers had good predictive the tumor post-treatment, indicating an increased tumoral T cell infiltra- power for classifying responders in patients with melanoma treated tion and activation by the treatment. with pembrolizumab. Finally, a 2L NSCLC classifier trained to classify re- Conclusions sponse groups from pembrolizumab-treated patients also identified The automated 5-plex IHC assays and digital pathology algorithms NSCLC responders with a high PPV from patients treated with pembro- developed in this study provide a robust tool for quantitative and lizumab in combination with an epigenetic drug. spatially resolved whole-slide characterization of the tumor-immune Conclusions contexture. Applying these tools in large-scale clinical investigations Collectively, these results suggest that a set of EpiSwitchTM bio- may provide better understanding of the response/resistance mecha- markers correlates with outcome on anti–PD-1/PD-L1 immunother- nisms to cancer immunotherapies. apies. Classifier signatures could be generated to work across treatment lines, indications, and combinations, and could be helpful Cellular Therapies for baseline patient stratification. P145 Acknowledgements Expanding Iovance’s tumor infiltrating lymphocytes (TIL) from core The authors would like to thank patients enrolled in the EMR000070-001 biopsies for adoptive T cell therapy using a 22-day manufacturing JAVELIN solid tumor trial for agreeing to consent usage of samples for process research purposes. This work was funded by Merck KGaA, Darmstadt, Michelle Abelson, PhD, Kenneth D'Arigo, Florangel Hilton, Maria Fardis, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany PhD, MBA, Cecile Chartier and Pfizer Inc., New York, NY, USA. Iovance Biotherapeutics, Inc., Tampa Bay, FL, United States Correspondence: Cecile Chartier (cecile.chartier@iovance.com) References Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P145 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- Background cer. Front Genet. 2016; 7:194. Iovance’s TIL products Lifileucel and LN-145 have demonstrated re- Ethics Approval markable clinical activity in melanoma and cervical cancer utilizing The protocol was approved by the institutional review board or independent Iovance’s proprietary 22-day manufacturing process and surgically ethics committee at each enrolling center. resected tumor lesions ~ 1.5-cm diameter [1, 2]. Using a core needle biopsy procedure to obtain tumor samples could allow for greater convenience of collecting the tumor from patients [3]. We asked whether a streamlined manufacturing process could be implemented P144 to produce therapeutically relevant TIL from multiple histologies Quantification of tumor-stroma-immune contexture by multiplex starting with a core biopsy. fluorescent immunohistochemistry and whole-slide digital image Methods analysis 1 1 2 Core biopsies obtained from 4 melanoma and 3 pancreatic, 2 breast, Adriana Racolta, PhD , Mehrnoush Khojasteh, PhD , Jennifer Giltnane , 1 1 1 2 ovarian, and 1 lung tumors were processed in vitro, using a 22-day Antony Hubbard, BS , Hongjun Zhang , Miriam Matei , Jessica 1 1 1 expansion method termed ‘Core process’. Core biopsy-derived TIL Baumann , Wenjun Zhang, MD, PhD , Tsu-Shuen Tsao, PhD , Hartmut 2 2 1 1 1 were assessed for expansion, phenotype (lineage, youth/differenti- Koeppen , Lisa Ryner , Xingwei Wang , Jim Martin , Auranuch Lorsakul , 1 1 1 1 ation, activation, and exhaustion markers), function (IFN-gamma and Ilya Ravkin , Smadar Shiffman , Lidija Pestic-Dragovich , Lei Tang, PhD , CD107a mobilization), and TCR repertoire. Yulei Wang, BA PhD 1 2 Results Roche Tissue Diagnostics, Tucson, AZ, United States; Genentech, South Iovance’s Core process successfully generated TIL products from all San Francisco, CA, United States tested samples. One to 2 cores yielded more than 10e9 T cells for 10 Correspondence: Yulei Wang (wang.yulei@gene.com) of the 12 preparations. Phenotypic analyses revealed no significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P144 differences in terms of T cell lineages and memory subsets, or ex- pression of activation, differentiation, and exhaustion markers when Background compared to Iovance’s current products. Core-derived TIL products Understanding response to immunotherapies in relation to tumor- responded to PMA and to anti-CD3 stimulations by inducing levels of immune contexture requires a paradigm shift from a single-marker CD107a mobilization and IFN-gamma secretion like those produced test towards multiplexed immunohistochemistry (IHC). Here we re- by TIL derived from excisional biopsies. Preliminary TCR sequencing port the development, early proof of concept of two fully automated data suggest that high-diversity products can be also be obtained 5-plex fluorescent multiplex IHC assays and accompanying digital from small samples, similar to what is obtained from TIL expansion. pathology algorithms. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 80 of 272 Conclusions observed compared with single transduced CAR T-cells (product A or This work demonstrates that the Iovance 22-day Core manufacturing B). Expression of the IL7R_CCR in both AUTO6NG and product A con- method is highly robust and that it is feasible to expand TIL to thera- ferred exogenous-cytokine-independent viability and homeostatic peutically relevant numbers from as little as 1 to 2 core biopsies from proliferation of modified T-cells, without causing autonomous T-cell multiple histologies with this method. Resulting products were shown growth. Furthermore, AUOTO6NG T-cells and product B but not to be phenotypically comparable to, and as potent as, products gener- product A proved resistant to both TGFb- and PD1/PDL1-mediated ated with Iovance’s process from excisional biopsy. Iovance anticipates immunosuppression in vitro due to the presence of dnTGFbRII and implementing this process in the clinic in the near future. dSHP2 in those genetically engineered CAR T-cells. Finally, intraven- ous delivery of AUTO6NG exhibited potent anti-tumour activity and References extended survival in NSG mice with established tumour burden. 1. Jazaeri AA, Zsiros E, Amaria RN, Artz AS, Edwards RP, Robert Michael Conclusions Wenham RM, et al. Safety and efficacy of adoptive cell transfer using These results demonstrate the feasibility, safety, and efficacy of autologous tumor infiltrating lymphocytes (LN-145) for treatment of AUTO6NG T-cells. The addition of IL7R_CCR, dnTGFbRII and dSHP2 recurrent, metastatic, or persistent cervical carcinoma. Clin Oncol. modules to the AUTO6NG product augment its functions by extend- 2019;37:15:2538 (suppl). ing T-cell persistence and rendering modified T-cells resistant to 2. Sarnaik A, Khushalani NI, Chesney JA, Kluger HM, Curti BD, et al. Safety TGFb- and PD1/PDL1-driven immune inhibition. and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients P147 who progressed on multiple prior therapies including anti-PD-1. J Clin Effect of chemotherapy on cellular kinetics of NKG2D-based CAR T- Oncol. 2019;37:15:2518 (suppl). cells in metastatic colorectal cancer patients 3. Ullenhag GJ, Sadeghi AM, Carlsson B, Ahlström H, Mosavi F, Wagenius G, Erik Marcelo Alcantar Orozco, Eytan Breman, MSc, Marie-Sophie Dheur, Tötterman TH, et al. Adoptive T-cell therapy for malignant melanoma pa- PhD, Fabian Borghese, PhD, Emilie Cerf, PhD, Nathalie Braun, Caroline tients with TILs obtained by ultrasound-guided needle biopsy. Cancer Lonez, PhD, Anne Flament, Frederic Lehmann, MD Immunol Immunother. 2012;61:725–732. Celyad, Mont-Saint-Guibert, Belgium Correspondence: Frederic Lehmann (flehmann@celyad.com) P146 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P147 AUTO6NG: Next generation GD2-targeting CAR T-cell therapy with improved persistence and insensitivity to TGFb and checkpoint Background inhibition for relapsed/refractory neuroblastoma Autologous and allogeneic Chimeric Antigen Receptor (CAR) T-cells are 1 1 1 Daniela Achkova, PhD , Adrian Zarzoso , Yusuf Demir , Fernando under thorough investigation to translate their success in B-cell malig- 1 1 1 1 Gallardo , Maria Stavrou , Marco Della Peruta , Saket Srivastava , Mathew nancies to other types of cancer. Previous studies associated the anti- 1 1 1 1 Robson , Shimobi Onuoha , Simon Thomas , Shaun Cordoba , Martin tumour effect of CAR T-cells to their long-term persistence. Most stud- 1,2 Pule ies use cyclophosphamide and fludarabine (CyFlu) preconditioning 1 2 Autolus Ltd, London, United Kingdom; University College London chemotherapy to facilitate CAR T-cell persistence. However, the effect Cancer Institute, London, United Kingdom of CyFlu preconditioning was rarely compared to other chemotherapies Correspondence: Martin Pule (m.pule@autolus.com) or to CAR T-cells alone. The THINK, SHRINK and ALLOSHRINK trials Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P146 evaluate the safety and clinical activity of NKG2D receptor-based CAR T-cells in metastatic colorectal cancer (mCRC) patients. THINK and Background SHRINK utilize autologous CAR T-cells, whereas ALLOSHRINK utilizes Neuroblastoma is the most common extracranial solid cancer in chil- allogeneic CAR T-cells. In THINK, CAR T-cells are injected without pre- dren with poor long-term survival in those with high-risk disease. A conditioning chemotherapy or after CyFlu. In SHRINK and ALLOSHRINK, currently ongoing phase I clinical study of GD2-targeted CART for re- FOLFOX chemotherapy is given before CAR T-cell injections. Herein we fractory/relapsed neuroblastoma (NCT02761915) shows activity present cellular kinetics results from these three trials. against disseminated disease without inducing on target/off tumor Methods toxicity. However, CART persistence was limited and clinical activity Whole blood samples were drawn at various timepoints from pa- transient and incomplete. tients receiving at least one injection of CAR T-cells. Peripheral blood Building on the GD2 CAR used in this study, we have developed a next mononuclear cells (PBMCs) were isolated by ficoll gradient centrifu- generation T-cell product candidate termed AUTO6NG. The AUTO6NG gation at a central laboratory designated by the Sponsor. Genomic product consists of 3 distinct populations of GD2-targeted CAR T-cells, DNA was isolated using a commercially available kit. Engraftment of produced by dual transduction of T-cells with two separate retroviral CAR T-cells was measured by digital droplet polymerase chain reac- vectors. The first vector directs the expression of a GD2-targeting CAR, tion (ddPCR) using transgene-specific primers and reported as trans- co-expressed with a constitutively signalling IL7 cytokine receptor gene copies per microgram of genomic DNA. Long-term persistence (IL7R_CCR) (product A), while the second vector is a tri-cistronic retro- of CAR T-cells was measured by calculating the area under the curve viral vector encoding the same GD2 CAR, co-expressed with dominant (AUC) using the linear trapezoidal rule. negative TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). Results dSHP2 confers resistance to inhibitory signals such as those from PD1. 35 mCRC patients have been treated in THINK (14), SHRINK (9) and Methods ALLOSHRINK (12). Preliminary results are available for 29 subjects. Cell Human T-cells were either dual transduced with both vectors yield- kinetics for subjects having received one injection of autologous CAR T- ing a mix of product A/B/A+B (AUTO6NG) or single transduced with cells show a seven-fold increase in mean peak levels of T-cell engraft- each vector individually giving raise to product A or B. Both single ment with CyFlu compared to FOLFOX. Mean AUC is four times higher and dual transduced CAR T-cells were extensively evaluated in vitro with CyFlu compared to FOLFOX. Peak levels of engraftment and per- for redirected lysis, cytokine secretion, T-cell proliferation and survival sistence observed with FOLFOX and without previous chemotherapy and resistance to immunosuppressive pathways (including TGFb and are similar. Additionally, allogeneic CAR T-cells exhibit a five-fold in- PD1/PDL1 inhibition) in co-culture assays with GD2-positive and crease in mean AUC and a ten-fold increase in mean peak levels com- negative tumour cell lines. Additionally, anti-tumour activity of pared to autologous cells with the same prior chemotherapy regimen. AUTO6NG was evaluated in vivo by intravenous administration in an Additional analyses will be presented during the congress. established neuroblastoma xenograft model in NSG mice. Conclusions Results Analyses of the initial 29 patients receiving either autologous or allo- AUTO6NG T-cells (product A/B/A+B) were highly potent in cytotox- geneic NKG2D-based CAR T-cells demonstrate that CyFlu enhances icity assays against GD2 positive tumour cell lines with no differences peak levels and persistence of adoptively transferred cells. FOLFOX Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 81 of 272 does not appear to influence engraftment or persistence of CAR T-cells. P149 Allogeneic CAR T-cells show higher peaks and time-averaged persist- Silencing PD-1 using self-delivering RNAi PH-762- to improve ence compared to autologous cells. Analysis of the results is ongoing. Iovance TIL effector function using Gen 2 manufacturing method 1 1 Ethics Approval Inbar Azoulay-Alfaguter, PhD , Michelle Abelson, PhD , Krit Ritthipichai, 1 1 1 2 The studies referred to in this abstract were approved by all relevant DVM, PhD , Kenneth D’Arigo , Florangel Hilton , Marcus Machin, BS , 2 2 1 1 ethical committees and authorities. Dingxue Yan , James Cardia , Maria Fardis, PhD, MBA , Cecile Chartier 1 2 Iovance Biotherapeutics, Inc., Tampa, FL, United States; Phio Pharmaceuticals, Tampa, FL, United States P148 Correspondence: Cecile Chartier (cecile.chartier@iovance.com) High affinity NK cells expressing a PD-L1 chimeric antigen receptor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P149 demonstrate anti-tumor activity in head and neck cancer through multiple distinct mechanisms Background 1 1 1 Yevtte Robbins , Jay Friedman, PhD , Sarah Greene , Kellsye Fabian, Adoptive T-cell transfer with tumor infiltrating lymphocytes (TIL) 1 1 2 2 PhD , Michelle Padget , John Lee, MD , Patrick Soon-Shiong, MD , is an investigational immunotherapy for advanced solid cancers. 3 2 2 1 Kayvan Niazi , Lennie Sender , Laurent Boissel , Jeffrey Schlom, PhD , Ongoing Phase II clinical trials of Iovance’s lifileucel and LN-145 1 1 James Hodge, PhD, MBA , Clint Allen, MD TIL products have demonstrated efficacy with ORRs of 38% and 1 2 NIH, Bethesda, MD, United States; NantKwest, Culver City, CA, United 44% in patients with melanoma and cervical cancer, respectively 3 4 States; Nantworks, Culver City, CA, United States; NIH/NIDCD, Bethesda, [1,2]. Anti-PD-1 therapy has been widely used as a first-line ther- MD, United States apy in several types of cancer. TIL infusion products from the pa- Correspondence: Clint Allen (clint.allen@nih.gov) tients previously treated with anti-PD-1 therapy still sustain PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P148 expression, especially the subset of tumor antigen-specific TIL [3]. Building on the therapeutic efficacy of PD-1 blockade, we rea- Background soned that intrinsic silencing of PD-1 in our TIL products, may A significant portion of head and neck cancers (HNCs) harbor gen- provide similar benefits to systemic administration of anti-PD-1 omic alterations that render them insensitive to T cell detection. For therapy, while decreasing the side effects associated with sys- these patients, natural killer (NK) cellular therapy may be an effective temic anti-PD-1 [3]. Self-delivering small interfering RNA (sd- complementary treatment approach. We studied the anti-tumor ac- rxRNA) is a chemically modified siRNA molecule, which has ability tivity of a novel, off the shelf, NK cellular therapy consisting of high to penetrate cell types with high knockdown efficiency of specific affinity NK cells engineered to express a chimeric antigen receptor target genes [4]. Furthermore, a knockdown approach yields a (CAR) targeting PD-L1 (PD-L1 t-haNKs). transient effect, which may prove a more favorable approach Methods when compared with permanent genetic modification. Here, we Irradiated (15 Gy) PD-L1 t-haNK cells were assessed for direct cytotox- tested the silencing efficiency of a PD-1-targeted sd-rxRNA, icity of five human and two murine HNC cell lines by real-time imped- termed PH-762, in TIL and its effect on TIL phenotype and ance analysis. PD-L1 knockout by CRISPR/Cas9 gene editing was function. performed in select cells to assess PD-L1-specific killing. Co-culture as- Methods says with PD-L1 t-haNKs and murine or human peripheral and tumor TIL from melanoma, breast cancer, lung cancer, H&N cancer, infiltrating leukocytes were performed to determine selective elimin- and sarcoma were expanded ex vivo with Iovance’s proprietary ation of cells. Wild-type C57BL/6 (B6) or NSG mice were engrafted with 22-day process in the presence of PH-762. Resulting TIL parental or PD-L1 knockout murine or human tumors and assessed for products were assessed for PD-1 knockdown, cell expansion tumor growth inhibition (TGI) following PD-L1 t-haNK treatment. and viability, phenotype (T-cell lineage, differentiation, activa- Results tion, and exhaustion), and effector functions (IFN-gamma PD-L1 CAR expression on PD-L1 t-haNKs was verified. PD-L1 t-haNKs induction). killed all human and murine HNC cell lines at low effector:target ra- Results tios. Killing of cells was significantly enhanced with increased PD-L1 Average silencing of the PD-1 levels was 85%. Sixteen of the 19 expression following IFN-γ pre-treatment. Baseline killing was par- tumors tested demonstrated >80% silencing at the surface of tially reversed and IFN-γ -enhanced killing was completely abrogated PH-762-treated TIL relative to control sd-rxRNA-treated TIL. The in PD-L1 knockout cells. Ex vivo co-culture of PD-L1 t-haNKs with per- remaining 3 samples had ~70% silencing efficiency. Expression ipheral and tumor infiltrating leukocytes from tumor bearing mice or of T-cell activation markers including 4-1BB and OX40 was sig- with peripheral leukocytes from HNC patients revealed selective elim- nificantly increased in TIL expanded with PH-762. Importantly, ination of PD-L1 high macrophages and myeloid derived suppressor other inhibitory and exhaustion molecules remained unaffected, cells (MDSC) but not lymphocyte subsets. Treatment of B6 mice bear- suggesting that compensatory mechanisms were not triggered ing murine oral cancers with PD-L1 t-haNKs in vivo resulted in ≥50% by PD-1 silencing. Functionally, PD-1 knockdown TIL displayed reduction in PD-L1 high macrophages and MDSC but no reduction in elevated IFN-gamma secretion when co-cultured with autolo- lymphocytes. Treatment of NSG mice bearing parental human HNC gous tumor cells, indicating improved effector function upon or B6 mice bearing parental murine oral cancer resulted in significant specific T-cell re-stimulation. TGI after PD-L1 t-haNK treatment. TGI was completely abrogated in Conclusions mice bearing PD-L1 knockout tumors. sd-rxRNA-mediated silencing of PD-1 with PH-762 in TIL was Conclusions highly efficient and generated TIL products with elevated PD-L1 t-haNKs mediated potent PD-L1-specific cytotoxicity against effector function, providing a strong rationale for clinical HNC cells and selectively eliminate immunosuppressive macrophages testing. and MDSC expressing high levels of PD-L1 from the periphery and tumor microenvironment. PD-L1 t-haNK monotherapy resulted in PD- Acknowledgements L1-specific TGI in xenograft and syngeneic models. These data pro- PH-762 was kindly provided by Phio Pharmaceuticals. vide the pre-clinical rationale for the clinical study of PD-L1 t-haNKs in solid tumors. Evidence that PD-L1 t-haNKs selectively eliminate im- References munosuppressive macrophages and MDSC support the clinical study 1. Sarnaik A. et al. Safety and efficacy of cryopreserved autologous tumor of PD-L1 t-haNKs as a monotherapy or in combination with treat- infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic ments designed to activate T cell immunity. melanoma patients who progressed on multiple prior therapies Ethics Approval including anti-PD-1. J Clin Oncol. 2019;37:2518-2518. The study was approved by the NIH Animal care and Use Committee, 2. Jazaeri A A, et al. Safety and efficacy of adoptive cell transfer using approval number 1464-18. autologous tumor infiltrating lymphocytes (LN-145) for treatment of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 82 of 272 recurrent, metastatic, or persistent cervical carcinoma. J ClinOncol. 2019;37:2538-2538. 3. Gros A, et al. PD-1 identifies the patient-specific CD8(+) tumor- reactive repertoire infiltrating human tumors. J Clin Invest. 2014;124:2246-2259. 4. Ligtenberg M A, et al. Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malig- nant Melanoma. Mol Ther. 2018;26:1482-1493. P150 1st-in-human CAR T clinical trial for metastatic breast cancers Cynthia Bamdad, PhD , Andrew Stewart, PhD, Pengyu Huang, PhD, Benoit Smagghe, PhD, Scott Moe, PhD, Tyler Swanson, Thomas Jeon, Danica Page, Ketan Mathavan, PhD, Trevor Grant, PhD, Rachel Herrup Minerva Biotechnologies, Waltham, MA, United States Correspondence: Cynthia Bamdad (cbamdad@minervabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P150 Background Minerva will open a 1st-in-human CAR T clinical trial for meta- static breast cancers at the Fred Hutchinson Center September, 2019. huMNC2-CAR44 targets a novel form of MUC1; no thera- peutic that targets this form has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 Fig. 1 (abstract P150). huMNC2-CAR44 T cells kill MUC1* have targeted the tandem repeat domains, which are cleaved positive tumors and shed from the surface of cancer cells. Cleavage and shed- ding of the tandem repeat domain increases as tumor stage in- creases. huMNC2-CAR44 targets the truncated extra cellular P151 domain of MUC1* (muk 1 star), also known as MUC1-C, which is Solid tumor cytotoxicity by natural killer cells expressing a HER2- the transmembrane cleavage product that remains after MUC1 directed chimeric antigen receptor enhanced by MyD88/CD40 (MC) is cleaved and the tandem repeat domain is shed from the can- Xiaomei Wang, PhD, Daniel Jasinski, PhD, Jan Medina, David Spencer, cer cells. The MNC2 antibody, which is the targeting head of PhD, Aaron Foster, PhD, Joseph Bayle, PhD the CAR, cannot bind to full-length MUC1. It binds to an ectopic Bellicum Pharmaceuticals, Houston, TX, United States epitope that is only unmasked by cleavage and release of the Correspondence: Aaron Foster (afoster@bellicum.com); Joseph Bayle MUC1 tandem repeat domain. MUC1* growth factor receptor is (jhbayle@bellicum.com) activated when onco-embryonicgrowth factorNME7ABdimer- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P151 izes its truncated extracellular domain. NME7AB and the huMNC2 antibody both competefor thesamebinding site, Background which is masked in full-length MUC1. The potent, innate anti-tumor cytotoxicity of natural killer (NK) cells Methods combined with their low risk of inducing graft-versus-host disease Monoclonal antibody MNC2 was selected because it recognizes have made NK cells an emerging platform for allogeneic, off-the- a conformational epitope within MUC1* that is created by cleav- shelf CAR-based cell therapies. However, adoptive transfers of NK age by MMP9, which is overexpressed in breast cancers and is cells have shown limited expansion and persistence which may im- an indicatorofpoorprognosis. The luminal edge of some nor- pact their ability to induce durable anti-tumor responses. Here, we mal tissues express a cleaved MUC1*-like form; however, on demonstrate that constitutive expression of a novel chimeric costi- normal tissues, MUC1 is cleaved by a different cleavage enzyme, mulatory protein, comprised of the signaling domains from MyD88 which alters the conformation of the truncated extra cellular and CD40 (MC) and secreted IL-15 dramatically improves the prolifer- domain and it is not recognized by the MNC2 antibody. ation and anti-tumor efficacy of HER2 CAR-redirected NK cells. Results Methods huMNC2-scFv recognizes 95% of breast cancers, across all subtypes, Human CD56-positive cells were enriched from PBMCs derived from wherein the average percent staining for each tissue specimen is healthy donors and activated with irradiated K562 cells in the presence ~80%. Despite this robust staining of cancerous tissues, huMNC2- of IL-15. NK cells were subsequently transduced with retroviral vector en- scFv showed almost no binding to normal tissues and no staining of coding inducible Caspase-9 (iC9), a HER2-specific CAR (HER2.ζ), MyD88/ critical organs. In vitro, huMNC2-CAR44 T cells killed cancer cells, but CD40 (MC) [1] and IL-15. Gene-modified NK cells were evaluated for ex- not non-cancer cells even if they expressed MUC1 or a cleaved pansion, cytotoxicity, cell phenotype and cytokine production, in vitro MUC1. In NSG mice (n>300), huMNC2-CAR44 T cells eliminated and in an HER2 positive OE-19 NSG mouse xenograft model. MUC1* positive tumors from implanted naturally occurring breast Results cancer cells. A single CAR T cell injection eliminated tumors for 100 NK cells were efficiently transduced (>50%) and demonstrated robust days; control animals had to be sacrificed at Day 20. Further, ex vivo expansion (150 fold, 14 days post-activation) in culture rela- huMNC2-CAR44 T cell mediated killing increased as MUC1* density tive to transduced NK cells. In coculture assays with HER2-expressing increased (Figure 1). OE19 and SKOV3 tumor cells, MC-enhanced CAR-NK cells showed po- Conclusions tent cytotoxicity with elevated expression of pro-inflammatory cyto- If successful, huMNC2-CAR44 could treat a wide variety of solid kines and chemokines including MIP1α, IFN-γ, and GM-CSF. In tumors. huMNC2-scFv binds to 95% of breast, 83% ovarian, 78% addition, NK cells expressing the iC9 safety switch could be rapidly pancreatic and 71% of lung cancers. ablated by treatment with 1 nM rimiducid to initiate apoptosis. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 83 of 272 animals engrafted with OE-19 tumor cells, iC9-CAR.ζ-MC-IL15 modi- Conclusions fied NK cells demonstrated significantly improved control of tumor Through the direct cytosolic delivery of antigen, we engineered expansion compared with control NK cells. unfractionated PBMCs to function as potent APCs. This strategy has Conclusions demonstrated significant potential to generate CD8+ T cell responses MyD88/CD40 and IL-15 enhance the proliferation and anti-tumor po- in both mouse and human systems and has been scaled for clinical tency of CAR-modified NK cells. Further, inclusion of the iC9 safety implementation. switch can be used to mitigate potential toxicities. These technolo- Ethics Approval gies have the potential to provide a potent, off-the-shelf allogeneic Human samples were supplied by an approved vendor and animal cell therapy to treat solid tumors. studies were conducted in accordance with SQZ Biotech's Animal Care Program and IACUC which operate according to principles set Reference forth in PHS Policy and the Guide for the Care and Use of Laboratory 1. Collinson-Pautz MR, Chang WC, Lu A, Khalil M, Crisostomo JW, Lin PY, Animals - 8th edition. Mahendravada A, Shinners NP, Brandt ME, Zhang M, Duong M, Bayle JH, Slawin KM, Spencer DM, Foster AE. Constitutively active MyD88CD40 costimulation enhances expansion and efficacy of chimeric antigen re- P153 ceptor T cells targeting hematological malignancies. Leukemia. 2019; Memory CD8+ T cells are more resistant to cancer stem cell (CSC) 33:2195-2207. suppression than effector CD8+ T cells and are more effective at Ethics Approval targeting CSC in a murine melanoma model This study was approved by Bellicum's IACUC and performed in its AAALAC 1 2 2 2 Brooke Bredbeck, MD , Shibin Qu , Alicia Kevelin , Ashley Pepple , Amy approved vivarium. 1 2 1 Felsted , Anutosh Ganguly , Clifford Cho, MD, FACS University of Michigan Medical School, Ann Arbor, MI, United States; P152 Ann Arbor VA Medical Center, Ann Arbor, MI, United States Antigen delivery to PBMCs by microfluidic squeezing primes anti- Correspondence: Clifford Cho (cliffcho@med.umich.edu) tumor immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P153 Matthew Booty, PhD, Kelan Hlavaty, Emrah Ozay, PhD, Carolyne Smith, PhD, Katherine Seidl, PhD, Howard Bernstein, MD, PhD, Armon Sharei, Background Scott Loughhead The ability to suppress immune reactivity is a defining hallmark SQZ Biotechnologies, Watertown, MA, United States of cancer [1-3]. Both the administration and disinhibition of Correspondence: Matthew Booty (matt.booty@sqzbiotech.com) CD8+ T cells, through adoptive immunotherapy and checkpoint Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P152 inhibition respectively, have yielded unprecedented responses in patients with advanced melanoma [4-8]. However, a majority Background of patients remain stubbornly unresponsive to T cell-based The presentation of sufficient antigen on major histocompatibility therapy [9,10]. A better knowledge of cancer-induced T cell sup- complex class I (MHC-I) is a potential barrier to generating potent pression is needed improve efficacy. Memory CD8+ T cells cancer immunizations. We use microfluidics-based squeezing to de- (Tmem) are more effective than effector CD8+ T cells (Teff) at liver antigen directly to the cytosol of target antigen presenting cells controlling melanoma growth after adoptive cell transfer (ACT) (APCs) – resulting in the enhanced presentation of antigen on MHC-I. in a murine melanoma model [11,12]. Melanoma cancer stem In addition to facilitating potent CD8+ T cell priming by professional cells (CSC) are primarily responsible for tumor growth and APCs, this approach can make unfractionated peripheral blood metastasis [13,14]. We hypothesized that Tmem are both more mononuclear cells (PBMCs) effective, unorthodox APCs capable of resistant to CSC suppression and more effective at targeting priming CD8+ T cell responses in mouse and human systems. CSC after ACT. Methods Methods Protein and peptide antigens were delivered to the cytosol of murine The B16F10 melanoma cell line was stably transfected to ex- splenocytes or human PBMCs by microfluidic squeezing. The re- press low levels of lymphocytic choriomeningitisvirus(LCMV) sponse to in vivo immunization was assessed by flow cytometry in a peptide antigen GP33 (B16GP33). Ly5.1+/C57BL/6 mice were in- series of experiments in mice. Tumor experiments were conducted fected with LCMV to isolate Teff and Tmem on post-infection with the TC-1 cell line, which expresses the viral antigens E6 and E7 days 8 or > 30, respectively. Ly5.2+/C57BL/6 mice were inocu- from human papilloma virus type 16 (HPV16). lated with subcutaneous B16GP33 tumors followed by either no Human PBMCs were loaded with synthetic long peptides (SLPs) con- treatment or ACT with Teff or Tmem on days 1 or 7. On day taining MHC-I restricted epitopes from cytomegalovirus (CMV) or 18-20, tumors were harvested for flow cytometric analysis HPV16. These PBMCs were co-cultured with epitope-reactive human (FACS) to characterize tumor-infiltrating lymphocytes (TIL) and responder CD8+ T cells, and interferon gamma production was quan- composition of melanoma CSC versus non-CSC (NCSC) based on tified to assess antigen-specific responses in vitro. expression of the CSC-specific marker aldehyde dehydrogenase Results (ALDH). In mice, we demonstrate that microfluidic squeezing enables delivery to Results all cell subsets within the spleen and that delivered protein antigen is Tumor inhibition was observed after ACT, with greatest treatment rapidly processed and presented on MHC-I. In vivo immunization using effect found after Tmem ACT (Figure 1). FACS analysis of CD8+ splenocytes squeezed with a HPV16-derived E7 SLP primes E7-specific re- TIL showed a predominant exhausted and non-activated pheno- sponses. Prophylactic immunization of mice implanted with TC-1 resulted type after Teff ACT; in contrast, CD8+ TIL exhibited a highly acti- in complete protection and these responses were durable, as mice were vated phenotype as well as superior endogenous CD8+ T cell protected upon TC-1 re-challenge. Therapeutic immunization following recruitment (Figure 2) after Tmem ACT. FACS analysis of tumor TC-1 implantation reduced tumor growth and extended survival com- cells after ACT demonstrated that ALDHhigh CSC fractions were pared to unimmunized mice (25 days vs 50 days). Following therapeutic markedly expanded after Teff ACT, but diminished after Tmem immunization, 85% of tumor infiltrating CD8+ T cells were found to be ACT (Figure 3). E7-specific compared to 3% in unimmunized mice. Conclusions In human cells, we demonstrate that squeezing of primary PBMCs en- Tmem-based ACT resulted in optimal tumor growth suppression, a ables delivery to all cell subsets. Delivery of CMV and HPV16 SLPs leads more activated TIL phenotype with superior CD8+ T cell recruitment, to presentation on MHC-I, as demonstrated by in vitro responses of and substantially stronger clearance of CSC compared to Teff ACT both CD8+ T cell clones and patient-derived memory populations. De- and controls. These observations suggest that use of Tmem may en- livery of CMV antigens at the manufacturing scale (~1 x 10^9 cells) also able cellular therapies to more effectively evade the suppressive ef- results in presentation and activation of CD8+ T cells. fects of melanoma while selectively targeting CSC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 84 of 272 References 1. Marincola F, Wang E, Herlyn M, et al. Tumors as elusive targets of T-cell- based active immunotherapy. Trends Immunol. 2003; 24:335-342. 2. Dunn GP, Old LJ, et al. The three E’s of cancer immunoediting. Ann Rev Immunol. 2004; 22:329-360. 3. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Ann Rev Immunol. 2007; 25:267-295. 4. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell trans- fer immunotherapy. Clin Cancer Res. 2011; 17:4550-4557. 5. Dudley MS, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy folloing non-myeloablative but lymphodepleting chemotehrapy for the treatemnt of patients with refractory metastatic melanoma. J Clin Oncol. 2005; 23:2346-2357. 6. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711-723. 7. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimu- mab or monotherapy in untreated melanoma. N Engl J Med. 2015; 373:23-34. 8. Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013; 369:122-33. 9. Brahmer JR, Tykodi SS, Chow LQM, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366:2455-2465. 10. Royal RE, Levy C, Turner K, et al. Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J Immunother. 2010; 33:828-33. Fig. 2 (abstract P153). CD8+ TIL exhibit a more activated 11. Contreras A, Sen S, Tatar AJ, et al. Enhanced local and systemic anti- phenotype after memory ACT melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice. Cancer Immunol Immunother. 2016; 65:601-611. 12. Contreras A, Beems MV, Tatar AJ, et al. Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive melanoma im- munotherapy in a mouse model. J Immunother Cancer. 2018; 6:41. 13. Maccalli C, DeMaria R. Cancer stem cells: perspectives for therapeutic targeting. Cancer Immunol Immunother. 2015; 64:91-97. 14. Pan Q, Li Q, Liu S, Ning N, Zhang X, Yu Y, Chang AE, Wicha MS. Concise review: targeting cancer stem cells using immunological approaches. Stem Cells. 2015; 33:2085-2092. Ethics Approval This study was approved by University of Michigan’s ethics board (IACUC), approval #1608-004. Fig. 3 (abstract P153). Memory CD8+ T cells target melanoma CSC P154 Development of an antigen-presenting bead kit for activation and expansion of human antigen-specific T cells Yelena Bronevetsky, PhD (yelena.bronevetsky@gmail.com) Berkeley Lights Inc, Alameda, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P154 Background Immunogenicity validation of peptide neoantigens represents a crit- ical bottleneck in the tumor antigen discovery process. Current bio- informatics platforms for antigen prediction are unsatisfactory, forcing researchers to screen many peptides per protein target in order to identify the few bona fide antigens. Standard immunogen- icity assays also cannot discriminate antigen-Human Leukocyte Anti- Fig. 1 (abstract P153). Memory CD8+ T cells vs effector CD8+ T gen (HLA) binding from T cell receptor (TCR) recognition, leading to cells in melanoma unnecessary screening of non-HLA binders in expensive and lengthy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 85 of 272 T cell reactivity assays that require large numbers of expensive pri- products were stimulated ex vivo with a T cell-specific superantigen. mary cells. Further, the use of donor-derived antigen-presenting cells Dogs received ACT of ex vivo-activated T cells followed by five sub- to assay T cell immunogenicity and expand rare, antigen-specific cutaneous IL-2 injections. Dogs were monitored for development of cells from the endogenous repertoire has inherent variability and a metastases via thoracic radiographs every three months. minimal degree of quality control. Berkeley Lights has developed an Results artificial antigen-presenting bead kit that expands antigen-specific T All 14 patients received autologous vaccinations. Due to early metas- cells from peripheral blood. tasis, 11 dogs received ACT. One dog did not receive adjuvant IL-2; Methods ten dogs completed the entire protocol. Toxicity was minimal after Peptide binding to HLA Class I and stability of the peptide-HLA com- pre-medicants (NSAID, antihistamine, and antiemetic) were instituted plex is assayed by loading peptides onto beads and staining with prior to ACT. With premedication, all toxicities were VCOG grade I/II. antibody. Following validation of peptide-HLA binding, primary CD8+ Median disease-free interval for all dogs was 213 days. Median sur- T cells from peripheral blood are stimulated by antigen-presenting vival time (MST) for all dogs was 415 days. Five dogs have survived beads twice over the course of two weeks. Frequencies of antigen- for over 621 days and are disease-free (Figure 1.) In addition, the re- specific T cells in the resulting cells is assayed by tetramer staining. sults included at least one dog with complete regression of distant Antigen-specific T cells can be loaded onto the Berkeley Lights (BLI) macroscopic metastasis. Lightning platform, a novel microfluidic platform that enables thou- Conclusions sands of single cell experiments in parallel. On the BLI Optoselect This immunotherapy protocol is safe and tolerable. Compared to chip, IFNg secretion and CD137 upregulation of antigen-specific T MST for historical amputation alone with or without adjuvant chemo- cells is assayed in response to antigenic stimulation. Following ana- therapy (MST of 307(1) and 134(2) days, respectively), a significant lysis, single cells can be exported for further analysis. survival benefit is noted in this group of patients. Further prospective Results studies are warranted to gain additional immunologic insight to the Berkeley Lights has developed an artificial antigen-presenting bead protocol, further improve disease response and survival, and evaluate that expands antigen-specific T cells from peripheral blood 10 times the translational impact this treatment could have in advancing hu- more effectively than autologous dendritic cells. This system allows man medicine. users to load peptides of choice onto magnetic beads and use them to assay peptide-HLA binding and stability, and to efficiently stimu- References late and expand antigen-specific T cells. Finally, in conjunction with 1. Phillips B, Powers BE, Dernell WS, Straw RC, Khanna C, Hogge GS, Vail the Berkeley Lights Lightning platform and the T cell Phenotype and DM. Use of single-agent carboplatin as adjuvant or neoadjuvant therapy Functional Analytics workflow, multiple functional parameters can be in conjunction with amputation for appendicular osteosarcoma in dogs. assayed from as few as 1000s of T cells, linking peptide-HLA binding J Am Anim Hosp Assoc. 2009; 45:33-8. and recognition to antigen-specific effector function. 2. Spodnick GJ, Berg J, Rand WM, Schelling SH, Couto G, Harvey HJ, Henderson RA, MacEwen G, Mauldin N, McCaw DL, Moore AS, Morrison W, Norris AM, O’Bradovich, J, O’Keefe DA, Page R, Ruslander D, Klausner J, Straw R, Thompson JP, Withrow SJ. Prognosis for dogs with appendicular P155 osteosarcoma treated by amputation alone: 162 cases (1978-1988). J Am Prospective translational study evaluating vaccine-enhanced Vet Med Assoc. 1992; 200(7):995-9. adoptive T cell therapy for treatment of osteosarcoma in Ethics Approval companion dogs The study was approved by University of Missouri’s IACUC, approval number 1 1 Tammie Wahaus, BSBA , Noe Reyes, DVM , Jeffrey Bryan, DVM, MS, PhD, 2 2 DACVIM-Oncology , Jeffrey Bryan, DVM, MS, PhD, DACVIM-Oncology , 3 2 Gary Wood, PhD , Brian Flesner, DVM, MS, DACVIM-Oncology , Lindsay 2 2 Donnelly, DVM, MS, DACVIM-Oncology , Debbie Tate, RVT, VTS 1 2 ELIAS Animal Health, Olathe, KS, United States; University of Missouri, Columbia, MO, United States; TVAX Biomedical, Olathe, KS, United States Correspondence: Tammie Wahaus (twahaus@eliasah.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P155 Background Canine osteosarcoma (OSA) is an aggressively metastatic primary bone malignancy with a 90% mortality rate. Many naturally occurring canine cancers are genetically and biologically similar to their human counterparts. All cancers express neoantigens and therefore are po- tentially susceptible to vaccine-enhanced adoptive T cell therapy. Syngeneic rodent studies demonstrated that metastases could be permanently eliminated with vaccine-enhanced adoptive T cell ther- apy. Canine OSA studies provide an excellent translational bridge be- tween experimental metastatic rodent cancer studies and metastatic human cancer clinical trials. We hypothesized that dogs with OSA could be safely treated at diagnosis with surgery, autologous cancer cell/P. acnes vaccination, adoptive T cell transfer (ACT) of ex vivo-acti- vated T cells, and low dose human interleukin-2 (IL-2) resulting in im- proved survival compared to carboplatin. We further hypothesized that significant efficacy would be achieved by treating dogs with in- tact immune systems and minimal residual disease [1,2]. Methods 14 client-owned cancer bearing dogs were enrolled in a one-arm prospective trial. Dogs were staged with bloodwork, limb and thor- acic radiographs, histopathology, and bone scans prior to amputation to remove the primary bone tumor. Autologous cancer cell/P. acnes Fig. 1 (abstract P155). Survival Analysis of Dogs vaccinations were administered intradermally weekly for three weeks. Completing Protocol Dogs underwent leukapheresis. Mononuclear white blood cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 86 of 272 P156 2. Hodge G, Barnawi J, Jurisevic C, Moffat D, Holmes M, Reynolds PN, Lung cancer sub-types exhibit differential susceptibility to natural Jersmann H, Hodge S. Lung cancer is associated with decreased killer cell cytotoxicity expression of perforin, granzyme B and interferon(IFN)-γ by infiltrating Jason Cahoon, BS, Shilan Dong, MS, Rafet Amoor, Donna Sonntag, MS, lung tissue T cells, natural killer (NK) T-like and NK cells. Clin Exp Immu- Alexander Spurrell, Rachit Ohri, PhD nol. 2014; 178:79–85. Enable Life Sciences, Worcester, MA, United States Correspondence: Rachit Ohri (rachit@enablelifesciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P156 Background Natural Killer (NK) cells hold great promise in immunotherapy, particularly for lung cancer [1]. However, there is a paucity of literature which organizes the susceptibility of various lung can- cer subtypes to NK cells. We evaluated the cytotoxicity (necrosis and apoptosis) of the NK cell line KHYG-1 (Effector) against cell- lines of 3 different subtypes of lung cancer i.e. H1975, H1703, A549 (Target). We also determined the levels of biomarkers rele- vant to NK cell activation and function [2] i.e. the cell-surface biomarker CD107a and 5 soluble biomarkers [Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha]. Methods Three lung cancer cell lines (H1975, H1703, A549) (ATCC, Vir- ginia) were plated at 100% confluency in 96-well plates (1.25 cells/cm^2, 3.2*10^5 cells/mL), while K562 cells (ATCC, Virginia), used as a positive control, were suspended in the plate wells at 3.2*10^5 cells/mL. KHYG-1 cells (JCRB, Japan) were added at a density of 6.4*10^6 cells/mL, at a 20:1 Effector:Target (E:T) ra- tio. Cells incubated for 5 hours at 5% CO2, 37 °C. Subsequently: [i] Cytotoxicity (necrosis and apoptosis) in target cells were quantified using flow cytometry with PerCP-Cy™5.5 Annexin V and Propidium Iodide (PI). [ii] CD107a expression on KHYG-1 cells was evaluated using Fig. 1 (abstract P156). Necrosis flow cytometry with PE-labeled anti-human CD107a Ab. [iii] Expression levels of 5 soluble biomarkers (Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha) were determined in the cell supernatants using Luminex. Results The cytotoxicity data suggests that for necrosis, all target cell- lines were significantly different (all p values < 0.0001) from each other in terms of susceptibility to the effector cells (A549 > H1703 > H1975 > K562) (Figure 1). K562 cells are significantly higher in late apoptosis than all three lung cancer cell lines (Figure 2). Amongst the 3 lung cancer cell-lines, the H1703 cell line is significantly higher in late apoptosis than H1975 and A549 cells (p-value < 0.05) (Figure 3). Although differences were seen in the necrotic and late apoptotic profiles of target cells, the CD107a expression on the KHYG-1 effector cells was similar across all co-cultures (Figure 4). The soluble biomarker data (Luminex) is being collected. Conclusions In conclusion, cell-lines corresponding to different lung cancer subtypes, i.e. A549 (Carcinoma), H1703 (Squamous Cell) and H1975 (Adenocarcinoma) exhibit significant differences in both their necrosis and late apoptosis susceptibility when co-cultured with NK cells. Such insight could be used to better guide NK cell based immunotherapy development. References 1. Aktaş O, Öztürk A, Erman B, Erus S, Tanju S, Dilege S. Role of Natural Killer Fig. 2 (abstract P156). Late Apoptosis Cells in Lung Cancer. J Clin Cancer Res Clin Oncol. 2018; 144:997-1003. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 87 of 272 Background Adoptive cell transfer (ACT) of tumor-targeted T cells has demonstrated encouraging clinical efficacy in some hematological cancers. However, in solid tumors, targeting a single antigen (e.g., CAR-T and TCR therap- ies) can lead to antigen escape and development of resistance. Further- more, although support provided by lymphodepletion or cytokine administration can enhance responses to ACT, these systemic treat- ments are often associated with significant toxicities. Torque’sSlipstream™ T cell manufacturing platform is a high-efficiency process for generating Deep-Primed™ T cells: polyclonal non- genetically engineered T cells that (1) are targeted against multiple tumor-specific antigens and (2) carry immunomodulating cytokine pay- loads to provide prolonged and locally directed immune support with- out systemic toxicities. The Slipstream™ process is designed to resolve the manufacturing challenge of generating high yields of early memory phenotype tumor-reactive T cells, which are associated with clinical benefit. Here, we show that the Slipstream™ process drives robust ex- pansion while preserving favorable memory characteristics of natural tumor-reactive T cells, and we demonstrate that Deep-Priming™ Tcells with Deep IL-15 or Deep IL-12 improves function. Methods Multi-targeted T cells (MTC) were comparatively generated from do- nors via either a first-generation process or the new Slipstream™ process that leverages ex vivo expansion conditions optimized for MTC production. T cell reactivity against tumor-associated antigens, memory, polyfunctionality, cytotoxicity, and response to Deep IL-15 Fig. 3 (abstract P156). Late Apoptosis minus K562 and Deep IL-12 were measured. The modularity of Slipstream™ was tested by training MTC against antigen cassettes including cancer or viral antigens and measuring reactivity against antigen subsets. Results Compared to a first-generation process, MTC generated with Slip- stream™ exhibited >20-fold improvement in antigen-specific reactiv- ity and a substantial improvement in the yield of memory-phenotype antigen-specific T cells including a 10-fold increase in Tcf1-positive cells. Furthermore, the Slipstream™ process yielded MTC with in- creased polyfunctionality and specificity as measured by cytokine production and TCR sequencing, respectively. Notably, T cells ex- panded using the Slipstream™ process showed potent cytotoxicity against human cancer cells as well as responsiveness to Deep IL-15 and Deep IL-12. The Slipstream™ process can also be adapted for simultaneous training of MTC against different antigens including virus-associated tumor antigens. Conclusions The Slipstream™ process is optimized to produce Deep-Primed™ MTC with substantive increases in characteristics associated with clinical efficacy: antigen reactivity, memory phenotype, and polyfunctionality. Modularity of the Slipstream™ process has been demonstrated by simultaneously training T cell clones reactive to cancer and virus- associated antigens, and Deep-Primed™ MTC with cell-associated Deep IL-15 or Deep IL-12 drives enhanced T cell function in vitro. P158 Suboptimal er stress induced autophagy regulates anti-tumor T cell response Fig. 4 (abstract P156). CD107a Expression Shilpak Chatterjee, Danh Tran, Kim Dosung, Satish Nadig, Carl Atkinson, Hongjun Wang, J. Alan Dieh, Shikhar Mehrotra, PhD, Paramita Chakraborty, PhD Medical University of South Carolina, Charleston, SC, United States P157 Correspondence: Shikhar Mehrotra (mehrotr@musc.edu) Optimized process for manufacturing Deep-Primed™ Tcells Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P158 creates product with improved functional characteristics and reactivity against multiple tumor-associated antigens Background Shawn Carey, PhD, Christine McInnis, PhD, Alicia Worthylake, Angela Endoplasmic reticulum (ER) stress induced by external or internal Forte, Elisabeth Brown, Darren Smith, Kate Sackton, PhD, Rosemary stimuli activates a number of well-orchestrated cellular signaling pro- Soucy, Tap Maniar, MD, Karsten Sauer, PhD, Thomas Andresen, PhD, cesses aimed to promote either cell apoptosis or to restore cellular Andy Rakestraw, PhD function and resolve the stress. In tumor microenvironment, induc- Torque Therapeutics, Cambridge, MA, United States tion of ER stress is known to dampen the antitumor activity of T cells Correspondence: Thomas Andresen (tandresen@torquetx.com) by reducing their mitochondrial function. However, if magnitude of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P157 ER stress governs the T cell fate and function is unknown. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 88 of 272 Methods Results We performed our study on B16 murine melanoma model and used Of the 12 peptides with high predicted score, we confirmed 7 (in- standard immunological techniques like flow cytometry, immunoblot cluding NY-ESO-1 antigen SLLMWITQC) strongly activate human pri- analysis, microscopy, real time PCR mary NY-ESO C259-expressing T cells. These off-target peptides Results include peptides with up to 7 amino acid changes (of 9 possible), Using melanoma antigen gp100 reactive T cells, we found that low which could not be predicted using the recognition motif as deter- level of ER stress enhances T cell stemness and promotes mitochondrial mined by alanine scans. biogenesis, whereas high level of ER stress triggers T cell death. More- Conclusions over, upon adoptive transfer, T cells treated with low dose ER stress in- Thus, this replacement scan assay determines the “TCR fingerprint” ducer are able to form long-lived memory in vivo, express reduced and, when coupled with the algorithm applied to the database of level of co-inhibitory molecule, and demonstrate superior anti-tumor human 9-mer peptides binding to HLA-A*02:01, enables identifica- immunity by increasing overall survival of B16 murine melanoma bear- tion of potential off-target antigens and the tissues where they are ing mouse. Mechanistically, we discovered that, upon ER insult at sub- expressed. This platform enables both screening of multiple TCRs to optimal level, a protective autophagy pathway is induced to promote identify the best candidate for clinical development and identifica- cell survival and maintain stemness through the protein kinase R-like tion of TCR-specific cross-reactive peptide recognition and consti- endoplasmic reticulum kinase (PERK)/ activating transcription factor-4 tutes an improved methodology for the identification of potential (ATF4)-dependent manner. Conversely, knockdown of PERK abrogates off-target peptides presented on MHC class I molecules. We used this autophagy activation, hampers mitochondrial biogenesis in response to platform and demonstrate screening of multiple TCRs targeting suboptimal ER stress, which in-turn compromises the antitumor func- tumor antigens. tion of melanoma antigen specific T cells. Furthermore, we demon- strated that blocking autophagy in T cells hampers T cell anti-tumor P160 activity. Lastly, T cells which initiates autophagic process due to sub- Engineered natural killer cells redirected against adenosinergic optimal ER stress show better potential to control tumor compared to immunometabolic suppression for the immunotherapy of lung those, that do not enter into the process carcinoma Conclusions Andrea Chambers, MS, Kyle Lupo, BS, Jiao Wang, PhD, Sandro Matosevic, Overall, these preclinical data highlights that, low level of ER stress PhD response is important for healthy cellular function and therapeutic- Purdue University, Lafayette, IN, United States ally, ER stress pathways can be manipulated in T cells in order to Correspondence: Sandro Matosevic (sandro@purdue.edu) regulate their antitumor potential. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P160 Ethics Approval The study was approved by Medical University of South Carolina‘s Background Ethics Board, approval number 2018-00628. NK cells are powerful effectors in cancer immunotherapy and have po- tential to treat various cancers; however significant challenges remain P159 in the treatment of solid tumors. Energy availability is compromised TCR fingerprinting and off-target peptide identification surrounding solid tumors and NK cell metabolic reprogramming can Armen Karapetyan, Chawaree Chaipan, Katharina Winkelbach, Sandra occur to inhibit NK effector functions [1]. Accumulation of adenosine in Wimberger, Jun Seop Jeong, Bishnu Joshi, Robert Stein, MD PhD, Dennis the tumor microenvironment (TME) from the activity of ectoenzymes Underwood, PhD, Eleni Chantzoura, PhD, Alvaro Yague, Jan Bergmann, CD39 and CD73 on cancer cells is one mechanism that leads to im- John Castle, PhD, Marc Van Dijk, PhD, Volker Seibert paired NK cell function. Our previously published data has established Agenus Inc, Lexington, MA, United States that the effects of TME adenosine on NK cells cause specific Correspondence: John Castle (john.castle@agenusbio.com); Marc Van reorganization of the cells’ metabolism and effector signatures to sup- Dijk (marc.vandijk@agentustherapeutics.com) press NK cell function, and the cytokine combination of IL-12/15 was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P159 hyperresponsive to adenosine [2]. One way to combat immunosuppres- sion induced by cancer-produced adenosine is to engineer NK cells to Background overcome this inhibition. To that end, we engineered NK cells to dir- Adoptive T cell therapy using patient T cells redirected to recognize ectly target CD73 by imparting NK-specific signaling to enhance anti- tumor-specific antigens by expressing genetically engineered high- tumor activity against CD73+ lung carcinoma. affinity T-cell receptors (TCRs) has therapeutic potential for melanoma Methods and other solid tumors. Clinical trials implementing genetically modified Peripheral blood-derived NK cells were isolated from healthy human TCRs in melanoma patients have raised concerns regarding off-target tox- donors and expanded using feeder cells. NK cells were electroporated icities resulting in lethal destruction of healthy tissue, highlighting the ur- using mRNA or transduced with lentivirus expressing the CD73- gency of assessing which off-target peptides can be recognized by a TCR. targeting construct which bears signaling domains derived from Methods FcγRIIIa. Engineered NK cells expressing the construct were tested for As a model system we used the clinically efficacious NY-ESO-1-spe- their killing ability against lung carcinoma A549 cells. The engineered cific TCR C259, which recognizes the peptide epitope SLLMWITQC NK cells were then adoptively transferred into a CD73+ lung cancer presented by HLA-A*02:01. We investigated which amino acids at xenograft into NSG mice. Circulating CD73-CAR NK cells were quanti- each position enable a TCR interaction by sequentially replacing fied for their expression of activating markers NKG2D, DNAM, and every amino acid position outside of anchor positions 2 and 9 with NKp30 and visualized using immunohistochemistry to determine infil- all 19 possible alternative amino acids, resulting in 134 peptides (133 tration into tumors, and mice were assessed for tumor growth. altered peptides plus epitope peptide). Each peptide was individually Results evaluated using three different in vitro assays: binding of the NY-ESO We showed NK cells can be efficiently redirected against CD73 to C259 TCR to the peptide, peptide-dependent activation of TCR- block the generation of immunosuppressive adenosine and rescue expressing cells, and killing of peptide-presenting target cells. To impaired NK cell anti-tumor immunity. Specifically, primary human represent the TCR recognition kernel, we defined Position Weight NK cells were successfully engineered to express the synthetic CD73- Matrices (PWMs) for each assay by assigning normalized measure- FCyRIIIa construct. Retargeted NK cells showed enhanced anti-tumor ments to each of the 20 amino acids in each position. To predict functions in vitro against CD73-expressing A549 cells. Engineered pri- potential off-target peptides, we applied a novel algorithm project- mary NK cells also showed promise in stunting CD73+ lung cancer ing the PWM-defined kernel into the human proteome, scoring NY- tumor growth for up to 3 weeks in vivo. Current and future studies ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 include evaluation of off target effects and local injection to further binding 9-mer peptides. evaluate infiltration of the NK cells in vivo. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 89 of 272 Conclusions Background The microenvironment of solid tumors is highly immunosuppressive Adoptive cancer antigen-specific T cell therapy currently comprised and adenosine has been shown to impair NK cell anti-tumor immun- of chimeric antigen receptor (CAR-) and T cell receptor (TCR) engi- ity. A novel anti-CD73 targeting construct using NK cell signaling neered T cells. Clinical results from CAR-T cells have demonstrated components has been developed and shown to prevent tumor promising results in treating leukemia, while TCR-engineered T cells growth of CD73+ lung carcinoma. which have the advantage of recognizing intracellular tumor anti- gens is still in very early development. References Methods 1. Chambers A *, Lupo K*, Matosevic S. Tumor-microenvironment-induced Here, we report the development of two CD4+ or CD8+ TCRαβ-KO immunometabolic reprogramming of natural killer cells. Front Immunol. reporter T cell lines for the screening and characterization of trans- 2018; 9:2517. genic TCRs. A TCRαβ-KO reporter T cell line was first developed by 2. Chambers A, Wang J, Lupo K, and Matosevic S. Adenosinergic knocking out the endogenous TCR α and β chains in the reporter T signaling alters natural killer cell functional responses. Front Immunol, cell line using CRISPR/Cas9 and the successful knockout is confirmed 2018; 9:2533. by phenotypic assays and TCR v chain locus sequencing. Ethics Approval Results The study was approved by Purdue University Institution's Review Board, We demonstrated that re-introduction of HA peptide-specific HA1.7 approval number 1804020540. TCR α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Furthermore, P161 the select expression of CD4 or CD8 variants in the TCRαβ-KO re- High-efficiency CAR-T cell manufacturing by improved scalable porter T cell line could enable the development of TCRs for both electroporation MHCI- and MHCII-restricted tumor antigen targets. Jian Chen, PhD, George Sun Conclusions Celetrix LLC, Manassas, VA, United States The CD4+ and CD8+ TCRαβ-deficient reporter T cells can serve as Correspondence: Jian Chen (jchen@celetrix.com) valuable tools for screening and characterization of neoantigen- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P161 specific TCRs Background CAR-T cells are currently manufactured for clinical use by infection of P163 human T cells with viral vectors containing the CAR gene. The Effect of common gamma-chain cytokines on myeloid-derived current viral CAR-T manufacturing process is lengthy and costly and suppressor cell and M2 macrophage suppressive function: electroporation has emerged as a promising alternative. However, Implications for cellular immunotherapy 1 2 2 2 clinical use of electroporation technology in CAR-T has been difficult Anna Cole, BA , Charlotte Rivas , Josue Pineda , Corrine Baumgartner , 2 2 and several clinical trials have met significant problems due to the Stephanie Fetzko , Robin Parihar 1 2 low transfection efficiency and/or high cell mortality. Rice University, Houston, TX, United States; Baylor College of Medicine, Methods Houston, TX, United States Our novel understanding of the electroporation mechanism revealed that Correspondence: Robin Parihar (rxpariha@texaschildrens.org) the current widely-used electroporation methods have significant mis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P163 takes in the physical design as well as electroporation buffer design. The first problem is the electroporation sample container design. It is well Background known that electrochemical reaction generates gas bubbles that are Immunotherapy using antigen-redirected lymphocytes such as harmful to the cells and there was no good solution to the problem. Here chimeric antigen receptor (CAR)-T or -NK cells in patients with solid we used a novel pressurization approach to largely eliminate the effect. tumors has shown poor efficacy. Cell therapies are hindered by im- Results munosuppressive cells such as inhibitory macrophages (M2s) and Combined with other improvements including electroporation myeloid-derived suppressor cells (MDSCs) that contribute to a highly buffer design and post-electroporation cell culture strategy, we suppressive tumor microenvironment (TME) [1]. Researchers have have been able to achieve over 80% plasmid transfection effi- armed redirected lymphocytes with the ability to secrete cytokines in ciency in unstimulated T cells and over 90% plasmid transfection hopes of promoting their proliferation and function in suppressive efficiency in stimulated T cells. The viability in survived cells is TMEs [2,3]. However, the effect of these cytokines on other immune over 95% measured by live/dead staining and the true survival cells within the TME, such as MDSCs and M2s, is unknown. rate measured by survived cell number is over 66%. The new Methods electroporation method can achieve over 90% in gene editing To determine how the human common gamma-chain cytokines, and the method is also widely applicable in electroporation of interleukin(IL)-2, IL-7, IL-15, and IL-21 affect human MDSCs and M2s, NK cells, DC cells and monocytes. we exposed ex vivo enriched M2s and MDSCs to each cytokine sep- Conclusions arately and assessed changes in MDSC and M2 phenotype and ability The new method is also scalable as billions of cells can be processed to dampen T-cell activation and proliferation. To further define in the large volume electroporation setting. Our method can poten- cytokine-induced changes in MDSC/M2 function in a more clinically tially eliminate the need for expensive cell expansion and virus pro- relevant system, we tested the ability of cytokine-exposed MDSCs/ duction altogether, therefore cutting the huge economic burden of M2s to impair CAR-T cell proliferation and anti-tumor activity in a CAR-T therapy. TME co-culture. As a clinical correlate, we assessed common gamma- chain cytokine receptor expression on MDSCs and M2s within neuro- blastoma and sarcoma patient tumors and tested the effects of cyto- P162 kine exposure on their suppressive capacity. Development of CD4+ and CD8+ TCRαβ-deficient bioluminescent Results reporter T cells for screening and characterization of neoantigen- Subsets of ex vivo enriched M2s and MDSCs expressed common specific TCRs gamma-chain cytokine receptors. MDSCs expressed receptors for IL-2 Zhi-jie Cheng, PhD, Jamison Grailer, PhD, Michael Slater, Pete Stecha, Jim (22%, avg. MFI=67, n=3), IL-7 (43%, avg. MFI=375, n=3), IL-15 (23%, Hartnett, Frank Fan, PhD, Mei Cong, PhD avg. MFI=310, n=3), and IL-21 (65%, avg. MFI=124, n=4); whereas Promega Corporation, Madison, WI, United States M2s expressed receptors for IL-2 (17%, avg. MFI=59), IL-7 (98%, avg. Correspondence: Zhi-jie Cheng (jey.cheng@promega.com) MFI=543), IL-15 (36%, avg. MFI=619), and IL-21 (91%, avg. MFI=296). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P162 Exposure of human MDSCs or M2s to IL-2, IL-7, IL-15, or IL-21 did not Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 90 of 272 alter their cell-surface phenotype. Exposure of these suppressive median survival of 24 days. Treatment with PM21-NK cells improved myeloid cells to IL-2, IL-7, and IL-15 did not change their ability to survival over untreated (p=0.0003) and PD-L1 alone (p=0.0002) suppress T-cell proliferation. In contrast, exposure of M2s and MDSCs groups having median survival of 40 days. Combination of PM21-NK to IL-21 increased their ability to suppress T-cell proliferation and ac- cells with anti-PD-L1 further improved of survival over the PM21-NK tivation (98% suppression by IL-21 exposed MDSCs vs. 72% suppres- cells alone group (48 days, p=0.042) with 25% of mice still remaining sion by control MDSCs; 98% suppression by IL-21 exposed M2 vs. in good health at day 58. 79% suppression by control M2 at a 2:1 T cell:MDSC/M2 ratio). Conclusions Conclusions These data support the use of anti-PD-L1 in NK cell therapy, regard- These results suggest that IL-21 increases the suppressive capacity of less of initial tumor PD-L1 status. PM21-NK cells can be used for human MDSCs and M2s. Ongoing experiments will define the mech- tumor treatment and to prime tumors to express PD-L1. The PD-L1 anisms by which IL-21 alters MDSC and M2 suppression and further induced upon NK cell treatment can serve as “universal targetable define the effect of IL-21 exposed MDSCs and M2s on tumor growth ligand” if used with humanized anti-PD-L1 antibodies to cause tumor and CAR-T cell therapeutic efficacy in vivo. killing by ADCC. References P165 1. Martinez M, Moon EK. CAR T cells for solid tumors: New strategies for Robust, reproducible and highly scalable manufacturing of P- finding, infiltrating, and surviving in the tumor microenvironment. Front. BCMA-ALLO1, an allogeneic CAR-T stem cell memory product for Immunol. 2019; 10:128. multiple myeloma, from numerous healthy donors 2. Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T Stacey Cranert, PhD, Maximilian Richter, PhD, Min Tong, MS, Leslie Weiss, cells enhance antitumor efficacy and overcome the tumor MS, Yening Tan, MS, Eric Ostertag, MD, PhD, Julia Coronella, PhD, Devon microenvironment. Sci Rep. 2017; 7:10541. Shedlock, PhD 3. Liu D, Song L, Wei J, Courtney AN, Gao X, Marinova E, Guo L, Heczey A, Poseida Therapeutics, San Diego, CA, United States Asgharzadeh S, Kim E, et al. IL-15 protects NKT cells from inhibition by Correspondence: Devon Shedlock (dshedlock@poseida.com) tumor-associated macrophages and enhances antimetastatic activity. J Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P165 Clin Invest. 2012; 122:2221-2233. Ethics Approval Background Tumor tissue use was approved by Baylor College of Medicine IRB study Autologous Chimeric Antigen Receptor (CAR) T cell therapy for re- protocol #26691, and samples were de-identified prior to laboratory lapsed/refractory Multiple Myeloma (MM), such as Poseida’s anti-B evaluation. cell maturation antigen (BCMA) product candidate, P-BCMA-101, have shown significant efficacy in the clinic. P-BCMA-101 is com- P164 prised of a high percentage of stem cell memory T cells (TSCM), Effect of NK cell treatment on PD-L1 expression and anti-PD-L1 resulting in a product that is much safer and potentially more dur- response able than other anti-BCMA autologous product candidates. However, Alicja Copik, PhD, Jeremiah Oyer, Sarah Gitto, Deborah Altomare, PhD individualized products have expensive and time-consuming manu- University of Central Florida, Orlando, FL, United States facturing and significant variability in input patient T cells characteris- Correspondence: Deborah Altomare (deborah.altomare@ucf.edu) tics. We are developing P-BCMA-ALLO1, an off-the-shelf anti-BCMA Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P164 allogeneic (allo) CAR-T product candidate manufactured from serial healthy donor material that circumvents many of the downsides of Background an individualized CAR-T product. PD-1 axis blockade therapies have shown success but responses are Methods limited to ~15% of cancer patients. These responses correlate with P-BCMA-ALLO1 is produced using two key platform technologies: presence of lymphocyte infiltrated, PD-L1 positive tumors. Strategies the nonviral piggyBac® (PB) DNA Modification System and the that increase PD-L1 expression may improve outcomes of PD-1 axis high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. blockade. PD-L1 on tumor cells is induced by IFNγ, secreted by NK The PB transposase mRNA and DNA encoding the PB-based cells. We developed a method for producing therapeutic quantities transgene are electroporated along with the components of the (>1,000 fold expansion within two weeks) of hyper-activated NK cells CC system needed to knockout (KO) the T Cell Receptor (TCR) with high anti-tumor cytotoxicity and enhanced IFNγ secretion. The and beta-2 microglobulin, thereby eliminating expression of Major utilizes particles from Plasma Membrane of K562 cells expressing Histocompatibility Complex (MHC) class I. The T cells are then ex- membrane bound IL21 (PM21-particles). Herein, the ability of PM21- panded using our proprietary "booster molecule.” The resulting particle expanded NK cells to induce PD-L1 expression on various tu- product demonstrates expression of the transgene in nearly all mors was tested in vitro and in vivo in ovarian cancer model. Fur- cells, and after a purification step, have eliminated all TCR expres- thermore, the effect of anti-PD-L1 on NK cell anti-tumor activity was sion and most MHC class I expression. tested in vitro and in vivo. Results Methods We have produced P-BCMA-ALLO1 at both research and near- NK cells were expanded with PM21-particles as described. For in vivo clinical scale from >35 donors with >97% manufacturing success. experiments, NSG mice were implanted with 1x10^6 SKOV-3 cells Efficiencies of TCR-KO ranged from ~50-90%, with final product i.p.. Mice were treated with 10^7 PM21-NK cells (n=6) or with vehicle always demonstrating >99% TCR-KO. T cell expansion varied from control (n=6) on days 8 and 13. Mice were sacrificed on day 20 to ~0.5-20 fold. At clinical production scale, this translates to up to collect tumors. Tumors were perfused and retrieved tumor cells were 250 doses of CAR-T per manufacturing run at a dose of 150x10e6 analyzed for PD-L1 expression while infiltrating immune cells were cells/patient. P-BCMA-ALLO1 demonstrated a high-percentage of phenotyped. TSCM cells (CD45RA+CD62L+CD45RO-). Furthermore, P-BCMA- Results ALLO1 generated from multiple donors demonstrated potent effi- PM21-NK treatment induced PD-L1 on >30% of tumor cells across cacy in the RPMI-8226 xenograft model in NSG mice, thus estab- multiple cell lines. PM21-NK cells are negative for PD-1 and addition lishing the feasibility of using serial individual donors in our of anti-PD-L1 had no effect on their cytotoxicity or cytokine produc- manufacturing process. tion. In in vivo experiment, PM21-NK cell treated mice had increased Conclusions PD-L1+ tumors vs. the untreated group (29.7% vs 14.5%, p<0.0001). In summary, these data demonstrate a robust, reproducible and Despite T-cell depletion, T-cells made up ~22% of hCD45+ events in highly scalable manufacturing process. Moreover, this production perfused tumors, 83% of which were Tregs. PM21-NK cells are PD-1-, process can be expanded for use with additional targets for treat- but are inhibited by Tregs. Untreated and anti-PD-L1 alone mice had ment of other heme or solid tumors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 91 of 272 P166 P167 ET140202 T-cell therapy for the treatment of liver cancer is built Improved efficacy leveraging CAR-T therapeutics that produce upon a novel antibody-T cell receptor (AbTCR) ARTEMIS™ T-cell tightly controlled, tumor proximal, immunomodulatory outputs platform Michon Pinnix, Krista McNally, Jay Danao, BS, Melissa Fardy, Rachel Jun Cui, PhD, Pengbo Zhang, Hongruo Yun, Yiyang Xu, Lucas Horan, Hovde, MS, Charlotte Davis, Nicole Grant, Dianna Lester-Zeiner, David PhD, Shaohua Xu, Sean Xu, Hong Liu Mai, Ben Wang, PhD, Gus Zeiner, PhD Eureka Therapeutics, Inc., Emeryville, CA, United States Chimera Bioengineering, Emeryville, CA, United States Correspondence: Hong Liu (hong.liu@eurekainc.com) Correspondence: Gus ZeinerGus Zeiner (gus@chimera.bio) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P166 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P167 Background Background The use of engineered T cells for the treatment of solid cancers remains Current chimeric antigen receptor (CAR) T cell therapies are clinically challenging. Recently, we developed a novel antibody-T cell receptor efficacious against several B cell malignancies, but are less effective (AbTCR) ARTEMIS™ T-cell platform, which combines antibody-based target at eliminating solid tumors. A key contributor to this observed lack recognition with gamma/delta TCR-based cellular activation [1]. In contrast of efficacy is the tumor microenvironment (TME) that is erected by to chimeric antigen receptors (CARs), the AbTCR forms a natural multi- solid tumors to impose immunosuppressive physical and chemical meric receptor with the endogenous CD3 complex, which feeds into a barriers to T cell function and survival. To break TME-driven immuno- network of signaling pathways that regulate T-cell activation. In addition, suppression, attempts have been made to arm CAR-T cells with the the inclusion of gamma/delta TCR chains within the AbTCR avoids the for- ability to produce immunomodulatory payloads that target features mation of mispaired receptors with unknown cross-reactivity, which is a of the TME. By their nature, these immunomodulators (e.g. Interleu- potential risk associated with current alpha/beta TCR-based therapies. kin 12) are frequently toxic when systemically delivered. Due to a Using the core design of the AbTCR ARTEMIS™ T-cell platform, we de- limited repertoire of existing programmable gene regulators, consti- veloped ET140202 for the treatment of hepatocellular carcinoma (HCC). tutive expression and systemic distribution of immunomodulators by ET140202 features an AbTCR targeting alpha-fetoprotein (AFP)peptide/ armed CAR-T cells is common. MHC complexes (specifically AFP158-166/HLA-A2) expressed on HCC Chimera Bioengineering has characterized a novel, post-transcriptional cancer cells. To optimize T-cell activation and expansion, the AbTCR is gene regulatory node that strictly governs effector outputs in human T co-expressed with a CD28-based co-stimulatory molecule engineered cells. This gene regulatory node, termed Gold, has been used to create to target Glypican 3 (GPC3) expressed on HCC cancer cells. enhanced CAR-T therapeutics that produce tightly controlled immuno- Methods modulatory outputs only upon tumor engagement. To test the specificity and potency of ET140202 T cells in vitro, Methods ET140202 T cells were co-incubated with either target-positive or Methods include: design and characterization of a proprietary bicis- target-negative cells. Lactate Dehydrogenase release was used to tronic GoldCAR lentiviral vector to simultaneously deliver both CAR quantify target cell lysis. CFSE assay was used to measure cell prolif- and IL12 transgenes; production of lentivirus and infection of primary eration. Expression of differentiation and exhaustion markers were human T cells; in vitro characterization of GoldCAR-T cell function determined by flow cytometry. The in vivo anti-tumor activity of utilizing cell based assays, immunoassays and flow cytometry; im- ET140202 T cells was tested in an AFP+/HLA-A2+ Hep G2 liver cancer plantation of subcutaneous xenograft tumors in NSG mice with xenograft model. We also engineered the same anti-AFP158-166/ tumor growth monitored by caliper and bioluminescent imaging HLA-A2 binding moiety onto a CD28-based CAR (AFP-CAR) and com- (BLI) to assess in vivo efficacy; and ex vivo analysis of blood, tumor pared AFP-CAR-T cells to ET140202 T cells in various assays. and lymphatic organs to characterize safety profile. Results Results ET140202 T cells specifically lysed AFP-positive tumor cells. Com- CD19 targeted GoldCAR-T cells delivering IL12 demonstrate im- pared to AFP-CAR-T cells, ET140202 T cells displayed enhanced proved efficacy over standard CD19 CAR-T cells in a subcutaneous in vitro cell killing and proliferation even after repetitive antigen Daudi B cell xenograft mouse model. These GoldCAR-T cells exhibit stimulations. ET140202 T cells also display a less exhausted surface comparable efficacy to CAR-T cells with constitutive IL12 expression, phenotype (e.g. lower PD-1 expression) and a higher percentage of but levels of pro-inflammatory cytokines in the peripheral blood are central memory T cells (CCR7+ CD45RA-) after antigen stimulation. In lower in the mice treated with GoldCAR-T cells. vivo, both intravenous and intratumoral single administration of Conclusions ET140202 T cells led to significant tumor growth inhibition. GoldCAR-T cells with tumor proximal IL12 delivery demonstrate en- Conclusions hanced efficacy over standard CAR-T cells and an improved safety ET140202 is built upon our novel AbTCR ARTEMIS™ T-cell platform, profile compared to CAR-T cells with constitutive IL12 expression. which was designed to harness the natural biology of T cells to fight The implications of this study point to an amplified CAR-T cell re- cancer. Both in vitro cellular assays and in vivo mouse studies sup- sponse in an immunosuppressive tumor microenvironment. port the safety and efficacy of ET140202 T cells. Whether these pre- Trial Registration clinical findings for AbTCR-based ET140202 T-cell therapy translate Not applicable into the clinical setting is currently being tested (clinicaltrial.gov, Ethics Approval NCT0399803). The animal study was conducted under the Institutional Animal Care and Use Committee (IACUC) of LumiGenics, LLC, 750 Alfred Nobel Reference Drive, Suite 103, Hercules CA 94547 1. Xu Y, Yang Z, Horan LH, Zhang P, Liu L, Zimdahl B, Green S, Lu J, Morales JF, Barrett DM et al. A novel antibody-TCR (AbTCR) platform combines P168 Fab-based antigen recognition with gamma/delta-TCR signaling to facili- Role and function of T-cell immunoglobulin– and mucin domain– tate T-cell cytotoxicity with low cytokine release. Cell Discovery 2018, containing (TIM)–3 receptor on natural killer cells in solid tumors 4(1):62. Tram Dao, Sandro Matosevic, PhD Ethics Approval Purdue University College of Pharmacy, West Lafayette, IN, United States All animal experiments were conducted according to protocols approved Correspondence: Sandro Matosevic (smatosev@purdue.edu) by their Institutional Animal Care and Use Committee (IACUC) and in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P168 accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Background Washington, DC, 1996) and the Policy on Humane Care and Use of Natural killer (NK) cells are part of the innate immune system, but are Laboratory Animals (Department of Health and Human Services, capable of participating in both innate and adaptive immune Bethesda, MD). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 92 of 272 responses due to their wide range of cytolytic activities, from de- P169 granulation, secretion of cytokines to antibody-dependent cell- Enriching non-genetically modified antigen specific marrow mediated cytotoxicity. These are possible due to the cells’ ability to infiltrating lymphocytes (MILs) to target HPV+ oropharyngeal recognize self and non-self-entities via the net signal generated from squamous cell carcinoma 1 1 2 their activating and inhibitory receptors upon engagement. One such Danielle Dillard , Vanessa Chan , Lakshmi Rudraraju, MS , Elizabeth 2 1 1 1 receptor is TIM-3, which is expressed on various lymphocytes. In T- DeOliveira , Amy Thomas , Ervin Griffin , Megan Heimann , Luca Biavati, 1 1 1 cells, TIM-3 is an exhaustion marker [1], but on NK cells, results are MD , Elizabeth Zawidzka , Marguerrita El Asmar , Drew Pardoll, MD, 1 1 3 1 conflicting in regards to its function as the receptor exhibits both ac- PhD , Kellie Smith, PhD , Carole Fakhry, MD , Ivan Borrello, MD tivating and inhibitory effects depending on disease type and activa- Johns Hopkins School of Medicine, Baltimore, MD, United States; 2 3 tion status [2-6]. WindMIL therapeutics, Baltimore, MD, United States; Otolaryngology, Methods Head and Neck Surgery, Baltimore, MD, United States NK cells were isolated from peripheral blood of healthy donors. Correspondence: Danielle Dillard (danielle.dillard@jhmi.edu) After expansion, they were co-cultured for 4 hours with glioblast- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P169 oma (U87) at effector:target (E:T) ratios of 2.5:1 and 10:1, and various receptors were screened by flow cytometry, including PD- Background 1, NKG2A, LAG-3, CD158b, CEAMCAM-1 and TIM-3. Then, expres- Human papillomavirus positive (HPV+) oropharyngeal squamous cell sion of TIM-3 was measured when in the presence of patient- carcinoma (HPV-OPSCC) accounts for ~80% of OPSCCs in the United derived primary glioblastoma cells (GBM43) and prostate cancer States. Although HPV-positivity confers improved survival relative to (PC3) for 4 hours. To determine the effect of TIM-3 expression, HPV(-) OPSCC, outcomes for metastatic HPV-OPSCC remain dismal. killing assay are being carried out by blocking TIM-3 on NK cells. High tumor infiltrating lymphocytes (TILs) are associated with better Further investigation is being performed by blocking one of TIM- outcomes in HPV-OPSCC and have the promise of a therapeutic role 3’s primary ligands, Galectin-9, on cancer cells to determine its based upon their intrinsic enhanced tumor specificity. However, not impact on NK cell cytotoxicity. Statistical analyses are completed all tumors possess TILs. To date, therapeutic expansions involve sur- in SAS JMP Pro 14. gical excision of the tumor, expansion of TILs with high-dose IL-2 for Results up to 4 weeks and clinical product success rate between 40-60%. We found that TIM-3 is significantly downregulated on primary hu- Marrow infiltrating lymphocytes (MILs) represent a novel and distinct man NK cells, in both frequency and surface density, when exposed T cell population obtained from the bone marrow (BM) of patients to solid tumor cells such as U87, GBM43 and PC3 at multiple E:T ra- that possess significant tumor-specificity over peripheral blood lym- tios. Unlike other inhibitory NK receptors, this downregulation was phocytes (PBLs). Although the early work was done in myeloma, the unique to TIM-3. However, it is not known why the downregulation unique nature of the BM microenvironment makes it a reservoir of occurs with solid tumors, and whether this change in expression af- antigen-experienced memory T cells in numerous solid tumors. As fects NK killing capacity. Here, we report the role of TIM-3 on NK cell such, we sought to determine whether HPV-specific MILs could be cytotoxicity against solid tumor cell lines and the role of Galectin-9 in identified and expanded ex vivo. mediating NK cell activity. Methods Conclusions We obtained bone marrow and peripheral blood from patients with We found that Tim-3 was significantly downregulated on NK cells in localized, HPV-OPSCC. A modified version of the MANAFEST assay response to solid tumor cells. Understanding the complex roles of was used to evaluate proliferation of peripheral and bone marrow- Tim-3 expression on NK cells allows us to better understand the nu- derived CD8+ T cells in response to HPV early 6 (E6) and early 7 (E7) anced immunomodulatory role of Tim-3 on NK cell anti-tumor re- peptides (HPVFEST) in a HPV-OPSCC patient. sponses, and provide a basis for the development of Results immunotherapies targeting impaired NK cell function in solid tumors T cell receptor sequencing and bioinformatic analysis of each peptide- stimulated culture revealed a markedly increased frequency of HPV- References specific T cells in MILs compared to peripheral blood. HPV-specific MILs 1. Sánchez-Fueyo A, Tian J, Picarella D, Domenig C, Zheng XX, Sabatos CA, showed a higher average TCR clone frequency relative to PBLs (p value Manlongat N, Bender O, Kamradt T, Kuchroo VK, Gutiérrez-Ramos JC, = 0.0037). Additionally, of the 5 highest expanded TCR clones for both Coyle AJ, Strom TB. Tim-3 inhibits T helper type 1-mediated auto- and compartments HPV-specific MILs possessed an increased average rep- alloimmune responses and promotes immunological tolerance. Nat resentative clone frequency (p value = 0.0001). To investigate general Immunol. 2003; 4:1093-101. responsiveness to shared tumor antigens, we incubated autologous BM 2. Ndhlovu LC, Lopez-Vergès S, Barbour JD, Jones RB, Jha AR, Long BR, with lysate from an HPV+ OPSCC and then added ex-vivo activated Schoeffler EC, Fujita T, Nixon DF, Lanier LL. Tim-3 marks human natural MILs and PBLs. Tumor specificity was defined as IFN훾 production in killer cell maturation and suppresses cell-mediated cytotoxicity. Blood. CD3 cells. MILs possessed an average of 50.73% IFN훾 production as 2012; 119:3734-43. compared to 0.11% in PBLs to HPV-OPSCC lysate. 3. Jost S, Moreno-Nieves UY, Garcia-Beltran WF, Rands K, Reardon J, Toth I, Conclusions Piechocka-Trocha A, Altfeld M, Addo MM. Dysregulated Tim-3 expression Collectively, these data indicate that HPV-specific T cells exist in the on natural killer cells is associated with increased Galectin-9 levels in HIV- BM of patients with localized disease and possess a greater clonoyt- 1 infection. Retrovirology. 2013; 10:74. pic frequency and functional tumor recognition compared to PBLs. 4. Gleason MK1, Lenvik TR, McCullar V, Felices M, O'Brien MS, Cooley SA, These data provide the rationale for developing this novel adoptive T Verneris MR, Cichocki F, Holman CJ, Panoskaltsis-Mortari A, Niki T, Hira- cell approach using MILs in this patient population. shima M, Blazar BR, Miller JS. Tim-3 is an inducible human natural killer Ethics Approval cell receptor that enhances interferon gamma production in response to This study was approved by Johns Hopkins University Institution Re- galectin-9. Blood. 2012; 119:3064-72. view Board, approval number IRB00128334 and NA_00028682. 5. Ju Y, Hou N, Meng J, Wang X, Zhang X, Zhao D, Liu Y, Zhu F, Zhang L, Sun W, Liang X, Gao L, Ma C. T cell immunoglobulin- and mucin- P170 domain-containing molecule-3 (Tim-3) mediates natural killer cell sup- Sequential anti-CD19, anti-CD22, and anti-CD20 autologous pression in chronic hepatitis B. J Heptatol. 2010; 52:322-9. chimeric antigen receptor T cell (CAR-T) therapies treating a child 6. da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo with relapsed refractory Burkitt lymphoma VK, Osman I, Bhardwaj N. Reversal of NK-cell exhaustion in advanced mel- Juan Du, MD, Yonghong Zhang anoma by Tim-3 blockade. Cancer Immunol Res. 2014; 2:410-22. Beijing Boren Hospital, Beijing, China Ethics Approval Correspondence: Yonghong Zhang (yhzhang58@126.com) This study was approved by Purdue Intuition’s Ethics Board, approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P170 number 1804020540. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 93 of 272 Background Results Currently, the prognosis of children with relapsed refractory(r/r) Burkitt lym- Using this method, we have retrieved multiple TCRs that confer specific phoma(BL) remains dismal . New therapies are exlored to achieve a higher recognition of a public neoepitope derived from a PIK3CA hotspot mu- remission rate such as immunotherapy for these patients .We have success- tation (H1047L). These TCRs are restricted by HLA-A*03:01, an allele fully treated a case by adopting sequential autologous chimeric antigen re- present in 20.5% of the North American population. Immune- ceptor T cell(CAR-T) therapies, targeting antigen CD19,CD22,and CD20. precipitation/tandem mass spectrometry analysis determined that the Methods endogenously processed and presented public neoepitope is a 9 amino An 8-year-old boy was studied,who presented with a mass on the acid sequence containing a His to Leu substitution at position 2. To right side of the neck and was diagnosed with BL by pathology. The understand the mechanistic basis for the immunogenicity of this public child was treated with standard chemotherapy but suffered from re- neoepitope, we generated x-ray crystallography structures of mutant lapse. Subsequently, anti-CD19, anti-CD22, and anti-CD20 autologous and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These stud- CAR-T cell treatments were sequentially administered. We observed ies revealed significant topologic overlap in the bound peptides. By the clinical manifestations and response to the three cycles of CAR-T contrast, the thermal and kinetic stability of the mutant peptide/HLA- treatments, values of peripheral CAR-T cells were also monitored and A*03:01 complex was significantly enhanced relative to the WT com- side effects were assessed. plex, as measured by differential scanning fluorimetry and fluorescence Results anisotropy assays. Peripheral blood T cells genetically engineered with The patient displayed no response to anti-CD19-CART treatment PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells .After CD-22 directed CART the patient got partial remission (PR),but in a HLA/mutation-specific manner, leaving HLA-mismatched or WT tar- relapse occurred quickly. Finally, after the use of anti-CD20 CAR-T cell get cells unperturbed. therapy, the child achieved complete remission (CR) and has cur- Conclusions rently achieved a 6-month event-free survival (EFS). During the CD19 These findings reveal for the first time the existence of an endogen- and CD20 CAR-T cell treatments, only mild cytokine release syn- ously processed and presented public neoantigen derived from a drome (CRS) were observed in the patient (grade 1) while he devel- PIK3CA hotspot mutation. These results open the possibility of target- oped a grade 3 CRS during CD22 CAR-T therapy, the symptoms ing this common driver oncogene using adoptively transferred and included fever and hypoxemia. genetically redirected T cells. Conclusions Ethics Approval Autologous CAR-T cell therapies targeting multplei tumor antigens The study was approved by Smita Chandran's Institution‘s Ethics could be novel and safe treatments for children with r/r BL. Board, approval number IRB 17-250. Consent Written informed consent was obtained from the patient for publica- P172 tion of this abstract and any accompanying images. A copy of the Shape and material properties increase artificial antigen written consent is available for review by the Editor of this journal. presenting cell effectiveness Savannah Est Witte, BS, Kaitlyn Calebrisi, Jordan Green, PhD P171 Johns Hopkins University, Baltimore, MD, United States T cell receptor gene therapy for a public neoantigen derived from Correspondence: Jordan Green (green@jhu.edu) mutated PIK3CA, a dominant driver oncogene in breast and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P172 endometrial cancers 2 1 3 3 Jiaqi Ma , Martin Klatt, PhD , Friederike Dundar, PhD , Paul Zumbo , Background 1 3 1 Matthew Femia , Doron Betel, PhD , David Scheinberg , Brian Baker, Particulate delivery of artificial antigen presenting cells (aAPCs) is a promis- 2 1 1 PhD , Christopher Klebanoff, MD , Smita Chandran, PhD ing cell-free strategy to initiate selective T cell stimulation for immunother- 1 2 MSKCC, New York, NY, United States; University of Notre Dame, Notre apy in vivo. While 2D aAPC strategies aim to optimize T cell proliferation Dame, IN, United States; Weill Cornell Medicine, New York, NY, United States and selection in vitro for subsequent cell therapy, it would be advanta- Correspondence: Christopher Klebanoff (klebanoc@mskcc.org); Smita geous to deliver “off-the-shelf” biodegradable aAPCs directly as an in vivo Chandran (chandrs1@mskcc.org) therapeutic. 3D aAPC effectiveness has been limited due to inefficiencies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P171 in stimulating T cells as well as rapid clearance of delivered particles. To improve bioavailability as well as increase particulate aAPC effectiveness, Background we have developed a soft, biodegradable, microparticle aAPC (Figure 1). “Public” neoantigens represent immunogenic epitopes encompassing To create a new platform technology for immunoengineering, material hotspot mutations in driver oncogenes that are also restricted by and shape are investigated as parameters for improving T cell stimulation. common HLA alleles. In contrast with patient-specific “private” Methods neoantigens, public neoantigens are conceptually attractive because One micron size particles were synthesized using a poly(ethylene gly- they are tumor-specific, clonally conserved, and shared across pa- col) diacrylate (PEGDA) or using a poly(lactic-co-glycolic acid) emulsion tients. Whether PIK3CA, the most common driver oncogene in breast method [1,2]. Anti-CD3 and anti-CD28 conjugated particles were incu- and endometrial cancer, can yield public neoepitopes that may be bated with primary mouse T cells and proliferation was quantified at 3 exploited for cancer immunotherapy is unknown. and 7 days. For macrophage uptake studies, particles were incubated Methods with macrophages at 37 °C to analyze particle uptake and 4 °C to evalu- We have developed a high-throughput, single-cell functional assay for ate binding of particles to cells. To synthesize soft, ellipsoidal aAPCs, a the discovery and retrieval of TCR alpha/beta gene sequences that con- novel thin-film stretching technique was developed where emulsified fer specific recognition of endogenously processed and presented pub- PEGDA droplets were frozen then cast into films and stretched [3]. lic neoantigens and not the corresponding wild type (WT) sequence. In Results this approach, donor-derived T cells are sensitized with autologous Protein conjugation efficiency and T cell proliferation were 10-fold antigen presenting cells (APCs) electroporated with RNA encoding higher for PEGDA particles than PLGA particles (Figure 2a-b). Uptake PIK3CA hotspot mutations. Expanded T cells are subsequently divided studies indicate a ~20-fold decrease in binding and uptake by the into paired daughter wells for short-term co-culture with APCs electro- PEGDA particles (Figure 2c). Ellipsoidal aAPCs stimulate T cells 3 porated with minigenes containing either mutant or WT PIK3CA se- times more effectively than spherical particles (Figure 3b,d). Uptake quences. Acutely re-stimulated T cells from paired wells are subject to studies indicate a ~10-fold decrease in nonspecific uptake of ellips- single-cell alpha/beta TCR VDJ and RNA sequencing. TCR alpha/beta oidal particles (Figure 3c). gene sequences associated with selective upregulation of TCR signaling Conclusions transcripts to mutant but not WT PIK3CA stimulation are subsequently Particle material and shape are significant factors in designing partic- cloned into retroviral vectors to confirm reactivity. ulates as biomimetic aAPCs for in vitro T cell stimulation. Ongoing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 94 of 272 work on further decoupling these parameters and optimizing in vivo efficacy has the potential to unleash a promising biomimetic plat- form technology for immunoengineering of T cells. References 1. Anselmo AC, Mitragotri S. Impact of Particle Elasticity on Particle-Based Drug Delivery Systems. Adv. Drug Deliv. Rev. 2017; 108:51–67. 2. Meyer RA, Sunshine JC. Biodegradable Nanoellipsoidal Artificial Antigen Presenting Cells for T Cell Activation. Small. 2016; 11:1519–1525. 3. Meyer RA, Meyer RS, Green JJ. An Automated Multidimensional Thin Film Stretching Device for the Generation of Anisotropic Polymeric Micro- and Nanoparticles. J. Biomed. Mater. Res. A. 2015; 103:2747–2757. Fig. 3 (abstract P172). See text for description P173 Case reports: Correlates of response following adoptive transfer of ADP-A2M4, affinity-enhanced T-cells targeting MAGE-A4, in synovial sarcoma 1 1 1 Svetlana Fayngerts, PhD , Zohar Wolchinsky , Shravani Shitole , Joana 1 1 1 Senra , Rebecca Dryer-Minnerly, PhD , Ruoxi Wang , Jean-Marc Navenot, 1 1 1 1 PhD , Olga Ochkur , Gareth Betts, PhD , Natalie Bath, MSc , Erin Van 1 1 1 1 Winkle , Tom Holdich , Malini Iyengar, PhD , Rafael Amado, MD , Marcus 2 3 1 1 Butler, MD , David Hong, MD , Alex Tipping, PhD , Samik Basu, MD , Indu Ramachandran, PhD 1 2 Adaptimmune, Philadelphia, PA, United States; Princess Margaret Cancer Centre, Toronto, Ontario, Canada; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Indu Ramachandran (indu.ramachandran@adaptimmune.com) Fig. 1 (abstract P172). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P173 Background ADP-A2M4 is a genetically engineered autologous affinity-enhanced receptor immunotherapy (SPEAR T-cells) directed towards a MAGE- A4 peptide expressed in the context of HLA-A*02 on tumor cells. Clinical responses with ADP-A2M4 have been reported in patients with advanced MAGE-A4+ synovial sarcoma (SS) tumors. Here, we describe intra-tumoral and peripheral correlates associated with clin- ical response and resistance in two patients with SS. Methods Transduced T-cell persistence was determined by qPCR in PBMCs. Serum cytokines were measured via a multiplexed electrochemiluminescence-based immunoassay (MSD). Immunohisto- chemistry for antigen and immune markers was performed on FFPE tumor biopsies collected from patients prior to and following ADP- A2M4 transfer. A digital PCR-based assay was performed on FFPE tumor biopsies to detect the presence of SPEAR T-cells in the tumor. T-cell cytotoxicity assays were performed in vitro using the IncuCyte® platform. Clinical responses were assessed by RECIST v1.1. Results In the first patient, the best overall response (BOR) following ADP- A2M4 treatment was a partial response. Multiple correlates previously shown to be associated with response were observed. The patient’s pre- and post-infusion tumor biopsies expressed high levels of MAGE-A4 protein. Post-infusion, high levels of persisting transduced cells were observed in peripheral blood. Additionally, the patient had a grade 2 CRS event associated with a high level of serum IFN-g and Fig. 2 (abstract P172). See text for description IL-15 induction. In the post-infusion tumor sample, a notable increase Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 95 of 272 in CD3+ T-cell infiltration, including SPEAR T-cells, was observed and simultaneously any potential TAA. RevTMs were able to effi- along with PD-L1 induction. ciently redirect RevCAR-T-cells specifically against different tumor tar- In the second patient, the BOR was stable disease; then the disease pro- gets. Moreover, we show that combinatorial targeting can be gressed. MAGE-A4 protein expression was lower prior to ADP-A2M4 infu- achieved using our RevCAR system. Here, dual-RevCAR-T-cells were sion, compared to the 1st patient. Minimal peripheral induction of IFN-g efficiently activated only after engagement by two RevTMs targeting and IL-15 was observed post-infusion along with a lower level of trans- the activating or costimulatory RevCAR and different TAAs. duced T-cell persistence, compared with the 1st patient. No CRS was re- Conclusions ported in this patient. Both patients’ manufactured products contained Taken together, we developed a switchable RevCAR platform showing transduced CD8+ T-cells capable of killing antigen-expressing targets high effectiveness, increased specificity, improved safety, easy control- in vitro. In the responding patient, effective target killing was observed in lability, and small size facilitating combinatorial tumor targeting. transduced CD8+ T-cells isolated from the tumor site post-infusion. Eight Ethics Approval additional SS patients have been treated, and we continue to analyze The study was approved by local authorities and the Ethics Board. biomarkers in these patients. Conclusions P175 Based on these two cases, we have identified some factors that may PD-L1: A side-effect of T cell engagement or a main player in MDS contribute to the anti-tumor activity of ADP-A2M4. High antigen ex- tumor immune evasion? pression levels, IL-15 and IFN-g cytokine induction, good engraft- 1 1 1 2 Valentina Ferrari, BA , Alison Tarke , Hannah Fields , Tiffany Tanaka , ment, tumor site trafficking, and cytolytic function of SPEAR T-cells 2 1 1 Rafael Bejar , Thomas Lane, MD , Antonella Vitiello, PhD , Maurizio may be associated with favorable responses in SS patients treated Zanetti, MD with ADP-A2M4. 1 2 PersImmune, San Diego, CA, United States; University Of California San Trial Registration Diego, San Diego, CA, United States NCT03132922 Correspondence: Antonella Vitiello (avitiello@persimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P175 P174 Combinatorial tumor targeting using a novel switchable RevCAR Background system Immune checkpoint inhibitors (ICIs) are being tested in myelodys- 1 1 1 Anja Feldmann, PhD , Anja Hoffmann , Ralf Bergmann , Liliana Loureiro, plastic syndromes (MDS) based on pre-clinical data suggesting that 1 2 1 1 PhD , Enrico Kittel-Boselli , Nicola Mitwasi , Stefanie Koristka , Justyna the relevant targets are expressed on tumor and immune cells. Here 3 1 1 1 Jureczek , Nicole Berndt , Claudia Arndt , Michael Bachmann we study both tumor cells and T cells from patients with higher-risk 1 2 Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; University MDS to assess the role of PD-L1. Hospital Carl Gustav Carus, Dresden, Germany; German Cancer Research Methods Center (DKFZ), Heidelberg, Germany Patients’ CD3+ control cells, CD34+ stem cells, and their autologous Correspondence: Anja Feldmann (a.feldmann@hzdr.de) MDS cell lines (MCLs) were analyzed by DNA and RNA sequencing to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P174 identify somatic variants present in the tumor cells and absent from the control cells. From all somatic variants identified, we generated Background and tested neopeptides in vitro for their ability to induce tumor- Although T-cells genetically modified to express chimeric antigen recep- specific T cell responses. A T cell killing assay was performed to as- tors (CARs) are successfully used to treat hematological malignancies, pa- sess which neopeptide-specific T cells were capable of mediating tients still suffer from several drawbacks of conventional CAR (cCAR) tumor cell lysis (Figure 1A). In parallel, tumor PD-L1 expression levels therapy. CAR-T-cells can cause severe to life-threatening adverse reac- were measured by flow cytometry before and after 24-hour incuba- tions like on-target, off-tumor toxicities which cannot be controlled in pa- tion with tumor-specific autologous T cells. As a control for back- tients. Moreover, cCAR therapy often fails to successfully affect solid ground tumor cell lysis and PD-L1 expression, tumor cells were also tumors and bears the risk to encourage tumor escape variants upon tar- incubated with CEF-specific T cells (CEF: CMV, EBV, and flu peptides). geting of only one single tumor-associated antigen (TAA). In order to Results overcome these problems, we have established a novel on/off-switchable Patients’ tumor cells did not express PD-L1 at baseline. Remarkably, RevCAR system facilitating combinatorial targeting strategies. after co-culture, PD-L1 expression on the tumor cells ranged from Methods 20% to 70% (Figure 1B). Tumor cells, when incubated with CEF- For combinatorial targeting one T-cell has to be modified with two separ- specific T cells, did not upregulate PD-L1, suggesting that PD-L1 ex- ate CARs recognizing different TAAs. The first CAR mediates the activation pression may be linked to target recognition by neoantigen-specific and the second CAR the costimulatory signal. In case of ‘AND’ gate tar- T cells (Figure 2). Interestingly, tumor cell lysis was independent of geting, dual-CAR-T-cells have to recognize both TAAs on the surface of PD-L1 expression on tumor cells (Figure 1C). Additionally, IFNg the target cells to get activated. However, such combinatorial targeting neutralization did not affect PD-L1 expression nor ability to lyse strategies are struggling with several challenges including the adjustment tumor cells (Figure 3). These data show that when tumor cells are in- of signal strength and affinity of both split CARs as well as the CAR size cubated with autologous tumor-specific T cells, the tumor cells up- limiting the number of transduced specificities. In order to overcome regulate PD-L1 expression yet do not escape lysis by T cells. these obstacles, our idea was to construct small RevCARs comprising only Since lysis of tumor cells may occur prior to their upregulating PD-L1, we a small peptide epitope as extracellular domain. By removing the extra- pre-incubated tumor cells with soluble IFNg prior to co-culture with T cells. cellular single-chain variable fragment (scFv) of cCARs, RevCARs avoid Tumor cells were 96% PD-L1+, and were lysed by tumor-specific T cells at tonic signaling induced by scFv dimerization. As RevCARs do not have an the same level of target cells that had not been treated with IFNg (Figure 4). extracellular antigen binding moiety, they cannot bind to any antigen Conclusions per se. Thus, actually they are switched off. Only in the presence of a bis- Collectively, these data lend support to the notion that, in this sys- pecific target module (RevTM), RevCAR-T-cells can be redirected to tumor tem, PD-L1 is not a main player in MDS tumor immune evasion, sug- cells and switched on. Finally, short-living RevTMs allow a repeatedly on/ gesting that tumor immune evasion might function in a PD-L1- off-switch and controllability of RevCAR-T-cells and furthermore a flexible independent way. They also suggest that cognate recognition of redirection of RevCAR-T-cells to any target. tumor cells by neoantigen-specific T cells can cause the upregulation Results PD-L1 on target cells via a yet to be identified mechanism. For proof of concept two small peptide epitopes were selected to Ethics Approval construct the respective RevCARs. Additionally, a series of different The study was approved by the University of California, San Diego's RevTMs was generated recognizing one of the two peptide epitopes Institutional Review Board, HRPP #161345. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 96 of 272 P176 Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) for metastatic uveal melanoma: feasibility of treatment using a product generated from the primary tumor Marie-Andree Forget, PhD, Cara Haymaker, PhD, Orenthial Fulbright, BS, Shawne Thorsen, BS, Esteban Flores, BS, Arely Wahl, MSc, Rene Tavera, BS, Benjamin Tintera, BS, Timothy Woody, Michelle Williams, Yun Shin Chun, Patrick Hwu, MD, Dan Gombos, Sapna Patel, MD, Rodabe Amaria, MD, Chantale Bernatchez MD Anderson Cancer Center, Houston, TX, United States Correspondence: Chantale Bernatchez (CBernatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P176 Background MD Anderson Cancer Center’s tumor-infiltrating lymphocyte (TIL) program has expanded TIL from tumor fragments of cutaneous metastatic melanoma using high dose IL-2 from over 900 patients with a growth success averaging 62% [1]. Surprisingly, this growth success plunges to 45% for metastatic uveal melanoma tumor fragments [2] and less than 20% from primary uveal tu- mors. The reason for this drop is unclear as uveal melanomas have an infiltration of CD8+TIL comparable to cutaneous melan- oma [2], which in theory makes it an attractive candidate for im- Fig. 1 (abstract P175). Ferrari, V SITC 2019 abstract slide 1 munotherapy. However, limited success observed with checkpoint therapy prompted us to explore ex vivo manipulation of the TIL. A previous report demonstrated the feasibility of TIL adoptive cell therapy for metastatic uveal patients using a TIL product ex- panded from metastases [3]. Since the primary and metastatic sites of uveal melanoma dis- play preserved gene mutations, indicating a potential shared antigen landscape, one could propose generating a TIL product from the primary tumor when the patient undergoes enucle- ation and to utilize this product for treatment at time of recurrence. Methods Given the challenge of propagating TIL from a primary uveal tumor in high dose of IL-2 only, we hypothesized that our new TIL3.0 method to propagate TIL from tumor fragments (1st phase of expansion), based on the 3-signals required for optimal activa- tion of a T-cell (TCR engagement, costimulation and cytokine ex- Fig. 2 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 posure) would enable TIL growth from a higher percentage of primary uveal tumors given the success obtained with metastatic sites [1]. This product can be banked and accessed later for treat- ment at recurrence. Results The TIL3.0 expansion platform was shown to be optimal for T-cell propagation allowing for successful expansion of TIL from primary uveal melanoma tumors in >90% of the cases (n=20). This expan- sion was rapid (less than 3 weeks) and consistently composed of CD8+CD3+TIL. This later observation is attributed to the use of the agonistic anti-CD137/4-1BB, Urelumab, as of costimulation sig- nal in our TIL3.0 method. The TIL3.0 method applied to primary tumors could be scaled and adapted for GMP. This process, followed by a rapid expan- sion protocol, was applied to treat the first metastatic uveal pa- tient with a TIL product generated from the primary tumor. The patient was infused with a total of 14.4 billion TIL with a viability of 99%. Conclusions This study demonstrates the feasibility of generating a TIL product from a primary uveal tumor to be used for treatment at recurrence. Trial Registration Fig. 3 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 NCT00338377 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 97 of 272 References Deletion of p50 in patient-derived marrow CD34+ cells and subse- 1. Tavera RJ, Forget MA, et al. Utilizing T-cell Activation Signals 1, 2, and 3 quent production of IMC for adoptive transfer may contribute to the for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over therapy of these and additional cancers, alone or with additional the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma. J immunotherapies. Immunother. 2018; 41:399-405. Ethics Approval 2. Qin Y, de Macedo MP, Reuben A, et al. Parallel profiling of immune The study was approved by the Johns Hopkins University Animal infiltrate subsets in uveal melanoma versus cutaneous melanoma unveils Care and Use Committee, protocol MO19M10. similarities and differences: A pilot study. OncoImmunology. 2017; 6:e1321187. 3. Chandran SS, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two- P178 stage, single-arm, phase 2 study. Lancet Oncol. 2017; 18:792-802. Dual inhibition of PI3Kdelta and PI3Kgamma to enhance Ethics Approval mitochondrial mass and ex vivo expansion of central and stem cell Institutional review board (IRB)-approved protocol# 2004-0069 memory T cells from CLL patients Christopher Funk, BS, , Shuhua Wang, MD, Alexandra Waller, BS, Lauren Fleischer, BS, Aditi Sharma, Harold Spencer, PhD, Vikas Gupta, MD, PhD, P177 Sruthi Ravindranathan, PhD, Mala Shanmugam, PhD, Christopher NF-kB p50-deficient immature myeloid cell (p50-IMC) adoptive Flowers, MD, MS, Edmund Waller, MD, PhD, FACP transfer slows the growth of murine prostate and pancreatic Emory University School of Medicine, Atlanta, GA, United States ductal carcinoma Correspondence: Edmund Waller (ewaller@emory.edu) Rahul Suresh, PhD, David Barakat, PhD, Theresa Barberi, PhD, Lei Zheng, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P178 PhD MD, Elizabeth Jaffee, MD, Kenneth Pienta, MD, Alan Friedman, MD Johns Hopkins University School of Medicine, Baltimore, MD, United Background States For chimeric antigen receptor T-cell (CAR T) therapy to treat Correspondence: Alan Friedman (afriedm2@jhmi.edu) chronic lymphocytic leukemia (CLL), recent work associates remis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P177 sions with infusion of sufficient non-exhausted memory CAR T, capable of oxidative phosphorylation [1]. Our work aims to Background modulate metabolic pathways during ex vivo expansion of T-cells NF-kB p50 binds DNA but, unlike p65, lacks a trans-activation do- for translation of these findings to clinical adoptive therapies. main and recruits co-repressors. Macrophages and dendritic cells Class I catalytic PI3K enzymes, such as PI3Kdelta and PI3Kgamma, lacking NF-kB p50 are skewed towards a pro-inflammatory pheno- regulate T-cell differentiation, regulatory T cell formation, and TCR type, with increased cytokine expression and enhanced T cell acti- signaling [2]. In this study, we hypothesized that pharmacological vation; additionally, murine melanoma, fibrosarcoma, colon inhibition of these pathways during ex vivo culture would in- carcinoma, and glioblastoma grow slower in p50-/- mice. Given crease populations of early memory CAR T with enhanced meta- these data, we evaluated efficacy of p50-deficient immature mye- bolic and survival potential. loid cells (p50-IMC) adoptively transferred into tumor-bearing hosts. Methods Immature cells were utilized to maximize tumor localization, and Healthy- and CLL- donor peripheral blood mononuclear cells pretreatment with 5-fluorouracil (5FU) was examined due to its po- were isolated and cryopreserved. Thawed cells were sorted for tential to impair marrow production of myeloid cells, to target CD3 expression prior to culture (or transduction) and expanded tumor myeloid cells, and to potentially release tumor neoantigens. in G-Rex plates using anti-CD3/CD28 beads, 30 U/mL Methods interleukin-2, and investigational drugs: idelalisib, duvelisib and WT-IMC or p50-IMC were generated by culturing lineage-negative ibrutinib for 9 days. marrow cells from WT or p50-/- mice in media containing TPO, SCF, Results and FL for six days followed by M-CSF for one day on ultra-low at- Class I catalytic enzymes in T-cells, PI3Kdelta and PI3Kgamma, tachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) exhibit domain homology (Figure-1A). Accordingly, maximum cell or K-Ras(G12D) pancreatic ductal carcinoma (PDC)-luciferase cells re- yields for both idelalisib (Figure 1B) and duvelisib (Figure 1C) ceived 5FU followed five days later by three doses of 1E7 IMC every occurred upon inhibition of both isoforms. Comparing doses of three to four days. Some groups also received four doses of anti-PD- duvelisib, idelalisib, and ibrutinib that yielded optimal T-cell 1 antibody twice weekly alone or with p50-IMC. expansion, we showed potent PI3Kdelta/gamma dual antagonism Results maximizes live T-cell yields (Figure 1D) out-performing PCa grew slower in p50-/- mice, and absence of host p50 led to interleukin-2-inducible kinase inhibition. PI3K antagonists in- prolonged survival of mice inoculated orthotopically with PDC. creased frequencies of cells expressing co-stimulatory molecules 5FU followed by p50-IMC slowed PCa and PDC tumor growth ~3- (Figure 2A-B). A dose-dependent increase in cells expressing FAS/ fold in contrast to 5FU followed by WT-IMC, 5FU alone, or p50- FAS-L (Figure 2C) and an increased expression of pro-survival IMC alone. Slowed tumor growth was evident for 93% of PCa tu- BCL-2 (Figure 2D) after anti-CD3/28 stimulation suggests the mors but only 53% of PDC tumors. In PCa, p50-IMC predomin- increase in live cells with a TSCM phenotype is likely due to en- antly generated tumor and draining lymph node F4/80+ hanced cell survival. macrophages, but also CD11b+F4/80-CD11c+ conventional den- Next,weconfirmed thepositiveeffectofdualPI3Kdelta/gamma dritic cells. A subset of tumor and nodal macrophages co- inhibition in expansion of T cells from CLL donors (Figure 3A-B). expressed Ly6C and MHCII and had reduced MR compared to PI3K antagonists increased frequencies of CD8 cells (Figure 3C) host macrophages, collectively indicating a pro-inflammatory and co-stimulatory molecule expressing cells (Figure 3D-E). Inter- phenotype. p50-IMC also produced a 5-fold increase in activated estingly, addition of idelalisib to T cell cultures resulted in a PCa tumor CD8 T cells, and antibody-mediated CD8 T cell deple- dose-dependent decrease in immune checkpoint molecules LAG- tion obviated slower tumor growth induced by 5FU followed by 3, Tim-3, and PD-1 (Figure 3F-J). Given the importance of T cells p50-IMC. Anti-PD-1 markedly slowed PCa growth but had little ef- with the stem cell memory (TSCM) phenotype in adoptive T-cell ficacy against PDC, whereas anti-PD-1 combined with p50-IMC therapy, T-cell differentiation was studied. PI3K antagonists in- slowed PDC tumor growth to prolong survival more effectively crease the frequency of early, T-memory, and effector memory than either alone in an initial experiment. cells (Figure 4A). Notably, the frequency of TSCM doubled (Figure Conclusions 4B). Lastly, PI3K antagonists significantly increased the mitochon- 5FU followed by p50-IMC slows the growth of murine prostate and drial mass (Figure 4C) within total CD3 (Figure 4D) and CD8 (Fig- pancreatic ductal carcinoma and depends upon CD8 T cell activation. ure 4E) subsets. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 98 of 272 Conclusions Dual inhibition of PI3Kdelta and PI3Kgamma modulates aspects of T-cell biology relevant to CAR T remissions. PI3Kdelta/gamma antagonism enhances CD27 expression and mitochondrial mass, decreases immune checkpoint expression, and enriches the TSCM phenotype. Acknowledgements The authors thank the patients and healthy volunteers for their blood donations. CR Funk is supported by a Howard Hughes Medical Research Fellowship. The authors thank Emory’s Clinical Lymphoma Research Team for requesting patient consent and aiding in sample collection. References 1. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 2018; 24:563-571. [2]2. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, and Abraham RT. Leading Edge The PI3K Pathway in Human Disease. Cell. 2017; 170:605-635. Ethics Approval The study was approved by Emory University's Institutional Review Committee, approval number 00057236. Fig. 3 (abstract P178). See text for description Fig. 1 (abstract P178). See text for description Fig. 4 (abstract P178). See text for description P179 Checkpoint Cbl-b siRNA-based APN401 adoptive cell therapy: superior efficacy & immune memory induction in murine hepatocellular carcinoma following APN401 monotherapy and synergism with anti-PD1 1 1 1 Anderson Gaweco, MD, PhD , Kathrin Thell , Maria Urban , Julia 1 1 2 3 Harrauer , Isabella Haslinger , Josef Penninger , Vincent Chung , 4 5 Anthony El-Khoueiry, MD , Carlos Becerra, MD 1 2 Apeiron Biologics, Vienna, Austria; University of British Columbia, 3 4 Vancouver, BC, Canada; City of Hope, Duarte, CA, United States; USC Norris Comprehensive Cancer Center, Los Angeles, CA, United States; Baylor University Medical Center, Dallas, TX, United States Correspondence: Anderson Gaweco (anderson.gaweco@apeiron- biologics.com) Fig. 2 (abstract P178). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P179 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 99 of 272 Background combination of immunomodulatory cytokines: IL12 and IL21. Upon The intracellular master checkpoint Cbl-b, an E3 ubiquitin ligase, administration, SENTI-101 innately homes to peritoneal tumors, se- negatively regulates the innate and adaptive anti-tumor immune re- cretes IL12 and IL21 in a localized and sustained fashion, and induces sponses. Selective cell-based targeting of Cbl-b not only induces a robust anti-tumor immune response. anti-tumor activity in vivo but also overrides immune regulation by Methods the PD-L1/PD-1 pathway in vitro [1]. Human APN401 is an autologous Two syngeneic pre-clinical models of disseminated peritoneal carcin- adoptive cellular therapy of ex vivo Cbl-b-silenced human PBMCs omatosis with distinct immune phenotypes were established by currently in a clinical Phase 1b multiple dose study demonstrating implanting cells in the peritoneal cavities of mice (CT26-fLUC = early clinical safety and tolerability in patients with advanced solid immune-inflamed; B16-F10-fLUC = immune-excluded) [3]. A library of tumors. Herein the preclinical Proof of Concept efficacy of murine over 50 murine MSC lines engineered to express immune effectors APN401 immunotherapy is established in the syngeneic mouse hepa- (cytokines, chemokines, growth factors), either individually or in com- tocellular carcinoma Hepa1-6 tumor rechallenge study. bination, was administered intraperitoneally and evaluated for anti- Methods tumor activity via bioluminescence and tumor weight measurements. Hepa1-6-C57/BL6-tumor bearing mice were treated for 19 days with Immune phenotype was characterized by flow-cytometry and multi- murine APN401, ex vivo silenced immune cells with Cbl-b specific plexed immunohistochemistry. siRNA, as monotherapy or in combination with anti-PD1 (clone Results RMP1-14) versus control siRNA on D7 post-inoculation. Mice were MSCs expressing the combination of IL12 and IL21 (SENTI-101) were se- later rechallenged on D29 with Hepa1-6 s.c. on the contralateral flank lected based on significant tumor-burden reduction and immune pro- in the absence of any further APN401 treatment. file changes in both syngeneic models. Notably, the combination Results outperformed each individual cytokine in extending survival (p=0.02). Significant tumor growth inhibition (TGI) was observed following 19 Intraperitoneal administration of SENTI-101 into tumor-bearing mice days of treatment with APN401 alone or in combination with anti- led to preferential co-localization with tumors (>10-fold higher vs. PD1 starting on D7 after s.c. inoculation (p<0.0001). Following rechal- normal tissues, p=0.001). Local concentrations of IL12 and IL21 were lenge, significant TGI of 86% (p<0.001) was observed in prior ~100-fold greater in the peritoneal space vs. serum (p=0.002). SENTI- APN401-treated mice versus control siRNA-treated mice. Mice that re- 101 treatment reduced tumor-burden more than 200-fold (p50% of ceived APN401 in combination with anti-PD1 prior to rechallenge the mice were tumor-free after 90 days, while control groups and demonstrate profound synergistic anti-tumor efficacy (p<0.001) ver- groups treated with anti-PD1 antibody had a median survival of 21 sus anti-PD1 alone with control siRNA. APN401 was well tolerated to 30 days. Surviving mice were able to reject newly implanted and APN401-treated mice were unremarkable with optimal body tumor cells, demonstrating anti-tumor immune memory. conditions. Anti-tumor effects of SENTI-101 are mediated by a multi-modal im- Conclusions mune response. The frequency of antigen-presenting cells in periton- APN401 monotherapy demonstrates striking anti-tumor efficacy in eal tumor-draining lymph nodes was more than doubled vs. controls the murine hepatocellular carcinoma Hepa1-6 model. The preclinical (p=0.01). This correlated with increased T-cell and B-cell tumor infil- synergistic effects of APN401 with anti-PD1 support its therapeutic trates forming tertiary-lymphoid structures, which are associated with utility as a combination therapy with immune checkpoint anti-PD1 improved prognosis in cancer [4]. T-cell activation markers (CD38, treatment. The significant TGI observed following tumor rechallenge IFNg, GranzymeB) were significantly increased locally. indicate that prior selective cell-based Cbl-b-silencing with APN401 Conclusions alone or in combination with anti-PD1 induced systemic and durable SENTI-101 induces localized immune-modulation, regulates multiple anti-tumor immune memory responses. These findings highlight the steps of the cancer immunity cycle, and results in durable anti-tumor potential promise of a selective adoptive cell-based Cbl-b silencing responses. These data warrant further development of SENTI-101 for by APN401 as a novel immunotherapy for cancer. the loco-regional treatment of advanced solid tumors. Reference References 1. Fujiwara M, Anstadt EJ, Clark RB. Cbl-b Deficiency Mediates Resistance to 1. Desai JP, Moustarah F. Cancer, Peritoneal Metastasis. StatPearls Programmed Death-Ligand 1/Programmed Death-1 Regulation. Front Publishing. 2019 [Updated 2019 Jun 30] Immunol. 2017 Jan 26;8:42. 2. Lengyel E. Ovarian Cancer Development and Metastasis. Am J Pathol. 2010; 177(3): 1053–1064 3. Mosely SI, Prime JE, Sainson RC, et al. Rational Selection of Syngeneic P180 Preclinical Tumor Models for Immunotherapeutic Drug Discovery. Cancer SENTI-101, an allogeneic cell product, induces potent and durable Immunol Res. 2017; 5(1):29-41 anti-tumor immunity in pre-clinical models of peritoneal 4. Sautès-Fridman C, Petitprez F, Calderaro J, Fridman WH. Tertiary carcinomatosis lymphoid structures in the era of cancer immunotherapy. Nat Rev Alba Gonzalez Junca, PhD, Gary Lee, PhD, Archana Nagaraja, PhD, Alyssa Cancer. 2019;19(6):307-325 Mullenix, Russell Gordley, PhD, Daniel Frimannson, PhD, Anissa Benabbas, Chen-Ting Lee, PhD, Tiffany Truong, Allison Quach, Mengxi Tian, Rishi Savur, Rowena Martinez, Alyssa Perry-McNamara, Don-Hong P181 Wang, PhD, Ori Maller, PhD, Dharini Iyer, PhD, Ashita Magal, Christina Multi-phenotype CRISPR-Cas9 screens identify p38 kinase as a Huynh, Carmina Blanco, Jack Lin, PhD, Brian Garrison, PhD, Philip Lee, target for adoptive immunotherapies PhD, Timothy Lu, MD, PhD, Sravani Mangalampalli Devikala Gurusamy, PhD, Suman Vodnala, Amanda Henning, Rigel Senti Biosciences Inc., South San Francisco, CA, United States Kishton, Tori Yamamoto, Arash Eidizadeh, Li Jia, Christine Kariya, Mary Correspondence: Gary Lee (gary.lee@sentibio.com) Black, Robert Eil, Douglas Palmer, Zhiya Yu, Jenny Pan, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P180 Madhusudhanan Sukumar, Shashank J. Patel, Nicholas Restifo, MD National Institutes of Health, Bethesda, MD, United States Background Correspondence: Nicholas Restifo (restifo@nih.gov) More effective therapies for disseminated peritoneal carcinomatosis, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P181 including high-grade serous ovarian cancer, remain a major medical need [1]. Although several treatments offer initial responses to local- Background ized disease, patients with disseminated peritoneal tumors face poor Adoptive T cell transfer immunotherapy (ACT) using tumor-infiltrating overall survival [2]. lymphocytes (TIL) and gene-modified T cells can induce complete and SENTI-101 is a novel therapeutic agent comprising allogeneic mesen- durable regression of metastatic human malignancies that are other- chymal stromal cells (MSCs) genetically modified to express a potent wise refractory to treatment. While successful T cell-based treatments Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 100 of 272 of patients with widely metastatic melanoma, synovial sarcoma, cholan- concentration of 1 x 106 cells/ml with high concentration of hIL-2 giocarcinoma and cancers of the breast, colon, and cervix have been and 5% UltraGRO® for 14 days in our original closed system. Then, reported in recent years, most patients with common epithelial cancers we confirmed the expression of surface markers, CD107a fail to respond to treatment. While several factors can contribute to the mobilization and cell-mediated cytotoxicity against various tumor efficacy of ACT, a major inherent limitation is the induction of termin- cells and normal cells with or without monoclonal antibody drugs ally differentiated phenotype coupled with the loss of proliferative cap- in vitro and antitumor effects against peritoneal dissemination model acity in TIL during current ex vivo expansion protocols. Individual gene using SKOV3 in vivo. knockout approaches for enhancing T cell-based cancer immunother- Results apies are low-throughput and can improve one desired function (T cell [Results and Discussion] Importantly, we’ve found that our GAIA-102 memory) at the expense of another equally important function (expan- exhibited CD3-/CD56bright/CD57- immature phenotype that could sion). Thus, there is significant interest in identifying T-cell intrinsic kill various tumor cells efficiently from various origins, including Raji negative regulatory circuits that limit their ability to expand robustly cells that was highly resistant to NK cell killing. More importantly, ex vivo, while dampening their terminal effector differentiation along massive accumulation, retention, infiltration and sphere destruction with the reduction of oxidative stress and genomic damage. by GAIA-102 were affected neither by myeloid-derived suppressor Methods cells nor regulatory T-lymphocytes. GAIA-102 was also effective To identify the T cell intrinsic negative regulatory circuits, we developed a in vivo to murine model of peritoneal dissemination of human ovar- multi-phenotype genetic screen to systematically target 29 major kinases ian cancer. screen to concurrently measure the impacts of individual gene knockouts Conclusions on T cell expansion, differentiation, oxidative stress and genomic stress. Thus, these findings indicate that GAIA-102 has a potential to be an Using CRISPR-Cas9-based gene perturbation combined with high- ‘upward compatible’ modality over CAR-T strategy, and would be a throughput flow cytometry, we developed and validated a multi-phenotype new and promising candidate for adoptive immunotherapy against screen, which identified Mapk14/p38 kinase as a target that improved all solid tumors. We now just started GMP/GCTP production of this new four phenotypes in CD8+ T cells. We used murine and human ex vivo T cell and powerful NK cells and first-in-human clinical trials in use of expansion models to validate the results from our genetic screen. GAIA-102 will be initiated on 2020. Results Ethics Approval Results from our genetic screen identified p38 kinase as a unique [Ethics Approval] Written informed consent was obtained from all multi-phenotypic regulator of cellular differentiation, oxidative, and healthy volunteers, in accordance with the Declaration of Helsinki. genomic stress while achieving improved cellular expansion. Further- Upon the approval of the institutional ethical committee (ap- more, pharmacological inhibition of p38 kinase in murine and human proval no. 29-315) of Kyushu University, peripheral blood samples ex vivo T cell expansion models validated the results from our gen- were collected from healthy volunteers. The animal experiments etic screen. Cells cultured in the presence of a p38 inhibitor had in- were reviewed and approved by the Institutional Animal Care creased capacity for cytokine production, specifically interferon-γ and and Use Committee of Kyushu University (approval nos. A30-234- demonstrated improved in vivo persistence. Additionally, cells cul- 0 and A30-359-0). tured in the presence of the p38 inhibitor demonstrated enhanced in vivo cell-expansion, tumor infiltration, and anti-tumor efficacy in P183 an immunocompetent tumor mouse model. Development of novel chimeric antigen receptor T cells for Conclusions immunotherapy of hepatocellular carcinoma This study establishes p38 inhibition in T cells as a potentially import- Yukai He, PhD, Leidy Caraballo Galva, Xiaotao Jiang ant strategy for improving ACT immunotherapy for cancer patients. Augusta University, Augusta, GA, United States Ethics Approval Correspondence: Yukai He (yhe@augusta.edu) All human samples were isolated in accordance with approved clin- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P183 ical protocols and in accordance with NIH institutional review board approval and informed consent from patients and healthy donors. Background Immunotherapy has a great potential for hepatocellular carcin- P182 oma (HCC). Several human glypican 3 (hGPC3)-specific chimeric GAIA-102: a new class NK cell-like phenotype manufactured in antigen receptor T cells (CARTs) are being tested for HCC. But, accordance with GMP/GCTP that can eliminate solid tumors most, if not all, are constructed from one monoclonal antibody Yui Harada, PhD, Yoshikazu Yonemitsu, MD, PhD (mAb). It is unknown whether targeting different epitopes of Kyushu University, Fukuoka, Japan hGPC3 will create more effective CARTs. Here, we aim to develop Correspondence: Yui Harada (rkfraile@med.kyushu-u.ac.jp) novel CARTs that target different regions of hGPC3. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P182 Methods BalB/C mice were immunized with hGPC3 protein. Hybridomas Background and mAbs were generated and characterized. Then, CARTs were [Background] Cancer immunotherapy has been established as a new built from the novel mAbs and their antitumor effect was therapeutic category since the recent success of immune checkpoint studied. inhibitors and a type of adoptive immunotherapy, namely chimeric Results antigen receptor-modified T cells (CAR-T). Although CAR-T demon- Twenty-two hGPC3-specific mAbs were identified by ELISA. Out of strated impressive clinical results, serious adverse effects (cytokine them, 14 bound HepG2 cells. Five mAbs were further character- storm and on-target off-tumor toxicity) and undefined efficacy on ized by immunohistochemical staining. Three of them (6G11, 8F8, solid tumors are important issues to be solved. We’ve developed a and 12D7) were found to specifically stain HCC tumor but not cutting-edge, simple, and feeder-free method to generate highly acti- adjacent normal tissues. The 3 mAb’s affinity were in the nano- vated and expanded human NK cells from peripheral blood molar range. 6G11 and 8F8 bound to hGPC3 epitope aa25-39 (US9404083, PCT/JP2018/018236, PCT/JP2019/012744), and have and aa463-496, respectively. No specific epitope was identified been conducting further investigation why our new type of NK cells, for 12D7 though it bound to the N-fragment (25-358aa). CARTs named as GAIA-102, are so effective to kill malignant cells. built from the 3 mAbs underwent expansion in response to Methods HepG2 cell stimulation. However, their effector function was sig- [Materials and Methods] Cryopreserved PBMCs purchased from nificantly different. 8F8 CARTs possessed the strongest effector HemaCare Corporation were mixed and processed by using LOVO function. 6G11 CARTs generated the greatest expansion, but with and CliniMACS® Prodigy (automated/closed systems). CD3+ and slightly weaker function. In contrast, 12D7 CART had the weakest CD34+ cells were depleted, and the cells were cultured at a effector function. Soluble hGPC3 did not activate CARTs, nor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 101 of 272 blocked CART activation by tumor cells. Adoptive transfer of 8F8 P185 and 6G11, but not 12D7, CARTs generated potent antitumor ef- T cell antigen presenting cell (tAPC) is a strategy to induce CAR T fects with complete regression of HCC xenografts, which corre- expansion in vivo in the absence of a tumor for on-target toxicity lated to their expansion in vivo. studies Conclusions Yen Ho, BS, Jon Jones, BA, Patrick Carlson, BS, Cyr de Imus, The three novel CARTs that target different hGPC3 regions pos- Rebekah Turk, Dina Alcorn, Kyle Kolaja, Ruth Salmon, PhD, Thomas sess significantly different effector function and antitumor effects. Long Adoptive transfer of CARTs targeting the hGPC3 N- or C-epitope Celgene Corporation, Seattle, WA, United States results in complete eradication of HCC xenografts. Correspondence: Thomas Long (thomas.long@junotherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P185 P184 Background Activating marrow infiltrating lymphocytes in hypoxia enhances Preclinical safety evaluation of chimeric antigen receptor (CAR) their efficacy in adoptive T-cell therapy T cells presents a number of unique challenges. One of those 1 1 1 1 Megan Heimann , Ervin Griffin , Luca Biavati, MD , Amy Thomas , challenges is the development ofaCynomolgus macaquetoxi- 1 1 2 Danielle Dillard , Elizabeth Zawidzka , Brianna Richardson , Robert cology model as a tool to understand potential on-target CAR T 1 2 3 1 Leone , Gregory Szeto , Kimberly Noonan, PhD , Ivan Borrello, MD cell toxicity. This is important for CAR T cell programs where Johns Hopkins University School of Medicine, Baltimore, MD, United the lead binder is cyno cross-reactive and the target has known States; University of Maryland Baltimore County, Baltimore, MD, United normal tissue expression conserved across species. Unlike many States; WindMIL Therapeutics, Baltimore, MD, United States mouse models, non-human primates lack target-expressing Correspondence: Ivan Borrello (iborrell@jhmi.edu) tumors that can drive CAR T cell activation and expansion; it Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P184 would be advantageous to recapitulate that expansion in the context of a toxicity model. Background Methods Marrow infiltrating lymphocytes (MILs) are a promising candidate for T cell antigen presenting cells (tAPCs) have been reported to adoptive cell therapy (ACT) due to their broader anti-tumor specifi- drive measurable CAR T cell expansion in Rhesus macaques [1]. city and persistence. These characteristics are due to intrinsic proper- The advantages of this strategy include the ease of manufactur- ties of the bone marrow (BM); known to be a reservoir for long-lived ing tAPCs in parallel to CAR T cells and co-engraftment of memory T cells. It has also been established that naïve and memory tAPCs with CAR T cells in hematological niches to ensure anti- T cells are metabolically quiescent, favoring oxidative phosphoryl- gen availability. The optimal tAPC dosing strategy to drive a ation (OXPHOS) over glycolysis, while effector T cells favor glycolysis strong and persistent CAR T cell activity has not been deter- to fuel their rapid proliferation. mined. Here, we generated human anti-CD19 CAR T cells Methods expressing firefly luciferase and human T cells expressing a We examined how activation and expansion of MILs in hypoxia truncated human CD19 (CD19t) lacking the intracellular domain could be used to better understand the inherent properties of as tAPCs. We then dosed mice either with CAR and tAPC con- the BM, to exploit these properties, and enhance the efficacy of currently, or with CAR first then followed by tAPCs three days MILs in ACT, especially when compared to that of peripheral later. blood lymphocytes (PBLs). By activating MILs in hypoxia, we can Results select for and/or alter the cells best suited to mount an effective (Figure 1) shows bioluminescent imaging (BLI) measurements anti-tumor response. that indicate concurrent and delayed tAPC dosing have similar Results CAR T cell expansion kinetics; however, the delayed tAPC Activation under hypoxic conditions alters MILs in several unique dosing exhibits a greater magnitude of CAR T cell expansion. In ways. MILs show greater overall expansion, enhanced tumor- both cases, the CAR T cell expansion occurs in a tAPC dose- specificity, and a unique metabolic profile—upregulating both dependent manner. Imaging shows that concurrent dosing OXPHOS and glycolytic machinery. This metabolic profile suggests leads to CAR T cell proliferation primary in the lungs, whereas that hypoxia-activated MILs possess properties of both effector delayed tAPC dosing leads to more systemic CAR T cell and memory cells. PBLs grown under the same conditions fail to expansion. In addition, flow cytometry data show a significant expand significantly and show no metabolic differences or specifi- depletion of tAPCs in the peripheral blood between day 7 and city. Following activation in hypoxia we have found that MILs day 14. have upregulated metabolism-related genes such as CPT1A and Conclusions GLUT1 and 3, as well as anti-apoptotic factors such as BCL2 and Follow-up studies delivering additional tAPC doses three, six, BCL2L1 at an RNA level. The post-expansion MILs product, using and nine days after CAR T cells show that repeated tAPC ad- intracellular staining and FACs analysis, shows an increase in ministration can significantly increase the CAR T cell exposure mitochondrial proteins, including TOMM20, CPT1a, and SDHa, in- over time compared to a single tAPC dose. Overall, these data creased mTOR signaling, and increased glycolytic machinery—HK2 demonstrate that tAPCs can be used to induce CAR T cell ex- and GLUT1. Additionally, while cell-cycle analysis with Ki67 and PI pansion in vivo in the absence of a tumor and will enable us shows that MILs are more resting at baseline and arrested in G0, to design a tAPC strategy for use in a Cynomolgus macaque upon activation in hypoxia they become more proliferative—mov- model to evaluate the safety of CAR T cell candidates. ing into S and G2/M phase—than normoxic MILs or PBLs and maintain this phenotype over time. This finding is supported by our RNAseq data showing a lack of transcriptional activity at baseline but a significant increase by day 3 of activation. Gene Reference expression analysis has also shown that there is a substantial in- 1. Berger C, Sommermeyer D, Hudecek M, Berger M, Balakrishnan A, crease in expression of IL2 and IL15Ra with a significant decrease Paszkiewicz PJ, Kosasih PL, Rader C, Riddell SR. Safety of targeting in the expression of IL10 transcripts. ROR1 in primates with chimeric antigen receptor-modified T cells. Conclusions Cancer Immunol Res. 2015;3(2):206-16. These findings suggest that hypoxia contributes to the unique Ethics Approval properties of MILs through modification of their metabolic profile All animal studies were conducted in accordance with protocols approved and is being uniquely employed to generate more effective MILs, by the Institutional Animal Care and Use Committee. and not PBLs, for adoptive cell therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 102 of 272 P187 Single-cell RNA sequencing and functional assessment of healthy donor- and cancer patient-derived T and CAR-T cells Zinkal Padalia, MS, Konstantinos Karagiannis, PhD, Brigid Mcewan, MA, Vahan Simonyan, PhD, Jonathan Terrett, PhD, Demetrios Kalaitzidis, PhD CRISPR Therapeutics, Cambridge, MA, United States Correspondence: Demetrios Kalaitzidis (d.kalaitzidis@crisprtx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P187 Background Autologous chimeric antigen receptor T (CAR-T) cell therapies have shown remarkable success in treating relapsed/refractory B-cell ma- lignancies. However, even in indications with high complete re- sponse rates, not all patients respond or have durable responses after CAR-T treatment. Furthermore, autologous CAR-T treatments have not yielded the same impressive outcomes in solid malignan- cies to date. A major limitation of autologous CAR-T therapy may be the dysfunctional state of a patient’s T cell populations used for manufacturing of a drug product. Allogeneic therapeutics can bypass this limitation by enabling the use of healthy donor starting material. Moreover, healthy donor material that exhibits specific T cell attri- butes can be selected for drug product manufacturing. Methods Fig. 1 (abstract P185). See text for description To identify attributes that can be associated with improved perform- ance of CAR-T cells we have characterized T cells from healthy do- nors as well as cancer patients, in particular from chronic P186 lymphocytic leukemia (CLL) patients as these have been described Evaluation of antigen-specific T-cell immunity at the single cell previously to be dysfunctional. level using large panels of DNA barcoded MHC multimers Results 1 2 2 Kivin Jacobsen, PhD , Dagmar Walter , Michael Stubbington , Katherine We show impaired function of cancer patient-derived CAR-T cells 2 1 1 2 Pfeiffer , Charlotte Halgreen , Liselotte Brix, PhD , Stephane Boutet , when compared to healthy donor-derived cells utilizing both in vitro Kivin Jacobsen, PhD and in vivo assays. We have performed single-cell RNA sequencing 1 2 Immudex, Copenhagen, Denmark; 10x genomics, Pleasanton, CA, (scRNA seq) on both starting material T cells and CAR-T cells from United States multiple healthy and CLL donors used in functional assays to uncover Correspondence: Liselotte Brix (lb@immudex.com) both gene expression and population differences associated with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P186 CAR-T cell performance. scRNA seq analysis revealed marked hetero- geneity among starting populations as well as CAR-T lots from the Background cancer patient-derived T cells. Identification of disease-specific T-cell epitopes is key to developing Conclusions novel cancer vaccines and immunotherapies. Profiling disease-specific Our analysis has allowed us to associate distinct cellular subpopula- T cells, emerging during an induced cellular immune response is im- tion and gene expression profiles with preclinical functional outputs. portant for understanding anti-tumor immunity and guide personalized therapy. The MHC dCODE™Dextramer® technology enables simultan- P188 eous screening of high numbers of T-cell specificities in the same sam- Enhanced anti-tumor activity of human placental CD34+ derived ple using MHC multimer-specific DNA barcodes and next generation natural killer cells in combination with ACY-241 for multiple sequencing as readout. Combining this technology with 10x Genomics myeloma immunotherapy Chromium single cell assay further enables the simultaneous analysis of Lin Kang, PhD, Xiaokui Zhang, Shuyang He, Vanessa Voskinarian-Berse, antigen specific T-cells, sequencing of the cognate T-cell receptors and Bhavani Stout, Valentina Rousseva, William van der Touw, James Edinger, single cell gene expression profiling. Robert Hariri Methods Celularity Inc., Warren, NJ, United States Panels of up to 50 dCODE™Dextramer® reagents were used for screening Correspondence: Xiaokui Zhang (xiaokui.zhang@celularity.com) antigen-specific T-cells in human blood samples. Single cell sequencing was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P188 performed on the isolated T-cell subpopulations, and their gene expression profile analyzed in combination with cell phenotype and TCR sequences. Background Results Celularity, Inc. is developing human placental CD34+ derived, off-the- The experiment generated a large dataset and we show one example of shelf, and allogeneic natural killer (PNK) cells for various hematologic how such a dataset can be analyzed to generate useful information. By com- malignancies and solid tumors. ACY-241 is an orally bioavailable and bining the gene expression profile, cellular phenotypes and Dextramer specifi- selective histone deacetylase (HDAC) 6 inhibitor in MM clinical devel- city we identified expanded populations of antigen-specific T cells in the opment. ACY-241 has been shown to sensitize MM cells to endogen- memory T cell compartment and characterized their individual TCR clono- ous NK cell killing [1]. Here, we investigated the potential types based on TCR sequence. pMHC-specific T-cells were also detected in augmentation of PNK mediated anti-MM activity by ACY-241 the naïve T cell compartment, showing a more diverse TCR sequence profile. treatment. Conclusions Methods This experiment demonstrates a novel method of exploring antigen- Placental CD34+ cells were cultivated in the presence of cytokines in- specific T-cell responses. Linking TCR sequences with pMHC specifi- cluding thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 city, cellular phenotypes and gene expression at this scale and reso- days to generate PNK cells. MM cell lines were treated with different lution provide a more comprehensive analysis of the antigen-specific doses (0, 0.1, 0.3, 1, 3, 10 and 30μM) of ACY-241 over 24h, 48h, or T cell response than previously available in a single workflow. Novel 72h. Cytotoxicity of PNK against different doses of ACY-241 pre- biomarkers and improved strategies of T cell based immunothera- treated MM cell lines was assessed by a PKH26/TO-PRO-3 FACS based peutic development will result from T cell analysis at this scale and assay. Ligands to NK activating receptors of ACY-241 treated MM resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 103 of 272 cells were evaluated by flow cytometry. The RPMI8226 subcutaneous- surface, as well as release of damage-associated molecular patterns ly(SubQ) xenografted NOD scid gamma (NSG) mouse model was including HMGB1 and ATP. Moreover, we show that TTFields treated used for in vivo efficacy study. cells promote phagocytosis by DCs, DCs maturation in vitro, and pro- Results mote immune cells recruitment in vivo. We also show that the com- ACY-241 treatment of MM cell lines for >48h resulted in significant bined treatment of TTFields plus anti-PD-1 led to a significant inhibition of cancer cell growth and decreased cell viability at doses decrease in tumor volume and significant increases in CD45+ tumor >10μM. In a dose dependent manner, ACY-241 further enhanced infiltrating cells in both tumor models. In the lung tumors, these infil- cytotoxicity of PNK against MM cells. In a 4h cytotoxicity assay at ef- trating cells, specifically macrophages and DCs, demonstrated upreg- fector to target (E:T) ratio of 10:1, relative to vehicle control, PNK (n= ulation of surface PD-L1 expression following short treatment 3 donors) showed increased cytotoxicity to ACY-241 pretreated MM duration. Correspondingly, cytotoxic T-cells isolated from these cell lines: RPMI8226 (16.3% to 34.9%), MM.1S (15.1% to 26.0%), OPM2 tumors have shown higher levels of IFN-γ production relative to (12.7% to 37.2%), and U266 (0% to 9.1%). Increased expression of li- untreated mice. In the colon cancer tumors, significant increases in gands to activating NK receptors, MIC A/B, CD56, CD54, and CD155 T-cell infiltration was observed following long treatment duration was detected from ACY-241 treated MM cells, suggesting that en- with TTFields plus anti-PD-1. gagement of NKG2D, CD11a or DNAM-1 of NK cells leads to en- Conclusions hancement of the anti-MM effect. Our results demonstrate the potential of TTFields therapy to induce In vivo anti-MM activity of PNK in combination with ACY-241 was ICD. We also demonstrate robust efficacy of concurrent application assessed in a RPMI8226 SubQ xenograft NSG model. Single intraven- of TTFields and anti PD-1 therapy in mouse models of cancer. These ous dosing of 1.0E7 PNK in combination with ACY-241 significantly data suggest that combining TTFields with anti-PD-1 might achieve reduced the tumor growth compared to vehicle control (P<0.001). tumor control by further enhancing antitumor immunity. Conclusions Our data demonstrated that enhanced in vitro anti-MM activity of Acknowledgements PNK in combination with ACY-241. In vivo efficacy of PNK in combin- The authors would like to thank Dr. Kenneth Swanson from Beth Israel ation with ACY-241 was further demonstrated in a RPMI8226 SubQ Deaconess Medical Center, and Dr. Ilan Volovitz from Tel Aviv Sourasky xenograft NSG model. Taken together, our results demonstrate the Medical Center for their helpful and constructive comments. synergistic effects of combining an HDAC inhibitor with an NK cell Ethics Approval therapy for anti-MM enhancement. Further development of a com- This study was approved by Novocure’s Ethics Board and by the Israel binatorial PNK and ACY-241 therapy for MM treatment is warranted. National Ethics Board; approval numbers 160816, 21015, IL-17-3-131 and IL- 19-1-38. Reference 1. Ray A, Das DS, Song Y, Hideshima T, Tai YT, Chauhan D, Anderson KC. P190 Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 anti- Single-day CAR manufacturing platform using mRNA and Flow body enhances anti-tumor immunity and cytotoxicity in multiple mye- Electroporation Technology loma. Leukemia. 2018 Mar;32(3):843-846. 1 1 1 1 Michael Kuo , Robert Keefe, PhD , Linhong Li, PhD , Angelia Viley , Mary 1 2 3 Loveras , Brian Mulhern , Melanie Hartsough , Claudio Dansky Ullmann, 4 1 P189 MD , Dhana Chinnasamy 1 2 Tumor Treating Fields (TTFields) induce immunogenic cell death MaxCyte Inc, Gaithersburg, MD, United States; Scilucent, Washington resulting in enhanced antitumor efficacy when combined with DC, United States; Hartsough Nonclinical Consulting, Gaithersburg, MD, anti-PD-1 therapy United States; MaxCyte, Cambridge, MA, United States 1 1 1 1 Noa Kaynan, PhD , Tali Voloshin , Shiri Davidi , Yaara Porat , Anna Correspondence: Dhana Chinnasamy (dhanac@maxcyte.com) 1 1 1 Shteingauz , Mijal Munster , Rosa Schnaiderman , Catherine Tempel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P190 1 1 1 1 1 Brami , Yaniv Alon , Einav Zeevi , Karnit Gotlib , Roni Blat , Orni Tal 1 1 1 1 Yitzhaki , Shay Cahal , Aviran Itzhaki , Eilon Kirson , Uri Weinberg, MD Background 1 2 1 1 PhD , Adrian Kinzel , Yoram Palti , Moshe Giladi MaxCyte has developed a rapid and potent cell therapy that uti- 1 2 Novocure Ltd., Haifa, Israel; Novocure GmbH, Munich, Germany lizes mRNA in transient Flow Electroporation (FEP) to produce Correspondence: Moshe Giladi (mgiladi@novocure.com) gene-modified cell products, termed CARMA™. This proprietary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P189 CARMA platform modifies peripheral blood mononuclear cells (PBMC) from apheresis to generate a cryopreserved drug product Background in a single-day manufacturing process using the cGMP-compliant, Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic closed MaxCyte GT® Transfection System, dramatically reducing treatment modality delivered via noninvasive application of low-intensity, the labor, facilities investment, and cost of raw materials typically intermediate-frequency, alternating electric fields. In this study we evalu- required for such products. The CARMA one-day manufacturing ated whether TTFields-induced cell death can be immunogenic and process using cGMP grade mRNA has the potential to therefore suitable for combination with anti-PD-1 therapy. revolutionize cell therapy strategies by significantly reducing the Methods wait time for patients receiving treatment. Cancer cells were treated with TTFields using the inovitro(TM) sys- Methods tem. Immunogenic cell death (ICD) was characterized by the expos- We report here the implementation of the CARMA platform to ure of calreticulin on the cell surface, secretion of ATP, and release of manufacture MCY-M11, a PBMC cell therapy product expressing HMGB1. For detection of ER stress, phosphorylation of eIF2α was an anti-mesothelin chimeric antigen receptor (Meso-CAR) de- assessed. TTFields effect on autophagy was evaluated using electron signed to target mesothelin-expressing solid malignancies. MCY- microscopy, and evaluation of LC3. Bone marrow derived dendritic M11 expresses the Meso-CAR in all cells in the PBMC preparation, cells (DCs) were co-incubated with TTFields treated cells and phago- which are processed and cryopreserved without the need for cytosis by DCs and DCs maturation were evaluated. The combination prior activation or selective expansion. MCY-M11 for clinical appli- of TTFields and anti-PD-1 was evaluated in short duration treatment cation is manufactured under the appropriate cGMP quality sys- protocol in orthotopic lung cancer model and long duration treat- tems and controls by MaxCyte at HCATS, a Contract Development ment protocol in subcutaneous colon cancer model. Analysis of infil- Manufacturing Organization (CDMO). Manufacturing release speci- trating cells was performed using flow cytometry. fications are preliminarily assigned, with multiple For Information Results Only (FIO) data points being accumulated during clinical produc- We demonstrate that cancer cells that die during TTFields application tion, while sufficient clinical data is being generated to establish exhibit ER stress leading to calreticulin translocation to the cell meaningful release criteria. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 104 of 272 Results lymph nodes. Given the plasticity of Th17 and Treg cells, we A total of 20 CARMA product development and engineering runs assessed FoxP3 expression within the cell product 10 days post were performed during the technology transfer campaign, with transfer and found that the frequency of FoxP3+ transferred cells analytical test methods and supply chain established. The viable was significantly heightened through IL-6 blockade. cell yield from pre-FEP to post-FEP samples averaged around Conclusions 92%. Meso-CAR expression in T and NK cell subsets in MCY-M11 IL-6 induced by Th17 cell therapy promotes an inflammatory ranged from 42-83% (average 73%) and 28-75% (average 59%), over regulatory phenotype in vivo permitting durable memory respectively. The anti-tumor bioactivity and target specificity of against tumors. The expansion of tumor-specific regulatory cells MCY-M11 was successfully established in vitro by demonstrating from the transferred product is enhanced in the absence of IL-6 antigen-specific cytotoxicity and inflammatory cytokine release in signaling. This work implies that the universal strategy of IL-6 co-culture assays with various mesothelin-expressing human inhibition for cytokine release syndrome may come at the ex- tumor cell lines. Increased survival and efficacy were also demon- pense of long-term efficacy for cell therapy approaches. strated in vivo using a human mesothelin expressing ovarian syn- Ethics Approval geneic mouse tumor model. All animal studies were approved by MUSC's IACUC committee, ap- Conclusions proval number 0488. The CARMA one-day manufacturing process using cGMP grade mRNA has the potential to revolutionize cell therapy strategies by significantly reducing the wait time for patients receiving treat- P192 ment. MCY-M11 is currently being tested in a first-in-human clin- Anti-HLA-G antigen receptor T-cells exhibit potent anti-tumor ical trial for advanced epithelial ovarian cancer and peritoneal effects against human solid tumors mesothelioma (ClinicalTrials.gov Identifier: NCT03608618). Alan Epstein, MD, PhD, Aida Kouhi, Aida Kouhi Trial Registration University of Southern California, Los Angeles, CA, United States NCT03608618 Correspondence: Alan Epstein (aepstein@usc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P192 P191 Background IL-6 fuels durable memory for Th17-mediated responses to tumors 1 1 1 HLA-G is highly expressed on human placenta during pregnancy and has Hannah Knochelmann, BS , Connor Dwyer, PhD , Aubrey Smith, BS , 1 1 1 been found to suppress the NK response to cells that lose their HLA and/ Megan Wyatt, MS , Guillermo Rangel RIvera , Jacob Bowers , Michelle 1 2 3 or beta2-microglobulin expression. [1-2]. In addition, except for pregnancy, Nelson, PhD , Gregory Lesinski, PhD, MPH , Zihai Li, MD, PhD , Mark 1 1 HLA-G is rarely expressed in normal adult tissues. Moreover, roughly 50% Rubinstein, PhD , Chrystal Paulos, PhD of human solid tumors lose their HLA expression to avoid detection by Medical University of South Carolina, Charleston, SC, United States; 2 3 the human immune system. [3] HLA-G is therefore an outstanding target Emory University, Atlanta, GA, United States; Ohio State University, for CAR T-cells since, like the placenta, it is up-regulated in HLA-negative Columbus, OH, United States tumors to suppress NK destruction. [4] We have successfully generated Correspondence: Hannah Knochelmann (knochelm@musc.edu) anti-HLA-G CAR T-cells to treat solid tumors that express HLA-G. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P191 Methods An anti-HLA-G CAR construct was generated by fusing anti HLA-G Background scFv to a second generation CAR containing the CD8α leader se- Accessibility of T cell transfer therapies for most patients is hin- quence, 4-1BB co-stimulation sequence, and CD3ζ signaling domain. dered by cost and time required for product development. Our The CAR vector was then fused with a lentivirus vector in-frame with lab has shown that shortening ex vivo expansion of Th17 cells the CAR backbone, and was used to transduce primary human CD3 licenses a proinflammatory cell product which induces cytokine positive T-cells. After transduction, expanded CAR-T cells were char- stormwithhighlevelsofsystemicIL-6intumor-bearing hosts. acterized for their ability to bind HLA-G antigen and HLA-G positive Despite potential toxicity, briefly expanded Th17 cells eradicate SKOV-3 cells (human ovarian cancer model) using flow cytometry. large established tumors in low doses and generate durable Results memory against tumor rechallenge, suggesting a therapeutic Expanded CAR-T cells were able to bind successfully both the benefit to the inflammatory state. Prior reports show that IL-6 HLA-G antigen and SKOV-3 cells in vitro. Expanded CAR-T cells promotes functional CD4+ T cell memory formation. Given that were then co-cultured with SKOV3-Luc cells and studied for their IL-6 is blocked clinically to manage cytokine release syndrome, epitope-driven cytotoxicity. Anti HLA-G CAR T-cells displayed we addressed the physiologic impact of IL-6 on efficacy and dur- dose-dependent cytotoxicity when co-cultured with tumor cells. ability of Th17 cell therapy. We have recently developed an in vivo model of ovarian cancer Methods that can be used for testing the efficacy of our CAR-T cells. In Th17 cells were expanded ex vivo using the TRP-1 transgenic this model, NSG mice are injected with 2 million SKOV3-Luc cells mouse model in which CD4+ T cells express a TCR that recog- intraperitoneally (ip). Seven-10 days after injection, tumors are nizes tyrosinase-related protein 1 on melanoma. Naïve CD4+ T visible when observed by bioluminescence imaging, at which cells were polarized to the Th17 phenotype and infused into time the treatment group will receive an ip injection of anti HLA- mice with B16F10 melanoma after a nonmyeloablative total G CAR-T cells. Since ovarian cancer rapidly metastasizes to the body irradiation (5 Gy) preparative regimen. Serum cytokine peritoneum, the aforementioned model should provide relevant levels were obtained by multiplex array and IL-6 signaling was clinical data that can be translated to patients, and like inhibited with antibodies targeting the IL-6R and neutralizing IL- hematopoietic cancers, will present antigen quickly after injection 6cytokine. of CAR T-cell to keep them stimulated and functional. Results Conclusions Acute IL-6 blockade post Th17 cell transfer did not impact the We are currently testing the efficacy of anti-HLA-G CAR-T cells primary response against melanoma nor the engraftment of Th17 in vivo using this ip model, and plan to show that HLA-G as a cells. However, blocking IL-6 abrogated long-term responses in- pan tumor target will provide selective and specific cell based creasing the frequency of tumor relapse upon secondary chal- therapy which may in the near future be clinically relevant for lenge and reduced survival. Mechanistically, IL-6 blockade chemotherapy resistant ovarian cancer and other tumors. reduced phosphorylation of STAT3 in transferred T cells associat- ing with diminished Bcl-2 expression. The CD4+ compartment Acknowledgements was reshaped by IL-6 blockade via promoting a greater frequency This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. of FoxP3+ Treg cells in the peripheral blood, tumor and draining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 105 of 272 References Immetacyte is assessing this approach in our current Phase I/II clinical 1. Apps R, Gardner L, Moffett A. A critical look at HLA-G. Trends Immunol. trial of TIL in ovarian cancer patients (EudraCT–2019-000106-30). 2008; 29:313–321. 2. Jurisicova A, Casper RF, MacLusky NJ, Mills GB, Librach CL. HLA-G expres- Acknowledgements sion during preimplantation human embryo development. Proc. Natl. This research was supported by IUK projects 133299 and 104468 Acad. Sci. U. S. A. 1996; 93:161–5. Ethics Approval 3. de Kruijf EM et al. HLA-E and HLA-G expression in classical HLA class I- This study was approved by the South Central Research Ethics Committee : negative tumors is of prognostic value for clinical outcome of early 19/SC/0355 breast cancer patients. J. Immunol. 2010; 185:7452–9. 4. Lin, A. & Yan, W.-H. Human Leukocyte Antigen-G (HLA-G) Expression in P194 Cancers: Roles in Immune Evasion, Metastasis and Target for Therapy. Use of stimulatory cells in conjunction with IL-12 and IL-18 Mol. Med. 2015; 21:782–791. augments NK cell expansion and transduction, drives a Ethics Approval memory phenotype, and improves in vitro and in vivo CAR This study was approved by the IRB of the University of Southern California NK activity protocol #HS-16-00029 on 2-29-16. Anmol Vohra, MS, Katherine Jamboretz, MS, Sasha Lazetic, Denise Gonzalez, Daofeng Liu, PhD, Ivan Chan, PhD, James Trager, PhD P193 Nkarta Inc, South San Francisco, CA, United States CoStAR (Costimulatory Antigen Receptor) enhancement of tumour Correspondence: James Trager (jtrager@nkartatx.com) infiltrating lymphocyte therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P194 Gray Kueberuwa, PhD, John Bridgeman, PhD, Martina Sykorova, Milena Kalaitsidou, Michelle Le Brocq, Robert Hawkins Background Immetacyte, Manchester, Manchester County, United Kingdom NK cells have been expanded on K562 stimulatory cells express- Correspondence: Robert Hawkins (r.hawkins@immetacyte.com) ing membrane-bound (mb) IL-15 and 41BBL for clinical use, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P193 can be genetically modified to express activating chimeric recep- tors [1,2,3]. Engineered NK cells targeting CD19 or ligands of Background NKG2D show in vitro and in vivo cytotoxicity against relevant The efficacy of TIL therapy is limited in some patients due to the tumor targets that can overcome endogenous resistance to NK failure of the cells to respond to tumour sufficiently or persist cells. NK cells activated in the presence of IL-12, IL-15 and IL-18 long enough to have a necessary anti-tumour effect. We have ad- develop cytokine induced memory-like phenotype and function; dressed this issue by developing Co-stimulatory receptors (Co- these cells have shown clinical promise [4]. Here we describe NK StARs) that provide enhanced signaling to tumour-specific T-cells cell function and phenotype achieved by combining the robust upon encountering tumour associated antigens. driven by K562-mbIL15-41BBL with the induction of a cytokine- Since tumour reactivity is determined by natural TCRs that have induced memory phenotype achieved after exposure to IL-12 and undergone thymic selection, this approach does not bear with it the IL-18. risks of other therapies targeting tumour antigens expressed on the Methods cell surface Healthy donor PBMC NK were expanded on K562-mbIL15-41BBL Methods stimulatory cells with IL-2 alone or with IL-2 plus IL-12 and IL-18 In order to identify optimal signalling domain for Co-StAR mole- (12-18). We compared NK cell expansion, cytokine secretion, cyto- cules, several iterations of our prototype receptor were synthe- toxicity against tumor lines at various time points, and persist- sised. The ability of each to enhance T-cell activation, ence in culture over 4 weeks. The expanded NK were transduced proliferation, secretion of cytokines and increase resistance to with CD19 and NKG2D CAR constructs, and the resulting cells apoptosis were assessed. evaluated for CAR expression, cytotoxicity and in vivo efficacy To explore if this approach has the potential of wide applicabil- against relevant cell lines. ity, we went on to assess targeting of two additional ovarian Results cancer tumour associated antigens. To achieve this, the antigen Addition of 12-18 to the K562-mbIL15-41BBL stimulatory cells im- binding moiety was exchanged and signaling domain kept proves NK expansion 2-3 fold [If we have a p value, it’s better to constant. give it:<br>‘…significantly improves NK expansion 2-3 fold (p<x) Results relative to that…’][Will put together later for the poster]relative We show that colorectal cancer specific Co-StAR significantly en- to that achieved using the stimulatory cell line alone, while NK hances the number of T-cells expressing IL2, TNFα,41BB,CD107a cell cytotoxicity is unchanged. IFNγ and TNFα production and and bcl-xL by factors ranging from 2-4 fold in model systems. transduction efficiency are also improved in this setting. Over 3 This shows an increase in activation, effector function and resist- weeks of culture following brief exposure to 12-18, NK cell ance to apoptosis. We also identified an optimal signaling do- phenotype changes, with an increased percentage of CD62L+ main that caused the greatest magnitude of enhancement for and NKG2C+ cells, and increased NKG2C expression per NK cell. the above factors. While the NKG2D-CAR driven cytotoxic activity is unchanged by Observations of Co-StAR enhancement were mirrored in model 12-18 at 14 days post-exposure, cytotoxic activity increases in systems for ovarian cancer, targeting two separate ovarian cancer these cells by day 21. In addition, this improved cytotoxicity at tumour associated antigens. day 21 is reflected by improved CD19-CAR driven in vivo activity In addition, stimulation assays showed that Co-StAR with optimal sig- against the CD19+ tumor target [Raji or Nalm6?]Nalm6 [Oops, naling domain increased T-cell proliferation over 3 weeks in compari- Nalm6, thx.] with an increased presence of circulating NK cells son to prototype Co-StAR, Co-StAR that binds an irrelevant target, or over 4 weeks in the mice. indeed, mock transduced T-cells. Conclusions Conclusions The data demonstrates that the addition of IL-12 and IL-18 to Our optimal Co-StAR provides a means to effectively deliver “signal K562-mbIL15-41BBL stimulatory cells during NK expansion main- 2” to T-cells. Enhancing activation, effector functions and resistance tains in vitro cytotoxicity and improves expansion, transduction, to apoptosis upon contact with target tumour cells. persistence, and in vivo efficacy. Further, IL-12 and IL-18 drive de- Since T-cell activation primarily requires “signal 1”, application to of velopment of a memory phenotype and more sustained NK cell Co-StAR to enhance TIL therapy, which works through natural, thymi- function over the course of several weeks. This activation system cally selected TCRs, provides a means to increase the activity of may allow for development of more robust and potent engi- tumour-reactive TIL without risking severe off-tumour side effects. neered NK cells for clinical use. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 106 of 272 References Conclusions 1. Lapteva N, Durett AG, Sun J, Rollins LA, Huye LL, Fang J, Dandekar V, These promising results infer that adoptive transfer of transgenic T Mei Z, Jackson K, Vera J, Ando J, Ngo MC, Coustan-Smith E, Cam- cells can offer an effective strategy to not only prevent further tumor pana D, Szmania S, Garg T, Moreno-Bost A, Vanrhee F, Gee AP, Roo- growth as rapamycin therapy does, but also to treat and even elimin- ney CM. Large-scale ex vivo expansion and characterization of ate arising tumors. This strategy might offer a cure for patients with natural killer cells for clinical applications. Cytotherapy. LAM, a disease that hits women in the prime of their lives. 2012;14(9):1131-1143 2. Chihaya I, Iwamoto S, Campana D. Genetic modification of primary Acknowledgements natural killer cells overcomes inhibitory signals and induces specific Studies supported by a DoD Tuberous Sclerosis Complex Research Program killing of leukemic cells. Blood. 2005; 106:376-383. Clinical Translational Research Award to CLP. 3. Yang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Ethics Approval Chimeric Receptor with NKG2D Specificity Enhances Natural Killer All animal experiments were approved by the Animal Care and Use Cell Activation and Killing of Tumor Cells. Cancer Res. Committee of Northwestern University and followed the institutional 2013;73(6):1777-1786 guidelines; protocol number IS00008259. 4. Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, Neal CC, Yu L, Oh ST, Lee YS, Mulder A, Claas F, Cooper MA, Fehniger TA. Cytokine-induced P196 memory-like natural killer cells exhibit enhanced responses against mye- The first step toward the universal cell therapy: Simultaneous loid leukemia. Sci Trans Med. 2016;8(357): 357ra123 removal of HLAs (Human leukocyte antigens) using CRISPR- Ethics Approval mediated quadruple genome editing in allogeneic T cells Animal studies were conducted and approved by the Explora IACUC Jeewon Lee, Ph D, Munkyung Kim, Joong Hyuk Sheen, Jihye Ryu, Yu committee. Young Kim, Okjae Lim MOGAM Institute for Biomedical Research, Yongin-si, Gyeonggi-do, Republic of Korea P195 Correspondence: Okjae Lim (blubelle@mogam.re.kr) Loss of function of the TSC1-TSC2 complex renders tumors eligible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P196 for GD3 CART therapy 1 1 1 Ancy Thomas , Saurav Sumughan , Zhussipbek Mukhatayev , Emilia 1 1 2 2 Background Dellacecca , Nicola Lancki , Levi Barse , Jesus Zamora-Pineda , Suhail 2 2 1 2 Chimeric antigen receptor (CAR) T cell therapy is the revolution- Akhtar , Maria Picken , Denise Scholtens , Daniel Dilling , Richard 3 1 ary treatment of choice for hematologic malignancies. Currently Junghans, PhD, MD , Caroline Le Poole 1 2 approved CAR T therapies require patients’ own immune cells, Northwestern University, Chicago, IL, United States; Loyola University, and this autologous T cell manufacturing process involves certain Maywood, IL, United States; Boston University, Boston, MA, United limitations primarily derived from the nature of individualized States therapy. Thus, engineering allogeneic donor cells to evade host Correspondence: Caroline Le Poole immune rejection is required for a broader clinical application of (caroline.lepoole@northwestern.edu) the therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P195 Methods In this study, we attempted to inhibit expression of both HLA I Background and II through the CRISPR/Cas9 gene editing system to reduce Benign tumors can arise from bi-allelic mutations in a single gene. In tu- allo-reactive immune rejection response. First, we screened 60 berous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), gRNAs targeting B2M and 60 gRNAs each targeting alpha chains tumors do not acquire additional mutations, and patients are not eli- of HLA-II molecules (DP, DQ and DR, respectively) to find gRNA gible for therapeutics that rely on neoantigen formation. However, the sequences efficiently ablate expression of HLA molecules on T affected gene is responsible for several predictable phenotypic cell surface. Next, we investigated whether the absence of HLA-I/ changes. As mTOR hyperactivity resulting from mutations in TSC1 or II expression in donor T cells could alleviate immune response TSC2 is associated with overexpression of some melanoma-associated from allogeneic responders using in vitro mixed lymphocyte reac- antigens, de novo expression of ganglioside D3 expression may render tion (MLR) assays. the resulting, benign tumors eligible for immunotherapy. Results Methods We have identified gRNA sequences highly efficient in targeting We probed the expression of GD3 in human TSC lesions of the lungs, B2M and alpha chains of HLA-II molecules without carrying off- kidneys, skin and brain by immunostaining and monitored anti-GD3 target effects. Selected gRNA sequences for HLA-II ablation cov- titers in serum by ELISA. Infiltration by NK cells and NKT was mea- ered the vast majority of each HLA-II alpha chain allele. HLA-I/II sured to look for natural responses to the cell surface antigen. We double negative T cells generated by simultaneous quadruple next isolated tumors cells from TSC2 heterozygote mice and con- genome editing with the selected gRNAs maintained their pheno- firmed loss of heterozygosity by genotyping before challenging types and cytotoxicity upon TCR stimulations compared to the groups of 10 SCID/beige mice in 2 repeat experiments, and subject- control cells treated with non-target gRNA. Furthermore, the MLR ing them to adoptive transfer by GD3-CART cells and measured assays showed that IFN- and TNF-α production in allo-responder tumor sizes over time. Similarly we treated groups of 8 ageing TSC T cells was significantly decreased in the absence of donor HLA-I mice >16 months of age by adoptive GD3 CART-cell transfer, and alone and was further diminished in response to HLA-I/II double measured surface tumor growth on internal organs. negative donor T cells compared with the control cells, implicat- Results ing prolonged survival of the adoptively transferred immune cells. We found consistent overexpression of GD3 in tissues from TSC pa- Conclusions tients compared to healthy controls. GD3 overexpression was not ac- In conclusion, we have identified novel gRNA sequences ablat- companied by an influx of NK(T) cells, and anti-GD3 titers were ing expression of HLA molecules on donor T cell surfaces to reduced rather than elevated in patients, supporting the concept dramatically reduce donor-derived allo-responses, establishing that slow growing tumors in TSC patients are not immunogenic. an essential cornerstone towardsthe universalTcell therapy. However, CART cells responsive to GD3, supplemented by IL-2, medi- Ethics Approval ated prolonged and significant anti-tumor responses in both immu- Human PBMCs were obtained from healthy volunteers by leukapher- nodeficient and immune competent hosts. The majority of TSC2 esis from the Samsung Medical Center (SMC) under IRB approval heterozygote mice treated by CAR T cells displayed no tumors at (SMC IRB no.2018-01-089). end point, versus all mice treated with untransduced T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 107 of 272 P197 we developed a novel modular universal CAR platform termed Uni- The development of an autologous neoantigen specific T cell CAR. UniCAR T-cells are exclusively activated in the presence of a tar- product from peripheral blood, NEO-PTC-01, through the ex-vivo get module (TM), which establishes the cross-link between antigen- induction protocol, NEO-STIM™ specific cancer cells and UniCAR T-cells in an individualized time- 1 1 1 Divya Lenkala, MS , Marit Van Buuren, PhD , Brian McCarthy , Jessica and target-dependent manner. The carbohydrate antigen sialyl-Tn 1 1 1 1 Kohler, PhD , Michael Nelson , Flavian Brown , Yvonne Ware, MS , (STn) is a particularly interesting target due to its expression in sev- 1 1 Yuting Huang, MS , Janani Sridar , Yusuf Nasrullah, MS, United eral types of cancer and absence in normal healthy tissues. Given the 1 1 2 Kingdom , Dewi Harjanto , Joost Van Den Berg, PharmD , Matthew small size of such TMs, they are rapidly eliminated and thus, possible 3 1 Goldstein, MD, PhD , Richard Gaynor, MD side effects and activation of UniCAR T-cells can be easily controlled 1 2 Neon Therapeutics, Cambridge, MA, United States; Netherlands Cancer by TM dosing. In late phases of treatment, TMs with extended half- Institute, Amsterdam, Netherlands; Tango Therapeutics, Cambridge, MA, life may play an important role by improving the eradication of re- United States sidual tumor cells. Correspondence: Marit Van Buuren Methods (mvanbuuren@neontherapeutics.com) In this work, a novel longer-lasting TM against STn was developed, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P197 characterized and compared to the previously developed short-lived anti-STn TM [1]. Short-lived TMs are composed of a tumor-specific Background binding moiety fused to the La peptide epitope (E5B9) which is rec- Neoantigens are tumor-specific antigens that have been shown to be ognized by UniCAR T-cells. In extended half-life TMs, these two com- important in the anti-tumor immune response. These antigens are ponents are fused via an Fc domain derived from the human IgG4 not subject to central immune tolerance and are therefore potentially molecule. Functional and pharmacokinetic properties were assessed more immunogenic than tumor-associated antigens. The goal of our using in vitro and in vivo assays. studies is to generate neoantigen specific T cell responses and per- Results form detailed characterization of the induced T cell responses to- The developed anti-STn IgG4-based TM efficiently activates and redi- wards these neoantigen targets to assess the applicability of the rects UniCAR T-cells to STn-expressing tumors in a highly efficient approach for adoptive cell therapy. target-specific and target-dependent manner, promoting the secre- Methods tion of pro-inflammatory cytokines, tumor cell lysis of breast and Patient-specific neoantigens were predicted using our RECON® bio- bladder cancer cells in vitro and of breast cancer cells in experimen- informatics platform, and the predicted high-quality neoantigens tal mice. A comparable or increased killing efficiency was obtained at were utilized in our proprietary ex-vivo stimulation protocol, NEO- a lower concentration range in comparison to the results obtained STIM to assess immunogenicity. NEO-STIM is used to prime, activate for the anti-STn scFv-based TM. Additionally, PET studies demon- and expand memory and de novo T cell responses from both the strate the specific enrichment of the anti-STn IgG4-based TM at the CD4+ as well as the CD8+ compartment. In-depth analysis was per- tumor site presenting a prolonged serum half-life compared to the formed to characterize the specificity, functionality (cytokine produc- scFv short-lived TM. tion and cytolytic capacity) and diversity of the induced T cell Conclusions responses through high throughput flow cytometric analysis. Taken together, these data demonstrate the effective and potential Results application of this CAR T-cell-derived modular system to target STn Here we present the successful induction of memory and de novo in different types of cancer using different TM formats. The use and CD8+ and CD4+ T cell responses in peripheral blood mononuclear combination of such molecules with different formats and half-lives cells isolated by leukapheresis from five melanoma patients using provides highly promising and customized tools for retargeting of NEO-STIM. We then extensively characterized these T cell responses UniCAR T-cells in a flexible, individualized and safe manner at differ- and show that these responses are functional, specific and have cyto- ent stages of treatment. lytic capacity. Conclusions Reference NEO-STIM is a novel platform to understand in detail the immuno- 1. Loureiro L R, Feldmann A, Bergmann R, Koristka S, Berndt N, Arndt C, genic potential of high-quality neoantigen-targets. Moreover, this Pietzsch J, Novo C, Videira P, Bachmann M. Development of a novel platform can be utilized to generate T cell products from peripheral target module redirecting UniCAR T cells to Sialyl Tn-expressing tumor blood for adoptive cell therapy for patients with a variety of solid cells. Blood Cancer J. 2018; 8(9): 81. tumors. Ethics Approval Ethics Approval All animal activities and procedures were performed in accordance with the The samples for the study were collected under ClinicalTrials.gov: protocols approved by the Institutional Review Board at Semmelweis NCT02897765 and N16NEON protocol University - Budapest, approval number PE/EA/50-2/2019. P198 P199 Short-lived and extended half-life target modules for redirecting Generation of functionally and phenotypically mature, allogeneic UniCAR T-cells against sialyl-Tn expressing cancer cells natural killer cells from human induced pluripotent stem cells 1 1 1 Liliana Loureiro, PhD , Anja Feldmann, PhD , Ralf Bergmann , Stefanie under chemically-defined, feeder- and serum- free culture 1 1 2 2 Koristka , Nicole Berndt , Nikolett Hegedüs , Domokos Máthé , Paula conditions 3 1 1 Videira , Michael Bachmann , Claudia Arndt Kyle Lupo, BS, Andrea Chambers, MS, Sandro Matosevic, PhD Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; Purdue University, Lafayette, IN, United States 2 3 Semmelweis University, Budapest, Hungary; Faculdade de Ciências e Correspondence: Sandro Matosevic (sandro@purdue.edu) Tecnologia/UNL, Caparica, Portugal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P199 Correspondence: Liliana Loureiro (l.loureiro@hzdr.de) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P198 Background While targeted immunotherapy with engineered natural killer (NK) Background cells has emerged as a promising approach for the treatment of solid The development of chimeric antigen receptors (CARs) has rapidly tumors, challenges in sourcing, processing, and genetically modifying emerged as a promising approach in cancer immunotherapy. None- blood-derived NK cells limit the potential for developing life-saving theless, drawbacks associated with CAR T-cell therapies include on- treatments for cancer patients. As an alternative, the use of induced target/off-tumor effects and cytokine release syndrome. Aiming an pluripotent stem cells (iPSCs) offers a promising approach to over- increased clinical safety while preserving the efficacy of such therapy, coming existing challenges faced when engineering NK cell-based Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 108 of 272 immunotherapies. However, approaches for generating NK cells from antigen-specificity, higher CD8:CD4 ratio and ability to persist long- iPSCs described so far have several shortcomings: they utilize sera or term [3]. Based on these differences, we hypothesized that MILsTM feeder layers to adapt iPSCs or culture hematopoietic progenitors, would provide a more robust platform for CAR-T therapy compared take months to complete, and rely on individualized, and thus highly to PBLs. We have previously shown that CAR-modified MILsTM (CAR- variable, iPSC reprogramming protocols, limiting their utility. MILsTM) demonstrate superior killing of tumor target cells in vitro Methods compared to CAR-T cells generated from patient-matched PBLs (CAR- We have generated NK cells from iPS cells using a novel feeder-free PBLs) [4]. In this study, we compared, at the single cell level, func- differentiation protocol starting from either centrally-validated and tionality of patient-matched CAR-MILsTM and CAR-PBLs following banked iPSC lines or iPSCs reprogrammed from donor fibroblasts. antigen-specific in vitro stimulation. Our protocol utilizes a two-step, entirely feeder-free procedure in- Methods volving hematopoietic progenitor generation followed by NK differ- CAR-MILsTM and CAR-PBLs engineered to express a BCMA-specific, 4- entiation. These differentiated NK cells have been characterized for 1BB/CD3z-signaling CAR were produced using cryopreserved lympho- inhibitory and activating marker expression, IFN-γ production, de- cytes from the bone marrow and blood of six patients with multiple mye- granulation, and cytotoxicity against a number of solid tumor targets, loma. CD4 and CD8 T cells isolated from the CAR-MILsTM and CAR-PBLs including primary patient-derived glioblastoma cells. Moreover, these products were stimulated with K562 cells transduced with either BCMA cells were expanded in culture and manipulated to generate a cyto- (K562-BCMA) or nerve growth factor receptor (K562-NGFR) at a ratio of toxic infusible cell therapy product. 1:2 for 20 hrs. After 20 hrs of co-culture, T cells were enriched and loaded Results into IsoCode chips containing ~12,000 microchambers pre-patterned iPS cells were differentiated into hematopoietic progenitor cells, with a 32-plex antibody array. Protein secretion from 1000-2000 single T yielding CD34+/CD45+ and CD34+/CD43+ cell populations at yields cells per product was detected by a fluorescence ELISA-based assay and consistent with results described in literature using feeder-based pro- single cell polyfunctional profiles analyzed using IsoPeak (IsoPlexis). tocols [1]. Following four weeks of NK cell differentiation, cells Results showed high expression of several NK cell maturation markers as CD4 and CD8 T cells from both CAR-MILsTM and CAR-PBLs demon- well as inhibitory and activating receptors (CD56+/CD3-, NKG2D, strated an antigen-specific increase in polyfunctionality (secretion of 2+ NKp30, NKp44, NKp46, DNAM-1, CD16, CD94/NKG2A, and CD158b). cytokines per cell) and polyfunctional strength index (PSI) in response iPSC-NK cells derived using our protocol were also similar to blood- to BCMA stimulation compared to NGFR control. When compared to derived NK cells in morphology, expansion rate, and functionality (in CAR-PBLs, CAR-MILsTM demonstrated increased polyfunctionality and terms of cytotoxicity and degranulation potential). By using centrally- increased PSI in both CD4 and CD8 T cells. The enhanced PSI in CAR- validated iPSC lines, we further demonstrate our ability of avoiding MILsTM was predominated by effector, stimulatory and chemoattrac- donor-specific reprogramming protocols. tive proteins associated with antitumor activity including Granzyme B, Conclusions IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and en- We developed a new protocol for the generation of NK cells from hanced secretion of these same proteins was reported to be associated iPSCs that is entirely feeder and serum-free and can be extended to with improved clinical responses in patients with Non-Hodgkin lymph- the use of validated iPSC lines avoiding donor and reprogramming oma treated with CD19-specific CAR-T therapy [5]. variability. iPSC-derived NK cells using our protocol exhibit character- Conclusions istics of mature blood-derived NK cells and powerful cytotoxicity Based on these data and the inherent antitumor properties of against solid tumor targets. Additionally, these cells offer the advan- MILsTM, we speculate that CAR-MILsTM would be more potent tage of increased expansion rates, improved ease of transfection and effective than currently approved CAR-T products derived while in the iPSC state, and are free of contaminating T-cells associ- from PBLs. ated with GvHD risk, overcoming many limitations of existing NK cell based immunotherapies. References 1. Borrello I and Noonan KA, Marrow-Infiltrating Lymphocytes – Role in Biol- Reference ogy and Cancer Therapy. Front Immunol. 2016 March 30; 7(112) 1. Bock A, Knorr D, and Kaufman D. Development, Expansion, and In Vivo 2. Noonan K.A., Huff C.A., Davis J., Lemas M. V., Fiorino S., Bitzan J., Ferguson Monitoring of Human NK Cells from Human Embryonic Stem Cells A., Emerling A., … Borrello I. Adoptive transfer of activated marrow- (hESCs) and Induced Pluripotent Stem Cells (iPSCs). Journal of Visualized infiltrating lymphocytes induces measurable antitumor immunity in the Experiments : JoVE 74 (2013): 50337. bone marrow in multiple myeloma. Sci. Transl. Med. 2015; 7: 288ra78. 3. Noonan KA, Rudraraju L, Hoyos V, Lutz E and Borrello I. Persistence of Non Gene-Modified Adoptively Transferred Marrow Infiltrating Lympho- P200 cytes (MILs) More Than Five Years Post Transfer. Blood 2016 128:4552. CART-engineered Marrow-infiltrating Lymphocytes (MILsTM) are 4. Lutz ER, Hoyos V, Rudraraju L, DeOliveira E, Jana S, Weiss I, Borrello IM, more polyfunctional than their matched peripheral blood Noonan K. Marrow-infiltrating Lymphocytes (MILs) provide a robust plat- counterparts form for CAR-T cell therapy. Blood 2018 132:3337. 1 1 1 Eric Lutz, PhD , Lakshmi Rudraraju, MS , Elizabeth DeOliveira , Srikanta 5. Rossi J, Paczkowski P, Shen Y, … Bot A. Preinfusion polyfunctional anti- 1 2 2 3 Jana , Jing Zhou, MD, PhD , Sean Mackay, MBA , Ivan Borrello, MD , CD19 chimeric antigen receptor T cells are associated with clinical out- Kimberly Noonan, PhD comes in NHL. Blood 2018 132(8):804-814. 1 2 WindMIL Therapeutics, Baltimore, MD, United States; Isoplexis, Ethics Approval Branford, CT, United States; Johns Hopkins University, Baltimore, MD, The study was approved by the Johns Hopkins University IRB. United States Correspondence: Eric Lutz (lutz@windmiltherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P200 P201 Comparison of phenotype and anti-tumor profile of CD19-CAR-T Background cells generated from either umbilical cord blood- or peripheral WindMIL Therapeutics is developing Marrow-infiltrating Lymphocytes blood-derived T lymphocytes 1 1 1 (MILsTM), a novel form of adoptive T cell therapy composed of bone Cristina Maccalli, PhD , Dhanya Kizhakayil, PhD , Shilpa Ravindran, BSc , 1 1 1 marrow-derived, patient-autologous, polyclonal CD4 and CD8 T cells Saad Rasool , Rebecca Mathew , Valentina Mattei , Monica Casucci, 2 1 1 1 [1]. Genetically unmodified MILsTM have demonstrated antitumor ac- PhD , Sara Deola, MD, PhD , Chiara Cugno, MD , Damien Chaussabel , 1 1 tivity in patients with multiple myeloma [2] and are being developed Sara Tomei, PhD , Christof von Kalle , 1 2 for several other tumor types. Distinguishing features of T cells from Sidra Medicine, Doha, Qatar; San Raffaele Scientific Institute, Milan, Italy bone marrow compared to T cells from peripheral blood lympho- Correspondence: Cristina Maccalli (cmaccalli@sidra.org) cytes (PBLs) include their memory phenotype, inherent tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P201 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 109 of 272 Background Background T lymphocytes expressing antigen-specific chimeric receptors (CARs) Natural killer (NK) cells are innate immune cells with a critical role in im- have been revealed as a powerful therapeutic approach for aggres- mune surveillance against cell transformation and tumor development. NK sive and refectory childhood and adult B cell malignancies. Umbilical cells express an array of unique activating and inhibitory receptors whose cord blood cells (UCB), with their unique capacity of broad leukocyte aggregate signaling determine activation of NK cell effector function. antigen (HLA)-matching, can represent an appealing starting material Adoptive transfer of NK cells has demonstrated the potential to induce an- for the generation of “off-the shelf” CAR-T cells to render this type of titumor responses in the clinic. Celularity has developed a platform for therapy accessible to a large number of cancer patients. generating cytotoxic NK cells from placental CD34+ cells (PNK cells) for Methods adoptive cancer immunotherapy. Although PNK cells demonstrate cytotox- CAR-T cells have been generated from either UCB (N=5, ALLCELLS, icity against diverse cancer cell types, their activating mechanisms are little USA) and peripheral blood lymphocytes (PBLs; N=2) from healthy do- characterized. In this study, we explore the contribution of specific signal- nors. In vitro enriched T cells have been transduced with CD19- ing pathways and upstream NK cell receptors involved in PNK cell cytotox- CD28z-CD3z and CD19-4-1BBz-CD3z encoding lentiviral vectors (LVs). icity against glioblastoma multiforme (GBM) cell targets. Deep phenotype characterization of these CAR-T cells has been per- Methods formed utilizing an in-house designed IF multiparametric (28 PNK cells were transcriptionally profiled using scRNAseq and qRT-PCR markers) panel. Functional assays have been performed to assess to identify candidate pathways regulating cytolytic function. Expression cytokine (IFN-gamma, IL2, IL-5 and IL-17), perforin and granzyme B of major receptors and intracellular signaling molecules were analyzed release (EliSpot or multicolor FluoroSpot) and cytotoxic activity (Del- using flow cytometry and western blot. PNK cell phenotype was com- fia assay) by CAR-T cells following the co-culture with CD19+ or pared to circulating NK cells. PNK cytotoxicity was evaluated against CD19- target cells. In addition, transcriptomic modular repertoire [1- GBM cell lines (LN-18 and U251) in xCELLigence platform and a de- 3] has been applied by parallel quantitative PCR using the high granulation assay using CD107a staining. The role of key signaling path- throughput BioMark HD platform to determine gene expression pro- ways driving PNK effector functions was analyzed in cytolysis assays file of CAR-T cells described above. using small molecule inhibitors of Src kinases, SYK, PLC-γ,PI3Kand Results MAP kinases, including JNK, p38 and ERK. Efficient LV transduction was achieved for UCB-T cells, although requir- Results ing higher MOI as compared to PBL (25 vs. 5; 66-80 vs. 70-88 % of PNK cells highly express genes mediating NK cell effector functions, includ- transduction, respectively). The frequency of CD4+ transduced T cells ing NCR1, NCR2, NCR3, KLRK1 and CD226. Flow cytometry demonstrated (45-59% of positive cells) was superior in UCB as compared to PBL (27- increased expression of NKp44 (99.7±0.2% vs. 69.3±5.2%), NKG2D (68.7± 36% of positive cells) while transduced CD8+ T cells were 18-20 and 7.9% vs. 44.6±5.8%) and GITR 99.7±0.2% vs. 15.0±3.2%) on PNK cells when 40-67%, respectively. CB-CAR-T cells were enriched of compared to circulating NK cells. PNK cells demonstrated strong cytolytic CD45RA+CCR7+CD27+CD62L+ T cells. These cells co-expressed ICOS activity against multiple GBM cell lines. While inhibitors of Src, SYK, PLC-γ, and 4-1BB but these molecules were not detectable on PBL-CAR-T cells. p38 and ERK did not modulate PNK cytotoxicity, inhibitors targeting JNK Markers associated with late differentiation/exhaustion of T cells, such and PI3K pathways significantly suppressed PNK cell cytotoxicity, specific- as LAG-3 and TIM-3, were found only on PBL-CAR-T cells. PD-1 was ally, 64.9±4.7% inhibition by SP600125 (JNK) and 25.2±3.3% inhibition by expressed at higher levels in CB- vs. PBL-derived CAR-T cells (15-40% Ly294002 (PI3K) on LN-18 cells; 100% inhibition by SP600125 and 45.5.5± and 40-60% in CD45RA+ and CD45RO+ T cells, respectively). 8.9% by Ly294002 on U251. JNK and PI3K inhibitors also reduced degranu- Antigen-specific reactivity was shown by CAR-T cells either isolated lation (70.6±3.2% by SP600125 and 58.4±3.6% by Ly294002). Furthermore, from UCB or PBL against acute lymphoblastic leukemia or EBV-B cells PI3K pathway controlled PNK cytokine production upon coculture with overexpressing CD19. U251 cell line, whereas JNK inhibition had minimal effect. Interestingly, differential gene expression profiles were assessed Conclusions through the comparison of UCB- vs. PBL-derived CAR-T cells and Our results demonstrate the importance of PI3K and JNK pathways in their co-culture with antigen-specific target cells. Genes differentially mediating PNK cytotoxicity to GBM cell line targets. These data com- expressed in CD19-CD28z-CD3z vs. CD19-4-1BBz-CD3z CAR-T cells bined with our receptor profiling on PNK cells establish the rationale were also found. for further investigating receptor-ligand interactions that directly Conclusions modulate PI3K and JNK activity. Taken together, these results proved that anti-tumor early differenti- ated/central memory CAR-T cells can be efficiently isolated from UCB P203 with distinctive phenotype as compared to PBL-CAR-T cells. Discovery and characterization of the first fully human Phosphopeptide Tumor Target-specific T cell receptor References 1 1 2 Xavier Michelet, PhD ,Eleni Chantzoura,PhD , Ekaterina Breous-Nystrom, PhD , 1 Chaussabel, D. and N. Baldwin. Democratizing systems immunology with 2 1 1 1 Alessandra Franchino ,RachelSmith , Daniel Pollacksmith ,Jan Bergmann , modular transcriptional repertoire analyses. Nat Rev Immunol, 2014, 1 2 2 2 Alvaro Sebastian Yague , Paisley Myers, PhD , Erin Jeffrey , Benjamin Wolf , 14:271-80; 2 1 1 Dennis Underwood, PhD ,Marc Van Dijk,PhD ,ArthurHurwitz, PhD 2 Altman MC., Rinchai D., Baldwin N., Whalen E., Garand M., Ahamed 1 2 Agentus Therapeutics, Lexington, MA, United States; Agenus, Basel, Kabeer B., et al. A Novel Repertoire of Blood Transcriptome Modules Switzerland Based on Co-expression Patterns Across Sixteen Disease and Physio- Correspondence: Arthur Hurwitz logical States. 2019, bioRxiv; doi: https://doi.org/10.1101/525709. (andy.hurwitz@agentustherapeutics.com) 3 Altman MC., Baldwin N., Whalen E., Al-Shaikhly T., Presnell S., Khaenam P., Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P203 et al. A Transcriptome Fingerprinting Assay for Clinical Immune Monitor- ing. 2019, bioRxiv; doi: https://doi.org/10.1101/587295. Background AgenTus Therapeutics is developing innovative adoptive cell therap- P202 ies to target a novel class of neoantigens called Phosphopeptide Mechanisms underlying human placental CD34+-derived natural Tumor Targets (PTTs). These post-translational modification-based killer cell cytotoxicity against glioblastoma neo-antigens arise in tumor cells through dysregulated kinase and Tanel Mahlakoiv, PhD, Bhavani Stout, Valentina Rousseva, Irene Raitman, phosphatase activities. PTTs represent one of the most promising cell Lin Kang, PhD, Robert Hariri, Xiaokui Zhang, William van der Touw therapy targets, as they are shared within and between cancer indi- Celularity, Warren, NJ, United States cations. Using a mass-spectrometry-based approach that analyzes Correspondence: William van der Touw MHC I-bound peptides, we have analyzed PTTs from several indica- (william.vandertouw@celularity.com) tions. This approach allows us to survey the TCR ligandome of tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P202 cells and healthy tissues. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 110 of 272 Phospho-ligandome analysis identified the phosphopeptide after injection, the alloreactive CD4 T-cells underwent marked expan- EPRpSPSHSM presented by HLA*B07:02+ cancer cells in a patient sion and produced higher levels of interferon-gamma compared to with Acute Myeloid Leukemia (AML). This phosphopeptide results syngeneic CD4 T-cells. This was accompanied by markedly increased from the phosphorylation of the Mixed Lineage Leukemia-1 (MLL1) infiltration of host macrophages within the tumors as early as four protein, a histone lysine methyl transferase that functions as a tran- hours after injection. These tumor-infiltrating macrophages secreted scriptional regulator and has been associated with tumorigenesis. higher levels of interleukin (IL)-1β, IL-12 and IL-23, which are critical for Methods inducing effector T-cell responses. Indeed, 24 hours after injection of + + Using proprietary platforms consisting of primary T cell expansion alloreactive CD4 T-cells, the infiltration of host effector CD8 T-cells from the central compartment and a mammalian display platform into tumors significantly increased, as evidenced by their production of containing TCR α and β chain libraries from the expanded T cells, we high levels of perforin and granzyme B. Furthermore, the melanoma B6 isolated the first fully-human PTT-specific TCR: agenT-04002. mice that survived alloreactive CD4 T-cell therapy developed host Results memory T-cells specific to the B16 melanoma and acquired complete Functional characterization demonstrated that target recognition by resistance to the tumor rechallenge. agenT-04002 is dependent on the phosphoseryl-moiety. Furthermore, Conclusions agenT-04002 shows potent cytotoxic activity against numerous human Results showed that immune reactions triggered by ex vivo-gener- hematologic tumor cell lines in vitro and AML tumor control in vivo in a ated alloreactive CD4 T-cells disrupt immunosuppressive tumor mi- mouse xenograft model. Activated T cells harboring the recombinant croenvironments and establish long-term host antitumor memory T- TCR display a pro-inflammatory phenotype in vitro and in vivo following cell responses. Our findings may help develop new strategies for sig- tumor challenge. Most importantly, when co-cultured with AML cancer nificantly enhancing the efficacy of cancer immunotherapy. cells from patients, agenT-04002 T cells specifically recognize and kill tumor cells while sparing healthy myeloid cells. Acknowledgements Conclusions This work was supported by JSPS KAKENHI Grant Numbers JP15K09659, and AgenTus is developing the next generation of TCRs by targeting a JP19K07754. unique class of neo-antigens with multi-cancer potential. Our data demonstrate feasibility, specificity, and potency of PTT-specific TCRs. References Targeting PTTs across diverse indications will enable us to have 1. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, Bader P, broader applicability of cellular therapies. MR. Verneris MR, Stefanski HE, Myers GD, Qayed M, Moerloose BD, Hiramatsu H, Schlis K, Davis KL, Martin PL, Nemecek ER, Yanik GA, Peters C, Baruchel A, Boissel N, Mechinaud F, Balduzzi A, Krueger J, June CH, Levine P204 BL, Wood P, Taran T, Leung M, Mueller KT, Zhang Y, Sen K, Lebwohl D, Ex vivo-activated allogeneic CD4 T-cells disrupt Pulsipher MA, Grupp SA. Tisagenlecleucel in Children and Young Adults immunosuppressive tumor microenvironment, and induce host with B-Cell Lymphoblastic Leukemia. NEJM. 2018; 378: 439-448. tumor-specific cytotoxic T-cells in mice 2. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, Brudno JN, Stetler- 1 1 1 Kazuhiro Mochizuki, MD, PhD , Shogo Kobayashi , Nobuhisa Takahashi , Stevenson M, Feldman SA, Hansen BG, Fellowes VS, Hakim FT, Gress RE, 1 1 2 3 1 Hideki Sano , Yoshihiro Ohara , Shin Mineishi, MD , Yi Zhang , Atsushi Kikuta and Kochenderfer JN. T cells expressing an anti–B-cell maturation antigen 1 2 Fukushima Medical University, Fukushima City, Japan; Penn State chimeric antigen receptor cause remissions of multiple myeloma. Blood. Cancer Institute, Hershey, PA, United States; Temple University, 2016;128:1688-1700. Philadelphia, PA, United States 3. Majzner RG, Mackall CL. Tumor Antigen Escape from CAR T-cell Therapy. Correspondence: Kazuhiro Mochizuki (mochi-k@fmu.ac.jp) Cancer Discov. 2018; 8: 1219–1226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P204 4. Bezu L, Kepp O, Cerrato G, Pol J, Fucikovag J, Spisekg R, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: Peptide-based vaccines in anticancer Background therapy. Oncoimmunol. 2018; 7: e1511506 (15 pages). Cancer immunotherapies that target tumor-specific or tumor- 5. Negrin RS. Graft-versus-host disease versus graft-versus-leukemia. associated antigens are promising treatments for patients with incur- Hematology Am Soc Hematol Educ Program. 2015; 2015: 225-230 able cancers [1,2]. However, relapses due to the loss of target anti- Ethics Approval gens challenge the success of these therapies [1,3]. Multitargeted Experimental protocols were approved by the Fukushima Medical immunotherapies, such as cancer vaccinations specific to multiple University’s committee on Use and Care of Animals; approval number cancer-associated peptides, are possible approaches. However, clin- 28054, 29039, and 2019048. ical studies have shown that they have limited efficacy with respect to the induction of objective responses [4]. The graft-versus-leukemia effect observed after allogeneic hematopoietic stem cell transplant- P205 ation (allo-HSCT) is another example of strong multitargeted antitu- Automated, closed bioreactors for T cell processing and dendritic mor immunity mediated by donor T-cells that recognize and react to cell-T cell co-culture multiple allo-antigens [5]. In the present study, we demonstrated a Lekhana Bhandary, BS, PhD, Andrew Kozbial, BS, PhD, Shashi Murthy, BS, novel approach for attaining alloreactive CD4 T-cell-induced multi- PhD targeted cancer immunity that does not utilize allo-HSCT. Northeastern University, Boston, MA, United States Methods Correspondence: Shashi Murthy (s.murthy@northeastern.edu) + + Cluster of differentiation (CD)4 and CD8 T-cells isolated form the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P205 spleen of BALB/c mice were separately activated in cultures by den- dritic cells (DCs) generated from the bone marrow of C57BL/6 (B6) Background mice. The resultant host-reactive donor T-cells were injected into B6 Functionally closed and affordable automated cell culture systems mice bearing pre-established B16 melanoma. Host T-cells activated are critical to the success of cell-based immunotherapy. Despite by syngeneic DCs were used as the control. major advances in these therapies, there are few systems available Results that are practical for use at both the pre-clinical and clinical stages. Whereas the intratumoral injection of host-reactive donor CD4 T-cells To address this need, we have designed a system called BATON elicited potent antitumor immunity against established B16 melanoma which is designed for optimal culture of both adherent and suspen- in an alloantigen-dependent manner, intratumoral injection of host- sion cell types (Fig. 1). Cells are cultured via continual perfusion and reactive donor CD8 T-cells or host-type syngeneic T-cells failed to in- the fluid flow loop also enables automated cell loading and harvest. duce antitumor responses. The number of injected donor-type host- This poster will describe two application areas, namely T cell expan- reactive CD4 T-cells diminished after tumor regression and did not in- sion relevant to autologous CAR-T and TCR therapies and dendritic duce graft-versus-host disease-like complications. Interestingly, early cell (DC)-T cell co-culture for neo-antigen-based T cell therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 111 of 272 Methods The T cell expansion capability of the BATON system was evaluated by seeding BATON cartridges each having a surface area of 40 cm2 and volume of 25 mL with 23 million PBMCs along with CD3/28 Dynabeads. Cells were continually perfused with Irvine Scientific Prime XV xeno- free T cell medium with 33 U/mL IL-2 for 9 days. For comparison, a simi- lar culture was performed in a G-Rex 6 well plate. For DC-T cell co- culture experiments enriched monocytes (MOs) were seeded into the BATON system at a seeding density of approximately 600k MOs/cm2 into two cartridges. Monocytes were differentiated into immature DCs by continually perfusing the seeded MOs for 6 days with CellGenix DC Medium supplemented with 350 U/mL IL-4 and GM-CSF (CellGenix). On Day 6, the DCs from one cartridge were harvested for flow cytometry. The other cartridge was drained without removal of the DCs and seeded with approximately 23 million PBMCs. This cartridge was then perfused with Irvine Scientific Prime XV xeno-free T cell medium with 33 U/mL IL-2. Cells were harvested following 7 days of co-culture. In addition to flow cytometry characterization, the cytotoxicity of the T cells was evaluated via co-culture with Jurkat cells. Results BATON achieved high levels of T cell expansion, comparable to G-Rex (Fig. 2-3) and harvested cells showed strong cytotoxic ability (Fig. 4). For DC-T cell co-culture experiments, the BATON system generated DCs from monocytes at high yield (27% of seeded monocytes converted into DCs) (Fig. 5A). Expansion of T cells from the seeded PBMCs was ro- Fig. 2 (abstract P205). See text for description bust, with 26-fold expansion achieved in 7 days (Fig. 5B). Harvested T cells showed strong cytotoxic ability relative to control (Fig. 6). Conclusions The BATON system is an effective platform for reagent- and DC- mediated T cell expansion. Acknowledgements Funding from the NSF via grant 1645205 is gratefully acknowledged. Fig. 1 (abstract P205). See text for description Fig. 3 (abstract P205). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 112 of 272 Fig. 4 (abstract P205). See text for description Fig. 6 (abstract P205). See text for description P206 Transmembrane and linker domain amino acid composition alters chimeric antigen receptor (CAR) membrane residence and may conceal detection of novel functional CAR formats 1 2 1 Dina Schneider, PhD , Virginia Hoglund, MS , Ying Xiong, PhD , Darong 1 1 2 Wu, MS , Boro Dropulic, PhD, MBA , Rimas Orentas, PhD Lentigen Technology, Miltenyi Biotec, Gaithersburg, MD, United States; Seattle Children’s Research Institute, Seattle, WA, United States Correspondence: Rimas Orentas (rimas.orentas@seattlechildrens.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P206 Background The relationship between the structure of the extracellular linker (L) and transmembrane (TM) domains, and CAR-T function has not been fully described. In previous studies we used L and TM domains de- rived from CD8. To better define amino acid sequences governing cell surface expression and anti-tumor activity, we altered the se- quence and length of these domains and tested the impact on CAR T biology. Methods TM domains from glycoproteins expressed on the T cell surface were aligned to CD8 and those with a high degree of similarity were used to create new CARs. In some constructs the extracellular sequence proximal to the membrane of those proteins (L) was also included. CAR function was tested using LV-transduced human T cells. Protein expression was analyzed by flow cytometry and western blot, in vitro function by cytokine release and cell-mediated cytolysis, and in vivo function in xenograft models. CAR protein expression was also ana- lyzed by immunofluorescent microscopy. Results Sequences from T cell-expressed CD antigens, the CD3 complex, acti- vation markers, and members of the tumor necrosis factor receptor superfamily (TNFRSF) were analyzed. Based on sequence conserva- tion we created new CARs expressing combinations of CD4 and CD8 Fig. 5 (abstract P205). See text for description TM domains, as well as TNFRSF9, TNFRSF16, and TNFRSF19 (CD137, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 113 of 272 NGFR, TROY/TAJ). All constructs were detected by western blot. Conclusions Strong T cell surface expression was seen for CD8L/CD4TM, CD8L/ These results demonstrate that 3D-ACT model using ex vivo ex- CD4TM, CD8L/TNFRSF19TM, and TNFRSF16L/TNFRSF16TM. Intermedi- panded TILs and 3D tumoroid models is an effective tool for the ate surface expression was seen for TNFRSF9L/TNFRSF9TM. Con- therapeutic assessment of autologous TILs and indicate that it can structs with TNFRSF19L/ TNFRSF19TM had very poor surface also be used to assess efficacy of other cellular therapy applications. expression. However, these “undetectable” CARs by flow cytometry Furthermore, implementation of this platform in the clinical studies had the highest level of cytotoxicity and cytokine release vs Raji may also allow determining the most effective combinatorial cellular lymphoma. Immunofluorescence studies with transduced T cells on therapy strategies for individual patients. their own, or in the presence of Raji target cells, demonstrated that TNFRSF19 sequence may mediate an intracellular residence profile. Association of CARs with the CD3 complex was also noted. P208 Conclusions Impact of combined blockade of PD1 and activation of CD137 on The production of CARs for clinical use generally requires detection tumor infiltration and tumor cell killing efficacy of TILs in an of the CAR protein on the cell surface. We found that high-activity ex vivo autologous 3D tumoroid model of NSCLC patient samples CAR-T constructs can be created using the linker and TM domains of Jenny Kreahling, PhD, Melba Page, PhD, Mibel Pabon, PhD, Vijayendra TNFRSF19, even though these constructs are expressed on the cell Agrawal, PhD, Soner Altiok, MD, PhD surface at low to undetectable levels. The mechanism by which these Nilogen Oncosystems, Tampa, FL, United States CARs are functionally active, while in a primarily intracellular state, is Correspondence: Soner Altiok (soner@nilogen.com) under investigation. Intracellular residence of CARs may be a novel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P208 mechanism to prevent undesired activation or T cell exhaustion and represents a novel locus of CAR-T activity control. Background Adoptive cell therapy (ACT) with TILs has been of growing interest as anti- cancer treatment in solid tumors. This therapy consists of the outgrowth P207 and expansion of tumor resident T cells from tumor material and their trans- A patient-driven ex vivo 3D tumor organoid model to assess fer back into the same patient to achieve tumor cell killing. However, exist- efficacy of tumor infiltrating T-cell adoptive cell therapy ence of intrinsic immune escape mechanisms may diminish the efficacy of Jenny Kreahling, PhD, Mibel Pabon, PhD, Melba Page, PhD, Vijayendra therapeutic applications of TILs. Here we describe an ex vivo patient derived Agrawal, PhD, Soner Altiok, MD, PhD 3D tumoroid platform utilizing powerful high content confocal imaging mo- Nilogen Oncosystems, Tampa, FL, United States dalities to monitor the impact of PD1 inhibition and CD137 activation on au- Correspondence: Soner Altiok (soner@nilogen.com) tologous TIL infiltration and ACT mediated tumor cell killing. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P207 Methods Human tumor samples were obtained with patient consent and rele- Background vant IRB approval. Fresh patient tumor samples were processed into Adoptive cell transfer (ACT) of ex vivo expanded tumor- tumoroids measuring 100-150 μm in size. For these studies, autolo- infiltrating lymphocytes (TILs) has shown promising therapeutic gous TILs were propagated from each tumor sample. TILs were fluo- efficacy in subsets of patients with several solid tumors including rescently labeled and incubated together with 3D tumoroids in the NSCLC. However, to improve the anti-tumor efficacy of TIL ACT in presence or absence of the PD1 inhibitor nivolumab and/or an agon- solid tumors it is critical to develop rational combination strat- ist anti-CD137 mAb urelumab. TIL infiltration into tumoroids and kill- egies and to identify biomarker(s) predictive of patients who ing of metabolically labeled tumor cells were quantified by advanced would respond favorably to TIL therapy. Here we describe a high confocal microscopy and a custom image analysis algorithm that was content imaging approach using a fresh tumoroid model with in- correlated with flow cytometry and cytokine profiling. tact tumor stroma for quantitative assessment of autologous TIL Results infiltration and target tumor cell killing. We show that nivolumab and urelumab treatments had significant Methods impacts on TIL infiltration in subsets of NSCLC tumoroids. Flow cy- All human tumor samples were obtained with patient consent and tometry analysis demonstrated treatment-mediated activation of TILs relevant IRB approval. For the ex vivo assays 3D tumoroids measuring accompanied by marked changes in the release of pro-and anti- 100-150 micron in size were prepared and cryopreserved during the inflammatory cytokine profiles. Furthermore, we documented the ef- process of ex vivo propagation of autologous TILs. Allogeneic periph- fect of TIL transfer and drug treatment on resident T-cells, Tregs and eral blood mononuclear cells (PBMCs) were used as control. Ex vivo myeloid cell populations within the tumoroids. No correlation was propagated TILs were fluorescently labeled and their growth and found between TIL activity and composition of propagated TILs or functional characteristics in the presence or absence of CD3/CD28 PD-L1 expression on tumor cells. tetramer were assessed via flow cytometry. High content confocal Conclusions analysis was used to quantify TILs infiltration into the tumoroids and This data suggests that combined blockade of PD1 and activation of target tumor cell killing using Nilogen’s 3D-ACT platform. Multiplex CD137 may enhance the therapeutic efficacy of TIL ACT in NSCLC. cytokine assays and flow cytometry analysis were performed to as- Overall, this study also shows that our 3D-ACT tumoroid model al- sess TIL activation upon exposure to tumoroids. lows comprehensive analysis of compensatory mechanisms and se- Results lection of rational combinatorial treatment using adaptive cellular We successfully prepared matched autologous TILs and unpropa- therapy with autologous TILs and likely with other types of cellular gated 3D tumoroids from NSCLC patient tumors. The characteristics therapies. of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved tumor organoids using a cus- tom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of P209 marker-specific cell number, cell viability and apoptosis in addition to Hijacking CAR-CD19 T cells to potently control Her2-positive solid structural and functional analysis of cells in intact 3D tumoroids. High tumors in vitro and in vivo through the use of unique and content confocal imaging analysis demonstrated that CD3/CD28 pre- selective bridging proteins activated TILs with increased activation phenotypes and enhanced Paul Rennert, PhD, Christine Ambrose, PhD, Fay Dufort, PhD, Lihe Dufort, pro-inflammatory cytokine release had marked infiltration into the Alyssa Birt, Thomas Sanford, Lan Wu, Roy Lobb, PhD 3D tumor organoids compared to untreated TILs and PBMCs. The Aleta Biotherapeutics, Natick, MA, United States data was correlated with quantitative tumor cell killing assessment Correspondence: Paul Rennert (paul.rennert@aletabio.com) for tumoroids. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P209 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 114 of 272 Background using antibodies directed against the scFv extracellular region. The Cell therapy success is limited by two critical issues. One is loss of anti- analytic panel included 29 cell surface, activation, exhaustion, and gen expression. This accounts for the ~50% relapse rate seen in CAR- cell-cell adhesion markers to identify/characterize lymphocyte sub- treated B cell malignancies. Solid tumors have highly variable antigen sets; and 9 intracellular markers to characterize functional status and expression and CARs targeting a single antigen fail as antigen-negative activation of signaling pathways. viSNE was used to visualize high- tumor cells escape, driving tumor resistance. dimensional data on a 2D map and quantify CyTOF data. A related issue is that most cell therapeutics fail to persist in the pa- Results tient. This is a particularly true of solid tumor treatment with CAR T Axi-cel products contained a median of 63% (range, 20-86%) trans- cells. The persistence failure may result from unproductive CAR-T inter- duced CAR (CAR+) T cells, and included relatively undifferentiated T cell action with the targeted tumor cell. subsets: a median of 0.4% and 52% of CD4 CAR+ cells were T stem cell We have developed CAR-CD19 T cells (CAR19s) that secrete bridging memory (SCM) and central memory (CM) cells, respectively, and 7% proteins to address these two critical issues. We leverage the ability and 34% of CD8 CAR+ cells were SCM and CM cells, respectively. CAR+ of CAR19s to persist independently of the target tumor cell while T cells in products had significantly higher expression of proliferation, simultaneously endowing these CARs with potent targeting technol- activation, and exhaustion markers (Ki67, CD25, HLA-DR, ICOS, OX-40, ogy. Here we illustrate this technology using the CD19-based bridg- Tim3, LAG3) and higher expression of cell-cell adhesion molecules ing protein that binds both EGFR and Her2. These data demonstrate (CD49d, CD29) compared with CAR-negative (CAR–)Tcells. On day 7, a that CAR19 T cells can be redirected to kill solid tumors in vivo. median of 11% of circulating T cells (range 0.6-58.4%) were CD4 CAR+ Methods and 3% (range 0.6-44.1%) were CD8 CAR+. Both CAR– and CAR+ T cells We cloned a highly stable CD19 extracellular domain (ECD) in frame with showed evidence of activation, but circulating CAR+ T cells expressed an anti-Her2 scFv to create, express and purify CD19-ECD-anti-Her2 bridg- higher levels of Ki67, 4-1BB, Tim3, PD-1, PD-L1, CXCR3, CD29, pZAP70, ing proteins. The sequence was also cloned downstream of a CAR19 do- pSTAT3 and pSTAT5, compared to CAR– Tcells. main and P2A cleavage site in a lentiviral vector. Transduced primary T Conclusions cells expressed the CAR19 and secreted the bridging protein. These CyTOF enables detailed characterization of CAR T cell products com- bridging protein formats were evaluated with in vitro cytotoxicity assays prising heterogeneous T cell subsets. Axi-cel comprises both trans- and Her2+ tumors in vivo. Finally, we added an anti-EGFR scFv sequence, duced and untransduced T cells at various stages of differentiation, creating an extremely potent multi-antigen targeting module. including SCM cells. CAR+ T cells showed higher expression of a broad Results range of proliferation and activation/exhaustion markers, compared to Incorporating a stabilized CD19 ECD in bridging proteins improved pro- CAR– cells, both in axi-cel products and in peripheral blood 7 days after tein expression, including from CAR19 T cells. CAR19 T cells secreting CAR T cell infusion. These data shed light on phenotypic and functional stabilized CD19-anti-Her2 bridging proteins were highly potent in vitro diversity of CAR T cells, pre- and post-infusion, influenced by the manu- and in vivo targeting CD19+ or Her2+ cells. An anti-EGFR scFv was facturing process, conditioning-related homeostatic cytokines and added to the CD19-ECD-anti-Her2 bridging protein. This novel multi- antigen-driven activation. Future studies may explore associations be- antigen targeting bridging protein supports highly potent cytotoxicity tween product composition and clinical outcomes. against single and dual antigen-expressing tumor cells while retaining Trial Registration intrinsic anti-CD19 activity, providing these unique CARs with a tumor- NCT02926833 independent and self-renewing antigen depot in CD19-positive normal B cells. For specific indications a third binding domain is added. P211 Conclusions Combining Deep™ IL-12 Primed and Deep™ IL-15 Primed T cells We have created a robust system for targeting multiple tumor anti- leverages complementary mechanisms to enhance anti-tumor gens simultaneously with a single CAR T cell. Further the use of activity CAR19s supports CAR-T cell persistence independently of tumor anti- Katharine Sackton, PhD, Elena Geretti, PhD, Pengpeng Cao, PhD, Shawn gen expression. This unique technology addresses critical issues in Carey, PhD, Xiaoyan Liang, Jonathan Nardozzi, PhD, Zishu Gui, Alicia cell therapy using a potent technology whose modular nature allows Worthylake, Glenn Leary, Becker Hewes, MD, Tap Maniar, MD, Jonathan for rapid program and pipeline development. Fitzgerald, PhD, Andy Rakestraw, PhD, Karsten Sauer, PhD, Douglas Jones, PhD, Thomas Andresen, PhD P210 Torque Therapeutics, Cambridge, MA, United States Deep phenotypic and functional analysis of transduced anti-CD19 Correspondence: Thomas Andresen (tandresen@torquetx.com) CAR T cells and untransduced T cells in patients treated with axi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P211 cel by single cell mass cytometry 1 1 1 Yohei Arihara, MD, PhD , Caron Jacobson, MD , Philippe Armand, MD , Background 2 2 2 John Rossi, MS , Nathalie Scholler, MD, PhD , Stuart Sievers, PhD , Interleukin-15 (IL-15) and Interleukin-12 (IL-12) play complementary 2 2 2 Edmund Chang, PhD , Mauro Avanzi, MD, PhD , Adrian Bot, MD, PhD , roles as immunomodulators. IL-15 induces T cell memory and supports Jerome Ritz, MD survival, activation and proliferation of CD8+ T and NK cells. IL-12 pro- Dana-Farber Cancer Institute and Harvard Medical School, Boston, motes T cell cytotoxicity and innate immune responses in the tumor MA,United States; Kite, a Gilead Company, Santa Monica, CA, Santa microenvironment. Both cytokines have been explored as cancer im- Monica, CA, United States munotherapies, but clinical success has been limited due to severe side Correspondence: Jerome Ritz (jerome_ritz@dfci.harvard.edu) effects. To limit systemic toxicities, Torque has developed its Deep- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P210 Primed™ T cell therapy technology. Multi-targeted T cells (MTC) specific for multiple tumor antigens are generated from patient apheresis. Cyto- Background kines are tethered to MTCs to support MTC persistence and activity fol- Axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T lowing adoptive transfer into patients, while limiting systemic cytokine cell therapy, has shown high efficacy in relapsed/refractory (R/R) dif- exposure. This study evaluates the combination of cytotoxic T lympho- fuse large B cell lymphoma (DLBCL). Axi-cel contains heterogeneous cytes (CTL) Deep-Primed with Deep™ IL-15 and Deep™ IL-12 to leverage populations of transduced and untransduced T cells. We used single their complementary biology for superior efficacy. cell mass cytometry (CyTOF) to analyze the impact of this heterogen- Methods eity on proliferation and expansion of these cells after infusion. CTLs reactive against MART-1 antigen were generated from healthy Methods donors (MART-1 CTLs). Next, expansion and cytotoxicity of MART-1 CyTOF examined CAR T cell products from 12 patients with R/R CTLs loaded with Deep IL-12, Deep IL-15 or both against MART-1 ex- DLBCL and peripheral blood mononuclear cells obtained 7 days after pressing SKMEL-5 melanoma cells were assessed. In addition, murine axi-cel infusion. We identified anti-CD19 CAR T cells (CAR+ cells) PMEL CD8+ T cells reactive against the B16-F10 melanoma antigen Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 115 of 272 gp100 were loaded with Deep IL-12, Deep IL-15 or both and evalu- Conclusions ated for in vitro expansion, activation and cytotoxicity against B16- Conclusions: These results indicate the viability of TIL ACT for refrac- F10 melanoma cells, as well as for anti-tumor activity in B16-F10 tory ovarian cancer by allowing for the large expansion of anti-tumor tumor-bearing mice. TIL in a short time and consistent manner. A Phase II clinical trial Results based on this work is currently open at UTMDACC to evaluate the Loading with Deep IL-15 promoted MART-1 CTL proliferation and feasibility of TIL ACT in recurrent or refractory OvCa (NCT03610490). preserved antigen reactivity over time. Deep IL-12 loaded MART-1 Ethics Approval CTLs displayed enhanced IFN-γ secretion and cytotoxicity, particularly Ethics approval: Ethical approval and tissue from surgical resections used at low effector:target ratios. Combination of MART-1 CTLs loaded to expand TIL were both obtained under a protocol (PA16-0912 and with Deep IL-12 and Deep IL-15 further enhanced T cell expansion, LAB02-188) approved by the Institutional Review Board of UTMDACC. IFN-γ secretion and cytotoxicity. Similarly, combination of murine PMEL T cells loaded with Deep IL-12 and Deep IL-15 resulted in per- P213 sistent T cell activation, improved memory, and enhanced cytotox- Conversion of peripheral blood mononuclear cells into tumor- icity over individually loaded T cells. Coadministration of Deep IL-12 specific cytolytic cell populations using tumor cells engineered and Deep IL-15 loaded PMEL T cells to B16-F10 melanoma-bearing with multiple immunomodulatory factors mice was well-tolerated, with minimal and reversible body weight 1 1,2 2 Joshua Keegan , James Lederer , Frank Borriello, MD, PhD , Nathan loss, and elicited superior anti-tumor activity. Schomer, MS Conclusions 1 2 BWH Harvard Medical School, Boston, MA, United States; Alloplex Modular tethering of Deep™ IL-12 and Deep™ IL-15 to T cells uniquely Biotherapeutics, Inc., Winchester, MA, United States leverages their complementary functions as immunomodulators to Correspondence: James Lederer (jlederer@alloplexbio.com); Frank maximize anti-tumor activity without notable toxicity in preclinical Borriello (fborriello@alloplexbio.com) models. A Phase I clinical trial of Deep IL-15 Primed MTCs (TRQ15-01) in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P213 solid cancers and lymphoma is enrolling (NCT03815682). Torque is initi- ating clinical evaluation of Deep IL-12 Primed MTCs (TRQ12-01), includ- Background ing a combination arm with TRQ15-01. Numerous studies and two recent clinical approvals have demon- strated the efficacy and safety of cellular immunotherapies to treat P212 diverse cancer subtypes. To date, the only approved cytotoxic cel- Potential clinical application of tumor-infiltrating lymphocyte lular therapies in the U.S. are T-cell based. However, ongoing clin- therapy for ovarian epithelial cancer prior or post-resistance to ical trials using gamma delta T cells and natural killer cells have chemotherapy suggested not only clinical efficacy, but also synergistic beneficial Donastas Sakellariou-Thompson, BS, Marie-Andree Forget, PhD, Emily effects when combined with checkpoint inhibitor antibody therap- Hinchcliff, MD, Joseph Celestino, Patrick Hwu, MD, Amir Jazaeri, MD, Cara ies. Our group developed novel immune stimulatory allogeneic Haymaker, PhD, Chantale Bernatchez tumor cells engineered with multiple immunomodulatory factors as MD Anderson Cancer Center, Houston, TX, United States a novel approach to generate heterologous tumor cell lytic popula- Correspondence: Cara Haymaker (chaymaker@mdanderson.org); tions of human peripheral blood mononuclear cells (PBMCs) for Chantale Bernatchez (cbernatchez@mdanderson.org) cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P212 Methods SK-MEL-2 cells obtained from the NIH were engineered to express a set Background of immunomodulatory proteins with the aim of expanding oncolytic cell Background: Epithelial ovarian cancer (OvCa) is the deadliest populations. Cells were engineered using lentiviral vectors prepared by gynecological cancer, and is estimated to account for almost 14,000 VectorBuilder, and sorted by flow cytometer for high expression of de- deaths in 2018. Traditional management of advanced stage OvCa in- sired immunomodulators. These engineered cells were then mixed with cludes tumor reductive surgery and adjuvant platinum-taxane chemo- freshly thawed human PBMCs and co-incubated for up to 14 days. Cells therapy, which results in high rates of initial complete response. were collected at time points during the incubation period for pheno- However, nearly 90% of patients recur and the 5-year survival rate for typic analysis using mass cytometry (CyTOF). Functional characterization late-stage disease is only 28. Immunotherapy has become a powerful of stimulated PBMCs was conducted using a cytotoxicity assay against treatment option for several solid tumor types. The presence of tumor- targets from several cancer subtypes. infiltrating lymphocytes (TIL) is correlated with better prognosis in ovar- Results ian cancer, pointing at the possibility to benefit from harnessing their CyTOF analysis of stimulated PBMCs revealed expansion of natural anti-tumor activity. The effectiveness of adoptive cell therapy (ACT) killer cells, gamma-delta T cells, and CD8 T cells by 8 days after with TIL has already been shown in metastatic melanoma with object- stimulation (Figure 1). All these populations expressed high levels of ive response rates of 40-50%. This preclinical study explores the feasibil- NKG2D and Granzyme B, suggesting widespread recognition of ity of transposing TIL ACT to OvCa using an improved culture method. tumor antigens and cytotoxic capability. In contrast, PBMCs that were Methods activated and expanded by CD3/CD28 activation beads showed less Methods: High-grade serous ovarian cancer (n=84), pre- or post- heterogenous expansion. PBMCs expanded with immunomodulatory chemotherapy and primary or metastatic, samples were accrued. TIL cell lines demonstrated potent cytotoxic activity towards both the were cultured using either high-dose IL-2 only, high-dose IL-2 with an parental cell line, as well as other melanoma and non-melanoma agonistic antibodies targeting 4-1BB (a41BB), or a combination of IL-2, cancer cell lines (Figure 2). As a control for specific cytotoxicity, both a41BB, and an agonistic anti-CD3 mAb. The cells were phenotyped autologous and allogeneic PBMCs were tested as targets and dis- using flow cytometry in the fresh tissue and after expansion. Tumor re- played no detectable cytotoxicity in our assay. activity was assessed against HLA-matched ovarian cancer cell lines via Conclusions IFN-γ ELISPOT. We developed a novel approach to expand immune cell populations Results from normal human PBMCs demonstrating anti-tumor activity Results: Ovarian cancer is highly infiltrated with CD8+ TIL that are pref- against multiple cancer cell types. This strategy is being developed erentially and robustly expanded with IL-2 and the two agonistic anti- to activate and expand PBMCs from cancer patients that will be used bodies. With a 95% success rate, the TIL are grown to ≥100x10^6 cells for autologous cellular immunotherapy. Taken together, the results in 2-3 weeks without over differentiation. In addition, the CD8+ TIL from this study demonstrate our ability to generate multiple popula- grown with this method showed HLA-restricted tumor recognition. TIL tions of cytotoxic effector cells from PBMCs, which should provide a growth and tumor recognition was independent of surgery site or straight-forward approach to generate clinically-relevant cells for chemotherapy exposure. adoptive cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 116 of 272 replace endogenous TCRs with the native neoTCR sequence in each edited T cell for expression at native TCR levels. Precision genome en- gineering of a colon cancer tumor cell line (SW620) was used to express the patient-specific neoantigen (COX6C-R20Q) at native levels. neoTCR- T cells were co-cultured with SW620 expressing the COX6C-R20Q muta- tion or the COX6C wild-type peptide. Functional readouts were T cell proliferation, cytokine secretion and tumor cell killing. Results Seven neoTCR clonotypes against the mutated COX6C peptide (COX6C-R20Q) presented in the context of HLA-A2 were cloned from imPACT-captured neoE-specific CD8 T cells. Primary human T cells were engineered with the 7 different TCR specificities against the COX6C-R20Q. Each of the seven candidate neoTCR-engineered T cells displayed specific cytotoxicity against tumor cells expressing en- dogenous levels of the COX6C-R20Q neoantigen. At 96 hours, using Effector to Tumor cell ratio (E:T) 1:1, 85-90% tumor elimination was observed (p < 0.000001 for each comparison). Significant tumor cell killing was detected with an E:T ratio as low as 1:5. neoTCR-T cells also proliferated and secreted interferon-gamma in response to co- culture with the relevant tumor target. Importantly, neoTCR-T cell ac- tivity was absent when co-cultured with tumor cells expressing wild type COXC6 protein. Conclusions Fig. 1 (abstract P213). See text for description These results demonstrate that the imPACT Isolation Technology used to capture antigen-experienced, neoE-specific T cells from the blood of patients with cancer authenticates that these neoE-HLA tar- gets are relevant for engineering neoTCR-T cells therapies. Lever- aging this approach, PACT is developing autologous personalized adoptive T cell therapy (NeoTCR-P1 product). A Phase 1 clinical trial to test NeoTCR-P1 T cells in subjects with solid tumors is currently ongoing (NCT03970382). References 1. Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, Urbinati F, Foy S, Hunter T, Tai A, Bethune M, Peng A, Dalmas O, Franzusoff A and Mandl S. Abstract 4783: Highly efficient, non-viral preci- sion genome engineering for the generation of personalized neoepitope- specific adoptive T cell therapies. Cancer Res 2019;79(13 Suppl). 2. Sennino B, Conroy A, Purandare B, Litterman A, Jacoby K, Moot R, Lu W, Nguyen D, Urbinati F, Foy S, Hunter T, Dalmas O, Bethune M, Park T, Peng S, Franzusoff A and Mandl S. Abstract 1433: NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with ‘youn- ger’ phenotypes that rapidly proliferate and kill target cells upon recogni- tion of cognate antigen. Cancer Res 2019;79(13 Suppl). Fig. 2 (abstract P213). See text for description 3. Peng S, Quach B, An D, Sandoval S, Bao R, Pan Z, Bethune M, Dalmas O, Yi M, Meadows C, Heeringa K, Guo L, Yuen B, Sorfleet J, Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, P214 Mandl S and Franzusoff A. Abstract 1435: An ultra-sensitive and high- T cells precision engineered to express neoepitope-specific TCRs throughput technology (imPACT) for the identification and isolation of cloned from a patient with colorectal cancer specifically target and intrinsic and emergent neoepitope-specific T cells from the peripheral kill relevant neoantigen-expressing tumor cells blood and TILs of cancer patients. Cancer Res 2019;79(13 Suppl). Barbara Sennino, PhD, Adam Litterman, John Gagnon, Andrew Conroy, Ethics Approval Bhamini Purandare, Zheng Pan, Dalmas Olivier, Kyle Jacoby, Songming Human samples in this study were procured from a commercial vendor who Peng, Alex Franzusoff, Stefanie Mandl, PhD collected them according to their established ethics policies. PACT Pharma, South San Francisco, CA, United States Correspondence: Alex Franzusoff (afranzusoff@pactpharma.com); Stefanie Mandl (smandl@pactpharma.com) P215 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P214 Depletion of CD45RA-positive cells potentiates the reactivation of EBV-specific T-cells from EBV positive lymphoma patients Background Sandhya Sharma, BSc, Naren Mehta, Kathan Parikh, RA, Cliona Rooney, Neoepitopes (neoE) derived from private tumor-exclusive mutations PhD represent compelling targets for personalized TCR-T cell therapy to Baylor College of Medicine, Houston, TX, United States eradicate tumor cells throughout the body. The imPACT Isolation Tech- Correspondence: Sandhya Sharma (me.sandhya@gmail.com) nology® is an ultra-sensitive and high-throughput process for the cap- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P215 ture of mutation-targeted CD8 T cells from patient blood. NeoTCRs of native sequence, cloned from the captured T cells, were evaluated for Background tumor-targeted functionality by non-viral precision genome engineer- Epstein-Barr virus (EBV)-positive lymphomas express viral type-2 latency ing of fresh human CD8 and CD4 T cells for neoTCR expression [1,2]. proteins (T2-Ags). Autologous EBV-specific T-cells (EBVSTs) directed to Methods T2-Ags have produced complete responses in ~50% lymphoma pa- NeoTCRs were isolated from the blood of a patient with colorectal can- tients[1]. However, in our ongoing clinical trial, we failed to generate cer using the imPACT Isolation Technology® [3]. Subsequently, healthy EBVSTs from 24% of the patients procured; manufacturing failures donor CD8 and CD4 T cells were precision genome engineered to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 117 of 272 associated with lack of T-cell expansion and T2-Ag-specificity. Further, Background in lines successfully expanded, many EBVST lines demonstrated low T2- Administration of Toll-like receptor agonists with adoptively trans- Ag specificity and some contained a high frequency of NK-cells. Our ferred T cells augment tumor immunity. However, systemic or local goal is to improve both the manufacturing success rate and clinical effi- administration of TLR agonists could heighten inflammation and lead cacy of EBVSTs. In EBV-exposed individuals, EBVSTs reside in CD45RA- to toxic side effects when coupled with CAR or TIL-based therapies. CD45RO+ memory compartment, while CD45RA-positive population in- Thus, we hypothesized that TLR agonists could be used ex vivo dur- cludes unwanted naïve T-cells, suppressive regulatory-T cells, and NK- ing T cell expansion and ultimately generate a cell product with en- cells[2,3]. We hypothesized that removal of CD45RA-positive cells from hanced anti-tumor properties for adoptive cell transfer therapy while PBMCs prior to EBV-antigen specific stimulation would improve EBVST reducing potential toxicity to patients. generation and specificity by eliminating competing naïve T-cells, while Methods reducing potentially inhibitory cells capable of inhibiting the outgrowth To test our hypothesis, we employed the Pmel-1 transgenic mouse of antigen-specific T-cells. We, therefore investigated the effects of se- model, in which CD8+ T cells harbor a TCR specific for the gp100 epi- lective depletion of CD45RA-positive cells from PBMCs prior to EBV T2- tope expressed on melanoma and healthy melanocytes. Pmel spleno- Ags stimulation. cytes were stimulated with hgp100 peptide in the presence or absence Methods of TLR9 agonist CpG (ODN-1668) and expanded in IL-2 for one week. T EBVSTs were generated from whole PBMCs and CD45RA depleted (RAD) cell phenotype was analyzed via flow cytometry and anti-tumor activity PBMCs and we measured proliferation by counting, T2-Ag specificity assessed in mice bearing B16F10 melanoma tumors. using IFN-gamma ELIspot assays and cell-phenotype by flow cytometric Results analysis. To compare their in-vivo efficacy, EBVSTs were adoptively trans- T cells expanded with CpG possessed a unique phenotype (IL-2Ralpha- ferred into immunodeficient mice bearing autologous EBV-transformed high, ICOS-high, CD39-low) and mediated more potent responses lymphoblastoid tumor cells and tumor clearance was evaluated. against melanoma in vivo than traditionally expanded T cells. Interest- Results ingly, this phenotype and anti-tumor function was dependent on the RAD-EBVSTs produced greater expansion of EBVSTs from PBMCs and presence of B cells at the start of culture, as their removal resulted in a decreased the frequency of NK cells, which dominated some of our pa- loss of this unique phenotype and anti-tumor efficacy in vivo. Con- tient lines. T2-Ag specificity increased by 3-5 fold as measured by versely, removal of CD4+ T cells, NK cells, dendritic cells, or macro- gamma-IFN release in response to T2-Ags stimulation in both healthy phages from culture did not ablate the phenotype or anti-tumor donors and patients. RAD-EBVSTs maintained antigen specificity over activity of CpG-generated T cells. The CpG-elicited T cell effects were multiple rounds of weekly T2-Ags stimulation. Phenotypic analysis dem- also dependent on the peptide-mediated interaction between the T onstrated decreased expression of exhaustion markers in RAD-EBVSTs. cell and APC in culture as activating with plate-bound or bead-bound Most importantly, this approach restored responsiveness to antigen antibody strategies resulted in a T cell population that was similar to stimulation in most unresponsive patients enabling the successful re- the ineffective vehicle treated cells both phenotypically and therapeut- activation and expansion of EBVSTs from lymphoma patients that failed ically. We further found that CpG-treated B cells expressed heightened previously. This advantage extends to VSTs with specificity for HPV, VZV levels of CD40, suggesting that induction of a CD40-CD40L axis be- and adenoviral antigens, suggesting that we have identified a general tween B and T cells may account for the generation of potent IL- approach for improving the activity of VSTs for immunotherapy. EBVSTs 2Ralpha-high, ICOS-high, CD39-low T cells. generated from RAD-PBMCs demonstrated more rapid tumor clearance Conclusions and superior control of metastatic tumors in our xenograft model. The TLR9 agonist CpG can be used in ex vivo culture to potentiate Conclusions an anti-tumor T cell product for ACT. The CpG-associated T cell This approach to the generation of VSTs is being translated to the phenotype (IL-2Ralpha-high, ICOS-high, CD39-low) and anti-tumor clinic for use in multiple clinical trials. In future, we aim to elucidate ability was dependent on the presence of B cells and a direct inter- the mechanisms underlying the inhibitory effects of the CD45RA action between the anti-tumor T cells and APC via peptide activation. positive population in the reactivation and expansion of EBVSTs. Post CpG stimulation B cells express heightened CD40, which may promote the B cell/T cell interaction and thus the generation of a po- References tent T cell product for ACT. 1. Bollard CM, Gottschalk S, Torrano V, et al. Sustained Complete Responses in Ethics Approval Patients With Lymphoma Receiving Autologous Cytotoxic T Lymphocytes This study was approved by the Institutional Animal Care & Use Com- Targeting Epstein-Barr Virus Latent Membrane Proteins. Journal of Clinical mittee of the Medical University of South Carolina, protocol number Oncology. 2016;32(8):798-808. doi:10.1200/JCO.2013.51.5304. 0488. 2. Krzywinska E, Cornillon A, Allende-Vega N, et al. CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity. Bobé P, ed. P217 PLoS ONE. 2016;11(4):e0150434. doi:10.1371/journal.pone.0150434. An NK cell line (PD-L1 t-haNK) engineered to target PD-L1 3. Krzywinska E, Allende-Vega N, Cornillon A, et al. Identification of Anti- efficiently kills tumor cells and myeloid derived suppressor cells tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With 1 1 1 Kellsye Fabian, PhD , Michelle Padget , Renee Donahue, PhD , Kristen Hematological Cancers. EBioMedicine. 2015;2(10):1364-1376. doi:10.1016/ 1 2 3 Solocinski, PhD , Clint Allen, MD , John Lee, MD , Patrick Soon-Shiong, j.ebiom.2015.08.021. 3 1 1 MD , Jeffrey Schlom, PhD , James Hodge, PhD, MBA Ethics Approval 1 2 NCI/NIH, Bethesda, MD, United States; NIDCD/NIH, Bethesda, MD, The study was approved by Baylor College of Medicine's Ethics Board, United States; NantKwest, Culver City, CA approval number H7634 and H7666 for human subjects. Animal protocol Correspondence: James Hodge (jh241d@nih.gov) AN5551. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P217 P216 Background CD8+ T cells break tolerance to tumors in a B cell-dependent The ability of natural killer (NK) cells to lyse tumor targets without manner via TLR9 signaling prior sensitization and without human leukocyte antigen (HLA)-re- Aubrey Smith, BS, Hannah Knochelmann, BS, Connor Dwyer, PhD, striction make them promising candidates for “off the shelf” cell- Megan Wyatt, MS, Guillermo Rangel Rivera, Jessica Thaxton, PhD, MSCR, based immunotherapy. Here we investigate the anti-tumor efficacy Mark Rubinstein, PhD, Bei Liu, MD MPH, Eric Bartee, PhD, Chrystal Paulos, of a novel NK cell platform, the PD-L1-targeted high-affinity NK (PD- PhD L1 t-haNK), which lacks killer inhibitory receptors (KIRs), carries a high Medical University of South Carolina, Mount Pleasant, SC, United States payload of granzyme and perforin granules, and has been designed Correspondence: Aubrey Smith (butchera@musc.edu) with a chimeric antigen receptor (CAR) to target PD-L1 expressing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P216 cells on cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 118 of 272 Methods Conclusions Frozen, irradiated (15 Gy) PD-L1 t-haNK cells were thawed and charac- The assay provides a sensitive, simple read-out to support discovery terized via flow cytometry and RNAseq. PD-L1 t-haNK lytic activity was research and/or QC lot release applications. assessed in vitro using MDA-MB-231, BT549, T47D, MCF7, SUM149, H460, H441, HCC4006, SW480, SW620, DU145, HTB1, CaSki, and CH22 P220 cell lines as targets in indium-based and flow cytometry-based killing Targeting neoantigens with immunotherapy: Are we limited to assays. The effect of pre-treating tumor targets with IFNγ on PD-L1 t- pre-existing autologous neoantigen-specific T-cells? haNK targeting was also examined. PD-L1 t-haNK cells were co-cultured Aline Bracher, Slavoljub Milosevic, Daniel Sommermeyer with human peripheral blood mononuclear cells (PBMCs) and purified Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany human myeloid derived suppressor cells (MDSCs) to investigate the ef- Correspondence: Daniel Sommermeyer fects of PD-L1 t-haNK on immune subsets. The therapeutic activity of (d.sommermeyer@medigene.com) PD-L1 t-haNK in vivo was studied by adoptive transfer of PD-L1 t-haNK Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P220 cells to NOD scid gamma (NSG) mice engrafted with parental MDA-MB- 231 (PD-L1+) and MDA-MB-231/PD-L1 CRISPR knockout (PD-L1-null). Background Results Several facts shape our considerations regarding the use of neoanti- Here, we show that irradiated PD-L1 t-haNK cells express PD-L1- gens as highly specific targets for immunotherapy of cancer. First, specific CAR, and high levels of perforin and granzyme B. PD-L1 t- adoptive T-cell therapy using TILs seems to be most successful if the haNKs lysed all 14 human tumor cell lines tested in vitro, and in- TILs include T-cells specific for antigens resulting from individual muta- creased cell lysis corresponded with increasing levels of PD-L1 ex- tions. Second, the success of checkpoint inhibitors is often correlated pression on tumor cells. Increasing PD-L1 expression on tumor cells with the mutational load of tumors. However, a mutation must fulfill through IFNγ treatment improved PD-L1 t-haNK-mediated lysis by up several criteria to be effective as a neoantigen that can be recognized to 100%. In vivo, adoptive transfer of PD-L1 t-haNK cells inhibited the by T-cells. Obviously, the mutation must lead to a novel amino acid se- growth of engrafted parental MDA-MB-231 but not PD-L1 null MDA- quence (e.g. single amino acid substitution, fusion- or frameshift- MB-231 tumors. Finally, when co-cultured with human PBMCs and sequence), and be located in a gene expressed in tumor cells. Further- purified human MDSCs expressing PD-L1, PD-L1 t-haNK cells prefer- more, a peptide spanning the new sequence needs to be efficiently entially lysed the MDSC population but not other PBMC subsets. processed and presented by HLA molecules. Finally, a T-cell response Conclusions must be triggered that can specifically recognize the mutated epitope. This study provides a rationale for the potential use of adoptively trans- Targeting neoantigens as patient-individualized epitopes requires ro- ferred irradiated PD-L1 t-haNK cells as a unique immunotherapeutic plat- bust processes for rational and rapid selection and validation of form against a range of human tumor types. In addition to lysing the neoantigens as T-cell targets. Currently, the most challenging step is tumor cells, MDSC killing may be a novel mechanism of action of PD-L1 t- predicting specific T-cell responses. Huge efforts have been made to haNK cells that may potentiate their impact especially in cancers where analyze the reactivity of patients’ T-cells against mutations. However, MDSC’s limit immune approaches. The safety and clinical benefit of PD-L1 this approach is limited to the T-cell repertoire present in patients at t-haNK cells in cancer patients are being assessed in ongoing clinical trials. the time of tumor resection or blood draw and might miss potential Ethics Approval potent T-cell responses that were lacking or no longer present in the The study was approved by the NCI IRB, NIH protocol 99-CC-0168. patient. In our opinion, only screening the T-cell repertoire of several healthy donors can answer the question if a specific mutation can P218 trigger T-cell responses. We present proof-of-concept data how we A novel, bioluminescent assay for the selective detection of target use our high-throughput T-cell receptor (TCR) platform technologies cell killing in mixed cultures and automated processes for fast and efficient screenings of T-cells Richard Somberg, PhD, Brock Binkowski, Aileen Paguio, Peter Stecha, isolated from several partially HLA-matched healthy donors. Chris Eggers, Braeden Butler, Michael Beck, Julia Gilden, PhD, Jey Cheng, Methods Mei Cong, PhD, Frank Fan, PhD Promising neoantigens were predicted and T-cells responses after Promega, Madison, WI, United States stimulation with antigen-presenting cells either transfected with Correspondence: Brock Binkowski (brock.binkowski@promega.com) minigene constructs or loaded with peptides were compared. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P218 Results Peptide stimulation triggered specific T-cells for most tested muta- Background tions, indicating that T-cell repertoires of healthy donors can Efforts to develop and commercialize cellular immunotherapies recognize neoantigens when they are forced to be presented. Also, would benefit from assays that selectively monitor target cell death with the clinically relevant approach using endogenously processed that are sensitive and easy-to-use. To address this, we have devel- antigen encoded by minigenes, specific T-cell responses against oped an approach to selectively quantify target cell death using a neoantigens presented on different HLAs were efficiently triggered, gain-of-signal assay format and bioluminescence read-out. although only against some of the tested epitopes. Methods Conclusions The method relies on the release of a HiBiT-tagged protein from tar- This screening strategy has the aim to develop future TCR-based get cells following cell lysis. HiBiT, an 11 a.a. peptide tag, binds to therapies and can be used for the identification of promising muta- cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconsti- tions for vaccination or as a source for TCRs for adoptive T-cell ther- tute NanoBiT Luciferase. Target cells are engineered to express a apy. Furthermore, generated data can subsequently improve HiBiT-tagged protein using either ectopic expression or CRISPR/Cas9 algorithms predicting the immunogenicity of neoantigens. to tag endogenous lactate dehydrogenase (LDH), and cell lysis is quantified by adding a detection reagent containing LgBiT and furi- P221 mazine substrate (no medium removal). Superior anti-tumor activity of metabolically enhanced bispecific Results antibody armed CAR-less Bionic T cells The signal is proportional to the amount of target cell death, and 1 2 1 Archana Thakur, PhD , John Scholler , Edwin Bliemeister , Carl June, measurements can be made using endpoint or kinetic formats. Cell 2 1 MD , Lawrence Lum, MD, DSc lines have low rates of spontaneous release and fusion proteins that 1 2 University of Virginia, Charlottesville, VA, United States; University of are stable in the extracellular medium, enabling assays up to 24 Pennsylvania, Philadelphia, PA, United States hours or more. The bright signal from NanoBiT Luciferase allows the Correspondence: Archana Thakur (at2fx@virginia.edu) use of low numbers of target cells per well (e.g. 2,500), and the assay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P221 can detect very low levels of target cell death (e.g. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 119 of 272 Background inevitably leads to excessive T cell differentiation ex vivo, and de- The major challenges of T cell-based therapies in solid tumors are creased T cell functionality and persistence in vivo. Importantly, T cell the metabolic insufficiency of T cells to exhibit effector functions and differentiation is tightly controlled by epigenetic mechanisms that persist in an altered metabolic landscape of the tumor microenviron- could be targeted therapeutically. The BET protein BRD4 has been re- ment (TME). To overcome these challenges, we developed the next ported to regulate expression of the transcription factor BATF that generation of chimeric antigen receptors-less (CAR-less) Bionic T cells drives CD8+ T cells towards a more effector-like phenotype, while for the treatment of solid cancers. We hypothesize that metabolically BRD4 inhibition by the drug JQ1 prevented this and thereby en- enhanced Bionic T cells armed with bispecific antibody (BiAb) will hanced in vivo T cell persistence and function in adoptive immuno- show enhanced anti-tumor responses and controlled off-tumor on- therapy models [1]. Ph-29089 is a chemically modified self-delivery target toxicity in solid cancer. RNA inhibitor containing an asymmetric duplex structure (≤ 15 base Methods pair duplex) and a single-strand phosphorothioate tail targeting the We engineered metabolically enhanced “Bionic T cells” (BTC) that are BET protein BRD4. PH-29089 is efficiently delivered to immune cells, devoid of CAR but contain a transmembrane and an intracellular do- including T cells, without the need for specialized formulations or main of a co-stimulatory molecule and TCR signaling domain of mechanical transfection as is observed with current RNAi’s. CD3z. T cells were transduced with CAR-less constructs without co- Methods stimulatory domain-FLAG-ζ (Control) or with co-stimulatory domain- Purified human CD8+ T cells were expanded using the rapid expan- FLAG-4-1BB-ζ, FLAG-CD28-ζ, FLAG-ICOS-ζ and FLAG-OX40-ζ, FLAG-27- sion protocol (REP) developed by the National Cancer Institute. Flow ζ and were tested for their hypoxic tolerance, anti-tumor activity, cytometry was used to study Ph-29089 for its ability to knock down cytokine production and exhaustion phenotype in the presence or BRD4 at the protein level in expanding T cells, and to determine T tumor targets. cell differentiation status during and immediately after ex vivo ex- Results pansion. Release of IFNγ by T cells cocultured with tumor cells was Our data show that hypoxia differentially affected BTC survival de- assessed by ELISA. pending on the co-stimulatory endodomain. Under normoxia Bionics Results with CD28ζ (28z) endodomain show 5% apoptosis versus 61% apop- Ph-29089 elicited a concentration dependent silencing of BRD4 pro- tosis under hypoxic (5% oxygen) condition. On the other hand, Bion- tein with an IC50 of 1-2 μM. The BRD4 silencing persisted at least 5 ics with 4-1BBζ showed only 13% apoptosis under hypoxic condition days post-treatment, whereas media and non-targeting control (NTC) suggesting enhanced hypoxic tolerance. HER2 and EGFR BiAb armed did not significantly affect BRD4 protein levels. Compared to un- BTC were tested against various low-high HER2 and EGFR expressing treated, NTC-treated and JQ1-treated CD8+ T cells, Ph-29089-treated cancer cell lines. Specific cytotoxicity of anti-HER2 BiAb (HER2Bi) and CD8+ T cells contained higher percentages of central memory and anti-EGFR BiAb (EGFRBi) armed BTC against MDA-MB-231, SK-BR-3, stem cell-like memory T cells, as determined by expression of BT-20, MiaPaCa-2 cell lines measured by real time cell analysis using CD45RA, CCR7, CD62L and CD95. Moreover, Ph-29089-treated CD8+ xCELLigence ranged between 75-100% at 2:1 E/T ratio at 72 hours. T cells displayed superior functionality, as indicated by enhanced Sequential killing by HER2Bi armed BTC followed by (f/b) EGFRBi IFNγ production when exposed to the allogeneic melanoma cell lines armed BTC showed efficient killing against target cells (86.2%) or A375 and ROAL. EGFRBi-BTC f/b HER2Bi-BTC (88.2%) compared to the killing by Conclusions HER2Bi-BTC (49.7%) or EGFRBi-BTC (43.5%) alone at low E/T ratio in These findings support the hypothesis that BRD4 silencing by Ph- the presence of 100 IU/ml IL-2 at 96 hours. Cytokine levels of IFN-γ, 29089 is a viable approach for expanding T-cells with superior anti- IL-15, IL-2R, and GM-CSF were significantly higher in culture superna- tumor potential for adoptive cell therapies. tants of tumor cells (SK-BR-3) and HER2Bi-BTC or EGFRBi-BTC co- cultures compared to the control condition with unarmed BTC. Reference Phenotypic data show increased expression of 4-1BB, ICOS and OX40 1. Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, on CD4+ and CD8+ T cells after short antigenic exposure and con- et al. BET bromodomain inhibition enhances T cell persistence and tinuous antigenic exposure. function in adoptive immunotherapy models. J Clin Invest. 2016 Conclusions 126(9):3479–94. Data show that armed BTC: 1) exhibit superior survival in hypoxic Ethics Approval condition; 2) can effectively kill multiple tumor targets in serial killing This study was carried out in accordance with the recommendations of assay; 3) cytokines and chemokines show immune modulating and Karolinska Institutet review board with written informed consent from all tumor killing profile. subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Stockholm Acknowledgements Regional Ethics Committee, with approval number (2011/143-32/1). This study was made possible by start-up funds for LGL from the University of Virginia. AT is a co-founder of NOVA Immune Platform. CJ is a co-founder P223 of T Immunity. LGL is a co-founder of Transtarget, Inc. and sits on the Withdrawn Scientific Advisory Board for Rapa Therapeutics. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P223 P222 Modulating BRD4 in T cells using self-delivery RNAi to improve P224 adoptive cell therapy of cancer 1 1 1 Case studies of sarcoma and MRCLS following treatment with NY- Jeroen Melief, PhD , Laura Van Leeuwe Kirsch, BSc , Esmeralda Hemme , 2 2 2 ESO-1 TCR T Cells (GSK3377794): correlates of predictable John Barrett, PhD , Simon Fricker, PhD , Gerrit Dispersyn, PhD , Rolf response characteristics Kiessling, MD, PhD 1 2 3 1 2 Brian Van Tine, MD, PhD , Sandra D'Angelo, MD , Alexandra Gyurdieva , Karolinska Institute, Stockholm, Sweden; Phio Pharmaceuticals, 3 3 3 3 Laura Johnson , David Turner , Jenna Tress , M. Phillip DeYoung , Yuehui Marlborough, MA, United States 3 3 4 Wu , Aisha Hasan, MBBS MD , Dejka Araujo, MD Correspondence: Rolf Kiessling (rolf.kiessling@ki.se) Washington University in St. Louis, St. Louis, MO, United States; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P222 Memorial Sloan Kettering Cancer Center, New York, NY, United States; 3 4 GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Background Center, Houston, TX, United States Ex vivo expansion of T cells for adoptive cell therapy (ACT) of cancer Correspondence: Brian Van Tine (bvantine@wustl.edu) is commonly done with cytokines and stimuli for efficient activation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P224 and expansion of large numbers of tumor-specific T cells. This Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 120 of 272 Background Background Genetically engineered NY-ESO-1 specific T cells (NY-ESO-1 T Cells; Chimeric Antigen Receptor (CAR) T cell therapy is a revolutionary GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a cancer treatment that genetically alters T cells to redirect and har- self-inactivating lentiviral vector to express an affinity-enhanced NY- ness their cancer killing potential. Currently FDA approved CAR T cell ESO-1-specific T-cell receptor. Phase 1 and 2 trials are evaluating products are autologous-based, requiring individualized blood apher- GSK3377794 in solid tumors and hematologic malignancies. This study esis and manufacture. Deriving patient-specific CAR T cell products is will review biomarker data for eight patients from two ongoing phase expensive, laborious, and time consuming, with logistical and regula- 1/2 pilot studies of GSK3377794 in synovial sarcoma (SS; NCT01343043; tory challenges. The success rate of autologous CAR T cell therapy is N=7) and myxoid/round-cell liposarcoma (MRCLS; NCT02992743; N=1) also limited by the urgency of acute and aggressive cancers, uncer- with prolonged response and stable disease (SD). tainty over T cell number, and intrinsic differences in T cell function- Methods ality. Generating CAR T cells from induced pluripotent stem cells Patients who were progression free ≥4 months following first infu- (iPSC) holds encouraging prospect for generating ‘off-the-shelf’ CAR sion were selected. All received the same lymphodepletion (30 mg/ T cell products and overcoming these challenges. iPSCs can prolifer- m2 x3D fludarabine, 600 mg/m2 x3D cyclophosphamide) followed ate almost infinitely while keeping their pluripotency and lineage by GSK3377794 infusion. Six patients with SS were eligible for second differentiation potential. However, the complexity of T cell develop- infusion and received higher-dose lymphodepletion (30 mg/m2 x4D ment and disturbance of T cell differentiation by CAR expression cre- fludarabine, 1800 mg/m2 x2D cyclophosphamide) before second in- ates a challenge for successful iPSC-derived CAR T cell generation. fusion. Pretreatment biopsies were analyzed for CD3 infiltration by Previously reported iPSC-derived CAR T cells showed innate like phe- RNAScope. Transduced cell persistence was measured by quantitative notypes with weak antigen-specific cytotoxicity and compromised PCR of transgene vectors peripheral blood mononuclear cell (PBMC) cytokine production [1]. DNA. Cytokine expression was measured by Meso Scale Discovery Methods immunoassay. PBMC phenotypes were characterized by flow In our current study, we generated iPSC lines from healthy donor T cells cytometry. by an integration-free method using iPSC reprograming episomal vec- Results tors. The iPSC cells were transduced with clinical grade lentivirus to ex- Five of seven patients with SS had SD for 17.8–105 weeks; two had press CD19-specific CARs (CD19CAR), sorted and colonized to generate partial response/complete response (PR/CR) per RECIST1.1. The pa- a homogeneous CAR+ iPSC cell bank. By using a 3D co-culture system, tient with MRCLS had PR per RECIST1.1 lasting 8.8 months. Six of we successfully generated iPSC-derived CD19CAR T cells. seven patients with SS received second infusion; 2/7 had SD, 3/7 had Results PR, 1/7 had CR. Immunohistochemistry revealed ≥50% of cells with The produced iPSC-derived CD19CAR T cells have a surface marker 2+/3+ NY-ESO-1 expression; this was maintained before second infu- phenotype (CD3+CD5+CD7+TCRalphabeta+CD8alphabeta+ and sion. Baseline tumor samples consistently showed 10 fold over first CD3+CD5+CD7+TCRalphabeta+CD4+) and gene expression signa- infusion; of these, there was one PR and one CR. Cytokine increases tures typical of natural T cells. These iPSC-derived CD19CAR T cells reflecting immune cell activation (eg, IFN gamma, IL-6, and IL-2R expanded robustly within two weeks (~100 fold), and showed potent alpha were observed 4–7 days after both infusions. Transduced T antigen-specific cytotoxicity against CD19+ parental tumor cells such cells within manufactured product showed increased expression of as NALM6 and Raji comparing to their CD19 knockout control cells. It activation markers (eg, CD28, ICOS, and CD40L) versus T cells from is intriguing that the in vitro cytotoxicity potency of iPSC-derived apheresis. In 2 patients, transduced CD8 cells primarily had T effector CD19CAR T cells was superior to conventional PBMC-derived CAR T memory RA+ (CD45RA+CCR7-) and T effector memory (CD45RA- cells generated from the same donor. These iPSC-derived CD19CAR T CCR7-) phenotype. In one, 34.3% transduced CD8 cells had T stem cells also demonstrated efficient degranulation activity and a Th1 cell memory (CD45RA+CCR7+) phenotype. cytokine profile (e.g. IFNgamma and TNFalpha when challenged with Conclusions CD19+ target cells. Furthermore, these cells demonstrated potent SS and MRCLS tumors initially show low immune cell infiltration. anti-tumor activity in vivo in a NSG mouse model using NALM6 as Upon GSK3377794 infusion, increased expression of activated im- target tumor. mune cell cytokines was observed in serum from selected patients. Conclusions Further analysis can provide insights into clinical response character- Our study demonstrates the feasibility of generating naturalistic istics and identification of predictive biomarkers. and functional CAR T cells from iPSCs, which may provide utility in the development of ‘off-the-shelf’ CAR T cell manufacturing Acknowledgements strategies. Medical writing assistance was provided by provided by Fiona Woodward and Chloe Stevenson of Fishawack Indicia Ltd, UK. These studies Acknowledgements (NCT01343043 and NCT02992743) were funded by GlaxoSmithKline (GSK). This research is supported by Mustang Bio. Inc. Trial Registration NCT01343043 and NCT02992743 Reference Ethics Approval 1. Themeli, M., et al., Generation of tumor-targeted human T lymphocytes This study was approved by the appropriate institutional review boards and from induced pluripotent stem cells for cancer therapy. Nat Biotechnol, independent ethics committees. 2013. 31(10): p. 928-33. Ethics Approval The study was approved by the COH Institutional Review Board (IRB) and P225 Office of Human Subjects Protection. Generating iPSC-derived CAR T cells with an endogenous T cell phenotype and conventional CAR T functionality 1 1 Zhiqiang (Daniel) Wang, PhD , Hellen McWilliams-Koeppen, MS , P226 1 1 1 1 Christian Huynh, BS , Hernan Reza , Vibhuti Vyas , Xiuli Wang, PhD , Iovance Gen2 TIL manufacturing process produces drug products 1 1 1 Wen-Chung Chang, MS , Julie Ostberg, PhD , Renate Starr, MS , Jamie that exhibit favorable quality attributes for adoptive cell transfer 1 1 1 1 1 Wagner , Brenda Aguilar, BS , Xiwei Wu , Jinhui Wang , Wei Chen , Chris across 5 solid tumor indications 2 2 1 1 Seet , Gay Crooks , Christine Brown, PhD , Stephen Forman, MD Seth Wardell, Maritza Lienlaf-Moreno, Lavakumar Karyampudi, Anand 1 2 City of Hope, Duarte, CA, United States; UCLA, Los Angeles, CA, United Veerapathran, PhD, Ian Frank, Michelle Blaskovich, BS, Kenneth Onimus, States Arvind Natarajan, Maria Fardis, PhD, MBA, Joe Wypych Correspondence: Zhiqiang (Daniel) Wang (zhwang@coh.org); Christine Iovance Biotherapeutics, Inc., Tampa, FL, United States Brown (cbrown@coh.org); Stephen Forman (sforman@coh.org) Correspondence: Joe Wypych (joe.wypych@iovance.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P225 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P226 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 121 of 272 Background P227 The Iovance Gen2 manufacturing process is a robust T-cell expansion Co-expression of the metabolic enzyme GOT2 with a GPC3- platform that produces a cryopreserved drug product after a 22-day targeted CAR-T overcomes challenges of the solid tumor manufacturing period. Gen2 represents a flexible closed cell produc- microenvironment, substantially improving therapeutic efficacy in tion process that is scalable to meet commercial demand. Drug prod- solid tumor xenografts ucts generated by this process display favorable quality attributes for Kathleen Whiteman, MS, Tapasya Pai, Eugene Choi, Taylor Hickman, Tyler adoptive transfer and the method is reproducible across 5 solid Johnson, Luke Barron, PhD, Taylor Friedman, Madaline Gilbert, Binzhang tumor indications at clinical scale. Shen, Seth Ettenberg, Kathleen McGinness, Greg Motz Methods Unum Therapeutics, Cambridge, MA, United States Methods to assess proliferation, phenotype, and function were ap- Correspondence: Greg Motz (greg.motz@unumrx.com) plied to in-process and final drug products generated with Gen2 at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P227 clinical scale to determine fit within the internally defined target product profile. TIL expansion was assessed through automated enu- Background meration of total and viable nucleated cells. Culture health was The metabolic demands of cancer cells in the solid tumor microenvir- assessed through cellular viability determined by DAPI exclusion. onment (TME) create an unfavorable T cell environment through deple- Immunophenotyping was performed to determine identity and pur- tion of critical nutrients and amino acids and accumulation of waste ity as well as relative levels of activation and differentiation of the products. This drives T cell dysfunction and inhibits the effectiveness of cell product. Cellular function was evaluated as the ability of the cell immunotherapies. To overcome these and other TME challenges, we product to secrete IFN-ɣ in response to CD3, CD28, and 4-1BB recep- developed the BOXR (bolt-on chimeric receptor) platform in which tor engagement. engineered T cells co-express both a chimeric-targeting receptor and a Results “bolt-on” transgene [1]. In a screen of 100+ genes for enhanced T cell Reported herein is the collective experience at Iovance for expansion function when co-expressed with an anti-glypican-3 (GPC3) CAR, we of TIL from five tumor types. The Iovance Gen 2 manufacturing identified the first candidate of our BOXR platform, BOXR1030, which process achieved doses comparable to lifileucel (LN-144, melanoma) co-expresses the transgene glutamic-oxaloacetic transaminase 2 and previously published methods across 5 primary tumor indica- (GOT2), a critical enzyme involved in mitochondrial metabolism. tions (Melanoma: mean 2.83 x 10e10 viable cells, n=82; Cervical: Methods mean 2.31 x 10e10 viable cells, n=53; Head & Neck: mean 5.82 x We compared functional and phenotypic readouts of second- 10e10 viable cells, n=12; Lung: mean 2.09 x 10e10 viable cells, n=3; generation GPC3 CAR-T cells with BOXR1030. Broad transcrip- Sarcoma: mean 1.12 x 10e10 viable cells, n=5). Quality attributes of tome profiling, metabolic characterization, and comprehensive drug products generated with Gen 2 were comparable across all 5 phenotypic assessments were performed; T cell proliferation and primary tumor indications evaluated in terms of T-cell purity, expres- cytokine production under TME-stress conditions (limiting nutri- sion of costimulatory molecules, and memory subsets. Gen 2 drug ents and hypoxia) were evaluated. In vivo, we assessed T cell products across all 5 additional indications continued to exhibit ro- anti-tumor activity, expansion and phenotype using GPC3- bust capacity to produce INF-ɣ upon reactivation, comparable to expressing solid tumor xenograft models in mice. lifileucel. Results Conclusions The addition of GOT2 had pleiotropic effects on BOXR1030 T The Iovance Gen2 manufacturing process allows for the rapid cells, improving multiple T cell functions relative to parent generation of clinical scale doses for patients in urgent need of GPC3 CAR-T cells. BOXR1030 CD4+ T cells had greater polyfunc- therapy. The cryopreserved drug product introduced critical logis- tionality relative to parent CAR-T. BOXR1030 showed improved tical efficiencies and flexibility in distribution that overcame trad- proliferation in vitro, including against TME-challenges. itional barriers to the commercialization of TIL therapy. Gen 2 BOXR1030 CD8+ T cells had a greater proportion of less differ- drug products exhibit favorable quality attributes for adoptive entiated CD27+ cells following production, and CD8+ T cells transfer including high levels of co-stimulatory molecules, and a evaluated ex vivo from xenograft tumors had substantially di- robust capability to secrete cytokine upon reactivation. These minished level of inhibitory receptors (PD-1, Tim-3) suggesting characteristics are reproducible across a broad range of solid resistance to exhaustion in the TME (Figure 1). Further, tumor indications at a high manufacturing success rate opening BOXR1030 was highly efficacious against GPC3-expressing solid the door for many more patients to benefit from this highly tumor models that resisted parental CAR-T therapy (Figure 2), beneficial therapy. [1,2,3] and activity was associated with improved T cell expansion and persistence in peripheral blood. References Conclusions 1. Donia M, Junker N, Ellebaek E, Andersen MH, Straten PT, Svane IM. Co-expression of a metabolic gene to enhance T cell function is Characterization and comparison of ‘Standard’ and ‘Young’ tumor a novel approach to cell therapy for solid tumors. BOXR1030 infiltrating lymphocytes for adoptive cell therapy at a Danish had substantially improved T cell phenotype and function in di- Translational Research Institution. Scand. J. Immunol. 2011;75:157–67. verse ways relative to the parent GPC3 CAR, and GOT2 con- 2. Besser MJ, Shapira-Frommer R, Treves AJ. et al. Clinical responses in a ferred superior activity against numerous TME challenges both phase II study using adoptive transfer of short-term cultured tumor infil- in vitro and in vivo. These results demonstrate that engineering tration lymphocytes in metastatic melanoma patients. Clin Cancer Res. of T cell immunometabolism is an effective and potent strategy 2010;16:2646–55. to overcome the challenges of the solid tumor microenviron- 3. Jin J, Sabatino M, Somerville R, Wilson JR, Dudley ME, Stroncek DF, et al. ment. IND-enabling studies with BOXR1030 are underway with Simplified method of the growth of human tumor infiltrating the expectation that BOXR1030 will be evaluated clinically in lymphocytes in gas-permeable flasks to numbers needed for patient the treatment of GPC3+ malignancies. treatment. J Immunother 2012;35:283–92. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 122 of 272 Reference P228 1. Barron L, Whiteman K, Gilbert M, Pai T, Snyder M, Fray M, Nelson A, Tumor infiltrating lymphocyte recognition of shared neoantigens Johnson T, Lakeman K, Shin J, Boomer R, Ettenberg S, McGinness K, from mutated DNA repair/remodeling proteins in a patient with Motz G. Select metabolic and costimulatory “bolt-on” transgenes metastatic pancreatic adenocarcinoma 1 1 enhance chimeric receptor-bearing T cell activity against solid tu- Ghanshyam Singh Yadav, PhD , Chetana Bhaskarla, PhD , Smriti 1 1 2 2 mors. J Immunother Cancer. 2018;6(Suppl 1):114:110-111, abstract Chaurasia, PhD , Joshua Tobin , Xinming Zhuo , Yinghong Pan , 2 1 P216. Annerose Berndt , Udai Kammula, MD, FACS 1 2 Ethics Approval University of Pittsburgh, Pittsburgh, PA, United States; UPMC Genome This study was approved by Unum Therapeutics’ Institutional Animal Care Center, Pittsburgh, PA, United States and Use Committee (IACUC); approval number 2016-04-004. Correspondence: Udai Kammula (kammulaus@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P228 Background Defective DNA repair, a hallmark of cancer, results in genomic instability and accumulation of genetic abnormalities in many malignancies. Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TIL) rep- resents a personalized cancer immunotherapy capable of targeting shared and private neoantigens resulting from tumor somatic mutations. We sought to interrogate TIL neoantigen reactivity in a tumor harboring a som- atic mutation in the DNA repair gene, ATM (Ataxia-Telangiectasia Mutated). Methods TIL cultures were generated from a surgically resected pancreatic cancer metastasis harboring a somatic ATM mutation. Anti-tumor re- activity of TIL culture was assessed by coculturing TIL with autolo- gous tumor cells and measurement of IFN-gamma release and upregulation of 4-1BB by flow cytometry. Tumor specific mutations were identified by whole genome sequencing (WGS). DNA fragments encoding the altered gene sequences were synthesized and expressed in autologous dendritic cells by RNA electroporation to en- able neoantigen reactivity screening. T cell receptor (TCR) sequencing was performed after single-cell sorting of tumor reactive TIL followed by primer specific PCR for TCR alpha and beta chains. Results Analysis of the pancreatic cancer TIL revealed high level reactivity against autologous tumor. Tumor WGS identified 141 somatic muta- tions (107 SNVs; 19 frameshifts; 15 other). Screening for neoantigen re- activity identified CD8+ T-cell responses against a missense mutation in Fig. 1 (abstract P227). See text for description ATM (23% of TIL) and a frameshift mutation in ARID1A (32% of TIL) but not against respective wild type gene products. TCR sequencing identi- fied a single unique TCR specific for ATM and ARID1A, respectively. Genes encoding the ATM specific TCR were retrovirally transduced into healthy donor T-cells and found to confer strong ATM mutation reactiv- ity without recognition of wild type ATM. Conclusions Over 50% of the TIL expanded from a patient with metastatic pancre- atic cancer were found to recognize neoantigens from either mu- tated ATM or ARID1A, which play a crucial role in DNA repair and chromatin remodeling. ACT using T-cells genetically engineered with these TCRs represents an attractive immunotherapy for patients har- boring these shared tumor mutations. Acknowledgements The study was supported by UPMC Immune Transplant and Therapy Center (ITTC). Ethics Approval This study was reviewed and approved by University of Pittsburgh Institutional Review Board. IRB#18010273. P229 The next generation “off-the-shelf” universal CAR for adoptive immunotherapy 1 1 1 1 Weichih Yang, PhD , Yun Ji , Xiaobing Luo , Huijuan Cui , Michael 1 1 1 1 1 1 Patrick , Yutian Wei , Shigui Zhu , Jiaqi Huang , Xin Yao , Yihong Yao , 2 2 Aibing Liang , Ping Li 1 2 Cellular BioMedicine Group, Gaithersburg, MD, United States; Tongji Hospital of Tongji University, Shanghai, China Correspondence: Yun Ji (yun.ji@cellbiomedgroup.com) Fig. 2 (abstract P227). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P229 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 123 of 272 Background antigen CD4+ and CD8+ responses and cancer-associated antigen- Adoptive immunotherapy using autologous T cells redirected with specific CD8+ T cell responses in vivo. We observe that prophylactic chimeric antigen receptors (CARs) has emerged as a powerful means and therapeutic AAC administration to mice strongly impedes tumor of treating cancer, such as B-cell malignancy. However, this approach growth and extends survival. Following therapeutic immunization, the is limited by the availability of autologous T cells especially for infant anti-tumor responses correlate with an over 10x increase in antigen- patients or patients undergoing multi rounds of chemotherapy. specific CD8+ tumor-infiltrating lymphocytes compared to untreated Methods mice. Finally, in an in vitro human system, we demonstrate that AACs Here we show that employment of the CRISPR-Cas9 system allows can be highly loaded with antigen and adjuvant using CellSqueeze®, highly efficient multiplex gene editing of T Cell Receptor Alpha Con- and that these AACs can be engulfed by human monocyte-derived stant and Beta-2-Microglobulin in primary human T cells, which is dendritic cells. intended to avoid graft-versus-host-disease and minimize the immuno- Conclusions genicity of transferred cells. Furthermore, redirecting the gene- In summary, these results indicate that antigen and adjuvant delivery engineered cells with a B cell maturation antigen (BCMA) CAR led to to APCs in vivo can effectively prime a potent anti-tumor response in their efficient destruction of BCMA+ tumor targets. To further improve mice and support the further study of SQZ AACs as an immunother- the efficacy of these universal BCMA CAR T cells, we use a strategy to apy for cancer treatment. generate the next generation universal CAR T cells by starting from naive precursors and producing the CAR T cells in conditions favoring T P231 memory stem (Tscm) cell expansion. Invariant natural killer T cells as an allogeneic cell therapy Results platform These Tscm enriched gene-engineered BCMA CAR T cells demonstrated Burcu Yigit, PhD, Xavier Michelet, PhD, Simon Yue, Darrian Moskowitz, superior activity compared to the conventional universal CAR T cells Mark Exley, Burcu Yigit, PhD based on their expansion, phenotype, IFN-gamma release, and cytotox- AgenTus Therapeutics, Lexington, MA, United States icity. An early phase clinical trial using this BCMA CAR in an autologous Correspondence: Burcu Yigit (burcu.yigit@agentustherapeutics.com) setting has demonstrated promising clinical readout (NCT03815383). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P231 Conclusions Therefore, we believe this next generation Tscm-enriched universal Background CAR T cells employing the same BCMA vector will provide another al- AgenTus Therapeutics is developing innovative allogeneic and ‘off- ternative choice for multiple myeloma patients in an “off-the-shelf” the shelf’ cell therapies by utilizing invariant natural killer T cells manner similar to other biological drugs. (iNKT) to target solid and liquid tumors. iNKT cells are innate-like lym- phocytes that bridge innate and adaptive immune responses. They P230 can be activated via their invariant T cell receptor recognizing lipid Activating antigen carriers for cancer therapy: preclinical immune antigens (e.g. alpha-Galactosylceramide) presented on CD1d mole- responses drive tumor regression cules, through NKG2D - NKG2D ligand interactions, and by cytokines. Defne Yarar, PhD, Amritha Ramakrishnan, Katarina Blagovic, PhD, Upon activation, large amounts of IFN-gamma production leads to Katherine Seidl, Howard Bernstein, MD, PhD, Armon Sharei recruitment and activation of T cells and NK cells. iNKT cells also SQZ Biotechnologies, Watertown, MA, United States exert potent direct cytolytic activity. While they are found in very low Correspondence: Defne Yarar (defne.yarar@sqzbiotech.com) numbers in human blood (~ 0.01% of T lymphocytes), some of their Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P230 unique properties make them valuable for cell therapy platforms. Due to their invariant antigen receptor, their ability to cause GvHD is Background minimal, and in fact, they have been demonstrated to suppress Productive activation of the immune system by antigen presenting GvHD in BMT settings. This facilitates the use of iNKT cells in an allo- cells (APCs) loaded ex vivo has proven to be challenging. To over- geneic cell therapy setting. In addition, iNKT cells are very efficient in come this, we have developed an approach that harnesses the nat- infiltrating solid tumors to exert their cytotoxic function and activate ural process of red blood cell (RBC) clearance from the body to other anti-tumor immune cells. activate the immune response in vivo. Using the CellSqueeze® micro- Methods fluidics platform, we have generated activating antigen carriers Due to low frequency of circulating iNKT cells, we have developed (AACs), engineered from RBCs, that are highly loaded with antigen and optimized a method to isolate and generate large numbers of and adjuvant and potently activate APCs in vivo. Here, we show that these cells in vitro for use in ‘off-the-shelf’ and allogeneic setting. AAC-mediated antigen and adjuvant targeting to APCs drives antigen Results presentation in vivo and primes potent anti-tumor T cell responses. We can achieve over 40,000-fold expansion of iNKT cells through Methods stimulation of the invariant TCR in less than 30 days. Importantly, To generate AACs, we loaded proteins or synthetic long peptide anti- after such massive expansion, iNKT cells retain their inherent cyto- gens together with adjuvants into murine or human RBCs with CellS- toxic capacity and cytokine production in response to tumor cells. queeze®. Following intravenous AAC injection into mice, we measured Conclusions AAC clearance kinetics from the blood and characterized the site and AgenTus is applying its proprietary Antigen Receptor platforms to cell type of AAC uptake. In addition, we quantified endogenous im- identify novel CARs and TCRs directed against tumor-specific anti- mune responses to AAC administration by flow cytometry. To deter- gens. We believe that through modification with tumor-targeted mine the ability of AACs to control subcutaneously implanted tumors, CARs and TCRs, iNKT cells will serve as potent allogeneic cell therapy we measured tumor growth rates in mice treated either prophylactic- vehicles. This should enable an ‘off-the-shelf’ approach for improving ally or therapeutically with AACs. Finally, to assess if AACs could be patient access to cell therapy. engulfed by antigen-presenting cells in a human system, we quantified in vitro uptake of human AACs by monocyte-derived dendritic cells P232 using flow cytometry and fluorescence microscopy. Characterization of ADCC resistance in multiple cancer types Results David Zahavi, MS, BS, Yongwei ZHANG, Sandra Jablonski, PhD, Louis We demonstrate that CellSqueeze® loads antigen and adjuvant into Weiner, MD AACs effectively. When administered into a mouse, these carriers are Georgetown University, Washington, DC, United States cleared from circulation within one hour and are engulfed by profes- Correspondence: Louis Weiner (weinerl@georgetown.edu) sional phagocytes in both the spleen and liver. Moreover, we find that Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P232 murine SQZ AACs processed with CellSqueeze® stimulate model Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 124 of 272 Background conjugated with H-2Kb OVA, m4-1BBL and mIL-12. Naïve OT1 Antibody-dependent cell-mediated cytotoxicity (ADCC) is an import- cells were transferred into tumor bearing mice followed by ant mechanism of action in targeted monoclonal antibody (mAb) mRBC-321 treatment. Dramatic dose-dependent expansion of cancer immunotherapy. The majority of patients who receive tar- OT1, comprised of functional effector and central memory pheno- geted mAbs develop resistance to therapy and there remains a great types, was observed in peripheral blood (9468-fold Tem; 146-fold need to understand resistance mechanisms. In vitro modeling of Tcm expansion) and secondary lymphoid organs (3323-fold Tem; ADCC provides an experimental system for uncovering tumor cell 725-fold Tcm expansion in spleen) on day 7. mRBC-321 treatment based immune resistance mechanisms. using an EG7.OVA tumor model resulted in tumor regressions in Methods 12/16 mice, 6 of which were cured, and increased survival (p Utilizing our in vitro model system of continuous selection pressure Conclusions with NK92-CD16V effector cells and the mAbs Cetuximab and Trastu- Based on these results, human RTX-321 expressing h4-1BBL, hIL-12, zumab we have generated three ADCC resistant cell lines from par- and HLA-A*02 with an HPV E7 peptide was generated to activate ental A431, SK-OV-3, and FaDu cells. and expand HPV E7-specific T cells. RTX-321 activated TCR signaling Results in an engineered HPV E7-specific TCR Jurkat line and stimulated 4- We show that the induction of ADCC resistance in all three cells lines 1BB and IL-12R signaling in respective reporter cell lines. These re- involves a loss of target cell adhesion properties required for the es- sults support the clinical development of RTX-321, which is currently tablishment of an immune synapse, NK cell activation, and target cell in IND-enabling studies for the treatment of HPV16+ advanced solid cytotoxicity. Remarkably, ADCC-resistant cells possess reduced cell tumors. surface expression of multiple proteins that contribute to intercellular interactions and immune synapse formation. We have termed the P234 loss of a selection of cell surface proteins which contributes to ADCC DAP10 and DAP12 signaling based CAR circumvents ligand- resistance Testudinidosis. This phenomenon is characterized by dys- dependent tonic signaling and mediates potent anti-tumor regulation of protein trafficking and subcellular localization of the cell response in vivo surface molecules. Additionally, ADCC resistant cell lines exhibit aber- Alan Epstein, MD, PhD, Long Zheng, Long Zheng rant IFN/STAT1 signaling. University of Southern California, Los Angeles, CA, United States Conclusions Correspondence: Alan Epstein (aepstein@usc.edu) Using multiple cell lines to model ADCC resistance has led to the dis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P234 covery of a shared mechanism of resistance across cancer types that may reveal potential therapeutic targets for combination Background immunotherapy. The Lym-1 antibody which targets a unique, discontinuous epitope on the HLA-Dr protein expressed in human B-cell lymphomas and leukemias de- P233 veloped in the late 1970’s by co-author ALE has been found to be clinically RTX-321, an allogeneic artificial antigen presenting red cell safe and effective as an I-131 radioimmunoconjugate [1-3]. Based upon therapeutic, expressing MHC I-Peptide, 41BBL and IL12, promotes these early clinical data, we have generated a humanized version (huLym- antigen-specific T cell expansion and anti-tumor activity in HPV16+ 1-B) to construct chimeric antigen receptors (CARs) using the single chain tumors variable fragment (ScFv) of huLym-1-B to treat Lym-1 positive tumors. Xuqing Zhang, PhD, Tiffany Chen Methods Rubius Therapeutics, Cambridge, MA, United States The antibody ScFv was ligated to a lentivirus vector in frame with Correspondence: Tiffany Chen (tiffany.chen@rubiustx.com) CAR backbone and CAR lentiviruses were produced and used to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P233 transduce primary CD3+ human T-cells. After transduction, we char- acterized the expansion, the effector function, and the immune- Background phenotypes of CAR-T cells. The in vivo efficacy of the CAR-T cells was Autologous CAR-T therapy has demonstrated efficacy in a small measured in a metastatic Raji lymphoma xenograft 8 days after the subset of hematological cancers. The wider adoption of antigen- iv injection of 1 million tumor cells in NSG mice. specific therapies has been limited by significant toxicity and a Results lack of effectiveness in solid tumors. Manufacturing is costly, huLym-1-BCAR was successfully transduced at an efficiency between time-consuming and difficult to scale. To address these limita- 50-80%. Surprisingly, the CAR transduced T-cells showed limited expan- tions, Rubius Therapeutics has genetically engineered red cells to sion, reaching approximately 8-fold expansion after 11 days of culture create an allogeneic artificial antigen-presenting cell (aAPC), compared to mock T-cells which had a robust 310 to 380-fold expan- called RTX-321, for the treatment of HPV16+ advanced solid tu- sion. In expanded CAR-T cells, over 28% of cells showed phosphory- mors. RTX-321 presents an HPV E7 peptide on major histocom- lated CD3z and increased PD-1 and LAG3 expression indicative of T-cell patibility complex I (MHC I [HPV]), a costimulatory signal (4-1BBL) exhaustion which was not detected in mock T-cells. Since Lym-1 recog- and a membrane-bound cytokine (IL-12) on the cell’ssurface to nizes a conformational epitope on HLA-Dr which may be weakly mimic the human immunobiology of T cell APC interactions. RTX- expressed by activated T-cells, we speculated that the huLym-1-BCAR 321 is designed to activate and expand tumor-specific T cells may stimulate the transduced T-cells to cause tonic signaling and ex- present within the patient and eliminates the need for manufac- pansion failure. This was confirmed by flow cytometry that detected turing patient-derived T cells. huLym-1-B but not huLym-1-Bmut, a huLym-1-B mutant that lost its Methods binding ability, binding on T cells. Meanwhile, neither huLym-1-Bmut As a proof of principle, red cells engineered to express mouse based CAR or huLym-1-BCAR with inactive CD3z domains showed de- MHC I H-2Kb loaded with OVA 257-264 peptide (H-2Kb OVA), creased expansion or increased CD3z phosphorylation. murine 4-1BBL (m4-1BBL) and murine IL-12 (mIL-12) were used to Conclusions stimulate OT1 cells. Although the proliferation issue did not compromise CAR-T cells’ ef- Results fector function, this would impose a potential challenge for large-scale Compared to cells expressing H-2Kb OVA or m4-1BBL alone, manufacture. We discovered that replacing the BB3z in huLym-1-BCAR these cells induced up to a 14-fold expansion of OVA antigen- with signaling domains from DAP10 and DAP12 addressed this prolifer- specific OT1 cells in vivo. These expanded cells displayed a mem- ation issue but enabled potent anti-tumor efficacy in vivo. In addition, ory phenotype and enhanced antigen-specific tumor killing of the expanded huLym-1-B DAPCAR-T cells enabled a lower dose of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate, injected CAR T-cells to induce durable control of metastatic lymphoma mRBC-321, was created using murine red cells chemically in mice with large tumor burdens. Further testing of DAP signaling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 125 of 272 sequences in other CAR T-cells may show that this change also im- suggesting myeloid cell proliferation. Finally, stable or partial re- proves the clinical efficacy of CAR T-cell therapy directed against other sponse to therapy tracked with either a dense T cell- or myeloid-cell antigens targeted in both hematopoietic and solid tumors. infiltrate while patients who progressed displayed low infiltrate levels. Acknowledgements Conclusions This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. In conclusion, immunotherapy associated skin rash contains a com- plex immune infiltrate, with increased cellular proliferation among References sites of dense infiltration. The highest levels of T cell or myeloid cell 1. Epstein AL, et al. Two new monoclonal antibodies, Lym-1 and Lym-2, re- infiltrate were seen in stable or responding patients, suggesting a active with human B-lymphocytes and derived tumors, with immunodi- link between the immune profile of skin rash and therapy response. agnostic and immunotherapeutic potential, Cancer Res. 1987; 47:830-840. Ethics Approval 2. DeNardo GL, et al. Low-dose, fractionated radioimmunotherapy for B-cell The study was approved by The Northwestern University Ethics Board maligancies using 131I-Lym-1 antibody. Cancer Biother Radio. 1998; 13:239-254. 3. Hu E, et al. A phase 1a clinical trial of LYM-1 monoclonal antibody ser- otherapy in patients with refractory B cell malignancies. Hematol Oncol. P236 1989; 7:155-166. Nivolumab related side effects based on patient-reported Ethics Approval outcomes: A multicenter study from real-life setting 1 1 This study was approved by the IRB of the University of Southern California, Canan Karadas , Zehra Gok Metin, Assoc Prof, PhD, RN , Nur Izgu, PhD, 1 2 2 protocol number HS-16-00029 approved 2-29-16, and by IACUC protocol RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, Checkpoint Blockade Therapy Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Correspondence: Canan Karadas (karadas.canan@gmail.com) P235 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P236 A dense, proliferative myeloid and T cell-rich, immune infiltrate characterizes immunotherapy-induced skin rash Background Cormac Cosgrove, PhD, Cory Kosche, Suyeon Hong, Caroline Le Poole, Immune checkpoint inhibitors provide an effective treatment option Jennifer Choi for patients with melanoma and other cancer types. Nivolumab, as Northwestern University, Chicago, IL, United States an immune checkpoint inhibitor, acts via blockade of the PD-1 recep- Correspondence: Cormac Cosgrove tor, and limits immune responses against tumors. As reported for (cormac.cosgrove@northwestern.edu) nivolumab, the anti-PD-1 antibodies can induce immune-related ad- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P235 verse events [1, 2]. Nivolumab related adverse effects compose of general, pulmonary, gastrointestinal, neurologic, skin, and infusion re- Background actions. These adverse effects may be life threatening, so following Checkpoint inhibitor immunotherapy is associated with a unique tox- of patients receiving nivolumab is essential for early diagnosis and icity profile, collectively known as immune related adverse events management. Therefore, this study aimed to examine nivolumab re- (irAEs). One of the earliest and most common irAEs is skin rash. Inter- lated adverse effects based on patient- reported outcomes (PROs) estingly, development of rash has been associated with improved from a real-life setting. survival, and is likely an early indicator of a successfully activated im- Methods mune response. More severe toxicities also occur, can affect nearly In total, 40 patients receiving first cycle of nivolumab were included every organ and are clinical justification for dose reduction or termin- from three hospitals located in Turkey. Patient Information Form and ation of immunotherapy. However, the mechanism underpinning Patient Monitoring Checklist-Nivolumab were used for data collection. rash development and its link to disease outcome or toxicities is not Patient Monitoring Checklist-Nivolumab included totally 46 questions: known. Thus, we examined the makeup of immunotherapy- (21-general adverse effects, 3-pulmonary, 8-gastrointestinal, 5- associated skin rash infiltrates with the goal of uncovering mechanis- neurological, 2-skin, and 7-infusion reactions) and two options in each tic insights behind rash development. item that evaluating presence of related adverse effect, as “yes” or “no”. Methods PROs were analyzed by frequency and percentages. Immunohistochemistry was used to describe the immune infiltrate in Results skin biopsies from healthy subjects and from a lesional site of patients The mean age of patients was 55.47±16.48 years, and the majority of who developed rash secondary to immunotherapy. Rash samples were them (82.5%) had diagnosed with cancer longer than one year. The obtained from 7 patients receiving α-PD1, α-CTLA4/α-PD1 combination cancer diagnosis composed of melanoma (52.5%), renal cell carcin- or α-PDL1/α-NKG2A combination. Acetone-fixed sections from frozen oma (17.5%), nasopharyngeal carcinoma (7.5%) and other cancer biopsies were stained for CD3, CD4, CD8, CD68, CD11c, CD1a, CD207, types (23.5%). Considering differences in terms of gender, only two or Ki67. Cell abundance in the dermis was compared among groups. items including 8th item related general adverse effects, quiring loss Results of hair, and 37th item evaluating changes in mental status, percep- : Rash samples showed significant enrichment of T cells (CD3 p= tion, judgement, and memory had a significant difference. Female 0.01), CD8 T cells (p=0.03) and dendritic cells (CD11c p=0.03) in the patients reported higher adverse effects than male regarding afore- dermis vs controls. More moderate enrichment of macrophages mentioned items (p (CD68) was observed in rash versus control samples while the abun- Conclusions dance of dermal Langerhans cells (CD1a or CD207) was comparable PROs related nivolumab in this study included fatigue, rash and itch- among groups. We observed more proliferating cells in the dermis of ing, and diarrhea as consisted with previous reports. A comprehen- rash vs control samples (Ki67 p=0.024), associated with areas of sive approach is needed to reduce and manage nivolumab related dense immune infiltration. Ki67 expression was highly correlated with adverse effects. both CD68 (r=0.89; p=0.012) and CD11c (r=0.786; p=0.048), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 126 of 272 References aware of the rare, debilitating, and possibly previously undescribed 1. Naidoo J, Page DB, Li BT, Connell LC, Schindler K, Lacouture ME, Wolchok paraneoplastic and autoimmune toxicities that may occur. JD. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint anti- bodies. Ann Oncol. 2015; 26:2375–2391. References 2. Spain L, Diem S, Larkin J. Management of toxicities of immune 1. Forster MD, Devlin MJ. Immune Checkpoint Inhibition in Head and Neck checkpoint inhibitors. Cancer Treat Rev. 2016; 44:51–60. Cancer. Front Oncol. 2018; 8:310. Ethics Approval 2. Pollack MH, Betof A, Dearden H. Safety of resuming anti-PD-1 in pa- The study was approved by clinical trials ethics committee of the University tients with immune-related adverse events (irAEs) during combined of Health Sciences Ankara Oncology Training and Research Hospital anti-CTLA-4 and anti-PD1 in metastatic melanoma. Ann Oncol. 2018; (decision number: 2018–06/52) and performed in accordance with the 29:250-255. Helsinki Declaration. 3. Bataller L, Wade DF, Graus F. Antibodies to Zic4 in paraneoplastic neurologic disorders and small-cell lung cancer. Neurology. 2004; 62. 4. Schwab KS, Kristiansen G, Schild HH. Successful Treatment of Refractory P237 Squamous Cell Cancer of the Head and Neck with Nivolumab and A case of anti-Zic4 antibody-mediated cerebellar toxicity induced Ipilimumab. Case Rep Oncol 2018;11:17–20. by dual checkpoint inhibition in HNSCC Consent Sunil Iyer, MD, Nidah Khakoo, MD, Gabriella Aitcheson, MD, Marina Consent was obtained from the patient for publication of this abstract. Kushnirsky, MD, Cesar Perez, MD University of Miami, Miami Beach, FL, United States Correspondence: Sunil Iyer (sunil.iyer@jhsmiami.org) P238 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P237 Longitudinal immune and genomic monitoring reveals signatures of response and Immune-related adverse events in cancer patients Background receiving checkpoint inhibitor therapy Combined checkpoint inhibition therapy targeting the PD-L1 and Shaheen Khan, PhD, David Gerber CTLA4 pathways has been a successful approach in the treatment of UT Southwestern Medical Center, Dallas, TX, United States metastatic melanoma, leading to its investigation in the treatment of Correspondence: David Gerber (david.gerber@utsouthwestern.edu) head and neck squamous cell carcinoma (HNSCC) with PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P238 sion [1]. Despite the potential for excellent responses, an increased rate of autoimmune neurological toxicity and paraneoplastic conditions has Background been observed [2]. We present the case of a patient with metastatic Despite the remarkable success of immune checkpoint inhibitor (ICI) HPV-positive HNSCC treated with ipilimumab/nivolumab who experi- therapy, a significant number of patients develop severe and unpredict- enced severe cerebellar ataxia, with a positive screen for the anti-Zic4 able immune-related adverse events (irAEs) affecting a wide variety of antibody, which has been associated with cerebellar degeneration in organs. Concerns over irAE have led to the exclusion of patients with small cell lung cancer (SCLC) and has not been reported in HNSCC [3]. autoimmune disease from ICI clinical trials. Role of host genetic and im- Results mune factors in mediating irAEs remain unclear and it is not clear if the A 40-year-old man diagnosed with HPV-positive HNSCC with meta- manifestations of irAEs is associated with response to therapy. static recurrence after radiation treatment of the initial tumor was Methods started on a clinical trial of a DNA-PK inhibitor. His disease pro- We used multi-faceted approach to identify blood-based biomarkers gressed, and given his PD-L1 tumor proportion score of 70% he was predictive of irAEs and response to ICI therapy. We characterized initiated on ipilimumab/nivolumab. After his second cycle, he pre- changes in host immune system in 200 patients receiving ICI therapy sented with sudden blurred vision and mild ataxia, which rapidly pro- at baseline and post-immunotherapy (100 with irAEs and 100 without gressed to severe ataxia and dysarthria. Autoimmune toxicity was irAEs). We assessed genetic predisposition to autoimmunity in these suspected; initial brain imaging and serum testing were unremark- patients using the Illumina GSA SNP array and via targeted resequen- able. While awaiting the results of complex autoimmune and para- cing of over 150 immunoregulatory loci including the HLA region. We neoplastic CSF testing, he was treated with multiple modalities in an evaluated serum levels of cytokines/chemokines, Antinuclear auto- escalating fashion with minimal improvement, including pulse-dose antibodies (ANA) and 124 autoantibodies and performed RNA se- corticosteroids, IVIG, and plasmapheresis. The paraneoplastic panel quencing and flow cytometry on peripheral blood mononuclear cells returned negative for common autoimmune culprits in cerebellar en- (PBMCs) at baseline and post immunotherapy in patients with and cephalopathy including anti-Hu and anti-Yo; however, anti-Zic4 was without irAEs. detected at borderline levels. Repeat MRI showed an enhancing le- Results sion in the cerebellum. Finally, rituximab was initiated, and the pa- Our preliminary data analysis identified signatures of autoantibodies tient is slowly improving. Notably, restaging scans show a mixed and cytokines correlating with response and toxicity. We also identi- response with resolution of previously extensive metastatic disease fied HLA haplotypes linked with autoimmunity in selected patients in the thorax, however with worsening osseous lesions. that developed immune-related adverse events. In this meeting, we Conclusions will present immune and genetic correlates of irAEs and response to We present a case of anti-Zic4-mediated cerebellar toxicity in the set- therapy in our patient cohort. ting of dual PD-L1/CTLA4 inhibition in the treatment of metastatic Conclusions HNSCC. Anti-Zic4 has been historically associated with cerebellar- In-depth analysis of immune and genetic datasets is currently under- predominant paraneoplastic neurological disorders in SCLC [3], and way. We hope that our studies can help identify blood-based bio- to our knowledge, has not been described in HNSCC. Although the marker signatures predictive of irAEs and/or response and reveal patient experienced an impressive partial response, he suffered novel insights into the mechanisms underlying irAE. Our current gen- grade 4 cerebellar neurotoxicity. Cases have demonstrated excellent etic data suggests further expanding the genetic studies in larger pa- clinical responses utilizing dual PD-L1/CTLA4 inhibition in HNSCC [4], tient population to identify the role of underlying genetic however high-grade adverse events have also been reported in this predisposition to autoimmunity in mediating irAEs. We hope our regimen’s more established use in metastatic melanoma [2]. Despite findings may ultimately help identify customize therapy, expand use the exciting advances in cancer immunotherapy, clinicians must be of immunotherapy and prevent toxicities. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 127 of 272 P239 Table 1 (abstract P239). ICI-arthritis patient characteristics (total n=372) Immune checkpoint inhibitor-associated arthritis: a systematic literature review of case series and case reports 1 2 3 Michael Tiongson, BA , Nilasha Ghosh, MD , Carolyn Stewart, BA , 2 2 2 Karmela Chan, MD , Bridget Gatto, MLIS , Anne Bass, MD 1 2 Albany Medical College, Nanuet, NY, United States; Hospital for Special Surgery, New York, NY, United States; Weill Cornell Medicine, New York, NY, United States Correspondence: Nilasha Ghosh (GhoshN@HSS.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P239 Background As immune checkpoint inhibitors (ICI) continue to revolutionize can- cer treatment, immune-related adverse events (irAE) are becoming more prevalent. Inflammatory arthritis occurs in approximately 4% of ICI-treated patients [1] but remains poorly characterized. We per- formed a systematic literature review to identify all reports of ICI- associated inflammatory arthritis in order to describe it phenotypic- ally and serologically. Methods PubMed, Embase and Cochrane databases were searched for pub- lications reporting musculoskeletal irAEs secondary to ICI treat- ment through the search date, May 31, 2019. Publications were included if they provided individual patient-level data regarding the pattern of joint involvement. Two reviewers screened all ab- stracts and full texts to extract demographics, clinical features, se- rologies, treatment data and outcomes. Descriptive statistics were used to summarize results. Results 4339 articles were screened, of which 67 were included (42 case reports, 15 case series, 10 retrospective chart reviews) encom- passing 372 patients (Table 1). Mean age was 63 +/- 11 years; 61% patients were male. The majority of patients had metastatic melanoma (57%) and were treated with anti-PD1 or anti-PDL1 P240 therapy (78%). Median time to onset of arthritis was 4 months Quantitative cell-based bioassays to advance immunotherapy (range: 1 day-53 months). 49% had polyarticular arthritis, 17% oli- programs targeting immune checkpoint receptors goarthritis, 3% monoarthritis, 10% arthralgia and 21% polymyalgia Vanessa Ott, PhD, Jamison Grailer, PhD, Jun Wang, Julia Gilden, PhD, rheumatica (PMR). 9% tested positive for rheumatoid factor (RF) Pete Stecha, Denise Garvin, Michael Beck, Jim Hartnett, Frank Fan, PhD, or cyclic citrullinated peptide (CCP) antibodies. 74% required cor- Mei Cong, PhD, Zhi-jie Jey Cheng ticosteroids and 45% required additional therapies, including 5% Promega, Madison, WI, United States requiring a TNF inhibitor. 63% of patients achieved control of Correspondence: Zhi-jie Jey Cheng (jey.cheng@promega.com) their musculoskeletal symptoms with treatment, and 32% were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P240 ultimately able to discontinue anti-rheumatic treatments. ICI were continued in 49%, transiently withheld in 11%, and permanently Background discontinued due to musculoskeletal irAEs in 13%. At last follow- The human immune system is comprised of a complex network of im- up, 27% had progression of their cancer. mune checkpoint receptors that are promising new immunotherapy tar- Conclusions gets for the treatment of a variety of cancers and autoimmune-mediated Half of reported ICI-associated arthritis cases have a polyarthritis disorders. Immunotherapies designed to block co-inhibitory receptors (e.g. (often in an RA distribution) but only 9% are seropositive. PMR is also PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of can- commonly seen. The vast majority of ICI-arthritis cases are in melan- cer. However, not all patients and tumor types respond to this approach. oma patients treated with anti-PD1/PDL1 therapy. Most patients re- This has resulted in broadening of immunotherapy research programs to spond to steroids alone but about half require additional anti- target additional co-inhibitory (e.g. LAG-3, TIM-3) and co-stimulatory (e.g. rheumatic agents. Further studies are needed to determine long- 4-1BB, GITR, OX40, ICOS) receptors individually and in combination. term musculoskeletal outcomes in these patients and the impact of Methods arthritis treatment on cancer survival. A major challenge in the development of biologics is access to quantita- tive and reproducible functional bioassays. Existing methods rely on pri- Reference mary cells and measurement of complex functional endpoints. These 1. Kostine M, Rouxel L, Barnetche T, Veillon R, Martin F, Dutriaux C, assays are cumbersome, highly variable and fail to yield data quality re- Dousset L, Pham-Ledard A, Prey S, Beylot-Barry M, Daste A. Rheum- quired for drug development in a quality-controlled environment. To ad- atic disorders associated with immune checkpoint inhibitors in pa- dress this need, we have developed a suite of cell-based functional tients with cancer—clinical aspects and relationship with tumour bioassays to interrogate modulation of immune checkpoint receptors indi- response: a single-centre prospective cohort study. Ann Rheum Dis. vidually (e.g.PD-1,CTLA-4,LAG-3, TIM-3, GITR, 4-1BB) and in combination 2018; 77:393-8. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 128 of 272 (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) that express luciferase reporters driven by response elements under the and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) precise control of mechanistically relevant intracellular signals. could serve as subpopulations relevant for patient stratification (Figure Results 2). We further validated these results using CyTOF wherein these same The bioassays reflect mechanisms of action for the drug candidates de- subpopulations were differentially abundant between responders and signed for each immune checkpoint receptor and demonstrate high spe- non-responders at the pre-therapy time-point. cificity, sensitivity and reproducibility. For example, using the PD-1 Conclusions blockade bioassay, TCR-mediated luciferase activity is recovered with Collectively, our results revealed that (1) PD-1 blockade-based anti-PD-1 and PD-L1 blocking Abs but not with unrelated control anti- treatment-induced gene expression profiling changes (increase cell bodies. Similarly, TCR and CD28-mediated luciferase activity is recovered proliferation, metabolism and activation) of CD8 T cells are detect- in the CTLA-4 blockade bioassay with an anti-CTLA-4 blocking antibody able as early as EOC1 in the PB; (2) specific subpopulations of plastic (ipilimumab), but not with unrelated control antibodies. In a PD-1+CTLA- CD8 T cells identified have the potential to serve as an actionable 4 combination bioassay, anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimu- biomarker to select AML patients most likely to benefit from such im- mab) blocking antibodies individually induced luciferase activity (3.0- and mune checkpoint therapies. These findings need to be confirmed in 5.6-fold, respectively), the combination of both antibodies resulted in a larger studies with αPD-1 based therapies in AML. synergistic 18-fold increase in luciferase activity. Similar antibody-induced Acknowledgements luciferase activity is observed in a panel of bioassays specific for agonist NIH (R01CA174385), CPRIT (RP180466), MRA Established Investigator Award antibodies, including GITR, 4-1BB, OX40 and CD40. This response can be to NV (509800), Welch Foundation (E1774), NSF (1705464), CDMRP enhanced following Fc receptor cross-linking, reflecting in vivo activity. (CA160591), and Owens foundation. We would like to acknowledge the Conclusions MDACC Flow Cytometry and Cellular Imaging Core facility for the FACS Cell-based reporter bioassays overcome the limitations of primary sorting (NCI P30CA16672), UH Seq-N-Edit core for RNA-sequencing service, cell-based assays for functional characterization of antibody and and Intel for the loan of computing cluster. other biologics drugs targeting individual or combination immune Trial Registration checkpoint receptors. Here we show a portfolio of mechanism of NCT02397720 action-based bioassays for co-inhibitory and co-stimulatory immune References checkpoint receptors that can be used for antibody screening, 1. Daver, N. et al. Efficacy, Safety, and Biomarkers of Response to Azacitidine characterization, potency and stability studies. and Nivolumab in Relapsed/Refractory Acute Myeloid Leukemia: A Nonrandomized, Open-Label, Phase II Study. Cancer Discov. 2019; 9: 370–383. P241 2. Corces, M. R. et al. Lineage-specific and single-cell chromatin accessibility Single-cell deconvolution identifies T-cell correlates of response to charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016; PD-1 blockade treatment in AML 48: 1193–1203. 1 1 2 2 Xingyue An, BS , Jay R Adolacion , Mansour Alfayez , Jairo Matthews , 3. Linsley, P. S., Speake, C., Whalen, E. & Chaussabel, D. Copy number loss of 2 2 1 2 Wilmer Flores , Steven Kornblau, MD , Arash Saeedi , Sreyashi Basu , the interferon gene cluster in melanomas is linked to reduced T cell 2 1 Naval Daver, MD , Navin Varadarajan, PhD infiltrate and poor patient prognosis. PLoS One. 2014; 9. 1 2 University of Houston, Houston, TX, United States; Univ. of Texas MD 4. Szczepanski, M. J. et al. Increased frequency and suppression by Anderson Cancer Center, Houston, TX, United States regulatory T cells in patients with acute myelogenous leukemia. Clin. Correspondence: Navin Varadarajan (nvaradarajan@uh.edu) Cancer Res. 2009; 15: 3325–3332.[5. Knaus, H. A. et al. Signatures of CD8+ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P241 T cell dysfunction in AML patients and their reversibility with response to chemotherapy. JCI Insight 2018; 3. Background Ethics Approval The combination of the αPD-1 (nivolumab) and hypomethylating agent All patients signed an informed consent form approved by the Institutional azacytidine demonstrated encouraging response in R/R acute myeloid Review Board (IRB) from The University of Texas MD Anderson Cancer leukemia (AML) patients, but the percentage of patients who achieved Center. The study was conducted in accordance with the Declaration of IWG 2016 responses was limited[1]. Early predictive biomarkers to facili- Helsinki. The study was approved by the IRB from University of Houston. tate future trials patient selection are desirable. A better understanding of T cells in AML pre-therapy and on-therapy should yield valuable in- Table 1 (abstract P241). See text for description sights on the treatment-induced anti-tumor response. Methods We performed RNA-sequencing on T cells from a cohort of AML pa- tients who were treated with azacytidine and nivolumab (Table 1). By leveraging subset definitions based on single-cell RNA-sequencing results from T cells of cancer patients, we implemented deconvolu- tion of our bulk T-cell RNA-sequencing data to obtain the relative abundance of different T-cell subsets (in-silico dissection). Results For validation purpose, we compared the gene expression of periph- eral blood (PB) T cells from AML patients and healthy donors (HD)[2,3]. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients[4,5] (Figure 1). Compared with HD T cell, circulating AML CD4 T cells consisted of a higher frequency of Treg[4]. PB CD8 T cells from AML patients were with a significantly lower frequency of naïve, and higher frequencies of effector and exhausted phenotypes[5]. Independent of the clinical responses, comparison of the pre-treatment CD8 T cells from bone marrow (BM) and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PB CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression changes in the AML circulating CD8 T cells, characterized by increased cell me- tabolism and cell proliferation. Deconvolution identified that pre- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 129 of 272 contribute to immune suppression in the tumor microenvironment, inhibiting angiogenic pathways may normalize the tumor vasculature and relieve immunosuppression to augment antitumor immunity, es- pecially with concurrent immune checkpoint inhibition. Therefore, we characterized lucitanib’s selectivity and investigated the antitu- mor efficacy and mechanisms of action of lucitanib combined with anti-PD-1 in nonclinical models. Methods The kinase inhibition profile of lucitanib was evaluated using func- tional biochemical assays. Kinase phosphorylation in cancer cells or mouse tissues was assessed by western blot. In vivo efficacy studies were conducted in syngeneic mouse models with monotherapy or combinations of lucitanib (10 mg/kg daily), DC101 (mouse VEGFR2 monoclonal antibody; 40 mg/kg every 2 days), or anti-PD-1 (5−10 mg/kg biweekly). Treated tumors were analyzed for gene and protein expression and immune composition. Results In vitro, lucitanib demonstrated selective and potent inhibition of the tyrosine kinases VEGFR1-3, PDGFRalpha/beta, FGFR1-3, CSF1R, DDR1, and RET. Lucitanib caused dose-dependent inhibition of VEGFR2 phosphorylation in vivo; one 10 mg/kg dose sustained inhibition for 12 hours. Compared with DC101, lucitanib significantly enhanced tumor growth inhibition and survival in MC38 colon tumor-bearing mice. Lucitanib combined with anti-PD-1 significantly increased anti- tumor activity relative to single agents and to DC101 plus anti-PD-1. Fig. 1 (abstract P214). See text for description Lucitanib-treated MC38 tumors exhibited gene expression changes beyond those observed with DC101 treatment. Across multiple syn- geneic mouse models, tumor growth was significantly inhibited 81%−98% by lucitanib combined with anti-PD-1 and 57%−88% by lucitanib alone. The combination significantly extended survival by 90% to >186% and 15% to >35% compared with vehicle or the best mono- therapy, respectively. Lucitanib combined with anti-PD-1 increased gene expression associated with T-cells, cytotoxic cells, and T-cell sig- naling in BR5FVB1-Akt ovarian tumors, relative to each monotherapy. However, lucitanib alone appeared sufficient to modulate innate and adaptive immunity-related gene expression in MC38 tumors. Additional tumor profiling and mechanism of action studies are ongoing. Conclusions Lucitanib, a potent and selective angiogenesis inhibitor, is differenti- ated from DC101 and displays enhanced antitumor activity in com- bination with PD-1 inhibition in multiple syngeneic models. Gene expression changes associated with tumor immune infiltration and increased antitumor immunity were observed in the combination- treated tumors and may contribute to the increased antitumor activ- ity. Results from these studies support the clinical development of the combination of lucitanib and immune checkpoint blockade as a potential treatment for patients with solid tumors. Ethics Approval The studies were conducted in accordance with the Shanghai Medi- cilon Inc.Guidelines for Use and Care of Animals or an approved IACUC protocol at Crown Biosciences. P243 Fig. 2 (abstract P214). See text for description Modulating the immunogenicity of low-mutation soft tissues sarcomas with epigenetic targeted therapy Himavanth Gatla, PhD, Maggie Phillips, Brian Ladle Johns Hopkins Medicine, Owings Mills, MD, United States P242 Correspondence: Brian Ladle (bladle@jhmi.edu) Combination of the angiogenesis inhibitor lucitanib with immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P243 checkpoint blockade augments anti-tumor activity in syngeneic models Background Rachel Dusek, PhD, Liliane Robillard, PhD, Thomas Harding, PhD, Andrew Sarcomas account for 13% of all cancers in young adults under the Simmons, PhD, Minh Nguyen age of 20. For recurrent and metastatic sarcomas, the very poor sur- Clovis Oncology, Inc., San Francisco, CA, United States vival rate despite surgery and chemotherapy warrants better sys- Correspondence: Rachel Dusek (rdusek@clovisoncology.com) temic therapies. As opposed to cancers that show good responses to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P242 immune checkpoint blockade which have high mutation burden, pediatric sarcomas present with low mutation burden, and a Background complete ineffectiveness of immune checkpoint blockade as a mono- Lucitanib is an anti-angiogenic, small molecule multi-tyrosine kinase therapy, suggesting poor immune system activation, and immune inhibitor that has demonstrated potent tumor growth inhibition in infiltration. multiple cancer xenograft models. Because angiogenic factors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 130 of 272 Methods types of DNA damage repair deficiencies and immune evasion have We have used a mutated Kras-driven murine sarcoma syngeneic been lacking. Hence, we have modeled the biology of ovarian cancer tumor model (KP Sarc) and GM-CSF-secreting whole cell tumor cell using patient-relevant mutational landscapes in an immune- vaccine approach (GVAX) to investigate if epigenetic targeted ther- proficient, syngeneic mouse model in order to help us identify the apy induced tumor associated antigens are capable of generating contribution of common driver mutations to the immune repertoire sustained anti-tumor immunity in the tumor microenvironment, and thus to responses of HGSOC tu- Results mors to immunotherapy. We show that sequential combination of epigenetic modifying drugs Methods - DNA methyl transferase inhibitor (decitabine) and HDAC inhibitor We hypothesize that the immune composition and gene expression (entinostat) significantly increases the expression of cancer testis an- signatures of the resulting tumors will vary based on the combin- tigens (CTAs), compared to either drugs alone. In addition, these ation of genetic alterations and the DNA repair proficiency of the drugs modify chemokine expression including increased expression transformed cells. To this end, we have engineered novel syngeneic of CXCL10. Significantly improved immune responses can be gener- mouse models from murine-fallopian-tube epithelium using CRISPR/ ated to decitabine and entinostat pre-treated KP sarc tumor cells as Cas9 technology. These tumors capture the most common combina- assessed by slowed tumor growth, increased T cell infiltrates, and in- tions of co-occurring mutations observed in HR-deficient and -profi- creased cytokine production. Furthermore, immune checkpoint cient patient samples. blockade therapy potentiates the tumor regression mediated by the Results combination of decitabine and entinostat more effectively than the To validate the DNA repair proficiency of the transformed cells, we regression mediated by either drugs alone. This suggests that com- measured Rad51 nuclear focus formation after ionizing radiation (IR) bination therapy induced antigenicity generates better anti-tumor and PARP inhibitor and DNA-damaging agent sensitivity. The HR- immune responses. Rechallenging the mice which rejected epigeneti- deficient cell lines had significantly fewer Rad51 nuclear foci and cally modified KP Sarc tumor formation with similarly treated KP Sarc were more sensitive to PARP inhibition in comparison to HR- cells did not result in tumor formation, whereas untreated KP Sarc proficient cells. Initial immune /stromal analysis using flow cytometry, cells grew uninhibited, suggesting that epigenetic therapy induced scRNA seq transcriptomic and immunofluorescence analysis re- tumor associated antigens are capable of generating a sustained vealed substantial differences in the myeloid and T-cell regulatory memory immune response. By depleting CD4 and CD8 T lympho- compartments between HR-proficient and -deficient primary and cytes, we show that epigenetic targeted therapy induced anti-tumor metastatic tumors and within the ascitic fluid. Preliminary results responses are mediated by both CD4 and CD8 T lymphocytes. Diffi- also suggest that inhibition of the DNA damage response (DDR), culty with in vivo treatment includes the myelosuppressive side ef- checkpoint kinase 1 (Chk1) in combination with immune check- fects of decitabine and entinostat which can inhibit T cell responses point inhibitors, potentiates antitumor effects and augments cyto- immediately after treatment. Proper sequencing of the drugs when toxic T-cell infiltration. given in vivo will be crucial to generate successful adaptive T cell re- Conclusions sponses to newly expressed antigens. Understanding the genetic basis of these complex cellular interac- Conclusions tions will be critical to better tailor combinations of existing targeted Epigenetic targeted therapy induced tumor associated antigens are treatments and immunotherapies in ovarian cancer to fight this dev- capable of generating sustained anti-tumor immune responses. astating disease. P244 P245 The genomic architecture of serous carcinomas shapes the tumor Durvalumab after concurrent chemoradiotherapy in inoperable microenvironment and modulates responses to targeted and stage III non-small cell lung cancer (NSCLC) – a German radiation immunotherapies oncology survey 1 2 3 4 Sonia Iyer, PhD , Shuang Zhang , Anniina Farkkila , Sean Smith , David Lukas Kaesmann, MD, Chukwuka Eze, Julian Taugner, Olarn 5 5 1 1 1 Pepin , Raghav Mohan , Tian Xia , Ferenc Reinhardt , Tony Chavarria , Roengvoraphoj, Claus Belka, Farkhad Manapov 1 6 3 7 Esmee Hoefsmit , Shailja Pathania , Yunlan Zhou , Kevin Elias , Benjamin University Hospital, Munich, Germany 8 1 Neel , Robert Weinberg, PhD Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) Whitehead Institute for Biomedical Research, Cambridge, MA, United Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P245 2 3 States; NYU Langone Health, New York, NY, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Institute of Background Technology, Cambridge, MA, United States; Massachusetts General Consolidation PD-L1 inhibition with durvalumab after platin-based Hospital, Boston, MA, United States; University of Massachusetts, Boston, concurrent chemoradiotherapy (CRT) has become the standard of MA, United States; Brigham and Women's Hospital, Boston, MA, United care in inoperable stage III non-small cell lung cancer (NSCLC) States; NYU-Langone Medical Center, New York, NY, United States based on the excellent PACIFIC trial results. Treatment recommen- Correspondence: Sonia Iyer (iyers@wi.mit.edu); Robert Weinberg dations need time for implementation in nationwide settings and (weinberg@wi.mit.edu) require the close interaction of different medical specialities. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P244 this nationwide survey, we questioned the distribution and clin- ical settings of durvalumab treatment after concurrent CRT, ob- Background served side effects of this treatment and summarize follow-up High-grade serous ovarian cancer (HGSOC) is the most frequent and management. most aggressive histologic subtype of ovarian cancer. The corner- Methods stone of the existing treatment of HGSOC is DNA-damaging chemo- We surveyed radiation oncology institutions in Germany via an an- therapy; however, practically all patients eventually develop the onymous online questionnaire sent by e-mail to all members of the progressive disease and the 5-year survival is only 40%. Immunother- German Radiation Oncology Society. apy would seem to be an attractive alternative treatment to chemo- Results therapy, yet existing immunotherapies perform poorly in ovarian We received a total of 255 responses (response rate: 18%). Of cancer, with only ~10% of patients responding to checkpoint block- which 203 (80%) were completed and returned and thus eligible ade. Why this is the case remains poorly understood and there is a for further evaluation. The respondents work in 87 different cities pressing need to understand the underlying biology of immune eva- and 44% in a private medical practice, 29% in university and sion in ovarian cancer. One critical area of interest is the role of hom- 22% in a general hospital. Responses of the same department ology dependent DNA repair (HR) in immune evasion. Unfortunately, were analysed for congruence. Durvalumab was implemented in the preclinical tools required to explore the relationship between the clinical routine by 143 (70%) respondents. Reasons for failed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 131 of 272 implementation in clinical practice were patient ineligibility, deci- months. All patients were alive at the time of evaluation. Four (25%) sion of medical oncologists or absence of updated German evi- patients have developed oligoprogression. Metastastic sites were dence review (S3-guidelines) regarding this treatment approach. bone, brain, adrenal gland and distant lymph nodes. Two patients re- Durvalumab was generally administered according to the re- ceived second-line chemotherapy after distant failure. Another two spondents by private oncological practices in 32%, general or received stereotactic body radiotherapy for all metastatic sites and university hospital in 57% and in the radiation oncology de- continued on durvalumab. Common toxicity during durvalumab was partment, which delivered the CRT in 11% of cases. Importantly, dermatitis (I-II° CTCAE) which occurred earliest after 2 cycles in 10 according to 36% of all respondents initial PD-L1 status was (65%) patients and pneumonitis II° CTCAE in 2 (13%) and III° CTCAE present in ≤30% of all patients. 82% of respondents have treated in 2 (13%) patient between 2-7 months after completion of CRT. In 1-15 patients with durvalumab and 14% of respondents >15 pa- total, 3 (19%) patients discontinued durvalumab treatment after a tients. Furthermore, no respondent had applied durvalumab in median of 4 months due to distant progression or unacceptable less than 14 days after the completion of CRT. 65 (46%)and 49 toxicity. (34%) respondents started durvalumab 14-28 days and later than Conclusions 28 days after CRT, respectively. The majority of respondents Durvalumab was well tolerated with reversible acute toxicity. 25% of (>80%) re-staged the patients with CT (thorax/upper abdomen) patients develop oligoprogression after a mean time of 5.5 months prior to durvalumab. Severe side effects requiring hospital admis- after the end of CRT. sion in more than 10% of all patients were reported by only 12% Ethics Approval of all respondents. The study was approved by the University Ethics Board, approval Conclusions number 17-230. Durvalumab was implemented in the multimodal treatment of inop- Consent erable stage III NSCLC and administered by the absolute majority of Written informed consent was obtained from the patient for publica- respondents. Low testing rates of PD-L1 at initial diagnosis were ob- tion of this abstract and any accompanying images. A copy of the served and should be considered a major barrier to universal adop- written consent is available for review by the Editor of this journal. tion and integration in the clinical work-flow. Durvalumab appears to be well tolerated. However, treatment-related side effects need to be P247 considered during and after multimodal therapy. TLR3-targeting combinatorial chemokine modulation sensitizes “Cold” tumors for the therapeutic effectiveness of immune Acknowledgements checkpoint inhibition We would like to thank the Board of the German Society for Radiation 1 2 Kathleen Kokolus, MS, PhD , Natasa Obermajer, PhD , Per Basse, MD, Oncology (DEGRO) for their approval and their office team for providing the 1 1 PhD , Pawel Kalinski, MD, PhD mailing list. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Ethics Approval UPMC Hillman Cancer Center, Pittsburgh, PA, United States The Board of the German Society for Radiation Oncology (DEGRO) approved Correspondence: Pawel Kalinski (Pawel.Kalinski@roswellpark.org) the survey. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P247 P246 Background Prospective evaluation of outcome and toxicity of durvalumab Immune checkpoint inhibition (ICI) has emerged as life-prolonging treatment after chemoradiotherapy in inoperable stage III non- and occasionally curative treatment for many cancer patients, but small cell lung cancer (NSCLC) patients their activity remains disappointing in many common tumors. ICI Lukas Kaesmann, MD, Julian Taugner, Chukwuka Eze, Olarn therapies are effective against “hot” tumors infiltrated with cytotoxic Roengvoraphoj, Claus Belka, Farkhad Manapov T lymphocytes (CTLs) but inefficient against “cold” tumors lacking University hospital, Munich, Germany CTLs. The importance of CTLs, availability of CTL targets and local ex- Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) pression of PD-L1 and PD-L2 (induced by CTL-produced IFNγ) in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P246 overall effectiveness of ICI remain controversial. We have observed that chemokine-modulatory (CKM) regimens combining TLR3 ligands Background with type-1 IFNs are up to 100-fold more effectiveness in inducing Consolidation PD-L1 inhibition with durvalumab after concurrent CTL attractants (CXCL9, CXCL10, CCL5) compared to either factor chemoradiotherapy (CRT) has become the standard of care in inoper- alone. Moreover, CKM suppresses local Treg attractants, and targets able stage III non-small cell lung cancer (NSCLC) based on the excel- tumor microenvironment (TME) rather than healthy tissues [1-3]. lent PACIFIC trial results. The aim of this prospective single center Thus, we tested whether local or systemic CKM treatments enhance study was to evaluate the outcome and toxicity of durvalumab treat- CTL infiltration in “cold” tumors and determined the feasibility of ment after CRT in a tertiary cancer center. short-term CKM to sensitize poorly immunogenic, αPD-1 resistant, tu- Methods mors to PD-1 blockade. We prospectively collected clinical characteristics, toxicity and out- Methods come of all patients with inoperable stage III NSCLC treated with dur- C57BL/6 mice inoculated with MC38 (colorectal) or ID8 (ovarian) can- valumab after CRT/RT since 9th November 2018. Toxicity was cer cells were treated starting on day three (low-stage disease) or collected using the Common Terminology Criteria for Adverse Events eight (late-stage disease). A two dose course of CKM (IFNα and rinta- version 5 before and during treatment. Re-staging after CRT and be- tolimod [2]) followed by three doses of αPD-1 in two distinct regi- fore the start of durvalumab consisted of a CT scan (thorax/upper ab- mens: 1) Sequentially (following CKM) or 2) Concurrent with CKM. domen). 18F-FDG-PET-CT was performed 3 months and CT 6 months Mice were monitored for intratumoral CTL, tumor growth and after start of consolidation treatment. survival. Results Results Data of 16 patients treated with durvalumab after CRT/RT were eval- We observed strong effectiveness of CKM in promoting intratumoral uated. Three patients (19%) were female and 13 (68%) male, median increases in CTL and PD-L1 expression in the TME. CKM aids in the age at treatment start was 64 years. 10 (53%) patients had T4 or T3 sensitization of the largely αPD1-resistant tumors to PD1 blockade. tumors, four (25%) patients had N3 and 9 (56%) N2 disease. 15 Pa- Both sequential and concurrent CKM allowed antitumor effectiveness tients had CRT with a medium radiation dose of 63.20 Gy and were of αPD-1, resulting in overall prolongation of survival and 30-100% treated with two concurrent cycles of platin-based chemotherapy. cures, depending on treatment initiation. Sensitizing tumors to αPD- One patient was treated with moderate hypofractionated radiother- 1 did not require intratumoral CKM administration and was observed apy without chemotherapy. Median follow-up was 7 (range:2-16) with systemic application at distant sites, consistent with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 132 of 272 preferential activation of tumor tissues by CKM observed in tumor 95% CrI: 0.42-2.73) and avelumab+axitinib (HR=1.49, 95% CrI: 0.76- explant model [3]. Although strong antitumor effects were seen in 2.96) over P+A but were not statistically significant. the absence of any vaccination component, a stronger effect could In the intermediate + poor IMDC risk group, P+A showed a significant OS be observed by vaccination with tumor-loaded dendritic cells. benefit over sunitinib (HR=0.52, 95% CrI: 0.37-0.74) and was favored over Conclusions the other two interventions evaluated, but not statistically significant We demonstrate that local or systemic CKM sensitizes mice with [cabozantinib (HR=0.65, 95% CrI: 0.38-1.11) and N+I (HR=0.79, 95% CrI: poorly immunogenic tumors for subsequent effectiveness of PD-1 0.53-1.18)]. P+A showed a significant PFS benefit over sunitinib (HR=0.67, blockade. Thus, promoting intratumoral CTL accumulation may be 95% CrI: 0.53-0.85) and was favored over N+I (HR=0.87, 95% CrI: 0.65- sufficient for therapeutic effectiveness of ICI against “cold” tumors 1.17), but not statistically significant. PFS benefit favored cabozantinib with low mutational load. Our data provides rationale for clinical test- (HR=1.40, 95% CrI: 0.85-2.31) over P+A, but was not statistically significant. ing of sequential regimens where short-term CKM is followed by rou- Conclusions tine ICI, limiting the inconvenience for patients and facilitating the The results of this analysis suggest that pembrolizumab+axitinib may inclusion of CKM into routine immunotherapy plans. have PFS and OS advantages over most alternative first-line treat- ment options for mRCC, irrespective of IMDC risk groups. Acknowledgements Funded by 1P01CA132714, Rustum Family Foundation and Institutional P249 Support. Time-dependent blood transcriptomic perturbations differentially associated with mono and combination checkpoint inhibitor References therapy 1. Muthuswamy R, Berk E, Junecko BF et al. Cancer Res. 2012; 72:3735-3743. 1 2 1 Darawan Rinchai, PhD , Emily Hinchcliff, MD , Wouter Hendrickx, PhD , 2. Theodoraki MN, Yernei S, Sarkar et al. Cancer Res. 2018; 78:4292-4302. 1 1 Jessica Roelands, Master , Damien Chaussabel , Davide Bedognetti, MD, 3. Obermajer H, Urban J, Wieckowski E et al. Nat Protoc. 2018; 13:335-357. 1 2 PhD , Amir Jazaeri, MD Ethics Approval 1 2 Sidra Medicine, Doha, Qatar; The University of Texas MD Anderson This study was approved by Roswell Park Comprehensive Cancer Center's Cancer Center, Houston, TX, United States IACUC (protocol 1398M). Correspondence: Amir Jazaeri (aajazaeri@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P249 P248 Pembrolizumab plus axitinib (P+A) versus other first-line (1L) Background systemic therapies for advanced/metastatic clear-cell renal cell The rationale for combination immunotherapy is based on presumed carcinoma (ccmRCC) by IMDC Risk Status – a network meta- additive or synergistic properties of each individual drug [1-2]. analysis (NMA) Understanding the molecular mechanisms modulated by a given 1 1 1 Ian McGovern, MPH , Rohan Shirali, MS , Andrew Simon, ScM , Yichen drug is critical to implement more efficient therapeutic approaches. 2 2 1 Zhong, PhD , Rodolfo Perini, MD , Maria Lorenzi, MSc , Oluwakayode We conducted a study to determine 1) whether response to anti- Adejoro, MD, MPH anti-CTLA4 monotherapy (Tremelimumab) or combination of anti- 1 2 Precision Xtract, Boston, MA, United States; Merck & Co. Inc., PDL1(Durvalumab) and anti-CTLA4 (Tremelimumab) therapy could be Kenilworth, NJ, United States measured via blood transcriptome profiling; and 2) whether differ- Correspondence: Oluwakayode Adejoro ences between both treatment groups could be observed. In blood (oluwakayode.adejoro@merck.com) transcriptomic correlative studies performed so far, samples have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P248 been collected at limited time points (i.e., before and after treat- ments), preventing the description of the kinetic and dynamic Background changes associated with specific treatment modalities [3]. The International Metastatic Renal Cell Carcinoma Database Consortium Methods (IMDC) risk group classification is an important prognostic factor for ef- An adaptively randomized phase II trial of sequential versus combination ficacy outcomes of first-line systemic treatment of advanced/metastatic administration of Tremelimumab and Durvalumab (MEDI4736) in patients mRCC. IMDC risk is predictive of outcomes including overall survival with recurrent platinum resistant ovarian, peritoneal or fallopian tube (OS), progression-free survival (PFS) and overall response rate (ORR). cancers at the M.D. Anderson Cancer Center. Peripheral blood samples Pembrolizumab in combination with axitinib showed superior and clin- were collected serially before treatment and at 6 time points post- ically meaningful improvements in OS, PFS and ORR versus sunitinib in treatment from patients receiving Tremelimumab, alone or in combin- subjects with untreated ccmRCC in the KEYNOTE-426 trial. This NMA ation with Durvalumab, administered every 28 days. A total of 91 samples synthesized evidence from randomized clinical trials (RCTs) to indirectly were analyzed. Time points include C1D01 (baseline), C1H12 (12 hours compare the relative treatment effects of P+A vs other therapies in sub- after treatment), C1D08 (cycle one day 8), C1D15 (cycle one day 15), jects with favorable and intermediate + poor IMDC risk groups. C2D01 (cycle 2 day one) and C3D01 (cycle 3 day one). Blood transcrip- Methods tome profiles were generated by RNA-seq (Illumina HiSeq4000) at Sidra Fixed-effect Bayesian NMA was conducted to determine the relative ef- medicine. A set of 382 transcriptional modules was used for the analysis ficacy of treatments. Hazard ratios (HRs) for PFS and OS were estimated of this dataset using a pre-defined framework [4-5]. A module is consid- with 95% credible intervals (CrIs). Analyses were conducted among ered to be “responsive” to the treatment when significant changes in subjects with favorable risk, and intermediate + poor risk disease. abundance are observed for a proportion of its constitutive transcripts Results that is greaterthatwhatcouldbeexpectedbychance[4-5]. Among subjects with favorable IMDC risk, the estimated HRs for OS Results favored P+A over the other 3 interventions evaluated [nivolumab+i- We identified changes in blood transcript abundance in both treatment pilimumab (N+I) (HR=0.53, 95% CrI: 0.18-1.60), sunitinib (HR=0.64, groups, the modular perturbation peaking at cycle 1 day 15 post- 95% CrI: 0.24-1.70) and pazopanib (HR=0.73, 95% CrI: 0.26-2.03)] but treatment (Figure 1). Perturbations of Cell cycle, Protein synthesis and none were statistically significant. For PFS, P+A had statistically sig- Gene transcription modules were observed in both groups. But import- nificant benefit over 2 out of 9 interventions evaluated [interferon- ant qualitative differences were observed as well. Most notably, abun- alpha (IFN) (HR=0.30, 95% CrI 0.15-0.60) and bevacizumab (B)+ tem- dance of gene sets associated with Interferon, Tumor necrotic factor, sirolimus (HR=0.41, 95% CrI 0.18-0.98)]. The results numerically fa- cytotoxic lymphocyte and erythroid signatures was specifically in- vored P+A over 5 out of 9 interventions evaluated [ranging from creased in patients receiving combination immunotherapy B+IFN (HR=0.50, 95% CrI 0.23-1.10) through, atezolizumab, pazopa- Conclusions nib, N+I, to sunitinib (HR=0.81, 95% CrI: 0.53-1.23)], but were not sta- We characterized differential, time-dependent, systemic perturbations tistically significant. PFS benefits favored B+atezolizumab (HR=1.07, associated with mono vs combination immune checkpoint blockade. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 133 of 272 The peak of this immune modulation is observed at day 15 after P250 treatment. The mechanistic and clinical relevance of these findings Combination treatment of the oral CHK1 inhibitor, SRA737 and remains to be explored in a larger group of patients low dose gemcitabine, enhances the effect of PD-L1 blockade by Trial Registration modulating the immune microenvironment in small cell lung NCT03026062 cancer Triparna Sen, PhD (triparnasen@gmail.com) References Memorial Sloan Kettering Cancer Center, New York, NY, United States 1. Naumann RW, Coleman RL. Management strategies for recurrent Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P250 platinum-resistant ovarian cancer. Drugs 2011; 71: 1397-412. 2. Davis A, Tinker AV, Friedlander M. "Platinum resistant" ovarian cancer: what is Background it, who to treat and how to measure benefit? Gynecologic oncology 2014; Small cell lung cancer (SCLC), the most aggressive form of lung cancer, 133: 624-31. shows poor response rates to immunotherapy targeting the pro- 3. Friedlander P, Wassmann K, Christenfeld AM, Fisher D, Kyi C, Kirkwood JM, grammed cell death protein 1 pathway (PD-(L)1). Our group previously Bhardwaj N, Oh WK. Whole-blood RNA transcript-based models can predict discovered that SCLC exhibits high expression of checkpoint kinase 1 clinical response in two large independent clinical studies of patients with (CHK1) and that the CHK1 inhibitor SRA737 activates the innate im- advanced melanoma treated with the checkpoint inhibitor, tremelimumab. mune STING pathway, demonstrating robust anti-tumor activity and J Immunother Cancer 2017 Aug 15;5(1):67. doi: 10.1186/s40425-017-0272-z. synergy in combination with anti-PD-L1 in an SCLC model. 4. Chaussabel D, Baldwin N. Democratizing Systems Immunology with Modular Methods Transcriptional Repertoires Analyses. Nat Rev Immunol. 2014 Apr;14(4):271–80. As SRA737 is being tested in SCLC patients in combination with low 5. Altman MC, Rinchai D, et al. A Novel Repertoire of Blood Transcriptome dose gemcitabine (LDG), we evaluated the efficacy and immune cor- Modules Based on Co-expression Patterns Across Sixteen Disease and relates (including macrophages associated with resistance to immune Physiological States. bioRxiv 525709; doi: https://doi.org/10.1101/525709 checkpoint blockade) of the SRA737+LDG regimen in combination Ethics Approval with anti-PD-L1 in an SCLC model. The study was approved by the MD Anderson Cancer Center Ethics Board, Results approval number IRB 5 IRB00006023 and Sidra Medicine's IRB, approval Trp53, Rb1 and p130 (RPP) triple knockout SCLC cells were implanted 1804022877 into the flank of B6129F1 immunocompetent mice. After the mice developed tumors, they were treated with single agents or various drug combinations. Anti-PD-L1 and LDG demonstrated minimal ef- fect on tumor growth as single agents and only a modest effect as a combination. Moderate to strong anti- tumor activity was however observed with SRA737 monotherapy which directly correlated with dosing intensity. The most profound and synergistic anti-tumor activity was observed when anti-PD-L1 was com- bined with the SRA737+LDG regimen, with all animals showing durable regressions. Analysis of tumor infiltrating immune cells at the end of this treatment regimen showed a dramatic induction of cytotoxic T- cells and a reduction of exhausted and regulatory T cells. Similarly, pro- inflammatory M1 type macrophages and dendritic cells were increased while immunosuppressive M2 type macrophages and MDSC cells were dramatically decreased. As monotherapy, the more dose intensive SRA737 schedule resulted in similar effects on lymphocytes when com- bined with anti-PD-L1. These effects are consistent with our previous data showing that SRA737 treatment leads to an induction of STING and type I interferon signaling in tumors, which is associated with the establishment of an anti-tumor immune microenvironment. Conclusions Our findings suggest that the combination of anti-PD-L1 with the SRA737+LDG regimen may represent the optimal implementation of these agents, leading to a dramatic anti-tumor activity accom- panied by the establishment of a strong anti-tumor immune microenvironment. Given that anti-PD-(L)1 drugs are approved but show limited efficacy in SCLC, our preclinical data provide a strong rationale for combining these agents with the SRA737+LDG regimen to enhance clinical response rates. P251 CTX-8371, a novel bispecific targeting both PD-1 and PD-L1, is more potent than combination anti-PD-1 and PD-L1 therapy and provides enhanced protection from tumors in vivo Diana Albu, PhD, Pearl Bakhru, Wilson Guzman, BS, Michael Ophir, Rachel McCrory, BS, William Carson, Jason Kong, Beata Bobrowicz, Pia Muyot, Amanda Oliphant, Dalton Markrush, Rachel Rennard, Cheuk Lun Leung, Sara Haserlat, Michael Schmidt, Jose Gonzalo, Bing Gong, Robert Tighe, BS, Diana Albu, PhD, Benjamin Wolf Compass Therapeutics, Cambridge, MA, United States Correspondence: Benjamin Wolf (benjamin.wolf@compasstherapeutics.com) Fig. 1 (abstract P249). Modular repertoire analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P251 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 134 of 272 Background Results Monoclonal antibody immunotherapies targeting immune check- From 10/2017 to 2/2019, 83 patients received I+N for mRCC and point receptors have shown great promise for a subset of cancer were included. Demographics are shown in Table 1. By International patients. However, novel combination therapies are still needed Metastatic RCC Database Consortium (IMDC) risk criteria [2], 20.5% to increase the benefit of cancer immunotherapy and bring it to were favorable, 61.4% intermediate, and 18.1% poor risk. 65% were broader patient populations. Here we describe the preclinical stage IV at diagnosis, 63.9% were untreated, and 16.9% patients had evaluation of CTX-8371, which combines PD-1 and PD-L1 target- prior nivolumab exposure. 77.1% of patients had clear cell pathology. ing in one bispecific, tetravalent molecule. 12/83 (14.4%) have sarcomatoid differentiation: 2 have an ongoing Methods response and 7 have died. At the data cutoff date, 44/83 (53%) pa- Our proprietary Stitchmabs®™ bispecific screening platform was used tients have progressed or died. Median PFS was 5.3 months (95% CI to conduct an unbiased screen of bispecifics comprised of various 3-8.5) (Figure 1). OS rates at 6, 12, and 18 months were 76.2%, 63.8%, checkpoint blocking antibodies. This screen yielded the surprising and 51.5%, respectively (Figure 2). Rates of best radiographic re- discovery that a bispecific containing both PD-1 and PD-L1 binding sponse were CR 4.8%, PR 22.9%, SD 18.1%, PD 32.5%, and unknown arms was more potent than the combination of parent monoclonal 21.7%. 44/83 (53%) patients experienced no adverse event (AE). 18/ antibodies. We then generated common light chain bispecifics con- 83 (21.7%) patients experienced a grade 3/4 AE (most commonly taining compatible anti-PD-1 and PD-L1 antibodies and used multiple diarrhea, n=7), 20/83 (24%) patients experiencing a grade 1-2 AE at in vitro assays to identify our lead, CTX-8371. Additional in vitro and worst (most commonly hypothyroidism, n=14), and one grade 5 AE oc- in vivo experiments confirmed CTX-8371’s reactivity across species, curred. 23/83 (27.7%) patients have died, with 10/83 (12.0%) patients in vivo anti-tumor effects, and the underlining mechanisms driving dying within 90 days of receiving the first dose of I+N. 4/83 patients its distinctive activity. have achieved a complete response. Two of these patients discontin- Results ued treatment at 11 and 12 months with a sustained response at 1 and We found that CTX-8371 binds to human and cynomolgus monkey 5 months, respectively. Three other patients remain off therapy for AEs PD-1 and PD-L1 targets with sub-nanomolar affinities and is cross- and have not progressed after 11, 5, and 3 months. reactive to mouse PD-1 and PD-L1. Compared to Keytruda®, CTX- Conclusions 8371 increased T cell activation and tumor cell killing in vitro, signifi- In our real-world cohort of mRCC patients, I+N has similar clinical effi- cantly delayed tumor growth, and prolonged survival in human cell cacy as previously described; however, the cohort is more frail, with transfer tumor models. Additionally, CTX-8371 demonstrated efficacy 16.9% of patients treated in the nivolumab refractory setting. Five in transplantable mouse syngeneic models. Investigation into the patients remain off therapy. This forms the basis for larger prospect- mechanisms responsible for the enhanced efficacy of CTX-8371 unex- ive treatment discontinuation trials (ie. Alliance A031704 phase 3 pectedly found that the bispecific causes a massive loss of PD-1 from trial) with prospective treatment discontinuation for complete re- the T cell surface, which was not observed in response to monoclo- sponse patients at 1-year. nal antibodies alone or combined. This robust PD-1 downregulation, potentially mediated through bridging together the T cell and tumor References cell, may explain the ability of CTX-8371 to reverse PD-1 suppression 1. Motzer RJ, Tannir NM, McDermott DF, et al. Nivolumab plus Ipilimumab more potently than standard blocking antibodies. versus Sunitinib in Advanced Renal-Cell Carcinoma. N Engl J Med. Conclusions 2018;378(14):1277-1290. Taken together, the results demonstrate that the bispecific, tetrava- 2. Heng DY, Xie W, Regan MM, et al. Prognostic factors for overall survival lent antibody CTX-8371 has increased potency in vitro and in vivo as in patients with metastatic renal cell carcinoma treated with vascular compared to clinical checkpoint blockade agents. Some of its effects endothelial growth factor-targeted agents: results from a large, multicen- are likely attributable to its unique mechanism of action, driving ro- ter study. J Clin Oncol. 2009;27(34):5794-5799. bust downregulation of cell-surface PD-1. Thus, CTX-8371 has the po- Ethics Approval tential to increase the number of patients that benefit from PD-1/PD- This study was approved by the Duke University IRB (#Pro00101984) L1 checkpoint blockade. P252 A retrospective study to evaluate real-world clinical outcomes in patients with metastatic renal cell carcinoma (mRCC) treated with Ipilimumab and Nivolumab Landon Brown, MD, Emily Kinsey, MD, Chester Kao, MD, Patrick Healy, Andrew Armstrong, MD, Megan McNamara, MD, Sundhar Ramalingam, MD, Michael Harrision, MD, Daniel George, MD, Tian Zhang, MD Duke University, Durham, NC, United States Correspondence: Landon Brown (landon.brown@duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P252 Background The combination of nivolumab at 3mg/kg plus ipilimumab at 1mg/ kg (I+N) followed by maintenance nivolumab has greatly improved outcomes in patients with intermediate or poor-risk untreated mRCC [1]. Real-world series of patients treated with this combination are scarce. In this retrospective analysis, we present a real-world experi- ence with this combination immunotherapy. Methods A search was performed to identify all mRCC patients treated in the Duke Cancer Institute network with I+N. An extensive chart review was conducted. Patient characteristics are summarized with descrip- tive statistics; Kaplan Meier analysis was performed for progression Fig. 1 (abstract P252). See text for description free survival (PFS) and overall survival (OS). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 135 of 272 Methods We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. Results We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young [3]. αPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibit- ing this property. Efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC dif- fered by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM-3 expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. Conclusions Treatment differences in aged versus young could depend on im- mune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. PD-L2 expression differences could be a mechanism for treatment differences. We are now identi- fying mechanisms for increased PD-L2 and contributions to αPD- L2 efficacy in aged, and testing TCSC effects on treatments (Fig- ure 1-3). Our work can improve cancer immunotherapy in aged Fig. 2 (abstract P252). See text for description hosts and further provide important insights even in young hosts. Table 1 (abstract P252). See text for description Acknowledgements South Texas MSTP training grant (NIH T32GM113896), TL1TR002647, R01 CA231325. References 1. Schildberg FA,Klein SR,Freeman GJ,SharpeAH. Coinhibitory Pathways in the B7-CD28 Ligand-Receptor Family. Immunity. 2016;44(5):955-72. 2. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 ther- apy. Nature. 2016;537(7620):417-21. 3. Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol. 2018;105:146-54. Ethics Approval All animal work was done under UTHSA Institutional Animal Care and Use Committee approved studies in compliance with the Guide for the Care and Use of Laboratory Animal Resources (published by National Research Council of the National Academies), Animal Welfare Act (AWA) (published by USDA), Public Health Service Policy on Humane Care and Use of Laboratory Animals (published by NIH) and US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Approval number: 20180021AR. P253 Distinct clinical and immunological responses to αPD-1, ⍺PD-L1 and ⍺PD-L2 immunotherapy in B16 melanoma in aged versus young hosts includes T-cell stem cell effects and PD-L2 expression differences Myrna Garcia, BS, Alvaro Padron, Yilun Deng, MD, PhD, Harshita Gupta, PhD, Aravind Kancharla, Tyler Curiel, MD University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Correspondence: Tyler Curiel (curielt@uthscsa.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P253 Background Aging is the biggest risk factor for cancer, yet little is known about can- cer immunotherapy effects. αPD-1 can block PD-L1 and PD-L2 while ⍺PD-L1 blocks PD-1 and CD80 [1]. A recent key finding in young hosts Fig. 1 (abstract P253). ⍺PD-L2 treats B16 melanoma in aged mice including humans is that melanoma response to αPD-1/αPD-L1 corre- but not young mice lates with CD8+TCF-1+ T cell stem cell (TCSC) generation [2]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 136 of 272 functional Fc as monotherapies or in a combination with anti-PD-1 was carried out in order to better understand the effects of anti-TIGIT anti- body with functional Fc at molecular level at different time points. Results Here we demonstrate using mouse tumor models that anti-mTIGIT antibodies require interactions with Fc gamma receptors on myeloid cells in the tumor microenvironment for effective anti-tumor re- sponse. Our observations reveal that the anti-mTIGIT therapeutic ef- fect is not achieved by depletion of intratumoral regulatory T cells, but instead is mediated by “reverse activating signals” through Fc gamma receptors on myeloid cells, inducing expression of various mediators such as cytokines, including TNF-alpha and IL-23, and che- Fig. 2 (abstract P253). PD-L2 expression is significantly higher in mokines, such as CXCL10 and CXCL11, thus generating the condi- aged mice when compared to young tions for potentially promoting immune infiltrates into the tumor microenvironment. In addition, up-regulation of co-stimulatory mole- cules, such as CD80, CD86, and CD40, has been observed, consistent with the heightened anti-tumor activity of Fc gamma receptor binding com- petent anti-mTIGIT antibodies. Furthermore, we discovered induction of a robust and persistent granzyme B and perforin response from the in vivo treatment of anti-mTIGIT antibody with a functional Fc, distinct from a predominantly interferon-gamma-driven anti-PD-1 blockade. Conclusions Our observations for the first time provide mechanistic insights into the requirement for Fc engagement of anti-mTIGIT monoclonal anti- bodies for effective anti-tumor activity in vivo which has implications for the various human antibodies of various isotypes are currently under intense clinical investigations. P255 Preclinical characterization and efficacy of MG1124, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy 1 1 1 Jae-Chul Lee, Master degree , Minkyu Hur , Hye-Young Park , Mi-Young 1 1 1 2 3 Oh , Hye-mi Nam , Hye In Yum , HyungSuk Choi , Jaehwan Kim , 3 1 Byoung Chul Cho, MDphD , Yangmi Lim MOGAM Institute for Biomedical Research, Yong-in, Korea, Republic of; 2 3 GC Pharma, Yongin-si, Korea, Republic of; Yonsei Cancer Center, Seoul, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P255 Fig. 3 (abstract P253). αPD-1, αPD-L2 and αPD-L1 elicit distinct Background TCSC that also differ by age CEACAM1 is one of the several immune checkpoint receptors expressed on T cells and NK cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction in- P254 duces downregulation of ZAP70 phosphorylation in response to T cell re- Requirement of Fc gamma receptor-mediated myeloid-cell ceptor (TCR) stimulation. CEACAM1 is also highly expressed on non-small activation for effective cancer immunotherapy with an anti-TIGIT cell lung cancer (NSCLC) and its expression is correlated with cancer pro- antibody gression and poor prognosis. We developed a fully human monoclonal Jin-hwan Han, PhD, Mingmei Cai, Jeffery Grein, Samanthi Perera, PhD, antibody MG1124, targeting human CEACAM1. Hongmei Wang, Mike Bigler, Roenna Ueda, Thomas Rosahl, Elaine Methods Pinheiro, PhD, Drake LaFace, Wolfgang Seghezzi, Sybil Williams, PhD T cell activation of MG1124 was determined by an NFAT-luciferase Merck Research Laboratories, South San Francisco, CA, United States reporter assay with CEACAM1 overexpressing Jurkat stable cells. Correspondence: Sybil Williams (sybil_williams@merck.com) Evaluation of the homophilic interaction of CEACAM1 or interaction Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P254 of CEACAM1 with CEACAM6 was performed by protein ELISA. In vitro efficacy of MG1124 was examined using an NK-mediated tumor cell Background killing assay. The anti-tumor efficacy of MG1124 alone or in combin- The molecule “T cell immunoreceptor with immunoglobulin and ITIM ation was studied in vivo in a humanized mouse model engrafted domain”, or TIGIT, has recently received much attention as a promis- with NSCLC patient-derived tumor xenografts. ing target in the treatment of various malignancies. In spite of the Results quick progression of anti-TIGIT antibodies into clinical testing both as Anti-CEACAM1 antibody MG1124 bound to CEACAM1 but not to other monotherapy and in combination with programmed death-1 (PD-1)– CEA family members. MG1124 blocked CEACAM1-CEACAM1 homophi- directed immune checkpoint blockade, the molecular mechanism be- lic interaction and CEACAM1-CEACAM6 heteropilic interation by bind- hind the observed therapeutic benefits remains poorly understood. ing to the N domain of CEACAM1. Especially CEACAM1-CEACAM1 Methods homophilic interaction induced downregulation of ZAP70 phosphoryl- Anti-Mouse TIGIT (mTIGIT) blocking antibodies of two distinct isotypes ation in response to TCR stimulation in a CEACAM1 overexpressing Jur- (mouse IgG1 with D265A mutation and mouse IgG2a) and TIGIT- kat stable cell line, which was rescued by MG1124 resulting in deficient mice were generated and used to demonstrate the require- augmentation of NFAT activity and IL-2 expression. NK cell-mediated ment of IgG-Fc gamma receptor interaction for effective anti-tumor re- tumor lysis was increased by MG1124 in a CEACAM1 expression- sponse in vivo studies. Gene expression profiling of whole tumors after dependent manner. In an NSCLC PDX-huNSG mouse model, MG1124 in vivo treatment of anti-mTIGIT antibody with functional or non- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 137 of 272 suppressed tumor progression as a monotherapy and combination Conclusions with pembrolizumab in a CEACAM1 high expressing model. In single Our data validate the therapeutic potential of providing IL-7 signals to mouse trial analysis, MG1124 suppressed tumor progression more than overcome PD-1 resistance. The bifunctional anti-PD1/IL-7 favors the T- 30% as monotherapy (53%, 10/19) as well as in combination (73%, 16/ cell effector over T-regulatory immune balance by stimulating effector 22) with pembrolizumab (5 mpk, 2qW). Moreover, PDXs of adenocarcin- and exhausted T-cells while disarming Tregs suppressive functions. oma origin with more than 50% of CEACAM1 expression were more ef- ficiently prohibited for progression with MG1124, suggesting the P257 potential therapeutic use of MG1124 in patients with NSCLC. Obesity is associated with diminished anti-PD-1-based Conclusions immunotherapy response rates in renal cancer MG1124, an anti-CEACAM1 antibody, blocked CEACAM1-mediated 1 1 1 Rachael Orlandella, BS , Shannon Boi , Justin Gibson, BS , William negative regulation and restored T/NK cell activities. MG1124 showed 1 2 2 1 Turbitt , Gal Wald , Lewis Thomas , Katlyn Norris , Lakshminarayanan effective anti-tumor activity in in vivo mouse models and its combin- 1 1 1 Nandagopal, MD , Peng Li, PhD , Eddy Yang, MD, PhD , Tatiana ation with PD-1 blockade further enhanced treatment efficacy. 1 1 Marquez-Lago, PhD , Lyse Norian, PhD MG1124 is a potential therapeutic candidate for immune checkpoint 1 2 University of Alabama, Birmingham, AL, United States; University of blockade in cancer therapy. Iowa, Iowa City, IA, United States Correspondence: Lyse Norian (lnorian@uab.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P257 P256 A novel bifunctional anti-PD-1 / IL-7 fusion protein potentiates Background effector function of exhausted T cell and disarms Treg suppressive Obesity is a regarded as a major risk factor for developing renal cell car- activity cinoma (RCC). Despite the success of anti-PD-1 checkpoint blockade in 1 1 1 Aurore Morello, PhD , Justine Durand , Caroline Mary , Virginie RCC, response rates remain low (20-30%). Recent studies have observed 1 1 1 2 Thepenier , Margaux Seite , Géraldine Teppaz , Nicolas Poirier that obesity is associated with heightened frequencies of PD-1+ CD8 T 1 2 OSE immunotherapeutics, Nantes, France; Poirier Household, Nantes, France cells [1] and favorable outcomes and responses to immunotherapy in Correspondence: Nicolas Poirier (nicolas.poirier@ose-immuno.com) melanoma [1, 2]. However, the effects of obesity on anti-tumor immun- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P256 ity and immunotherapeutic efficacy in renal cancer remain unknown. Methods Background PD-1 expression on tumor-infiltrating CD8 T cells from treatment-naive Despite the clinical success of anti-PD(L)1 therapies, most patients re- RCC subjects with (BMI >30 kg/m2) or without (BMI < 30 kg/m2) obes- main unresponsive or fail to develop a durable response. We explored ity (n = 18) was determined via flow cytometry. In a separate retro- a second generation of PD-1 antibody by fusing IL-7 cytokine to the Fc spective study, outcome data were queried for RCC patients with (BMI portion. IL-7 is an optimal target for immunotherapy to preferentially >30 kg/m2) or without (BMI < 30 kg/m2) obesity that were treated with stimulate effector T-cell (Teff) functions over regulatory T-cells (Treg), anti-PD-1 as standard of care and had at least 6 months of follow-up due to the differential expression of IL-7R. Moreover, It has been pub- (n=58). Overall survival (OS) was analyzed using Kaplan-Meier methods lished that PD-1 blockades increase IL-7R expression and improve IL-7 and Cox proportional hazards regression after controlling for patients’ signaling in exhausted T-cells rationalizing our combinatorial approach. age, sex, and number of prior treatments. For murine studies, BALB/c Methods mice were randomized to and maintained on either standard chow or Proliferation (H3 thymidine), IFN-γ, IL-7R signaling (pSTAT5) and NFAT (PD- high-fat diet for 20 weeks to generate age-matched lean or diet- 1 bioassay, Promega) assays were tested to determine anti PD-1/IL-7 effi- induced obese (DIO) mice. Mice were then given an orthotopic renal cacy on naïve and/or exhausted-like T-cells. For the suppressive assay, CD4 tumor challenge with syngeneic Renca cells and treated with an anti- Treg and autologous CD8 Teff were co-cultured (1:1) and proliferation was PD-1-based combination immunotherapy or saline. assessed on Day 5. Tumor infiltrating T cells (TILs) were isolated from orthotopic tumor-bearing mice (Hepa1.6, LLC-1,AK7) and subjected to IL-7 Results ex vivo, IL-7R signaling pSTAT5 was determined by flow cytometry. Obesity was associated with reduced frequencies of intratumoral PD- Results 1highCD8+ T cells in treatment-naive murine and human renal tumors. Our anti PD-1/IL-7 bispecific antibody efficiently blocks the PD-1/PD- Although the majority (73%) of lean mice responded to immunotherapy, L1 and PD-L2 interactions and the PD-1-mediated inhibitory signal DIO mice exhibited a reduced response rate (44%). Lean and DIO re- (pSHP1). Importantly, we observed that the IL-7 portion synergizes sponders exhibited favorable ratios of activated CD8+ T cells to myeloid- with the anti-PD-1 to enhance TCR mediated signaling (NFAT). Al- derived suppressor cells (MDSC), reduced PD-1 expression on CD8+ T though IL-7R expression on T cells decrease over repeated antigen cells, and elevated concentrations of CCL5 in renal tumors. Neutralization stimulation, we demonstrated that IL-7 still efficiently activate par- of CCL5 in lean immunotherapy-treated mice yielded a reduced response tially and fully-exhausted human T-cells (pSTAT5) and maintain their rate (43%), unfavorable ratios of activated CD8+ T cells to MDSCs, and di- proliferation capacity. We next characterized sensitivity of TILs to IL-7 minished IFNg secretion from intratumoral CD8+ T cells. The translational in multiple orthotopic mouse models. In PD-1 sensitive tumor (Meso- relevance of our murine findings was reflected in metastatic RCC patients, thelioma), only 10% of TILs express IL-7R whereas in PD-1 resistant as patients with obesity had a trending reduction in OS following stand- model (Hepatocarcinoma and Lung carcinoma), 40-60% of TILs (CD4 ard of care nivolumab (p= 0.06) and a 10.2 month reduction in OS. and CD8) express IL-7R and respond to IL-7 stimulation ex vivo as Conclusions measured by pSTAT5 signaling. These data suggest that the anti PD- Our data suggest that obesity is associated with reduced responses to 1/IL-7 bispecific can reactivate TILs that are resistant to PD-1 therapy. anti-PD-1 based immunotherapies in the context of renal cancer. Contin- Knowing that Tregs have a key suppressive function, we also ex- ued study of this critical issue is needed to better inform patient care. plored the possibility that the anti-PD-1/IL-7 fusion protein affect Treg functions. In a human Treg/Teff coculture assay, we observed Acknowledgements that the anti PD-1/IL-7 molecule abrogate the Treg capacity to inhibit Financial support was provided by NIH grant R01CA181088 to LAN; proliferation and IFN-gamma secretion of CD8+ Teff. Moreover, IL-7 CPCTP T32 fellowship #T32CA047888 to RMO; CPCTP R25 fellowship and the anti-PD-1/IL-7 does not stimulate Treg proliferation, in con- #R25CA047888 to SKB; CMDB T32 fellowship #T32GM008111 to JTG; and trast to IL-2 and IL-15 cytokines. Oncology T32 fellowship #T32CA183926 to WJT. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 138 of 272 References 1. McQuade, J.L., et al., Association of body-mass index and outcomes in patients with metastatic melanoma treated with targeted therapy, immunotherapy, or chemotherapy: a retrospective, multicohort ana- lysis. Lancet Oncol, 2018. 19(3): p. 310-322. 2. Wang, Z., et al., Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): p. 141-151. Ethics Approval Human subject studies were approved by the UAB IRB (protocol # X151013003); murine studies were approved by UAB IACUC (protocol #20233). P258 Combination of NK Cells and anti-PD-L1 Ab with ADCC Fig. 1 (abstract P258). See text for description enhances the anti-tumor effects in PD-L1 high cancer cells 1 2 1 Ji-Eun Park, BS , Bhumsuk Keam, MD, PhD , Ha-ram Park , Soyeon Kim, 3 2 2 2 PhD , Chan-Young Ock, MD, PhD , Miso Kim , Tae Min Kim, MD, PhD , P259 2 2 Dong-Wan Kim, MD PhD , Dae Seog Heo Hydrogel-enabled intratumoral co-delivery of anti-PD-1 antibody Seoul National University Cancer Research Institute, Seoul, Korea, and adenosine deaminase in a mouse model of renal cell Republic of; Seoul National University Hospital, Seoul, Korea, Republic carcinoma of; Biomedical Research Institute, SNUH, Seoul, Korea, Republic of Ketki Velankar, MS, Ngoc Pham, BS, Wilson Meng, PhD, Ellen Gawalt, Correspondence: Bhumsuk Keam (bhumsuk@snu.ac.kr) Nathan Schueller Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P258 Duquesne University, Pittsburgh, PA, United States Correspondence: Wilson Meng (meng@duq.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P259 Although Programmed cell death-1 (PD-1)/ Programmed death-ligand 1 (PD-L1) inhibitors showed remarkable antitumor activity, a large por- Background tion of cancer patients do not response to PD-1/PD-L1 inhibitors even It is hypothesized that tumor resistance to anti-PD-1 monoclo- in the PD-L1 high tumor. Most of PD-L1 inhibitors were modified in FcR nal antibodies is due in part to the accumulation of adenosine binding site to prevent antibody-dependent cellular cytotoxicity (ADCC) (ADO) generated in the tumor microenvironment (TME). ADO against PD-L1 expressing non-tumor cells. IMC-001, developed by impairs the activation and proliferation of effector T cells while ImmuneOncia, is a fully human PD-L1 recombinant monoclonal anti- expanding regulatory T cell (Treg) population, which is in- body that did not modify FcR binding and preserved ADCC. Therefore, versely related to the overall survival of cancer patients, includ- IMC-001 would be synergistic with NK compared to other PD-L1 mono- ing those with renal cell carcinoma (RCC). We propose to clonal antibodies (mAbs). We evaluate anti-tumor efficacy of IMC-001 develop an injectable system by which ADO are degraded in and NK cells against several PD-L1 high cancer cell lines through ADCC. the TME in order to enhance the efficacy of anti-PD1 treat- Methods ment. To this end, we have developed a hydrogel to co-deliver PD-L1 expression was measured by flow cytometry. Standard 51Cr-release anti-PD-1 antibody with adenosine deaminase (ADA), which ca- and CD107a degranulation assays were performed to evaluate the in vitro tabolizes ADO. The hydrogel contains a bioaffinity module ADCC efficacy of 3 groups: control, anti-PD-L1 Ab without ADCC (atezolizu- (named “Z15_EAK”) to retain the anti-PD-1 antibody in tumors mab), anti-PD-L1 Abs with ADCC (IMC-001, Anti-hPD-L1-hIgG1 [atezolizu- while limit the diffusion of ADA in TME for extended durations. mab with wild type FcR binding site, hPD-L1mab]). Various cancer cell We have previously shown that Z15_EAK hydrogel can retain lines were used as target cells, including head and neck squamous carcin- IgG at subcutaneous injection site for at least two weeks [1]. oma (HNSCC), lung cancer, stomach cancer, ovarian cancer, bladder cancer The expectation is that persistent co-localization of anti-PD-1 and lymphoma cell lines. 51Cr-release assay was performed using NK-92- and ADA in the TME will expand Th1 T cells and reduce Treg CD16 as an effector cell with effector to target ratio (E:T) of 30:1. CD107a in draining lymph nodes (DLN) and systemic lymphoid tissues. degranulation assay was performed using peripheral blood mononuclear This postulation was tested in an immunocompetent mouse cells (PBMC) from healthy donors with E:T ratio of 1:1. PBMC was activated model of RCC. by IL-15 and grouped by CD16 V158F genotyping individually. Methods Results A mouse RCC cell line (RENCA) was cultivated for in vitro assays and The expression of PD-L1 is high in several cell lines including SNU- in vivo inoculation into BALB/c mice. Beginning three days after 1076 (8.8±1.3), FaDu (15±0.1), HN31 (21.8±1.1) and H1975 (11.3±1.7). tumor inoculation, the hydrogel loaded with an anti-PD-1 IgG anti- NK cell cytotoxicity in PD-L1 high cell lines was more potent in IMC- body and ADA was injected subcutaneously in the peri-tumoral re- 001 or anti-hPD-L1-hIgG1 compared to control treatment or atezoli- gion for three doses three days apart. DLN, spleen, and tumors were zumab. The PD-L1 high or PD-L1 low tumor cell specific lysis was de- collected for flow cytometric analysis and ELISA measurements. tected by 51Cr-release assay in control group (isotype and Results atezolizumab) vs. and anti-PD-L1Ab with ADCC groups (IMC-001 vs. After three doses, DLN in mice received the hydrogel loaded with Anti-hPD-L1-hIgG1) (Figure 1). Besides, in CD107a degranulation anti-PD-1 antibody and ADA were five times larger than those in assay, activated PBMC cytotoxicity was increased when target cells mice received saline control (Figure 1). In addition, the lymph nodes are opsonized by anti-PD-L1 Abs with ADCC. NK cells that character- in treated mice contained fewer CD4+CD25+FoxP3+ Treg cells com- ized with CD16 high affinity genotype (V/F) are more enhancing pared to controls (Figure 2). After ex vivo re-stimulation with RENCA ADCC than (F/F) low affinity genotype. Two genotyped (V/F vs. FF) for expansion, lymphocytes in treated mice exhibited higher NK cell lysis induced by IMC-001 in FaDu cells was 39.3% vs. 12.8%. interferon-gamma levels than controls, indicating elevated Th1 Conclusions phenotype in the DLN. Anti-PD-L1 Abs with ADCC, such as IMC-001, enhanced the cytotoxic Conclusions activity of NK cells on various PD-L1 high cell lines. This study provides The preliminary data indicate that the local delivery of anti-PD- rationale that NK92-CD16 or NK cell immunotherapy for PD-L1 high 1 and ADA with the hydrogel shifted the local T cell popula- tumor through combination with ADCC preserved anti-PD-L1 Ab. tion toward an effector phenotype (Th1) while limiting the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 139 of 272 Treg expansion. An extended co-localization of anti-PD-1 and P260 ADA in the TME not only modulates immune events in the Characterization of AB154, a humanized, non-depleting α-TIGIT local lymphoid tissues but can also enhance the anti-tumor re- antibody undergoing clinical evaluation in subjects with advanced sponse systemically. Furthermore, the localized delivery reduces solid tumors off-target toxicities of anti-PD-1 antibody. Alejandra Lopez, BSc, Joanne Tan, PhD, Amy Anderson, PhD, Akshata Udyavar, PhD, Nell Narasappa, MSC, Susan Lee, PhD, Daniel DiRenzo, Reference PhD, Kristen Zhang, BS, Hema Singh, Sharon Zhao, Kimberline Gerrick, 1. Pham, N.B., et al. Toward reducing biomaterial antigenic potential: a Adam Park, Lisa Seitz, MA, Nigel Walker, PhD, Matthew Walters, PhD miniaturized Fc-binding domain for local deposition of antibodies. Biomaterials Arcus Biosciences, Inc., Hayward, CA, United States Science. 2019: 7(3): 760-772. Correspondence: Joanne Tan (jtan@arcusbio.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P260 The animal study was approved by Duquesne University's Ethics Board, approval number 180604. Background TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). CD226 is an activating receptor found on NK cells, monocytes and a subset of T cells. TIGIT and CD226 are paired receptors that compete for shared ligands CD155 and CD112, which are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression, whereas binding of the same ligand to CD226 promotes immune activation. AB154, designed to lack FcɣR binding, blocks human TIGIT with minimal risk of depleting intra-tumoral antigen-experienced CD8+ T cells. Methods Translational studies quantifying TIGIT, CD226 and CD155 expression in vari- ous tumor types and normal tissues were performed using flow cytometry, immunohistochemistry (IHC) and by mining publicly available RNASeq data- sets. TIGIT occupancy (RO) and Ki-67 levels from Ph1 dose escalation cohorts were quantified by flow cytometry. Downstream transcriptional effects of TIGIT/CD155 interaction in CD8+ T cells and Treg were assessed using Nano- string®. AB154, AB154 modified to restore wild-type (wt) IgG1 effector func- tion or to display enhance FcɣR binding via Fc mutations, were used in functional assays and antibody-dependent cell cytotoxicity (ADCC) studies. Results AB154, regardless of IgG1 variant, effectively abrogated the TIGIT- mediated inhibitory effects on activated T cells. In contrast, only non- depleting AB154 lacked ADCC activity in mixed cultures containing NK cells and activated T cells. Data assembled from TCGA, confirmed by flow cytometry as well as IHC, identified multiple tumor types bearing high TIGIT and CD155 expression. In particular, antigen-experienced T cells isolated from late stage head and neck squamous cell carcinoma tumors express higher levels of TIGIT and PD-1 than of CD226. Levels of Fig. 1 (abstract P259). See text for description TIGIT expression in this subset was equivalent, if not higher, than intra- tumoral Treg. Preliminary results from our Phase-1 dose escalation study demonstrated near complete target engagement by AB154 in T cells, NK cells, and NKT cells, coupled with concomitant increases in Ki- 67 expression within the aforementioned subsets. Conclusions Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented here provide: 1) rationale for clinical development of a non-depleting a- TIGIT blocking antibody (AB154), 2) evidence of AB154-related immune activation in subjects with advanced solid tumors, 3) evidence supporting AB154 as a rational combination partner with a-PD-1 (AB122). Trial Registration NCT03628677 P261 Recruitment of CD103+ DCs via tumor stroma-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy John-Michael Williford, PhD, Jun Ishihara, PhD, Ako Ishihara, Aslan Mansurov, BChen, Tiffany Marchell, Melody Swartz, PhD, Jeffrey Hubbell University of Chicago, Chicago, IL, United States Correspondence: Jeffrey Hubbell (jhubbell@uchicago.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P261 Background Checkpoint inhibitor antibody (CPI) therapy has demonstrated signifi- cant clinical benefit in a number of tumor types. Unfortunately, certain Fig. 2 (abstract P259). See text for description tumor characteristics, such as the lack of immune cell infiltration, often Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 140 of 272 correlate with poor responses to CPI therapy. Studies have identified C- P262 C Motif Chemokine Ligand 4 (CCL4) as a key molecule necessary for Schweinfurthins Cause Rapid Induction of Ecto-calreticulin Expression the recruitment of cross-presenting, CD103+ dendritic cells (DCs) to the Raymond Hohl, MD, Jeffrey Neighbors, PhD, Ruoheng Zhang, MBBS tumor; tumors lacking CCL4 expression exhibit a “cold tumor” pheno- Penn State College of Medicine, Hershey, PA, United States type and respond poorly to immunotherapy [1]. Based on these results, Correspondence: Raymond Hohl (rhohl@pennstatehealth.psu.edu) we hypothesized that tumor-targeted CCL4 could enhance immune cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P262 infiltration into the tumor and synergize with CPI therapy. Methods Background We generated a fusion protein comprised of CCL4 and a collagen bind- Our previous study demonstrated that schweinfurthin analogs im- ing domain (CBD) derived from von Willebrand factor, a tumor-stroma prove anti-PD-1 immunotherapy in a murine melanoma model by in- targeting strategy developed in our lab [2]. Anti-tumor efficacy studies ducing sustainable in vivo anti-tumor immunity.[1] We speculate that were performed in mouse syngeneic models, including B16F10 melan- the induction of immunogenic cell death (ICD) contributes to these oma, EMT6 breast cancer, and PyMT breast cancer. Flow cytometry was effects. The release of immunogenic danger-associated molecular employed to evaluate the tumor immune infiltrate. patterns (DAMPs) from tumor cells during ICD directly activate anti- Results cancer immunity.[2] Cell surface exposure of calreticulin (ecto-CRT) is Utilizing exposure of collagen in leaky tumor vasculature due to its disor- the major determinative DAMP because it stimulates cancer cell dered structure, we observed that intravenous (i.v.) infusion of CBD-CCL4 phagocytosis by dendritic cells and further activates anti-cancer im- fusion proteins, but not native CCL4, can enhance infiltration of CD103+ munity.[3] Phosphorylation of eukaryotic translation inhibition factor DCs, CD8+ T cells, and natural killer cells and slow B16F10 tumor growth eIF2α, an indicator of ER stress, is highly correlated to CRT exposure when combined with CPI therapy consisting of anti-cytotoxic T- on the cell surface in some forms of ICD.[4] Previous studies of the lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand schweinfurthin family of compounds have shown that they increase 1 antibody (PD-L1) (Figure 1, A-B) Further analysis showed strong correla- the phosphorylation of eIF2α and have complex effects on lipid tions between the presence of CD103+ DCs and CD8+ T cells and tumor homeostasis.[5] We hypothesize that schweinfurthin analogs enhance regression. Similarly, in the EMT6 breast cancer model, tumor-targeted CRT exposure during induction of ICD via eIF2α phosphorylation to CCL4 in combination with CPI, but not native form CCL4, enhanced recruit- improve anti-cancer immunity. ment of CD103+ DCs, CD8+ T cells, and led to a reduction in tumor Methods growth. To confirm the importance of CD103+ DCs in mediating anti- B16.F10 murine metastatic melanoma cells were cultured with increas- tumor responses, we utilized Batf3 knockout mice bearing B16F10 tumors; ing concentrations of the schweinfurthin analog, TTI-3114 or vehicle for in this instance, anti-tumor efficacy of CPI + CBD-CCL4 was completely lost. 24 hours, followed by measurement of ecto-CRT expression by flow cy- Efficacy studies in PyMT breast cancer models highlighted the therapeutic tometry. Kinetics of ecto-CRT exposure was also assessed. To determine benefit of CBD-CCL4 delivery (Figure 1C); CPI therapy alone led to the importance of lipids in this process, studies were carried out with complete tumor remission in only 10% of mice, whereas combination normal or charcoal-stripped lipid-free media. In addition, the role of therapy of CPI + CBD-CCL4 cured 50% of the treated mice (Figure 1D). eIF2α phosphorylation in TTI-3114-induced CRT exposure was evalu- Conclusions ated using western blots for total and phosphorylated eIF2α with thap- These results highlight the utility of recruiting CD103+ DCs to the sigargin acting as a positive control for the induction of ER stress. tumor to improve the efficacy of CPI therapy. This engineered chemo- Results kine delivery strategy demonstrates significant translational potential by targeting the tumor stroma following systemic administration. TTI-3114 induces rapid surface calreticulin exposure on B16.F10 murine melanoma cells in a concentration-dependent manner, References starting at 30nM. 1. Spranger S, Gajewski T. Impact of oncogenic pathways on evasion of Lipid depletion sensitizes melanoma cells to TTI-3114-induced antitumor immune responses. Nat Rev Cancer. 2018; 18:139-147. calreticulin exposure. 2. Ishihara J et al. Targeted antibody and cytokine cancer immunotherapies TTI-3114 causes eIF2α phosphorylation before CRT exposure in through collagen affinity. Sci Transl Med. 2019; 11:eaau3259. melanoma cells, indicating potential ER stress response. Conclusions Based on the above results, we hypothesize that schweinfurthins in- duce CRT exposure on the cell surface likely by promoting a form of ER-stress, and this effect is augmented by lipid depletion. Future ex- periments will investigate the function of various lipids in schweinfurthin-induced CRT exposure and subsequent immunogenic cell death. We will also explore the exact mechanisms which are re- sponsible for the CRT cell surface exposure phenomena. References 1. Kokolus, K. M. et al. Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity. Oncoimmunology 8, 1–13 (2019). 2. Zhou, J. et al. Immunogenic cell death in cancer therapy: Present and emerging inducers. J. Cell. Mol. Med. 4854–4865 (2019). doi:10.1111/ jcmm.14356 3. Obeid, M. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61 (2007). 4. Bezu, L. et al. eIF2α phosphorylation: A hallmark of immunogenic cell death. Oncoimmunology 7, 1–3 (2018). 5. Kuder, C. H. et al. Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification. Mol. Fig. 1 (abstract P261). See text for description Pharmacol. 82, 9–16 (2012). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 141 of 272 P263 either alone or in combination with other immune checkpoint therap- Targeting EZH2 enhances antigen presentation, antitumor ies. We hypothesized that an antibody with a unique binding mode immunity and circumvents anti-PD-1 resistance in head and neck could activate T cells in an Fc effector-less format. Hence, we developed cancer 7A5, a CD137 agonist monoclonal antibody which potentially has a 1 2 1 Liye Zhou, PhD , Ravindra Uppaluri, MD, PhD , Tenny Mudianto, BS , ligand-like structural binding mode and demonstrated that it effectively 1 1 Xiaojing Ma, PhD , Rachel Riley, BS engages the CD137 receptor in preclinical studies. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Methods Women's Hospital/DFCI, Boston, MA, United States 7A5 was identified from a human Fab phage display library screen Correspondence: Ravindra Uppaluri and engineered to an IgG1 Fc effector null antibody. Solid phase (ravindra_uppaluri@dfci.harvard.edu) binding assays with recombinant CD137 protein and cell-based as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P263 says in CD137 expressing cells were used to evaluate binding and functional activity in vitro. To assess agonist activity, 7A5 was tested Background in NF-kB luciferase reporter, PBMC co-stimulation and Treg suppres- Anti-programmed death-1 (PD-1) receptor-based therapeutics im- sion assays. To determine antitumor activity in vivo, human tumor prove survival in recurrent head and neck squamous cell carcinoma xenograft mouse models (NSG mice harboring human NCI-H292 or (HNSCC) patients but many do not benefit due to a low response HCC827 NSCLC tumors) reconstituted with human PBMCs or T cells, rate. Multiple mechanisms of immunoevasion have been identified in were used. Treatments included 7A5 monotherapy and the combin- HNSCCs including in the antigen presentation machinery. Herein, we ation with anti-PD-L1 antibody in these models. identified enhancer of zeste homolog 2 (EZH2) as a therapeutic tar- Results get in HNSCCs that enhanced tumor cell antigen presentation and In this study, we characterized 7A5, a fully human IgG1 Fc effector subsequently sensitized resistant tumors to anti-PD-1 therapy. null monoclonal antibody. We showed 7A5 binds CD137 and the Methods binding epitope overlaps with the CD137 ligand binding site. 7A5 en- EZH2 regulation of antigen presentation was defined using EZH2 in- gages the CD137 receptor and activates signaling independent of hibitors (GSK126 and EPZ6438) in human and mouse HNSCC cell cross-linking or Fc effector function. It binds to activated primary T lines. Mechanistic dissection of EZH2 in regulation of antigen presen- cells and leads to T cell stimulation in cell-based assays. Monother- tation was investigated using flow cytometry, qRT-PCR, ELISA and apy with 7A5 inhibits tumor growth in humanized mouse models chromatin-immunoprecipitation assays. EZH2 deficient cell lines were and this activity is enhanced when combined with a PD-L1 antagon- generated using CRISPR-CAS9. GSK126 and anti-PD-1 blocking anti- ist antibody. Furthermore, changes to the intra-tumoral immune body were used in testing combinatorial therapy in vivo. gene expression signature in response to 7A5 is highly suggestive for Results a mechanism of enhanced T cell infiltration and activation. EZH2 expression was negatively correlated with antigen processing Conclusions machinery (APM) pathway components in HNSCC TCGA datasets. In summary, CD137 antibody 7A5 represents a differentiated agonist EZH2 inhibition resulted in significant upregulation of MHC class I ex- with preclinical biological properties that support its further develop- pression in both human and mouse HNSCC lines and increased anti- ment as an anti-cancer immunotherapy. gen presentation in mouse models. This increased antigen presentation on the tumor cell by EZH2 inhibitors or CRISPR medi- P265 ated EZH2 deficiency, increased antigen specific CD8+ T cell prolifer- Immune checkpoint inhibitors induce response in a dose- ation, IFNγ production and tumor cell cytotoxicity. Mechanistically, dependent manner while their immune related adverse events are EZH2 inhibition reduced the histone H2K27me3 modification on the dose-independent, a meta-analysis β-2-microglobulin promoter to regulate antigen presentation. Finally, 1 2 1 Osama Rahma, MD , Joshua Reuss, MD , Anita Giobbie-Hurder, MS , in an anti-PD-1 resistant model of HNSCC, combination EZH2 inhib- 3 4 5 Ghazaleh Razavi, MD , Pooja Mehra, MD , Seema Gupta , Rawad Elias, ition with anti-PD-1 suppressed tumor growth at least partially due 6 5 MD , Samir Khleif, MD to the upregulation of antigen presentation capacity of tumor cells. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; John Hopkins, Conclusions Baltimore, MD, United States; Georgia Cancer Center, Augusta, GA, Our results demonstrated that targeting EZH2 enhanced antigen presen- United States; University of Virginia, Charlottesville, VA, United States; tation and circumvented anti-PD-1 resistance. Thus, combining EZH2 tar- 5 6 Georgetown University, Washington, DC, United States; Hartford geting with anti-PD-1 may increase therapeutic susceptibility in HNSCC. Healthcare, Hartford, CT, United States Correspondence: Samir Khleif (snk48@georgetown.edu) P264 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P265 Characterization of a human CD137 (4-1BB) receptor binding monoclonal antibody with differential agonist properties that Background promotes antitumor immunity Despite the expansion of Immune Checkpoint Inhibitor (ICI) indi- Helen Kotanides, Rose Marie Sattler, Maria Lebron, Carmine Carpenito, cations, the relationship between ICI dose-escalation and toxicity PhD, Juqun Shen, Jingxing Li, David Surguladze, Jaafar Haidar, Colleen or response has not been established. To understand this correl- Burns, Leyi Shen, Ivan Inigo, BS, Anthony Pennello, Amelie Forest, MSc, ation, we performed a meta-analysis of all available clinical trials Xinlei Chen, Darin Chin, Andreas Sonyi, Michael Topper, Lauren Boucher, investigating ICIs. Prachi Sharma, Yiwei Zhang, Douglas Burtrum, Ruslan Novosiadly, Dale Methods Ludwig, Gregory Plowman, Michael Kalos We searched PubMed and abstracts presented at (inter)national Eli Lilly and Company, Indianapolis, IN, United States meetings for trials (T) using FDA-approved ICIs including ipilimumab, Correspondence: Helen Kotanides (helen.kotanides@lilly.com) atezolizumab, nivolumab, and pembrolizumab. The reported rates of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P264 treatment-related grade 3-5 adverse events (G3-5AEs), immune- related adverse events (irAEs), and response were collected. For each Background ICI, comparisons of incidence rates between doses or diseases were CD137 (4-1BB) is a member of the TNFR receptor superfamily that plays based on marginal, exact generalized linear models. a key role in mediating immune response through costimulatory sig- Results nals that promote T cell proliferation, survival and memory. CD137 A total of 74T (7469 patients (pts)) published between 1/2010 – 1/2017 agonism has the potential to reinvigorate potent antitumor immunity were included (15T-ipilimumab (1058 pts), 30T-nivolumab (2281 pts), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 142 of 272 29T-pembrolizumab (4130 pts)) (Figure 1). For ipilimumab, the overall incidence of G3-5AEs was 34%. A significant 27% reduced risk of G3- 5AEs was seen with 3 mg/kg compared to 10 mg/kg (p=0.002) (Figure 2). However, there was no relationship observed between dose of ipili- mumab and incidence of irAEs or response to therapy (Figure 3). With nivolumab, the overall incidence of G3-5AEs was 20.1%. Incidence of G3-5AEs was significantly lower in NSCLC, with risk reductions of 24- 38% when compared to RCC or melanoma (p≤0.05). No dose-toxicity relationship was seen for G3-5AEs or irAEs (Figure 4). In both melanoma (6T) and NSCLC (7T), a dose-response association was observed, with significantly decreased odds of response of 17% and 64% for 1mg/kg compared to 3mg/kg in melanoma and NSCLC, respectively (Figure 5,6) with no further increase in response for doses above 3 mg/kg. This as- sociation was not observed in RCC (Figure 7). For pembrolizumab, the overall incidence of G3-5 AEs was 13.3%. Risk of G3-5AEs was 17% lower in melanoma than in NSCLC (p=0.03). No dose-toxicity relation- ship was seen for G3-5AEs or irAEs (Figure 8). In melanoma (7T), 2mg/ kg every 3 weeks (q3w) had 22% decreased odds of response com- pared to 10mg/kg (q2w) (p=0.01) (Figure 9). For NSCLC (5T), no dose- response relationship was noted (Figure 10). Conclusions We found no correlation between dose of ipilimumab and odds of G3-5iAEs or response. For pembrolizumab and nivolumab, no dose-toxicity correlation was seen but a dose-response correlation was observed suggesting that, for the PD-1 inhibitors, efficacy ap- Fig. 2 (abstract P265). Bootstrap analysis for G3-4 AEs pears to be dose-dependent while toxicity does not. Accordingly, of ipilimumab future clinical trial design of ICIs should use a dose escalation method with a primary objective of identifying an effective dose rather than a maximum tolerated dose. Acknowledgements Merck and BMS for providing input Fig. 1 (abstract P265). Consort Diagram of Literature Search Fig. 3 (abstract P265). Bootstrap analysis for ORR for ipilimumab Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 143 of 272 Fig. 6 (abstract P265). Bootstrap analysis for ORR for nivolumab Fig. 4 (abstract P265). Bootstrap analysis for G3-4 AEs in NSCLC for nivolumab Fig. 5 (abstract P265). Bootstrap analysis for ORR for nivo Fig. 7 (abstract P265). Bootstrap analysis for ORR for nivolumab in melanoma in RCC Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 144 of 272 Fig. 10 (abstract P265). Bootstrap for ORR for pembrolizumab in NSCLC Fig. 8 (abstract P265). Bootstrap for incidence of G3-4 AE P266 in pembrolizumab Evaluation of immunomodulatory receptor/ligand expression on matched human biospecimens Shawn Fahl (shawn.fahl@dls.com) Discovery Life Sciences, Huntsville, AL, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P266 Background The integration of immunomodulatory receptor signaling is crucial for the activation status of responding T cells, and modulation of these re- ceptors, and their ligands, may be of therapeutic benefit. Indeed, recent breakthroughs in checkpoint inhibitor therapies, and in particular those that target the PDL1/PD1 interaction, have demonstrated success in nu- merous oncological indications. Understanding the expression of these receptors and their cognate ligands within the complex cellular archi- tecture of solid tumors will be fundamentally important to the design on the next-generation of immunotherapies. Methods Bulk RNASeq analysis of primary human tumor tissue revealed the expression of numerous co-stimulatory (LIGHT/HVEM, 41BB/41BBL, OX40/OX40L, GITR/GITRL) and co-inhibitory (Lag3, VISTA, PVR/PVRL2/ TIGIT, Tim3/Galectin-9) receptors and ligands within the tumor micro- environment. Using multiparametric flow cytometry, we have profiled the expression of these immunomodulatory receptors and their re- spective ligands on the major cellular components of the tumor microenvironment and correlated it with expression on cellular sub- sets within matched peripheral blood. P267 Phenotyping of TIGIT pathway members may be used for cancer selection in the clinical application of anti-TIGIT antibody EOS884448 Noemie Wald, PhD, Julia Cuende, PhD, Marjorie Mercier, Florence Nyawouame, MSc, Margreet Brouwer, MSc, Erica Houthuys, PhD, Gregory Driessens, PhD, Veronique Bodo, PhD, Catherine Hoofd iTeos Therapeutics, Gosselies, Belgium Correspondence: Gregory Driessens Fig. 9 (abstract P265). Bootstrap for ORR for pembrolizumab (gregory.driessens@iteostherapeutics.com) in melanoma Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P267 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 145 of 272 Background The complement system consists of a network of more than 50 dif- TIGIT is a T cell co-inhibitory receptor that drives tumor cell mediated ferent plasma and membrane associated proteins. It is a part of the immunosuppression. Predominantly expressed on CD4+ Tregs, CD8+ T innate immune system and plays a key role in host defense against and NK cells in healthy individuals, TIGIT is further upregulated in these pathogens as well as in tissue homeostasis. The anaphylatoxin C5a is cells in cancer patients. In patients, it is frequently co-expressed with formed upon cleavage of C5 during the process of complement acti- exhaustion markers such as PD-1. DNAM-1/CD226, a co-stimulatory re- vation. C5a is the most potent chemoattractant and induces recruit- ceptor, is expressed on NK and T cells and competes with TIGIT for ment and activation of different immune cells to inflamed tissue, PVR/CD155 binding, but with a lower affinity. Since cancer cells express among which are neutrophils, eosinophils, monocytes, basophils, and high level of CD155 and because TIGIT expression is increased on TILs, mast cells. C5a binds to the seven transmembrane-spanning recep- the TIGIT pathway represents a major mechanism for immunosuppres- tors C5aR1 (CD88) and C5aR2 (C5L2). Blocking C5aR1 thus appears as sion within the tumor. We developed EOS884448, an antagonist anti- a potent mean to control the myeloid suppressive cells in the TME. TIGIT antibody, to prevent TIGIT-mediated immunosuppression in can- In this context we developed IPH5401, a fully human blocking anti- cer patients. C5aR1 monoclonal antibody that prevents binding to C5a. Methods Results To support selection of indications for clinical application of We first explore further the expression profile of C5aR1 in the TME EOS884448, we used flow cytometry and immunohistochemistry both at mRNA and protein levels in several solid cancer indications (IHC) to characterize peripheral and tumoral expression of TIGIT, showing various levels of infiltration by C5aR1 positive immune cells. CD155 and CD226 in healthy or cancer donors. Then, we demonstrated that IPH5401 can block activation and migra- Results tion of Human neutrophils and macrophages in vitro. TIGIT is expressed on multiple immune subsets in healthy donors. Simi- Conclusions lar analysis on matched PBMCs and TILs from 15 cancer donors Altogether, these results support our ongoing multi-center, open highlighted the overexpression of TIGIT on cells from those samples. label, dose-escalation and dose expansion Phase I/II clinical trial Interestingly, ex vivo polyfunctional analysis of cytokine production (STELLAR-001) evaluating the safety and efficacy of IPH5401 in com- demonstrated immunosuppression of TIGIT+ TILs versus their TIGIT- bination with durvalumab, an anti-PD-L1 immune checkpoint inhibi- counterparts. Among PBMCs and TILs assessed by flow cytometry, tor, as a treatment for patients with advanced solid tumors. tumor-infiltrating Tregs exhibit the highest TIGIT expression (frequency of positive cells and receptor density). This finding was confirmed by P269 IHC on tumor samples, supporting the potential value of an ADCC- IL-15 together with TIGIT blockade reverses PVR-mediated NK cell competent antibody targeting preferentially tumor-infiltrating Tregs. dysfunction in melanoma Finally, intrinsic expression of TIGIT on tumor cells was detected on 1 1 1 Joe-Marc Chauvin, PhD , Mignane Ka , Ornella Pagliano , Carmine several haematological malignancies, opening the potential for 1 1 1 2 Menna , Quanquan Ding , Cindy Sander , Jiajie Hou , Soldano Ferrone, EOS8844488 to directly kill tumor cells in addition to its activities to re- 1 1 1 MD, PhD , Diwakar Davar, MD , John Kirkwood, MD , Robert Johnston, invigorate immunity. The expression of the TIGIT ligands CD155 and 3 3 2 1 PhD , Alan Korman, PhD , Mark Smyth , Hassane Zarour, MD CD226 co-receptor were also analysed by IHC in tissues (n=284-307) 1 2 University of Pittsburgh, Pittsburgh, PA, United States; QIMR Berghofer from 9 cancer indications. CD155 is mostly expressed by tumor cells, Medical Research Institute, Queensland, Australia; Bristol-Myers Squibb, ranging from a median of 2% of CD155high tumor cells for cervix to Redwood City, CA, United States 50% for pancreatic cancer. CD155 expression is highest in pancreatic, Correspondence: Joe-Marc Chauvin (chauvinj@upmc.edu) prostate, kidney, gastric and colon cancers. CD226 is detected on im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P269 mune cells infiltrating tumors. The median percentage of tissue area positive for CD226 ranged from 0.07% in head and neck to 0.98% in Background gastric cancer, with gastric, lung and renal cancer showing the highest Natural Killer cells (NKs) play a critical role in tumor immunosurveil- CD226 expression. lance. Multiple activating and inhibitory receptors regulate NK cell- Conclusions mediated tumor cytotoxicity. The inhibitory receptor TIGIT and its Together, these findings strongly support the relevance of targeting counter-receptor CD226 exert opposite effects on NK cell function, TIGIT with an ADCC-competent antibody and provide a method to with TIGIT blockade reinvigorating NK cell-mediated tumor reactivity. select cancer types that may benefit from treatment with Whether and how the manipulation of the TIGIT/CD226/PVR axis may EOS884448. reactivate NK-cell mediated antitumor activity in melanoma patients Ethics Approval (MPs) has not yet been thoroughly evaluated. The study was approved by UCL‘s Ethics Board, approval number Bio- Methods bank2019/09MAI/005 Flow cytometry was used to evaluate the phenotype and function of NKs in the periphery (cNKs) and tumor sites (TiNKs) in MPs. CD226 P268 mRNA was evaluated by RT-PCR. CD226 and TIGIT internalization was IPH5401 anti-human C5aR antibody targets suppressive myeloid evaluated on isolated cNKs by Imagestream analysis. Melanoma lung cells in the TME metastasis tumor models in WT and TIGIT-/- mice were used to Joanna Fares, Léa Simon, Caroline Soulas, Marion Loivet, Elodie Bonnet, evaluate in vivo effects of TIGIT and CD226 blockades on NK- Luciana Batista, Romain Remark, PhD, Cécile Bonnafous, Robert Zerbib, mediated tumor control with or without IL-15 treatment. MSc, Mathieu Bléry Results Innate Pharma, Marseille, France In sharp contrast with CD8+ T cells, TiNKs downregulated both TIGIT Correspondence: Robert Zerbib (robert.zerbib@innate-pharma.fr) and CD226 expression as compared to cNKs. TiNKs exhibited de- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P268 creased expression of activation markers, lytic potential and melan- oma killing capacity as compared to cNKs in MPs. Membrane-bound Background PVR, but not soluble PVR, triggered CD226 internalization and deg- A hallmark mechanism of synergy in immunotherapy is the elimin- radation, leading to NK dysfunction. IL-15 stimulation increased ation of immunosuppressive cells, such as myeloid cells and neutro- CD226 and TIGIT expression levels and NK cell function, and TIGIT phils, to allow for the reactivation of effector cells. These blockade further increased TiNK proliferation and function against immunosuppressive cells are indeed associated with poor prognosis MHC class-I deficient tumors as compared to IL-15 or TIGIT blockade in many cancer types as well as resistance to checkpoint blockade. alone. TIGIT blockade promotes tumor antigen-specific CD8+ T cells From a therapy perspective, we aimed to specifically target the re- proliferation independently of NK cells. TIGIT blockade impeded me- cruitment of these major mediators of pro-tumoral inflammation into tastasis in mice in a CD226-dependent manner and only in presence the tumor microenvironment (TME). of IL-15. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 146 of 272 Conclusions P271 Membrane-bound PVR plays a critical role in the tumor microenviron- Anti-SIRPalpha antibodies exert anti-tumor activity in both CD47- ment by modulating TIGIT/CD226 expression and the function of dependent and CD47-independent manners TiNKs. IL-15 together with TIGIT blockade, counteracts PVR-mediated Hongtao Lu, PhD, Xiaofeng Niu, PhD, Qinglin Du, PhD, Jingfeng Yu, TiNK dysfunction in melanoma, and prevent metastasis occurrence in Roumei Xing, Yanfen Hu, Jinfeng Zhao, Fengli Wang, Zhihao Wu, PhD, mice in a CD226 dependent manner. Our findings support the devel- Yangsheng Qiu, Hongtao Lu, PhD opment of novel combinatorial immunotherapy with IL-15 and TIGIT Elpiscience Biopharma, Shanghai, China blockade to promote NK cell-mediated destruction of MHC class I- Correspondence: Hongtao Lu (hongtaolu@elpiscience.com) deficient melanoma, which are refractory to CD8+ T cell-mediated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P271 immunity and PD-1 blockade. Background Signal-regulatory protein alpha (SIRPα), is an inhibitory receptor P270 expressed on myeloid cells and dendritic cells. Ligation of CD47 Expression and clinical significance of the CD47/SIRPα pathway as to SIRPα delivers a “don’teat me” signal to suppress phagocyt- a candidate immunotherapy target in non-small cell lung cancer osis. Tumor cells frequently overexpress CD47 to evade (NSCLC) macrophage-mediated destruction. Currently, agents targeting 1 1 1 Shruti Desai, PhD , Franz Villarroel-Espindola , Patricia Gaule, PhD , Adam CD47 have proceeded to clinical trials and demonstrated promis- 1 2 2 Ducler , Marisa Peluso, MS , Benjamin Lee, MD PhD , Kurt Schalper, MD, ing anti-tumor results. However, these agents have been associ- 1 2 PhD , Pamela Holland, PhD ated with hemolytic anemia and thrombocytopenia. In addition, School of Medicine, Yale University, New Haven, CT, United States; universal expression of CD47 causes antigen sink, which leads to Surface Oncology, Cambridge, United States reduced efficacy. We therefore consider targeting SIRPα to Correspondence: Kurt Schalper (kurt.schalper@yale.edu) achieve an improved efficacy with a better safety profile. We Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P270 have developed 2 classes of anti-SIRPα antibodies: CD47-SIRPα interaction “blocker” and “non-blocker”. Both groups of antibody Background functionally stimulate phagocytosis of multiple cancer cell types Immunostimulatory therapies have revolutionized the treatment of by macrophages. NSCLC. Multiple studies show that activation of the CD47/SIRPα path- Methods way can mediate cancer immune evasion by blocking phagocytic ac- Using SIRPα extracellular domain (ECD), SIRPα overexpression tivity of macrophages. Although early stage clinical trials blocking stable cell line and plasmid encoding SIRPα as immunogens, this pathway are ongoing, the expression, tissue distribution and clin- anti-SIRPα antibodies were generated by traditional hybridoma ical significance of the CD47 axis in NSCLC remains unknown. technology. Pan-allele/SIRP family homologue binding proper- Methods ties, and species cross-reactivity profile were evaluated by ELISA Using control tissue samples/cell line transfectants, we validated anti- and FACS. In vitro function activity was determined by phago- bodies to reliably detect CD47 and SIRPα protein in FFPE tissue and cytosis assay. In vivo safety profile was assessed in hCD47/ standardized a multiplexed quantitative immunofluorescence panel for hSIRPα double knock-in mice. Lead clone was humanized via simultaneous measurement of DAPI, pan cytokeratin, CD8/CD47/SIRPα. CDR grafting and back mutation screening. Stress tests were We used this panel to interrogate two retrospective NSCLC Yale cohorts carried out to evaluate the developability of candidate represented in tissue microarrays (#1: n=297 and #2: n=175). Cohort #3, antibody. n=139 adenocarcinomas with mutation testing were also analyzed. We Results studied the levels of the targets, tissue distribution, association with A panel of anti-SIRPα antibodies that stimulate phagocytosis of clinicopathologic/molecular variables and survival. multiple cancer cells were developed. One lead clone (B4) was se- Results lected from the “Blocker” group based on ranking of in vitro bind- Predominant tumor CD47 expression (cytoplasmic/membranous) was ing/function properties. Subsequently, ES004-B4 was identified as recognized in 82 -88% of cases in the NSCLC cohorts. SIRPα protein the candidate antibody after humanization, affinity maturation, was detected in 94- 98% of cases located in the stroma. Elevated ex- and property characterization. It recognized pan-allele human pression of CD47/SIRPα was significantly associated with high CD8+ SIRPα with high affinity (KD hSIRPα V1/V2, 0.86nM/1.43nM). ES004- tumor infiltrating lymphocytes in the cohorts. The targets showed no B4 was shown to stimulate phagocytosis of multiple cancer cell consistent associations between major clinicopathologic variables. types. Another lead antibody (N4) was selected from the CD47/ Lung adenocarcinomas with KRAS mutation showed significantly SIRPα interaction “non-blocker” group. ES004-N4 also induced lower levels of CD47 than EGFR mutated or EGFR/KRAS WT tumors. strong phagocytosis of multiple cancer cell types by macrophages, Although individual CD47/SIRPα levels were not prominently associ- albeit it did not disrupt CD47/SIRPα interaction, suggesting an ated with outcome, their co-localization in stroma was positively as- unique mode of action. ES004-B4 and ES004-N4 didn’t trigger sociated with longer 5-year overall survival. hemagglutination and had no negative impact on T cell prolifera- Conclusions tion in vitro. No severe hemolytic anemia and thrombocytopenia CD47 and SIRPα are frequently expressed in NSCLCs with higher were observed in hCD47/hSIRPα double knock-in mice treated levels in CD8+/T-cell inflamed tumors, suggesting adaptive upregula- with 10mg/kg of each antibody, suggesting a low safety risk tion of this pathway associated with anti-tumor immune pressure. in vivo. CD47 levels are associated with major oncogenic signaling events in Conclusions lung adenocarcinomas. Localized measurement of stromal CD47/ In summary, we have developed 2 anti-SIRPα antibodies with “Best- SIRPα co-expression in intact tumor specimens could provide valu- in-Class” potential: 1) CD47/SIRPα interaction “Blocker” ES004-B4, 2) able information about the pathway activation. CD47/SIRPα interaction “Non-Blocker” ES004-N4. Both antibodies Ethics Approval greatly enhance macrophage-mediated tumor cell destruction, pos- All tissues were used after approval from the Yale Human Investiga- sibly through different mechanisms of action. We are currently ad- tion committee protocol #9505008219 which approved the patient vancing the development of ES004-B4 and ES004-N4 into clinical consent forms or waiver of consent. candidates. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 147 of 272 P272 P273 SRF231, a fully human high-affinity anti-CD47 antibody, exerts Blockade of glyco-immune checkpoint using EAGLE to potentiate potent preclinical antitumor activity through engagement of the anticancer immunity Fc receptor (FcR), CD32a Lizhi Cao, Jenny Che, Abhishek Das, PhD, Sujata Nerle, Wayne Gatlin, Marisa Peluso, MS, Kshama Doshi, PhD, Caroline Armet, BS, Li Zhang, Robert Leblanc, Zakir Siddiquee, Hui Xu, Karl Normington, PhD, MBA, Rachel O'Connor, BS, Matthew Rausch, PhD, Jonathan Hill, PhD, Weiguo Yao, James Broderick, Li Peng, PhD Benjamin Lee, MD PhD, Pamela Holland, PhD, Vito Palombella, PhD, Palleon Pharmaceuticals, Waltham, MA, United States Alison Paterson, PhD Correspondence: Li Peng (lpeng@palleonpharma.com) Surface Oncology, Inc., Cambridge, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P273 Correspondence: Marisa Peluso (mpeluso@surfaceoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P272 Background The glyco-immune checkpoint (Siglec/sialogylcan) axis has recently Background emerged as a new mechanism of cancer immune evasion. We have CD47 is a transmembrane protein that acts as a “Don’t Eat Me” signal previously described a bifunctional antibody-sialidase fusion platform to evade immune recognition. It is overexpressed in multiple cancer named EAGLE (Enzyme-Antibody Glyco-Ligand Editing) to inhibit this subtypes and is associated with poor prognosis. Several anti-CD47 axis by selectively removing the terminal sialic acids of sialoglycans molecules designed to antagonize the CD47 axis are being tested in on tumor cells. Using a bacterial sialidase for proof-of-concept stud- the clinic. Preclinical characteristics and antitumor mechanisms of ies, we have shown that EAGLE leads to enzyme-dependent robust the investigational agent SRF231, a fully human antibody targeting monotherapy efficacy with complete regressions and immune mem- CD47, are described. ory in syngeneic mouse tumor models. Methods Methods SRF231 monovalent affinity and binding properties were evaluated Since the bacterial sialidase poses immunogenicity concerns, we by Surface Plasmon Resonance (SPR) technology and in vitro agglu- engineered and optimized a human sialidase for EAGLE overcoming tination assays. Directly labeled SRF231 was used to profile tumor vs the low expression yield and poor stability of human sialidases. We normal cell binding. SRF231-mediated antitumor activity was confirmed the antitumor activity of human sialidase-containing assessed in tumor using macrophage coculture systems designed to EAGLE in vitro and in vivo using coculture of cancer cells and primary evaluate the impact of SRF231 on tumor cell phagocytosis, cell death, human immune cells and immunocompetent tumor models. Further- and cell depletion. Receptor occupancy (RO)/activity relationships more, we explored and identified predictive and correlative pharma- and dependency on FcR were also assessed. A xenograft tumor codynamic (PD) biomarkers to EAGLE treatment in preclinical tumor model was used to characterize the pharmacokinetic (PK)/pharmaco- models. dynamic (PD)/tumor exposure/efficacy relationship of SRF231 follow- Results ing single dose administration. The expression yield of human sialidase was improved by ~400 fold Results compared to the wild type through protein engineering, which en- SRF231 is a fully human, high-affinity anti-CD47 antibody with a slow ables the production of human sialidase-based EAGLE. We con- off-rate and binding mode that does not lead to agglutination of structed EAGLE-408, consisting of the engineered human sialidase patient-derived red blood cells or tumor cells. Increased binding to and anti-HER2 antibody trastuzumab, and confirmed its robust desia- several tumor vs normal cells is observed with SRF231. Analyses lylation efficiency using various HER2-expressing tumor cells in vitro. probing the relationship between FcR and SRF231 activity revealed EAGLE-408 enhanced macrophage-mediated phagocytosis of tumor that SRF231 leads to antitumor activity through both phagocytosis cells in vitro. EAGLE-408 also demonstrated enzyme-dependent and cell death mechanisms in a manner largely dependent on the monotherapy efficacy with complete regressions and immune mem- activating receptor FcγRIIa (CD32a), predominantly expressed by ory in syngeneic EMT6-HER2 mouse tumor models. Furthermore, in myeloid cells. Additionally, SRF231-mediated antitumor activity is the PD study, we observed EAGLE-408 treatment enhanced CD8+ T retained in longer-term assay conditions and in washout conditions. cell infiltration into tumors, increased CD8+ T cells in the draining Moreover, submaximal SRF231 RO is sufficient for maximal phagocyt- lymph nodes, and augmented NK cells and myeloid cells in osis induction in vitro. In a B-cell lymphoma xenograft model, a sin- circulation. gle dose of SRF231/mouse yields tumor stasis out to 21 days with Conclusions submaximal tumor exposure. Antitumor activity is associated with an In summary, EAGLE with engineered human sialidase offers a increase of host macrophage infiltration and cytokine induction sug- new immunomodulatory approach to overcome resistance to gestive of an innate immune response. current immunotherapies by effectively inhibiting Siglec/sialogly- Conclusions can axis in the tumor microenvironment. SRF231 is a high affinity, CD47-targeting antibody that delivers an ac- tivating signal to myeloid cells via CD32a and displays favorable pre- P274 clinical characteristics regarding its RO/tumor exposure/efficacy Antibody targeting of tumor associated macrophages in relationship. SRF231 is currently being evaluated in a Phase 1 clinical pancreatic cancer and melanoma remodel the tumor trial [NCT03512340] in advanced solid tumors and lymphomas. Un- microenvironment and revives immune targeting of tumor cells derstanding these PK/PD/tumor exposure/efficacy relationships, as Dhifaf Sarhan, PhD (dhifaf.sarhan@ki.se) well as the role of target vs FcR affinity, offers guidance on the devel- Karolinska Institute, Stockholm, Sweden opment of CD47 antagonists for patients with cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P274 Ethics Approval Mice were used in compliance with protocols approved by the IACUC Background of Mispro Biotech Services, Cambridge, MA (#2017-03-21SUR-1), Immunotherapy for cancer has revolutionized clinical practice and Charles River Accelerator and Development Lab, Cambridge, MA enabled cures for previously lethal cancers. However, the clinical re- (#CR-008), Charles River Laboratories, Worcester, MA (#I023), or MI sponses are variable and highly influenced by immune regulatory Bioresearch, Ann Arbor, MI (VUF #26). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 148 of 272 compartments in the tumor microenvironment (TME). This is espe- tumor types. Immunotherapeutic agents have a mechanism of action cially true for pancreatic cancer (PC) where clinical trials aiming to re- distinct from chemotherapy and are being used across a broad array cover T cell anti-tumor activity have been disappointing. Thus, in PC of tumor types. A standardized, universal scoring system for patho- and other cancers there is a clinical need for alternative treatments logic response that encompasses features characteristic for immuno- We have previously shown that antibodies targeting the scavenger therapy and spans tumor types is needed. receptor MARCO expressed on tumor-associated macrophages Methods (TAMs), reduces tumor growth and impair metastasis in murine can- Hematoxylin and eosin-stained slides from neoadjuvant surgical re- cer models. Here we investigated targeting of the scavenger receptor sections and on-treatment biopsies were assessed for features of MARCO on human TAMs in pancreatic ductal adenocarcinoma immune-related pathologic response (irPR). 258 specimens from pa- (PDAC) and hypothesized that targeting this receptor will remodel tients with 11 tumor types as part of ongoing clinical trials for anti- the suppressive environment and relive the anti-tumor responses to PD-1 were evaluated. An additional 98 specimens from patients re- increase the efficacy of immunotherapy. ceiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types. Methods Results To test our hypothesis, analysis of MARCO gene expression data Common irPR features (immune activation, cell death, tissue repair, from the Human Protein Atlas (HPA) project was performed in- regression bed) were present in all tumor types reviewed, including vestigating pancreatic tumors (n=176), as these consist of up to melanoma, non-small cell lung, head and neck squamous cell, Merkel 80% stroma, compared with healthy pancreatic tissues (n=171). In cell, and renal cell carcinoma, amongst others. Features were consist- vitro, cytokine differentiated macrophages alternatively cultured ent across primary tumors, lymph nodes, and distant metastases. with PC cell lines under hypoxia and normoxia conditions were Specimens from patients treated with anti-PD-(L)1 in combination co-cultured with cytotoxic cells to mimic their interaction in the with another agent also exhibited irPR features. TME. Later, macrophages were treated with anti-MARCO Abs and Conclusions their phenotype and function were examined prior and following irPR features are consistent across tumor types and treatment set- interaction with immune effector cells. Subsequently, anti-MARCO tings. Standardized, pan-tumor immune-mediated pathologic re- ab anti-tumor efficacy was tested in vitro and in vivo in PC and sponse criteria (irPRC) are defined and associated specimen-handling melanoma models. considerations are described. Future, prospective studies are merited Results to validate irPRC in larger datasets and to associate pathologic fea- We found a 30-fold increase in MARCO expression in pancreatic tu- tures with long-term patient outcomes. mors compared to healthy tissues. Also, a significant (p=0.03) correl- Ethics Approval ation between high expression and decreased survival was noted in The study was approved by the Johns Hopkins University Institu- pancreatic cancer patients. Furthermore, pancreatic cancer cell lines tional Review Board. induced MARCO expression on macrophages and dedifferentiated them towards myeloid-derived suppressor cells (MDSC). This ef- fect was amplified by hypoxic condition. Notably, MARCO+ MDSC P276 in contrast to control monocytes and macrophages suppressed T- Efficacy of PD-1/PDL-1 Immune Checkpoint Inhibitors in and Natural Killer (NK) cell anti-tumor activities, which was re- Elderly Patients with Non-Small-Cell Lung Cancer; A Subgroup versed by treatment with anti-human MARCO Abs. In addition, Meta-analysis of Randomized Controlled Trials targeting TAMs with anti-MARCO Abs, abolished their anti- 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD inflammatory phenotype in vitro and in vivo and normalized their 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of metabolism towards pro-inflammatory. Moreover, in B16 melan- Medicine at Mount Sinai, New York, NY, United States; King Edward oma tumor model the anti-MARCO Ab mediated an anti-tumor Medical University, Lahore, Pakistan; The University of Texas, effect that was dependent on NK cells and their TRAIL-mediated Houston, TX, United States killing mechanism. Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P276 Thus, our study reveals a novel interaction between TAMs and cyto- toxic cells as a result of TAM targeting with monoclonal Ab demon- Background strating a promising approach as immunotherapy that remodel the PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as immune TME. an efficacious drug class for the treatment of non-small-cell lung can- Ethics Approval cer (NSCLC). The efficacy of PD-1/PD-L1 ICPI therapy in the elderly The study was approved by Institutional Ethics Board, approval num- (patients age ≥65 and ≥75) has not been thoroughly investigated. ber Dnr 2013.977-31.1. The aim of this study was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy in elderly patients with NSCLC. Methods P275 A systematic review of the literature to identify randomized con- Pan-tumor pathologic scoring of response to PD-(L)1 blockade trolled trials (RCTs) which reported overall survival (OS) and progres- Julie Stein, Evan Lipson, MD, Tricia Cottrell, MD, PhD, Patrick Forde, sion free survival (PFS) of elderly patients with NSCLC who were MD, Robert Anders, MD, PhD, Ashley Cimino-Mathews, Elizabeth randomized to receive PD-1/PDL-1 ICPIs or docetaxel/investigator’s Thompson, MD PhD, Mohamad Allaf, MD, Mark Yarchoan, Josephine choice chemotherapy. The hazard ratios (HR) of OS and PFS in elderly Feliciano, MD, Elizabeth Jaffee, MD, Drew Pardoll, MD, PhD, Suzanne patients (along with their 95% confidence intervals; CI) were ex- Topalian, MD, Janis Taube, MD, MSC tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Johns Hopkins University School of Medicine, Baltimore, MD, United 1 versus chemotherapy stratified by patient age (≥65 and ≥75). A States random effects model was employed only when there was significant Correspondence: Janis Taube (jtaube1@jhmi.edu) heterogeneity among studies (>40%, as assessed by I-squared). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P275 Results Screening of 15,092 studies yielded four RCTs (two with patients age Background ≥75) which enrolled a total of 2,429 patients. No significant difference Pathologic response assessment of tumor specimens from patients in PFS with PD-1/PDL-1 treatment versus chemotherapy was found receiving systemic treatment provide an early indication of thera- among patients aged ≥65 (HR 0.8, 95% CI 0.53-1.06, I2 78.30%) or pa- peutic efficacy and predict long-term survival. Grading systems for tients aged ≥75 (HR 1.1, 95% CI 0.43-1.77,I2 0.00%). Patients aged ≥65 pathologic response were first developed for chemotherapy in select had an improved OS with PD-1/PD-L1 ICPI treatment versus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 149 of 272 chemotherapy (HR 0.67, 95% CI 0.51-0.84, I2 62.50%). Patients aged 1 versus chemotherapy stratified by the degree of PD-L1 expression. ≥75 did not show any significant difference in OS when treated with A random effects model was employed only when there was signifi- PD-1/PD-L1 versus chemotherapy (HR 1.02, 95% CI 0.35-1.69,I2 0.00%). cant heterogeneity among studies (>40%, as assessed by I-squared Results Conclusions Screening of 15,092 studies yielded four RCTs (two reporting only Patients aged ≥65 show an improved OS with PD-1/PD-L1 therapy. How- PD-L1 expression ≥50%) which enrolled a total of 2,429 patients. An ever, NSCLC patients ≥75 do not show a significant difference in OS or improved PFS with PD-1/PDL-1 treatment versus chemotherapy was PFS when treated with PD-1/PD-L1 ICPI versus chemotherapy (Figure 1). found among patients with PD-L1 expression ≤1% (HR 0.78, 95% CI This may be a consequence of aging and its impact on individuals’ cap- 0.57-0.98, I2 25.30%) ≥1% (HR 0.62, 95% CI 0.46-0.78, I2 0.00%), ≥5% ability to mount an anti-tumor response. Further studies assessing the ef- (HR 0.46, 95% CI 0.31-0.6, I2 0.00%), and ≥10% (HR 0.44, 95% CI ficacy of PD-1/PD-L1 ICPI use in patients ≥75 are warranted. 0.29-0.58, I2 0.00%). Similarly, an improved OS was observed with PD-1/PDL-1 treatment versus chemotherapy was found among patients with PD-L1 expression ≥1% (HR 0.7, 95% CI 0.52-0.87, I2 0.00%), ≥5% (HR 0.54, 95% CI 0.38-0.69, I2 0.00%), and ≥10% (HR 0.51, 95% CI 0.35-0.68, I2 0.00%), but not with PD-L1 expression of ≤1%, ≤5%, and ≤10% (figure 1). No significant difference in OS and PFS was observed with PD-1/PD-L1 treatment versus chemotherapy among patients with ≥50% PD-L1 expression. Of the two RCTs which reported ≥50% PD-L1 expression, one used Nivolumab (and reported a negative outcome in OS and PFS) whereas the other used Pembrolizu- mab (and reported a favorable OS and PFS). Conclusions NSCLC patients who express PD-L1 have an improved OS and PFS with PD-1/PD-L1 ICPI use versus chemotherapy. However, high PD-L1 expression (≥50%) may not translate into a better response to all PD- 1/PD-L1 ICPIs (Figure 1). Further studies are needed to assess the intra-class efficacy of different PD-1/PD-L1 ICPIs in NSCLC patients with a high PD-L1 expression. Fig. 1 (abstract P276). OS and PFS in Elderly Patients with NSCLC P277 Utility of PD-L1 Expression in Non-Small-Cell Lung Cancer Patients Treated with PD-1/PDL-1 Immune Checkpoint Inhibitors; A Subgroup Meta-analysis of Randomized Controlled Trials 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States; King Edward Medical University, Lahore, Pakistan; The University of Texas, Houston, TX, United States Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P277 Background Fig. 1 (abstract P277). OS and PFS Stratified by PD-L1 Expression PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an efficacious drug class for the treatment of non-small-cell lung can- cer (NSCLC) evident by findings of multiple randomized controlled P278 trials (RCTs). The utility of PD-L1 expression analysis in treatment PD-1/PDL-1 immune checkpoint inhibitors in smokers with non- planning and prognosis remains questionable. The aim of this study small-cell lung cancer; a subgroup meta-analysis of randomized was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemo- controlled trials therapy in patients with NSCLC stratified by PD-L1 expression 1 2 3 4 Faisal Ali , Bibek Pannu , Maryam Hussain , Arafat Farooqui , Taha Methods 1 1 5 Alrifai , Phyo Myint , Syed Jafri, MD A systematic review of the literature to identify RCTs which reported 1 2 Saint Joseph Hospital, Chicago, IL, United States; John H Stroger Jr overall survival (OS) and progression free survival (PFS) of patients Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount with NSCLC who were randomized to receive PD-1/PDL-1 ICPIs or do- Sinai, New York, NY, United States; King Edward Medical University, cetaxel/investigator’s choice chemotherapy and underwent PD-L1 ex- Lahore, Pakistan; The University of Texas, Houston, TX, United States pression analysis. The hazard ratios (HR) of various degrees of PD-L1 Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) expression (along with their 95% confidence intervals; CI) were ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P278 tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 150 of 272 Background P279 PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an effi- PDL-1 is highly expressed in ovarian germ cell tumor and cacious drug class for the treatment of non-small-cell lung cancer (NSCLC) associated with cancer stem cells population expressing CD44 evident by findings of multiple randomized controlled trials (RCTs). How- Salmah Alamri, Miral Mashhour, Kholoud Alwosaibai, PhD ever, the efficacy of PD-1/PDL-1 ICPIs in patients with NSCLC who are King Fahad Specialist Hospital, Dammam, Saudi Arabia current or former smokers remains to be debated. The aim of this study Correspondence: Kholoud Alwosaibai (kh20978@gmail.com) was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P279 in patients with NSCLC who were active or former smokers Methods Background A systematic review of the literature to identify RCTs which reported Immunotherapy using checkpoint inhibitors have proposed beneficial overall survival (OS) and progression free survival (PFS) among effects for some types of cancer such as lung cancer and melanoma. former or active smokers with NSCLC who were randomized to re- However, using PD-1/PDL-1 inhibitors to treat ovarian cancer was lim- ceive PD-1/PDL-1 ICPIs or docetaxel or investigator’s choice chemo- ited and further studies are needed to identify the patients that will therapy. The hazard ratios (HR) of these subgroups (along with their benefit from this treatment. In this study we have explored predictive 95% confidence intervals; CI) were extracted to compute a pooled biomarkers in ovarian cancer that might associate with checkpoint (HR) to report the efficacy of PD-1/PDL-1 versus chemotherapy in treatment outcome. For the first time, we have investigated the role of former or active smokers with NSCLC. A random effects model was PDL-1 expression in the tumor microenvironment cells that includes employed only when there was significant heterogeneity among immune cells and cancer stem cells in different types of ovarian cancer. studies (>40%, as assessed by I-squared) Methods Results 66 surgical samples of different types of ovarian cancer have been Screening of 15,092 studies yielded six RCTs which enrolled a total of collected from pathology department. IHC staining has been per- 3,584 patients. PFS did not differ significantly with PD-1/PDL-1 treat- formed using PD-L1 IHC 22C3 pharmDx to detect PDL-1, CD8 and ment or chemotherapy among non-smokers (HR 1.33, 95% 0.80-1.80; I2 CD4 to detect tumor infiltrating lymphocyte (TIL), and CD44, CD117 20.6%) and former or active smokers (HR 0.83, 95% CI 0.63-1.04; I2 and OCT3/4 to detect stem cell markers. 67.5%). Survival analysis revealed that former or active smokers tended Results to have an improved OS when treated with PD-1/PDL-1 inhibitors ver- We found that 47% of ovarian cancer patients express PDL-1. The expres- sus chemotherapy (HR 0.72, 95% CI 0.54-0.90; I2 70.5%), whereas OS sion of PDL-1 have been detected in different types of ovarian cancer in- did not differ significantly among non-smokers when treated with PD- cluding, serous carcinoma, germ cell tumor, endometrioid. The majority 1/PDL-1 inhibitors versus chemotherapy (HR 0.76, 95% CI 0.48-1.04; I2 (73%) of germ cell tumor tissues express PDL-1 whereas serous cancer 0.0%) and endomitrioid express PDL-1 in 46% and 50% of the cancer tissue, re- Conclusions spectively. However, PDL-1 protein was undetectable in some histological NSCLC patients who are former or active smokers have an improved type of ovarian cancer such as granulosa tumor and mucinous tumor. OS when treated with PD-1/PDL-1 ICPIs, though the PFS does not dif- Also we determined the expression levels of TIL in the ovarian cancer fer significantly (Figure 1). This may be a consequence of a higher tissue that either PDL-1 positive or negative. We found that 81% of mutation burden among smokers. Further research into the inter- ovarian cancer samples have TIL that express CD8 and 92% of these action between carcinogens, toxins and proinflammatory substances ovarian cancer samples are associated with PDL-1 expression.TIL that of smoking and ICPIs are warranted. express CD4 have been found in 66% of all ovarian cancer samples and 77% of these samples are associated with PDL-1 expression. Further, we have studied the association between PDL-1 expression and cancer stem cell markers such as CD44, CD117, OCT 3/4. We found that all PDL-1 positive samples are expressing CD44 and also, we found strong association between CD117 expression and PDL-1 expression. Conclusions Immunotherapy treatment using PDL-1 inhibitor could be considered for ovarian cancer patients that expressing PDL-1 particularly, germ cell ovarian cancer. In addition, PDL-1 expression is strongly associated with CD44. Inhibiting PDL-1 using immunotherapy might downregulate stem cell populations and decrease the chemotherapy resistance and recurrence that derived by stem cell residual in the cancer tissue. Ethics Approval The study was approved by the IRB at King Fahd Specialist Hospital- Dammam. approval number ONC0340 P280 Impact of tumor mutational burden on overall survival in patients with non-small cell lung cancer treated with immunotherapy 1 2 1 Mark Awad, MD PhD , Navin Mahadevan , Andrew Polio , Natalie 1 1 1 1 Vokes , Elizabeth Aguilar , Biagio Ricciuti, MD , Giuseppe Lamberti, MD , 1 1 1 Gonzalo Recondo, MD , Giulia Leonardi , Anika Adeni , Pasi Janne, MD 1 1 1 1 PhD , Eliezer Van Allen, MD , Adem Albayrak, MS , Renato Umeton , Lynette Sholl 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Women’s Hospital, Boston, MA, United States Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P280 Background In non-small cell lung cancer (NSCLC), tumor mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint in- hibitors, but the optimal TMB cutpoint associated with a survival benefit Fig. 1 (abstract P278). OS and PFS in Smokers and Non-Smokers remains unclear. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 151 of 272 Methods We collected clinicopathologic data from patients with NSCLC se- quenced by the OncoPanel NGS platform at the Dana-Farber Cancer Institute. The relationship between TMB and clinical outcomes after treatment with immune checkpoint inhibitors was investigated in the subset of patients treated with immunotherapy. Results Among 2690 patients identified, median TMB was significantly higher Fig. 3 (abstract P280). See text for description among current smokers compared to former (P<0.0001) and never smokers (P<0.0001) and among squamous tumors compared to smoking-related nonsquamous tumors (P=0.01) (Figure 1). Patients without oncogenic drivers and those harboring BRAF or KRAS mutation had the highest median TMB (10.6, 11.1 and 9.8 mutations/megabase [mut/Mb], respectively), while tumors with ROS1, MET, RET and ALK al- terations had the lowest median TMB (6.7, 6.1, 5.3, 5.3 mut/Mb, respect- ively) (Figure 2). Among patients treated with immunotherapy (N=489), a recursive partitioning algorithm identified an optimal TMB cut-off for PFS and OS of 18.5 mut/Mb, which represents the 88th percentile for TMB in this cohort. Baseline clinicopathologic characteristics were well- balanced between patients with a TMB of ≥18.5 and <18.5 mut/Mb (Table 1). Patients with a TMB of ≥18.5 mut/Mb had a significantly Table 1 (abstract P280). See text for description higher response rate (43.3% vs. 17.5%, P<0.0001), a longer median progression-free survival (8.2 vs. 2.7 months, HR:0.52 [95%CI:0.38-0.72], P<0.0001), and a longer median overall survival (20.7 vs. 10.2 months, HR:0.55 [95%CI:0.38-0.79], P=0.001) compared to those with a TMB < 18.5 mut/Mb (Figure 3). After adjusting for performance status, smoking history, and PD-L1 expression, a TMB of ≥18.5 mut/Mb was associated with a significantly longer PFS (HR:0.56 [95%CI: 0.41-0.78], P =0.001) and OS (HR: 0.57 [95%CI: 0.40-0.82], P =0.003) in multivariate analysis. Conclusions Tumor-only NGS identifies clinical and genomic correlates of high TMB in NSCLC. Patients with a TMB ≥88th percentile are most likely to experi- ence a survival benefit when treated with immune checkpoint inhibitors. Fig. 1 (abstract P280). See text for description Fig. 2 (abstract P280). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 152 of 272 P281 L1+ tumors, do not benefit. The magnitude of immune activation Durable response after immunotherapy discontinuation: a promoted by PD-1/PD-L1 axis blockade can be further enhanced multicenter real-life experience through concomitant T-cell co-stimulation such as that achieved 1 2 3 Maria Bassanelli, MD, PhD , Diana Giannarelli, MD , Marco Russano , through CD137 agonism; however, clinical applications of such an 2 4 5 Fabiana Letizia Cecere , Maria Rita Migliorino , Silvana Giacinti , Viola approach may be limited by toxicity associated with the systemic ef- 4 6 5 4 Barucca , Emilio Bria , Enzo Maria Ruggeri , Fabio Calabrò , Alain fects of CD137 agonists. Here we characterize PD-L1 and CD137 7 3 1 Gelibter , Daniele Santini , Anna Ceribelli tumor expression supporting the development of a PD-L1xCD137 1 2 San Camillo de Lellis Hospital, Rieti, Italy; Regina Elena National Cancer bispecific molecule that provides PD-1 axis blockade coupled with 3 4 Institute, Rome, Italy; University Campus Bio-Medico, Rome, Italy; San tumor-targeted CD137 co-stimulation. Camillo Forlanini Hospital, Rome, Italy; Belcolle Hospital, Viterbo, Italy; Methods 6 7 Fondazione Pol. Universitario A.Gemelli, Rome, Italy; Policlinico In situ hybridization (ISH) and multicolor flow cytometry was per- Umberto I, Rome, Italy formed to characterize PD-L1 and CD137 expression in tumor biop- Correspondence: Maria Bassanelli (maria.bassanelli@yahoo.it) sies. A PD-L1xCD137 bispecific molecule (PD-L1xCD137) was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P281 constructed based on PD-L1 blocking mAbs and CD137-engaging mAbs and was evaluated for binding to respective antigens. Its func- Background tional activity was evaluated in CD3 or SEB-driven T-cell activation Immune checkpoint inhibitors (ICIs) have significantly improved over- systems, MLR assays and tumor microenvironment models. Anti- all survival (OS) in several cancer types. Unlike chemotherapy, the tumor activity in vivo was evaluated in combination with tumor tar- optimum treatment duration with ICIs is not clearly established [1,2] geted anti-CD3 based bispecific DART® molecules. Methods Results We conducted an observational, retrospective analysis of 46 consecu- ISH revealed expression of PD-L1 in significant proportion of surgi- tive patients (pts) with advanced solid tumors who discontinued cally resected carcinomas; noteworthy, many such tumors contained immune-based therapies for any reason except progressive disease. CD137+ immune infiltrate adjacent to PD-L1+ cells. Moreover, The aim of this study was to assess the outcome and the antitumor ex vivo co-incubation of tumor and immune cells in the presence of activity of ICIs after treatment discontinuation. Treatment-free sur- CD3-based bispecifics or Fc-enhanced antibodies further induces PD- vival (TFS) was defined as the time from interruption of immunother- L1 and CD137 expression. PD-L1xCD137 binds and blocks PD-L1, re- apy for any reason except progressive disease to start of subsequent versing PD-1-mediated T-cell inhibition equipotently to the effect of anticancer therapy or best supportive care or death. Median OS, pro- approved PD-L1 benchmark mAbs; it also binds CD137, but absent gression free survival (PFS), TFS and the 95% confidence interval (CI) clustering supported by PD-L1+ cells, fails to induce CD137 signaling. were estimated with the Kaplan -Meier method In the presence of PD-L1-expressing cells, however, PD-L1xCD137 Results drives CD137 activation and immune cell co-stimulation. Robust T- 46 pts (median age 68 years [range 41-86]; male: 65.2%) with advanced cell activation and cytokine secretion was induced by PD-L1xCD137, cancer (n.39 non-small-cell lung cancer, n.15 renal cell carcinoma and with significantly greater activity than that observed with the com- n.2 melanoma) were treated with ICIs, as clinically indicated, at eight bination of PD-L1 blocking and CD137 agonistic mAbs. Notably, Italian institutions: 44 pts received programmed death 1 (PD-1) inhibi- when combined with tumor targeted immunotherapies, PD- tors (n.31 nivolumab, n.13 pembrolizumab) and 2 pts programmed L1xCD137 supports enhanced activation of effector cells and anti- death ligand 1 (PD-L1) (n.1 durvalumab, n.1 atezolizumab). A median of tumor activity. 8 cycles were administered [range 1 to 52]. 36 pts discontinued ICIs Conclusions due to toxicities (diarrhoea, pneumonitis, hepatotoxicity)and 10 pts for These data show that an investigational PD-L1xCD137 bispecific can reasons non immune-related. The median PFS from the beginning of switch on CD137 co-stimulation in a PD-L1-dependent fashion. While ICIs was 12.4 months (mo) [95% CI: 8.2-16.6] and the median OS was tumor adaptive resistance via PD-L1 induction promotes tumor im- 20.0 mo (95% CI: 11.8-28.2). Median PFS from ICIs completion was 5.0 mune escape, PD-L1xCD137 can exploit the checkpoint ligand up- mo [95% CI: 2.7-7.3] and median OS was 16.1 mo (95% CI: 5.4-26.8). Me- regulation by contributing a co-stimulatory signal in addition to dian TFS was 7.4 (95% CI: 5.8-8.9) mo checkpoint blockade. PD-L1xCD137 provides a potential therapeutic Conclusions approach to overcome limitations of existing PD-1/PD-L1-targeting This study shows the durable cancer-specific immune response in pts strategies either as monotherapy or in combination with comple- with advanced cancer even after stopping the ICIs for any reason ex- mentary immune based therapeutic modalities, such as CD3 based cept progressive disease bispecifics or Fc-enhanced mAbs. References P283 1. Kim PS, Ahmed R. Features of responding T cells in cancer and chronic Generation and characterization of a PD-1 resistant mouse tumor infection. Curr Opin Immunol 2010; 22: 223–230. model 2. Schadendorf D, Hodi FS, Robert C et al. Pooled analysis of long-term sur- Marie Bernardo, PhD, Tatiana Tolstykh, Yu-An Zhang, Dinesh Bangari, Hui vival data from phase II and phase III trials of ipilimumab in unresectable Cao, PhD, Joon Sang Lee, PhD, Natalia Malkova, Jack Pollard, PhD, or metastatic melanoma. J Clin Oncol 2015; 33: 1889–1894. Fangxian Sun, Dmitri Wiederschain, Timothy Wagenaar Sanofi Oncology Research, Cambridge, MA, United States P282 Correspondence: Timothy Wagenaar (timothy.wagenaar@sanofi.com) Tumor-targeted T-cell activation via an investigational PD-L1 x Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P283 CD137 bispecific molecule Alexey Berezhnoy, PhD, Ling Huang, Daorong Liu, Jennifer DiChiara, Background Jonathan Li, PhD, Douglas Smith, PhD, Jill Rillema, BS, Valentina Immune checkpoint blockade elicits durable anti-cancer responses Ciccarone, PhD, James Tamura, PhD, Ralph Alderson, PhD, Gundo in the clinic, however a large proportion of patients do not bene- Diedrich, Ezio Bonvini, PhD, Paul Moore, PhD fit from treatment. Several mechanisms of innate and acquired MacroGenics, Inc, Rockville, MD, United States resistance to checkpoint blockade have been defined and include Correspondence: Paul Moore (moorep@macrogenics.com) mutations of MHC I and IFNg signaling pathways. However, such Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P282 mutations occur in a low frequency of patients and additional mechanisms have yet to be defined. In an effort to better under- Background stand acquired resistance to checkpoint blockade, we generated Blockade of the PD-1/PD-L1 axis can improve outcome in a variety of a mouse tumor model exhibiting in vivo resistance to anti-PD-1 cancers; yet, many patients, including subsets of patients with PD- antibody treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 153 of 272 Methods CD8+ T cells in the blood of E2 supplemented huNOG mice. Tumor A PD-1 resistant mouse tumor model was generated by serially pas- immunohistochemistry showed low number of TILs and very low ex- saging MC38 tumors in mice treated with anti-PD-1. The resistant pression of programmed death-ligand 1 (PD-L1). No clear anti-tumor tumor line was characterized using a range of molecular techniques. effects were observed with pembrolizumab treatment compared to Results the isotype control group. MC38 tumors acquired resistance to PD-1 blockade following serial Conclusions in vivo passaging. Lack of sensitivity to PD-1 blockade could not be In conclusion, estrogen supplementation was needed to support attributed to dysregulation of PD-L1 or B2M expression, as both were orthotopic ER+ breast cancer growth. However, estrogen decreased expressed at similar levels in parental and resistant cells. Similarly, survival of female huNOG mice. Estrogen had immunomodulatory ef- IFNg signaling and antigen processing and presentation pathways fects and induced adverse effects including anemia. Due to these were functional in both parental and resistant cell lines. Unbiased deleterious effects and decreased number of immune cells, no direct gene expression analysis was used to further characterize potential conclusions can be drawn for the possible anti-tumor effects of pem- resistance mechanisms. RNA-sequencing revealed substantial differ- brolizumab in this orthotopic ER+ breast cancer model. Caution ences in global gene expression with PD-1 resistant tumors display- should be taken when evaluating efficacy of immunotherapies in ing a marked reduction in expression of immune-related genes hormone-dependent preclinical cancer models, and using ER+ breast relative to parental MC38 tumors. Transcriptomic data revealed that cancer models where tumor growth is supported by local microenvir- PD-1 resistant tumors exhibit reduced immune infiltration across onment, such as in bone metastasis models, should be considered. multiple cell types, including T and NK cells, while pathway analysis Ethics Approval identified activation of two major tumor promoting signaling path- This study was approved by the National Animal Experiment Board ways in PD-1 resistant tumors. Pharmacological inhibition of these in Finland; license number ESAVI-2331-04 10 07-2017. pathways in combination with PD-1 blockade inhibited tumor growth and extended the survival of mice bearing resistant tumors. P285 Conclusions Integrated molecular characterization of primary resistance This study describes a novel PD-1 resistant mouse tumor model and mechanisms to immune checkpoint blockade in advanced non- underscores the importance of two well defined signaling pathways small cell lung carcinoma (a-NSCLC) to response to immune checkpoint blockade. 1 2 2 Benjamin Besse, MD PhD , Caroline Fraslon, PhD , Hui Cao, PhD , Joon 2 2 2 Sang Lee, PhD , Stephanie Malyszka, MS , Marielle Chiron, PhD , Anne 2 2 1 P284 Caron, PhD , Souâd Naimi, PhD , Laura Mezquita, MD , David Planchard, 1 1 1 Pitfalls in preclinical development of immunotherapies for ER+ MD, PhD , Jean-Yves Scoazec, MD , Ludovic Lacroix, PharmD , Etienne 1 1 breast cancer: estrogen as an immunomodulator potentially Rouleau, MD , Naima Imam-Sghiouar, PhD , Aurélien Marabelle, MD 1 1 1 2 influencing pembrolizumab efficacy in a breast cancer model in PhD , Eric Angevin, MD , Christophe Massard, MD , Jack Pollard, PhD 1 2 humanized mice Gustave Roussy Cancer Center, Villejuif, France; Sanofi, Vitry sur Seine, 1 1 1 2 Tiina Kähkönen , Mari Suominen , Jenni Mäki-Jouppila , Emrah Yatkin , France 3 3 1 1 Philip Dube , Azusa Tanaka , Jussi Halleen , Jenni Bernoulli, PhD Correspondence: Caroline Fraslon (caroline.fraslon@sanofi.com) 1 2 Pharmatest Services, Turku, Finland; University of Turku, Turku, Finland; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P285 Taconic Biosciences, Rensselaer, NY, United States Correspondence: Jenni Bernoulli (jenni.bernoulli@pharmatest.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P284 Reinvigoration of anti-tumor immunity via immune checkpoint block- ade (ICB) has transformed outcomes in a-NSCLC. However, a majority Background of patients are innately resistant to ICB, and a better understanding Immunotherapies have the potential to improve outcomes in triple- of the resistance mechanisms may guide the development of new negative breast cancer patients but evidence is less consistent in es- treatment strategies and patients therapies. trogen receptor -positive (ER+) patients. To advance preclinical devel- Methods opment and understand the effects of immunotherapies against ER+ Biopsies performed immediately before treatment with single agent breast cancer, we aimed to establish a novel orthotopic ER+ breast ICB in patients with a-NSCLC (MATCH-R trial [NCT02517892]) were an- cancer model in humanized mice and to study efficacy of pembroli- alyzed. The stromal microenvironment and immune context was zumab in the model. characterized via an integrated analysis of whole transcriptome Methods (RNA-seq), whole exome sequencing (WES), and immunohistochemis- Female CIEA NOG® (NOG) mice and NOG mice engrafted with human try (IHC) of CD3, CD8, FOXP3 and PDL1. Specifically, the immune con- CD34+ hematopoietic stem cells (huNOG, Taconic Biosciences) were text and the relative abundance of 10 immune and stromal cell types implanted with 5 μg/day estradiol (E2)-releasing implants, and one were assessed with integrated IHC and Cell Populations-counter week later inoculated with ER+ MCF-7 human breast cancer cells into (MCP-counter) [1] analysis of the RNA-seq. Somatic mutations and the mammary fat pad. One group of huNOG mice did not receive E2 Tumor Mutation Burden (TMB) were evaluated. The transcriptional implants. Orthotopic tumor growth was followed by caliper mea- state of the tumor and its microenvironment was assessed by GSVA surements. At study week 2, the E2 supplemented mice were strati- analysis [2] of the MSigDB collection [3]. Patient’s outcome was asso- fied to receive human IgG4 isotype control or pembrolizumab (5 ciated to molecular data. Primary resistance to ICB was defined as PD mg/kg, i.p., Q5D) until the end of the study. The study was termi- in the first radiological examination, or a median PFS inferior to 3 nated at study week 7 and tumors were processed for histological months. and immunohistochemical analysis of tumor infiltrating lympho- Results cytes (TILs). Changes in blood cell counts were assessed by flow cy- Thirty-one patients with adenocarcinoma were enrolled: Median age tometry and hematology. was 60 (34-80), 13 were female, 28 were smokers, 10 were re- Results sponders, and 21 were non-responders. Median tumor cellularity was ER+ orthotopic breast tumors grew only in the presence of E2 sup- 60% (30%-90%). plement. However, general condition of huNOG mice started to de- Responders had higher TMB and immune infiltration compared to crease after 3 weeks on E2 supplementation and their survival was non-responders. Non-responders could be divided into two classes: decreased compared to huNOG mice without E2 supplement. those with equal infiltration to responders “hot tumors” and those Hematological analysis indicated that estrogen decreased the levels with less infiltration “cold tumors”. Neutrophil infiltration was associ- of white and red blood cells, hemoglobin and hematocrit that were ated with resistance to ICB in both “hot” and “cold” resistant tumors. further decreased by concomitant pembrolizumab treatment. Flow Mutations in the IFN-γ and/or KRAS/STK11/KEAP pathways were asso- cytometry analysis confirmed lower numbers of CD3+, CD4+ and ciated to ICB resistance. Increased activation of hypoxia-response, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 154 of 272 transforming growth factor (TGF-β) and MYC pathways from the partial responders and non-responders (high expression), thereby GSVA analysis were also associated to ICB resistance. TGF-β pathway suggesting that the identified target is relevant to the clinical activation was associated to the “cold tumor” ICB-resistant tumor situation. class. Conclusions Conclusions The mAb is currently under preclinical development as a novel im- ICB sensitivity was associated to TMB, IFN-γ pathway mutation, and munotherapeutic antibody to overcome resistance to anti-PD-1 im- immune infiltration. We have further refined our understanding of mune checkpoint blockade. the primary ICB resistance mechanisms in that there are both “hot” and “cold” non-responsive tumors, which suggests that different References therapeutic approaches be may be required in these two subtypes, 1. Perrot I, Michaud HA, Giraudon-Paoli M, et al. Blocking Antibodies i.e. targeting the TGF-β pathway in the “cold” non-responding tumor Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Im- class. We are continuing to increase our baseline cohort size and will mune Responses in Combination Cancer Therapies. Cell Rep. 2019 include post-treatment biopsies collected with this protocol. May 21;27(8):2411-2425.e9. doi: 10.1016/j.celrep.2019.04.091 2. Hugo W, Zaretsky JM, Sun L et al. Genomic and Transcriptomic References Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. 1. Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, Selves J, Cell. 2016 Mar 24;165(1):35-44. doi: 10.1016/j.cell.2016.02.065. Epub Laurent-Puig P, Sautès-Fridman C, Fridman WH, de Reyniès A. Estimating 2016 Mar 17. the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol. 2016; 17(1):218-238. P287 2. Hänzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for A novel bispecific checkpoint inhibitor antibody to preferentially microarray and RNA-seq data. BMC Bioinformatics. 2013;14:7-22. block PD-1 and LAG-3 on dysfunctional TILs whilst sparing Treg 3. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, activation Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 1 1 2 Laura Codarri Deak , Patrick Weber, Mr , Stefan Seeber, Dr , Mario Perro, 2011; 27(12):1739-40. 1 1 1 1 Dr , Xavier Miot, Mr , Heidi Poulet, Mrs , Iryna Dekhtiarenko, Dr , Ethics Approval 3 3 4 Matthias Füth , Henry Kao, Dr , Christine McIntyre, Dr , Francesca This study was approved by Gustave Roussy Scientific Board, the French 3 1 1 Michielin, Dr , Christian Klein, Dr rer nat , Pablo Umana, PhD , Lin-Chi National Health Agency and Ethical Committee (ID-RCB : 2014-A01147- 5 2 3 Chen, Dr , Christoph Markert, Dr , Merlind Mücke, Dr 40) 1 2 Roche Innovation Center Zurich, Zurich, Switzerland; Roche Innovation Center Munich, Munich, Germany; Roche Innovation Center Basel, Basel, P286 Switzerland; Roche Innovation Center Welwyn, Welwyn, United In vivo genetic screens in PD-1 resistant mouse models identified Kingdom; Roche Innovation Center New York, New York, NY, United modulators of anti-PD1 response with relevance to States pembrolizumab-treated human metastatic melanoma Correspondence: Laura Codarri Deak (laura.codarri_deak@roche.com) Aurélie Docquier, PhD, Cécile Déjou, Gilles Alberici, Armand Bensussan, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P287 PhD, Jeremy Bastid, Nathalie Bonnefoy, PhD OREGA Biotech, Ecully, France Background Correspondence: Jeremy Bastid (jeremy.bastid@orega-biotech.com) Check point inhibitors targeting PD-1 have shown unprecedented Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P286 clinical efficacy in several cancer indications and therefore have, revolutionized the standard of care. However, despite this ad- Background vancement in cancer immunotherapy, only ~20-30% of the pa- We previously reported the identification of CD39 as a regulator of tients derive durable benefit from such a treatment. One of the anti-PD-1 response in combination with oxaliplatin in resistant suggested reasons for this limited success is the expression/acti- models using our screening approach [1]. vation of compensatory inhibitory pathways such as LAG-3 on Methods tumor-reactive T cells. These pathways compensate for the loss Similar approach was used to identify novel targets that modulate of function of PD-1 upon its blockade. Therefore, it is envisioned anti-PD-1 response. Here we report the discovery of a novel potent that simultaneous antagonism of PD-1 and LAG-3 receptors regulator of anti-PD-1 therapy in MCA205 fibrosarcoma and B16K1 would overcome this adaptive resistance mechanism and allow a melanoma syngeneic mouse models harboring no or low response more profound reinvigoration of dysfunctional tumor-reactive T rates to PD-1 immune checkpoint inhibitor. cells. Conversely, a recent report has highlighted that blockade of Results LAG-3 on regulatory T cells (Tregs) increases their suppressive Remarkably, the Knock-Out (KO) of the identified target had limited, function and, therefore may off-set its benefit on the reinvigor- if any, impact on tumor growth in various mouse models. Whereas ation of dysfunctional tumor-reactive T cells. the mouse models used are broadly resistant to anti-PD1 therapy, Methods treatment in the KO background markedly improved the efficacy of We therefore developed a 1+1 PD1-LAG3 bispecific antibody (BsAb) anti-PD-1 mAb by increasing the response rates and by increasing with a 10-20 fold higher affinity for PD-1 than for LAG-3, allowing an the rate of complete vs partial responses, which translated into im- avidity driven selectivity gain to PD-1 and LAG-3 double positive T proved mouse survivals. We generated various human-mouse cross- cells. reactive blocking antibodies to the target, including a humanized Results mAb. The neutralizing antibodies mimicked the KO phenotype and Hence, PD1-LAG3 BsAb is assumed to have the following advantages markedly improved the response to anti-PD1 therapy in preclinical over monospecific and other bispecific aPD/L1 and aLAG-3 anti- mouse models. The mechanism of action is being investigated. We bodies: 1) improved targeting to dysfunctional T cells rather than found that the expression of target within the tumor induces an im- Tregs due to the selectivity gain and different expression patterns of munosuppressive tumor immune microenvironment by upregulating PD-1 and LAG-3 on these two T cell types, 2) reduced internalization, several immunoregulatory cytokines and chemokines. Interestingly, 3) Fc silent-mediated resistance to drug-shaving by macrophages. we re-analyzed transcriptomic data from 28 metastatic melanoma tu- Conclusions mors prior to anti-PD-1 pembrolizumab therapy [2] to validate our These characteristics translated in a significant increase in 1) in vitro target and related signaling pathways. We found a stepwise in- T cell effector functions even in the presence of Tregs, and 2) in vivo creased expression of our target, its receptor and the identified tar- tumor control/eradication in mouse models compared to combin- gets of the pathway from complete responders (low expression) to ation of monospecific anti-PD1 and anti-LAG3 antibodies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 155 of 272 P288 P289 CCR5+CTLA4+ Treg cell subset characterizes renal tumors (RCC) Macrophages modulate patient response to immune checkpoint immunosuppressed by PD1 blockade inhibition in a novel lung tumour explant model 1 1 1 1 1 Agathe Dubuisson , Agathe Dubuisson , Charles Bayard , Sebastien Lauren Evans, BSc, MSc , Kate Milward, DPhil , Richard Attanoos, BSc, MB 1 1 1 1 2 1 1 Lofek , Nicolas Voisin , Séverine Mouraud , Delphine Bredel , Sandrine BS, FRCPath , Aled Clayton, PhD , Rachel Errington, PhD , Zsuzsanna 1 1 1 2 1 Susini , Mathieu Rouanne, MD , Hervé Beaumert , Bastien Parier , Tabi, PhD 1 1 1 2 Aurélien Marabelle, MD PhD , Laurence Zitvogel, MD, PhD , Mélodie Cardiff University, Cardiff, United Kingdom; University Hospital Wales, 1 1 Bonvalet , Mélodie Bonvalet Cardiff, United Kingdom 1 2 Gustave Rousy, Villejuif, France; APHP, Le Kremlin Bicetre, France Correspondence: Lauren Evans (Evansl57@cardiff.ac.uk) Correspondence: Mélodie Bonvalet (bonvaletmelodie@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P289 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P288 Background Background The tumour microenvironment (TME) consists of a dynamic interplay A small subset of patients undergoing PD1 blockade therapy develop between the tumour and stroma. Various stroma-resident and a hyper-progressive disease (HPD), corresponding to an acceleration tumour-infiltrating immune cells are associated with pro-tumour ac- of tumor growth rate [1]. However, the mechanisms underlying HPD tivity. Whilst immunotherapies may trigger anti-tumour immune re- are unknown, and the identification of predictive biomarkers remains sponses, anti-inflammatory M2-like tumour-associated macrophages an unmet clinical need. We aimed at better understanding the mech- (TAMs) within the TME present an obstacle for effective treatment anism of hyper-progression following nivolumab treatment by exam- [1]. Pre-clinical research has predominantly used mouse models to ining the tumor immune infiltrate of fresh RCC using an ex vivo represent the in vivo TME. Unfortunately, such models do not faith- model system. fully replicate the human immune system therefore, providing an in- Methods adequate measure of immunotherapy response. Human tumour- Fifteen fresh primary RCC were processed according to methods pre- derived explants maintain the original 3D tumour architecture and viously reported [2] and stimulated for 3 days with nivolumab. At combination of multiple cell types. Therefore, we established an baseline, we monitored the phenotype of the immune infiltrate by ex vivo tumour explant model of non-small cell lung cancer (NSCLC) flow cytometry. After 3 days of culture, we measured 26 soluble fac- incorporating TAMs, to determine their role in immunotherapeutic tors which were released into the supernatant. We then examined response. the relationship between baseline phenotype and functional immune Methods reactivity to nivolumab, considering variations of at least 2 independ- Tumour explants (ca. 1mm³) were generated from fresh tumour tis- ent soluble factors in the range of sue. Autologous CD14 peripheral blood mononuclear cells (PBMCs) Results were added to explants for 48h followed by flow cytometry pheno- Nivolumab induced a strong inhibition of soluble factor release in 5 out typing using a 7-marker macrophage panel (CD14; CD64; PDL-1; of 15 RCC. The decreased soluble factors were IL1RA (3 out of 5 tumors CD163; CD206; CD23; CD200R). The functional contribution of macro- (3/5)), IFNg, CXCL10, IL10, G-CSF, GM-CSF (2/5), IL-4, IL-5, IL-6, IL-8, IL-9, phages to explant-mediated immunosuppression was assessed + + CCL4, CCL5, PDGFbb (1/5). In these hypo-sensitive tumors (HS), nivolu- through measuring IFNγ/TNFα production from CD4 /CD8 T cells by mab induced a significant decrease of IL-4, IL-8, G-CSF (p<0.05). flow cytometry. T cells, in the presence of explants, were stimulated Conclusions with a viral peptide pool and incubated for 6 days ±250μg/mL Atezo- The TME of HS renal tumors that are immunosuppressed by nivolu- lizumab, prior to intracellular cytokine staining. Culture supernatants mab display high levels of the CCR5 ligands and CCR5+ CTLA4+ Treg were collected and the Th1/Th2 cytokine profile determined using a cells, compared to NHS tumors. As reported by Kamada et al. [3], dis- LEGENDplex bead-based immunoassay (BioLegend). Transcriptomic tinct Treg subset might be involved in the deleterious effects of nivo- analysis was performed on patient tumour tissue and peripheral lumab observed in HPD. Prospective clinical studies should blood using the NanoString PanCancer IO360 gene expression panel. determine if nivolumab-associated HPD is caused by a pre-existing Results pool of highly immunosuppressive CCR5+ CTLA4+ Treg cells. Tumour explants significantly promoted M2-like macrophage differ- entiation and suppressed T cell activity ex vivo. The PDL-1 inhibitor, Acknowledgements Atezolizumab significantly improved T cell function in some patients This work was supported by Bristol-Myers Squibb. and reduced explant-mediated immunosuppression, particularly in the presence of macrophages. This suggests that response or resist- References ance to anti-PDL-1 therapies may be partially TAM dependent. The 1. Champiat S, Dercle L, Ammari S, Massard C, Hollebecque A, Postel-Vinay differential effect of Atezolizumab observed in the presence of S, Chaput N, Eggermont A, Marabelle A, Soria JC, Ferté C. Hyperprogres- patient-derived tumour explants indicates the potential of this model sive Disease Is a New Pattern of Progression in Cancer Patients Treated in predicting immunotherapy responses. Ongoing transcriptomic by Anti-PD-1/PD-L1. Clin Cancer Res. 2017;23(8):1920-1928. analysis of lung cancer tissue aims to reveal associations between 2. Jacquelot N, Roberti MP, Enot DP, Rusakiewicz S, Ternès N, Jegou S, the TME and T cell function. Variations in the cytokine secretion pro- Woods DM, Sodré AL, Hansen M, Meirow Y, Sade-Feldman M, Burra A, file of tumour explant co-cultures are also being studied under differ- Kwek SS, Flament C, Messaoudene M, Duong CPM, Chen L, Kwon BS, An- ent experimental conditions. derson AC, Kuchroo VK, Weide B, Aubin F, Borg C, Dalle S, Beatrix O, Conclusions Ayyoub M, Balme B, Tomasic G, Di Giacomo AM, Maio M, Schadendorf D, Using the tumour explant model, we found that alleviation of Melero I, Dréno B, Khammari A, Dummer R, Levesque M, Koguchi Y, Fong explant-mediated immunosuppression by Atezolizumab may be L, Lotem M, Baniyash M, Schmidt H, Svane IM, Kroemer G, Marabelle A, macrophage dependent. This model could be used to predict patient Michiels S, Cavalcanti A, Smyth MJ, Weber JS, Eggermont AM, Zitvogel L. response to anti-PDL-1 immunotherapy, and explore combination Predictors of responses to immune checkpoint blockade in advanced therapies. Ongoing research aims to improve immunotherapy re- melanoma. Nat Commun. 2017 ;8(1):592. sponses through reprogramming TAMs using immune-modifying 3. Kamada T, Togashi Y, Tay C, Ha D, Sasaki A, Nakamura Y, Sato E, Fukuoka drugs. S, Tada Y, Tanaka A, Morikawa H, Kawazoe A, Kinoshita T, Shitara K, Sakaguchi S, Nishikawa H. PD-1+ regulatory T cells amplified by PD-1 Acknowledgements blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A. Study supported by research funds from Cardiff University, School of 2019 May 14;116(20):9999-10008. Medicine. We would like to thank the Wales Cancer Bank and Lung Ethics Approval Multidisciplinary Research Group for their ongoing involvement in patient The study was approved by the Comité de Protection des Personnes Ile de recruitment and sample acquisition, and to the patients who donated their France III Ethics Board, approval number 2016-A00732-49. samples for our research. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 156 of 272 Reference P291 1. Cassetta L, Kitamura T. Targeting Tumor-Associated Macrophages as a Po- Driving T cell dysfunction in vitro for rational immunotherapy tential Strategy to Enhance the Response to Immune Checkpoint Inhibi- design tors. 2018;6. Matthew Hancock, Cailin Joyce, PhD, Thomas Horn, Simarjot Pabla, Ethics Approval Benjamin Duckless, Andrew Basinski, Dhan Chand, PhD, Jeremy Waight, Ethical approval for this project was provided through the Wales Cancer PhD, Mariana Manrique, PhD, Nicholas Wilson, Alex Duncan, PhD, Bank (WCB). The WCB has ethics approval as a Research Tissue Bank from Jennifer Buell, PhD, David Savitsky, PhD, Lukasz Swiech, John Castle, PhD the Wales Research Ethics Committee 3, reference 16/WA/0256 (previous Agenus Inc, Lexington, MA, United States approval references – 06/MRE09/26 and 11/WA/0279). This approval Correspondence: John Castle (john.castle@agenusbio.com) covers the collection of samples (including consent), processing and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P291 storing samples across multiple collection and storage sites. The approval also allows release of anonymised samples to researchers carrying out Background cancer related activity, following successful application approval from the Immune checkpoint blockade (ICB) elicits durable responses in some WCB External Review panel (project 17/016). Sample collection of blood cancer patients, but novel targets and combination approaches are and tissue, from non-small cell lung cancer patients undergoing surgical needed to address resistance and broaden clinical benefit. Here, we resection, was performed at the University Hospital of Wales, Cardiff. present a system for characterizing mechanisms of T cell dysfunction in the tumor microenvironment (TME) and apply the system to un- cover novel approaches to ICB resistance. P290 Methods Interferon gamma production by regulatory T cells is necessary for We developed a long-term human co-culture system comprised of response to cancer immunotherapy primary T cells and cancer cells that enables controlled differentiation Angela Gocher, PhD, Dario Vignali, PhD, Creg Workman, PhD, Sanah of naïve T cells to effector, memory and dysfunctional states. We lon- Handu gitudinally monitored T cell effector functions, protein and RNA ex- University of Pittsburgh, Pittsburgh, PA, United States pression across states and single cells. Finally, we challenged the Correspondence: Dario Vignali (dvignali@pitt.edu) system with PD1 antibody to uncover biomarkers and mechanisms Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P290 of therapeutic resistance. Results Background T cells in our system become activated and then gradually progress Our lab has shown that murine regulatory T cells (Tregs) produce the to a terminally dysfunctional state driven by multiple cancer antigen cytokine interferon gamma (IFNγ) during anti-PD1 therapy and that exposures. T cell cytotoxicity is maintained over several antigen ex- Treg response to IFNγ is necessary for tumor eradication by anti-PD1 posures before sharply decreasing whereas cytokine secretion begins therapy [1]. However, the role of this cellular source of IFNγ in the to decrease with only one prior antigen exposure. The expression of tumor microenvironment (TME) and how Treg derived IFNγ dictates known T cell regulators and novel factors is altered over the time response to other cancer immunotherapies has yet to be studied. In course, with known factors reflecting previous observations in vivo. addition, it has been shown that IL-12-induced production of IFNγ is Anti-PD1 prolongs cytotoxic capacity but T cells eventually fail to re- necessary for response to anti-PD1 therapy [2]. However, it has yet to spond. Single cell mapping in the presence of anti-PD1 reveals an ex- be determined whether Tregs are a key responder to IL-12 during panded population of T cells that co-express PD1, TIGIT and cancer immunotherapy. Thus, elucidating the interplay of IL12, IFNγ activation markers. Consistent with this, the combination of PD1 and and Tregs in the TME is essential for maximizing efficacy and minim- TIGIT blockade enhances cytotoxicity relative to monotherapies. izing the clinical resistance to current cancer immunotherapies. Conclusions Methods These findings demonstrate the utility of our system to deeply inter- Our lab has generated two novel murine models that allow for Treg- rogate therapeutic response and resistance. Moreover, its scalability restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/ and modularity enable genome-scale screening to discover novel tar- LFoxp3Cre-YFP). These murine models were validated and used to gets that could enhance antitumor activity of both natural and adop- assess the contribution of Treg-derived IFNγ on the growth of syn- tively transferred T cells. geneic MC38 (colon adenocarcinoma) and B16 (melanoma) tumors and response to a various cancer immunotherapies (checkpoint blockade, vaccination and tumor-specific antibodies). Flow cytometry P292 was conducted on tumor and control non-draining lymph nodes to Tissue site of tumor growth dictates anti-tumor immunity and phenotype the immune infiltrate in response to immunotherapy. response to checkpoint blockade Results Brendan Horton, PhD, Stefani Spranger, PhD Mice with Tregs either unable to generate IFNγ (IfngL/LFoxp3Cre- Massachusetts Institute of Technology,, Cambridge, MA, United States YFP) or respond to the IFNγ-inducing cytokine IL-12 (Il12rb2L/ Correspondence: Stefani Spranger (spranger@mit.edu) LFoxp3Cre-YFP) were unable to eradicate tumors in response to im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P292 munotherapy. In addition, preliminary results suggest that Il12rb2- deficient Tregs may be more suppressive as indicated by an increase Background in tumor growth compared to control. An emerging area of clinical importance is differential responsiveness Conclusions to checkpoint blockade immunotherapy across different tissues sites These data suggest that IFNγ producing Tregs are necessary to shift of tumor growth, leading to partial responses and ultimately cancer- the balance from an immunosuppressive TME to one that favors the related deaths [1, 2]. However, tissue-specific anti-tumor immune re- reinvigoration of the anti-tumor response generated by current can- sponses are not well understood. We used mouse models to deter- cer immunotherapeutic agents. Future studies will determine mine how anti-tumor immune responses differ across tissue sites and whether the capacity of Tregs to produce IFNγ can predict response how this relates to immunotherapy efficacy. to immunotherapy. Methods Mice were inoculated subcutaneously or intravenously with syngen- References eic KP lung cancer cells, then treated with anti-CTLA-4 and anti-PD- 1. Overacre-Delgoffe A, Chikia M. Interferon-γ Drives Treg Fragility to Pro- L1 antibodies, and analyzed for tumor burden. Response to check- mote Anti-tumor Immunity. Cell. 2017; 169: 1130-1141. point blockade was correlated with tumor-infiltrating T cells (TIL) and 2. Garris C, Arlauckas S. Successful Anti-PD1 Cancer Immunotherapy Re- systemic immune responses using flow cytometry and immuno- quires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL- histochemistry. Mice were also tested for their ability to generate sys- 12. Immunity. 2018; 49: 1148-1161. temic and protective immunity against a second tumor challenge. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 157 of 272 Results ~500 cancer causing genes) as the number of nonsynonymous coding Comparing lung and subcutaneous tumors, we observed striking dif- mutations per megabase and the optimal cut-off for TMBhigh was de- ferences within the TIL compartment. Lung tumors contained more termined using the 98th percentile of newly-diagnosed tumors and TIL with higher expression of PD-1. Despite this, lung tumors did not segmented linear regression analysis. Patients were stratified by histo- respond to anti-CTLA-4 + anti-PD-L1, while subcutaneous tumors did. molecular subtype, IDH mutation, 1p/19q co-deletion, TMB, and ICB TIL expanded in subcutaneous tumors after immunotherapy, while treatment under clinical trials and expanded access. Overall (OS) and lung tumors had no change in the number of TIL. We further tested progression-free (PFS) survival were estimated by the Kaplan-Meier for concomitant immunity and found that subcutaneous KP tumors method and evaluated using multivariable Cox regression. generated an immune response that could protect against a second Results tumor. However, KP lung tumors failed to generate a protective sys- We identified 1,223 HGG patients with genomics, including 64 temic immune response. These results were reproduced using a pan- hypermutated tumors. Overall the cohort consisted of 79% newly- creatic cancer cell line, indicating that tumors growing in the lung diagnosed and 21% recurrent gliomas and subclass distribution generated weaker systemic immune responses than subcutaneous was 75% IDH-wildtype glioblastomas/anaplastic astrocytomas,16% tumors. Protective concomitant immunity required CD8+ T cells, and IDH-mutant glioblastomas/anaplastic astrocytomas, and 6% 1p/19- to a lesser extent CD4+ T cells, but not NK cells. Therefore, we inves- codeleted anaplastic oligodendrogliomas. Hypermutated HGG tigated differences in the generation of systemic, antigen-specific were predominantly seen in the setting of recurrence (18% of re- CD8+ T cell responses between subcutaneous and lung KP tumors. current HGGs vs 2% of newly diagnosed, 5% overall incidence). Using KP.SIY tumors, elispot assays found that lung tumors gener- Comparisons of biomarkers and genomics in hypermutated versus ated fewer antigen-specific T cells in the spleen. Transferred 2C T non-hypermutated HGGs showed distinct characteristics in glioma cells proliferated less in the spleens of lung tumor-bearing hosts than subtypes with implications for differential mechanisms of in hosts with subcutaneous tumors, even though proliferation in TMBhigh acquisition in HGGs with the most important biomarkers draining lymph nodes was similar. Consistently, KP.SIY lung tumors of differential hypermutation risk being MGMT promoter methyla- generated weaker concomitant immunity than subcutaneous KP.SIY tion (22% of methylated vs. 6% of unmethylated cases, p=0.02) tumors. and IDH1/2 mutation (25% of IDH-mutant vs. 12% of IDH- Conclusions wildtype HGGs, p=0.007). The tissue site of tumor growth determines the number and pheno- 129 (11%) of HGGs (mostly IDH-wildtype GBMs/AAs) received PD-1/ type of TIL, the generation of systemic immunity, and the response PD-L1 ICB therapy, including 13% (n=8) of hypermutated HGGs. to checkpoint blockade. Response to immunotherapy correlates not Immunohistopathologic assessment of tumor responses and immune with the number of TIL, or their phenotype, but with the ability of infiltrates was conducted and correlated with the genomic profiling. the host to mount a systemic anti-tumor CD8+ T cell response. Because the majority of ICB-treated cases were IDH-wildtype HGGs, this subset was further evaluated in analyses which were risk- References adjusted by age, sex, histomolecular subgroup, MGMT promoter 1. Khoja, L., et al., Patterns of response to anti-PD-1 treatment: an explora- methylation, and prior therapy (i.e. RT/TMZ/CCNU/bevacizumab). Cor- tory comparison of four radiological response criteria and associations relations between TMB, molecular genotypes, and clinical response with overall survival in metastatic melanoma patients. Br J Cancer, 2016. to ICB in HGGs will be presented. 115(10): p. 1186-1192. Conclusions 2. Lee, J.H.J., et al., Metastasis-specific patterns of response and progression Our study represents the largest set of genomically characterized gli- with anti-PD-1 treatment in metastatic melanoma. Pigment Cell Melan- omas and gliomas with hypermutation with data related to re- oma Res, 2018. 31(3): p. 404-410. sponses to PD-1/PD-L1 ICB therapy. Our data support systematic TMB Ethics Approval characterization and clinical molecular genotyping to assist therapy- The animal work in these experiments was reviewed and approved by the decision making in gliomas and provide foundational data essential MIT Committee on Animal Care. The approved animal protocol number to future clinical trial designs of immunotherapeutics. for this work is 0117-012-20. Ethics Approval This study was approved by the Partners HealthCare (#2015P002352) and Dana-Farber Cancer Institute (10-417) institutional review boards. P293 Tumor mutational burden (TMB) and genomic predictors of clinical outcomes to PD-1/PD-L1 checkpoint blockade in high-grade P294 gliomas Long survival associated with receipt of anti-CTLA-4 in patients 1 1 1 1 Bryan Iorgulescu, MD , Mehdi Touat , Yvonne Li , Liam Spurr , Gareth with metastatic melanoma from acral lentiginous, mucosal and 1 1 1 1 Grant , William Pisano , Mary Jane Lim-Fat , Eudocia Lee , Lakshmi uveal primary tumors 1 2 2 3 1 Nayak , E Chiocca , Raymond Huang , Andrew Cherniack , Patrick Wen , Nicholas Klemen, MD, Melinda Wang, BS, Kelly Olino, MD, James Clune, 1 4 1 1 Ahmed Idbaih , Franck Bielle , David Reardon, MD , Keith Ligon MD, Stephan Ariyan, MD, Charles Cha, MD, Sarah Weiss, MD, Harriet 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Kluger, MD, Mario Sznol, MD Women's Hospital, Boston, MA, United States; Broad Institute, Yale University School of Medicine, New York, NY, United States Cambridge, MA, United States; Sorbonne Université, Paris (UPMC), Paris, Correspondence: Mario Sznol (mario.sznol@yale.edu) France Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P294 Correspondence: David Reardon (david_reardon@dfci.harvard.edu); Keith Ligon (Keith_Ligon@dfci.harvard.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P293 Metastatic melanoma from acral lentiginous, mucosal and uveal pri- mary tumors responds poorly to checkpoint inhibitors (CPI), poten- Background tially due to a low burden of immunogenic neoantigens [1-7]. Long- High tumor mutational burden (TMB) is an emerging biomarker for term outcomes of these patients after treatment with CPI have not predicting response to PD-1/PD-L1 immune checkpoint blockade been established. (ICB) in a spectrum of cancer patients, however, its clinical value and Methods therapeutic implications in high-grade gliomas (HGG) is not yet We performed a retrospective review of a single institutional experi- established. ence using antibodies against CTLA-4, PD-1 and PD-L1 for patients Methods with stage IV melanoma. Primary tumor histology was categorized as We retrospectively reviewed all HGGs at our institutions from 2013- cutaneous, acral, mucosal or uveal. Patients with unknown primary 2018 that underwent genomic characterization. TMB was determined were excluded. We measured overall survival (OS) after the first dose from clinical targeted exome next-generation sequencing (DFCI-Profile, of CPI using the Kaplan-Meier method. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 158 of 272 Results We treated 428 patients with metastatic melanoma from 2007-2019. Pri- mary tumors were cutaneous in 283 (66%) patients, unknown in 55 (13%), acralin22(5%), mucosalin38(9%)and uvealin30(7%)(Table 1).Mucosal patients had a slight female preponderance. The proportion staged M1c was higher in mucosal and uveal patients. Patients with cutaneous primary tumors had median OS after CPI of 45 months, compared with 17 months for acral (P = 0.047), 18 months for mucosal (P = 0.003) and 12 months for uveal (P < 0.001)(Figure 1). Five-year survival for cutaneous, acral, mucosal and uveal patients was 46%, 34%, 21% and 22% respectively. Next we combined the patients with acral, mucosal and uveal melan- oma (n = 90) and performed survival analysis stratified by the first type of CPI treatment. Median OS after anti-PD-1 or anti-PD-L1 was 9 months, compared with 18 months after anti-CTLA-4 (P = 0.010) and 20 months after combination therapy with anti-CTLA-4 plus anti-PD-1 (P = 0.003)(Figure 2). While 21 of 31 (68%) patients treated with anti- CTLA-4 later were treated with anti-PD-1, only 5 of 18 (28%) patients Fig. 1 (abstract P294). Overall survival stratified by histology treated with anti-PD-1 later received anti-CTLA-4 (P = 0.02). There were 21 patients who survived at least three years after CPI, all of whom were treated with anti-CTLA-4 with or without anti-PD-1. Of the 10 patients with actual five-year survival, 3 had complete re- sponses while the other 7 all required local and/or regional therapies to control progressive disease (Table 2). Conclusions Long survival in patients with metastatic melanoma from acral, mu- cosal and uveal primary tumors was uniformly associated with re- ceipt of anti-CTLA-4. Our experience shows that while acral, mucosal and uveal melanomas have worse outcomes than cutaneous melan- oma, with an aggressive multidiscliplinary approach five-year survival is still possible for 25-32% of these patients. References 1. Luke JJ, Callahan MK, Postow MA, Romano E, Ramaiya N, Bluth M, et al. Clinical activity of ipilimumab for metastatic uveal melanoma. Cancer. 2013;119:3687–95. 2. Algazi AP, Tsai KK, Shoushtari AN, Munhoz RR, Eroglu Z, Piulats JM, et al. Clinical outcomes in metastatic uveal melanoma treated with PD-1 and PD-L1 antibodies. Cancer. 2016;122:3344–53. 3. D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy Fig. 2 (abstract P294). Overall survival stratified by treatment and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis. JCO. 2017;35:226–35. 4. Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for Patients With Advanced Mucosal Melanoma. The Table 2 (abstract P294). Characteristics of five-year survivors Oncologist. 2013;18:726–32. 5. Bello DM, Chou JF, Panageas KS, Brady MS, Coit DG, Carvajal RD, et al. Prognosis of acral melanoma: a series of 281 patients. Ann Surg Oncol. Springer US; 2013;20:3618–25. 6. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. Nature Publishing Group; 2012;44:1006–14. 7. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al. Mutational heterogeneity in cancer and the search for new cancer- associated genes. Nature. 2013;499:214–8. Ethics Approval The study was approved by the Yale-New Haven Hospital Institutional Review Board, approval number 2000021595. Table 1 (abstract P294). Demographics and first treatment P295 The impact of metastatic sites on checkpoint inhibitor outcomes in patients with cutaneous and unknown primary melanoma Penina Krieger, MPhil, Francisco Sanchez-Vega, Nikolaus Schultz, Alexander Shoushtari, MD Memorial Sloan Kettering Cancer Center, Bronx, NY, United States Correspondence: Penina Krieger (peninakrieger@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P295 Background Clinical biomarkers of response to Programmed Death-1 (PD-1) based therapies are sorely needed in melanoma. The American Joint Committee on Cancer (AJCC) 8th Edition Staging system is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 159 of 272 derived from the pre-checkpoint inhibitor era and divides patients into locally advanced or soft tissue disease (M0/M1a), lung metastases (M1b), other visceral metastases (M1c), and brain metastases (M1d). It is unclear whether this prognostic classification remains valid in the mod- ern therapeutic era. The number of metastatic sites influences out- comes with BRAF-MEK therapy [1], but its prognostic importance with PD-1-based therapy is unknown. Methods All patients with melanoma who had prospective tumor molecu- lar profiling at a single center (MSK-IMPACT) and subsequently received frontline PD-1 blockade as single agent (Nivolumab or Pembrolizumab) or combination therapy with Ipilimumab (combo) were included. Demographic and clinical data were collected, in- cluding metastatic sites present at time of PD-1 therapy. Overall survival (OS) and time to treatment failure (TTF) were calculated from the onset of PD-1 therapy using Kaplan-Meier methodology. The impact of specific metastatic sites on TTF and OS was determined using Cox Proportional Hazards Regression Models. Tumor mutational burden (TMB) was calculated as previously described [2]. Results Fig. 1 (abstract P295). See text for description 309 patients received frontline PD-1 monotherapy (n=179) or PD-1 combo (n=130). Overall survival varied by AJCC stage (p<0.0001, Fig- ure 1); M1b and M0/M1a groups had similar OS (median=NR, p = 0.27) followed by M1c (median=39 mo) and M1d (median=28 mo). Patients with 3+ metastatic sites had worse median OS than those with 0-2 metastatic sites (45 vs 39 mo, p<0.0001, Figure 2). Among patients with M1c disease, those with bone or liver me- tastases had worse median OS than those without them (39 vs NR mo, Liver HR=2.4, p=0.036, Bone HR=2.6, p=0.021, Figure 3). Among patients with M1d disease, those with liver metastases had shorter TTF than those without when treated with PD-1 com- bination therapy (HR=2.4, p=0.044). There was no significant dif- ference in median TMB by AJCC M stage at treatment or by site of metastasis. Conclusions AJCC 8th edition M1b disease has a similar prognosis to M0/M1a dis- ease in the era of PD-1-based therapy. The presence of liver or bone metastases at time of PD-1 therapy portends worse OS and TTF even within M1c and M1d disease. Patients with 0-2 metastatic sites live longer than those with 3+ sites. These clinical observations are not explained by differences in TMB. Future trials of PD-1 blockade in ad- vanced melanoma should account for these differences. Fig. 2 (abstract P295). See text for description Acknowledgements Research reported in this abstract was supported by the National Cancer Institute of the National Institutes of Health under Award Number R25CA020449 and by National Cancer Institute Cancer Center Core Grant P30CA008748, Kravis Center for Molecular Oncology. The content is the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1. Long G, Grob J, Nathan P, Ribas A, Robert C, Schadendorf D, Lane S, Mak C, Legenne P, Flaherty K, Davies M. Factors predictive of response, disease progression, and overall survival after dabrafenib and trametinib combination treatment: a pooled analysis of individual patient data from randomised trials. Lancet Oncol. 2016; 17:1743–54. 2. Samstein RM, Lee CH, Shoushtari AN, Hellmann MD, Shen R, Janjigian YY, Barron DA, Zehir A, Jordan EJ, Omuro A, Kaley TJ, Kendall SM, Motzer RJ, Hakimi AA, Voss MH, Russo P, Rosenberg J, Iyer G, Bochner BH, Bajorin DF, Al-Ahmadie HA, Chaft JE, Rudin CM, Riely GJ, Baxi S, Ho AL, Wong RJ, Pfister DG, Wolchok JD, Barker CA, Gutin PH, Brennan CW, Tabar V, Mel- linghoff IK, DeAngelis LM, Ariyan CE, Lee N, Tap WD, Gounder MM, D’An- gelo SP, Saltz L, Stadler ZK, Scher HI, Baselga J, Razavi P, Klebanoff CA, Yaeger R, Segal NH, Ku GY, DeMatteo RP, Ladanyi M, Rizvi NA, Berger MF, Riaz N, Solit DB, Chan TA, Morris LGT. Tumor mutational load predicts sur- vival after immunotherapy across multiple cancer types. Nat Genet. 2019; 51:202–206 Ethics Approval The study was approved by Memorial Sloan Kettering's Ethics Board, Fig. 3 (abstract P295). See text for description approval number 18-244. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 160 of 272 P296 BMI, irAE and gene expression signatures predict resistance and survival to immune-checkpoint inhibition in renal cell carcinoma 1 1 1 2 Brian Labadie, MD , Ping Liu, PhD , Riyue Bao, PhD , Michael Crist, MD , 3 4 5 Ricardo Fernandes, MD , Laura Ferreira Freire , Scott Graupner , Andrew 5 4 3 6 Poklepovic, MD , Ignacio Duran , Saman Maleki Vareki , Arjun Balar, MD , Jason Luke, MD, FACP 1 2 University of Chicago, Chicago, IL, United States; NYU School of Medicine, New York, NY, United States; Western University, London, 4 5 Ontario, Canada; HUMV, Santander, Spain; Virginia Commonwealth University, Richmond, VA, United States; New York University, New York, NY, United States; University of Pittsburgh, Pittsburgh, PA, United States Correspondence: Jason Luke (lukejj@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P296 Background Treatment with immune-checkpoint inhibition (ICI) has changed the treatment paradigm in ccRCC however many do not respond to these treatments and no reliable molecular biomarker exists to pre- dict response to ICI in individual patients. Clinical variables may cor- relate with lack of response to treatment (primary resistance) or clinical benefit. Methods Via an international multi-institution collaboration, clinical charac- Fig. 1 (abstract P296). Kaplan-Meier curves depicting teristics from patients with ccRCC treated with anti-PD-1/L1 ther- survival outcomes apy were collected. Patients with primary resistance (defined as progression on initial computed tomography scan) were com- pared to patients with clinical benefit. Multivariable analysis was performed to identify factors associated with improved time to progression or death. The Cancer Genome Atlas Kidney Renal P297 Clear Cell Carcinoma cohort (TCGA-ccRCC) was examined for the A semi-mechanistic platform model to capture individual animal correlation between gene expression patterns, clinical factors, and responses to checkpoint inhibitors in a syngeneic mouse model 1 2 1 2 2 survival outcomes. Lin Lin, PhD , Alison Betts , Carissa Young , Wendy Qiao , Jatin Narula , 2 2 2 2 Results Peter O’Brien , Derek Bartlett , Andrea Hooper , Jason Williams , John 1 1 1 1 Of 90 patients, 38 (42.2%) had primary resistance and 52 (57.8%) Burke , Joshua Apgar , Lore Gruenbaum, PhD , Fei Hua, PhD 1 2 had clinical benefit. Compared with the cohort of patients with Applied BioMath, LLC, Concord, MA, United States; Pfizer Worldwide initial benefit, primary resistance was more likely to occur in pa- R&D, Cambridge, MA, United States tients with worse ECOG performance status (p=0.03), earlier stage Correspondence: Fei Hua (fei.hua@appliedbiomath.com) at diagnosis (p=0.04), no prior nephrectomy (p=0.04) and no Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P297 immune-related adverse events (irAE) (p=0.02). In the entire co- hort, improved overall survival was significantly correlated with Background lower International Metastatic RCC Database Consortium risk Syngeneic mouse models have been widely employed in preclinical score (p=0.02) and lower neutrophil:lymphocyte ratio (p=0.04). In discovery of checkpoint inhibitors as they enable study of drug im- patients with clinical benefit, improved progression free survival pact on the intact immune system. However, the interpretation of was significantly associated with increased BMI (p=0.007) and irAE such studies remains challenging partly due to the large variability in occurrence (p=0.02) while improved overall survival was signifi- individual animal responses to drug treatment. cantly correlated with overweight BMI (BMI 25-30)(p=0.03) and no Methods brain metastasis (p=0.005). In the TCGA-ccRCC analysis, higher ex- In this work, we describe the generation of a model platform that pression of angiogenesis gene signature was found to be corre- captures essential aspects of the pharmacokinetics, cellular and lated with lower neoplasm histologic grade and better survival (p tumor growth effects of murine surrogates of two checkpoint thera- < 0.05). Angiogenesis and T cell-inflamed gene signatures were peutic antibodies, anti-PD1 and anti-CTLA4, in the CT26 syngeneic inversely correlated in tumors of high T cell-inflamed gene ex- tumor model. The model describes individual animal responses with pression (p=0.008), a pattern not observed in non-T cell-inflamed regard to drug exposure, key intra-tumoral cell kinetics and tumor tumors. (Figure 1) volume changes and provides biologically plausible explanations for Conclusions the observed differences between good and poor responders to Identification of BMI, performance status and prior nephrectomy as treatment with anti-PD1 or anti-CTLA4. predictors of response to PD1/L1 in ccRCC may help inform treat- Results ment selection. The inverse association of angiogenesis gene signa- We used the model to predict the antibody dose-response relation- tures with ccRCC histologic grade highlight opportunities for ships for individual animals and to identify dose thresholds above adjuvant combination VEGFR2 TKI and ICI. which complete tumor elimination can be achieved in good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 161 of 272 responders. In contrast, our models predict that poor responders demonstrated higher expression of angiogenesis-related profiles and would not achieve complete response even with much higher drug increased CD31 expression. Furthermore, Pbrm1 deficient Renca sub- doses. The parameters in our model that impact the response in cutaneous tumors in mice demonstrated longer latency but more re- poor responders are not drug-related. This finding suggests that sistance to programmed death-1 (PD-1) blockade. Analysis of the immune-cell related barriers have to be crossed in order to achieve a IMmotion150 cohort revealed that ccRCC patients with PBRM1 muta- therapeutic response in these animals - possibly via combination tions were associated with decreased immune infiltrates and a re- therapy. duced response rate to atezolizumab monotherapy or combination In addition, we identified the net tumor cell doubling rate, one po- therapy with bevacizumab. tential parameter that contributes to individual variability in response Conclusions to treatment, as the most sensitive biological parameter determining Pbrm1 and PBRM1 loss reduced IFN gamma-STAT1 signaling. Pbrm1 tumor volume changes upon treatment with anti-PD1 or anti-CTLA4. inactivation was associated with a less immunogenic tumor micro- Measuring individual animal tumor cell growth characteristics may environment in animal and human tissue samples. Response to PD- help with the experimental design and qualification of animals for L1 blockade is reduced in patients with PBRM1 mutations in the studies (in addition to absolute tumor volume), and thereby reduce IMmotion 150 study. This study forms a framework for future mech- inter-animal variability and enhance the interpretability of study re- anistic and clinical studies on the interaction between genomic fea- sults, especially in combination with a model such as the one pre- tures in RCC and response to immunotherapy. sented here. Conclusions Acknowledgements This model platform can be adapted to capture and compare check- We acknowledge the TCGA Research network. This work was supported by point drug effects in different syngeneic tumor models. Moreover, it funding from DOD grant W81XWH-17.1.0307, DOD grant CA160728P1, UT can be expanded to add additional drug mechanisms and can serve MD Anderson Cancer Center CCSG grant 5 P30 CA016672 (Biostatistics as a tool to inform the experimental design of mouse studies. shared resource group) and the Adopt-a-Scientist Foundation. Ethics Approval The animal protocols (2018-0376) were approved by Institutional Animal P298 Care and Use Committee (IACUC) of The Health Science Center, Texas A&M PBRM1 loss defines distinct tumor phenotype associated with University. Human subject protocol (2007-0511) was approved by immunotherapy resistance in renal cell carcinoma Institutional Research Board at M.D. Anderson Cancer Center. 1 1 1 1 Xiande Liu, PhD , Wen Kong , Christine Peterson , Daniel McGrail , Anh 1 1 1 1 Hoang , Xuesong Zhang , Truong Lam , Patrick Pilie, MD , Haifeng Zhu, 1 2 2 3 PhD , Kathryn Beckermann , Scott Haake , Sevinj Isgandrova , Margarita P299 3 1 2 Martinez-Moczygemba , Nidhi Sahni , W. Kimryn Rathmell , Eric Jonasch, Neuropilin-1 is a T cell memory checkpoint limiting long-term MD tumor immunity 1 2 1 2 3 MD Anderson Cancer Center, Houston, TX, United States; Vanderbilt Chang Liu, PhD , Ashwin Somasundaram, MD , Sasikanth Manne , 3 1 1 1 University Medical Center, Nashville, TN, United States; Texas A&M Angela Gocher, PhD , Andrea Szymczak-workman , Kate Vignali , Daniel 2 1 1 Health Science Center, Houston, TX, United States Normolle, PhD , Robert Ferris, MD, PhD , Tullia Bruno, PhD , E. John 3 1 1 Correspondence: Eric Jonasch (ejonasch@mdanderson.org) Wherry, PhD , Creg Workman, PhD , Dario Vignali, PhD 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P298 University of Pittsburgh, Pittsburgh, PA, United States; UPMC Hillman Cancer Center, Pittsburgh, PA, United States; University of Pennsylvania, Background Philadelphia, PA, United States Polybromo-1 (PBRM1), encoding a mammalian specific subunit of the Correspondence: Dario Vignali (dvignali@pitt.edu) switch/sucrose non fermenting (SWI/SNF) chromatin remodeling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P299 complex, is the second most frequently mutated gene in clear cell renal cell carcinoma (ccRCC). Data thus far on the effect of PBRM1 Background loss on immune responsiveness are inconsistent. The impact of Robust CD8+ T cell memory is essential for long-term protective im- PBRM1 mutation on response to immunotherapy in patients with munity but is often impaired in cancer due to T cell exhaustion, which renal cell carcinoma (RCC) has become a topic of intense debate. causes a loss of memory precursors. Immunotherapy via checkpoint RCC-specific mechanistic and large-scale clinical data are needed to blockade does not effectively reverse this defect in the majority of pa- precisely further characterize the influence of PBRM1 loss on re- tients, potentially underlying disease relapse. Resistance mechanisms sponse to immunotherapy. that underlie poor CD8+ memory development remain unknown. Methods Methods An immunocompetent murine RCC model was applied to investigate The development of post-surgical tumor immunity [1] was interrogated in the response to anti-PD-1 therapy. Multiple human RCC datasets the CD8+ T cell-restricted Neuropilin-1 (Nrp1)-deficient mice (E8ICreNrp1L/ (TCGA, IMmotion150 and ICGC), a murine pre-malignant dataset and L), by surgically removing the primary B16F10 tumor followed by re- a Renca tumor dataset were used to perform gene signature enrich- challenge 30- or 60-days post resection. The synergy between CD8-specific ment analysis (GSEA). Immunohistochemistry and Multiplex Opal Im- Nrp1 deficiency and anti-PD1 blockade was investigated with the MC38 munofluorescence were performed to assess immune cell infiltration. tumor model. In a competitive setting, the Nrp1–/– and Nrp1+/+ pMel-T Real-time PCR, western blot, ELISA and chromatin immunoprecipita- cells [2] were co-transferred into the same host (CD45.1) followed by tion (ChIP) were used to study the activity of the interferon gamma gp100-B16 tumor inoculation, where the in vivo long-term persistence of signaling pathway. the donor cells was assessed. The transcriptomic modulation by Nrp1 defi- Results ciency was studied using bulk population RNA sequencing (bpRNAseq) on Pbrm1 knockout in murine RCC Renca cells impaired the binding of the pMel-T cells (Nrp1–/– vs. Nrp1+/+) recovered from various phase of brahma-related gene 1 (BRG1), the adenosine-triphosphate- in vivo activation (effector, memory and recall). Lastly, the physiological rele- dependent enzyme subunit of the SWI/SNF complex, to the promoter vance of NRP1 expression on CD8+ T cells in cancer patients was studied of IFN gamma receptor 2 (Ifngr2) and reduced Ifngr2 expression. in a cohort of peripheral blood leukocyte (PBL) samples from treatment- PBRM1/Pbrm1 deficiency impaired IFN gamma-induced phosphoryl- naïve patients with head and neck squamous cell carcinoma (HNSCC). ation of Janus kinase 2 (JAK2) and STAT1, and the subsequent ex- Results pression of downstream target genes involved in tumor The E8ICreNrp1L/L mice exhibited substantially enhanced protection from microenvironment (TME) modulation, such as Cxcl9, Icam1, Irf1, and tumor re-challenge, despite unchanged primary tumor growth. Enhanced Stat1 itself. In both human and murine RCC tumors, PBRM1/Pbrm1 responsiveness to anti-PD1 immunotherapy was also observed. NRP1 was loss was associated with lower expression of immune-related profiles co-expressed with multiple inhibitory receptors (IRs) on CD8+ T cells and and reduced T cell infiltration. PBRM1/Pbrm1 loss tumors also restrained memory differentiation and development by repressing an Id3- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 162 of 272 dependent transcriptional program. NRP1 was also highly expressed on medications received before recruitment to INT-NA had significant the exhausted CD8+ T cells found in the HNSCC patients and negatively relevance in the outcome. Naïve patients or previously challenged associated with the size of memory T cell pool and disease prognosis. by Immunotherapy had the best clinical benefit compared to Conclusions those receiving first line BRAF/MEK inhibitors (RR 40% vs 10%; p= These data reveal NRP1 as a unique “immune memory checkpoint” 0.001). Again, after disease progression treatment with target ther- with a mode of action that is distinct from other immune check- apy in naïve and immunotherapy previously treated patients, points. NRP1 blockade may promote the establishment of long-term showed a different beneficial pattern compared to patients re- T cell memory that is essential for durable anti-tumor immunity. ceived BRAF/MEK inhibitors before immunotherapy. The clinical parameters correlated with an increase of clinical benefit were References genus (female vs male), age (older [+60] vs younger), LDH score 1. Zhang, P., et al., Induction of postsurgical tumor immunity and T-cell mem- (normal vs high and very high), neutrophil/lymphocyte ratio (ele- ory by a poorly immunogenic tumor. Cancer Res, 2007. 67(13): p. 6468-76. vated vs normal). The introduction of an algorithm including the 2. Overwijk, W.W., et al., Tumor regression and autoimmunity after reversal of a clinical variables above mentioned could define four predictable functionally tolerant state of self-reactive CD8+ T cells. J Exp Med, 2003. 198(4): cohorts of benefit with a 95% of accuracy. p. 569-80. Conclusions Ethics Approval From the real-life analysis, we generated a simple algorithm that All animal experiments were performed in the American Association for the might drive clinical decision. Our finding clearly showed how previ- Accreditation of Laboratory Animal Care-accredited, specific-pathogen-free ous treatment impacted outcome: patients treated with iBRAF/iMEK facilities in Division of Laboratory Animal Resources, University of Pittsburgh after treatment with ICI showed better clinical outcome respect pa- School of Medicine (UPSOM). Animal protocols were approved by the tients treated with iBRAF/iMEK before treatment with ICI. Institutional Animal Care and Use Committees of University of Pittsburgh. Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) Acknowledgements electing to undergo treatment were offered the option to participate in the The study was supported by the Institutional Project "Ricerca Corrente" of University of Pittsburgh Cancer Institute (UPCI) protocol for research. All Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy patients signed an informed consent that was approved by the Institutional Review Board (IRB) of the University of Pittsburgh. References 1. Balar AV, Weber JS. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother 2017; 66(5):551–564.2. P300 2. Larkin J, Minor D, D’Angelo S et al. Overall survival in patients with Real World data analysis related to metastatic melanoma patients advanced melanoma who received nivolumab versus investigator’s treated with immunotherapy from 2012 to 2018 at Istituto choice chemotherapy in CheckMate 037: a randomized, controlled, Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy open-label phase III trial. J Clin Oncol 2018; 36(4): 383–390. Gabriele Madonna, Medical Biotechnology (LS) , Mariaelena Capone, 3. Schachter J, Ribas A, Long GV et al. Pembrolizumab versus ipilimumab 1 1 1 1 MD , Marilena Tuffanelli , Marcello Curvietto, PhD , Miriam Paone, PhD , for advanced melanoma: final overall survival results of a multicentre, 1 1 1 Assunta Esposito, PhD , Antonio Sorrentino , Marco Palla , Luigi randomised, open-label phase 3 study (KEYNOTE-006). Lancet 2017;390: 1 1 1 Scarpato , Domenico Mallardo, MD , Ester Simeone, MD , Antonio 1853–1862.4. 1 2 2 2 Grimaldi, MD , Kristina Viktorsson , Lisa Villabona , Rolf Lewensohn , 4. Larkin J, Chiarion-Sileni V, Gonzalez R et al. Combined nivolumab and ipi- 2 1 Giuseppe Masucci, MD, PhD , Paolo Antonio Ascierto, MD limumab or monotherapy in untreated melanoma. N Engl J Med2015; Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, Naples, Italy; 373: 23–34.5. Karolinska Institutet, Stockholm, Sweden 5. Robert C, Long GV, Brady B et al. Nivolumab in previously untreated Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) melanoma without BRAF mutation. N Engl J Med 2015; 372(4):320–330. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P300 6. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbé C, et al: Guidelines for the evaluation of immune therapy activity in solid tumors: Background immune-related response criteria. Clin Cancer Res 2009, 15:7412–20. Immuno checkpoint inhibitors (ICI) have improved the prognosis for Ethics Approval patients with advanced malignancy [1-5]. Their real-life application The study was approved by the internal ethics board of the Istituto may give different outcome compared to the benefit presented by Nazionale Tumori IRCCS Fondazione “G. Pascale” in Napoli Italy, approval clinical trials as the inclusion and exclusion criteria might be selective number of registry 33/17 and give overoptimistic survival rates. Here we present the analysis of cutaneous metastatic melanoma patients treated with check-point inhibitors at Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” P301 of Napoli Italy (INT-NA). Expression of tumor matrix metalloproteases ADAM10 and Methods ADAM17 correlates with low PD-L1 protein-to-mRNA ratio in We investigated retrospectively, from 2012 to 2018, 578 stage IV multiple tumors, predicting poor outcomes melanoma patients received ipilimumab, pembrolizumab or nivo- Aaron Mansfield, MD, Jacob Orme, MD PhD, Roxana Dronca, MD, lumab as monotherapy at the INT-NA. Ipilimumab was adminis- Haidong Dong, MD, PhD tered intravenously at the dosage of 3 mg/kg every 3 weeks for Mayo Clinic, Rochester, MN, United States four doses, pembrolizumab at the dosage of 200 mg every 3 Correspondence: Jacob Orme (orme.jacob@mayo.edu) weeks and nivolumab at the dosage of 3 mg/kg every 2 weeks Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P301 until disease progression or unacceptable toxicity appeared. Dis- ease evaluation was performed at baselineand then every 12 Background weeks until progression or the discontinuation of treatment ac- ADAM10 and ADAM17 portend poor prognosis in many malig- cording to the Response Evaluation Criteria in Solid Tumors nancies [1]–[6]. We previously showed these proteases cleave Pro- (RECIST 1.1) [6]. Survival analysis was performed using the Kaplan grammed death-ligand 1 (PD-L1) from tumors in soluble form Meier method and with the log-rank test. Cox regression was (sPD-L1) [7]. sPD-L1 engages immune cell Programmed death 1 used in the univariate and multivariate analysis. The results were (PD-1) to inhibit tumor immunity. It is unknown how broadly this considered significant if p mechanism occurs in solid malignancies. We hypothesized (1) Results ADAM10 and/or ADAM17 may be elevated in tumors with low Patients treated at INT-NA with nivolumab and pembrolizumab PD-L1 protein despite high PD-L1 (CD274) mRNA and (2) this low showed comparable better clinical benefit toward patients treated tumor PD-L1 protein-to-mRNA ratio may predict lower overall with ipilimumab (RR 44.5% vs 20.7%; p=0.01). The anti-tumoural survival. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 163 of 272 Methods immunity – a resistance mechanism to PD-1 checkpoint blockade We queried the Cancer Genome Atlas (TCGA) for all solid tumors in melanoma,” in CRI-CIMT-EATI-AACR International Cancer Confer- with Level 3 reverse phase protein array (RPPA) PD-L1 protein ence, 2018. levels and RNA-seq sequence per million mapped fragments Ethics Approval (FPKM) PD-L1 (CD274), ADAM10, and ADAM17 mRNA levels. We Lab studies involving human subjects are approved by Mayo Clinic‘s calculated a PD-L1 protein-to-mRNA ratio for each sample. Groups Institutional Review Board (IRB), approval number 15-000934. of high and low PD-L1 protein-to-mRNA ratios were evaluated for (1) ADAM10 and ADAM17 expression and (2) overall survival by Cox proportional hazards modeling, adjusting for age and stage at diagnosis. Results Tumor samples demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly more ADAM10 and/or ADAM17 in 23 of 25 cancer types (Table 1). Cox proportional hazards ratios for Table 1 (abstract P301). See text for description death in each group were calculated and reported as a forest plot including hazard ratios and 95% confidence intervals for overall survival for each cancer subtype adjusted for patient age and tumor stage (Figure 1). Patients with tumors demonstrating low PD-L1 protein-to-mRNA ratio experienced significantly worse outcomes in 8 of 25 tumor types and improved outcomes in 2 tumor types (Table 2). Conclusions In this work we report that reduced human PD-L1 protein-to-mRNA ratios are associated with (1) high ADAM10 and/or ADAM17 expres- sion and (2) poor outcomes in multiple cancers. We previously showed in multiple cell lines that ADAM10 and ADAM17 cleave PD- L1 from the surface of tumor cells [7]. Our results suggest that ADAM10 and/or ADAM17 may cleave PD-L1 to cause a low PD-L1 protein-to-mRNA ratio in these tumors. This process may explain poorer survival of patients with low PD-L1 protein-to-mRNA ratios in some cancers. Our findings may explain why some tumors that do not have detect- able PD-L1 expression on immunohistochemistry respond to PD-(L)1 inhibitor therapy given the solubilization of PD-L1 and its subsequent systemic negative regulatory effects on T cells. Further, ADAM10/ ADAM17 inhibition may prevent PD-L1 shedding and sensitize tu- mors to therapy. While this work is correlative in nature, studies ex- ploring this mechanism of tumor immune system evasion are ongoing. Table 2 (abstract P301). See text for description Acknowledgements The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. References 1. M. Uhlen et al., “A pathology atlas of the human cancer transcriptome.,” Science, vol. 357, no. 6352, p. eaan2507, Aug. 2017. 2. P. C. Buchanan et al., “Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17.,” J. Biol. Chem., vol. 292, no. 15, pp. 6339–6351, Apr. 2017. 3. M. E. Powers, H. K. Kim, Y. Wang, and J. Bubeck Wardenburg, “ADAM10 Mediates Vascular Injury Induced by Staphylococcus aureus α- Hemolysin,” J. Infect. Dis., vol. 206, no. 3, pp. 352–356, 2012. 4. S.-S. Ni, J. Zhang, W.-L. Zhao, X.-C. Dong, and J.-L. Wang, “ADAM17 is overexpressed in non-small cell lung cancer and its expression correlates with poor patient survival,” Tumor Biol., vol. 34, no. 3, pp. 1813–1818, Jun. 5. Y.-Y. Wang, Z.-Y. Ye, L. Li, Z.-S. Zhao, Q.-S. Shao, and H.-Q. Tao, “ADAM 10 is associated with gastric cancer progression and prognosis of patients,” J. Surg. Oncol., vol. 103, no. 2, pp. 116–123, Feb. 2011. 6. B. You, Y. Shan, S. Shi, X. Li, and Y. You, “Effects of ADAM10 upregulation on progression, migration, and prognosis of nasopharyngeal carcinoma,” Cancer Sci., vol. 106, no. 11, pp. 1506–1514, Nov. 2015. 7. J. J. Orme et al., “Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 164 of 272 “responders” and “non-responders” to ICI drugs and is also being used to screen co-stimulatory agonists, peptide biologics and other drug classes. Conclusions Taken together, our data indicate that the recall antigen potency assay we established is highly potential to screen the drug candidates for im- mune checkpoint inhibitor and enhancer drug candidates. P303 PD-1 checkpoint blockade in advanced melanoma patients: Neutrophils, NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates Yago Pico de Coaña, Maria Wolodarski, MD, Irene van der Haar Àvila, Takahiro Nakajima, Stamatina Rentouli, Andreas Lundqvist, PhD, Giuseppe Masucci, MD, PhD, Johan Hansson, Rolf Kiessling, MD, PhD Karolinska Institute, Stockholm, Sweden Correspondence: Yago Pico de Coaña (yago.pico.de.coana@ki.se) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P303 Background Blockade of the PD-1 receptor has revolutionized the treatment of meta- static melanoma, with significant increases in overall survival and a dra- matic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of benefitting patients is limited and there is a need for predictive biomarkers and a deeper mechanistic ana- lysis of the cellular populations involved in a clinical response. Methods Fig. 1 (abstract P301). See text for description With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients undergoing treatment with pem- P302 brolizumab (n=7) or nivolumab (n=30) treatment at Karolinska Uni- A reversible T cell exhaustion-like in vitro assay to screen versity Hospital. Blood samples were collected from patients at the candidate drugs following time points: Before treatment and at the time of the sec- 1 2 2 Wushouer Ouerkaxi , Eden Kleiman , Pirouz Daftarian ond and fourth doses. Peripheral blood mononuclear cells (PBMCs) 1 2 MBL International, Woburn, MA, United States; JSR Lifesciences, were isolated by density gradient centrifugation and stained for flow Sunnyvale, CA, United States cytometric analysis within two hours of sample collection. Correspondence: Pirouz Daftarian (pdaftarian@jsrlifesciences.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P302 Two distinct cellular populations were inversely correlated with survival: Neutrophils, and Monocytic myeloid derived suppressor cells (MDSCs). Background Furthermore, overall survival and progression free survival were also T cells undergo different layers of suppression, with a spectrum of events found to be inversely correlated with the activation status of NK cells. that may overlap such as dysfunction, anergy, unresponsiveness, toler- Finally, PD-L1 expression in different monocytic subsets was signifi- ance, and exhaustion. Many factors have been attributed to this including cantly increased in patients with shorter progression free survival and multiple co-inhibitory receptor surface expression, altered transcription was correspondingly correlated inversely with overall survival. factor expression, epigenetic rewiring and dysregulated metabolism. Anti- Conclusions gen persistence is necessary for driving TEX maintenance in both the Our results suggest that cellular populations other than T cells can chronic viral infection setting and cancer. In addition to persistent antigen be critical in the outcome of PD-1 blockade treatment. Specifically, exposure, tumor-infiltrating lymphocytes (TILs) within the tumor micro- the frequencies of activated NK cells and monocytic MDSCs are in- environment (TME) encounter numerous tumor-mediated immunosup- versely correlated with survival and clinical benefit and their role as pressive metabolic byproducts, suppressive cytokines, hypoxia and predictive biomarkers should be further evaluated. cellular debris which converge to suppress T cell function and uniquely Ethics Approval alter is transcription factor profile. These suppressed or dysfunctional T The protocol was approved by the local Ethics Committee and the In- cells are incapable of mounting an optimal anti-tumor response in part stitutional Review Board at Karolinska Institute (approval number due to lack of fitness in competing for glucose and oxygen. Our team 2015/1862-32) and all patients provided written informed consent in has utilized the recall antigen potency assay as a tool for function-based accordance with the Declaration of Helsinki. screening of immune checkpoint inhibitor (ICI) drug candidates. Methods P304 A recall antigen potency assay has been used as a tool for function- DNA damage response gene alterations are associated with high based screening of immune checkpoint inhibitor (ICI) drug candidates. In tumor mutational burden and clinical benefit from programmed this assay, healthy human PBMCs are stimulated with peptide(s) derived death 1 axis inhibition in non-small cell lung cancer from either viruses or tumor proteins and grown in culture for one week. 1 1 1 Biagio Ricciuti, MD , Gonzalo Recondo, MD , Renato Umeton , Giuseppe Day 4 supernatants are functionally assayed by ELISA for IFN-γ secretion 1 2 2 1 Lamberti, MD , Mizuki Nishino , Lynette Sholl , Michael Cheng , Mark and cells are assayed on day 7 by flow cytometry for CD8+ or CD4+ T cell Awad, MD PhD expansion by using a single or cocktail of pMHC tetramers. 1 2 Dana Farber Cancer Institute, Boston, MA, United States; Brigham and Results Women’s Hospital, Boston, MA, United States We have shown that in roughly 30% of donor PBMCs, ICI drugs such as Correspondence: Biagio Ricciuti (biagio_ricciuti@dfci.harvard.edu) pembrolizumab are able to boost both IFN-γ secretion and antigen-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P304 recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory re- Background ceptor downstream signaling is ameliorated with ICI drugs releasing these DNA damage response (DDR) gene alterations are associated with in- T cells from the repressive effects of these co-inhibitory receptors. Further, creased tumor infiltrating lymphocytes, higher genomic instability, this assay has the potential to screen for donors who are in vitro Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 165 of 272 and higher tumor mutational burden (TMB) in cancer. Whether DDR Table 1 (abstract P304). See text for description alterations are associated with benefit from immune-checkpoint in- hibitors (ICIs) in non-small cell lung cancer (NSCLC) is unknown. Methods Clinicopathologic and genomic data were collected from patients (pts) with advanced NSCLC at the Dana-Farber Cancer Institute (DFCI) treated with PD-(L)1 inhibitors. Targeted next-generation sequencing (NGS) by OncoPa- nel was used to determine DDR gene mutation status and TMB. Patients were categorized based on the presence or absence of deleterious DDR gene alterations in a panel of 53 DDR genes. All loss-of-function alterations in DDR genes (including nonsense, frameshift, or splice site) were classified as pathogenic. Missense mutations were evaluated using the Catalogue of Somatic Mutations in Cancer (COSMIC) [1], and ClinVar databases [2], as well as the PolyPhen-2 (Polymorphism Phenotyping v2) functional predic- tion tool [3]. Missense mutations were classified as pathogenic if annotated as pathogenic by either COSMIC or ClinVar and damaging by Polyphen-2. Because only tumor tissue was sequenced, common single nucleotide poly- morphisms (SNPs) were filtered if present at ≥0.1% in Genome Aggregation Database (gnomAD) version 2.1.1 [4]. Clinical outcomes to immunotherapy were evaluated according to DDR mutation status. Results Among 256 pts with successful NGS who received ICIs, 134 (52.3%) were identified as having deleterious DDR mutations (DDR-positive). DDR-positive and DDR-negative groups were well balanced in terms of baseline clinico- pathological characteristics (Table 1). The median TMB was significantly higher in the DDR-positive group compared to the DDR-negative group (12.17 vs 8.36 mutations/megabase, P< 0.0001), as well as among never smokers (9.40 versus 5.70 mut/Mb, P = 0.035, Figure 1B). Compared to DDR-negative pts (N=122), DDR-positive pts had a significantly higher ob- jective response rate (28.6% vs 16.4%, P=0.025, Figure 2A), longer median progression-free survival (4.2 vs 2.2 months, HR: 0.64 [95%CI: 0.49-0.84], P= 0.001, Figure 2B) and overall survival (17.5 vs 9.9 months, HR: 0.60 [95%CI: 0.43-0.82], P=0.002, Figure 2C) with PD-(L)1 therapy. DDR-positive status was associated with significantly longer PFS (HR: 0.70 [0.51-0.95], P=0.024) and OS (HR: 0.61 [95%CI: 0.43-0.85], P=0.004) in multivariate analysis (Table 2). Conclusions Deleterious DDR alterations are frequent in NSCLC and are associated with higher TMB and improved clinical outcomes in NSCLC pts treated with PD-1 axis inhibition. References 1. Forbes SA, Beare D, Boutselakis H, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic acids res. 2017; 45:D777-D783. 2. Landrum MJ, Lee JM, Benson M, et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic acids res. 2016; 44:D862-D868 3. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat. Methods. 2010; 7:248 4. https://gnomad.broadinstitute.org Fig. 1 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 166 of 272 Table 2 (abstract P304). See text for description Response to Pembrolizumab and tumor microenvironment composition is associated with IL8 expression in a head and neck squamous-cell carcinoma cohort 1 2 2 1 Arun Khattri, PhD , Jason Reeves , SuFey Ong , Riyue Bao, PhD , Arya 2 1 2 Bahrami, PhD , Yi-Hung Carol Tan, PhD , Andrew White, BSc , Michael 2 2 2 Bailey , Heather Brauer, PhD , Sarah Warren, PhD , Joseph Beechem, 2 3 PhD , Tanguy Seiwert, MD 1 2 University of Chicago, Chicago, IL, United States; NanoString Technologies, Seattle, WA, United States; Johns Hopkins University, Baltimore, MD, United States Correspondence: Tanguy Seiwert (tseiwert@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P305 Background A subset of head and neck squamous-cell carcinomas are known to re- spond to immune checkpoint inhibitors. To better understand response to therapy in these tumors, a cohort of samples treated with Pembrolizumab was examined to determine if specific cell types are associated with re- sponse to intervention by combined profiling of standard bulk expression assays, ISH staining, and spatially resolved multiplexed protein analysis. Methods RNA was extracted from archival FFPE samples (n = 107) col- lected prior to therapeutic intervention and profiled using the NanoString® nCounter® PanCancer IO 360™ panel (Research Use Only). Gene expression signatures were calculated for immune cell subsets, as well as the Tumor Inflammation Signature (TIS; Ayers 2017 JCI). Individual gene expression and signatures were compared to patient outcome. Subsequently, expression of IL8 was validated by RNAScope in a subset of responders and non- responders (n = 9). To determine whether the IL8 staining pat- tern observed was consistent with specific cell types, a set of six tumor samples (three IL8+ and three IL8-) were further character- ized by multiplexed protein expression analysis on the GeoMx™ digital spatial profiling (DSP) platform to quantitate expression of 40 antibodies. IL8 staining was used to guide DSP selection of re- gions of interest (ROI) within the tumors that were either IL8+ or IL8-. Protein expression was specifically measured from tumor or stromal areas based on Pan-cytokeratin immunofluorescence. Results Initial analysis of the head and neck cohort found that previously reported signatures of response, including TIS, were not associ- ated with patient outcome in this cohort. In contrast, IL8 expres- sion was observed to be highest in patients with progressive disease. Further investigation demonstrated that IL8 expression was most specifically associated with neutrophil markers/expres- sion signatures. DSP profiling confirmed that tumor and stromal segments from IL8+ regions were associated with high expression of CD66B and ARG1 and lower expression HLA-DR consistent with neutrophil/granulocytic MDSCs presence. Furthermore, these re- gions were shown to have lower expression of T-cell markers in- cluding CD3, CD8 and CD4. Conclusions These results demonstrate that, in addition to previously reported biomarkers, IL8 expression and neutrophil presence may be related to response to checkpoint therapy in head and neck cancers. De- creased T-cell marker expression in IL8+ regions may reflect de- creased response in the larger cohort. Ethics Approval The study was approved by the University of Chicago‘s Ethics Board, approval number 8980 and 16-1269 P306 Innate immune cells play a role in therapy resistance to anti-PD1 in Hu-mice melanoma model Raj Somasundaram, PhD, Meenhard Herlyn, DVM PhD P305 The Wistar Institute, Philadelphia, PA, United States Correspondence: Raj Somasundaram (shyam@wistar.org) Fig. 2 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P306 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 167 of 272 Background constituted the majority of cases in both groups. The overall re- Immune checkpoint inhibitor therapy is rapidly emerging as a front- sponse rates of the CPI+M group were significantly higher than those line treatment option for many solid tumors. However, only a third of treated with CPI only (75% vs 53.3%, p = 0.05). melanoma patients respond to immune checkpoint blockade. Cur- Conclusions rently available mouse models have many short comings and are un- The data from this chart review shows an apparent benefit with respect able to address the basis of therapy resistance and immune non- to overall response rates in patients treated with M and CPIs. The cohorts responsiveness that are observed in patients. remain small in this study, but the data is significant. These results align Methods well with data from mouse studies and two small retrospective human Our laboratory has developed a novel humanized mouse melanoma studies in melanoma [2] and lung cancer [3], hence, demonstrating an in- model. Immuno-deficient NSG mice were reconstituted with human creased immune response and higher response rates. Currently, we are CD34+ cells and after 8-12 weeks, mice are fully reconstituted with hu- expanding our cohort number and data-set as we gained access to a big- man innate and adaptive immune cells. Humanized mice were then ger data warehouse. More prospective data are awaited from an ongoing challenged with HLA-matched melanoma cells and the functional abil- phase Ib (UMIN000028405), and phase II (NCT03800602, NCT03048500) ity of human immune cells to restrict tumor growth was monitored. trials that should shed more light on the effects of metformin on Results immuno-oncologic therapies. Restricted tumor growth was observed in humanized mice indicating in vivo sensitization of human immune cells to melanoma. References In therapy studies, tumor-bearing humanized mice treated with anti- 1. Scharping N, Menk A, Whetstone R, Zeng X, Delgoffe G. Efficacy of PD-1 PD-1 showed restricted tumor growth. Anti-PD-1 therapy resulted in blockade is potentiated by metformin-induced reduction of tumor hyp- enhanced infiltration of T-cells that correlated with tumor response. oxia. Cancer Immunol Res. 2017;5(1):9-16. MassCyTOF studies was performed using a panel of immune markers 2. Afzal M, Mercado R, Shirai K. Efficacy of metformin in combination with to understand the mechanism of therapy non-responsiveness in immune checkpoint inhibitors (anti-PD-1/anti-CTLA-4) in metastatic some tumors. Results indicated downmodulation of HLA-class I mole- malignant melanoma. J Immunother Cancer. 2018;6(1):64. cules and increased presence of mast cells cells in the tumor region. 3. Afzal M, Dragnev K, Sarwar T, Shirai K. Clinical outcomes in non-small-cell In tumor-bearing mice, combination of therapy drugs targeting c- lung cancer patients receiving concurrent metformin and immune kit+ mast cells and anti-PD1 caused complete regression of tumor le- checkpoint inhibitors. Lung Cancer Manag. 2019:1-12 (online publication). sions. Tumor free mice were able to reject freshly challenged melan- Ethics Approval oma cells indicating the presence of memory T-cell responses. The study was approved by Louisiana State University Health Science Center Conclusions of Shreveport’s Institutional Review Board separately for each site, IRB Our results suggest that humanized mouse melanoma model can be numbers are STUDY00000891, and STUDY00001017. explored further to understand the therapy resistance mechanisms to immune-based treatments. Further, model will be useful for devel- P308 oping new therapeutic strategies for treating melanoma patients. The impact of obesity on the response rates of checkpoint inhibitor (CPI) cancer immunotherapy 1 2 P307 Philip Haddad, MD, MPH , David Sommerhalder, MD The impact of metformin (M) on the response rates of checkpoint Overton Brooks VA Medical Center/LSUHSC-S, Shreveport, LA, United inhibitors (CPI) States; Louisiana State University Health Science, Bossier City, LA, United 1 2 Philip Haddad, MD, MPH , David Sommerhalder, MD States 1 2 Overton Brooks VA/ LSUHSC, Shreveport, LA, United States; Louisiana Correspondence: Philip Haddad (haddad8838@msn.com) State University Health Science, Bossier City, LA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P308 Correspondence: Philip Haddad (haddad8838@msn.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P307 Background With durable responses, improved clinical benefit, and relatively fewer Background toxicities, CPIs targeting cytotoxic T-lymphocyte-associated protein 4 Immunotherapies in oncology have brought significant change and (CTLA4), programmed death-1 (PD-1), and its ligand (PD-L1) have estab- hope to the field over the last several years. Responses, however, re- lished themselves as essential components of cancer immunotherapy main unpredictable and low in most cases. One of the aspects thought across multiple cancer types. Obesity is a known risk factor for several to be limiting immunotherapies is the tumor microenvironment which cancer types. It is associated with increased progression and cancer- favors tumor growth and immunosuppression. It has been hypothe- related death. This is thought to be the result of inflammaging and PD- sized that dysregulated tumor metabolism creates a hypoxic tumor 1 mediated immune suppression. Recently, two large retrospective microenvironment which acts as a barrier to antitumor immunity. Met- studies found that obesity conferred a survival advantage for cancer formin has been shown to reduce oxygen consumption and subse- patients treated with CPIs which may be independent of sex [1,2]. How- quently reduce the microenvironment hypoxia, leading to improved ever, the mechanistic explanation of this observed obesity paradox, as- response rates of checkpoint inhibitors in murine in vitro and in vivo suming it is real, has been the subject of many scientific conjectures. models [1]. We performed a retrospective review to evaluate response These studies focused on the impact of obesity on CPI overall survival rates in our patients who were treated with CPI+M. which can be affected by many confounding variables. Instead, we ex- Methods plored the effect of obesity on CPI response rates. We reviewed all adult cancer patients who were treated with a CPIs. Pa- Methods tients treated with M and CPIs were compared to those who were We retrospectively reviewed every cancer patient that received CPIs at treated with CPIs only. All tumor types and all CPI drugs were included. Overton Brooks VA Medical Center (OBVAMC) between 2015 and 2019. The primary endpoint was overall response rate, which included stable Patient’s BMI scores at the beginning of CPI therapies were calculated. disease, partial response, and complete response. Additional data was Based on the WHO definition, the patients were grouped according to captured for subgroup analysis. Patients were excluded if they had never their BMIs into overweight and obese (Group A) versus normal and received the treatment, or if they were never assessed for response. underweight (Group B). Our primary outcome of interest was defined as Results the presence or absence of CPI response. Patients who attained stable As of this date, 144 patients had been included in the study data-set. disease, partial response, and complete response were categorized as re- Of those, 24 patients were treated with M and CPIs and 120 patients sponders. Those who progressed on CPI were labeled as non-responders. received CPIs only. Both groups were comparable with respect to Thesignificanceofthe associationbetween the grouped BMI categories sex. However, the M+CPI group was slightly older. Lung cancer and the occurrence of any response was analyzed statistically. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 168 of 272 Results Results Between 2015 and 2019, 65 patients were treated with CPIs and had In the neoadjuvant ICB cohort, increasing age correlated with better documented responses. Both groups were comparable with respect to response to immunotherapy (OR= 0.91, P=0.039) and remained a sig- age, sex, race, and types of CPIs. Lung cancer constituted the majority nificant predictor of response in the multivariable model (OR=0.88, of cases in both groups. Head and neck cancers were more prevalent P=0.03) after adjusting for sex, tumor site, stage, prior therapies, and in Group B while renal and bladder cancer and melanoma were more toxicity; similar results were observed in the OpACIN neoadjuvant prevalent in Group A. Group B had a significantly higher response rate trial study (NCT02977052). Tumor mutational load was positively cor- (80% vs 50%, p=0.01). Furthermore, a higher response rate was ob- related with age (r=0.4) but did not reach statistical significance (P= served between normal BMI and overweight patients (p=0.001) and 0.1). Differential gene expression analysis revealed genes associated normal BMI and obese patients (p=0.06). No difference in response with MHC class II antigen expression (HLA-DQB1, HLA-DRB1) as sig- rates was observed between underweight and obese patients. nificantly overexpressed in younger patients upon treatment with Conclusions ICB. Furthermore, MHC class II regulator interferon-gamma signaling This is the first report to show a detrimental effect of overweight was also observed upregulated in younger patients (P=0.03). PD-1 ex- and obesity on CPI response rates in a retrospective cohort of pression (by IHC) was lower in older patients upon treatment with non-selected consecutive cancer patients in a real-world clinical ICB (r=-0.47, P=0.035). In keeping with human cohort, mice injected setting. with Yumm1.7 melanoma cells showed significant increase in im- mune cell populations expressing MHC class II antigen in the young References mice versus aged mice (P=0.0048). 1. McQuade J, et al. Association of body-mass index and outcomes in pa- Conclusions tients with metastatic melanoma treated with targeted therapy, immuno- Increased age was associated with improved outcomes in melanoma therapy, or chemotherapy: a retrospective, multicohort analysis. Lancet patients receiving neoadjuvant ICB, similar to results in stage IV. The Oncol. 2018;19(3):310-322. mechanisms behind this association are likely multifactorial, and may 2. Xu H, Cao D, He A, Ge W. The prognostic role of obesity is independent relate in part to MHC class II antigen expression mediated immune of sex in cancer patients treated with immune checkpoint inhibitors: A evasion in younger patients, though further studies are needed to pooled analysis of 4090 cancer patients. Int Immunopharmacol. delineate contributing factors. 2019;74:1-10. Trial Registration Ethics Approval NCT02519322 The study was approved by the Louisiana State University Health Science Ethics Approval Center of Shreveport Institutional Review Board, IRB number This trial was approved by the MD Anderson Cancer Center Institu- STUDY00001017. tional Review Board. The trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and with adherence to the Good Clinical Practice guidelines, as defined by the Inter- P309 national Conference on Harmonization. This protocol was conducted Older age predicts better outcome to neoadjuvant immune with compliance with all relevant ethical regulations. checkpoint blockade in metastatic melanoma Consent 1 2 1 Rohit Thakur , Stephen Douglass, PhD , Beth Helmink, MD PhD , Rodabe Written informed consent was obtained from all participants. The MD 1 1 1 Amaria, MD , Hussein Tawbi, MD, PhD , Jennifer McQuade , Eliza Anderson Data Safety Monitoring Board reviewed the data at 12- 3 1 4 Rozeman , Elizabeth Burton , Sangeetha Reddy, MD, MSci , John month increments. 5 3 6 Wherry , Christian Blank, MD PhD , Georgina Long , Jeffrey Gershenwald, 1 1 1 MD , Michael Davies, MD, PhD , Michael Tetzlaff, MD PhD , Ashani 7 1 Weeraratna , Jennifer Wargo, MD, MMSc P310 1 2 MD Anderson Cancer Center, Houston, TX, United States; The Wistar Optimal priming prevents the induction of dysfunctional CD8 T- Institute, Philadelphia, PA, United States; The Netherlands Cancer cells in subprimed conditions, reversing resistance to anti-PD-1 Institute, Amsterdam, Netherlands; UT Southwestern Medical Center, Vivek Verma, PhD, Rahul Nandre, PhD, Jose Lopez, Seema Gupta, PhD, Houston, TX, United States; University of Pennsylvania, Philadelphia, PA, Samir Khleif, MD 6 7 United States; Melanoma Institute Australia, Sydney, Australia; Johns Georgetown University Medical Center, Washington, DC, United States Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Samir Khleif (snk48@georgetown.edu) Correspondence: Jennifer Wargo (JWargo@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P310 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P309 Background Background Suboptimal-priming of lymphocytes by low-affinity antigens is a mechan- There is a growing appreciation for studying the impact of host and ism for prevention of generation of strong immune responses against environmental factors on response to immune checkpoint blockade self-antigens. However, we recently found that the suboptimally-primed (ICB). Recently it was reported that older age correlated with better CD8 cells have a pre-disposition to develop a dysfunctional phenotype outcome in stage IV melanoma patients treated with ICB. We exam- that is marked by expression of CD38 on PD1+CD8+T-cells. Interestingly, ined the association of age with response to ICB and anti-tumor im- the number of these dysfunctional cells were significantly increased upon munity in melanoma patients treated neoadjuvantly (NCT02519322) PD-1 blockade in these suboptimally-primed CD8 cells that served as a and interrogated underlying mechanisms in a murine model. reason for resistance to anti-PD-1 therapy. On the other hand, anti-PD-1 Methods blockade of optimally-primed CD8 cells did not generate these dysfunc- Tumor samples were obtained from neoadjuvant ICB trial patients tional cells and led to cell activation and generation of effector functions pre-treatment, on-treatment and on-surgery. Transcriptome profil- [1]. Hence, here we investigated the effect of optimal-priming on revers- ing was performed using Illumina NextSeq platform and whole ing the resistance to anti-PD-1 therapy. exome sequencing using Illumina HiSeq 2500 platform. Univari- Methods able and multivariable analyses were performed using logistic re- Mice were inoculated with TC-1 cells (a mouse lung epithelial cell- gression modeling. Immune profiling was performed by line, expressing human papillomavirus-specific E7-peptide) in the Immunohistochemistry (IHC). The effects of age on anti-tumor im- presence or absence of concomitant priming with gp100 peptide, a munity were examined by implanting 2x105 Yumm1.7 cells sub- non-cognate tumor vaccine. Seven days later mice were treated with dermally in young (8wks) and aged (>12months) male mice. anti-PD-1 either alone or followed by combination of tumor-specific Tumors were harvested after 30 days of growth and immune sur- E7-peptide vaccine+anti-PD-1. Tumor growth rates, mice survival, face markers were analyzed by flow cytometry. and immune responses in the TME were estimated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 169 of 272 Results results in anti-tumor efficacy. TPST-1495 is a first-in-class, orally avail- We found that in suboptimally-primed TC-1 tumor-bearing mice, able, small molecule, selective dual antagonist of the human PGE2 anti-PD-1 treatment did not show any anti-tumor effects. Therefore, receptors EP2 and EP4, currently under development by Tempest. to check if the optimal priming of CD8 T-cells could reverse this re- Methods sistance, we vaccinated mice with gp100 at the time of tumor im- The effects of TPST-1495 as monotherapy or in combination with plantation with TC-1 cells. We found that compared to suboptimally- anti-PD1 were evaluated in the syngeneic mouse colon models CT26 primed mice, anti-PD-1 treatment of primed-mice resulted in signifi- and Apcmin/+. The mechanism of anti-tumor immunity of TPST-1495 cant retardation of tumor growth and a prolonged mice survival. was evaluated using in vitro primary dendritic cell (DC) differentiation Interestingly, tumor-specific vaccination (E7-peptide) of these and activation assays. Characterization of in vitro differentiated im- primed-mice at the time of anti-PD-1 treatment further enhanced the mune cells or tumor infiltrating lymphocytes were performed using therapeutic efficacy of PD-1 blockade. Moreover, in gp100-primed- flow cytometry. ELISA was used for measurement of cytokine mice we found a significant reduction in the number of PD-1+CD38hi production. dysfunctional cells compared to suboptimally-primed mice. The num- Results ber of these dysfunctional cells was further reduced upon anti-PD-1 Treatment with TPST-1495 reversed PGE2 immune suppression treatment of primed-mice. In addition, the priming state also affected in vitro and in vivo compared to antagonism of EP4 alone or all 4 EP the functionality of CD8 T-cells. Although gp100 alone prevented the receptors. TPST-1495 prevented PGE2 inhibition in vitro of DC differ- induction of these dysfunctional cells, the functionality of CD8 T-cells entiation and activation from human donor monocytes; single EP2 or was only increased when anti-PD-1 was given subsequent to priming EP4 antagonists were sub-optimal in this assay. Significantly, combin- with gp100 peptide. ation with EP1 and/or EP3 antagonists reversed the effect of dual Conclusions EP2 and EP4 blockade on PGE2 immune suppression, suggesting that Here we demonstrate that optimal priming of CD8 cells reverses re- COX-2 inhibition is not optimal for blocking the effects of PGE2. TPST- sistance to anti-PD-1 therapy. The suboptimal-priming of the CD8+ T- 1495 induced potent anti-tumor immune responses and significant cells induces higher numbers of dysfunctional PD-1+CD38hi CD8+ T- tumor regression as a monotherapy in two different murine tumor cells and their frequency further increases upon anti-PD-1 therapy, treatment models of colon cancer, CT26 and Apcmin/+. CT26 tumors leading to therapeutic failure. Since in most tumors, T-cells are analyzed from mice treated with TPST-1495 alone revealed a significant suboptimally-primed [2,3], our mouse data demonstrate the import- increase of infiltrating effector T cells. TPST-1495 combination with anti- ance of appropriately primed T-cells in responding to anti-PD-1 PD1 synergistically inhibited CT26 tumor progression. treatment. Conclusions TPST-1495 is a differentiated highly potent selective dual antagonist References of EP2 and EP4 that overcomes prostaglandin-mediated immune 1 Verma, V. et al. PD-1 blockade in subprimed CD8 cells induces dysfunc- suppression and promotes anti-tumor efficacy. tional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat Immunol, doi:10.1038/s41590-019-0441-y (2019). P312 2 Vonderheide, R. H. The Immune Revolution: A Case for Priming, Not Ipilimumab treatment immunophenotypic changes are associated Checkpoint. Cancer Cell 33, 563-569, doi:10.1016/j.ccell.2018.03.008 (2018). with progression of disease with sequential nivolumab therapy in 3 Vonderheide, R. H., Domchek, S. M. & Clark, A. S. Immunotherapy for metastatic melanoma Breast Cancer: What Are We Missing? Clin Cancer Res 23, 2640-2646, 1 1 2 David Woods, PhD , Andressa Sodre Laino, PhD , Aidan Winters , Jason doi:10.1158/1078-0432.CCR-16-2569 (2017). 1 1 1 Alexandre , Jeffrey Weber, MD, PhD , Pratip Chattopadhyay 1 2 NYU Langone Health, New York, NY, United States; UCSF, San P311 Francisco, CA, United States Dual antagonism of prostaglandin receptors EP2 and EP4 by TPST- Correspondence: Pratip Chattopadhyay 1495 suppresses tumor growth and stimulates anti-tumor (Pratip.Chattopadhyay@nyulangone.org) immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P312 1 2 2 Chan Whiting, PhD , Kim Fischer, PhD , Bryan Laffitte, PhD , Lisa 2 2 2 Rahbaek, PhD , Nick Stock, PhD , Davorka Messmer, PhD , Austin Chen, Background 2 2 2 1 PhD , Traci Olafson , Natalie Nguyen , Amanda Enstrom, PhD , Derek Nivolumab (nivo) and ipilimumab (ipi) combination immunotherapy has 1 1 3 Metzger , Brian Francica , Dingzhi Wang, PhD , Raymond Dubois, PhD, a ~60% response rate in metastatic melanoma patients. However, the im- 3 1 2 1 MD , Ginna Laport, MD , Peppi Prasit, PhD , Thomas Dubensky, PhD pact of these therapies on immune cell phenotypes and the relationship 1 2 Tempest Therapeutics, San Francisco, CA, United States; Inception of those changes to patient outcomes remains under-investigated. Sciences, San Diego, CA, United States; Medical University of South Methods Carolina, Charleston, SC, United States High dimension flow cytometry on baseline and at week 13 (e.g. Correspondence: Brian Francica (bfrancica@tempesttx.com) after initial nivo or ipi therapy) was performed for peripheral blood Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P311 samples from 33 metastatic melanoma patients receiving sequential nivo-ipi or the reverse sequence. We used a novel computational ap- Background proach to analyze the data through semi-comprehensive Boolean Progression of diverse malignancies is promoted by elevated levels gating in which immune cell lineages (e.g. CD3+CD4+) were evalu- of Prostaglandin E2 (PGE2). High PGE2 levels results from dysregula- ated for all possible combinations for up to 15 markers. tion of Cyclooxygenase-2 (COX-2), the enzyme that produces this Results lipid. PGE2 stimulates tumor cell proliferation, survival, evasion and 3,844 measured immunophenotypes were significantly altered post-nivo, metastasis along with host angiogenesis. PGE2 suppresses anti-tumor and 7,133 immunophenotypes were altered post-ipi. The frequency of immunity through inhibiting the function of critical immune effectors 584 immunophenotypes were significantly changed in both treatments, such as NK and T cells, and M1 macrophages, while promoting the with 59 of those changing in opposing directions. In the nivo-ipi cohort, activity of suppressive immune cells including myeloid derived sup- 260 baseline and 662 post-nivo immunophenotypes were significantly as- pressor cells, M2 macrophages, and regulatory T cells. PGE2 signals sociated with response and survival (outcomes). In the ipi-nivo cohort, 432 through a family of four homologous E-prostanoid (EP) G-coupled re- baseline and 668 post-ipi immunophenotypes were associated with out- ceptors, known as EP1, EP2, EP3 and EP4; which,are activated via dis- comes. Two highly similar immunophenotypes associated with outcomes tinct signal transduction pathways. Published literature and overlapped between the cohorts, CD14+CD11C+CD33+CD15-CD19-PDL1- experimental results presented here demonstrate that selective an- PDL2+CD163+GAL9-CD80-CD86-41BBL+CD40+OX40L+ cells. While lower tagonism of both EP2 and EP4 receptor signaling, but not EP1 and levels of these cells were associated with response and improved survival EP3, effectively overcomes PGE2-mediated immune suppression and in nivo-ipi treated patients, lower levels were associated with better Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 170 of 272 outcomes in the ipi-nivo treated patients. Of the 3,844 immunopheno- Specifically, these data show that changes in secretome production types altered post-nivolumab, 100 were also associated with ipi-nivo re- potential of T-cells after treatment with the PD1 blocking antibody sponse. Of these 100, 97% were altered in a manner positively associated nivolumab are associated with metastatic melanoma patient out- with response (e.g. upregulated by nivolumab and higher in responders). comes. We identified a signature of secreted molecules that were as- For example, nivo upregulated CD4+CD45RO-CCR7+ frequencies, which sociated with patient outcomes, providing rationale for targeting were associated with response and longer survival in ipi-nivo treated pa- these molecules to increase the efficacy of nivolumab. Work is under- tients. Of the 7,133 immunophenotypes altered post-ipi, 110 were also as- way to validate the observed associations in an independent set of sociated with nivo-ipi response. Of these, 95% were altered in a manner patient samples. negatively associated with response. This includes ipi associated upregula- Ethics Approval tion of a population of CD4+CD38+CD39+CD127-GARP- cells that are The study was approved by NYU Langone Health's IRB, approval negatively associated with coutcomes in nivo-ipi treated patients as well number S16-00035. as downregulation of a CD4+CD127+CD45RO+CD95+CCR7+ population of cells positively associated with outcomes. P314 Conclusions Alterations of DNA damage response signaling in the development These results demonstrate that nivo and ipi altered the peripheral im- of antibody-dependent cellular cytotoxicity (ADCC) resistance mune landscape in distinct ways. While immunophenotypic changes Louis Weiner, MD, Yongwei Zhang, Dalal Aldeghaither, David Zahavi, MS, BS post-nivo favored response in ipi-nivo treated patients, changes associ- Lombardi Cancer Center, Washington, DC, United States ated with ipi treatment favored progression with ipi-nivo. These data Correspondence: Louis Weiner (weinerl@georgetown.edu) suggest that the immunophenotypic impact of ipi alters the immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P314 landscape in a manner that may impair a subsequent response to nivo. These data also highlight several novel immune cell populations that Background are associated with both treatment effects and patient outcomes. Accumulating evidence has shown that DNA damage response Ethics Approval (DDR) is closely associated with immune response. Innate im- The study was approved by NYU Langone Health's IRB, approval mune responses, such as Natural killer (NK) cell-mediated killing, number S16-00035. are dependent on the DDR essential kinases Ataxia telangiectasia mutated (ATM) or ATM- and RAD3-related (ATR) [1]. However, P313 DNA damage inducing agents activate the NF-kappaB-regulated Single-cell secretome assessment of metastatic melanoma patient interferon immune response pathway [2]. Moreover, DDR inhib- peripheral T-cells reveals a pharmacokinetic signature of patient ition resulting from DNA repair deficiency or cell cycle check- response to nivolumab therapy point inhibition can potentiate efficacy of antibody-based David Woods, PhD, Andressa Sodre de Castro Laino, Daniel Freeman, immunotherapy, such as immune checkpoint blockade and ADCC Jeffrey Weber, MD, PhD, Pratip Chattopadhyay [3-5]. However, the mechanistic role of DNA damage response NYU Langone Health, New York, NY, United States signalinginthe developmentofimmunotherapy resistance re- Correspondence: David Woods (David.Woods@nyumc.org) mains unclear. A NK cell-mediated cetuximab-dependent killing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P313 in vitroADCCmodel wasusedtostudy this involvement. Our previous studies have suggested a loss of cell surface adhesion Background molecules in ADCC resistant cells [6]. Here we study the alter- Therapies targeting T-cell co-inhibitory molecules (e.g. PD1) have dem- ations of DNA damage response signaling in the process of onstrated unprecedented efficacy in the treatment of metastatic melan- ADCC resistance development. oma. However, not all patients respond to checkpoint inhibition, so Methods there is an unmet need to identify mechanisms of resistance/response. A431, a human epidermoid carcinoma cell line, develops resist- Methods ance to cetuximab and NK92-CD16v cell-mediated killing after Using IsoLight, a platform for assessing the secretion of 32 analytes 35 challenges of continuous exposure. Surviving cells following at single-cell resolution, we evaluated previously frozen peripheral each ADCC challenge were collected and studied for protein ex- blood T-cells from six responding and six progressing patients (ac- pression by western blot, immunofluorescence and flow cytome- cording to RECIST 1.1 criteria) treated with nivolumab. Baseline and try. Neutral comet assay was used to measure the level of DNA week 13, post-treatment CD4+ and CD8+ T-cell samples were double strand breaks. Fluorescence-based cytotoxicity methods assessed for each patient. T-cells were stimulated with CD3 and were used to determine the activity of ADCC. Apoptosis was CD28 activating antibodies overnight and subsequently placed on measured by Annexin-V-PI staining. Small interference RNA capture chips for 20 hours. Approximately 400 single-cell events were (siRNA) were used to knockdown gene expression. assessed for each sample. Results Results Levels of gammaH2AX, a marker of DNA damage signaling, CD4+ T-cells from progressing patients, compared to those from were significantly increased with exposure to ADCC, but no in- responding patients, had significantly (p<0.05) higher mean pro- creased DNA breaks were detected in ADCC resistant cells. p53, duction of IL-17F post-nivolumab and an increased proportion of phosphorylated-p53 and signal transducer and activator of tran- cells secreting IL-13, RANTES, IL-6, soluble CD137, TNF, MIP1a and scription 1 (STAT1) were also enhanced during the process of MIP1b relative to baseline. Using an elastic net machine learning ADCC resistance development. Interestingly, phosphorylated- algorithm with cross validation, we assessed the ability of a STAT1 reached a peak prior to the emergence of ADCC resist- manually curated list of analytes to predict patient outcomes. ance and then decreased until cells became entirely resistant. Delta values (post-treatment minus baseline values) were used for There was less apoptosis induction, no caspase activation, less 15 parameters. A receiver operating characteristic with an area induction of gammaH2AX and no activation of p53 in response under the curve of 0.898 was achieved. The most important fea- to ADCC in resistant cells. Inhibition of p53 or STAT1 by siRNA, tures in these models were the percentage of CD4+ T-cells ex- and ATM/ATR inhibitors enhanced ADCC activity in A431 cells pressing IL-6, MIP1a and soluble CD137 along with the but not in ADCC resistant cells.TP53knockdown activated percentage of CD8+ T-cells expressing IL-13. STAT1 in A431 cells, but reduced activation of STAT1 in ADCC Conclusions resistant cells. These results demonstrate the ability of single-cell, high-dimension Conclusions technologies coupled with machine learning to reveal complex asso- DNA damage response signaling was altered during the develop- ciations between immune cell function and clinical outcomes. ment of ADCC resistance, which is involved in ADCC activity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 171 of 272 regulation and might become be a signature of cell sensitivity in PD1/PD-L1 therapies. PD-L2 expression is observed in multiple tumor response to immunotherapy. Further DDR-related multiplex mech- types, and animal studies suggest PD-L2 may be involved in T-cell anisms are being investigated. suppression. However, the role of PD-L2 expression in the TME and the role as a predictive biomarker in immunotherapy is not as well References understood as PD-L1. Here, we investigated the expression of PD-L2 1. Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathway in multiple cellular components of the TME and its potential impact regulates innate immune system ligands of the NKG2D receptor. Nature. on the efficacy of PD-L1 blockade in cancer patients. 2005;436(7054):1186-90. Methods 2. Brzostek-Racine S, Gordon C, Van Scoy S, Reich NC. The DNA damage re- Single-cell RNA sequencing (scRNAseq) was performed using 10X sponse induces IFN. J Immunol. 2011;187(10):5336-5345. genomics for two commercial NSCLC samples and ~4,500 cells were 3. SenT,Rodriguez BL,ChenL,Corte CMD, Morikawa N, Fujimoto J, clustered by their expression pattern using shared nearest neighbor. Cristea S,Nguyen T, Diao L, Li L, Fan Y, Yang Y, Wang J, Glisson BS, Gene expression data for lung adenocarcinoma (LUAD) and squa- Wistuba II, Sage J, Heymach JV, Gibbons DL, Byers LA. Targeting mous carcinoma (LUSC) in TCGA were analyzed. Baseline tumor tran- DNA Damage Response Promotes Antitumor Immunity through scriptomes were profiled for 97 1L+ NSCLC patients treated with PD- STING-Mediated T-cell Activation in Small Cell Lung Cancer. Cancer L1 inhibitor durvalumab (NCT01693562). PD-L1 and PD-L2 immuno- Discov. 2019,9(5):646-661. staining was performed on baseline samples in CP1108. Gene expres- 4. Fenerty KE, Padget M, Wolfson B, Gameiro SR, Su Z, Lee JH, sion signatures for macrophage, fibroblast, dendritic cells, cancer Rabizadeh S, Soon-Shiong P, Hodge JW. Immunotherapy utilizing associated fibroblast (CAF) and interferon gamma were curated in- the combination of natural killer- and antibody dependent cellu- house or adopted from previous studies. lar cytotoxicity (ADCC)-mediating agents withpoly (ADP-ribose) Results polymerase (PARP) inhibition. J Immunother Cancer. 2018,6(1):133- In scRNAseq data, PD-L2 mRNA was found in over 10% of macro- 136. phage and fibroblast cells, and 5. Germano G, Lamba S, Rospo G, Barault L, Magrì A, Maione F, Russo M, Conclusions CrisafulliG, Bartolini A, Lerda G, Siravegna G, Mussolin B, Frapolli R, PD-L2 mRNA expression is mainly in immunosuppressive cellular Montone M, MoranoF, de Braud F, Amirouchene-Angelozzi N, Marsoni S, components of the TME in NSCLC, including macrophage and cancer D'Incalci M, Orlandi A,Giraudo E, Sartore-Bianchi A, Siena S, Pietrantonio associated fibroblast cells and low PD-L2 expression in PD-L1 high F, Di Nicolantonio F,Bardelli A. Inactivation of DNA repair triggers neoan- NSCLC patients may associate with improved OS. tigen generation and impairstumour growth. Nature. 2017,552(7683):116- Trial Registration 120. NCT01693562 6. Aldeghaither DS, Zahavi DJ, Murray JC, Fertig EJ, Graham GT, Zhang Ethics Approval YW,O'Connell A, Ma J, Jablonski SA, Weiner LM. A Mechanism of This study was conducted according to the Declaration of Helsinki Resistance to Antibody-Targeted Immune Attack. Cancer Immunol Res. and approved by the independent ethics committee/institutional re- 2019;7(2):230-243. view board at each participating center, with informed consent ob- tained from all patients. P316 MG1131, a novel TIGIT-targeted monoclonal antibody, induces T cell activation and anti-tumor immune response and suppresses Treg cell activity 1 1 2 1 Hyemi Nam, MS , Hye-Young Park , Eun Jung Song , Eunhee Lee , Hye 1 1 1 1 1 In Yum , Munkyung Kim , Jeewon Lee, Ph D , So Jung Lim , Okjae Lim , Yangmi Lim MOGAM Institute for Biomedical Research, Yongin-si, Korea, Republic of; GC Pharma, Yongin-si, Gyeonggi-do, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P316 Background Fig. 1 (abstract P314). Enhancement of DDR signaling T cell immunoreceptor with Ig and ITIM domain (TIGIT) is a co- pathway molecules inhibitory receptor expressed on CD8+ T cells, CD4+ T cells, NK cells, and regulatory T cells (Treg). TIGIT binds two ligands PVR (CD155) and PVRL2 (CD112) and these ligands are expressed by P315 T cells, APCs, and tumor cells. As malignancies progress, PVR The expression of programmed death ligand 2 (PD-L2) in over-expressed in tumor cells interacts with TIGIT expressed on immunosuppressive tumor microenvironment of non-small cell tumor infiltrating lymphocytes (TIL) and suppresses TIL activity lung cancer (NSCLC) and its potential association with by sending an inhibitory signal to immune cells, which is an im- immunotherapy 1 2 2 2 mune escape mechanism in cancer. In cancer, TIGIT blockade Qu Zhang, PhD , Stefan Bentink , Vinay Pawar , Farzad Sekhavati, PhD , 1 1 1 results in improved effector CD8+ T cell and NK cell function as Keith Steele, DVM, PhD , Jason Hipp , Song Wu, PhD 1 2 well as decreased Treg-cell-mediated suppression. Therefore, we AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, developed MG1131, a novel anti-TIGIT antibody, to modulate Germany the tumor microenvironment towards a more effective anti- Correspondence: Qu Zhang (zhangq@medimmune.com) cancer response. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P315 Methods TIGIT-targeting antibody candidates were screened out of a Background phage display library. The TIGIT antigen binding affinity and PVR In the tumor microenvironment (TME), programmed death-1 receptor blocking of anti-TIGIT antibodies were evaluated through both (PD-1), combined with its ligands PD-L1 and PD-L2, play an important protein-based and cell-based assays. Functional consequences of suppressive role in the immune response to cancer. PD-L1 expression MG1131 were determined using a cell-based reporter assay for T has been shown to be a key predictive biomarker of response to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 172 of 272 cell activation, a Treg cell functional assay and an NK-mediated group were hypertension (32%), anemia (29%), colitis/enterocolitis tumor killing assay. Treg cells and NK cells were isolated from (26%), and nausea/vomiting (25%). Median survival was signifi- healthy donor PBMC. cantly longer in the IO group relative to the CT group (18.6 Results months and 15.1 months, respectively; adjusted HR 0.73, 95% CI We screened out a few of the clones which stood out showing 0.66-0.81, Figure 1). high affinity binding to TIGIT and significant blocking of the Conclusions TIGIT-PVR interaction. Among the tested antibodies, MG1131 was Among the veteran population in real-world settings, patient identified to have the strongest affinity for human TIGIT and sig- characteristics were similar for 2L IO or CT therapy, with the ex- nificant blocking activity, resulting in competition with PVR in a ception of age and geographic region. Rates of common AEs dose dependent manner. Furthermore, MG1131 was cross-reactive were as expected. Our findings indicate improved survival among with cynomolgus monkey TIGIT, but not with mouse TIGIT. Our patients receiving IO versus CT in the 2L setting. More in vitro efficacy data demonstrated that MG1131 significantly en- population-based studies are needed to confirm these findings in hances T cell activation and NK-mediated tumor killing activities other healthcare settings. in a PVR-dependent manner and MG1131 induces IFN-γ secretion and proliferation of CD8+ T cells by inhibiting Treg suppressive Acknowledgements function. The team would like to thank Daniel Lane, PharmD, PhD, MBA for his Conclusions support of this study. We developed an anti-TIGIT antibody, MG1131, with pronounced in- Ethics Approval hibitory activity on the TIGIT-PVR signaling axis. In this study, This study was approved by the Durham VA Institutional Review Board (IRB MG1131 significantly enhanced T cell activation and NK-mediated #02009). tumor killing activity, and efficiently suppressed Treg cell function. Therefore, MG1131 is a potential candidate for cancer immunotherapy. P317 Utilization of second-line immuno-oncology agents and associated health outcomes among united states veterans with advanced non-small cell lung cancer 1 2 3 Mina Allo, PharmD, MPH , Lin Gu, MS , Vishal Vashistha, MD , Ashlyn 2 2 2 Press, MPH , Michael Kelley, MD , Christina Williams, PHD, MPH 1 2 Bristol-Myers Squibb, Princeton, NJ, United States; Durham Veterans Affair, Duke Cancer Ins, Durham, NC, United States; Dept of Medicine, Duke University, Durham, NC, United States Correspondence: Mina Allo (mina.allo@bms.com); Christina Williams (christina.williams4@va.gov) Fig. 1 (abstract P317). KM curve of overall survival of patients in 2L Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P317 Background Studies describing treatment safety, effectiveness and patterns of use are needed to evaluate the real-world impact of immuno-oncology P318 (IO) in advanced non-small cell lung cancer (NSCLC) relative to Large-scale evaluation of concordance of genomic scores in whole chemotherapy (CT). This retrospective cohort analysis assessed exome sequencing and Foundation Medicine comprehensive utilization of IO and CT agents and associated outcomes in second- genomic platform across cancer types 1 1 1 1 line (2L) treatment among stage IV NSCLC patients receiving care in Deepti Aurora-Garg , Andrew Albright, PhD , Ping Qiu, PhD , Yongjin Li , 1 2 1 1 the Veterans Affairs (VA). Xiaoqiao Liu , David Fabrizio, PhD , Lixin Lang , Jared Lunceford, PhD , Methods Razvan Cristescu, PhD 1 2 The VA Corporate Data Warehouse (CDW) oncology database was Merck & Co., Inc., Kenilworth, NJ, United States; Foundation Medicine, used to determine survival and 14 common adverse events (AE) Cambridge, MA, USA, Cambridge, MA, United States of adult patients with stage IV NSCLC diagnosed from 2012 to Correspondence: Deepti Aurora-Garg (deepti.aurora-garg@merck.com) 2017 who received systemic non-targeted (ALK, EGFR) therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P318 within 120 days of diagnosis and were followed until death or end of the study period in June 2019. Descriptive statistics were Background used to summarize treatment and AE occurrence. Kaplan-Meier Whole exome sequencing (WES) is a comprehensive method to methodology and multivariate Cox regression were used to evalu- evaluate the clinical relevance of DNA molecular characteristics, in- ate survival. cluding pan-exomic genomic scores (eg, tumor mutational burden Results [TMB] and homologous recombination deficiency–loss of heterozy- We identified 1655 patients who received 2L therapy, with 42% gosity [HRD-LOH]) and individual alterations (eg, BRCA1/2). Although (n=695) receiving IO monotherapy (nivolumab, pembrolizumab, comprehensive targeted genomic panels are available to measure atezolizumab, and durvalumab), 56.5% (n=935) receiving CT only, TMB and HRD-LOH, including FoundationOne® CDx (F1CDx), imple- and 1.5% (n=25) receiving IO+CT (not included in the current mentation of WES as a diagnostic approach in clinical practice can analysis due to limited sample). Greater than 99% of 2L IO users be challenging. To assess the feasibility of translating findings using used CT only in 1L setting, and >96% of 2L CT only group used WES as an exploratory tool into a practical diagnostic device such as CT in 1L (~ 3% used IO monotherapy, 0.6% IO+CT). Median age F1CDx, we evaluated the concordance of genomic scores (TMB and was 67 vs. 65 years in the IO and CT groups, respectively (p= HRD-LOH) and single-gene alterations between WES and F1CDx in a 0.006). No statistically significant differences between the IO and large pan-tumor data set. CT groups were observed by sex (~97% male), race (~77% White), Methods ethnicity (~98% non-Hispanic), smoking history (~95% current/ This analysis used solid tumor samples from patients with advanced former smoker), or histology (~58% adenocarcinoma). The most disease who received pembrolizumab monotherapy in the second common AEs in the IO group were dyspnea (50%), colitis/entero- line or later during single-arm clinical trials. WES and F1CDx (Dx1 colitis (40%), and anemia (28%); most common AEs in the CT baitset) were used to analyze samples from 436 patients across 22 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 173 of 272 tumor types. Spearman rank-order correlation and linear regression June 2019. Patient Information Form, Brief Fatigue Inventory (BFI), were used to determine concordance and cutoff equivalence for Functional Living Index-Cancer (FLIC), and Dermatology Life Quality TMB and HRD-LOH, each calculated by both WES [1] and F1CDx Index (DLQI) were used for data collection at 1st, 2nd, 3rd, and 4th (Foundation Medicine proprietary pipeline QSR_F1Dx_v1.0.3). cycles of nivolumab. Descriptive statistics, Mann Whitney U and Results Friedman tests were utilized for data analysis. Using WES and F1CDx, high concordance was observed in the pan- Results tumor assessment of TMB (Spearman correlation, 0.7; n=413) and The majority of the patients was male (60%), and the mean age of HRD-LOH (Spearman correlation, 0.5; n=364). When individual indica- patients was 54.10±18.88 years. The mean time of diagnosis for pa- tions were considered, the concordance was further improved for in- tients with melanoma was 40.50±47.84 months (range 2-168). There dications with higher distribution medians. TMB concordance was were no significant differences between patients’ BFI, FLIC, and DLQI higher when restricted to non–small cell lung cancer (Spearman cor- total scores in terms of age and gender (p>.05). The mean scores of relation, 0.8; n=38). HRD-LOH concordance was higher when re- BFI scores were 4.15±2.90 at the 1stcycle, and 3.75±2.98 at the 4th stricted to ovarian cancer (Spearman correlation, 0.7; n=54) and cycle; FLIC scores were 88.15±9.69 and 95.26±12.07; and DLQI scores breast cancer (Spearman correlation, 0.6; n=80). Regression analysis were 2.60±6.30 at 1.90±3.11, respectively. Considering the changes of TMB using both platforms identified F1CDx (Foundation Medicine within time in terms of all scale scores, no significant differences proprietary pipeline QSR_F1Dx_v1.0.3) TMB cutoffs of 10 and 13 mu- were found in BFI (p=.29), and DLQI (p=.49). With regard to FLIC tations/megabase to correspond to WES TMB of ~150 and ~175 mu- scores a significant difference was found from the 1st to the 4th tations/exome, respectively. Assessment of BRCA1/2 deleterious cycle of nivolumab (p=.05). mutations also demonstrated agreement between WES and F1CDx, Conclusions with 305 of 309 (98.7%) samples showing agreement; 282 samples The present study may be the first effort to evaluate changes in BFI, showed wild-type status by both methods and 23 samples showed FLIC and DLQI scores during nivolumab treatment in patients with mel- mutant status by both methods. anoma from the 1st to the 4th cycle. The study findings revealed that Conclusions improvements in BFI, and DLQI scores following nivolumab, even not The high level of concordance between WES and F1CDx suggests statistically significant. Lastly supporting literature [3,4], significant in- that molecular biomarker discoveries, including clinically relevant cut- crease has been found in FLIC scores with nivolumab treatment. offs and molecular epidemiology findings evaluated on the transla- tional WES platform, may be translated successfully in the diagnostic References setting. To our knowledge, this is the first evaluation of concordance 1. Dine J, Gordon R, Shames Y, Kasler MK, Barton-Burke M. Immune checkpoint of genomic scores performed in the context of clinical trial data inhibitors: an innovation in immunotherapy for the treatment and manage- across many indications and in a large data set. ment of patients with cancer. Asia Pac J Oncol Nurs. 2017; 4(2):127-135. 2. Shepherd FA, Douillard JY, Blumenschein GR. Immunotherapy for non- Reference small cell lung cancer: Novel approaches to improve patient outcome. J 1. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for Thorac Oncol. 2011; 6(10): 1763-1773. PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362. pii: 3. Ramirez RA, Lu J, Thomas KE. Quality of life for non-small cell lung cancer eaar3593. patients in the age of immunotherapy. Transl Lung Cancer Res. 2018; Ethics Approval 7(Suppl 2):149-152. The studies in which patient samples were collected were approved by an 4. Schadendorf D, Larkin J, Wolchok J, Hodi FS. Chiarion-Sileni V, Gonzalez independent ethics committee before being initiated at each site. R, Wagstaff J. Health-related quality of life results from the phase III Check Mate 067 study. Eur J Cancer. 2017; 82: 80-91. Ethics Approval P319 The study was approved by clinical trials ethics committee of the University Changes in fatigue severity, health related, and dermatology of Health Sciences Ankara Oncology Training and Research Hospital related quality of life in melanoma patients receiving nivolumab: (decision number: 2018–04/52) and performed in accordance with the Preliminary results of a prospective study from real-life experience Helsinki Declaration. 1 1 Canan Karadas , Nur Izgu, PhD, RN , Zehra Gok Metin, Assoc Prof, PhD, 1 2 2 RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD P320 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Novel in vivo preclinical humanized models for the evaluation of Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, human specific immune checkpoint inhibitors 4 1 2 1 1 Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Anya Avrutskaya , Fabienne Sonego , Jacob Hauser , Emily O’Koren , 1 2 2 1 1 Correspondence: Canan Karadas (karadas.canan@gmail.com) Robin Ball , Gaëlle Martin , Julie Chaix , Thi Bui , Ian Belle , Elizabeth 1 1 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P319 Reap , Patrick Fadden, PhD , Chassidy Hall , Kader Thiam , Paula Miliani de Marval 1 2 Background Charles River Discovery, Wilmington, MA, United States; Genoway, lyon, Previous studies have reported that nivolumab, as an immune check- France point inhibitor, may enhance survival, reduce therapy related toxic- Correspondence: Paula Miliani de Marval ities and improve quality of life (QoL) among melanoma patients (paula.milianidemarval@crl.com) compared with traditional cytotoxic chemotherapy [1,2]. However, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P320 nivolumab is frequently associated with fatigue, increased risk of skin toxicity and autoimmune-related adverse events. Thus, patients may Background experience important changes in daily living activities, health related In the last few years, there has been an increasing demand for suitable pre- QoL, and skin integrity during nivolumab treatment. In view of this clinical mouse models for evaluating the efficacy of checkpoint inhibition- issue, studies are needed evaluating these important changes related based cancer immunotherapies. During tumor progression, immune cells to nivolumab, concurrently. Therefore, this study aimed to investigate can become unresponsive and evade immune surveillance upon chronic fatigue severity, health related QoL, and dermatology related QoL in activation and expression of the programmed cell death protein-1 (PD-1) melanoma patients receiving nivolumab. the ligand PD-L1 on tumor cells or expression of the T lymphocyte associ- Methods ated antigen 4 (CTLA4) in T-cells cells resulting in tumor immune-tolerance. A total of 20 patients, scheduled to receive first dose of nivolumab, We have previously demonstrated that murine anti-PD-1, anti-PD-L1 and in three leading hospitals located in Ankara, was included in this de- CTLA-4 blockade can effectively enhance immune normalization and re- scriptive, prospective and multicenter study. All the patients received activate the antitumor response against multiple syngeneic tumor models. at least four cycle of nivolumab infusion between October 2018 and While these models proved instrumental for evaluating murine immune- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 174 of 272 checkpoint inhibitors (ICI), there is a clear need for additional mouse models Conclusions to evaluate the efficacy of ICI specific for human targets. While heterogeneous patient characteristics may have influenced Methods the results of this study, the trends observed suggest favorable To address this need, we describe the development of humanized PD- outcomes in patients with aNSCLC treated with IO monotherapy 1 and CTLA-4 knock-in (KI) mouse models. The main advantage of in the 1L setting. Further research should explore whether this is these models is that human PD-1 or CTLA-4 proteins are expressed in related to a predominance of patients with high PD-L1 expression the context of a fully functional immune system. To validate these among those who received IO monotherapies. TTD for IO-based models we evaluated the response to pembrolizumab or ipilimumab in therapies in this real-world setting appears to be shorter than a colorectal carcinoma and a glioblastoma preclinical tumor models. PFS reported in previous trials, indicating an unmet need may re- Results main and needs to be explored. However, these results could be We observed significant tumor growth inhibition and growth delay in the influenced by effects of informative censoring or other underlying MC38 tumor model with either monotherapy, but not when treated with clinical factors. Additionally, future studies should investigate dif- the murine counterparts: anti-PD-1 (clone RPM1-14) or CTLA-4 (clone 9H10). ferences in the tolerability profiles of 1L regimens, as well as To extend our validation studies to other tumor models, we implanted how treatment sequences contribute to outcomes. GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembro- Acknowledgements lizumab compared to both the control group and the group treated with This study was funded by Merck KGaA, Darmstadt, Germany, as part of an alliance murine anti-PD-1 antibody. Furthermore, we found that pembrolizumab between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. and ipilimumab therapy results in enhanced effector functions of CD4+and CD8+ T cells associated with increased expression of Granzyme B. Reference Conclusions 1. Blumenthal GM, Gong Y, Kehl K, et al. Analysis of time-to-treatment dis- In summary, the results shown here underscore the value of resour- continuation of targeted therapy, immunotherapy, and chemotherapy in cing to humanized knock-in (KI) mouse models as tools to evaluate clinical trials of patients with non-small-cell lung cancer. Ann Oncol. human specific immune-checkpoint based therapeutics alone and in 2019;30(5):830-8. combination with other agents. Ethics Approval The study was reviewed and granted exception and waiver of consent by the US Oncology, Inc. Institutional Review Board. P321 Real-world clinical outcomes among patients with advanced non- small cell lung cancer who initiated first-line regimens 1 2 3 3 Eric Nadler, MD , Bhakti Arondekar, PhD , Kathleen Aguilar , Jie Zhou , Table 1 (abstract P321). See text for description 2 4 4 Jane Chang , Xinke Zhang , Vivek Pawar 1 3 Texas Oncology, Medical Oncology, Dallas, TX, United States; Pfizer Inc., New York, NY, United States; McKesson Life Sciences, The Woodlands, TX, United States; EMD Serono, Inc., Billerica, MA, United States Correspondence: Eric Nadler (eric.nadler@USONCOLOGY.COM) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P321 Background While clinical trials have demonstrated the clinical benefit of immuno-oncology (IO) regimens for the treatment of advanced non- small cell lung cancer (aNSCLC), either in combination or as mono- therapies, limited research has evaluated clinical outcomes with these therapies in a real-world setting. This retrospective observa- tional study evaluated time to treatment discontinuation (TTD) and overall survival (OS) in patients with aNSCLC receiving care in US community oncology clinics. Previous research suggests that TTD is a pragmatic real-world efficacy endpoint, as TTD and progression-free survival (PFS) are associated across different types of therapy in NSCLC clinical trials [1], therefore TTD was explored in this study. Methods Patients with aNSCLC who initiated first-line (1L) treatment with sys- temic chemotherapies, targeted therapies, or IO regimens in the US Oncology Network between 3/1/15 and 8/1/18 were included in the study population. Electronic health record data for these patients was captured through 2/1/19. Descriptive analyses were performed to assess baseline characteristics and treatment patterns, and the Kaplan-Meier method was used to evaluate TTD and OS from the start of 1L treatment. Results In total, 7,746 patients were included in this analysis (Table 1): 5,859 (75.6%) initiated 1L systemic chemotherapies, 656 (8.5%) targeted therapies, 907 (11.7%) IO monotherapies, and 324 (4.2%) IO combin- ation regimens (with chemotherapies or targeted therapies). Of these, 51.8%, 50.3%, 21.7%, and 17.6%, respectively, proceeded to a subsequent treatment following 1L discontinuation. Median TTD ranged from 2.0 months (95% CI 1.9-2.1) in patients who received systemic chemotherapies to 3.5 months (95%CI: 2.8-4.2) in patients who received IO monotherapies (Figure 1). Similarly, median OS was longest in patients who received IO monotherapies (19.9 months Fig. 1 (abstract P321). See text for description [95%CI: 16.6-24.1]; Figure 2). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 175 of 272 Conclusions We describe the development of a very high affinity antagonistic TIGIT antibody, COM902, that is currently in preclinical development. Co-expression of TIGIT with PVRIG in TILs and their non-redundant in- hibitory effects on T cell activation suggest a potential therapeutic advantage in clinical combinations targeting both pathways. Towards this end we are planning a trial that will eventually incorporate com- binations of COM902 with the anti-PVRIG antibody, COM701. P323 IPH5301, a CD73 blocking antibody targeting the adenosine immunosuppressive pathway for cancer immunotherapy Ivan Perrot, Caroline Denis, PhD, Marc Giraudon-Paoli, Severine Augier, Rachel Courtois, Diana Jecko, Violette Breso, Thomas Arnoux, Nicolas Gourdin, PhD, Romain Remark, PhD, Cecile Bonnafous, Ariane Morel, PhD, Eric Vivier, Yannis Morel, PhD, Pascale Andre, Carine Paturel, PhD Innate Pharma, Marseille, France Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P323 Fig. 2 (abstract P321). See text for description Background CD73 is an extracellular ectonucleotidase highly expressed by tumoral P322 or stromal cells in the tumor microenvironment. By inducing tumor cell COM902, a novel therapeutic antibody targeting TIGIT augments T death, conventional anti-cancer therapies induce extracellular release cell function and the activity of PVRIG pathway blockade in vitro of adenosine triphosphate (ATP), which is degraded by CD39 into ad- and in vivo enosine monophosphate (AMP) and then by CD73 into adenosine, an Maya Kotturi, BS, PhD, Eran Ophir, PhD, Sarah Whelan, PhD, Spencer inhibitor of immune response. Blockade of CD73-mediated degradation Liang, Kathryn Logronio, BS PhD, Kyle Hansen, BS, Zoya Alteber, PhD, of AMP may therefore stimulate anti-tumor immunity across a wide Mark White, BS, PhD range of tumors through preventing the production of adenosine. Compugen, South San Francisco, CA,United States IPH5301 is a humanized effector-silent IgG1 monoclonal antibody that Correspondence: Eran Ophir (erano@cgen.com) selectively binds to and inhibits the activity of both membrane-bound Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P322 and soluble human CD73. IPH5301 is designed to enhance anti-tumor immune responses by inhibiting the enzymatic activity of CD73 in the Background tumor microenvironment, thus releasing tumor-infiltrating lymphocytes TIGIT is a coinhibitory receptor that is highly expressed on tumor in- from adenosine-mediated suppression. Here, we described the expres- filtrating lymphocytes (TILs), including effector and regulatory (Treg) sion of CD73 in several human solid tumors, characterized IPH5301 CD4+ T cells, effector CD8+ T cells, and NK cells. Engagement of antibody properties and its efficacy in vitro. TIGIT with its cognate ligand PVR directly suppresses lymphocyte ac- Methods tivation. TIGIT and PVR are broadly expressed in different types of CD73 expression was assessed by immunochemistry on cohorts of solid tumors, suggesting that TIGIT-PVR signaling may be a dominant solid tumors i.e breast, ovarian, lung, melanoma, pancreatic and head immune escape mechanism for cancer. Utilizing COM902, a thera- and neck cancer. In vitro efficacy of IPH5301 was evaluated (1) in hu- peutic antibody targeting TIGIT, we demonstrate that co-blockade of man T cell proliferation assays; and (2) in enzymatic assays with lym- TIGIT and a new checkpoint inhibitor, PVRIG, augments T cell re- phocytes and serum from healthy donors and human CD73-knock-in sponses in vitro and in vivo. (huCD73KI) mice. To get more insight into the mechanism of action Methods of IPH5301, CD73-IPH5301 complex was analyzed using electron mi- Multi-color flow cytometry analysis of dissociated tumors was used croscopy and the crystal structure of IPH5301 Fab in complex with to quantify TIGIT and PVRIG expression on TILs. Membranous PVR CD73 ectodomain was determined. and PVRL2 expression was characterized by immunohistochemistry. Results The ability of COM902 to promote T cell responses in vitro, alone Whereas inter-patient variability was observed in all tested indications, and in combination with an anti-PVRIG antibody, COM701, was eval- CD73 expression was always detected mainly on tumor cells and did not uated in a primary TIL assay. To examine the in vivo effects of TIGIT correlate with the expression of CD39 or PD-L1. In vitro IPH5301 effi- blockade with COM902 a chimeric antibody with the constant region ciently restored T cell proliferation and blocked adenosine-mediated sup- of mouse IgG1 was generated. The anti-tumor activity of the chimeric pression of Tcellproliferationinamixedlymphocytereactioninadose- COM902 antibody in combination with an anti-mouse PVRIG anti- dependent manner. IPH5301 did not induce CD73 down-modulation and body was assessed in the mouse CT26 colon carcinoma model. did not directly activate B cells. Furthermore, IPH5301 efficiently blocked Results CD73 enzymatic activity in human serum and whole blood as well as in COM902 is a fully human antibody that binds TIGIT with high affinity serum and splenocytes from huCD73KI mice. Finally, we showed that and specificity and disrupts the binding of TIGIT to PVR. This anti- IPH5301 contrains CD73 in an intermediate inactive form. body binds to TIGIT on human CD8+ T cells with higher affinity than Conclusions tested benchmark antibodies. In dissociated tumor samples, TIGIT ex- These results indicate that IPH5301 blocks CD73 with a differentiated pression was highest on TILs in endometrial, head and neck, kidney mechanism of action compared to benchmarked anti-CD73 clinical and lung tumors, and directly correlated with PVRIG expression. Ex- candidates and support the clinical development of IPH5301 for can- cept for breast tumors, PVR was moderately to highly expressed in cer immunotherapy, potentially in combination with chemotherapy all tumor types examined, while PVRL2 expression was highest in or immune checkpoint inhibitors. prostate, ovarian, liver and endometrial tumors. Combination of COM902 and COM701 resulted in enhanced CD3+ TIL activity Acknowledgements in vitro. Furthermore, the combination of chimeric COM902 and anti- The research leading to CD73 results were obtained within the TumAdoR PVRIG resulted in significant CT26 tumor growth inhibition and en- collaborative consortium that received funding from the European hanced overall survival, which was comparable to the combination Community's Seventh Framework Program (FP7/2007-2013) under grant of chimeric COM902 and anti-PD-L1. agreement n°602200. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 176 of 272 Clinical Trial Completed outcomes in other cancer types and in randomized settings will pro- vide additional insight into their prognostic or predictive character. P324 Pan-tumor analysis of the association of cancer and immune Acknowledgements biology-related gene expression signatures with response to Joanne E Tomassini for writing support and Sheila Erespe for editorial pembrolizumab monotherapy support, both employees of Merck & Co., Inc. 1 1 Razvan Cristescu, PhD , Michael Nebozhyn, PhD , Chunsheng Zhang, Trial Registration 1 1 1 1 PhD , Andrew Albright, PhD , Julie Kobie, PhD , Lingkang Huang , Qing NCT01295827, NCT01866319, NCT02335411; NCT02335424; NCT01848834; 1 1 1 1 1 Zhao, MD PhD , Anran Wang , Hua Ma , Andrea Webber , Petar Jelinic , NCT02255097; NCT02447003; NCT02674061; NCT02853344 1 1 1 1 Mohini Rajasagi , Sandra Souza , Raluca Predoiu , Z. Alexander Cao , 1 1 1 1 Junshui Ma , Michael Morrissey , Clemens Krepler, MD , Stephen Keefe , References 1 1 1 Jonathan Cheng, MD , Vassiliki Karantza , Sukrut Shah , Rodolfo Perini, 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-gamma-related mRNA pro- 1 2 3 4 MD , Antoni Ribas, MD, PhD , Petros Grivas, MD, PhD , David Cescon , file predicts clinical response to PD-1 blockade. J Clin Invest 1 1 1 Terrill McClanahan, PhD , Alexandra Snyder, MD , Mark Ayers, PhD , 2017;127:2930-2940. 1 1 Jared Lunceford, PhD , Andrey Loboda, PhD 2. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for 1 2 Merck Inc., Boston, MA, United States; University of California, Los PD-1 checkpoint blockade-based immunotherapy. Science Angeles, Los Angeles, CA, United States; University of Washington, 2018;362:eaar3593. Seattle, WA, United States; Princess Margaret Cancer Centre, Toronto, 3. Ayers M, Nebozhyn M, Cristescu R, et al. Molecular Profiling of Cohorts of Canada Tumor Samples to Guide Clinical Development of Pembrolizumab as Correspondence: Razvan Cristescu (razvan_cristescu@merck.com) Monotherapy. Clin Cancer Res 2019;25:1564-1573. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P324 Ethics Approval The clinical trials included in this analysis were approved by the appropriate Background ethics committees at each participating study center. RNA sequencing (RNASeq) data on baseline tumor biopsies from pa- tients in pembrolizumab monotherapy studies were used to explore Table 1 (abstract P324). See text for description potential relationships between key biological gene expression signa- tures and objective response rate (ORR) in the trials. Methods A canonical set of 10 consensus signatures representative of key tumor biology and tumor microenvironment (TME) elements beyond the 18- gene T-cell inflamed gene expression profile (GEP [1]) was defined using the independent Merck-Moffitt and TCGA databases [2,3], exter- nal to any pembrolizumab trial and prior to relating RNASeq data to clinical outcomes from studies evaluated. These signatures (Angiogen- esis, Hypoxia, Glycolysis, Proliferation, MYC, RAS, gMDSC, mMDSC, Stroma/EMT/TGFβ, WNT) were evaluated in the trial dataset blinded to clinical outcome, to test the association with ORR (RECIST 1.1; where re- sponse=PR or CR). Studies with available RNASeq data (N=1188) in- cluded: KN001/KN006-Melanoma (N=476; pembrolizumab-treated and ipilimumab-naïve), KN052-urothelial (N=186), KN012/KN055-HNSCC (N= 147; HPV-negative by whole exome sequencing), KN086-TNBC (N=132), KN059-Gastric (N=92), and KN427-RCC (N=78), KN100-Ovarian (N=77). Pan-cancer logistic regression analysis of ORR for consensus signatures included terms adjusting for cancer type, ECOG performance status, and the T-cell inflamed GEP, an approach equivalent to evaluating asso- ciation between ORR and the residuals of consensus signatures after detrending them for their relationship with the T-cell inflamed GEP and cancer type. Testing of the 10 pre-specified consensus signatures for P325 negative association (except Proliferation with a hypothesized positive- Tumor Infiltrating Lymphocytes (TILs) in triple-negative breast association) with ORR was adjusted for multiplicity. cancer: High Immunoscore is associated with pathological CR in Results patients receiving neoadjuvant chemotherapy. 1 2 3 4 Covariance patterns of the 11 signatures (including GEP) in Merck- Bernardo Rapoport, MD , Simon Nayler , Jerome Galon, Dr , T Mlecnik , 1 1 4 5 Moffitt and TCGA showed highly concordant co-expression patterns Teresa Smit , Jacqui Barnard-Tidy , Aurelie Fugon , Marine Martel , 6 2 in the RNASeq data from pembrolizumab trials. As anticipated, the T- Ronald Anderson, Professor , Carol Benn cell inflamed GEP demonstrated the strongest association with ORR The Medical Oncology Centre of Rosebank, Johannesburg, South Africa; 2 3 to pembrolizumab. Beyond the positive association seen with T-cell Prof., Johannesburg, South Africa; LABORATORY OF INTEGRATIVE inflamed GEP, three other RNA signatures, Angiogenesis, mMDSC CANCER IMMUNOL, France, France; Luminy Biotech enterprises 163 Ave and Stroma/EMT/TGFβ, exhibited negative associations at the 0.05 de Lu, Marseille, France; HalioDx, Marseille, France; level after adjusting for multiple testing (Table 1). Immunology,University of Pretoria, Pretoria, South Africa Conclusions Correspondence: Teresa Smit (pharmacist@rapoport.co.za) Pan-cancer testing of exploratory gene expression signatures using Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P325 the RNASeq platform in 1188 patients from single-arm pembrolizu- mab trials suggests that features beyond interferon gamma-related Background T-cell inflammation may be relevant to response to anti-PD1 mono- The presence of high levels of tumor infiltrating lymphocytes (TILs) has therapy and may define other axes of tumor biology as rational can- been associated with better prognosis in early triple-negative breast didates for pembrolizumab combinations. These features cancer (TNBC). Immunoscore is a prognostic tool, which categorizes the (Angiogenesis, mMDSC and Stroma/EMT/TGFβ) have been previously densities of spatially positioned CD3 and CD8 cells in both invasive hypothesized to represent immune-suppressive axes with potential margins (IM) and the center of the tumor (CT) yielding a five-tiered clas- negative impact on immunotherapy efficacy. Future evaluation of sification (0–4). High immunoscores have been reported to be associ- the association of these signatures with response and survival ated with improved outcomes in patients with colorectal cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 177 of 272 Methods low initial immunity, characterized as 0.05. Furthermore, patients with We performed the Immunoscore in a cohort of 103 breast cancer low initial immunity who received the DNA-based vaccine showed (BC) patients previously receiving neo-adjuvant chemotherapy. There improved immunogenicity towards the unvaccinated extracellular were triple-negative (TNBC)=53, Luminal=32, Her2+=18 who received domain of HER2 at 1 month and 6 months, p treatment with anthracycline and/or taxane- and/or trastuzumab- Conclusions based neo-adjuvant chemotherapy. Pre-treatment tumor samples A comparison of DNA-based and peptide-based vaccines targeting were immune-stained for CD3 and CD8 T-cell markers. Quantitative HER2 intracellular domain epitopes revealed a more robust and long- analysis of the immune cells was carried out using a computer- lasting immunogenic response with the DNA-based vaccine while assisted image analysis in different tumor locations. maintaining an excellent safety profile. Additionally, immune re- Results sponses to extracellular domain regions of HER2 demonstrate the The pathological complete response (pCR) rate of the entire cohort intra-epitope spreading potential in the DNA-based vaccine. was 44%. On univariate analysis factors associated with higher pCR included primary tumor size (T1=43.48% vs. T2=52.31% vs. T3+T4 Acknowledgements 6.67%, Chi2=10.3201, p40=56.86% vs.15-39=40.54% vs. A special thanks to the Boyd Scholarship for financial assistance during my T-cell density subsets (CD3, CD8) and Immunoscore were significantly research. higher in TNBC compared to non-TNBC patients. Receiver-operating Trial Registration characteristic (ROC) curve analysis was used to determine the opti- Clinicaltrials.gov: NCT00436254 and NCT0034310 mal cut-off points for CD3 and CD8. A high density of CD3 (> than 800mm2) and CD8 (> than 400mm2) positive T-cells in the CT was References associated with higher pCR (CD3 CT:60% vs.25%, p=0.00035 and CD8 1. Datta J., et. al. Progressive loss of anti-HER2 CD4+ T-helper type 1 re- CT: 64% vs.27%, p=0.00016). Analysis of CD3 (> than 1400mm2) (CD3 sponse in breast tumorigenesis and the potential for immune restoration. IM:63% vs.19%, p=0.0001) and CD8 densities in the IM (> than Oncoimmunology. 2015;4(10):e1022301. 500mm2) was also significantly associated with pCR (CD8 IM:63% vs. 2. Mittendorf EA, et. al. Clinical trial results of the HER-2/neu (E75) vaccine 15%, p=0.00003). High immunoscore (24/38 pts (63%)) vs. intermedi- to prevent breast cancer recurrence in high-risk patients: from US Military ate (17/48 pts (35%)) vs. low (4/17 pts (24%)) was significantly associ- Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer. ated with pCR (p=0.00674). In a logistic regression model, Ki-67 (p 2012;118(10):2594-602. Conclusions 3. Disis, M.L., et al., Generation of T cell immunity of the HER-2/neu protein The results of this study show a significant prognostic and potentially after active immunization with HER-2/neu peptide based vaccine. J. Clin predictive role for the Immunoscore and Ki-67 in BC patients, particu- Onc, 2002;20(11): 26424-2632 larly in the TNBC subset. 4. Disis, M.L., et al., Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based Acknowledgements vaccine. Clin Cancer Res, 1999;5(6):1289-97. Dr Ronwyn van Eeden 5. Disis ML, et. al. A phase I trial of the safety and immunogenicity of a Ethics Approval DNA- based vaccine encoding the HER-2/neu (HER2) intracellular domain Ethics Approval was obtained from Pharmaethics SA and University of PTA, in subjects with HER2+ breast cancer. ASCOAnnual Meeting. 2014; approval no 517/2017 6. Disis ML, et. al. Phase II study of a HER-2/neu (HER2) intracellular domain Consent (ICD) vaccine given concurrently with trastuzumab in patients with newly Written informed consent was obtained from the patient for publication of diagnosed advanced stage breast cancer. SABCC. 2009; this abstract and any accompanying images. A copy of the written consent 7. Aurisicchio L., Ciliberto G. Genetic cancer vaccines: Current status and is available for review by the Editor of this journal. perspectives. Expert Opin. Biol. Ther. 2012;12:1043–1058. P326 P327 A retrospective analysis of DNA plasmid and peptide-based Evaluation of PD-L1 and cutoff selection to define a predictive vaccine therapy in treatment of HER-2/neu+ breast cancer biomarker for pembrolizumab monotherapy in esophageal cancer 1 1 1 Aaron Stewart, BS , William Gwin, MD , Mary Disis, MD, FACP , Jennifer using KEYNOTE-180 1 2 1 1 Childs , James Dai , Doreen Higgins, RN, BSN, OCN , Angela Kask Mary Savage, Serafino Pantano, Qi Liu, Jared Lunceford, PhD, Peter Kang, 1 2 University of Washington, Seattle, WA, United States; Fred Hutchinson Pooja Bhagia, MBBS, MD, Kenneth Emancipator, MD Cancer Research Center, Seattle, WA, United States Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United States Correspondence: William Gwin (wrgwin@medicine.uw.edu) Correspondence: Kenneth Emancipator Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P326 (kenneth.emancipator@merck.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P327 Background Patients with HER-2/neu+ overexpressing breast cancer often lose Background immunity toward the HER2 antigen [1]. Vaccines are capable of indu- Interim analysis of the KEYNOTE-180 study (NCT02559687) was used cing a cytotoxic T lymphocyte immune response toward overexpress- to establish a relationship between PD-L1 expression levels and ob- ing antigens, leading to targeted tumor destruction [2-4]. Clinical jective response rate (ORR) in patients with esophageal cancer whose trials have explored peptide-based and DNA-based vaccines as pos- disease progressed after ≥2 lines of therapy and to select a PD-L1 sible vehicles for vaccine delivery, though a direct comparison of cutoff for further validation in the randomized setting (ie, KEYNOTE- safety and immunogenicity has not been previously studied[5-6]. We 181). hypothesize that the DNA-based vaccine will produce superior im- Methods munogenicity due to stable plasmid persistence within the tissue KEYNOTE-180 was a single-arm, open-label, phase 2 study of pem- leading to prolonged HER2 immune response[7]. brolizumab in patients with previously treated, advanced esophageal Methods cancer. Pembrolizumab 200 mg was given intravenously every 3 We retrospectively analyzed adverse events and ELISpot data from weeks. The primary objective was ORR. Patients were required to 104 patients treated with vaccines targeting the intracellular domain provide a tumor sample for retrospective analysis of biomarkers, of HER2/neu using either DNA or peptide fragments. which may predict response to pembrolizumab. PD-L1 expression Results was measured using the PD-L1 IHC 22C3 PharmDx assay and evalu- Adverse event profiles of the 104 patients analyzed were similar with ated using a combined positive score (CPS). CPS is the ratio of PD- no reported grade 3, 4, or 5 events. There was no significant effect L1–expressing cells (tumor cells, lymphocytes, macrophages) to vi- on left ventricular ejection fraction (p=0.88 and p=0.59). Patients with able tumor cells. Testing for a relationship between CPS and ORR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 178 of 272 (per RECIST v1.1 by central review) was conducted using logistic re- Background gression. Cutoff selection proceeded by joint evaluation of the ORR BL-8040 (BL), a CXCR4 antagonist, increases T cell entry into the enrichment profile, the sensitivity and specificity profile (receiver op- bloodstream and thereafter into tumor in humans. We hypothesized erating characteristic analysis), the prevalence of patients with late- that BL in combination with pembrolizumab (P) may thereby pro- line esophageal cancer selected by the cutoff, and trends in overall mote efficacy in metastatic pancreatic cancer (mPC). survival (OS) by Kaplan-Meier (KM) curves. Methods Results Methods: This phase IIb open label study enrolled patients with There were 8 responders among 105 patients with available PD-L1 progression after at least one prior chemotherapy for mPC. Two results at the time of interim analysis (March 1, 2017). CPS was statis- weeks of single agent BL (1.25 mg/kg) was followed by 3-week tically significantly associated (P=0.03) with probability of response. A cycles of P (200 mg IV d1) plus BL (d1,4,8,11). Biopsies for tumor cutoff of CPS 1 did not show enrichment of ORR, whereas higher cut- biology were performed before treatment, after BL monotherapy offs did (Table). CPS 10 showed >3-fold enrichment in ORR above (optional), and after BL/P combination versus below the cutoff. Although higher cutoffs indicated further Results enrichment for ORR, attendant drops in sensitivity and prevalence As of July 2019, 20 pts enrolled; 15 were evaluable for the pri- occurred. Separation between KM OS curves for CPS ≥10 versus CPS mary endpoint of radiologic response. Baseline characteristics: Conclusions median age 66, 10M/10F, median 2 prior lines of therapy (range CPS was useful for identifying patients who responded to pembroli- 1-3). Best overall response includes 1 PR, 2 SD, 12 PD yielding zumab monotherapy. Through the evaluation of several clinical utility 21.4% disease control (1PR+2SD). Median TTP was 2 months over- dimensions, CPS 10 was chosen for further validation in the random- all and 7 months for the PR/SD pts. Median OS was 7 months ized setting based on its ability to enrich for ORR and simultaneously overall and 12 months in PR/SD pts. The combination was well to preserve sensitivity. The preservation of sensitivity, along with tolerated with most AEs being injection site discomfort. Five pa- prevalence, was considered particularly important because of the tients experienced grade 3/4 toxicities. Grade 3 toxicities included safety profile of pembrolizumab and the paucity of treatment op- HTN (n = 1), Alk Phos (n = 1), N/V (n = 2), ascites (n = 1), dys- tions in this patient population. pnea (n = 1), and abd pain (n = 2). One pt had grade 4 dyspnea. Trial Registration Paired biopsies have been analyzed for six patients (1 PR 2 SD 3 ClinicalTrials.gov, NCT02559687 PD). Patients with PR/SD had, at baseline, trends towards greater Ethics Approval T cell, especially cytotoxic CD8+ T cell counts within the tumor The study and the protocol were approved by the Institutional Re- niche than patients with PD (T cells: 188-627 cells/mm2 vs 7-41 view Board or ethics committee at each site. cells/mm2, Cytotoxic CD8+ T cells: 18-137 cells/mm2 vs 0-2 cells/ Consent mm2). The PR patient demonstrated an increase in cytotoxic All patients provided written informed consent to participate in the CD8+ T cell number in the tumor niche and reduction in the clinical trial. stroma following treatment. Additional molecular profiling data from multiplex IF will be available at the meeting. Conclusions Table 1 (abstract P327). See text for description This combination of immunotherapy with pembrolizumab plus BL-8040, without cytotoxic chemotherapy, shows clinical activity in patients with pancreatic cancer even in this heavily pretreated population. The combination was well tolerated. We noted a trend towards greater CD8+ T cell infiltrate at baseline within the tumor cell niche among patients who demonstrated clinical bene- fit. OS for all comers was longer than expected in this heavily pretreated patient group suggesting that P + BL might have a salutary effect on survival time if used earlier in the disease course. Trial Registration NCT02907099 Ethics Approval This study was approved by the M.D. Anderson Institutional Review Board, approval number 2016-0410. P329 PolyPEPI1018 off-the shelf vaccine as add-on to maintenance P328 therapy achieved durable treatment responses in patients with A phase IIB study of Pembrolizumab plus BL-8040 in metastatic microsatellite-stable metastatic colorectal cancer patients (MSS pancreatic cancer: Clinical outcomes and biological correlates 1 1 1 mCRC) David Fogelman, MD , Michael Overman, MD , Robert Wolff , Milind 1 1 1 2 1 2 1 Joleen Hubbard, MD , Chiara Cremolini, MD , Rondell Graham, MD , Javle, MD , Shubham Pant, MBBS , Gauri Varadhachary , Rachna Shroff , 1 1 2 1 1 Roberto Moretto, MD , Jessica Mitchell, CNP , Jaclynn Wessling , Eniko Ignacio Wistuba, MD , Carmelia Barreto, PhD , Renganayaki 1 3 3 3 3 3 3 Toke , Zsolt Csiszovszki, PhD , Orsolya Lőrincz , Levente Molnár , Eszter Pandurengan, MS , Sandesh Subramanya, PhD , Debora Ledesma, PhD , 4 4 4 3 3 3 3 3 Somogyi , Mónika Megyesi , Kata Pántya , József Tóth , Péter Páles , Abi Vainstein-Haras, MD , Ella Sorani, PhD , Tzipora Lustig , Osnat 4 4 5 6 3 2 1 István Miklós , Alfredo Falcone, MD , Joleen Hubbard, MD Kashtan , Yosi Gozlan , Steven Townson, PhD , Jeanne Fahey, PhD , 1 1 2 Mayo Clinic, Rochester, MN, United States; Azienda Ospedaliera James Yao, MD 1 2 3 Universitaria Pisana, Pisa, Italy; Treos Bio, Budapest, Hungary M.D. Anderson Cancer Center, Houston, TX, United States; University of Correspondence: Joleen Hubbard (joleenhubbard@gmail.com) Arizona, Tuscon, AZ, United States; MD Anderson Cancer Center, 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P329 Houston, TX, United States; Bioline Rx, Modi'in, Israel; Merck & Co., Inc., Shoreline, WA, United States; Merck & Co., Inc, Boston, MA, United Background States PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine against CRC, Correspondence: David Fogelman (dfogelman@mdanderson.org) containing 12 immunogenic epitopes derived from 7 conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P328 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 179 of 272 cancer antigens frequently expressed in mCRC based on the analysis tologous cell products. Whilst feasible in such relatively small of 2,931 biopsies. Here we report the results of the phase I study of patient populations, delivering an autologous product to large PolyPEPI1018 vaccine as an add-on to maintenance therapy in MSS cohorts of patients is likely beyond the current logistical cap- mCRC patients. abilities. In the phase 1 alloSHRINK study, we tested the first-in- Methods class non-gene edited allogeneic CAR T-cell therapy, CYAD-101, 11 patients with MSS mCRC in the first-line setting were vaccinated administered concurrently with chemotherapy, for the treatment with PolyPEPI1018 just after the transition to maintenance therapy of metastatic colorectal cancer (mCRC). The NKG2D-based CAR with a fluoropyrimidine and a targeted agent (bevacizumab). (Part A: of CYAD-101 targets eight ligands present at high frequencies n= 5, single dose, 12 weeks follow-up; Part B: n= 6, 3 doses, Q12W). in mCRC, not only on tumor cells but also cells from the tumor Primary endpoints were safety and immunogenicity. Multiple analysis microenvironment, and co-express a T-cell receptor (TCR) inhi- of vaccine-induced immune responses in blood and tumor were per- biting molecule (TIM) that interferes with TCR signaling in an at- formed. Both immune response and clinical benefit were predicted tempt to avoid the main issue of allogeneic T-cell therapy, the using the autologous HLA-genotype determined from patient’s saliva graftversushostdisease (GvHD). sample. Methods Results The alloSHRINK study (NCT03692429) evaluates the safety and The vaccine was well tolerated; most common side effects were tran- clinical activity of multiple infusions of CYAD-101, administered sient skin reactions and flu-like syndrome. No vaccine-related SAE oc- concurrently with standard of care FOLFOX chemotherapy, in pa- curred. 90% of patients had vaccine-specific CD8+ T-cell responses of tients with non-resectable mCRC who received prior chemother- memory-effector type against at least 2 of the 7 vaccine antigens, 5 apy lines (i.e. rechallenge population). Three dose-levels (DL; on average. Vaccine specific CD4+ T-cell responses were detected in 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated all patients. Ex vivo CD8+ T cell responses of effector type were de- through a 3+3 design. tected in 71% of patients, as well as increased fractions of CRC- Results reactive, polyfunctional, circulating CD8+ and CD4+ T cells in pa- In total 12 patients have been enrolled in the dose escalation tient’s PBMC after vaccination. Among the 11 patients 3 patients had segment, now completed (3 at DL1, 3 at DL2 and 6 at DL3). At objective tumor response according to RECIST v1.1, one of them re- the time of submission, only data from the first two DLs were ceived a single dose and 2 of them received 3 doses. For the Part B available. At DL1 and DL2, there was no report of dose-limiting of the study, the Objective Response Rate (ORR) was 33% (2/6) and toxicity (DLT) and no patient experienced Grade ≥ 3 related ad- the Disease Control Rate (DCR) was 67% (4/6). Notably, one patient verse events (uncleaned database). No clinical evidence of GvHD experienced complete tumor shrinkage on 2 of 3 target lesions and has been recorded. Best overall response ≥ 3monthsinclude 1 partial response on 1 lesion after 25 weeks of treatment, qualifying partial response and 3 stable disease over the first 6 patients for curative surgery. Median duration of disease control was 9 (DL1 and 2). At DL1 and 2, preliminary data show a dose- months (95%CI 6.3-11.5) (mPFS not reached during the study). The dependent effect on the cell kinetics and control of the host- 10 month PFS was 50% (3/6). Predicted vaccine antigen-specific versus-graft response against CYAD-101 cells as evidenced by the CD8+ T cell responses were confirmed in vitro with a PPV of 79% (p= similar levels of CYAD-101 engraftment after 2nd and 3rd 0.01). Predicted multiantigenic immune responses tend to correlate infusions. with both PFS and tumor volume reduction. Conclusions Conclusions As of August 2019, no GvHD has been observed following infu- Treatment with PolyPEPI1018 vaccine and maintenance therapy was sions of non-gene edited allogeneic CAR T-cells to mCRC pa- safe, well-tolerated, and demonstrated evidence of immunological tients at the first two DLs, with preliminary signals of clinical and clinical activity in MSS mCRC tumors. In addition predicted multi- activity. The study will have reached protocol-specified end- antigenic immune responses indicated treatment benefit, which sup- points for analysis at the time of presentation and safety, clin- ports further development of a companion diagnostic together with ical and cell engraftment will be presented. The results from the vaccine. this study, in comparison with a study evaluating the autolo- Trial Registration gous analog of CYAD-101 in mCRC will provide critical informa- NCT03391232 tion to support the development of CAR-T therapy in solid Ethics Approval tumors. This study was approved by Mayo Clinic Institutional Review Board Trial Registration and by Central Ethics Committee, Italy (Protocol number: OBERTO- NCT03692429 101). Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics P330 Boards and authorities. Results from the completed dose-escalation of the alloSHRINK phase I study evaluating the allogeneic NKG2D-based CAR T- cell therapy CYAD-101 in metastatic colorectal cancer patients P331 1 1 2 Hans Prenen, MD , Marika Rasschaert, MD , Alain Hendlisz, MD , Leila Results from the completed dose-escalation phase I SHRINK study 2 3 3 Shaza, MD , Erik Alcantar-Orozco, MD, PhD , Emilie Cerf, PhD , Florence evaluating the autologous NKG2D-based CAR T-cell therapy CYAD- 3 3 3 4 Renard , Caroline Lonez, PhD , Anne Flament , Jeroen Dekervel, MD , 01 in metastatic colorectal cancer patients 4 1 1 1 Eric Van Cutsem, MD, PhD Leila Shaza, MD , Alain Hendlisz, MD , Ahmad Awada, MD, PhD , Jean- 1 2 2 3 University Hospital Antwerp (UZ Antwerp), Edegem, Belgium; Luc Canon, MD , Javier Carrasco , Eric Van Cutsem, MD, PhD , Jeroen 2 3 3 4 4 Institut Jules Bordet, BRUSSELS, Belgium; Celyad, Mont-Saint- Dekervel, MD , Erik Alcantar-Orozco, MD, PhD , Florence Renard , Emilie 4 4 4 4 Guibert, Belgium; University Hospital Leuven (UZ Leuven), Leuven, Cerf, PhD , Caroline Lonez, PhD , Anne Flament , Marc Van den Eynde, 5 5 Belgium MD , Jean-Pascal Machiels, MD, PhD 1 2 Correspondence: Caroline Lonez (clonez@celyad.com) Institut Jules Bordet, Brussels, Belgium; Grand Hôpital de Charleroi Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P330 (GHdC), Charleroi, Belgium; University Hospital Leuven (UZ Leuven), 4 5 Leuven, Belgium; Celyad, Mont-Saint-Guibert, Belgium; Cliniques Background Universitaires Saint Luc, Brussels, Brussels, Belgium Current success of chimeric antigen receptor T-cell (CAR-T) ther- Correspondence: Caroline Lonez (clonez@celyad.com) apy in hematological malignancies has been achieved using au- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P331 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 180 of 272 Background Methods Chimeric antigen receptor T-cell (CAR-Ts) therapies have yet to demon- The antigen-specific T cell response in the patients who received a strate positive results in the context of solid tumors largely due to the vaccine targeting the prostate cancer antigen, TARP (TCRγ alternate lack of suitable target antigens. NKG2D-based CARs target 8 stress ligands reading frame protein)[1] for biochemically recurrent (D0) prostate notably expressed to a very high frequency across the metastatic colorec- cancer on NCT00972309 was assessed. Patients with HLA-A0201 re- tal cancer (mCRC) patient population. The autologous NKG2D-based ceived vaccination at weeks 3, 6, 9, 12 and 15 following 1:1 CAR-T therapy CYAD-01 achieved stable disease in several patients with randomization between a vaccine consisting of TARP peptides, mon- mCRC when given as a monotherapy in a multiple injection setting with- tanide ISA 51 VG and GM-CSF versus autologous dendritic cell (DC) out any other supportive therapy (THINK study). In the SHRINK phase 1 pulsed with TARP peptides. Both vaccines used two types of pep- study, CYAD-01 was given concurrently with FOLFOX chemotherapy. tides; wild type (WT) TARP 27-35 (TARP2735) and epitope-enhanced Methods TARP 29-37 peptides (TARP2937-9V). The peripheral blood mono- The SHRINK phase 1 study (NCT03310008) evaluated the safety and clin- nuclear cells were collected at baseline and study weeks following ical activity of multiple infusions of CYAD-01, administered concurrently vaccination to be stored in liquid nitrogen until analysis. Cells were with FOLFOX chemotherapy in mCRC patients. Three dose-levels (DL; thawed and stimulated in vitro with TARP peptides with cytokine 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated through support for multicolor flow cytometry. a 3+3 design in two different mCRC patient populations: (i) resectable Results liver dominant mCRC with FOLFOX chemotherapy as 1st line treatment CD8+ T cell subsets were analyzed for association with the dis- (i.e. neoadjuvant population), and (ii) non-resectable mCRC with prior ease response in 5 patients each who were responders and non- chemotherapy lines for mCRC including FOLFOX and/or FOLFIRI (i.e. re- responders whereas response was defined as slowing of PSA challenge population). slope log value as previously published by Wood et al [3]. The Results proportion of PD1-expressing CD8+ cells showed statistically im- The three DL have been completed with 9 patients in total (3 at each portant differences from baseline with an increase in responders DL), without any report of dose-limiting toxicity (DLT). Only 1 patient and a decrease in non-responders at week 12 after stimulation experienced Grade 3 related adverse event (AE) and no patient expe- with TARP 2735 (p= 0.016), TARP 2937 (p=0.032) and TARP 2937- rienced Grade 4 related AE (uncleaned database as of August 2019). 9V (p= 0.016) peptides. Other CD8+ subsets with granzyme A, Best overall response ≥ 3 months includes 1 partial response and 6 IFN-γ, IL-2, TNFα and perforin positive cells were investigated. stable disease out of 9 patients. Preliminary data show a dose- CD8+TNFα+ cells at week 12 (p=0.032) and CD8+perforin+ cells dependent effect on the cell kinetics. The study will have reached at week 18 (p=0.032) stimulated with TARP 2937 also showed protocol-specified endpoints for analysis at the time of presentation. statistically large differences with an increase in responders and a Conclusions decrease in non-responders. Early data show preliminary signs of clinical activity with the concurrent Conclusions administration of CYAD-01 and FOLFOX chemotherapy in the present Antigen-specific CD8+ T cells from responders as defined by reduced SHRINK study. Safety, clinical and translational research data (cell engraft- PSA slope log showed statistically greater activation as assessed by ment) will be presented. The results from this study, in comparison with expression of activation marker PD-1 than those from non- the results from a similar Phase I study evaluating the allogeneic analog responders after the vaccination in a first-in-human trial targeting of CYAD-01 in mCRC patients (i.e. CYAD-101), will provide critical informa- TARP. The antigen-specific T cell response to the vaccine peptides is tion to support the development of CAR T-cell therapy in solid tumors. an immune correlate of vaccine-induced protection and may be used Trial Registration to guide the ongoing study NCT02362451 that does not have HLA NCT03310008 restrictions. Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics Acknowledgements Boards and authorities. This work was supported by the Center for Cancer Research, National Cancer Institute, National Institue of Health. Trial Registration P332 NCT00972309 Cancer vaccine against prostate cancer antigen TARP induces antigen-specific CD8+ T cells with upregulation of activation marker References PD1 in patients with decreased PSA velocity in D0 prostate cancer 1 1 2 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein Hoyoung Maeng, MD , Brittni Moore , Lauren Wood, MD , Seth 1 1 1 expressed in prostate and breast cancer cells derived from an alternate Steinberg , Katherine McKinnon, MS , Masaki Terabe, PhD , Purevdorj 1 1 1 reading frame of the T cell receptor gamma chain locus. Proc Natl Acad Olkhanud , Ira Pastan, MD , Jay Berzofsky, MD, PhD 1 2 Sci U S A. 2000;97(17):9437–9442. National Cancer Institute, Bethesda, MD, United States; PDS 2. Oh S, Terabe M, Pendleton CD et al. Human CTLs to wild-type and en- Biotechnology, Berkeley Heights, NJ, United States hanced epitopes of a novel prostate and breast tumor-associated pro- Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) tein, TARP, lyse human breast cancer cells. Cancer Res 2004;64(7):2610- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P332 3. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated Background with slowing in PSA velocity and decreasing tumor growth rates in With the success of immune checkpoint inhibitors, anti-tumor im- patients with StageD0prostatecancer. Oncoimmunology. munity is at the focus of cancer therapy. The pursuit of the mechan- 2016;5(8):e1197459. ism to boost anti-tumor T-cell responses is critical to improve the Ethics Approval suboptimal response rate to checkpoint inhibitors. Cancer vaccines The study was approved by the National Cancer Institute Ethics Board can be used to induce such tumor-specific T-cell responses as one of assigned a local number 09C0139, approval number P08397.” the solutions to low responses to checkpoint inhibitors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 181 of 272 P333 Ethics Approval Timed anti-tumor vaccination during chemotherapy induces This study was approved by the Central Committee of Human Investigations strong T-cell immunity and prolonged survival of late stage and by the ethical board of the Leiden University Medical Center (LUMC): cervical cancer patients EudraCT 2013-1804-12. 1 1 Marij Schoenmaekers-Welters, PhD , Marij Welters, PhD , Cornelis Melief, 2 3 1 MD, PhD , Ignace Vergrote , Judith Kroep, MD, PhD , Gemma Kenter, 4 5 6 7 P334 MD,PhD , Nelleke Ottevanger , Wiebren Tjalma , Hannelore Denys , 1 8 8 Concurrent cetuximab (CTX) and nivolumab (NIVO) in patients Mariette van Poelgeest, MD, PhD , Hans Nijman , Anna Reyners , Thierry 9 9 10 1 1 with recurrent and/or metastatic (R/M) head and neck Velu , Frederic Goffin , Roy Lalisang , Nikki Loof , Sanne Boekestijn , 2 2 2 squamous cell carcinoma (HNSCC): Safety results of a phase I/II Willem Jan Krebber , Leon Hooftman , Sonja Visscher , Brent 11 2 5 study Blumenstein, PhD , Richard Stead , Winald Gerritsen , Sjoerd van der 1 1 2 3 Christine Chung, MD , Marcelo Bonomi, MD , Conor Steuer, MD , Burg, PhD 1 2 1 1 1 1 Michael Schell , Jiannong Li , Matthew Johnson , Caitlin McMullen ,J. Leiden University Medical Center, Leiden, ZA, Netherlands; ISA 3 1 1 1 1 Trad Wadsworth , Krupal Patel , Julie Kish, MD , Jameel Muzaffar, MD , Pharmaceuticals, Leiden, Netherlands; University Hospital Leuven, 4 1 4 3 Kedar Kirtane , James Rocco , Nabil Saba, MD Leuven, Belgium; Center for Gynecological Oncology, Amsterdam, 5 1 2 Moffitt Cancer Center, Tampa, FL, United States; Ohio State University Netherlands; Nijmegen University Medical Center, Nijmegen, 6 7 3 Medical Center, Columbus, OH, United States; Emory University, Atlanta, Netherlands; University Hospital Antwerp, Antwerp, Belgium; University 8 4 GA, United States; Ohio State University, Columbus, OH, United States Hospital Gent, Gent, Belgium; University Medical Center Groningen, Correspondence: Christine Chung (christine.chung@moffitt.org) Groningen, Netherlands; Chirec Cancer Institute, Maastricht, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P334 Netherlands; University Medical Center Maastricht, Maastricht, Netherlands; Trial Architecture Consulting, Washington, DC, United Background States Use of anti-Programmed Death-1 (anti-PD-1) inhibitors is a Correspondence: Marij Schoenmaekers-Welters (M.J.P.Schoenmaekers- standard of care for patients (pts) with R/M HNSCC, but only Welters@lumc.nl) limited numbers of pts achieve long term clinical benefits. Im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P333 proving its efficacy and maintaining low toxicity profile are critical in combination strategies. We report the safety results Background of a phase I/II trial of CTX and NIVO in pts with R/M HNSCC. High-risk human papilloma virus type 16 (HPV16) is the major cause Methods of inducing cervical cancer. The oncoproteins E6 and E7 are respon- Pts were treated with CTX 500 mg/m2 IV on Day (D) -14 as a lead-in sible for the cancer development and therefore targeted by the followed by CTX 500 mg/m2 IV and NIVO 240 mg/m2 IV on D1 and therapeutic synthetic long peptide (SLP) vaccine ISA101. Monother- D15 every 28-D cycle (C). Pts with CTX infusion reaction or who did apy induced HPV16 E6/E7-specific T cells and was clinically effective not receive C1D1 for any reason were considered to be non- in half of the HPV16-SLP vaccinated patients with HPV16+ high- evaluable and were replaced. The toxicities with possible, probable, grade premalignant lesions of the vulva. However, in HPV16+ cervical and definite attribution were included in treatment-related adverse cancer patients additional measures need to be taken as the vaccine- events (TRAEs) and immune-related adverse events (IRAEs) analyses. induced T cells encounter an immunosuppressive milieu in the tumor NIVO dose reduction was not allowed. microenvironment. Therefore, in the current study the ISA101 vaccin- Results ation is combined with standard-of-care chemotherapy. For the phase I cohort, 3 pts were enrolled. No dose limiting toxic- Methods ities were observed during 4 weeks of observation period after C1D1, Late stage cervical cancer patients (n=77) were treated 3 times with and no dose reduction was required. An additional 44 pts were en- ISA101 with a 3-week interval in a single arm dose escalation study rolled, and 2 pts were non-evaluable. A total of 45 pts were analyzed. testing 4 different doses of ISA101 and with the addition or not of The median age was 64 (range 24-77), with 37 males and 8 females. pegylated IFN alpha (PegIntron). The start of ISA101 vaccination was The ECOG performance status at baseline was 0 (9 pts, 20%), 1 (33 at day 15 after the second cycle of standard-of-care chemotherapy, pts, 73.3%), and 2 (3 pts, 6.7%). The primary sites were oral cavity 10 which consisted of carboplatin (AUC6)/paclitaxel (175mg/m2). Blood (22%), oropharynx 24 (53%), hypopharynx 3 (7%), larynx 6 (13%), and samples taken during the study were subjected to a set of comple- unknown primary 2 (4%). The p16 status was positive 22 (49%), mentary immune assays to determine the vaccine-induced T-cell negative 11 (24%), and unknown 12 (27%). The p16 status of the responses. subsite, oropharynx, was positive 20 (83%) and negative 4 (17%). The Results smoking status was current 6 (13%); former 27 (60%) and never 12 In 43% of the 72 evaluated patients an objective clinical response (27%) with median pack years of 20 (range 0-185). Prior chemother- was observed. Carboplatin/paclitaxel depleted myeloid suppressive apy was given in 44 (98%). Prior radiotherapy was given in 36 (80%). cells (p The most common grade 3 TRAEs occurring >2% were fatigue 5 Conclusions (11%) and rash-acneiform 2 (4.4%). The only grade 4 TRAE was CTX Our study demonstrates that chemotherapy combined with immuno- infusion reaction in 1 (2.2%). The most common grade 3 and 4 IRAEs therapy, in this case HPV16-SLP vaccination, can be exploited to ef- occurring >2% were fatigue 2 (4.4%). No grade 5 TRAEs or IRAEs oc- fectively treat HPV16+ cervical cancer patients and warrants curred. TRAEs led to CTX dose reduction in 4 (9%) of pts: infusion re- confirmation in a randomized controlled trial. action, diarrhea, hypomagnesemia, fatigue (one each). Conclusions Acknowledgements The combination of CTX and NIVO is well tolerated and remains to This work was financially supported by the Dutch Cancer Society grant 2009-4400 be an option for future studies. (to C.J.M. Melief and S.H. van der Burg). ISA Pharmaceuticals sponsored the trial. Ethics Approval Trial Registration The study was approved by Advarra CIRBI, approval number 00000971 ClinicalTrials.gov NCT02128126. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 182 of 272 P335 Surprisingly, FOXP3+, and CD68+ staining cells were positively as- The tumor immune microenvironment and its association with pCR sociated with pCR. Additional analyses currently being conducted in NRG Oncology/NSABP B-52: Quantification of PD-1, PD-L1, CD8, to assess during treatment biopsies and the spatial relationships FOXP3, and CD68 by multiplex fluorescent-immunohistochemistry between different immune markers may provide further mechan- 1 1 1 1 1 Marion Joy, PhD , Ying Wang , Rim Kim , Nan Song , Ashok Srinivasan , istic insights. 1 1 2 1 Huichen Feng , Corey Lipchik , Reena Cecchini , Samuel Jacobs , Joseph 2 3 4 5 Costantino , Sandra Swain , Eleftherios Mamounas , Priya Rastogi , Acknowledgements 6 7 2 8 Soonmyung Paik , C. Kent Osborne , Norman Wolmark , Peter Lucas , U10CA180868; U24CA196067; UG1CA189867; Genentech, BCRF 9 1 Mothaffar Rimawi , Katherine Pogue-Geile Trial Registration 1 2 NRG Oncology/NSABP, Pittsburgh, PA, United States; NRG Oncology/ NCT02003209 NSABP and the University of Pittsburgh, Pittsburgh, PA, United States; Ethics Approval NRG Oncology/NSABP and Georgetown Lombardi Comprehensive Chesapeake IRB: Samples are exempt based on the Determination for NSABP Cancer Center, Georgetown University Medical Center, Washington, Foundation, Inc. Protocol TB-2 “NSABP TB-2: Comprehensive Survey of DC, United States; NRG Oncology/NSABP and Orlando Health, UF Prognostic and Predictive Markers for Breast and Colon Cancer” Health Cancer Center, Orlando, FL, United States; NRG Oncology/ (Pro00005069). All patients provided written informed consent to the NSABP NSABP and the University of Pittsburgh Cancer Institute, Pittsburgh, B-52 Clinical Study, which was reviewed and approved by the NCI CIRB. No PA, United States; NRG Oncology/NSABP and Yonsei University personal identifiable information is included. College of Medicine, Pittsburgh, PA, United States; NRG Oncology/ NSABP and The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States; NRG Table 1 (abstract P335). See text for description Oncology/NSABP and the University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; NRG Oncology/NSABP The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States Correspondence: Katherine Pogue-Geile (katherine.pogue- geile@nsabp.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P335 Background The NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test whether addition of estrogen deprivation (ED) would improve pCR rate in HER2+/ER+ breast cancer patients treated with docetaxel, carboplatin, trastuzumab, and pertuzu- mab (TCHP). A numerical increase in pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. B-52 provided the opportunity to explore potential predictive markers of pCR to possibly guide new treatment strategies. We examined the tumor immune microenvironment (TME) of B-52 baseline biopsy tumors to determine its associ- ation with pCR. Methods Pretreatment (N=238) biopsies were assessed for CD8, FOXP3, P336 CD68, PD-L1, and PD-1, with multiplex fluorescent immunohisto- Impact of cytokine release syndrome on cardiac function following chemistry (mf-IHC) utilizing the Vectra® Quantitative Pathology CD19 CAR-T cell therapy in children and young adults with acute Imaging System and inForm® Advanced Image Analysis software. lymphoblastic leukemia 1 1 2 Tumor and stromal regions were defined with a panCK antibody Amita Kulshrestha , Haneen Shalabi, Do , Vandana Sachdev , Douglas 2 2 3 4 (included in the same multiplex) and annotated by a patholo- Rosing , Stanislav Sidenko , Crystal Mackall, MD , Brandon Wiley , Daniel 5 6 gist. Digital quantitation of all markers was assessed in the Lee , Nirali Shah 1 2 tumor and stromal regions (defined by panCK). In our pre-specified, National Institutes of Health, Bethesda, MD, United States; National CTEP-approved analysis, we tested the association of PD-L1 in tumor+- Heart, Lung and Blood Institute, Bethesda, MD, United States; Stanford, stroma with a cut-point optimized by ROC curves (Table1). In explora- Stanford, CA, United States; Mayo Clinic, Rochester, MN, United States; 5 6 tory analyses, we also tested a clinically meaningful PD-L1 cut-point of University of Virginia, Charlottesville, VA, United States; National Cancer >1%; other markers were tested for associations with pCR using chi- Institute, Kensington, MD, United States square tests and a median cut-off (Table 1). Correspondence: Nirali Shah (nirali.shah@nih.gov) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P336 Based on our pre-specified analysis, total PD-L1 (assessed in both stroma+tumor) was positively associated with pCR across Background trial arms (45% v 30%, p=0.038). PD-L1 was not significantly as- Cytokine release syndrome (CRS) is the main toxicity of CAR-T cell sociated with pCR with a clinically utilized cut-off of >1% within therapy, which may require hemodynamic support. The impact of the stromal-immune cell compartment (CD8+FOXP3+CD68). Sur- CRS on cardiac function has not been well described. prisingly, in both stromal and tumor cell compartments, CD68 Methods was positively associated with pCR when the two arms are eval- We report on cardiac toxicity seen in children and young adults with uated together. When the treatment arms are examined separ- ALL treated on our phase I trial of CD19 CAR-T cell therapy (clinical- ately, CD68 in the stromal compartment correlates positively trials.gov NCT01593696). All patients had a baseline echocardiogram. with pCR. FOXP3 was also positively correlated with pCR in the Cumulative anthracycline exposure was calculated from prior expos- stromal cell compartment across arms and in the TCHP+ED arm. ure. Additional studies included increased frequency of echocardio- PD-1 and CD8 were not significantly associated with pCR. grams upon ICU transfer, and serial troponin and proBNP. Conclusions Results B-52 showed a positive association of PD-L1 expression with pCR From July 2012 to March 2016, 52 patients, with a median age of 13.4 based on our pre-specified analysis but the extremely low cut-off years (range, 4.2-30.3) were treated on-study; 23 underwent at least (0.05%) makes the clinical utility of this observation doubtful. one prior allogeneic stem cell transplantation. CRS was seen in 37/52 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 183 of 272 (71%), which was grade 3-4 CRS in 8 subjects (21.6%). The median prior anthracycline exposure was 205 mg/m2 (range, 70-620 mg/m2) in doxorubicin equivalents. The median baseline LV ejection fraction (LVEF) was 62% (range 52%-71%). The median LV global longitudinal strain (GLS), at baseline was abnormal: -17 (range, -14 to -24, n=35). ICU transfers occurred in 20 patients, 11 of whom required vasoactive hemodynamic support, with 5 necessitating more than 1 pressor. Seven patients received tocilizumab and 4 patients received steroids. Six (16%) patients developed cardiac dysfunction, amongst whom 4 had grades 3-4 CRS. (Figure 1) Severe cardiac dysfunction, (LVEF < 30%) was seen in 3, with one patient developing cardiac arrest with subse- quent full recovery following placement of an intra-aortic balloon pump, steroids, and tocilizumab. In 2 of these patients, anthracycline exposures exceeded > 360 mg/m2. All but 2 patients had full resolution of cardiac dysfunction by day 28 post CAR. Troponin elevations were seen in 4 of 6 patients with low LVEF. In a limited cohort of patients with pre/post pro-BNP, pro-BNP was higher during CRS, with the high- est levels correlating with more severe cardiac dysfunction. (Figure 2) Conclusions Patients with higher-grade CRS are more likely to experience significant cardiac side effects from CAR T-cell therapy. In most cases, resolution to near baseline was seen coinciding with resolution of CRS, with most having near complete resolution by day 28 post infusion. Implementation of more frequent echocardiogram monitoring and incorporation of BNP and troponin into daily laboratory panel may help to identify those at highest risk of severe cardiac Fig. 2 (abstract P336). Changes in BNP pre and post-CAR infusion dysfunction at an earlier time point, allowing for earlier interven- tion in CRS to potentially limit acute cardiac toxicity. P337 Acknowledgements Survival prolongation by dendritic cell vaccination in combination This research was supported by the Intramural Research Programs of the Center with OK-432, gemcitabine and/or S-1 in patients with advanced of Cancer Research, National Cancer Institute, NIH and the Clinical Center. pancreatic cancer Trial Registration Masahiro Ogasawara, MD, PhD, Shuichi Ota, MD PhD The clinical trial is registered at clinicaltrials.gov NCT01593696 Sapporo Hokuyu Hospital, Sapporo, Japan Ethics Approval Correspondence: Masahiro Ogasawara (ogasawara@hokuyu-aoth.org) This study was approved by the National Cancer Institute Institutional Review Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P337 Board. Background Pancreatic cancer is the most fatal human cancer, with a 5-year overall survival rate of less than 5%. In the current study, we have evaluated the clinical and the immunological responses in patients with advanced pancreatic cancer who received dendritic cell (DC) vaccination in combination with a toll like receptor (TLR) 4 agonist, OK-432 and chemotherapeutic agents, gemcitabine (GEM) and/or S-1. Methods Twenty three patients (13 males, 10 females; aged 37-83 years, me- dian 64 year old) with advanced pancreatic cancer refractory to standard treatment were treated with DC vaccination in combination with OK-432, GEM and/or S-1 from 2012 to 2013 at Sapporo Hokuyu Hospital. Autologous DCs were generated by culturing adherent mononuclear cells with interleukin-4 and granulocyte-macrophage colony stimulating factor. DCs were then loaded with synthetic pep- tides derived from cancer antigens such as Wilms’ tumor 1 (WT1) and MUC1 following maturation by prostaglandin E2 and OK-432. Peptide-loaded mature DCs and OK-432 were administered intrader- mally every 2 weeks, 7 times. The induction of vaccine-induced T cell responses was monitored by using HLA-tetramer and ELISPOT assays. Results The treatment was well tolerated and none of the patients experi- enced more than grade 3 adverse events during the treatment period. Of 23 patients, 1 had partial response (PR), 8 had stable dis- ease (SD) and 14 had progressive disease after one course of vaccin- ation. The median overall survival (OS) was 9.6 months. Survival of patients achieving PR or SD (responders) was longer than those who did not respond to the treatment (non-responders) (median OS; 18.0 Fig. 1 (abstract P336). Change in Ejection Fraction Post CRS Onset vs 5.8 months). An HLA-tetramer assay showed an increase in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 184 of 272 positivity of WT1-specific CD8+ T cells in both responders and non- Results responders after vaccination. However, the increment in the positivity 792 patients were enrolled (intention-to-treat [ITT] population), includ- was remarkable in responders in comparison with non-responders; ing 529 with PD-L1+ tumors (primary analysis population) and 263 with 46.3 and 10.7 fold in responders and non-responders, respectively. PD-L1− tumors. (n=263). At data cut-off (March 4, 2019) in the PD-L1+ Similarly, an ELISPOT assay showed marked increase in spot-positive population, median duration of follow-up for OS was 35.4 months in cells in responders. The median OS in patients showing the positivity the avelumab arm (n=264) and 34.7 months in the docetaxel arm (n= in both assays was longer than those who were positive in either 265); study treatment was ongoing in 25 (9.5%) vs 0 patients, and 17 assay or who were negative in both assays; a median OS was 18.4 (6.4%) vs 74 (27.9%) had received a posttreatment checkpoint inhibitor months, 9.7 months and 4.7 months, respectively, suggesting a cor- (CPI), respectively. 2-year OS rates with avelumab vs docetaxel in differ- relation between an immune response and a clinical outcome. ent PD-L1+ subgroups are shown (Table 1). Of patients with PD-L1+ tu- Conclusions mors alive at 2 years, 67% had received a posttreatment CPI in the DC vaccination combined with a conventional chemotherapy in pa- docetaxel arm compared with 13% in the avelumab arm. In patients tients with advanced pancreatic cancer was demonstrated to be safe with PD-L1+ tumors who had an objective response with avelumab (50 and can elicit immune responses against tumor antigens, which was [18.9%]) or docetaxel (28 [10.6%]), median duration of response (DOR; correlated with clinical effects. investigator assessed) was 19.1 months (95% CI: 10.8-34.8) vs 5.7 Ethics Approval months (95% CI: 4.1-8.3), and proportions with a response lasting ≥6 This study was approved by the Ethics and Internal Review Board at months were 86.0% (95% CI: 72.9%-93.1%) vs 48.1% (95% CI: 28.7%- Sapporo Hokuyu Hospital, approval number 131111.08 65.2%), respectively. Safety profiles of avelumab and docetaxel were similar to those in previous analyses. Conclusions P338 Updated data from JAVELIN Lung 200 showed that although avelu- 2-year follow-up from JAVELIN Lung 200, an open-label, mab did not significantly prolong OS vs docetaxel in the primary randomized, phase 3 study of avelumab vs docetaxel in patients confirmatory analysis, 2-year OS rates were doubled with avelumab with platinum-treated advanced non-small cell lung cancer vs docetaxel in higher PD-L1+ subgroups, and median DOR was >12 (NSCLC) months longer with avelumab vs docetaxel. 1 2 3 Fabrice Barlesi, MD, PhD , Mustafa Özgüroğlu , Johan Vansteenkiste , 4 5 6 7 David Spigel , James Yang , Hidenobu Ishii , Marina Garassino , Filippo Acknowledgements 8 9 10 11 de Marinis , Aleksandra Szczesna , Andreas Polychronis , Ruchan Uslu , This study was funded by Merck KGaA, Darmstadt, Germany, as part of an 12 13 14 Maciej Krzakowski , Jong-Seok Lee , Luana Calabro, MD , Osvaldo alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, 15 16 17 17 Aren Frontera , Barbara Ellers-Lenz , Marcis Bajars , Mary Ruisi , NY, USA. Keunchil Park Trial Registration Aix-Marseille University, Assistance Publique - Hôpitaux de Marseille, NCT02395172 Livon, France; Cerrahpaşa Medical Faculty, Istanbul University, Leuven, Ethics Approval 3 4 Belgium; University Hospital KU Leuven, Leuven, Belgium; Sarah The study protocol was approved by institutional review boards and ethics Cannon Research Institute, Nashville, TN, United States; National Taiwan committees at each institution. The study was done in accordance with the University Hospital, Taipei, Taiwan, Province of China; Kurume University trial protocol, Good Clinical Practice guidelines, and the Declaration of School of Medicine, Kurume, Japan; Fondazione IRCCS Istituto Helsinki. All patients provided written informed consent. Nazionale dei Tumori, Milan, Italy; Istituto Europeo di Oncologia, Milan, 9 10 Italy; Regional Lung Disease Hospital, Otwock, Poland; Mount Vernon Cancer Centre, Northwood, Middlesex, United Kingdom; Ege University 12 Table 1 (abstract P338). See text for description Hospital, Izmir, Turkey; Centrum Onkologii-Instytut Im. M. Skłodowskiej- Curie w Warszawie, Warszawa, Poland; Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea, Republic of; University Hospital of Siena, Siena, Italy; 15 16 Instituto Nacional del Cáncer, Santiago, Chile; Merck KGaA, Darmstadt, Germany; EMD Serono Inc, Billerica, MA, United States; Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence: Fabrice Barlesi (Fabrice.BARLESI@ap-hm.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P338 Background P339 Avelumab, a human IgG1 anti–PD-L1 monoclonal antibody, is ap- Survival is improved by antigen-specific cytotoxic T lymphocytes proved as monotherapy for metastatic Merkel cell carcinoma and (CTL) responses after treatment with the vaccine Tedopi in HLA-A2 platinum-treated urothelial carcinoma in various countries, and in positive advanced non-small cell lung cancer (NSCLC) patients 1 2 combination with axitinib to treat advanced renal cell carcinoma in Benjamin Besse, MD PhD , Enriqueta Felip, MD PhD , Giuseppe 3 4 5 6 the United States. In the JAVELIN Lung 200 study, avelumab did not Giaccone , Rafal Dziadziuszko , Elisabeth Quoix, MD , Werner Hilgers , 7 8 9 10 significantly prolong overall survival (OS) vs docetaxel in patients Federico Cappuzzo , Christophe Borg , Jordi Remon , Nicolas Poirier , 10 10 11 with platinum-treated PD-L1+ NSCLC (primary objective); however, Dominique Costantini , Bérangère Vasseur , Santiago Viteri 1 2 prespecified exploratory analyses showed longer OS with avelumab Gustave Roussy, Villejuif, France; Vall d'Hebron University Hospital, vs docetaxel in patients with higher PD-L1+ tumors. We report up- Barcelona, Spain; Georgetown University, Washington, DC, WA, United 4 5 dated data from JAVELIN Lung 200. States; Medical University of Gdańsk, Gdańsk, Poland; Nouvel Hôpital Methods Civil, Strasbourg, France; Institut Sainte Catherine, Avignon, France; 7 8 Patients with stage IIIB/IV or recurrent NSCLC and disease progres- AUSL Romagna, Ravenna, Italy; Centre Hospitalier Universitaire, 9 10 sion following platinum-doublet chemotherapy were randomized 1:1 Besançon, France; CIOCC- Barcelona, Barcelona, Spain; OSE to avelumab 10 mg/kg Q2W or docetaxel 75 mg/m2 Q3W. The pri- Immunotherapeutics, Nantes, France; University Hospital Dexeus, mary endpoint was OS; the primary analysis population was patients Barcelona, Spain with PD-L1+ tumors (≥1% tumor cell expression; PD-L1 IHC 73-10 Correspondence: Benjamin Besse (benjamin.besse@gustaveroussy.fr) pharmDx assay). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P339 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 185 of 272 Background Tedopi (OSE2101) is a multiple epitope vaccine restricted to HLA-A2 positive patients (45%), targeting five tumor-associated antigens (TAA) frequently expressed in solid tumors: carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER-2/neu), melanoma-associated antigen type 2 and 3 (MAGE2 and MAGE3), and p53. Tedopi is composed by 2 wild type and 7 chemically modi- fied peptides to increase HLA-A2 or T cell receptor (TCR) affinity. A pan-DR epitope (PADRE) of helper T-lymphocyte (HTL) has been added to increase the Cytotoxic T Lymphocyte (CTL) responses. In previously treated advanced NSCLC patients, Tedopi showed a strong CTL immune response, which correlated with overall survival (OS) [1]. The aim of the current translational study was to explore the predict- ive effect of the epitope type and number of epitopes on OS. Methods Fig. 1 (abstract P339). Overall survival in patients with 3-6 vs 0-2 Out of 64 previously treated HLA2+ advanced NSCLC patients en- CTL responses rolled in a phase II trial testing the efficacy of Tedopi (1mL subcuta- neously Q3W for 6 cycles, then Q8W for the reminder year 1 and Q12W up to year 2), 33 patients were assessed for epitope-specific cytotoxic response and HTL responses using an interferon gamma P340 enzyme-linked immunosorbent assay. Leukapheresis was performed Region-focused deep survival learning on PD-L1 stained tissue at baseline, at week 9, 18 and 30 for immunogenicity assays. Predict- samples for data-driven stratification of durvalumab-treated ive analyses of OS were performed using Cox regression. NSCLC patients Results 1 1 1 Nicolas Brieu, PhD , Ansh Kapil , Armin Meier, PhD , Keith Steele, DVM, Patients were stage IV (64%), or locally advanced stage IIIb (36%). 2 2 1 PhD , Marlon Rebelatto, DVM, PhD, DACVP , Guenter Schmidt, PhD Eleven patients were assessed for all 10 epitopes, and 33 for 6 se- 1 2 Definiens AG, Munich, Germany; AstraZeneca, Gaithersburg, MD, lected epitopes (2 CEA, 1 HER-2, MAGE2, MAGE3, PADRE). Median United States survival was 30 months. Correspondence: Nicolas Brieu (nbrieu@definiens.com) There was at least one CTL response to one vaccine epitope in >90% Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P340 of patients. Eight epitopes were highly immunogenic (from 55% to 91%), while HER-2 wild type and one p53 analogue shown a lower Background response (respectively 36% and 9%). The selection of metastatic non-small cell lung cancer (NSCLC) pa- In patients evaluated for 6 selected epitopes, the best cut-off of num- tients that are likely to respond to an anti-PD-L1 checkpoint mono- ber of CTL responses to discriminate OS were 1-6 versus 0, 2-6 vs 0-1 therapy can be guided by the visual assessment by pathologists of or 3-6 vs 0-2. All of three were statistically significant. As an example, the Tumor Cell (TC) score on PD-L1 stained tissue samples [1]. Deep patients with CTL responses to 3-6 epitopes (n=23) had a median OS learning approaches have recently enabled the computer-based rep- of 38 months compared to 15 months in patients (n=10) with CTL re- lication of this visual TC score [2,3] and of its ability to predict overall sponses to 0-2 epitopes (HR=0.39; p=0.04) (Figure 1). CTL response survival (OS) [4]. Because these methods try to reproduce as close as to HER-2 analogue, MAGE3, PADRE and one p53 analogue were pre- possible the visual scoring methodology, they are built on extensive dictive of better OS. prior hypotheses (e.g. definition of cell positivity, score and cut-off) Conclusions and do not enable the data-driven discovery of novel stratification In NSCLC patients, survival was significantly prolonged in patients immu- rules. We present here a novel region-focused end-to-end deep- nized to epitope specific Tedopi vaccine. HER-2, MAGE3, PADRE and p53 learning approach that enables the data-driven generation of survival were identified as vaccine predictive epitopes for prolonged survival. risk heatmaps and the stratification of patients into two risk groups. Methods Acknowledgements On a subset (N=151) of core needle biopsies and tissue resections from We thank François Montestruc and Constant Josse (eXYSTAT, Malakoff, the NCT01693562 clinical trial (NSCLC), epithelium regions are automat- France) for the statistical analysis ically segmented within the manually delineated tumor area [3]. A patch-based convolutional neural network (CNN) is trained on selected Reference patches in a two-fold pre-validation procedure to maximize a log partial 1. Barve M, Bender J, Senzer N, Cunningham C, Greco FA, McCune D, Steis likelihood derived from the Cox proportional hazards model [5,6]. To R, Khong H, Richards D, Stephenson J, Ganesa P, Nemunaitis J, Ishioka G, avoid a disproportionately large number of patches from tissue resec- Pappen B, Nemunaitis M, Morse M, Mills B, Maples PB, Sherman J and tions, a random subset of up to 10K patches is selected for each patient Nemunaitis JJ. Induction of Immune Responses and Clinical Efficacy in a within the segmented regions. The overall survival risk is predicted and Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, aggregated by mean on the detected epithelium regions only. Patients in Metastatic Non-Small-Cell Lung Cancer. J Clin Oncol. 2008;26(27):4418– are finally stratified based on the cohort median of the resulting aggre- gated risk scores. For baseline comparison, the same steps are repeated Ethics Approval considering the complete delineated tumor area instead of the sole The study protocol and its related documents (including the patient segmented epithelium regions. information and informed consent form) received approval from the Institutional Review Board (IRB), and the Competent Authority prior to Results study initiation. The proposed epithelium-focused and data-driven survival CNN Consent yields similar patient stratification (HR=0.525, p=0.003) as obtained Each patient gave his/her written informed consent prior to study with 25% cut-off on visual (HR=0.574, p=0.01) or automated (HR = enrolment. 0.539, p=0.004) TC score (Figure 1), while releasing prior hypotheses Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 186 of 272 on PD-L1 region positivity, score methodology, and cut-off value. As P341 expected on durvalumab-treated patients, high and low risks are as- Activation of toll-like receptors via PS-targeting monoclonal sociated with low and high PD-L1 staining respectively. No relevant antibodies risk groups are identified if the analysis is performed on the full Rolf Brekken, PhD (rolf.brekken@utsouthwestern.edu) tumor area instead. UT Southwestern Medical Center, Dallas, TX, United States Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P341 Our results suggest, for the first time on core needle biopsies and tis- sue resections, (i) the ability of end-to-end deep survival learning to Background directly learn relevant patient stratification as well as (ii) the neces- Multifocal immune suppression in the tumor microenvironment is a sity, in case of small patient cohorts, to restrict the analysis to auto- major underlying cause for the limited efficacy of immune check- matically detected meaningful regions. point blockade. Persistent immune suppression prevents the devel- opment of a robust T cell response to tumor specific antigens that is References required for effective downstream immune checkpoint blockade. An 1. Rebelatto et al., Development of a programmed cell death ligand-1 im- underappreciated but significant contributor to immune suppression munohistochemical assay validated for analysis of non-small cell lung in tumors is the exposure of the membrane phospholipid phosphati- cancer and head and neck squamous cell carcinoma, Diagnostic Path- dylserine (PS) on the surface of tumor cells and tumor-derived micro- ology 2016 vesicles. PS is recognized by receptors on immune cells where it 2. A. Kapil et al., Deep Semi Supervised Generative Learning for Automated triggers the secretion of immune suppressive cytokines, prevents the Tumor Proportion Scoring on NSCLC Tissue Needle Biopsies, Scientific differentiation of myeloid-derived suppressor cells (MDSCs) and in- reports, 2018 hibits dendritic cell (DC) maturation; events that prevent a productive 3. A. Kapil et al., DASGAN - Joint Domain Adaptation and Segmentation for anti-tumor T cell response. Bavituximab, a chimeric monoclonal the Analysis of Epithelial Regions in Histopathology PD-L1 Images, arXiv antibody (mAb) that targets PS and inhibits PS-mediated im- preprint arXiv:1906.11118, 2019 munosuppressive signaling, drives immune activation by reducing 4. N. Brieu et al., Deep learning-based PD-L1 tumor cell (TC) scoring im- the levels of MDSCs, by polarizing tumor-associated macrophages proves survival prediction compared to pathologists on durvalumab- towards an immune stimulatory phenotype and by promoting treated NSCLC patients, SITC 2018. the maturation of dendritic cells (DCs). Bavituximab and other PS- 5. P. Mobadersany et al., Predicting cancer outcomes from histology and targeting mAbs (2aG4 and 1N11) bind to PS via beta-2 glycopro- genomics using convolutional networks, PNAS 2018. tein 1 (β2GP1). β2GP1, an abundant serum glycoprotein, was re- 6. A. Meier et al., End-to-end learning to predict survival in patients with cently identified as a novel component of innate immunity via gastric cancer using convolutional neural networks, Annals of Oncology activation of Toll-like receptors (TLRs). (ESMO), 2018. Methods Ethics Approval Human β2GP1. Monoclonal-antibodies: Bavituximab and 1N11. qPCR, For the Phase 1/2 durvalumab trial (NCT01693562), the study protocol was WB, IP, ICC/IHC, CD, TEM, MST and ELISA reviewed and approved by the Institutional Review Board of the Results participating centers and informed consent was obtained from all We investigated whether the innate immune changes induced by patients. PS-targeting mAbs is mediated in part by β2GP1-induced activation of TLRs in myeloid cells. β2GP1 has a closed conformation that pre- vents its interaction with PS and cell surface receptors. However, interaction of β2GP1 with LPS can induce an open conformation of the protein that allows interaction of β2GP1 with PS and cell surface receptors, including the TLRs. We found through circular dichroism and transmission electron microscopy that the PS-targeting anti- bodies also induce conformational changes in β2GP1, shifting it to an open conformation. In addition, we demonstrate that PS-targeting mAb-mediated dimerization of β2GP1 stimulated pro-inflammatory polarization of bone marrow-derived macrophages (BMDMs) and in- duced TLR2 signaling. Finally, we investigated the expression of a newly characterized TLR-induced transcription factor, Spi-C in bone marrow progenitor cells after stimulation with TLR specific agonists or PS-targeting mAbs +/- β2GP1. Conclusions These studies demonstrate that the PS-targeting mAbs stimulate Spi- C expression in a β2GP1-dependent manner. Overall these data sup- port that one mechanism of innate immune activation induced by PS-targeting mAbs is through TLR2 stimulation on myeloid cells. Fu- ture studies are focused on validating these results in vivo using Tlr- deficient and β2gp1-deficient mice. Trial Registration NCT01999673, NCT03139916, NCT03519997 Ethics Approval All animal experiments were done according to the Animal Research Center (ARC) at UT Southwestern Medical Center. All clinical trials Fig. 1 (abstract P340). See text for description were conducted in accordance with all Federal and State Laws. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 187 of 272 P342 P343 Long term outcomes of a phase I study with UV1, a second Initial results from a Phase II study (TACTI-002) in non-small cell generation telomerase based vaccine, in patients with advanced lung cancer, or head and neck cancer patients receiving non-small cell lung cancer eftilagimod alpha (LAG-3 fusion protein) and pembrolizumab 1 2 2 1 2 3 Wenche Rasch, PhD , Paal Brunsvig, MD PhD , Martha Nyakas, MD , Clau Julio Peguero, MD , Enriqueta Felip, MD PhD , Bernard Doger , Margarita 2 2 2 4 5 6 7 Reisse, MD , Jon Amund Kyte , Hedvig Vidarsdotter Juul , Steinar Majem , Enric Carcereny , Tim Clay , Pawan Bajaj , Matthew Krebs, MD 1 3 2 8 9 Aamdal , Gustav Gaudernack, PhD , Else Marit Inderberg PhD , Frederic Triebel, MD, PhD 1 2 1 2 Ultimovacs ASA, Oslo, Norway; Oslo University Hospital, Oslo, Norway; Oncology Consultants, Houston, TX, United States; Vall d’ Hebron 3 3 Ultimovacs ASA, Prof. Emeritus, Oslo University Hospital, Oslo, Norway Institute of Oncology, Barcelona, Spain; Fundación Jimenez Díaz, Correspondence: Wenche Rasch (wenche.rasch@ultimovacs.com) Madrid, Spain; Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P342 Institut Català d'Oncologia Badalona, Barcelona, Spain; St John of God Subiaco Hospital, Perth, Australia; Tasman Health Care, Queensland, Background Australia; The University of Manchester and The Christie NHS A first generation hTERT vaccine (GV1001) showed evidence of clin- Foundation Trust, Manchester, United Kingdom; Immutep, Orsay, France ical efficacy in patients with advanced non-small cell lung cancer Correspondence: Frederic Triebel (ftriebel@immutep.com) (NSCLC). We have now tested a second generation hTERT vaccine, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P343 UV1. This vaccine is designed to give high population coverage and is composed of three synthetic long peptides containing multiple Background epitopes identified by epitope spreading data from long-term survi- Eftilagimod alpha (efti; previously IMP321) is a recombinant LAG-3 Ig vors who participated in previous hTERT vaccination trials. fusion protein that binds to MHC class II molecules to mediate anti- Methods gen presenting cell (APC) and CD8 T-cell activation. The stimulation Eighteen non-HLA-typed patients with stage III/IV NSCLC with no evi- of the dendritic cell network and subsequent T cell recruitment at dence of progression after prior treatments, were enrolled in a phase the tumor site with efti may lead to stronger anti-tumor CD8 T cell I dose-escalation study of UV1 vaccination with GM-CSF as adjuvant, responses than observed with pembrolizumab alone. Combining an evaluating safety, immune response, and long term clinical outcome. APC activator with an immune checkpoint inhibitor (ICI) aims to in- The present study also aimed to provide a rationale for combining crease efficacy without additional toxicity. We hereby report initial re- UV1 vaccine with PD-1/PD-L1 blockade. sults of stage 1 of this phase II trial (NCT03625323). Results Methods Treatment with GM-CSF and UV1 was well tolerated with no serious The study is based on a Simon's optimal two-stage design, with ob- adverse events observed. All patients experienced one or more ad- jective response rate (ORR) as primary endpoint. Secondary end- verse events, the majority grade 1, such as injection site reactions points include progression free survival and overall survival. Blood and fatigue. Seventeen patients were evaluable for tumor response; for PK/PD assessments and anti-drug antibody evaluation is col- 15 patients had stable disease as best response, while 2 patients had lected. During the first stage of the study, patients (pts) are recruited progressive disease. The median progression free survival (PFS) was into each of three indications: A: 1st line, PD-X naïve NSCLC; B: 2nd 12.3 months and the median overall survival (OS) was 28.2 months. line, PD-X refractory NSCLC; C: 2nd line PD-X naïve HNSCC. Additional The OS at 3 years was 44%. None of the 7 long-term surviving pa- patients (N2) will be recruited for each part if the pre-specified tients (median survival 4.96 years, range 4.04-5.51) have received threshold for ORR is met. In total 109 patients are planned to be en- checkpoint blockade therapy after UV1 vaccination. UV1-vaccination rolled. Eftilagimod alpha is administered as 30 mg subcutaneous in- induced specific T helper 1 (Th1) immune responses in the majority jection every 2 weeks for the first 8 cycles and every 3 weeks for the (67%) of patients. Both immune responses and OS were dose related. 9 following cycles. Pembrolizumab is administered at a standard Conclusions dose of 200 mg intravenous infusion every 3 weeks for maximum 2 The highest dose of UV1 (700 μg) resulted in the highest proportion years. The study was approved by all relevant ethics committees and of immune responses. These responses occurred more rapidly and institutional review boards. were stronger compared to lower doses and the patients in this Results group had a 3-year OS of 83%. This, together with the safety and Between 05 March and 24 July 2019, 27 pts were enrolled and clinical outcome data, favours 700 μg as the preferred UV1 dose in treated in the study. The mean age was 67 (range 53-84) and 74% this patient population. These results provide a rationale for further were male. The ECOG PS was 0 in 59% of the pts and 1 in 41% of clinical studies in NSCLC with UV1 vaccination in combination with the pts. The treatment has been well tolerated with the most com- immune checkpoint blockade. mon AEs being cough (9%), dyspnea (9%), diarrhea (6%) and asthe- nia (5%). Eleven treatment related SAEs were reported in ten pts. Acknowledgements Thirteen (13) pts of part A are evaluable (data cut-off 24th July 2019) We thank all the patients for their participation in the study for efficacy. The vast majority had only one post-baseline tumor as- Trial Registration sessment. Four pts of 13 (31%) had a partial response and six (46%) The clinical trial UV1/hTERT-L was performed with NoMA approval and is pts had stable disease according to iRECIST at data cut-off. registered with Clinicaltrials.gov on February 11, 2013 (NCT01789099). Conclusions Patients provided written informed consent to participate. Enrollment started Thirty (30) mg efti s.c. every 2 weeks in combination with standard on April 8, 2013. dose of pembrolizumab is safe and shows encouraging antitumor Ethics Approval activity. The study was approved by the institutional protocol board, the regional Trial Registration Ethical Committee (REC 2012/1114, EudraCT 2012- 001852-20) and the EudraCT: 2018-001994-25 Norwegian Medicines Agency (NOMA) and the study was registered at NCT: 03625323 clinicaltrials.gov (NCT01789099). Ethics Approval Consent The study was approved by Advarra IRB (US), approval number N/A, Individual patient consent was not applicable as no information in this approval date: 13/08/2018; London - Harrow Research Ethics Com- abstract/poster can be categorized as identifiable. mittee (UK), approval number 18/LO/1889; Instituto de Investigación Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 188 of 272 Hospital 12 de Octubre (Spain), approval number 18/376; Belberry 3.3mos), or overall survival (p=0.40, overall mOS 11.4 mos); therefore, HREC (Australia), approval number 2018-08-636; St John of God the study closed to enrollment at interim analysis. PD-L1 expression Health Care (Australia), approval number 1450. was not associated with response (p=0.52). IF demonstrated an asso- ciation between intratumoral CD4+/PD1+/Ki67+ cells and response (p=0.02). P344 Conclusions A randomized multi-center phase 2 study of combined PD-L1/ We did not observe a benefit adding targeted radiotherapy concur- CTLA-4 inhibition with or without radiation in non-small cell lung rently with combined PD-L1/CTLA-4 therapy in a PD-(L)1 inhibitor re- cancer patients who progressed on PD-(L)1 directed therapy: fractory NSCLC population. However, across cohorts PD-L1/CTLA-4 ETCTN 10021 was generally tolerable and led to response/disease control in some 1 2 3 Arta Monjazeb, MD, PhD , Anita Giobbie-Hurder, MS , Ana Lako , Mark patients, including responses >6 months. Multiplex IF suggests tumor 3 4 5 6 Awad, MD PhD , Ryan Gentzler, MD , Carrie Lee , Joleen Hubbard , infiltration by Ki-67+/PD-1+ CD4 T cells is associated with response 7 8 9 James Abbruzzese, MD , Salma Jabbour, MD , Nataliya Uboha , Kevin and worthy of further investigation. Additional correlative genomic 10 11 12 13 Stephans , Jennifer Johnson, MD , Haeseong Park , Liza Villaruz, MD , and immune analyses are planned. 3 14 15 Katrina Kao , Elad Sharon, MD, MPH , David Raben, MD , Raymond Trial Registration 3 14 14 Mak , Howard Streicher, MD , Helen Chen, MD , Mansoor Ahmed, ClinicalTrials.gov Identifier: NCT02888743 14 16 3 PhD , Scott Rodig, MD, PhD , F. Stephen Hodi, MD , Jonathan Ethics Approval Schoenfeld, MD, MPH This study was approved by the NCI Central IRB. 1 2 UC Davis Cancer Center, Sacramento, CA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; University of Virginia, Charlottesville, Table 1 (abstract P344). See text for description VA, United States; University of North Carolina, Chapel Hill, United 6 7 States; Mayo Clinic, Rochester, United States; Duke, Durham, United States; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States; University of Wisconsin, Madison, WI, United States; 10 11 Cleveland Clinic, Cleveland, United States; Jefferson Medical Center, Philadelphia, PA, United States; Washington University in St. Louis, St. Louis, United States; University of Pittsburgh Medical Center, Pittsburgh, PA, United States; National Institutes of Health, Bethesda, MD, United States; University of Colorado, Greenwood Village, CO, United States; Brigham and Women's Hospital, Boston, MA, United States Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P344 Background Preclinical data support combined PD-L1/CTLA-4 inhibition and sug- gest synergy between PD-L1/CTLA-4 inhibition and targeted radi- ation via enhanced systemic anti-tumor immune responses. We aimed to evaluate combined PD-L1/CTLA-4 inhibition in NSCLC pa- tients who progressed on prior PD-(L)1 inhibitors and determine whether high- or low-dose radiation could increase objective re- sponses outside the radiation field. Methods ETCTN 10021 is a multicenter randomized phase 2 study evaluating the addition of repeated low-dose fractionated radiotherapy (0.5 Gy BID x 2 days) or hypofractionated radiation (8 Gy x 3) concurrently P345 with PD-L1/CTLA-4 inhibition (durvalumab 1500mg/tremelimumab PARP inhibition when combined with PD-L1 inhibition has a 75mg q4w for 4 cycles followed by durvalumab monotherapy) in suppressive effect on T cells in patients with relapsed or recurrent NSCLC patients progressive on prior PD-(L)1 inhibitors (intervening small cell lung cancer therapy allowed). Patients were randomized 1:1:1 to durvalumab/tre- Nobuyuki Takahashi, MD, PhD, Vinodh Rajapakse, Min-Jung Lee, Akira melimumab alone or with low-dose or hypofractionated radiother- Yuno, Sunmin Lee, Sehyun Kim, Rasa Vilimas, Samantha Nichols, Jane apy. The primary endpoint was objective response per RECIST v1.1 Trepel, Anish Thomas excluding irradiated lesions with planned interim analysis. Correlative National Cancer Institute, Bethesda, MD, United States analyses were performed on tissue obtained following progression Correspondence: Anish Thomas (anish.thomas@nih.gov) on prior PD-(L)1 inhibitors using PD-L1 immunohistochemistry and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P345 multiplex immunofluorescence (IF) evaluating CD8, CD4, PD1, Ki67, and cytokeratin in tandem. Background Results Poly ADP-ribose polymerase (PARP) inhibition increased PD-L1 ex- We randomized 78 patients (26 per each of 3 arms) who received >= pression, augmented cytotoxic T-cell infiltration and potentiated the 1 cycle of study therapy between August 2017 and March 2019 anti-tumor effect of PD-L1 blockade in small cell lung cancer (SCLC) across 18 sites. Patients received PD-(L)1 inhibitors for a median of 1 in vivo [1]. Yet in clinical studies, PARP inhibitor plus PD-L1 inhibitor cycles (range 1-5) prior to enrollment; 68% had prior radiation. Treat- did not improve responses in relapsed or recurrent SCLC patients ment related adverse events (TRAE) of any grade were observed in compared to historical controls of PD-L1 inhibitor alone [2, 3]. Given 53 subjects (68%), and grade ≥3 events in 18 subjects (23%), includ- the role of PARPs in activating inflammatory gene expression [4], we ing 1 grade 5 respiratory failure. Response rate across all cohorts investigated the effects of PARP inhibition plus PD-L1 blockade on were 10% (n=8, 95% exact CI: 5%-19%), and disease control 19% (n= the adaptive immune system in SCLC patients. 15, 95% exact CI: 11-30%). Median duration of response was 10.3 Methods months (95% CI: 1.4 months – not reached). Response and disease NCT02484404 SCLC cohort is an open label phase 2 study evaluating control weren’t significantly different between arms (Table 1, p=0.99/ the combination of durvalumab (1500 mg iv, Q4W) and olaparib (300 0.52, respectively), nor was time to progression (p=0.88, overall mTTP Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 189 of 272 mg BID) in patients with relapsed or recurrent SCLC [2]. Peripheral Preliminary efficacy from a cohort of NSCLC patients who re- blood lineages (pretreatment [C1D1], 2 weeks [C1D15] and 6 weeks ceived prior immune checkpoint therapy was previously pre- [C3D1] after treatment) were serially assessed by flow cytometry. sented (SITC-2017); baseline tumor samples were collected to Results assess mechanisms of resistance to prior therapy. Here we de- 20 patients were evaluated. Activated Ki67+ HLA-DR+ T cells sig- scribe the baseline tumor microenvironments (TMEs) of 2 co- nificantly decreased post-treatment (median [interquartile range] horts of patients, melanoma and NSCLC, who experienced on C1D1 and C3D1: 3.5% [2.2–5.9] vs. 2.1% [1.8–3.3], p=0.033 progressive disease on prior anti–PD-(L)1 treatment before among CD4+ T cells; 2.6% [1.8–5.1] vs. 1.4% [1.0–2.3], p=0.002 starting bintrafusp alfa administration. among CD8+ T cells). Activated Ki67+ PD-1- T cells also signifi- Methods cantly decreased post-treatment (1.9% [1.2–3.9] vs. 1.3% [1.1–2.4], Fresh tumor biopsies from melanoma (n=29) and NSCLC (n=64) pa- p=0.020 among CD4+ T cells; 3.2% [2.0–5.0] vs. 2.4% [2.0–2.7], p= tients refractory or resistant to prior anti–PD-(L)1 therapy were proc- 0.025 among CD8+ T cells). By contrast, exhausted Ki67- TIM-3+ essed for FFPE and subjected to RNA sequencing. Based on the CD8+ T cells significantly increased post-treatment (0.8% [0.5–1.3] mechanism of action of bintrafusp alfa, genes and gene signatures vs. 1.2% [0.8–2.0], p=0.002). PD-1 expression on regulatory related to immune and TGF-β pathways were evaluated and com- Foxp3+ CD25+ T cells (Treg) and effector regulatory CD45RA- pared between tumor types. Foxp3hi T cells (eTreg) significantly increased post-treatment (me- Results dian [IQR] of mean fluorescence intensity [MFI] ratio on C1D1 Melanoma had elevated CD8+ T-cell gene signatures but low levels and C3D1: 2.2 [1.6–2.8] vs. 3.1 [2.2–3.4], p=0.002 among Treg; 2.4 of predicted tumor neo-antigens. NSCLC had elevated gene signa- [1.7–2.8] vs. 3.4 [2.3–4.2], p=0.002 among eTreg). CTLA-4 expres- tures suggesting increased infiltration of inhibitory immune cells and sion on non-regulatory Foxp3- CD4+ T cells (non-Treg) also in- gene signatures related to the TGF-β pathway. Lastly, certain mesen- creased post-treatment (0.18 [0.16–0.20] vs. 0.21 [0.18–0.23], p= chymal markers were expressed at a higher level in melanoma com- 0.007). pared with NSCLC—in particular, the gene encoding vimentin, which Conclusions is associated with metastasis and poor prognosis, was 4-fold higher The combination of olaparib and durvalumab resulted in significant in melanoma. decrease in peripheral blood activated T cells, whereas exhausted T Conclusions cells and inhibitory markers on Treg, eTreg and non-Treg cells signifi- The results suggest that this cohort of melanoma patients may cantly increased. These findings contrast with the expected changes have resisted prior immune therapy due to a low neo-antigen under PD-L1 inhibitor treatment alone. These paradoxical changes of count and/or a mesenchymal phenotype. In contrast, the mecha- immune subsets likely reflect the anti-inflammatory effect of olaparib, nisms of resistance in NSCLC patients in this study may have which may have attenuated the antitumor immune efficacy of been related to high levels of inhibitory immune and TGF-β path- durvalumab. way genes. Collectively, these results provide novel insights into Trial Registration TMEs of melanoma and NSCLC in patients refractory or resistant NCT02484404 to anti–PD-(L)1 agents. Ethics Approval References This study was performed with IRB (#15C0179) and FDA approval 1. Sen T, Rodriguez BL, Chen L, Corte CMD, Morikawa N, Fujimoto J, et al. and is registered with Clinicaltrials.gov on August 7, 2015 Targeting DNA Damage Response Promotes Antitumor Immunity (NCT02517398). Patients provided written informed consent to through STING-Mediated T-cell Activation in Small Cell Lung Cancer. Can- participate. cer Discov. 2019;9(5):646-61. 2. Thomas A, Vilimas R, Trindade C, Erwin-Cohen R, Roper N, Xi L, et al. Dur- P347 valumab in Combination with Olaparib in Patients with Relapsed SCLC: Immunomodulation in tumor and peripheral blood following Toca Results from a Phase II Study. J Thorac Oncol. 2019. 511 & Toca FC treatment in patients with solid tumors 3. Krebs M, Ross K, Kim S, De Jonge M, Barlesi F, Postel-Vinay S, et al. P1.15- 1 2 3 4 Gerald Falchook, MD , Jordi Rodon , Shree Venkat , Arthur Donahue , 004 An Open-Label, Multitumor Phase II Basket Study of Olaparib and 4 3 5 Peder Horner , Amber Thomassen , William Accomando , Maria Durvalumab (MEDIOLA): Results in Patients with Relapsed SCLC. J Thorac 5 5 5 5 Rodriquez-Aguirre , Cornelia Bentley , Daniel Hogan , Derek Ostertag , Oncol. 2017;12(11):S2044-S5. 5 5 5 5 Sharon Yavrom , Thian Khoeh , Douglas Jolly, PhD , Harry Gruber, MD , 4. Rosado MM, Bennici E, Novelli F, Pioli C. Beyond DNA repair, the 5 3 Jolene Shorr , Jaime Merchan immunological role of PARP-1 and its siblings. Immunology. 1 2 Sarah Cannon Research Institute, Denver, CO, United States; MD 2013;139(4):428-37. Anderson Cancer Center, Barcelona, Spain; University of Miami, Miami, Ethics Approval FL, United States; Diversified Radiology of Colorado, Denver, CO, United The trial was conducted under a National Cancer Institute Center for States; Tocagen Inc., San Diego, CA, United States Cancer Research–sponsored investigational new drug application Correspondence: Gerald Falchook (gerald.falchook@sarahcannon.com) with institutional review board approval; approval number 15-c- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P347 Background P346 Toca 511 (vocimagene amiretrorepvec) is a cancer-selective, gamma- Comparison of TMEs from melanoma and NSCLC patients retroviral replicating vector encoding yeast cytosine deaminase, an refractory or resistant to anti–PD-(L)1 therapies enzyme that converts 5 fluorocytosine (5-FC) into 5-fluorouracil in George Locke, Cherie Taglienti, PhD, Laureen S. Ojalvo, Christoph the tumor microenvironment. Preclinical models indicated that Toca Helwig, MSc, Alex Rolfe, Olaf Christensen, Isabelle Dussault, PhD 511 and 5-FC treatment kills dividing cancer and nearby immunosup- EMD Serono Research & Development, Billerica, MA, United States pressive cells, leading to T-cell priming and antitumor immune activ- Correspondence: Isabelle Dussault (isabelle.dussault@emdserono.com) ity [1]. A Phase 3 trial of Toca 511 & Toca FC (extended-release 5-FC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P346 for treatment of recurrent high grade glioma is ongoing, following Phase 1 observations of prolonged survival and durable complete re- Background sponses in some patients [2]. Bintrafusp alfa (M7824), an innovative first-in-class bifunctional Methods fusion protein composed of the extracellular domain of the This Phase 1b, single-arm, multicenter study (Toca 6) was designed TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb to investigate immunological changes following Toca 511 & Toca FC blocking PD-L1, has shown evidence of clinical activity in treatment in patients with advanced solid tumors. Patients received phase 1 studies of patients with advanced solid tumors. intravenous (IV) Toca 511 for 3 days (Week 1), underwent biopsy of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 190 of 272 metastatic tumor (Week 2), were dosed with oral Toca FC (Weeks 5 Background and 6), underwent follow-up biopsy (~Week 9), and then repeated The anti-tumor responses to immune checkpoint inhibitors (ICI) is oral Toca FC every 4-6 weeks. Longitudinal peripheral blood mono- limited in malignancies such as pancreatic adenocarcinoma (PA), nuclear cell (PBMC) samples were immunophenotyped by flow cy- head and neck squamous cell carcinoma (HNSCC) and castration re- tometry, and tumor biopsies were analyzed by sistant prostate cancer (CRPC) through establishment of inaccessible immunohistochemistry (IHC). hypoxic regions [1,2]. Under hypoxic conditions, evofosfamide (EVO) Results releases the alkylating agent Br-IPM, which decreases hypoxia, re- A total of 21 patients with a median 4 lines of prior chemotherapy duces density of MDSC, restores T cell infiltration and increases were enrolled (17 colorectal cancer, 2 sarcoma, 1 each non-small cell tumor antigen presentation [3]. Across syngeneic models, EVO dem- lung and pancreas cancer). PBMC results suggest T-cell shifts from onstrates strong therapeutic cooperativity with ICI [4]. naïve to effector phenotypes, CD4+ memory T-cell expansion, and/or Methods B-cell increases after Toca FC in 41% of patients with pre- and post- A phase 1, dose-escalation trial using 3+3 design was conducted to Toca FC blood samples. Following treatment with Toca FC, some pa- determine the safety, tolerability and activity of EVO in combination tients showed marked changes in tumor infiltrating immune popula- with ipilimumab (IPI) for the treatment of 4 tumor types: metastatic tions assessed by IHC, including decreases in CD11b+ myeloid cells, or locally advanced PA, HPV negative HNSCC, ICI-refractory melan- Tregs, and exhausted T-cells, and increases in CD8+ T-cells. In oma and CRPC (NCT03098160). The study drugs (EVO, IPI) were given addition, IV Toca 511 led to viral expression in tumor, which was de- at the following doses respectively: level 1 (400mg/m2, 3mg/kg), creased post-Toca FC. Treatment has been generally well tolerated, level 2 (480mg/m2, 3mg/kg), level 3 (560mg/m2, 3mg/kg), level 4 with no related Grade 4 adverse events. At data cut-off, 9 patients (640mg/m2, 3mg/kg). EVO was administered on days 1 and 8 in cy- were alive (median follow-up 10.4 months); median overall survival cles 1 and 2. IPI was administered on day 8 of each 3 week cycle for was 9.6 months (95% CI 6.3, 16.4). A patient receiving concomitant a maximum of 4 doses after which retreatment was allowed in those panitumumab had a partial response. with irCR/ irPR/ irSD or irPD anytime after study initiation. Tumor re- Conclusions sponse was assessed using irRECIST. Change from baseline in periph- Results suggest Toca 511 infects metastatic tumor following IV eral blood and tumor tissue immune and hypoxia parameters were administration, and subsequent Toca FC induces tumor and im- evaluated as potential biomarkers of activity for this combination. munosuppressive cell killing. Preliminary analyses indicate Toca Results 511 & Toca FC treatment may be associated with T-cell mediated Twenty-one patients with a median age of 67 years were enrolled in immune activity in peripheral blood and metastatic tumor, con- the study, of whom a majority had CRPC (n=11) followed by PA (n= sistent with the immunologic mechanism of action observed in 7), melanoma (n=2) and HNSCC (n=1). Three patients were enrolled preclinical models. Preliminary clinical data suggest a signal of ac- at level 1 and six in level 2, 3 and 4 each. The most common any tivity in these heavily pretreated patients warranting further grade adverse events were rash (n=17), anemia (n=16) and investigation. leukopenia (n=12). The most common grade 3 adverse events were Trial Registration transaminitis (n=4), lymphopenia (n=3) and anemia (n=3). One pa- NCT02576665 tient required treatment discontinuation and 3 required EVO dose re- duction for toxicities. Out of 18 patients with measurable disease at References baseline, three (16.7%) had PR (2 with CRPC and 1 with HNSCC) and 1. Mitchell LA, Lopez Espinoza FL, Mendoza D, et al. Toca 511 gene transfer twelve (66.7%) had SD. The best responses were observed at dose and treatment with the prodrug, 5-fluorocytosine, promotes durable anti- level 3 (Figure 1). Reduced hypoxic exposure of myeloid stroma, cor- tumor immunity in a mouse glioma model. Neuro Oncol. 2017;19:930- relating with reduced suppressive polarization was observed. 939. Conclusions 2. Cloughesy TF, Landolfi J, Vogelbaum, et al. Durable complete responses No new or unexpected safety signals were observed with combined in some recurrent high-grade glioma patients treated with Toca 511 + EVO and IPI. The combination showed evidence of activity in heavily Toca FC. Neuro Oncol. 2018;20:1383-1392. pretreated refractory solid tumors. Dose expansion is planned at EVO Ethics Approval 560mg/m2 and IPI 3mg/kg. This study was approved by the institutional review boards of University of Miami Hospitals and Clinics, The University of Texas MD Anderson References Cancer Center, and Sarah Cannon Research Institute at HealthONE. 1. Blank CU, Haanen JB, Ribas A, et al: CANCER IMMUNOLOGY. The "cancer immunogram". Science 352:658-60, 2016 2. Chouaib S, Noman MZ, Kosmatopoulos K, et al: Hypoxic stress: obstacles P348 and opportunities for innovative immunotherapy of cancer. Oncogene A phase 1 dose escalation study to evaluate the safety and 36:439-445, 2017 tolerability of evofosfamide in combination with ipilimumab in 3. Duan JX, Jiao H, Kaizerman J, et al: Potent and highly selective hypoxia- advanced solid malignancies activated achiral phosphoramidate mustards as anticancer drugs. J Med 1 1 1 Aparna Hegde, MD , Priyamvada Jayaprakash, PhD , Elizabeth Sumner , Chem 51:2412-20, 2008 1 1 1 1 Di Nguyen , Hira Zain , Sarina Piha-Paul, MD , Daniel Karp , Jordi 4. Ai M, Budhani P, Sheng J, et al. Tumor hypoxia drives immune 1 1 1 Rodon , Shubham Pant, MBBS , Siqing Fu, MD, PhD , Ecaterina suppression and immunotherapy resistance. J Immunotherapy of Cancer 1 1 1 Dumbrava, MD , Timothy Yap, MD PhD , Vivek Subbiah, MD , Priya 2015;3(Suppl 2):P392. 1 2 2 2 Bhosale, MD , Jack Higgins, PhD , Eric T.Williams , Thomas F. Wilson , Ethics Approval 1 1 1 Funda Meric-Bernstam, MD , Michael Curran, PhD , David Hong, MD The study was approved by University of Texas MD Anderson Cancer The University of Texas MD Anderson Cancer Center, Bee Cave, TX, Center's Ethics Board, approval number IRB00000121. United States; Molecular Templates, Austin, TX, United States Consent Correspondence: Michael Curran (MCurran@mdanderson.org); David Written informed consent was obtained from the patient for publication of Hong (dshong@mdanderson.org) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P348 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 191 of 272 to reduce α-SMA expression vs RT, suggesting that bintrafusp alfa can reduce RT-induced fibrosis, presumably via TGF-β blockade. Conclusions Collectively, these preclinical findings support the clinical develop- ment of bintrafusp alfa and RT combination therapy and support the rationale for a clinical trial investigating bintrafusp alfa in combin- ation with chemoradiation (CRT) in stage III non-small cell lung can- cer (NSCLC; NCT03840902). In addition, the enhanced efficacy seen in multiple murine models supports the broad application of this combination for treatment of additional cancer indications. Ethics Approval This study was approved by the Institutional Animal Care and Use Committee at EMD Serono, Inc.; approval number [17-008]. P350 Clinical signal/profile in a phase I study of T cCell receptor (TCR) affinity-enhanced specific T cells (TAEST) in advanced cancer patients 1 1 2 3 Yi Li, PhD , Zhaosheng Han, PhD , Xing Zhang, MD , Jian Zhang , 4 1 2 Chengzhi Zhou , Haiping Gong , Desheng Weng, MD , Jianchuan Xia, 2 5 4 3 PhD MD , Johnson Lau , Shiyue Li , Weiliang Zhu Fig. 1 (abstract P348). See text for description 1 2 Guangdong Xiangxue Life Sciences, Ltd., Guangzhou, China; Sun Yat- sen University Cancer Center, Guangzhou City, Guangdong Provi, Peoples Republic of China; Zhujiang Hospital, Guangzhou, China; 4 5 Guangzhou Institute of Respiratory Healt, Guangzhou, China; Axis Therapeutics Ltd., Hongkong, Hong Kong P349 Correspondence: Shiyue Li (lishiyue@188.com) Effects of bintrafusp alfa (M7824) and radiation combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P350 therapy on antitumor activity, immune response, and radiation- induced fibrosis in multiple cancer models Background Yan Lan, MD, Chunxiao Xu, PhD, Huakui Yu, Guozhong Qin, Bo Marelli, T-cell triggering thresholds can be improved by engineered TCR with Jin Qi, Rachel E. Fontana, Amit Deshpande, George Locke, Alex Rolfe, enhanced binding affinity. TAEST for NY-ESO-1 was designed for po- Molly H. Jenkins, Joern-Peter Halle, Kin-Ming Lo tentially better efficacy and good safety profile. EMD Serono Research & Development, Billerica, MA, United States Methods Correspondence: Yan Lan (yan.lan@emdserono.com) Preclinical: Determined the TCR affinities. Expression of CD3, CD4, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P349 CD8, and TCR were traced by antibodies/tetramer. Specificity/efficacy in vitro/in vivo and TAEST infiltration in tumor/lymph node were Background evaluated. Clinical: Phase I study – 14 advanced cancer patients were We recently reported the enhanced preclinical antitumor activity of bin- treated with TAEST. trafusp alfa (M7824), an innovative first-in-class bifunctional fusion pro- Results tein composed of the extracellular domain of the TGF-βII receptor (a Preclinical: TAEST had higher affinity to its antigen vs wild type T- TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In phase 1 cells and with great expression (80-90% positive engineered TCR-T and 1b expansion studies in patients with advanced solid tumors, bin- cells), with ~5-6X more CD8+ over CD4+ cells. There was great trafusp alfa showed early evidence of clinical activity. Bintrafusp alfa is in vitro and in vivo efficacy with strong evidence of tumor specific a particularly rational combination partner for radiation therapy (RT) be- TAEST infiltration. Clinical: Stage I - TAEST alone was dosed in 3 cause RT induces expression of TGF-β, which can promote epithelial-to- NSCLC patients with demonstrated safety and stable disease (SD) ob- mesenchymal transition (EMT), fibrosis, and metastasis, and the expres- served for 28-165 days (OS: 77-308 days). Stage II - lymphodepletion sion of PD-L1. Furthermore, the induction of abscopal effects requires was added to TAEST in 11 patients (NSCLC 4, thyroid CA 1, liver the combination of RT with immunotherapy in mouse models, and CA 1, breast CA 1, colon CA 1, melanoma CA 1, synovial sarcoma abscopal responses have been reported in patients receiving RT in 1,and fibrotic sarcoma 1,) and treated with 0.8-2.15x1010 TAEST combination with an immune checkpoint inhibitor. cells: the synovial sarcoma patient had PR (> 70% tumor size re- Methods duction) with > 12 months duration; the breast CA patient had a The combination of bintrafusp alfa and RT was compared with bin- > 40% tumor shrinkage with healing of skin metastatic ulcers trafusp alfa monotherapy or RT alone in MC38 colorectal carcinoma, during Rx; Two other patients (liver, thyroid CA) showed SD but GL261-luc2 glioma, and 4T1 breast cancer murine models. Antitumor significant tumor necrosis (>50%) with symptomatic relief of local activity was evaluated via tumor growth, survival, and lung metasta- pain. Three NSCLC patients had SD with 59-188 days (Survival ses. Enzyme-linked immune absorbent spot (ELISpot) and immuno- 129-392 days). The fibrotic patient had SD for 87 days (Survival: histochemistry were used to measure the function and infiltration of 273 days); the melanoma patient had SD with 105 days (Survival: CD8+ T cells and the quantity of α-SMA, a marker of cancer- 176 days); last 2 patients ( NSCLC 1, colon CA 1) had PD at 14- associated fibroblasts. Gene expression signature scores of different 16 days post infusion ( OS: 92-129 days) . pathways were calculated from targeted RNAseq analysis. The treatment was tolerated well with fever (12/14), chills (4/14), neu- Results tropenia (5/14), thrombocytopenia(1/14), diarrhea(2/14), chest pain The combination therapy enhanced antitumor activity in all three mur- (1/14), and skin rash (3/14) observed. Expected cytokine response, ine models, increased tumor-specific and tumor-infiltrating CD8+ T cells TCR-gene detection/persistence (>60 days), were also observed in in the MC38 and 4T1 models, respectively, and potentiated an abscopal the patients above (particularly, >362 day for synovial sarcoma effect in secondary MC38 tumors. In the 4T1 model, combination ther- patient). apy decreased lung metastases vs either monotherapy and decreased the expression of EMT and VEGF pathway gene signatures vs RT. Ex- Conclusions pression of α-SMA significantly decreased with bintrafusp alfa mono- (1) TAEST, with its enhanced TCR binding affinity, is safe and toler- therapy in this model, whereas it significantly increased with RT able in a clinical phase I study; (2) TAEST exhibits encouraging effi- monotherapy. However, the combination with bintrafusp alfa was able cacy (DCR: 85.7%, 12/14) with a near CR for synovial sarcoma patient Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 192 of 272 (duration >12 months), and marked tumor necrosis with two more Conclusions patients (liver CA, thyroid CA); (3) Lymphodepletion pretreatment ap- The discordance between disease control per irRECIST and RECIST peared to be critical for efficacy/cytokine response/persistence of suggests that for approximately one in 12 patients, irRECIST is a bet- TAEST cells. ter indicator of clinical benefit from ICI treatment than RECIST. How- ever, overall no stronger association was observed between OS and irPFS compared with between OS and PFS. Thus, neither RECIST nor Acknowledgements irRECIST showed a clear advantage for predicting OS for clinical deci- The National key R&D Program of China, 2016YFC1303404; The Sciences and sions or regulatory purposes. Technology Program of Guangzhou, No. 201704020220. Trial Registration Acknowledgements ClinicalTrials.gov Identifier: NCT03159585; NCT03029273; NCT03462316 This study was funded by Merck KGaA, Darmstadt, Germany, as part of an Ethics Approval alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, The study of bone sarcoma and soft tissue sarcoma was approved by Sun NY, USA. Yat-sen University Cancer Center, approval number B2017-023-01. Trial Registration The study of NSCLC was approved by The First Affiliated Hospital of All trials were registered at clinicaltrials.gov, trial numbers NCT01772004 and Guangzhou Medical University, approval number 2016 No.63. NCT02155647. The study of multiple solid tumor was approved by Zhujiang Hospital of Ethics Approval Southern Mediacl University, approval number 2017-ZLZX-001. The trials were approved by the institutional review board or independent Consent ethics committee at each participating center. Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. P352 Workflow for Immune Monitoring during Clinical Trials by using unsupervised high dimensional augmented intelligence assisted P351 analysis Association between response assessment using RECIST and Alessandra Metelli, PhD, Carsten Krieg, PhD, Luis Cardenas, BS irRECIST in 1765 patients with advanced solid tumors treated with Medical University of South Carolina, Charleston, SC, United States avelumab monotherapy 1 2 1 Correspondence: Carsten Krieg (KriegC@musc.edu) Juliane Manitz , Peter Eggleton , Marcis Bajars , Oliver Bohnsack, MD, 3 4 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P352 PhD, MBA , James Gulley, MD, PhD, FACP EMD Serono Research and Development Institute, Inc, Billerica, Background Massachusetts, United States; Merck KGaA, Darmstadt, Germany, Following checkpoint inhibitors, which significantly improved cancer Darmstadt, Germany; PAREXEL Informatics, Berlin, Germany, Berlin, treatment, an increasing number of combination therapeutics is be- GERMANY; Center for Cancer Research, National Cancer Institute, ing tested in clinical trials. To find possible clinical or biological corre- National Cancer Institutes of Health, Bethesda, MD, United States lates of response or intervention, high throughput multi-omic Correspondence: Juliane Manitz (juliane.manitz@emdserono.com) approaches are necessary to catch all features of possible immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P351 responses during treatment. Methods Background To this aim, we present a portfolio of multi-omic approaches includ- A subset of patients receiving immune checkpoint inhibitor (ICI) ing high-dimensional mass cytometry (CyTOF) and single cell se- treatment may have unconventional response patterns, such as pseu- quencing in combination with unsupervised machine-learning doprogression, which can be classified as best overall response (BOR) bioinformatics to perform in depth characterization of immune re- of progressive disease (PD) by Response Evaluation Criteria in Solid sponses during clinical (immuno)therapy. The analysis is data driven, Tumors (RECIST) v1.1; therefore, immune-related (ir) response criteria, can be adapted to high throughput approaches and can model arbi- irRECIST, have been proposed. This analysis reports the differences in trary trial designs. response assessment by RECIST v1.1 and irRECIST and their associ- Results ation with overall survival (OS) in patients with advanced solid tu- We here show three proof of concept projects using biobanked per- mors treated with avelumab monotherapy (anti–PD-L1). ipheral blood mononuclear cells (PBMCs). In the first study, 51 pa- Methods tients with stage IV melanoma before and after 12 weeks of anti-PD- Data from patients with metastatic or locally advanced solid tumors 1 therapy were analyzed. We observed a clear T cell response on (n=1677, data cutoff, February 15, 2017) enrolled in the phase 1, therapy. The most evident difference in responders before therapy open-label JAVELIN Solid Tumor trial (NCT01772004), and data from was an enhanced frequency of CD14+ CD16+HLA-DRhi classical patients with metastatic Merkel cell carcinoma with disease progres- monocytes. We validated our results using conventional flow and sion after prior chemotherapy (n=88, data cutoff, March 24, 2017) en- found a clear correlation of enhanced monocyte frequencies before rolled in part A of the phase 2 open-label JAVELIN Merkel 200 trial therapy initiation with clinical response such as lower hazard and ex- (NCT02155647) were pooled. Patients with castration-resistant pros- tended progression-free and overall survival. In a second study, we tate cancer from the JAVELIN Solid Tumor study were excluded. All used CyTOF to monitor immune response in 21 non-small cell lung patients received avelumab 10 mg/kg every 2 weeks by intravenous cancer (NSCLC) patients that initially responded and then progressed infusion. BOR, disease control rate, and progression-free survival under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 (PFS) were evaluated. Concordance of disease control rates, Kaplan- plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a Meier, landmark OS, and correlation analyses were performed. response in the CD8+ T cell compartment was observed. Unexpect- Results edly, our high dimensional unbiased analysis was able to detect and A total of 1765 patients were included. All patients had ≥3 months characterize a strong expansion of innate tumor-reactive effector NK of follow-up. The pooled data set included 12 tumor types. The dis- cells starting around day 4 of therapy. In our third unpublished study cordance between the tumor assessment criteria for disease control we were able to identify neutrophils as predictors of outcome in lung rate was 8.3% (n=147), i.e. complete response + partial response + cancer patients. stable disease (SD) per irRECIST and PD + not evaluable per RECIST; Conclusions most patients (n=135) had a BOR of PD by RECIST and irBOR of SD Taken together, our unbiased artificial intelligence-driven immune by irRECIST. The Kaplan-Meier analysis according to Wolchok et al ex- workflow is an extraordinary instrument to monitor immune re- hibited clear separation of the respective (dis)concordant subgroups. sponses during (immuno)therapy and serves as a novel approach for The rank correlations between OS and PFS and OS and irPFS were therapeutic target identification. 0.73 (95% CI, 0.70-0.75) and 0.75 (95% CI, 0.72-0.78), respectively. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 193 of 272 Trial Registration P354 ClinicalTrials.gov Identifier: NCT02523469 Exploring correlates of clinical and immune response to cancer Ethics Approval immunotherapy using FAUST, a novel unbiased cell population This study was approved by the MUSC and Zurich institutional review discovery method, in whole blood flow cytometry board. Steven Fling, PhD, Nirasha Ramchurren, PhD, Leonard D'Amico, Martin Cheever, MD, Evan Greene, Greg Finak, Raphael Gottardo, PhD Fred Hutchinson Cancer Research Center, Seattle, WA, United States P353 Correspondence: Steven Fling (sfling@fredhutch.org) Phase 1 pilot study of RRx-001 + nivolumab in advanced Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P354 metastatic cancer (PRIMETIME) 1 2 1 3 Corey Carter, MD , Bryan Oronsky, MD PhD , Mary Quinn , Jane Trepel , Background 4 5 Nacer Abrouk, PhD , Jeff Skinner, MD The purpose of this study is to describe an immune monitoring ap- 1 2 EpicentRx, Inc., La Jolla, CA, United States; EpicentRx Inc, La Jolla, CA, proach that combines multi-parameter whole blood flow cytometry 3 4 United States; NIH, Bethesda, MD, United States; Clinical Trials with an automated, unbiased, cell population evaluation method to Innovations, Mountain View, CA, United States; Walter Reed National Mil facilitate discovery of informative correlative biomarkers. The Cancer Medical Center, Bethesda, MD, United States Immunotherapy Trials Network (CITN) coordinates multi-center can- Correspondence: Mary Quinn (mquinn@epicentrx.com) cer immunotherapy trials, wherein multiparameter flow cytometry is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P353 performed in real time on longitudinally collected whole blood samples. Background Methods RRx-001 is a minimally toxic small molecule that downregulates We recently reported results from two CITN multi-center clinical trials. CD47 and repolarizes tumor associated macrophages (TAMs) as well We also reported a non-parametric method for unbiased cell popula- as normalizes aberrant tumor perfusion. On the premise that the tion discovery that annotates cell populations with biologically inter- interaction between a CD-47 downregulator like RRx-001 and an pretable phenotypes through a new procedure called Full anti-PD-1 inhibitor like nivolumab may serve to activate both arms of Annotation Using Shape-constrained Trees (FAUST). We used FAUST the immune system, a phase 1 pilot study was undertaken to deter- to analyze extensive flow cytometry data in these two CITN clinical mine the safety and feasibility of RRx-001 and nivolumab in patients trials and demonstrate that candidate biomarkers can be associated with advanced cancer and no standard options. with clinical outcome. Here we compare flow cytometry data ana- Methods lyzed both by conventional manual gating strategies as well as by This single arm, open-label pilot study (NCT02518958) called PRIME- the FAUST method. TIME was designed to evaluate the safety profile of RRx-001 and Results nivolumab in patients with advanced malignancies and no other We highlight the value of FAUST in identifying predictive biomarkers of standard therapeutic options. A 3+3 trial design was used to estab- clinical responses to immunotherapy within fresh whole blood. By com- lish safety of the combination at each dose level and guide the deci- bining whole blood flow staining with FAUST, our results demonstrate sion to escalate dose. RRx-001 is infused once weekly while the ability to capture important minor cell subpopulations, including nivolumab is given at 3mg/kg once every 2 weeks. The RRx-001 start- within the CD8 T cell compartment, that otherwise are missed by man- ing dose was 2 mg IV weekly with 4 dose level escalations up to 16 ual gating. Manual gating can be biased and limited to characterizing mg IV weekly. From January 2015 to November 2015, twelve patients cell populations considered a-priori to be significant. received treatment for only 4 cycles (total 12 weeks) with the com- Conclusions bination due to unavailability of nivolumab, which was not supplied Our results emphasize the unique value of performing flow cytome- to the Sponsor. Treatment-emergent (all cause, TEAEs) and try in multi-center trials using fresh whole blood which preserves the treatment-related (TRAEs) adverse events that occurred within 16 minor cell populations identified by FAUST which may be lost or weeks of the first dose of RRx-001 and nivolumab were characterized compromised by standard cryopreservation methods. according to CTCAE v4.03. Results Twelve patients received >1 dose of RRx-001 and nivolumab. One P355 discontinuation occurred due to pneumonitis and one to volun- Multicenter, open-label, phase 1 study of DSP-7888 Dosing tary withdrawal after a post-procedural infection. There were no Emulsion (DSP-7888) in patients with advanced malignancies 2 3 4 5 DLTs. The main adverse event related to RRx-001 was infusion re- Morris Groves , Aaron Hansen , Wael Harb , Kelly Curtis, MD , Erina 6 7 7 8 action (33.3%). The main adverse event related to the combin- Koga-Yamakawa , Makoto Origuchi , Zhonggai Li , Jose Iglesias , Walid 9 1 ation was pseudoprogression manifested by larger tumors in Shaib, MD , Alexander Spira, MD, PhD, FACP 1 2 patients that were symptomatically improved (25%). The most Virginia Cancer Specialists, Fairfax, VA, United States; Texas Oncology- common immune-related treatment-emergent AEs were pneu- Austin Midtown, Austin, TX, United States; UHN Princess Margaret monitis (8.3%), and hypothyroidism (8.3%). The objective response Cancer Centre, Toronto, Canada; Horizon Oncology Research, LLC, rate at 12 weeks was 25% and the disease control rate (DCR) Lafayette, IN, United States; Syneos Health, Phoenix, AZ, United States; consisting of > SD was 67% by Response Evaluation Criteria in Sumitomo Dainippon Pharma Co., Ltd, Cambridge, MA, United States; 7 8 Solid Tumors (RECIST) version 1.1 25% of the patients progressed Boston Biomedical, Inc., Cambridge, MA, United States; Former on the combination. employee, Boston Biomedical, Inc., Cambridge, MA, United States; Conclusions Emory University, Atlanta, GA, United States The combination of RRx-001 and nivolumab was safe and well- Correspondence: Alexander Spira (Alexander.Spira@USOncology.com) tolerated with preliminary evidence of anti-cancer activity. Further Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P355 analyses with a larger sample size will be required to confirm the ac- tivity of the combination and to determine the optimum schedule Background for RRx-001 and nivolumab. DSP-7888, a cancer vaccine composed of 2 synthetic peptides de- Ethics Approval rived from Wilms’ tumor 1 (WT1) protein, may induce WT1-specific The study was approved by all the revelant Institution‘s Ethics cytotoxic T-lymphocytes (CTLs) and helper T-lymphocytes–mediated Boards. immune responses against WT1-expressing tumors. This dose- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 194 of 272 escalation study (NCT02498665) evaluated DSP-7888 in patients with infiltrating leukocyte (TIL) analysis. CTL, IgG, and TIL were mea- recurrent or progressive advanced malignancies, despite receiving sured by ELISPOT assay, Luminex assay, and IHC (CD8 positive), standard therapy, or in patients intolerant to standard therapy or for respectively. whom no standard of therapy existed for their malignancy. The pri- Results mary objectives were safety, tolerability, and identification of the rec- Seventeen patients were enrolled in 9mg (n=10) and 27mg (n= ommended phase 2 dose (RP2D). Secondary and exploratory 7) groups; the median age was 65 years, and 53% of the pa- objectives included overall survival and WT1-specific CTLs induction. tients had ECOG PS 1. There was no serious adverse drug reac- Methods tion (ADR) in any patient. All ADRs were of grade 1 or 2, with Patients who failed or were intolerant to prior lines of treatment and the most frequent being dermatological injection site reaction, tested positive for HLA-A*02:01, HLA-A*02:06, or HLA-A*24:02 re- in 7/10 (70%) and 6/7 (86%) patients and pyrexia, in 1/10 (10%) ceived escalating doses of intradermal (ID) or subcutaneous (SC) and 2/7 (29%) for the 9mg and 27mg groups, respectively. The DSP-7888 in a rolling study design: 3.5, 10.5, or 17.5 (ID only) mg best overall response was stable disease, in 2/10 (20%) and 2/7 every week for 4 weeks, then every 1–2 weeks for 6 weeks, and every (28%) patients. One patient with cancer of unknown origin re- 2–4 weeks thereafter until progression or other discontinuation event ceived prolonged administration (over 10 months) of the 9mg was met. Dose-limiting toxicities (DLTs) were evaluated over days 1– dose.Inthe 9mgand 27mggroups, antigenspecific IgG was 29. The dose at which ≤1 of 6 patients had a DLT was eligible to be augmented in 9/10 (90%) and 7/7 patients (100%), antigen spe- the RP2D. WT1-specific CTL inductions were assessed by HLA Tetra- cific CTL was detected in 2/10 (20%) and 3/7 patients (43%), mer with peripheral blood. and TIL counts were increased in 2/3 (67%) and 3/4 patients Results (75%), respectively. Twenty-four patients received ID (3.5 mg, n=4; 10.5 mg, n=3; 17.5 Conclusions mg, n=3) or SC DSP-7888 (3.5 mg, n=9; 10.5 mg, n=5). The most fre- TAS0313 demonstrated safety, tolerability, and immunological re- quent adverse event (AE) was injection site reaction (ISR; n=15; sponses in patients with advanced solid tumors in the 9mg and 62.5% [ID: 100% of patients, SC: 36%]); all were grade 1 or 2. No DLT 27mg groups. A phase II part, evaluating the efficacy of combin- was observed. ID DSP-7888 10.5 mg was determined to be the dose ation therapy with pembrolizumab in patients with urothelial car- level for further study based on the RP2D identified in a phase 1/2 cinoma and monotherapy in glioblastoma patients, is currently study of DSP-7888 in patients with myelodysplastic syndrome underway. (NCT02436252). Four patients (ID 17.5 mg, n=1; SC 3.5 mg, n=1; SC Trial Registration 10.5 mg, n=2) had stable disease, 16 had progressive disease, and 4 JapicCTI-183824 were not evaluable. Twenty-one patients were evaluable for WT1- Ethics Approval specific CTL detection. In evaluable patients, WT1-specific CTL induc- The study was approved by National Cancer Research Center Central tion was observed in 6 of 9 ID patients (66.7%) and 5 of 12 SC pa- Hospital’s Ethics Board, approval number T4499. tients (41.7%). Consent Conclusions Written informed consent was obtained from the patients for publi- DSP-7888 was well tolerated, with no DLTs, in patients with ad- cation of this abstract. A copy of the written consent is available for vanced malignancies, supporting further evaluation of DSP-7888. The review by the Editor of this journal. 10.5 mg ID dose was identified as a dose level and route of adminis- tration for further evaluation. P357 Phase I/II clinical and immune responses for locally advanced or P356 metastatic pancreatic cancer using anti-CD3 x anti-EGFR bispecific First-in-human study of the cancer peptide vaccine, TAS0313, in antibody armed T cells (BATs) 1 1 2 1 patients with advanced solid tumors: phase I dose finding part Lawrence Lum, MD, DSc , Tri Le , Minsig Choi , Archana Thakur, PhD , 1 1 3 1 results Matthew Reilly , Paul Kunk, MD , Abhinav Deol, MD , Karen Ballen, MD , 1 1 1 Noboru Yamamoto, MD, PhD, Jun Sato, MD, PhD, Satoru Iwasa, MD, Tamila Kindwall-Keller, DO , Dana Schalk , Ewa Kubicka , Manley Huang, 1 3 3 3 4 PhD, Kan Yonemori, MD, PhD, Takafumi Koyama, MD, Kenji Tamura, MD, PhD , Philip Philip, MD , Hussein Aoun , Gregory Dyson, PhD , Qin Liu , PhD, Toshio Shimizu, MD, PhD, Syunsuke Kondo, Shigehisa Kitano Anthony Shields, MD PhD 1 2 National Cancer Center Hospital, Tokyo, Japan University of Virginia, Charlottesville, VA, United States; Stony Brook Correspondence: Toshio Shimizu (tosshimi@ncc.go.jp) University, Stony Brook, NY, United States; Karmanos Cancer Institute, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P356 Wayne State U, Detroit, MI, United States; Wistar Institute, Philadelphia, PA, United States Background Correspondence: Lawrence Lum (lawrenceglum@cs.com) TAS0313 is a cancer vaccine cocktail containing three long peptides, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P357 with a total of 12 cytotoxic T lymphocyte (CTL) epitope peptides. These peptides were derived from eight cancer-associated antigens Background that are highly expressed in various cancers. We report the results of Chemotherapy for locally advanced pancreatic cancer (LAPC) and a phase I part examining the tolerability, safety, potential efficacy, metastatic pancreatic cancer (MPC) has poor responses and survival and immunological responses of 9 mg and 27 mg TAS0313 in pa- rates. Retargeting anti-CD3 activated T cells (ATC) by arming them tients with advanced solid tumors. with anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) makes ATC Methods into specific cytotoxic T lymphocytes (EGFR BATs). Targeting pancre- The enrolled patients had ECOG PS 0-1 and at least one of the atic cancer cell lines induces cytokine secretion, proliferation, cyto- following HLA types: HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, toxicity, and inhibits tumor growth. We present 5 phase I and 13 -A*24:02, -A*31:01, or -A*33:03. Emulsified TAS0313 solution with phase II patients for a total of 18 evaluable patients out of 21 who an immunological adjuvant (Montanide ISA-51) was subcutane- underwent apheresis. ously administered on Days 1, 8, and 15 of Cycles 1 and 2 and Methods on Day 1 of Cycle 3 or later in 21-day cycles until disease pro- In the phase I, LAPC or MPC patients at Karmanos Cancer Institute gression, or unacceptable toxicity.. Tolerability was assessed in at (KCI) on Protocol #2011-025, in a dose escalation, were given 10, 20, least six patients during the first cycle. Tumor response was eval- and 40 x 10^9 BATs/infusion weekly for 3 weeks, followed by a uated using RECIST v1.1. Blood samples were collected pre- and booster infusion 3 months later if patients were stable or better. post-treatment for the analysis of antigen specific CTL and IgG. There were no dose limiting toxicities, and all infusions were given in Optional serial tumor biopsies were performed for tumor the outpatient setting. In the phase II portion, 13 PC patients at KCI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 195 of 272 (NCT02620865) and University of Virginia (NCT03269526) received Methods twice weekly infusions of 10 x 10^9 BATs/infusion over 4 weeks for a Patients with advanced SS were enrolled to cohorts based on NY-ESO-1 total of 80 x 10^9 EGFR BATs. expression (Cohort 2, low; Cohort 4, high) determined by immunohisto- Results chemistry. Treatment response (RECIST v1.1), safety (CTCAE v4.0), and Eighteen patients were evaluable. Four patients were stable at 6.1, GSK3377794 persistence in transduced PBMCs (transgene vector copies 6.5, 5.3, and 39 months. Two patients developed complete responses measured by qPCR) were assessed. Progression-free survival (PFS) was (CR) when chemotherapy was restarted after their BATs infusions. Pa- defined as the interval between first infusion and first documented dis- tient IT20104 was stable for 1 year on capcitabine, developed “pseu- ease progression or death. Safety was monitored throughout. The study doprogression,” achieved a CR after restarting capcitabine, and was was not designed/powered for cohort comparison. off therapy until 54 months after enrollment when relapse occurred. Results The median overall survival is 14.8 months with a time to progres- As of April 2019, 50 patients were enrolled (N=13 Cohort 2; N=15 Co- sion of 6.6 months. Specific cytotoxicity mediated by peripheral hort 4). Table 2 summarizes response outcomes by Cohort. Median PFS blood mononuclear cells (PBMC) peaked at 31% two weeks after the (95% CI) was 13.1 weeks (7.9, 13.9; Cohort 2) and 22.4 weeks (11.3, 26.6; third infusion, and IFN-γ EliSpots rose from Cohort 4). Median peak (range) persistence of ~64,712 DNA copies/μg Conclusions (13,364–197,546) occurred in Cohort 2 first week post-infusion versus EGFR BATs infusions were safe and induced specific adaptive anti- ~16,468 DNA copies/μg(163–131,175) in Cohort 4. No significant cor- tumor responses. This phase I/II study strongly suggests that multiple relation was observed between peak persistence and best overall re- infusions of EGFR BATs may provide a survival benefit in patients sponse in either cohort (p>0.05). Grade 3/4 adverse events occurring in with pancreatic cancer, and that BATs therapy may increase the ef- ≥40% of patients in both cohorts were leukopenia, neutropenia, fectiveness of subsequent chemotherapy, which will drive the design anemia, thrombocytopenia, lymphopenia, and hypophosphatemia. of future combination trials of BATs and other modalities of therapy. Conclusions Cohorts 2 and 4 showed similar ORRs; more durable responses were Acknowledgements observed in Cohort 4, with prolonged DoR, duration of stable dis- These studies were made possible thanks to philanthropy from Karmanos Cancer ease, and PFS. Peak persistence of GSK3377794 was higher in Cohort Institute and start-up funds for LGL from the University of Virginia. LGL and MH 2, likely due to higher lymphodepletion, but this did not correlate are co-founders of Transtarget, Inc. LGL is a member of the Scientific Advisory with response, unlike data previously reported in other cohorts. Fur- Board for Rapa Therapeutics. AT is a co-founder of NOVA Immune Platform. ther development in SS will be based on previously reported data Trial Registration from Cohort 1. Protocol #2011-025; NCT02620865; NCT03269526 Ethics Approval Acknowledgements These studies were approved by the Karmanos Cancer Institute / Wayne Medical writing assistance was provided by provided by Fiona Woodward State University IRB, approval numbers 2011-25 and 2015-100, and the and Victoria Hunter of Fishawack Indicia Ltd. This study (NCT01343043) was University of Virginia IRB, approval number HSR 19236. funded by GlaxoSmithKline. Trial Registration NCT01343043 P358 Ethics Approval Phase 1 trial of NY-ESO-1-specific adoptive T-cell therapy with This study was approved by the appropriate institutional review boards and GSK3377794 in patients with advanced synovial sarcoma independent ethics committees. 1 2 3 Sandra D'Angelo, MD , George Demetri , Brian Van Tine, MD, PhD , 4 5 6 7 Mihaela Druta , John Glod, MD , Warren Chow , Jenna Tress , M. Phillip 7 7 7 7 7 DeYoung , Aisha Hasan, MBBS MD , Yuehui Wu , David Turner , Ran Ji , 7 8 Alexandra Gyurdieva , Dejka Araujo, MD Table 1 (abstract P358). See text for description Memorial Sloan Kettering Cancer Center, New York, NY, United States; 2 3 Dana-Farber Cancer Institute, New York, NY, United States; Washington University in St. Louis, St. Louis, MO, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; National Cancer Institute, Bethesda, MD, United States; City of Hope Comprehensive Cancer Center, Duarte, 7 8 CA, United States; GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P358 Background Genetically-engineered NY-ESO-1 specific T-cells (NY-ESO-1 T-Cells; GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a self-inactivating lentiviral vector to express affinity-enhanced NY-ESO-1- specific T-cell receptors (TCRs). Ongoing phase 1 and 2 trials are evalu- ating GSK3377794 in solid tumors and multiple myeloma. Study NCT01343043 (208466) is a phase 1 clinical trial assessing GSK3377794 in patients with previously treated, advanced metastatic synovial sar- coma (SS), stratified into 4 cohorts (Table 1). Of 12 patients receiving GSK3377794 infusion in Cohort 1, responses were observed in 6 pa- tients (1 complete response [CR]/5 partial responses [PR]), with an over- all response rate (ORR) of 50% (95% confidence interval [CI]: 0.21–0.79). Median progression-free survival (PFS) was 15.2 weeks (95% CI: 7.6– 37.9); median duration of response (DoR) was 30.9 weeks (95% CI: 14– 72). As of October 15, 2018, median overall survival (OS) was 105 weeks (95% CI: 37–NA). This abstract reports data from Cohorts 2 and 4. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 196 of 272 Table 2 (abstract P358). See text for description manageable adverse events. Our study shows feasibility of tracking phenotypic profiles of adoptively transferred tumor-antigen-specific T cells in patients and derive association between adoptive T cell sta- tus and clinical read-outs. Ethics Approval The study was approved by Mie University Ethics Board, approval number H2018-092. P360 The influence of Durvalumab/Tremelimumab Combination Therapy on Sarcomas Immune Microenvironment profile in a phase II clinical trial (NCT02815995) 1 1 1 Edwin Parra, MD, PhD , Carmelia Barreto, PhD , Ruth Salazar, MD , Cara 1 1 1 1 Haymaker, PhD , Heather Lin , Carmen Behrens, MD , Mei Jiang , Luisa 1 1 1 Solis, MD , Krishna Pandurenga, MS , Sandesh Subramanya, PhD , Young 2 1 1 1 Kim, PhD , Chantale Bernatchez , Jack Lee, PhD , Taylor Tate , Teresa 1 1 1 Simmons , Alexander Lazar, MD, PhD , Wei-Lien Wang , Zachary Cooper, 3 3 3 PhD , Jaime Rodriguez-Canales, MD , Jean Soria, MD , Anthony Conley, 1 1 1 MD , Ignacio Wistuba, MD , Neeta Somaiah, MD, MBBS 1 2 MD Anderson Cancer Center, Houston, TX, United States; Translational Molecular Pathology, Houston, TX, United States; AstraZeneca, Gaithesburg, MD, United States Correspondence: Edwin Parra (erparra@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P360 Background To determine the tumor microenvironment (TME) changes after the combination of durvalumab/tremelimumab treatment, longitudinal P359 sarcoma tissue collections were obtained and analyzed for in-depth Tracking and profiling of NY-ESO-1 TCR-transgenic T cells upon immunoprofiling. adoptive transfer in patients with NY-ESO-1-expressing solid Methods tumors: in vivo differentiation associated with response 1 1 1 Sixty-two patients were enrolled and 36 paired samples (Liposarco- Michael Fehlings , Alessandra Nardin, DVM , Faris Kairi , Evan Newell, 2 3 3 3 ma,LS=6; Angiosarcoma,AS=2; Leiomyosarcoma,LMS=2; Osteo/Chon- PHD , Yoshihiro Mihayara , Shinichi Kageyama , Hiroshi Shiku, MD 1 2 drosarcoma,OS/CS=3/1; Undifferentiated Pleomorphic Sarcoma,UPS= immunoSCAPE, Singapore, Singapore; Fred Hutchinson Cancer 3; Alveolar Soft Part Sarcoma,ASPS=8; Synovial Sarcoma,SS=3; Chor- Research Center, Singapore, Singapore; Mie University School of doma,C =2; and other types,OT=6), were evaluable for TME changes Medicine, Tsu, Japan and correlated with clinical benefit (PR or SD). All patients were Correspondence: Hiroshi Shiku (shiku@clin.medic.mie-u.ac.jp) treated with durvalumab/tremelimumab every 4 weeks for four cy- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P359 cles and then continued durvalumab every 4 weeks for up to 1 year. Biopsies were collected prior to-and- during treatment (Wk6). Malig- Background nant cells (MCs) PD-L1+ was studied by immunohistochemistry. NY-ESO-1 is highly expressed in the majority of synovial sarcomas as Tumor-infiltrating-lymphocytes (TILs), and macrophages were interro- well as other solid tumors and may be an effective target for T cell- gated by multiplex immunofluorescence, Figure-1. The combination based therapies. We conducted a clinical study of adoptive transfer of three T-cell phenotypes (CD3+,CD3+CD8+ and CD3+CD8+GZB+) of lymphocytes transduced with NY-ESO-1-specific TCR in refractory greater than the median density as higher TILs (TILs+) was used to cancer patients with preconditioning (TBI-1301). define “inflamed” tumors and ≤ than the median of 1 or 2 of these Methods as lowest TILs (TILs–) to define “non-inflamed” tumors, through base- High-dose of 5 billion autologous transduced and expanded lympho- line to Wk6. To characterize patterns of the TME and changes be- cytes, consisting of >96% T cells, was transferred into 6 patients, three of tween baseline and Wk6 we stratified the tumors in four groups whom with synovial sarcoma. Longitudinal PBMC samples were obtained using an approach similar as Teng's criteria (1). for immunomonitoring. We used high-dimensional mass cytometry and Results combined a 36-antibody panel with a multiplexed combinatorial Overall, all the phenotype median densities increased from baseline to peptide-MHC tetramer staining approach to longitudinally track and Wk6, Table-1. PR or SD was observed in 17/36 with paired samples, phenotypically characterize adoptively transferred HLA-A*02:01-NY-ESO-1 47% (3 LS, 1 LMS, 1 OS, 7 ASPS, 1 SS, 2 C, and 2 OT). Five ASPS showed transgenic TCR T cells 14, 28, and 56 days after treatment. PR and 2 SD out of 8 cases. We categorized as inflamed tumor 1 LPS, 1 Results AS, 1 LMS, 1 OS, and 5 ASPS at baseline. Interestingly 1 AS, 1 OS, 1 CS, Three out of 6 patients with tumors expressing >75% NY-ESO-1 experi- 1ASPS, 1 SS and 2 OT defined as non-inflamed tumors at baseline chan- enced an objective clinical response (PR) and had cytokine-release syn- ged to inflamed tumors at Wk6 (Figure-2) and from those, OS and ASPS drome (CRS) with high-levels of IL-6 and MCP-1 that could be managed showed SD and PR, respectively. Finally, 4/17 inflamed tumors showed with tocilizumab. The infusion products had variable percentages of SD and 3/17 PR. Furthermore, CD3+CD8+CD45Ro+ increase in the in- naive, TEMRA and EM CD8+ cells, with the three clinical responders flamed tumors at Wk6 than non-inflamed tumors (P=0.005). The most having the highest proportion of EM T cells. NY-ESO-1 TCR transgenic T frequent TME pattern detected at baseline and Wk6 was the immuno- cells could be detected in the circulation of 5 out of 6 treated patients, logical ignorance (TILs-PD-L1-, 61% and 47%, respectively). Interestingly, with frequencies peaking at day 14 and day 28; specific T cells were un- adaptive immune resistance pattern changed from baseline to Wk6 detectable in all patients by day 56. In responders, a substantial num- (TILs+PD-L1+, 14% and 22%, respectively), Figure-3. ber of circulating NY-ESO-1-specific CD8+ T cells showed a phenotypic Conclusions profile consistent with antigen-experience, activation and differenti- Combination of durvalumab/tremelimumab influenced the TME in ation into an effector phenotype 28 days post transfusion. the selected sarcomas cohorts. The immunologic score assessment in Conclusions this longitudinal collection demonstrates the capability to distinguish Adoptive transfer of NY-ESO-1 TCR-transgenic T cells has shown signs non-inflamed vs inflamed tumors and relate it with a clinical benefit, of efficacy in patients with high NY-ESO-1 tumor expression, with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 197 of 272 showing the value of use these markers as possible immune prog- nostic markers in sarcomas. Trial Registration This trial is registered with ClinicalTrials.gov (NCT02815995) References 1. Teng MW, Ngiow SF, Ribas A, Smyth MJ. Classifying Cancers Based on T- cell Infiltration and PD-L1. Cancer Res. 2015;75(11):2139-45. Ethics Approval The study was approved by MD Anderson Institution Ethics Board, Clinical Trail number NCT02815995 Fig. 3 (abstract P360). See text for description Table 1 (abstract P360). See text for description P361 Molecular and immunologic profiling of CD8+ T cell responses in patients receiving a multiple antigen-engineered dendritic cell vaccine 1 2 2 Juraj Adamik, PhD , Patricia Santos, PhD , Samuel Du, BS , Lazar 2 1 3 Vujanovic, PhD , Timothy Howes , Sarah Warren, PhD , Andrea 2 2 1 Gambotto, MD , John Kirkwood, MD , Lisa Butterfield, PhD Parker Institute for Cancer Immunotherapy, San Francisco, CA, United States; University of Pittsburgh, Pittsburgh, PA, United States; NanoString Technologies, Seattle, WA, United States Correspondence: Lisa Butterfield (lbutterfield@parkerici.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P361 Background Despite the immunogenicity and safety profile of dendritic cell (DC) vaccines, the importance of vaccine-induced antigen-specific T cell responses is unclear across clinical trials, and therapeutic efficacy re- mains low with limited clinical responses. Our comprehensive characterization of T cell responses, cell-intrinsic and soluble immune checkpoint molecules and immune-related gene expression profiles reveal novel insights into CD8+ T cells specific for melanoma- associated antigens (MAA) from patients who received autologous DC engineered to express three full length melanoma antigens: tyro- sinase, MART-1 and MAGE-A6 [1]. Methods MAA-specific T cell responses were examined by standardized IFNγ Fig. 1 (abstract P360). See text for description ELISPOT assays at baseline, day 43 (post DC vaccines) and d89 (post observation or IFNα). Luminex was used to detect serum checkpoint and costimulatory molecules, and whole blood flow cytometry was used to quantify PBMC subsets. Targeted mRNA and protein expres- sion analyses in circulating lymphocytes and melanoma tumor sam- ples were performed using NanoString nCounter platform (RUO). Results The majority of the 35 patients were successfully vaccinated, and the total vaccine-induced T cell responses were higher among those exhibiting a favorable clinical outcome. Patients who received check- point blockade treatment prior to DC vaccination had higher base- line MAA-specific CD8+ T cell responses, yet they did not respond more strongly to the vaccine. Two patients who received checkpoint blockade post-DC vaccine showed very strong amplification of their MAA-specific T cells. Molecular profiling in circulating lymphocytes and tumor biopsies showed that elevated PD-1 and CTLA-4 protein levels and gene expression signatures representing checkpoint sig- naling, interferon response and T-cell exhaustion were associated with unfavorable clinical outcome. Gene signatures showing positive correlation with PD-1 protein expression included CD28-dependent PI3K-AKT signaling, the IL12/STAT4 pathway and pan-semaphorin re- ceptor interactions. CTLA-4 protein levels correlated with type I inter- feron response and NOTCH signaling genes. Interestingly, B cell receptor pathways negatively correlated with PD-1 expression, while gene signatures downstream of T cell receptor activation and IL-2 signaling were negatively correlated with CTLA-4 expression. Serum levels of PD-1 and PD-L2 were inversely correlated and post-vaccine Fig. 2 (abstract P360). See text for description serum levels PD-L2 correlated with decreased circulating Treg and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 198 of 272 favorable outcome in patients, suggesting that it may serve as a bio- (22.4%). Median follow-up was 21.2 months (range, 14.9-36.6). marker of clinical response. The ORR was 39.7% (95% CI: 30.7%-49.2%), including 19 patients Conclusions (16.4%) with a CR and 27 (23.3%) with a PR. In patients with PD- Collectively, our study shows that specific checkpoint molecular path- L1+ (n=21 [18.1%]) or PD-L1− (n=87 [75.0%]) tumors, ORRs were ways are critical for vaccine outcomes and for the activation of anti- 61.9% (95% CI: 38.4%-81.9%) and 33.3% (95% CI: 23.6%-44.3%), tumor responses in melanoma patients. Comprehensive profiling of respectively. Median DOR was 18.2 months (95% CI: 11.3 months- MAA-specific T cell responses suggests that DC-vaccine immunization not estimable). 35 patients had a response lasting ≥6months followed by immune checkpoint blockade may be an optimal se- (durable response rate, 30.2% [95% CI: 22.0%-39.4%]). 6- and 12- quential therapy to improve antitumor immunity in melanoma. month PFS rates were 41% (95% CI: 32%-50%) and 31% (95% CI: Trial Registration 23%-40%), respectively. Median OS was 20.3 months (95% CI: FDA IND #15044 and NCT01622933. 12.4 months-not evaluable), and the 12-month OS rate was 60% (95% CI: 50%-68%). In PD-L1+ and PD-L1− subgroups, 12-month Reference OS rates were 71% (95% CI: 47%-86%) and 56% (95% CI: 45%- 1. Butterfield LH, Vujanovic L, Santos PM, Maurer DM, Gambotto A, Lohr J, 66%), respectively. Treatment-related adverse events (TRAEs) of Li C, Waldman J, Chandran U, Lin Y, Lin H, Tawbi HA, Tarhini AA, any grade occurred in 94 patients (81.0%), including grade ≥3 Kirkwood JM. Multiple antigen-engineered DC vaccines with or without TRAEs in 21 (18.1%). No treatment-related deaths occurred. IFNa to promote antitumor immunity in melanoma. JITC. 2019; 7:113. Conclusions Ethics Approval Updated results from JAVELIN Merkel 200 confirm that first-line ave- The clinical trial was fully approved by the Univ. Pittsburgh PRC and IRB lumab treatment was associated with durable responses, a clinically (PRO12010416, #09–021). meaningful OS benefit, and an acceptable safety profile in patients with mMCC. P362 Acknowledgements First-line avelumab treatment in patients with metastatic Merkel This study was funded by Merck KGaA, Darmstadt, Germany, as part of an cell carcinoma: primary analysis after ≥15 months of follow-up alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, from JAVELIN Merkel 200, a registrational phase 2 trial NY, USA. 1 2 3 Sandra D'Angelo, MD , Celeste Lebbé , Laurent Mortier , Andrew Brohl, Trial Registration 4 5 6 7 8 MD , Nicola Fazio , Jean-Jacques Grob , Natalie Prinzi , Glenn Hanna , Registered at www.clinicaltrials.gov, NCT02155647 9 10 11 12 Jessica Hassel, MD , Felix Kiecker , Barbara Ellers-Lenz , Marcis Bajars , Ethics Approval 12 13 Meliessa Hennessy, MPH , Paul Nghiem, MD, PhD The trial was conducted in accordance with international good clinical Memorial Sloan Kettering Cancer Center, New York, NY, United States; practice standards and approved by the independent ethics committee at 2 3 Saint Louis Hospital, Paris, France; Lille Hospital–Claude Huriez Hospital, each participating center Lille Cedex, France; H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States; European Institute of Oncology (IEO), IRCCS, Milan, Italy; Aix-Marseille University, AP-HM Hospital, Marseille, P364 France; Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; A gp100 targeting TCR-based soluble T cell engaging bispecific 8 9 Dana-Farber Cancer Institute, Boston, MA, United States; Heidelberg induces mobilisation and activation of peripheral T cells in patients University Hospital, Heidelberg, Germany; Charité Universitätsmedizin with metastatic melanoma Berlin, Campus Charité Mitte, Berlin, Germany; Merck KGaA, Darmstadt, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc Germany; EMD Serono Research and Development Institut Inc, PhD, Mariantonella Vardeu, Jacob Hurst, PhD, Cheryl McAlpine, MSN Billerica, MA, United States; University of Washington Medical Center at Immunocore Ltd, Abingdon, United Kingdom South Lake Union, Seattle, WA, United States Correspondence: Sion Lewis (Sion.Lewis@immunocore.com) Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P364 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P362 Background Background ImmTAC® (immune-mobilizing monoclonal TCRs Against Cancer) Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine car- molecules are a new class of bispecific therapeutics, consisting of cinoma with a poor prognosis. In the pivotal phase 2 JAVELIN Merkel a high affinity TCR fused to an anti-CD3 single-chain variable 200 trial (NCT02155647), avelumab, a human anti–PD-L1 monoclonal fragment (scFv) T cell-activating moiety [1]. Tebentafusp (gp100 antibody, yielded durable responses in patients with metastatic MCC antigen specific ImmTAC) can elicit a polyfunctional T cell re- (mMCC) who had received prior chemotherapy (part A), and a high sponse and has demonstrated monotherapy activity in advanced objective response rate (ORR) in an initial subgroup treated in the metastatic melanoma [2-5]. Clinical benefit was associated with a first-line metastatic setting (part B), leading to regulatory approval reduction in peripheral CXCR3+ T cells and concurrent increase in worldwide. Here we report the primary analysis of JAVELIN Merkel serum CXCL-10 [6, 7]. The chemokine receptor CCR5 is under- 200 part B after ≥15 months of follow-up in the full patient stood to drive T cell extravasation and potentialy synsergise with population. CXCR3 to promote tumour infiltration [8, 9]. Therefore the aim of Methods this study was to investigate the effect of tebentafusp on the dy- Eligible patients had no prior systemic therapy for mMCC and were namics of T cell mobilisation and activation in metastatic melan- enrolled irrespective of biomarker status; PD-L1+ status was defined oma patients. as ≥1% expression in tumor cells (PD-L1 IHC 73-10 pharmDx assay). Methods All patients received avelumab 10 mg/kg IV every 2 weeks. The pri- HLA-A2+ patients with metastatic melanoma were enrolled on a mary endpoint was durable response, defined as objective response first-in-human, multicentre, Phase I/II, open-label, dose-finding (complete response [CR] or partial response [PR] per RECIST v1.1, ad- study (NCT01211262). Immunophenotypic analysis was under- judicated by independent endpoint review committee) lasting ≥6 taken to assess baseline levels and pharmacodynamic changes months. Secondary endpoints included best overall response, dur- in peripheral immune subsets. PBMC samples were analysed by ation of response (DOR), progression-free survival (PFS), overall sur- flow cytometry from patients at baseline (n=38) and on- vival (OS), and safety. treatment (n=22) over the first dosing cycle, and from age/sex- Results matched healthy control subjects (n=18). Data is reported on T At data cut-off on May 2, 2019, 116 patients had been treated cell populations, markers of activation (CD25) and function with avelumab. Median treatment duration was 5.5 months (CCR5, Ki67) and represented as mean ± standard deviation of (range, 0.5-35.4), and treatment was ongoing in 26 patients subset frequency or percentage change from baseline, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 199 of 272 relationships with overall survival (OS) assessed by univariate vaccine adjuvants to support T cell responses to peptide vaccines. Cox proportional hazards model. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC and/ Results or low-dose metronomic cyclophosphamide (mCy) would be safe Tebentafusp induced T cell extravasation within 24hrs (p and would support strong and durable CD4+ T cell responses in T cells from patients on-treatment exhibited an increase in activation combination with an incomplete Freund’s adjuvant (IFA). marker expression and an expansion of memory and effector T cell Methods subsets (p<0.05). An adaptive design based upon toxicity and durable immune re- Conclusions sponse (dRsp) was used to assign participants with resected stage Tebentafusp administratation induces the rapid extravasation of che- IIA-IV melanoma to one of four study regimens, including a vaccine mokine receptor expressing T cells and the expansion and activation comprising 6 melanoma peptides restricted by Class II MHC (6MHP), of memory T cells. The association between clinical benefit and base- administered in an emulsion with IFA (Montanide ISA-51), with or line levels of peripheral immune subsets may aid our mechanistic un- without the TLR3 agonist polyICLC and with or without systemic derstanding of its anti-tumour activity in metastatic melanoma mCy. Toxicities were recorded (CTCAE v4). T cell responses were mea- patients. sured in peripheral blood lymphocytes (PBL) and in vaccine-site Trial Registration draining lymph node (sentinel immunized node, SIN) with IFNγ ELI- NCT01211262 spot assay ex vivo. Serum antibody responses to 6MHP were mea- sured by ELISA, and changes in circulating regulatory T cells were References assessed by flow cytometry. 1. Lowe KL, et al. Cancer Treat Rev. 2019. Results 2. Boudousquie C, et al. Immunology 2017;152:425–38. Forty-eight eligible patients were enrolled and treated. Following an 3. Middleton M, et al. Presented at ASCO 2016. Abstract 3016. adaptive design, early safety data and T cell response data favored en- 4. Carvajal R, et al. Presented at SITC 2017. Abstract P208. rollment on arm D. At study conclusion, total enrollment was 3, 7, 6, 5. Middleton M, et al. Presented at ASCO 2019. Abstract 9523. and 32 individuals for arms A-D, respectively. Treatment-related dose- 6. Middleton M, et al. Presented at ASCO 2019. Abstract 9530 limiting toxicities (DLTs) were observed in 1/7 (14%) patients on arm B 7. Mullins et al, Cancer Res 2004; 64, 7697-7701 and 2/32 (6%) on arm D, with no treatment arm exceeding the DLT 8. Hong M et al, Cancer Res 2011; 71, 22:6997-7009 25% threshold for early stopping. Strong and durable T cell responses 9. Harlin, Cancer Res 2009;69(7):3077–85: to 6MHP were detected ex vivo in 0%, 29%, 50%, and 50% of patients Ethics Approval enrolled on arms A-D, respectively (Table 1). IgG antibody responses This study was approved by following institutions’ Ethics Boards: were also induced and were greatest for arms C and D (Figure 1). Circu- lating regulatory T cell frequencies were not altered by use of mCy. Oxfordshire Research Ethics Committee; 10/H0604/47, Approved Conclusions June 4, 2010. Combination vaccine adjuvants with IFA, polyICLC, and mCy were Mary Crowley Cancer Research Center; MCMRC IRB # 12-06, Ap- well-tolerated. The dRsp rate for arm D (IFA + polyICLC + mCy) of proved March 16, 2012. 50% (90% CI: [34,66]) exceeded the 18% dRsp rate (90% CI: [11,26]) Human Investigation Committee, Yale University; HIC Protocol # from prior experience with 6MHP in IFA alone. The regimen with IFA 1302011504, Approved March 22, 2012. + pICLC alone also showed promise for enhancing T cell and anti- IntegReview; Protocol No IMCgp100/01, Approved November 13, body responses. Addition of mCy does not alter circulating T reg fre- 2013. quencies but shows some promise as a systemic vaccine adjuvant. Western Sydney Local Health District; HREC2012/7/4.1 (3552) AU RED HREC/12/WMEAD/237, Approved on October 24, Acknowledgements 2012. We thank the Cancer Research Institute/ Ludwig Institute for Cancer Western Institutional Review Board; Panel 1, Study Num 1147687, Research for providing the polyICLC used in the vaccines. Funding was WIRB Pro Num 20141184, Approved July 15, 2014. provided by NCI R01 CA178846 (CLS); 5K25CA181638 (NW); P30 CA044579 Memorial Sloan Kettering Cancer Center, Institutional Review (Biorepository and Tissue Research Facility, Office of Clinical Research, and Board; Protocol # 14-152, August 28, 2014. Biostatistics Shared Resource). Trial Registration The clinical trial Mel63 is registered with Clinicaltrials.gov (NCT02425306). P365 Ethics Approval A trial to evaluate the immunogenicity and safety of a melanoma The clinical trial Mel63 was performed with IRB (#17860) and FDA approval helper peptide vaccine plus incomplete Freund’s adjuvant, (IND #10825). cyclophosphamide, and polyICLC (Mel63) 1 1 1 Craig Slingluff, MD , Gina Petroni, PhD , Kimberly Chianese-Bullock, PhD , 1 1 1 1 Nolan Wages, PhD , Walter Olson, PhD , Kelly Smith , Lynn Dengel, MD , 1 2 1 Anna Dickinson , Caroline Reed , Elizabeth Gaughan, MD , William 1 1 1 1 Grosh, MD , Varinder Kaur, MD , Nikole Varhegyi , Mark Smolkin , 1 1 Table 1 (abstract P365). T cell responses to 6MHP Nadejda Galeassi , Donna Deacon, BS 1 2 University of Virginia, Charlottesville, VA, United States; Emory University School of Medicine, Atlanta, GA, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P365 Background Cancer vaccines require adjuvants to induce effective and durable protective immunity. However, there is no consensus on optimal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 200 of 272 assay. Circulating antibody (Ab) responses to overlapping NY-ESO-1 peptides were detected by ELISA. Tumor biopsies obtained pre- treatment and day 85 were evaluated for immune infiltrates by multi- spectral immunofluorescence histology. Results Target enrollment was 27; study closed early for slow enrollment. Eight patients enrolled and were treated (Table 1). All had ≥ 1 treat- ment emergent adverse event (TEAE); most common (≥50%): rash, fatigue, injection site reaction, pruritus, and diarrhea. Two patients had Gr3 TEAEs related to IPI but not to vaccine. There were no DLTs. Best responses: SD (n=4); PD (n=4). T cell responses to NY-ESO-1 were detected in 6 of 8 (75%) patients.[1] Both patients without T cell response had PD as best response. Ab responses were detected in 7/8 (88%) patients (Table 1). The patient without Ab response had PD as best response. The breadth of Ab responses to NY-ESO-1 was greater for patients with SD than those with PD (p = 0.02). Evaluation of TME of 5 patients revealed increases in proliferating (Ki67+) CD8 T cells, decreases in RORγt+ CD4+ T cells (Figure 1). Interestingly, there were increases in density of CD8+ and CD4+ cells for those with SD (n=3), but decreases for those with PD (n=2, not shown). Conclusions T cell responses and Ab responses to NY-ESO-1 were induced in most patients and were evident ex vivo, suggesting that IPI may have en- hanced the T cell responses to NY-ESO-1 protein and OLP4. Inte- grated T cell and antibody responses were associated with tumor control. Preliminary data of the TME suggests increased activating and proliferating T cells after vaccination plus IPI, especially in pa- tients with tumor control. Acknowledgements The trial was supported by the Ludwig Institute for Cancer Research, the Fig. 1 (abstract P365). Antibody responses to 6MHP Cancer Research Institute, and by the National Institutes of Health, including support from the University of Virginia Cancer Center Support Grant (NIH/NCI P30 CA44579:, Clinical Trials Office, Biorepository and Tissue Procurement Facility, Flow Cytometry Core, and Biomolecular Core Facility). P366 Earlier presentation of results of this clinical trial [1] has been expanded in A phase 1 study of NY-ESO-1 vaccine + ipilimumab (ipi) in patients the present abstract with additional biologic correlates. with unresectable or metastatic melanoma 1 2 3 Trial Registration Craig Slingluff, MD , Hassane Zarour, MD , Michael Postow, MD , Philip 4 5 1 This trial was registered at ClinicalTrials.gov (NCT01810016). Friedlander, MD PhD , Craig Devoe, MD , Ileana Mauldin, PhD , Kelly 1 6 Smith , Mary Macri, BSc 1 2 Reference University of Virginia, Charlottesville, VA, United States; University of 1. Slingluff CL Jr, Zarour HM, Postow MA, Friedlander P, Devoe CE, Macri M, Pittsburgh Cancer Center, Pittsburgh, PA, United States; Memorial Sloan Ryan A, Venhaus R, Wolchok J. J Clin Oncol. 2018; 36(suppl; abstr e15175). Kettering Cancer Center, New York, NY, United States; Mount Sinai Ethics Approval Medical Center, New York, NY, United States; Northwell Health Cancer The study was approved by each institution's Ethics Board, with approval Institute, Lake Success, NY, United States; Ludwig Institute for Cancer numbers: IRB#12-253 (Memorial Sloan Kettering), HS#13-00471 (Mount Research, New York, NY, United States Sinai), IRB#14-133B (Northwell Health), MOD13030240-02/ Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) PRO13030240(University of Pittsburgh), and HRS#16347(University of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P366 Virginia), and to the FDA with IND 10369. Background Ipilimumab (IPI) is an approved immunotherapy for advanced melan- oma. It can enhance immunity to cancer-testis antigen NY-ESO-1. Vac- Table 1 (abstract P366). Enrollment, immune and clinical responses cines with NY-ESO-1 protein or NY-ESO-1 overlapping long peptides (OLP4) have enhanced immunity when administered with Montanide ISA-51 (Montanide) and/or Poly-ICLC (pICLC) adjuvants. This trial assessed safety, immunogenicity, clinical responses (irRC), and effects of IPI + NY-ESO-1 vaccines on the tumor microenvironment (TME). Methods This Phase 1, open-label study enrolled patients among 3 arms: IPI (3 mg/kg i.v. q3 wks x 4) + NY-ESO-1 protein + pICLC + Montanide (Arm A); IPI + NY-ESO-1 OLP4 + pICLC + Montanide (Arm B); and IPI + NY-ESO-1 OLP4 + pICLC (Arm C). Patients had measurable NY-ESO- 1+ tumors. Treatments were administered days 1, 22, 43, 64. Circulat- ing T cell responses were assessed by ex vivo IFN-gamma ELIspot Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 201 of 272 Conclusions Seviprotimut-L is very well tolerated. Subgroup efficacy analyses identi- fied two populations who may benefit from Seviprotimut-L: those with AJCC stage IIB/IIC melanoma and those under age 60. These data support proceeding to the definitive final part of the MAVIS phase III trial testing seviprotimut-L for stage IIB/C patients, in particular those under age 60. Acknowledgements We acknowledge the support of all investigators and clinical coordinators responsible for enrolling patients to this trial. Trial Registration This trial was registered at ClinicalTrials.gov: NCT01546571. References 1. Bystryn JC, Zeleniuch-Jacquotte A, Oratz R et al. Double-blind trial of a Fig. 1 (abstract P366). See text for description polyvalent, shed-antigen, melanoma vaccine.[see comment]. Clinical Can- cer Research 2001; 7: 1882-1887. P367 2. Dorshkind K, Swain S. Age-associated declines in immune system devel- A multicenter, double-blind, placebo-controlled trial of opment and function: causes, consequences, and reversal. Curr Opin seviprotimut-L polyvalent melanoma vaccine in post-resection Immunol 2009; 21: 404-407. melanoma patients at high risk of recurrence 1 2 3 Ethics Approval Craig Slingluff, MD , Brent Blumenstein, PhD , Karl Lewis, MD , Robert 4 5 The study was approved by the Ethics Board at each participating institution (IRB#), as Andtbacka, MD, CM, FACS, FRCSC , John Hyngstrom, MD , Mohammed 6 7 8 follows: University of Virginia Hospital (16223); Anschutz Cancer Pavilion, UC Milhem, MBBS , Svetomir Markovic, MD, PhD , Omid Hamid, MD , Leonel 9 10 11 Denver(1134601); Huntsman Cancer Institute, / Univ of Utah Health Care (55911); Hernandez-Aya, MD PhD , Tawnya Bowles, MD , Prejesh Philips, MD , 12 13 14 University of Iowa Hospitals and Clinics (1133782); Mayo Clinic Cancer Center / Joel Claveau, MD , Sekwon Jang, MD , Jose Lutzky, MD, FACP , Anna 15 16 Mayo Clinic Rochester (12-002308); The Angeles Clinic and Research Institute Bar, MD , Peter Beitsch, MD 1 2 (1196134); Washington University School of Medicine (201205056); Intermountain University of Virginia, Charlottesville, VA, United States; Tri Arc Medical Center (1024288); University of Louisville (15.0039); CHU de Quebec, Consulting, Washington, DC, United States; University of Colorado, L'Hotel Dieu de Quebec (MP-20-2015-2318); Inova Melanoma and Skin Cancer Aurora, CO, United States; Seven and Eight Biopharmaceuticals, Salt Center (1152774); Mount Sinai Medical Center (12-16-H-03); Oregon Health and Lake City, UT, United States; Huntsman Cancer Institute/ Univ of Utah, Science University (IRB00011848); Cancer Solutions (1197259). Salt Lake City, UT, United States; University of Iowa Hospitals and Clinics, Iowa City, IA, United States; Mayo Clinic Rochester, Rochester, MN, United States; The Angeles Clinic & Research Institute, Los Angeles, Table 1 (abstract P367). Enrollment and adverse events CA, United States; Washington University School of Medicine, Saint Louis, MO, United States; Intermountain Medical Center, Murray, UT, United States; University of Louisville, Louisville, KY, United States; 12 13 CHU de Quebec, L'Hotel Dieu de Quebec, Quebec, Canada; Inova Melanoma and Skin Center, Fairfax, VA, United States; Mount Sinai Medical Center, Miami Beach, FL, United States; Oregon Health and Science University, Portland, OR, United States; Cancer Solutions, Dallas, TX, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P367 Background Seviprotimut-L is a vaccine prepared from antigens shed by 3 human mel- anoma cell lines, administered with alum. Prior formulations showed prom- ising immunogenicity for T cell and antibody responses and improved survival in a small phase II clinical trial[1]. Part B1 of MAVIS (Melanoma Anti- gen Vaccine Immunotherapy Study, a three part, Phase III clinical program), was a multicenter, double-blind, placebo-controlled trial to assess the effi- cacy of seviprotimut-L, with the primary endpoint of relapse-free survival (RFS) in patients at high risk of recurrence after definitive surgical resection. Methods For MAVIS Part B1, patients with AJCC v7 stage IIB-III cutaneous melan- oma, after surgical resection, age 18-75, ECOG PS 0-1, were randomized 2:1 to seviprotimut-L 40 mcg or placebo, administered subcutaneously every 2 weeks x 5, then monthly x 4, then every 3 months to month 24. Patients were stratified by stage (IIB/C, IIIA, IIIB/C). Target enrollment was 325. The study was powered for assessment of RFS, with target hazard ratio (HR) of 0.625, one-sided alpha of 0.10, and power 80%. Results 347 patients were randomized, and arms were well-balanced. Treatment- emergent adverse events (AEs) were similar for seviprotimut-L and placebo patients (Table 1). By intent-to-treat (ITT) analysis, RFS was not significantly enhanced for seviprotimut-L in the full study population, but trended slightly higher (Figure 1A). Analysis of subgroups based on pre-planned stratification suggested enhanced RFS for seviprotimut-L among Stage IIB/IIC patients (HR 0.59, 95% CI[0.33,1.07], Figure 1B). Age has been identified as a cause of de- creased immune competence[2]; thus, outcomes were assessed as a function of age as an effect modifier. Figures 1C and 1D show all randomized patients Fig. 1 (abstract P367). Clinical outcome (Figure 1C) and Stage IIB/IIC subset (Figure 1D) by arm and age split at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 202 of 272 Clinical Trial In Progress 3. Bernhardt SL, Gjertsen MK, Trachsel S, et al. Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose es- P368 calating phase I/II study. Br J Cancer. 2006;95:1474-1482 ZI-H04 - A novel MHC class II restricted TCR based cellular therapy targeting hTERT to treat solid tumours Jens-Peter Marschner, MD, Mona Welschof, PhD, Miguel Forte, Eva P369 Kristine Klemsdal, Sylvie Pollmann, Namir Hassan Feasibility of a phase I personalized adoptive T-cell therapy in Zelluna, Seeheim-Jugenheim, Germany patients with relapsed/refractory solid tumors 1 3 Correspondence: Jens-Peter Marschner Apostolia Tsimberidou, MD, PhD , Ali Mohamed , Stephen Eck, MD, 4 4 1 1 (jenspeter.marschner@zelluna.com) PhD , Harpreet Singh , Patrick Hwu, MD , Cassian Yee, MD , Borje Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P368 Andersson, MD, PhD 1 2 MD Anderson Cancer Center, Houston, TX, United States; MD Background Anderson, Houston, TX, United States; Immatics Biotechnology, Chimeric Antigen Receptor T-cells (CAR-T) are highly effective in the Tubingen, Germany; Immatics, Houston, TX, United States treatment of some hematological malignancies but solid tumors re- Correspondence: Apostolia Tsimberidou (atsimber@mdanderson.org) main a challenge for cellular therapies. A few T-cell Receptor T- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P369 cells (TCR-T) have been investigated in solid tumors. To our knowledge, there is only one published study and one case re- Background port using MHC Class II restricted TCRs targeting MAGE-A3 and Adoptive cellular therapy (ACT) is limited in solid tumors due to lack of NY-ESO-1, respectively [1,2]. suitable immunotherapy targets with high specificity and frequent re- lapse following immunotherapy to single targets often associated with Methods loss of target expression in the tumor. ACTolog® is a personalized, ZI-H04 represents a novel approach of TCR based therapies. multi-targeted ACT approach in which autologous T-cell products are Autologous T-cells from patients are genetically modified by manufactured against the most relevant tumor target peptides for indi- lentiviral transduction to express the TCR targeting hTERT in vidual patients whose tumors are positive against predefined targets. the context of the MHC Class II allele, HLA-DPB1*04:01. The Methods TCR was isolated from a pancreatic cancer patient who experi- Patients with advanced metastatic cancers and HLA-A*02:01 enced clinical benefit following a peptide-based cancer vaccin- phenotype, undergo a tumor biopsy. Patients whose tumors ex- ation against hTERT [3]. The TCR clone responded to press >1 of 8 cancer targets undergo leukapheresis. Autologous T autologous tumor and preclinical data demonstrate that ZI-H04 cells are primed against the expressed ACTolog targets in the exhibits high sensitivity to hTERT peptide as well as recogni- presence of IL-21 followed by HLA tetramer-guided cell sorting tion of processed antigen. Furthermore, specificity analysis sup- and rapid expansion. Patients who meet criteria for treatment re- ports the safety of the TCR-T. The restricted combined ceive lymphodepletion with Fludarabine 40 mg/m2 i.v. and Cyclo- expression of hTERT plus HLA class II on normal cells limits the phosphamide 500 mg/m2 i.v. (Days, -6 to -3). T-cells are infused potential for on-target off tumor toxicity. A first-in-human on Day 0, followed by low-dose of IL-2 for 14 days (www.clinical- study is designed to treat patients with relapsed/refractory trials.gov NCT02876510). solid tumors lacking an option of further treatments. Patients Results must be tested positive for HLA-DPB1*04:01 and the tumors From 7/2017 to 7/2019, 203 patients signed an informed consent to must express hTERT. Adequate organ function and lab parame- participate in the study, 91 had HLA-A*02:01 phenotype, 52 had a ters are required. CNS involvement, autoimmune diseases, in- tumor biopsy and 34 patients underwent leukapheresis. To date, 9 fections and immunosuppressive medication are main exclusion patients have received treatment (median age, 38 yrs; range, 25-58 criteria. Primary objectives are safety and tolerability. Part 1 of yrs; 2 men and 7 women; breast cancer, 3; sarcoma, 3; ovarian can- the study, starting in 2020, will be a dose finding part, Part 2 cer, 1; nasopharyngeal, 1; anal carcinoma, 1; median time from diag- a dose extension part with 5 cohorts. Prior to adoptive cell in- nosis 4 years, range, 2-18 years; median number of prior therapies 6, fusion patients will receive a low dose conditioning regimen range 3-12). Very high ACTolog cell doses could be administered. Pa- consisting of 2 x 600 mg/m² cyclophosphamide followed by 3 tients received a median of 2 target-specific ACTolog products (range x 25 mg/m² fludarabine. Patients will be observed for safety, 1-3). Treatment was overall well tolerated. The most common ad- efficacy and exploratory biomarkers. verse events were cytopenias and cytokine release syndrome. All pa- Conclusions tients are alive to date. At 6 weeks, restaging imaging studies ZI-H04 is a novel TCR-T with potentially favourable characteristics. demonstrated stable disease in all patients. One patient with squa- The TCR was isolated from an hTERT vaccinated pancreatic cancer mous cell carcinoma of the anus treated with T cells directed to patient that experienced clinical benefit. A lower probability of COL6A3, exon 6, and PRAME had 26% decrease in tumor measure- off-target activity is expected since no engineering was done to ments at week 6 associated with high T-cell frequencies at 2 weeks the TCR. The MHC Class II restriction provides the possibility to but her disease subsequently progressed. Another patient with naso- induce a multi-pronged immune response including antigen pharyngeal cancer treated with COL6A3 tumor stroma-specific T cells spreading as demonstrated in a case report using an MHC Class had resolution of tumor associated pain and has not required further II TCR-T in melanoma [2]. More than 50% of patients are HLA- treatment for 11 months. A recent tumor biopsy demonstrated nec- DPB1*04:01 positive and the expression rate of hTERT is >80% in rotic cells and no tumor cells could be identified. many tumors. Therefore, a substantial population may benefit Conclusions from ZI-H04 treatment. ACTolog IMA101 is well-tolerated and no safety issues have been noted to date. The study is ongoing. References Trial Registration 1. Lu Y-C, Parker LL, Lu T, et al. Treatment of patients with metastatic cancer www.clinicaltrials.gov NCT02876510 using a major histocompatibility complex class II-restricted T-cell receptor Ethics Approval targeting the cancer germline antigen MAGE-A3. J Clin Oncol. The study was approved by MD Anderson's IRB. 2017;35:3322-3329 Consent 2. Hunder NN, Wallen H, Cao J, et al. Treatment of metastatic melanoma Written informed consent was obtained from the patient for publica- with autologous CD4+ T cells against NY-ESO-1. N Engl J Med. tion of this abstract and any accompanying images. A copy of the 2008;358:2698-703 written consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 203 of 272 P370 Trial Registration The positive correlation between baseline absolute eosinophil NCT02791334 count (AEC) in blood and clinical benefit to PD-(L)1 inhibition monotherapy Reference 1 1 1 Anna Szpurka, PhD , Danni Yu, PhD , Michelle Carlsen , Antoine Tanizaki, Junko, Koji Haratani, Hidetoshi Hayashi, Yasutaka Chiba, Yasushi 2 3 4 Hollebecque, MD , Hyun Cheol Chung, MD, PhD , Amita Patnaik , Nakamura, Kimio Yonesaka, Keita Kudo, et al. "Peripheral Blood 5 6 7 Johanna Bendell, MD , Antoine Italiano, MD , Yung-Jue Bang, MD PhD , Biomarkers Associated with Clinical outcome in Non–Small Cell Lung 8 9 10 Chia-Chi Lin, MD, PhD , Marcus Butler, MD , Timothy Yap, MD PhD , Cancer Patients Treated with Nivolumab." Journal of Thoracic Oncology 11 11 María José de Miguel, MD , María José de Miguel, MD , Jean-Pascal 13, no. 1 (2018/01/01/ 2018): 97-105. 12 13 14 Machiels, MD, PhD , Marc Peeters, MD, PhD , Wu-Chou Su, MD , Ethics Approval 15 1 1 Victor Moreno, MD , Yumin Zhao, PhD , Erik Rasmussen, PhD , Xiaojian The study included multiple investigator sites, and some of these had Xu, MD approval dates instead of approval numbers. The following are listed 1 2 Eli Lilly and Company, Indianapolis, IN, United States; University of Paris showing approving committee followed by approval date or approval # Sud, Villejuif, France; Yonsei University College of Medicine, Seoul, (if available): IntegReview, 15Jun2016; IntegReview, 21June2016; MD Korea, Republic of; South Texas Accelerated Research Therape, San Anderson Office of Protocol Approval IRB, 26May2016; Princess Margaret Antonio, TX, United States; Sarah Cannon Research Institute, Nashville, Cancer Centre, University Health Network Research Ethics Board, 16-5824 6 7 TN, United States; Institut Bergonié, Bordeaux, France; Seoul National (initial approval date 01Feb2017); Comite de protection des Personnes University Hospital, Seoul, Korea; National Taiwan University Hospital, (CPP), EudraCT number 2016-000440-33. Taipei, Taiwan, Province of China; Princess Margaret Cancer Center, Toronto, Canada; The University of Texas, Houston, TX, United States; 11 12 START-HM Sanchinarro, Madrid, Spain; jean- pascal.machiels@uclouvain.be, Brussels, Belgium; Antwerp University Hospital,, Antwerp, Belgium; National Cheng Kung University Hospital, Tainan, Taiwan, Province of China; START Madrid-FJD, Madrid, Spain Correspondence: Danni Yu (yu_danni@lilly.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P370 Background Anti-PD-(L)1 immunotherapies have increased the response rate in certain cancer subtypes however, some patients who may have clin- ical benefit are not identifiable with existing predictive biomarkers. Research is ongoing to identify routinely available blood and clinical markers to predict response to PD-(L)1 therapies. In this study, we ex- Fig. 1 (abstract P370). See text for description plored absolute eosinophil count (AEC) as a biomarker in patient’s re- sponse to PD-(L)1 treatment. Methods P371 This is a phase 1a/1b study of an anti-PD-L1 antibody (LY3300054) Cytokine Microdialysis for real-time immune monitoring in administered alone or in combination with other agents in patients Glioblastoma patients undergoing Checkpoint Blockade 1 2 2 with advanced refractory solid tumors. Eligible patients were ≥18 John Lynes, MD , Victoria Sanchez , Anthony Nwankwo , Gifty 2 2 2 2 years old, had ECOG status ≤1 and had at least 1 measurable lesion Dominah , Xiang Wang, MS , Isac Kunnath , Samantha Dill , Gretchen 2 2 2 2 2 per RECIST v1.1. We assessed the association of AEC with confirmed Scott , Christi Hayes , Tianxia Wu , Marta Penas-Prado , Jing Wu , Eric 2 2 2 2 best overall response (BORc). The AEC cutoff 0.155 (10^9/L) maxi- Burton , John Heiss , Christopher Hourigan, MD, PhD , Mark Gilbert , mized the difference in ORR, similar to previous reports (Tanizaki etal, Edjah Nduom, MD 1 2 2018, JTO [13] e85-e86) . The impact of AEC status was demonstrated Georgetown University, Bethesda, MD, United States; National by a 3-dimensional waterfall plot depicting the best change in tumor Institutes of Health, Bethesda, MD, United States size and overall survival (OS). Correspondence: Edjah Nduom (edjah.nduom@nih.gov) We also tested the correlation between AEC and OS/proxy-progres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P371 sion-free survival (PFS; time to next treatment, TTNT) in NSCLC pa- tients (n=455) who received anti-PD-1 therapy with same cutoff from Background an independent Flatiron database. Glioblastoma is the most common primary malignancy of the brain, Results with a dismal prognosis. Immunomodulation via checkpoint inhibition As of 8 December 2017, 30 patients (MSI-H: n=22, M: n=8) were has provided encouraging results in non-CNS malignancies, but predic- treated. There were no deaths due to adverse events. Two patients tion of responders has proven to be challenging in glioblastoma pa- in MSI-H cohort experienced grade 3 treatment-related AEs (TRAEs): tients. OBJECTIVES: To determine the proportion of patients who have diarrhea (n=1, 4.5%), blood creatinine phosphokinase increased (n=1, a measurable increase of interferon gamma levels in brain tumor tissue 4.5%), and hyponatraemia (n=1, 4.5%). No grade 3 events were re- after their first dose of nivolumab; to evaluate the safety of using brain ported in M cohort, and no grade 4/5 TRAEs were reported in either tumor microdialysis to monitor for immune response; to evaluate the cohorts. There were no TRAEs leading to discontinuation of study safety of the combination of anti-programmed death 1 (PD-1) and anti- treatment. Preliminary efficacy data in MSI-H cohort showed ORR of lymphocyte activation gene 3 (LAG-3) checkpoint inhibition in recurrent 36% [CR in 1 pt (5%)(ovarian), PR in 7 pts (32%)(small intestine glioblastoma patients. adenocarcinoma [1 pt], endometrial [3 pts], colon [3 pts])], DCR in Methods 64% [SD in 6 pts (27%)]; mPFS was 7.39 months (95% CI 1.7, NR). In The study design is a single-center, nonrandomized phase 1 clinical the M cohort, DCR was 63% [PR in 1 pt (13%), SD in 4 pts (50%)]. As trial. Up to 20 adult patients with recurrent glioblastoma will be en- of data cut-off, 16 pts (53%) remain on treatment. Preliminary bio- rolled with the goal of 10 patients completing the trial over an antici- marker analysis, including but not limited to, PD-L1 and CD8 expres- pated 18 months. Patients will undergo biopsy; placement of sion and circulating markers will be presented. microdialysis catheters and lumbar drains; treatment with anti-PD-1 Conclusions checkpoint inhibition; comprehensive immune biomarker collection; LY3300054 was well-tolerated and demonstrated antitumor activity tumor resection; and then treatment with anti-PD-1 and anti-LAG-3 in patients with MSI-H solid tumors; combination expansions are checkpoint inhibition until progression (Figure 1). Three patients ongoing. have undergone all study procedures (Figures 2 and 3). There have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 204 of 272 been no serious adverse events related to the research surgical pro- cedure, nor during the microdialysis portion of the trial. Enrollment is ongoing. EXPECTED OUTCOMES: We expect interferon gamma levels to in- crease in the brain as measured via microdialysis in treated patients. Based on published reports, microdialysis in this patient population is expected to be safe, and anti-LAG-3 and anti-PD-1 combined will likely have a similar side effect profile to other checkpoint inhibitor combinations. The failure of recent trials of immune therapies in glioblastoma un- derscores the need to appropriately measure response in the treated tissue. This trial may provide insight on indicators of which patients will respond to immune therapy. Acknowledgements Funding and support came from: Intramural Research Program of the Na- tional Institute of Neurological Disorders and Stroke Trial Registration Clinicaltrials.gov: NCT03493932 (Registration Date: April 11, 2018) Fig. 3 (abstract P371). See text for description Ethics Approval Institutional Approvals: National Institutes of Health Combined Neurosciences Internal Review Board number - 18-N-0077 P372 Circulating immune cell biomarkers predict response to immune checkpoint inhibitor therapy in metastatic breast cancer Jin Sun Bitar, MD, Colt Egelston, PhD, Susan Yost, Wanqiu Hou, Padam Simran, Paul Frankel, PhD, Mina Sedrak, Jana Portnow, MD, Joanne Mortimer, MD, Christina Yeon, MD, Arti Hurria, MD, Aileen Tang, Norma Martinez, Peter Lee, MD, Yuan Yuan City of Hope, Duarte, CA, United States Correspondence: Yuan Yuan (yuyuan@coh.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P372 Background The role of immune checkpoint PD-1/PD-L1 inhibitor (ICI) in breast cancer (BC) is being investigated in clinical trials. Preclinical evidence Fig. 1 (abstract P371). See text for description strongly supports the synergistic effects of CDK4/6 inhibitor and ICI [1]. A phase II trial is testing the safety and efficacy of the combin- ation of letrozole, palbociclib and pembrolizumab in patients with hormone receptor positive (HR+) BC (NCT02778685). Currently, there is no well-defined circulating biomarker to predict response to ICI. Methods Peripheral blood mononuclear cells (PBMC) were collected at day 1 of cycles 1 (pre-treatment), 2, 4, 6 and 8. The comprehensive characterization of circulating immune cell composition was per- formed using 15-color flow cytometry. Results Preliminary analysis included 9 patients with the following responses by RECIST 1.1: 1 complete response, 4 partial response, 2 stable disease, and 2 progressive disease. Higher baseline frequencies of CD4+ effector memory (p=0.01) and CD8+ CD45RA+ effector memory cells (p=0.01) were observed in patient responders. Additionally, patient responders demonstrated higher fre- quencies of T cells expressing KLRG1, a marker of effector T cell differenti- ation, on both CD4+ (p=0.001) and CD8+ T cells (p=0.004) at baseline. An increase in the frequency of circulating CXCR5+ CD8+ T cells (p=0.01) at cycle 2 was identified in all treated patients and an increase in CCR10+ CD8+ T cells (p=0.03) was detected at cycle 2 in patient responders indi- cating changes in T cell trafficking. Finally, a shift in myeloid cell compos- ition from predominantly classical to non-classical monocytes was observed in patient responders between baseline and cycle 2 (p=0.007). Conclusions High baseline levels of both CD4+ and CD8+ effector T cells indicate the necessity for a pre-existing favorable T cell composition in check- point blockade responders. Temporal changes in T cell trafficking mole- Fig. 2 (abstract P371). See text for description cules and shifts in myeloid cell composition over the course of therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 205 of 272 indicate potential changes in T cell priming and regulation. Further ana- 2. Bernstein V, Ellard SL, Dent SF, et al. A randomized phase II study of lysis is currently ongoing to understand correlates of systemic immune weekly paclitaxel with or without pelareorep in patients with metastatic changes and changes in the tumor microenvironment. breast cancer: final analysis of Canadian Cancer Trials Group IND.213. Breast Cancer Res Treat 2018;167:485-93. Trial Registration 3. Nuciforo, P., Pascual, T., Cortés, J., Llombart-Cussac, A., Fasani, R., Paré, L., NCT02778685 … Holgado, E. (2018). A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2- Reference positive breast cancer treated with chemo-free dual HER2 blockade. An- 1. Goel S, DeCristo MJ, Watt AC, et al. CDK4/6 inhibition triggers anti- nals of Oncology. https://doi.org/10.1093/annonc/mdx647 tumour immunity. Nature. 2017;548(7668):471–475. Ethics Approval Ethics Approval This study was approved by the Spanish Health Authority, protocol number The study was approved by City of Hope National Cancer Center‘s Ethics 2018-003345-42. Board, approval number 16058 P373 A window-of-opportunity study of pelareorep in early breast cancer (AWARE-1) 1 2 3 4 Luis Manso , Patricia Villagrasa , Nuria Chic , Juan Cejalvo , Yann 5 5 6 3 Izarzugaza , Blanca Cantos , Salvador Blanch , Manel Juan , Blanca 3 7 8 7 Gonzalez , Rita Laeufle , Gerard Nuovo , Grey Wilkinson, PhD , Matt 7 3 3 2 2 Coffey , Azucena Gonzalez , Patricia Galvan , Laia Paré , Jordi Canes , 9 3 6 Xavier Gonzalez , Aleix Prat, MD PhD , Joaquín Gavilá 1 2 Hospital Universitario 12 de Octubre, Madrid, Spain; SOLTI Breast Cancer Research Group, Barcelona, Spain; Hospital Clinic, Barcelona, 4 5 Spain; Hospital Clínico, Valencia, Spain; Hospital Universitario, Madrid, Spain; Instituto Valenciano de Oncología (IVO), Valencia, Spain; 7 8 Oncolytics Biotech Inc., San Diego, CA, United States; Ohio State University, Columbus, OH, United States; Hospital Universitari, Sant Cugat del Vallés, Spain Fig. 1 (abstract P373). See text for description Correspondence: Aleix Prat (ALPRAT@clinic.cat) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P373 Background P374 Pelareorep is an intravenously delivered (IV) unmodified oncolytic reo- TCR repertoires from peripheral blood correlate with prognostic virus. Clinical studies have demonstrated that IV delivered pelareorep can response in TNBC cancer vaccine immunotherapy replicate in tumor tissue and promote an inflamed tumor phenotype Sadanand Vodala, PhD, Andrew Nguyen, PhD, Noe Rodriguez, Peter characterized the recruitment of CD8+ T cells and upregulation of PD-L1 Sieling, Charles Vaske, Jon Van Lew, Kayvan Niazi, John Lee, MD, Patrick [1]. Consistent with pelareorep’s role in promoting adaptive anti-tumor Soon-Shiong, MD, FRCS, FACS, Shahrooz Rabizadeh immunity, a randomized phase 2 study in metastatic breast cancer dem- ImmunityBio, Inc, Culver City, CA, United States onstrated a statistically significant improvement in overall survival when Correspondence: Kayvan Niazi (kayvan.niazi@immunitybio.com) pelareorep was combined with paclitaxel [2]. We hypothesize that pelar- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P374 eorep mediated anti-tumor immune responses, such as those mediated by T cells, represent a novel strategy for the control or elimination of Background tumor cells in breast cancer. Specifically, in the preoperative setting for TNBC is an aggressive, heterogeneous, and high-grade subtype that rep- early breast cancer, we examined if pelareorep in combination with anti- resents 10-20% of breast carcinomas. Recently, TECENTRIQ and Abraxane PD-L1 therapy, atezolizumab, and other breast cancer therapies offers were approved for the treatment of PD-L1+ unresectable locally ad- clinical benefit in terms of CeLTIL score, a metric for quantifying tumor vanced or mTNBC suggesting a role for immunotherapy in the treatment cellularity (Cel) and tumor-infiltrating lymphocytes (TIL) [3]. of this disease. Growing evidence suggests that chemotherapeutic agents Methods and immunotherapies synergize in patients. Cancer vaccines are also a This exploratory, non-randomized, window of opportunity study, will promising option for TNBC due to the discovery of neo-antigens and evaluate the safety and effect of pelareorep ± atezolizumab on the tumor tumor associated antigens that mobilize anti-tumor T cells. microenvironment in 38 women with early breast cancer. Patients will re- Methods ceive study treatment for ~21 days prior to definitive surgery or neoadju- Here, we characterize T cell receptor repertoires in patients enrolled in a vant chemotherapy. Five cohorts will be examined (Figure 1): Cohort 1: Phase Ib/2 clinical trial (NCT03387085) following treatment by a regimen HR+/HER2-neg (10 patients), pelareorep + letrozole. Cohort 2: HR+/HER2- intended to induce a synchronized, multi-compartment, anti-tumor im- neg (10 patients): pelareorep + letrozole + atezolizumab. Cohort 3: TNBC mune response by mitigating the immune-suppressive effects of the (6 patients): pelareorep + atezolizumab. Cohort 4: HER2+/HR+ (6 pa- tumor microenvironment and activating immunogenic tumor cell death. tients): pelareorep + trastuzumab + atezolizumab. Cohort 5: HER2+/HR- The trial combined metronomic low-dose chemotherapy, SBRT, an allo- (6 patients): pelareorep + trastuzumab + atezolizumab. CelTIL, viral repli- geneic NK cell line expressing high affinity CD16, yeast and adenoviral cation, and other immune-based biomarkers will be used to examine tumor-associated antigen vaccines, an IL15RαFc super-agonist, check- treatment-related changes within the tumor microenvironment. Blood point inhibition, and an anti-angiogenic agent in a manner predicted to and tumor tissue biopsies will be collected at screening, Day 3 (after maximize cytotoxic T-cell mediated immunological recognition of tumor pelareorep but before atezolizumab), and at surgery (Day ~21). cells. Tumor associated antigens included adenoviral vector-based CEA, Trial Registration MUC1, brachyury, and yeast-based brachyury and CEA vaccines. Blood Spanish clinical studies registry: 2018-003345-42 was collected pre- and post-treatment and target lesion analysis was per- formed using irRC and Recist1.1. Total RNA from PBMCs was used to gen- References erate sequencing libraries from each longitudinal sample. Using NGS, 1. Samson A, Scott KJ, Taggart D, et al. Intravenous delivery of oncolytic TCR-α and -β CDR3s were clonotyped and tracked over serial blood reovirus to brain tumor patients immunologically primes for subsequent draws. Additionally the Shannon-Wiener Diversity Index (SWDI) was calcu- checkpoint blockade. Sci Transl Med 2018;10. lated for each time point. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 206 of 272 Results (T+CTX) versus 1 experimental arm (M+CTX) + either MGA012 or Patient samples showing consistent positive responses by irRC/Recist1.1 MGD013, depending on the interim analysis from part 1; with 250 pa- showed emergence and persistence of new TCR clones post-induction. tients each. The primary efficacy endpoint for cohort A (both parts) is This was further reflected in acute surges in SWDI. Furthermore, a high ORR per RECIST 1.1; for cohort B part 2 it is overall survival. SWDI at baseline and post-treatment indicated clinical benefit suggest- ing an inverse correlation between disease severity and peripheral rep- Acknowledgements ertoire diversity. A TNBC super responder showed dramatic increases in The authors thank all the patients, their families, and the entire staff who are mean SWDI index from 74 prior treatment to 1177 at first biopsy post participating in this trial. Professional medical writing support was provided treatment (34% decrease by irRC and 26% by Recist1.1 analysis) and by Meredith Rogers, MS, CMPP, of The Lockwood Group (Stamford, achieved an index as high as 3516 in a subsequent biopsy (83% and Connecticut, USA), in accordance with Good Publication Practice (GPP3) 64% decrease by irRC and Recist 1.1 respectively). guidelines, with funding by MacroGenics, Inc. (Rockville, MD, USA). Trial Registration Conclusions NCT number to come Our findings strongly suggest that peripheral blood TCR repertoires are prognostic indicators/biomarker for TNBC cancer vaccine immunotherapy References and for T cell-based immunotherapy in general. Taken together, these re- 1. Nordstrom JL, Gorlatov S, Zhang W, et al. Anti-tumor activity and toxico- sults strongly indicate activation and expansion of anti-tumor T cell clones kinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with en- following combination therapy. Further functional studies will expand our hanced Fcγ receptor binding properties. Breast Cancer Res. 2011;13:R123. understanding of T cell based cancer vaccine immunotherapy in TNBC. 2. Nordstrom JL, Muth J, Erskine CL, et al. High frequency of HER2-specific immunity observed in patients (pts) with HER2+ cancers treated with margetuximab (M), an Fc-enhanced anti-HER2 monoclonal antibody P375 (mAb). J Clin Oncol. 2019;37(suppl; abstr 1030). Margetuximab combined with anti-PD-1 (MGA012) or anti-PD-1/LAG-3 3. Catenacci DVT, Limet KH, Uronis HE, al. Antitumor activity of (MGD013) +/- chemotherapy in first-line therapy of advanced/metastatic margetuximab (M) plus pembrolizumab (P) in patients (pts) with HER2+ gastroesophageal junction (GEJ) or gastric cancer (GC) 1 2 2 advanced HER2+ (IHC3+) gastric carcinoma (GC). J Clin Oncol. Daniel Catenacci, MD , Minori Rosales , Jon Wigginton, MD ,HyunCheol 3 4 5 6 7 2019;37(suppl 4; abstr 65). Chung, MD, PhD , Harry Yoon ,Lin Shen , Yoon-Koo Kang ,MarkusMoehler 1 2 4. Fuchs CS, Doi T, Jang RW, et al. Safety and efficacy of pembrolizumab University of Chicago, Chicago, IL, United States; MacroGenics, Inc. monotherapy in patients with previously treated advanced gastric and Rockville, MD, United States; University College of Medicine, Seoul, gastroesophageal junction cancer: phase 2 clinical KEYNOTE-059 trial. Korea, Republic of; Mayo Clinic Cancer Center, Rochester, MN, United 5 6 JAMA Oncol. 2018;4:e180013. States; Beijing Cancer Hospital, Beijing, China; Asan Medical, Seoul, 5. Kang YK, Boku N, Satoh T, et al. Nivolumab in patients with advanced Korea, Republic of; University Medical Center Mainz, Mainz, Germany gastric or gastro-oesophageal junction cancer refractory to, or intolerant Correspondence: Daniel Catenacci (dcatenac@bsd.uchicago.edu) of, at least two previous chemotherapy regimens (ONO-4538-12, ATTR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P375 ACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;390:2461-2471. Background Ethics Approval Trastuzumab (T), a monoclonal antibody (mAb) targeting the human Each investigator’s institutional review/ethics board approved the study. epidermal growth factor receptor 2 (HER2) is the standard of care pal- liative first-line therapy for advanced HER2+ GEJ and GC patients. Mar- getuximab (M) is an Fc-engineered anti-HER2 mAb targeting the same P376 HER2 epitope, but with higher affinity for both 158V (high binding) and NOUS-209: A phase I, first-in-human, multicenter, open-label study 158F (low binding) alleles of the activating Fc receptor CD16A. Even of Nous-209 genetic vaccine for the treatment of microsatellite more, M coordinately enhanced both innate and adaptive immunity, in- unstable solid tumors cluding antigen-specific T-cell responses to HER2 [1,2]. Programmed cell Anna Gorrasi, PhD, Elisa Scarelli, Maria Teresa Catanese, Cinzia Traboni, death receptor 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) are Paola Antonini, Denis Brkic both T-cell checkpoint molecules that suppress T-cell function. MGA012 Nouscom Srl, Rome, Italy (INCMGA00012) is a humanized, hinge-stabilized, IgG4κanti-PD-1 mAb Correspondence: Elisa Scarelli (booking@nouscom.com) blocking binding of PD-L1 or PD-L2 to PD-1. MGD013 is a humanized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P376 Fc-bearing bispecific tetravalent protein that concomitantly binds to PD-1 and LAG-3, inhibiting their respective ligand-binding. We previ- Background ously reported that a chemotherapy (CTX)-free regimen consisting of MSI-H tumors are caused by a defective DNA mismatch repair M+PD-1 blockade was well tolerated in GEJ/GC patients, and induced a (dMMR) system that leads to the accumulation of mutations within 30% objective response rate (ORR) [3]. This was 2- to 3-fold greater than microsatellite regions. Insertions or deletions (indels) in microsatel- in historical controls with checkpoint inhibitors alone [4,5]. This lites of coding regions can result in the synthesis of tumor-specific registration-directed trial investigates the efficacy, safety, and tolerabil- frameshift peptides (FSPs). FSPs are considered safe and potent ity of M+checkpoint inhibition ± CTX in metastatic/locally advanced, neoantigens because they are not expressed in the normal human treatment-naïve, HER2+ GEJ/GC patients. proteome. We selected shared FSPs among patients with MSI cancers Methods with the aim of developing an off-the-shelf vaccine for the cure of This adaptive open-label phase 2/3 study includes 2 cohorts. In the first MSI tumors. 209 FSPs were assembled into 4 artificial genes and single arm, CTX-free cohort A, M+MGA012 is evaluated in HER2+ (im- cloned into 4 Great Apes Adenoviral (GAd) and 4 Modified Vaccinia munohistochemistry [IHC] 3+) and PD-L1+ (excluding microsatellite in- Ankara (MVA) vectors to generate a viral vectored vaccine called stability high) patients. After 40 patients are evaluated for response/ Nous-209. We showed that treatment of tumor-bearing mice with safety, 60 more patients will be enrolled if the threshold for study con- GAd/MVA-based neoantigen vaccines synergizes with Checkpoint In- tinuation is met. In the randomized cohort B, HER2+ (IHC 3+ or IHC 2+/ hibitors (CPI), resulting in a 3-fold-increase of cured animals over CPI fluorescent in situ hybridization+) patients, are enrolled irrespective of monotherapy. PD-L1 status. Part 1 randomizes patients to 1 of 4 arms (50 patients Methods each): control arm (T+CTX) or 1 experimental arm (M+CTX; A Phase-I, FIH study was designed to evaluate the safety, tolerability, M+CTX+MGA012; M+CTX+MGD013). CTX is investigator’s choice of and immunogenicity of Nous-209 genetic polyvalent vaccine in XELOX or mFOLFOX-6. Part 2 (pick-the-winner) consists of the control Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 207 of 272 combination with the licensed programmed death receptor-1 (PD-1)- Conclusions blocking antibody pembrolizumab and to detect any preliminary evi- The combination of vactosertib plus pembrolizumab was tolerable dence of anti-tumor activity. Nous-209 is administered intramuscu- with no additional safety concern. The activity of this combination in larly, with a heterologous prime/boost regimen composed of 1 prime CRC and GC patients will be further evaluated in the Dose Expansion with the mixture of 4 GAd vectors (GAd20-209-FSP) and 3 boosts part of the study. Clinical trial information: NCT03724851 with 4 MVA vectors (MVA-209-FSP). The target population includes Trial Registration adult patients with unresectable or metastatic dMMR or MSI-H colo- NCT03724851 rectal cancer (CRC), gastric, and gastroesophageal (G-E) junction Ethics Approval tumors. The study was approved by Ethics Board from Asan Medical Center, The study is composed of two sequential cohorts. In the first cohort Samsung Seoul Hospital, Severance Hospital, Seoul National Univer- (dose escalation), the Recommended Phase 2 Dose (RP2D) will be sity Bundang Hospital, and National Cancer Center, with approval established. In the second part (dose expansion), additional patients number 2018-1215, SMC 2018-07-146-006, 4-2018-0728, B-1808/487- will be evaluated to consolidate the safety of the RP2D and establish 003 and NCC2019-0042, respectively. the immunogenicity of the vaccination. NOUS-209 IND has been cleared by the US Food and Drug Adminis- P378 tration (FDA). The trial will be enrolling up to 30 patients at US clin- Phase II study of combination ipilimumab, nivolumab, and ical sites. Preliminary results from the study are expected in early panitumumab in patients with KRAS, NRAS, and BRAF wild-type (WT) microsatellite stable (MSS) metastatic colorectal cancer (mCRC) 1 2 3 P377 Michael Lee, MD , Patrick Loehrer, MD , Iman Imanirad, MD , Stacey 4 5 1 Safety and anti-tumor activity of the transforming growth factor β Cohen, MD , Kristen Ciombor, MD , Cheryl Carlson, MD,PhD , Hanna 1 1 receptor I kinase inhibitor, vactosertib, in combination with Sanoff, MD , Autumn McRee, MD pembrolizumab in patients with metastatic colorectal or gastric Univ of North Carolina at Chapel Hill, Chapel Hill, NC, United States; 2 3 cancer Indiana University, Indianapolis, IN, United States; Moffitt Cancer 1 2 3 4 Keun-Wook Lee, MD , Young Suk Park, MD, PhD , Joong Bae Ahn , Sun Center, Tampa, FL, United States; University of Washington, Seattle, WA, 3 4 4 5 5 Young Rha , Hark Kyun Kim , Park Young Lee , Min-Hee Ryu , Jeeyun United States; Vanderbilt University, Nashville, TN, United States 2 6 6 6 Lee, MD, PhD , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kiim , Tae Correspondence: Michael Lee (michael_s_lee@med.unc.edu) Won Kim, MD, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P378 Seoul National University Bundang Hospit, Seongnam, Korea, Republic 2 3 of; Samsung Medical Center, Seoul, Korea, Republic of; Severance Background Hospital, Seoul, Korea, Republic of; National Cancer Center, Kyunggi-do, Panitumumab is an IgG2 monoclonal antibody (mAb) targeting the Korea, Republic of; Asan Medical Center, Seoul, Korea, Republic of; epidermal growth factor receptor (EGFR) and is a standard therapy Medpacto, Inc, Seoul, Korea, Republic of for patients with KRAS, NRAS, and BRAF WT mCRC. Preclinical data Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) shows that anti-EGFR mAbs require functional innate and adaptive Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P377 immunity to mediate efficacy. Anti-EGFR therapy causes a tumor- specific adaptive immune response and immunogenic apoptosis Background [1,2], and anti-EGFR antibodies require functional T cells for in vivo Vactosertib is a highly selective and potent inhibitor of transforming efficacy [3]. However, resistance to anti-EGFR therapy inevitably de- growth factor beta (TGF-β) receptor type 1. Recent studies have re- velops and is associated with increased regulatory T cells expressing vealed that inhibition of TGF-β signaling reverses immunosuppres- CTLA-4 [4] and activated immunosuppressive macrophages with up- sive tumor microenvironment and poor responses to cancer regulation of PD-L1 [5]. Thus, resistance to anti-EGFR antibody ther- immunotherapy. To date, antitumor efficacy by immune check point apy is associated with increased expression of both CTLA-4 and PD- inhibitors in colorectal or gastric/gastroesophageal cancer as mono- L1. We hypothesized that treatment with ipilimumab (anti-CTLA-4) therapy is known to be limited. A combination of TGF-β and PD-1 in- and nivolumab (anti-PD-1) synergizes with panitumumab to signifi- hibition may induce immune restoration and improve antitumor cantly improve the response rate in patients with KRAS, NRAS, and responses. We are reporting Dose Finding part of Phase 1b/2a study BRAF WT MSS mCRC. evaluating the combination of vactosertib plus pembrolizumab in Methods metastatic colorectal cancer (CRC) or diffuse gastric cancer (GC). LCCC1632 is a multicenter, single-arm, phase II clinical trial with a Methods pre-specified safety run-in of panitumumab, ipilimumab, and nivolu- Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and have mab in KRAS/NRAS/BRAF WT, MSS mCRC (NCT03442569). Eligible pa- no prior exposure to immunotherapy including anti-CTLA-4, anti-PD-1, tients must have received 1-2 prior lines of therapy and no prior anti-PD-L1, and TGFβR1 kinase inhibitors. The primary objective is to as- anti-EGFR or immune checkpoint inhibitor therapy. A 6-subject safety sess the safety and the recommended dose of vactosertib given 5 days run-in was treated with ipilimumab 1 mg/kg IV q6wk, nivolumab 240 on/2 days off in combination with pembrolizumab 200 mg every 3 mg IV q2wk, and panitumumab 6 mg/kg IV q2wk and observed for weeks. The Dose Finding part starts with vactosertib 200 mg BID plus 12 weeks for dose-limiting toxicities (DLTs), followed by expansion pembrolizumab. Secondary objectives include characterization of vac- into a Simon’s two stage phase II trial, with 26 more subjects enrolled tosertib pharmacokinetics and anti-tumor activity by response rate. in the first phase and 56 total subjects planned. The primary end- Results point is response rate defined by RECIST 1.1. Secondary endpoints in- As of July 8, 2019, among 10 patients enrolled to 200 mg BID cohort, 6 clude response rate by irRECIST, progression-free survival (PFS), were with CRC and 4 diffuse type GC. Median age was 51 (range 31-71), overall survival (OS), and duration of response. Correlative studies in- 50% were male, median number of previous lines of chemotherapy was 4 clude Consensus Molecular Subtype analysis by archival tissue and (range 2-6). All patients were immune checkpoint inhibitor naïve. No dose assessment of peripheral immune cell activation. limiting toxicity was reported. Common adverse events (AE) were anorexia Results (33%), fatigue (33%), abdominal pain (33%), and fever (33%). There were 3 Within the 12-week DLT period, only one grade 3-4 toxicity (grade 3 serious adverse events (SAE) reported; bilirubin elevations (1), pleural effu- increased lipase) and no DLTs were observed. The most common sion (1), and ileus (1). All SAEs were not related to the study drugs. One pa- grade 1-2 treatment-related adverse events within the DLT period in- tient (1/6) with microsatellite stable (MSS) metastatic CRC achieved partial cluded acneiform rash, hypomagnesemia, decreased lymphocyte response. Biomarker data will be presented at the meeting. count, anemia, nausea, vomiting, hypothyroidism, fatigue, cough, oral Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 208 of 272 mucositis, and elevated AST. No dose modifications were required. CD73 levels will derive greater benefit from CD73 inhibition. Immu- Five of the 6 subjects (83%) had disease control (1 unconfirmed par- nohistochemistry analyses were performed on serial sections of tial response, 4 stable disease) at 12 weeks. tumor tissue to correlate protein and gene expression. Conclusions Results The combination of panitumumab, ipilimumab, and nivolumab was The HV study enrolled more than 50 participants, randomized 3:1 (ac- well-tolerated, without unexpected toxicities encountered in the tive: placebo). AB680 exhibited a good safety profile and displayed a safety run-in cohort. The response rate and disease control rate dem- long half-life following a 30-60 minute intravenous (IV) infusion, con- onstrate early signs of clinical activity. Enrollment in the phase II ex- sistent with the intended Q2W dosing schedule in cancer patients. pansion is ongoing. Doses were identified that provided maximal inhibition of peripheral Trial Registration AMP-ase activity. Our bioinformatics analyses identified tumors that ClinicalTrials.gov Identifier: NCT03442569 have high CD73 expression relative to TNAP and identified pan-RAS mutations that correlate with upregulated CD73 and poor prognosis. References Conclusions 1. Garrido G, Rabasa A, Sanchez B, et al. Induction of immunogenic AB680 is the first potent and selective small-molecule CD73 inhibitor apoptosis by blockade of epidermal growth factor receptor activation to be tested in humans. This first-in-human study demonstrates that with a specific antibody. J Immunol 2011;187:4954-66. AB680 is well tolerated and has optimal PK/PD to support its contin- 2. Pozzi C, Cuomo A, Spadoni I, et al. The EGFR-specific antibody cetuximab ued evaluation in cancer patients. combined with chemotherapy triggers immunogenic cell death. Nat Trial Registration Med 2016;22:624-31. ClinicalTrials.gov NCT03677973 3. Garrido G, Lorenzano P, Sanchez B, et al. T cells are crucial for the anti- Ethics Approval metastatic effect of anti-epidermal growth factor receptor antibodies. The study was approved by Bellberry Limited Ethics Board, approval Cancer Immunol Immunother 2007;56:1701-10. number 2018-08-673 4. Jie HB, Schuler PJ, Lee SC, et al. CTLA-4(+) Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cyto- toxicity and Correlate with Poor Prognosis. Cancer Res 2015;75:2200-10. P380 5. Pander J, Heusinkveld M, van der Straaten T, et al. Activation of tumor- Phase 1b/2 study of BXCL701, a small molecule inhibitor of promoting type 2 macrophages by EGFR-targeting antibody cetuximab. dipeptidyl peptidases, with bempegaldesleukin (bempeg, NKTR- Clin Cancer Res 2011;17:5668-73. 214) and avelumab (anti-PD-L1) in unresectable or metastatic Ethics Approval pancreatic cancer 1 1 2 3 The study was approved by the Institutional Review Board of the University of North Louis Weiner, MD , Benjamin Weinberg, MD , Stina Singel , Cedric Burg , 3 2 3 Carolina at Chapel Hill (IRB number 17-1832) and by the IRB of each subsite. Diane Healey , Jonathan Zalevsky, PhD , Chetan Lathia , Willem 2 4 2 Overwijk, PhD , Cristian Massacesi , Joyce Acbay , John MacDougall, 3 3 PhD , Vincent O'Neill P379 Georgetown Lombardi Comprehensive Cancer Center, Washington, DC, Phase 1 safety study in healthy volunteers of AB680, a small- United States; Nektar Therapeutics, San Francisco, CA, United States; 3 4 molecule inhibitor of CD73 and rationale for combination therapy BioXcel Therapeutics, New Haven, CT, United States; Pfizer, New York, in patients with gastrointestinal malignancies NY, United States Devika Ashok, PhD, Irene Luu, Akshata Udyavar, PhD, Lixia Jin, Lijuan Fu, Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) Elaine Ginn, Ken Lawson, Jenna Jeffrey, PhD, Manmohan Leleti, PhD, Jay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P380 Powers, PhD, Eric Connor, Andy Pennell, Daniel DiRenzo, PhD, Dana Piovesan, MSc, Joanne Tan, PhD, Amanda Garofalo, Wade Berry, BA, Background Matthew Walters, PhD, Steve Young, PhD, Fangfang Yin, PhD, Dominic Treatment of pancreatic cancer continues to have poor outcomes with Lai, Lisa Seitz, MA currently available therapies including checkpoint inhibitors. BXCL701 Arcus Biosciences, Hayward, CA, United States (talabostat, previously PT100) is an orally administered, small molecule Correspondence: Dominic Lai (dlai@arcusbio.com) inhibitor of dipeptidyl peptidases (DPP) specifically DPP4, DPP8 and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P379 DPP9. Inhibition of DPP8 and DPP9 triggers a process in macrophages called pyroptosis leading to innate immune proinflammatory stimula- Background tion of the tumor microenvironment[1,2,3]. BXCL701 also inhibits fibro- Extracellular adenosine, present at high concentrations in the tumor blast activation protein (FAP) releasing the FAP-mediated block of T-cell microenvironment (TME), suppresses immune function. The enzymes migration into the tumor[4]. In syngeneic animal models, significant ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phos- tumor responses were observed when BXCL701 was used with check- phatase (TNAP) catalyze extracellular conversion of adenosine mono- point inhibition[2]. Bempegaldesleukin (bempeg, NKTR-214) is a CD122- phosphate (AMP) into adenosine. Inhibition of CD73 eliminates a preferential interleukin-2 (IL-2) pathway agonist being investigated for major pathway of adenosine production in the TME and can reverse its potential to leverage the clinically validated IL-2 pathway and select- adenosine‐mediated immune suppression. Here we present the first ively stimulate an immune response, without overacting the immune results from a Phase 1 healthy volunteer (HV) study of AB680, a po- system. Bempeg has demonstrated robust anti-cancer activity when tent, reversible and selective small-molecule CD73 inhibitor. This used with checkpoint inhibition in multiple murine tumor models and placebo-controlled HV study assessed the safety, tolerability, pharma- recently in multiple human cancers[5,6]. Avelumab is a checkpoint in- cokinetic (PK) and pharmacodynamic (PD) profile of AB680. hibitor that binds PD-L1 resulting in the release of immune inhibitory Methods effects of this pathway thereby restoring immune responses, including Male or female healthy volunteers aged 18-55 with a body mass anti-cancer immune responses. In a syngeneic mouse model of pancre- index of 18-30 kg/m2 were eligible for enrollment in the atic cancer (Pan02), the triple combination demonstrated potent anti- AB680CSP0001 study (NCT03677973). Escalating doses of AB680 were cancer activity, including long-lasting anti-cancer immunity[7]. These re- evaluated in a single ascending dose (SAD) and repeat dosing study. sults provide therapeutic rationale for testing of this combination in pa- Post dosing, participants were admitted for evaluation and serially tients with pancreatic cancer. assessed for adverse events. Blood samples were collected at various Methods timepoints to elucidate the PK and PD profiles of AB680. AB680 This is an open-label, multicenter study to determine the safety and effi- plasma concentrations were determined using LC-MS/MS and PD ef- cacy of the triple combination therapy of BXCL701, bempeg and avelu- fects were evaluated by monitoring AMP-ase activity in serum. Linear mab. Patients with pancreatic cancer should have received at least 1 line models were used to predict tumor up-regulation of CD73 in mul- of gemcitabine-based therapy and no more than 2 lines of chemother- tiple tumor types with the assumption that tumors expressing higher apy for unresectable or metastatic disease, received no prior anti-PD-1/ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 209 of 272 PD-L1, IL-2 based or other T-cell directed anti-cancer therapy, and have observed when BXCL701 was used in combination with checkpoint in- ECOG 0-1. Patients must agree to biopsy of metastatic disease. Part 1 hibition[2]. Therefore, it is believed BXCL701 mediated activation of the (Phase 1b), is the 3+3 dose escalation phase designed to evaluate the innate immune system via macrophage pyroptosis inflames the cancer safety of escalating doses of BXCL701 with bempeg and avelumab. Part microenvironment and t-SCNC might become responsive to checkpoint 2 (Phase 2) will begin once the recommended combination dose is de- inhibition combined with BXCL701. termined. A Simon two-stage design will be used in Phase 2, initially en- Methods rolling 13 patients. If 2 or more responses are observed, the cohort will This is an open-label, multicenter study in patients with progressive, expand to 34 patients. The primary efficacy parameter is objective re- metastatic castration resistant prostate cancer (CRPC) as defined by sponse by RECIST 1.1. The study will also assess other parameters meas- PCWG3. Patients should have received at least 1 line of systemic uring clinical benefit and mechanistic effects on the immune system therapy and no more than 2 lines of cytotoxic chemotherapy for and tumor microenvironment. The study is not yet recruiting in the US. CRPC, received no prior anti-PD-1/PD-L1 or other T-cell directed anti- Trial Registration cancer therapy, and have ECOG 0-2. Patients in Phase 2 must also Pending have evidence of SCNC,NEPC by central pathology and agree to bi- opsy of metastatic disease. Phase 1b, is the 3+3 dose escalation References phase designed to evaluate the safety of 0.4 mg and 0.6mg BXCL701 1. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP QD on days 1 to 14 of 21-day cycle plus fixed dose pembrolizumab inhibitor, and immune checkpoint inhibitors discovered using AI and Big 200 mg administered IV on day 1 every 21 days to determine the Data analytics. Cancer Research 2017;77(13 Suppl):Abstract nr 2629. recommended dose for phase 2. A Simon’s two-stage design will be 2. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- used in Phase 2 and initially 15 patients with SCNC/NEPC will be en- caspase-1-dependent monocyte and macrophage pyroptosis. Nature rolled. If more than 2 responses are observed, then the cohort will Chemical Biology. 2017;13(1):46-53 expand to 28 patients. Primary efficacy parameter is the composite 3. Okondo M, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the response defined as achieving 1 or more of the following: • Objective Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 response by RECIST 1.1 • CTC conversion from > 5/7.5 mL to < 5/7.5 4. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- mL per Veridex assay by Week 12 • Greater than 50% PSA decline rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. from baseline by Week 12. The study is open in the US with expan- 2015;75(14):2800-2810. sion to the UK underway. 5. Charych D, Khalili S, Dixit V et al. Modeling the receptor pharmacology, Trial Registration pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically- NCT03910660 controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. EUDRACT:2018-003734-32 PLOS ONE 2017; 12(7): e0179431. 6. Diab A, Tannir N, Bernatchez C, et al. A phase ½ study of a novel IL-2 References cytokine, NKTR-214, and nivolumab in patients with select locally ad- 1. Aggarwal R, Huang J, Alumkal JJ, et al. Clinical and Genomic vanced or metastatic solid tumors. J Clin Oncol. 2017;35(suppl):e14040. Characterization of Treatment-Emergent Small-Cell Neuroendocrine Pros- 7. Rastelli L, Gupta S, Jagga Z, et al. Efficacy and immune modulation by tate Cancer: A Multi-institutional Prospective Study. J Clin Oncol. 2018; BXCL701 and dipeptidyl peptidase inhibitor, NKTR-214 a CD122-biased 36(24): 2492-2505 immune agonist with PD1 blockage in murine pancreatic tumors [ab- 2. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP stract]. J Clin Oncol. 2018;36(suppl):3085 inhibitor, and immune checkpoint inhibitors discovered using AI and Big Ethics Approval Data analytics [abstract]. In: Proceedings of the American Association for This study was approved by Institution Review Boards or Ethics Committees Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. affiliated with participating institutions Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2629. 3. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- caspase-1-dependent monocyte and macrophage pyroptosis. Nature P381 Chemical Biology. 2017;13(1):46-53 Phase 1b/2 study of BXCL701, a small molecule inhibitor of 4. OkondoM, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the dipeptidyl peptidases (DPP), with pembrolizumab, (anti-PD-1) Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 monoclonal antibody, in small cell neuroendocrine prostate cancer 5. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- (SCNC, NEPC) rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. 1 2 2 Diane Healey , Rahul Aggarwal, MD , Rahul Aggarwal, MD , Vincent 2015;75(14):2800-2810. 1 1 1 3 O'Neill , Cedric Burg , Diane Healey , Jiaoti Huang , Johann De Bono, 6. cBioPortal version 1.4.3 (dataset accessed on 9th March, 2017) 4 2 MD , Eric Small Ethics Approval 1 2 BioXcel Therapeutics, New Haven, CT, United States; UCSF Helen Diller The study was approved by Institution Review Boards or Ethics Committees Family Comprehensive Cancer Center, San Francisco, CA, United States; affiliated with participating institutions. Duke University School of Medicine, Durham, NC, United States; Institute for Cancer Research Royal Marsden NHS Foundation Trust, London, United Kingdom P382 Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) KEYNOTE-365 cohort D: phase 1b/2 study of pembrolizumab plus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P381 abiraterone acetate and prednisone in metastatic castration- resistant prostate cancer 1 2 3 Background Leonard Appleman, MD, PhD , Josep Piulats , Nataliya Mar , José 4 5 6 7 Treatment emergent Small Cell Neuroendocrine Prostate Cancer (t-SCNC) Arranz , Anthony Joshua, MD , Tina Mayer , Neal Shore, MD , Haiyan Wu, 8 8 9 is an aggressive with poor survival outcomes on standard therapies given PhD , Charles Schloss , Evan Yu 1 2 for metastatic castration-resistant disease[1 ]. BXCL701 (talabostat previ- UPMC Hillman Cancer Center, Pittsburgh, PA, United States; Catalan ously PT100) is an orally administered, small molecule inhibitor of dipepti- Cancer Institute, Barcelona, Spain; UC Irvine Medical Center, Orange, CA, dyl peptidases (DPP) specifically DPP4, DPP8 and DPP9 triggering Uunited States; Hospital General Universitario Gregorio Marañon, macrophage cell death via pyroptosis resulting in proinflammatory stimu- Madrid, Spain; St. Vincent’s Hospital Sydney, Sydney, NSW, Australia; lation of the innate immunity pathway[2,3,4]. BXCL701 also inhibits fibro- Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United blast activation protein (FAP) releasing the FAP-mediated block of T-cell States; Carolina Urologic Research Center, Myrtle Beach, SC,United 8 9 migration into the tumor[5]. FAP, DPP8 and DPP9 are expressed and acti- States; Merck & Co. Inc., Kenilworth, NJ, United States; University of vated in neuroendocrine CRPC[6]. Correlation is robust between the ex- Washington, Seattle, WA, United States pression of PD-L1 and the targets of BXCL701, particularly FAP, DPP8 and Correspondence: Leonard Appleman (applemanlj@upmc.edu) DPP9[2]. In syngeneic animal models, significant tumor responses were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P382 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 210 of 272 Background Background For patients with metastatic castration-resistant prostate cancer HPN424 is a PSMA-targeting T cell engager derived from the TriTAC (mCRPC), additional therapeutic options are needed to improve over- platform (Tri-specific T Cell-Activating Construct). PSMA is a well- all outcomes and to delay the use of chemotherapy. In early-phase validated antigen specific to prostate epithelial cells upregulated trials, pembrolizumab, an anti–PD-1 antibody, showed some activity upon malignant prostate cancer with limited expression in normal as monotherapy in heavily pretreated patients with mCRPC. Cohort D tissues. HPN424 is a recombinant polypeptide of ~50kDa containing of the nonrandomized, multicohort, open-label, phase 1b/2 three humanized antibody-derived binding domains, targeting PSMA KEYNOTE-365 (NCT02861573) study has been designed to evaluate (for tumor binding), albumin (for half-life extension) and CD3 (for T the safety and efficacy of pembrolizumab combined with abiraterone cell engagement). It has been engineered to be a small, globular pro- acetate and prednisone in patients with mCRPC who have not re- tein to enable efficient exposure in solid tumor tissue with prolonged ceived chemotherapy for mCRPC. half-life and excellent stability under physiological conditions. Methods HPN424 binds monomerically to CD3 and PSMA, minimizing non- Adults (≥18 years) with histologically or cytologically confirmed pros- specific T-cell activation. These features are designed to increase the tate cancer, without small cell histology, and who experience pro- therapeutic index compared to earlier generations of T cell engagers gression ≤6 months before screening and have an ECOG PS score of by minimizing off-target toxicities. HPN424 mediates potent target 0 or 1 are eligible. Patients must be chemotherapy naïve for mCRPC tumor cell killing in a PSMA-specific manner in vitro and in xenograft and must not have received second-generation hormonal therapy for models in the presence of T cells, demonstrated at very low antigen mCRPC or must not have experienced failed treatment with enzaluta- densities. Consistent with its mechanism of action (MOA), tumor cell mide or become intolerant to enzalutamide for mCRPC. Patients will killing is accompanied by T cell activation, cytokine induction, and T receive pembrolizumab 200 mg IV every 3 weeks, abiraterone acet- cell expansion. ate 1000 mg once daily, and prednisone 5 mg twice daily. Responses Methods will be radiographically assessed every 9 weeks during year 1 and This is a Phase 1, open-label, multicenter, dose escalation and dose every 12 weeks thereafter. Pembrolizumab treatment will continue expansion study to evaluate the safety, tolerability, clinical activity, for up to 35 cycles (approximately 2 years) or until disease progres- and pharmacokinetics of HPN424 in adult patients with meta- sion, unacceptable toxicity, or patient/physician decision to withdraw. static castrate-resistant prostate cancer (mCRPC) who have pro- Patients who discontinue 1 of the 2 drugs in the combination be- gressed on the prior regimen (per PCWG3 criteria) and received cause of drug-related adverse events can continue with the other at least 2 prior systemic therapies approved for mCRPC. HPN424 combination partner. All patients who discontinue treatment will be is administered once weekly as one-hour IV infusion by single- monitored until trial completion. Primary end points are prostate- patient cohorts until either a Grade ≥2 adverse event (AE) that is specific antigen (PSA) response rate, defined as a PSA decrease of possibly related to HPN424 is observed or an estimated thera- ≥50% from baseline measured on 2 occasions at least 3 weeks apart peutic dose level has been reached. Then a conventional 3+3 de- for confirmation, objective response rate (ORR) per RECIST v1.1 by sign is implemented. Dose escalation will continue until a blinded independent central review (BICR), and safety. Secondary recommended phase 2 dose (RP2D) is determined. In dose ex- end points include time to PSA progression, ORR based on Prostate pansion, up to 18 patients receive HPN424 at the established Cancer Working Group 3 (PCWG3)–modified RECIST v1.1 assessed by RP2D. Additional expansion cohorts may be added. Patients may BICR, duration of response based on RECIST 1.1 and PCWG3-modified continue weekly HPN424 treatment as long as they are receiving RECIST 1.1 assessed by BICR, radiographic progression-free survival clinical benefit. Primary endpoints are number and severity of based on PCWG3-modified RECIST 1.1 assessed by BICR, and overall DLTs following treatment with escalating doses of HPN424 during survival. Recruitment began in December 2018 and will continue escalation, and overall response rate (per PCWG3 criteria) in dose until ~100 patients are enrolled. expansion. Secondary endpoints include AEs, preliminary anti- Ethics Approval tumor activity, pharmacokinetic and pharmacodynamic parame- The study and the protocol were approved by the Institutional Re- ters based on the proposed MOA of HPN424. view Board or ethics committee at each site. Trial Registration Consent NCT03577028 All patients provided written informed consent to participate in the Ethics Approval clinical trial. This study was approved by each participating institution's Institu- tional Review Board. P383 Withdrawn P385 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P383 Pembrolizumab plus enzalutamide versus placebo plus enzalutamide for metastatic castration-resistant prostate cancer: phase 3 KEYNOTE-641 study 1 2 3 4 Julie Nicole Graff , Joseph Burgents , Li Wen Liang , Arnulf Stenzl P384 Knight Cancer Institute, Oregon Health & Science University, Portland, A phase I dose escalation and expansion study of HPN424, a OR, United States; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, 3 4 PSMA-targeting T cell engager, in patients with advanced prostate United States; MSD, China, Beijing, China, Beijing, China; University of cancer refractory to androgen therapy Tuebingen Medical School, Tuebingen, Germany, Tübingen, Germany 1 2 3 Johanna Bendell, MD , Mark Stein, MD , Johann de Bono, MD , Richard Correspondence: Julie Nicole Graff (graffj@ohsu.edu) 4 4 4 Austin, PhD , Sue Hirabayashi , Che-Leung Law, PhD , Bryan Lemon, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P385 4 4 5 PhD , Holger Wesche, PhD , Aaron Weitzman, MD FACP , Lawrence Fong, MD Background Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, Treatment options for patients with metastatic castration-resistant 2 3 United States; Columbia University, New York, NY, United States; Royal prostate cancer (mCRPC) are noncurative, and life expectancy is only Marsden Hospital and The Institute of Cancer Research, Sutton, United about 3 years. Enzalutamide is an androgen receptor inhibitor used Kingdom; Harpoon Therapeutics, South San Francisco, CA, United for the treatment of patients with mCRPC. Pembrolizumab is a pro- States; Weitzman Consulting Group, Los Altos Hills, CA, United States; grammed death 1 (PD-1) inhibitor with antitumor activity as mono- University of California, San Francisco, San Francisco, CA, United States therapy in mCRPC. Results of clinical studies have shown that the Correspondence: Johanna Bendell (jlee@samornbiosciences.com) mechanisms of action of pembrolizumab and enzalutamide may be Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P384 synergistic. In the phase 1b/2 KEYNOTE-365 (NCT02861573) study, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 211 of 272 antitumor activity of pembrolizumab plus enzalutamide was ob- [1]. To further assess the economic impact of NIVO+IPI associated served in mCRPC patients pretreated with abiraterone acetate. Also, with TFS, this study compared healthcare costs among untreated in a single-arm, phase 2 study (NCT02312557) of patients who pro- intermediate/poor-risk aRCC patients with different lengths of TFS. gressed on enzalutamide, some patients had profound anticancer re- Methods sponse when pembrolizumab was added to enzalutamide that lasted This study used individual patient data from the NIVO+IPI arm in Check- years. KEYNOTE-641 (NCT03834493) is a randomized, double-blind, Mate 214 (database lock, August 6, 2018; minimum follow-up, 30 phase 3 trial to evaluate efficacy and safety of pembrolizumab plus months). TFS is defined as the time from last dose of NIVO+IPI to the start enzalutamide versus placebo plus enzalutamide for patients with of subsequent systemic therapy or death, whichever occurs first. All inter- mCRPC. mediate/poor-risk aRCC patients who received NIVO+IPI and provided Methods consent were classified into 3 cohorts based on the length of TFS: cohort Approximately 1200 patients will be randomly assigned 1:1 to receive 1 remained on NIVO monotherapy maintenance; cohort 2 had TFS ≤6 enzalutamide 160 mg/day plus pembrolizumab 200 mg Q3W or months; and cohort 3 had TFS >6 months. Patient characteristics and enzalutamide 160 mg/day plus placebo. Treatment will be stratified overall survival from randomization were described for the 3 cohorts. per prior abiraterone acetate treatment (yes/no), metastases (bone Monthly costs from randomization to last known date alive, including only/liver/other), and prior docetaxel treatment for metastatic study treatment costs, all-cause grade 3/4 adverse event costs, terminal hormone-sensitive prostate cancer (yes/no). Adults (≥18 years) with care costs, and subsequent treatment costs were compared between co- histologically or cytologically confirmed prostate cancer and mCRPC hort 3 versus cohort 1 and cohort 3 versus cohort 2 using Wilcoxon rank- who experienced biochemical or radiographic progression are eli- sum tests. All costs were adjusted to 2019 United States dollars. gible. Patients who received chemotherapy for mCRPC, checkpoint Results inhibition, or any treatment with a second-generation androgen re- Of the 420 eligible patients, 16.4% (N=69) remained on NIVO mono- ceptor inhibitor (eg, enzalutamide, apalutamide, or darolutamide) are therapy maintenance, 60.2% (N=253) had TFS ≤6 months, and 23.3% excluded. Patients intolerant of or experiencing progression with (N=98) had TFS >6 months by the end of patient follow-up. Patient prior abiraterone acetate therapy are included. Patients must have characteristics were mostly similar between cohort 3 versus cohort 1 or ECOG PS 0/1, adequate organ function, and tissue for biomarker ana- 2. By definition, all patients (100%) in cohort 1 were alive at the end of lysis. Responses will be assessed by CT/MRI and radionuclide bone follow-up. The survival probabilities were 94% for cohort 3 and 60% for imaging per PCWG-modified RECIST v1.1 every 9 weeks during the cohort 2 by 18 months and 83% for cohort 3 and 39% for cohort 2 by first year and every 12 weeks thereafter. Treatment will continue with 30 months. Patients with TFS >6 months had significantly lower enzalutamide plus pembrolizumab/placebo until radiographic disease monthly costs ($8,318) compared with those who never discontinued progression, unacceptable toxicity, or consent withdrawal, with a NIVO+IPI ($16,374) and those with TFS ≤6 months ($22,811) (Figure 1). maximum of 2 years of treatment for the pembrolizumab/placebo Conclusions component of the combination. Dual primary end points are overall This retrospective healthcare cost assessment of 3 patterns of patient survival and radiographic progression-free survival by blinded inde- outcomes during and after treatment with NIVO+IPI suggests an eco- pendent central review. The key secondary efficacy end point is time nomic value of achieving prolonged TFS (>6 months). Thus, manage- to subsequent anticancer therapy or death. Additional secondary ment strategies that would lead to prolonged TFS could be end points include objective response rate, duration of response, beneficial both clinically and economically. prostate specific antigen (PSA) response rate, PSA-undetectable rate, time to PSA progression, time to pain progression, and time to radio- Acknowledgements graphic soft tissue progression. Safety and tolerability will also be Writing support was provided by Analysis Group, Inc. and editorial support reported. was provided by Parexel, funded by Bristol-Myers Squibb. Trial Registration Trial Registration ClinicalTrials.gov; NCT03834493 NCT02231749. Ethics Approval The study and the protocol were approved by the Institutional Re- Reference view Board or ethics committee at each site. 1. McDermott DF, Rini BI, Motzer RJ, Tannir NM, Escudier B, Consent Kollmannsberger CK, Hammers HJ, Porta C, George S, Donskov F, Gurney All patients provided written informed consent to participate in the HP. 874P Treatment-free interval (TFI) following discontinuation of first- clinical trial. line nivolumab plus ipilimumab (N+ I) or sunitinib (S) in patients (Pts) with advanced renal cell carcinoma (aRCC): CheckMate 214 analysis. Ann Oncol. 2018; 29(suppl 8):mdy283.083. P386 Ethics Approval Economic benefits associated with treatment-free survival of This trial was approved by the institutional review board or ethics committee immuno-oncology agents among untreated patients with at each site. intermediate/poor-risk advanced or metastatic renal cell carcinoma 1 2 3 Michael Harrison, MD , Meredith Regan, PhD , Michael Atkins, MD , 4 4 4 Sumati Rao, PhD , Shuo Yang, PhD , Jennifer Johansen, PharmD, BCPS , 5 5 5 5 Ella Du, MESc , Chenyang Gu , Ela Fadli , Keith Betts, PhD , David McDermott, MD 1 2 Duke Cancer Institute, Chapel Hill, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Georgetown Lombardi Comprehensive Cancer, Washington, DC, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Analysis Group, Inc, Los Angeles, CA, United States; Beth Israel Deaconess Medical Center, Milton, MA, United States Correspondence: Michael Harrison (Michael.Harrison@Duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P386 Background Nivolumab plus ipilimumab (NIVO+IPI) was associated with signifi- cantly longer treatment-free survival (TFS) compared with sunitinib in intermediate/poor-risk patients with previously untreated advanced Fig. 1 (abstract P386). See text for description or metastatic renal cell carcinoma (aRCC) in the CheckMate 214 trial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 212 of 272 P387 Trial Registration A multicenter, open-label, exploratory platform study to evaluate https://clinicaltrials.gov/ct2/show/NCT03835533 biomarkers and immunotherapy combinations for the treatment of patients with metastatic castration-resistant prostate cancer References (PORTER) 1. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin 1 2 3 Leo Nissola, MD , Karen Autio, MS, MD , Nina Bhardwaj, MD, PhD , AV, et al. Signatures of mutational processes in human cancer. Nature 3 4 5 Matthew Galsky, MD , Kristopher Wentzel, MD , Vanessa Lucey , Cheryl 2013;500(7463):415–21. Beer TM, Kwon ED, Drake CG, Fizazi K, Logothetis 1 1 1 Selinsky, PhD , Christopher Perry , Christopher Cabanski, PhD , Ari C, Gravis G, et al. Randomized, Double-Blind, Phase III Trial of Ipilimumab 1 1 6 7 Bitton , Justin Fairchild , Christine Horak, PhD , Jeffrey Skolnik, MD , Versus Placebo in Asymptomatic or Minimally Symptomatic Patients With 8 6 1 Michael Yellin, MD , Ute Dugan, MD, PhD , Ramy Ibrahim, MD , Metastatic Chemotherapy-Naive Castration-Resistant Prostate Cancer. J Lawrence Fong, MD Clin Oncol 2017;35(1):40–7. Parker Institute for Cancer Immunotherapy, San Francisco, CA, United 2. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, 2 3 States; Memorial Sloan Kettering Cancer Center, New York, NY; The et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) Mount Sinai Hospital, New York, NY, United States; The Angeles Clinic & in refractory solid tumors: safety, clinical activity, pharmacodynamics, and Research Institute, Los Angeles, CA, United States; Cancer Research immunologic correlates. J Clin Oncol 2010;28(19):3167–75. Institute, New York, NY, United States; Bristol-Myers Squibb, 3. Di Lorenzo G, Buonerba C, Kantoff PW. Immunotherapy for the treatment Lawrenceville, NJ, United States; Inovio Pharmaceuticals, Plymouth of prostate cancer. Nat Rev Clin Oncol 2011;8(9):551–61. Drake CG. Meeting, PA, United States; Celldex Therapeutics, Hampton, NJ, United Prostate cancer as a model for tumour immunotherapy. Nat Rev States; University of California San Francisco, San Francisco, CA, United Immunol States 2010;10(8):580–93. Correspondence: Leo Nissola (lnissola@parkerici.org) 4. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P387 et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. Background 5. Flammiger A, Bayer F, Cirugeda-Kühnert A, Huland H, Tennstedt P, Simon Metastatic castration resistant prostate cancer (mCRPC), the lethal R, et al. Intratumoral T but not B lymphocytes are related to clinical out- form of prostate cancer, has shown limited benefit from immune come in prostate cancer. APMIS 2012;120(11):901–8. checkpoint inhibition as monotherapy, with two randomized phase 3 6. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. trials with ipilimumab failing to show a survival benefit, and a large VISTA is an inhibitory immune checkpoint that is increased after phase 2 trial with pembrolizumab demonstrating an overall response ipilimumab therapy in patients with prostate cancer. Nat Med rate (ORR) of 3-5%. Clearly novel combinations are needed and a 2017;23(5):551–5. deeper understanding of immune resistance. 7. Graff JN, Alumkal JJ, Drake CG, Thomas GV, Redmond WL, Farhad M, Using a multi-arm, multi-stage platform design, the PORTER study et al. Early evidence of anti-PD-1 activity in enzalutamide-resistant pros- will adaptively test multiple immunotherapeutic combinations to ac- tate cancer. Oncotarget 2016;7(33):52810–7. Kantoff PW, Higano CS, tivate the innate and adaptive immune systems. Coupled with deep Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel- immune biomarker profiling, this design will enable rapid insights Timmunotherapy for castration-resistant prostate cancer. N Engl J Med into the immune responses for each combination, providing data for 2010;363(5):411–22. potential larger definitive trials, while generating hypotheses for new 8. Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den Eertwegh AJM, cohorts. et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after Methods docetaxel chemotherapy (CA184-043): a multicentre, randomised, PORTER is an open-label, non-randomized, exploratory platform double-blind, phase 3 trial. Lancet Oncol 2014;15(7):700–12. study designed to assess the safety and antitumor activity of multiple 9. Lee P, Gujar S. Potentiating prostate cancer immunotherapy with immunotherapy combinations in participants with mCRPC who have oncolytic viruses. Nat Rev Urol 2018;15(4):235–50. received prior secondary androgen inhibition therapy. Each cohort 10. Lopez-Bujanda Z, Drake CG. Myeloid-derived cells in prostate cancer pro- has a two-stage design (initial n = 15, expansion n = 15) with a deci- gression: phenotype and prospective therapies. J Leukoc Biol sion to expand based on the safety, clinical activity, and biomarker 2017;102(2):393–406. results observed in the initial stage. 11. Martin AM, Nirschl TR, Nirschl CJ, Francica BJ, Kochel CM, van Cohort A is open and recruiting, testing the combination of bempe- Bokhoven A, et al. Paucity of PD-L1 expression in prostate cancer: In- galdesleukin (“BEMPEG”, NKTR-214; a CD-122 preferential IL-2 path- nate and adaptive immune resistance. Prostate Cancer Prostatic Dis way agonist) with nivolumab (PD-1 inhibitor), postulating that this 2015;18(4):325–32. will increase PD-L1 expression, intratumoral T and NK cells, and in- Ethics Approval duce an IFN gamma signature. The study was approved by WIRB‘s Ethics Board, IRB Tracking Number: Cohort B will combine CDX-301 (Flt3L), poly-ICLC (PAMP-adjuvant), nivolumab and stereotactic body radiation therapy, in 1-5 metastatic sites, inducing immunogenic cell death, mobilizing and activating dendritic cells increasing tumor antigen presentation, and overcom- P388 ing adaptive immune resistance in mCRPC. Pembrolizumab plus docetaxel and prednisone for enzalutamide- Cohort C will evaluate INO-5151, a DNA vaccine encoding PSA, PSMA, or abiraterone acetate–pretreated patients with metastatic and IL-12 delivered via intramuscular electroporation, in addition to castration-resistant prostate cancer: phase 3 KEYNOTE-921 study 1 2 3 CDX-301 and nivolumab. This is a multi-pronged approach to Neal Shore, MD , Daniel Petrylak, MD , Mostefa Bennamoun , Raffaele 4 5 6 6 7 mobilize and activate dendritic cells, stimulate anti-tumor CD8 T cells, Ratta , Josep Piulats , Ben Li , Charles Schloss , Karim Fizazi and circumvent adaptive immune resistance. Carolina Urologic Research Center, Myrtle Beach, SC, USA, Myrtle Beach, Inclusion criteria include: histologically-confirmed mCRPC that is SC, United States; Smilow Cancer Hospital at Yale University, New measurable or non-measurable by Prostate Cancer Clinical Trials Haven, CT, USA, New Haven, CT, United States; Institut Mutualiste Working Group 3 and progressing despite secondary androgen re- Montsouris, Paris, France, Paris, France; Hopital Foch, Suresnes, France, ceptor signaling inhibitor therapy. Suresnes, France; Catalan Cancer Institute, Barcelona, Spain, Barcelona, The primary endpoint: safety, as assessed by the incidence and se- Spain; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United verity of adverse events. Secondary endpoints: Composite ORR (PSA States; Gustave Roussy, Villejuif, France, Villejuif, France reduction >50%, confirmed CR or PR per RECIST 1.1, or change in cir- Correspondence: Neal Shore (nshore@gsuro.com) culating tumor cell (CTC) from >5 cells/7.5 ml to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P388 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 213 of 272 Background (HD) IL-2 regimen has demonstrated superior overall response rate Docetaxel is an established treatment for patients with metastatic (ORR), depth and durability of response versus lower dose alterna- castration-resistant prostate cancer (mCRPC). Pembrolizumab is a tives. Resistance mechanisms exploited by tumors may play a domin- programmed death 1 inhibitor that was found to have antitumor ac- ant role in limiting the effectiveness of T-cell mediated cancer tivity as monotherapy in mCRPC. In the phase 1b/2 KEYNOTE-365 therapies. The PD-1/PD-L1 interaction is a major pathway hijacked by study (NCT02861573), docetaxel plus pembrolizumab and prednisone RCC tumors to suppress immune control. Antibody-mediated PD1 had activity in patients treated with abiraterone acetate or enzaluta- blockade with pembro results in spontaneous and durable regres- mide for mCRPC, warranting further evaluation of this treatment sions for a subset of RCC tumors (SS Tykodi et al., ASCO 2019, ab- combination. KEYNOTE-921 (NCT03834506) is a randomized phase 3 stract #4570). PD1 blockade has entered clinical practice for trial to evaluate the efficacy and safety of pembrolizumab plus doce- advanced RCC as both a front-line and salvage therapy option. A fa- taxel and prednisone in chemotherapy-naïve patients who were pre- vorable safety profile for PD1 blockade has encouraged exploration viously treated with enzalutamide or abiraterone acetate for mCRPC of novel immuno-oncology combinations. and experienced progression while on therapy. Methods Methods Methods: This is an investigator-initiated, phase I trial of IL-2 plus Approximately 1000 patients will be randomly assigned 1:1 to receive pembro in patients with advanced, clear cell RCC. The study will use docetaxel 75 mg/m2 every 3 weeks (Q3W) plus prednisone/prednis- a 3 + 3 trial design to test three IL-2 dose levels in combination with olone 5 mg twice daily (BID) and pembrolizumab 200 mg Q3W or do- pembro given every 3-weeks at 200 mg flat dosing. Cohorts will re- cetaxel 75 mg/m2 Q3W plus prednisone/prednisolone 5 mg BID plus ceive subcutaneous IL-2 given once daily, 5 days per week for 6 placebo Q3W. Treatment will be stratified per previous treatment with weeks (250,000 U/kg week 1, 125,000 U/kg weeks 2-6); or IV bolus a next-generation hormonal agent (abiraterone acetate or enzaluta- dosing at 72,000 U/kg or 600,000 U/kg (HD IL-2) every 8 hours to a mide) and metastases (bone only, liver, other). Adult (≥18 years) maximum of 14 doses on week 1 and 4 of a 12-week treatment patients with chemotherapy-naïve histologically or cytologically con- course. Patients with stable or responding disease and without firmed mCRPC who experienced progression while receiving androgen treatment-limiting toxicity can receive up to 3 courses of therapy. deprivation therapy (or postbilateral orchiectomy) within 6 months be- The HD IL-2 cohort will enroll an additional 9 patients to gain further fore screening were eligible. Patients must have experienced progres- insight into anti-tumor efficacy. The primary objective is to evaluate sion after ≥8weeks (≥14 weeks for those with bone progression) or safety and tolerability for IL-2 plus pembro. The secondary objective become intolerant after ≥4 weeks of abiraterone acetate or enzaluta- is to assess antitumor activity by RECIST 1.1 for ORR, disease control mide treatment (but not both) with androgen-deprivation therapy in rate, and progression free survival. Exploratory endpoints will include the chemotherapy-naïve mCRPC state. Patients must have ECOG PS 0 pretreatment tumor analysis for PD-L1 expression, and quantitation or 1, adequate organ function, and tissue for biomarker analysis. Re- of regulatory T cell frequency in peripheral blood and tumor sponses will be assessed by CT or MRI and radionuclide bone imaging microenvironment. perProstateCancer Working Group–modified RECIST v1.1 by blinded Trial Registration independent central review (BICR) Q9W during the first year and Q12W Trial Registration: ClinicalTrials.gov, NCT03260504 thereafter. Treatment will continue with docetaxel and prednisone for Ethics Approval up to 10 cycles and with pembrolizumab for up to 35 cycles or until Ethics Approval: This study was approved by the Fred Hutchinson radiographic disease progression, unacceptable toxicity, or consent Cancer Research Center Institutional Review Board, approval number withdrawal. Primary end points are radiographic progression-free sur- 9611. vival by BICR and overall survival. The key secondary efficacy end point is time to initiation of subsequent anticancer therapy or death. Add- P390 itional secondary end points include prostate-specific antigen response Pembrolizumab plus olaparib vs enzalutamide or abiraterone in rate (decline of ≥50% from baseline, measured on 2 occasions at least patients with metastatic castration-resistant prostate cancer who 3 weeks apart), time to PSA progression, and objective response rate experienced progression on chemotherapy: phase 3 KEYLYNK-010 and duration of response per PCWG-modified RECIST 1.1 as assessed study by BICR. Safety and tolerability will also be reported. 1 2 3 4 5 Evan Yu , Se Hoon Park , Yi-Hsiu Huang , Mostefa Bennamoun ,Lu Xu , Trial Registration 5 6 Jeri Kim , Emmanuel Antonarakis, MD ClinicalTrials.gov, NCT03834506 1 2 University of Washington, Seattle, WA, United STates; Samsung Medical Ethics Approval Center, Seoul, South Korea, Seoul, Korea, Republic of; Taipei Veterans The study and the protocol were approved by the Institutional Re- General Hospital, Taipei, Taiwan, Taipei, Taiwan, Province of China; view Board or ethics committee at each site. 4 5 Institut Mutualiste Montsouris, Paris, France, Paris, France; Merck & Co., Consent Inc., Kenilworth, NJ, USA, Kenilworth, United States; Johns Hopkins All patients provided written informed consent to participate in the University, Baltimore, MD, United States clinical trial. Correspondence: Evan Yu (evanyu@u.washington.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P390 P389 A phase I trial of Interleukin-2 (IL-2) and Pembrolizumab (Pembro) Background Combination Therapy for patients with advanced renal cell The docetaxel-pretreated, metastatic castrate-resistant prostate can- carcinoma cer (mCRPC) disease state remains an unmet need for new therapeu- Scott Tykodi, MD, PhD, Johanna Whitney, Sumia Dakhil, Eleanor Bergren, tics. The programmed death 1 (PD-1) inhibitor pembrolizumab and Vivian Nguyen, Samantha Kiriluk, Shailender Bhatia, MD, John Thompson, the polyadenosine diphosphate ribose polymerase (PARP) inhibitor ola- MD parib have some independent antitumor monotherapy activity for University of Washington, Seattle, WA, United States mCRPC. In patients with mCRPC who were unselected for homologous Correspondence: Scott Tykodi (stykodi@fredhutch.org) recombination deficiency (HRD), promising activity was seen with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P389 combination of pembrolizumab and olaparib in the phase 1b/2 KEYNOTE-365 study (NCT02861573), warranting further investigation in Background this population. KEYLYNK-010 (NCT03834519) is a randomized, open- Background: Cellular immune responses play a key role modulating label, phase 3 trial to evaluate the efficacy and safety of pembrolizu- renal cell carcinoma (RCC) progression. IL-2 (aldesleukin) is a potent mab plus olaparib in molecularly unselected enzalutamide-pretreated growth and differentiation factor for T and NK cells with anti-tumor or abiraterone acetate–pretreated patients with mCRPC whose disease activity for advanced RCC across a broad dose range. A high dose progressed on or after taxane chemotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 214 of 272 Methods adjunctive therapy after completion of standard optimal therapy. Key Approximately 780 patients will be randomly assigned 2:1 to receive eligibility criteria are a diagnosis of stage III or IV primary epithelial pembrolizumab 200 mg intravenously Q3W plus olaparib 300 mg or- ovarian cancer, successful establishment of a short-term cancer cell ally twice daily or abiraterone acetate 1000 mg orally once daily plus line, a successful leukapheresis collection of monocytes, and a Kar- prednisone/prednisolone 5 mg orally twice daily (for enzalutamide- nofsky Performance Status of 70 or greater at the time of pretreated patients) or enzalutamide 160 mg/day orally (for abirater- randomization, which takes place shortly after completion of primary one acetate–pretreated patients). Arms will be stratified per prior therapy. Tumor is collected at the time of surgery from which a treatment (abiraterone acetate/enzalutamide) and presence of meas- short-term cell line is derived. Dendritic cells are produced by incu- urable disease (yes/no). Eligible patients (≥18 years) must have histo- bating peripheral blood monocytes in the presence of GM-CSF and logically confirmed mCRPC, experienced progression while receiving IL-4. The antigen source is a lysate of irradiated tumor cells from the androgen deprivation therapy within 6 months before screening, cell culture. Six to seven months after tumor collection, after comple- previously received treatment with abiraterone acetate or enzaluta- tion of concurrent surgery and primary systemic therapy, patients are mide (but not both), and previously received treatment with chemo- stratified by whether they have detectable residual disease, and then therapy (1 prior docetaxel-based regimen). Patients must have an randomized 2:1 to receive the dendritic cell vaccine or autologous ECOG PS of 0 or 1, adequate organ function, and tumor tissue suit- monocytes. Both are admixed with GM-CSF and injected subcutane- able for biomarker analysis. Responses will be assessed by CT/MRI ously at weeks 1, 2, 3, 8, 12, 16, 20, and 24 for up to eight doses. The and radionuclide bone imaging per Prostate Cancer Working Group objective is to achieve a 50% reduction in the risk of death in the (PCWG)–modified RECIST v1.1 by blinded independent central review vaccine arm. (BICR) Q9W during the first year and Q12W thereafter. Treatment will Results continue with up to 2 years of pembrolizumab (35 cycles) and ola- Cell line success rate for submitted tumor samples is 22/22 with 1 in parib or abiraterone acetate/enzalutamide until radiographic disease progress. A satisfactory leukapheresis product has been obtained for progression, unacceptable toxicity, or consent withdrawal. Primary 14/14 patients, but was repeated for 2. 12 of a planned 99 patients end points are overall survival and radiographic progression-free sur- have been randomized. 11 have started treatment; 7 have completed vival. The key secondary efficacy end point is time to initiation of all 8 doses, 1 discontinued early for disease progression, 3 are cur- subsequent anticancer therapy. Other secondary end points are ob- rently in treatment. A total of 77 doses have been administered. No jective response rate and duration of response per PCWG-modified significant toxicity directly attributed to the vaccine has been RECIST v1.1 by BICR, time to prostate-specific antigen progression, reported. time to first symptomatic skeletal event, and safety and tolerability. Conclusions Prognostic or predictive molecular biomarkers (eg, genomic HRD sta- Although logistically complex, this patient-specific vaccine approach tus, microsatellite instability) and patient-reported outcomes will be is feasible, and has been well-tolerated. [NCT02033616] explored. Trial Registration Trial Registration ClinicalTrials.gov NCT02033616 ClinicalTrials.gov, NCT03834519 Ethics Approval Ethics Approval This study was approved by the Western Institutional Review Board The study and the protocol were approved by the Institutional Re- 20171661 view Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the P392 clinical trial. Phase 1 combination study of the CHK1 inhibitor prexasertib (LY2606368) and anti-PD-L1 antibody LY3300054, in patients with high-grade serous ovarian cancer and other advanced solid tumors P391 1 1 1 1 Khanh Do , Claire Manuszak , Sarah Kelland , Allison Powers , Adrienne Randomized phase II trial of autologous dendritic cells loaded with 1 2 1 Anderson , Alona Muzikansky , Andrew Wolanski , Mariano Severgnini, autologous tumor cell antigens from self-renewing cancer cells in 1 1 1 MSc , Geoffrey Shapiro, MD, PhD , Khanh Do, MD patients with newly diagnosed stage III or IV ovarian cancer 1 2 1 2 1 Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Lisa Abaid, MD , Richard Friedman, MD, PhD , John Brown, MD , Alberto 1 1 3 4 General Hospital, Boston, MA, United States Mendivil, MD , Tiffany Beck, MD , Bradley Corr , Leslie Randall , James 5 6 6 6 Correspondence: Khanh Do (Khanh_Do@dfci.harvard.edu) Mason , Candace Hsieh , Gabriel Nistor, MD , Robert Dillman, MD, FACP 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P392 Hoag Hospital, Newport Beach, CA, United States; Disney Family Cancer Center, Burbank, CA, United States; University of Colorado, Background Aurora, CO, United States; University of California Irvine, Orange, CA, Ovarian cancers are characterized by defects in DNA damage repair United States; Scripps Green & Memorial Hospitals, La Jolla, CA, United and high levels of replication stress, creating susceptibility to inhibition States; AiVita Biomedical, Inc., Irvine, CA, United States of checkpoint kinase 1 (CHK1). CHK1 inhibition results in intratumoral Correspondence: Robert Dillman (bob@aivitabiomedical.com) DNA damage that can drive T cell infiltration, as well as PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P391 sion. Combined CHK1 inhibition and immune checkpoint blockade therefore has the potential to enhance T cell activation against tumors. Background Methods Despite recent advances, the 5-year survival rate for patients who We conducted an open-label phase 1 study of prexasertib-mediated present with stage III or IV ovarian cancer remains less than 40%. CHK1 inhibition combined with LY3300054-mediated PD-L1 blockade Standard therapy includes surgical debulking and neoadjuvant and/ following a 3+3 design evaluating 3 administration schedules: lead-in or adjuvant combination chemotherapy, with or without bevacizu- of LY3300054 alone (Arm A), lead-in of prexasertib alone (Arm B), mab, with or without intraperitoneal therapy in certain stage III pa- and combined LY3300054 and prexasertib at outset (Arm C). Both tients, and increasingly the use of PARP inhibitors. So far advanced agents were administered on days 1 and 15 of a 28-day cycle. The ovarian cancer has been relatively refractory to anti-checkpoint ther- MTD was defined as the highest dose level at which less than one- apy, presumably because of limited host anti-tumor immune re- third of at least 6 patients experienced a DLT during C0+C1. Flow cy- sponses. Adjunctive treatment with an effective vaccine could tometry of peripheral blood mononuclear cells was performed for increase immune responses and improve survival. analysis of T cell subsets. Plasma cytokine and chemokine analyses Methods were conducted using the Luminex platform. Patients enrolled to the This randomized phase II trial was approved by Western IRB. AV- currently ongoing expansion phase of the study undergo mandatory OVA-1, patient-specific dendritic cell vaccines loaded with autologous tumor biopsies during C0 and on C1D16 after the combination. tumor antigens from self-renewing cancer cells, is administered as an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 215 of 272 Results 41-77) with ECOG 0-1 and a median of 4 prior lines of systemic treat- Fifteen patients have been treated in the dose escalation phase. The ment (ranging from 1 to 8). All 12 patients received platinum-based combination of both agents is tolerable at the RP2D with prexasertib treatment prior to study entry. No DLTs were observed. The most fre- at 105mg/m2 IV on days 1 and 15 in combination with LY3300054 at quent AEs regardless of relationship to study drug were fatigue (7 700mg flat dosing. Two DLTs occurred, including febrile neutropenia patients), nausea (7 patients) and UTI (5 patients). Overall, 7 patients (Arm C) and prolonged grade 4 neutropenia lasting > 5 days (Arm B). had AEs of > grade 3, 4 of which were considered related to study Most common drug-related adverse events included leukopenia, drug by the investigator. Pharmacodynamic assessments included neutropenia, thrombocytopenia, and anemia. Confirmed partial re- immune phenotyping at the periphery where immune activation was sponses have been observed in 2 patients with CCNE1-amplified observed following AGEN2034 treatment. In this subset of recurrent HGSOC ongoing for 9 and 10 months, respectively. One additional ovarian cancer patients, 1 of 12 patients developed a durable partial CCNE-1 amplified HGSOC patient has had prolonged SD for 11 response (42 wks) at the lowest dose level (1 mg/kg), 8 patients months. Preliminary data on T-cell subset analysis and cytokine pro- demonstrated at least stable disease lasting 8.7 -65.7 weeks, with 5 file show immune modulatory effect of prexasertib, confirming of them meeting the DCR criteria of at least 12 weeks of duration. proof-of-mechanism. Four patients demonstrated progressive disease at the first on treat- Conclusions ment tumor evaluation. Full-dose prexasertib in combination with immune checkpoint block- Conclusions ade is tolerable and has preliminary clinical activity in patients with AGEN2034, a PD-1 inhibitor, is well tolerated with no DLTs observed HGSOC with durable responses. An expansion cohort in this popula- at all dose levels evaluated. The clinical activity and safety observed tion is currently being enrolled utilizing schedule B. Comprehensive in the recurrent ovarian cancer subset were consistent with the over- characterization of the immune microenvironment will be performed all Phase 1 study population. Biomarker evaluations (including PD-L1 in paired tumor biopsies, with attention to pharmacodynamic proof- status) are ongoing. of-mechanism endpoints, including T cell infiltration and PD-L1 ex- Ethics Approval pression and their correlation with the induction of DNA damage. 20170314 - IRB tracking for Copernicus Additionally, immune signatures will be correlated with genomic pro- Consent file and response duration. Written informed consent was obtained from the patient for publica- Ethics Approval tion of this abstract and any accompanying images. A copy of the This study was approved by Dana-Farber Cancer Institute's Ethics written consent is available for review by the Editor of this journal. Board. Consent P394 Written informed consent was obtained from the patient for publica- A Phase 1 study of INCMGA00012, a PD-1 inhibitor, in patients tion of this abstract and any accompanying images. A copy of the with advanced solid tumors: Preliminary results for patients with written consent is available for review by the Editor of this journal. advanced cervical cancer (POD1UM-101) 1 2 3 Janice Mehnert, MD , Luis Paz Ares, MD, PhD , Joanna Pikiel, Dr n med , 4 5 6 P393 Udai Banerji, PhD , Anna Kryzhanivska, MD , Nehal Lakhani, MD, PhD , 7 8 Single agent activity of a novel PD-1 inhibitor AGEN2034 in Sebastian Ochsenreiter, Dr med , Tobias Arkenau, MD, PhD , Nawel 9 9 9 recurrent ovarian cancer:Subset analysis of phase I dose escalation Bourayou, MD , Deanna Kornacki, PhD , Chuan Tian, PhD , Thomas 9 10 NCT03104699 study Condamine, PhD , Itziar Gardeazabal González 1 1 2 3 1 David O'Malley , John Hays , Charles Drescher , Jasgit Sachdev , Wilberto Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United 4 5 6 7 2 3 Nieves-Neira , Breelyn Wilky , Marylin Huang , Kathleen Moore , Waldo States; Hospital Universitario, Madrid, Spain; Szpitale Pomorskie Sp. z 8 8 8 8 4 Ortuzar , Anna Wijatyk , Hagop Youssoufian , Remigiusz Kaleta, MD , o.o., Gdansk, Poland; Royal Marsden NHS Foundation Trust, Sutton, 8 8 8 5 Inbal Sapir , Christopher Dupont, PhD , Irina Shapiro , Debra Richardson, United Kingdom; Regional Clinical Oncology Center, Ivano-Frankivsk, 7 6 7 MD Ukraine; START Midwest, Grand Rapids, MI, United States; Charité 1 8 The James Cancer Center Hospital, Columbus, OH, United States; Comprehensive Cancer Center, Berlin, Germany; Sarah Cannon Institute, 2 3 9 Swedish Cancer Institute, Seattle, WA, United States; HonorHealth London, United Kingdom; Incyte Corporation, Wilmington, DE, United 4 10 Research Institute, Scottsdale, AZ, United States; Northwestern States; Hospital Vall d’Hebron, Barcelona, Spain University, Stroger Hospital, Chicago, IL, United States; University of Correspondence: Janice Mehnert (mehnerja@cinj.rutgers.edu) Colorado Anschultz Medica, Aurora, CO, United States; Sylvester Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P394 Comprehensive Cancer Center, Miami, FL, United States; Stephenson Cancer Center, Oklahoma City, OK, United States; Agenus Inc, Background Lexington, MA, United States Background Correspondence: Christopher Dupont INCMGA00012 is an investigational humanized, hinge-stabilized IgG4 (Christopher.Dupont@Agenusbio.com) monoclonal antibody that binds to PD-1. At all doses tested, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P393 INCMGA00012 has an acceptable tolerability profile with no dose- limiting toxicities or maximum tolerated dose. A dose of 3 mg/kg Background Q2W was initially selected for the tumor specific cohorts. Multiple AGEN2034 is a novel, fully human monoclonal immunoglobulin G4 fixed doses were also examined in this study. The 500 mg Q4W and (IgG4) antibody, designed to block PD-1 from interacting with its li- 375 mg Q3W doses are selected for further development based on gands PD-L1 and PD-L2 with high affinity. The overall objective of favorable pharmacokinetics and safety. the study was to assess safety, MTD, and pharmacokinetic (PK) and Methods pharmacodynamic (PD) characteristics of AGEN2034 monotherapy in Methods patients with advanced, refractory malignancies. The initial expansion phase contained 4 tumor-specific cohorts Methods (endometrial [unselected], cervical, soft tissue sarcoma, and non- Between April 2017 - April 2019, 50 patients with advanced solid tu- small cell lung) treated for up to 2 years. Eligible patients presented mors were enrolled in a phase 1 dose escalation study treated with with a histologically proven, unresectable locally advanced or meta- infusion of AGEN2034 every 2 weeks at the dose range of 1-10 mg/ static tumor, ECOG performance status (PS) ≤1, disease progression kg. Within the study population a subset of patients with heavily pre- during or following ≤5 prior treatments, measurable disease per treated recurrent epithelial ovarian cancer was identified. RECIST v1.1, and no prior treatment with immune checkpoint inhibi- Results tors. The primary endpoint is safety (using CTCAE v4.03 grading). Twelve patients with recurrent epithelial ovarian cancer were en- Confirmed best overall response rate and duration of response were rolled in the Phase I dose escalation. Median age was 58 years (range evaluated by RECIST v1.1 (investigator's assessment). Treatment past Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 216 of 272 progression was allowed for patients experiencing clinical benefit. A recent study of enoblituzumab combined with pembrolizumab Preliminary safety and efficacy results for patients with unresectable showed this combination is feasible and well tolerated with minimal locally advanced or metastatic cervical cancer are presented. additive toxicity [5]. While studies of monotherapy pembrolizumab in Results this population report responses below 17% [6], the overall response Results rate of PD-1/PD-L1 inhibitor-naïve patients (post platinum) receiving As of 24 APR 2019, 35 patients with cervical cancer were treated with 3 enoblituzumab plus pembrolizumab was 33% (6/18) including 1 con- mg/kg INCMGA00012. Median age was 51 (29-81) years, 88.6% were firmed CR and 5 confirmed PRs [5]. This suggests a cooperative white, 48.6% had an ECOG PS of 1. All patients were pretreated with at mechanism and provides a rationale for further development of this least 1 prior platinum-based chemotherapy for recurrent or advanced combination in patients with recurrent/metastatic SCCHN. disease, 91.4% were treated with radiotherapy, and 62.9% underwent MGA012 (also known as INCMGA00012) is an investigational anti-PD-1 surgery. Median drug exposure was 4.4 (0.03-16.0) months. Fourteen monoclonal antibody with a tolerable safety profile and efficacy signal patients (40.0%) experienced Grade (G) 3/4 AEs regardless of causality. consistent with other agents in its class [7] demonstrated in early studies. Seven patients (20.0%) had immune-related AEs (colitis [G2, n=1; G3, Methods n=2], infusion-related reaction [G1, n=1; G3, n=1], diarrhea [G1], hyper- This is a Phase 2/3, randomized, open label study in first-line treatment of thyroidism [G2], and maculopapular rash [G3]). Three patients with col- patients with R/M SCCHN not curable by local therapy (Figure 1). We itis discontinued study treatment. No treatment-related deaths hypothesize that combining enoblituzumab and PD-1 inhibition (with or occurred. Confirmed responses per RECIST v1.1 were observed in 6/31 without chemotherapy) will improve objective response rates and OS (19.4%) response evaluable patients, with 1 patient having a confirmed compared to pembrolizumab/chemotherapy in patients in R/M SCCHN. CR. Median duration of response was not reached as 5/6 patients re- Approximately 200 patients will be randomized in a 1:1:1:1 ratio to main on treatment (10.3, NE months). An additional 12 patients had one of four treatment arms to select the preferred enoblituzumab stable disease for an overall disease control rate of 58.1% (18/31). combination treatment for further evaluation based primarily on Conclusions ORR. In subsequent Phase 3 portion, the selected enoblituzumab/ Conclusions MGA012 regimen (with or without chemotherapy) will be compared INCMGA00012 has been generally well tolerated with evidence of to pembrolizumab and chemotherapy with an endpoint of OS. significant and durable antitumor activity in platinum-refractory cer- Trial Registration vical cancer. These data support further development of To be registered on clinicaltrials.gov INCMGA00012 in cervical cancer. Trial Registration References NCT03059823, 2017-000865-63 1. Siegel R, Naishadham D, and Jemal A, Cancer statistics, 2013. CA Cancer Ethics Approval J. Clin, 2013. 63(1): p. 11-30. The study was approved by institutional review boards or independ- 2. Price KA and Cohen EE, Current treatment options for metastatic head ent ethics committees of participating institutions. and neck cancer. Curr Treat Options Oncol, 2012. 13(1): p. 35-46. 3. Keynote 048. 1200/JCO.2019.37.15_suppl.6000 Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019) 6000-6000. P395 4. Collins M, Ling V, and Carreno BM, The B7 family of immune-regulatory li- Phase 2/3 open-label trial of enoblituzumab in combination with gands. Genome Biol, 2005. 6(6): p. 223. MGA012, with and without chemotherapy, in the treatment of 5. Aggarwal C et al, Open-Label, Dose Escalation Study of Enoblituzumab in patients with recurrent or metastatic head and neck squamous cell Combination with Pembrolizumab in Patients with Select Solid Tumors. carcinoma 1 2 1 33rd Annual Meeting of The Society for Immunotherapy of Cancer Wash- Fernanda Arnaldez , Charu Aggarwal, MD MPH , Scott Currence , Jan 1 1 3 ington, DC, USA November 7–11, 2018 Baughman, MPH , Paul Moore, PhD , George Blumenschein, MD , Jon 1 4 6. Cohen EE, Harrington KJ, Tourneau C, Dinis J, Licitra L, Ahn M, et al., Wigginton, MD , Robert Ferris, MD, PhD 1 2 Head and Neck Cancer, Excluding Thyroid. ESMO, 2017. 28. MacroGenics, Inc., Rockville, MD, United States; Abramson Cancer 3 7. Mehnert J et. At. 33rd Annual Meeting of The Society for Center, Philadelphia, PA, United States; MD Anderson Cancer Center, 4 Immunotherapy of Cancer Washington, DC, USA November 7–11, 2018 Houston, TX, United States; UPMC Hillman Cancer Center, Pittsburgh, Ethics Approval PA, United States Each institution will obtain Ethics Board approval prior to enrollment Correspondence: Fernanda Arnaldez (farnaldez@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P395 Background Squamous cell carcinoma of the head and neck (SCCHN) accounts for >500,000 new cases and nearly 300,000 deaths annually worldwide as of 2012 [1]. Patients with recurrent/metastatic (R/M) SCCHN have a poor prognosis with median overall survival (OS) of Enoblituzumab is an investigational Fc-modified monoclonal antibody that binds B7-H3, which is over-expressed in a wide range of cancers including SCCHN [4], but not in most normal tissues. It has increased affinity for the acti- vating FcγR IIIA (CD16A) and decreased affinity for the inhibitory FcγRIIB (CD32B). The engineered Fc domain confers enoblituzumab with target-specific antibody-dependent cellular cytotoxicity in vitro and anti-tumor activity in preclinical studies, and in vivo and clinical data suggest that Fc-optimized antibodies such as enoblituzumab can en- gage both innate and adaptive immunity as mediators of anti-tumor activity [6]. Enoblituzumab was well tolerated in a Phase 1 monother- Fig. 1 (abstract P395). Study Schema apy trial with no maximum tolerated dose defined up to 15 mg/kg. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 217 of 272 P396 Key secondary endpoints include occurrence and severity of treatment- A phase 2 efficacy and safety trial of ADU-S100 and emergent adverse events and changes from baseline in safety assess- pembrolizumab in adults with head and neck cancer ments. This trial is currently in the recruitment phase. 1 2 3 Ezra Cohen, MD , Robert Ferris, MD, PhD , Douglas Adkins, MD , Dan 2 4 5 Zandberg, MD , Arkadiusz Dudek, MD, PhD , Matthen Mathew , Lara References 6 7 8 Dunn, MD , Juneko Grilley-Olson, MD , Ammar Sukari, MD , Rebecca 1. Chen PL, Roh W, Reuben A, et al. Analysis of Immune Signatures in 9 10 11 Redman, MD , Julie Bauman, MD, MPH , John Kaczmar, MD , Lisle Longitudinal Tumor Samples Yields Insight into Biomarkers of Response 12 13 14 15 Nabell, MD , Nabil Saba, MD , Eric Nadler, MD , Young Kim, MD , and Mechanisms of Resistance to Immune Checkpoint Blockade. Cancer 16 17 17 Ranee Mehra, MD , Nitya Nair, PhD , Somayeh Honarmand, MS , Discov 2016;6:827-37. 17 18 Richard Cutler Jr. , Barbara Burtness, MD 2. Gajewski TF, Fuertes MB, Woo SR. Innate immune sensing of cancer: University of California at San Diego, La Jolla, CA, United States; clues from an identified role for type I IFNs. Cancer Immunol University of Pittsburgh Medical Center, Pittsburgh, PA, United States; Immunother 2012;61:1343-7. Washington University of St. Louis, St. Louis, United States; 3. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the HealthPartners Regions Cancer Care Center, St. Paul, MN, United States; tumor microenvironment. Nat Immunol 2013;14:1014-22. Columbia University Medical Center, New York, United States; 4. Gajewski TF, Woo SR, Zha Y, et al. Cancer immunotherapy strategies Memorial Sloan-Kettering Cancer Center, New York, NY, United States; based on overcoming barriers within the tumor microenvironment. Curr University of North Carolina at Chapel Hill, Chapel Hill, NC, United Opin Immunol 2013;25:268-76. States; Wayne State University School of Medicine, Detroit, MI, United 5. Woo SR, Corrales L, Gajewski TF. The STING pathway and the T cell- 9 10 States; University of Louisville, Louisville, United States; The University inflamed tumor microenvironment. Trends Immunol 2015;36:250-6. of Arizona Cancer Center, Tucson, United States; MUSC Hollings Ethics Approval Cancer Center, Charleston, SC, United States; University of Alabama at This study was approved or is currently under review by an institutional Birmingham, Birmingham, United States; Emory University, Atlanta, GA, review board at each site. United States; Baylor Charles A. Sammons Cancer Center, Dallas, United States; Vanderbilt University School of Medicine, Nashville, P397 United States; University of Maryland, Greenebaum Comprehensive Interim analysis of the combination of durvalumab and cetuximab Cancer Center, Baltimore, United States; Aduro Biotech, Berkeley, CA, in a phase II trial of patients with recurrent and metastatic head United States; Yale University School of Medicine, New Haven, CT, and neck squamous cell carcinoma United States 2 1 1 1 Shuchi Gulati, MD , Sarah Palackdharry , Layne Weatherford , Sarah Wilson , Correspondence: Richard Cutler Jr. (rcutler@aduro.com) 1 1 1 3 1 Shireen Desai , Aubrey Steele ,KashifRiaz , Vinita Takiar ,Trisha Draper Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P396 1 2 University of Cincinnati, Cincinnati, OH, United States; University of Cincinnati Cancer Institute, Cincinnati, OH, United States; University of Background Cincinnati/Barrett Cancer, Cincinnati, OH, United States Immune checkpoint inhibitors such as the PD-1 blocking antibody Correspondence: Shuchi Gulati (gulatisi@ucmail.uc.edu) pembrolizumab have demonstrated marked improvements in duration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P397 of response and long-term survival over standards of care (SOC) in head and neck squamous cell carcinoma (HNSCC) and other cancers. Background However, the significant percentage of patients who are nonresponsive Cetuximab ( IgG1 isotype monoclonal antibody) monotherapy is con- to these immunotherapies (primary resistance) or experience disease sidered standard of care therapy for recurrent and metastatic head relapse following an acquired immune resistance mechanism (second- and neck squamous cell carcinoma (HNSCC).[1] Cetuximab results in ary resistance) [1] highlights the need for new therapies. As tumor re- NK cell mediated ADCC and inhibition of the EGFR signaling path- sponsiveness to immunotherapy may depend, in part, on the way.[1] NK cell activation increases secretion of plasma transforming immunophenotype of the tumor microenvironment (TME) [2-5], one ex- growth factor β (TGFβ) and interleukin 10 (IL-10), resulting in in- ploratory approach to establish, re-establish, or enhance active immune creased expression of PD-1 on T cells and PD-L1 expression on tumor surveillance conditions within the TME is to inject innate immune mod- cells. Blocking the PD-1/PD-L1 check-point receptor pathway in- ulators directly into the tumor to promote an adaptive tumor-specific creases the cytotoxic response of NK cells in mice.[2] Therefore, it immune response. ADU-S100 (MIW815) is a novel synthetic cyclic di- was hypothesized that cetuximab and PD-1/PD-L1 blockade would nucleotide that activates the stimulator of interferon genes (STING) be synergistic. Here we report our findings on the combination of pathway within the TME leading to activation of tumor-resident APCs cetuximab with a PD-L1 inhibitor, durvalumab on T cells, NK cells and priming of tumor antigen specific CD8+ T cells. Direct activation of and cytokines from a phase II open-label single site clinical trial in STING via intratumoral injection of ADU-S100 (MIW815) has been HNSCC patients with recurrent or metastatic disease. (NCT03691714.) shown to overcome active tolerance mechanisms through stimulation Methods of resident leukocyte populations. Preclinical models indicate that sur- Interim analysis includes a total of 15 enrolled patients. Using vival and local tumor shrinkage were significantly enhanced when flow cytometry and Luminex, we evaluated the immune cell phe- ADU-S100 (MIW815) was administered with an anti-PD-1 antibody, sug- notypes and cytokine profiles of peripheral blood in patients be- gesting the PD-1 blockade may act synergistically with concomitant fore and after treatment with the combination of cetuximab and STING activation. In phase 1 trials, tumor shrinkage and durable re- durvalumab with respect to overall response rate (ORR). sponses have been observed after treatment with S100 alone or in Results combination with a PD-1 inhibitor. The primary objective of this trial is Fourteen patients who received at least 2 cycles of treatment were to evaluate the clinical efficacy of intratumoral ADU-S100 (MIW815) included in the interim analysis. Median age was 66 years (range 47- when administered in combination with pembrolizumab. 75), majority of patients were male (79%). Eight patients (57%) had Methods received 1 line of prior chemotherapy, while 3 (21%) had received 2 This open-label, multicenter phase 2 clinical trial (NCT03937141) aims to prior chemotherapies. Seven patients (50%) had received prior im- enroll 33 adults with PD-L1 positive, recurrent or metastatic HNSCC for munotherapy. Of the 7 patients who had next generation sequen- which pembrolizumab is indicated as SOC in the first-line setting. Patients cing completed, 1 was PDL1 positive (14%), all had MSI-high tumors with at least one lesion that is accessible for repeat intratumoral injection (100%) and all had TP53 mutations (100%). One patient achieved a and can provide tumor tissue for eligibility determination and biomarker partial response, and 3 were noted to have stable disease; overall re- analyses will receive intravenous infusions of pembrolizumab (200 mg) at sponse rate (ORR) was noted as 27%. No grade 3/ 4 adverse events Day 1 and intratumoral injections of ADU-S100 (MIW815) (800 mcg/le- attributable to study drugs were reported. Results from peripheral sion) at Day 1 and 8 in 21-day dosing cycles up to 35 cycles, or until cri- blood flow cytometry analyses showed an increase in cytokine pro- teria for treatment discontinuation are met. The primary endpoint is the ducing NK cells and CD3+ T cells in all responders. Luminex assay objective response per Response Evaluation Criteria in Solid Tumors v1.1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 218 of 272 revealed that all responders had a drop in their GM-CSF levels, and an increase in their TNF-alpha and CXCL-10 levels. Conclusions The combination of cetuximab and durvalumab results in a pro- tumorigenic profile with a modest ORR. References 1. Ferris, R. L. et al. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation. Cancer Treat. Rev. 63, 48–60 (2018). 2. Hsu, J. et al. Contribution of NK cells to immunotherapy mediated by PD- 1/PD-L1 blockade. J. Clin. Invest. 128, 4654–4668 Ethics Approval The study was approved by University of Cincinnati's Ethics Board, approval no. FWA #: 000003152 P398 miRNA-A and programmed death ligand 1 (PD-L1) expression in oral squamous cell carcinoma 1 2 2 2 Hong Hyung, MD PhD , Yoon Ho Ko , Lee Hee JIn , Sang Hoon Jeon 1 2 Catholi, Seoul, Korea, Republic of; Catholic Universtiy, Uijeongbu-si, Korea, Republic of Correspondence: Yoon Ho Ko (koyoonho@catholic.ac.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P398 Background Overexpression of PD-L1 in cancer cells is involved not only in the im- Fig. 1 (abstract P398). A and B. See text for description mune evasion but also in cancer progression. Increasing evidence indi- cates that dysregulation of miRNA(miR)s contributes to the pathogenesis of oral squamous cell carcinoma (OSCC). Here, we identified miR-C that regulate the expression of PD-L1 and elucidated whether miR-C affects chemotherapy responsiveness via regulating PD-L1 expression in OSCC. Methods To further verify the role of miRNAs on PD-L1 in OSCC, we carried out the functional study in human head and neck cancer cell line CAL27 and YD8. After transfection with scrambled miRNA-A, B, C (Scr), miRNAs-A, B, C for 48 h, PD-L1 mRNA and PD-L1 protein levels were assessed by RT-qPCR and western blot analysis. To perform EGFP reporter assay, cells were transfected with miRNA-C and re- porter constructs, containing the putative PD-L1 3’-UTR target sites, along with a control vector, EGFP levels were assessed by western blotting. Cell viability was assessed after treatment with 5-FU for 72 h using by MTT solution. GAPDH mRNA and its protein level were used for normalization and as a loading control. Results We investigated various miRs that were negatively correlated with PD-L1 in The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset and could recognize PD-L1 3'-UTR by analyzing TargetScan. Three miRs (miR-A, miR-B, miR-C) were identi- fied which had not been reported to be as associated with PD-L1 be- fore. EGFP reporter assay of only miR-C out of three miRs showed a decrease in the relative PD-L1 expression. This would indicate that only miR-C can recognize target sites in the 3’-UTR of PD-L1 mRNA in OSCC cells. Overexpression of miR-C induced the decrease of PD-L1 mRNA and protein (Figure 1A,1B). The sensitivity of CAL27 and YD8 cells to 5-FU was increased when miR-C was overexpressed (Figure 2). Also, the level of cleaved PARP, one of the apoptotic markers, was increased according to miR-A overexpression (Figure 3). Conclusions Our data suggest that miR-C can regulate PD-L1 expression by tar- geting PD-L1 mRNA, and our present findings shed new light on the complex regulation of PD-L1 in human tumors, and on miR-C in can- Fig. 2 (abstract P398). See text for description cer immuno-based therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 219 of 272 Trial Registration NCT03246958, NCT03341936, NCT03425331, NCT02971956, NCT03075527, NCT02635061 Ethics Approval The present studies were reviewed and approved by the Dana- Farber/Harvard Cancer Center (DF/HCC) institutional review board (Boston, Massachusetts, USA) and all were performed in accordance with relevant guidelines and regulations. Consent Written informed consent was obtained from all subjects prior to par- ticipation in these studies. Informed consent by patients to DF/HCC protocol 02-180 enabled collection of clinical and demographic data, and genomic characterization. P400 Sitravatinib and Nivolumab for resectable Oral cavity squamous cell carcinoma Window of opportunity study (SNOW) 1 1 2 Marc Oliva Bernal, MD , Douglas Chepeha, MD , Amy Prawira, MD , Fig. 3 (abstract P398). See text for description 1 1 3 Anna Spreafico, MD PhD , Scott Bratman, MD , Tina Shek, MD , John De 1 3 1 1 Almeida, MD , Ivan Yeung, MD , Aaron Hansen , Andrew Hope, MD , 1 1 P399 David Goldstein, MD , Ralph Gilbert, MD , Doug Vines, BSc, MRT(N), 3 1 1 1 Instructive conclusions from performing immune correlatives on IO CNMT , Patrick Gullane , Dale Brown, MD , Ilan Weinreb, MD , Bayardo 1 4 4 trials: a meta-analysis of patient tumor and blood samples Perez-Ordoñez, MD , Trevor Pugh, PhD , Pamela Ohashi, PhD , Ben 4 1 5 Patrick Lizotte, PhD, Megan Cavanaugh, Melissa Jean, Cloud Paweletz, Wang, PhD , Jonathan Irish, MD , Hirak Der-Torossianh, MD , Isan Chen, 5 1 PhD MD , Lillian Siu, MD Dana-Farber Cancer Institute, Boston, MA, United States Princess Margaret Cancer Centre, University of Toronto, Toronto, Correspondence: Cloud Paweletz Canada; The Kinghorn Cancer Centre, St Vincent’s Hospital, Darlinghurst, (CloudP_Paweletz@DFCI.HARVARD.EDU) Australia; Princess Margaret Cancer Centre, University of Toronto; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P399 Quantitative Imaging for Personalized Cancer Medicine, TECHNA Institute, University Health Network., Toronto, Canada; University of Background Toronto, Toronto, Canada; Mirati Therapeutics, San Diego, CA, United Blood-based immune phenotyping provides a cost-effective, States minimally invasive, longitudinal, and logistically convenient Correspondence: Lillian Siu (lillian.siu@uhn.ca) assay that may provide two critical pieces of information for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P400 cancer patients receiving immunotherapy: 1.) if the therapeutic is working as intended, and 2.) if there will be any benefit to Background the patient. Sitravatinib is a receptor tyrosine kinase inhibitor that blocks TAM Methods and VEGF family of receptors. Based on non-clinical findings, it is pre- Our group has performed multi-parameter flow cytometric immune- dicted to increase M1 macrophage response and decrease immuno- profiling on hundreds of blood and tumor samples from patients suppressive Tregs and MDSCs in the tumor microenvironment. treated with immunotherapy. We have compiled this dataset of im- Sitravatinib combined with nivolumab showed a safe toxicity profile mune correlatives from patients receiving immune checkpoint block- and promising antitumor activity in non-small cell lung cancer pa- ade enrolled on Dana-Farber clinical trials in the following cancers: tients (pts) progressing on anti-PD-1 agents [1]. The CheckMate-358 thyroid cancer (tumor n = 24; blood n = 180) , head & neck squa- study revealed that preoperative nivolumab was safe and active in mous cell carcinoma (tumor n =18; blood n = 118) , mesothelioma oral cavity squamous cell carcinoma (OCSCC) [2]. We hypothesize (tumor n = 41) , non-small cell lung cancer (tumor n = 34) , and that preoperative sitravatinib and nivolumab have synergistic im- gastric-esophageal cancer (tumor n= 55). munogenic and antitumor effects in OCSCC. Results Methods Our meta-analysis of blood and tumor flow-based immune profil- SNOW is an investigator-initiated, single-center, non- ing confirms that tumor tissue remains the benchmark for deter- randomized, window-of-opportunity study evaluating pre- mining therapeutic efficacy to immune checkpoint blockade. operative sitravatinib and nivolumab in pts with resectable, However, our profiling of blood has produced several previously untreated OCSCC. Pts with T2-4a, N0-2 or T1 generalizable findings: 1.) IO-relevant markers are generally (>1cm)-N2 tumors as per AJCC 8th edition, ECOG >/=1, ad- expressed at very low levels by circulating T cells and only subtly equate organ function and no autoimmune disorders are eli- change after treatment, 2.) the abundance of different leukocyte gible. Figure 1 summarizes study design and treatment. lineages is also largely static, 3.) serial blood profiling at time- Primary objective is to evaluate the immune and pharmaco- points later than three weeks after initiation of treatment are dynamic effects of the treatment combination. Secondary ob- minimally informative, 4.) some immune parameters significantly jectives are: (a) safety, including rate of treatmen-related correlated with therapeutic efficacy are simply indicative of adverse events (TRAEs), surgery completion within the immune-related adverse events, which are historically associated planned window and postoperative complications; (b) antitu- with improved therapeutic efficacy and also easy to diagnose mor activity, including clinical and pathologic responses; rate without immune correlatives, and 5.) it is impossible to delineate of pathological extranodal extension (ENE) and positive mar- between reinvigorated or activated tumor-specific circulating T gins; (c) pharmacokinetics/pharmacodynamics of sitravatinib cells and global reinvigoration or activation of, for instance, by- alone and combined with nivolumab. Correlative studies in- stander T cells without more sophisticated and costly TCR clude: immune biomarkers by multiplex immunohistochemis- deconvolution. try, tumor and blood immunophenotyping; tumor genome Conclusions and transcriptome analyses; intratumoral hypoxia changes We conclude that blood-based immune phenotyping can be, in using 18FAZA-PET. Preliminary results as of June 30th, 2019 some contexts, highly informative, but invites over-analysis. are reported. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 220 of 272 Results P401Neoadjuvant Seven out of the 12 planned evaluable pts have been en- and adjuvant pembrolizumab plus standard of care (SOC) in rolled: 1 pt is currently undergoing study treatment and thus patients with resectable, locally advanced head and neck excluded from this analysis. Median follow-up: 19.5 weeks. All squamous cell carcinoma (HNSCC): the phase 3 KEYNOTE-689 pts completed study treatment and had surgery within the study 1 2 3 planned window. None required sitravatinib dose reduction/ Ravindra Uppaluri, MD, PhD , Nancy Lee, MD , William Westra , Ezra 4 5 6 hold or nivolumab delay. No G3/G4 TRAEs occurred pre- Cohen, MD , Robert Haddad , Stephane Temam , Christophe Le 7 8 9 10 surgery. One pt had G3 neck infection and G3 bleeding from Tourneau , Rebecca Chernock , Sufia Safina , Arkadiy Klochikhin , 11 12 13 the tracheostomy site 11 days post-surgery, both resolved Amichay Meirovitz , Irene Brana, MD , Joy Yang Ge , Ramona Swaby, 13 13 8 and deemed possibly related to study drugs. Tumor reduction MD , Cecilia Pinheiro , Douglas Adkins, MD as per investigator’s assessment was observed in all pts. Five Dana-Farber Cancer Institute and Brigham and Women’s Hospital, pts had pathological downstaging, including 1 complete Boston, MA, United States; Memorial Sloan Kettering, New York, NY, pathological response (Table 1); all pts had clear margins and USA, Sylmar, CA, United States; Icahn School of Medicine, New York, no ENE. All pts received standard of care postoperative radio- NY, USA, New York, United States; University of California San Diego, La therapy based on clinical stage. None required postoperative Jolla, CA, USA, La Jolla, CA, United States; Dana-Farber Cancer Institute chemotherapy. All pts are alive with no recurrence to date. and Brigham and Women's Hospital, Boston, MA, United States; 6 7 Conclusions Gustave Roussy, Villejuif, France, Villejuif, France; Institut Curie, Paris, These preliminary results suggest that preoperative sitravatinib France, Paris & Saint-cloud, France; Washington University School of and nivolumab is a safe and active combination in OCSCC. On- Medicine, St. Louis, MO, United States; Republican Dispensary of going biomarker and tumor immunophenotyping analyses will Tatarstan MoH, Kazan, Russia, Kazan, Russian Federation; Yaroslavl be presented Regional Clinical Oncology, Ulitsa Chkalov, Yaroslavl, Russia, Yaroslavl, Russian Federation; Hadassah-Hebrew University Medical Center, Acknowledgements Jerusalem, Israel, Jerusalem, Israel; Hospital Vall d’Hebron, Barcelona, The authors would like to thank patients and their families for their Spain, Barcelona, Spain; Merck & Co., Inc., Kenilworth, NJ, USA, participation and Mirati Therapeutics for drug supply and their support of Kenilworth, NJ, United States this study. Correspondence: Ravindra Uppaluri Trial Registration (ravindra_uppaluri@dfci.harvard.edu) NCT03575598 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P401Neoadjuvant References Background 1. Leal et al. ESMO Meeting 2018, Abstract 1129O. Neoadjuvant and adjuvant pembrolizumabshowedevidenceof 2. Ferris et al. ESMO Meeting 2017, Abstract LBA46. pathological response (PR) and acceptable safety in patients Ethics Approval with high-risk, resectable, locally advanced (LA) HNSCC in phase This study was approved by the University Health Network Research Ethics 2 studies (NCT02296684 and NCT02641093). KEYNOTE-689 Board (Study number: 18-5537) on July 12th 2018. (NCT03765918), a randomized, open-label, phase 3 trial, will as- sess efficacy and safety of neoadjuvant pembrolizumab and ad- juvant pembrolizumab plus SOC in patients with previously untreated, resectable LA HNSCC. Methods Eligible patients are adults with newly diagnosed, resectable HNSCC (stage III oropharyngeal p16-positive disease [T4 (N0-N2), M0]; stage III/IVA oropharyngeal p16 negative; or stage III/IVA larynx or hypo- pharynx or oral cavity, independent of p16 status) [1] and ECOG per- Fig. 1 (abstract P400). SNOW study design and treatment plan formance status 0 or 1. Patients will be randomly assigned 1:1 to arms A and B, with randomization stratified by primary tumor site (oropharynx/oral cavity vs larynx vs hypopharynx), tumor stage (III vs Table 1 (abstract P400). Tumor downstaging following study IVA), and PD-L1 status defined by tumor proportion score 50% treatment (TPS≥50% vs TPS Trial Registration ClinicalTrials.gov, NCT03765918 Reference 1. American Joint Committee on Cancer. AJCC Cancer Staging Manual, Eight Edition. Amin MB, ed. Chicago, IL: American College of Surgeons; Ethics Approval The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 221 of 272 P402 BELINDA : A phase 3 study evaluating the safety and efficacy of tisagenlecleucel versus standard of care in adult patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma 1 3 4 5 Michael Bishop, MD , Ian Flinn, MD , Peter Borchmann , Ulrich Jaeger , 6 7 8 9 Jason Westin, MD , Nada Hamad , Duncan Purtill , Richard Greil , 10 11 12 13 Simone Thomas , Takanori Teshima , Hideo Harigae , Carlos Garcia , 14 15 16 17 Pere Barba , Abhinav Deol, MD , Paul Shaughnessy , Jessie Gu , 18 17 17 Giovanna Andreola , Marcela Martinez Prieto , Lida Pacaud , Stephen Schuster, MD 1 2 University of Chicago, Chicago, IL, United States; Univeristy of Chicago, Chicago, IL, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University Hospital of Cologne, Cologne, Germany; 5 6 Medical University of Vienna, Vienna, Austria; University of Texas, MD Anderson Cancer Center, Houston, TX, United States; St Vincent’s Hospital, Sydney, Australia; Fiona Stanley Hospital, Murdoch, Australia; 9 10 Paracelsus Medical University, Salzburg, Austria; University Hospital Fig. 1 (abstract P402). See text for description Regensburg, Regensburg, Germany; Hokkaido University, Hokkaido, Japan; Tohoku University Graduate School of Med, Miyagi, Japan; 13 14 University Hospital "12 de Octubre", Madrid, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain; Karmanos Cancer Institute, P403 Wayne State University, Detroit, MI, United States; Texas Transplant Serum soluble CD25 may predict the early therapeutic response in Institute, San Antonio, TX, United States; Novartis Pharmaceuticals pediatric patients with B-cell non-Hodgkin's lymphoma(B- Corporation, East Hanover, NJ, United States; Novartis Pharma AG, Basel, NHL)during autologous chimeric antigen receptor T cell(CAR-T) Switzerland: Abramson Cancer Center, University of Pennsylvania, therapy Philadelphia, PA, United States Jing Guo, Yonghong Zhang, Professor, Juan Du, MD Correspondence: Michael Bishop Beijing Boren Hospital, Beijing, China (mbishop@medicine.bsd.uchicago.edu) Correspondence: Yonghong Zhang (yhzhang58@126.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P402 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P403 Background Background Around one-third of aggressive B-cell non-Hodgkin lymphoma (NHL) CAR-T therapies have been widely employed in B-NHL.Immune acti- patients will not respond to, or will relapse or progress after frontline vation induced by CAR-T therapies includes significant changes of treatment; >50% of treatment failures occur within one year. Progno- some inflammatory cytokines. In this study we analyzed the changes sis is particularly poor in these patients, regardless of salvage chemo- of serum cytokine levels in 12 pediatric patients with B-NHL during therapy and autologous hematopoietic stem cell transplant (auto- CAR-T therapy, so as to explore the relationship between serum cyto- HSCT). Novel therapies are therefore needed for refractory or early- kine levels and early treatment response. The association between relapsed patients with NHL. cytokine levels and cytokine-release syndrome(CRS) grade was also Methods investigated. BELINDA (NCT03570892) is a randomized, open-label, multicenter, Methods phase 3 study to compare the safety and efficacy of two treat- 12 B-NHL pediatric patients aging from 4 to 14 in stage II to IV ac- ment strategies: tisagenlecleucel with standard of care (SOC) cording to st.jude stage were enrolled .Each patient received 1 to 3 immunochemotherapy followed by auto-HSCT in adult patients rounds sequential CAR-T treatment, consisting of CD19, CD20, CD22 with aggressive B-cell NHL whose disease relapsed or progressed CAR-T treatment. A total of 18 rounds CAR-T were performed, includ- after frontline immunochemotherapy. Eligible patients are aged ing 12 CD19, 3 CD20 and 3 CD22. Early tumor response were evalu- ≥18 years, have histologically confirmed aggressive B-cell NHL re- ated on day 15, 30 and 60 of each round of CAR-T and early side lapsed/refractory to frontline therapy containing rituximab and effect known as CRS were observed and graded. Serum samples anthracycline within one year of last dose, and are eligible for were collected at baseline and on day 3,7,11,15,20,30,60 of each auto-HSCT. Patients are apheresed prior to enrollment and ran- round of CAR-T. Levels of Interleukin-6 (IL-6), soluble CD25 (sCD25) domized 1:1 to receive tisagenlecleucel (Arm A) or SOC (Arm B) and interferon-γ (IFN-γ) interleukin-10 (IL-10), tumor necrosis factor- (Figure 1). Randomization is stratified by remission duration (re- α(TNF-α)were measured by ELISA(enzyme-linked immunosorbent fractory or relapse assay). We analyzed the cytokine changes in different treatment out- Trial Registration comes, explored the predictive value of different cytokines using NCT03570892 ROC curve .The relationship between cytokine level and CRS grade Ethics Approval was also analyzed. Tumor response was assessed per RECIST 1.1. The The study is done in accordance with the principles of Good Clin- ROC curve take” response “and “no response” as the outcome indica- ical Practice, the Declaration of Helsinki, and all local regulations. tors ,stable disease (SD) and progressive disease (PD)were defined as The study protocol and all amendments were reviewed and ap- “no response”, complete response(CR) and partial response(PR )were proved by independent ethics committees or institutional review defined as “response”. Adverse Event (AE) grade categorization is ac- boards for each center. All patients provided written informed cording to CTCAE 4.0. consent. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 222 of 272 Results with B cell malignancies for which there are no effective therap- There were statistically significant differences in sCD25 values among ies that induce durable remissions. different treatment responses (P<0.01), with an average decrease in SD patients and an average increase in PR patients ,it is speculated P405 that sCD25 may play a predictive role in treatment response. The CASSIOPEIA: A phase 2 study evaluating efficacy and safety of ROC curve analysis shows that sCD25 had a predictive effect on the tisagenlecleucel in first-line therapy for high-risk pediatric and response to treatment, and the AUC was 0.719,95%ci =(0.516, 0.922) young adult patients with B-ALL who are MRD positive at the EOC ,excluding 0.5, indicating that the difference is statistically significant. 1 1 Shannon Maude, MD, PhD , Hunger Stephen, MD , Jochen Buechner, In this study, other cytokines were not found to be predictive 2 1 3 4 5 MD , Stephen Grupp, MD , Susana Rives , Andre Baruchel , John Levine , markers of therapeutic response. Besides,Spearman rank correlation 6 7 8 9 Joerg Krueger , Theodore Laetsch , Marianne Ifversen , Aiesha Zia , coefficient showed that there was a positive correlation between 10 10 11 Jaclyn Davis , Eric Bleickardt , Mignon Loh sCD25 and CRS grade (r=0.693,P=0.001). University of Pennsylvania; Children’s Hospital of Philadelphia, Conclusions Philadelphia, PA, United States; Oslo University Hospital, Oslo, Norway; sCD25 may be a useful predictive marker for early response of CAR-T 3 4 Sant Joan de Deu Hospital, Barcelona, Spain; Hôpital Robert and level of sCD25 are correlated with CRS grade. Clinical trial Debré&Université de Paris, Paris, France; Mount Sinai School of information:ChiCTR18000144 Medicine, New York, NY, United States; The Hospital for Sick Children, Toranto, Canada; UT Southwestern Medical Center and Child, Dallas, TX, P404 United States; Copenhagen University Hospital Rigshospi, Copenhagen, 9 10 Developing canine CART-19 to fully leverage comparative Denmark; Novartis Pharma AG, Basel, Switzerland; Novartis oncology and inform human clinical trials Pharmaceuticals Corporation, East Hanover, NJ, United States; Kumudhini Haran, MS, Ailian Xiong, Enrico Radaelli, Patrick Savickas, University of California, San Francisco, CA, United States Avery Posey, Donald Siegel, Nicola Mason, BVet Med PhD Correspondence: Shannon Maude (maude@email.chop.edu) University of Pennsylvania, Springfield, PA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P405 Correspondence: Nicola Mason (nmason@vet.upenn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P404 Background Survival is compromised for patients with high-risk (HR) B-cell acute Background lymphoblastic leukemia (B-ALL) who have a poor response to first- CD19 specific chimeric antigen receptor T cell (CART-19) therapy has line chemotherapy. A Children’s Oncology Group (COG) phase-3 resulted in unprecedented durable clinical responses in adult and study for HR B-ALL, AALL0232, showed poor 5-year disease free sur- pediatric patients with B-cell malignancies. However, poor quality of vival (DFS) of 39% in patients with minimal residual disease (MRD) patient T cells, failed persistence, reduced effectiveness within an im- ≥0.1% at end of induction (EOI) and MRD ≥0.01% at the end of con- munosuppressive microenvironment and target antigen loss, repre- solidation (EOC) [1]. The objective of this trial is to determine the effi- sent some of the challenges to improving CART19 efficacy. cacy and safety of tisagenlecleucel in pediatric and young adult Furthermore, correlative biomarkers that predict CART-19 response patients with de novo HR B-ALL who received first-line treatment remain elusive. and remain MRD-positive after the EOC therapy. Pet dogs spontaneously develop B-NHL and B cell leukemias that Methods share oncogenic pathways and similar immunosuppressive features CASSIOPEIA (NCT03876769) is a phase-2, single-arm, global, multicen- to human B cell malignancies. Therefore, they provide an immuno- ter, open-label study being conducted in collaboration with COG. Pa- logically intact, parallel patient population in which to evaluate next tients aged 1-25 years with de novo National Cancer Institute generation CAR T cell strategies and combination approaches that defined HR B-ALL (presenting white blood count >50,000/μL or over address current CART19 challenges. Previously, we have demon- the age of 10 years) who are in first complete remission (CR1) but re- strated the ability to generate functional CD20-targeting canine CAR main MRD-positive (≥0.01% by flow cytometry determined at a cen- T cells. Their use in client owned animals with B-NHL can lead to the tral reference laboratory) at EOC are eligible. Prior to screening, development of canine anti-mouse antibody (CAMA) formation and patients will complete a standard of care first-line 4-drug induction, target antigen escape. To address these issues and provide a parallel MRD assessment at EOI, a Berlin-Frankfurt-Münster phase-1b consoli- reagent that can inform human CAR T cell strategies, we have devel- dation, and MRD assessment at EOC. Eligible patients undergo leuka- oped a fully canine CD19 targeting CAR and confirmed its function pheresis either at the EOI or EOC. Prior to tisagenlecleucel infusion, in vitro against CD19 expressing targets. patients will receive interim maintenance including high-dose Methods methotrexate. Following lymphodepleting chemotherapy, patients We employed a canine scFv phage display library to isolate canine receive a single infusion of tisagenlecleucel based on body weight; CD19-specific scFvs following 3-4 rounds of panning against the sol- 0.2-5.0x10^6 chimeric antigen receptor (CAR)-positive viable T-cells uble extracellular domain of canine CD19. Twelve unique scFvs were per kg in patients ≤50 kg or 0.1-2.5x10^8 CAR-positive viable T-cells isolated and their binding to soluble canine CD19 and cell surface in patients >50 kg. Patients may receive a second infusion based on expressed CD19 was confirmed by ELISA and flow cytometry respect- B-cell recovery and MRD status. Efficacy will be assessed at day 29, ively. One of the highest binding candidates was cloned into a fully then every 3 months for the first year, every 6 months for the second canine CD28ζ CAR in a pMX retroviral plasmid. Retroviruses pseudo- year, then yearly until the end of study. The primary outcome is 5- typed with both RD114 and VSV-G envelope proteins were generated year DFS rate by local investigator assessment, defined as the time using standard protocols and used to transduce primary canine T from tisagenlecleucel infusion to morphologic relapse, occurrence of cells activated using anti-canine CD3/CD28 beads in the presence of secondary malignancy or death from any cause, whichever occurs RetroNectin®. Successful transductions of canine T cells were ob- first. Secondary outcomes include percentage of patients in remis- tained with 45% of T cells expressing CAR on their cell surface by sion without allogeneic transplantation at 1 year, MRD negativity at flow cytometry. Canine CART-19 cells demonstrated antigen-specific month 3, overall survival, cellular kinetics, and safety. The primary proliferation and cytokine production in vitro. We now aim to per- analysis of DFS will be undertaken when 40 DFS events are observed form a pilot study to evaluate the safety and efficacy of this ap- or 6 years after first-patient-first-treatment, whichever occurs later. proach in canine patients with relapsed, refractory B-NHL. This work The estimated enrollment for this study is 160 patients (with 140 in- will serve the dual purpose of enabling pet dogs with spontan- fused). The study is currently enrolling patients in the U.S., Europe eous B cell malignancies to accelerate application of next gener- and Canada. ation CART cell therapies into the human clinics as well as Trial Registration provide much needed immunotherapeutics for canine patients NCT03876769 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 223 of 272 Reference 2. Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, 1. Borowitz MJ, Wood BL, Devidas M et al. Prognostic significance of Sadelain M. CAR T cell-induced cytokine release syndrome is mediated minimal residual disease in high risk B-ALL: a report from Children's On- by macrophages and abated by IL-1 blockade. Nat Med. 2018 cology Group study AALL0232. Blood. 2015;126(8):964-971. Jun;24(6):731-738. Ethics Approval Ethics Approval The study is done in accordance with the principles of Good Clinical UCLA IRB #19-000604 Practice, the Declaration of Helsinki, and all local regulations. The study protocol and all amendments were reviewed and approved by P407 independent ethics committees or institutional review boards for each Phase 3 KEYNOTE-937: adjuvant pembrolizumab versus placebo in center. All patients provided written informed consent. patients with hepatocellular carcinoma and complete radiologic response after surgical resection or local ablation 1 2 3 4 P406 Masatoshi Kudo, MD, PhD , Andrew Zhu , Arndt Vogel , Thomas Yau , 5 6 6 6 Interleukin-1 blockade to prevent severe immune effector cell- Jian Zhou , Erluo Chen , Usha Malhotra , Abby Siegel , Ann-Lii Cheng, associated neurotoxicity syndrome; Trial in progress MD PhD Caspian Oliai, MD, Anna Crosetti, John Timmerman, MD Kindai University School of Medicine, Osaka, Japan, Osaka-Sayama, UCLA, Los Angeles, CA, United States Japan; Massachusetts General Hospital Cancer Center, Harvard Medical Correspondence: John Timmerman (jtimmerman@mednet.ucla.edu) School, Boston, MA, United States; Medizinische Hochschule, Hannover, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P406 Germany, Hannover, Germany; University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong PRC; Zhongshan Hospital, Fudan Background University, Shanghai, China, Shanghai, China; Merck & Co., Inc., CAR T-cell therapy targeting CD19 is a promising new treatment for Kenilworth, NJ, USA, Kenilworth, NJ, United States; National Taiwan relapsed/refractory B-cell lymphomas and leukemias. However, se- University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan vere grade 3 neurotoxicity (immune effector cell-associated neuro- Correspondence: Masatoshi Kudo (m-kudo@med.kindai.ac.jp) toxicity syndrome, or ICANS) is seen in up to one-third of patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P407 Recently, preclinical animal studies of human CD19 CAR T-cell therap- ies have shown that while IL-6 and IL-1 receptor antagonists could Background prevent cytokine release syndrome (without impairing anti-tumor ef- For patients with hepatocellular carcinoma (HCC) who are undergo- ficacy), only IL-1 blockade could prevent neurotoxicity [1,2]. The re- ing potentially curative surgical resection or local ablation, 5-year re- combinant IL-1 receptor antagonist Anakinra was used successfully currence rates are up to 50%-80%; there is no standard of care for to avert lethal neurotoxicity in mice. Anakinra crosses the blood brain adjuvant treatment. The programmed death 1 inhibitor pembrolizu- barrier and has been shown to be safe and efficacious in rheumatologic mab is approved for the treatment of patients with HCC previously conditions driven by high levels of monocyte lineage-associated IL-1 in- treated with sorafenib. There is no direct evidence of benefit with cluding rheumatoid arthritis and neonatal onset multisystem inflamma- pembrolizumab in the HCC adjuvant setting, but a favorable benefit/ tory disease, for which it is FDA approved. We are conducting the first risk profile is anticipated based on data from other indications. human trial of Anakinra to treat ICANS in B-cell lymphoma patients KEYNOTE-937 (NCT03867084) is a randomized, double-blind, phase 3 treated with anti-CD19 CAR T-cells. The trial has been approved by the trial to examine the safety and efficacy of adjuvant pembrolizumab U.S. FDA under an investigator-sponsored IND, and Anakinra is supplied versus placebo in patients with complete radiologic response after by the agent’s manufacturer (Sobi Pharmaceuticals). surgical resection or local ablation of HCC. Methods Methods Patients with diffuse large B-cell lymphoma receiving standard of Eligible patients are aged ≥18 years and have confirmed HCC, complete care CAR T-cells are eligible for enrollment. The primary objectives radiologic response after complete resection or local ablation, Eastern are to: 1) Evaluate the effectiveness of IL-1 blockade in reducing the Cooperative Oncology Group performance status of 0, and class A incidence and duration of severe ICANS in participants receiving anti- Child-Pugh score. Patients with past or ongoing HCV or controlled HBV CD19 CAR T-cells, 2) Assess the safety of Anakinra in CAR T-cell pa- are eligible if they meet certain criteria. Patients (N=~950) will be ran- tients, 3) Measure cytokines (including IL-1, IL-6, IL-15, TNF-alpha, domly assigned 1:1 to receive pembrolizumab 200 mg or placebo every interferon-gamma) and nitric oxide in the serum and CSF of treated 3 weeks and stratified by geographic region, prior local therapy (resec- patients prior to and during CAR T-cell therapy for correlation with tion vs ablation), recurrence risk, and alpha-fetoprotein level at diagno- ICANS events, and 4) Determine the tumor response rate in compari- sis. Treatment will continue for up to 17 cycles (~1 year) or until son to historical controls. Upon development of grade 1 ICANS, or documented disease recurrence, unacceptable toxicity, or investigator/ grade 3 CRS (which is often followed by ICANS), participants will re- patient decision to withdraw. Dual primary end points are recurrence- ceive Anakinra 100 mg subcutaneously every 6 hours for at least 12 free survival (RFS) and overall survival. Secondary end points are safety, doses, or until ICANS returns to grade 1 in participants who develop tolerability, and quality of life. Exploratory end points include distant grade 2 neurotoxicity. Patients will be continuously evaluated for tox- metastases–free survival (DMFS); time to recurrence (TTR); and gen- icity, and assessed for overall tumor response by day 120 with PET/ omic, metabolic, and/or proteomic biomarkers. RFS, DMFS, and TTR will CT scanning. Thirty-six participants will be treated in this multi- be assessed radiographically by the investigator and/or by subsequent center trial, at four centers within the University of California biopsy and confirmed by blinded independent central review. Adverse Hematologic Malignancies Consortium (UC Los Angeles, UC San events (AEs), graded per National Cancer Institute Common Termin- Francisco, UC San Diego, and UC Davis). The trial is powered to ology Criteria for Adverse Events version 4.0, will be recorded up to 30 detect a 50% reduction in the rate of severe ICANS compared to days after last dose (90 days for serious AEs). historical rates. Trial Registration ClinicalTrials.gov, NCT03867084 References Ethics Approval 1. Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito The study and the protocol were approved by the Institutional Re- F, Ponzoni M, Doglioni C, Cristofori P, Traversari C, Bordignon C, Ciceri F, view Board or ethics committee at each site. Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and Consent IL-6 are differentially required for cytokine-release syndrome and neuro- All patients provided written informed consent to participate in the toxicity due to CAR T cells. Nat Med. 2018 Jun;24(6):739-748. clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 224 of 272 P408 P409 LEAP-002: phase 3 study of first-line lenvatinib plus A case report of personalized neoantigen peptide vaccine in pembrolizumab for patients with advanced hepatocellular treating patients with biliary tract cancer 1 2 1 3 3 carcinoma Fang Yong , Fan Mo , Jiawei Shou , Huimin Wang , Lin Chen , Shanshan 1 2 3 4 1 1 1 4 Josep Llovet , Masatoshi Kudo, MD, PhD , Ann-Lii Cheng, MD PhD , Zhang , Hongsen Li , Weidong Han , Hongming Pan , Shuqing Chen 4 5 6 7 1 2 Richard Finn , Peter Galle , Shuichi Kaneko, MD PhD , Tim Meyer , Sir Run Run Shaw Hospital, Hangzhou, China; Vancouver Prostate 8 9 10 10 3 Shukui Qin , Corina Dutcus , Erluo Chen , Leonid Dubrovsky , Abby Centre, UBC, Hangzhou, China; Hangzhou Neoantigen Therapeutics Co., 10 11 4 Siegel , Andrew Zhu Hangzhou, China; Zhejiang University, Hangzhou, China 1 2 Icahn School of Medicine at Mount Sinai, New York, NY, USA; Kindai Correspondence: Shuqing Chen (chenshuqing@zju.edu.cn) University School of Medicine, Higashiosaka, Japan, Osaka-Sayama, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P409 Japan; National Taiwan University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan; David Geffen School of Medicine at UCLA, Los Background Angeles, CA, USA, Los Angeles, CA, United States; University of Mainz Despite recent advance in immune checkpoint blockade therapies in Medical Center, Mainz, Germany, Mainz, Germany; Kanazawa University cancer, the overall response rate is still low in malignancy treatment. Hospital, Kanazawa, Japan, Kanazawa, Japan; University College London Arisen from tumor somatic mutations, neoantigens provide tumor Cancer Institute, London, United Kingdom, London, United Kingdom; specific targets for developing personalized cancer vaccines, further 8 9 Jinling Hospital, Nanjing, China, Nanjing, China; Eisai Inc., Woodcliff eliciting strong T cell-mediated immune response. A single-arm, Lake, NJ, USA, Woodcliff Lake, NJ, United States; Merck & Co., Inc., open-labelled, investigator-initiated clinical study was carried out to Kenilworth, NJ, USA, Kenilworth, NJ, United States; Massachusetts examine the safety and efficacy of personalized peptide vaccine General Hospital Cancer Center, Harvard Medical School, Boston, MA, (iNeo-Vac-P01). Total of 22 patients with solid tumors had been en- USA, Boston, MA, United States rolled in the trial from Feb 7th, 2018 to May 31st, 2019. A biliary tract Correspondence: Josep Llovet (Josep.Llovet@mountsinai.org) cancer patient achieved unique neoplastic changes. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P408 Methods The 63-year-old male, initially diagnosed with intrahepatic biliary Background tract cancer in 2013, was treated with surgical excision in Jun. 2013 Lenvatinib, a multikinase inhibitor, is approved for first-line treatment and postoperative chemotherapy in Apr. 2014 respectively. Both of unresectable hepatocellular carcinoma (HCC). Pembrolizumab, a tumor recurrence and metastases were confirmed with CT scan in programmed death 1 inhibitor, is approved for second-line treatment Nov. 2017. And then, he was treated with 6 cycles of PD-1 antibody of advanced HCC in patients previously treated with sorafenib. The in a clinical trial and dropped out due to disease progression. Under phase 1b KEYNOTE-524 trial showed that lenvatinib plus pembrolizu- his consent, his biopsy and blood samples were obtained for whole mab was well tolerated and demonstrated promising antitumor ac- exome sequencing (WES), RNA sequencing (RNA-seq), and neoanti- tivity in patients with unresectable HCC. LEAP-002 (NCT03713593) is gen identification [1-4]. Finally the total of 7 peptides were synthe- a phase 3 study to evaluate the safety and clinical benefit of lenvati- sized and pooled into 2 groups. nib plus pembrolizumab in patients with previously untreated ad- On Mar 22th, 2018, he started to receive iNeo-Vac-P01 subcutane- vanced HCC. ously (s.c.). The injection sites were the two upper arms. He was Methods scheduled to receive vaccinations with GM-CSF as adjuvant on Eligible patients are aged ≥18 years and have confirmed HCC, East- day 1, 4, 8,15 and 22 (i.e.priming phase),aswellas 6 subse- ern Cooperative Oncology Group performance status (ECOG PS) 0 or quent boosters [5-8]. 1, Barcelona Clinic Liver Cancer stage C or stage B disease not amen- Results able to locoregional or curative therapy, class A Child-Pugh score ≤7 After the last booster vaccination, a grade 3~4 allergic reaction days before study day 1, and ≥1 measurable lesion (per RECIST v1.1 (under NCI-CTCAE 4.03) happened, while clinical manifestations were by blinded independent central review [BICR]). Past or ongoing HCV nausea, vomiting and rash. The treatment-relating allergic reaction infection and controlled HBV are allowed. Patients will be randomly maybe result from peptide-specific antibody accumulation, however, assigned 1:1 to receive oral lenvatinib 12 mg (body weight [BW] ≥60 this hypothesis needs experimental validation by enzyme-linked im- kg) or 8 mg (BW 400 ng/mL); and ECOG PS (0 vs 1). Tumor imaging munosorbent assay. The CT scans indicated an evident increase of will be performed every 9 weeks. Dual primary end points are tumor size at 5th month, and a surprisingly decrease of tumor size at progression-free survival (PFS), assessed per modified RECIST v1.1 by 8th month, implying a pseudo-progression. The duration of stable BICR, and overall survival. Secondary end points are objective re- disease was 14.5+ months, and he had been keeping progression- sponse rate (ORR), duration of response (DOR), disease control rate free since then. The results of IFN-γ ELISPOT assay shows that the (DCR), and time to progression (TTP) per RECIST v1.1 by BICR, efficacy neoantigen peptides stimulated highest number of IFN-γ spots and outcomes (PFS, ORR, DOR, DCR, and TTP) per modified RECIST v1.1 induced significant peptide-specific T-cell response. TCR sequencing by BICR, pharmacokinetics, and safety. Exploratory end points are ef- demonstrated the evident increase of peripheral T cells with three ficacy outcomes evaluated per RECIST v1.1 and iRECIST assessed by TCRs (data unshown). the investigator. Adverse events (AEs), graded per National Cancer In- Conclusions stitute Common Terminology Criteria for Adverse Events version 4.0, The preliminary results demonstrated that iNeo-Vac-P01 treatment will be monitored throughout the treatment period and for 90 days was feasible and safe, and can prolong progression-free survival and after the last dose (120 days for serious AEs). overall survival. Trial Registration ClinicalTrials.gov, NCT03713593 Acknowledgements Ethics Approval The authors would like to gratitude all the patients who participated in the The study and the protocol were approved by the Institutional Re- trial and their families, as well as the Sir Run Shaw clinical site. This study was view Board or ethics committee at each site. funded by Hangzhou Neoantigen Therapeutics Co. Consent Trial Registration All patients provided written informed consent to participate in the This trial had been registration on ClinicalTrials.gov, the identifier number clinical trial. was NCT03662815. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 225 of 272 References dose finding part. Secondary objectives include characterization of 1. Chen F, Zou Z, Du J, Su S, Shao J, Meng F, Yang J, Xu Q, Ding N, Yang Y vactosertib pharmacokinetics and anti-tumor activity by response et al. Neoantigen identification strategies enable personalized rate (RECIST v1.1). immunotherapy in refractory solid tumors. J Clin Invest. 2019; 130. Results 2. Hundal J, Carreno BM, Petti AA, Linette GP, Griffith OL, Mardis ER, Griffith As of July 18, 2019, 13 patients were enrolled to the study (7 in 100 M. pVAC-Seq: A genome-guided in silico approach to identifying tumor mg BID cohort and 6 in 200 mg BID cohort). Median age was 66 neoantigens. Genome Med. 2016; 8(1):11. (range 45-76), 62% were male, median number of previous lines of 3. Ng AWR, Tan PJ, Hoo WPY, Liew DS, Teo MYM, Siak PY, Ng SM, Tan EW, chemotherapy was 4 (range 2-8). All patients were PD-L1 less than Abdul Rahim R, Lim RLH et al. In silico-guided sequence modifications of 25% by SP263 antibody assay. At 100 mg BID cohort, no dose limit- K-ras epitopes improve immunological outcome against G12V and G13D ing toxicity was observed. The most frequently reported adverse mutant KRAS antigens. PeerJ. 2018; 6:e5056. events (AE) were skin rash (30.8%), nausea (23.1%), and pruritis 4. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, Zhang W, Luoma A, (23.1%). There were 3 serious adverse events (SAE) reported; pleural Giobbie-Hurder A, Peter L et al. An immunogenic personal neoantigen effusion (1), skin eruption (1), and empyema (1), and no patients with vaccine for patients with melanoma. Nature. 2017; 547(7662):217-221. reported cardiotoxicity. Among 7 tumor response evaluable patients, 5. Gjertsen MK, Buanes T, Rosseland AR, Bakka A, Gladhaug I, Soreide O, best responses to treatment were SD in 3 patients; 5.9%, 10.4%, and Eriksen JA, Moller M, Baksaas I, Lothe RA et al. Intradermal ras peptide 26.4% decreases from baseline. Biomarker data will be presented at vaccination with granulocyte-macrophage colony-stimulating factor as the meeting. adjuvant: Clinical and immunological responses in patients with pancre- Conclusions atic adenocarcinoma. Int J Cancer. 2001; 92(3):441-450. The combination of vactosertib plus durvalumab has been tolerated 6. Keskin DB, Anandappa AJ, Sun J, Tirosh I, Mathewson ND, Li S, Oliveira G, thus far with no safety concerns; the study is ongoing. The anti- Giobbie-Hurder A, Felt K, Gjini E et al. Neoantigen vaccine generates tumor activity of this combination in patients with advanced NSCLC intratumoral T cell responses in phase Ib glioblastoma trial. Nature. 2019; will be further explored. Clinical trial information: NCT03732274 565(7738):234-239. Trial Registration 7. Weden S, Klemp M, Gladhaug IP, Moller M, Eriksen JA, Gaudernack G, NCT 03732274 Buanes T. Long-term follow-up of patients with resected pancreatic can- Ethics Approval cer following vaccination against mutant K-ras. Int J Cancer. 2011; The study was approved by Ethics Board of Severance Hospital (ap- 128(5):1120-1128. proval number 4-2018-0892) and National Cancer Center (approval 8. Kirner A, Mayer-Mokler A, Reinhardt C. IMA901: a multi-peptide cancer number NCC2019-0057) vaccine for treatment of renal cell cancer. Hum Vaccin Immunother. 2014; 10(11):3179-3189. P411 Ethics Approval Treating advanced non-small lung cancer (NSCLC) patients after This study was approved by the institutional review board and independent checkpoint inhibitor treatment failure with a novel combination of ethics committee of Sir Run Run Shaw Hospital, Zhejiang University Viagenpumatucel-L (HS-110) plus nivolumab School of Medicine; approval number 20180109-9. 1 1 Daniel Morgensztern, MD , Saiama Waqar, MD , Lyudmila Bazhenova, Consent 2 3 4 MD , Rachel Sanborn, MD , Lori Mcdermott, RN, MSc , Jeff Hutchins, Written informed consent was obtained from the patient for publication of 4 5 6 PhD , Luis Raez, MD, FACP, FCCP , Corey Langer, MD , Roger Cohen, this abstract and any accompanying images. A copy of the written MD consent is available for review by the Editor of this journal. Washington University School of Medicine, St. Louis, MO, United States; 2 3 Moores Cancer Center, La Jolla, CA, United States; Earle A. Chiles P410 Research Institute, Portland, OR, United States; Heat Biologics, Tampa, Safety and anti-tumor activity of the transforming growth factor β FL, United States; Memorial Cancer Institute, Pembroke Pines, FL, United receptor I kinase inhibitor, vactosertib, in combination with States; Perelman School of Medicine, Philadelphia, PA, United States durvalumab in patients with advanced non-small cell lung cancer Correspondence: Lori Mcdermott (lmcdermott74@yahoo.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P411 1 2 2 Ji-Youn Han, MD, PhD , Kyoung-Ho Pyo , Jea Hwan Kim , Chun-Feng 2 3 3 3 Xin , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kim , Byoung Chul Cho, Background 2 2 MDphD , Byoung Chul Cho, MDphD Viagenpumatucel-L (HS-110) is an allogeneic cellular vaccine derived 1 2 National Cancer Center, Goyang-si, Korea; Severance Hospital, Seoul, from a human lung adenocarcinoma cell line transfected with the Korea, Republic of; Medpacto, Inc, Seoul, Korea, Republic of gp96-Ig fusion protein that functions as an antigen chaperone for Correspondence: Byoung Chul Cho (cbc1971@yuhs.ac) cross presentation and dendritic cell activation. DURGA is a multi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P410 cohort study evaluating the combination of HS-110 and anti-PD-1 monoclonal antibodies in patients with advanced NSCLC. We report Background on Cohort B, which enrolled patients with progressive disease (PD) TGF-β signaling is known to be associated with poor response to single- after receiving a minimum of 4 months of treatment with a check- agent immune checkpoint inhibitors by immunosuppressive microenviron- point inhibitor (CPI) at any time prior to study entry. ment through strong epithelial-mesenchymal transition (EMT) induction. Methods Combined inhibition of immune checkpoint and TGF-β signal is anticipated Patients with previously treated NSCLC received weekly HS-110 (1 X as a promising therapeutic strategy because these two key pathways have 107 cells) intradermally for 18 consecutive weeks and nivolumab IV independent and complementary immunosuppressive functions. We are 240 mg every 2 weeks, followed by nivolumab maintenance until reporting the Dose Finding part of a Phase 1b/2a study evaluating the tumor progression or intolerable toxicity. Tissue was tested at base- combination of vactosertib, a highly selective and potent TGF-β inhibitor, line for PD-L1 expression (≥ 1% or < 1%) and tumor infiltrating lym- with durvalumab in patients with advanced non-small cell lung cancer phocytes (TILs). TIL high was defined as >10% CD8+ lymphocytes in (NSCLC) who progressed following platinum-based chemotherapy. the tumor stroma. The primary endpoint was objective response rate Methods (ORR) by RECIST 1.1. Secondary endpoints included ORR and clinical Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and benefit rate using iRECIST, progression-free survival (PFS), overall sur- have no prior exposure to immune checkpoint inhibitors, or TGFβ R1 vival (OS) and adverse events (AEs). kinase inhibitors. The primary objective is to assess the safety and Results the recommended dose of vactosertib given 5 days on/2 days off in As of March 2019, 56 patients were enrolled and evaluated for efficacy. combination with durvalumab 1500 mg every 4 weeks. Two dose The median number of prior treatment lines was 2 [range 1 to 6]. Seven levels of vactosertib (100 mg BID and 200 mg BID) were tested in the patients (13%) achieved partial response and 26 patients (46%) had Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 226 of 272 stable disease. Median PFS and median OS were 3.2 months and 11.8 Hossein Borghaei et al., Nivolumab versus Docetaxel in Advanced months, respectively. Immune ORR and clinical benefit rate by iRECIST Nonsquamous Non–Small-Cell Lung Cancer. The New England Journal of were 14% and 61%, respectively. Patients experiencing injection site re- Medicine. 2015; 373:1627-1639 actions (ISR) had improved PFS (3.7 vs 1.8 months; HR 0.21, p =0.0021) Achim Rittmeyer et al., Atezolizumab versus docetaxel in patients with and improved OS (12 vs 5 months; HR 0.16, p=0.0005) compared to previously treated non-small-cell lung cancer (OAK): a phase 3, open- those without ISR. 96% of patients experienced at least one adverse label, multicentre randomised controlled trial. The Lancet. 2017; 389 event, and 92% of all AEs were grade 1 or 2. The most common AEs (10066): 255-265 were fatigue (34%), hypocalcemia (18%), cough (16%) and diarrhea and Ethics Approval dyspnea (14% each). There were four grade 4 events: QTc prolongation, The last amendment was approved in June 2019 by WIRB and Copernicus stroke, pericardial tamponade, and hyponatremia, none of which were IRB, approval number 420160463 deemed related to treatment. There were no grade 5 AEs. Conclusions P413 The combination of HS-110 and nivolumab is well tolerated, and A Phase 1b/2 study of galunisertib in combination with nivolumab does not appear to increase the incidence of immune-related AEs as in solid tumors and NSCLC compared to CPI monotherapy. Patients continue to be enrolled into 1 2 3 Ernest Nadal, MD, PhD , Mansoor Saleh, MD , Santiago Ponce Aix, MD , this cohort. Data suggest that re-challenging the immune system 4 5 6 Maria Ochoa de Olza , Sandip Patel, MD , Scott Antonia, MD, PhD , with nivolumab and HS-110 after CPI treatment failure restores re- 7 7 7 Yumin Zhao, PhD , Ivelina Gueorguieva, PhD , Michael Man, PhD , sponsiveness and clinical benefit for some patients. 7 7 8 7 Shawn Estrem, PhD , Emin Avsar , Wen Hong Lin , Karim Benhadji , 7 9 1 Susan Guba , Inmaculada Ales Diaz , Ernest Nadal, MD, PhD Acknowledgements 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; University of Thank you to the Investigators, their staff, and the patients and family Alabama, Birmingham, AL, United States; Hospital 12 de Octubre, members that made this research possible. Madrid, Spain; Hospital Universitario Vall d'Hebron, Barcelona, Spain; Trial Registration 5 6 University of California, La Jolla, CA, United States; H. Lee Moffitt NCT 02439450 Cancer Center and, Tampa, FL, United States; Eli Lilly and Company, Ethics Approval Indianapolis, IN, United States; Bristol-Myers Squibb, New York, NY, This study was approved by Advarra IRB, Western IRB, Washington University United States; Hospital Universitario Regional de Mala, Malaga, Spain IRB, Cleveland Clinic IRB, UCSD IRB, Providence Portland IRB, NYU Winthrop Correspondence: Ernest Nadal (esnadal@iconcologia.net) IRB, Baptist Health Louisville IRB, Lifespan IRB, and US Oncology IRB. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P413 P412 Background Validation of a single-blinded (patients only) study design for the TGF-β promotes immune suppression. In this study, both TGF-β and prevention of premature patient consent withdrawal in the PD-1 were targeted in patients with advanced refractory solid tumors immuno-oncology trial DUBLIN-3 and recurrent/refractory NSCLC using galunisertib, an oral small mol- Ramon Mohanlal, MD, PhD, MBA, Huang Lan, PhD ecule inhibitor of TGF-β receptor I, in combination with nivolumab, a BeyondSpring Pharmaceuticals, Inc., New York, NY, United States monoclonal antibody that binds PD-1. Correspondence: Ramon Mohanlal Methods (rmohanlal@beyondspringpharma.com) This is a Phase 1b/2 open-label study. Eligible patients were ≥18 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P412 years old, had ECOG status ≤1, and were treatment-naive for anti-PD- 1/PD-L1, or TGFβ R1 kinase inhibitor. Patients had advanced solid tu- Background mors that were refractory to standard systemic therapy (Phase 1b). Patients (pts) generally prefer immunotherapy (IO) over chemother- NSCLC patients (Phase 2) were required to have received prior apy (Chemo) in clinical trials and may prematurely withdraw consent platinum-based treatment. Phase 2 portion of the trial evaluated the if allocated to Chemo. This may impact study outcome (Barlesi Lan- safety of 150 mg BID galunisertib administered on a 14 days on, 14 cet Onc 2018). Due to pts awareness of their treatment allocation in days off dosing schedule in combination with nivolumab given at 3 unblinded IO trials, ‘premature’ consent withdrawal (thus before re- mg/kg Q2W. Efficacy, pharmacokinetics (PK) and pharmacodynamic ceiving first dose of study drug) is consistently and significantly (p data were also evaluated. Methods Results ‘Premature’ pts consent withdrawal rate was calculated for the Plin/ 15 patients were enrolled in Phase 1b and 25 in Phase 2. No dose- Doc (n=174) and Doc (n=181) arms in DUBLIN-3 (NCT02504489) limiting toxicities were observed in the Phase 1 portion of the study. around the time of the first pre-planned Interim Analysis (IA). In the Phase 2 NSCLC cohort, the most frequent treatment-related Results grade 3 AEs included immune-related encephalitis, diarrhea, fatigue, ‘Premature’ consent withdrawal rate in DUBLIN-3 was 1.1 % for Doc ALT/ AST/GGT increase, blood alkaline phosphatase increase, abdom- and 2.3 % for Plin/Doc (p=0.53; NS). Premature consent withdrawal inal distension, cutaneous rash (n=1 each), and cholestasis (n=2) that rate of the Doc arm was significantly (p resolved or were resolving at the time of data cutoff. Two deaths on Conclusions treatment (multi-organ failure and myocardial infarction), both unre- A single-blinded design (for pts only) is effective in preventing pre- lated to study treatment, were observed. 6 (24%) patients had con- mature and imbalanced patient consent withdrawal. This finding firmed partial response (PR) and 4 (16%) had stable disease; 1 may have relevance for the design of future IO trials. A second pre- patient had confirmed PR after initial pseudo-progression. Among planned IA for DUBLIN-3 to evaluate OS is projected for end 2019. the 6 responders, 5 had low or negative PD-L1 expression (≤50%). Trial Registration Median PFS was 5.26 months (95% CI: 1.77, 9.20) and median OS was NCT02504489 11.99 months (95% CI: 8.15, NR). Phase 1b PK data showed rapid ab- sorption (1-3h) and elimination of galunisertib within 48h. Additional References biomarker data including tumor mutational burden and gene- Fabrice Barlesi et al., Avelumab versus docetaxel in patients platinum-treated expression data will be presented. advanced non-small-cell lung cancer (JAVELIN Lung 200): an open-label, Conclusions randomised, phase 3 study. The Lancet. 2018; 19 (11): 1468-1479 Combination treatment of galunisertib at the RP2D of 150 mg BID Roy S Herbst et al., Pembrolizumab versus docetaxel for previously treated, for 14 days on 14 days off schedule with nivolumab 3 mg/kg Q2W PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a was well tolerated. Preliminary efficacy was observed in a subset of randomised controlled trial. The Lancet. 2016; 387 (10027): 1540-1550 patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 227 of 272 Trial Registration Conclusions NCT02423343 Preliminary data suggest the combination of pepinemab plus avelu- Ethics Approval mab is well tolerated and shows initial signals of antitumor activity in The study was performed in accordance with the Declaration of patients with IO failure. We will present updated clinical response Helsinki and was approved by ethics committees in multiple investi- data, as well as additional immunophenotyping of tissue biopsies, in- gator sites. cluding but not limited to activated T cells, regulatory T cells, DCs, monocytes, macrophages, and importantly myeloid-derived suppres- sor cells (MDSCs). P414 Interim results from CLASSICAL-Lung, a phase 1b/2 study of Acknowledgements pepinemab (VX15/2503) in combination with avelumab in All of the CLASSICAL-Lung investigators, site staff, and patients advanced non-small cell lung cancer patients Trial Registration 1 2 2 Michael Shafique, MD , Terrence Fisher, PhD , Elizabeth Evans, PhD , NCT03268057 2 2 2 John Leonard, MD PhD , Desa Rae Pastore , Crystal Mallow, BS , Ernest 2 3 4 5 Smith, PhD , Andreas Schroeder, MD, PhD , Kevin Chin , Joseph Beck , References 6 7 Megan Baumgart, MD , Ramaswany Govindan, MD , Nashat Gabrail, 1. Evans EE et al. Antibody blockade of semaphorin 4D promotes immune 8 9 10 MD , Jonathan Goldman, MD , Rachel Sanborn, MD , Alexander Spira, infiltration into tumor and enhances response to other 11 12 13 MD, PhD, FACP , Nagashree Seetharamu , Yanyan Lou, MD , Aaron immunomodulatory therapies. Cancer Immunol Res. 2015; 3: 689-701 13 2 2 Mansfield, MD , Maurice Zauderer, PhD , Terrence Fisher, PhD 2. Clavijo PE et al. Semaphorin4D inhibition improves response to immune 1 2 Moffitt Cancer Center, Tampa, FL, United States; Vaccinex, Inc, checkpoint blockade via attenuation of MDSC recruitment and function. 3 4 Rochester, NY, United States; Merck KGaA, Darmstadt, Germany; EMD Cancer Immunol Res. 2019; 7(2):282-291. Serono, Rockland, MA, United States; Highlands Oncology Group, Ethics Approval Fayetteville, AZ, United States; University of Rochester, Rochester, NY, This protocol and its amendments were approved by the appropriate IRBs at United States; Washington University School of Medicine, St. Louis, MO, each site. 8 9 United States; Gabrail Cancer Center, Canton, OH, United States; UCLA Medical Center, Paramus, NJ, United States; Earle A. Chiles Research Institute, Portland, OR, United States; Virginia Cancer Specialists, Fairfax, P415 VA, United States; Northwell Health, New York, NY, United States; Tumor Treating Fields (TTFields, 150 kHz) concurrent with Mayo Clinic, Jacksonville, FL, United States standard of care treatment for stage 4 non-small cell lung cancer Correspondence: Terrence Fisher (tfisher@vaccinex.com) (NSCLC) in Phase 3 LUNAR Study Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P414 Ori Farber, Moshe Giladi, Ze'ev Bomzon, Eilon Kirson, Uri Weinberg, MD PhD Background Novocure Ltd., Haifa, Israel Despite progress of immune checkpoint blockade therapies, many Correspondence: Uri Weinberg (weinberg@novocure.com) patients with non-small cell lung cancer (NSCLC) do not receive dur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P415 able clinical benefit from these agents, and even in those who do re- spond initially, acquired resistance and tumor recurrence can Background develop. Pepinemab is an IgG4 humanized monoclonal antibody tar- Tumor Treating Fields (TTFields) are a non-invasive, anti-mitotic treat- geting semaphorin 4D (SEMA4D, CD100). In vivo preclinical studies ment that disrupts the formation of the mitotic spindle and disloca- demonstrated antibody blockade of SEMA4D promoted immune infil- tion of intracellular constituents. TTFields plus temozolomide tration and reduced function and recruitment of immunosuppressive significantly extended survival in newly diagnosed glioblastoma. Effi- myeloid cells within the tumor [1,2]. Importantly, preclinical combina- cacy of TTFields in NSCLC has been shown in preclinical models, and tions of anti-SEMA4D with various immunotherapies enhanced T cell safety in combination with pemetrexed in a pilot study. In the Phase infiltration and activity, as well as durable tumor regression. 3 LUNAR study [NCT02973789], we investigated if the addition of Methods TTFields to immune checkpoint inhibitors or docetaxel increases The CLASSICAL-Lung clinical trial evaluates the combination of overall survival (OS). pepinemab with anti-PD-L1 antibody avelumab to couple benefi- Methods cial modifications of the immune microenvironment via pepine- Trial Design: mab with immune activation via checkpoint inhibition. This Patients (N=534), with squamous or non-squamous NSCLC, are strati- ongoing phase 1b/2, open label, single arm, first-in-human com- fied by their selected standard therapy (immune checkpoint inhibitors bination study is designed to evaluate the safety, tolerability and or docetaxel), histology and geographical region. Key inclusion criteria efficacy of the combination in patients with advanced (stage IIIB/ are disease progression, ECOG 0-2, no electronic medical devices in the IV) NSCLC, including a dose escalation cohort and expansion co- upper torso, and absence of brain metastasis. TTFields (150 kHz) are ap- horts consisting of 1) 17 immunotherapy-naïve patients and 2) 33 plied to the upper torso for at >18 hours/day until progression in the patients whose tumors progressed during or following immuno- thorax and/or liver. The primary endpoint is superiority in OS between therapy (IO failure). patients treated with TTFields in combination with the standard of care Results treatments versus standard of care treatments alone. Key secondary The combination was well tolerated with no concerning safety sig- endpoints compare the OS in patients treated with TTFields and doce- nals identified to date. No patient experienced a treatment-related taxel versus docetaxel alone, and patients treated with TTFields and im- adverse event leading to permanent treatment discontinuation or mune checkpoint inhibitors vs those treated with immune checkpoint death and the most frequent related AEs were grades 1 or 2 fatigue, inhibitors alone. An exploratory analysis will test non-inferiority of pyrexia, or chills. Interim analysis focused on the IO failure cohort TTFields with docetaxel compared to checkpoint inhibitors alone. Sec- which included 22 evaluable patients. Two patients experienced a ondary endpoints include progression-free survival, radiological re- partial response (PR) with 49% and 37% tumor reduction on study sponse rate, quality of life based on the EORTC QLQ C30 questionnaire. following acquired resistance to prior treatment with pembrolizu- The sample size is powered to detect a HR of 0.75 in TTFields-treated mab. In addition, stable disease of at least 8 weeks was observed in patients versus control group. In January 2019, an independent Data 11 patients and 4 patients have remained on study for ≥20 weeks. Monitoring Committee (DMC) performed a review of the LUNAR trial Analysis of pre- and on-treatment lung biopsies demonstrated no or data collected to that point. The DMC concluded that no unexpected low tumor burden detected in 2 patients with PR, and interestingly safety issues could be found in patients treated with the combination no detectable tumor was observed in the biopsies from 3 of 4 pa- of immune checkpoint inhibitors and TTFields, and recommended to tients with stable disease. continue the LUNAR study as planned. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 228 of 272 Trial Registration P417 NCT02973789 ATLAS™ identifies relevant neoantigens for therapeutic anti-tumor vaccination and may serve as a biomarker for efficacy of immunotherapy of solid tumors P416 Parul Agnihotri, Tulin Dadali, Parul Agnihotri, PhD A phase 1 dose escalation with expansion study to evaluate the Genocea Biosciences Inc, Cambridge, MA, United States safety, tolerability, pharmacokinetics, and efficacy of AMV564 in Correspondence: Tulin Dadali (tulin.dadali@genocea.com) subjects with advanced solid tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P417 1 1 2 Raghad Abdul Kairm, MD , Anthony Tolcher , Victoria Smith , Sterling 2 2 2 Eckard, PhD , Jeanmarie Guenot , Patrick Chun Background NEXT Oncology and Texas Oncology, San Antonio, TX, United States; Mutation-derived neoantigen cancer vaccines are promising as Amphivena Therapeutics, Inc., South San Francisco, CA, United States next generation cancer therapies. However, the success of vaccin- Correspondence: Patrick Chun (pchun@amphivena.com) ation is dependent on the ability to identify the right neoanti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P416 gens for vaccine inclusion, which remains a critical challenge. Computationally-identified neoantigens do not necessarily gener- Background ate immunogenic responses. Recently, we reported interim im- Overcoming the suppressive tumor microenvironment is a major munogenicity results from the ongoing GEN-009 personalized challenge in immune therapy. The critical cellular effectors of immunotherapy Phase 1/2a clinical trial (NCT03633110). For GEN- the suppressive tumor microenvironment are the myeloid- 009, ATLAS, an ex vivo, cell-based assay selects neoantigens for derived suppressor cells (MDSC). MDSC are elevated in both the vaccine inclusion based on a patient’s own pre-existing T cell re- tumor microenvironment and periphery in cancer patients and sponses. The interim results revealed that vaccination elicited T are associated with immune dysfunction, repression of anti- cell responses to over 98% of administered peptides. Here, we tumor immunity and poor response to immunotherapy. MDSC explore the relationship between ATLAS readouts and immuno- secrete a variety of immunosuppressive factors that directly in- genicity outcomes in the same subjects. hibit both the cytolytic activity and proliferative capacity of Methods anti-tumor T cells. AMV564 is a bivalent, bispecific antibody that Antigens were profiled by expressing each mutation, identified by engages both CD3 and CD33. Preferential binding of AMV564 to whole exome sequencing, as individual clones in E. coli which regions of high CD33 density enables the selective targeting of are subsequently processed by each subject’sown antigenpre- MDSC. Data from both ex vivo studies [1] and an ongoing clin- senting cells and presented to autologous CD4+ or CD8+ T cells. ical trial in acute myeloid leukemia (AML) support the ability of Antigen-specific responses were determined based on cytokine AMV564 to selectively deplete monocytic and granulocytic secretion in the supernatant after overnight incubation. GEN-009, MDSC while sparing monocytes and neutrophils. composed of 4 pools of 1-5 unique ATLAS-identified neoantigen- Methods specific peptides combined with Hiltonol® was administered to AMV564-301 is an open label, phase 1, multicenter, dose- each subject. Both ex vivo and ten day in vitro stimulated Fluoro- escalation with expansion trial of AMV564 in patients with ad- Spot assays were performed on unsorted PBMC and CD4- and vanced solid tumors for which no recognized standard curative CD8-sorted T cells at baseline and 50 days post vaccination to therapy options are available. The key objectives of the dose- identify peptides to which T cells from the vaccinated patients escalation stage of the study are to characterize the safety and responded. tolerability of AMV564 and identify a maximum tolerated dose Results (MTD) or a recommended phase 2 dose (RP2D) for further study. In the first cohort of six patients, ATLAS identified neoantigens In the dose expansion stage of the study, the safety and toler- by recalling both stimulatory and inhibitory neoantigen-specific T ability of AMV564 will be further characterized in addition to cell responses. One subject, who had a greater proportion of in- evaluating the preliminary efficacy of AMV564. Other objectives hibitory to stimulatory responses detected, progressed prior to include characterization of AMV564 pharmacokinetics, pharmaco- vaccination while no vaccinated patients have experienced pro- dynamics, and immunogenic potential. gressive disease. Compared to NetMHCPan results, more than half Approximately 90 patients with locally advanced or metastatic of the ATLAS-identified neoantigens were not predicted. More- solid tumors will be enrolled. The Dose Escalation Stage will in- over, the predicted epitopes did not result in better immunogen- clude up to approximately 40 patients, depending on the dose icity outcomes post-vaccination than the non-predicted ATLAS- at which the MTD/RP2D is determined, and approximately 50 identified neoantigens. Comprehensively profiling T cell responses additional patients will be enrolled in the Expansion Stage. over time shows consistency of results in patients with no evi- AMV564 will be administered once daily as a subcutaneous in- dence of disease. jection for 14 days in each 21-day cycle. Patients will be treated Conclusions until disease progression, unacceptable toxicity, or withdrawal Neoantigens selected by immune response data from ATLAS and of consent. included in the GEN-009 vaccine were immunogenic and many Trial Registration were not algorithm-predicted, confirming that ATLAS identifies NCT pending relevant neoantigens. ATLAS will be useful for profiling epitope spread in tumor-bearing subjects post-vaccination. The proportion Reference of inhibitory to stimulatory neoantigen-specific responses may be 1. Cheng P, Eksioglu E, Chen X, Wei M, Guenot J, Fox J, List A, Wei S. a biomarker of immunotherapy success. Combination of GEN-009 Immunodepletion of MDSC by AMV564, a novel tetravalent bispecific with standard-of-care checkpoint blockade therapy is currently CD33/CD3 T cell engager restores immune homeostasis in MDS in vitro. ongoing. Blood. 2017;130:51. Trial Registration Ethics Approval ClinicalTrials.gov NCT03633110 This study will be approved by the Institutional Review Board (IRB) or Ethics Approval Independent Ethics Committee (IEC) at each participating institution The study was approved by Western Institutional Review Board, ap- prior to patient enrollment. proval number 1-1078861-1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 229 of 272 P418 paired peripheral blood analysis revealed a treatment-related in- Intratumoral IL-12 plus pembrolizumab combination therapy in crease of KLRG1+/CD127- SLECs as well as a treatment-related reduc- treatment refractory solid tumors: a safety and biomarker analysis tion of MDSCs in the periphery predominantly in responding Pablo Fernandez-Penas, MD, PhD , Matteo Carlino, MBBS, PhD, BMedSC, patients. 1 2 3 4 5 F , Victoria Atkinson, MD , Melinda Telli , Rohit Joshi , Sajev Thomas , Conclusions 6 4 7 Katy Tsai, MD , Rachel Roberts-Thomson , Andrew Haydon, MBBS PhD , TAVO + pembrolizumab continues to be well tolerated in patients 8 9 10 11 Andrew Mant , Tom Van Hagen , Katharine Cuff , Bianca Devitt , Igor with advanced solid tumors and peripheral blood analyses demon- 12 13 14 Puzanov, MD, MSCI, FACP , Marcus Butler, MD , Catalin Mihalcioiu , strates both local and most importantly, systemic signals of IL-12 me- 15 16 17 Hatem Soliman, MD , John Hyngstrom, MD , Mecker Moller , Gregory diated anti-tumor immunity in the absence of clinical signs of 18 19 20 Daniels, MD, PhD , Eric Whitman, MD, FACS , Erica Browning, BS , systemic IL-12 exposure. Thus, TAVO acts as an in situ vaccine to po- 20 20 20 20 Reneta Hermiz , Lauren Svenson , Jack Lee , Donna Bannavong , tentiate the anti-tumor activity of pembrolizumab with a favorable 20 20 20 20 Jendy Sell , Kellie Malloy , David Canton, PhD , Christopher Twitty , toxicity profile. 6 6 Adil Daud, MBBS MD , Alain Algazi, MD Trial Registration 1 2 Westmead Hospital, University of Sydney, Westmead, Australia; Princess NCT03132675; NCT03567720 Alexandra Hospital, University of Queensland, Woolloongabba, Australia; Ethics Approval Stanford University Medical School, Stanford, CA, United States; These studies were approved by the appropriate ethics committees. 4 5 Adelaide Oncology and Haematology, Adelaide SA, Australia; UF Consent Health Cancer Center, Orlando Health, Orlando, FL, United States; Written informed consent was obtained from the patient for publica- University of California San Francisco, San Francisco, CA, United States; tion of this abstract and any accompanying images. A copy of the 7 8 The Alfred Hospital, Melbourne, Australia; Box Hill Hospital, Box Hill, written consent is available for review. 9 10 Victoria, Australia; St. John of God Hospital, Subiaco, Australia; Princess Alexandra Hospital, Woolloongabba QLD, Australia; Eastern Health P419 Clinical School, Box Hill, VIC, Australia; Roswell Park Cancer Institute, A phase 1/2 study of GB1275, a novel CD11b modulator, as Buffalo, NY, United States; Princess Margaret Cancer Centre, Toronto, monotherapy and with an anti-PD-1 antibody in specified Canada; McGill University Health Centre, Montreal, QC, Canada; 15 16 advanced solid tumors or with chemotherapy in metastatic Moffitt Cancer Center, Tampa, FL, United States; Huntsman Cancer pancreatic cancer (mPDAC) Institute and Hospital, Salt Lake City, UT, United States; Sylvester 1 2 3 Johanna Bendell , Drew Rasco, MD , Wungki Park, MD , Lei Zhou, MD, Comprehensive Cancer Center, University of Miami, Miami, FL, United 18 4 4 4 4 MS , Anna Galkin, PhD , Debbie Slee, PhD , Laura Carter, PhD , David States; University of California San Diego, La Jolla, CA, United States; 19 20 4 4 4 4 Nickle, PhD , Rebecca Tran, MS , Jack Li, PhD , Beatrice Ferguson, MS , Atlantic Health System, Morristown, NJ, United States; OncoSec 4 5 3 Jakob Dupont, MD , Vineet Gupta, PhD , Eileen O'Reilly Medical Inc., San Diego, CA, United States 1 2 Sarah Cannon Research Institute, Nashville, TN, United States; The Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) START Center for Cancer Care, San Antonio, TX, United States; Memorial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P418 Sloan Kettering Cancer Center, New York, NY, United States; Gossamer Bio, San Diego, CA, United States; Rush University Medical Center, Background Chicago, IL, United States Intratumoral inflammation, including IL-12 expression and intratu- Correspondence: Johanna Bendell (ttobore@samornbiosciences.com) moral T cell infiltration, is a prerequisite for response to anti-PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P419 therapies. Previously, we demonstrated that enhanced intratumoral IL-12 expression via injection of plasmid IL-12 (tavokinogene telse- Background plasmid; TAVO) followed by electroporation (IT-tavo-EP) can increase Tumor influx of CD11b-expressing Myeloid-Derived Suppressor Cells TIL infiltration, ratios of CD8+ T cell:suppressive immune subsets, and (MDSCs) and M2 Tumor-Associated Macrophages (TAMs) creates an IFN-gamma gene signatures, converting weakly immunogenic tumors immunosuppressive tumor microenvironment that is associated with into highly inflamed, immunologically active lesions that regress with resistance to anti-PD-1 antibody therapy [1, 2, 3]. GB1275 is a novel, anti-PD-1 antibody therapy. Here, we present further support for our first-in-class, CD11b modulator that in vivo led to reduced MDSCs hypothesis that local IT-tavo-EP induces local and systemic immune and TAMs at the tumor site, repolarized M2 immuno-suppressive modulation with minimal systemic toxicity. TAMs towards an M1 phenotype, and subsequently increased tumor Methods infiltration of activated CD8+ T cells [4]. In combination settings with Melanoma (KEYNOTE-695) and mTNBC (KEYNOTE-890) patients were an anti-PD-1 antibody or chemotherapy, these immunomodulatory treated every three weeks with IT-tavo-EP on days 1, 5, and 8 of effects translated into potent anti-tumor effects and prolonged sur- every odd numbered cycle. Clinical toxicity was assessed at 3-week vival in orthotopic PDAC models [4]. We hypothesize that GB1275 ad- intervals and graded by CTCAE v4. In addition, pre- and post- ministration can alleviate myeloid cell-mediated immunosuppressive treatment tumor biopsies and peripheral blood samples were interro- effects and improve cancer treatment outcomes. gated for treatment-related changes in the frequency of CD8+ TIL Methods and other key IL-12-driven peripheral immune cell populations. In This is an open-label, first-in-human study consisting of a Phase 1 particular, we examined circulating short-lived effector T cells (SLECs, Dose Escalation phase with Regimen A using GB1275 monotherapy KLRG1+/CD127-), which are induced by IL-12 exposure, and granulo- and Regimen B using GB1275 with an anti-PD-1 antibody in pts with cytic myeloid derived suppressor cells (gMDSCs or PMN-MDSCs), pancreatic, esophageal, gastric/GEJ, triple negative breast, castration which serve a regulatory function, inhibiting effective anti-tumor im- resistant prostate, or Microsatellite Stable Colorectal Cancer (MSS mune responses. CRC) and Regimen C (GB1275 with Nab-paclitaxel + Gemcitabine Results (Nab-P+Gem)) in mPDAC, followed by a Phase 2 Expansion phase 62 patients were assessed including 46 patients with anti-PD-1 with three cohorts planned: newly diagnosed stage IV mPDAC, MSS antibody-refractory melanoma, and 16 patients with chemotherapy- CRC and PD-L1+ gastric/GEJ cancer. The study starts with Regimen A refractory mTNBC. TAVO in combination with pembrolizumab was with Regimen B starting after the completion of the first few cohorts well tolerated with only 2 of 46 (4.3%) patients from KEYNOTE-695 of Regimen A. Regimen C will start when Regimen A is completed. (cellulitis and presyncope) and 1 of 16 (6.3%) from KEYNOTE-890 Key Inclusion Criteria: Age ≥18 years, histologically confirmed locally (acute renal failure) experiencing grade 3 treatment-related adverse advanced/metastatic tumor specified, ECOG 0-1, prior immunother- events (TRAEs). Paired biopsies were available from both advanced apy is permissible in Dose Escalation phase for Regimen A and B, but melanoma patients and mTNBC patients. Flow cytometry on fresh bi- not for the expansion or Regimen C. Key Exclusion Criteria: untreated opsies from the KEYNOTE-695 revealed significant increases in CD8+ or symptomatic CNS metastasis, received prior myeloid targeting T cells after 1 cycle of treatment. Despite previous data demonstrat- agent or other prohibited medications, history of clinical significant ing non-detectable circulating IL-12 levels after treatment with TAVO, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 230 of 272 cardiovascular disease. Pts with active autoimmune disease requiring consistent with those expected from the poly-ICLC adjuvant alone, systemic therapy will be excluded from Regimen B. Primary objec- and no DLTs. ATLAS results show high interpatient variability as de- tives for the Dose Escalation phase are to determine the MTD/RP2D scribed previously. In an interim analysis of patients, vaccination has and PK profile of GB1275 monotherapy and in combination with an generated both CD8 and CD4 T cell responses measured by ex vivo anti-PD-1 antibody, and safety in combination with Nab-P+Gem. The fluorospot (Table 1). Ten-day in vitro stimulation (IVS) fluorospot as- primary objective for the Basket Expansion phase is to assess says confirm even broader immune responses. Overall, T cell re- efficacy. sponses were measured to 98% of administered peptides. Statistical Considerations: 3+3 design for the Dose Escalation Phase Conclusions and Simon’s 2-stage design for Expansion Phase. AEs graded per GEN-009 is a neoantigen vaccine that targets tumor specific immune CTCAE v5.0, responses per RECIST v1.1. The study is open for recruit- antigens recognized by the individual patient’s lymphocytes and ment and clinical trial registration on clinicaltrials.gov is pending likely expressed by tumor cells. Immunogenicity data show that (NCTxxxxx). ATLAS can, with very high frequency, identify relevant neoantigens and exclude putatively deleterious (immune inhibitory) antigens. References Clinical vaccination together with Standard of Care PD-1 blockade- 1. Fleming, V., Hu, X., Weber, R., Nagibin, V., Groth, C., Altevogt, P., et al. based regimens is in progress. Targeting myeloid-derived suppressor cells to bypass tumor-Induced im- Trial Registration munosuppression. Front Immunol. 2018; 9: 398. ClinicalTrials.gov NCT03633110 2. Kumar, V., Patel, S., Tcyganov, E. and Gabrilovich, D. I. The nature of Ethics Approval myeloid-derived suppressor cells in the tumor microenvironment. Trends The study was approved by Western Institututional Review Board, ap- Immunol. 2016; 37(3): 208-220. proval number 1-1078861-1. 3. Mantovani, A., Sozzani, S., Locati, M., Allavena, P. and Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized Table 1 (abstract P420). See text for description M2 mononuclear phagocytes. Trends Immunol. 2002; 23(11): 549-555. 4. Panni R., Herndon J., Zuo C., et al. Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. Sci Transl Med. 2019; 11: eaau9240. Ethics Approval The study was approved by the local IRB at each participating study site. P420 Broad immunogenicity from GEN-009, a neoantigen vaccine using ATLAS™, an autologous immune assay, to identify immunogenic and inhibitory tumor neoantigens 1 2 3 Roger Cohen, MD , Melissa Johnson, MD , Przemyslaw Twardowski, MD , P421 4 5 6 Mark Stein, MD , Ulka Vaishampayan, MD , Maura Gillison, MD, PhD , Lisa Phase 1 study of the safety, tolerability and preliminary anti-tumor 7 7 7 McNeil, PhD , Louisa Dowal, PhD , James Foti, PhD , Parul Agnihotri, activity of COM701 monotherapy in patients with advanced solid 7 7 7 7 PhD , Daniel DeOliveira, PhD , Manish Jain, MS , Jessica Price , Richard tumors 7 7 7 1 2 3 Hernandez , Arthur DeCillis, MD , Narinderjeet Singh, MS, MBA , Thomas Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD , Ryan 7 7 4 5 5 Davis, MD , Jessica Flechtner, PhD Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 1 2 6 7 7 University of Pennsylvania, Philadelphia, PA, United States; Sarah Adam ElNaggar, MD , John Hunter, PhD , Judy Olweny , Adeboye 3 7 8 9 Cannon Research Institute, Nashville, TN, United States; John Wayne Adewoye, MD , Bartosz Chmielowski, MD, PhD , Dale Shepard, MD PhD , 4 10 11 6 Cancer Institute, Duarte, CA, United States; Columbia University Medical Manish Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD , Drew 5 5 Center, New York, NY, United States; Karmanos Cancer Institute, Detroit, Rasco, MD 6 1 2 MI, United States; MD Anderson Cancer Center, Houston, TX, United The MD Anderson Cancer Center., Houston, TX, United States; The 7 3 States; Genocea, Cambridge, MA, United States University of Chicago, Chicago, IL, United States; Sarah Cannon Correspondence: Thomas Davis (tom.davis@genocea.com) Research Institute/TN Oncology, Nashville, TN, United States; 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P420 Massachusetts General Hospital, Needham, MA, United States; The START Center for Cancer Care, San Antonio, TX, United States; West Background Cancer Center and Research Institute, Memphis, TN, United States; 7 8 Tumor-specific neoantigens provide personalized targets for im- Compugen USA Inc, South San Francisco, CA, United States; University munotherapy. Vaccines against epitopes predicted by in silico ap- of California Los Angeles, Los Angeles, CA, United States; Cleveland proaches very rarely induce CD4+ and CD8+ ex vivo T cell responses Clinic, Cleveland, OH, United States; START - Midwest Cancer Center, regardless of formulation. ATLAS selects neoantigens for vaccine in- Chicago, IL, United States; Columbia University Medical Center, New clusion using ex vivo screening of all patient-specific mutations to York City, NY, United States identify pre-existing CD4+ or CD8+ T cell responses and to exclude Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) inhibitory peptides that may suppress immunity and potentially ac- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P421 celerate tumor progression. Preliminary data suggest that the inhibi- tory peptide profile may predict tumor response to immunotherapy. Background Methods COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of GEN-009-101 is a phase 1/2a study testing safety, immunogenicity poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It in- and clinical activity in immune responsive tumors (NCT03633110). hibits the binding of PVRIG with its ligand, PVRL2. PVRIG is a member After next-generation tumor sequencing and ATLAS testing of au- of the DNAM/TIGIT signaling axis regulating the activity of T/NK-cells. tologous leukocytes, each personalized vaccine is created using up In preclinical experiments we have demonstrated that PVRIG inhib- to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC. ition alone and in combination with anti-PD-1 and/or TIGIT blockers The immunogenicity pilot includes 8 patients in remission (NED), leads to activation of T cells in the tumor microenvironment generat- who received a course of GEN-009 monotherapy. ing an anti-tumor immune response and tumor growth inhibition [1]. Results Although ICI revolutionized cancer treatment, there is an urgent Eight patients have participated and reached the primary immuno- need to develop treatments for patients who are refractory or relapse genicity readout at day 50 (some data pending). The 24 doses given after treatment with ICI. We hypothesized that COM701 will be safe, across all patients have induced only grade 1/2 adverse events tolerable and demonstrate preliminary anti-tumor activity. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 231 of 272 Methods inhibits the binding of PVRIG with its ligand, PVRL2. Nivolumab is an A phase 1a, dose-escalation of COM701 monotherapy utilizing a hybrid anti-PD-1 antibody approved in patients with several malignancies accelerated and 3+3 study design was conducted to determine safety, [2]. PVRIG is a member of the DNAM/TIGIT signaling axis regulating tolerability, to assess the pharmacokinetics (PK), pharmacodynamics, to the activity of T/NK-cells. PD-1 inhibitors play an important role in determine the recommended phase 2 dose and to evaluate preliminary this axis by modulating DNAM activation [3]. In preclinical experi- anti-tumor activity of COM701. Patients with performance status ECOG 0- ments we have demonstrated that PVRIG inhibition alone and in 1 and advanced solid tumors who failed standard of care treatments combination with anti-PD-1 leads to activation of T cells in the tumor were eligible for inclusion. Prior ICIs were permissible. COM701 0.01, 0.03, microenvironment generating an anti-tumor immune response and 0.1, 0.3, 1, 3 and 10 mg/kg IV every 3 weeks were administered until pro- tumor growth inhibition [1]. Although ICI revolutionized cancer treat- gression, intolerable toxicity or investigator or patient discretion. Adverse ment there is an urgent need to develop treatments for patients events were reported per CTCAE v4.03 and anti-tumor activity was evalu- who are refractory or relapse after treatment with ICI. We hypothe- ated using RECIST v1.1. Dose-limiting toxicities (DLTs) were evaluated sized that COM701 will be safe and tolerable and demonstrate pre- within a 21-day window. Data cutoff date was August 09, 2019. liminary antitumor activity as monotherapy and in combination with Results nivolumab in patients with advanced solid tumors. A total of 13 patients were enrolled and treated during dose escalation Methods of COM701, including 6 patients with metastatic colorectal cancer (CRC), This is a phase 1 study with single patient cohorts and 3+3 study design 5 with microsatellite stable status (MSS) and 1 unknown. Patients were of COM701 in escalating doses as monotherapy IV Q3 weeks and in com- heavily pretreated with a median of 7 prior anticancer therapies (range 2- bination with nivolumab 360mg IV Q3 weeks. Key Inclusion Criteria: Age 15). No DLTs have been reported up to 10 mg/kg COM701 dose level. ≥18 years, histologically confirmed advanced solid tumor, performance The most frequent toxicities were fatigue (8%), abdominal pain (6%). status ECOG 0-1, prior anti-PD-1, anti-PD-L1, anti-CTLA-4, OX-40, CD137 Likely immune-related adverse events: elevated TSH and rash were ob- treatments are permissible. Key Exclusion Criteria: Active autoimmune dis- served in 2 patients. Overall 7/13 patients (54%) maintained best re- ease requiring systemic therapy in the last 2 years, symptomatic intersti- sponse of stable disease (SD) ≥12 weeks (13.6 – 43 weeks), including 5/6 tial or inflammatory lung disease, untreated or symptomatic central (83%) of patients with CRC. Five patients continue on study treatment. nervous system metastases. Primary objectives: to evaluate the safety Peripheral PVRIG receptor occupancy (≥90%) was demonstrated at and tolerability of COM701 monotherapy and in combination with nivo- ≥1mg/kg dose of COM701 and PK profile supports Q3 weekly dosing. lumab measured by the incidence of adverse events and dose-limiting Conclusions toxicities (21-day window), to evaluate the pharmacokinetics of COM701, COM701 monotherapy demonstrates an acceptable safety and toler- and to identify the maximum tolerated dose and/or the recommended ability profile with preliminary anti-tumor activity in a patient popula- dose for expansion as monotherapy and in combination with nivolumab. tion that had received multiple prior anti-cancer therapies. Updated Secondary objectives: to characterize the immunogenicity and prelimin- data will be presented at the conference. ary antitumor activity of COM701 in combination with nivolumab. Statis- Trial Registration tical Considerations: AEs will be reported as per CTCAE v4.03 and tumor Clinical trial identification: NCT03667716. responses will be evaluated per RECIST v1.1. Analyses of objectives are descriptive and hypothesis generating. Reference Results 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody At the time of submission no DLTs have been observed up to dose targeting the novel immune checkpoint PVRIG, for the treatment of level 7 of COM701 monotherapy and dose level 1 of COM701 in cancer. J Clin Oncol. 2017; (suppl; abstr 3074) combination with nivolumab 360mg IV Q3 weeks. Ethics Approval Conclusions The study was approved by each site's ethics board. Assessment of safety and tolerability is ongoing for all patients. Up- Consent dated results will be presented at the congress. Written informed consent was obtained from the patient for publication of Trial Registration this abstract and any accompanying images. A copy of the written Clinical trial identification: NCT03667716. consent is available for review by the Editor of this journal. References 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody P422 targeting the novel immune checkpoint PVRIG, for the treatment of Phase 1 study of COM701 monotherapy and in combination with cancer. J Clin Oncol. 2017; (suppl; abstr 3074) nivolumab in patients with advanced solid tumors 2. Nivolumab package insert. http://packageinserts.bms.com/pi/pi_opdivo.pdf. 1 2 3 Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD ,Ryan Accessed 07/22/2019. 4 5 5 Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 3. Wang B, Zhang W et al., Combination cancer immunotherapy targeting 6 7 7 Adam ElNaggar, MD ,JohnHunter, PhD ,JudyOlweny , Adewoye Adewoye, PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory 7 8 9 MD , Bartosz Chmielowski, MD, PhD ,DaleShepard,MDPhD ,Manish phenotype. Sci. Immunol. 2018; Nov 2:3(29). 10 11 6 5 Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD ,DrewRasco,MD Ethics Approval The University of Texas MD Anderson Cancer Center, Houston, TX, The study was approved by the Investigational Review Board/Ethics United States; The University of Chicago, Chicago, IL, United States; Committee of the participating sites. Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United States; Massachusetts General Hospital, Harvard Medical School, Needham, MA, United States; The START Center for Cancer Care, San P423 Antonio, TX, United States; West Cancer Center and Research Institute, SURPASS trial design: A phase 1 dose escalation trial to assess Memphis, TN, United States; Compugen USA Inc, South San Francisco, safety and efficacy of ADP-A2M4CD8 in HLA-A2+ patients with CA, United States; University of California Los Angeles, Los Angeles, CA, MAGE-A4+ tumors 9 10 2 3 4 United States; Cleveland Clinic, Cleveland, OH, United States; The David Hong, MD , Marcus Butler , Melissa Johnson, MD , Tanner 5 1 1 START-Midwest Center for Cancer Care, Chicago, IL, United States; Johanns , Francine Brophy , Rebecca Dryer-Minnerly, PhD , Trupti 11 1 1 1 Columbia University Medical Center, New York City, NY, United States Trivedi, MS , Rafael Amado, MD , Paula Fracasso, MD, PhD 1 2 Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) Adaptimmune, Philadelphia, PA, United States; MD Anderson Cancer Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P422 Center, Houston, TX, United States; Princess Margaret Cancer Centre, Toronto, Canada; Sarah Cannon, Nashville, TN, United States; Background Washington University in St. Louis, St. Louis, MO, United States COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of Correspondence: Paula Fracasso (paula.fracasso@adaptimmune.com) poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P423 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 232 of 272 Background tumors are presented. The study (NCT03548467) was approved by ADP-A2M4CD8 specific peptide enhanced affinity receptor (SPEAR) T- Central Ethics Committee in Heidelberg, Germany. cells are genetically engineered to target MAGE-A4+ tumors in the Methods context of HLA-A*02. ADP-A2M4CD8 are autologous CD4+ and CD8+ Patients with melanoma, NSCLC, clear RCC, urothelial cancer or SCCHN T-cells that express a high affinity MAGE-A4-specific T-cell receptor who did not reach complete responses after >12 weeks of immune (TCR) and an additional CD8α co-receptor. The ADP-A2M4 TCR is be- checkpoint inhibitor (CPI) therapy as standard of care were eligible. ing explored in a pilot study (NCT03132922), where clinical responses After patient-specific vaccine production, patients receive up to 14 vac- have been observed and the TCR has been well tolerated in doses cinations as intramuscular jet injections over a one-year period. CPI up to 10 × 10^9 transduced cells. Because CD4+ T-cells have a weak treatment continued. CT/MRI scans were performed according to hospi- effector function in response to class I antigens, a CD8α co-receptor tals’ routine. Immune responses were assessed by IFN-γ ELISpot. was genetically engineered alongside the TCR in ADP-A2M4CD8, to Results increase TCR binding avidity and enhance the polyfunctional re- At July 22, 2019 data cut-off, 15 patients (9 RCC, 4 SCCHN, 1 Melan- sponse of engineered CD4+ T-cells against MAGE-A4+ tumors. This oma, 1 NSCLC) had received ≤ 11 VB10.NEO vaccinations. Most com- approach is intended to widen the immune response to the tumor mon AEs were injection site reactions which all subsided within days. and improve depth and durability of clinical responses. 6 patients reported ≥ Grade 3 AEs, the most frequent ones were Methods injection-related hypertensive episodes normalizing within hours. In This phase 1, dose-escalation, open-label trial (SURPASS Trial) will the 4 patients assessed with low TMB (2 RCC, 2 SCCHN) strong T-cell characterize safety, tolerability, and antitumor activity across multiple responses towards 63% of selected neoepitopes were observed after tumor types. Patients who are HLA-A*02+ with MAGE-A4+ advanced 3-6 vaccinations. An amplification of existing neoepitope-specific T- esophageal, esophagogastric junction, gastric, head and neck cancers, cells (average of 250-fold increase) as well as de novo responses non-small cell lung, ovarian and urothelial carcinoma, melanoma, myx- were observed suggesting that VB10.NEO increases both the breadth oid/round cell liposarcoma, or synovial sarcoma and who meet all other and strength of the immune responses. inclusion criteria are eligible. Up to 30 patients will be enrolled. 10 patients were evaluable with >1 scan after VB10.NEO start (after Following apheresis, T-cells are isolated, transduced with CD8α_- being on CPI for 9-32 months). 4 patients (3 low TMB, 1 medium MAGE-A4c1032TCR, and expanded. Prior to ADP-A2M4CD8 infusion, TMB) started VB10.NEO with progressive disease (PD) development, patients will receive lymphodepletion consisting of fludarabine (30 of which 3 showed as stable disease (SD) in target lesions after vac- mg/m2/day x 4 days) and cyclophosphamide (600 mg/m2/day x 3 cination (followed up to 7 months), one developed PD after 5 days). During dose escalation, patients will be treated in one of three months. New lesions were detected in 2 patients. 6 patients (5 low ADP-A2M4CD8 dose groups. The initial dose of ADP-A2M4CD8 will TMB, 1 medium TMB) had SD at VB10.NEO start, 5 remained SD be 0.8 × 10^9 - 1.2 × 10^9 to be escalated to 1.2 × 10^9 - 3 × 10^9 (followed up to 9 months), while one had a best target lesion reduc- and then to 3.0 × 10^9 - 6.0 × 10^9 transduced cells in a modified 3 tion of 40%. Updated data will be presented. + 3 dose escalation scheme. Patients will be monitored for dose- Conclusions limiting toxicities (DLTs). Once the tolerability and safety of the lym- Vaccinations with VB10.NEO in addition to CPI were well tolerated. phodepletion regimen and cell dose has been demonstrated, the VB10.NEO induces strong T cell responses towards personalized dose range will increase to a maximum of 10 × 10^9 transduced neoepitopes; both novel T cell specificities and amplification of pre- cells in the expansion phase. Disease will be assessed per RECIST existing T cell responses were observed. Clinical signs of effect on v1.1 by CT/MRI at weeks 4, 8, 16, and 24, and every 3 months for 2 tumor size will continuously be monitored in the trial and early signs years, then every 6 months up to 15 years or until progression. Blood are promising. and tumor biopsy samples will be obtained pre- and post-infusion to Trial Registration evaluate safety, monitor persistence of transduced T-cells, and iden- NCT03548467 tify tumor-intrinsic correlates of response or resistance to therapy. Ethics Approval Trial Registration The study was approved by Central Ethics Committee in Heidelberg. NCT04044859 Ethics Approval P425 This trial is under review by the local institutional review boards for Phase 1b study of GX-I7, a long-acting interleukin-7, evaluating the each proposed study center. safety, pharmacokinetics and pharmacodynamics profiles in patients with advanced solid cancers 1 1 2 P424 Minkyu Heo , Minkyu Heo , Tae Won Kim, MD, PhD 1 2 Preliminary safety, efficacy and immunogenicity results from a Genexine, Inc, New York, NY, United States; Asan Medical Center, phase 1/2a study (DIRECT-01) of cancer neoantigen DNA vaccine Seoul, Korea VB10.NEO in patients with locally advanced or metastatic solid Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P425 1 2 3 1 Jürgen Krauss , Angela Krackhardt , Elke Jaeger , Anja Williams , Reza 3 4 4 4 Rafiyan , HEDDA WOLD, MSc , LIsa Gerner , Monika Sekelja , Agnete Background 4 4 4 Fredriksen, PhD , Karoline Schjetne , Mads Axelsen , Agnete Fredriksen, Cancer and treatment-related lymphopenia is associated with 4 4 PhD , Hedda Wold, MSc higher mortality in patients with various oncologic malignancies. 1 2 NCT, University Hospital Heidelberg, Heidelberg, Germany; Klinicum Interleukin-7(IL-7), a homeostatic cytokine of T lymphocytes, plays rechts der Isar, TUM, Munich, Germany; Krankenhaus Nordwest, a critical and non-redundant role in T cell development and Frankfurt, Germany; Vaccibody AS, OSLO, Norway homeostasis of mature T lymphocytes. IL-7 is a potent amplifier Correspondence: Hedda Wold (hwold@vaccibody.com) of naïve and memory T cells, thereby correcting T cell deficiency Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P424 and contributing to immune reconstitution. This may result in sig- nificant clinical benefit when combined with lymphopenia- Background inducing radiation/chemotherapy or immunotherapy where anti- Generation of potent neoantigen-specific T-cell responses has shown tumor effects are mediated by T cells. promising preclinical efficacy as well as clinical responses, especially Methods in patients with high tumor mutational burden (TMB). VB10.NEO is a A phase 1b study was conducted to assess the safety, pharmacokin- DNA vaccine with intrinsic adjuvant designed for delivery of 20 per- etics and pharmacodynamics of single-agent GX-I7(human IL-7 fused sonalized neoepitopes to antigen presenting cells. Preliminary results to the half-life extension hyFcTM) administered intramuscularly q3w from the ongoing phase 1/2a study treating patients with solid to advanced solid cancer patients who have no available effective Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 233 of 272 treatments (n=21). The dose escalation phase followed the 3+3 de- (NCT03416335). Primary objectives are to evaluate DSP-0509 safety/ sign of GX-I7 doses ranged from 60 to 1,200 μg/kg. Adverse events, tolerability, determine the maximum tolerated dose (Part A mono- PK, and subset analysis of peripheral blood monocytes(PBMCs) were therapy), and identify DSP-0509 monotherapy recommended phase evaluated. 2 dose (RP2D) and the RP2D of DSP-0509 in combination with pem- Results brolizumab for future studies. Secondary objectives include pharma- GX-I7 was well tolerated without DLT and cytokine release syn- cokinetics and antitumor activity. drome. Injection site reactions were the most common Methods treatment-emergent adverse events, which were Grade1 or 2 and Eligible patients are aged ≥18 years with advanced solid tumors (Part resolved. GX-I7 was slowly but steadily absorbed with a Tmax A and C) or melanoma or head and neck squamous cell carcinoma range of 12-48 hours with delay in higher doses. Following GX-I7 with acquired immune checkpoint inhibitor resistance (grouped high administration, up to 4-fold increase in absolute lymphocyte or low CD8+ cell density in tumor tissue; Part B). In Part A, approxi- count(ALC) were demonstrated. Importantly, the number of vari- mately 21–30 patients will be enrolled in each of the monotherapy ous subsets(naïve, TEM, TCM and TEMRA) of both CD4+ and and combination arms. DSP-0509 will be given as a constant rate IV CD8+ T cells was in a greater magnitude than that of ALC, infusion over 3 minutes at a fixed dose. Five provisional dose levels coupled with enhanced expression of Ki-67 peaked at day 7. of DSP-0509 may be tested, with approximately 3–6 patients at each Among T cell subsets, increase in naïve CD4+ and CD8+ T cells level (3 escalation levels at target doses of 0.3, 1, and 3 mg; 2 de- was most prominent. IL-7 receptor alpha(CD127) expression was escalation levels at target doses of 0.6 and 1.8 mg). During induction reduced during the first week, and recovered to baseline after treatment, patients will receive 5 doses of DSP-0509 every week for 4 2~3 weeks post GX-I7 administration. CCR5 expression in both weeks on days 1, 8, 15, 22, and 29 followed by every 2-weeks until CD4+ and CD8+ T cells increased transiently in a dose dependent discontinuation. In the combination arm, pembrolizumab will be ad- manner, suggesting GX-I7 promotes migration of T cell to tumor ministered IV at 200 mg every 3 weeks (Q3W). Dose limiting toxicities environment. No apparent increases in the number of other im- will be monitored within the first 6 weeks of dosing. The mono- and mune cells (NK cell, monocytes, B cells) were observed. combination DSP-0509 RP2Ds will be determined using a Bayesian Conclusions logistic regression model. In Part B, approximately 20–40 patients will A 3-week interval repeated IM administration of GX-I7 appears to receive DSP-0509 at the RP2D using the same dosing schedule as be well tolerated in dose range of 60 – 1,200 μg/kg in advanced Part A. In Part C, approximately 3–6 patients will be treated using the solid cancer patients. Following GX-I7 administration, dose- RP2D with the same induction treatment schedule as Part A, but dependent increase of ALC and T cell subsets(not Treg) were ob- followed by Q3W maintenance dosing. This study is currently recruit- served. These findings suggest that GX-I7 can be an excellent ing patients. combination partner for chemo-radiation, cancer vaccines and im- mune checkpoint inhibitors such as anti-PD-1/PD-L1 antibodies, P427 by increasing T lymphocytes and thereby contributing to en- A phase 1 study of IMC-001, novel anti-PD-L1 antibody, in patients hanced anti-tumor effects. with advanced solid tumors Trial Registration 1 1 Bhumsuk Keam, MD, PhD , Tae Min Kim, MD, PhD , Do-Youn Oh, MD, ClinicalTrials.gov Identifier: NCT03478995 1 1 2 PhD , Chan-Young Ock, MD, PhD , Won Ki Kang, MD, PhD , Yeon Hee Ethics Approval 2 2 3 Park, MD, PhD , Jeeyun Lee, MD, PhD , Ji Hye Lee, MD , Yun Jeong The study was approved by the Severance Hospital, Asan Medical 3 2 Song, MD , Young Suk Park, MD, PhD center, and Catholic Medical Center Institutional Review Board, proto- 1 2 Seoul National University Hospital, Seoul, Korea, Republic of; Samsung col number GX-I7-CA-003. Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, Republic of; ImmuneOncia Therapeutics Inc., Gyeonggi, South P426 Korea A first-in-human phase 1, multicenter trial of toll-like receptor Correspondence: Young Suk Park (pys27hmo@skku.edu) (TLR) 7 agonist DSP-0509 as monotherapy and in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P427 with pembrolizumab in adult patients with advanced solid tumors 2 3 4 Jared Weiss, MD , Anthony Olszanski, MD, RPh , Jordan Berlin, MD , Background 5 5 5 5 Makoto Origuchi , Zhonggai Li , Bella Ertik , Hongliang Cai , Daniel IMC-001 is a fully human IgG1 monoclonal antibody that binds to hu- 5 6 7 1 Clancy , Jose Iglesias , Vivek Subbiah, MD , Shadia Jalal, MD man PD-L1 and mediate the antibody-dependent cell-mediated cyto- Indiana University School of Medicine, Indianapolis, IN, United States; toxicity. The main objectives of this study were to evaluate the 2 3 UNC School of Medicine, Chapel Hill, NC, United States; Fox Chase safety, pharmacokinetics, and pharmacodynamics of IMC-001 in pa- Cancer Center, Phildelphia, PA, United States; Vanderbilt University tients with advanced solid tumors. Additional objectives were to ex- Medical Center, Nashville, TN, United States; Boston Biomedical Inc, plore the anti-tumor activity and identify the maximum tolerated Cambridge, MA, United States; Former employee, Boston Biomedical dose (MTD) of IMC-001. Inc, Cambridge, MA, United States; University of Texas, Houston, TX, Methods United States This is a phase 1, open-label study of IMC-001 in patients with meta- Correspondence: Shadia Jalal (sjalal@iu.edu) static or advanced solid tumors. IMC-001 was administered intraven- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P426 ously every two weeks with a standard 3+3 dose-escalation design until disease progression or unacceptable toxicity. Dose limiting tox- Background icity (DLT) window was defined as 21 days from the first dose. Ad- DSP-0509 is a TLR7 agonist designed to have high water solubility verse events (AEs) were assessed using CTCAE v4.03, and tumor allowing for intravenous (IV) administration and has rapid elimin- response was assessed by the Response Evaluation Criteria In Solid ation, partially due to excretion via organic anion transporting pep- Tumors, version v1.1. tide transporters. In preclinical models, DSP-0509 suppressed tumor Results volume and lung metastasis versus vehicle control. Furthermore, Fifteen patients (8 Male, 7 Female; Median age: 58 [range 39-69]) DSP-0509 in combination with anti–programmed cell death protein 1 were included in 5 dose escalation cohorts, dose ranging from 2 (PD-1) antibody suppressed tumor growth versus vehicle, DSP-0509 to 20 mg. Of the 15 subjects, 5 colorectal cancers, 3 biliary tract alone, or anti–PD-1 antibody alone. This 3-part dose escalation (Part cancers and 2 thymic cancers were included. No DLT was ob- A), dose expansion (Part B), and maintenance dose schedule evalu- served and the maximum tolerated dose was not reached. Most ation (Part C) study will investigate DSP-0509 alone or in combin- common AEs were decreased appetite, pyrexia, and cough. No ation with PD-1 inhibitor pembrolizumab (Part A only) Grade 4 or 5 treatment emergent AEs were reported during the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 234 of 272 study and no TEAE or serious AE led to treatment discontinuation Acknowledgements or death. There were no infusion-related reactions during this This study was funded by OSE Immunotherapeutics (the sponsor of the study. Two grade 2 serious AEs suspected to be related to IMC- study) in collaboration with Boehringer Ingelheim. 001 were seen in one subject at 2mg/kg cohort. Over the dose Trial Registration range 2 to 20 mg/kg IMC-001, AUC ,AUC ,and C gen- This study is registered under ClinicalTrials.gov Identifier: NCT03990233 0-14d 0—∞ max erally appeared to increase in a dose proportional manner for each step of dose escalation. Efficacy evaluation is ongoing and Reference will be presented in the future. 1. Gauttier V, Pengam S, Durand J, Morello A, Conchon S, Vanhove B and Conclusions Poirier N. Selective SIRPa blockade potentiates dendritic cell antigen IMC-001 demonstrated a favorable safety profile up to 20mg/kg cross-presentation and triggers memory T-cell antitumor responses. Can- given IV every 2 weeks in patients with advanced solid tumors. cer Res 2018;78(13 Supplement): abstr 1684. doi:10.1158/1538- Trial Registration 7445.AM2018-1684. Clinical trial identification : NCT03644056 Ethics Approval Ethics Approval The study protocol and its related documents (including the patient This study was approved by Institutional Review Board; approval information and informed consent form) received approval from the Ethics number SMC 2018-01-007-001 and H-1801-042-913. Committees, and the Competent Authority prior to study initiation. Each patient gave his/her written informed consent prior to study enrolment. P428 A phase 1 study evaluating BI 765063, a first in class selective P429 myeloid SIRPa inhibitor, as stand-alone and in combination with BI A phase 1/2a dose escalation and expansion study of HPN536, a 754091, a PD-1 inhibitor, in patients with advanced solid tumours mesothelin-targeting T cell engager, in patients with advanced 1 2 3 Nuria Kotecki, MD , Philippe Cassier , Jean-Pierre Delord, MD , Stéphane cancers expressing mesothelin who have failed standard therapy 4 1 2 3 1 2 2 Champiat , Christiane Jungels , Armelle Vinceneux , Iphigenie Korakis , Erika Hamilton, MD , Richard Austin, PhD , Sue Hirabayashi , Che-Leung 5 6 6 2 2 2 Richard Huhn , Nicolas Poirier , Dominique Costantini , Bérangère Law, PhD , Bryan Lemon, PhD , Holger Wesche, PhD , Debra Richardson, 6 4 3 Vasseur , Aurélien Marabelle MD 1 2 1 Institut Jules Bordet, Brussels, Belgium; Centre Léon Bérard, Lyon, Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United 3 4 2 3 France; IUCT, Oncopole, Toulouse, France; Gustave Roussy, Villejuif, States; Harpoon Therapeutics, South San Francisco, CA, United States; Sarah 5 6 France; Boehringer Ingelheim, Ridgefield, CT, United States; OSE Cannon Research Institute/Stephenson Cancer Center at the University of Immunotherapeutics, Nantes, France Oklahoma Health Sciences Center, Oklahoma City, OK, United States Correspondence: Nuria Kotecki (nuria.kotecki@bordet.be) Correspondence: Erika Hamilton (ehamilton@tnonc.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P428 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P429 Background Background Signal Regulatory Protein α [SIRPα] is a polymorphic protein, strongly HPN536 is a mesothelin-targeting T cell engager derived from the TriTAC expressed on myeloid suppressive cells. BI 765063 (OSE172), a hu- platform (Tri-specific T Cell-Activating Construct). Mesothelin (MSLN) is a manized IgG4 monoclonal antibody (mAb), is a selective antagonist tumor antigen overexpressed in malignant mesothelioma, ovarian carcin- of SIRPα/CD47 interaction, it does not bind to SIRPɣ, known to assist oma, pancreatic carcinoma, lung cancer, and triple negative breast cancer T cell co-stimulation and migration. BI 765063 strongly binds V1 al- with limited expression in normal tissues. HPN536 is a recombinant poly- lele, one of the 2 major functional allele of SIRPα expressed in more peptide of ~50kDa containing three humanized antibody-derived binding than 80% of general population and Asian (in 60%). domains, targeting mesothelin (for tumor binding), albumin (for half-life ex- Anti-tumor effect was shown in various in vivo cancer models using tension) and CD3 (for T cell engagement). It has been engineered to be a the validated anti-mouse SIRPα mAbs surrogate, as single agent. The small, globular protein to enable efficient exposure in solid tumor tissue effect was more pronounced in combination with T checkpoint inhib- with prolonged half-life and excellent stability under physiological condi- itors [1]. BI 765063 mechanism of action includes promotion of tions. HPN536 binds monomerically to CD3 and MSLN, minimizing non- tumor-antigen-presentation while preserving T-cell activation and in- specific T-cell activation. These features are designed to widen the thera- crease tumor phagocytosis. peutic index compared to earlier generations of T cell engagers by minimiz- The trial plans to assess the safety profile and preliminary efficacy of ing off target toxicities. HPN536 mediates potent target tumor cell killing in BI 765063, a first in class myeloid check point inhibitor antagonist of a MSLN-specific manner in vitro and in xenograft models in the presence of SIRPα on myeloid cells. T cells. Consistent with its mechanism of action (MOA), tumor cell killing is Methods accompanied by T cell activation, cytokine induction, and T cell expansion. This study comprises a dose escalation (step 1) to determine the Methods Dose-Limiting Toxicities, Maximum Tolerated Dose (MTD), and Rec- This is a Phase 1/2a, open-label, multicenter, dose escalation and ex- ommended Phase 2 Dose (RP2Ds) of BI 765063 monotherapy and pansion study to evaluate the safety, tolerability, clinical activity, and with BI 754091, and dose-confirmation expansion cohorts (step 2). pharmacokinetics of HPN536 in adult patients with advanced cancers In Step 1, ascending dose of BI 763063 once every 3 weeks intraven- expressing mesothelin who have failed standard available therapy. This ously (iv) using a Bayesian approach with overdose control are tested. study will be divided into 2 parts: Dose Escalation (Part 1) and Expan- When MTD determined, BI 763063 will be tested with BI 754091, a PD- sion (Part 2). Eligible patients with ovarian cancer will be enrolled in 1 mAb inhibitor. In step 2, 2 parallel randomized, non-comparative Dose Escalation. Dose expansion will include patients with ovarian can- mono and combination cohorts will further confirm the RP2Ds and as- cer, pancreatic carcinoma and mesothelioma. HPN536 is administered sess the safety and preliminary efficacy (RECIST 1.1 and iRECIST). once weekly as one-hour IV infusion by single-patient cohorts until ei- Patients ≥ 18 years, PS:0-1, with advanced solid tumor who failed or ther a Grade ≥2 adverse event (AE) that is possibly related to HPN536 is are not eligible to standard therapy will be included. V1/V1 and V1/ observed or an estimated therapeutic dose level has been reached. V2 patients (central testing) are evaluated in separate cohorts in step Then a conventional 3+3 design will be implemented. Dose escalation 1. In step 2, a selected population of V1/V1 patients with advanced- will continue until a recommended phase 2 dose (RP2D) is determined. stage cancers (e.g. non-small cell lung cancer, triple negative breast In dose expansion, up to 20 patients per group receive HPN536 at the cancer, or gastro-intestinal cancers) will be included. established RP2D based on a Simon 2-stage design. Patients may con- Pharmacokinetics (PK), SIRPα receptor occupancy (RO) and a compre- tinue weekly HPN536 treatment cycles until disease progression. Pri- hensive translational program (in blood and tumour) will assess PK/ mary endpoints are number and severity of DLTs following treatment PD profile and biomarkers of activity. with escalating doses of HPN536 during escalation, and overall re- A total of 116 (56 instep1 and60 in step2) patients willbe enrolled. sponse rate (by RECIST v1.1 for ovarian and pancreatic, mRECIST v1.1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 235 of 272 for mesothelioma) in dose expansion. Secondary endpoints include with a PD-1 inhibitor. After defining the MTD and/or RP2D, additional AEs, preliminary anti-tumor activity, pharmacokinetic and pharmacody- subjects may be enrolled at the respective dose schedules to further namic parameters based on the proposed MOA of HPN536. evaluate safety, pharmacodynamic effects, and anti-tumor activity Trial Registration Trial Registration NCT03872206 NCT04009681 Ethics Approval This study was approved by each participating institution's Institu- Table 1 (abstract P430). See text for description tional Review Board. P430 Open-label, multicenter phase 1/2 dose escalation and expansion study of THOR-707 as a single agent and in combination with a PD-1 inhibitor in adult subjects with advanced or metastatic solid tumors 1 2 3 David Luo , Raghad Abdul-Karim, MD , Arun Azad, MD , Joanna Bendell, 4 5 6 7 MD , Hui Gan, MBBS PhD , Filip Janku, MD, PhD , Shiraj Sen, MD, PhD , 8 9 1 1 Tira Tan, MBBS , Judy Wang, MD , Lisa Schechet , Lauren Baker, PhD , 1 10 Joseph Leveque, MD , Tarek Meniawy, MBBS FRACP 1 2 Synthorx Inc, La Jolla, CA, United States; NEXT Oncology, Texas Oncology, San Antonio, TX, United States; Peter MacCallum Cancer Centre, Melbourne, Austrailia; Sarah Cannon Research Institute, Nashville, TN, United States; Austin Hospital, Melbourne, Victoria, Australia; University of TX, MD Anderson Cancer Center, Houston, TX, United States; Sarah Cannon Research Institute at HealthONE, Houston, TX, United States; National Cancer Centre Singapore, Toronto, Canada; 9 10 Florida Cancer Specialists, Sarasota, FL, United States; Linear Clinical Research, Nedlands, WA, Australia Correspondence: Lauren Baker (lbaker@synthorx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P430 Background Recombinant interleukin-2 (aldesleukin), an approved immunotherapy in metastatic melanoma and renal cell carcinoma, can induce complete durable responses in some patients. The anti-neoplastic properties of IL-2 are mediated by activation of effector memory T cells and newly recruited naïve CD8+ T cells against the tumor. The widespread use of P431 IL-2 has been limited due to its high affinity bias for the IL-2 receptor Semi-mechanistic PK and target-occupancy modeling to support alpha chain (IL-2R⍺) on regulatory CD4+ T cells, leading to immunosup- dose justification for anti-PD-L1 clinical candidate CK-301 (TG- pression and, eosinophilic recruitment and activation on innate lymph- 1501) in oncology patients oid cells in the vascular endothelium causing vascular leak syndrome 1 1 2 3 Lin Lin, PhD , James Hilbert , Leonid Gorelik , Jian-Ping Tang , James (VLS). THOR-707 is a recombinant human IL-2 variant that is site- 4 1 1 1 Oliviero , Joshua Apgar , Lore Gruenbaum, PhD , John Burke specifically pegylated, providing a “not alpha” pharmacologic profile 1 2 Applied BioMath, LLC, Concord, MA, United States; Fortress Biotech, designed to prevent engagement of IL-2R⍺, thereby providing an im- Waltham, MA, United States; TG Therapeutics, New York, NY, United proved safety profile while still promoting newly recruited and effector States; Checkpoint Therapeutics, New York, NY, United States memory T cell anti-tumor activity In preclinical studies, eosinophilia was Correspondence: Lore Gruenbaum (lgruenbaum@gothamtx.com) not observed at a doses 10-fold higher than the dose responsible for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P431 eliciting maximal expansion of peripheral CD8+ T cells. Based on these findings, a first-in-human study of THOR-707 was started in June 2019. Background Methods Mathematical modeling was used in conjunction with in vitro, pre- This open-label, multicenter, dose escalation and expansion study in clinical and clinical data to facilitate dose selection of CK-301 (also adult subjects with advanced or metastatic solid tumors will evaluate known as TG-1501), an anti-PD-L1 monoclonal antibody (mAb), for THOR-707 as a single agent and in combination with a PD-1 inhibitor. ongoing and future clinical trials in oncology patients. Study objectives are to define the maximum tolerated dose (MTD) and/ Methods or recommended phase 2 dose (RP2D) of THOR-707 as single agent and A semi-mechanistic pharmacokinetic/target-occupancy (PKTO) model in combination with a PD-1 inhibitor; and to evaluate the overall safety was developed to predict pharmacokinetics (PK) of CK-301 at steady state and tolerability as well as, pharmacokinetics, pharmacodynamics, and and its tumor target occupancy (TO) under various dosing regimens. The preliminary anti-tumor activity. The study will be conducted in 3 parts. model captures the interactions between CK-301, PD-L1, soluble PD-L1 and PD-1 in 3 compartments: tumor, circulation (central) and other tissues (per- Part 1 will evaluate THOR-707 as a single agent across different dos- ipheral). The model was calibrated with CK-301 PK data from the first 5 pa- ing schedules (e.g., dosing every 2 weeks [Q2W] or 3 weeks [Q3W]). tients in a clinical study, CK-301-101, and PK data from published Phase 1 Part 2 will evaluate THOR-707 (Q3W) in combination with a PD- studies of 3 marketed anti-PD-L1 mAbs: atezolizumab, avelumab and durva- 1 inhibitor. lumab. Additionally, the model incorporated experimentally determined Part 3: Dose expansion will begin after the RP2D for THOR-707 as binding affinities for the 3 marketed anti-PD-L1 mAbs and CK-301. a single agent or in combination with a checkpoint inhibitor has Results been determined and will enroll selected populations (e.g., spe- Using the PKTO model, plasma Ctrough values and tumor TO of CK-301 at cific tumor types, treatment history, and/or biomarker profile). steady-state with 800 and 1200 mg q2w or q3w were projected. The TO of CK-301 were compared with predicted steady-state Ctrough TOs of atezoli- Between 50-100 subjects may be enrolled in the dose escalation phase zumab, avelumab and durvalumab at their marketed doses. The steady- to determine the MTD and/or RP2D as a single agent and in combination state Ctrough values of CK-301 are predicted to give >99% tumor TO for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 236 of 272 patients with a nominal or a 10-fold greater than nominal PD-L1 tumor bur- P433 den. This is similar to predicted TO for atezolizumab and durvalumab. The Initial results of the phase 1 portion of an ongoing phase 1/2 PKTO model was used to simulate PK and TO of CK-301 in 1000 virtual pa- study of RP1 as a single agent and in combination with nivolumab tients. The simulations predicted that, at 800 and 1200 mg q2w or q3w, in patients with solid tumors 1 2 3 ≥93.0% of patients with a nominal PD-L1 tumor burden or ≥80.1% of pa- M Middleton, MD PhD , Joseph Sacco , Jaime Merchan , Amber 3 4 5 tients with 10-fold higher than nominal PD-L1 tumor burden would have a Thomassen , Brendan Curti, MD , Ari VanderWalde, MD, MPH, MBioeth , 2 1 >99% tumorTOatsteady-stateCtrough. Anna Olsson-Brown, MBChB (Hons), BSc (Hons) , Francesca Aroldi , Nicos 6 5 7 Conclusions Fotiadis , Scott Baum , Howard Kaufman, MD, FACS , Kevin Harrington, At the proposed CK-301 dosing regimens of 800 and 1200 mg q2w or MD 1 2 q3w, a >99% TO is expected throughout the dosing interval. Relative to University of Oxford, Childrey, United Kingdom; University of Liverpool, atezolizumab and durvalumab treatments, similar percentages of pa- Liverpool, United Kingdom; University of Miami, Miami, United States; 4 5 tients would possibly benefit from CK-301 treatment. Providence Medical Center, Portland, OR, United States; West Cancer Center, Germantown, TN, United States; The Institute of Cancer Research, London, United Kingdom; Replimune, Woburn, MA, United States P432 Correspondence: Howard Kaufman (Howard.Kaufman@replimune.com) A phase 1 dose-escalation study of safety, tolerability, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P433 pharmacokinetics (PK) of ABBV-368 monotherapy and combination in patients (pts) with locally advanced or metastatic solid tumors Background 1 2 3 Christophe Le Tourneau , Wu-Chou Su, MD , Ki Chung, MD , Patricia Background: RP1 is an enhanced-potency oncolytic HSV-1 expressing 4 5 6 LoRusso, DO , Chia-Chi Lin, MD, PhD , Fabrice Barlesi, MD, PhD , Her- a fusogenic glycoprotein (GALV-GP R-) and GM-CSF which is being 7 8 9 Shyong Shiah , Eric Angevin, MD , Alexander Spira, MD, PhD, FACP , tested in a Phase 1/2 clinical trial in ~150 patients with a range of 10 11 Amita Patnaik, MD FRCP(C) , John Powderly, MD, CPI , Dimitrios solid tumors (NCT03767348). 12 13 14 Colevas , Helen Chew, MD , Maulik Patel, PharmD, PhD , Stacie Methods 14 14 14 14 Lambert , Yan Li , Daniel Da Costa , Martha Blaney, PharmD , Methods: The objectivesweretodefinethesafety of RP1 aloneand with 14 15 Michael McDevitt, MD, PhD , Philippe Cassier nivolumab, determine the recommended phase 2 dose (RP2D), and in 1 2 Institut Curie, Paris & Saint-cloud, France; National Cheng Kung University 30 patient phase 2 cohorts, assess efficacy in melanoma, non-melanoma Hospital, Tainan, Taiwan, Province of China; GHS Cancer Institute, skin cancer, urothelial carcinoma and MSI-H tumors. Initial phase 1 re- Spartanburg, SC, United States; Yale Cancer Center, New Haven, CT, United sults will be reported where patients were treated by intra-patient dose States; National Taiwan University Hospital, Taipei, Taiwan, Province of China; escalation of RP1 (up to 10mL of 104-108PFU/mL) by intratumoral injec- 6 7 Aix Marseille Univ Hôpitaux de Marseille, Livon, France; Taipei Medical tion into a single tumor Q2W up to 5 times followed by 12 patients University, Taipei, Taiwan, Province of China; Gustave Roussy, Villejuif, France; dosed 8 times at the RP2D combined with nivolumab (240mg Q2W for 9 10 Virginia Cancer Specialists Research Ins, Fairfax, VA, United States; South 4 months from the second RP1 dose, then 480 mg Q4W for 20 months). Texas Accelerated Research Thera, San Antonio, TX, United States; Carolina Clinically accessible lesions were directly injected, with imaging guidance BioOncology Institute, Huntersville, NC, United States; Stanford University, for deep/visceral lesions. Pre- and on-treatment tumor biopsies were ob- 13 14 Stanford, United States; UC Davis, Sacramento, United States; Abbvie Inc, tained for biomarker analysis. Viral shedding and anti-HSV antibody titers North Chicago, IL, United States; Léon Bérard Cancer Center, Lyon, France were also monitored. Correspondence: Michael McDevitt (michael.mcdevitt@abbvie.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P432 Results: 22 heavily pretreated patients with advanced tumors were enrolled into the dose-rising phase with largely low-grade adverse Background events, including febrile and other constitutional symptoms, local in- ABBV-368 is a humanized anti-OX40 monoclonal antibody. OX40 flammation and erythema. No clear differences were seen between is a member of the TNF receptor superfamily, which exerts its ac- superficial and visceral dosing. RP1 was detected at the injection site tion via activating T effector cells and inhibiting the suppressive and in blood for up to 14 days (next injection), suggesting virus repli- capacity of T reg cells. Preclinical data have shown ABBV-368 an- cation. All HSV seronegative patients seroconverted after three injec- titumor activity in animals. tions. Biological activity was demonstrated including tumor necrosis Methods and shrinkage, with extended clinical benefit and delayed (post-treat- This is a multicenter, phase 1, dose-escalation study (NCT03071757) ment termination and initial PD) systemic reduction in multiple tu- in pts (≥18 years; Eastern Cooperative Oncology Group performance mors in two patients (ipilimumab/nivolumab-refractory melanoma status 0–2) with locally advanced or metastatic solid tumors. The and chemotherapy-refractory cholangiocarcinoma) without interven- study consisted of 3 parts: 1) dose escalation (DE1); 2) cohort expan- ing treatment. The RP2D was selected as up to 10mL of 106PFU/mL sion (CE2); and 3) imaging substudy (IA3). Specific inclusion criteria followed Q2W by multiple doses of 107PFU/mL. Twelve evaluable pa- and dosing schedules for each cohort are shown in the table (Table tients (6 direct injection, 6 image-guided) were then enrolled into 1). In DE1, the primary endpoints are safety, tolerability, and PK of the phase 1 expansion combined with nivolumab. This demonstrated ABBV-368 monotherapy to establish the maximum tolerated dose or tolerability and clinical activity, including complete and partial re- reach the maximally administered dose; the secondary endpoint is sponses in patients with chemotherapy-refractory cutaneous squa- preliminary antitumor activity. Preliminary results for DE1 have been mous carcinoma, and ipilimumab/nivolumab-refractory melanoma. reported (Powderly et al. ESMO 2018). For CE2, the primary end- Treatment remains ongoing, and current data will be presented, in- points are safety, tolerability, and PK of ABBV-368 monotherapy and cluding biomarker data (CD8, PD-L1 staining and Nanostring analysis in combination with ABBV-181 (a humanized anti-programmed cell from tumor biopsies). death 1 monoclonal antibody), and to establish the recommended Conclusions phase 2 dose; the secondary endpoint is preliminary antitumor activ- Conclusions: The Phase 1 clinical data supports the safety and effi- ity of ABBV-368 monotherapy and combination therapy. The primary cacy of RP1 alone and when combined with nivolumab, including endpoints of the IA3 part are safety and tolerability. For all cohorts, demonstration of abscopal anti-tumor effects in patients refractory to AEs will be assessed according to the NCI CTCAE v4.03; response will prior checkpoint inhibitors. The Phase 2 portion of this clinical trial is be assessed Q2 months (mo) for 12 mo, and Q3 mo thereafter, as open in the US and the UK. per the immunotherapy Response Evaluation Criteria in Solid Tumors Trial Registration (iRECIST), and RECIST v1.1. As of 12 Jul 2018, enrollment into DE1 NCT03767348 was completed and CE2 has started. Ethics Approval Trial Registration The study was approved by applicable institutional review or ethics NCT03071757 boards. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 237 of 272 P434 2. Piccione et al. Preclinical and initial phase I clinical characterization of A phase 1/1b study to evaluate the humanized anti-CD73 CPI-006: an anti-CD73 monoclonal antibody with unique immunostimula- antibody, CPI-006, as a single agent, in combination with CPI-444, tory activity. Presented at Society for Immunotherapy of Cancer Meeting; and in combination with pembrolizumab in adult patients with November 7-11, 2018; Washington, DC, USA: Abstract P205. advanced cancers Ethics Approval 1 1 1 1 Mehrdad Mobasher , Richard Miller , Brian Munneke , Deborah Strahs , The study was approved by Western IRB, approval number 1-1066703-1. 1 1 1 Gabriel Luciano , Emily Piccione, PhD , Suresh Mahabhashyam , Jaime 2 3 4 Merchan , John Powderly, MD, CPI , Lauren Harshman, MD , Minal 5 6 7 8 Barve , Walter Stadler, MD , Patricia LoRusso, DO , Melissa Johnson , 9 10 Abhishek Tripathi, MD , Sumanta Pal, MD , Ben Markman, MBBS 11 12 13 FRACP , Jason Luke, MD, FACP , Thomas Marron, MD PhD 1 2 Corvus Pharmaceuticals Inc, Burlingame, CA, United States; University of Miami, Miami, United States; Carolina BioOncology Institute, Huntersville, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Mary Crowley Cancer Research Center, Dallas, TX, United States; University of Chicago Comprensive Cancer Center, Chicago, IL, United States; Yale University School of Medicine, New Haven, CT, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University of Oklahoma, Stephenson Cancer Center, Oklahoma City, OK, United States; City of Hope, Duarte, CA, United 11 12 States; Monash Health, Melbourne, Australia; UPMC, Pittsburgh, PA, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States Correspondence: Mehrdad Mobasher (mmobasher@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P434 Background CD73 expression is elevated in tumors and contributes to increas- ing levels of immunosuppressive adenosine in the tumor micro- environment. CD73 knockout mice exhibit reduced tumor growth and resistance to experimental metastasis. Inhibition of CD73 ac- tivity with an anti-CD73 antibody blocks adenosine production, Fig. 1 (abstract P434). See text for description shown to inhibit tumor growth in syngeneic models. Dual inhib- ition of CD73 and A2aR improves anti-tumor immune responses in mouse tumor models[1]. CPI-006 is a humanized IgG1 Fc gamma receptor binding-deficient anti-CD73 antibody that has a P435 dual mechanism of action. It blocks CD73 catalytic activity and A window of opportunity trial using intratumoral injection of adenosine production. In addition, it has immunomodulatory ac- glatiramer as an immune modulator in patients with resectable tivity on CD73 positive immune cells including B cells, T cells and head and neck and cutaneous squamous cell cancer antigen presenting cells. CPI-006 relieves adenosine-mediated im- Ghulam Rehman Mohyuddin, MD, Joaquina Baranda, MD, Andres Bur, munosuppression in vitro as a single agent and in combination Lisa Shnayder, Kiran Kakarala, Terry Tsue, Prakash Neupane, Gregory Gan, with ciforadenant[2]. CPI-006 is now being investigated in this Joshua Mammen, Daniel Aires, Sufi Thomas, Stephen Williamson, Nelli Phase 1/1b multicenter, open label trial as single agent (SA), in Lakis, Rashna Madan, Prabhakar Chalise, Scott Weir, Andrew Godwin, combination with ciforadenant, an oral, small molecule, selective Greg Reed, Cory Berkland A2aR antagonist and in combination with pembrolizumab, an Kansas University Medical Center, Kansas City, KS, United States anti-PD1 indicated for the treatment of patients across a number Correspondence: Joaquina Baranda (gioncol@gmail.com) of malignancies (NCT03454451). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P435 Methods Up to 462 subjects will be enrolled at approximately 35 sites in the Background US, Canada and Australia. Eligible patients with: non-small cell lung Immunotherapy using checkpoint inhibition improves outcome of cancer (NSCLC), renal cell carcinoma cancer (RCC), urothelial bladder patients with melanoma, lung, bladder, microsatellite instability-high cancer, cervical cancer, colorectal cancer, ovarian cancer, pancreatic and other tumors[1]. However, systemic administration of immuno- cancer, prostate cancer, head and neck cancer, triple-negative breast therapy may have limited activity in some tumors partly due to fail- cancer, endometrial cancer, select sarcomas and non-Hodgkin lymph- ure of activated T cells to migrate to tumor[1]. Intratumoral injections oma (NHL) who are relapsed, refractory or intolerant to 1 to 5 stand- (ITI) may allow high concentration of immunostimulatory products lo- ard therapies; aged ≥ 18 yo; with adequate organ function and cally while using small amounts of drugs. This may also facilitate mul- measurable disease. Study details is presented in Figure 1. tiple combination therapies and avoid systemic off-target toxicities. The primary objective of the dose escalation is to assess safety/ tolerabil- By capitalizing on existing data and experience, repurposing ap- ity, MTD or MDL of CPI-006 SA, in combination with ciforadenant and proved drugs for cancer represents an opportunity to rapidly ad- with pembrolizumab in ascending dose levels. Secondary objectives are vance promising therapies. to evaluate the PK of CPI-006 as SA or in combinations and analyze po- Glatiramer acetate is an agent commonly used for multiple scler- tential predictive biomarkers. In dose escalation, the primary objective is osis[2]. It acts as an immunomodulator and has essentially no sys- to assess the safety and tolerability of CPI-006 SA and in combinations in temic bioavailability but exhibits a high prevalence of injection site patients with selected advanced cancers. Secondary endpoints include reactions[2]. It upregulates the activity of natural killer cells in efficacy; PK of CPI-006 as SA and in combinations and to evaluate the re- leukemia cell lines [3], suggesting potential for immunostimulatory lationship between biomarkers and clinical activity. effect for ITI. For percutaneously accessible tumors for which the standard of care is surgical resection without any neoadjuvant ther- References apy, there exists a window of opportunity where ITI of glatiramer can 1. Young et al. Co-inhibition of CD73 and A2AR adenosine signaling im- be performed before surgery. proves anti-tumor immune responses. Cancer Cell. 2016; 30(3):391-403. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 238 of 272 Methods blood pressure, and must have provided a tumor sample evaluable for This is a proof-of-concept, investigator-initiated, window of opportunity PD-L1; and must not have immunodeficiency, active central nervous trial in subjects with percutaneously accessible head and neck or cuta- system metastases (except in GBM cohort), or be on steroid therapy neous squamous cell cancer who are to undergo surgery. Subjects will (patients with GBM may be on dexamethasone ≤2 mg/day orally or receive glatiramer 40 mg by ITI 3 times a week prior to surgery. Sub- equivalent and stable for 5 days at the time of enrollment). Patients will jects will receive at least one dose and up to 3 doses of glatiramer. receive lenvatinib 20 mg daily and pembrolizumab 200 mg every 3 About 10 eligible patients will be included in this trial. Safety data will weeks (Q3W) for ≤2 years, or until confirmed disease progression (may be collected. Tumor tissue at the time of diagnosis and at the time of continue lenvatinib if receiving clinical benefit), unacceptable toxicity, surgery will be collected and compared for biomarkers. Primary end- or study withdrawal. Patients with confirmed complete response may point is safety. Secondary endpoint is effect of ITI of glatiramer on bio- discontinue after ≥24 weeks combination therapy and ≥2pembrolizu- marker levels. Pre- and post-treatment tumor samples will be tested mab doses after initial complete response date. Tumor imaging will using an immunology panel that profiles immunology genes and pro- occur at baseline, Q9W (or for GBM patients: Q6W until 18 weeks, then teins including major classes of cytokines, interferons, KIR family, and Q9W) for the first 54 weeks, Q12W until week 102 (~2 years), and TNF-receptor. Tumors samples will also be evaluated for the Ki-67 pro- Q24W thereafter using RECIST v1.1/RANO by investigator assessment in liferative index and for caspase-3 and cleaved caspase-3 immunoex- the initial cohorts and by BICR after cohort expansion. Primary end- pression. Paired T-test or the Wilcoxon signed rank test will be used to points are objective response and safety (adverse events graded using assess the changes in the variables. We hypothesize that this approach NCI CTCAE v4.0, and discontinuation due to adverse events). Secondary will break the immunosuppressive tumor microenvironment as evi- endpoints include disease control, duration of response, progression- denced by an increase in inflammatory cytokines, chemokines, and im- free survival, and overall survival. Initially, approximately 180 patients mune cell infiltration. Decline in Ki-67 and increase in caspase-3 may be will be enrolled (30/cohort; each cohort may be expanded to 100 after a signal of anti-tumor activity. planned interim analysis). Enrollment is ongoing at 44 sites in 10 coun- Trial Registration tries across North America, South America, Europe, Asia, and Australia. NCT03982212 Acknowledgements References Writing support was provided by Shilpa Aggarwal, PhD, of C4 MedSolutions, 1. Whiteside, T.L., et al., Emerging Opportunities and Challenges in Cancer LLC (Yardley, PA, USA), a CHC Group company, funded by Eisai Inc. and Merck Immunotherapy. Clin Cancer Res, 2016. 22(8): p. 1845-55. Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. 2. Wingerchuk, D.M. and J.L. Carter, Multiple sclerosis: current and emerging Legal Entity Responsible for the Study: Eisai Inc. and Merck Sharp & Dohme disease-modifying therapies and treatment strategies. Mayo Clin Proc, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA 2014. 89(2): p. 225-40. 3. Maghazachi, A.A., K.L. Sand, and Z. Al-Jaderi, Glatiramer Acetate, Dimethyl Funding Source Fumarate, and Monomethyl Fumarate Upregulate the Expression of Funding for this research was provided by Eisai Inc. and Merck Sharp & CCR10 on the Surface of Natural Killer Cells and Enhance Their Chemo- Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. taxis and Cytotoxicity. Front Immunol, 2016. 7: p. 437. Trial Registration Ethics Approval NCT03797326 This study was approved by Kansas University Medical Center Institutional Ethics Approval Review Board, approval number: HSC00144030 An independent institutional review board or ethics committee approved the protocol at each study site, and the trial is being conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. P436 Phase 2 study of lenvatinib plus pembrolizumab in previously treated patients with solid tumors: LEAP-005 P437 1 2 3 4 5 Ravit Geva , Seock-Ah Im , Zarnie Lwin , Susan Weil , Lei Xu , Anne Disease-related biomarkers are associated with extended 5 5 6 Morosky , Kevin Norwood, MD , Hyun Cheol Chung, MD, PhD progression free survival after treatment with NEO-PV-01 in 1 2 Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; Seoul National combination with anti-PD1 in patients with metastatic cancers 3 1 2 University Hospital, Seoul, Korea, Republic of; University of Queensland, Patrick Ott, MD, PhD , Ramaswamy Govindan, MD , Aung Naing, MD, 4 3 4 5 6 Queensland, Australia; Eisai Inc., Woodcliff Lake, NJ, United States; FACP , Terence Friedlander, MD , Kim Margolin, MD , Jessica Lin, MD , 5 6 7 8 Merck & Co., Inc., Kenilworth, NJ, United States; Yonsei University Nina Bhardwaj, MD, PhD , Matthew Hellmann, MD , Mark Awad, MD 1 9 9 9 College of Medicine, Seoul, Korea, Republic of PhD , Amy Wanamaker , Lisa Cleary , Michael Rooney , Julian Scherer, 9 9 9 9 Correspondence: Ravit Geva (ravitg@tlvmc.gov.il) PhD , Meghan Bushway , Melissa Moles , Zakaria Khondker , Richard 9 9 9 9 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P436 Gaynor, MD , Lakshmi Srinivasan, PhD , Andrew Chi , Joel Greshock , Siwen Hu-Lieskovan, MD, PhD 1 2 Background Dana Farber Cancer Institute, Boston, MA, United States; Washington Lenvatinib (multiple receptor tyrosine kinase inhibitor of vascular endo- University, Saint Louis, MO, United States; MD Anderson Cancer Center, thelial growth factor receptors 1–3, fibroblast growth factor receptors 1– Houston, TX, United States; University of California San Francisco, San 4, platelet-derived growth factor receptor α,RET,and KIT) andanti–PD-1 Francisco, CA, United States; City Of Hope, Duarte, CA, United States; 6 7 inhibitor pembrolizumab have shown clinical benefit as monotherapies Massachusetts General Hospital, Boston, MA, United States; Mt. Sinai across multiple cancers. In preclinical studies, lenvatinib plus PD-1 block- Medical Center, New York, NY, United States; Memorial Sloan Kettering ade improved antitumor activity vs either agent alone. LEAP-005 Cancer Center, New York, NY, United States; Neon Therapeutics, (NCT03797326) evaluates the efficacy and safety of lenvatinib plus pem- Cambridge, MA, United States; Huntsman Cancer Institute, Los brolizumab in patients with previously treated selected advanced tumors. Angeles, CA, United States Methods Correspondence: Joel Greshock (jgreshock@neontherapeutics.com) This global, open-label, phase 2 study enrolls patients ≥18 years with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P437 the following previously treated histologically/cytologically confirmed advanced tumors: triple negative breast, ovarian, gastric, colorectal Background (non-MSI-H/pMMR), glioblastoma multiforme (GBM), or biliary tract (ex- Neoantigens arise from mutations in cancer cell DNA and are import- cluding ampulla of Vater). Patients must have progressed on or since ant targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a last treatment; have measurable disease per RECIST v1.1 (modified to personal neoantigen vaccine of up to 20 peptides designed by the follow ≤5 target lesions/organ [10 total]) or the RANO criteria (GBM RECON® bioinformatics platform using patient neoantigen and HLA only), assessed locally and confirmed by blinded independent central profiles. Here we report biomarker correlates of clinical benefit for review (BICR); ECOG performance score 0–1, adequately controlled NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 239 of 272 with nivolumab in anti-PD1 naïve metastatic melanoma, NSCLC and antigen recall studies (Hanson 2018) showed that soluble vopra- bladder cancer patients (NCT02897765). telimab stimulated a polyfunctional cytokine response only in Methods CD4 T cells that were ICOS hi, further supporting the hypothesis Patients received 12 weeks of nivolumab monotherapy (240 mgs that vopratelimab induces activation and proliferation of CD4 T Q2W), then NEO-PV-01 in a prime-boost format spanning 12 weeks, effector cells only after an initial priming event induces an ICOS nivolumab continued for up to 2 years. The primary objective was hi CD4 T cell phenotype. Furthermore, in melanoma patients safety, secondary objectives were overall response rate (ORR), treated with ipilimumab, a sustained increase in the frequency of progression-free survival (PFS), and overall survival. Comprehensive ICOS-positive CD4 T cells correlated with clinical benefit and sur- comparisons of baseline and serial molecular and immunological vival (Carthon 2010). In contrast, emergence of these ICOS hi cells characteristics between patients with vs. without durable PFS were has not been noted with PD-1/PD-L1 inhibitors (Hanson 2018). performed for all tumor cohorts. We hypothesized that the combination of vopratelimab with ipili- Results mumab will enhance the presence and functionality of ICOS hi A total of 34 melanoma, 27 NSCLC and 21 bladder cancer patients re- CD4 T effector cells, thereby potentially increasing the likelihood ceived nivolumab therapy, of which 27, 18 and 15 initiated vaccine of clinical benefit. respectively. The median follow up time was 13.4, 12.0 and 14.7 Methods months for melanoma, NSCLC and bladder cancer respectively. No This open label, multi-center, phase 2 study will evaluate efficacy, treatment-related serious adverse events were noted. The median safety, PK, and exploratory pharmacodynamics of vopratelimab in PFS for the melanoma cohort was not reached (95% CI: 3.3, NE), and combination with ipilimumab in adult patients with non-small cell the ORR was 47%. The median PFS in both the NSCLC and bladder lung cancer or urothelial cancer who have been previously cohort was 5.6 months (95% CI’s: 2.3, 8.7; 2.0, 8.1 respectively) with treated with PD-1/PD-L1 inhibitors. We expect to enroll approxi- ORR’s of 22% and 24% respectively. RECON tumor neoantigen abun- mately 200 evaluable subjects in total. Primary endpoint is ORR. dance was predictive of durable PFS in melanoma patients. Analyses Secondary endpoints include safety and tolerability, PFS, OS as of pre-treatment peripheral TCR repertoires reveal a more clonal T well as PK/PD. cell population in melanoma patients with extended PFS. Other fac- Trial Registration tors that associated with durable PFS included the abundance of B ClinicalTrials.gov NCT03989362 cells and CD8+ T cells in the tumor microenvironment. Finally, across Ethics Approval cohorts, longitudinal tumor biopsies from patients with extended Study was approved by the applicable Institution Ethics Boards PFS showed higher rates of initial pathologic responses after vaccin- ation vs. biopsies from patients with shorter PFS, suggesting vaccine- P439 related anti-tumor responses in this subset. Phase 1 first in human study of programmed cell death receptor- Conclusions 1(PD-1) inhibitor monoclonal antibody (mAb) JTX-4014 in adult NEO-PV-01 in combination with nivolumab is safe and leads to post- subjects with advanced refractory solid rumor malignancies vaccine immune and pathologic responses, indicating further clinical 1 2 Kyriakos Papadopoulos, MD , Gerald Falchook, MD , Nehal Lakhani, MD, evaluation is warranted. The association of baseline disease charac- 3 4 4 4 4 PhD , Gosia Riley , Jian Xu, PhD , Johan Baeck , Gilad Gordon , Elizabeth teristics with prolonged PFS suggests future patient enrichment 4 5 Trehu, MD , Judy Wang, MD strategies. 1 2 START, San Antonio, TX, United States; Sarah Cannon Research Inst. at Trial Registration Healthone, Denver, CO, United States; START-Midwest, Grand Rapids, NCT02897765 MI, United States; Jounce Therapeutics, Cambridge, MA, United States; Ethics Approval Florida Cancer Specialists - SCRI, Sarasota, FL, United States This trial has been approved by all institutional Review Boards of Correspondence: Kyriakos Papadopoulos every clinical trial site involved with this study. (Kyri.Papadopoulos@startsa.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P439 P438 Phase 2 Multicenter Trial of ICOS agonist vopratelimab and a Background CTLA-4 inhibitor in PD-1/PD-L1 inhibitor experienced adult JTX-4014 is a fully human mAb consisting of 2 identical hinge- subjects with Non-small Cell Lung Cancer or Urothelial Cancer stabilized immunoglobulin gamma 4 (IgG4, S228P) heavy and two (EMERGE) identical kappa (Igκ) light chains, that specifically binds to PD-1. The 1 1 2 Russell Pachynski, MD , Ramaswamy Govindan, MD , Ellen Hooper, MD , mechanism of action of JTX-4014 is to block the interaction of PD-1 2 2 2 Christopher Harvey, PhD , Amanda Hanson , Sean Lacey, MA , Rachel with its ligands, PD-L1 and PD-L2, and augment anti-tumor T-Cell ac- 2 2 2 2 McComb , Courtney Hart , Haley Laken , Johan Baeck , Elizabeth Trehu, tivity. This Phase 1 trial objectives were to evaluate the safety and MD tolerability of the drug along with its maximum tolerated dose (MTD) Washington University School of Medicine, St. Louis, MO, United States; and recommended Phase 2 dose. Jounce Therapeutics, Cambridge, MA, United States Methods Correspondence: Russell Pachynski (rkpachynski@wustl.edu) Key inclusion criteria included age ≥18 yrs, histologically or cytologic- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P438 ally confirmed extracranial solid tumor refractory to at least one prior line of therapy, no concurrent anticancer treatment, no prior anti-PD- Background 1 or anti-PD-L1 therapy, no requirement for selection based on PD- ICOS is a costimulatory molecule upregulated on activated T cells. L1 expression, no history of immune-mediated conditions, and ad- Vopratelimab (JTX-2011) is an IgG1 ICOS agonist monoclonal anti- equate renal, hepatic, and bone marrow function. The trial was a body known to activate and proliferate primed CD4 T effector standard 3+3 design with 5 fixed dose levels ranging from 80 mg cells in vitro, with established preclinical efficacy in multiple Q3wk to 1200 mg Q3wk given by IV infusion. In addition, there was tumor models. In the Phase 1/2 ICONIC trial (NCT02904226), one arm of 800 mg Q6wk. vopratelimab in patients with advanced solid tumors (Yap 2019) Results has shown to be safe and well tolerated as monotherapy and in 18 patients were enrolled in the trial (10 males, 8 females) with an combination with nivolumab. The ICONIC study showed no correl- average age of 66.3 yrs. Tumor types included ovarian (n=4), salivary ation between tumor reductions and ICOS and PD-L1 levels in gland, sarcoma, prostate and mesothelioma (n=2 each). The max- pre-treatment tumor samples by IHC. However, emergence of imum administered dose was 1200mg; MTD was not reached. There peripheral blood ICOS High (hi) CD4 T effector cells following were no deaths, no dose limiting toxicities. One treatment-related treatment with vopratelimab +/- nivolumab was associated with serious adverse event of pneumonitis occurred after the second dose tumor reductions and improved PFS and OS. In addition, ex vivo at 1200 mg Q3wk. Adverse events occurring in > 15% of patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 240 of 272 included fatigue, anemia, AST increased, dizziness, and tumor pain. 1 SA arm and 3 combination arms in which TPST-1120 is combined Only fatigue was noted as related in more than one patient (all with nivolumab, docetaxel or cetuximab. The RP2D of TPST-1120 to Grade 1 and 2). Grade 3 related AEs included increase alkaline phos- proceed to DEx will be determined by safety and biomarkers includ- phatase and pneumonitis. At time of data cutoff, median number of ing analysis of FAO/PPARα gene expression in the peripheral blood doses administered was 3 (range 1-11). Preliminary investigator and in tumor biopsies. The DEx arms will follow a 2-stage expansion assessed antitumor activity included: confirmed partial response (PR) design. This trial began accrual in May 2018 at U.S sites and is cur- in 1 patient with salivary gland carcinoma, unconfirmed PR in 1 pa- rently enrolling into the Monotherapy/Dose Escalation cohort. Expan- tient with ovarian cancer (both PD-L1+ by IHC) and best response of sion cohorts are projected to open in early 2019. The total sample stable disease in 6 patients. Systemic exposure of JTX-4014 increased size is up to 338 pts. dose proportionally; mean terminal half-life ranged from 11 to 17 Trial Registration days. JTX-4014 pharmacokinetics was comparable to other approved NCT03829436 anti-PD-1 mAbs. No anti-drug antibodies were observed. Ethics Approval Conclusions This study is being conducted in accordance with Good Clinical Prac- JTX-4014 is well-tolerated and appears to have similar qualities to tice and the Helsinki Declaration and has been approved by the known anti-PD-1 inhibitors in terms of pre-clinical and clinical charac- Western IRB/Copernicus Group, tracking # 20190182. teristics. Antitumor activity was observed in the difficult to treat population enrolled. Phase 2 testing JTX-4014 is planned. P441 Trial Registration ARTISTRY-2: a phase 1/2 study of subcutaneously administrated NCT03790488 ALKS 4230 as monotherapy and in combination with Ethics Approval pembrolizumab in patients with advanced solid tumors The study was approved by the relevant Institutions' Ethics Board 1 2 3 John Powderly, MD, CPI , Bradley Carthon, MD, PhD , Marc Ernstoff, MD , 4 5 6 Anthony Olszanski, MD, RPh , Stephen Liu, MD , Kelly Curtis, MD , 7 7 7 7 P440 Yangchun Du, PhD , Lei Sun, PhD , Emily Putiri, PhD , Yan Wang, PhD , 7 7 8 Phase 1/1b multicenter trial of TPST-1120, a peroxisome Heather Losey, PhD , Bruce Dezube, MD , Ulka Vaishampayan, MD 1 2 proliferator-activated receptor alpha (PPARα) antagonist as a Carolina BioOncology, Huntersville, NC, United States; Emory University, single agent (SA) or in combination in subjects with advanced Atlanta, GA, United States; Roswell Park Comprehensive Cancer Center, cancers Buffalo, NY, United States; Fox Chase Cancer Center, Phildelphia, PA, 1 2 2 5 John Powderly, MD, CPI , Saurin Chokshi, MD , Johanna Bendell, MD , United States; Georgetown University, Washington, DC, United States; 3 3 4 6 7 Leisha Emens, MD, PhD , Jason Luke, MD, FACP , Brian Francica , Chan Syneos Health, Phoenix, AZ, United States; Alkermes, Inc., Waltham, 4 4 4 8 Whiting, PhD , Thomas Dubensky, PhD , Ginna Laport, MD MA, United States; Barbara Ann Karmanos Cancer Institute, Detroit, MI, 1 2 Carolina BioOncology, Huntersville, NC, United States; Sarah Cannon United States Research Institute, Nashville, TN, United States; University of Pittsburgh, Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Pittsburgh, PA, United States; Tempest Therapeutics, South San Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P441 Francisco, CA, United States Correspondence: Thomas Dubensky (tdubensky@tempesttx.com); Background Ginna Laport (glaport@tempesttx.com) ALKS 4230 is an engineered fusion protein of circularly permuted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P440 interleukin-2 (IL-2) and IL-2 receptor α (IL-2Rα) designed to selectively bind the intermediate-affinity IL-2R for selective expansion of natural Background killer (NK) and CD8+ T cells (Figures 1 and 2). Compared with recom- Tumor cells initially rely on glucose consumption via aerobic glyco- binant human IL-2, ALKS 4230 exhibited enhanced pharmacokinetic lytic pathways. However, as tumor cells proliferate and metastasize in and selective pharmacodynamic properties in mice, resulting in im- an increasingly hypoxic tumor microenvironment (TME), tumors in- proved antitumor efficacy [1]. Intravenous dosing of ALKS 4230 is be- creasingly utilize fatty acid oxidation (FAO) as glucose stores are de- ing studied in the ARTISTRY-1 trial of patients with advanced solid pleted. FAO supports both tumor growth and suppressive immune tumors (NCT02799095), which has more than 50 patients enrolled cells in the TME, facilitating tumor progression. PPARα is a ligand- to date [2]. Here, we present a study investigating ALKS 4230 ad- activated nuclear transcription factor which regulates lipid metabol- ministered subcutaneously. Potential advantages of subcutaneous ism, FAO and inflammation. TPST-1120 is a first in class, oral, selective dosing over intravenous include: (i) lower peak serum drug con- PPARα antagonist that blocks transcription of PPARα target genes centrations with a prolonged exposure profile, which may result leading to a metabolic shift from FAO to glycolysis. Antagonism of in a milder safety profile and improved tolerability; (ii) lymphatic FAO in the TME leads to direct killing of tumor cells dependent on absorption, which may facilitate direct immunologic effects; and FAO and facilitates the cytotoxicity of effector cells. Preclinical studies (iii) a more convenient dosing schedule than daily inpatient intra- with various tumor models demonstrate efficacy of TPST-1120 as venous dosing. monotherapy and in combination with anti-PD1 antibodies and Methods chemotherapy. TPST-1120 has an IC50 of 0.04 nM with a >35 fold se- ARTISTRY-2 (NCT03861793) is a phase 1/2 study of ALKS 4230 ad- lectivity over other PPAR isoforms. ministered subcutaneously as monotherapy and in combination Methods with pembrolizumab in patients with advanced solid tumors. The We have initiated a phase 1/1b multicenter, open label trial to evalu- study will be conducted in 2 parts. In the first part (dose escal- ate TPST-1120 as a SA and in combination (combo) with other sys- ation; phase 1), multiple ascending doses of ALKS 4230 will be temic therapies including nivolumab, an anti-PD1 monoclonal administered subcutaneously every 7 days (q7d) or every 21 days antibody; docetaxel, a cytotoxic chemotherapeutic agent and cetuxi- (q21d) during a 6-week lead-in period. Injection site locations will mab, an anti-EGFR monoclonal antibody. The objectives are to 1) include the back of the arm, the thigh, or the abdomen. If the evaluate safety and tolerability of continuous dosing of TPST-1120 2) patient has tolerated ALKS 4230 monotherapy treatment, combin- identify a recommended phase 2 dose (RP2D) 3) evaluate efficacy, ation therapy with pembrolizumab (200 mg) administered as an and 4) evaluate PK/PD parameters. Eligibility criteria: 1) patients with intravenous infusion over 30 minutes q21d will be added to the advanced non-small cell lung, hepatocellular, renal cell, triple- ongoing ALKS 4230 regimen. In the second part (dose expansion; negative breast, urothelial, pancreatic, gastro-esophageal, castration- phase 2), ALKS 4230 will be administered subcutaneously at the resistant prostate, head and neck, or MSS colorectal cancer, or chol- selected recommended phase 2 dose (RP2D) and dosing schedule angiocarcinoma, or sarcoma; and 2) 1-5 prior therapies for metastatic from phase 1 in combination with pembrolizumab in 5 tumor- disease. This phase 1/1b adaptive design is composed of Dose Escal- specific cohorts of patients with non-small-cell lung cancer, small- ation (DEs) and Dose Expansion (DEx) cohorts. DEs consist of 4 arms, cell lung cancer, hepatocellular carcinoma, squamous cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 241 of 272 carcinoma of the head and neck, and squamous cell carcinoma Background of any tissue origin. Additional eligibility criteria for the study in- T-cell activation requires effective co-stimulation along with T-cell re- clude Eastern Cooperative Oncology Group performance status of ceptor (TCR) engagement. CD80 provides a well-characterized costi- 0 to 1 and adequate bone marrow, liver, and kidney function. mulatory signal by binding to CD28 on the surface of T cells. Outcomes include RP2D, safety, pharmacokinetics/pharmaco- Following activation, T cells upregulate CTLA4 on the cell surface dynamics, immunogenicity, and antitumor activity. Efficacy end- which binds to CD80 with higher affinity than CD28, disrupts effect- points include overall response rate, disease control rate, duration ive CD80-CD28 signaling, and inhibits T-cell activation. FPT155 is a of response, time to response, and progression-free survival and novel CD80 extracellular domain-Fc fusion protein that directly in- overall survival at 6- and 12-month milestones. duces T-cell activation and cytokine production by binding to CD28 and de-represses endogenous CD80-CD28 activity in the tumor Acknowledgements microenvironment by binding to CTLA4. FPT155 has potent efficacy The study is sponsored by Alkermes, Inc. Medical writing and editorial in syngeneic preclinical tumor models, including some that are not support was provided by Parexel and funded by Alkermes, Inc. responsive to agents targeting PD-1. FPT155 is not a superagonist as Trial Registration it requires separate, concurrent TCR engagement. ClinicalTrials.gov NCT03861793 Methods FPT155 is being investigated in a multi-center, open-label, first- References in-human phase 1 trial. The dose escalation portion of the trial 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of is currently enrolling patients with advanced solid tumors that ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic have progressed after treatment with available therapies. A tumor models alone and in combination with immune checkpoint minimum anticipated biological effect level (MABEL) based ap- inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. proach was used to select the initial dose in humans. Patients 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, receive a fixed dose of FPT155 every three weeks with single- McDermott DF, et al. Intravenous administration of ALKS 4230 as patient accelerated titration cohorts through the first four dose monotherapy and in combination with pembrolizumab in a phase I levels of 0.07, 0.21, 0.7 and 2.1 mg and a standard 3+3 dose- study of patients with advanced solid tumors. J Clin Oncol. escalation design for the subsequent 7, 21, 42, and 70mg dose 2019:37(Suppl). Abstract TPS2649. levels. The primary objective of the phase 1a portion of the trial Ethics Approval is to determine the recommended dose and evaluate the safety This study was approved by Ethics and Institutional Review Boards (IRBs) at and tolerability of FPT155. all study sites; IRB reference numbers 20182543 (Western IRB), 00006731 Results (Roswell Park Comprehensive Cancer Center), STUDY00000056 As of June 17, 2019, 7 patients have been treated on study with (Georgetown University, MedStar Health Research Institute). FPT155 doses ranging from 0.07-7mg; median age was 58 years, 57% had ECOG PS 1 and median number of prior therapies was 4 (range: 2-8). To date, no dose-limiting toxicities or ≥Grade 3 treatment- emergent adverse events (TEAEs) from causes other than disease progression have been reported. There have been no serious adverse events or ≥grade 3 TEAEs attributed to FPT155 and the only TEAE at- tributed to FPT155 in more than one patient has been fatigue (Gr1, Gr2; 1 pt each). 2/7 patients continue on treatment. Conclusions FPT155 as monotherapy has been well tolerated to date. Enrollment Fig. 1 (abstract P441). ALKS 4230 is a fusion of IL-2 and IL-2Rα of accelerated titration cohorts is complete with dose-escalation in 3+3 cohorts ongoing. Trial Registration ACTRN12618001955202 Ethics Approval The study was approved by IRBs at all participating study sites. P443 Expansion cohorts of non-small cell lung cancer (NSCLC) and castration resistant prostate cancer (CRPC) in COSMIC-021, a phase 1b study of cabozantinib plus atezolizumab 1 1 2 Nick Salgia, PhD , Sumanta Pal, MD , Santiago Ponce Aix, MD , Yohan 3 4 5 6 Loriot , Robert Dreicer , Ulka Vaishampayan, MD , Toni Choueiri , Patrick Fig. 2 (abstract P441). Cell activation by IL-2 and ALKS 4230 7 8 8 9 10 Schöffski , Giri Ramsingh , Amy Liu , Farah Lim , Joel Neal, MD, PhD , Neeraj Agarwal, MD 1 2 City of Hope, Duarte, CA, United States; University Hospital 12 de Octubre, Madrid, Spain; Institut de Cancérologie Gustave Roussy, Villejui, P442 France; University of Virginia School of Medicin, Charlottesville, VA, A phase 1 study of FPT155, a first-in-class CD80 extracellular United States; Karmanos Cancer Institute, Detroit, MI, United States; domain-Fc fusion protein, in patients with advanced solid tumors 2 3 4 5 6 7 Dana-Farber Cancer Institute, Boston, MA, United States; Leuven Jermaine Coward , Hui Gan, MBBS PhD , James Kuo , Michael Millward , 6 7 7 7 8 Cancer Institute, Leuven, France; Exelixis, Alameda, CA, United States; Gary Richardson , Wei Deng , Siddhartha Mitra , Maike Schmidt , Hong 7 8 1 9 10 Barts Cancer Institute, London, United Kingdom; Stanford University Xiang, PhD , Lisa Horvath , Amy Prawira, MD 1 2 11 Medical Center, Standford, CA, United States; Huntsman Cancer St. Vincent’s Hospital Sydney, Darlinghurst, Australia; Icon Cancer Institute, Salt Lake City, UT, United States Centre, Brisbane, Australia; Olivia Newton-John Cancer Center, Correspondence: Nick Salgia (jennifer.humbert@fishawack.com) Melbourne, Victoria, Australia; Scientia Clinical Research, Randwick, 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P443 Australia; Linear Clinical Research, Nedlands, Australia; Cabrini Hospital, Malvern, Australia; Five Prime Therapeutics, Inc, South San Francisco, Background CA, United States; Chris O’Brien Lifehouse, Camperdown, Australia Cabozantinib inhibits tyrosine kinases involved in tumor growth, Correspondence: Amy Prawira (amy.prawira@svha.org.au) angiogenesis, and immune regulation, including MET, VEGFR, RET, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P442 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 242 of 272 ROS1, and TAM family kinases (TYRO3, AXL, MER). Encouraged by with minimal toxicities at the dose levels studied (1×108 HPVSTs/m2) preclinical and clinical studies that suggested that cabozantinib pro- so far. However, most patients remained with disease after infusion. motes an immune-permissive environment, the safety and efficacy of A great challenge for HPVST therapy is to generate more specific and cabozantinib or cabozantinib in combination with atezolizumab are potent HPVSTs as ~30% of our HPVST products failed the potency being evaluated in the COSMIC 021 phase 1b study (NCT03170960) criterion, evaluated by γ-IFN ELISpot assay. The aim of this work was in solid tumors including NSCLC and CRPC. Cabozantinib has demon- to increase the potency and success rate of HPVST manufacturing. strated clinical activity as monotherapy in advanced NSCLC and in Methods previously treated CRPC [1,2]. Here we provide updated trial details The current manufacturing strategy uses peripheral blood mono- for expansion cohorts of NSCLC and CRPC patients. nuclear cells (PBMCs) as starting material for the enrichment and ex- Methods pansion of HPVSTs, in the presence of dendritic cells and cytokines. The dose-escalation stage of this global, open-label trial is com- Either low frequency or anergy of HPVSTs, even in HPV-exposed do- pleted; in the expansion stage, 20 combination cohorts are being en- nors, impede growth, and manufacturing failure is largely attributed rolled at the recommended dose of cabozantinib 40 mg QD PO + to non-specific T cell outgrowth. To overcome this problem, we eval- atezolizumab 1200 mg Q3W IV. uated CD45RA depletion of PBMCs to remove the bulk of non- NSCLC cohorts include patients with: (1) nonsquamous (nsq)NSCLC specific cells (naïve T cells and natural killer (NK) cells). The CD45RA with prior immune checkpoint inhibitor (ICI) therapy (anti–PD-1 or fraction also contains B-cells and T regulatory cells that may inhibit anti–PD-L1); (2) nsqNSCLC without prior systemic anticancer therapy specific outgrowth. We also evaluated the use of an HLA-negative for metastatic disease; (3) EGFR-mutant nsqNSCLC with prior EGFR- universal lymphoblastoid cell line (uLCL), developed in our center, as targeting therapy. An additional exploratory cohort will assess cabo- a co-stimulatory cell line to rapidly expand the cells while maintain- zantinib monotherapy (60 mg) in nsqNSCLC patients with prior ICI ing HPV specificity. therapy. Results CRPC cohorts include patients with: (1) metastatic CRPC adenocarcin- HPVSTs manufactured using CD45RA negative PBMC populations as oma with measurable disease and prior enzalutamide and/or abira- starting material consistently displayed overarchingly higher specifi- terone therapy; (2) high-risk (measurable visceral metastasis or city than HPVSTs manufactured from PBMCs. Interestingly, uLCLs not prostate-specific antigen doubling time of only supported exponential growth of HPVSTs, but increased their The study allows an initial enrollment of 30 patients in each cohort HPV specificity, opening the possibility of producing sufficient with potential for expansion per recommendation by the Study Over- HPVSTs for higher dose levels. We reported successful production sight Committee. Based on preliminary efficacy per RECIST v1.1 and using PBMC from HPV-exposed healthy donors and cancer patients, safety, the original cohorts of nsqNSCLC with prior ICI therapy and and greatly improved HPVST specificity in all. metastatic CRPC adenocarcinoma with measurable disease and prior Conclusions enzalutamide and/or abiraterone therapy are being expanded to 80 These changes will be incorporated in our HPVST manufacturing patients each. protocol with the goal of improving the anti-tumor activity of our The primary endpoint of the expansion stage is the objective re- product. sponse rate for each cohort. Exploratory objectives include correl- ation of tumor and plasma biomarkers and immune cell profiles with References clinical outcome. 1. Centers for Disease Control and Prevention https://www.cdc.gov/cancer/ Trial Registration hpv/index.htm NCT03170960 2. Forastiere AA, Ang KK, Brizel D, et al. Head and neck cancers. J Natl Compr Canc Netw. 2008; 6:646–695. References 3. 11. Greer BE, Koh WJ, Abu-Rustum N, et al. Cervical cancer. J Natl Compr 1. Smith DC, Smith MR, Sweeney C, Elfiky AA, Logothetis C, Corn PG, Canc Netw. 2008; 6:14–36 Vogelzang NJ, Small EJ, Harzstark AL, Gordon MS, Vaishampayan UN. J Ethics Approval Clin Oncol. 2013; 31:412-419. This study was approved by Baylor College of Medicine Institutional Review 2. Drilon A, Rekhtman N, Arcila M, Wang L, Ni A, Albano M, Van Board; approval number H7634, H7666 and HESTIA and HESTIA IND. Voorthuysen M, Somwar R, Smith RS, Montecalvo J, Plodkowski A. Lancet Oncol. 2016; 17:1653-60. P445 Preliminary results of a Phase 1 trial with a personalized P444 neoantigen vaccine (ADXS-NEO) in advanced and refractory cancer The quest for highly potent human papillomavirus-specific T patients 1 2 3 Lymphocytes for Adoptive Immunotherapy of HPV-associated Frank Tsai, MD , Jonathan Goldman, MD , Marc Matrana , Sumitra 4 4 4 4 malignancies Sheeri , John Heyburn , Megan Parsi , Andres Gutierrez, MD PhD , Joel 1 2 2 2 2 Pei Yun Teo, PhD , Sandhya Sharma, BSc , Alex Salyer , Dimitrios Hecht , Joel Hecht 2 2 2 3 1 Wagner , Benjamin Shin , Sachin Thakar , Li-Chun Huang , Shian Jiun Honor Health Virginia Piper Cancer Care, Scottsdale, AZ, United States; 3 2 2 2 Shih , Carlos Ramos , Cliona Rooney, PhD UCLA Jonsson Comprehensive Cancer Center, Paramus, NJ, United 1 3 4 Tessa Therapeutics/ Baylor College of Medicine, Houston, TX, United States; Ochsner Cancer Center, New Orleans, LA, United States; Advaxis 2 3 States; Baylor College of Medicine, Houston, TX, United States; Tessa Inc, Princeton, NJ, United States Therapeutics, Singapore, Singapore Correspondence: Joel Hecht (JRHecht@mednet.ucla.edu) Correspondence: Cliona Rooney (crooney@bcm.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P445 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P444 Background Background ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based im- The human papillomavirus is linked to 42,700 new cases of cancers munotherapy. This vaccine is a bioengineered Lm vector that se- each year [1]. While many HPV-associated cancers can be eradicated cretes an antigen-adjuvant fusion protein consisting of up to 40 by multimodal therapies, recurrent diseases have dismal prognosis unique (personal) neoantigens and a truncated fragment of listerioly- [2,3]. HPV-positive tumors express viral antigens (E6 and E7) that are sin O (tLLO), which has adjuvant properties. Preliminary clinical and recognized by HPV-specific T cells (HPVST). We are evaluating the immunogenicity results from two dose-levels of ADXS-NEO mono- adoptive transfer of ex vivo expanded autologous HPVSTs for the therapy evaluated in the ongoing Phase 1 trial are herein reported. treatment of HPV-positive cancers in a phase I clinical trial (HESTIA). Methods To date, 12 patients have been treated and promising outcomes ADXS-NEO-02 is a phase 1 dose-escalation study of ADXS-NEO mono- have been attained- 1 complete response and 1 partial response, therapy in subjects with advanced and refractory metastatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 243 of 272 microsatellite stable-colon cancer (MSS-CRC), metastatic squamous was also examined. Furthermore, the possibility of combination treatment histology head and neck cancer, and metastatic non-small cell lung of TAS-116 and anti-PD-1 mAb were investigated in animal models cancer (NSCLC). Manufacturing of ADXS-NEO starts with whole ex- Results ome sequencing of each pt-matched normal and tumor samples to TAS-116 significantly reduced Treg cells, particularly effector Treg cells in detect genetic alterations in the coding regions of the genome both peripheral blood and the TME, resulting in augmentation of tumor followed by its production under GMP specifications. ADXS-NEO is in- antigen-specific CD8+ T cells. STAT5, one of the HSP90 client proteins fused intravenously every 3 weeks until disease progression or limit- that is important for Treg cell development, maintenance and function ing toxicity. Main endpoints include safety, tolerability, preliminary was degraded by TAS-116, thereby reducing FoxP3 expression in effector efficacy and immune-correlative data. Treg cells. TAS-116 augmented tumor antigen-specific CD8+ T cells in Results animal models with reduction of Treg cells in the TME. Additionally, com- The turnaround time for manufacturing ADXS-NEO has consistently bination treatment with PD-1 blockade exhibited a far stronger antitumor been 7-8 weeks from biopsy to first dose. Two pts treated at 1X109 effect than either treatment alone. Moreover, in a phase I trial, the com- CFU (dose level 1) experienced dose limiting toxicities (i.e., Gr 3 hyp- bination of an anti-PD-1 mAb and TAS-116 exhibited a notable clinical ef- oxia ± Gr 3 hypotension) within 4 hours of completing the infusion ficacy in patients with microsatellite-stable (MSS) colorectal cancer of the second dose. These acute adverse events were manageable accompanied by effector Treg cell reduction in the TME. and reversible with tocilizumab and/or steroids. A de-escalated dose Conclusions of 1X108 CFU, has been found to be safe, tolerable and immuno- We propose a novel concept to control eTreg cells by targeting a genic in a cohort of 3 pts. ADXS-NEO at both doses induced: 1) acti- Treg cell-critical signaling pathway and the potential as a combin- vation and proliferation of CD4+ / CD8+ T cells; 2) neoantigen- ation therapy with PD-1 blockade. specific T cell responses -including hotspot mutations- after 1 week Trial Registration of the initial priming dose in pooled ELISPot analysis and 3) T cell re- UMIN000032801 sponses to neoantigens found in the pts’ tumor, but not included in Ethics Approval the construct (i.e., antigen spreading). Deconvolution ELISPot data This study was approved by the institutional review boards of the Na- from the first MSS-CRC pts. analyzed, showed T cell responses to tional Cancer Center and was conducted in accordance with ethical 90% of the targets in the ADXS-NEO construct. Two out of 4 initial guidelines, including the Declaration of Helsinki.All mouse experiments pts treated had stable disease. were approved by the Animals Committee for Animal Experimentation Conclusions of the National Cancer Center, Japan, and Taiho Pharmaceutical Co. Ltd. A safe and tolerable dose of ADXS-NEO monotherapy has been established (1X108 CFU) which elicited fast and broad antitumor im- P447 munity, including T cell responses to neoantigens and antigen ALKS 4230, an engineered IL-2 fusion protein, in monotherapy spreading. Enrollment in a combination therapy arm with pembroli- dose-escalation and combination therapy with pembrolizumab in zumab is due to start in 4Q2019. patients with solid tumors: ARTISTRY-1 trial Trial Registration 1 2 Ulka Vaishampayan, MD , Jameel Muzaffar, MD , Vamsidhar Velcheti, MD, NCT03265080 3 4 5 FACP , Christopher Hoimes, DO , Lucy Gilbert, MD , David McDermott, Ethics Approval 6 7 8 9 MD , Anna Spreafico, MD PhD , Quincy Chu, MD , Kelly Curtis, MD , This clinical tria has been performed in accordance with the Declar- 10 10 10 Yangchun Du, PhD , Harald Mackenzie, MB , Lei Sun, PhD , Emily ation of Helsinki and has been approved by appropriate ethics com- 10 10 10 Putiri, PhD , Heather Losey, PhD , Bruce Dezube, MD , Marc Ernstoff, mittee at UCLA LA, Ochsner Cancer Center LA and Honor Health MD Virginia G Piper Cancer Care AX. Barbara Ann Karmanos Cancer Institute, Detroit, MI, United States; 2 3 Moffitt Cancer Center, Tampa, FL, United States; Perlmutter Cancer P446 Center, NYU Langone Health, New York, NY, United States; UH A novel regulatory T Cell-Targeted Immunotherapy by targeting Cleveland Medical Center, Cleveland, OH, United States; Cedars Cancer their crucial signal by HSP90 inhibitors Center, Montreal, QC, Canada; Beth Israel Deaconess Medical Center, Ayaka Tsuge, MD, Yosuke Togashi, MD, PhD, Kohei Shitara, MD, Hiroyoshi Milton, MA, United States; Princess Margaret Cancer Centre, Toronto, Nishikawa, MD, PhD ON, Canada; Cross Cancer Institute, University of Alberta/Alberta Health National Cancer Center, Kashiwa, Japan Services, Edmonton, AB, Canada; Syneos Health, Phoenix, AZ, United 10 11 Correspondence: Hiroyoshi Nishikawa (hnishika@east.ncc.go.jp) States; Alkermes, Inc., Waltham, MA, United States; Roswell Park Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P446 Comprehensive Cancer Center, Buffalo, NY, United States Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P447 Cancer immunotherapy, particularly immune checkpoint inhibitors opened a new era of cancer therapy. Yet, the clinical efficacy is limited Background due to the complexed immune suppressive mechanisms in the tumor ALKS 4230 is an engineered fusion of IL-2 and IL-2Rα designed to se- microenvironment (TME). Regulatory T (Treg) cells, an immune suppres- lectively expand NK and CD8+ T cells (Figures 1 and 2). In preclinical sive subset of CD4+ T cells, are abundant in tumor tissues and play a key studies, ALKS 4230 exhibited enhanced pharmacokinetic and select- role as an immune suppressive mechanism in the TME via inhibiting ef- ive pharmacodynamic properties with improved antitumor efficacy fective antitumor immunity. While various Treg cell-targeted reagents is relative to IL-2 [1]. under development, none of them have not been translated into the Methods clinic due to the difficulty of specific Treg cell depletion in the TME. The ARTISTRY-1 (NCT02799095) is a phase 1/2 study investigating major obstacle to develop effective Treg cell-targeted reagents was the ALKS 4230 as monotherapy and in combination with pembrolizu- lack of molecules specifically expressed by Treg cells in the TME. We mab in adults with advanced solid tumors [2]. For monotherapy therefore focused on the specific signal(s) used in Treg cells in the TME. dose escalation, ALKS 4230 is administered intravenously over 30 Methods minutes once daily for 5 days every 14 or 21 days. For combin- We focused on HSP90 inhibitor, TAS-116 as a Treg cell regulator, especially ation therapy, the same regimen of ALKS 4230 is administered terminally-differentiated effector Treg cells. Peripheral blood mononuclear with pembrolizumab every 21 days in cohorts based on tumor cells (PBMCs) were treated with TAS-116, and the changes in T cell popula- type, prior anti-PD-1 therapy, and rollover from monotherapy. tions including Treg cells were analyzed. In addition, we explored the Outcomes include the monotherapy recommended phase 2 dose mechanism(s) of Treg cell reduction using PBMCs and FoxP3+ T cell lines. (RP2D), safety, pharmacodynamics, and antitumor activity (RECIST The effect of TAS-116 on tumor antigen (NY-ESO-1)-specific CD8+ T cells 1.1). Results of the completely enrolled cohorts of dose-escalation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 244 of 272 phase and of combination therapy in anti-PD-1-unapproved tu- mors as of June 21, 2019, are presented. Results For dose escalation, 36 patients received ALKS 4230 monotherapy ≤6 μg/kg/d. Maximum tolerated dose has not been reached. Most fre- quent adverse events (AEs), regardless of relationship, were pyrexia (75%) and chills (72%); the majority were grades 1 or 2. Grade ≥3 AEs related to ALKS 4230 occurred in 11 patients (31%) and were mainly transient leukopenia. One death from aspiration pneumonia was considered unrelated to ALKS 4230 by the investigator. ALKS 4230 induced dose-dependent increases in circulating NK and CD8+ T cells with minimal, non-dose-dependent effects on regulatory T Fig. 2 (abstract P447). ALKS 4230 structure and activity cells (Tregs). At 3 and 6 μg/kg/d, 8 of 14 patients with evaluable scans had stable disease. One patient with heavily pretreated pancre- atic adenocarcinoma had prolonged stable disease with 6+ months of monotherapy; CA19-9 decreased from 2571 U/mL (pretherapy) to P448 673 U/mL (nadir). Data from 20 patients enrolled in the combination Phase I study of Veliparib and Nivolumab in adults with refractory therapy cohort of PD-1-unapproved tumors indicate no new toxic- advanced solid tumor and lymphoma ities; 7 of 11 patients with evaluable scans had stable disease or bet- Young Kwang Chae, MD, Pedro Viveiros, MD, Sheetal Kircher, Valerie ter. One patient (ovarian cancer) had confirmed partial response; CA- Nelson, Aparna Kalyan, Devalingam Mahalingam 125 normalized from a peak of 282 to 24.5 U/mL after 2 months of Northwestern University, Chicago, IL, United States therapy. Correspondence: Young Kwang Chae (ychae@nm.org) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P448 ALKS 4230 is a promising agent with acceptable tolerability and pre- liminary clinical benefit. It selectively expanded CD8+ T cells and NK Background cells with minimal Treg expansion. The intravenous monotherapy Tumors with high mutation burden often respond to immunother- RP2D was established as 6 μg/kg/d. Safety and pharmacodynamic apy. Veliparib, a Poly (ADP-ribose) polymerase (PARP) inhibitor carries data enabled selection of the 3 μg/kg dose for initial evaluation in anti-neoplastic activity by accumulating DNA damage, possibly en- combination with pembrolizumab. hancing the effect of checkpoint inhibitors. Here we report safety and preliminary efficacy of veliparib and nivolumab combination Acknowledgements from the dose escalation phase of [NCT03061188] clinical trial. The authors would like to thank all the patients who are participating in this Methods study. The study is sponsored by Alkermes, Inc. Medical writing and editorial We conducted a phase I study of veliparib in combination with nivo- support was provided by Parexel and funded by Alkermes, Inc. lumab in chemo-refractory stage IV/ unresectable solid cancer pa- Trial Registration tients. Nivolumab 240mg IV day 1 and 15 q28 days for 4 cycles; ClinicalTrials.gov NCT02799095 480mg IV q28 days Cycle 5 onwards, combined with veliparib start- ing 300mg PO bid 3+3 dose escalation until maximum tolerated References dose (MTD) was established. Treatment continued until disease pro- 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of gression or limiting toxicity. Primary objective was establishing MTD ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic for veliparib, defined as the highest dose causing dose-limiting tox- tumor models alone and in combination with immune checkpoint icity (DLT) in < 2 of 6 patients. Secondary objective was to assess inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. safety, tolerability and early efficacy of combination. 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, Results McDermott DF, et al. Intravenous administration of ALKS 4230 as Nine patients with adequate end-organ function and performance monotherapy and in combination with pembrolizumab in a phase I status were enrolled. Tumor types included colon cancer (n = 3) and study of patients with advanced solid tumors. J Clin Oncol. pancreatic cancer (n = 2). Four patients (44%) had BRCA-related som- 2019:37(Suppl). Abstract TPS2649. atic mutations (BRCA1, BRCA2, ATM and BRIP1). Most common ad- Ethics Approval verse events categorized as possibly or probably related to treatment This study was approved by Ethics and Institutional Review Boards (IRBs) at were fatigue (n = 6, 67%), anemia (n = 5, 56%), nausea (n = 4, 44%) all study sites; IRB reference numbers 16-229 (Dana-Farber Cancer and diarrhea (n = 3, 33%). Grade 3 and 4 events were anemia (n = 3, Institute), MOD00003422/PH285316 (Roswell Park Comprehensive Cancer 33%), fatigue and alkaline phosphatase elevations (n = 2, 22% each), Center), 20160175 (Western IRB), i15-01394_MOD23 (New York University AST, ALT elevations, low platelet count, hypokalemia and hyperten- School of Medicine), STUDY20190090 (Cleveland Clinic), 0000097 sion (n = 1, 11% each). One patient experienced DLT, grade 3 fatigue, (ADVARRA). at dose level 2 (400 mg BiD) and the MTD was established as 400mg bid. Disease control rate after 24 week follow up was 11%. Five pa- tients presented disease progression (56%). One patient withdrew consent at Cycle 3 and another developed limiting fatigue at Cycle 3, both had stable disease (SD). A patient succumbed due to complica- tions of disease before first assessment. One patient with refractory metastatic pancreatic carcinoma harboring a BRIP1 L680fs*9 muta- tion had SD after a 35-week follow-up. Median progression-free sur- vival and overall survival were 9 and 25 weeks, respectively. Conclusions The recommend phase 2 dose of Veliparib is 400mg bid when com- bined with Nivolumab. The side effect profile is on par with the ones Fig. 1 (abstract P447). ALKS 4230 structure and activity previously described for veliparib and nivolumab in monotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 245 of 272 We are expanding the cohort to now include tumors harboring DNA Effects on tumor infiltrating immune cells and molecular signatures repair defects. in correlative biomarker analyses provide insights to ALX148’s mech- Trial Registration anism as a myeloid checkpoint inhibitor. NCT03061188 Ethics Approval Acknowledgements The study was approved by Northwestern University Ethics Board. We would like to thank all of the participating patients and their families as STU00204250. well as site research staff. Trial Registration ClinicalTrials.gov identifier NCT03013218. P449 Pharmacodynamic biomarker characterization of ALX148, a CD47 References blocker, in combination with established anticancer antibodies in 1. Kauder et al., ALX148 blocks CD47 and enhances innate and adaptive patients with advanced malignancy antitumor immunity with a favorable safety profile. PLOS ONE. 2018 1 2 3 Hong Wan, PhD , Laura Chow, MD , Justin Gainor, MD , Nehal Lakhani, 13(8): e0201832 4 5 6 MD, PhD , Hyun Chung, MD, PhD , Keun-Wook Lee, MD , Jeeyun Lee, 2. Lahkani et al., A phase 1 study of ALX148, a CD47 blocker, alone and in 7 8 9 MD, PhD , Patricia LoRusso, DO , Yung-Jue Bang, MD PhD , Stephen combination with established anticancer antibodies in patients with 10 11 1 12 Hodi , Wells Messersmith, MD , Philip Fanning, PhD , Pierre Squifflet , advanced malignancy and non-Hodgkin lymphoma. Journal of Clinical 1 1 1 1 Feng Jin , Tracy Kuo , Sangeetha Bollini , Jaume Pons, PhD , Sophia Oncology 2018 36:15_suppl, 3068-3068 Randolph, MD, PhD 3. Lahkani et al., A phase 1 study of ALX148: CD47 blockade in combination 1 2 ALX Oncology, Burlingame, CA, United States; University of with anticancer antibodies to bridge innate and adaptive immune Washington, Seattle, WA, United States; MGH Cancer Center, Boston, responses for advanced malignancy. Journal for ImmunoTherapy of MA, United States; START Midwest, Grand Rapids, MI, United States; Cancer 2018 6 (Suppl 1):114. Abstract 335. 5 6 Yosei Cancer Center, Seoul, Korea, Republic of; Seoul University 4. Chow et al., A phase I study of ALX148, a CD47 blocker, in combination Bundang Hospital, Seongnam, Korea, Republic of; Samsung Medical with established anticancer antibodies in patients with advanced Center, Seoul, Korea; Yale Cancer Center, New Haven, CT, United States; malignancy. Journal of Clinical Oncology 2019 37:15_suppl, 2514-2514. 9 10 Seoul National University Hospital, Seoul, Korea; Dana Farber Cancer Ethics Approval Center, Boston, MA, United States; University of Colorado Cancer The study was approved by institutional review boards or independent Center, Aurora, CO, United States; International Drug Development ethics committees of participating institutions (approval numbers on file Institute, Brussels, Belgium at ALX Oncology). Correspondence: Hong Wan (Hong@alxoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P449 P450 Background A phase I/IIa, open-label, dose-escalation and expansion study to CD47 is a myeloid checkpoint upregulated by tumor cells to investigate the safety, tolerability, pharmacokinetics and evade immune destruction. ALX148 (A) is a fusion protein com- pharmacodynamics of TJ107 in Chinese patients with advanced prised of a high affinity CD47 blocker linked to an inactive hu- solid tumors 1 1 1 1 1 1 man immunoglobulin Fc region [1]. We have previously shown in Jin Li , Ye Guo , Wei Peng , Junli Xue , Wei Zhao , Xiaoxiao Ge , Liqiong 1 1 1 1 2 the first-in-human clinical trial, that ALX148 is well tolerated in Xue , Wenbo Tang , Li Zhou , Min Zhang , Bingshi Guo , Liping Wang, 2 2 2 2 combination with trastuzumab (T) or pembrolizumab (P) with no MD , Jiyuan Guo , Feifei Cui, PhD , Haiyun Suo 1 2 maximum tolerated dose (MTD) identified [2, 3]. Antitumor activ- Shanghai East Hospital, Shanghai, China; I-Mab Biopharma, Shanghai, ity of ALX148 in combination with T or P was observed in pa- China tients with advanced gastric/ gastroesophageal junction (G/GEJ), Correspondence: Jin Li (lijin@csco.org.cn) head and neck squamous cell carcinoma (HNSCC) and non-small Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P450 cell lung cancer (NSCLC) [4]. The objective of this exploratory analysis was to characterize tumor infiltrating immune cells and Background molecular signatures from tumor biopsies and peripheral blood TJ107, an immuno-oncology agent also known as Hyleukin, is a samples obtained from this trial. T cell amplifier comprising a homodimer of engineered human Methods interleukin-7 (IL-7) fused with Genexine’s proprietary long-acting Patients with HER2-positive malignancy (including G/GEJ cancers pro- platform hybrid Fc. IL-7 is a critical homeostatic factor for T gressed on T + fluoropyrimidine and platinum-based therapy) re- cells, acting on T cells to increase their number, diversity and ceived A+T. Patients with advanced malignancy including NSCLC functionality. TJ107 could play a pivotal role in reconstitution [checkpoint inhibitor (CPI)-resistant/refractory or PD-L1 tumor propor- and reinvigoration of T cell immunity in cancer patients, provid- tion score (TPS) ing unique opportunities for immuno-oncology combination Results strategies. The aim of this study (NCT04001075) is to determine Eighty-two patients received A+T (n=30) or A+P (n=52) as of the safety, tolerability and PKPD profile of TJ107 in Chinese can- April 18, 2019. In dose expansion cohorts (N=60), anticancer ac- cer patients. tivity was observed in response-evaluable patients [G/GEJ (n= Methods 18) 4PR, 5SD; HNSCC (n=19) 3PR, 6SD and NSCLC (n=18) 3SD]. This ongoing study is to evaluate the safety, tolerability, PK profile, Near complete CD47 TO was maintained throughout the dosing and anti-tumor activity of TJ107 in patients with advanced solid tu- interval. No dose-dependent changes were apparent in mors who failed standard therapy. Patients receive TJ107 every 4 peripheral lymphocyte populations. Preliminary results from weeks by intramuscular (IM) injection. Dose escalation is aided by a paired biopsies (n=15) demonstrated increased tumor-associated 3+3 scheme from 240μg/kg to 1200μg/kg. A dose expansion cohort macrophages and lymphocytes in both intra-tumoral and peri- is being planned after the RP2D is determined. Safety is assessed by tumoral regions after treatment with ALX148 combinations. monitoring AEs and the associated grades per NCI CTCAE v5.0. Gene expression signatures of tumor inflammation and immune Tumor response will be assessed per RECIST v1.1. Samples will be cell subsets are being investigated. Results will be updated at collected for PK, PD, ADA, immunophenotyping and TCR repertoire presentation. analysis. Conclusions Results ALX148 demonstrates excellent tolerability with objective responses Three patients with colorectal cancer were enrolled in the first observed in patients with advanced G/GEJ cancer and HNSCC that cohort (240μg/kg).TJ107 was well tolerated and no DLTs were re- have progressed on prior systemic and HER2-targeted therapies. ported during the first cycle at this dose level. The preliminary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 246 of 272 PK results shows that TJ107 was rapidly absorbed and reached limiting toxicities (injection-site reaction [ISR], lymph node pain; serum peak concentration around 24 hours post-dose. TJ107 was ISR). Among all treated patients, the most common adverse slowly cleared from the body and remained detectable in serum events (AEs) were ISR (89.5%), fatigue (39.5%), nausea (28.9%), until Day 14 post-dose. A substantial increase in absolute constipation, and decreased appetite (23.7% each). Nine (23.7%) lymphocyte count (ALC) from baseline was observed and peaked patients had MEDI5083-related ≥G3 events, most commonly ISR. around 3 to 4 weeks post first dose. FACS analysis revealed in- Six (15.8%) patients discontinued due to a MEDI5083-related AE. creases in CD3+, CD4+ and CD8+ T cells. The numeric increase in There were 4 (10.5%) deaths due to AEs (sequential cohort, T cells is consistent with increased Ki67 expression on Day 8 post MEDI5083 5mg, n=2 and MEDI5083 7.5mg, n=1; concurrent co- first dose. There were no notable changes in B cells, monocytes, hort, MEDI5083 4mg, n=1), 3 unrelated to treatment and 1 pos- NK cells, neutrophils, nor Tregs, as expected. sibly related to MEDI5083. The maximum tolerated dose for Conclusions MEDI5083 was 5mg. In the response evaluable population (N=36), Preliminary results from this trial suggest that TJ107 activated a PR was observed (head and neck squamous cell carcinoma; se- IL-7 pathway and expanded T cells in cancer patients in a simi- quential cohort, MEDI5083 3mg; time to response 5.7 months) lar way to data previously reported in healthy subjects. TJ107 and 11 (30.6%) patients had SD (sequential cohort, n=7; concur- exhibits a promising safety and tolerability profile in cancer pa- rent cohort, n=4). Six patients had SD ≥24 weeks. The ORR (95% tients under current dose. These findings support further clinical CI) was 2.8% (0.1–14.5%). MEDI5083 showed dose-dependent investigation. pharmacological activity in the mobilization of peripheral blood B Trial Registration cells, and induced measurable increases in activated proliferative Investigate the Safety, Tolerability, Pharmacokinetics and Pharmaco- Ki-67+ CD8+ T cells in peripheral blood. dynamics of TJ107 in Chinese Patients With Advanced Solid Tumors. Conclusions ClinicalTrials.gov Identifier: NCT04001075 Subcutaneous administration of MEDI5083 caused high rates of injec- Ethics Approval tion site reactions. The toxicity profile does not support further devel- The study was approved by the Ethics Committee Shanghai East Hos- opment of the subcutaneous formulation of this drug. pital's, approval number 2018 (058). Trial Registration ClinicalTrials.gov NCT03089645 Ethics Approval P451 This study was approved by the Institutional Review Board/Independ- First-in-human study of CD40 agonist MEDI5083 in advanced solid ent Ethics Committee at each investigational site participating in the tumors with durvalumab administered sequentially or concurrently study 1 2 3 Ben Tran, MBBS FRACP , Mark Voskoboynik , Johanna Bendell, MD , 4 5 Martin Gutierrez, MD , Charlotte Lemech, MBBS BSc(med) MD(res) , 6 6 7 Daphne Day , Sophia Frentzas , Ignacio Garrido-Laguna , Chris P452 8 8 8 8 DelNagro , Fujun Wang , Charles Ferte, MD, PhD , Mayukh Das , SPEARHEAD-1 trial design: A phase 2, single arm, open-label Benedito Carneiro, MD clinical trial of ADP-A2M4 SPEAR T-cells in patients with advanced 1 2 Peter MacCallum Cancer Center, Melbourne, Australia; Nucleus synovial sarcoma or myxoid/round cell liposarcoma 3 1 2 3 4 Network, Melbourne, Australia; Sarah Cannon Research Institute, Dejka Araujo, MD , Jean-Yves Blay , Sandra Strauss , Claudia Valverde , 4 5 5 5 Nashville, TN, United States; Hackensack University Medical Center, Erin Van Winkle , Malini Iyengar, PhD , Rafael Amado, MD 5 1 2 Hackensack, NJ, United States; Scientia Clinical Research, Sydney, MD Anderson Cancer Center, Houston, TX, United States; Leon Berard, 6 7 3 Australia; Monash Medical Centre, Clayton, Australia; Huntsman Cancer Lyon, France; University College London Hospitals, London, United 8 4 Institute, Salt Lake City, UT, United States; AstraZeneca, San Francisco, Kingdom; Vall D'Hebron University Hospital, Barcelona, Spain; 9 5 CA, United States; The Warren Alpert Medical School, Providence, RI, Adaptimmune, Philadelphia, United States United States Correspondence: Erin Van Winkle (erin.vanwinkle@adaptimmune.com) Correspondence: Mayukh Das (mayukh.das@medimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P452 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P451 Background Background ADP-A2M4 specific peptide enhanced affinity receptor (SPEAR) T-cells MEDI5083 is a homodimeric fusion protein of three single-chain are genetically engineered to target MAGE-A4+ tumors in the con- CD40L domains linked to an immunoglobulin G fragment text of HLA-A*02. MAGE-A4 has been described as having high ex- crystallizable domain, and activates the CD40 pathway to promote pression in synovial sarcoma (SS) and myxoid/round cell liposarcoma immune responses. This first-in-human study evaluated the safety (MRCLS). In recent studies [1, 2], immunohistochemistry (IHC) ana- and clinical activity of MEDI5083 given sequentially or concurrently lyses showed that 82% of SS samples and 68% of MRCLS samples with the PD-L1 antibody durvalumab in patients with advanced solid expressed MAGE-A4. A pilot study (NCT03132922) of ADP-A2M4 in- tumors. duced clinical responses in patients with SS. Methods Methods Eligible patients with metastatic or recurrent tumor types progressing This phase 2, open-label trial (SPEARHEAD-1 Trial) will evaluate the on or refractory to prior therapy were enrolled in multiple cohorts of efficacy, safety and tolerability of ADP-A2M4 SPEAR T-cells. Patients MEDI5083 (3mg, 4mg, 5mg, 6mg, and 7.5mg) administered subcuta- who are HLA-A*02+ (excluding A*02:05, and A*02:07 and A*02 null neously (SC) Q2W for 4 doses. In the sequential-treatment cohort, as sole A*02 alleles), who have advanced/metastatic SS or MRCLS MEDI5083 was followed by a 4-week wash-out, then durvalumab who have received prior chemotherapy, and have MAGE-A4 expres- 1500 mg intravenously (IV) Q4W. In the concurrent-treatment cohort, sion assessed by IHC at ≥2+ in ≥ 30% of tumor cells, and who meet MEDI5083 was administered with concurrent durvalumab 1500 mg IV all other inclusion criteria are eligible for treatment. Up to 60 patients Q4W for 2 doses, followed by durvalumab 1500 mg IV Q4W. The pri- will be treated. mary endpoint was safety. Secondary endpoints included pharmaco- Following apheresis, T-cells are isolated, transduced with MAGE- kinetics, immunogenicity, and efficacy based on investigator- A4c1032TCR, and expanded. Prior to infusion, patients will receive assessed RECIST V1.1. lymphodepletion consisting of fludarabine (30 mg/m2/day x 4 days) Results and cyclophosphamide (600 mg/m2/day x 3 days). Patients will re- As of June 30, 2019, 38 patients were treated; 29 received se- ceive 1 – 10 × 10^9 transduced T-cells. Futility analysis will be con- quential treatment (MEDI5083 3mg, n=4; 4mg, n=4; 5mg, n=18; ducted after 15 patients are dosed and have been followed for at 7.5mg, n=3) and 9 patients received concurrent treatment least 4 months from the time of T-cell infusion. An independent Data (MEDI5083 3mg, n=3; 4mg, n=6). Two patients (sequential cohort, Safety Monitoring Board will review ongoing safety and benefit:risk MEDI5083 5mg and 7.5mg) had MEDI5083-related G3/4 dose- during the interventional phase of the study. Disease will be assessed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 247 of 272 by independent review per RECIST v1.1 by CT/MRI at weeks 4, 8, 12, Results 16, 24, and every 2 months thereafter until confirmed disease pro- The primary objective of sub-study 2 is to evaluate GSK3377794 gression. Once disease progression is established, patients will enter efficacy by overall response rate per RECIST v1.1 (central inde- the long-term follow-up phase of the study, with visits every 6 pendent review). Secondary objectives include: time to and dur- months through Year 5, and annually thereafter for Years 6-15. ation of response; disease control rate; progression-free survival; Trial Registration overall survival; potential immune response to GSK3377794; safety NCT04044768 and tolerability. Exploratory objectives include: correlation of T- cell persistence with safety, clinical responses, and phenotype of References infused T cells. Impact on quality of life and daily functioning will 1. Iura K, et al. Cancer-testis antigen expression in synovial sarcoma: NY- also be assessed. ESO-1, PRAME, MAGEA4, and MAGEA1. Human Pathology 2017a; 61:130- Conclusions 139. Based on the encouraging clinical activity of GSK3377794 observed 2. Iura K, et al. MAGEA4 expression in bone and soft tissue tumors: its utility in earlier trials, this larger clinical trial is being initiated to establish as a target for immunotherapy and diagnostic marker combined with and further discern the efficacy and safety of GSK3377794 in this NY-ESO-1. Virchow Archiv 2017b;471:383–392. biomarker-selected metastatic SS patient population. This innovative Ethics Approval Master Protocol study design permits evaluation of GSK3377794 This trial is under review by the institutional review board of the trial sites treatment in other NY-ESO-1+ tumor types in HLA A*02+ patients within separate sub-studies. P453 Acknowledgements Autologous T cells with NY-ESO-1-specific T-cell receptor Medical writing assistance was provided by Fiona Woodward of Fishawack (GSK3377794) in HLA-A*02+ previously-treated and -untreated Indicia Ltd, UK, funded by GlaxoSmithKline (GSK). This study (NCT03967223) advanced metastatic/unresectable synovial sarcoma: A master was funded by GSK. protocol study design Trial Registration 1 2 3 4 Sandra D'Angelo, MD , Jean-Yves Blay , Warren Chow , George Demetri , NCT03967223 5 6 7 Fiona Thistlethwaite, MD, PhD , Michael Wagner , David Loeb , Steven 8 9 10 Attia , Albiruni Razak , John Haanen, MD PhD , Aisha Hasan, MBBS References 11 11 11 11 11 MD , Julia Billiard , Laura Pearce , Yuehui Wu , Ran Ji , Laura 1. Riedel RF, Jones RL, Italiano A, et al. Systemic anti-cancer therapy in syn- 11 11 11 12 Johnson , Chandra Srinath , Aiman Shalabi , Sandra Strauss , ovial sarcoma: A systematic review. Cancers 2018; 10:E417. 4 13 1 Katherine Thornton , Crystal Mackall, MD , William Tap , Brian Van Tine, 2. Vlenterie M, Litière S, Rizzo E, et al. Outcome of chemotherapy in MD, PhD advanced synovial sarcoma patients: Review of 15 clinical trials from the Memorial Sloan Kettering Cancer Center, New York, NY, United States; European Organisation for Research and Treatment of Cancer Soft Tissue 2 3 Centre Léon Bérard, Lyon, France; City of Hope Comprehensive Cancer and Bone Sarcoma Group; setting a new landmark for studies in this Center, Duarte, CA, United States; Dana-Farber Cancer Institute, Boston, entity. Eur J Cancer 2016; 58:62–72. MA, United States; The Christie NHS Foundation Trust, Manchester, Ethics Approval United Kingdom; Fred Hutchinson Cancer Research Center, Seattle, WA, This Master Protocol will be conducted under approval by the appropriate United States; Montefiore Medical Center, New York, NY, United States; institutional review boards and independent ethics committees. 8 9 Mayo Clinic in Florida, Jacksonville, FL, United States; Princess Margaret Cancer Centre, Toronto, Canada; Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, Netherlands; GlaxoSmithKline, Collegeville, PA, United States; University College London Hospitals, London, United Kingdom; 13 14 Stanford University, Stanford, CA, United States; Washington University in St. Louis, St. Louis, MO, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P453 Background Synovial sarcoma (SS) comprises ~5%–10% of soft-tissue sarcomas [1]. Anthracycline-based chemotherapy is a 1st-line treatment in advanced metastatic/unresectable disease, but response rates are low Fig. 1 (abstract P453). Study Design Methods A clinical trial is underway utilizing a Master Protocol design allowing investigation of GSK3377794 in multiple tumor types (NCT03967223). The first 2 sub-studies are single-arm trials evaluating treatment in previously-untreated (sub-study 1) and previously-treated (sub-study P454 2) HLA A*02+ patients with NY-ESO-1+ metastatic SS. Sub-study 1 is Induction of serum CXCL10 by tebentafusp, a gp100-CD3 a pilot study evaluating efficacy as 1st-line treatment (N=10). Sub- bispecific fusion protein, was associated with survival in uveal study 2 plans to enroll 55 patients with metastatic/locally advanced melanoma in a Phase I/II Study unresectable SS who have progressed following anthracycline-based 1 2 2 Marcus Butler, MD , Brandon Higgs , Cheryl McAlpine, MSN , Joseph chemotherapy. Inclusion criteria include: ≥10 years of age; measur- 3 4 5 Sacco , Jessica Hassel, MD , Shaad Abdullah, MD , Koustubh Ranade, able disease; adequate organ function; ECOG performance status 0– 5 6 PhD , Richard Carvajal, MD 1. Exclusion criteria include: CNS metastases; clinically significant sys- 1 2 Princess Margaret Cancer Centre, Toronto, Canada; Immunocore, Ltd, temic illness; prior gene therapy with integrating vector or NY-ESO-1- Oxfordshire, United Kingdom; Clatterbridge Cancer Centre, Liverpool, specific T cells, vaccine or targeting antibody; prior autoimmune dis- United Kingdom; University Hospital Heidelberg, Heidelberg, Germany; ease or allogeneic hematopoietic stem-cell transplant. Patients will 5 6 Immnuocore, Ltd, Rockville, MD, United States; Columbia University undergo eligibility screening; leukapheresis and manufacture of Medical Center, New York, NY, United States GSK3377794; lymphodepletion and infusion of GSK3377794 followed Correspondence: Brandon Higgs (brandon.higgs@immunocore.com) by safety follow-up and disease assessments; and a 15-year follow-up Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P454 under a separate protocol (Figure 1). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 248 of 272 Background P455 Tebentafusp (formerly IMCgp100) is a TCR–anti-CD3 bispecific fusion A randomized phase 2 study of neoadjuvant talimogene protein targeting melanocyte-expressed gp100 antigen; Phase I/II laherparepvec (T-VEC) plus surgery vs surgery for resectable stage clinical studies showed monotherapy activity in metastatic melanoma IIIB-IVM1a melanoma: 2-year primary analysis of recurrence-free including uveal (UM) [1]. Historical 1-year survival (OS) rate for 2L survival (RFS) 1 2 3 metastatic UM is ~35% [2]. In a phase I clinical study of tebentafusp Reinhard Dummer, MD , David Gyorki , John Hyngstrom, MD , Adam 4 5 6 7 in cutaneous and 19 UM patients, serum IFNg-induced chemokines, Berger, FACS, MD , Robert Conry, MD , Lev Demidov , Anjali Sharma , 7 7 7 8 particularly CXCL10, were induced by tebentafusp and high induced Sheryl Treichel , Kevin Gorski , Abraham Anderson, PhD , Mark Faries , levels correlated with OS and tumor reduction. In a subsequent Merrick Ross, MD 1 2 phase I/II trial (NCT02570308) of 2L UM, we sought to confirm this University Hospital of Zurich, Zurich, Switzerland; Olivia Newton-John and increase mechanistic understanding with blood mRNA analysis. Cancer Centre, Melbourne, Australia; University of Utah Huntsman Methods Cancer Inst, Salt Lake City, UT, United States; Rutgers Cancer Institute of NCT02570308 was conducted in HLA-A*0201+ patients with ad- New Jersey, New Brunswick, United States; University of Alabama vanced 2L UM; this exploratory analysis focused on further investiga- School of Medicine, Birmingham, AL, United States; N.N. Blokhin Russian tion of an initial 40 patient cohort [3]. Intra-patient escalation dosing Cancer Research Ce, Moscow, United States; Amgen Inc, Thousand regimen used low initial dosing at Cycle1, Day1 (C1D1, 20 mcg) and Oaks, United States; John Wayne Cancer Institute, Santa Monica, United C1D8 (30 mcg). From C1D15, 19 patients received between 54-73 States; University of Texas MD Anderson Cancer, Houston, TX, United mcg , 22 patients received 68 mcg (expansion phase). Sera from 18 States and 22 patients in escalation and expansion phases, respectively Correspondence: Reinhard Dummer (Reinhard.Dummer@usz.ch) were profiled pre-treatment and post first and third dose with 11 im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P455 mune markers (Luminex); whole blood from 19 escalation phase pa- tients was analysed for gene expression (NanoString). Low/High Background groups were defined at the median for OS and Mann-Whitney tests Risk of recurrence and death after resection of stage IIIB-IVM1a mel- were used for time contrasts. anoma is high. In the previous 1-year interim analysis, T-VEC plus sur- Results gery demonstrated a pathological complete response rate of 22.8% In an updated analysis of rash (on-target, off-tumor toxicity), 31 of 40 and improved RFS compared to upfront surgery (Dummer et al, patients with Grade 2+ rash had 1-year OS rate ~77%; 1-year OS in ASCO 2019). Here, we present results from the primary 2-year RFS remaining patients was ~35%. Tebentafusp induced a transient re- and biomarker analyses (CT.gov identifier: NCT02211131). sponse in cytokines, reaching maximal changes at 8-24 hours post Methods first and third doses. High levels of CXCL10 induced at first dose cor- Patients with resectable stage IIIB-IVM1a melanoma, ≥ 1 injectable cuta- related with improved OS (HR=0.37 95%CI=[0.15, 0.89]); high induced neous, subcutaneous, or nodal lesions ≥10 mm, and no systemic treat- CXCL9 levels also trended with OS (HR=0.52 95%CI=[0.21,1.3]). ment 3 months prior were randomized 1:1 to 6 doses/12 wks of CXCL9/CXCL10 transcripts increased at the same time points, as did neoadjuvant T-VEC followed by surgery during weeks 13-18 (Arm 1) signatures for neutrophils, type I IFN, and eosinophils (folds>1.5, p<- versus surgery during weeks 1-6 (Arm 2). T-VEC was given at standard 2, p<-1.5, p dosing until surgery, no remaining injectable tumors, or intolerance. Conclusions The primary analysis estimated a between-group difference in 2-yr RFS In this exploratory analysis, high CXCL10 levels induced by tebenta- on the intent-to-treat set. RFS event was defined as the first local, re- fusp associated with improved OS in UM. Tebentafusp reduced CD8+ gional, or distant recurrence or death due to any cause after surgery. and CD4+ gene signatures in the blood and induced systemic cyto- Per protocol, patients who withdrew prior to surgery or had an R1 or kine and gene expression responses, consistent with T cell redirec- R2 resection were counted as an RFS event at randomization. An add- tion and immune activation. Patients who develop tebentafusp- itional analysis calculated RFS from randomization to the date of first induced Grade 2+ rash appear to have better survival than those post-surgery event regardless of surgical margin status. who do not. Results Trial Registration 150 pts were randomized (76 arm 1, 74 arm 2). Median (range) NCT02570308 follow-up time was 31.2 (0.1–49.9) months. 75% in Arm 1 and 93% in Arm 2 had surgery as planned. In the per protocol analysis, 29.5% of References patients in Arm 1 and 16.5% of patients in Arm 2 remained recur- 1. Middleton MR. J Clin Oncol 37, 2019 (suppl; abstr 9523) rence free (HR: 0.75, P=0.07). In the additional analysis, 50.5% of pts 2. Rantala ES, Hernberg M, Kivelä TT. Overall survival after treatment for in Arm 1 and 30.2 % in Arm 2 remained recurrence free (HR: 0.66, P= metastatic uveal melanoma: a systematic review and meta-analysis. Mel- 0.038). 2-year overall survival rates were 88.9% in Arm 1 and 77.4% anoma Res. 2019 Jan 16. doi: 10.1097/CMR.0000000000000575. in Arm 2 (HR: 0.49, P=0.050). In arm 1, T-VEC treatment resulted in a 3. Sato T DOI: 10.1200/JCO.2017.35.15_suppl.9531 Journal of Clinical 3-fold increase (P Oncology 35, no. 15_suppl (May 20 2017) 9531-9531. Conclusions Ethics Approval Neoadjuvant T-VEC improved 2-year RFS and OS in resectable stage This study was in accordance with the Declaration of Helsinki and was IIIB-IVM1a melanoma. T-cell influx and PD-L1 upregulation after T- approved by all IRBs/ethics committees from each clinical sites VEC treatment support a role for the adaptive immune system con- participating in the study. Specific approval numbers can be provided sistent with the mechanisms of action. Additional biomarker results upon request. including clinical correlations will be presented at the congress. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 249 of 272 Trial Registration Ethics Approval CT.gov identifier: NCT02211131 The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Reference Consent 1. Dummer R, et al. Presented at The American Society of Clinical All patients provided written informed consent to participate in the clinical Oncology; May 31–June 3, 2019; Chicago IL, USA. J Clin Oncol 37, 2019 trial. (suppl; abstr 9520) Ethics Approval P457 The study was approved by participating institutions' Ethics Board. Randomized phase II neoadjuvant study: PD-1 inhibitor TSR-042 vs. combination PD-1 inhibitor TSR-042 and Tim-3 inhibitor TSR- P456 022 in borderline resectable stage III or oligometastatic stage IV KEYNOTE-630: phase 3 study of adjuvant pembrolizumab versus melanoma 1 2 2 placebo in patients with high-risk, locally advanced cutaneous Zahra Kelly, DO , Yana Najjar, MD , Hassane Zarour, MD , John Kirkwood, 2 3 2 4 squamous cell carcinoma MD , Suthee Rapisuwon, MD , Hong Wang , Marc Ernstoff, MD , Joseph 1 2 2 5 2 6 Jessica Geiger , Gregory Daniels, MD, PhD , Ezra Cohen, MD , Joy Yang Drabick, MD, FACP, FIDSA , Diwakar Davar, MD , Mohan Bala 3 3 3 1 Ge , Burak Gumuscu, MD PhD , Ramona Swaby, MD , Anne Lynn University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 4 2 3 Chang University of Pittsburgh, Pittsburgh, PA, United States; Georgetown 1 2 4 Cleveland Clinic, Cleveland, OH, United States; University of California, University, Washington, DC, United States; Roswell Park Comprehensive 3 5 San Diego, La Jolla, CA, United States; Merck & Co., Inc., Kenilworth, NJ, Cancer Center, Buffalo, NY, United States; Penn State Cancer Institute, 4 6 United States; Stanford University Medical Center, Stanford, CA, United Palmyra, PA, United States; Tesaro, Inc, Wltham, MA, United States States Correspondence: Diwakar Davar (davard@upmc.edu) Correspondence: Jessica Geiger (GEIGERJ@ccf.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P457 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P456 Background Background Neoadjuvant PD-1 blockade produces pathological responses in Despite undergoing current standard-of-care surgical resection and ~30% of patients (pts) with high-risk resectable melanoma (MEL) adjuvant radiotherapy, ~20% of patients with high-risk, locally ad- with durable relapse-free benefit, and increased circulating acti- vanced cutaneous squamous cell carcinoma develop local recurrence vated CD8+ T cells (1,2). TIM-3 is an inhibitory immune check- within 5 years [1]. Recent data show effective antitumor activity and point and mediates immune escape; blockade of which produces acceptable safety of programmed death 1 inhibitors in patients with anti-tumor immune responses and synergizes with anti-PD-1 (3– locally advanced or metastatic cutaneous squamous cell carcinoma. 5). TSR-042/dostarlimab is an IgG4 humanized monoclonal anti- KEYNOTE-630 (NCT03833167), a randomized, double-blind, placebo- body that binds with high affinity to PD-1, inhibiting binding to controlled phase 3 trial, will evaluate the efficacy and safety of adju- PD-L1 and PD-L2. TSR-042 has been studied in patients with ad- vant pembrolizumab in patients with high-risk locally advanced or vanced non-small cell lung (NSCLC) and endometrial cancers with metastatic cutaneous squamous cell carcinoma. promising results (6). TSR-022 is an IgG4-k isotype humanized Methods monoclonal antibody that binds with high affinity to TIM-3, thus Patients with high-risk locally advanced cutaneous squamous cell enhancing T cell activity. TSR-042/TSR-022 combination has been carcinoma who have undergone surgical resection and radiother- studied in a phase I/II study that demonstrated promising efficacy apy will be randomly assigned 1:1 to intravenous pembrolizumab of combination in PD-1 refractory melanoma and NSCLC (7). We (400 mg every 6 weeks) or placebo for up to 9 cycles (~1 year) hypothesized that neoadjuvant therapy with TSR-042/TSR-022 or until disease recurrence, unacceptable toxicity, or investigator combination may improve pathologic response rates compared to or patient decision to withdraw. Randomization will be stratified TSR-042 monotherapy in high-risk resectable MEL. by extracapsular extension (yes vs no), cortical bone invasion (yes Methods vs no), and prior systemic therapy (yes vs no). Eligible patients Pts with regionally advanced (stage IIIB-D) or oligometastatic are adults with histologically confirmed locally advanced cutane- (stage IVA-B) melanoma who have yet to undergo definitive sur- ous squamous cell carcinoma with ≥1 high-risk features at the gery are eligible. Primary endpoints are rate of major pathologic primary site of malignancy and macroscopic resection with or response (MPR), safety, and incidence of dose-limiting toxicities without microscopic positive margins who completed adjuvant (DLT). Secondary endpoints are radiographic response, relapse- radiotherapy, were disease free ≤28 days from randomization, free survival (RFS) and overall survival (OS). Pathological response and have Eastern Cooperative Oncology Group performance sta- will be assessed depending on residual volume of tumor (RVT) tus 0 or 1. The primary efficacy end points are investigator- using prior cutoffs: 0% (complete response, pCR); 0%<RVT< assessed and biopsy-confirmed recurrence-free survival. Secondary RVT50% (non-response, pNR) (8–10). Sample size is 28 patients end points are overall survival, health-related quality of life, and per arm. Patients will receive neoadjuvant therapy for 6 weeks safety. To assess treatment response, radiographic imaging will prior to planned surgery. Surgery will occur 1-3 weeks after com- be performed at least every 12 weeks in year 1, then every 6 pletion of pre-operative therapy. After surgery, subjects will re- months until the end of year 5. All patients meeting crossover or ceive additional maintenance TSR-042 for approximately 48 weeks retreatment criteria at first disease recurrence may receive pem- (Figure 1). Sample size provides 80% power to detect an im- brolizumab 400 mg every 6 weeks for up to 18 cycles. Adverse provement of 30% upon MPR rate of 30% with anti-PD-1 events will be recorded until 30 days (90 days for serious adverse monotherapy. events) after study end and will be graded per NCI CTCAE v4.0. Enrollment of ~570 patients is planned, and recruitment is on- References going in 18 countries. 1. Amaria, R. N. et al. Neoadjuvant immune checkpoint blockade in high- Trial Registration risk resectable melanoma. Nature Medicine 24, 1649–1654 (2018). ClinicalTrials.gov, NCT03833167 2. Huang, A. C. et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nature Medicine 25, 454–461 Reference (2019). 1. Porceddu SV, Bressel M, Poulsen MG, et al. Postoperative concurrent 3. Fourcade, J. et al. Upregulation of Tim-3 and PD-1 expression is asso- chemoradiotherapy versus postoperative radiotherapy in high-risk cuta- ciated with tumor antigen–specific CD8+ T cell dysfunction in melan- neous squamous cell carcinoma of the head and neck: the randomized oma patients. The Journal of Experimental Medicine 207, 2175–2186 phase III TROG 05.01 trial. J Clin Oncol. 2018;36:1275-1283. (2010). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 250 of 272 4. Chauvin, J.-M. et al. TIGIT and PD-1 impair tumor antigen–specific CD8+ signals and tumor antigens that can be captured and processed by T cells in melanoma patients. Journal of Clinical Investigation 125, 2046– IT co-administered CD1c (BDCA-1)+ myDC, reinvigorating the cancer 2058 (2015). immunity cycle. 5. Woo, S.-R. et al. Immune Inhibitory Molecules LAG-3 and PD-1 Synergis- Methods tically Regulate T-cell Function to Promote Tumoral Immune Escape. Patients with advanced melanoma who failed standard therapy were Cancer Research 72, 917–927 (2012). eligible for IT injections of ≥1 non-visceral metastasis with T-VEC 6. Moreno, V. et al. Abstract CT053: Preliminary safety, efficacy, and PK/PD (10^6 PFU/mL; max total volume of 4 mL) on day 1 followed by IT in- characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 jection of autologous, non-substantially manipulated CD1c (BDCA-1)+ monoclonal antibody, TSR-042, in patients with recurrent or advanced myDC on day 2. Injection of T-VEC (10^8 PFU/mL; max total volume NSCLC and MSI-H endometrial cancer. Cancer Research 78, CT053–CT053 of 4 mL) was repeated on day 21 and every 14 days thereafter. Pa- (2018). tients were treated with 0.5x10^6, 1x10^6, or 10x10^6 CD1c (BDCA- 7. Davar, D. A phase 1 study of TSR-022, an anti-TIM-3 monoclonal anti- 1)+ myDC in cohort-1, -2, and -3, respectively. Primary objectives body, in combination with TSR-042 (anti-PD-1) in patients with colorectal were safety and feasibility. Repetitive biopsies of treated lesions were cancer and post-PD-1 NSCLC and melanoma. (2018). performed. 8. Cottrell, T. et al. Pathologic Features of Response to Neoadjuvant Anti- Results PD-1 in Resected Non-Small Cell Lung Carcinoma: A Proposal for Quanti- In this ongoing trial, 2 patients were treated in cohort-1, 2 patients in tative Immune-Related Pathologic Response Criteria (irPRC). Annals of on- cohort-2, and 3 patients in cohort-3. Patients received a median of 6 cology : official journal of the European Society for Medical Oncology (range 3-10) injections of T-VEC. All patients are evaluable for re- (2018). doi:10.1093/annonc/mdy218 sponse. The best overall tumor response (according to iRECIST) was a 9. Stein, J. et al. Major pathologic response on biopsy (MPRbx) in CR (pathologic CR) and one PR (confirmation pending; pathologic CR patients with advanced melanoma treated with anti-PD-1: evidence of treated lesions). Both patients were treated in cohort-3 and had for an early, on-therapy biomarker of response. Annals of oncology : previously progressed on anti-PD-1 checkpoint inhibition, and one official journal of the European Society for Medical Oncology 30, patient also on anti-CTLA-4 therapy. Adverse events include G1 fever 589–596 (2019). in 4 patients, G1-2 flu-like symptoms in 5 patients, transient G1-2 10. Tetzlaff, M. et al. Pathological assessment of resection specimens after local pain and redness at the injection-site in 3 patients, and G1 neoadjuvant therapy for metastatic melanoma. Annals of oncology : gastrointestinal symptoms in 4 patients. The patient with CR devel- official journal of the European Society for Medical Oncology 29, 1861– oped an asymptomatic G3 eosinophilia during treatment; the patient 1868 (2018). with PR developed a transient purpuric rash at the site of skin metas- tases after the first treatment. Multiplexed immune-profiling (Ultivue) of baseline and on-treatment tumor biopsies is ongoing. Conclusions IT co-injection of autologous CD1c (BDCA-1)+ myDC with T-VEC is feasible and tolerable and resulted in encouraging early signs of anti- tumor activity in patients with immune checkpoint inhibitor refrac- tory melanoma who received high dose CD1c (BDCA-1)+ myDC. Trial Registration ClinicalTrials.gov: NCT03747744 Ethics Approval This study was approved by the Ethics Board of Universitair Zieken- huis Brussel. Consent Written informed consent was obtained from the patient for publica- tion of this abstract and any accompanying images. A copy of the written consent (NL,FR) is available for review by the Editor of this journal. P459 New generation chimeric antigen receptor T-Cell Therapy Fig. 1 (abstract P457). See text for description (CoupledCAR) induces high rate remissions in solid tumor Chengfei Pu, Lei Xiao Innovative Cellular Therapeutics, Shanghai, China Correspondence: Lei Xiao (xiaolei@ictbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P459 P458 Background A phase I clinical trial on intratumoral administration of Conventional CAR T cell therapy showed weak CAR T expansion in autologous CD1c (BDCA-1)+ myeloid dendritic cells plus patients, thus achieved no or little response for treating solid tu- talimogene laherparepvec (T-VEC) in patients with advanced mors. Here, we generated "CoupledCAR" T cells including an anti- melanoma TSHR CAR molecule. Compared with conventional CART cells, "Cou- Julia Katharina Schwarze, MD, MSc, Gil Awada, Louise Cras, Inès Dufait, pledCAR" T cells successfully improved the expansion of CART cells Ramses Forsyth, Ivan Van Riet, Bart Neyns more than 100 times and enhanced CAR T cells’ migration ability, UZ Brussel, Brussels, Belgium allowing the CAR T cells to resist and infiltrate the tumor microenvir- Correspondence: Julia Katharina Schwarze onment and killed tumor cells. (juliakatharina.schwarze@uzbrussel.be) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P458 We designed a “CoupledCAR” lenti-vector containing a scFv targeting hTSHR. Patient‘s CD3 T cells were isolated and transduced with the Background lentivirus. Then,transduction efficiency was evaluated. After infusion, Intratumoral (IT) myeloid dendritic cells (myDC) play a pivotal role in peripheral blood samples were collected to analyze expansion and initiating antitumor immune responses and re-licensing of anti-tumor cytokine release. The evaluation of response level for patients were cytotoxic T-lymphocytes within the tumor microenvironment. IT in- performed at month 1,month 3,and month 6 by PET/CT. jection of the oncolytic virus T-VEC leads to the release of maturation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 251 of 272 Results and documented D0 prostate cancer whose PSADT is between 3 and To verify the effect of “CoupledCAR” T cells on solid tumors, we have 15 months are eligible. The participant should have normal organ func- completed several clinical trials for different solid tumors, including tions, ECOG 0-1. Patient should not be on any other cancer treatment two patients with thyroid cancer. Immunohistochemistry (IHC) results at enrollment. The primary objective is to assess the PSA slope log showed thyroid stimulating hormone receptors (TSHR) were highly change at week 24 and 48 compared to baseline. Secondary objectives expressed in thyroid cancer cells. In vitro co-culture experiments are 1) to assess the safety of ME-TARP DC vaccines 2) To characterize showed TSHR CAR T cells specifically recognized and killed TSHR- cellular and humoral immune responses associated with vaccination. positive tumor cells. Animal experiments showed TSHR CAR T cells Results inhibited the proliferation of TSHR-positive tumor cells. Therefore, we The lead-in safety cohort (N=6) completed the treatment without designed "CoupledCAR" T cells expressing a binding domain against DLT and we are currently enrolling the randomized arms (N=66). TSHR. Further,we did clinical trials of two group patients that were Conclusions successfully treated using conventional TSHR CAR T cells and "Cou- Multi-Epitope TARP Peptide Autologous Dendritic Cell Vaccination in pledCAR" T cells, respectively. In the group using conventional Men with Stage D0 Prostate is safe and open for further accrual in TSHR CAR T cells, patients showed weak cell expansion and less mi- randomized arms to compare vaccine versus placebo. gration ability. In the group using TSHR "CoupledCAR" T cells, pa- tients showed rapid expansion of CAR T cells and killing of Acknowledgements tumor cells. One month after infusion (M1), the patient was evalu- This study is supported by the Center for Cancer Research, National Cancer ated as PR(Partial Response): the lymph node metastasis disappeared, Institute, National Institute of Health. and thoracic paratracheal tumors decreased significantly. Three Trial Registration months after infusion (M3), the patient was evaluated as a durable NCT02362451 response, and the tumor tissue was substantially smaller than M1. Further, two patients with colorectal cancer were enrolled in this References trial and infused "CoupledCAR" T cells. One patient achieved PR and 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein the other one achieved SD. expressed in prostate and breast cancer cells derived from an alternate Conclusions reading frame of the T cell receptor gamma chain locus. Proc Natl Acad “CoupledCAR” T cells can effectively promote expansion, migration Sci U S A. 2000;97(17):9437–9442. and killing ability of CAR T cells in patients with thyroid cancer. “Cou- 2. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated pledCAR” T cell technology is a technological platform, which may with slowing in PSA velocity and decreasing tumor growth rates in be used to treat other cancer types. Next, we are recruiting more pa- patients with StageD0prostatecancer. Oncoimmunology. tients for clinical trials using “CoupledCAR” T cells. 2016;5(8):e1197459. Ethics Approval The study was approved by National Cancer Institute Ethics Board, approval P460 number P121083 and assigned a local number 15C0075. A randomized, placebo-controlled phase II study of multi-epitope TARP peptide autologous dendritic cell vaccination in men with stage D0 prostate cancer P461 1 3 1 Hoyoung Maeng, MD , Lauren Wood, MD , Seth Steinberg , David Phase 1b study of INCMGA00012, a programmed cell death-1 (PD- 2 1 1 Stroncek, MD , Masaki Terabe, PhD , Jay Berzofsky, MD, PhD 1) inhibitor, in combination with chemotherapy in patients with 1 2 National Cancer Institute, Bethesda, MD, United States; National advanced solid tumors (POD1UM-105) 3 1 2 2 Institute of Health, Bethesda, MD, United States; PDS Biotechnology, David Planchard, MD, PhD , Jill Bowman , Nawel Bourayou, MD 1 2 Berkeley Heights, NJ, United States Gustave Roussy Institute, Villejuif, France; Incyte Corporation, Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) Wilmington, DE, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P460 Correspondence: David Planchard (david.planchard@gustaveroussy.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P461 Background TARP is a 58 amino acid protein expressed in normal and malignant Background prostate tissue [1]. TARP is highly expressed in 95% of prostate cancers Background: The combination of PD 1/programmed death-ligand 1 including all Gleason types and in both castration sensitive (CSPC) and (PD-L1) checkpoint inhibitors with chemotherapy has proven clinic- castration resistant prostate cancer (CRPC). In the pilot study of 1st gen- ally meaningful efficacy and manageable safety profile based on sev- eration TARP peptide-pulsed autologous dendritic cell vaccination eral randomized trials in treatment-naive advanced non-small cell (TARP DC vaccine, NCT00972309, N= 41) utilizing TARP WT 27-35 and lung cancer (NSCLC) [1,2,3] and encouraging data are emerging in epitope enhanced EE29-37-9V in HLA-A*0201 positive men with stage unresectable advanced malignant pleural mesothelioma (MPM) [4]. D0 prostate cancer (PSA biochemical recurrence) published by Wood et INCMGA00012 is an investigational humanized immunoglobulin G4 al, the vaccine was found to be immunogenic and safe [2]. TARP vaccin- (IgG4) monoclonal antibody against human PD-1. In the phase 1 ation was also associated with a decreased slope log PSA in more than POD1UM-101 study, INCMGA00012 has demonstrated acceptable tol- 70 % of the patients at 24 and 48 weeks and a decrease in calculated erability with clinical activity observed in multiple tumor types, in- tumor growth rate constant. Standard of care in D0 prostate cancer cluding a confirmed objective response rate (by Response Evaluation ranges from watchful waiting, salvage radiation and anti-androgen Criteria in Solid Tumors [RECIST] version1.1) of 19% in an interim ana- therapy without strong evidence to support one or the other. In the lysis of a cohort of patients with platinum-refractory NSCLC [5]. The current study of multi-epitope (ME)-TARP DC vaccines, 5 overlapping POD1UM-105 trial aims to investigate INCMGA00012 in combination 18-20-mer peptides encompassing the full sequence of TARP are added with standard-of-care chemotherapy regimens in patients with ad- to 1st generation TARP peptides to pulse the autologous DCs. The use vanced NSCLC or MPM. of synthetic long peptides can increase the chance of a durable multi- Methods valent anti-TARP response. Methods: POD1UM-105 is a phase 1b, global, multicenter study, in pa- Methods tients with histologically or cytologically confirmed advanced/meta- This is a single-blinded, randomized, placebo-controlled phase II study static NSCLC or unresectable MPM and regardless of PD-L1 expression, in men with Stage D0 prostate cancer. Men with a PSADT between 3 not previously treated with systemic therapy. Key eligibility criteria in- and 15 months will be randomized 2:1 to receive a ME TARP DC vac- clude no prior systemic treatment (except for patients [with known sen- cine or a monocyte placebo. HLA restriction is not required. Patients sitizing mutations] who have disease progression on or following an will receive a total of 6 doses of vaccine or a placebo, 20e6 cells/dose approved targeted tyrosine kinase inhibitor, or chemotherapy com- intradermally every 3 weeks. Males older than 18 with adenocarcinoma pleted > 6 months before enrollment), no prior checkpoint inhibitor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 252 of 272 therapy, measurable or nonmeasurable disease by RECIST version 1.1, Combination Immunotherapies and Eastern Cooperative Oncology Group performance status ≤1. P462 Patients will be assigned to 1 of 4 treatment groups (12-24 patients ImmTAC®-chemotherapy combination: A preclinical evaluation each), and will receive INCMGA00012 every 3 weeks for up to 2 years shows potential benefits in combination with standard doses of chemotherapy agents (4 to 6 Filipa Bravo-Lopes, Nora Rippaus, Kristina Petrovic, Francesca Amicarella, cycles) (Treatment Group A: gemcitabine/cisplatin; Group B: peme- Adam Taylor, Laure Humbert, Rupert Kenefeck, Adel Benlahrech, BSc trexed/cisplatin; Group C: pemetrexed/carboplatin; Group D: pacli- PhD taxel/carboplatin) (Figure 1). Immunocore Ltd, Abingdon, United Kingdom The primary study objective is to evaluate safety, tolerability (DLTs), and Correspondence: Rupert Kenefeck (rupert.kenefeck@immunocore.com); determine a recommended phase 2 dose of INCMGA00012 in combin- Adel Benlahrech (adel.benlahrech@immunocore.com) ation with chemotherapy. Secondary objectives include determining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P462 preliminary clinical activity (measured by objective response rate, dur- ation of response, and disease control rate) and pharmacokinetics. Ex- Background ploratory objectives include assessment of additional efficacy measures ImmTAC molecules are bispecific T cell redirectors comprised of an (progression-free survival and overall survival), and relevant biomarkers. affinity‐enhanced TCR recognizing tumour antigen in the context of HLA molecules and a T cell engaging anti‐CD3 domain. Paclitaxel is a Acknowledgements chemotherapy drug which stabilises microtubules and it is widely This study is sponsored by Incyte Corporation (Wilmington, DE). used in combination with platinum-based chemotherapies against Trial Registration multiple indications. Building on the recent evidence that chemo- NCT03920839 therapy positively combines with immunotherapy [1], this study was designed to evaluate the potential of combining Paclitaxel with References ImmTAC-mediated T cell activation. 1. Gandhi L, Rodriguez-Abreu D, Gadgeel S, et al. Pembrolizumab plus Methods chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. In vitro assays to assess T cell-mediated lysis, T cell expansion, 2018;378:2078-2092. and cytokine release in response to ImmTAC-mediated T cell re- 2. Langer CJ, Gadgeel SM, Borghaei H, et al. Carboplatin and pemetrexed direction were performed with a range of ImmTAC concentrations with or without pembrolizumab for advanced, non-squamous non-small- and/or Paclitaxel. T cell-mediated lysis of antigen positive cells cell lung cancer: a randomised, phase 2 cohort of the open-label was monitored in real time while Granzyme A and B, IL-2, IL-8, KEYNOTE-021 study. Lancet. 2016;17:1497-1508. IL-10, IFN-γ, TNF-α,MIP-1α, IP-10, and MIG were measured in cell 3. Socinski MA et al. Atezolizumab for first-line treatment of metastatic non- culture supernatants. T cell expansion was measured by flow squamous NSCLC. N Engl J Med. 2018;378:2288-2301 cytometry. 4. Nowak AK, Lesterhuis WJ, Hughes BGM, et al. DREAM: a phase II study of Results durvalumab with first line chemotherapy in mesothelioma─first results. J ImmTAC molecules were highly effective at redirecting T cells to Clin Oncol. 2018;36(suppl):8503 proliferate, produce pro-inflammatory cytokines, chemokines and 5. Mehnert JM, Joshua AM, Lakhani N, et al. First-in-human phase 1 study of granzymes, and to lyse antigen positive targets. ImmTAC- INCMGA00012 in patients with advanced solid tumors: interim results of the co- Paclitaxel combination showed substantially enhanced lysis of tar- hort expansion phase. J Immunother Cancer. 2018;6(suppl 1):115. Abstract P669. get cells compared to either agent alone (in both magnitude and Ethics Approval kinetics). With 100 pM ImmTAC and 25 nM Paclitaxel, the median The study was approved by institutional review boards or independent cytolysis area under the curve using the compounds in combin- ethics committees of participating institutions. ation was 6269 compared to 3782 for Paclitaxel alone or 3196 for ImmTAC alone. Additionally, the time it took to lyse 80% of target cells decreased from 84 hours for ImmTAC alone to 51 hours when in combination with Paclitaxel. Despite enhanced kill- ing, some reduction of cytokine release and T cell proliferation were observed when ImmTAC and Paclitaxel were combined. Not- ably, cytokine secretion and T cell proliferation were restored, and improved killing maintained by pre-treating target cells with Paclitaxel. Similarly, pre-treatment of effector T cells with Pacli- taxel did not impact their ability to kill and proliferate in re- sponse to ImmTAC. Conclusions These in vitro studies show enhanced ImmTAC-mediated tumour cell lysis with Paclitaxel when administered either in combination with ImmTAC or sequentially. These data support the premise that Pacli- taxel positively combines with ImmTAC and provide strong rationale for further clinical investigation. Reference 1. Liu SV, Camidge DR, Gettinger SN, Giaccone G, Heist RS, Hodi FS, Ready NE, Zhang W, Wallin J, Funke R, Waterkamp D, Foster P, Iizuka K, Powderly J. Long-term survival follow-up of atezolizumab in combination with platinum-based doublet chemotherapy in pa- tients with advanced non-small-cell lung cancer. Eur J Cancer. 2018;101:114-122. Fig. 1 (abstract P461). See text for description Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 253 of 272 P463 melanoma efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine, correlating Immunotherapy combinations for betel-nuts related HNSCC: one with increases of T-cells and CD8α+ DCs institutional experiences in Taiwan Methods Jo-Pai Chen, MD, Ruey-Long Hong, MD, PhD, Wei-Chen Lu, MD Utilizing the B16F10 syngeneic mouse melanoma model, vaccina- National Taiwan University Hospital, Taipei, Taiwan, Province of China tions are administered by intramuscular electroporation with CpG ad- Correspondence: Ruey-Long Hong (rlhong@ntu.edu.gov.tw) juvant three times at one-week intervals beginning five days post Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P463 lethal tumor implantation. Aza is given intraperitoneally at 1mg/kg, and IFNα therapy is given in a series of one high followed by three Background low doses, as noted. Tumor sizes, growth, and survival were all Betel-nuts chewing might contribute to (1) strong inflammation, in- assessed. Tumor-infiltrating lymphocytes (TILs) were assessed by vasion, and angiogenesis; (2) poor response to traditional therapeis. stimulating the purified lymphocyte fraction of tumors with vaccine In the 1st line setting of R/M HNSCC, EPF offers survival benefit and antigens followed by intracellular cytokine staining flow cytometry. immune checkpoint inhibitor(anti-PD1 monoclonal antibody), like Dendritic Cells were assessed by flow cytometry. nivolumab or pembrolizumab, has already brought survival benefit in Results the 2nd line treatment. We demonstrate that the addition of IFNα and Aza treatments to Methods mice vaccinated with the MIP-3α-Gp100-Trp2 vaccine led to signifi- From 2016 to early 2019, 30 R/M HNSCC patients receiving cantly reduced tumor burden and overall increases in mouse survival, immunotherapy-containing regimens in Yun-lin Branch of National increasing median survival by 39% over vaccine. Importantly, this in- Taiwan University Hospital were reviewed. crease in efficacy was dependent on the presence of all three com- Results ponents, as vaccine plus IFNα or vaccine plus Aza did not differ These patients consisted of 1 HPV and 29 non-HPV; 18 pembrolizu- significantly from vaccine alone. The addition of Aza and IFNα to the mab and 12 nivolumab; 10 with afatinib(6 pembrolizumab & 4 nivo- vaccine increased T-cell tumor infiltration, altered the proportion of lumab); 5 with bevacizumab; 6 with chemotherapy. The objective CD8+T-cells, and increased CD8α+ DC infiltration. response rate was 47%(14/30) and clinical benefit was 80%(24/30). Conclusions 16 patients were still under use(4 afatinib with pembrolizumab; 4 afa- Efficient targeting of antigen to immature dendritic cells with a tinib with nivolumab). 1 patient under afatinib and pembrolizumab chemokine-fusion vaccine offers a potential alternative approach presented hyperprogression but then got pCR after bevacizumab to classic and dendritic cell-based vaccines currently undergoing combined with strong chemotherapy. 1 patient under low dose nivo- clinical investigation. Combining this approach with IFNα and Aza lumab in 20 mg biweekly with oral metronomic cyclophosphamide treatments significantly improved vaccine efficacy, with efficacy had mild tumor response. 1 patient under nivolumab with high dose correlating with changes in TILs and tumor infiltrating DCs. Fur- ifosfamide developed nephritis. 1 patient had rapid skin metastasis ther potential therapy optimization currently undergoing investi- over previous radiation fields after pembrolizumab, bevacizumab, gation offers promise for this line of investigation to become a and chemotherapy. 3 patients under afatinib & pembrolizumab de- novel melanoma therapy. veloped autoimmune cholestasis(2 also with pneumonitis). Afatinib & Ethics Approval nivolumab had similar efficacy but less toxicity. 10 pateints receiving All procedures performed in studies involving animals were in ac- afatinib combined with anti-PD1(8 faliling EPF, 7 with pleural/pericar- cordance with the ethical standards of the IACUC of the Johns Hop- dial/skin metastasis. 5 rapid progression within 3 months after defin- kins University under Protocols #MO16H147 and MO19H319. ite CCRT) had 70% response rate(7/10) and 90% clincial benefit(9/10). Post-progression use of anti-PD1 with other treatments were seen in P465 4 patients(esp. 1 with nivolumab & ipilimumab for sarcomatous Immune-mediated mechanisms involved in the synergistic anti- change). 3 patients got benefits and had longer survival. tumor efficacy of NHS-IL12 combined with the class I HDAC Conclusions inhibitor entinostat Immunotherapy-containing combinations are of clinical significance Kristin Hicks, PhD, Yohei Ozawa, MD, PhD, Karin Knudson, PhD, Jeffrey in refractory betel-nuts related HNSCC in Taiwan. Afatinib has several Schlom, Sofia Gameiro, PharmD, PhD immuno-modulatory effects. In high risk patients(pleural/pericardial/ National Cancer Institute, NIH, Bethesda, MD skin metastasis failing EPF and rapid progression within 3 months Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) after definite CCRT) in Taiwan, afatinib with anti-PD1 may be a good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P465 option to avoid hyperprogression. Earlier use, well biomarkers, best combinations, and optimal sequencing will be future goals. Background Combining epigenetic agents with immunotherapy has shown P464 clinical promise. Preclinically, we and others have shown that the IFN-α and 5-Aza-2’-deoxycytidine enhance the anti-tumor efficacy class I histone deacetylase (HDAC) inhibitor entinostat can po- of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by tentiate the anti-tumor efficacy of immunotherapies. Mechanistic- affecting T-cell and Dendritic Cell recruitment into tumor ally, this is partially mediated by tumor MHC Class I upregulation, James Gordy, PhD, Richard Markham, BS MD impairing regulatory CD4+ T cells (Tregs) and monocyte derived Johns Hopkins Bloomberg School of Public, Baltimore, MD, United States suppressive cells (MDSCs) and/or enhancing CD8+ T cells and Correspondence: Richard Markham (rmarkha1@jhu.edu) granzyme B levels in the tumor microenvironment (TME). In these Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P464 studies, we observed that entinostat induced murine tumor necrosis. Background Methods The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic Therefore, we investigated if this treatment-induced necrosis cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated anti- could be targeted by NHS-IL12, an IL-12 NHS76 conjugate that gens have shown improved efficacy and immunogenicity, compared binds free DNA in regions of tumor death/necrosis. NHS-IL12 has to vaccines lacking the chemokine. To optimize the therapy, our la- been shown to have significant anti-tumor efficacy in preclinical boratory has added agents designed to further enhance the T cell ac- murine models and has been demonstrated to be safe in pa- tivating function of DCs and overcome immunoregulatory tients. We hypothesize that increasing the pro-inflammatory, im- mechanisms of the tumor microenvironment. Here, we report that mune stimulatory cytokine IL-12 in the TME will synergize with the combination of type-I interferon therapy (IFNα) with 5-Aza-2’- the entinostat-mediated immune effects to induce significant deoxycitidine (Aza) profoundly enhanced the therapeutic anti- tumor control. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 254 of 272 Results paclitaxel was 54.5% (6/11), the median PFS was 6.4 months (95% CI In the EMT6 breast cancer murine model, administration of NHS- 3.7 to 9.2 months), and the median OS was 14.1 months (95% CI 3.2 IL12 at the onset of entinostat-induced necrosis synergized to to 25.0 months). produce significant anti-tumor efficacy, including resolving well- Conclusions established tumors and enhancing survival. These studies are Our preliminary data suggest that carboplatin and paclitaxel often in- now being extended to additional murine models of solid carcin- duce objective responses in patients with prior treatment with anti- omas. Examination of immune subsets in the TME by flow cytom- PD1/PD-L1 antibodies and that the clinical outcomes compare favor- etry revealed that the combination increased infiltration of ably to those seen in checkpoint inhibitor naïve patients. If con- granzyme B+ CD8+ T cells and M1-like CD38 expressing tumor firmed, these data suggest that there is no “opportunity cost” for macrophages. Currently, we are using depletion studies to assess administering immunotherapy in the first line. the relative contribution of these immune subsets to the anti- Ethics Approval tumor efficacy. Further, we are examining the effect of this com- Approval by the UCSF IRB has been requested and will be obtained bination on CD8+ T cell function and macrophage polarization, before this work is presented. phenotype, and function. Conclusions Overall, the combination of NHS-IL12 and entinostat is showing en- P467 couraging results in a preclinical solid tumor model, providing a ra- ALPN-202, a conditional CD28 costimulator and dual checkpoint tionale to examine this combination clinically. inhibitor, enhances the activity of multiple standard of care modalities Katherine Lewis, PhD, Mark Maurer, BS, Sherri Mudri, BS, Kayla Susmilch, Acknowledgements MS, Fariha Ahmed-Qadri, MS, Chelsea Gudgeon, BS, Steven Levin, PhD, The authors thank Curtis Randolph for excellent technical assistance. Martin Wolfson, BS, Stacey Dillon, PhD, Kristine Swiderek, PhD, Stanford Peng, MD, PhD Alpine Immune Sciences, Seattle, WA, United States P466 Correspondence: Katherine Lewis Carboplatin and paclitaxel after anti-PD1 or anti-PDL1 therapy: A (katherine.lewis@alpineimmunesciences.com) retrospective study in patients with squamous cell carcinoma of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P467 the head and neck Audrey Humphries, BS, Madeleine Welsh, BA, Alain Algazi, MD Background UCSF, San Francisco, CA, United States Checkpoint inhibitors targeting the PD-1 axis have transformed Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) cancer treatment. However, objective response rates remain low, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P466 suggesting that novel therapeutics and/or combination treat- ments are needed. At the same time, non-immuno-oncology Background therapeutic approaches such as chemotherapy remain standard Historically, cytotoxic chemotherapy has been the first line stand- of care for many malignancies. ALPN-202 is a variant CD80 ard of care for patients with RM-SCCHN. More recently, a ran- vIgD™-Fc fusion that mediates PD-L1-dependent CD28 costimula- domized phase 3 clinical trial demonstrated improved overall tion and inhibits the PD-L1 and CTLA-4 checkpoints. This novel survival in RM-SCCHN patients receiving pembrolizumab in the mechanism of action provides potent single agent immunomodu- first line versus those receiving the prior standard carboplatin, latory activity in mouse tumor models, and thus has the potential 5FU, cetuximab. Additional studies have suggested that sequen- to complement other therapeutic modalities, such as checkpoint cing of therapies impacts outcomes. Several small case series inhibitors or chemotherapies. have described clinical outcomes in patients treated with chemo- Methods therapy after immunotherapy, but these have often included pa- Mice were implanted subcutaneously with human (hu) PD-L1- tients treated with a variety of cytotoxic regimens such that transduced MC38 colon carcinoma and B16-F10 melanoma cell lines. comparison to historical controls is difficult. Here we present clin- Once measurable tumors were established, mice were treated with ical outcomes in patients treated with carboplatin and paclitaxel anti-mouse checkpoint (i.e. PD-1 or CTLA-4) blocking monoclonal immediately following disease progression on anti-PD1 or anti- antibodies (mAbs) or oxaliplatin, a platinum-based chemotherapeutic PDL1 therapy. agent, alone or in combination with ALPN-202, to evaluate compati- Methods bility of the novel ALPN-202 protein with existing cancer therapies. A chart review was performed to identify all patients with RM- Anti-tumor responses were evaluated by serial tumor volume measure- SCCHN, including tumors originating in the oral cavity, oropharynx, ments and RNA-Seq analysis of tumors isolated from treated mice. hypopharynx, larynx, and sinuses treated with an anti-PD1 or anti- Results PDL1 antibody immediately followed by carboplatin and paclitaxel Anti-PD-1, anti-CTLA-4, or oxaliplatin alone were only modestly ef- through December 2018. Patients were required to have both pre- fective as monotherapy in huPD-L1+ MC38 tumor-bearing mice, treatment and post-treatment imaging associated with treatment while ALPN-202 has potent anti-tumor activity in this model. with both immunotherapy and chemotherapy or documented death When the checkpoint inhibitors or chemotherapy were adminis- from disease after administration of at least 1 cycle of chemotherapy. tered in combination with ALPN-202, significantly greater reduc- Anti-PD1 / PDL1 treatment history, p16 IHC (for OPC), and baseline tions in tumor growth over time were observed than with any of PD-L1 IHC (where available) were assessed. The best overall response these agents alone (Figure 1 and Figure 2,respectively).Further- was assessed by RECIST v1.1 and PFS and OS were determined using more, ALPN-202 was extremely effective (92% tumor growth in- the Kaplan-Meier method. hibition) in improving the anti-tumor activity of anti-PD-1 mAb in Results mice bearing huPD-L1+ B16-F10 tumors, a tumor that is known A total of 11 patients meeting the inclusion criteria were identified to be poorly immunogenic and treatment-recalcitrant (Figure 3). including 5 patients with p16+ SCC of the oropharynx, and 3 with RNA-Seq analysis of tumors from the MC38 studies was per- SCC of the oral cavity. 5 patient has detectable staining for PD-L1 at formed to explore in-depth the mechanisms, including enhance- baseline, 2 did not, and 3 patients had no PD-L1 IHC available. 6 pa- ment of T cell effector transcript expression, that play a role in tients receive pembrolizumab alone or in combination, 4 received the ability of ALPN-202 to provide anti-tumor immunity and to durvalumab, and 1 patient was treated with nivolumab. The median enhance the activity of checkpoint inhibitors and the chemother- duration of prior PD1 / PDL1 exposure prior to chemotherapy was apeutic oxaliplatin. 2.7 months (range 1.4 to 18.2 months). The BORR to carboplatin and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 255 of 272 Conclusions P468 ALPN-202 demonstrates potent anti-tumor efficacy as monother- Targeting the ICOS pathway in combination with apy and significantly improves the anti-tumor activity of other chemotherapy to enhance T-cell mediated anti-tumor immune only modestly effective treatment modalities, such as checkpoint- responses only blockade mAbs and chemotherapy. ALPN-202 has the poten- Tamer Mahmoud, PhD, Jeffrey Riggs, Madhu Ramaswamy, PhD, Leigh tial to be significantly effective as a monotherapy, and its com- Hostetler, Brian Naiman, Ilyssa Ramos, Sean Turman, Fernanda Pilataxi, patibility with checkpoint inhibitors and chemotherapeutics Christopher Morehouse, MD, Alex Alfaro, Norman Peterson, Ryan suggest versatility in its potential to improve outcomes in the Fleming, Nazzareno Dimasi, Kyle Kuszpit, Ronald Herbst, Gianluca frontline setting alone and/or in combination with standard of Carlesso, PhD care of multiple cancer types. A first-in-human clinical study with AstraZeneca, Gaithersburg, MD, United States ALPN-202 is in preparation. Correspondence: Gianluca Carlesso (carlessog@medimmune.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P468 The study was approved by the vivarium's International Animal Care and Use Committee (IACUC), IR# 17-01. Background ICOS (Inducible T-cell Costimulator) is a member of the CD28 superfamily that is detected on activated and memory T lym- phocytes. Treatment with Immune checkpoint inhibitors (ICI) in clinical studies has shown that expansion of ICOS-expressing T cells is associated with positive patient outcome. Preclinical studies have validated the rationale for targeting ICOS and the clinical investigation of ICOS agonist antibodies (mAbs) com- bined with ICI is under way. To enhance tumor immunogenicity and increase the response rate to immunotherapy, we examined the efficacy of combination therapy of ICOS mAbs with stand- ard of care chemotherapy using a colorectal tumor syngeneic animal model. Methods For the microarray analysis, freshly isolated normal primary hu- man T cells were stimulated with sub-optimal concentration of Fig. 1 (abstract P467). See text for description anti-CD3 mAb with either ICOS, OX40 mAbs, or GITRL-FP for 4 hours. ¬In-vitro proliferation and cytokine secretion assays were performed using primary human T cells stimulated with anti- CD3 mAb and ICOS mAb in the presence of different inhibitors. For in-vivo studies, CT26 tumor cells were subcutaneously injected into BALB/c mice and ICOS or control mAbs were ad- ministered intraperitoneally (ip) or in combination with an intra- venous injection of 5-Fluorouracil (5-FU) at day 10 post tumor implantation. For the biodistribution study, CT26 tumor-bearing mice were dosed ip with 89Zr-labeled anti-ICOS or control mAb and imaged using positron emission tomography (PET)/SCAN. For the depletion study, CT26 tumor-bearing mice were injected ip with either CD4 or CD8 mAb twice a week starting one day before tumor implantation, whereas sphingosine-1 phosphate re- ceptor (S1PR) modulator (FTY720) was orally administered daily starting at day 9 post-implantation. Tumor growth was assessed three times a week. Results Fig. 2 (abstract P467). See text for description Compared to other T-cell agonists, in-vitro co-stimulation of human T cells with ICOS mAb lead to enhanced metabolic T cell reprogramming, activation of the PI3K/mTOR pathways, and secretion of IFN-gamma and IL-10. Immuno-PET scan imaging analysis revealed ICOS expression across tumor and secondary lymphoid tissues. ICOS mAb and 5-FU combination therapy on CT26 tumor-bearing mice resulted in a significant increase in anti-tumor responses compared to single-arm treatment or con- trol groups. Moreover, the efficacy of either anti-ICOS or 5-FU monotherapy or combination required cytotoxic T cell activity and was dependent on the S1P-mediated egress of immune cells from peripheral lymph nodes. Conclusions Collectively, the results of these studies show that ICOS agonism in combination with chemotherapy may provide an effective therapeutic option for the activation of T cells in solid tumor Fig. 3 (abstract P467). See text for description indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 256 of 272 P469 4. Cappello P, et al. Vaccination with ENO1 DNA prolongs survival of Chemotherapy enhances effector T cell responses to tumor genetically engineered mice with pancreatic cancer. Gastroenterology. associated antigens in human and mouse pancreatic cancer 2013;144:1098–106. 1 1 1 Giorgia Mandili, PhD , Claudia Curcio, PhD ,SaraBulfamante, MS , Ethics Approval 1 1 2 Laura Follia, MS , Daniele Giordano, MD , Rossella Spadi, MD ,Maria The study was approved by the local research ethical committee 2 1 Antonietta Satolli, MD , Paola Cappello, PhD MS , Francesco Novelli, (Azienda Ospedaliera Città della Salute e della Scienza di Torino, PhD Turin) and investigations were performed according to the Helsinki 1 2 University of Turin, Turin, Piedmont, Italy; Azienda Ospedaliera Declaration principles. Universitaria Città della Salute e della Scienza di Torino, Turin, Piedmont, Italy Correspondence: Francesco Novelli (franco.novelli@unito.it) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P469 P470 Impact of treatment-induced necrosis in the anti-tumor Background efficacy of Entinostat combined with the immunocytokine Pancreatic Ductal Adenocarcinoma (PDA) is one of the most NHS-IL12 lethal cancer, both for lack of effective screening method and Yohei Ozawa, MD, PhD, Kristin Hicks, PhD, Karin Knudson, PhD, Jeffrey resistance to chemotherapy (CT). Immunotherapy (IT) trials with Schlom, Sofia Gameiro, PharmD, PhD immune check-point inhibitors did not achieve significant gain National Cancer Institute, NIH, Bethesda, MD, United States of survival yet [1]. However, some CT agents, such as gemcita- Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) bine (GEM), have several immune modulating effects [2] and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P470 starting from the hypothesis that more immunogenic antigens can be induced by CT treatment, its ability to increase the sus- Background ceptibility of PDA to IT was evaluated. Tumor necrosis resulting from hypoxia, inflammation, or ab- Methods normal angiogenesis is associated with poor outcome in sev- Sera from 28 PDA patients before and after CT (BCT and ACT eral solid malignancies. However, theroleoftreatment- respectively) were profiled by serological proteome analysis induced necrosis is still controversial. The class I HDAC inhibi- (SERPA) [3]; the recognized TAAs were identified by mass spec- tor entinostat (Syndax) has been shown to promote significant trometry and confirmed by ELISA. The proliferation, phenotype tumor control in combination with immunotherapies through and cytokine production of T cells were evaluated on patients’ immune-mediated mechanisms. In addition, we observed that PBMCs from the same cohort after in vitro stimulation with entinostat induces tumor necrosis in murine solid tumors. We four selected TAAs (ENO1, G3P, K2C8 and FUBP1). Mice that hypothesize that entinostat-induced necrosis could be tar- spontaneously develop PDA (KC) were treated with GEM prior geted by NHS-IL12 (M9241, Merck KGaA), an IL-12 NHS76 con- of DNA vaccination against ENO1 [4]. Tumor lesions, immune jugate designed to bind free DNA in regions of tumor death/ infiltration and the titer of TAAs-specific antibody were evalu- necrosis, therefore promoting a synergistic anti-tumor effect. ated. TAAs-specific IFNγ production from splenocytes was NHS-IL12 has demonstrated significant anti-tumor efficacy in analyzed. preclinical solid tumor models and has been safely adminis- Results tered to cancer patients. The number of TAAs recognized by IgG in PDA patients’ sera, Methods as well as their ability to induce a complement dependent cyto- The amount of necrosis entinostat induced over time was exam- toxicity against PDA cells, was increased in ACT sera. Some ined in three distinct murine solid tumor models: MC38 (colon), identified TAAs showed a positive correlation between the in- 4T1 (triple-negative breast) and EMT6 (breast). Necrosis was crease of ACT antibody titer and longer patients’ survival. An in- evaluated by histological analysis of H&E staining and a ter- creased T cell TAAs-specific proliferative response after CT and minal deoxynucleotidyl transferase dUTP nick end labeling the evaluation of IFNγ/IL10 ratio was detected, revealing that (TUNEL) assay. To assess if NHS-IL12 could bind to necrotic ATC T cells shifted TAAs-specific responses from regulatory to tumor regions, tumor sections from mice treated with PBS or effector one. After stimulation with TAAs, in mostly patients the entinostat were incubated in vitro with NHS-IL12. The binding ratio between CD8 and CD4 Treg cells was increased in ATC T and distribution of NHS-IL12 in the tumor was then examined cells. The role of CT to enhance the TAAs-specific adaptive re- by immunofluorescence staining usingananti-humansecondary sponse prompt us to exploit its effect in combination with the antibody. DNA vaccination. Of clinical relevance, KC mice treated with Results GEM prior of ENO1 DNA vaccination displayed smaller tumor le- Entinostat promoted tumor control in all three tumor models, sions together with an increase of tumor-infiltrating CD4 and albeit to different degrees. The onset of entinostat-induced ne- CD8, in comparison to mice vaccinated or GEM-treated only. crosis was observed after two weeks of continuous dosing. In Furthermore, CT increased specific antibodies and IFNγ- EMT6 tumors, areas of tumor necrosis identified via H&E stain- producing T cells in vaccinated mice not only against ENO1, the ing also displayed DNA fragmentation measured by the TUNEL target TAA of vaccination, but also against G3P, suggesting an assay. Of note, in the entinostat-treated tumors there were antigen spreading effect of the combinatory treatment. areas of tumor death/necrosis with increased immune cell infil- Conclusions tration identified by H&E staining. Furthermore, initial immuno- Overall these data indicated that in pancreatic cancer CT effect- fluorescence studies indicate that NHS-IL12 binds to these areas ively ameliorates T cell responses against TAA and it might be in the entinostat-treated tumors in vitro. These preliminary find- reconsidered to render them suitable targets for IT. ings are being confirmed utilizing specimens from mice treated with all the agents. Additionally, in the EMT6 model, entinostat and NHS-IL12 syner- References gized to produce significant anti-tumor efficacy, including 1. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients resolving well-established tumors and enhancing survival. The with advanced cancer. N Engl J Med. 2012;366:2455–65. combination therapy promoted tumor infiltration of CD8 T cells 2. Cappello P, et al. Next generation immunotherapy for pancreatic and M1-like macrophages. Ongoing correlative studies in tumor cancer: DNA vaccination is seeking new combo partners. Cancers. specimens are examining the spatial distribution between 2018;10(2):51 entinostat-induced tumor necrosis, NHS-IL12 binding, and CD8 3. Tomaino B, et al. Autoantibody signature in human ductal pancreatic and M1 infiltration. adenocarcinoma. J Proteome Res. 2007;6:4025–31. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 257 of 272 Conclusions Journal of Clinical Oncology, ASCO, 2017 35, e14544. Overall, our results suggest that entinostat-induced necrosis may 2. Pankaj Gaur et al. CXCR4 antagonist (BL-8040) to enhance antitumor promote a tumor microenvironment conducive to NHS-IL12 bind- effects by increasing tumor infiltration of antigen-specific effector T-cells. ing, resulting in significant anti-tumor efficacy. These results in- Journal of Clinical Oncology, ASCO, 2018, 36, 73. form a rationale to examine this combination in the clinic for 3. Manuel M. Hidalgo et al. Evaluation of pharmacodynamic (PD) patients with solid carcinomas. biomarkers in patients with metastatic pancreatic cancer treated with BL- 8040, a novel CXCR4 antagonist. Acknowledgements Journal of Clinical Oncology, ASCO, 2018 36, 88-88. The authors thank Curtis Randolph for his excellent technical assistance. Ethics Approval The study was approved by the Hebrew University Ethics Board, approval number 18-15644-4. P471 Combination of BL-8040, anti PD-1 and chemotherapy significantly reduced pancreatic tumor growth and changed the balance between CD4+/FOXP3+ cells and CD8+ cells in the tumor Amnon Peled, PhD (peled@hadassah.org.il) Hadassah University Hospital, Jerusalem, Israel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P471 Background Cancer cells shape the tumor microenvironment (TME) to support their growth by recruiting immune suppressing cells such as T regulatory cells, as well as inhibiting the recruitment and activa- tion of effector CD8+ T cells. In this study, we investigated the effect of combining the CXCR4 antagonist BL-8040 [1, 2], anti PD- 1 immune checkpoint inhibitor and chemotherapy of Irinotecan, Fluorouracil and Leucovorin) IFL (on pancreatic tumor immune cell composition and growth. Methods The effect of BL-8040, anti PD-1 and IFL on tumor growth and im- mune cell constitution was assessed using the syngeneic Panc02 tumor mouse model. Tumors were established by s.c. injection . The Fig. 1 (abstract P471). Tumor growth accumulation of immune cells in the TME was assessed by immuno- histochemical staining for CD8, CD4, Foxp3, and CD69. Results Treatment of tumors with anti PD-1 or BL-8040 alone, had no effect on tumor growth, whereas, IFL treatment had significant effect on tumor growth (67% inhibition). Combination of anti PD-1 + IFL, had no significant better effect on tumor growth compared to IFL (p<0.09), whereas, BL-8040 + IFL, had a signifi- cantly better effect on tumor growth compared to IFL (p<0.04). Moreover, IFL + BL-8040 + anti PD-1, had a highly significantly better effect on tumor growth, compared to IFL alone (p<0.004) (Figure 1). The triple combination treatment (TCT), further re- duced tumor growth, compared to chemotherapy alone, by 58%.Inthe TCT, 3out of 8micedid notdevelop tumoratall, compared to 1 mouse that did not develop tumor in the IFL alone group. The TCT did not significantly change the number of CD8+ T cells accumulating in the tumor but increased their activation status (Figure 2A,B). Only in the TCT, the CD8+ T cells Fig. 2 (abstract P471). A, B, C. See text for description were larger in size inside the tumor parenchyma (p<0.01, Fig. 2C) and expressed CD69. Interestingly, we found that tumors treated with the TCT, had significantly reduced numbers of CD4+ and CD4+, Foxp3+ cells (Figure3). Conclusions TCT reduced significantly the number CD4+ and CD4+FOXP3+ cells and increased the numbers of activated CD8+, CD69+ cells in the TME. We hypothesize, that the ability of BL-8040 to modulate the TME may allow better activation of immune effector cells, contributing to the effect of chemotherapy and immunotherapy on tumor growth .A Phase IIa, multicenter, open label trial in patients with metastatic pancreatic cancer (the COMBAT study cohort II, [3]) is currently ongoing to as- sess the effect of the triple combination with BL-8040, +Pem- brolizumab and ILF on disease progression. References 1. Michal Abraham et al. Effect of BL-8040, high-affinity CXCR4 antagonist, on T-cell infiltration, tumor growth, and synergy with immunomodula- Fig. 3 (abstract P471). See text for description tory agents. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 258 of 272 P472 Background STAT3 ASO and Cisplatin combination sensitizes protected tumor While immune checkpoint blockade has revolutionized cancer care, microenvironments to checkpoint-mediated therapy many patients remain refractory to checkpoint inhibition. Converting Theresa Proia, PhD, Maneesh Singh, PhD, Nanhua Deng, Minwei Ye, checkpoint-refractory tumors into checkpoint-responsive tumors is a Frank McGrath, Douglas Ferguson, Simon Barry major challenge for cancer immunotherapy. Interleukin-12 (IL-12) is a AstraZeneca, Waltham, MA, United States potent cytokine that holds potential to reshape the immune environ- Correspondence: Simon Barry (simon.t.barry@astrazenea.com) ment in solid tumors. Its clinical utility, however, has been limited by Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P472 severe toxicities both from systemic administration and from expres- sion by adoptively transferred gene engineered T cells. We report Background here that adoptive cell therapy (ACT) with T cells carrying surface- Chemotherapy-immunotherapy (chemo-IO) combinations are being tethered DeepTM IL-12 overcomes these challenges, enhances T cell explored in the clinic. Immune responses mediated by PD-1 or PD-L1 therapeutic efficacy, activates immune cells in the tumor and over- inhibition may be enhanced by the immunogenic effects of cytotoxic comes resistance to checkpoint blockade. agents, which can increase tumor antigens. Chemo-IO combination Methods strategy relies on drug dose and schedule optimization, to minimize Activity of Deep IL-12 Primed T cells were evaluated in B16-F10 melan- direct T cell killing with chemotherapy, enhance antigen presenta- oma tumors, a cell line resistant to checkpoint inhibition. Mouse PMEL tion, and promote T cell activation. CD8 T cells, which are reactive against the B16-F10 melanoma antigen Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that se- gp100, were surface-tethered with Deep IL-12 and evaluated for anti- lectively targets STAT3, a master regulator of immune suppression, tumor activity in mice bearing B16-F10 melanoma. We additionally and is currently in Ph 1/2 clinical trials in combination with an anti- evaluated effects in the tumor of Deep IL-12 Primed T cells and PMEL T PD-L1 antibody, durvalumab. In preclinical tumor models, we demon- cells alone or co-administered with IL-12. Further studies evaluated strated that mouse surrogate STAT3 ASO remodels the suppressive combinations of Deep IL-12 Primed T cells with checkpoint inhibition. tumor microenvironment to enhance cytotoxic T cell activity in com- Results bination with anti-PDL1 (Singh et al. SITC 2019). ACT of Deep IL-12 Primed PMEL T cells significantly improved tumor Methods growth inhibition and overall survival of B16-F10 tumor bearing mice We sought to maximize the therapeutic benefits of chemo-IO by compared to PMEL T cells alone or combined with systemic co- dose and schedule optimization of adding cisplatin to STAT3 ASO administration of IL-12. In the tumor, Deep IL-12 repolarized im- and anti-PDL1. In patients, cisplatin dose ranges from 50mg/m2- munosuppressive monocytic myeloid-derived suppressor cells (M- 100mg/m2. We modeled relevant preclinical doses in the range of 5- MDSC) into an immune-activating antigen-presenting cell (APC) 10 mg/kg based on mouse plasma exposure, and proceeded with 5 phenotype. Consistent with an anti-tumor role for the repolarized M- mg/kg to represent a low clinical therapeutic (~60mg/m2) dose. MDSC, administration of an antibody to deplete these cells reduced Using the immunogenic MC38 model, various schedules of chemo-IO the efficacy of Deep IL-12 Primed T cells. Further evaluation revealed were explored; (1) Cisplatin priming on day 3 (to increase antigens), high expression of the checkpoint ligand PD-L1 on the repolarized followed by STAT3 ASO/anti-PDL1 on day 7. (2) STAT3 ASO pre- M-MDSC. To test the hypothesis that this limits efficacy of Deep IL-12 treatment on day 3 (to remodel the suppressive tumor microenviron- Primed T cell ACT, Deep IL-12 Primed T cells were co-administered ment), followed by cisplatin/anti-PDL1 on day 7. (3) All 3 agents with PD-L1 blockade. This further improved anti-tumor efficacy, dosed simultaneously on day 7 post implant. resulting in durable long-term responders. Efficacy was further im- Results proved by repeat dosing of Deep IL-12 Primed PMEL T cells. Cisplatin treatment in MC38-tumor bearing mice resulted in variable Conclusions tumor growth inhibition, with one tumor regression. Flow cytometry Our data demonstrates that ACT with tumor-specific T cells carrying analysis confirmed that cisplatin treatment had no detrimental effect surface-tethered Deep IL-12 repolarizes suppressive M-MDSC in the on T cell number or functionality, and showed a trend of increased tumor, enhances anti-tumor efficacy, and synergizes with checkpoint CD11b+/Ly6C+ dendritic cells, providing confidence to explore effi- inhibition in a checkpoint refractory cancer model. Torque is apply- cacy of the triplet combination. ing this approach to develop a novel adoptive cell therapy for can- Regardless of schedule, we observed 20% complete response rate in cer, Deep IL-12 Primed multi-targeted T cells (TRQ12-01), which is the triplet combinations, compared with 0 complete responses in expected to start clinical evaluation in 2019. any other treatment group; but interestingly, schedule 1 required dose holidays. Flow cytometry analysis of the triplet compared with P474 vehicle revealed enhanced CD4 T cell functionality (1.6x increase Efficacy and toxicity evaluation of anti-human CD47 and SIRPa IFNγ, p<0.001, and 1.2x increase IL-2, p=0.001), and enhanced NK antibodies in genetically humanized B-hSIRPa/hCD47 mice functionality (1.3x increase Granzyme B+, p<0.01; 4.3x increase TNFα, Frank An, Yanan Guo, Jie Xiang, Chaoshe Guo p<0.001). Biocytogen, Boston, MA, United States Conclusions Correspondence: Jie Xiang (info@biocytogen.com); Chaoshe Guo Collectively, we have generated data to support the combination of (info@Biocytogen.com) low-dose chemotherapy and immunotherapy to enhance the anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P474 tumor immune response. As chemo-IO combinations are being ex- plored in the clinic, it will be important to optimize dose and sched- Background ule to minimize toxicity and maximize therapeutic benefit. CD47 is a transmembrane protein expressed ubiquitously on the sur- face of human cells, including. SIRPa, one of the binding partners of P473 CD47, is a member of the signal-regulatory-protein (SIRP) family which Adoptive transfer of Deep IL-12 Primed T cells increases sensitivity is expressed on many myeloid cells including phagocytic cells. Engage- to PD-L1 blockade for superior efficacy in checkpoint refractory ment of SIRPa by CD47 elicits the “do not eat me” signal to prevent tumors phagocytosis of “self” cells by macrophages. Anti-CD47 and anti-SIRPa 1 1 2 Gulzar Ahmad, PhD , Jonathan Nardozzi, PhD , Lars Petersen, PhD , antibodies that interfere with the CD47-SIRPa interaction have demon- 2 2 1 Esben Christensen, MSc , Ditte Jaegher , James Suchy, PhD , Karsten strated promise in clinical trials as new class of therapeutics. Despite 1 1 1 Sauer, PhD , Douglas Jones, PhD , Thomas Andresen, PhD such exciting potential, side effects associated with CD47/SIRPa block- 1 2 Torque Therapeutics, Cambridge, MA, United States; Denmark ade such as anemia may represent a significant toxicity concern. There- Technical University, Kgs. Lyngby, Denmark fore, evaluation of both efficacy and toxicity of human CD47/SIRPa Correspondence: Thomas Andresen (tandresen@torquetx.com) antibody candidates emerges as one of most actively investigated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P473 areas in immuno-oncology in recent years. However, lack of animal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 259 of 272 models that enable expedient testing of anti-human CD47 or anti- further increased with loss of both PD1 and LAG3, as a result of en- human SIRPa antibodies in vivo has been a limiting factor for CD47/ hanced proliferation (Ki67/BrdU) – a phenotype demonstrated to be SIRPa antibody development. intrinsically regulated in the co-adoptive transfer system. Although Methods expression of TIM3, TIGIT and 2B4 IRs that normally co-express with To accelerate direct efficacy and toxicity testing of anti-human CD47 PD1 are maintained, CD8+ TIL isolated from Pdcd1L/L E8ICre.GFP and SIRPa antibodies, Biocytogen has generated the double human- and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP show increased functionality ized mice, B-hSIRPa/hCD47, where the human extracellular domains (IFNg, TNFa and GzmB release) by flow cytometry. Moreover, CD8+ of SIRPa and CD47 replace their respective murine counterparts. TIL polyfunctionality is evident with PD1/LAG3 loss that was largely Homozygous B-hSIRPa/hCD47 mice express humanized but not the driven by effector and chemoattractive secretions by analysis with a wild type mouse SIRPa and CD47. Further, we also created two triple 28-plex single-cell cytokine response panel (Isoplexis). humanized mouse strains, B-hPD1/hSIRPa/hCD47 and B-hPD-L1/ Conclusions hSIRPa/hCD47, where humanized PD1 and PD-L1 extracellular do- Overall, these data suggest that PD1 and LAG3 synergize to have a mains replace their mouse counterparts, respectively, in the B- dominant effect on CD8+ TIL and limit antitumor immune effects, as hSIRPa/hCD47 background. intrinsic removal of both IRs results in reduced B16-F10 tumor Results growth which has a substantive impact on the development of sys- We present here that B-hSIRPa/hCD47 mice were successfully used temic anti-tumor immunity. These results are encouraging for the for screening anti-human CD47 and anti-human SIRPa antibodies for continued development of LAG3 targeting agents in the clinic, which efficacy and toxicity in tumor models of the engineered MC38- would hopefully yield improved clinical responses in combination hCD47 cell line that expresses human CD47 in MC38 cells. Anti- with anti-PD1. human CD47 and anti-human SIRPa antibodies were efficacious in controlling MC38-hCD47 tumor growth in B-hSIRPa/hCD47 mice. Var- P476 ied toxicity profiles were observed in terms of body weight loss, The combination of a STING agonist with cytokines results in blood cell counts, and blood liver enzyme levels in anti-human CD47 robust anti-tumor effects in autochthonous tumor models antibody treatments. Anti-human PD-1 and anti-human CD47 anti- 1 1 1 2 Cristina Blaj, PhD , Yingjoy Li , Allen Chen , Anthony Descien, PhD , bodies showed single agent and combination anti-tumor effect in B- 2 2 3 Brian Francica, PhD , Sarah McWhirter , Lora Picton , K. Christopher hPD1/hSIRPa/hCD47 mice. So did anti-human PD-L1 and anti-human 3 1 Garcia , David Raulet, PhD CD47 antibodies in B-hPD-L1/hSIRPa/hCD47 mice. 1 2 University of California Berkeley, Berkeley, CA, United States; Aduro Conclusions Biotech Inc, Berkeley, CA, United States; Stanford University School of Taken together, we have validated three double and triple human- Medicine, Stanford, CA, United States ized CD47/SIRPa mouse models and demonstrate that these human- Correspondence: Cristina Blaj (cristina.blaj@berkeley.edu) ized mice are useful tools in facilitating development of therapeutics Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P476 targeting human CD47/SIRPa. Background P475 Cancer immunotherapies based on immune checkpoint blockade are PD1 and LAG3 converge to limit polyfunctionality and systemic highly effective, but only in a limited number of tumor types. Even in immunity transplanted preclinical models, monotherapy rarely results in cures. 1 2 2 Lawrence Andrews, PhD , Sasikanth Manne , E. John Wherry, PhD , Creg Moreover, preclinical subcutaneous models appear to be more respon- 1 1 Workman, PhD , Dario Vignali, PhD sive to therapies than preclinical carcinogen or genetically engineered 1 2 University of Pittsburgh, Pittsburgh, PA, United States; University of mouse (GEM) autochthonous models of cancer, or human tumors. To Pennsylvania, Philadelphia, PA, United States extend immunotherapy to a broader range of tumor types, our strategy Correspondence: Dario Vignali (dvignali@pitt.edu) is to rationally design combination immunotherapies with the potential Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P475 to boost innate and adaptive immune responses and overcome im- munosuppressive environments characteristic of human tumors. Our Background regimen includes a stimulator of interferon genes (STING) agonist, cyto- Targeting PD1 has yielded clinical success across a variety of tumor kines and checkpoint inhibitors. types, yet a significant proportion of patients remain unresponsive to Methods treatment. Thus, overcoming inhibitory receptor (IR)-mediated toler- Autochthonous tumors induced by a carcinogen or in GEM models, ance is essential to improve immunotherapeutic responses. Co- as well as subcutaneous models derived from the GEM models, were expression of PD1 and LAG3 on CD8+ tumor-infiltrating T cells (TIL) is treated with combination immunotherapies. We used flow cytometry, associated with an exhausted phenotype, exemplified by a severe de- immunofluorescence, Luminex assays and qPCR to characterize the fect in cytokine production, cytolytic activity and inability to proliferate. immune cell infiltration, activation status, receptor expression and se- In a number of murine tumor models, dual PD1/LAG3 blockade syner- cretion of cytokines in response to therapy. gistically limits tumor growth greater than targeting PD1 alone, yet the Results relative and synergistic contributions of PD1 and LAG3 on CD8+ T cells Intratumoral injection of cyclic dinucleotides (CDNs, specifically ADU- in preventing effective anti-tumor immunity is unknown. S100, a STING agonist) resulted in ~20% stable regressions of estab- Methods lished transplanted tumors and immunity to re-challenge in a sub- To understand the cellular and mechanistic basis for PD1/LAG3 syn- cutaneous sarcoma model. The same protocol resulted in significant ergy, conditional knockin mice “surgically dissect” Pdcd1 and/or Lag3 tumor growth delay in autochthonous GEM models and a carcinogen floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. To allow model. Antibody-mediated depletion studies revealed that natural for intrinsic analysis of PD1 and/or LAG3 on antigen-specific CD8+ T killer (NK) cells as well as CD4 and CD8 T-cells played an important cells, these mice have been generated as a pmel-1 transgenic back- role in mediating the anti-tumor effects. The combination of CDNs ground, with each mutant strain uniquely congenically marked for use and an IL-2 superkine resulted in synergistic anti-tumor efficiency in in a co-adoptive transfer system allowing analysis of PD1 and/or LAG3- all models tested. deficient CD8+ T cells, and controls, in the same host. Autochthonous tumors are clinically more relevant, as they resemble Results the tumor resistance signature observed in human cancers. Our sar- Mice with CD8+ T cells deficient in PD1 or PD1 and LAG3 (Pdcd1L/L coma models represent excellent platforms to dissect the differences E8ICre.GFP and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP, respectively) show between the refractory GEM models and the more responsive trans- attenuation of B16-F10 tumor growth with improved survival, com- planted tumors. Flow cytometry and immunofluorescence analysis pared to LAG3-deficient mice (Lag3L/L-yfp E8ICre.GFP) and controls revealed the prevalence of tumor associated macrophages in the (E8ICre.GFP). CD8+ TIL frequency is increased with loss of PD1, and GEM models, consistent with the established role of macrophages in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 260 of 272 promoting an immunosuppressive environment. Additionally, we dis- played by TGFb1 in this model. Based on these promising data, we covered a number of differentially secreted cytokines that might play have generated novel humanized 13A1 variants with in-vitro charac- a role in the anti-tumor response. The roles of macrophages and cy- teristics similar to the parental antibody. tokines are being tested. Conclusions Conclusions Isoform-specific blockade of active TGFb1 with antibody 13A1 is as The combination of cyclic dinucleotides with an IL-2 superkine efficacious or better than pan-TGFb blockade in enhancing the anti- produced strong antitumor effects in transplanted and autoch- tumor efficacy of PD-L1 checkpoint therapy in mice with established thonous tumor models and may translate into an efficacious ap- EMT6 tumors. Humanized 13A1 antibodies that maintain the in-vitro proach to treat human cancers. Our studies provide indications features of the biologically active parental murine 13A1 antibody are for additional targets that might modulate the tumor microenvir- therefore attractive clinical candidates for combination therapy with onment to enhance antitumor responses and synergize to en- checkpoint antibodies, especially in patients with low response rates hance immunotherapy effects. to current anti-checkpoint mono-therapies. Ethics Approval The study was approved by the University of California Berkeley Insti- References tutional Animal Care And Use Committee, approval number AUP- 1. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, de 2015-1-8058-1. Gramont A. Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther. 2015;147:22-31; Colak S. and Ten Dijke P. Targeting TGFb signaling in cancer. Trends Cancer 2017; 3(1):56-71.; Tauriello, D.V.D., Palomo-Ponce P477 S., et al. TGFb drives immune evasion in genetically reconstituted colon Isoform-specific blockade of active TGFb1 with mAb 13A1 cancer metastasis. Nature, 2018; 554:538-543 enhances the efficacy of PD-L1 checkpoint therapy in a EMT6 2. Poniatowski LA, Wojdasiewicz P, Gasik R, Szukiewicz D. Transforming mouse tumor model growth factor beta family: Insight into the role of growth factors in 1 2 2 Matteo Brioschi , Jacques Van Snick, PhD , Catherine Uyttenhove , regulation of fracture healing biology and potential clinical applications. 3 4 5 Pamela Cheou , George Coukos, MD, PhD , Gerd Ritter , Steven Dunn, Mediators Inflamm. 2015; 2015:137823 PhD 3. Uyttenhove C, Marillier RG, Tacchini-Cottier F, Charmoy M, Caspi RR, UNIL/Ludwig Cancer Research - Lausanne, Epalinges, Switzerland; Damsker JM, Goriely S, Su D, Van Damme J, Struyf S, Opdenakker G, Van 2 3 Ludwig Cancer Research - Brussels, Brussels, Belgium; University of Snick J. Amine-reactive OVA multimers for auto-vaccination against cyto- Louvain, Brussels, Belgium; CHUV/Ludwig Cancer Research - Lausanne, kines and other mediators: perspectives illustrated for GCP-2 in L. major Epalinges, Switzerland; Ludwig Cancer Research, New York, NY, United infection. J Leukoc Biol. 2011 Jun; 89(6):1001-1007 AND Brioschi M., States Cheou P., van Snick, J., Uyttenhove C., Coukos, G., Ritter, G., Dunn, S. Jour- Correspondence: Steven Dunn (steven.dunn@chuv.ch) nal for ImmunoTherapy of Cancer 2018, 6(Suppl 1):115 Abstr. Nr. 482 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P477 4. Gupta A, Budhu S, Giese R, van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Isoform specific TGF-β inhibition in combin- Background ation with radiation therapy as a novel immune therapeutic ap- TGFb is a highly pleiotropic cytokine implicated in tumor escape proach to cancer therapy. Journal for ImmunoTherapy of Cancer and progression. Targeting TGFb has recently emerged as an ex- 2017; 5 (suppl 2):87 Abstract 326 AND Gupta A, Budhu S, Giese R, citing new approach to overcome TGFb-mediated resistance to van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Target- checkpoint cancer immunotherapy [1]. In humans, three isoforms ing specific TGF-β isoforms in combination with radiation therapy of TGFb (TGFb1, -2 and -3) have been shown to individually drive leads to differential antitumor effects in mouse models of cancer. context-dependent physiological and phenotypic responses [2]. Cancer Res. 2018; 78(13 suppl): Abstract 4716 Most current therapeutic TGFb reagents do not adequately distin- 5. Mariathasan S., Turley S.J. et al. TGFb attenuates tumor response to PD-L1 guish among the three TGFb isoforms and could give rise to on- blockade by contributing to exclusion of T cells. Nature, 2018; 554:544- target off-tumor toxicity, undesirable inflammatory adverse events or lack of activity. To address this unmet need for more selective therapeutic reagents, we generated a panel of murine antibodies with mono-isoform specificity for TGFb1 (13A1) or TGFb3 (1901) P478 and successfully humanized these antibodies, carefully maintain- SEMA4D antibody blockade overcomes mechanisms of immune ing their selective specificity and neutralization potency [3]. The suppression and combination immunotherapy including TGFβ murine TGFb isoform-specific antibodies enhanced anti-tumor effi- blockade promotes efficient tumor regression 1 1 1 cacy in-vivo in B16 melanoma and 4T1 breast cancer models [4]. Terrence Fisher, PhD , Crystal Mallow, BS , Holm Bussler, PhD , Sebold 1 1 1 2 We now expanded our in-vivo studies into the immune-exclusion- Torno, BS , Desa Rae Pastore , Alan Howell, MS , Luis Ruffolo, MD , 2 2 1 1 type tumor model EMT6, in which a pan-specific TGFb antibody Nicholas Ullman , Brian Belt, JD , Joe Bucukovski , Christine Reilly, BS , 2 1 2 was shown to overcome TGFb mediated resistance to PD-L1 Benjamin Dale , Ernest Smith, PhD , David Linehan, MD , Maurice 1 1 checkpoint therapy [5]. Zauderer, PhD , Elizabeth Evans, PhD 1 2 Methods Vaccinex, Rochester, NY, United States; University of Rochester, Efficacy of the murine TGFb1 antibody 13A1 and a pan-TGFb anti- Rochester, NY, United States body (1D11) in combination with anti-mPD-L1 were evaluated in Correspondence: Elizabeth Evans (eevans@vaccinex.com) established orthotopic EMT6 murine breast carcinomas. Antibody Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P478 humanization was performed using molecular engineering, combin- ing framework grafting, competitive screening and selective back Background mutations guided by assaying for TGFb neutralization potency in Despite progress of immune checkpoint blockade therapies, resist- TMLEC reporter cells. Further humanized 13A1 variants with distinct ance mechanisms including myeloid suppression and upregulation kinetic properties were generated by error-prone mutagenesis and of TGFβ signaling prevent durable clinical benefit in many cancer pa- selective library screening. tients. Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody pro- Results motes immune infiltration, reduces immunosuppression, and We found that isoform-specific neutralization of active TGFb1 with enhances T cell activity in the tumor microenvironment (TME), result- mAb 13A1 was highly efficacious in overcoming the low efficacy of ing in increased tumor control in preclinical models when combined PD-L1 checkpoint mono-therapy, enhancing control of tumor growth with various immunotherapies [1,2]. Clinical trials of immune check- and increasing survival of mice with established EMT6 tumors. Fur- point inhibitors (ICI) in combination with pepinemab (VX15/2503), a ther, 13A1 appeared at least as potent as the pan-TGFb antibody humanized anti-SEMA4D antibody, are currently underway in several when combined with anti-PD-L1, highlighting the dominant role cancer indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 261 of 272 Methods Background Activity of anti-SEMA4D antibody in combination with immune Tumor necrosis factor receptor 2 (TNFR2) is central to immune bal- checkpoint inhibitors and TGFβ blockade was evaluated in preclinical ance control in humans and mice. We created human-directed anti- mouse tumor models. Ongoing clinical trials of immune checkpoint TNFR2 antibodies as a therapeutic approach in cancer, with positive inhibitors (ICI) in combination with pepinemab include: (i) a Phase findings [1]. We have now identified a murine-directed surrogate an- 1b/2a combination trial of pepinemab with avelumab in ICI naïve or tagonistic antibody to TNFR2. This antibody shares traits identified in ICI refractory or relapsed NSCLC (CLASSICAL-Lung) (NCT03268057, N= our human-directed antibodies as critical to limiting regulatory T cell 65); (ii) neoadjuvant integrated biomarker trials in patients with (Treg) expansion and activating T effector (Teff) cells. Here we metastatic melanoma (NCT03769155, n=36), metastatic colorectal, present data on this surrogate anti-TNFR2 antibody in two syngeneic pancreatic (NCT03373188, n=32) and head and neck (NCT03690986, mouse models of colon cancer. n=36) cancers treated with pepinemab in combination with nivolu- Methods mab or ipilimumab. Gene expression and immunohistochemical ana- We studied the therapeutic effects of solo and combined immuno- lysis are employed to evaluate changes in immunophenotype as well therapy using the murine-directed anti-TNFR2 antibody in CT26 and as TGFβ-induced effects on TME and tumor progression. MC38 colon tumor models. We compared the new mouse surrogate Results anti-TNFR2 antibody therapy, a commercially available anti-PD1 ther- Anti-SEMA4D antibody enhanced tumor regression when combined apy, and anti-TNFR2/anti-PD1 combination immunotherapy. Mice with antibodies targeting CTLA-4, PD-1, PD-L1, LAG3, and TGFβ in sev- were dosed bi-weekly (100ug/mouse antibody). Antigen-specific CD8 eral preclinical models. For example, anti-SEMA4D plus anti-TGFβ treat- and Treg infiltrates were also studied. ment resulted in maximal tumor growth delay (TGD) of 239% (p<0.01) Results and 10/15 complete tumor regressions (CR) (p<0.05), compared to 10% In the CT26 model, anti-TNFR2 antagonism alone or co-treatment TGD and 0/13 CR with single agent anti-TGFβ or 29% TGD and 1/10 CR with anti-PD1 and anti-TNFR2 was highly efficacious (55-62% of mice with anti-SEMA4D alone in MC38 colon carcinoma model. SEMA4D cured). Anti-PD1 alone was less efficacious (25% cured). In the MC38 blockade reversed expression of genes related to EMT. Additionally, the model, therapy with anti-TNFR2 alone showed some efficacy (20% combination of anti-SEMA4D, folfirinox, and ICI improved survival in cured) and anti-PD1 alone had the least efficacy (10% cured), but KP2-tumor bearing mice, a KPC-derived pancreatic adenocarcinoma anti-PD1 in combination with anti-TNFR2 yielded the best overall sur- model of immune exclusion, myeloid suppression and active TGFb sig- vival (70% cured). Sequential antibody dosing with anti-PD1 followed naling. In clinical trials, pepinemab was well-tolerated and analysis of by anti-TNFR2 yielded no synergy. In contrast, sequential treatment pre- and on-treatment biopsies revealed increased CD8:FoxP3 ratios with anti-TNFR2 first followed by anti-PD1 or the combination of and reduced presence of myeloid derived suppressor cells within TME. anti-TNFR2 plus anti-TNFRF2 showed synergy and highest efficacy. Conclusions Anti-TNFR2 therapy was distinct from anti-PD1 therapy in showing SEMA4D antibody blockade modulates the TME to enhance anti- pronounced regulatory Treg depletion and enhanced Teff infiltration tumor immunity and combination therapies further enhance anti- in the tumor microenvironment, demonstrating in vivo specificity for tumor activity and overcome important resistance mechanisms. Pre- disease-causing cells only in the tumor. liminary data suggest the combination of pepinemab plus immune Conclusions checkpoint therapy is well tolerated and shows initial signals of anti- Anti-TNFR2 immunotherapy provides benefits in two colon cancer tumor activity in patients. Ongoing analysis of various therapeutic models, both as a single agent and when administered in combin- combinations and immunophenotyping of tissue biopsies will shed ation with anti-PD1. Anti-PD1 before anti-TNFR2 was associated with light on mechanism of action of SEMA4D antibody blockade in sev- poor outcomes for survival, histology and lack of long-term cure, eral combination therapies. suggesting that non-specific unleashing of the immune system with anti-PD1 destroys the tumor microenvironment specificity of anti- Acknowledgements TNFR2. These results highlight the value of anti-TNFR2 antagonism We would like to thank the clinical and research teams at Emory University, in vivo in mouse tumor models as solo therapy or as a combination including Doctors Greg Lesinsky, Christina Wu, Conor Steuer, Nabil Saba, therapy, administered first or concurrently with anti-PD1. This study Michael Lowe, Ragini Kudchadkar, and Brian Olson. We also extend gratitude of new immunotherapy combinations highlights the need to test to the Avelumab team at EMD Serono, as well as clinical investigators and both single agent therapy as well as the sequencing of combination their teams related to the CLASSICAL-Lung trial. therapy as new agents are brought forward to the clinic. Trial Registration NCT03268057 References NCT03769155 1. Torrey H, Butterworth J, Mera T, et al.Targeting TNFR2 with antagonistic NCT03373188 antibodies inhibits proliferation of ovarian cancer cells and tumor- NCT03690986 associated Tregs. Sci Signal. 2017;10(462). pii: eaaf8608. Ethics Approval References Mice were tested and monitored for tumor growth by either Champions 1. Evans EE, et al. Antibody Blockade of Semaphorin 4D Promotes Immune Oncology (Hackensack, NJ) or a third-party pharmaceutical company in Infiltration into Tumor and Enhances Response to Other accordance with their animal welfare guidelines. Immunomodulatory Therapies. Cancer Immunol Res. 2015;3(6):689-701. 2. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith ES, Zauderer M, Evans P480 EE, Allen CT. Semaphorin4D inhibition improves response to immune Heterologous prime-boost vaccination safely enhances antitumor checkpoint blockade via attenuation of MDSC recruitment and function. immunity to the colorectal antigen GUCY2C Cancer Immunol Res. 2019;7(2):282-291 John Flickinger, BS, Robert Carlson, Jagmohan Singh, Trevor Baybutt, BS, Elinor Leong, Alicja Zalewski, Amanda Pattison, Jeffrey Rappaport, Joshua P479 Barton, Scott Waldman, Adam Snook, PhD Evaluation of a TNFR2 antibody with and without anti-PD-1 Thomas Jefferson University, Philadelphia, PA, United States therapy in two murine colon cancer models Correspondence: Adam Snook (adam.snook@jefferson.edu) Katie Case, Lisa Tran, Hui Zheng, Michael Yang, Denise Faustman, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P480 PhD Massachusetts General Hospital/Harvard Medical School, Charlestown,, Background MA, United States The transmembrane receptor guanylyl cyclase C (GUCY2C) is an Correspondence: Denise Faustman (faustman@helix.mgh.harvard.edu) emerging target for colorectal cancer immunotherapy. Recently, an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P479 adenovirus-based vaccine against GUCY2C was tested in a phase I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 262 of 272 clinical trial where it was found to safely induce GUCY2C- Although paradoxically, obesity has been found to improve re- specific immune responses [1]. However, GUCY2C immune re- sponse to immunotherapy in a subgroup of patients with mel- sponses following immunization wane over time and optimal anoma [4,5]. GUCY2C immunity may require multiple GUCY2C vaccinations. Methods Moreover, repeated vaccination utilizing adenovirus-based Wildtype C57BL/6femalemicewererandomizedto ahigh-fat vectors is hindered by the production of adenovirus-specific (60%) or low-fat standard chow (14%) diet for 16 weeks to gen- antibodies following first vaccination. For this reason, we have erate diet-induced obese (DIO) or age-matched lean controls, generated a recombinant strain of Listeria monocytogenes respectively. Animals were then challenged with the syngeneic secreting GUCY2C (Lm-GUCY2C) to boost GUCY2C immune E0771 mammary carcinoma cell line. Tumor outgrowth was responses. These studies assess the immunogenicity, thera- quantified by caliper measurements, bioluminescent imaging peutic efficacy, and safety of a heterologous prime-boost (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, immunization utilizing adenovirus and Listeria monocytogenes and endpoint tumor weights. Once tumors were palpable, ani- vectors. mals were randomized to receive no therapy or immunotherapy Methods consisting of intratumoral CpG co-administered with non- T-cell responses following vaccination were assessed by IFNy ELISpot. replicative adenovirus (Ad) encoding murine TNF-related apop- Tumor protection following vaccination was assessed by challenging tosis inducing ligand (TRAIL; AdT). Whole tumor immunogenetic mice with a luciferase-expressing CT26 colorectal cancer cell line. Lu- gene expression profiles were evaluated using nanoString and minescence following luciferin injection and overall survival were immune populations were assessed via multi-parameter flow cy- quantified. Safety was assessed by histopathologic evaluation of tometry. T cell cytokine production was evaluated via flow cy- known GUCY2C-expressing tissues following vaccinations. tometry following ex vivo CD3/CD28 stimulation. Results Results Construction of Lm-GUCY2C was validated by GUCY2C western DIO mice had significantly increased body weights at tumor blot on J774A.1 macrophage cells infected with Lm-GUCY2C. challenge versus lean controls (45 versus 25 grams, p <0.0001) Optimal GUCY2C immunogenicity was achieved utilizing All methodologies demonstrated that obesity significantly in- adenovirus-GUCY2C to ‘prime’ GUCY2C immune responses with creases primary mammary tumor outgrowth and alters cellular Lm-GUCY2C to ‘boost’ and was found to be superior to homolo- and immunogenetic profiles within the tumor microenviron- gous administration using either adenovirus-GUCY2C or Lm- ment. Notable alterations include significant reductions in the GUCY2C vectors. Similarly, anti-tumor studies found heterol- frequency of CD4+ T cells, CD8+ T cells, and CD19+ B cells; ogous administration of adenovirus-GUCY2C and Lm-GUCY2C to with a simultaneous increase in the frequency of myeloid- be superior to homologous administrations. Importantly, histo- derived suppressor cells (MDSCs). Following immunotherapy ad- pathologic evaluation of mice following heterologous prime- ministration, lean animals controlled tumor growth whereas DIO boost revealed no toxicity. animals experienced progressive tumor growth. Despite these Conclusions differential tumor outcomes, both lean and DIO animals dis- Heterologous prime-boost vaccination utilizing adenovirus and played robust intratumoral effector CD8+ T cell accumulation Listeria vectors expressing the tumor antigen GUCY2C demon- and ex vivo function. In contrast, immunotherapy reduced the strate superior immunogenicity and antitumor efficacy over intratumoral accumulation of monocytic and granulocytic MDSCs homologous immunization with either vector, a strategy that only in lean animals. Both MDSC populations persisted in the can be translated to colorectal cancer patients. tumors of animals with DIO, resulting in less favorable effector CD8+ T cell to MDSC ratios. Acknowledgements Conclusions The authors thank the Center for Cell and Gene Therapy, Baylor College of Our data implicate obesity as a causal factor in impairing im- Medicine for assistance in adenovirus vaccine manufacturing. munotherapeutic efficacy in a pre-clinical model of breast can- cer, potentially via accumulation of MDSCs. Our data suggest Reference that clinical investigation and consideration is needed for fac- 1. Snook AE, BaybuttTR, XiangB,Abraham TS,Flickinger JC,HyslopT, tors such as body composition and body mass index when Zhan T, Kraft WK, Sato T, and Waldman SA. Split tolerance permits treating breast cancer patients with immunotherapy. safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J. Immunother Cancer. 2019;7, 104. Acknowledgements Ethics Approval This study was supported in part by NIH-NIGMS training grant T32GM008111 Studies were approved by the Thomas Jefferson University IACUC (Protocol # to JTG. 01956). References P481 1. Incio, J., et al., Obesity promotes resistance to anti-VEGF therapy in breast Obesity impairs immunotherapeutic efficacy in pre-clinical breast cancer by up-regulating IL-6 and potentially FGF-2. Sci Transl Med, 2018. cancer 10(432). Justin Gibson, BS, Rachael Orlandella, BS, William Turbitt, Robert Sorge, 2. Sheng, X., et al., Adipocytes Sequester and Metabolize the PhD, Lyse Norian, PhD Chemotherapeutic Daunorubicin. Mol Cancer Res, 2017. 15(12): p. 1704- University of AlabamaBirmingham, AL, United States 1713. Correspondence: Lyse Norian (lnorian@uab.edu) 3. Lehuede, C., et al., Adipocytes promote breast cancer resistance to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P481 chemotherapy, a process amplified by obesity: role of the major vault protein (MVP). Breast Cancer Res, 2019. 21(1): p. 7. Background 4. McQuade, J.L., et al., Association of body-mass index and outcomes in Obesity has long been known to worsen prognosis and survival patients with metastatic melanoma treated with targeted therapy, im- for breast cancer patients. Recent reports further indicate that munotherapy, or chemotherapy: a retrospective, multicohort analysis. obesity negatively impacts response to targeted anti-VEGF ther- Lancet Oncol, 2018. 19(3): p. 310-322. apy [1] and efficacy of chemotherapeutics [2,3]. However, no 5. Wang, Z., et al., Paradoxical effects of obesity on T cell function during studies have yet investigated the impact of obesity on re- tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): sponse to immunotherapy in the context of breast cancer. p. 141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 263 of 272 P482 P483 STACT: A novel therapeutic platform that delivers TLR enhanced GVAX elicits tumor-specific tissue resident memory immunomodulatory payloads to tumor-resident myeloid cells After T cells independent of T cell priming IV dosing and demonstrates potent anti-tumor efficacy in Michael Korrer, Young Kim, MD, PhD, David Taylor preclinical studies Vanderbilt University Medical Center, Nashville, TN, United States Laura Glickman, PhD, Christopher Rae, PhD, Alexandre Iannello, PhD, Correspondence: Young Kim (young.j.kim@vanderbilt.edu) Anastasia Makarova, PhD, Haixing Kehoe, MS, John Faulhaber, Bill Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P483 Hanson, Christopher Thanos, PhD Actym Therapeutics, Inc, Berkeley, CA, United States Background Correspondence: Christopher Thanos (cthanos@actymthera.com) GVAX, a genetically modified whole cell vaccine, is proposed to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P482 work by recruiting and activating antigen-presenting cells which then traffic to the draining lymph node and elicit tumor-specific Background Tcells.Inmouse models GVAX haslittle therapeutic benefit as Many experimental therapies developed to promote proper T- a monotherapy, but when combined with a Toll-like receptor cell infiltration in immune-excluded tumors are too toxic for sys- (TLR) 4 adjuvant, TLR Enhanced GVAX (TEGVAX) significantly re- temic administration, which will be required in a metastatic dis- duces tumor burden. Paradoxically, the increased therapeutic ease setting. These include innate targets such as STING and benefit of TEGVAX corresponds with a decrease in delivery of TLR agonists, co-stimulatory receptor agonists, and type I/II tumor antigen to the draining lymph node. In order to improve cytokine receptor combinations. To address these limitations, the efficacy of the TEGVAX platform, it is critical to understand we have engineered a highly attenuated, microbial-based im- the mechanism by which it induces anti-tumor immune re- munotherapy platform called STACT (S. Typhimurium Attenuated sponses. Since TEGVAX requires T cells to work, yet significantly Cancer Therapy). Upon IV administration, the microbe traffics to reduces antigen delivery to the draining lymph node, we and enriches in the tumor microenvironment. There, it is specif- hypothesize that TEGVAX functions independent of lymph node ically phagocytosed and lysed by tumor-resident myeloid cells, priming. enabling efficient delivery of plasmids encoding immunomodu- Methods latory payloads. Using our proprietary platform, we have gener- B16-mOVA cells injected s.c. into B6 mice. TEGVAX (1e6 B16- ated multiple systemically-administered therapies that target mOVA + 1e5 B78H1-GM Irradiated cells + 20ug MPLA) injected several well-characterized, yet intractable immune pathways. s.c. into opposite flank 5 days after tumor injection. 10ug daily Characterization of STACT microbes encoding constitutively ac- FTY720 i.p. on day 4. tive STING variants (STACT-STING) and IL-2 (STACT-IL2) are pro- Results vided as examples. To determine if TEGVAX alters the priming of CD8 T cells, we Methods performed longitudinal studies of OT-1 CD8 T cell proliferation The STACT platform strain has been engineered using precision in vivo comparing TEGVAX to GVAX. We found that GVAX in- genome modifications for enhanced tolerability, reduced im- duced rapid proliferation of OT-1 cells, whereas TEGVAX failed to munosuppressive inflammation, and tumor specificity. STACT- induce OT-1 cell proliferation as shown by FACS and in vivo im- mediated delivery of immunomodulatory proteins in primary aging. We then determined if TEGVAX required myeloid or NK mouse and human cells was confirmed by in vitro functional as- cells to reduce tumor burden. We found that TEGVAX reduced says. STACT strains were evaluated in vivo for tumor-specific en- tumor burden in NK depleted, but not myeloid cell depleted richment, payload delivery, tolerability, and therapeutic efficacy mice. These results raised the question if priming in the draining following IV administration in several subcutaneous syngeneic lymph node was required for therapeutic efficacy of TEGVAX. To tumor studies. determine this, we performed tumor growth studies with mice Results administered FTY720, which sequesters circulating T cells in STACT was found to be 100,000-fold enriched in tumors, relative lymph nodes. We demonstrated that TEGVAX significantly re- to spleen, after tail vein injections in mice. Flow cytometry duced B16-mOVA tumor growth even in the presence of FTY720 staining revealed that STACT does not infect stromal or tumor treatment, suggesting T cell priming was not required. Immune cells and is specifically targeted by tumor-resident myeloid cells phenotyping of TEGVAX treated mice, showed a significant in- (TAMs, DCs, and monocytes). STACT is rapidly phagocytosed crease in tumor infiltrating tissue-resident memory (Trm) T cells. and then destroyed by these cells, delivering its plasmid DNA Conclusions contents encoding immunomodulatory protein expression cas- Our results demonstrate that combining TLR4 agonist with GVAX settes. We have measured highly efficient heterologous gene (TEGVAX) completely alters the immune response to vaccination. transfer and protein expression within primary mouse and hu- TEGVAX does not prime naïve T cells nor require trafficking of T cells man M2 macrophages treated with STACT, at levels comparable from LN to tumor to function. We observed an increased number of to DNA transfection. Therapeutically relevant levels of IL-2 were Trm CD8 T cells infiltrating the tumor leading us to conclude that measured in the tumor microenvironment of STACT-IL2 treated TEGVAX is functioning by eliciting a tumor-specific Trm T cell re- mice several weeks after dosing. For STACT-STING, significant sponse independent of lymph node priming. tumor growth inhibition, including complete tumor regressions Ethics Approval were observed, and the therapy was well tolerated. Immune The study was approved by Vanderbilt University Animal care and correlates were consistent with on-target expression in the use board tumor microenvironment, and the anti-tumor effect was adap- tive immune mediated. P484 Conclusions CB-708, an orally bioavailable small molecule inhibitor of CD73 STACT is a highly attenuated, microbial-based therapeutic platform with immunostimulatory and anti-tumor activity engineered to deliver immunomodulatory payloads, alone or in com- Clarissa Lee, PhD, Deepthi Bhupathi, Roland Billedeau, Jason Chen, Lijing bination, to phagocytic cells of the solid tumor microenvironment Chen, Rosalyn Dang, Matthew Gross, Tony Huang, Weiqun Li, PhD, Yong after systemic administration. The goal of STACT therapy is to pro- Ma, Andrew MacKinnon, Gisele Marguier, MS, Silinda Neou, MS, mote immune-mediated tumor clearance of T-cell excluded solid tu- Francesco Parlati, PhD, Natalija Sotirovska, MS, Sandra Spurlock, Timothy mors and elicit durable anti-tumor immunity. Stanton, Susanne Steggerda, PhD, Jing Zhang, Winter Zhang, Jim Li Ethics Approval Calithera Biosciences, South San Francisco, CA, United States All animals were used according to protocols approved by an Institu- Correspondence: Jim Li (jli@calithera.com) tional Animal Care and Use Committee and maintained in specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P484 pathogen-free conditions in a barrier facility. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 264 of 272 Background Methods High adenosine (ADO) in the tumor microenvironment suppresses CT26 tumor measurement seven days after treatment was used to the immune response against cancer cells by inhibiting immune ef- define tumors as KD033-surrogate responders (decreasing tumor vol- fector functions and promoting the development of immunosuppres- umes), non-responders (no change or increasing tumor volumes) and sive cells. Extracellular ADO can be generated from ATP released by non-targeted IL-15 best responders. RNA was isolated from these tu- cells undergoing stress or death through the combined actions of mors and analyzed using the Nanostring PanCancer IO 360 Gene Ex- the ectonucleotidases CD39 (ATP to AMP) and CD73 (AMP to ADO). pression Panel for immune cell responses. Combination therapy with Inhibition of ADO production via CD73 is a promising therapeutic ap- genes identified through Nanostring analysis was evaluated in a proach for the treatment of cancer. tumor model where KD033-surrogate monotherapy showed minimal Methods efficacy such as 4T1, an aggressive breast carcinoma murine model We developed CB-708, a potent and selective small molecule inhibi- involving spontaneous metastases to other organs. 4T1 cells were tor of CD73. The potency of CB-708 was evaluated against recombin- injected into the mammary gland of Balb/c mice and grown to 100 ant CD73 and CD73-expressing cells using a malachite green assay. mm3 prior to treatments. Tumors and metastasis nodules in the lung Selectivity against related ectonucleotidases was also assessed. Inhib- were evaluated. ition of CD73 in plasma was measured using LC/MS to assess conver- Results sion of 15N5-AMP into 15N5-ADO. Reversal of AMP-mediated Transcriptional analysis showed that CTLA-4 was one of the top immune suppression of human CD8+ T cells was determined by genes that was differentially upregulated after KD033-surrogate treat- measuring T cell activation in the presence of exogenous AMP. T cell ment in comparison to non-targeting IL-15. In the 4T1 tumor model, proliferation was assayed by flow cytometry and cytokine levels were monotherapies of both KD033-surrogate or anti-CTLA-4 did not have measured by ELISA. The EG7 and CT26 syngeneic tumor models were any effect on 4T1 tumor growths; however, the combination therapy used to assess the therapeutic effect of CB-708. with single dose of KD033-surrogate and repeat dose of anti-CTLA-4 Results showed a decrease in the average number of lung metastases and a CB-708 potently and completely inhibited soluble human CD73 (IC50 significant tumor growth inhibition compared to vehicle-treated = 170 pM) and cell-bound human CD73 (IC50 = 210 pM), but did not animals. inhibit human CD39, ENTPD2, or ENTPD3. CB-708 retained high po- Conclusions tency in the presence of whole human plasma (IC50 = 380 pM) and Analysis of murine tumors treated with KD033 surrogate in vivo re- reversed AMP-mediated suppression of human CD8+ T cell prolifera- sulted in combination strategies, including KD033 in combination tion and production of IFNγ and granzyme B in vitro. Oral administra- with CTLA-4, that can be exploited in targeting resistant and refrac- tion of CB-708 was well-tolerated in tumor-bearing mice, resulted in tory cancers. Based on the therapeutic activity and improved safety sustained exposure above mouse plasma IC50, and exhibited single- of the fusion protein, Kadmon plans to initiate clinical studies of agent tumor growth inhibition in syngeneic tumor models including KD033 in 2019. established EG7 tumors. Efficacy in the EG7 model was dependent Ethics Approval on CD8+ T cells and was correlated with pharmacodynamic inhib- Animal studies were conducted for Kadmon by Crown Bioscience Inc. ition of CD73. Enhanced tumor growth inhibition was observed when with approved SOP and IACUC protocol. CB-708 was combined with checkpoint inhibition (anti-PD-L1) or with chemotherapy (oxaliplatin, doxorubicin, docetaxel) in the EG7 model. P486 Conclusions Releasing the break on T cell activation through novel small CB-708 is an orally bioavailable and highly potent small molecule in- molecule inhibition of HPK1 hibitor of CD73. CB-708 reverses the immunosuppressive effects of Minhui Shen, Gayathri Bommakanti, PhD, Deanna Mele, PhD, Neil AMP-derived ADO in vitro and in vivo and has anti-tumor activity. Grimster, PhD CB-708 is expected to enter clinical development in 2019. AstraZeneca, Waltham, MA, United States Correspondence: Deanna Mele (Deanna.Mele@astrazeneca.com) P485 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P486 Synergistic efficacy of anti-PD-L1/IL-15 fusion protein in combination with anti-CTLA-4 antibody in a murine orthotopic 4T1 Background breast carcinoma model Loss of immune surveillance is required for cancer cell growth and 1 2 2 2 Stella Martomo, PhD , Dan Lu, MA , Jeegar Patel , Zhanna Polonskaya , metastasis, tumors co-opt suppressive mechanisms to evade detec- 2 2 Xenia Luna , Kevin McCracken tion by the immune system. The immune system is equipped with 1 2 Kadmon Corporation, New York, NY, United States; Kadmon, New York, multiple feedback mechanisms to limit inflammation in order to pre- NY, United States vent autoimmunity, tumors activate these pathways to escape im- Correspondence: Stella Martomo (stella.martomo@kadmon.com) munity. HPK1 (hematopoietic progenitor kinase 1) is a negative Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P485 regulator of T cell activation, the kinase activity limits T cell signaling and tumoricidal cytokine production. Recent literature has shown in- Background activation of the kinase function of HPK1 prevents tumor progression Administration of immune checkpoint inhibitors anti-PD1/PD-L1 have in murine tumor models [1]. led to durable objective responses in select cancers. However, a sub- Methods stantial number of patients fail to respond or become resistant to these We used lentiviral delivered shRNAs and CRISPR/Cas9 technology to therapies. We have generated a therapeutic fusion protein (KD033) by delete HPK1 in Jurkat cells and primary human T cells, respectively. combining a proprietary high affinity anti-human-PD-L1 (or anti- Jurkat T cells (wt/KO) were activated with anti-CD3 antibody and cell murine-PD-L1, (KD033-surrogate)) antibody with human IL-15. Initial as- lysates were assessed by western blot to examine signaling events sessment of this fusion antibody showed enhanced tolerability relative downstream of the T cell receptor (TCR). Primary human T cells or to a non-targeted IL-15 fusion protein in addition to its potent anti- CRISPR/Cas9 KO T cells were activated in vitro using anti-CD3/anti- tumor activity. In the CT26 murine colorectal tumor model, a single CD28 antibodies in the presence or absence of HPK-1 inhibitors +/- dose of KD033-surrogate consistently resulted in antitumor response prostaglandin E2 (PGE2). Cell viability and numbers were assessed by that included tumor clearance and long-term tumor-free survival. Initial flow cytometry. Cytokines were quantified by ELISA. analysis of KD033-surrogate treatment showed robust adaptive and Results cytotoxic immune gene signatures in tumors leading to tumor inhib- We present novel findings demonstrating that the role of HPK1 is ition and memory responses. We further analyzed tumors from KD033- conserved in human Jurkat cells as well as in primary human T cells. surrogate responders and non-responders to evaluate possible thera- Loss of HPK1 enhanced T cell receptor signaling in Jurkat cells. In peutic combinations to broaden the response of KD033. CRISPR/Cas9 KO HPK1 primary human T cells, TCR activation resulted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 265 of 272 in enhanced cytokine secretion and proliferation concomitant to a 100% lethality by 60 days, but 3 doses of AgN2a 4P leads to 100% decrease in pSLP76, the target molecule phosphorylated by HPK1. survival at 50 days after both allogeneic and syngeneic BMT. Consistent with the KO phenotype, our HPK1 inhibitors resulted in Conclusions enhanced T cell activation, cytokine secretion and proliferation. In Co-culture of NK cells with an engineered costimulatory vaccine is an addition, the inhibitors were able to rescue T cells from PGE2 medi- effective strategy to induce apoptosis of neuroblastoma tumor cells ated suppression. In vivo studies are currently underway to examine by increasing NK-mediated cytokine production and cytotoxicity, and anti-tumor activity of these compounds in various syngeneic models. enhances anti-tumor effects after BMT. Usage of cell-based vaccines Conclusions after BMT could be an effective strategy to augment NK cell activity In summary our small molecule inhibitors of HPK1 could enhance against neuroblastoma. anti-tumor immunity through increased T cell function overcoming suppressive signals in the tumor microenvironment and thus Acknowledgements broaden the response to check point inhibitors for cancer AgN2a 4P was a gift from Dr. Bryon Johnson at Medical College of immunotherapy. Wisconsin. This work was supported by grants from the St. Baldrick’s – Stand up to Cancer Pediatric Dream Team Translational Research Grant SU2C- Acknowledgements AACR-DT-27-17, NCI/NIH R01 CA215461, American Cancer Society Research Minhui Shen1, Gayathri Bommakanti1, Kevin Xu1, Kun Song1, Rob Ziegler1, Scholar grant RSG-18-104-01-LIB, Hyundai Hope on Wheels and the MACC Jason Kettle2, Adelphe Mfuh1 Jason Sheilds2, Neil Grimster1, Lisa Drew1, Fund (C.M.C). We would like to thank the UWCCC core facilities, who are Stephen Fawell1, Deanna A. Mele1 supported in part through NCI/NIH P30 CA014520. Stand Up to Cancer is a 1AZ Discovery Early Oncology, Boston, MA, 2AZ Discovery Early Oncology, division of the Entertainment Industry Foundation. Research Grants are Cambridge UK, MA administered by the American Association for Cancer Research, the Scientific Partner of SU2C. Reference Ethics Approval 1. Hernandez S, Qing J et al. The Kinase Activity of Hematopoietic The study was approved by University of Wisconsin-Madison Animal Care Progenitor Kinase 1 is Essential for the Regulation of T cell Function and Use Committee, approval number M005915. Cancer. Cell. 2018; 25: 80-94. P488 P487 IPH5201, a blocking antibody targeting the CD39 Combining an engineered costimulatory vaccine with NK cells immunosuppressive pathway, unleashes immune responses in induces an anti-tumor effect against murine neuroblastoma combination with cancer therapies 1 1 1 1 in vitro and after bone marrow transplant in vivo Pascale Andre , Ivan Perrot , Caroline Denis, PhD , Marc Giraudon-Paoli , 1 1 1 1 Nicholas Mohrdieck, BS, Paul Bates, Sean Rinella, Katharine Tippins, Severine Augier , Rachel Courtois , Diana Jecko , Thomas Arnoux , 1 1 2 Christian Capitini, MD Violette Breso , Nicolas Gourdin, PhD , Nadia Luheshi, PhD , Ariane 1 1 1 1 University of Wisconsin-Madison, Madison, WI, United States Morel, PhD , Yannis Morel, PhD , Eric Vivier , Carine Paturel, PhD 1 2 Correspondence: Christian Capitini (ccapitini@pediatrics.wisc.edu) Innate Pharma, Marseille, France; AstraZeneca, Milton, Cambridge, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P487 United Kingdom Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P488 High risk neuroblastoma remains a challenge to cure with only 50% survival, despite multi-modality treatment. Natural killer (NK) cells Background have been previously shown to have activity versus neuroblastoma CD39 is an extracellular ectonucleotidase highly expressed in the but have not been consistently successful in clinical trials. NK cell ac- tumor microenvironment, by stromal cells and some immune in- tivation via co-culture with a vaccine engineered to express CD54, filtrating cells. CD39 contributes to the production of adenosine, CD80, CD86, and CD137L, called AgN2a 4P, was studied to investi- an inhibitor of immune response, via sequential hydrolysis of ad- gate NK cells’ ability to induce cytotoxicity of murine neuroblastoma enosine triphosphate (ATP) and adenosine diphosphate into ad- tumor cells in vitro and in vivo. enosine monophosphate, which then is degraded into adenosine Methods by CD73 enzyme. In contrast, ATP has immune-stimulatory activ- NKs and irradiated AgN2a 4P were co-cultured in ratios of 1 (NKs):0.5 ity through promoting dendritic cell (DC) maturation. Blockade of (AgN2a 4P) and 1:1, and compared to NK only and AgN2a 4P only CD39-mediated degradation of ATP may therefore stimulate anti- controls, with all groups receiving IL-15/IL-15Ralpha, and then ana- tumor immunity across a wide range of tumors by preventing lyzed by flow cytometry, multiplex cytokine analysis, and cytotoxicity production of immunosuppressive adenosine and by promoting in vitro after 1, 3, 5, 7, and 9 days. To study the efficacy of in vivo accumulation of immunostimulatory ATP in the tumor microenvir- vaccination with AgN2a 4P after bone marrow transplant (BMT), onment. IPH5201 is a humanized monoclonal antibody that se- C57BL/6 or B6AJ recipients were lethally irradiated, followed by trans- lectively binds to and inhibits the activity of both membrane- plantation of T-cell depleted C57BL/6 donor bone marrow on day +0. bound and soluble human CD39. Here, we explored the efficacy BMT recipients were then treated with the AgN2a 4P vaccine for 2 of IPH5201 in vitro and in vivo in immunocompetent human versus 3 weekly doses, and with or without adoptive transfer of CD39 knockin (huCD39KI) mouse model in combination with im- donor NK cells to accelerate immune reconstitution. All recipients mune checkpoint inhibitor. were then challenged with NXS2 neuroblastoma tumor, and followed Methods for tumor growth and survival. In vitro, efficacy of IPH5201 was evaluated (1) on the phenotypic Results changes and stimulatory potential of monocyte-derived DC, (2) on The NK:AgN2a 4P co-culture at 1:0.5 and 1:1 increases Ly49A+ NKs the inflammasome pathway by assessing interleukin-1b secretion from 6% to 21%, and Ly49D+ NKs from 3% to 30% from day +0 to from in vitro-derived M1 macrophages, and (3) on T cell proliferation. day +9. pSTAT1 activation remains consistently high between 80%- HuCD39KI mice were characterized for the expression and function 98%, and pSTAT3 activation remains 20%-50%, across the co-culture of human CD39. To assess CD39 blockade in vivo, a mouse IgG1 ver- period. NK cells release increased levels of IFN-gamma and IL-6 at sion of IPH5201 was produced (moIPH5201), which contained key the co-cultured ratios of 1:0.5 and 1:1, and CXCL1 at the 1:1 ratio, as point mutations in the Fc region to abrogate Fc receptor interactions. compared to the NK with IL-15/IL-15Ralpha controls. The NK:AgN2a Antitumor efficacy of CD39 blockade was assessed in huCD39KI mice 4P ratios of 1:0.5 and 1:1 induce significantly increased apoptosis of grafted with mouse tumor cells not expressing mouse CD39. Neuro2a neuroblastoma cells than NK cells with IL-15/IL-15Ralpha HuCD39KI mice were treated with blocking anti-human CD39 Ab, ei- alone. In vivo, injection of 2 doses of AgN2a 4P vaccine leads to ther alone or in combination with a blocking anti-mouse PD-L1 Ab. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 266 of 272 Results the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and As hypothesized, in vitro IPH5201 enhanced the phenotypic 5 mg/kg induced significant tumor growth inhibition (55% and 57%, maturation and the activation of DCs and macrophages by inhi- respectively) as compared to vehicle control or Fc-silenced huIgG1 biting ATP hydrolysis. IPH5201 also efficiently restored T cell controls. Additionally, 2 out of 9 animals in the 5 mg/kg MCLA-145– proliferation in a dose-dependent manner to the levels ob- treated group had complete tumor regression. MCLA-145 increased served in the absence of ATP addition. Thus, IPH5201 preserved the number of infiltrating CD8+ T cells, as well as the percentage of extracellular ATP, thereby promoting the activation of DCs and central memory CD8+ T cells. macrophages and limiting adenosine accumulation and its im- Conclusions munosuppressive effect on T cells. Furthermore, moIPH5201 MCLA-145 is currently undergoing clinical development mice treatment efficiently inhibits membrane and soluble hu- (NCT03922204). man CD39, from huCD39KI mice ex vivo without mediating CD39-expressing cell depletion in vivo. Finally, blockade of P490 CD39 potentiates the anti-tumor efficacy of anti-PD-L1 Ab Antibody derived from an elite responder to checkpoint inhibitor monotherapy. therapy relieves immunosuppression by tumor associated Conclusions macrophages Together these data indicate that blocking CD39 in conjunction with Randi Simmons, Siddarth Chandrasekaran, Melissa Conerly, Tyrel Smith, PD-1/PD-L1 checkpoint inhibitors provides increased anti-tumor effi- Sam Lam, Jacqueline Pham, Ray Fox, Darbie Whitman, Meghan Zuck, cacy and support the rational for assessing this combination in clin- Sara Carbonetti, Kamal Puri, PhD ical trials. Oncoresponse Inc, Seattle, WA, United States Correspondence: Kamal Puri (kpuri@oncoresponseinc.com) P489 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P490 Identification and characterization of MCLA-145 (CD137 x PD-L1): a bispecific antibody that requires PD-L1 binding to activate CD137 Background 1 1 1 Simon Plyte , Cecile Geuijen , John de Kruif , Pieter Fokko van Loo, Tumor-associated macrophages (TAM)s in the tumor microenviron- 1 1 1 1 PhD , Paul Tacken , vanessa Zondag-vander Zande , Rinse klooster , ment (TME) contribute to tumor immune evasion by suppressing 1 1 1 1 hans van Maaden , Erik Rovers , steef engels , floris franzen , abdul anti-tumor immune responses and by promoting a tumorigenic mi- 1 1 1 2 basmeleh , willem bartelink , Mark Throsby , Patrick Mayes , Horacio lieu. High infiltration of immunosuppressive myeloid cells generally 2 2 2 2 2 Nastri , shaun stewart , jing zhou , steve wang , Chen-yen Huang , predicts unfavorable prognosis. Reduction or repolarization of sup- 2 2 2 2 thomas codamine , ashwini kularni , yao bin lui , arpita mondal , leslie pressive myeloid cells is an attractive strategy to enhance clinical re- 2 2 2 2 2 hall , soeon kim , marina martinez , shaun o'brien , edmund moon , sponses to immune checkpoint inhibitor (CPI) therapy. Cancer steven albelda patients who achieved durable response to CPI therapy (elite re- 1 2 Merus, Utrecht, Netherlands; Incyte Research Institute, Wilmington, DE, sponders) may harbor antibodies that contribute to clinical response United States by promoting an anti-tumor TME. Correspondence: Simon Plyte (simon.plyte@merus.nl) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P489 B cells derived from elite responders were cloned and screened for IgG antibodies binding to myeloid derived suppressor cells. Background Hits were prioritized based on myeloid binding profiles and their CD137 (4-1BB) is costimulatory receptor on T and NK cells that re- variable-regions sequenced, cloned, and expressed as recombin- quires clustering to elicit its effects on target cells and enhance adap- ant IgG1. Cloned antibodies underwent further characterization to tive immune responses against tumors. The development of CD137 evaluate their ability to reverse the immunosuppressive effects of targeted agents for cancer therapy has been hampered by on-target myeloid cells in assays modelling the TME. Primary human mono- off-tumor toxicity in the case of agonistic monospecific antibodies, or cytes and T cells were used to interrogate antibody-dependent limited antitumor activity in the case of Fcγ-mediated crosslinking of immunomodulatory responses in vitro. A humanized mouse mAbs. model was used to evaluate the anti-tumor activity of the lead Methods antibody, OR2805. To address the issues of toxicity and efficacy, we have identified a Results highly selective and potent CD137xPD-L1 bispecific antibody, MCLA- The target of OR2805 is highly expressed on TAMs and M2-like 145. Collections of common light chain Fabs recognizing CD137 and macrophages. OR2805 does not bind to other hematopoietic PD-L1 were produced based on antibody panels from immunized cells nor a panel of human primary non-immune cells. The anti- MeMo® mice. Unbiased, combinatorial, functional screening was then body stains positively on M2-like TAMs from primary human performed on a large and diverse panel of CD137xPD-L1 bAbs to lung tumor samples. OR2805 treatment reduces expression of identify those for which CD137 mediated activation is dependent on cell-surface markers associated with tumor-promoting M2c-like the presence of PD-L1 on a neighboring cell macrophages. In co-culture assays, OR2805 relieves the suppres- Results sive effect of M2 macrophages and resulted in increased T cell Both the CD137 and PD-L1 Fab arms block the interaction with their activation and proliferation, upregulation of T cell activation respective ligands as demonstrated in competition flow cytometry or markers, and enhanced T cell-mediated tumor cell killing. Ad- ELISA assays, respectively. MCLA-145 drives transactivation of CD137 ministration of OR2805 in humanized NSG-SGM3 mouse tumor in the vicinity of cells expressing PD-L1 and the degree of CD137 ag- models resulted in approximately 50% reduction in A549 tumor onistic activity in T cells correlated with the expression level of PD-L1 growth and a 60% reduction in H1975 tumor growth. In this on neighboring cells. CD137 signaling was induced by MCLA-145 in model, OR2805 treatment significantly increased the proportions multiple primary human immune cell assays and reversed T cell sup- of human CD8+ T cells and human CD11b+ myeloid cells in the pression mediated by M2 macrophages or Tregs, in vitro. In one hu- spleen as well as significantly enhanced expression of activation manized mouse tumor model, human T cells expressing NY-ESO markers (ICOS, OX-40) by human CD8+ T cells. specific TCR were adoptively transferred to mice bearing A549 tu- OR2805 reduces TAM-mediated immunosuppression and enhances mors, which expressed NY-ESO antigen and human PD-L1. MCLA-145 anti-tumor immune responses. OR2805 treatment induces robust treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) anti-tumor activity in lung cancer xenograft models in humanized as compared to T cell only–treated mice. In the tumors of MCLA- mice. This data justifies further development of OR2805 as anti- 145–treated mice, the percentage of NY-ESO specific CD8+ T cells cancer therapy in combination with other CPI treatments. OR2805 were significantly increased compared with controls. In a second has the potential to increase the number of patients who may bene- model, mice engrafted with human CD34+ cells were implanted with fit from current CPI therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 267 of 272 P491 References Preclinical development of a novel TNFRSF25 agonist antibody, 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. PTX-35, for cancer immunotherapy combinations Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 Matthew Seavey, PhD, Jayalakshmi Miriyala, MS, Jason Rose, MS, Vikas 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg Tahiliani, PhD, Patrick Dillon, PhD, Elena Gorovits, PhD, Jeff Hutchins, expansion in mice by TNFRSF25 prevents allergic lung inflammation. J PhD, Rahul Jasuja, PhD Clin Invest.; 120(10):3629-3640 Heat Biologics, Inc., Durham, NC, United States 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed Correspondence: Matthew Seavey (mseavey@heatbio.com) M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P491 That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell Background responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Tumor Necrosis Factor Receptor Super Family 25 (TNFRSF25), 5. Schreiber T.H., et al.; (2014). Comparative combination cancer also known as Death Receptor 3 (DR3), is preferentially immunotherapy with vaccination and TNFRSF stimulation. Society of expressed by activated and antigen-experienced T-cells. Immunotherapy of Cancer (SITC) Conference, Poster: https:// TNFRSF25 is a potent costimulatory molecule, similar to OX40, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ 4-1BB and GITR. PTX-35 was developed as a humanized, affinity heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf matured, IgG2 mAb against TNFRSF25 for use with current can- 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ cer immunotherapy options, including cancer vaccines. All regulatory T cells and the severity of acute graft-versus-host disease. pharmacology and IND-enabling PK and toxicology has been Blood 128 (24), 2846-2858 completed for PTX-35 [1-6]. Methods Previous proof-of-concept studies were completed elsewhere. P492 Pharmacology studies described here were completed with a sur- A new generation anti-TGFβ antibody, SAR439459, relieves rogate antibody, mouse-IgG1-PTX-35 (mPTX-35). Regulatory T-cell immunosuppression and improves anti-tumor efficacy of PD1 expansion studies were completed with Foxp3-RFP+ transgenic blockade mice (FIR). CD8+ T-cell expansion studies were conducted with Rita Greco, Hongjing Qu, Joachim Theilhaber, Gary Shapiro, Richard adoptively transferred OVA-specific, TCR-transgenic, CD8+ T-cells Gregory, PhD, Christopher Winter, Natalia Malkova, Lily Pao, Mikhail Levit, (OT-1). Human PTX-35 was tested in 28-day mouse, and 2-week Alexei Protopopov, Jack Pollard, PhD, Tun Tun Lin, MD, Dmitri and 8-week non-human primate, toxicology studies. Human, Wiederschain, Sharad Sharma, PhD mouse, and monkey, in vitro, tissue-cross reactivity tests were Sanofi, Cambridge, MA, United States also performed to check species cross-reactivity. Human PTX-35 Correspondence: Sharad Sharma (sharad.sharma@sanofi.com) was also tested in a human PBMC stimulation assay, in vitro, to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P492 check for impact on proliferation and cytokine release. Background Results TGFβ is a potent immunosuppressive cytokine that acts on TNFRSF25-engagement in mice expanded antigen specific CD8+ multiple cell types of the innate and adaptive arms of the im- T-cells when given in the context of vaccination (6 and 19-fold mune system within the tumor microenvironment. Emerging increase in CD8+ T-cells in blood over vaccination alone at peak data implicates role of TGFβ in tumor immune evasion, resist- and boost, respectively), and Tregs were expanded in vaccine ance to cancer therapy and poor prognosis. We report preclin- absence in FIR animals (2-fold increase in CD4+ Foxp3+ T-cells ical data on SAR439459, a new pan anti-TGFβ antibody, which in blood).The MABELand NOEL,inFIR mice,using mPTX-35, is capable of neutralizing all active isoforms of human and was determined to be 0.01 mg/kg and 0.001 mg/kg, respect- murine TGFβ. ively. The 2-week PTX-35 treatment of cynomolgus monkeys confirmed findings in mice, that treatment results in the expan- Methods sion of Teff and Treg cell subsets. Intravenous bolus injection Cancer patient databases were analyzed for various gene signa- once every 2-weeks over an 8-week period was well tolerated tures. In vitro experiments were performed to examine the effi- giving a NOAEL of 100 mg/kg. Species cross-reactivity was ob- cacy of SAR439459 in preventing TGFβ-mediated suppression of served for mouse, human and monkey tissues. No adverse primary human T and NK cells. To evaluate anti-tumor efficacy, events were recorded for the 28-day mouse toxicology study. MC38 and EMT6 mouse tumor models were treated with Testing PTX-35 in a human PBMC, anti-CD3 proliferation assay, SAR439459, anti-PD1, or combination. MC38 model was used to in vitro, showed that TNFRSF25-engagement provided the ne- examine immune cell functions ex vivo. cessary costimulation to drive cellular division at sub-optimal Results concentrations of anti-CD3 (2-fold increase), providing the ne- TGFβ was found to be upregulated in cancer patients and its cessary in vitro, human proof-of-concept data, which was con- increased activation correlated with reduced overall survival (OS) sistent across four different human blood donors. in PD-1 refractory cancer patients. SAR439459 blocked TGFβ- Conclusions mediated suppression of human T and NK cells. TGFβ impaired PTX-35 is a potent costimulatory agonist targeting a novel pathway the activity of anti-PD1 mediated T cell response, while that can work in concert with cancer vaccines. Due to the ability of SAR439459 restored this activity. In MC38 and EMT6 tumor PTX-35 to possibly stimulate both pro-inflammatory and anti- models, treatment with SAR439459 resulted in anti-tumor efficacy; inflammatory pathways, depending on treatment context, thera- the combination of SAR439459 with anti-PD1 enhanced this activ- peutic modulation can provide numerous opportunities for cancer ity and resulted in complete tumor regression and elicited a pro- and inflammatory diseases. longed anti-tumor response. Conclusions Acknowledgements This data demonstrates that combination of SAR439459 with anti- Pelican Therapeutics would like to thank the Cancer Prevention and PD1 generates a strong anti-tumor immune response and im- Research Institute of Texas (CPRIT) that helped fund these studies. We would proves anti-tumor efficacy in several tumor models. The preclin- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for ical data presented here formed the basis for the ongoing clinical mouse TL1A-Ig and advice, both located at the University of Miami. investigation of SAR439459 in cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 268 of 272 P493 Background High-dimensional analysis delineates modulation of myeloid and Heat (Heat) Biologics has developed a next generation cellular vac- lymphoid compartments with STAT3 ASO and PDL-1 combination cine platform that incorporates a tumor antigen chaperone (gp96-Ig) therapy in a tumor cell line and a host of over-expressed cancer associated Theresa Proia, PhD, Maneesh Singh, PhD, Larissa Carnevalli, PhD, Gayathri neoantigens. Viagenpumatucel-L (HS110), a human lung adenocar- Bommakanti, PhD, Nanhua Deng, Matthew Griffin, Lukasz Magiera, Adina cinoma cell line, stably transfected to express gp96-Ig, is being tested Hughes, Laura Prickett, Patricia McCoon, PhD, Corinne Reimer, Simon in a phase 1/2 clinical trial (NCT#02439450) for NSCLC. Heat has re- Barry, PhD cently developed HS130, an allogeneic cell-based vaccine, designed AstraZeneca, Waltham, MA, United States to secrete tumor-associated antigens along with a costimulatory mol- Correspondence: Theresa Proia (theresa.proia@astrazeneca.com); ecule, OX40L. Preclinical results of mouse HS130 (mHS130) in com- Gayathri Bommakanti (gayathri.bommakanti@astrazeneca.com) bination with mouse HS110 (mHS110) has shown a potent anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P493 tumor effector and memory CD8+ T cell response, followed by tumor regression. In our current study, we further characterized the role of Background mHS110 and mHS130 in combination with an agonist TNFRSF25 STAT3 is a ubiquitously expressed transcription factor and master monoclonal antibody, PTX35. PTX35 is a potent stimulator of effector regulator of immune suppression in the tumor microenvironment and memory CD8+ T cell responses, which taken-together with (TME). Danvatirsen, a therapeutic antisense oligonucleotide (ASO) HS110 and HS130 has the potential of treating human cancers [1-7]. that selectively targets STAT3, has shown clinical benefit alone and in Methods combination with durvalumab (anti-PDL1) and is currently in Phase To study expansion, contraction, and maintenance of tumor-specific 1/2 clinical studies. CD8+ T cell responses, mHS110 and/or mHS130, in combination with Methods different doses of mouse-IgG1-PTX35 (mPTX35) was administered to To gain mechanistic insight into the therapeutic response induced C57BL/6 mice that were adoptively transferred with syngeneic OVA- by mouse surrogate STAT3 ASO in the CT26 syngeneic mouse tumor specific T cells (OT-I). Mice were then challenged with murine melan- model, we have used two complementary forms of high-dimensional oma tumors (B16F10-OVA) to characterize the tumor-specific immune profiling; mass cytometry (CyTOF) and flow cytometry. We supported cells in the periphery, spleen, and tumor-microenvironment that the in vivo mouse findings with in vitro studies in human macro- were involved in tumor regression. phages treated with danvatirsen. Results Results Combination of mPTX-35 with mHS-110 and mHS130 increased the Multidimensional immune profiling studies provided key mechan- expansion of tumor-specific CD8+ T-cells, in a mPTX-35 dose- istic observations: (1) Robust reduction of total STAT3 protein in dependent manner. This cellular expansion was significantly higher myeloid lineage cells, including an 80% reduction in macro- in the 1 mg/kg dose of mPTX-35 and far exceeded the additive value phages and 50% reduction in dendritic cells, but not in CD8+ T of mPTX-35, mHS130, and mHS110 treatment alone. Systemic admin- cells. (2) In the combination treatment arm, STAT3 ASO treatment istration of mPTX35, in combination with mHS110 and mHS130, led promoted a two-fold reduction of intratumoral immunosuppres- to a significant increase in the expansion of activated CD8+ T cells in sive macrophages, doubling of iNOS positive activated macro- the blood and stimulated activation of KLRGhi IL7Rlo short-lived ef- phages and enhanced proliferation and IFNγ production from fector cells (SLECs). Importantly, this combination resulted in higher tumor antigen specific T cells. (3) The tumor-associated mono- frequencies of tumor infiltrating lymphocytes (TILs), which enhanced cyte/macrophage compartment is highly complex and dynamic regression of established B16F10-OVA tumors and increased overall and displays a spectrum of activation states ranging from a pre- survival. dominantly anti-inflammatory phenotype (F4/80+ CD206+ IL4r+ Conclusions MerTK+; six fold higher) in progressively growing control tumors These results strongly suggest that mPTX35 synergizes with mHS110 to a predominantly proinflammatory phenotype (F4/80+ iNOS+ and mHS130 to amplify activated tumor-specific CD8+ T cells, pro- CCR2+; three fold higher) in responding tumors from combination gram a strong memory response, and allow for tumor regression. treated groups. The combinations of these three treatments in the clinic may trans- In vitro, human macrophages were highly sensitive to danvatirsen late into an efficacious approach to treating human cancers. treatment, with an IC50 of 60nM for total STAT3. Human ‘M2-like’ macrophages were generated in the presence of IL10 and M-CSF and Acknowledgements treated with danvatirsen, which promoted an increase in IFNγ, IL-12, Pelican Therapeutics would like to thank the Cancer Prevention and and TNFα, as well as increased CD80/CD86 expression, consistent Research Institute of Texas (CPRIT) that helped fund these studies. We would with polarization from a suppressive phenotype to a pro- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for inflammatory phenotype. mouse TL1A-Ig and advice, both located at the University of Miami. Conclusions Our data support the hypothesis that STAT3 reduction in the myeloid References lineage results in activation of macrophages entering the TME and 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. enhanced effector T cell responses in combination with checkpoint Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 inhibition. Our ongoing work is focused on exploring the effects of 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg STAT3 reduction in other key immune cells in which we have ob- expansion in mice by TNFRSF25 prevents allergic lung inflammation. J served robust knockdown including Tregs, endothelial cells, and Clin Invest.; 120(10):3629-3640 CAFs. 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 P494 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell A novel TNFRSF25 agonist, PTX35, synergizes with Gp96-Ig/OX40L- responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Ig to enhance effector and memory anti-tumor CD8+ T cell 5. Schreiber T.H., et al.; (2014). Comparative combination cancer responses and delay tumor growth immunotherapy with vaccination and TNFRSF stimulation. Society of Vikas Tahiliani, Patrick Dillon, PhD, Jayalakshmi Miriyala, MS, Jason Rose, Immunotherapy of Cancer (SITC) Conference, Poster: https:// MS, Anh Trinh, Rahul Jasuja, PhD, Jeff Hutchins, PhD, Matthew Seavey, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ PhD heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf Heat Biologics, Inc., Durham, NC, United States 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ Correspondence: Matthew Seavey (mseavey@heatbio.com) regulatory T cells and the severity of acute graft-versus-host disease. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P494 Blood 128 (24), 2846-2858 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 269 of 272 7. FrommG,deSilva S, GiffinL,Xu X,Rose J,Schreiber TH.Gp96-Ig/ P496 Costimulator (OX40L, ICOSL, or 4-1BBL) Combination Vaccine Im- Inhibition of autophagy enhances multifunctional genetically- proves T-cell Priming and Enhances Immunity, Memory, and Tumor engineered NK cell-based immunotherapy of glioblastoma Elimination. Cancer Immunol Res. 2016;4(9):766-778. doi:10.1158/2326- Jiao Wang, PhD, Sandro Matosevic, PhD 6066.CIR-15-0228 Purdue University, West Lafayette, IN, United States Correspondence: Sandro Matosevic (sandro@purdue.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P496 P495 Secondary immune resistance mechanisms induced by therapeutic Background cancer vaccines which prevent tumor regression and foster Despite aggressive treatments, the median life expectancy for GBM recurrences patients is only around 15 months, highlighting the need for new Sjoerd Van der Burg, PhD, Elham Beyranvand Nejad, Camilla Labrie, therapeutic approaches. NK cells are showing potential for immuno- Suzanne van Duikeren, Ing, Ramon Arens, PhD, Thorbald van Hall, PhD, therapy of GBM. However, NK cells struggle to cross the blood brain Sjoerd van der Burg, PhD barrier (BBB) and infiltrate into GBM [1]. Moreover, the immunosup- Leiden University Medical Center, Leiden, Netherlands pressive tumor microenvironment (TME) impairs NK cell activity, for Correspondence: Sjoerd van der Burg (shvdburg@lumc.nl) instance due to adenosine-mediated downregulation of NKG2D ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P495 pression [2]. Methods Background We developed an innovative NK cell-based immunotherapy for Immunotherapy may induce complete tumor regressions but often GBM that targets multiple “checkpoints” simultaneously, by tumors partially regress followed by tumor recurrence. Here, we fo- combining 1) multifunctional NK cells which consist of a cleav- cused on the underlying mechanisms. able scFv targeting CD73 alongside dual chimeric antigen recep- Methods tors directed against GD2 and NKG2D ligands and 2) inhibition The TC-1 mouse tumor model in which different formulation and ap- of autophagy in GBM cells to sensitize them to NK cell lysis and plication of an HPV16 SLP vaccine results in full cure or tumor recur- promote NK cell infiltration into GBM via the secretion of GBM- rence and therapy resistance after initial full tumor regression. specific chemoattractants. Tumors, spleens and lymph nodes were analyzed by mass- and flow- Results cytometry. Mice were treated with antibodies to PD-1, PD-L1, OX-40, We have designed and synthesized a multifunctional CAR con- 4-1BB, NKG2A and TGFβ. Cell-sorted tumor cells were RNA se- struct that expresses an anti-CD73 scFv which is cleavable by quenced. Immune parameters were assessed in 5-10 mice, survival GBM-associated proteases, and a dual CAR that enables NK cells analyses were performed on at least 10 mice per group. All experi- to avoid antigen escape common to GBM (Figure 1A). We have ments were performed 2-3 times. generated engineered NK-92 or primary human NK cells that effi- Results ciently express the construct, from which the anti-CD73 scFv Optimal vaccination resulted in about 7% circulating tumor- could be functionally released via uPA treatment (Figure 1B and specific CD8+ T cells and complete cure of all mice, whereas sub- C). Engineered NK cells showed a significantly higher in vitro abil- optimal vaccination led on average to 1.7% tumor-specific T cells ity to kill patient-derived GBM43 targets (Figure 1D and E). To and tumor regression followed by recurrence in all mice. Neither target autophagy, BECN1- GBM43 cells were generated, and their booster vaccinations, which increased the numbers of circulating in vivo subcutaneous growth in RAG-1-/- mice validated the crit- tumor-specific type 1 cytokine-producing CD4+ and CD8+ T cells ical role of autophagy in GBM onset and progression (Figure 1F). (p<0.01), nor the co-administration of (combinations of) anti- We further showed that targeting autophagy inhibited the bodies against PD-1, PD-L1, 4-1BB, or OX-40 prevented tumor re- in vitro proliferation of GBM43 itself (Figure 1G), sensitized GBM currence or improved survival after vaccination. Immune escape to NK cell lysis (Fig. 1H), and induced elevated chemokine secre- was intrinsic to the tumor cells as the direct reinjection of ex- tion (CCL5), which in turn increased NK cell migration across the vivo cell-sorted recurrent tumor cells into groups of 10 naïve BBB using an in vitro BBB model (Figure 1I). hosts did not result in any response to vaccination while in all Conclusions cases the reinjected ex-vivo cell-sorted nontreated tumor cells We have generated multifunctional NK cells that can target mul- displayed vaccine-induced tumor regression followed by relapse tiple “checkpoints” at once showing improved cytotoxicity against (p<0.01). Ex-vivo analyses of escaped tumor cells showed no al- GBM through increased resistance to the immunosuppressive terations in the expression of MHC-I or the E7 tumor-antigen or TME via adenosinergic CD73 blockade and the ability to avoid their sensitivity to tumor-specific CTL mediated killing. RNA se- antigen escape by GBM via dual CARs. We have also found that quencing on bulk sorted (CD45-) tumor cells from non-treated blocking the autophagy pathway in GBM displayed potent syn- (n=4) and escaped (n=4) tumors, revealed a specific vaccine- ergy with NK cell-mediated immunotherapy. Based on these re- induced downregulation of the TNF- and P53-signaling pathways sults, to achieve combined therapeutic effects in vivo, we are and upregulation of TGFβ signaling. Indeed, more TGFβ positive currently evaluating this immunotherapy in an orthotopic GBM fibroblasts surrounded the escaped tumors (p<0.05). Recurrent tumors mouse model. Taken together, this approach provides a promis- displayed strongly reduced numbers of infiltrated CD8+ T cells (p< ing platform for the combination treatment of GBM with engi- 0.001), whereas this was not the case for escaped tumor cells- neered NK cells. reinjected tumors. However, both types of escaped tumors displayed lower numbers of tumor-infiltrating Ly6C+MHCI-II+ inflammatory mac- rophages (p<0.01). TGFβ-blockade delayed but did not prevent relapses References to recur (p=0.07). The use of inflammation inducing chemotherapy rein- 1. Kmiecik J, Zimmer J, Chekenya M. Natural killer cells in intracranial stalled the infiltration of tumors with inflammatory myeloid cells after neoplasms: presence and therapeutic efficacy against brain tumours. J vaccination, prevented relapse and reinstalled sensitivity of escaped tu- Neurooncol. 2014 ;116(1):1-9. mors to therapeutic vaccination (p=0.01). 2. Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting Conclusions enhances immunotherapy of CD73+ solid tumors with piggyBac- The sequential clinical phases during non-curative immunotherapy engineered chimeric antigen receptor natural killer cells. J Immunother may involve several distinct secondary escape mechanisms. Cancer. 2018;6(1):136. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 270 of 272 not show tumor growth inhibition in this model, DRP-104 significantly inhibited tumor growth and the combination further enhanced efficacy, illustrated by extended survival for both DRP-104 alone (50 days) and combination (96 days) treatment groups compared to vehicle (33days) and anti-PD-L1 alone (33days). Combination treatment also resulted in long term durable cures in 50% of the mice. Conclusions DRP-104 treatment results in dramatic remodeling of the tumor micro environment, leading to enhanced function of multiple immune cells distinct from activities obtained by anti-PD-1 Ab. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti- tumor efficacy including long-term durable cures even in checkpoint in- hibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in com- bination with PD-1/PD-L-1 checkpoint inhibitors. P498 Blockade of PD-1 and LAG-3 on CD8+ T cells, induced by vaccination, elicits superior anti-tumor efficacy Christopher Zahm, PhD, Douglas McNeel, MD, PhD, Laurne Delmastro UW Carbone Cancer Center, Madison, WI, United States Correspondence: Douglas McNeel (dm3@medicine.wisc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P498 Fig. 1 (abstract P496). See text for description Background T cell immune checkpoint receptors (ICR) and their ligands have emerged as a major mechanism by which tumors avoid immune de- tection. ICR blockade targeting PD-1/PD-L1 and/or CTLA-4 have revo- P497 lutionized the treatment of many cancer types. However, not all DRP-104, a novel broad acting glutamine antagonist, induces cancers respond to ICR blockade, in large part mediated by the pres- distinctive immune modulation mechanisms and synergistic ence or absence of tumor infiltrating CD8+ T cells. We have focused efficacy in combination with immune checkpoint blockade on tumor vaccines as a means to increase the number of tumor- Yumi Yokoyama, PhD, Michael Nedelcovych, PhD, Robert Wild, PhD specific CD8+ T cells. We have previously demonstrated that activa- Dracen Pharmaceutical, New York,, NY, United States tion of CD8+ T cells by vaccination leads to increased expression of Correspondence: Robert Wild (rwild@dracenpharma.com) specific ICR, and that blockade of these ICR with vaccination leads to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P497 better anti-tumor response than either alone. Differences in expres- sion of specific ICR, notably PD-1 and LAG-3, appeared dependent on Background presentation of antigen by professional versus non-professional APC, Glutamine is an essential amino acid for rapidly proliferating cancer hence we hypothesized that blockade of both of these ICR with vac- cells, thus depriving the same fuel from immune cells and contributing cination should be superior to either alone. to tumor immune evasion. DRP-104 was designed as a novel prodrug Methods of the broad acting glutamine antagonist 6-Diazo-5-oxo-L-norleucine In these studies we directly assessed the expression of PD-1, LAG-3, (DON). DRP-104 is inert in its prodrug form, affords high levels of CTLA-4, and TIM-3 on CD8+ T cells following activation in the pres- plasma and gastro-intestinal (GI) tissue stability; has high tumor cell ence or absence of professional APC. Next, we transferred these cells permeability and preferential tumor versus plasma/GI tissue distribution into tumor bearing mice, alone or in combination ICR blocking anti- for DON. Here we sought to (1) compare immunological modulation of bodies, to directly evaluate their anti-tumor efficacy. Finally, we im- DRP-104 to anti-PD-1Ab, and (2) evaluate the combination effect of munized tumor-bearing HLA-A2-transgenic mice with different anti- DRP-104 with PD-1/PD-L1 checkpoint inhibitors. tumor DNA vaccines that have previously been shown to elicit CD8+ Methods T cells preferentially expressing either PD-1 or LAG-3, and used each Immunomodulatory effects of DRP-104 as a single agent and com- vaccine alone or in combination with ICR blockade. bination with anti-PD-1Ab was evaluated in the CT26 mouse colon Results carcinoma model by flow cytometry and Luminex assay. In vivo anti- We found that PD-1, LAG-3, CTLA-4 and TIM-3 are all increased on tumor efficacy of combination with anti-PD-1/PD-L1Ab was evaluated CD8+ T cells after activation by professional APC, however LAG-3 in CT26 and H22 hepatocellular carcinoma models. alone was increased on CD8+ T cells activated in the absence of pro- Results fessional APC (Figure 1). When these cells were adoptively trans- DRP-104 treatment showed broad immune cell modulation effects in- ferred into tumor bearing mice, LAG-3 blockade improved the anti- cluding increased T, NK, and macrophages; while anti-PD-1Ab affected tumor efficacy of CD8+ T cells activated without APC, and PD-1 mainly CD8+T cells. Cytokine modulation in tumor and plasma revealed blockade improved the anti-tumor efficacy of CD8+ T cells activated that DRP-104 decreased pro-tumorigenic cytokines such as VEGF and by APC (Figure 2). Immunization with different DNA constructs [1-3] KC(IL-8) while anti-PD-1Ab showed either no change or slight increase in combination with ICR blockade led to improved anti-tumor re- in these cytokines. CT26 bearing mice treated with anti-PD-1Ab alone, sponses, however combining LAG-3 blockade with PD-1 blockade DRP-104, and the combination showed tumor growth inhibition at day showed no benefit over PD-1 blockade alone (Figure 3). 12 of 48%, 90%, and 94%, respectively. Median survival days were 31.5, Conclusions 36, and 56 days, respectively (vehicle; 17.5 days). Notably 9 mice These data support our previous finding that PD-1 blockade improves treated with combination of anti-PD-1 with DRP-104 were tumor free at the efficacy of CD8+ T cells activated by vaccination. In this model, we end of the experiment (day 77) and 100% of these mice rejected a detected no additional benefit to concurrent LAG-3 blockade. The role of CT26 tumor re-challenge. In the H22 model, mice were treated with ei- other ICR in limiting anti-tumor immunity, and strategic blockade of these ther anti-PD-L1 Ab, DRP-104, or combination. While anti-PD-L1Ab did receptors following T-cell activation, is an area of active investigation. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 271 of 272 References 1 Smith, H. A., Rekoske, B. T. & McNeel, D. G. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses. Vaccine 32, 1707-1715, doi:10.1016/j.vac- cine.2014.01.048 (2014). 2 Rekoske, B. T., Smith, H. A., Olson, B. M., Maricque, B. B. & McNeel, D. G. PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization. Cancer Immunol Res 3, 946-955, doi:10.1158/2326-6066.CIR-14-0206 (2015). 3 Colluru,V. T., Zahm,C.D.&McNeel,D.G.Mini-intronicplasmid vac- cination elicits tolerant LAG3+ CD8 T cells and inferior anti-tumor re- sponses. OncoImmunology, 0-0, doi:10.1080/2162402X.2016.1223002 (2016). Fig 3 (abstract P498). Some vaccines only effective when combined with ICR blockade P499 Nano-Pulse Stimulation in combination with the TLR 7/8 agonist, resiquimod, synergizes to eliminate murine melanoma through innate and adaptive immune responses Joel Benjamin, PhD, Amanda McDaniel, BA, Kristin von Rothstein, Sasha Farina, Bruce Freimark, PhD, Richard Nuccitelli, MS, PhD Pulse Biosciences, Inc, Hayward, CA, United States Correspondence: Richard Nuccitelli (rnuccitelli@pulsebiosciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P499 Fig. 1 (abstract P498). Priming with professional APCs leads to Background ICR expression Nano-Pulse Stimulation (NPS) is a non-thermal treatment modality that provides high amplitude electrical energy pulses in the nanosec- ond range that is focal and directly acts on cellular structures and membranes to initiate regulated cell death. Previous work has shown that NPS induces release of tumor antigen and stimulates an in situ anti-tumor immune response [1, 2, 3]. The TLR 7/8 agonist resiqui- mod (RES) has been used as an immune adjuvant in previous cancer vaccine treatments in murine models to aid in antigen processing and presentation [4]. We have evaluated the combination of NPS and RES treatment to inhibit tumor growth and induce innate and adaptive immune responses. Methods The B16-F10 melanoma in C57BL/6j mice was used to investigate the potential combined effects of NPS and RES. B16-F10 tumor cells (2x10e5) were injected intradermally on the left flank and treated with NPS 5 days after inoculation. RES (50 μg) was then dosed in multiple combinations and timing to assess optimal tumor cell elimination and immune stimulation from the combin- ation treatment. Tumor efficacy (volume) was measured twice per week. Immune biomarkers included flow cytometry and IHC of T cell and myeloid immune cells from tumors, draining lymph nodes and spleens. Results Low energy NPS and up to 3 doses of RES as monotherapies partially inhibit tumor growth. However, combination of NPS with 24-hour, post treatment of RES resulted in complete regression in a large frac- tion of treated animals. This efficacy is concomitant with increased antigen-specific and memory CD8 populations in the spleen and lymph node, as well as an increase in certain innate immune cell Fig. 2 (abstract P498). APC-induced ICR signaling compromised populations. Tumor regressions showed no sign of regrowth 90 days anti-tumor response after treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 272 of 272 Conclusions Methods Compared to monotherapy treatments, the combination of NPS and Three models of colon tumorigenesis were used to conduct the RES treatments induce stronger tumor growth inhibition which per- present study (IACUC#3408), which included a colitis (AOM/DSS –in- sists as long-term tumor regression. Persistent tumor regression by duced), a spontaneous (APCMin-driven), and a syngeneic (engrafted combination treatment is associated with increases in B16F10 with MC-38 or CT-26 cells) model. Several mutant mice were used in antigen-specific and memory CD8 populations, as well as increases in these models including PARP-1-/-, PARP-1-/+ APCMin/+, APCMin/ innate immune populations with potential for antigen presentation. +PARP-1-/-+/−, APCMin/+PARP-1-/+ as well as WT mice. Mice were These data support a mechanism by which NPS combined with randomized and assigned to the different experimental groups. TLR7/8 agonists activates an enhanced immune response. Some groups of mice were administered olaparib, anti-mouse PD-1 antibodies, or a combination of the two agents. Mice were scarified References according to the requirements of each model and tumor and tissues 1. Nuccitelli R, Berridge JC, Mallon Z, Kreis M, Athos B, Nuccitelli P. were collected for the analysis. MDSCs were generated by incubating Nanoelectroablation of murine tumors triggers a CD8-dependent inhib- bone marrow cells with GM-CSF, G-CSF, and IL-6. Tumor MDSCs were ition of secondary tumor growth. PLoS ONE. 2015; 10(7): e0134364 1-17. generated by enzymatic digestion of MC-38-engrafted tumors 2. Nuccitelli R, McDaniel A, Anand S, Mallon Z, Berridge JC, Uecker D. Nano- followed by positive selection. The suppression assay was performed Pulse Stimulation is a physical modality that can trigger immunogenic by co-cultured with CD3/CD28-stimulated CFSE-labeled T cells and tumor cell death. J Immunotherapy Cancer. 2017; 5:32 DOI 10.1186/ proliferation was assessed by FACS. s40425-017-0234-5. Results 3. Skeate JG, DaSilva DM, Chavez-Juan E, Anand S, Nuccitelli R, Kast W.M. Here, we show that partial PARP-1 inhibition via gene heterozygosity or Nano-Pulse Stimulation induces immunogenic cell death in human a moderate olaparib dose was sufficient to protect against colitis- or papillomavirus-transformed tumors and initiates an adaptive immune re- APCMin-mediated intestinal tumorigenesis, while extensive inhibition sponse. 2018; PLoS ONE. 13(1): e0191311 via gene knockout or a high olaparib dose was ineffective or aggra- 4. Caisova V, Vieru A, Kumzakova Z, Glaserova S, Husnikova H, Vacova N, vated the burden despite anti-inflammatory effects and promotion of a Krejcova G, Padoukova L, Jochmanova I, Wolf KI, Chmelar J, Kopecky J, tumor-suppressive microenvironment. A sub-IC50 metronomic dose of Zenka J. Innate immunity-based cancer immunotherapy: B16-F10 murine olaparib or PARP-1 heterozygosity was also sufficient to block tumori- melanoma model. 2016; BMC Cancer.16(1); 940; DOI:10.1186/s12885-016- genesis in syngeneic colon cancer models by modulating the suppres- 2982-x. sive function, but not differentiation or intratumoral migration, of myeloid-derived suppressor cells (MDSCs). These effects occurred through a reduction of arginase-1, iNOS, and COX-2 expression but in- P500 dependently of PARP-1-trapping on chromatin. Interestingly, the metro- Targeting PARP-1 with metronomic therapy as a new approach to nomic olaparib dose increased the intratumoral numbers, but not modulate MDSC function and enhance anti-PD1 immunotherapy in percentages, of CD8+ T cells. Adoptive transfer of WT bone marrow- colon cancer derived MDSCs abrogated the protective effects of PARP-1 heterozy- 1 1 1 Salome Valentina Ibba , Mohamed Ghonim , Abdelmetalab Tarhuni , gosity against the tumor burden. A metronomic olaparib dose was 1 1 1 Matthew Dean , Hamid Boulares, PhD , Augusto Ochoa, MD , Youssef highly synergistic with anti-PD1-based immunotherapy leading to al- 1 1 1 1 Errami , Ali Elbahraway , Ilyes Benslimane , Dorota Wyczechowska , Luis most complete eradication of tumors on mice. 1 2 1 Del Valle, MD , Amir Al-Khami , Hanh Luu Conclusions Louisiana State University-Health Science Center, New Orleans, LA, Our results support a paradigm-shifting concept that expands the United States; Tanta University, Tanta, Egypt utility of PARPi and encourage testing metronomic dosing of PARPi Correspondence: Hamid Boulares (hboulr@lsuhsc.edu) to enhance efficacy of check-point inhibitor-based immunotherapies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P500 not only in cancer of the colon but also that of other tissues ultim- ately benefiting a large proportion of cancer patients. Background Ethics Approval PARP inhibitors (PARPi) have important anti-tumor effects in BRCA- IACUC#3408 defective cancers but efficacy requires their use at/near maximum- tolerated-doses to achieve trapping of the enzyme on damaged Publisher’s Note chromatin. However, the benefits of targeting non-DNA repair as- Springer Nature remains neutral with regard to jurisdictional claims pects of PARP with low metronomic doses have not been explored. in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1

Journal for ImmunoTherapy of Cancer , Volume 7 (1) – Nov 6, 2019

34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1

Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 https://doi.org/10.1186/s40425-019-0763-1 MEETING ABSTRACTS Open Access 34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1 National Harbor, MD, USA. 6-10 November 2019 Published: 6 November 2019 About this supplement These abstracts have been published as part of Journal for ImmunoTherapy of Cancer Volume 7 Supplement 1, 2019. The full contents of the supplement are available online at https://jitc.biomedcentral.com/articles/supplements/volume-7-supplement-1. Please note that this is part 1 of 2. Results Poster Presentations Preliminary cohort of 10 patients includes 4 with recurrence at a median Biomarkers, Immune Monitoring, and Novel of 2.4 years and 6 with no recurrence at a median of 12 years post-LT. Technologies We found that patients with recurrence post-LT have significantly higher densities of MPO+ PMNs compared to those with no recurrence. This dif- P1 ference is primarily driven by PMNs located within the peritumoral Peritumoral neutrophil infiltration predicts recurrence of stroma (Median [interquartile range [IQR] 2.46 [1.99 - 2.92] vs 1.23 [0.723 hepatocellular carcinoma following liver transplantation -1.78], p=0.019). Intratumoral PMN infiltration was not associated with re- 1 1 1 1 Marc Najjar, MD , Michael Ross , Ayush Srivastava , Robyn Gartrell, MD , currence (Median [IQR] 0.91 [0.59 - 1.20] vs 1.33 [0.56 – 1.90], p=0.308). 1 1 1 Emanuelle Rizk, BA , Olivia Perez , Evan Lieberman , Charles Drake, MD, Moreover, density of CD3, both intratumoral and peritumoral, did not cor- 1 1 1 1 PhD , Ladan Fazlollahi , Helen Remotti , Elizabeth Verna , Karim relate with recurrence, nor did the tissue-derived NLR. Further, we found 2 1 1 Halazun , Jean Emond , Yvonne Saenger, MD that the tissue-derived NLR did not correlate with NLR in blood. 1 2 Columbia University Medical Center, New York, NY, United States; Weill Conclusions Cornell Medicine, New York, NY, United States Higher densities of peritumoral PMNs are associated with post-LT Correspondence: Marc Najjar (mn2594@cumc.columbia.edu) HCC recurrence. Evaluation of TME using qmIF can be used to predict Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P1 recurrence in post-LT HCC. Further, tissue based analysis of PMNs does not correlate with serum NLR allowing potential for composite Background biomarkers. As this is preliminary, further analysis is underway and Hepatocellular carcinoma (HCC) is the most common liver malig- will be validated on the larger cohort of patients. nancy and the 5th cause of cancer-related mortality worldwide. Though previous studies have found that serum neutrophil-to- Reference lymphocyte ratio (NLR) is predictive of survival post liver transplant 1. Gartrell RD, Marks DK, Hart TD, et al. Quantitative Analysis of Immune (LT), peritumoral neutrophil (PMN) infiltration in the tumor micro- Infiltrates in Primary Melanoma. Cancer Immunol Res 2018;6:481-93. environment (TME) of HCC has not been thoroughly investigated yet. In this study we sought to evaluate tissue based PMN infiltration in HCC post LT using quantitative multiplex immunofluorescence (qmIF), previously used to study the TME of several other tumor types[1]. Methods A database of 634 patients was created at Columbia University Irving Medical Center (CUIMC) including adult patients with available clin- ical follow up who underwent liver transplantation (LT) for HCC be- tween 1998 and 2018. We evaluated a preliminary cohort of 10 patients using qmIF, excluding patients with viral hepatitis. FFPE tumor sections were pre-selected by a GI pathologist. Slides were stained using qmIF for MPO (PMNs), CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), HLA-DR (immune activation), and Hep- Par1 (hepatocytes/tumor). Multiplex images were visualized using Vectra (Akoya) and processed using inForm (Akoya). Data was ana- lyzed using R Studio for concatenation, density, nearest neighbor Fig. 1 (abstract P1). Quantitative multiplex immunofluorescence and statistical analysis. Serum NLR was calculated using complete images of HCC blood counts collected prior to LT(Figure 1). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 2 of 272 P2 determined via flow cytometry. Analyte secretion was determined Single-cell RNAseq analysis of the effects of cryopreservation on from supernatant using Milliplex MAP Human CD8+ T-cell Panel. primary tumor tissue Results Shawn Fahl (shawn.fahl@dls.com) We detected pembrolizumab binding to T-cells in a dose dependent Discovery Life Sciences, Huntsville, AL, United States manner and an increase in the activation marker CD69 on T-cells fol- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P2 lowing tumor cell and pembrolizumab treatment in three of four pa- tients tested. We devised an initial E:T optimization screen to identify Background a patient-specific ratio which renders our subsequent therapy re- The tumor microenvironment is a complex mixture of multiple cell sponse profiling highly personalized. CD3+CD8+ T-cell mediated types, and numerous therapeutic interventions have been developed tumor cell death and enhanced killing was detected in the presence targeting distinct aspects of this environment. Tumor tissue samples of pembrolizumab. Immune cell infiltration as well as therapy related are an integral part of identifying and understanding potential thera- cell death was observed in our 3D microtumors. Altered patient spe- peutic targets within the tumor microenvironment of multiple cancer cific cytokine secretion was measured when the cultures were indications. As early biomarker discovery is often hindered by the lo- treated with pembrolizumab and significantly correlated with pem- gistical demands of sourcing fresh human tumor tissue, cryopre- brolizumab induced reduction of microtumor growth rates. served dissociated tumor cell suspensions provide a viable Conclusions alternative for accessing multiple, highly-annotated tumor samples The data generated from these two complex 3D in vitro models al- for complex studies. Previous evaluations of cryopreservation on vi- lows us to better understand immune responses to autologous able tumor tissue have relied on flow or mass cytometry which, while tumor cells and checkpoint blockade. Our models are therefore ideal powerful, are limited in the number of targets that can be analyzed. and complimentary for preclinical testing of new I/O agents as well Single cell gene expression can analyze the expression of signifi- as patient response predictions to I/O based therapies. cantly more targets and provide a clearer picture on the effects of Ethics Approval cryopreservation on the cellular composition of the tumor. Tissue was acquired with approval from Prisma Health's Institutional Methods Review Board, PRO# 00069834. Multiple unique primary tumor samples were dissociated to the single-cell level and profiled by flow cytometry. These single cell sus- P4 pensions were subsequently subjected to single cell RNASeq using Novel immune competent murine glioblastoma models derived the 10X Genomics platform prior to, and immediately following, cryo- T2 L/L L/L L/L from Nestin-CreER ; Quaking ; P53 ; PTEN mice preservation. Data was subsequently analyzed to determine how 1 1 2 Chao-Hsien Chen, MD , Renee Chin, MS , Genevieve Hartley, PhD , cryopreservation impacted the cellular composition of the tumor 1 2 2 Cheng-En Hsieh, MD , Rishika Prasad, MS , Takashi Shingu, PhD , David microenvironment. 2 2 2 Hong, MD , Jian Hu, PhD , Michael Curran, PhD The University of Texas MD Anderson Cancer Center UTHealth P3 Graduate School of Biomedical Sciences, Houston, TX, United States; Predicting patient response to checkpoint blockade therapy using The University of Texas MD Anderson Cancer Center, Houston, TX, in vitro 3D cultures United States Kathryn Appleton, PhD, Ashley Elrod, Qi Jin Guo, Dennis Ruder, Tessa Correspondence: Michael Curran (MCurran@mdanderson.org) DesRochers, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P4 KIYATEC, Inc., Greenville, SC, United States Correspondence: Tessa DesRochers (tessa.desrochers@kiyatec.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P3 The widely used glioblastoma multiforme (GBM) model GL261 is highly immunogenic and readily cured by checkpoint blockade limit- Background ing its use for pre-clinical modeling of immunotherapy for human Knowledge of immune responses that correlate with clinical outcome GBM [1,2]. We developed four novel murine immunocompetent glio- T2 is essential for the development of strategies to harness a patient’s blastoma stem cell (QPP) lines derived from Nestin-CreER Quaking L/L L/L L/L immune system to eradicate cancer. Pre-clinical platforms that recap- (QKI) ; P53 ; PTEN mice, reflecting a common set of alterations itulate the immune response in the context of cancer are necessary in patients [3-5]. The four QPP cell lines are syngeneic to C57BL/6J for adequate understanding and detection of clinical efficacy, how- mice and exhibit distinct responses to T-cell checkpoint blockade. ever, the technology to accurately test immuno-oncology (I/O) ther- Methods apy response is lacking. Despite the value animal models provide in The differential responsiveness of each QPP line was assessed a pre-clinical setting, they lack matched patient tumor and immune through analysis of tumor growth in the brain versus the flank in un- cell interactions. To address this shortcoming, we developed in vitro treated, αPD-1, or αCTLA-4 treated mice. The impact of tumor gen- 3D tissue models that maintain autologous patient tumor cells and omic landscape on responsiveness at each site was measured immune cells for the testing and prediction of immune cell re- through whole exome sequencing. To understand cellular factors sponses. We hypothesize that these 3D tissue models will recapitu- modulating responsiveness of these GBM lines to checkpoint block- late the patient tumor microenvironment and detect response to I/O ade, the immune microenvironments of sensitive (QPP7) versus re- agents. sistant (QPP8) lines were compared in the brain using high Methods parameter flow cytometry. Drivers of flank sensitivity versus brain re- Tumor cells and T-cells were obtained from seven melanoma patient sistance were also measured for QPP8. biopsies and screened for PD-L1 and lymphocyte populations prior Results to incorporation into 3D culture. Effector cell to Tumor cell (E:T) QPP GBM lines demonstrate a range of sensitivities to CTLA-4 and optimization assays were conducted with expanded T-cells at differ- PD-1 blockade when implanted on the flank ranging from complete ent densities and co-cultured at different time points with tumor sensitivity (QPP7) to complete resistance (QPP4). In the brain, QPP7 cells. Viability was measured using CellTiter-Glo® 3D. T-cell response remains sensitive to both antibodies, but QPP4 and QPP8 fail to re- was determined using flow cytometry following 24-hour co-culture spond to blockade of either checkpoint (Figure 1). Analysis of the with tumor cells. Microtumors were established using a biologically QPP8 immune infiltrate in skin reveals enhanced ratios of CD8s to inert scaffold and extracellular matrix components. Microtumor via- Treg and myeloid suppressors in response to checkpoint blockade; bility was determined using PrestoBlue and T-cell infiltration was however, none of these benefits manifest in the brain QPP8 except a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 3 of 272 very specific increase in CD8s relative to granulocytic suppressors (Figure 2). Brain-implanted QPP8 reacts adaptively to checkpoint blockade by upregulating PD-L1 expression across its myeloid stroma. In contrast, immune-responsive QPP7 does not induce PD-L1 and shows markers of enhanced CD8 T cell fitness. Consistent with these observations, genomic analysis reveals a higher mutation dens- ity in QPP7 versus the other QPP lines. Using checkpoint-insensitive QPP4/8, we have now identified agonists of the Stimulator of Inter- feron Genes (STING) pathway as highly promising therapeutics for treating these tumors in the brain. Conclusions We have developed novel syngeneic models of GBM with relevant genetics and immune sensitivities relative to human disease. Through comparing T cell checkpoint blockade sensitive versus in- sensitive variants of these QPP lines, and through comparing variant sensitivity dictated by site of implantation, we have begun to identify the genetic and cellular components that govern immunotherapeutic sensitivity of GBM. References 1. Reardon DA, Omuro A, Brandes AA, Rieger J, Wick A, Sepulveda J, Phuphanich S, de Souza P, Ahluwalia MS, Lim M, Vlahovic G, Sampson J (2017) OS10.3 Randomized Phase 3 Study Evaluating the Efficacy and Safety of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: CheckMate 143. Neuro-Oncology 19: iii21-iii21. 2. Reardon DA, Gokhale PC, Klein SR, et al. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model. Cancer Immunol Res. 2016;4(2):124-135. 3. Hu J, Ho AL, Yuan L, et al. From the Cover: Neutralization of terminal differentiation in gliomagenesis. Proc Natl Acad Sci U S A. 2013;110(36):14520-14527. Fig. 2 (abstract P4). Immune landscape of QPP8 TME in 4. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic different niches landscape of glioblastoma. Cell. 2013;155(2):462-477. 5. Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating P5 endolysosomal degradation. Nat Genet. 2017;49(1):75-86. Laminar Wash™ AUTO system: a reliable walk-away sample Ethics Approval preparation solution for better TIL recovery without centrifugation All experiments were conducted according to protocols approved by the 1 1 2 2 Ira Kim , Melvin Lye , Roberta Zappasodi, PhD , Isabell Schulze , University of Texas MD Anderson Cancer Center Institutional Animal Care 3 1 1 Christoph Eberle, PhD , Chyan Ying Ke , Kong Leong Cheng , Ih Chin and Use Committee. 1 1 2 1 Kon , Royce Pek , Taha Merghoub, PhD , Namyong Kim, PhD 1 2 Curiox Biosystems, Boston, MA, United States; MSKCC, New York, NY, United States; Charles River Laboratories, Worcester, MA, United States Correspondence: Namyong Kim (namyong@curiox.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P5 Background The naturally occurring tumor infiltrating lymphocytes (TILs) exist in a complex microenvironment containing the extracellular matrix, blood vessels, and stromal and endothelial components in addition to vari- ous immune cells. While a growing number of preclinical mouse models is under development, the heterogeneity of the cell compos- ition in a solid tumor poses considerable technical challenges in iso- lating and characterizing the TILs for the downstream analysis. One common problem with TILs preparation occurs during solid tumor dissociation, whereby the TILs are left in a mixture with tissue debris and dead cells in suspension. Consequently, a preparation of autolo- gous TILs often requires further costly and laborious processing such as density gradient centrifugation, immune cell sorting and enrich- ment, and dead cell and debris removal in combination with multiple centrifugation steps. We introduce a novel Laminar Wash™ technol- ogy, which can help overcome these technical challenges. Methods We performed pilot studies on syngeneic (MC38, CT26, Cloud- manS91, 4T1) and humanized mouse tumor models as well as with human PBMCs and tumor biopsies using the Laminar Wash™ technol- ogy. Briefly, we evaluated various functional parameters of TILs such as polyfunctional CD8+ T cell responses and glucose update effi- ciency (2-NBDG) as well as conducting a side-by-side comparison of the TIL recovery rate and immunophenotypic characteristics of lymphoid and myeloid subsets on the Laminar Wash™ and the Fig. 1 (abstract P4). Orthotopic QPP survival and immune sensitivity centrifugation-based systems. In addition, the cell retention rate, cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 4 of 272 viability, debris removal, epitope preservation and the overall pro- detectable in the periphery starting at 2 weeks with stable levels (up cessing time were assessed and compared. Furthermore, we intro- to 40% of live cells to 8 weeks) with increasing T effector memory duce a complete walk-away approach to sample preparation that cells over the course of study. All tumors evaluated had high levels eliminates operator-based variability while significantly enhancing re- of TIL as measured by flow cytometry and immunohistochemistry. producibility and consistency of downstream analysis. Costimulatory and inhibitory molecules evaluated on CD4 and CD8 Results included 4-1BB, TIM-3, LAG-3, OX-40, as well as PD-1, which was Our data demonstrate that the Laminar Wash™ method resulted in expressed on both peripheral blood cells and in TIL. Tumor growth higher cell retention and viability, more clearly defined immune sub- kinetics was unaltered by PBMC humanization through a 5-week sets, a lowered background signal, and an enhanced yield of the TILs study window. from freshly dissociated tumor samples compared to the Conclusions centrifugation-based counterparts. The Laminar Wash™ system can PBMC-humanized NSG-B2M mice may represent a model for evaluat- effectively remove the floating debris in suspension while keeping ing of IO therapeutics with a long study window due to the lack of the live cells unperturbed, allowing the cell surface architecture and xGVHD. While PBMC engraftment kinetics are donor dependent, simi- epitopes better retained for improved downstream analysis with flow lar phenotypes are observed and T cell subsets expressing several cytometer. Additionally, the Laminar Wash™ AUTO system offers a relevant therapeutic targets, including PD-1 are present. This model completely automated sample processing solution for dissociated may permit a rapid in vivo method to study checkpoint blockade tumor samples, simplifying and expediting cell preparation with en- and other T-cell-directed IO therapeutics. hanced consistency and reproducibility. Ethics Approval Conclusions The study was approved by Champions Oncology's Institutional Ani- Laminar Wash™ results in healthy, viable, and well defined popula- mal Care and Use Committee (IACUC). tion of TILs, while improving the overall quality of data. The AUTO station provides an automated, centrifuge-free, and walk-away work- P7 flow for dissociated tumor samples for cytometry-based assays. Development of a natural killer (NK) ImmunoGraft platform for the evaluation of the pharmacodynamics of immuno-oncology Acknowledgements therapeutics Laminar Wash™ results in healthy, viable, and well defined population of TILs, 1 1 2 Bhavana Verma, PhD , Bruce Ruggeri , Jon Weidanz, PhD , Amy Wesa, while improving the overall quality of data. The AUTO station provides an PhD automated, centrifuge-free, and walk-away workflow for dissociated tumor 1 2 Champions Oncology, Rockville, MD, United States; Abexxa Biologics, samples for cytometry-based assays. Arlington, TX, United States Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); P6 Amy Wesa (awesa@championsoncology.com) Development of a peripheral blood mononuclear cells (PBMC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P7 ImmunoGraft platform to evaluate the pharmacodynamics of Immuno-oncology therapeutics Background Bhavana Verma, PhD, Bruce Ruggeri, Amy Wesa, PhD Harnessing NK cell anti-cancer cytotoxicity has gained interest as a Champions Oncology, Rockville, MD, United States therapeutic strategy, and consequently improved preclinical models Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); supporting the translation of NK cell–mediated therapies to the clinic Amy Wesa (awesa@championsoncology.com) are desired. Reproducible models with human NK engraftment into Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P6 immunodeficient mice co-engrafted with cell line-derived xenograft or patient-derived xenograft tumor models have been lacking due to Background an inability to support NK cell engraftment and persistence. Here we Humanized immune system (HIS) mouse models enable in vivo stud- evaluated IL-15-NOG mice for the engraftment and sustained survival ies in the context of the human immune cells with a human tumor of both ex vivo expanded and primary human NK cell isolates for es- and are critical for the development of next generation immune- tablishing models that engraft effectively with both human NK cells oncology (IO) agents. Humanization of immunodeficient mice and a PDX or CDX tumor. through the adoptive transfer of normal adult PBMC leads to rapid Methods engraftment of human T cells to study immune-modulatory agents NK cells from normal adult peripheral blood mononuclear cells in the context of human tumor xenografts, but is limited by the de- (PBMC) donors (N=3) were expanded using two different commer- velopment of xenogeneic graft-versus host disease (xGVHD). In this cially available kits and evaluated for NK phenotype, expansion rates study, we evaluated the engraftment of PBMC in β2microglobulin and yields. Titrated doses of ex vivo expanded NK cells were adop- null super-immunodeficient mice NSG-B2M mice, that lack MHC Class tively transferred into IL-15-NOG mice for human chimerism, and the I on host tissues. A cell line-derived xenograft model (CDX) co- persistence and survival of NK cells and their immunophenotype engrafted with PBMC (PBMC-ImmunoGraft) was characterized for were assessed. In separate studies, naïve NK cells enriched from humanization, tumor infiltrating leukocytes (TIL) phenotype and PBMC were also evaluated for NK cell persistence and expansion tumor response to checkpoint inhibitors. in vivo. To establish an NK ImmunoGraft, NK cells were engrafted in Methods xenograft tumor bearing mice and tumor growth kinetics were PBMC from healthy donors (N=7) were implanted and engraftment characterized. in peripheral blood was assessed by flow cytometry. T cell memory Results phenotypes were assessed over time in a small cohort, and costimu- Donor dependent NK expansion was observed ex vivo, with 28 to latory and inhibitory T cell subsets were evaluated at the terminal 50-fold expansion by two weeks. NK cells expanded ex vivo were time point in blood and secondary lymphoid organs. Next, NSG-B2M CD3-CD16+CD56± and varied based on the expansion kit utilized. mice were co-implanted with MDA-MB-231 breast cancer cell line s.c, Nearly all CD45+ cells in circulation were NK cells, and these peaked humanized with PBMC and tumor growth kinetics were monitored. by week 2, and were maintained for up to 10 weeks in IL-15-NOG Efficacy studies evaluating check point inhibitors are currently mice. Primary NK cells engrafted with slower kinetics, with peak ongoing. abundance at 3-4 weeks. NK cells expressed granzyme B, and further Results functional studies are in progress. For all NK cell populations, cell Successful PBMC engraftment without xGVHD was observed in NSG- density-dependent engraftment was observed with a largely stable B2M mice up to 8 weeks, in contrast with MHC Class I expressing im- NK phenotype observed across the study. In the absence of any munodeficient mice that developed xGVHD within 4-5 weeks. Dose therapeutic treatment, NK cell persistence and expansion in vivo did and donor- dependent chimerism was observed. T cells were not inhibit tumor xenograft growth kinetics in IL-15-NOG mice Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 5 of 272 Conclusions sensitivity and as such could be utilized to assess efficacy of the IL-15-NOG mice support the survival and persistence of human NK monoclonal antibody administered. There are also potential applica- cells from both ex vivo expanded and naïve NK cells, suggesting the tions related to rapid drug screening using the patient-derived xeno- universality of this platform for human NK engraftment. Our prelimin- graft model. Our future plans are focused on adapting these ary studies support IL-15 NOG mouse model as a suitable system for solutions to the characterization of immune checkpoint inhibitor evaluation of NK cellular therapies or NK cell-modulating therapies in therapeutics in standard-of-care FFPE tissues obtained from patients the context of patient-derived or cell-line derived xenograft (PDX or undergoing immunotherapy. CDX) mouse models Ethics Approval P9 The study was approved by Champions Oncology's Institutional Ani- Immune checkpoint biomarkers in hepatocellular carcinoma (HCC): mal Care and Use Committee (IACUC). Assessment of PD-L1 and tumour mutation burden in tumour samples from clinical patients 1 1 1 1 P8 Hisani Horne, PhD, MPH , Young Lee , Todd Creasy , Rebecca Fish , 1 1 2 1 Monoclonal antibody detection from formalin-fixed paraffin- Jonathan Cairns , Paul Scorer , Janine Feng , Marietta Scott, PhD , Mark 1 1 1 embedded tumor tissues using Fab-selective proteolysis nSMOL Gustavson , Aleksandra Dudek-Madej , Craig Barker , Nicholas 1 1 2 2 coupled with liquid chromatography and triple quadrupole mass Holoweckyi , Rebecca Halpin , Peiyi Wang , Quinea Lassiter , Xiaoling 2 2 1 1 spectrometry Xia , Mohammed Abdelwahab , Weimin Li , Alejandra Negro , Jill 1 1 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Noriko Iwamoto, PhD , Walker 2 2 2 1 2 Yoshinobu Koguchi, MD, PhD , John Cha , Brian Piening, PhD , Eric Tran, AstraZeneca, Gaithersburg, MD, United States; Roche Tissue 2 2 2 2 PhD , Hong-Ming Hu, PhD , Bernard Fox, PhD , William Redmond, PhD Diagnostics, Oro Valley, United States 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Correspondence: Hisani Horne (hisani.madison@astrazeneca.com) Cancer Center, Portland, OR, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P9 Correspondence: Takashi Shimada (tashimada@shimadzu.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P8 Background Programmed cell death ligand-1 (PD-L1) expression and tumour Background mutation burden (TMB) have been shown to be predictive of re- With the development of immune checkpoint inhibitors, the focus of sponse to anti-PD-1/PD-L1 immunotherapies in various cancers. cancer therapy is shifting to immunotherapy. Our purpose is to de- The prevalence and distribution of PD-L1 expression and/or velop the drug efficacy index by rapid analysis of antibodies accumu- tumour mutations in HCC and correlation with clinical characteris- lating in cancer tissue using liquid chromatography and mass tics are poorly understood. A better understanding of these bio- spectrometry (LC-MS/MS) and by characterization of the antibody markers may help inform appropriate patient selection strategies distribution in the tumor microenvironment. Using a novel proteoly- in HCC. sis method in which antibody molecules are collected on a 100 nm Methods resin pore and trypsin is immobilized on a 200 nm nanoparticle sur- PD-L1 expression was evaluated on tumour cells (TC), immune cells face, we have developed a method for physicochemically limiting (IC), or combined TC and IC using the VENTANA PD-L1 (SP263) Assay trypsin access to antibody and identifying the structural specificity of in three independent HCC sample sets: 2 from commercial tissue complementarity-determining regions while minimizing extra pep- banks (n = 500 and n = 2417) and 1 from patients enrolled in tides and protease without depending on the type of antibody. NCT02519348, encompassing a wide range of stage and grade of dis- Using this method to detect antibodies from formalin-fixed paraffin- ease. NCT02519348 is a phase 2 study evaluating safety, efficacy out- embedded (FFPE) tumor tissues, we aim to develop novel diagnostics comes of durvalumab with or without tremelimumab in advanced that can aid in therapeutic dosing and predicting responses to HCC. TMB was assessed in tissue by whole exome sequencing in a antibody-based therapies. subset of 70 patients from NCT02519348. Methods Results To demonstrate the feasibility of these approaches, the human At a cut-off date of Feb 28, 2019, 282/335 (84.2%) patients en- breast and epidermoid carcinoma cell lines SKBR3 and A431 were in- rolled in NCT02519348 were successfully evaluated for PD-L1 cubated with either trastuzumab and cetuximab, which bind to (Table 1). Significant expression was seen in ICs relative to TCs. erbB2 and EGFR, respectively. FFPE cell blocks were then prepared Patients in NCT02519348 showed higher PD-L1 expression in TCs and proteins were extracted from 8 μm sections after deparaffiniza- than commercial cohorts. In a univariate analysis using 1% cut tion and decrosslinking. The extracted proteins were subjected to offs, higher TC (but not IC or combined TC and IC) PD-L1 expres- the Fab-selective proteolysis nSMOL, and the signature peptides of sion was associated with patients with HCV infection (p=0.003). each antibody, IYPTNGYTR for trastuzumab and SQVFFK for cetuxi- Of 70 study patients tested, 55 were evaluable for TMB (median mab, were detected via triple-quadrupole LC-MS/MS. SCID mice were 2.59, range 0.46 - 5.61 Mut/Mb). Among patients with available subcutaneously implanted with BT474 cells and 5 days later were in- TMB data there was no observed correlation between TMB and fused with 10 mg/kg or 20 mg/kg trastuzumab. 24 h after administra- PD-L1 expression. tion, tumor and other tissues were harvested and FFPE block were Conclusions prepared for trastuzumab quantitation in FFPE tissues. PD-L1 expression was observed in both TC and ICs in HCC, with the Results latter being more prevalent. Viral status and disease stage may im- As a result of the pretreatment protocol using the cell block, the con- pact PD-L1 expression in this setting, but further work is needed to ditions of deparaffinization, decrosslinking, and protein extraction confirm this. TMB and PD-L1 appear to identify distinct patient sub- were optimized. Mass spectra of the signature peptides from trastu- sets in HCC. zumab and cetuximab could be detected using 20,000 cells. This Trial Registration condition was also applied to xenograft tissue and the degree of NCT02519348 trastuzumab accumulation was detected in FFPE tumor tissue in a Ethics Approval dose-dependent manner. The study (NCT02519348) is performed in accordance with eth- Conclusions ical principles that have their origin in the Declaration of We show that these approaches can be utilized to quantify antibody Helsinki and are consistent with ICH/GCP, and applicable regula- concentrations in typically-challenging FFPE specimens with good tory requirements. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 6 of 272 Table 1 (abstract P9). See text for description different subsets, compared to DGM, potentially allowing more as- says to be executed from the same blood sample. Overall, this tech- nology has the potential to transform cell separation by automating a variable and labor-intensive processes, and therefore has utility in applications that require consistent cell quality and functionality. Table 1 (abstract P10). See text for description P10 Table 2 (abstract P10). See text for description Inertial microfluidics enables highly consistent separation and concentration of leukocytes from human peripheral blood for downstream B-cell and T-cell functional assays Sarah Mickool, Eric Smith, Aleksander Jonca, Gustavo Arnal, Mary Vincent Larcom, Melanie Scully, Peng Megn Kou, PhD, Nitin Kulkarni, Kyle Smith MicroMedicine, Inc., Waltham, MA, United States Correspondence: Kyle Smith (kyle@micromedicine.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P10 Background Cell separation plays a vital role in research and clinical settings for the development and monitoring of cutting-edge therapies. Despite its labor-intensiveness and variability, density gradient P11 centrifugation-based method (DGM) has remained the primary Early detection of breast cancer (BCa) through MDSC and method of upstream cell isolation for decades due to a lack of viable lymphocyte immunophenotyping: from manual gating to pattern alternatives. This is problematic as DGM is a non-scalable, manual recognition neural networks 1 1 1 process. To address this lack of innovation, we have developed an George Dominguez, PhD , John Roop , Alexander Polo, BS , Anthony 1 2 1 automated Microfluidic System based on inertial focusing that en- Campisi, BS , Dmitry Gabrilovich, MD/PhD , Amit Kumar, PhD 1 2 ables label-free white blood cell (WBC) separation and concentration Anixa Biosciences, San Jose, CA, United States; The Wistar Institute, from 3-75mL of whole blood in short timescale with high Philadelphia, PA, United States consistency, providing reliable sample preparation for downstream Correspondence: George Dominguez (george@anixa.com) functional assays. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P11 Methods WBCs were isolated from 15% ACD-A anticoagulated peripheral hu- Background man blood using the Microfluidic System or DGM. Cell number, via- Myeloid-derived suppressor cells (MDSCs) are contributors in sup- bility, and immune phenotype were evaluated by hematology porting tumor progression and escape [1,2]. Studies have quantified analyzer and flow cytometry. To assess B-cell function, cells were MDSCs to detect tumor development, monitor progression, and/or cryopreserved post separation, thawed, and stimulated with IL-2 and predict therapeutic responses [3, 4]. Here, we compared several ma- R848, followed by Human IgG and IgM ELISPOT. To assess T-cell func- chine learning (ML) approaches to analyze flow cytometry data to tion, thawed cells underwent bead-based granulocyte depletion and detect breast cancer (stage I/II) through manual gating and hyper- stimulation with CEF peptide pool, followed by Human IFNγ ELISPOT. voxelation of cell events. Results Methods The prototype Microfluidic System consistently processed 40mL of We used standard multiparametric flow cytometry techniques to anticoagulated blood in approximately 20 minutes with minimal measure myeloid-derived suppressor cell (MDSC), myeloid, and hands-on time as opposed to 60–90 minutes for DGM with signifi- lymphocyte cell populations found in the peripheral blood of 99 cant hands-on time. While DGM collects only peripheral mononuclear biopsy-confirmed early stage BCa patients and 88 healthy donor fe- cells (PBMCs), the System isolates the total WBC population and may male (HDF) controls. Manual gating was performed to generate be beneficial for immunophenotyping. As shown in Table 1, the gated values, and raw flow cytometry data were transformed using Microfluidic System consistently provided improved WBC or PBMC HyperVOX to generate hypervoxelated cytometry event counts. The recovery, viability, purity, RBC depletion, and platelet depletion as ML algorithms used were: support vector machine (SVM), Bayes SVM, compared to DGM. Immune phenotyping shows that the Microfluidic Ensemble SVM, k-nearest neighbor (kNN), and pattern recognition System also consistently resulted in improved recovery of lympho- neural network (PRNN). All algorithms were trained using data from cyte subsets, including CD19+, CD3+, CD4+, and CD8+ cells (Table 2). 64 BCa patients and 69 HDF controls. Predictions were evaluated B-cell and T-cell functionality were found to be equivalent between using the performance of each trained ML algorithm on 35 early the two cell isolation methods based on IgM/IgG and IFNγ secretion, stage BCa patients and 19 HDF that were not used for training (hold- respectively. With the improved cell recovery using the Microfluidic out test set). System, more target cells from the same blood sample may be col- Results lected for downstream assays. Using manually gated counts, the resulting accuracies were: SVM = Conclusions 75.4%, Bayes SVM = 71.3%, Ensemble SVM = 65.6%, and kNN = The Microfluidic System offers a faster, more reliable method than 69.7%. Using hypervoxelated event counts, the resulting accuracies DGM for upstream cell separation from whole blood. The System were: SVM = 78.7%, Bayes SVM = 77.1%, Ensemble SVM = 57.4%, consistently recovers more cells, including functional lymphocytes of kNN = 67.2%, and PRNN = 92.6%. Hypervoxelated data analyzed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 7 of 272 using PRNN resulted in the highest accuracy with a sensitivity of regulated; q-value > 0.05 and log2 fold change > 0.58). In addition to the 91.4% and a specificity of 94.7%; the resulting AUC = 0.9098 (95%CI acute phase proteins (e.g. CRP and SAA1) which were previously verified = 0.8031 to 1.000). Additionally, we tested 26 samples collected from to be elevated in subjects with NSCLC, partial least squares discriminant patients with confirmed ductal carcinoma in situ (DCIS) using hyper- analysis helped identify additional proteins that are differentially voxelated counts with a PRNN. Even though they are clinically expressed between the sample groups. Most relevant to immune func- deemed as pre-cancerous (stage 0), 18 out of 26 (AUC = 0.8421; tion was CLC (Galectin-10), which was elevated in NSCLC samples and 95%CI = 0.7163 to 0.9679) were classified as BCa suggesting utility has been identified as key component supporting the suppressive func- for detecting the existence of even a non-invasive cancerous lesion. tion of Tregs.[1] Furthermore, F13A1 was suppressed in the NSCLC sam- Conclusions ples which is known to be associated with macrophage activation. Although further study is needed, we believe that using PRNN with Conclusions MDSC immunophenotyping, in conjunction with other known clinical 162 proteins were identified as candidate biomarkers and reflect the risk factors, would allow for clinicians to make a more informed diag- host immune response via acute phase response signaling, innate im- nosis and treatment recommendation when screening and for mune response, and other proinflammatory stimuli. Several of these recommending subsequent interventions for early stage breast markers have been linked to patient outcomes and poor prognosis. cancer. Reference References 1. Kubach, J., et. al.; Blood 2007 110:1550-1558 1. Kumar V, Patel S, Tcyganov E, Gabrilovich D. The nature of myeloid- derived suppressor cells in the tumor microenvironment. Trends Immu- P13 nol. 2016; 37:208-220. Immunomodulatory effects of Interleukin 2 in the circulation of 2. Marvel D, Gabrilovich D. Myeloid-derived suppressor cells in the tumor melanoma patients and the added impact of VEGF inhibition with microenvironment: expect the unexpected. J Clin Invest. 2015; 125:3356- Ziv-aflibercept 1 2 3 Arjun Khunger, MD , Ghanashyam Sarikonda , Paul Frankel, PhD , Jenn 3. Elliott L, Doherty G, Sheahan K, Ryan E. Human tumor-infiltrating myeloid 2 2 2 2 Tsau, PhD , Zeni Alfonso, PhD , Jane Gao, MS , Anil Pahuja, BSc , cells: phenotypic and functional diversity. Front Immunol. 2017; 8:86. 2 2 2 Christine Vaupel, PhD , Naveen Dakappagari , Shabnam Tangri, PhD , 4. Okla K, Wertel I, Wawruszak A, Bobinski M, Kotarski J. Blood-based ana- Ahmad Tarhini, MD, PhD lyses of cancer: circulating myeloid-derived suppressor cells – is a new 1 2 Memorial Hospital West, Pembroke Pines, FL, United States; Navigate era coming? Crit Rev Clin Lab Sci. 2018. BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA, United Ethics Approval 3 4 States; City of Hope, Duarte, CA, United States; Emory University and The study was approved by the Virtua Oncology (#20161), University of Winship Comprehensive Cancer center, Atlanta, GA, United States Pennsylvania (#826544), and Cooper Health (#17-174) IRBs. Correspondence: Ahmad Tarhini (tarhiniaa@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P13 P12 Deep characterization of the depleted plasma proteome in Background subjects with NSCLC using data independent acquisition mass Interleukin 2 (IL-2) plays a key role in antitumor immunity by en- spectrometry reveal host immune response mechanisms hancing survival of antitumor cytotoxic T lymphocytes and nat- Nicholas Dupuis, PhD, Linda Sensbach, Sebastian Müller, Lukas Reiter ural killer (NK) cells and promoting proinflammatory cytokines, Biognosys AG, Schlieren, Switzerland that can lead to durable responses in patients with melanoma. Correspondence: Nicholas Dupuis (nicholas.dupuis@biognosys.com) High levels of vascular endothelial growth factor (VEGF) are asso- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P12 ciated with non-response to IL-2 and combination biotherapy with Ziv-aflibercept (inhibitor of the VEGF pathway) and high- Background dose IL-2 may lead to improved antitumor efficacy. Mechanistic Measurement of circulating biomarkers in cancer has proven utility studies utilizing peripheral blood of melanoma patients treated for early detection, differential diagnosis, and predicting pre- with this biotherapy may illuminate the underlying mechanisms treatment response to therapy. More recently, circulating proteomic of immune susceptibility and resistance [1]. biomarkers for pre-treatment prediction of therapeutic response Methods have received additional attention due to the heterogeneous re- Patients with stage III or stage IV inoperable melanoma were sponses to immunotherapies. To develop a greater understanding of treated with high-dose IL-2 alone or in combination with Ziv- the circulating plasma proteome in subjects with cancer we have op- aflibercept in a phase 2 clinical trial [1] (NCI8628; Tarhini et al. timized a depleted plasma proteomic workflow, based on label-free Cancer. 2018). Peripheral blood mononuclear cells (PBMC) from data independent acquisition (DIA) mass spectrometry, and applied it treated patients (N=89) on this trial were tested at baseline (be- to plasma from subjects with late stage NSCLC. This approach pro- fore initiating systemic immunotherapy), and 6-weeks (following vides a deep and unbiased description of the plasma proteome and immunotherapy initiation). High complexity (14-color) flow cytom- the dysregulated biological pathways associated with lung cancer. etry designed to detect key immunological biomarkers such as Methods myeloid-derived suppressor cells (MDSCs), regulatory T cells Plasma samples from subjects with Stage III-IV non-small cell lung (Tregs), proliferating T-cells, PD-1 and TIM3 expression on T-cells, cancer (NSCLC, n = 15) and age matched healthy donors (n = 15) and differentiation of T-cells into Th1, Th2 or Th17 phenotype were depleted of 14 high abundance proteins using MARS Hu-14 were used to evaluate the correlation between immunological spin columns (Agilent). All samples were prepared for mass spectro- biomarker expression and efficacy. Statistical significance was de- metric acquisition using two-hour gradients on a C18 column termined using ANOVA or paired student’st-test. coupled online to a Thermo Scientific Q Exactive HF-X operated in Results DIA mode. Targeted data extraction was performed using Spectro- Treatment with high dose IL-2 resulted in significant immune activa- naut (Biognosys) with a hybrid library approach. Statistical analysis tion as detected by significant increases in both proliferating CD4+ was conducted to identify disease associated biomarker candidates (p<0.0001) and CD8+ (p<0.0001) T-cells at 6-weeks post-treatment in and pathway analysis highlights dysregulated biological functions. both treatment arms in addition to increase in Tregs (CD4+ CD25+ Results Foxp3+ T-cells; p<0.0001). Addition of VEGF inhibition showed a gen- A comprehensive protein spectral library was created containing 1,827 eral trend towards decrease in classical monocytes (CD14+ CD16-; p= unique proteins. In DIA acquisition, in total 1,304 proteins were quantified 0.0769) as well as Th17 cells (defined as CD45RA- CCR6+ CXCR3- across all samples (1,105 average per sample). Univariate statistical testing CCR4+; p=0.0597). In patients receiving combination therapy, a identified 162 dysregulated proteins (125 up-regulated and 37 down- higher proportion of subjects experienced CBR (Clinically Beneficial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 8 of 272 Response = CR+PR+SD) compared to monotherapy and this CBR cor- P15 related with a decrease in CD4+ ICOS+ (p=0.0219), classical mono- Microsatellite instability detection with cell-free DNA next- cytes (CD14+ CD16-; p=0.0141), Th17 cells (CD45RA- CCR6+ CXCR3- generation sequencing CCR4+; p=0.0445) as well activated CD4+ T-cells (CD4+ CD38+ HLA- Ariane Lozac’hmeur, MS, Jason Perera, PhD, Denise Lau, PhD, Aly Khan, DR+; p=0.0285). PhD, Ariane Lozac’hmeur, MS Conclusions Tempus Labs, Chicago, IL, United States VEGF inhibition with Ziv-aflibercept adds significant immunomod- Correspondence: Ariane Lozac’hmeur ulatory effects when combined with IL-2. Further correlative ana- (ariane.lozachmeur@tempus.com) lyses determining the effect of combination therapy on Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P15 progression-free survival and identifying predictive biomarkers of therapeutic efficacy are ongoing and will be presented at the Background meeting. Microsatellite instability is a clinically actionable genomic indication for cancer immunotherapy. In microsatellite instability-high (MSI-H) tumors, Acknowledgements defects in DNA mismatch repair (MMR) can cause a hypermutated This United States (U.S.) National Cancer Institute (NCI)-sponsored study was phenotype where alterations accumulate in the repetitive microsatellite initiated by the California Cancer Consortium under N01 contract NO1-CM- regions of DNA. MSI detection is typically performed by subjecting 2011-00038. Laboratory correlatives were supported by Navigate BioPharma. tumor tissue (“solid biopsy”) to clinical next-generation sequencing or Trial Registration specific assays, such as MMR IHC or MSI PCR. Circulating cell-free tumor https://clinicaltrials.gov/ct2/show/NCT01258855 DNA (cfDNA) testing (“liquid biopsy”) is rapidly emerging as a less inva- sive method for cancer detection and monitoring disease progression. Reference Here, we explore the possibility of detecting MSI in cfDNA and develop 1. Tarhini AA, Frankel P, Ruel C, Ernstoff MS, Kuzel TM, Logan TF, et al. NCI a novel cfDNA MSI detection assay with high specificity. 8628: A randomized phase 2 study of ziv‐aflibercept and high‐dose Methods interleukin 2 or high‐dose interleukin 2 alone for inoperable stage III or The Tempus cfDNA targeted panel contains 39 highly informative IV melanoma. Cancer. 2018;124(22):4332-41. microsatellite loci previously used by the clinically validated Tempus Ethics Approval xT 595-gene panel. For each microsatellite locus, we identified all se- The study was initiated after approval by the ethics committee at the quencing reads that mapped to the corresponding microsatellite re- participating sites and was conducted in accordance with the gion and quantified the number of repeat units contained within the Declaration of Helsinki. sequencing read. Next, three distinct summary statistics were calcu- lated to characterize the distribution of the number of repeat units for each locus. Finally, using 54 labeled patient samples (17 MSI-H, P14 37 microsatellite stable) sequenced with the Tempus cfDNA panel, a Validation of dendritic cell and natural killer cell signatures for k-Nearest Neighbor (k-NN) classifier was trained to classify each locus clinical biomarker development for a new sample. Patient samples with more than 50% unstable loci Bolan Linghu, PHD, Pei Zhang, PhD, Marylens Hernandez, Mingchao Xie, were classified as MSI-H. PhD, Christine Barbon, Srimathi Srinivasan, Deanna Russell, MS, Anna Results Coenen-Stass, Deanna Mele, PhD, Patricia McCoon, PhD, Jonathan Dry, We validated the ability of our model to detect MSI on a new inde- Ben Sidders, Kris Sachsenmeier, PhD pendent validation dataset. MSI-H status was detected in 6 patient AstraZeneca, Waltham, MA, United States samples. In 3 of these patients (2 colorectal, 1 skin cancer), abnormal Correspondence: Ben Sidders (benjamin.sidders@astrazeneca.com); Kris MMR IHC confirmed the detected MSI-H status. In the other 3 pa- Sachsenmeier (kris.sachsenmeier@astrazeneca.com) tients (1 colorectal, 1 non-small cell lung cancer, and 1 endometrial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P14 cancer), MSI-H status was confirmed by our clinically validated solid tumor MSI assay. Furthermore, the reliability of the model was vali- Background dated in 10 technical replicates from 2 MSI-H patients in our training Quantification of immune cell abundance using gene signatures from dataset. The results were 100% concordant with all 10 replicates clas- mRNA profiling has the potential to inform clinical studies of cancer sified as MSI-H. immunotherapy. However, few of the signatures reported in previous Conclusions studies have been validated therefore the concordance of signature These results demonstrate the ability of our assay to detect MSI in scores with corresponding immune cell abundance is unknown. cfDNA with high specificity, providing a transformative opportunity Methods to report a clinically actionable insight alongside other somatic To tackle this challenge we designed a two-stage validation strategy. changes detected from cfDNA. Firstly we validate signatures computationally using previously pub- lished datasets. Secondly we generate expression profiling data from an immune cell spike-in experiment with human PBMCs. As a proof P16 of concept experiment, we implemented the method to validate two Circulating immunological biomarkers for predicting response to gene signatures for CD141+ dendritic cells (DC) and CD56+ natural neoadjuvant chemotherapy in TNBC patients killer (NK) cells. Charlotte Milton, PhD, Thanussuyah Alaguthurai, Atousa khiabany, Mres, Results Sheeba Irshad, MD PhD We demonstrate gene signatures for both CD56+ NK and CD141+ Kings College London, London, United Kingdom DC cell types show high and significant agreement to the corre- Correspondence: Sheeba Irshad (sheeba.irshad@kcl.ac.uk) sponding immune cell abundance. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P16 Conclusions This work establishes a starting point for validating gene signatures Background through an approach that is tractable yet recapitulates real-world Triple negative breast cancer (TNBC) accounts for 10-20% of breast variability we might expect in clinical use. cancer and is associated with particularly poor prognosis. Patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 9 of 272 are commonly treated with neoadjuvant chemotherapy (NAC) and decisions. In this work, we perform whole plasma and plasma- response to treatment is a strong predictor of overall survival. Re- derived exosome proteomic profiling to construct a predictive model cently, the ability of chemotherapeutics to stimulate an anti-tumour of immunotherapy response and toxicity, and to glean further bio- immune response has been appreciated as an important mechanism logic insight into the mechanisms underlying resistance to ICB. of action; possibly contributing to the elimination of distant micro- Methods metastatic disease by resetting of the attenuated functional immun- Whole plasma was analyzed in a cohort of 55metastatic melanoma ity. In TNBCs, higher levels of tumour-infiltrating lymphocytes correl- patients receiving anti-PD1 antibodies (MGH IRB #11-181) at baseline, ate with response to NAC and high intra-tumoral levels of immune- and on-treatment at 6 week and 6 month time-points. Exosomes related genes, including those associated with type I interferon re- were analyzed in 15 of these patients for all time-points. Proteomic sponses, and the presence of CD8+ cytotoxic T lymphocytes, correl- analysis was performed using an innovative multiplex proximity ex- ate with improved disease outcome. tension assay that enabled detection of more than 1000 proteins Methods simultaneously. A linear mixed model with maximum likelihood esti- The underlying hypothesis of this study is that phenotypic profiling mation for model parameters was used to analyze differences be- of peripheral blood cells have the potential to inform clinical deci- tween patient groups, and significant differences were determined sions and help predict therapeutic response, with lower costs and after Benjamini and Hochberg multiple hypothesis correction. higher compliance than serial tumour biopsies, due to their minimal Results invasiveness. Whilst significant research efforts have been made to Between plasma baseline and on-treatment time-points, 67 differen- assess circulating markers such as circulating tumour cells and circu- tially expressed proteins were identified including markers of inflam- lating tumour DNA as potential biomarkers; understanding the evolv- mation such as PD1, CXCL9, CXCL10, CXCL11, IL10, CCL3 and TNFR2. ing peripheral “immunological status” of TNBC patients on NAC is Exosome samples had a distinct protein signature over the treatment warranted. period compared to plasma, including differential expression of We therefore set out to analyse serial blood samples from TNBC pa- CXCL16, CCL18, CCL20, and IL6, among others. 41 proteins were dif- tients receiving NAC to monitor the changes in the peripheral im- ferentially expressed in plasma between ICB responders and non- mune response through deep analysis of functional and phenotypic responders including several inflammatory proteins such as CD28, immune markers. We investigated (1) whether chemotherapy affects TNFb, MCSFRa and IL8, and others implicated in melanoma resist- the immune phenotype; and (2) whether a defined peripheral blood ance, such as MIA and ERBB2. Similarly, exosome revealed a distinct immune phenotypic profile relates to treatment response. protein signature between responders and non-responders com- Results pared to plasma consisting of CXCL9, CXCL13, CXCL16, CCL19, CD8a, Here we present preliminary results from 10 TNBC patients receiving GZMA and CD5 expression. Whereas plasma proteins reflected a NAC. Analysis of 39 PBMC populations using mass cytometry by myeloid signature, exosome proteins reflected a lymphoid signature, time-of-flight (CyTOF), highlighted phenotypic changes in B cell pop- suggesting that the two compartments may capture elements of dif- ulations in response to treatment, in particular a dramatic increase in ferent immune processes. Integrating data from both plasma and circulating regulatory B cells (CD19+CD24+CD38+) post- exosome proteomics, we applied machine learning tools to build a chemotherapy (5.4% and 46.2% of B cells pre- and post- predictor of response. Further analysis to look for predictors of tox- chemotherapy, respectively, p=0.0004). We also detected an increase icity is currently underway. in expression of exhaustion markers (CD38+CD39+) on CD8+ T cells Conclusions which was associated with poor response to chemotherapy (0.8 and Overall, our work suggests that plasma and exosome protein signa- 2.7 fold increase from baseline in exhausted CD8+ T cells in patients tures are distinct and may reflect unique immunological processes. with pathological complete response and residual disease, respect- Proteomic analysis of these compartments may be an effective way ively, p=0.008). for non-invasive liquid biopsy to predict ICB response. Conclusions We now plan to integrate these data with Luminex profiling of 36 P18 serum cytokines, mass spectrometry analysis of circulating exosomes Liquid biopsy protein biomarkers to predict responses and and clinicopathological and standard of care blood monitoring. elucidate resistance to cancer immunotherapy Taken together, this study aims to provide a comprehensive analysis 1 2 3 Arnav Mehta, MD PhD , Marijana Rucevic, PhD , Gyulnara Kasumova , of the utility of immune monitoring to understand TNBC patient re- 2 2 3 Emmett Sprecher , Lina Hultin Rosenberg , Dennie Frederick , Ryan sponse to NAC. 3 3 3 3 Sullivan, MD , Nir Hacohen , Keith Flaherty , Genevieve Boland Ethics Approval 1 2 MGH and Broad Institute, Boston, MA, United States; Olink Proteomics, The study was approved by NRES Committee London - Chelsea, ap- Watertown, MA, United States; MGH, Hanover, MA, United States proval number 13/LO/1248. Correspondence: Marijana Rucevic (m.rucevic@olink.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P18 P17 Role of plasma-derived exosome in monitoring immunotherapy Background response and toxicity The response of metastatic melanoma to anti-PD1 is heterogeneous. 1 2 3 Arnav Mehta, MD PhD , Gyulnara Kasumova , Alvin Shi , Lina Hultin We performed proteomic profiling of patient plasma samples to 4 4 2 2 Rosenberg , Emmett Sprecher , Dennie Frederick , Ryan Sullivan, MD , build a predictor of immunotherapy response and uncover biological 2 1 2 Keith Flaherty , Nir Hacohen , Genevieve Boland , Marijana Rucevic, insights underlying primary resistance. PhD Methods 1 2 MGH and Broad Institute, Boston, MA, United States; MGH, Hanover, An initial cohort comprised 55 metastatic melanoma patients re- 3 4 MA, United States; MIT, Cambridge, MA, United States; Olink ceiving anti-PD1 (Pembrolizumab or Nivolumab) at Massachu- Proteomics, Uppsala, MA, United States setts General Hospital (MGH), and 116 additional patients Correspondence: Arnav Mehta (nawi214@gmail.com) comprised a validation cohort. Plasma samples were collected Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P17 baseline and on-treatment, at 6 weeks and 6 months’ time- points, and profiled for 1000 proteins by a multiplex Proximity Background Extension Assay (PEA, by Olink Proteomics). A subset of patients Immune checkpoint blockade (ICB) has revolutionized the treatment had single-cell RNA-seq (Smart-Seq2 protocol) performed on of many solid tumors, including metastatic melanoma. Despite recent tumor tissue. Group differences and treatment effects were eval- successes, many patients fail to respond or are overcome by severe uated using linear mixed models with maximum likelihood esti- toxicities that limit further treatment. To date, there are no non- mation for model parameters, and Benjamini and Hochberg invasive predictors of response and toxicity that can guide treatment multiple hypothesis correction. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 10 of 272 Results immunohistochemical staining of Sema4D of the tumor associated At the baseline, 6 differentially expressed proteins were identified inflammatory cells (TAIs). between responders (R) and non-responders (NR) whereas im- Results mune suppression marker ST2 and IL-6 were found significantly sSema4D levels in plasma of HNSCC and the autoimmune individuals higher among NR. Kaplan-Meier survival curves stratified by the (p=0.18, independent-samples Mann-Whitney test), were not statisti- baseline differentially expressed proteins were highly predictive cally significantly different, but sSema4D levels were significantly of overall survival (OS) and progression-free survival (PFS). At 6- higher in the HNSCC and the autoimmune groups compared to weeks on-treatment time point, 80 proteins were found differen- healthy donors (p<0.001 for both comparisons). Three histological tially expressed between R and NR including several proteins im- patterns of tumor inflammation were defined according to the extent plicated in primary or acquired resistance (IL8, MIA, TNFR1 of stromal inflammation and TAIs infiltrate into the tumor islands. among others). Several 6-weeks differentially expressed proteins First; the inflamed type (TAIs infiltrated the tumor cells), second the were highly predictive of survival (ICOSL, IL8, MIA). Furthermore, TAIs excluded type (inflamed stroma but TAIs did not infiltrate the 160 significantly differentially expressed (DE) proteins were identi- tumor islands and/or were excluded by a thin peri-tumoral fibromyx- fied across the treatment period majority of which are reflective oid zone) and third as deserted ( minimal to no TAIs in the peri- of immune activation under the pressure of the immunotherapy. tumroal stroma or the tumor islands). The paired tumor tissue and Analysis of single-cell RNA-seq data of tumor tissue from a subset blood samples collected at the same time point, showed that high of these patients revealed that gene expression of most proteins levels of sSema4D in plasma, correlated directly with TAIs excluded predictive of response were enriched among tumor myeloid cells, histological pattern of tumor inflammation (p= 0.04). with the remainder of proteins being reflective of exhausted T Conclusions cell states. Our data presents a novel role of Sema4D as a soluble immune bio- Conclusions marker that can read in real time the histological pattern of tumor in- These results unveil a putative role of myeloid cells within the flammation. This opens new avenues for personalized immunotherapy tumor microenvironment in anti-PD1 response or primary resist- and HNSCC patient stratification. ance. Whole plasma proteomic profiling of anti-PD1 treated pa- tients revealed DE proteins between R and NR that may enable a References liquid biopsy to predict anti-PD1 response. Importantly, we dem- 1. Derakhshandeh R, Sanadhya S, Lee Han K, Chen H, Goloubeva O, Webb onstrate the relationship of serum biomarkers to OS and PFS and TJ, Younis RH. Semaphorin 4D in human head and neck cancer tissue are currently attempting to build machine learning classifiers as and peripheral blood: A dense fibrotic peri-tumoral stromal phenotype. predictors of response to checkpoint therapy leveraging early and Oncotarget. 2018; 9:11126-11144. late on-treatment time points. 2. Younis RH, Han KL, Webb TJ. Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid- Derived Suppressor Cells. J Immunol. 2016; 196:1419-29. P20 3. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith E, Zauderer M, Evans Semaphorin 4D in peripheral blood of head and neck squamous EE, Allen CT. Semaphorin4D Inhibition Improves Response to Immune- cell carcinoma reads the histological pattern of tumor Checkpoint Blockade via Attenuation of MDSC Recruitment and Function. inflammation in real time Cancer Immunol Res. 2019; 7:282-291. Ioana Ghita, Manar Elnaggar, Risa Chaisuparat, John Papadimitriou, 4. Ayers M, et al. IFN-gamma-related mRNA profile predicts clinical response Joshua Lubek, Rania Younis, BDS, MDS, PhD, Soren Bentzen, PhD to PD-1 blockade. J Clin Invest. 2017; 127:2930–40 University of Maryland, Baltimore, MD, United States Ethics Approval Correspondence: Rania Younis (ryounis@umaryland.edu) The study was approved by University of Maryland institutional review board, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P20 Institutution‘s Ethics Board, approval number (HCR-HP-00073603) Background There is an urgent need for immune biomarkers that can monitor P21 the status of inflammation of cancer patients. Soluble biomarkers Activation Profiling of tumor infiltrating CD8+ T cells reveals CTLA- represent a convenient prognostic and diagnostic method. Sema- 4 mean fluorescence intensity correlates with response in phorin 4D (Sema4D) is a glycoprotein that can function as a trans- treatment naïve melanoma membrane protein or a cleaved soluble form (sSema4D), that we Lauren Levine, MD, Katy Tsai, MD, James Lee, MD, Clinton Wu, BS, Kelly previously detected in peripheral blood [1]. The role of Sema4D as Mahuron, MD, Alain Algazi, MD, Michael Rosenblum, MD PhD, Adil an inflammatory mediator in several pathological aspects and its role Daud, MD in tumor immune suppression [2,3], highlights its significance as a University of California, San Francisco, San Francisco, CA, United States molecule to be further investigated for translational potential. The Correspondence: Adil Daud (daudai@gmail.com) objective of this work was to investigate the level of sSema4D in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P21 plasma in relation to the histological pattern of tumor inflammation of head and neck squamous cell carcinoma (HNSCC) patients in real Background time. Background: Activation markers such as PD-1 and PDL-1 as well as Methods tumor mutation burden and IFN-gamma gene expression profiling Under University of Maryland institutional review board approval and have been explored as markers for response in melanoma and in upon patient consent, we obtained paired peripheral blood and other cancers. PD-1 inhibition activates checkpoint positive cytotoxic tumor tissue of thirty-nine HNSCC patients, collected at the same T lymphocytes (cpCTLs) inducing tumor regression. We have previ- time point to allow for real time correlative analysis. Thirty eight pa- ously demonstrated that baseline peCTL frequency predicts response tients of classic autoimmune conditions, thirteen allergy patients, to anti–PD-1 monotherapy and combination CTLA4/PD-1 blockade in seven osteoarthritis patients and thirty-one healthy donors were in- metastatic melanoma. We evaluated the frequency of this CD8+ T cell cluded as controls. The level of Sema4D in plasma was detected subset at baseline and after immunotherapy treatment and evaluated using tailored direct ELISA assay. The histological pattern of tumor in- the utility of the intensity of expression activation marker expression flammation [4] was analyzed by three pathologists using the as a surrogate for tumor response as assessed by flow cytometry. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 11 of 272 Methods proportional hazard modeling to associate gene expression and We identified 490 patients with melanoma biopsied pre and post signature scores with the clinical annotations. PD-1 therapy and available for analysis. Of these 148 patients Results had unresectable stage III or stage IV melanoma and were treat- In the PanCancer IO 360 analysis, genes and signatures are compared ment naïve and started PD-1 therapy following biopsy. An add- to clinical annotations through heat maps, volcano plots, forest plots, itional 61 patients were identified with PD-1 resistant melanoma. box plots, waterfall plots, swim lane plots, Kaplan Myer plots, scatter Approximately 2 × 106 cells were stained with anti-hCD3, anti- plots, and the IO 360 wheel plot. The report is delivered in an HTML hCD8, anti-hCD45, anti-CD4 , anti-Foxp3, anti–hCTLA-4 (14D3), format that provides interactive visualizations, quality control, and anti–PD-1 , anti–HLA-DR, anti–PD-L1, and LIVE/DEAD Fix- able downloadable results. Data are analyzed individually and as part of Aqua Dead Cell Stain (Life Technologies). Data were acquired by larger treatment groups. an LSRFortessa (BD Biosciences) and analyzed using FlowJo soft- Conclusions ware (Tree Star, Inc.). Objective Responses were evaluated by The PanCancer IO 360 assay is a tool for characterizing transcriptional RECIST 1.1, CR/PR were classified as “responders” and SD/PD as patterns associated with tumor-immune interactions that can be ap- “non-responders.” plied across a wide range of cancer types. Gene signatures enable ro- Results bust characterization of immune activity from small sample cohorts, : cpCTL percentage correlated with response. The mean cpCTL was and the report simplifies the interpretation of results. This combin- 27.1% for treatment naïve responders (R), 16.52% for treatment naïve ation enables researchers to have insight into clinically relevant biol- non-responders (NR) and 8.59% for PD-1 resistant patients post treat- ogy that will ultimately lead to help drive the immune-oncology ment (ANOVA p=0.0003 for R/NR, 801 (ANOVA p=0.0002). field. Conclusions PD-1 progressive patients are significantly depleted in cpCTL even References compared to treatment naïve non-responders, suggesting that add- 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-γ-related mRNA profile pre- itional T cell influx may be needed for effective checkpoint blockade dicts clinical response to PD-1 blockade. J Clin Invest. 2017;127(8):2930- in these patients. In treatment naïve melanoma, CD8+ activation as shown by CTLA-4 MFI has an optimal range along the activation- 2. Danaher P, Warren S, Dennis L, et al. Gene expression markers of Tumor dysfunction spectrum, and strongly correlates with response to PD-1 Infiltrating Leukocytes. J Immunother Cancer. 2017;5:18. checkpoint therapy. 3. Danaher P, Warren S, Lu R, et al. Pan-cancer adaptive immune resist- ance as defined by the Tumor Inflammation Signature (TIS): results Acknowledgements from The Cancer Genome Atlas (TCGA). J Immunother Cancer. We gratefully acknowledge the patients who participated in this study 2018;6(1):63. Ethics Approval The study was approved by UCSF's Ethics Board approval number 138510 P23 High dimensional immune monitoring of peripheral blood samples P22 from breast cancer patients using mass cytometry (CyTOF) 1 1 2 Transcriptomic characterization of immune response within Jose Villasboas, MD , Kaitlyn McGrath, MS , El-ad David Amir, PhD , 1 1 1 diverse tumor environments using the NanoString® nCounter® Roberto Leon-Ferre, MD , Matthew Goetz, MD , Judy Boughey, MD , 1 1 1 PanCancer IO 360™ assay Jody Carter, MD, PhD , Krishna Kalari, PhD , Liewei Wang, MD, PhD , 1 1 Jessica Perez, PhD, Lei Yang, David Henderson, PhD, Heather Brauer, Vera Suman, PhD , Richard Weinshilboum, MD , Stephen Ansell, MD, PhD, Sarah Warren, PhD PhD 1 2 NanoString, Seattle, WA, United States Mayo Clinic, Rochester, MN, United States; Astrolabe Diagnostics, Fort Correspondence: Sarah Warren (swarren@nanostring.com) Lee, NJ, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P22 Correspondence: Jose Villasboas (Villasboas@mayo.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P23 Background The efficacy of immune response in solid tumor settings is driven Background by many factors including the biology of the tumor, the immune CyTOF produces high dimensional single cell data allowing simultan- system, and the microenvironment. The Tumor Inflammation Sig- eous monitoring of multiple immune cell subsets. This enables nature (TIS) is an 18-gene Research Use Only (RUO) signature that characterization of the immune system in normal and disease states. measures the presence of a preexisting immune response on the We developed a standardized pipeline to study human peripheral nCounter platform and enriches for response to pembrolizumab blood mononuclear cells (PBMCs) of cancer patients. Here we detail [1]. We have incorporated TIS into the PanCancer IO 360 panel, a our process and present early findings on a cohort of 40 patients 770-gene RUO expression assay containing 48 additional signa- with early-stage triple-negative breast cancer (TNBC) treated with tures of tumor-immune biology. To accompany this panel, we neoadjuvant chemotherapy. have created analysis software that associates the gene expres- Methods sion and signature scores with annotations of the samples to Thirty commercially-available metal-tagged antibodies were opti- characterize the immune system, tumor, and stroma within the mized to identify major cell subsets using a 4-point titration scheme. tumor microenvironment to give insight into underlying biology Replicates of cryopreserved PBMCs from a pool of 4 healthy donors of response to treatment, disease progression, survival, and other were created for panel titration and used as longitudinal references. sample characteristics. We studied 40 cryopreserved PBMCs from patients with TNBC. We Methods stained samples individually using standard protocol, barcoded over- The PanCancer IO 360 assay relies on gene signatures to describe night during DNA intercalation, and pooled for acquisition. Debar- biological processes, measure the presence of 14 different im- coded output data was normalized on a per-batch basis to the mune cell populations, or report the expression of key thera- median intensity of EQbeads. We uploaded files to an automated peutic targets. Data from The Cancer Genome Atlas (TCGA) was platform for unbiased processing. Patient-level meta-data was added used for signature training and development. Signatures are ei- to experiment matrix to determine differential abundance of immune ther single genes, weighted linear sums of multiple genes with subsets across clinical and pathological groups. coregulated expression, or algorithms to determine under- Results expression of genes in a coregulated pathway [2,3]. The analysis We required 7 rounds of titration to optimize antibody concentra- software leverages differential expression analysis and Cox tions. Data was collected on over 23 million live single-cell events Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 12 of 272 (Table 1) assigned to 31 canonical populations (Figure 1A). The me- Table 1 (abstract P23). See text for description dian frequencies of main populations were: B cells (11.9%), T-CD4+ cells (34.3%), T-CD8+ cells (11.7%), NK cells (8.6%), and monocytes (11.3%). At the profiling level, 76 subsets were agnostically identified, with B, T, NK, and monocytes broken down into 10, 32, 8, and 13 subsets respectively. Activated (CD38+CD161+) CD16+NK cells (Fig- ure 1B) were more prevalent in TNBC samples (median 5.2%, range 0.5%-11.9%) compared to normal blood (median 0.76%, range 0.1%- 2.4%). A population with phenotype suggestive of myeloid derived suppressor cells (LineagenegHLA-DRLowCD66b+CD24+CD16+; Figure 1C) was also more prevalent in TNBC samples (median 1.2%, range P24 0.1%-17.3%) compared to normal blood (median 0.6%, range 0.2%- Molecularly guided digital spatial profiling for highly multiplexed 1.0%). These populations demonstrated opposite association trends analysis of gene expression with spatial and single cell resolution when patients were stratified by clinical outcomes. Activated NK cells 1 2 2 Anushka Dikshit, PhD , Chris Merritt, PhD , Jamie Rose Kuhar , Karen were more frequent in patients achieving pathological complete re- 2 2 1 1 Nyugen , Kristina Sorg , Bingqing Zhang , Courtney Anderson, PhD , sponse while MDSC-like cells were more frequent in those with re- Xiao-Jun Ma sidual disease (Figures 1D-1E). 1 2 Advanced Cell Diagnostics, Newark, CA, United States; NanoString Conclusions Technologies, Seattle, WA, United States We demonstrated the feasibility of a complete pipeline for deep phe- Correspondence: Xiao-Jun Ma (xiao-jun.ma@bio-techne.com) notyping of cryopreserved PBMCs in cancer patients. Our approach Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P24 identified rare cell subsets using an unbiased analysis tool, linking specific populations to opposite clinical outcomes. High dimensional Background immune monitoring is feasible and should be applied to study the The tumor microenvironment (TME) is a network of complex inter- immune system of cancer patients at large. actions between the tumor and surrounding immune cells. Im- munotherapies including immune checkpoint blockade have Acknowledgements demonstrated therapeutic efficacy and durable responses for sev- This work was part of the Mayo Clinic Cancer Immunome Project which is eral tumor types, however most patients are nonresponsive or de- supported by the Wohlers Family Foundation. Samples were obtained in velop resistance to such immunotherapies. To identify new collaboration with investigators from the Mayo Clinic Breast Cancer Genome predictive biomarkers to better stratify patients, it is essential to Guided Therapy (BEAUTY) study. The BEAUTY study is funded in part by the comprehensively characterize the immune cells within the TME at Mayo Clinic Center for Individualized Medicine; Nadia’s Gift Foundation; John the molecular level. Traditional methods to assess gene expression P. Guider; the Eveleigh Family; George M. Eisenberg Foundation for Charities; in tissues lack either spatial information or sensitivity/specificity. To generous support from Afaf Al-Bahar; and the Pharmacogenomics Research address this, we have developed a novel workflow combining the Network (PGRN). Other contributing groups include the Mayo Clinic Cancer single molecule and single cell visualization capabilities of the RNA- Center and the Mayo Clinic Breast Specialized Program of Research scope in situ hybridization (ISH) assay with the highly multiplexed Excellence (SPORE). spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler Ethics Approval (DSP) RNA assays (Research Use Only). The study was reviewed approved by the Mayo Clinic Institutional review Methods board (IRB). The fully automated RNAscope Multiplex Fluorescent assay was used to visually identify CD3E (T-cell)-enriched regions and CD19 and CD20 (B-cell)-enriched regions within FFPE human lung cancer tis- sues. Using the GeoMx DSP, 10 CD3E-enriched regions of interest (ROI) and 10 CD19-enriched ROI were spatially profiled for 78 genes related to immune-oncology research. The RNAscope Multiplex Fluor- escence assay was used again to visually confirm the differentially expressed genes between the T and B-cell-enriched regions with sin- gle cell resolution. Results To show a workflow combining RNAscope molecularly guided visualization and GeoMx DSP profiling is feasible, we confirmed that both assay protocols are compatible. We then examined con- cordance between GeoMx DSP and RNAscope ISH data, demon- strating that RNAscope and GeoMx DSP data can be obtained on the same section. To test the full automated workflow, we com- pared the differentially expressed genes within the T cell and B cell-enriched ROI. The RNAscope assay confirmed that, while the expression of the immunoregulatory molecules CTLA4, PD-L1, PD- 1, and ICOSLG were detected in both ROI, the CD3E (T-cell)- enriched ROI demonstrated significantly higher expression of these checkpoint markers. Compared to the CD19-enriched ROI, the CD3-enriched ROI also showed increased inflammatory signa- ture, demonstrated by elevated levels of cytokines and chemo- kines such as CCL5, CXCL9 and IFNG. Conclusions We present a robust workflow that overcomes the historical limita- tions of ISH and IHC by combining high resolution imaging with high plex profiling. With this workflow, the RNAscope ISH technology can molecularly guide the GeoMx DSP to precisely profile ROI while retaining the morphological context of heterogenous tumors. Fur- Fig. 1 (abstract P23). High dimensional immune monitoring of thermore, RNAscope assays can be used to confirm GeoMx DSP- breast cancer PBMCs identified gene expression signatures at single cell resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 13 of 272 P25 4. Borbulevych OY, Santhanagopolan SM, Hossain M, Baker BM. TCRs used A conserved MART-1 T cell receptor motif is predictive of in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens responses to checkpoint blockade via distinct mechanisms. J Immunol. 2011;187(5):2453-2463. 1 2 2 Ariel Isser, BS , Tatsuya Yoshida , Junya Ichikawa , Jeffrey Weber, MD, Ethics Approval 2 3 PhD , Jonathan Schneck, MD, PhD The protocol was approved by the NYU Institutional Review Board, i16-01975 1 2 Johns Hopkins University, Baltimore, MD, United States; New York School of Medicine, New York, NY, United States; Johns Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Jeffrey Weber (jeffrey.weber@nyulangone.org); Jonathan Schneck (jschnec1@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P25 Background Since the introduction of checkpoint blockade inhibitors for cancer immunotherapy, numerous studies have sought to identify bio- markers predictive of patient response [1]. However, the relevance of antigen-driven responses to the tumor has yet to be investigated. To address this question, we examined T cell responses to MART-1, an antigen overexpressed in melanoma cells and a target for melanoma clinical trials that have had variable degrees of success. We hypothe- sized that features of patients’ MART-1 CD8+ T cell repertoires could predict their response to checkpoint blockade. Methods To understand the MART-1 T cell repertoire, MART-1 CD8+ T cells were expanded from HLA-A2+ melanoma patients and healthy do- nors using artificial antigen presenting cells (aAPC) or peptide-pulsed dendritic cells. Tetramer positive cells were sorted after 14-22 days and CDR3β sequenced. Motif analysis based on sequence homology was performed using the Immunomap algorithm by clustering 11,252 unique MART-1 CDR3β sequences from 33 samples and 20 donors, including five nivolumab responders and five non- responders [2]. Fig. 1 (abstract P25). See text for description Results No significant difference in the frequency of MART-1 expanded T cells was seen between healthy donors and melanoma patients with or P26 without checkpoint therapy. There was no immunodominant Vβ gene Murine T cell phenotype and function in a single-well format: a usage and limited clonotype overlap between donors. However, se- novel, multiplexed and high-throughput assay workflow using the quence homology showed extensive overlap between donors, driven iQue platform by two clusters present in 60% and 80% of samples at average frequen- Veronica Bruce, PhD, Caroline Weldon, John O'Rourke, Veronica Bruce, cies of 10% and 14%, respectively. These clusters were homologous to PhD each other as well as the DMF4 T cell receptor (TCR), one of the first Sartorius, Albuquerque, NM, United States clinically used genetically engineered T cells, with a known crystal struc- Correspondence: John O'Rourke (John.ORourke@Sartorius.com) ture [3,4]. The core region of these clusters contained a conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P26 amino acid motif that was identical to contact residues between the DMF4 TCR and MART-1 peptide bound to HLA-A2. The motif identified Background from the core region of these clusters was highly conserved across Immunotherapy is an actively growing arena in oncotherapeutics re- samples, present almost exclusively in the junctional region between search and development. In this context, whether testing CAR-T cells, the D and J genes of the CDR3β, and encoded by a diverse range of checkpoint inhibitors, or novel bispecific antibodies, the ultimate nucleotides, all evidence of selective pressure. Despite its conservation, goal is to modulate the immune system to harness its tumor killing the frequency of this motif was nearly six times lower in pre-therapy power. T cells play a critical role in immune-regulated clearance of samples expanded from non-responders compared to responders (40% both liquid and solid tumors. Upon antigenic stimulation and activa- vs. 7%, p=0.0045, Figure 1). tion, T cells rapidly expand, secrete cytokines, and differentiate to Conclusions various functional subsets (e.g. effector T cells, memory T cells). On Since the frequency of the identified MART-1 TCR motif is significantly the other hand, suppression of T cells (i.e. exhaustion) leads to im- lower in non-responders compared to responders, it could potentially mune escape and the spread of tumor cells. Mouse models remain be used as a biomarker to predict response of HLA-A2+ melanoma pa- the most commonly used animal system for in vivo and in vitro can- tients to checkpoint blockade prior to the onset of therapy. cer biology research and drug discovery. As researchers move for- ward to either better understand the role of T cells in cancer biology References or to develop novel immunotherapies, there is a need for improved 1. Zappasodi R, Wolchok JD, Merghoub T. Strategies for Predicting methods to quickly gather comprehensive data on T cell biology in Response to Checkpoint Inhibitors. Curr Hematol Malig Rep. this model. To address this, we demonstrate a multiplexed, high- 2018;13(5):383-395. throughput, robust assay workflow capable of measuring multiple 2. Sidhom J-W, Bessell CA, Havel JJ, Kosmides A, Chan TA, Schneck JP. murine T cell biology endpoints quickly and reproducibly in a single- ImmunoMap: A Bioinformatics Tool for T-Cell Repertoire Analysis. Cancer well format. Immunol Res. January 2017; 6(2):151-162. Methods 3. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of Tumor-Infiltrating In our workflow, stimulated mouse T cells were assayed in a 96-well Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with plate using fluorescent antibodies against CD3, CD4, CD8, CD69, Metastatic Melanoma. N Engl J Med. 1988;319(25):1676-1680. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 14 of 272 CD44, CD62L, and PD-1, QBeads for cytokine detection, and markers the tumor tissue upon drug treatment. The impact of different for cell viability and proliferation. Data were acquired on the iQue3 immuno-oncology drug treatments ex vivo on TME will be discussed. technology (VBR configuration) and analyzed on a plate-based level Application of this platform in the clinical studies may also allow de- using the integrated ForeCyt software. termining the most effective combinatorial therapeutic strategies for Results individual patients. Our assay workflow enabled simultaneous evaluation of viability, in- terrogation of helper and cytotoxic T cells for markers of activation P28 and exhaustion, and identification of key memory subsets. Prolifera- Mass spectroscopy-based highly multiplexed super-resolution tion and secreted cytokines (IFN-gamma and IL-2) were also quanti- imaging method for fine details of tumor microenvironment fied. Data analysis and visualization of multiple endpoints was monitoring and tumor-immune cell interactions streamlined and performed in real time using the ForeCyt software. 1 1 1 2 Yunhao Bai, BS , Bokai Zhu , Michael Angelo, MD, PhD , Yongxin Zhao , Conclusions 1 1 1 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Garry Nolan, PhD The assay was completed in four hours, including data analysis. This 1 2 Stanford University, Stanford, CA, United States; Carnegie Mellon workflow saves the end user’s time and resources by combining mul- University, Pittsburgh, PA, United States tiple experiments into a single, multiplexed workflow, and helps Correspondence: Sizun Jiang (sizunj@stanford.edu); Xavier Rovira Clave minimize subject-to-subject variability. Altogether, our workflow al- (xrovira@stanford.edu); Garry Nolan (gnolan@stanford.edu) lows for easy phenotype and functional profiling of murine T cells in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P28 a single-well format while generating actionable results in a matter of hours. Background In tumor microenvironment, tumor-immune interactions are indi- P27 cated by cell surface proteins such as T cell receptor (TCR) and PD- Functional 3D-plEX quantitative multiplex immunofluorescence L1. The key workhorse for studying these cellular interactions is via platform to assess IO drug impact on tumor microenvironment in imaging; conventional imaging methods are limited by the number ex vivo treated intact 3D-tumor organoids of fresh patient tumor of channels and the spatial resolving capabilities. tissue A new modality of imaging, Multiplexed Ion Beam Imaging (MIBI) Jenny Kreahling, PhD, Vijayendra Agrawal, PhD, Melba Page, PhD, Mibel [1,2], can resolve >40 parameters simultaneously in biological sam- Pabon, PhD, Soner Altiok, MD, PhD ples. MIBI can current attain single cell resolutions but has difficulties Nilogen Oncosystems, Tampa, FL, United States in resolving fine subcellular features. Here, we present Expansion Correspondence: Soner Altiok (soner@nilogen.com) MIBI (ExMIBI), which combines a physical expansion of a biological Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P27 sample with the MIBI imaging method. ExMIBI will be critical for the scientific community to obtain previously inaccessible insights into Background the fine details of tumor microenvironment and cancer-immune cell The tumor stroma consists of various components of the tumor interactions, and promises to unravel fundamental insights in patient microenvironment including tumor cells, fibroblasts, immune cells immunotherapy responses. and the extracellular matrix. Spatial organization and dynamic inter- Methods play of the complex cell-to-cell interactions play an important role Expansion microscopy (ExM) [3,4] is a technique that can physical ex- in cellular phenotypes that can result in permanent alterations in pansion of biological specimens 4 to 10 folds through polymer cellular functions and response to oncology as well as immuno- chemistry, three-color fluorescent imaging of cellular features with oncology drug treatments. While informative, conventional 2D an apparent lateral resolution of 70 nm in diffraction-limited confocal tumor dissociated models do not maintain the stromal- microscopes has been achieved. However, the expanded gel is fragile stoichiometry of the tumor microenvironment, lacking vital support and contains up to 99.9% water, which limits its usage in imaging mechanisms necessary to accurately assess ex-vivo tumor cell via- method that requires high vacuum condition. We explored a way to bility and immune-cell activation after drug treatment. Here, we de- collapse the tissue-containing gel on a complementary charged sub- scribe a functional quantitative multiplex immunofluorescence strate to achieve a vacuum-compatible gel that can be imaged by platform, 3D-plEX, to quantify drug-mediated changes in tumor im- the MIBIscope, with lateral resolution <100 nm. Various methods for mune microenvironment and tumor cell viability in intact 3D tumor sample charging removal are systematically tested for imaging a organoids of patient tumor samples. non-conductive gel in MIBI. Methods Results All patient tumor samples were obtained with patient consent We have established a robust method, ExMIBI, that allows ExM and relevant IRB approval. Unpropagated live 3D tumoroids hydrogels to be compatible with the high vacuum imaging condi- measuring 100-150 micron in size were prepared from fresh pa- tions of the MIBI. This method can achieve 40 parameters. A vali- tient tumors using a proprietary technology, pooled together to dated panel of MIBI compatible antibodies, focusing on the immune represent tumor heterogeneity and equally distributed to differ- system, is being tested and established for ExMIBI in FFPE tissues ent treatment groups including nivolumab, ipilimumab, atezolizu- (Figure 1). mab and urelumab singly or in different combinations. Cell Conclusions media was collected for multiplex cytokine release assay. Tumor- The combination of ExM and MIBI, termed ExMIBI, permits highly oids were fixed and embedded for multiplex immunofluorescence multiplexed super resolution imaging of tissue samples. We will now studies. In addition to tumor cell killing, treatment-mediated be able to map previously inaccessible, finer details of the tumor changes in TME was analyzed in each treatment group side-by- microenvironment. The application of ExMIBI to dissect cellular im- side using multiplex immunofluorescence markers including CD4, mune interactions, in their spatial biological context, will allow a bet- CD8, FoxP3, CD68, Pan-CK, PD-L1 and Ki67. ter understanding into the basic principles of our immune system in Results healthy and disease states. Our results demonstrated that 3D-plEX platform using clinically rele- vant intact, uniformly sized tumoroids of fresh patient tumor tissue is Acknowledgements highly versatile and reliable approach to quantify drug-mediated We thank Matt Newgren for tireless technical support on the MIBI changes in cellular composition and spatial organization of the tumor instrument. This research has received advice and help from Prof. Michael immune microenvironment. Angelo, Prof. Sean Bendall and their research group. B.Z. is supported by the Conclusions Stanford Graduate Student fellowship. S.J is supported by a Stanford Dean’s Combination of this approach with multiplex cytokine release assay Fellowship and the Leukemia & Lymphoma Society Career Development allows a comprehensive understanding of dynamic changes within Program. X.R.-C. is supported by a long-term EMBO fellowship. This work was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 15 of 272 supported by grants from the FDA, NIH, Parker Institute for Cancer separate tumor and stromal compartments, detect tumor/stroma Immunotherapy, the Bill and Melinda Gates Foundation, as well as the margins, and identify leukocytes in immunostained tissues were im- Rachford and Carlota A. Harris Endowed Professorship to G.P.N. plemented in each tissue analyzed. Resulting image markups of cell detection and biomarker expression measured by image analysis References were reviewed by an MD pathologist for acceptance. Tissues not 1. Angelo M, Bendall SC, Finck R, et al. Multiplexed ion beam imaging of meeting acceptance criteria were re-analyzed until acceptable to the human breast tumors. Nature Medicine. 2014;20(4):436–42. reviewing pathologist. 2. Keren L, Bosse M, Marquez D, et al. A Structured Tumor-Immune Micro- Results environment in Triple Negative Breast Cancer Revealed by Multiplexed We demonstrate the synergistic value of layered image analysis algo- Ion Beam Imaging. Cell. 2018;174(6):1373-87.E19. rithms which provide context to biomarker expression in NSCLC tis- 3. Chen F, Tillberg PW, Boyden ES. Expansion Microscopy, Science. sues. Samples were grouped in to immune desert, excluded, and 2015;347(6221):543-8. inflamed phenotypes based on total leukocyte and CD8 expression 4. Tillberg PW, Chen F, Piatkevich KD, et al. Protein-retention expansion mi- patterns in the tumor, stroma, and margin. PD-L1 expression was croscopy of cells and tissues labeled using standard fluorescent proteins scored based on percentages of tumor and stromal expression, as and antibodies. Nature Biotechnology. 2016;34(9):987–92. well as digital representations of common PD-L1 scoring paradigms. Additionally, samples were stratified by PD-L1 patterns of constitu- tive, induced, immune, or ignorant expression. Conclusions Digital image analysis of IHC stained tissues creates comprehensive tissue biomarker profiles that are useful in assessment of tumor and immune interactions in IO drug development and patient stratifica- tion. Complex algorithms that utilize AI and machine learning can be overseen by MD pathologists to create clinically acceptable digital analysis solutions. References 1. Rimm DL, Han G, Taube JM, et al. A Prospective, Multi-institutional, Pathologist-Based Assessment of 4 Immunohistochemistry Assays for PD- L1 Expression in Non–Small Cell Lung Cancer. JAMA Oncol. 2017;3(8):1051–1058. 2. Hendry S, Salgado R, Gevaert T, et al. Assessing Tumor-infiltrating Lym- phocytes in Solid Tumors: A Practical Review for Pathologists and Pro- Fig. 1 (abstract P28). The workflow and sample images of ExM-MIBI posal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcin- oma In Situ, Metastatic Tumor Deposits and Areas for Further Research. P29 Adv Anat Pathol. 2017;24(5):235–251. Comprehensive image analysis of immunostained NSCLC tissues 3. Taube JM, Galon J, Sholl LM, et al. Implications of the tumor immune provides necessary context for immune oncology biomarker microenvironment for staging and therapeutics. Mod Pathol. profiling 2018;31(2):214–234. Charles Caldwell, PhD, Jenifer Caldara, BS, Will Paces, BS, Kelsey Weigel, 4. Silva MA, Ryall KA, Wilm C, Caldara J, Grote HJ, et al. PD-L1 immunostain- PhD, Roberto Gianani, MD ing scoring for non-small cell lung cancer based on immunosurveillance Flagship Biosciences, Westminster, CO, United States parameters. PLOS ONE 2018; 13(6): e0196464 Correspondence: Charles Caldwell (ccaldwell@flagshipbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P29 P30 Background Deep spatial profiling of the immune landscape of MSI and MSS Manual pathology assessments of Immunohistochemistry (IHC) colorectal tumors markers in immune oncology (IO) is often challenging and results Sarah Church, PhD, Jason Reeves, Daniel Zollinger, Jill McKay-Fleisch, can be highly variable[1,2]. Measuring biomarker presence in IO must Andrew White, BSc, Michael Bailey, Arya Bahrami, PhD, Chris Merritt, take in to account both immune and tumor environments and pro- PhD, Margaret Hoang, Sarah Warren, PhD, Joseph Beechem, PhD vide contextual information on the interaction between tumor and NanoString Technologies, Everett, WA, United States immune biomarker landscapes [3]. Due to the complex nature sur- Correspondence: Sarah Church (schurch@nanostring.com) rounding tissue biomarker interpretation in IO, digital image analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P30 (IA) solutions have been developed that layer complex artificial intelligence (AI) and machine learning algorithms to obtain full tissue Background biomarker profiles necessary for drug development and patient In colorectal cancer (CRC) there have been many recent advances in stratification[4]. immune-related biomarkers that are both prognostic and predictive Here, a comprehensive tissue analysis solution is presented in mono- of response to immunotherapy. Microsatellite instability (MSI)/mis- plex PD-L1 and CD8 stained slides that includes precise digital bio- match repair deficiency (dMMR) is present in ~15-20% of CRCs and marker scoring in tumor and stromal compartments, recapitulation of corelates with increased immunogenic mutations that often augment common scoring paradigms, analysis of biomarker expression at the lymphocyte infiltration into the tumor microenvironment (TME). Add- tumor/stroma interface (margin), and quantification, scoring, and itionally, location of tumor infiltrating T-cells in two areas of the TME, spatial localization of leukocytes in the tumor and stroma. Aggrega- the tumor center (CT) and invasive margin (IM) has also been shown tion of all cellular and biomarker data generates tissue phenotypes to be prognostic and predictive of response to immunotherapy. Here that characterize the IO landscape of each tissue. we use multiplexed protein and RNA digital spatial profiling to elicit Methods the immune landscape of MSI-MSS characterized CRC tumors. Serial sections of 20 NSCLC samples were IHC stained for PD-L1 and Methods CD8 expression. Stained slides were scanned at 20x magnification Forty-eight CRC tumors were analyzed for gene expression (GX) and analyzed using Flagship Biosciences’ image analysis solutions. using the NanoString® nCounter® PanCancer IO 360™ Research Use Image analysis algorithms which quantify biomarker expression, Only (RUO) Gene Expression Panel and assessed for 48 cell typing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 16 of 272 and biological signatures, including MMR loss/MSI predictor and the Polaris® with pre-defined acquisition parameters. Scans were un- Tumor Inflammation Signature (TIS). A subset of 18 CRC tumors (6 mixed and analyzed with inForm® software using a pre-configured al- MSI-TIS-hi, 6 MSS-TIS-hi, 6 MSS-TIS-lo) was selected for analysis with gorithm tailored to the PD1/PD-L1 Lung Cancer Panel Kit. Spatial the RUO GeoMx™ Digital Spatial Profiler (DSP) using 40 antibodies, analyses and visualizations were performed using the phenoptr and 84 or 1,600+ in situ probes. Selection of regions of interest (ROIs) in phenoptrReports R-based packages and custom scripts. two locations, CT and IM were guided by staining with fluorescent Results markers (CD45, CD3, pan-CK, DNA). 300-600 μM diameter circle ROIs The pre-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer Panel were selected, and in some cases segmented by pan-CK+/pan-CK-. Kit was able to visualize the panel targets (PD-L1, PD-1, CD8, CD68, Results FoxP3, and Cytokeratin) across the variety of lung cancer samples in Using whole tissue GX, we first confirmed MSI/dMMR characterization the TMA. Cell phenotyping and spatial analyses revealed core-to-core and TIS status of 48 CRC tumors using PanCancer IO 360 signatures. variations in cell densities and proximities among different markers. We selected 18 tumors within this cohort based on TIS status to fur- Measurement of the dynamic range of PD-L1 expression across dif- ther dissect the location-dependent immune contexture of the TME, ferent cores also revealed the improved sensitivity in PD-L1 detection with a particular emphasis on differentiating MSI-TIS-hi and MSS-TIS- provided by unmixing. hi CRCs. DSP confirmed loss of dMMR markers (MSH2/MLH1) and Conclusions identified an increased amount of potentially suppressive macro- The end-to-end Phenoptics staining, imaging, unmixing, and spatial phages (CD163+PD-L1+) in MSI-TIS-hi versus MSS-TIS-hi tumors. Seg- analysis workflow described here provides a robust and sensitive mentation of ROIs based on tumor versus stroma (pan-CK+/-) platform for exploring the immune landscape within the tumor identified samples with high proportions of tumor-invading TILs. microenvironment. These samples were then further profiled using probes against 1600+ mRNA targets revealing distinct pathways related immune cell Reference orientation within the TME. 1. Lu S, Stein JE, Rimm DL, et al. Comparison of Biomarker Modalities for Conclusions Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Here we show the use of novel high-plex spatial profiling to profile Review and Meta-analysis. JAMA Oncol. Published online July 18, 2019. location and pathways in the TME of MSI and MSS CRC tumors. doi:10.1001/jamaoncol.2019.1549 These findings elicit unique biology related to the location and sig- naling of immune cells, which have the potential to unveil targets for P32 therapeutic combinations. Differential immune contexture of human colorectal carcinomas with mismatch repair deficiency (MSI-H) and increased DNA P31 damage responses (DDR) 2 2 2 Applying multispectral unmixing and spatial analyses to explore Shruti Desai, PhD , Venkata Nagineni , Micaela Morgada , Aravind 2 2 1 2 tumor heterogeneity with a pre-optimized 7-color immuno- Kalathil , Ila Datar , Charles Fuchs, MD, MPH , Patricia LoRusso, DO , 2 2 oncology workflow Ranjit Bindra , Kurt Schalper, MD, PhD 1 2 Carla Coltharp, PhD, Bethany Remeniuk, PhD, Chichung Wang, Rachel Yale University, New Haven, CT, United States; Yale University, School Schaefer, Linying Liu, Glenn Milton, Victoria Duckworth, Michael McLane, of Medicine, New Haven, CT, United States Peter Miller, Yi Zheng, Carla Coltharp, PhD Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Akoya Biosciences, Hopkinton, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P32 Correspondence: Yi Zheng (YZheng@akoyabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P31 Background Tumor cells accumulate deleterious genomic alterations through sus- Background tained mutagenic exposure and defective DNA repair. Approximately The tumor microenvironment hosts a myriad of cellular interactions 15% of human colorectal carcinomas (CRCs) display mismatch repair that influence tumor biology and patient outcomes. Multiplex im- deficiency (MSI-H) associated with increased somatic mutations and munofluorescence (mIF) provides the ability to investigate a large sensitivity to immune checkpoint blockers. Advanced tumors can number of these interactions in a single tissue section, and has been harbor additional DNA-repair alterations with functional/therapeutic shown to outperform other testing modalities for predicting re- implications. Increased double strand DNA breaks have been re- sponse to immunotherapies [1]. ported across solid tumors and can be detected by changes in Multispectral imaging (MSI) improves the capabilities of mIF by pro- Serine139-phosphorylated histone H2AX (γH2AX). We studied the im- viding the ability to spectrally unmix fluorescence signals. This in- mune composition of human CRCs with MSI-H and elevated DDR. creases the number of markers that can be probed in the same scan Methods and allows for separation of true immunofluorescence signals from Using multiplexed quantitative immunofluorescence (QIF), we stud- tissue autofluorescence background. ied the level of major adaptive and innate immune markers in a Here, we apply MSI to explore spatial interactions observed in lung retrospective collection of 265 stage I-IV CRCs from Yale represented cancer samples using an end-to-end translational workflow based on in tissuemicroarrays. We used previously validated QIF panels includ- the PhenopticsTM platform. The workflow includes a pre-optimized ing the markers DAPI, cytokeratin, γH2AX, CD3, CD4, CD8, CD20, PD- 7-color staining panel kit along with a pre-configured analysis algo- L1, CD15, myeloperoxidase (MPO), IL-8, Ki-67, granzyme-B (GZB), rithm for cell phenotyping. Beta-2 microglobulin (B2M), HLA-class I and HLA-class II. The MSI sta- Using tissue microarrays (TMA), we demonstrate the heterogeneity of tus was determined using clinical-grade immunohistochemistry de- spatial interactions observed among different lung cancer samples tection of MLH1, MSH2, MSH6 and PMS2. We analyzed the and the improved sensitivity of detection afforded by unmixing association between localized measurement of markers and with multispectral scans. major clinicopathologic variables/survival. Methods Results A lung cancer TMA was created using the 3DHistech TMA Master II From 252 evaluable cases, 12.1% were classified as MSI-H. Relative to from five formalin-fixed paraffin-embedded lung cancer tissue blocks. MSS tumors, MSI-H CRCs showed significantly higher levels of PD-L1, The TMAs were stained using the Opal Polaris 7-Color PD1/PD-L1 lower CD20 and non-significant increases in CD3, CD4, CD8, T-cell Ki- Lung Cancer Panel Kit on the Leica BOND RXTM automated stainer 67 and T-cell GZB. MSI-H cases displayed lower tumor-cell B2M and using the associated preloaded Opal 7-Color Panel Kit protocol. increased stromal HLA-class II expression. MSI-H tumors also showed Whole slide MOTiFTM multispectral scans were acquired on Vectra significantly higher levels of IL-8 and MPO+ cells than MSS Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 17 of 272 counterpart. The level of γH2AX was comparable between MSI-H and clinicopathologic associations were found. γH2Ax was not prognostic MSS malignancies. Cases with increased tumor-cell γH2AX (> cohort as single marker. However, elevated simultaneous expression of median) showed significantly higher levels of PD-L1 and all studied γH2Ax and CD3 was associated with longer 5-year overall survival in lymphocyte markers than cases with lower γH2AX. In addition, these the immunotherapy-naïve cohorts. In patients treated with PD-1 axis tumors displayed significantly higher T-cell proliferation, mild in- blockers, elevated baseline γH2Ax/CD3 was associated with a clear creases in T-cell GZB and higher levels of HLA-class I/class II proteins. trend toward longer survival but did not reach statistical significance. The levels of IL-8 and MPO+ cells were comparable across the γH2AX Conclusions groups. The DNA repair and immune markers were variably associ- Active DDR as measured by tumor-cell γH2Ax expression occurs in a ated with 5-year overall survival in the cohort. high proportion of human NSCLCs and is associated with T-cell in- Conclusions flamed tumors. Despite their association with smoking, lung adeno- DNA repair deficiency defines human CRCs with distinct innate and carcinomas harboring activating mutations in KRAS display lower adaptive immune contexture. While mismatch repair deficiency is as- DDR markers than EGFR mutant or EGFR/KRAS wild type malignan- sociated with mild/moderate intratumor T-cell responses and prom- cies. Collectively, our results support the use of combination therapy inent myeloid cell features; elevated DDR display prominent adaptive targeting DDR and immunostimulatory therapies in a fraction of immunity and unaltered myeloid-cell changes. Our data indicate that NSCLC. MSI-H and DDR phenotypes are independent features in human CRC Ethics Approval and this could be used to design optimal therapeutic strategies. All tissues were used after approval from the Yale Human Investiga- Ethics Approval tion committee protocol #9505008219 which approved the patient All tissues were used after approval from the Yale Human Investiga- consent forms or waiver of consent. tion committee protocol #9505008219 which approved the patient consent forms or waiver of consent. P34 A fully optimized end-to-end solution for I/O multiplex P33 immunofluorescence staining using Opal Polaris 7-Color PD1/PD- DNA damage response (DDR) is associated with increased adaptive L1 Panel Kits for lung cancer and melanoma anti-tumor responses and PD-L1 expression in human non-small Yi Zheng, Rachel Schaefer, Linying Liu, Glenn Milton, Carla Coltharp, cell lung cancer 2 2 1 PhD, Victoria Duckworth, MS, Michael McLane, Peter Miller, MS Shruti Desai, PhD , Aravind Kalathil , Roy Herbst, MD, PhD , Ranjit 2 1 2 Akoya Biosciences, Hopkinton, MA, United States Bindra , Patricia LoRusso, DO , Kurt Schalper, MD, PhD 1 2 Correspondence: Peter Miller (pmiller@akoyabio.com) Yale University, New Haven, CT, United States; Yale University, School Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P34 of Medicine, New Haven, CT, United States Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P33 Understanding cellular heterogeneity and spatial relationships be- tween biomarkers within the tumor microenvironment (TME) is a key Background component to translational research in immuno-oncology. Multiplex Tumor cells accumulate genomic alterations as a consequence of immunofluorescence (mIF) on formalin-fixed, paraffin-embedded sustained mutagenic events and defective DNA repair mechanisms, (FFPE) tissue is the multiparameter assay most frequently chosen collectively called DNA damage response (DDR). Targeting DDR path- across all current I/O clinical trials, as it allows for quantitative assess- ways can induce synthetic lethality and prominent anti-tumor re- ment of these relationships in situ. Running medium to large scale sponses in neoplasms with DNA repair deficiency. In addition, translational studies on FFPE tissue demands an assay that is repro- increased DNA damage could favor anti-tumor immune responses by ducible, quantitative, easy-to-use, and standardized, yet still allows increasing the neo-antigenic load and T-cell recognition. Despite its for flexibility when detecting differentially expressing biomarkers therapeutic implications, the frequency and significance of DDR alter- across samples. In this study, we demonstrate a fully developed, flex- ations in human non-small cell lung cancer (NSCLC) remains poorly ible, end-to-end workflow solution for tissue biomarker discovery by understood. applying miF in lung cancer and melanoma. This newly developed Methods Phenoptics™ solution provides an integrated MOTiF™ workflow in- Using irradiated cell line preparations and expression controls, we cluding primary antibodies and image analysis algorithms enabling a standardized a multiplexed quantitative immunofluorescence more comprehensive and specific TME analysis with minimal user (mQIF) panel for simultaneous and localized measurement of optimization. DAPI (all cells), cytokeratin for tumor epithelial cells (AE1/AE3, Methods DAKO), γH2AX to map active DNA damage/repair responses FFPE samples from human lung cancer and melanoma were stained (JBW301, Millipore), CD3 for T-lymphocytes (Rabbit polyclonal, using Opal Polaris 7-Color PD1/PD-L1 Lung Cancer and Melanoma DAKO) and PD-L1 (E1L3N, CST) in formalin-fixed paraffin- Panel Kits. Staining was performed on the Leica BOND RX™ auto- embedded (FFPE) tissue samples. We used this panel to interro- mated stainer with the pre-loaded MOTiF protocol. Multispectral gate 4 retrospective NSCLC cohorts from Yale represented in tis- scans were acquired on Vectra Polaris® with pre-optimized acquisi- sue microarray format including immunotherapy-naïve cases tion parameters and analyzed with a pre-configured phenotyping al- (Cohort#1: n=297 and #2:n=175); lung adenocarcinomas tested gorithm in inForm®. Spatial analyses and visualizations were for major oncogenic mutations (Cohort #3, n=139); and baseline performed in R using phenoptr and phenoptrReports. NSCLC samples from patients treated with immune checkpoint Results blockers (Cohort #4, n=84). We analyzed the levels of the markers This simplified end-to-end solution results in better quantification of in different tumor tissue compartments and their association with cancer-immune interactions by providing: major clinicopathological variables. Results Detectable nuclear tumor-cell γH2Ax was recognized in 37-58% of  Well-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer NSCLCs. Elevated tumor-cell γH2Ax expression was consistently asso- and Melanoma Panel Kits. Along with the pre-loaded Leica ciated with smoking history, increased intratumor CD3+ T-cells and BOND RX automation protocol, we provide a staining workflow PD-L1 protein expression across the cohorts. The level of γH2Ax was with pre-defined primary antibody concentration, fixed staining significantly lower in KRAS mutant lung adenocarcinomas than in order, and Opal™ dye-antibody pairs, leaving Opal concentra- EGFR mutant or EGFR/KRAS wild type tumors. No additional tions as a flexible dial. Recommended image acquisition Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 18 of 272 parameters on the Vectra Polaris® that significantly simplify lymphoid aggregates. Micro-regions were picked and sequenced. visualization of multiple markers via multispectral isolation. Pre- Hierarchical clustering and differential expression analysis differ- configured image analysis algorithms that make quantitative entiated the three micro-region types and revealed tumor- and T analysis at a per-cell and per-slide level streamlined and cell-specific expression signatures. CIBERSORT demonstrated the standardized. presence of T cell-associated transcriptomic profiles in lymphoid aggregates and in TIL-containing micro-regions that were propor- tional to the number of T cells retrieved. Aligned RNA-seq reads Conclusions were further analyzed via TraCeR to identify TCR α and β chain The 7-Color PD1/PD-L1 panel kits utilizing MOTiF whole slide scan- sequences from retrieved TILs. ning enable visualization of multiple biomarkers at the whole slide Conclusions level, revealing distribution patterns and their spatial context across These data establish the potential of combining multi-parameter IF the entire tissue section. With these new assays, we have demon- microscopy with highly focused RNA sequencing as a powerful tool strated an easy-to-use yet comprehensive end-to-end Phenoptics re- for investigation and biomarker discovery for immuno-oncology. search workflow. We have radically simplified the Opal method and facilitated the development and optimization of translational multi- plex fluorescent assays by providing pre-defined staining conditions P36 while still giving researchers the flexibility to balance signals based The complexity of myeloid-derived suppressor cells in non-small on their tissue samples. Complementary pre-configured phenotyping cell lung cancer: A combinatorial multiplex IHC and flow cytometry provides researchers faster access to quantitative data across study approach samples. 1 2 1 1 Amanda Finan, PhD , Muriel Smet , Maroua Tliba , Manon Motte , Jean- 1 1 1 Philippe Coton , Domenico Lazzaro , Renaud Burrer 1 2 Histalim, Montpellier, France; Barc Lab, Ghent, Belgium P35 Correspondence: Renaud Burrer (rburrer@histalim.com) Pick-Seq®: a spatial analysis tool for immuno-oncology biomarker Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P36 discovery utilizing multi-parameter imaging and RNA sequencing of tissue micro-regions Background 1 1 1 Nolan Ericson , Rebecca Podyminogin , Jennifer Chow, PhD , Yu-An Lung cancer is the most common cause of cancer-related deaths 2 2 2 2 1 Chen , Jia-Ren Lin , Zoltan Maliga , Peter Sorger , Kyla Teplitz , Melinda worldwide with non-small cell lung cancer (NSCLC) representing the 1 1 1 Duplessis, PhD , Eric Kaldjian, MD , Tad George, PhD gross majority of the cases. The immune microenvironment of NSCLC 1 2 RareCyte, Seattle, WA, United States; Harvard Medical School, Boston, is diverse with many players that can impact tumor development MA, United States and clinical outcomes. In particular, myeloid-derived suppressor cells Correspondence: Tad George (tgeorge@rarecyte.com) (MDSC) are important components of the immunosuppressive net- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P35 work that can hinder the activity of T cells, natural killer cells, and dendritic cells. MDSC in the blood may represent prognostic markers Background for NSCLC patients and for monitoring a patient’s response to im- Pick-Seq is a novel workflow uniquely enabled by the RareCyte Cyte- munotherapies. There is a gap in the relevance of MDSC within the Finder® Instrument that combines visualization of multiple protein tissue context due to limitations with conventional immunohisto- markers with investigation of gene expression from selected micro- chemistry. Multiplex immunofluorescence offers a technical advan- regions on tissue slides, providing spatial and contextual investiga- tage by allowing the detection of co-expression and spatial tion of tumors and their microenvironment. organization of multiple targets within a preserved tissue architecture Methods on a single slide. Frozen breast carcinoma and formalin-fixed, paraffin-embedded ton- Methods sil sections were stained by multi-parameter immunofluorescence (IF) We have developed the multiplex immunofluorescence for markers of T cells, B cells, and cytokeratin. Slides were imaged Histoprofile-MDSC panel to identify monocytic MDSC (M-MDSC) with the CyteFinder® Instrument and 40 μm micro-regions were re- and polymorphonuclear MDSC (PMN-MDSC) in situ. Five human trieved with the integrated CytePicker® Retrieval Module. RNA was NSCLC tissue samples were investigated by multiplex immunofluor- isolated and whole transcriptome amplified (SMART-seq v4), followed escence and H&E staining. After multispectral acquisition, the MDSC by Nextera XT library preparation, sequencing on Illumina MiSeq, and populations were evaluated with the imaging software HALO. gene expression analysis. Differentially expressed genes were se- Paired peripheral blood was analyzed for circulating M-MDSC by lected to create a Pick-Seq-informed IF staining panel to confirm flow cytometry. RNA expression results. Cell compositions of each micro-region were Results deconvolved with CIBERSORT. The development and verification of the multiplex panel are pre- Results sented. The NSCLC subtype of the samples was determined by a Tonsil micro-regions from one T cell zone and two adjacent follicles pathologist from the H&E sections. Monocytes, neutrophils, M-MDSC, were retrieved for RNA sequencing. Transcriptomic analysis con- and PMN-MDSC were evaluated in the five tissue samples. The neu- firmed increased expression of B cell markers in follicles and T cell trophils, monocytes, and M-MDSC in the peripheral blood could be markers in the T cell zone. CIBERSORT analysis revealed distinct cellu- assessed by flow cytometry. A varying distribution of the cell popula- lar compositions between T cell zones and the B cell follicles. tions in the lung tissue and the peripheral blood of the different Principle component analysis of gene expression found that micro- NSCLC subtypes can be appreciated. The two approaches are regions retrieved from the two follicles clustered independently from compared. each other, and from the T cell zone micro-regions. Differential ex- Conclusions pression analysis between the adjacent follicles revealed distinct pat- We present an in-depth combined approach for MDSC investigation terns of CD21 expression, a marker which was not present in the in lung tissue and the peripheral blood of NSCLC patients. The ap- original IF staining panel. Subsequent staining confirmed differential proaches presented here demonstrate the power of multiplex immu- protein expression of CD21, indicating that only one follicle con- nohistochemistry and flow cytometry in the identification and tained a germinal center. In breast carcinoma, ROI were identified for quantification of multiple immune cell populations with a limited micro-region retrieval that included tumor cells, tumor cells with quantity of patient sample and the potential application of this interspersed tumor infiltrating lymphocytes (TIL), or adjacent method in both preclinical and clinical studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 19 of 272 P37 8. Otvos B, Silver DJ, Mulkearns-Hubert EE, et al. Cancer Stem Cell-Secreted ImmunoPET imaging of glioma-infiltrating myeloid cells using Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Sup- Zirconium-89-labeled anti-CD11b antibody pressor Cell Function and Facilitates Glioblastoma Immune Evasion. Stem Alexandra Foster, BS, Rajeev Kumar, Shubhanchi Nigam, Lauren McCarl, Cells. 2016; 34:2026–2039. Robert Edinger, Ian Pollack, Carolyn Anderson, Wilson Edwards, Gary 9. Meyer C, Cagnon L, Costa-Nunes CM, et al. Frequencies of circulating Kohanbash, Alexandra Foster, BS MDSC correlate with clinical outcome of melanoma patients treated with University of Pittsburgh, Pittsburgh, PA, United States ipilimumab. Cancer Immunol Immunother. 2014; 63:247–257. Correspondence: Gary Kohanbash (gary.kohanbash2@chp.edu) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P37 The study was approved by University of Pittsburgh's Institutional Animal Care and Use Committee (IACUC). Background Gliomas are the most common primary central nervous system tumor, P38 with malignant gliomas causing significant morbidity and mortality. Sensitive methodologies for tracking T cell immunotherapy by MRI Thirty percent of a glioma’s cellular mass may be attributed to immuno- 1 2 1 2 Brooke Helfer, PhD , Deanne Lister , Charles O'Hanlon III , Eric Ahrens , suppressive and pro-tumoral tumor-associated myeloid cells (TAMCs), Brooke Helfer, PhD primarily myeloid-derived suppressor cells (MDSCs) and tumor- 1 2 Celsense, Inc, Pittsburgh, PA, United States; UCSD, La Jolla, CA, United associated macrophages (TAMs) [1-4]. Multiple preclinical studies and States clinical trials have attempted to target these cells; however, monitoring Correspondence: Brooke Helfer (brooke@celsense.com) responses to these therapies remains a challenge. Quantifying TAMCs Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P38 within gliomas using an antibody-based tracer for non-invasive posi- tron emission tomography (immunoPET) may allow for better patient Background stratification, monitoring of treatment efficacy, and ultimately improve Cancer immunotherapies have made a great progress and hold much survival rates [5-9]. Integrin CD11b is a cellular marker expressed on the promise in the treatment of cancer. Specifically, in the case of B-cell surface of TAMCs frequently used to identify macrophages and micro- malignancies (such as Acute Lymphoblastic Leukemia, or ALL), CAR glia. We therefore hypothesized that radiolabeled anti-CD11b antibody (chimeric antigen receptor) and TCR (T-cell receptor) therapies have (Ab) could be used for immunoPET imaging of TAMCs in a preclinical demonstrated encouraging clinical results. As we begin to target solid orthotopic syngeneic glioma model. tumors with TCR and CAR T-cells, the hurdle of being able to select a Methods suitable target and achieve successful cellular delivery/homing to the The human/mouse cross-reactive anti-CD11b Ab (clone M1/70) was site of disease remains. With this in mind, being able to visualize a rap- conjugated with p-NCS-Bz-DFO chelator and radiolabeled with 89Zr idly dividing cellular population is another obstacle to consider. for PET imaging with specific activity of 2 μCi/μg. PET/CT imaging, Methods with or without a blocking dose of anti-CD11b Ab, was performed in Here we demonstrate the application of two clinically applicable per- mice bearing established orthotopic syngeneic GL261 gliomas. Flow fluorocarbon (PFC) tracers, one commercially available and a next- cytometry and histology in tissues collected from post-imaging bio- generation magnetic resonance imaging (MRI) probe called FETRIS. distribution validated targeting of CD11b+ TAMCs. Both of these agents enable the migration and persistence of cellular Results therapies to be noninvasively imaged by 19F MRI, while the FETRIS Standard uptake values (SUV) indicated significant 89Zr-anti-CD11b reagent adds additional detection sensitivity. Ab uptake in the tumor ipsilateral right brain (SUVmean = 2.6 ± 0.24) Results compared to contralateral left brain (SUVmean = 0.6 ± 0.11). Blocking Using a general T-cell expansion protocol, we show that adding a cellular with 10-fold lower specific activity 89Zr-anti-CD11b Ab reduced the label does not alter the viability or release characteristics of T cells. By SUV in right brain with (SUVmean = 0.11 ± 0.06). Spleen and lymph pairing the PFC signal with conventional proton MRI from the same im- nodes also showed high uptake, while bone and muscle showed low aging session, the images are able to be overlaid, allowing cells to be uptake. Biodistribution analysis confirmed these results. Additionally, traced to their anatomical location. With nominal exogenous fluorine nat- no uptake was observed in the brain of non-tumor bearing mice that urally present in tissue, labeled cells appear with little background. received 89Zr-ant- CD11b. Flow cytometry with QuantiBRITE Fluores- Conclusions cence Quantitation Kit demonstrated that the majority of tumor- Images of both reagents show the detection and sensitivity of the infiltrating immune cells expressed CD11b at an average of 54,076 method and how they can be applied to monitor the distribution of CD11b molecules per cell in GL261. cells over time. Conclusions Imaging TAMCs with 89Zr-labeled anti-CD11b Ab may be feasibility for preclinical studies, patient stratification, and monitoring of immunotherapy. P39 References Looking beyond the assay: Comparison of multiplex chromogenic 1. Gabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate and fluorescent immunohistochemistry for standardized immune immune cells resemble M0 macrophage phenotype. JCI insight. 2016; oncology profiling in non-small cell lung carcinoma patients 1 2 2 1(2). Ana Hidalgo Sastre, PhD , Lorenz Rognoni, PhD , Monika Baehner , 2 2 2 2 2. Kennedy BC, Showers CR, Anderson DE, et al. Tumor-associated macro- Marco Testori , Jessica Chan , Andreas Spitzmüller , Nicolas Brieu, PhD , 3 3 3 phages in glioma: friend or foe? J oncol; 2013. 2013. Bonnie Phillips, PhD , Katir Patel, PhD , Sean Downing, PhD , Alex 4 4 2 3. Kohanbash G, Okada H. Myeloid-derived suppressor cells (MDSCs) in gli- Haragan , John Field , Florian Leiss, PhD 1 2 3 omas and glioma-development. Immunolo invest. 2012; 41:658–679. Definiens, Munich, Germany; Definiens AG, Munich, Germany; Ultivue, 4. Lapa C, Linsenmann T, Lückerath K, et al. Tumor-associated macrophages Cambridge, MA, United States; Liverpool University Hospital, Liverpool, in glioblastoma multiforme-a suitable target for somatostatin receptor- United Kingdom based imaging and therapy? PloS one. 2015; 10. Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) 5. Kohanbash G, McKaveney K, Sakaki M, et al. GM-CSF Promotes the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P39 Immunosuppressive Activity of Glioma-Infiltrating Myeloid Cells through Interleukin-4 Receptor-α. Cancer Res. 2013; 73:6413–6423. Background 6. Raychaudhuri B, Rayman P, Huang P, et al. Myeloid derived suppressor cell Given the heterogeneity of tumors and the variety of potential bio- infiltration of murine and human gliomas is associated with reduction of markers in immune oncology, there is a need for quantitative standard- tumor infiltrating lymphocytes. J Neurooncol. 2015; 122:293–301. ized assays to reliably assess the immune status of a patient’stumor to 7. Okada H, Kohanbash G, Zhu X, et al. Immunotherapeutic approaches for be able to extract the true biological information across cohorts. Here, glioma. Crit rev immunol. 2009; 29:1–42. two different tissue-based approaches have been compared: multiplex Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 20 of 272 immunofluorescence (mIF) and multiplex chromogenic immunohisto- P40 chemistry (mIHC). Independently of the technique used, assay reproduci- Tumour immunity signatures to expand current diagnostic bility and standardized quantification of staining intensity are a approaches in mismatch repair deficient cancers in the context of prerequisite for obtaining consistent results. Using a cohort of non-small Lynch Syndrome through InSituPlex technology and Tissue cell lung carcinoma (NSCLC) patients, we identified patterns of immune Phenomics integration 1 2 3 cell infiltration that were comparable, independent of the assay applied. Ryan Hutchinson, Fellow , Armin Meier, PhD , Bonnie Philips ,Katir 3 3 3 1 Methods Patel, PhD , Sean Downing, PhD , Karan Sharma , Julia Como ,Simin 1 2 4 1 Formalin-fixed paraffin-embedded (FFPE) true consecutive slides from 7 Daneshvar , Gillian Livock , Ingrid Winship , Christophe Rosty ,Mark 5 2 1 NSCLC resections were stained with a multiplex chromogenic panel (in- Jenkins ,GunterSchmidt , Daniel Buchanan ,RyanHutchinson, cluding CD3, PD-L1, CD68, CD8, PD-1) at Mosaic Laboratories [1] and with Fellow the UltiMapper kits (I/O PD-L1 and I/O PD-1) from Ultivue. mIHC scans Victorian Comprehensive Cancer Centre, Melbourne, Australia; 2 3 were acquired with an Aperio AT Turbo scanner (Leica), while mIF scans Definiens AG, Munich, Germany; Ultivue, Cambridge, MA, USA, Boston, 4 5 were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss) both as whole MA, United States; Royal Melbourne Hospital, Melbourne, Australia; The slide images. mIHC and mIF images were co-registered, and Definiens University of Melbourne, Melbourne, Australia custom algorithms for digital image analysis were applied [2,3]. Correspondence: Daniel Buchanan (daniel.buchanan@unimelb.edu.au) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P40 Densities of immune cell populations and their locations in different com- partments (invasive margin vs tumor center and tumor epithelium vs Background tumor stroma) were measured (Figure 1). For instance, CD3 cell density Deficiency in the mismatch repair (dMMR) can result from had a Pearson correlation of 0,91 and a Spearman correlation of 0,89 be- inherited mechanisms (Lynch Syndrome (LS)) or from somatic tween both assays (mIHC vs mIF). Differentiation between tumor epithe- inactivation caused by hypermethylation of the MLH1 gene lium and tumor stroma was based on a histology-driven deep learning promoter (MLH1 methylated). A third subtype of dMMR colo- approach for mIHC and on pan Cytokeratin for mIF (Figure 1). rectal cancer (CRC) and endometrial cancer (EC) have neither Conclusions LS nor MLH1 promoter methylation and are referred to as sus- By applying mIHC and mIF in true consecutive tissue slides we re- pected Lynch syndrome (SLS). There remains a knowledge gap trieved the information of tumor immune cell infiltrates that was as to whether the tumour microenvironment (TiME) is different consistent across the different assays and distinguished it from infor- between LS, MLH1-methylated and SLS dMMR CRC and EC. The mation that is specific to either of the assays. We believe that being aimofthisstudy wastocharacterise and identify immune pat- able to relate across staining techniques could help pathologists and terns within the TiME that may enhance the current clinical research centers draw conclusions across cohorts that were stained triaging of LS, SLS and MLH1-methylated subtypes of dMMR with the same markers but with different assays. CRC and EC. Methods References Ten FFPE samples from seven individuals were studied: CRC (N=5; 1. Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of 1xLS, 2xMLH1 methylated, 1xSLS and 1x proficient MMR (pMMR)) inflammatory cell patterns and densities using multiplex and EC (N=2; 1xLS and 1xpMMR) and where available adjacent nor- immunohistochemistry immuno-oncology assays [abstract]. In: Proceed- mal tissue (N=5: 3xcolon and 2xendometrium) were characterized ings of the AACR-NCI-EORTC International Conference: Molecular Targets using the Ultivue UltiMapper I/O portfolio (InSituPlex) on a Leica and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia Bond autostainer and digitally acquired using the Zeiss Axio Scan Z1. (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl): Abstract nr B069. We evaluated CD3, CD8, CD11c, CD20, CD45RO, CD68, CD163, Gran- 2. Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated zyme B, Ki-67, MHC II, PD-1, PD-L1, and pan-cytokeratin. Tissue phe- quantification of whole-slide multispectral immunofluorescence images to nomic approaches were developed to spatially characterize, quantify identify spatial expression patterns in the lung cancer microenvironment. immune cell patterns and visualize heterogeneity within the TiME SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. (Figure 1). 3. Brieu, Nicolas & Meier, Armin & Kapil, Ansh & Schönmeyer, Ralf & Gavriel, Results Christos & Caie, Peter & Schmidt, Günter. (2019). Domain Adaptation- InSituPlex technology enabled the visualization of the based Augmentation for Weakly Supervised Nuclei Detection. heterogenous infiltration and co-localization patterns across LS Ethics Approval dMMR CRC (Figures 2&3). Tissue phenomic approaches demon- Ethical approval was granted by the Liverpool Research Ethics Committee, strated the following; within the SLS category the colon cancer reference number 97/141. had higher mean areas of intraepithelial (IE) PD-L1 (6% vs. 2%), CD8 (18% vs. 8%) and CD68 (28% vs. 12%) compared to the pMMR EC. The MLH1 methylated tumour with a high tumour mu- tation burden (33.84 mutations/MB) had a higher mean area of IE PD-L1 (8% vs. 2%) and CD8 (30% vs. 5%) while the tumour with low TMB had a higher mean area of IE CD68 (15% vs. 8%). Within LS, the EC had a higher mean area of IE PD-L1 compared to the CRC (14% vs. 0%), in contrast the CRC had a higher IE CD8 area (27% vs. 10%) (Figures 4&5). Conclusions This study evaluated the immune contexture within inherited and sporadic subtypes of dMMR CRCs and ECs, highlighting dif- fering immune infiltration patterns and phenomic densities. In- tegration of multiplex technologies and Tissue Phenomics can enhance the understanding of the dMMR TiME and with poten- tial utility in clinical triaging and to inform immune-oncology clinical trials. Acknowledgements We thank the investigators and participants of the ACCFR and ANGELS Fig. 1 (abstract P39). Consecutive slides from NSCLC resection studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 21 of 272 Fig. 2 (abstract P40). TiME regions of immune infiltration Fig. 1 (abstract P40). Phenotypes Fig. 3 (abstract P40). TiME regions of immune infiltration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 22 of 272 P41 Multiplexed Imaging for the simultaneous detection of nucleic acids and proteins to dissect the tissue immune landscape and microenvironment of viral diseases 1 1 2 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Chi Ngai Chan, PhD , Bokai 1 1 1 1 Zhu , Yunhao Bai, BS , Marc Bosse, PhD , David McIlwain, PhD , Sean 1 1 2 Bendall, PhD , Michael Angelo, MD, PhD , Jacob Estes, PhD , Garry Nolan, PhD 1 2 Stanford University, Stanford, CA, United States; Oregon Health and Sciences University, Stanford, CA, United States Correspondence: Jacob Estes (estesja@ohsu.edu); Garry Nolan (gnolan@stanford.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P41 Background Multiplexed Ion Beam Imaging (MIBI) is a novel imaging modal- ity capable of resolving >40 parameters simultaneously in bio- logical samples. Here, we developed viralMIBI, a highly sensitive method capable of detecting down to single copies of nucleic acids, in addition to protein epitopes. ViralMIBI en- ables the functional dissection of the immune landscape in viral driven diseases, such as that of tumor viruses (HBV, EBV, LCV) and others (HIV, SIV, Zika, Ebola). The combination of vir- alMIBI and cutting-edge cell neighborhood analytical methods will be paramount to better understand the immunological host-pathogen interactions for viral diseases, revealing insights into virus-induced immunodeficiency as well as virus-driven cancers. Methods To allow for the sensitive detection of nucleic acids, we took advantage of a customized branched DNA amplification method that can be easily adapted to a variety of multiplexed Fig. 4 (abstract P40). TiME regions of immune infiltration imaging platforms. Formalin-Fixed and Paraffin-Embedded (FFPE) tissue samples from Rhesus macaque animal models for a number of viral diseases were processed for viralMIBI nucleic acid and protein marker detection. Imaging was performed with the MIBIscope, a secondary ion mass spectrometry based device. Results We have established a robust method for highly multiplexed nucleic acid and protein epitope detection in FFPE tissue samples. As a proof of concept, we were able to detect down to single integrated copies of SIV. The establishment and validation of a Rhesus macaque spe- cific antibody panel allowed for the in-depth characterization of cel- lular identities at the single-cell level, while maintaining their tissue geopositions. Conclusions ViralMIBI enables the MIBI to achieve highly sensitive nucleic acid detection, in addition to its multiplexed protein capabil- ities. Here, we leveage this method for the detection of various viral pathogens. ViralMIBI is also applicable to other targets, such as genomic amplifications frequently seen in cancers, or gene expression studies. The ability to image >40 parameters in tissue samples will vital for a better understanding of im- mune regulation of diseases, such as the establishment of viral related cancers as well as latent tissue reservoirs of pathogens. These discoveries can then be translated to better immuno- therapy treatments against viral driven diseases. Acknowledgements We thank Matt Newgren for tireless technical support on the MIBI instrument, Rachel Finck, Xiao-Jun Ma and Bingqing Zhang for helpful discussions. S.J was supported by a Stanford Dean’s Fellowship and the Leukemia & Lymphoma Society Career Development Program. X.R.-C. was supported by a long-term EMBO fellowship. This work was supported by grants from the FDA, NIH, Parker Institute for Cancer Immunotherapy, the Bill and Melinda Gates Foundation, as well as Fig. 5 (abstract P40). TiME regions of immune infiltration the Rachford and Carlota A. Harris Endowed Professorship to G.P.N. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 23 of 272 P42 An integrated multiplexing approach for the immunoprofiling of the tumor microenvironment of ovarian granulosa cell tumors 1 2 1 1 Juncker-Jensen, PhD , Tyvette Hilliard , Nicholas Stavrou , Erinn Parnell , 1 1 1 1 Judy Kuo , Eric Leones , Flora Sahafi , Josette William, PhD, MD , Sharon 2 1 Stack , Anna Juncker-Jensen 1 2 NeoGenomics, Aliso Viejo, CA, United States; University of Notre Dame, South Bend, IN, United States Correspondence: Anna Juncker-Jensen (anna.juncker- jensen@neogenomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P42 Background Ovarian granulosa cell tumors (GCTs) are rare tumor accounting for 2-5% of all ovarian cancers. The main current treatment for GCT is surgery, however a subset require chemotherapy for residual and re- current disease. GCT malignancies are often low-grade, however a clinical characteristic of these tumors is a tendency for late recur- rence which is the most critical factor for GCT death. As the onset of recurrence is unpredictable, future research should focus on identify- ing both biomarkers for prognosis prediction, as well as targets that could help guide clinical trials in the development of targeted ther- apies for this rare indication. As GCTs are rare tumors making tissue Fig. 1 (abstract P41). Validation of viralMIBI: Detection of SIV in availability very limited, we used a dual multiplexing approach in infected tissue order to maximize the data output from a total of 14 FFPE tumor samples (6 primary tumors, and 8 recurrent tumors). Methods For protein multiplexing we have used MultiOmyx™, an immunofluores- cence (IF) multiplexing assay utilizing a pair of directly conjugated Cya- nine dye-labeled (Cy3, Cy5) antibodies per round of staining (Figure 1). Each round of staining is imaged and followed by dye inactivation en- abling repeated rounds of staining and deactivation, while deep learn- ing based cell classification algorithms identify positive cells for each biomarker. We generated a 15-marker panel consisting of CD3, CD4, CD8, FoxP3, CD68, CD163, HLA-DR, CD34, CTLA-4, PD-1, PD-L1, Ki67, vimentin, S100, and Pan Cytokeratin. For the gene expression analysis RNA was extracted from the adjacent 10 μm section and then analyzed using the Nanostring nCounter assay, specifically the 770 gene PanCan- cer Immune Panel. Hybridization, purification and immobilization and counts were based on manufacturer’sprotocol. Results On protein level we confirmed previous findings that ovarian GCTs are so-called “cold” tumors, with a very low density of T cell infiltra- tion. When we analyzed the presence of macrophages in the tumor microenvironment however, we found a 113% increase in TAM dens- ity in recurrent tumors compared to primary tumors. When searching for markers differentially expressed between primary and recurrent tumors we detected 4 genes in our PanCancer immune panel that were either significantly down-regulated (CCND3 or TOLLIP), or up- regulated (MAP3K and TNFSF4) in recurrent tumors. TNFSF4 encodes the protein OX40L, and interestingly a high expression of its receptor OX40 has previously been shown to be indicative for response to chemotherapy in recurrent ovarian cancer [1]. Conclusions We have used a dual multiplexing approach on both gene and pro- tein level in order to immunoprofile the tumor microenvironment of ovarian rare granulosa tumors. Reference 1. Ramser M, Eichelberger S, Däster S, Weixler B, Kraljević M, Mechera R, Tampakis A, Delko T, Güth U, Stadlmann S, Terracciano L, Droeser RA, Singer G. High OX40 expression in recurrent ovarian carcinoma is Fig. 2 (abstract P41). Detection of single integration events of SIV indicative for response to repeated chemotherapy. BMC Cancer. with viralMIBI 2018;18:425-433. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 24 of 272 custom panel of more than 25 markers on the PBMC samples and acquired the images using a Keyence benchtop microscope. Results The CODEX system was used to generate highly multiplexed im- mune profiles of human PBMC samples using an optimized cus- tom panel of CODEX antibodies. The images were processed using the CODEX Software Suite and cell phenotypes were clus- tered and annotated using the Multiplexed Analysis Viewer (MAV). Antibody specificity and panel performance were evalu- ated by assessing co-expression and mutually exclusive expres- sion of relevant immune markers with the CODEX analysis pipeline. Conclusions Simultaneous analysis of tens of markers in blood or plasma sam- ples can have several applications in the discovery of cellular bio- markers, immune monitoring and drug discovery and development. This preliminary study shows the compatibility of the CODEX system with cell suspensions for highly multiplexed, single-cell analysis and offers a more cost-effective method for immune profiling of blood samples. P44 Solar-IHC: Cell-to-cell distances in the tumour immune microenvironment of Hepatocellular Carcinoma has the potential Fig. 1 (abstract P42). Immunofluorescent overlay image of GCT to prognosticate survival 1 2 2 recurrent tumor Matthew Leong, NA , Toh Han Chong , Choo Su Pin , Kiat Hon Lim 3 2 2 4 Tony , Joycelyn Lee , David Wai , Poh Sheng Joe Yeong , Jin Miao Chen, PhD Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, Singapore; National Cancer Centre Singapore, Singapore, Singapore; Singapore General Hospital, Singapore, P43 Singapore, Singapore; Department of Anatomical Pathology, Singapore A novel platform for highly multiplexed, single-cell imaging of cell General hospital, Singapore, Singapore, Singapore; Singapore suspensions Immunology Network, Agency of Science, Technology and Research, 1 2 1 1 Anum Khan , Won-Mean Lee , Jon Mulholland , Dhananjay Wagh , John Singapore, Singapore, Singapore 1 2 Coller , Gabriel Mercado Correspondence: Jin Miao Chen (Chen_Jinmiao@immunol.a- 1 2 Stanford University, Palo Alto, CA, United States; Akoya Biosciences, star.edu.sg) Menlo Park, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P44 Correspondence: Won-Mean Lee (wmlee@akoyabio.com); Gabriel Mercado (gmercado@akoyabio.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P43 Hepatocellular carcinoma (HCC) is a lethal cancer, being the fourth leading cause of cancer-associated mortality worldwide Background due to its low five-year survival and high reoccurrence rates [1], Analyzing populations at the single cell level has become increas- and identifying indicators of prognosis is key in developing novel ingly important in the study of cancer and autoimmune disorders treatments and improving survival of HCC patients. With the ad- due to high levels of population heterogeneity and rare cell phe- vent of digital pathology, the immune-architecture of solid tu- notypes that can drive disease pathogenesis and progression. mours has become a central interest of cancer research and has Until recently, characterizing protein markers on single cells was been studied for the development of predictive and diagnostic limited to a handful of markers due to the technical and logis- applications. Here, we have assessed if intercellular Euclidean dis- tical challenges of flow cytometry platforms. New advancements tances in the tumour immune microenvironment can possibly be in single cell analysis technologies have enabled researchers to used to predict patient prognosis. study more than 30 parameters per cell. But these platforms are Methods expensive and require significant panel design, thereby limiting In this study, biopsies were taken from 110 HCC patients who access and usability. Here we demonstrate the use of the recently underwent surgical resection. The solar-IHC pipeline involves ar- launched CODEX System to generate an in-depth immune profile ranging the liver biopsies into tissue arrays and subsequently of human PBMC samples. studying them using automated multiplex immunohistochemistry/ Methods immunofluorescence (mIHC/IF) protocol developed in Singapore The CODEX® System is an affordable, benchtop instrument that General Hospital, with biomarkers Ecadherin, CD3, CD8, CD103 integrates with existing fluorescence microscopes and enables and PD1 [2], followed by image analysis software inForm version highly multiplexed imaging of over 40 markers in fresh frozen 2.4.2. and FFPE tissue samples. The CODEX technology uses a DNA- Results based barcode library to label antibodies and iterative cycles of Ecadherin was adopted as the tumour cell marker, and 26 im- adding and removing cognate dye-labeled oligonucleotides to mune cell phenotypes are defined by variable levels of immune reveal the staining of three markers per cycle. Data acquisition markers CD8, CD103, and PD-1 (as shown in Table 1) Dimension- is fully automated by the CODEX instrument. We tested a ality reduction and unsupervised clustering of the distances Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 25 of 272 between tumour and immune cell phenotypes showed distinct P45 clusters of patients with significant differences in clinical out- Multiplex immunofluorescence staining, whole slide imaging, and comes. Long cell-cell distances between immune cell phenotypes spatial phenotyping of T-cell exhaustion, regulatory T cells, and and tumor cells was associated with an improved overall survival myeloid-derived suppressor cells in tumor FFPE samples 2 3 1 1 (p-value = 0.02) and disease-free survival (p-value = 0.01), while Kyla Teplitz , Katir Patel, PhD , Michael Tomac, MS , Kate Lillard, PhD , 3 1 the opposite was true for short cell-cell distances between im- Mael Manesse, PhD , Anne Hellebust, PhD 1 2 mune cell phenotypes and tumor cells. This was observed in the Indica Labs, Inc., Alcester, United Kingdom; Rarecyte, Inc, Seattle, WA, analysis of all CD8+, CD8-, CD103+, CD103-, PD1+ and PD1- cells, United States; Ultivue, Inc, Cambridge, MA, United States possibly due to the suppressive immunomodulatory effect Ecad- Correspondence: Kate Lillard (kate@indicalab.com) herin+ tumour cells has on neighbouring immune cells [3]. Fur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P45 thermore, machine learning enabled the prediction of clusters with considerable accuracy, with a K-fold cross-validation of 91%. Background This indicates a strong association of cell-cell distances with pa- The immune cell milieu that comprises the tumor microenviron- tient survival, and a robust reproducibility of distance pattern- ment (TME) is highly heterogeneous and complex. Depending based predictors. on biological interactions and functional state, immune cell Conclusions populations can either promote or suppress tumor progression. In this study, our data suggests that the analysis of intercellular CD8+ T cells, for example, are the primary mediators of anti- distances has the potential to be used as a prognostic indicator tumor immunity; however, they are often ineffective either be- in HCC. Coupled with next generation machine learning tech- cause they are unable to infiltrate the tumor or because they niques, this novel approach to cell-to-cell distance analysis has become functionally exhausted [1,2], Pathologically activated the potential to be an easily implementable algorithm to predict myeloid-derived suppressor cells (MDSCs) which infiltrate the patient prognosis in HCC. This bioinformatics approach can also tumor are also associated with tumor progression [3,4]. Multiple be utilized in the analysis of other biomarkers and cancer types, biomarkers are required to accurately identify these individual and this brings exciting prospects for the future of cancer immune cell types and their functional states. In this work, we research. employ advanced multiplexing techniques to observe biologic- ally and functionally distinct T cell and MDSC populations and References to quantify their density and distribution within the TME of sev- 1. McGlynn KA, Petrick JL, London WT. Global epidemiology of eral tumor types. hepatocellular carcinoma: an emphasis on demographic and regional Methods variability. Clinics in liver disease. 2015;19(2):223-38. UltiMapper assays were used to perform multiplex immunofluor- 2. LimJCT,Yeong JPS, LimCJ, OngCCH,WongSC, Chew VSP, escence on multiple tumor FFPE samples (lung, colorectal, breast). Ahmed SS, Tan PH, Iqbal J: An automated staining protocol for Three multilpex panels were run in this study: UltiMapper PD-1 seven-colour immunofluorescence of human tissue sections [CD3, CD45RO, PD-1, CK/Sox1], UltiMapper T-reg[CD4, CD8, FoxP3, for diagnostic and prognostic use. Pathology. 2018 CK, Sox10], and UltiMapper MDSC[CD11b, CD14, CD15, HLA- Apr;50(3):333-341 DR].FFPE slides were stained using the BOND RX autostainer from 3. Nagl S, Haas M, Lahmer G, Büttner-Herold M, Grabenbauer GG, Fietkau R, Leica Biosystems and scanned on the CyteFinder® II HT Instru- et al. Cell-to-cell distances between tumor-infiltrating inflammatory cells ment from RareCyte, Inc. This instrument performs high-speed, have the potential to distinguish functionally active from suppressed in- whole-slide scanning in the 5 channels used in the UltiMapper flammatory cells. Oncoimmunology. 2016;5(5):e1127494. Kits and outputs an open source, stitched, pyramidal TIFF. Image Ethics Approval analysis was conducted using HALO 3.0 software to perform cell This study was approved by the Institutional Review Board (IRB), approval phenotyping, proximity analysis, image registration, and density number 2014/590/B. mapping. Results Cell counts for relevant phenotypes were obtained for each panel to identify exhausted T cells, T-regs, cytotoxic T cells, M-MDSCs, and Table 1 (abstract P44). See text for description PMN-MDSCs. Spatial analysis was employed to map the degree of T- cell infiltration and exhaustion correlating to T-reg and MDSC expres- sion in the tumor microenvironment. Conclusions Here we present a workflow for tackling the complexity of the tumor immune microenvironment by leveraging high-quality multiplex panels, high-speed whole-slide imaging, and quanti- tative spatial analysis. UltiMapper assays used in this study (PD-1, T-reg, and MDSC) were able to identify single-cell phe- notypes through co-localization and negative selection of markers. Using HALO image analysis, cell populations were enumerated and quantified to measure the level of T-cell ex- haustion caused by T-cell regulation and myeloid-derived im- mune cell suppression. References 1. Fridman WH, Zitvogel L, Sautes-Fridman C, Kroemer G. The immune con- texture in cancer prognosis and treatment. Nat. Rev. Clin. Oncol. 2017; 14:717–734. 2. Thommen DS, Schumacher TN. T Cell Dysfunction in Cancer. Cancer Cell. 2018; 33:547-562. 3. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 2012; 12:253–268. 4. Kumar V, Patel S, Tcyganov E, Gabrilovich DI. The Nature of Myeloid- Derived Suppressor Cells in the Tumor Microenvironment. Trends Immu- nol. 2016; 37:208–220. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 26 of 272 P46 medicine but without the complex workflow, safety, and half-life limi- Same-slide multiplex immunofluorescence and brightfield tations. In this study, we compared the behavior of monocytes histological staining as a new research tool for fast and loaded with nanoparticles in vitro and nanoparticles injected intra- comprehensive pathology assessment of the tumor venously and subsequently taken up by phagocytic cells (in situ load- microenvironment ing) and imaged using MPI the differences in biodistribution and Mael Manesse, PhD, Douglas Wood, PhD, Heike Boisvert, PhD, Sean migration of monocytes in in naïve and tumor-bearing and naïve Downing, PhD, Mael Manesse, PhD (control) mice. Ultivue, Cambridge, MA, United States Methods Correspondence: Mael Manesse (mael.manesse@ultivue.com) Twenty mice were implanted with 300,000 4T1 tumour cells in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P46 4th mammary fat pad. CD11b+ mouse monocytes were harvested using EasySep® Mouse CD11b positive selection kit II (StemCell Tech- Background nologies). The isolated monocytes were prelabeled with Vivotrax® Innovative and efficient translational research tools enabling a better (100 μg/mL). On day 7 post-implantation, either 5 million prelabeled understanding of the tumor and its microenvironment are a keystone cells or free Vivotrax (6 mg/kg) were intravenously injected into nor- of the development of digital pathology. Current immunohistochem- mal or tumor-bearing mice for in vitro or in situ targeting experi- istry (IHC) methods limit the depth of information from a single tis- ments (N=5 mice for all four groups). 3D MPI images using a sue sample to a single target in the case of chromogenic staining, or MOMENTUM MPI system (Magnetic Insight) were acquired 1, 4, 7 to sample morphology and general cell identification in the case of and 10 days after injection. MicroCT images (CT120, Trifoil Imaging) hematoxylin and eosin staining (H&E). True phenotyping requires the were acquired and co-registered using VivoQuant (Invicro). Tumors, use of a single section, as serial sections may not contain the same liver, spleen and draining lymph nodes were then harvested, imaged, cells, especially small immune cells such as T-cells. Multiplex im- fixed, and stained with Prussian blue and analyzed for iron contents. munofluorescence (mIF) methods have been established to provide Results insights into a wide number of markers of interest and their spatial Tumor-bearing mice showed a significant accumulation of nanoparti- context in a single sample. Here, we demonstrate a new research ap- cles for both the in situ and in vitro targeting methods, although the proach combining multiplexed detection of protein markers with time and amount of accumulation was different. For both experi- standard H&E pathology review in tumor samples, in a streamlined, ments, nanoparticles were predominately detected in the expanding single-day sample-to-answer workflow. margins of the tumor. For the in vitro labeled monocytes, accumula- Methods tion was rapid, with the maximum accumulation being at 24 hours InSituPlex technology was used to perform multiplex immunofluores- post-injection, while for the in situ labeled cells, accumulation was cence staining of formalin-fixed, paraffin-embedded (FFPE) samples slower. from human tonsil and primary tumor biopsies on the Leica Biosys- Conclusions tems BOND RX autostainer. The tissues were then imaged in five dis- By combining the sensitivity, specificity as well as accurate quantita- tinct fluorescent channels (DAPI, FITC, TRITC, Cy5, Cy7) before being tion potentials of MPI, information can be obtained on labeled stained using standard H&E protocols and imaged again. Fluorescent monocytes and their biodistribution in tumour models. Other cells and brightfield whole-slide images were acquired on a ZEISS AxioS- can also be labeled (dendritic cells, MDSCs, NKs, and T cells) and this can.Z1 slide scanner. Images of the same tissue section were co- information can be utilized to better understand the factors influen- registered and fused into a single image for analysis using Indica cing immune cell migration in and around tumors. Labs HALO software. Results P48 The InSituPlex technology enables deep phenotyping of immune Turning ‘cold’ tumours ‘hot’: Guided magnetic hyperthermia for cells through colocalization and co-expression of multiple protein tumour immune stimulation markers in tumor samples. Phenotypic information was then overlaid 1 1 2 1 Patrick Goodwill , Daniel Hensley , Zhi Wei Tay , Elaine Yu , James with the H&E image of the same section to facilitate identification 1 1 1 1 Mansfield, Msc , Blayne Kettlewell , Ryan Orendorff , Kyle Fields , Steve and immuno-profiling of specific cells in the tumor and its environ- Conolly ment. The fused images were also analyzed to provide cell counts, 1 2 Magnetic Insight, Alameda, CA, United States; University of California distance mapping, and expression levels of each of the markers. Berkeley, Berkeley, CA, United States Conclusions Correspondence: James Mansfield (jim@jmansfield.com) In this work, we present a new modality for pathology research with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P48 a convenient workflow that enables fast tissue review and deep immuno-profiling and phenotyping of the tumor via fusion of H&E Background and mIHC staining of the same tissue section. Cancer immunotherapy is now the “fifth pillar” of cancer thera- peutics [1]. Although hugely successful, there are limitations. In P47 many studies, less than half the patients are responsive to ther- Imaging cancer immunology: Systemic tracking of immune cells apy. One hypothesis is that refractory tumours are immunologic- in vivo with magnetic particle imaging ally ‘cold’– i.e., there are insufficient immune cells in the tumour 1 1 2 James Mansfield, Msc, Gang Ren , Jeff Gaudet , Yanrong Zhang , Sara for the therapy to be efficacious [2]. Thus, methods to stimulate 2 2 1 Ghobadi , Max Wintermark , Patrick Goodwill an immunogenic response in solid tumours to improve immuno- 1 2 Magnetic Insight, Alameda, CA, United States; Stanford University, Palo therapy efficacy are desirable. Alto, CA, United States Hyperthermia is known to induce a local immunogenic response, Correspondence: James Mansfield (jim@jmansfield.com) making it a potential adjunct to radiation and immune therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P47 One hyperthermia method is Magnetic Fluid Hyperthermia (MFH), which is based on electromagnetic heating of magnetic nanoparti- Background cles (MNPs) [3,4]. , However, poor control of heating localization and The rapid growth of research into immuno-oncology research has magnitude have prevented MFH’s widespread clinical adoption. fueled a need to track be able to determine the location of a variety Magnetic Particle Imaging (MPI) is an emerging tracer imaging tech- of immune cells systemically and in solid tumors. However, existing nique that directly detects and quantitates superparamagnetic iron- methods for cell tracking that have generally been insufficient. Mag- oxide nanoparticles with exceptional contrast and high sensitivity at netic Particle Imaging (MPI) is a novel tomographic molecular im- millimeter-scale resolutions [5]. MPI’s contrast is similar to nuclear aging technique that can be used to non-invasively track iron-oxide medicine, but without the complex workflow, safety, and half-life lim- tagged immune cells in 3D in vivo, with contrast similar to nuclear itations of a radioactive tracer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 27 of 272 Methods conventional Treg and CTL populations in GIT tissue sections Here we describe how MPI and MFH can be combined to pro- from IBD patients versus normal individuals by multiplex duce spatially localized heating and accurate control of heating immunofluorescence. magnitude. Spatial localization is achieved using a unique Methods mechanism, magnetic localization. Localization is effected by Conventional Treg are typically defined as lymphocytes with a CD3+/ using a strong magnetic field gradient to produce a “field-free CD4+/CD25+/FoxP3+ immuno-phenotype. This complex antigenic region” (FFR) where nanoparticles are heated, while nanoparti- signature has made it difficult to definitively label Treg populations cles outside the FFR are quenched and do not heat. The use of in tissue sections by immunohistochemistry. We combined a 5-plex an FFR thus enables millimeter-scale control over which MNPs (CD3, CD4, CD8α, CD25, FoxP3) immunofluorescence assay using Ulti- are heated [6-8]. vue InSituPlex® multiplex technology with image analysis using Results Indica Labs HaloTM software to identify, localize and enumerate: 1) MPI is first used to quantitate the MNPs prior to heating, to enable total CD3+ T cells, 2) CD8α+ cytotoxic T lymphocytes (CTL) and 3) treatment planning and prediction of the heating dose. MFH can CD3+/CD4+/CD25+/FoxP3+ conventional Treg in formalin-fixed then be induced in target regions of interest located anywhere in paraffin-embedded (FFPE) sections of GIT from patients with UC and the body while avoiding regions containing MNPs that should not be CD versus controls. Using this approach, we were able to definitively heated, such as the liver or lymph nodes. identify and enumerate these immune cell populations on single Conclusions FFPE tissue sections from each specimen. Combined MPI-MFH enables new treatment workflows that ex- Results ploit spatially localized MFH and accurate control of heating We found greater Treg and CTL cell densities (cells/mm2) in colon magnitude. These workflows may resemble image-guided radi- from CD and UC patients versus controls and higher densities of Treg ation therapy or image-guided high-intensity focused ultra- and lower densities of CTL in small intestine from patients with CD sound. Combined MPI-MFH also prevents damage to nearby versus controls. healthy tissue while enabling new applications such as targeted Conclusions immunogenic stimulation. MPI-MFH also enables new heat- The Ultivue InSituPlex© assay was capable of discretely localizing actuation applications involving systemic injection of MNPs conventional Tregs and CTL in human tissues. This multiplex platform followed by local targeting such as local release of a drug [9] could be used to simultaneously localize Tregs and CTL in FFPE surgi- (break thermally labile bonds/nanocarriers) without requiring ac- cal resections and biopsies of neoplastic tissue as well. tive chemical targeting. While currently only available for small animal use, its underlying physics does not prevent its transla- References tion to human sizes 1. Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, Sung JJY, Kaplan GG. References Worldwide incidence and prevalence of inflammatory bowel disease in 1. Zaidi, N; Jaffee, E. J Clin Invest (2018). the 21st century: a systematic review of population-based studies. Lancet. 2. Sharma, P. et al. Curr Opinion Immunol (2016). 2018; 390:2769-78. 3. Jordan, A. et al. Journal of Magnetism and Magnetic materials, (1999). 2. Corridoni D, Arseneau KO, Cominelli F. Inflammatory bowel disease. 4. Latorre, M. Puerto Rico health sciences journal, 28(3) (2009). Immunol Lett. 2014; 161:231-5. 5. Gleich, B. & Weizenecker, J. Nature 435,1214–1217 (2005). 3. van Herk EH, Te Velde AA. Treg subsets in inflammatory bowel disease 6. Murase, K. Physica Medica, 29(6), 624-630 (2013). and colorectal carcinoma: Characteristics, role, and therapeutic targets. J 7. Hensley, D. Physics in Medicine & Biology, 62(9), 3483 (2017). Gastroenterol Hepatol. 2016; 31:1393-404. 8. Tay, Z. ACS nano, 12(4), 3699-3713 (2018). 4. Yamada A, Arakaki R, Saito M, Tsunematsu T, Kudo Y, Ishimaru N. Role of 9. Liu, J. F., Small, 14(44), 1802563 (2018). regulatory T cell in the pathogenesis of inflammatory bowel disease. World J Gastroenterol. 2016; 22:2195-205. Ethics Approval P49 Human tissues were obtained from the National Disease Research Use of Ultivue InSituPlex® multiplex immunofluorescence to Interchange (NDRI) with support from NIH grant U42OD11158. Tissues localize and quantify regulatory T lymphocytes in formalin-fixed were collected for research purposes under IRB-approved informed paraffin-embedded human tissue sections consent and collection procedures and provided to Pfizer in accordance 1 1 2 Shawn O'Neil, DVM, PhD , Renee Huynh , Courtney Hebert , Jamie with applicable government regulations and guidelines. 2 2 1 1 Buell , Sean Downing, PhD , John Jakubczak, PhD , Yutian Zhan, MS 1 2 Pfizer, Cambridge, MA, United States; Ultivue, Inc., Cambridge, MA, United States P50 Correspondence: Shawn O’Neil (llospo@gmail.com) Rapid high-plex staining and simultaneous imaging for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P49 immunophenotyping of tissue sections 1 2 1 Benjamin Pelz, PhD , Daniel Migliozzi , Diego Dupouy , Anne-Laure 3 3 2 Background Leblond , Alex Soltermann , Martin Gijs 1 2 The inflammatory bowel diseases ulcerative colitis (UC) and Lunaphore Technologies, Lausanne, Switzerland; École Polytechnique Crohn’s disease (CD) are chronic, relapsing inflammatory disorders Fédérale de Lausanne, Lausanne, Switzerland; Universitätsspital Zürich, of the gastrointestinal tract (GIT) that affect millions of individuals Zurich, Switzerland worldwide [1]. The pathogenesis of these disorders is thought to Correspondence: Benjamin Pelz (benjamin.pelz@lunaphore.com) involve dysregulation of mucosal immune homeostasis in the GIT Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P50 in response to environmental factors in genetically susceptible in- dividuals [2]. Regulatory T cells (Treg) are CD4+ T lymphocytes Background that play a central role in peripheral immune tolerance, actively The tumor microenvironment plays a vital role in cancer development. inhibiting inflammation upon antigenic stimulation. There are two Multiplex immunostainings allow studying the interaction of different cell major populations of Treg: conventional Treg and TR1 cells [3]. types in the tumor microenvironment using a single tissue slide. Though Conventional Treg arise from the thymus (tTreg) or can be in- several techniques are available to perform high-plex stainings, they re- duced in the periphery (pTreg); both tTreg and pTreg constitu- quire intensive manual handling, are highly time consuming or not com- tively express FoxP3 and CD25 (IL-2Rα). An imbalance in patible with tissue sections on standard microscope slides. Here we conventional Treg and effector T cells in the GIT microenviron- present a fully automated microscope integrated method for rapid high- ment is thought to play a part in the pathogenesis of inflamma- plex sequential fluorescent immunostaining and imaging of tissue tory bowel disease (IBD) [4]. Thus, we sought to quantify sections. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 28 of 272 Methods P51 Formalin-fixed, paraffin-embedded tissue sections underwent manual dewax- Phenotypic and spatial analysis of inter- and intra-tumor ing and antigen retrieval step. All subsequent steps of staining, antibody elu- heterogeneity using multiplexed ion beam imaging tion and imaging were automated on the microscope integrated (MIBI) 1 2 2 microfluidic device. A single tissue section was stained sequentially for 24 dif- Jason Ptacek, PhD , Robert Johnson, PHD , Joann Palma, PHD , Jay 1 1 1 1 1 ferent immunophenotyping and tissue structural markers. Each staining cycle Tarolli , Rachel Finck , Murat Aksoy , Yi Zhang , Jessica Finn , Jason consisted of incubation of the tissue section with a pair of mouse and rabbit Ptacek, PhD 1 2 primary antibodies, followed by the corresponding fluorescently labelled sec- Ionpath, Inc, Menlo Park, CA, United States; AbbVie, North Chicago, IL, ondary antibodies and DAPI. The section was imaged after each staining United States cycle and subsequently eluted before staining the next pair of markers. Correspondence: Jessica Finn (jessica.finn@ionpath.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P51 Our microscope integrated microfluidic system allowed automated 24-plex staining with conventional primary and fluorescently labelled secondary Background antibodies in less than five hours, including image acquisition steps. The Elucidating both the cell types present in the tumor microenvir- microfluidic tissue processor enabled fast fluidic exchange and thereby re- onment and the spatial relationship between immune and sulted in reduced staining time down to 10-12min per marker. Integration cancerous cells is at the forefront of immunotherapy research. of a window into the microfluidic chip allowed direct tissue imaging under To address this, MIBI has been developed to image up to 40 the microscope avoiding the removal and mounting of the slide. Protocol markers at single cell resolution. optimization resulted in a high signal to background noise ratio for each Methods marker and complete elution of antibodies from the previous staining step. Staining of 10 NSCLC formalin-fixed paraffin embedded (FFPE) A comparison of a 10-plex staining with standard chromogenic stainings tissue sections was performed similarly to traditional IHC ex- on sequential sections showed high concordance for the stained area on cept that a panel of 20 metal labeled antibodies were stained tonsil as well as lung cancer tissue sections (Figure 1). simultaneously. The tissue was imaged at subcellular resolution Conclusions using an ion beam and time-of-flight secondary ion mass spec- With the microscope integrated microfluidic system, it is possible to trometry (ToF-SIMS). The masses of detected species were then perform fast multiplex stainings including image acquisition without assigned to target biomolecules given the unique label of each the need to handle the tissue slide. Moreover, due to the sequential na- antibody and multi-step processing and segmentation were ture of the system it would be easily possible to further increase the performed to create images of the TME and enable quantitative number of markers in the multiplex staining. We foresee this technique metrics of different cell subsets. to greatly facilitates the execution of high-plex stainings and thereby Results the discovery of novel tumor-microenvironment interactions. Control samples imaged at study start and end showed con- sistent marker quantification (inter-run R2>0.99), indicating MIBI staining and acquisition is reproducible and robust. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. Highly expressed nuclear, membrane, and cytoplasmic markers were utilized in conjunc- tion to accurately determine cell boundaries in tissue images. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relation- ships between cells of different types. At the highest level, cells were classified as positive for markers that are indicative of immune and tumor cells based on measured intensities of marker expression, such as CD45+ and keratin+ cells in epi- thelial cancers, respectively. Co-expression of markers were used to classify immune cells into subsets, including T cells and macrophages (Figure 1). Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. Conclusions MIBI offers high-parameter capability, at sensitivity and reso- lution uniquely suited to understanding the complex tumor im- mune landscape, including the spatial relationship of immune and tumor cells and the expression of immunoregulatory proteins. Fig. 1 (abstract P50). Automated microfluidics-assisted multiplexing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 29 of 272 Conclusions Anti-ILT3 mAb treatment induced a conversion from intra- tumoral immune suppression to activation in a SK-MEL-5 hu- NSG tumor model. CyTOF in anti-ILT3 drug discovery holds promise to effect a paradigm-shift in our ability to under- stand MOA and evaluate the impact of therapeutic interven- tions that can accelerate biomarker discovery and drug development. The anti-ILT3 mAb activity in human immune cells in vitro and tumor efficacy in vivo is presented in a companion poster. Ethics Approval The study was approved by Merck Institutional Animal Care and Use Committee, approval number 2022-200518-FEB. P53 Pixelwise H-score: a novel digital image analysis-based metric to quantify membrane biomarker expression from IHC images Amy-Jackson Fisher, Pamela Whalen, Cory Painter, Pamela Vizcarra, Eric Powell, MD, Sripad Ram, PhD Pfizer, Inc., San Diego, CA, United States Correspondence: Sripad Ram (sripad.ram@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P53 Fig. 1 (abstract P51). Cell segmentation and classification Background Immunohistochemistry (IHC) assays play a central role in evaluat- ing biomarker expression in tissue sections for diagnostic and re- P52 search applications. Manual scoring of IHC images, which is the CyTOF in anti-ILT3 mAb drug discovery - humanized tumor model current standard of practice, is based on qualitative criteria and selection and in vivo PD/biomarker exploration are known to have several shortcomings in terms of reproduci- Yujie Qu, MD, Alan Byford, Caniga Michael, Ying Huo, Barbara Joyce- bility and scalability to large scale studies. While digital image Shaikh, BS, Laurence Fayadat-Dilman, Veronica Juan, Carl Mieczkowski, analysis (DIA) based approaches hold significant promise to over- Laura Bald, Jeanne Baker, Michael Meehl, Scott Pruitt, MD, PhD, Stephen come these limitations, current DIA methods pose several chal- Alves, Lily Moy, Philip Brandish, PhD, Jie Zhang-Hoover, Jie Zhang- lenges that have limited their widespread use in analyzing Hoover clinical samples. Merck, Boston, MA, United States Methods Correspondence: Jie Zhang-Hoover (jie.zhang-hoover@merck.com) We introduce a novel DIA metric, the pixelwise H-score (pix H- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P52 score), that quantifies biomarker expression from whole-slide scanned IHC images. Pix H-score is unique in that it does not rely Background on the detection of individual cells or the delineation of subcellu- ILT3 on human monocytic myeloid cells is linked to immune tol- lar compartments (e.g. nucleus and cell membrane) which are ne- erance in transplantation and immune suppression in cancer. cessary for traditional scoring algorithms such as the H-score. All Anti-ILT3 mAb is being developed as a cancer immunotherapy to DIA metrics are calculated using either commercially available reverse the suppression and increase T cell activation. To evaluate (HALO, Visiopharm) or open-source (QuPath) digital pathology the effect of an anti-ILT3 mAb in vivo, we sought to select an ap- software packages. propriate humanized tumor model and identify immune activa- Results tion signatures in the model that are associated with the We compute the pix H-score, the ATM score [1] and the trad- treatment efficacy. itional H-score [2] from IHC images for several biomarkers includ- Methods ing PD-L1. Our results show that the pix H-score exhibit tight Humanized tumor models were generated by subcutaneously concordance to multiple orthogonal measurements such as mRNA implanting Panc 08.13 or SK-MEL-5 tumor cells in NSG mice levels and pathologist score, and provide consistently better per- engrafted with human cord-blood CD34+ hematopoietic stem cells formance over other DIA metrics. (hu-NSG). Tumor-bearing mice were treated with a human-mouse Conclusions chimeric anti-ILT3 mAb. Single cell mass cytometry (CyTOF) that sim- We anticipate that the new metric introduced here will be ultaneously quantifies over 40 cell surface and intracellular markers broadly applicable to quantify biomarker expression from a was used to phenotype tumor infiltrating cells (TILs) in these tumor wide variety of IHC images. Although not shown here, the new models. metric can also be applied to immunofluorescence images. Results Moreover, these results underscore the benefit of digital image The CyTOF phenotyping of untreated mice showed an overall im- analysis-based approaches which offer an objective, reprodu- mune suppressive environment in the tumor in both Panc 08.13 and cible and highly scalable strategy to quantitatively analyze IHC SK-MEL-5 hu-NSG models. ILT3 expression was detected in both images. models. However, the levels of ILT3 expression on CD14+ myeloid cells and percentage of CD14+ myeloid cells among TILs were higher in SK-MEL-5 compared to Panc 08.13 tumors, which led to the selec- References tion of the SK-MEL-5 model for further exploration. Anti-ILT3 mAb 1. Choudhury KR, Yagle KJ, Swanson PE, Krohn KA, Rajendran JG, A Robust treatment in the SK-MEL-5 hu-NSG model increased activation of Automated Measure of Average Antibody Staining in CD14+ myeloid sub-populations in TILs by viSNE CyTOF clustering Immunohistochemistry Images. J Histochem Cytochem. 2010; 58: 96-107. analysis. Furthermore, the treatment increased levels of CD69 and 2. Hatanaka Y, Hashizume K, Nitta K, Kato T, Itoh I, Tani Y, Cytometrical HLA-DR expression on CD4+ T cells, while reducing the percentage image analysis for immunohistochemical hormone receptor status in of naïve CD4+ T suppressor cells in CD45+ TILs. breast carcinomas. Pathol Int. 2003; 53: 693-699. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 30 of 272 P54 P55 Development of a 9-color immunofluorescence assay using Combining the best of two worlds: Transfer of multiplex tyramide signal amplification and multispectral imaging for immunofluorescence images from non-small cell lung carcinoma patients high-throughput studies on FFPE tissue sections into pseudo multiplex chromogenic immunohistochemistry images 1 2 1 1 Bethany Remeniuk, PhD, Carla Coltharp, PhD, Kristin Roman, MS, Lorenz Rognoni, PhD ,Ana HidalgoSastre, PhD ,Linda Brützel , Philipp Wortmann , 1 1 1 3 Chichung Wang, Clifford Hoyt, MS Monika Baehner ,Marco Testori ,Jessica Chan , Bonnie Phillips, PhD , Katir Patel, 3 3 4 4 1 Akoya Biosciences, Hopkinton, MA, United States PhD , Sean Downing, PhD , Alex Haragan ,JohnField , Florian Leiss, PhD 1 2 3 Correspondence: Clifford Hoyt (choyt@akoyabio.com) Definiens AG, Munich, Germany; Definiens, Munich, Germany; Ultivue, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P54 Cambridge, MA, United States; Liverpool University Hospital, Liverpool, United Kingdom Background Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) In cancer research, advancing our understanding of the under- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P55 lying mechanisms driving disease progression is key to devel- oping new therapeutic regimens and improving patient Background outcomes. Over the past several years, multiplex immunofluor- One of the biggest challenges in multiplex chromogenic IHC (mIHC) is to escence (mIF) has played a vital role in elucidating novel accurately identify and quantify double positive cells. Multiplex immuno- immune-tumor interactions and identifying targets of interest fluorescence (mIF) instead, allows for visualization of plenty of biomarkers for drug discovery and development. at once with true co-localization. However, visualizing tissue morphology Emerging studies utilizing mIF have revealed complex cell-to- in mIF images can be challenging and the vast color combinations over- cell interactions within the tumor microenvironment (TME), whelming. Pathologists are key to retrieve biological information from however, greater interrogation of the biology comprising multiplex assays and provide annotations for assay validation. To support these interactions, including cellular composition and pathologist analysis, resections of non-smallcelllungcarcinoma (NSCLC) functional status, require higher levels of multiplexing. With patients were stained with mIF and displayed as pseudo mIHC images. the rapidly increasing number of available multiplexing ap- Additionally, consecutive slides were stained with a mIHC panel. PD-L1 proaches, there is an inherent tradeoff between capability positive macrophages from the pseudo mIHC images were quantified and throughput. and compared to the readouts identified in the real chromogenic IHC. In this study, we demonstrate a streamlined workflow to Methods develop and optimize a 9-color assay on the Leica BOND RX™ 7 formalin-fixed paraffin-embedded (FFPE) resections from NSCLC patients autostainer. This methodology offers an optimal balance be- were stained using Ultivue’s UltiMapper I/O PD-L1 kit and I/O PD-1 kit and tween multiplexing and sample throughput to facilitate research whole image scans were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss). and support translational studies on whole formalin-fixed Consecutive slides were stained with a multiplex chromogenic panel (in- paraffin-embedded (FFPE) tissue. cluding CD68, CD8, PD-1) at Mosaic Laboratories (1) and scanned with an Aperio AT Turbo scanner (Leica). Images were analyzed using an automated Methods workflow for quantitative multiplex image analysis developed at Definiens Forthe9-colorassay,Opal™ fluorophores were used on serial (2). Afterwards, mIF images were converted into pseudo mIHC images. Pa- sections of lung cancer FFPE tissue. The panel was designed on thologists annotated double positive macrophages for CD68 and PD-L1 on Akoya’s Mantra 2 semi-automated multispectral microscope, both images. Results were compared with automatically detected double which allows for rapid analysis of staining performance. Once positive cells and across assays. In addition, pathologists qualitatively optimized, multispectral images were acquired on both the assessed visual similarity of real and artificial chromogenic images. Mantra 2 and Vectra Polaris of the same tissue regions and an- Results alyzed to show equivalence between the platforms. Cell counts, Pathologists annotated double positive macrophages for CD68 and densities, and spatial parameters were generated using Akoya’s PD-L1 markers on both images (mIF and pseudo mIHC). Results were inForm imageanalysissoftwareand theRscript package compared with those obtained using artificial intelligence to auto- phenoptrReports, which produces quick, summarized outputs of matically detect double positive cells and across assays. In addition, the image analysis data. These same analyses were also used pathologists qualitatively assessed visual similarity of real and artifi- to evaluate reproducibility of all markers when run in a high- cial chromogenic images (Figure 1). throughput process. Conclusions Results Transferring mIF into pseudo mIHC images helps to combine the advan- Dynamic range of measured per cell signals for all markers had tages from both approaches: true colocalization of biomarkers whilst main- a median of 200:1. Agreement between the Mantra 2 and taining tissue morphology, facilitating visual evaluation of digital images Vectra Polaris-based measurements was generally >95% when by pathologists. This technology could be used to complement research, comparing cellular expression signals and cell counts based on clinical routine diagnostic, drug development and biomarker discovery. cell phenotyping classifiers. Cross talk was undetectable after spectral unmixing despite significant spectral overlap inherent References in a 9-color assay. Reproducibility across three batches of five (1) Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of serial sections of lung cancer tissue was generally <10% coeffi- inflammatory cell patterns and densities using multiplex immunohistochemistry cient of variation for all markers in the assay, supporting a immuno-oncology assays [abstract]. In: Proceedings of the AACR-NCI-EORTC high-through process of approximately 20 resection samples per International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct day. 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Conclusions Suppl): Abstract nr B069. We have successfully established a standardized process for 9- (2) Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated color multiplexing that offers a balance between elucidating the quantification of whole-slide multispectral immunofluorescence images to intricate cellular biology driving disease progression and thera- identify spatial expression patterns in the lung cancer microenvironment. peutic responsiveness within the TME while simultaneously SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. providing a practical and reliable assay that can be imple- Ethics Approval mented to support translational, high-throughput studies in clin- Ethical approval was granted by the Liverpool Research Ethics Committee, ical research. reference number 97/141. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 31 of 272 using matched 3,3′-Diaminobenzidine chromogenic and single bio- marker fluorescent controls tonsil tissue, which were validated using tumor samples from patients with high grade glioma. We characterized the spatial arrangement of myeloid subpopulations, ex vivo and corre- lated the changes in spatial orientation, quantity and localization of cells to the tumor. We determined that dynamic re-arrangement of myeloid cells can be observed under pressure of immunotherapy within the tumor, and confirm both a time-dependent and dose- dependent effect of oHSV-1 on this immune cell modulation. Conclusions These data suggest a unique, multiplexed approach to study spatial ar- rangement of myeloid and T-cell populations and their spatial distribu- tion within tumors under basal growth conditions or in the presence of anticancer immunotherapies, which may implicate the activity of mye- loid cells with treatment responses. These findings could impact per- sonalized cancer immunotherapy for patients receiving care. Ethics Approval The samples were collected under IRB approval. Consent The samples were collected under written patient consent for publi- cation of this abstract. P57 An ex-vivo human system elucidates a role for natural killer cells in the anticancer effect of drug combinations in triple negative breast cancer Fig. 1 (abstract P55). mIF and pseudo mIHC 1 2 2 Aaron Goldman , Douglas Best , Saravanan Thiyagarajan , Misti Jain, 2 2 1 2 PhD , Basavaraja Shanthappa , Munisha Smalley, PhD , Hans Gertje, BS , Aaron Goldman P56 1 2 Brigham and Women's Hospital; Mitra Biotech, Woburn, MA, United States Interrogating the effect of oncolytic Herpes simplex virus-1 on Correspondence: Aaron Goldman (goldman1@mit.edu) spatial arrangement of myeloid cells in glioblastoma multiforme Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P57 using an ex vivo human system and multiplex immunohistochemistry 1 2 2 Background Munisha Smalley, PhD , Misti Jain, PhD , Saravanan Thyiagarajan , Emily 3 3 4 2 Response and resistance to cancer therapy relies on the presence of ac- Alonzo , Katherine Crosby , Douglas Best , Hans Gertje, BS , Basavaraja 2 5 5 1 tive immune cells in the tumor microenvironment, which recalibrate Shanthappa , Ralph Pulchalski , Charles Cobbs , E. Antonio Chiocca , 1 1 1 the body’s own defense largely by modulating exhaustion of cytotoxic Sean Lawler, PhD , Aaron Goldman , Munisha Smalley 1 2 lymphocytes including T cells and natural killer (NK) cells. However, Brigham and Women's Hospital, Woburn, MA, United States; Mitra there is a critical gap in our understanding for the role of immune cells Biotech RxDx, Bangalore, India; Cell Signaling Technologies, Danvers, to drive response or resistance to drugs and immunotherapies at the MA, United States; University of Birmingham, Birmingham, United individual patient level. This is primarily due to limitations in complex Kingdom; Swedish Neuroscience Institute, Seattle, WA, United States tumor-immune interfaces that exist in many current tumor models. Correspondence: Aaron Goldman (goldman1@mit.edu) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P56 Here, we deployed an ex-vivo human system that uses an explant of native, patient-derived solid tumors including autologous immune cells. Background Utilizing biopsied tumor tissue from patients diagnosed with triple- The role of myeloid cell populations within a tumor and their contribu- negative (ER- PR- HER2-) breast cancers (TNBC, N=7), we studied drug- tion to effective cancer immunotherapy is emerging with considerable induced cell death (cleaved caspase-3) and spatial heterogeneity of NK interest. However, assessing the role of intratumoral myeloid cells cells (CD3-CD56+PanCK-) using multiplex immunohistochemistry under therapy pressure has it’s challenges. Here, we implemented a (mIHC). Spatial orientation of cells in the microenvironment, including multiplex immunohistochemistry (mIHC) panel and a human ex vivo proximity of NK to tumor and NK cell density within regions of the system to interrogate key myeloid subsets as they affiliate with infiltra- tumor vs. stroma were performed using HALO-based quantitative ana- tion and activation of an emerging immunotherapy for glioblastoma lyses. Finally, we deployed in-vitro co-culture studies using 3-D TNBC multiforme (GBM) – oncolytic Herpes simplex virus-1 (oHSV-1). mIHC organoids and human-derived NK cells (NK-92MI). combined with advancements in digital pathology and machine learn- Results ing algorithms have enabled identification, quantification and spatial First, we report the ability of the ex-vivo human system to retain the orientation of multiple cell types in a single field of view (FOV). spatial orientation and total population of natural killer cells and T- Methods cells over the course of a 72h explant culture. Next, using Spearman Cell Signaling Technology antibodies (CD3e,D7A6E™, ID: 85061), (CD68, correlation analyses and principal component analysis (PCA), we de- D4B9C, ID: 76437), (CD11c, D3V1E, 49420), (MHC Class II (HLA-DRB) LGII- termined that drug response to both immunotherapy and conven- 612.14), (Pan-Keratin, C11, 4545) were optimized for mIHC staining, tional cancer drugs, indicated by high incidence of cleaved caspase-3 using a tyramide signal amplification approach to pin-point, in a single after drug pressure, is directly associated with changes to the tumor- FOV: intratumoral T-cells, defined by CD3e+; macrophages, defined by NK cell proximity and density of NK cells within the tumor bed vs. CD68 and conventional dendritic cells defined by CD11c+MHCII +, in the stroma. Finally, using the 3-D tumor organoid cultures with NK relation to the surrounding tissue architecture defined by pan cytokera- cells, we determined that activity and tumor cytolysis by NK cells is tin. These biomarkers were integrated with incidences of oHSV-1 infil- hampered through cancer cell-activated cytokines, which diminish tration and replication (via expression of green fluorescent protein). expression of activating biomarkers including NKG2D/C. Results Conclusions First, we confirmed an optimized protocol for treating GBM ex vivo Taken together, these results provide a method to study the spatial with oHSV-1 such that tissue viability, infiltration and replication of arrangement of immune cells in an entirely human system, which the virus are optimal. The staining of the mIHC panel was optimized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 32 of 272 can be perturbed with anticancer drugs to reliably influence the ex- P59 pression and growth patterns of immune cells. We further demon- Highly consistent automated multiplex immunofluorescence for strate that this strategy can help to guide in-vitro studies to further immunoprofiling of solid tumors in clinical trials: assay validation elucidate mechanisms of action of drugs, which influence response study using multispectral imaging and digital analysis 1 2 2 vs. resistance via immune cell activity. Michael Surace, PhD , Lorenz Rognoni, PhD , Farzad Sekhavati , Andrew 2 2 2 1 Ethics Approval Fisher, PhD , Andreas Spitzmueller , Sara Batelli, PhD , Karma Dacosta , 2 3 4 Anonymous breast cancer tissue samples were collected under IRB Vinay Pawar , Clifford Hoyt , Edwin Parra, MD, PhD , Jaime Rodriguez- approval with due written consent from each patient. Canales, MD 1 2 AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, 3 4 Germany; Akoya Biosciences, Hopkinton, MA, United States; UT - MD P58 Anderson Cancer Center, Houston, TX, United States Brain MRI performed within 4 weeks of PD-1 inhibitors as a Correspondence: Jaime Rodriguez-Canales potential prognostic marker for non-small cell lung cancer (rodriguezcanalesj@medimmune.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P59 Ammar Sukari, MD, Misako Nagasaka, MD, Seongho Kim, PhD, Tahmida Chowdhury, Natasha Robinette, MD Background Karmanos Cancer Institute, Detroit, MI, United States Novel multiplex immunofluorescent (mIF) platforms have been devel- Correspondence: Ammar Sukari (sukaria@karmanos.org) oped for immunoprofiling of solid tumors to understand the tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P58 microenvironment and to identify biomarkers for immunotherapy. One of these methods employ IF and tyramide signal amplification (TSA) to Background generate between 4 to 9-marker multiplex panels analyzed with multi- PD-1 inhibitors aim to re-instate the natural anti-cancer immune- spectral imaging. Although this method can provide reliable data, they mediated cytotoxicity. Although PD-1 inhibitors are now consid- can show variability in consistency depending on the markers [1], ered part of standard of care treatment in advanced metastatic which affects the reliability of mIF for use in clinical trials. The goal of NSCLC [1], little is known about the effects of PD-1 inhibitors on this study was to develop and validate a highly consistent mIF method asymptomatic central nervous system (CNS) metastases. We hy- for its use in clinical trials in the pharma and academic environments. pothesized that early MRI brain imaging due to the development Methods of neurological signs and symptoms following the initiation of A mIF panel for the analysis of carcinomas was optimized using an PD-1 inhibitor may help delineate a subset of NSCLC patients automated stainer (Leica) and automated multispectral scanner (Po- with asymptomatic and undiagnosed CNS metastases prior to ini- laris, Akoya Biosciences). The markers included keratins (AE1/AE3), tiation of therapy and may predict for worse outcomes. CD68, PD-L1, PD1, CD8 and Ki67. Each primary antibody was first per- Methods formed on standard chromogenic IHC according to previously vali- Data from NSCLC patients who received at least one dose of PD- dated protocols. The mIF panel was developed with a secondary 1 inhibitors between September 2013 through the data cut-off of antibody detection and TSA, using specific fluorophores for each May 2017 were captured from our institution’s pharmacy data- marker. Once optimized, the miF panel was tested on serial sections base. The primary objective was to describe the characteristics of of formalin-fixed human tonsil controls and six non-small cell lung patients with MRI brain being performed within 4 weeks of the carcinomas, including replicates, together with standard IHC staining first dose of PD-1 inhibitors and the secondary objectives were of each individual marker for comparison. A set of three repeats on estimation of progression free survival (PFS) and overall survival different days for each mIF with all cases was performed to test (OS) for the same population. consistency and reproducibility of the mIF method. All slides were Results digitally scanned and analyzed using Automated Definiens Insights 140 NSCLC patients received at least one dose of PD-1 inhibitors Platform with custom algorithms (Definiens AG, Munich, Germany), prior to data cut-off. Median age was 64 (range: 24-86). 83 (59%) comparing the cell populations in the serial section slides between were male. 64 (46%) were treated on a clinical trial. There were standard IHC and mIF, and between mIF repeated rounds. The data 92 (66%) adenocarcinoma, 41 (29%) squamous cell carcinoma was statistically analyzed using Pearson’s correlations. (SCC) and 7 (5%) poorly differentiated NSCLC. 84 (40%) had a Results pre-PD1 inhibitor MRI brain performed and 25 (18%) had been Using an automated workflow, the mIF data compared with standard diagnosed with baseline CNS metastases. 128 (91%; Group 1) did IHC showed correlations between 0.83 to 0.99. The data from the not have an MRI brain performed within 4 weeks of starting PD-1 three rounds of mIF performed on different days showed correlations inhibitors, while 12 (9%; Group 2) patients did. 9 out of 12 pa- between 0.89 and 0.99. The marker that showed the lowest correl- tients had new or worsening CNS metastases. Of the 9, 1 had ation was CD68 (r=0.83), the possible cause was the difficulties on WBRT, 1 had gamma knife, 1 went onto hospice while a decision cell segmentation due to morphologic irregularity shown by macro- was made to monitor imaging and symptoms in 6 patients. The phages. Overall our present data showed a much higher consistency median PFS was 5.28 months (95% CI, 3.90 to 8.03) and 1.75 than our previously published results using a non-automated mIF months (95% CI, 1.08 to NE) for Group 1 and Group 2, respect- protocol, which correlations ranged from 0.17 to 0.87[1]. ively. The median OS was not reached (95% CI, 15.38 to NE) and Conclusions 5.77 months (95% CI, 2.85 to NE) for Group 1 and Group 2, Our data demonstrates that using an automated workflow including respectively. automated staining, scanning and analysis, and with properly vali- Conclusions dated IHC markers, mIF becomes a highly consistent methodology In this retrospective analysis, patients who had MRI brain within and it is compatible for its use with clinical trial tissue specimens. 4 weeks of starting PD-1 inhibitors had worse outcomes. Trial Registration Not applicable Reference 1. NCCN Clinical Practice Guidelines in Oncology. Non-Small Cell Lung Can- Reference cer. Version 5. 2019- June 7, 2019. https://www.nccn.org/professionals/ 1. Parra ER, Uraoka N, Jiang M, Cook P, Gibbons D, Forget MA, Bernatchez physician_gls/default.aspx#site, last accessed 7/30/2019. C, Haymaker C, Wistuba II, Rodriguez-Canales J. Validation of multiplex Ethics Approval immunofluorescence panels using multispectral microscopy for immune- The study was approved by the Wayne State University Institution's Ethics profiling of formalin-fixed and paraffin-embedded human tumor tissues. Board, approval number 062616M1E. Sci Rep. 2017; 7:13380-13391. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 33 of 272 P60 P61 Combining transcriptomic immune population inference with Segmentation and classification of single cells using multiplexed automated digital masking of H&E images finds immune effectors ion beam imaging preferentially distribute within stroma regions Jay Tarolli, Rachel Finck, Murat Aksoy, Yari Sigal, Noah Newgren, Jessica 1 2 2 Christopher Szeto, PhD , Mustafa Jaber, PhD , Liudmila Beziaeva , Kevin Finn, Jason Ptacek, PhD 1 1 1 Kazmierczak , Steve Benz , Shahrooz Rabizadeh IONpath, Menlo Park, CA, United States 1 2 ImmunityBio, Santa Cruz, CA, United States; NantOmics, Culver City, Correspondence: Jay Tarolli (jay@ionpath.com) CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P61 Correspondence: Steve Benz (Steve.Benz@nantomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P60 Background When studying the tumor microenvironment, knowing not only Background the types of immune cells present but also the spatial distribu- Multiple methods to characterize immune-cell populations in tion and relationship of these immune cells to other immune and tumor microenvironment (TME) are being assessed as potential tumor cells provides crucial information. In the past, techniques biomarkers of immunotherapy response. These include manual used to analyze these spatial relationships have been limited by pathological assessment of lymphocyte infiltration, immunohisto- the number of biomarkers that could be simultaneously mea- chemical (IHC) staining for specific adaptive response markers sured. Recently, with the development of multiplexed ion beam such as CD8, and more recently transcriptomic-based deconvolu- imaging (MIBI), 40+ biomarkers can be simultaneously measured tions of immune populations such as xCell and TIMER. Here we in a single scan [1]. By probing with an ion beam, tissue sections combined digital masking using deep-neural nets with transcrip- can be imaged at a spatial resolution on the same order of mag- tomic deconvolution to infer where immune-subpopulations may nitude as light based techniques, providing subcellular resolution. reside in the TME. This combination of multiplexed biomarker measurements and Methods subcellular spatial resolution enables segmentation of the image An unselected set of 187 clinical samples from the ImmunityBio data- into individual cells, making possible subsequent cell type classifi- base were analyzed. Each had H&E stained diagnostic slides with cation and quantification. pathologist-annotated tumor regions, as well as deep whole- Methods transcriptomic sequencing (>200M reads). Deep neural networks pre- Samples of placenta, lung, tonsil, lymph node, thymus, and liver viously trained on TCGA slide images were used to generate digital were imaged with MIBI. Segmentation of these images was per- spatial masks for 3 characteristics: tumor-content, lymphocytes, and formed in two steps. First, a MaskRCNN [2] model was trained stroma. Patients were scored based on the presence of intratumoral to utilize multiplexed MIBI data to predict the location of cell lymphocytes (iTIL) and stromal lymphocytes (sTILs). Immune subpop- instances in a MIBI image for a single class of objects by learn- ulations were inferred from RNAseq expression of published ing features from a set of nuclear, cytoplasmic, and membrane immune-cell-specific genesets [1,2], as was Wnt-signaling level [3]. markers. The centroids of each predicted cell instance were Significant associations between immune subpopulations and level used as seed points and, after manual refinement of these seed of infiltration were analyzed. points, watershed segmentation was performed to determine Results boundaries between instances. Both the summed intensity of a Manually annotated positive tumor regions were accurately digitally marker as well as a weighted cell score which accounts for the masked as >83% tumor or lymphocyte. Wnt signaling was strongly spatial distribution of a marker’s expression throughout a cell associated with overall stromal content (Rho=0.47, p<0.0001). Strong instance were calculated and were used for cell type anti-correlation was observed between levels of sTILs and iTILs classification. (Rho=-0.42, p<0.0001), and remained significant when including Results overall stroma area as a covariate. Digital lymphocyte masks some- Cell population and densities were calculated for a number of what correlated with RNAseq-based deconvolution of lymphocyte different cell types, including T cells, B cells, and macrophages classes (Rho=0.30, p=0.0001) in line with reports from others [4], based on a combination of one or more coexpressed biomarkers however this decreased when comparing lymphocyte count within present within segmented cells. Figure 1 shows an example FOV annotated tumor regions only (Rho=0.17, p=0.03), despite high con- with several cell types classified in a single image. Expression of cordance of lymphocyte counts within and outside of annotated re- immunoregulatory proteins including PD-1 and PD-L1 were quan- gions overall (Rho=0.82, p<0.0001). RNAseq-based lymphocyte levels tified and assigned to specific cell types. Finally, nearest neighbor were more associated with sTILs than iTILs (Rho=0.19 vs. -0.28, p< distances between various cell types were determined to 0.01 respectively). characterize the spatial organization of cell populations within Conclusions each tissue image. Adaptive response effectors such as NK and T-cells are found Conclusions more resident in surrounding stromal tissue than infiltrating The ability to characterize the many different cell types within tumor tissue. Increased Wnt/B-catenin signaling in stromal re- the tumor microenvironment is made possible by the highly mul- gions, reported by others as immunosuppressive, may sequester tiplexed nature of MIBI data, the subcellular spatial resolution of immune effectors and aid in immune escape. the image data, and downstream analysis tools, including com- puter vision approaches, which enable cell segmentation, classifi- References cation, and spatial analysis. 1. Bindea G, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 2013; 39:782-795. References 2. Danaher P, et al. Gene expression markers of tumor infiltrating 1. Keren L, Bosse M, Marquez D, Angoshtari R, Jain S, Varma S, Yang S, leukocytes. J Immunother Cancer. 2017; 5:18. Kurian A, Van Valen D, West R, Bendall S, Angelo M. A Structured 3. Slattery ML,et al. Expression of Wnt-signaling pathway genes and Tumor-Immune Microenvironment in Triple Negative Breast Cancer their associations with miRNAs in colorectal cancer. Oncotarget. 2018; Revealed by Multiplexed Ion Beam Imaging. Cell. 2018; 174:1373- 9:6075. 1387. 4. Pai SG, et al. Wnt/beta-catenin pathway: modulating anticancer immune 2. He K, Gkioxari G, Dollár P, Girshick R. Mask R-CNN. IEEE International Con- response. J Hematol Oncol. 2017; 10:101. ference on Computer Vision (ICCV). 2017; 2961-2969. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 34 of 272 combinations for a seven-color panel. Using this method, the number of slides was reduced to three per target (18) plus con- firmation seven-color slides resulting in a panel containing CD3, CD8, CD68, Cytokeratin, FOXP3 and PD-L1 (Figure 2). Conclusions Multiplex IF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interaction/co-localization of multiple targets within a single tissue specimen. MIF panels can take eight or more weeks to optimize, however, researchers can save time and resources using validated antibodies and this antibody order guide. Fig. 1 (abstract P62). See text for description Fig. 1 (abstract P61). See text for description Table 1 (abstract P62). See text for description P62 Bringing the tumor microenvironment into focus: Simplified development of seven-color multiplex immunohistochemistry- immunofluorescence (mIF) panels Melissa Whiteman, PhD, Eric McIntush, PhD, Mike Spencer Bethyl Laboratories, Montgomery, TX, United States Correspondence: Mike Spencer (mspencer@bethyl.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P62 Background For the advancement of immunotherapeutics, the need to under- stand the tumor microenvironment has never been more press- ing. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers. This is critical in instances where sample is limited, such as a tumor bi- opsy or other clinical specimen. The applications of mIF are nu- merous, and span clinical, translational, and basic research applications. A seven-color mIF can take eight weeks, or more, to develop. Herein, we describe a simplified, faster approach. Methods FFPE human tissue was stained with PathPlex™ Panel 4 IHC vali- dated primary antibodies (Bethyl Laboratories [A810-004]), mouse or rabbit HRP-conjugated secondary antibodies (Bethyl Laborator- ies [A90-116P, A120-501P]) and detected using Opal™ Polaris 7- color IHC kit fluorophores (Perkin Elmer [NEL861001KT]). Primary antibody order was optimized utilizing tissue microarray serial sections, and three slides per target by staining after the first, third, or sixth heat-induced epitope retrieval (HIER). All three slides were imaged using the same exposure time and analyzed Fig. 2 (abstract P62). See text for description for target/nucleus counts, signal intensity, and background. Fi- nally, the order was tested in the seven-color mIF and compared to single stain for confirmation. Whole slide scans were gener- ated using the Vectra Polaris® and analyzed using InForm® image analysis package. P63 Results Clinical assay development and validation of multiplex Development time of a seven-color mIF was reduced using IHC immunofluorescent (mIHC) marker panel for evaluation of tumor validated antibodies and the optimized dilution. Antibody order infiltration myeloid cells in FFPE tissue sections 1 1 2 was guided by results of three slides stained after first, third or Lan Yi, PhD , Jonathan Juco, MD , Ashhad Mahmood, MD , Omar 1 1 1 sixth HEIR. The ratio of target staining/DAPI nuclear counts, aver- Laterza , Charo Garrido , Lan Yi, PhD 1 2 age intensity and overall background predicts the optimal order Merck & Co., Inc, Hillsborough, NJ, United States; Diagnostic Pathology of staining. Some targets reveal larger average area staining, Services, LLC, Skillman, NJ, United States higher intensity and lower background when stained last, for ex- Correspondence: Omar Laterza (omar_laterza@merck.com); Charo ample FOXP3 (Table 1, Figure 1), while the inverse may be true, Garrido (charo.garrido@merck.com) or no effect for other targets. There are 720 possible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P63 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 35 of 272 Background staining pattern of more than 20 parameters. The high-parameter anti- Myeloid-derived suppressor cells (MDSCs) are a group of leuco- body panel used was optimized with human FFPE tonsil tissues. Human cytes with myeloid origin and immune-suppressive function. lung cancer FFPE tissues were then analyzed with this same panel. Cell Ample recent evidence supports key contributions of MDSC to clustering using the MAV software identified tens of cell types within tumor progression through immune-mediated mechanisms. the tumor tissues. Immune cell sub-types were mapped onto the ori- MDSCs include two major subsets based on their phenotypic and ginal image data to assess infiltration and spatial associations. morphological features: polymorphonuclear (PMN)-MDSC and Conclusions monocytic (M)-MDSC. However, these cells remain less studied Unlike other cyclic IF approaches involving multiple antibody staining than T lymphocytes as their phenotypical, morphological and and stripping steps, the CODEX platform involves a single initial functional heterogeneity generate confusion in investigation and staining step and subsequent gentle and relatively fast manipulation analysis of their role. of the tissue thereafter. This provides a superior workflow and pre- Methods vents tissue degradation. CODEX data from various normal and can- With the progresses on multiplex IHC assay technology, we are now cer human FFPE tissue types is shown here with corresponding able to develop multiplex immunofluorescent marker panels to single-cell analysis of key tissue features. Overall, the CODEX platform evaluate the expression and localization of the main subpopulations is an accessible and versatile technology for high parameter, spatial of myeloid cells in the tumor microenvironment. profiling of tissue specimens. Results We have developed and validated CD14, CD66b, CD163 and P65 MHCII (HLA-DR) IHC multiplex marker panel to evaluate the main Mutation-targeted T cell responses in blood from patients with populations of myeloid cells, along with their activation status. solid tumors prior to treatment and which evolve with clinical After completing the validation for individual markers in a single benefit from anti-PD-1 therapies chromogenic IHC platform, we optimized the incorporation of 1 2 1 1 Benjamin Yuen, PhD , Fangfang Yin, PhD , Duo An, PhD , Boi Quach , each marker into the multiplex platform. In parallel, a multiplex 1 2 1 Linlin Guo, PhD , Joanne Tan, PhD , Songming Peng, PhD , Zheng Pan, image analysis algorithm (APP) was generated and validated to 1 1 1 1 PhD , Olivier Dalmas, PhD , Robert Bao, PhD , Kyle Jacoby, PhD , Barbara quantify each subpopulation of myeloid cells in the tumor area. 1 1 2 Sennino, PhD , Stefanie Mandl, PhD , Matt Walters, PhD , Juan Jaen, Last, fit for purpose analytical validation, including sensitivity, spe- 2 1 1 PhD , Alex Franzusoff, PhD , Benjamin Yuen, PhD cificity and precision was successfully carried out. 1 2 PACT Pharma, South San Francisco, CA, United States; Arcus Conclusions Biosciences, Hayward, CA, United States This validated assay is currently being used to support multiple on- Correspondence: Songming Peng (speng@pactpharma.com) going and future clinical trials. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P65 P64 Background Highly multiplexed single-cell spatial analysis of FFPE tumor T cells targeting tumor-exclusive neoepitopes (neoE) have been pos- tissues using CODEX® tulated to represent the primary mediators of clinical benefit for pa- Jessica Yuan, PhD, Gajalakshmi Dakshinamoorthy, Joseph Kim, Sejal tients with solid tumors treated with immunotherapies. Identifying Mistry, Nadya Nikulina, Roya Bashier, Cassandra Hempel, Maria Elena and tracking these T cells in patients can help to understand the Gallina, Julia Kennedy-Darling, Jessica Yuan, PhD mechanism for immune checkpoint inhibitor therapies, as well as Akoya Biosciences, Menlo Park, CA, United States provide new therapeutic candidates for personalized adoptive cell Correspondence: Julia Kennedy-Darling (j.kennedy@akoyabio.com) therapies. However, this has been hampered by the low frequency of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P64 neoE-specific T cells in peripheral blood. To this end, we demonstrate the use of the imPACT Isolation Technology®, an ultra-sensitive high- Background throughput technology, to capture neoE-specific CD8 T (neoE-T) cells Characterizing the complexities of the tumor microenvironment is elem- from peripheral blood. In addition, this technology can be utilized to ental to understanding disease mechanisms. The spatial relationships be- quantify and monitor neoE-T cells longitudinally during therapy. We tween infiltrating immune cells and the remodeling of the cellular matrix show here preliminary data applying the imPACT technology to clin- is widely recognized as a key component to defining tumor heterogen- ical trial samples for the characterization of mutation-targeted T cell eity. Current methodologies for analyzing the spatial dimension in tissues, responses from patients associated with clinical benefit. like traditional immunofluorescence (IF) and immunohistochemistry (IHC), Methods are limited to a few parameters at a time, restricting the scope of identifi- Peripheral blood mononuclear cells (PBMC) from patients with non- able cells. Conversely, single-cell technologies like mass cytometry and small cell lung cancer (NSCLC) and treated with combinations con- NGS-based tools provide multiplexing capabilities, but at the expense of taining an anti-PD-1 antibody were analyzed. Briefly, tumor-exclusive the associated spatial information. Here, we present the analysis of hu- neoE-HLA target candidates were predicted and barcoded snare li- man lung cancer FFPE tissues with CODEX using a panel of more than 20 braries comprising personalized neoE-HLA reagents were produced markers targeting the tumor microenvironment. for capture of neoE-specific CD8+ T cells from PBMCs. Longitudinal Methods analysis of neoE-T cells responses throughout the duration of treat- The CODEX technology, developed by Akoya, is comprised of a fluidics ment was performed to obtain valuable information on neoTCR se- instrument that interfaces with existing microscope hardware, as well as quences and neoE-T cell quantification & phenotype. a suite of reagents and associated control and analysis software. The Results CODEX technology involves labeling antibodies with oligonucleotide- A baseline neoE-specific CD8 T cell profile was identified in all sub- based Barcodes followed by a single staining step. Around 40 parameters jects prior to treatment. Among NSCLC subjects exhibiting objective can be measured within a single tissue through fully-automated, iterative responses to therapy, some neoE-T cell clones identified at baseline cycles of adding and removing corresponding dye-conjugated Reporters. persist in the blood and/or diversify in clonality over the course of Here, we apply this technology using a panel of antibodies targeting im- treatment. In some circumstances, new neoE-T cell clones have mune, cancer and other architectural features to measure cell subsets in emerged on treatment with anti-PD-1. Furthermore, phenotype ana- cancer FFPE tissues. Image data is processed using the CODEX analysis lysis suggested the neoE-T cells captured from blood have been acti- pipeline, including clustering, annotation and mapping of cell types to vated, indicating previous encounter with their respective neoE-HLA the original image data with the Multiplexed Analysis Viewer (MAV). targets. Results Conclusions The CODEX technology was used to ascertain complex cellular niches The imPACT technology was used to assess the phenotype & quan- and spatial associations between multiple cell types based on the tity of neoE-specific T cells in blood of trial participants over time. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 36 of 272 This approach revealed the evolution of mutation-targeted T cell re- Conclusions sponses in participants with clinical benefit and may prove to be a Future plans include analyzing the remainder of patients enrolled in powerful tool to provide mechanistic understanding of immune re- the trial and the utilization of CD8+ and MDSC-specific CyTOF panels sponses associated with clinical benefit. These data support further to further classify the aforementioned subpopulations to further elu- testing of the neoE-T cell capture technology, with the potential to cidate this relationship. This methodology offers insight into the pro- uncover the identity of neoE-specific T cells pre-existing in the blood gression of vaccine-induced patient immune responses and exhibits of patients and the evolution of immune attack to cancer. promise as a platform that may be extrapolated to other Ethics Approval immunotherapies. The study was approved by the institutional review boards or ethics committees of the participating sites in Arcus Biosciences’ clinical References studies. 1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW. The 2016 World Health Organization Classification of Tumors of the Cen- P66 tral Nervous System: A summary. Acta Neuropathol. 2016; 131:803– A novel mass cytometry-based immunomonitoring platform for characterizing the peptide vaccine-induced immune response of 2. Chheda Z, Kohanbash G, Okada K, Jahan N, Sidney J, Pecoraro M, Yang X, HLA-A*0201+ patients with K27M+ diffuse midline gliomas Carrera D, Downey K, Shrivastav S, Liu S, Lin Y, Lagisetti C, Chuntova P, 1 1 1 Jared Taitt, BA , Payal Watchmaker, PhD , Takahide Nejo, MD, PhD , Neil Watchmaker P, Mueller S, Pollack I, Rajalingam R, Carcaboso A, Mann M, 2 1 1 Almeida , Kaori Okada , Sabine Mueller, MD PhD , Hideho Okada, MD, Sette A, Garcia C, Hou Y, Okada H. Novel and shared neoantigen derived 1 1 PhD , Jared Taitt, BA from histone 3 variant H3.3K27M mutation for glioma T cell therapy. J University Of California, San Francisco, San Francisco, CA, United States; Exp Med. 2017; 215:141-157. George Washington University, Washington DC, United States Ethics Approval Correspondence: Sabine Mueller (sabine.mueller@ucsf.edu); Hideho The study was approved by UCSF IRB #: 16-20574 Okada (hideho.okada@ucsf.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P66 P67 Background Use of a regional integrated health record data network to identify Diffuse midline glioma, including diffuse intrinsic pontine glioma patients who received checkpoint therapy following cancer (DIPG) constitutes up to 20% of pediatric brain cancer and has a me- diagnosis as a foundation for exploring immunotoxic events dian survival of 9-10 months. Given the proximity of DIPG to paren- Theresa Walunas, PhD, Carlos Galvez, Saya Jacob, Jeffrey Sosman, MD, chymal regions that play vital homeostatic functions, surgical Abel Kho resections are often restricted in size and scope, leaving irradiation Northwestern University, Chicago, IL, United States and chemotherapy as the primary management options. The on- Correspondence: Theresa Walunas (t-walunas@northwestern.edu) going development of immunotherapy has shown significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P67 promise in many fields, including that of gliomas. Genetic studies revealed that greater than 70% of DIPG cases harbor an amino Background acid substitution from lysine (K) to methionine (M) at position 27 Immune related adverse events (irAE) occur in >80% of patients of histone 3 variant 3 (H3.3). We previously identified a novel receiving immune checkpoint inhibitors (ICI). Currently, most HLA-A*02:01-restricted neoantigen epitope encompassing the data about the incidence of irAE comes from clinical trials with H3.3K27M mutation. Accordingly, we have implemented a pilot restrictive eligibility requirements. With the wide use of ICI ther- vaccine through the Pacific Pediatric Neuro-Oncology Consortium apy as standard of care for many cancers, it is important to as- (PNOC). sess incidence of irAE in a general patient population. The Methods Chicago Area Patient Reported Outcomes Research Network Twenty-nine newly diagnosed DIPG patients who are HLA-A2+ and (CAPriCORN) is a clinical data research network containing med- H3.3K27M+ underwent radiation therapy, and then received the ical records for >9.5M patients who receive care in 11 institu- H3.3K27M peptide vaccine and tetanus toxoid (TT) peptide emulsified in tions spanning diverse patient populations and healthcare Montanide in combination with poly-ICLC every 3 weeks for a total of 24 settings [1]. Using CAPriCORN, we wanted to determine whether weeks. Our objective is to characterize vaccine-induced H3.3K27M-spe- we could identify a large, diverse cohort of patients who re- cific CD8+ T-cell and myeloid-derived immunosuppressive subpopula- ceived ICIs as a foundation for exploring the incidence of irAE tions in peripheral blood mononuclear cells utilizing a novel H3.3K27M- in a real-world data source. specific dextramer-based mass cytometry (CyTOF) method [1,2]. Methods Results We identified all patients within CAPriCORN who were 19-88 Through this approach, the temporal expansion of vaccine-reactive years old, had a diagnosis for an ICI-approved cancer, and re- CD8+ T-cells was observed in all patients who completed a minimum ceived an ICI from 1/1/2011 through 12/31/2018. Clinical experts of 24 weeks on the study (n = 4). Simultaneously, this expansion was identified the International Classification of Disease 9 and 10 not observed in 4 of 5 patients who withdrew from the regimen due codes used to document cancer diagnosis and the RxNorm [2] to progression. These T-cells were clustered on a tSNE plot using ca- codes for each ICI documented as a medication ordered in the nonical CD8+ T-cell activation markers and further classified by their medical record (Table 1). The query was developed against the expression profiles, revealing distinct effector memory, central mem- PCORnet Common Data Model version 4.1 [3], validated on the ory and transitional effector subpopulations. Chronological monitor- Northwestern University site node in CAPriCORN and distributed ing of these groups indicates the time course-dependent to all CAPriCORN sites. Six of 9 sites returned counts. Data was development and persistence of vaccine-reactive exhausted and ef- centrally aggregated and stratified by age, race, sex and therapy. fector memory CD8+ T-cells in 3 of the 4 initial patients analyzed. All data are aggregated counts. Furthermore, an analogous clustering and phenotyping approach Results was used for myeloid cells, allowing for the identification of myeloid- As shown in Table 2, we identified 6,541 patients within CAPri- derived suppressor cell (MDSC) subpopulations. A comparative ana- CORN who received ICI therapy for cancer. 45% are female, 75% lysis revealed a positive correlation between two monocytic myeloid- identify as white, 13% African American, 2% Asian and 1% Native derived suppressor cell (M-MDSC) subpopulations and progression- American, and 86% are 51-83 years of age. The most well repre- free survival. sented cancers were Non-small Cell Lung Carcinoma (50%) and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 37 of 272 Metastatic Melanoma (18%). Overall, 67% received anti-PD1 ther- P68 apies, followed by combination ICI therapies, anti-PDL1 and anti- Innate inflammatory pathways are associated with TIL growth and CTLA4, though usage varied within cancer types. response to adoptive immunotherapy 1 1 1 Conclusions Suhendan Ekmekcioglu, PhD , Dai Ogata, MD, PhD , Caitlin Creasy, MS , 1 1 1 Our results demonstrate that a large cohort of cancer patients who Marie Forget, PhD , Sun-Hee Kim, PhD , Jason Roszik, PhD , Mike 3 1 1 have received ICI therapy can be identified in an integrated medical Spencer , Patrick Hwu, MD , Elizabeth Grimm, PhD , Chantale 1 1 record data environment that spans 11 institutions in a major urban Bernatchez, PhD , Suhendan Ekmekcioglu, PhD center. This population is racially diverse, represents both sexes, a The Univeristy of Texax MD Anderson Cancer Center, Houston, TX, wide range of ages and includes all cancer types approved for ICI United States; Bethyl Laboratories, Inc, Montgomery, TX, United States therapy as of 2018. This real-world cohort will be an effective founda- Correspondence: Suhendan Ekmekcioglu tion on which to explore the incidence of irAE, particularly rare irAE (sekmekcioglu@mdanderson.org); Chantale Bernatchez that require large sample sizes to investigate. (cbarnatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P68 Acknowledgements The investigators would like to acknowledge the CAPriCORN network for Background query support. This work was supported by Patient Centered Outcomes Immune infiltration of T cells (TIL) into the melanoma microenviron- Research Institute (PCORI) CDRN-1306-04737. ment has been associated with improved survival for some patients, and also has been exploited to grow TIL in vitro for adoptive therapy. References However, prognostic significance of immune infiltrating cells in melan- 1. Kho AN, Hynes DM, Goel S, et al. CAPriCORN: Chicago Area Patient- oma and other tumors remains a relatively new concept, and markers Centered Outcomes Research Network. J. Am Med Inform Assoc. 2014; related to suppressive versus active functional TIL remain unclear. We 21:607-611. previously reported that in Stage III melanoma patients’ tumors, posi- 2. https://pcornet.org/download/pcornet-common-data-model-v4-1- tive expression of CD74 together with low or absent Macrophage Mi- specification-5-may-2018/?wpdmdl=1919&refresh= gration Inhibitory Factor (MIF) associates with favorable prognosis [1]. 5d42596fab3e11564629359 Methods 3. https://www.nlm.nih.gov/research/umls/rxnorm/ From an ongoing clinical trial using TIL intended for adoptive immuno- Ethics Approval therapy, we have studied the melanoma patient tumors specimens The study was approved under the CAPriCORN IRB, CHAIRb: Research Protocol (FFPE) from 20 patients whose autologous TIL lines grew to sufficient #14120201 “CAPriCORN Clinical Data Research Network Master Protocol”. number for possible use clinically. We also examined another 20 sets of melanoma tumor from which the TIL did not grow or not grow well. We analyzed the differences in the two groups of tumors (40 total FFPE) for Table 1 (abstract P67). See text for description CD74 regulated pathway features and inflammatory marker expression. Results CD74 regulated markers included CD44, MIF, and downstream in- flammatory targets including inducible Nitric Oxide Synthase (iNOS) and Nitrotyrosine (NT). Our findings confirm our previous report in that tumor CD74 expression significantly associates with favorable OS and PFS (both, p=0.0038) and provides new data that in this set of patients the CD74 also correlates with best irRC of TIL treated pa- tients. New findings include that the NT expression in tumor cells as- sociated with poor TIL growth (p=0.014), as well as lack of clinical response to TIL treatment (p=0.02). We have also found that tumor cell-derived MIF and iNOS expression correlate with unfavorable prognosis for both OS and PFS (p=0.016 and 0.018, respectively). Conclusions We have identified the protein expression of CD74, MIF and of iNOS as providing survival information, and proposed that CD74+/MIF-/iNOS- to- gether be considered to form a "signature" of good prognosis in general melanoma outcomes as well as TIL growth and favorable responses for these patients. Use of this signature for selecting patients for entry into TIL and possibly other immunotherapy trials, as well as research on the differential pathways of IFN-γ signaling in melanoma appear as important areas for future mechanistic research to improve patient outcome. Table 2 (abstract P67). See text for description Reference 1. Ekmekcioglu S, Davies MA, Tanese K, Roszik J, Shin-Sim M, Bassett RL Jr, Milton DR, Woodman SE, Prieto VG, Gershenwald JE, Morton DL, Hoon DS, Grimm EA.Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and Its Expression Associates with Favorable Sur- vival for Stage III Melanoma. Clin Cancer Res. 2016 Jun 15;22(12):3016-24. P69 Highly multiplexed single cell spatial analysis of the tumor microenvironment in lymphoma 1 1 2 Monirath Hav, MD, PhD , Anthony Colombo , Erik Gerdtsson , Mohan 2 2 2 2 Singh , Denaly Chen , Imran Siddiqi, MD PhD , James Hicks , Peter Kuhn, 2 1 1 PhD , Akil Merchant, MD , Akil Merchant, MD 1 2 Cedars Sinai Medical Center, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States Correspondence: Akil Merchant (akil.merchant@cshs.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P69 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 38 of 272 Background Diffuse large B cell lymphoma (DLBCL) being the most subtype of non-Hodgkin lymphoma. Despite evidence of expression of PDL-1 on lymphoma cells, less than 10% of DLBCL patients re- spond to PD1 therapy [1]. We hypothesize that a better characterization of spatial architecture of the tumour microenvir- onment (TME) in lymphoma will help explain differences in re- sponses to PD1/PDL-1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cy- tometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [2]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for rele- vant immunophenotypes, the spatial architecture of those pheno- types and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype (Figure 1A). Immune cell represented 33% of the cells represented by CD4 (36%), CD8 (30%), macro- phages (26%) and TREG (8%) (Figure 1B). Immune cell infiltra- tion in individual tumor samples ranged from 7% to 75% with marked heterogeneity. (Figure 1C-D. Analysis of immune Fig. 1 (abstract P69). See text for description marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure 1E) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macro- phages, and tumor cells (Figure 2A). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure 2B). Meta-clusters 2, 6, 8 and 11 were the 4 most dominant patterns of CD8 spatial interaction in the TME. Each CD8 spatial interaction pattern is dis- tinctive with case to case heterogeneity (Figures 2C-D). Risk as- sessment analyses of spatial clusters 1, 2 and 4 (“hazardous”)had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 (“protective”) (Figure 2E). In the “protective” spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREG pheno- types, with opposite in “hazardous” areas (Figures 3A-B). TIM-3 expression was high both on T cells and tumor cells in the “haz- ardous” neighborhoods. Conclusions Our novel approach to spatial analysis of the immune architecture re- veals clinically relevant insights into the TME. References 1. Ansell, S. M. et al. Nivolumab for Relapsed/Refractory Diffuse Large B-Cell Lymphoma in Patients Ineligible for or Having Failed Autologous Trans- plantation: A Single-Arm, Phase II Study. J. Clin. Oncol. 37, 481–489 (2019). 2. Giesen, C. et al. highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. methods | 11, 417 (2014). Ethics Approval Fig. 2 (abstract P69). See text for description The study was approved by USC IRB, approval number HS10-260 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 39 of 272 of PD-L1+ immunoreactivity in epithelial/tumor cells, increased im- munoreactivity for PD-L1 in CD68+ cells, and a closer relationship be- tween intra-epithelial and stromal CD8+ cells with PD-L1+ cells. In addition, areas in CRC and CD heavily infiltrated by immune cells or with tertiary lymphoid structures contained clusters of PD-L1+ cells that were negative for both CD68 and pan-Cytokeratin. Most of the CD3+ cells in non-lesional CD were PD-1 negative, except around ter- tiary lymphoid structures. In contrast, a greater percentage of CD3+ cells were also PD-1+ in CRC, and more so in lesional CD tissue. Conclusions The Ultivue UltiMapper multiplex fluorescence immunohisto- chemistry platform was effective in characterizing the PD-L1/PD- 1 axis and T cell exhaustion environment in FFPE tissue, in part due to the ability to clearly identify more complex immune cell phenotypes than traditional multiplex IHC. The application of the UltiMapper assays demonstrated many similarities between marker and cell type distribution between lesional colonic CD and CRC. P71 Refining tumor mutation burden values using variant expression Shannon Bailey, PhD, Muhammad Ekram, PhD, Jim Lund, Jeffrey Gulcher WuXi NextCODE Genomics, Arlington, MA, United States Correspondence: Jeffrey Gulcher (jgulcher@wuxinetcode.com) Fig. 3 (abstract P69). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P71 Background P70 Tumor mutation burden (TMB) is used as a surrogate marker for the Tissue-based characterization of T cell exhaustion in inflammatory neoantigen load of a tumor, and many studies have shown that TMB bowel disease and colorectal cancer using multiplex IHC predicts the success of immune-oncology (IO) treatments for cancers, 1 1 1 Marina Bleck, PhD, Diane Mierz , Ania Mikucki , Marie Marcher , Sidharth such as anti-PD-1 or anti-CTLA4 therapy. While IO treatment of pa- 1 1 2 Kerkar, MD , Gerald Nabozny, PhD , Sean Downing, PhD , Alexander tients with high TMB has led to success and excitement in the field, 1 1 Klimowicz, PhD , Alexander Klimowicz, PhD not all high TMB patients respond to IO treatment. TMB can be de- 1 2 Boehringer Ingelheim, Ridgefield, CT, United States; Ultivue, termined using next-generation sequencing, and both panel and Cambridge, MA, United States whole-exome DNA sequencing have been used to measure the mu- Correspondence: Alexander Klimowicz tation load of a tumor. (alexander.klimowicz@boehringer-ingelheim.com) Because all genes are not expressed in every cell, inclusion of the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P70 mutations found in unexpressed genes may confound the utility of TMB to predict neoantigen load. In this study, we explore improving Background TMB by taking into account both DNA mutation and RNA expression T cell exhaustion and the PD-L1/PD-1 checkpoint axis has been ex- with the hypothesis that using both criteria will lead to a TMB bio- tensively characterized in peripheral blood mononuclear cells and in marker that correlates better with neoantigen load. human tumor tissues. This has provided a better understanding of Methods the role this pathway plays in tumor immunology and of its clinical We examined DNA and RNA sequencing data for different cancer types in utility in predicting responsiveness to checkpoint inhibitor therapies. The Cancer Genome Atlas (TCGA) to determine refined TMB values. The T cell exhaustion is not only associated with tumor progression, but data were assessed to identify mutations with and without expression. has recently been associated with better prognosis and milder course Results of disease for a number of autoimmune and autoinflammatory disor- We found that a significant faction mutations included in a standard ders. We set out to characterize and contrast the T-cell exhaustion TMB calculations reside in genes that are not expressed, and this frac- environment between colonic Crohn’s disease (CD) and colorectal tion varies significantly among samples. A corrected TMB that incorpo- cancer (CRC). We applied the Ultivue UltiMapper multiplex fluores- rates gene expression is likely to be a better predictor of neoantigen cence IHC platform to capture complex immune cell phenotypes and load. In addition, our group has previously identified TCGA samples provide a more in depth characterization than traditional IHC. with allele specific expression in which up to 25% of the tumor muta- Methods tions are not expressed despite significant expression of the gene. The Commercially sourced FFPE surgical resections from n=5 colonic CD pa- fraction of unexpressed mutations is much higher in some cancer tients (matched lesional and non-lesional tissue) were compared to n= types. Adding this correction to the TMB calculation, including only var- 5 CRC tumor resections (3 hot and 2 cold tumors) using the Ultivue Ulti- iants that are expressed in the tumor in the TMB calculation, changes Mapper multiplex fluorescence immunohistochemistry platform. Two the average TMB values found among different cancer types, and cor- UltiMapper kits were used to evaluate the T cell environment in these rects the TMB in individual samples. This refined TMB value provides a tissues: UltiMapper I/O PD-L1 panel included the markers CD8, CD68, biomarker that reclassifies samples scored as high TMB to low TMB and PD-L1, and pan-Cytokeratin/Sox10; UltiMapper I/O PD-1 panel included is likely to better predict response to IO treatment. the markers CD3, CD45RO, PD-1, and pan-Cytokeratin/Sox10. All assays Conclusions were stained on Leica BOND RX autostainers. Whole-slide images were As our results suggest, adding RNA sequencing can be used to im- acquired on a ZEISS Axio Scan.Z1 slide scanner. Image analysis was per- prove the TMB biomarker to better separate treatment groups. The formed using Indica Labs HALO software. initial analysis was performed with TCGA exome data and is now be- Results ing extended to refine TMB values generated from gene panels in- Contrasted to non-lesional CD tissues, several similarities were ob- cluding TSO 500. We are also evaluating differences in tumor- served between CD lesional tissue and CRC, including the presence infiltrating lymphocytes in based on the refined TMB value. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 40 of 272 P72 attractive targets for immunotherapy. Identifying the full comple- Comprehensive and accurate prediction of presented neoantigens ment of peptides derived from a protein presented on a major class-I using ImmunoID NeXT and advanced machine learning algorithms HLA restriction provides a vital step toward increasing the speed and Dattatreya Mellacheruvu, PhD, Rachel Pyke, Charles Abbott, PhD, Nick viability of many immunotherapeutic strategies. Advances in next- Phillips, Rena McClory, John West, MBA, Richard Chen, Sean Boyle, PhD, generation sequencing (NGS) and single-cell technologies have en- Dattatreya Mellacheruvu, PhD abled the accurate capture of somatic mutations accumulated by a Personalis Inc., Menlo Park, CA, United States tumour, yet a significant hurdle remains how this information can be Correspondence: Sean Boyle (sean.boyle@personalis.com) utilized for immunotherapeutic benefit. Identifying which somatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P72 mutations produce neoantigens is crucial in providing the link be- tween genetic change and immunological impact. Background Methods Comprehensive detection of potential neoantigens and accurate pre- Directly identifying potential neoantigens using mass spectrometry diction of their MHC presentation are critical prerequisites for selecting offers a significant improvement over traditional approaches based neoepitopes that can be used for creating personalized cancer vac- on prediction. However, the relatively high sample requirement of cines. However, prediction models developed using in-vitro MHC- this approach inherently limits the depth of analysis that can be per- peptide binding assays cannot model upstream presentation machin- formed, with a significant risk that low abundance neoantigens are ery, such as proteasome cleavage and peptide loading. Advances in not detected. immuno-affinity purification followed by mass spectrometry (IP-MS) By integrating multiomics data from over 1000 experiments in 200 have enabled direct detection of MHC-bound peptides and can there- immortalised cell lines, we have generated a database of over two mil- fore be used for modelling native MHC-peptide presentation. Further, lion unique HLA-peptide sequences that offers near total coverage of genetically engineered cell lines that express a single HLA allele enable the protein-coding genome. Our comprehensive HLA class-I peptide unambiguous HLA-peptide assignment. Here, we present an overview atlas has been used as a reference tool to aid direct identification of of our MHC presentation prediction framework based on a large collec- neoantigens by targeted mass spectrometry, to probe indirectly for the tion of such mono-allelic cell lines and discuss its utility in conjunction presence of neoantigens, and to explore how many common driver with ImmunoID NeXT, our commercially available exome scale DNA mutations associated with cancer interact with the immunopeptidome. and RNA sequencing and analytics platform specifically designed to en- Results able the development of immuno-therapies. We have identified hundreds of neoantigens directly by mass spec- Methods trometry and found that mutated proteins follow the same pattern Mono-allelic cell lines were generated from K-562 null-HLA parental of antigen processing and presentation as their unmutated equiva- cells by transfecting each of the selected alleles. Cells were grown, lents. As a result, our HLA peptide atlas offers significant value in pre- screened for surface expression, lysed and immuno-affinity purified dicting the likelihood of a somatic mutation creating a neoantigen. using a column coated with HLA class I (W6/32) antibody. Peptides Comparing predicted neoantigens with those directly identified by were gently eluted and analyzed using LC-MS/MS. Peptide-to-spectrum mass spectrometry, we show effective prioritization of mutations by assignment was performed and filtered at 1% false discovery rate. accurately predicting the presence and relative abundance of neoan- Results tigens. Applying this process toward the five most commonly mu- The training data for our MHC presentation prediction framework were tated genes in cancer reveals a marked bias toward mutations that generated using a large collection of genetically engineered mono- either act negatively or are in ‘quiet’ areas of the immune landscape. allelic cell lines, encompassing approximately 60 HLA Class I alleles that As all mutated peptides contain novel amino acid sequence, and are are frequently present across various populations. The resulting hence able to elicit an immune response, this ability to convert ‘po- immuno-peptidomics data were comprehensive and of high quality - tential’ into ‘actual’ is crucial in establishing a mechanism for identify- the peptide yields were high (median of approx. 1600 unique peptides ing false positive results observed in cell-based assays. per allele) and the dominant motifs were in agreement with published Conclusions motifs. Our prediction framework is based on multiple modelling algo- An integrative multiomics approach to neoantigen identification rithms, including a multi-layer neural network, and uses proprietary and has delivered a powerful reference for developing novel standard features such as peptide sequence, peptide length, binding immunotherapies pocket sequence and abundance (measured by transcripts per million). We created allele-specific and pan-allele models and evaluated them P74 on an independent hold-out dataset. Both our allele-specific and pan- Integrating CD8 and CD4 effector neo-epitope content with allele models had superior performance compared to other public regulatory T cell epitope exclusion is a superior prognostic tools, with a higher precision across a range of recall (sensitivity) values. biomarker for bladder cancer patient compared to their tumor Conclusions mutation burden Our integrated pipeline for neoepitope discovery, which includes the 1 2 1 Guilhem Richard, PhD , Randy Sweis, MD , Matthew Ardito, BA , comprehensive profiling of putative neoantigens using ImmunoID 2 1 3 Tzintzuni Garcia , Leonard Moise, PhD , Michael Princiotta, MS, PhD , NeXT and accurate and sensitive prediction of MHC presentation of 3 1 3 Dominique Bridon , William Martin, BA MD , Gad Berdugo, MSc, MBA , such neoantigens across all HLA Class I alleles (using our pan-allelic 4 4 1 Arjun Balar , Gary Steinberg , Anne de Groot, MD models) enables the effective generation of neoepitopes that are crit- 1 2 EpiVax, Inc., Providence, RI, United States; University of Chicago, ical for developing personalized cancer vaccines. Chicago, IL, United States; EpiVax Oncology, New York, NY, United States; NYU Langone Health, New York, NY, United States P73 Correspondence: Gad Berdugo (gberdugo@epivaxonco.com) Large scale multiomics reveals a marked bias in driver mutations Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P74 toward areas not reliably presented to the immune system Alex Powlesland, PhD, Michael Cundell, PhD, Floriana Capuano, PhD, Background Brandon Higgs, David Lowne, BS, Ricardo Carreira, PhD We hypothesized that neo-epitope-based prediction using an ad- Immunocore Ltd, Abingdon, United Kingdom vanced in silico T cell epitope screening system (Ancer™) may better Correspondence: Alex Powlesland (alex.powlesland@gmail.com) identify patients with improved prognosis than tumor mutation bur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P73 den. Analysis of genomic data derived from the muscle-invasive bladder cancer (BLCA) cohort of The Cancer Genome Atlas (TCGA) Background database for CD4, CD8, and Treg neo-epitopes was performed to de- The repertoire of HLA-peptides presented to the immune system termine whether Ancer™ would improve prognostic stratification which derive from cancer-associated, viral, and mutated proteins are compared to tumor mutational burden (TMB). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 41 of 272 Methods BLCA patient mutanomes (n=412) were retrieved from the TCGA and evaluated with Ancer™, an innovative and automated neo-epitope screening platform that combines proprietary machine learning- based HLA I and HLA II neo-epitope identification tools with removal of inhibitory regulatory T cell epitopes for neo-epitope ranking and personalized cancer vaccine design. BLCA patients were separated based on median TMB or neo-epitope burdens. We investigated the effect of integrating both CD8 and CD4 neo-epitope burdens as most mutanome pipelines exclusively focus on the identification of Class I neo-epitopes. Overall survival was analyzed using the Kaplan-Meier method and differences analyzed by log-rank testing. Results Compared to low TMB, high TMB was significantly associated with improved survival (p = 0.0001, difference of 38.5 months in median survival, Figure 1). Improved differentiation of median survival times was obtained when separating patients based on their Class I neo- epitope content, as estimated by Ancer™ (p < 0.0001, difference of 59.8 months in median survival). Adding Class II neo-epitope burden further increased separation of OS times, showcased by a 69.6-month increase in median survival for BLCA patients with both high CD8 and high CD4 neo-epitope contents compared to other patients (p = 0.0001). Since we discovered that Class II neo-epitopes can induce in- hibitory responses, we further evaluated whether the screening of these detrimental sequences could improve our analysis. Upon iden- tifying Class II neo-epitopes likely to induce T effector (Teff) re- Fig. 2 (abstract P74). See text for description sponses, we found that the median survival of patients with high CD8 and high CD4 Teff contents was extended by nearly 4 months to 73.4 months compared, to the remainder of the cohort (p < 0.0001, Figure 2). P75 Conclusions targetSCAPE and ultraSCAPE: Simultaneous identification and deep Our analysis suggests that optimal host-immune recognition of profiling of human antigen-specific T cells and other immune cell CD8+, CD4+, and Treg epitopes plays a key role in cancer sur- subsets by mass cytometry 1 1 1 vival. While defining CD8 neo-epitope burden enhanced associa- David Roumanes, PhD , Faris Kairi , Alessandra Nardin, DVM , Evan 2 1 tions with OS, the inclusion of CD4 Teff neo-epitope burden Newell, PHD , Michael Fehlings 1 2 substantially helped identify long-term survivors. These results immunoSCAPE, Cambridge, MA, United States; Fred Hutchinson suggest that defining the number of true neo-epitopes using Cancer Research Center, Singapore, Singapore Ancer™ may represent a novel prognostic or predictive Correspondence: Alessandra Nardin biomarker. (alessandra.nardin@immunoscape.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P75 Background During clinical trial immune monitoring, especially in the field of im- munotherapy, it is critical to collect in-depth phenotypic information from multiple immune cell populations in order to assess the biological activity of the immunotherapy, to identify biomarkers of response or progression, and/or to identify new drug targets. However, patient sam- ples, for example peripheral blood mononuclear cells (PBMC) or tissues, are often only available in small amounts and current methods face limitations in either depth of analysis and/or cell throughput. Methods In order to identify therapy-relevant antigens and to facilitate a concur- rent in-depth characterization of cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling cap- abilities of cytometry by time of flight (CyTOF) and a unique method- ology allowing the identification and characterization of rare antigen- specific T-cell subsets (targetSCAPE). By implementing a new technol- ogy (ultraSCAPE) that combines flow and mass cytometry together with a combinatorial live cell barcoding strategy, we further increased the high-dimensional phenotyping capacities to over 100 different marker molecules through simultaneous in-depth profiling of up to three add- itional immune cell subsets from the same sample. Results We isolated 4 different immune cell populations from a single sample and combined 3 different phenotypic panels consisting of 35 makers each together with a combinatorial tetramer multiplex and phenotyp- ing panel for deep profiling of myeloid cells, NK cells, B cells and T cells. We demonstrate the potential of this novel immuno-phenotyping method, by tracking virus-specific T cells while simultaneously charac- Fig. 1 (abstract P74). See text for description terizing 4 immune cell subsets with over 100 distinct phenotypic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 42 of 272 markers from a single sample, which is currently impossible employing P77 modern flow cytometers or classical mass cytometry methods. Comprehensive profiling of tumor-immune interaction in anti-PD-1 Conclusions treated melanoma patients reveals subject-specific tumor escape With its ability to provide an unprecedented picture of the immune mechanisms 1 1 1 1 status within a single sample, including T cell specificity information Charles Abbott, PhD , Eric Levy, PhD , Rachel Pyke , Rena McClory , 2 1 1 and in depth profiling of relevant immune cell subsets, ultraSCAPE in Sekwon Jang, MD , Richard Chen, PhD , Sean Boyle, PhD 1 2 combination with targetSCAPE can provide detailed insights on the Personalis, Menlo Park, CA, United States; Inova, Fairfax, VA, United effects of immunotherapy on the immune cell population. Informa- States tion learned from in-depth immune phenotyping of several immune Correspondence: Sean Boyle (sean.boyle@personalis.com) cell subsets such as T, NK and myeloid cell subsets can be leveraged Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P77 for the development of novel diagnostics, for biomarker discovery and for monitoring therapeutic strategies in immunotherapy. Background Checkpoint inhibitor therapy has demonstrated meaningful antitu- mor activity for many patients, though the majority fail to achieve P76 complete response. Thus, it is of particular interest to identify bio- Development of immunopeptidomic platform for human leucocyte markers and mechanisms that promote positive response to im- antigens class I using microflow liquid chromatography and munotherapy. In the present study, we apply our comprehensive quadrupole time-of-flight mass spectrometry tumor immunogenomics platform (ImmunoID NeXT), integrating data 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Yoshinobu Koguchi, MD, from the tumor, tumor microenvironment and immune system to 2 2 2 2 PhD , John Cha , Brian Piening, PhD , Eric Tran, PhD , Hong-Ming Hu, create a comprehensive biological signature of patient response to 2 2 2 PhD , Bernard Fox, PhD , William Redmond, PhD therapy. 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Methods Cancer Center, Portland, OR, United States We characterized the immunogenomics of 52 unresectable, stage III/ Correspondence: Takashi Shimada (tashimada@shimadzu.com) IV melanoma patients who underwent anti-PD-1 therapy to assess Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P76 factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 12 months. Background For each patient, a single paired FFPE tumor and normal blood sam- The highly complex population of peptides associated with human ple was collected and profiled using Personalis’ ImmunoID NeXT plat- leucocyte antigens (HLA) is the human immunopeptidome. Compre- form; an augmented exome/transcriptome platform and analysis hensive characterization of the immunopeptidome is key in predict- pipeline, which produces comprehensive tumor mutation informa- ing immunotherapeutic responses by evaluating targets of T cell tion, gene expression quantification, neoantigen characterization, interaction and in developing the next generation of cancer im- HLA typing and LOH, TCR repertoire profiling and tumor microenvir- munotherapies. Mass spectrometry (MS) is a technology that holds onment profiling. Tumor molecular information was then analyzed significant promise for untargeted and complete identification of the together with clinical outcome. immunopeptidome. MS acquisition is mainly used an electrospray Results ionization (ESI) combined with nanoflow liquid chromatography (LC). Comprehensive profiling demonstrated that elevated pretreatment However, the analysis time and retention reproducibility could be is- neoantigen burden was predictive of response to PD-1 blockade, and sues. Therefore, we tried to develop an MS platform that can ensure significantly associated with progression-free survival. Additionally, the coverage while increasing throughput using a microflow LC. we observed increased response to anti-PD-1 therapy in patients Methods with elevated pretreatment TCR clonality. Patients with high neoanti- HLA class I complexes were purified from A431 cell lysate by W6/32 im- gen burden and TCR clonality that failed to achieve complete re- munoaffinity. Purified HLA peptides were eluted with 5% formic acid. sponse revealed potential resistance mechanisms to anti-PD-1 The peptides were fractionated by 10 kDa ultrafiltration. HLA peptides therapy. Specifically, we identified two patients with high expression were separated with L-column2 ODS (0.3x150 mm) using a trap-elute of IDO1 or CTLA4, which may facilitate PD-1-independent immune protocol of microflow LC (Nexera-Mikros) and quadrupole time-of-flight escape. Additionally, we found two patients with antigen presenta- MS (LCMS-9030). Flow rate was set at 5 μl/min with a gradient of aceto- tion machinery (APM) mutations. The first patient had independent nitrile in 0.1% formic acid for 18 min. MS/MS spectra were acquired HLA-A and HLA-B mutations, likely leading to loss of surface expres- using a data-dependent manner of top-10 precursor intensities. The sion of the proteins. In the second APM mutation patient we ob- precursor scan was first set from 400 to 600 Da. The charge states of served a high frequency (80% AF) frameshift variant in B2M, which precursors were set between 1 to 4, and MS/MS scan was from 200 to potentially prevents proper HLA class I folding and antigen presenta- 1200 Da. The data were analyzed by Mascot proteome server and tion. These APM mutations suggest reduced neoantigen presentation PEAKS sequencing software on SwissProt database. The mass toler- in these patients, which are probable mechanisms for tumor escape. ances of precursors and fragments were set at 0.05 Da and 0.3 Da. Min- By integrating neoantigen burden, HLA-LOH and APM mutational imal peptide length was set to 8 amino acids. data into a corrected neoantigen burden, we were able to increase Results the predictive strength of this biomarker. An initial round of optimizations was performed to establish optimal Conclusions parameters for immunopeptidome identifications. Using tryptic pep- In summary, our comprehensive cancer immunogenomic analyses tides from A431 lysate, we optimized the 50-100 msec repeat of MS/ demonstrate that genomic and immune profiling of pretreatment pa- MS scanning, top-10 of data-dependent acquisitions per scan, 50 Da tient samples can identify biomarkers and resistance mechanisms to scan range, 35V±10V spread of collision electrode voltage, and 3.0 kV immune checkpoint blockade, suggesting the potential efficacy of of electrospray voltage. These parameters were then applied for these as an integrated biomarker to optimize anti-PD-1 therapy pa- identification of HLA-associated peptides from A431 cells. From this, tient selection. we identified 4,217 MS/MS and 801 sequences from 34,042 spectra. Conclusions From these data, we demonstrate that similar sensitivity can be suffi- P78 ciently achieved with microflow platform as has been demonstrated Optimization of tumor nutation burden measurement in FFPE DNA preciously for nanoflow LC-MS. This has significant advantages in Janice Au-Young, PhD, Iris Casuga, PhD, Vinay Mittal, Dinesh Cyanam, terms of throughput, instrument maintenance, and widespread ap- MS, Elaine Wong-Ho, Fiona Hyland, Seth Sadis, Warren Tom, PhD plicability. Our future directions are to determine whether cancer Thermo Fisher Scientific, South San Francisco, CA, United States neoepitopes identified by these approaches may be recognized and Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) therapeutically targeted by patient T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P78 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 43 of 272 Background demonstrated [1]. Solid tumors systemically reprogram the lung Tumor mutation burden (TMB) measures the number of somatic mutations unique immune environment, dominated by intravascular neutro- and is a positive predictive factor for response to immune-checkpoint in- phil functions [2], to colonize this site. The concept of ‘oligopro- hibitors in multiple cancer types. While whole exome sequencing (WES) is gression’ has recently received mounting attention, due to its thegoldstandardfor TMBmeasurement,itisnot practicalfor routineuse. relevance and because it represents an interesting in vivo model TMB values measured using targeted sequencing have been shown to to study TIME, although the specific mechanisms of oligometa- have good correlation with WES. However, during FFPE preservation, DNA static process are relatively underinvestigated [3, 4]. may undergo cytosine deamination, resulting in false C>T substitutions Methods and elevated TMB values. We have assessed the effect of DNA damage RNA sequencing was performed on a retrospective collection of tis- andrepaironTMB values usingthe Oncomine TumorMutationLoad sue samples from primary renal cell carcinoma, melanoma, and Assay (OTMLA), a targeted next generation sequencing assay. NSCLC and paired lung oligometastases of untreated patients (Figure Methods 1). Enrichment of tumor-related pathways and transcripts that reflect We measured TMB from 37 FFPE colon, lung, endometrial and gastric tu- the enrichment of immune cell subsets was assessed by single sam- mors usingthe OTMLApanel on IonGeneStudiowith20ngofinput DNA ple gene set enrichment analysis. Differentially expressed genes be- from tumor only samples. The informatics workflow utilizes a custom vari- tween primary tumors versus the corresponding lung metastases ant calling and germline variant filtering algorithm to accurately estimate were used for pathway analysis. Neutrophils extracellular traps (NETs) somatic variants in tumor tissue. In parallel, TMB was measured by Whole were revealed by immunofluorescence, assessing extracellular DNA Exome Sequencing (WES) targeting 50Mb using 100ng of tumor and and citrullinated H4 histone co-localization and/or myeloperoxidase matched normal DNA on a HiSeq X instrument. We examined factors [5]. Autophagy was assessed the CYTO-ID® kit [5]. that affect OTMLA measurements: Deamination signature, degree of de- Results amination and allele ratio identify DNA samples with high levels of dam- While tumor-related pathway enrichment differed mostly according age due to FFPE preservation. A Uracil-DNA glycosylase (UDG) repair step to the primary tumor histology, perturbations of immune-regulatory was introduced to eliminate damaged targets and improve usable TMB pathways was observed during oligoprogression in the lung. Decon- values of DNA from FFPE tumor tissue. At the variant level, samples with volution of immune cell subpopulations identified increased imma- high deamination scores were analyzed dynamically as a function of al- ture dendritic cells and reduced T cell abundance in oligometastatic lele frequency to study TMB values for correlation with WES. lesions. Strikingly, a large proportion of differentially modulated path- Results ways were “immune” rather than “cancer-cell”-related. Core analysis OTMLA TMB values showed good correlation with WES-derived TMB; confirmed that the main transcriptomic network that is affected dur- however ~10% of tumor DNA samples had high TMB and deamination ing disease progression is immune-based, centered on a cross-link values outside the expected range. These samples were included as a between innate and adaptive immunity. Specifically, it was associ- subset of samples tested with and without the UDG repair step. UDG ated with decreased HLA, iCOS, IL-9, and IL-17 pathway activity and treatment decreased TMB and deamination scores, resulting in higher downregulation of interferon signaling. During progression, we ob- correlation with WES TMB values. Some samples with very high de- served coherent modulation of transcripts associated with NET gen- amination scores were unable to be rescued; however, TMB values in eration, related to upregulation of key autophagic genes, to samples with low deamination and minimal damage were not affected. competition of the HMGB1 molecule with CXCL12 and CXCR4 and Conclusions RAGE receptor (AGER) activation. Accordingly, NET expression was We show that deaminated cytosine bases can be enzymatically re- strikingly more abundant in lung metastases than in primary tumors. moved by treatment with UDG. In a subset of FFPE samples tested, Conclusions UDG treatment was demonstrated to reduce the OTMLA estimated Our results identify evident molecular mechanisms associated with SNP proportion consistent with deamination. This results in consist- suppression of the immune milieu during disease oligoprogression in ent and effective reduction of C>T artifacts without affecting true the lung across different tumors. They include innate-adaptive im- variants and can provide TMB values in a biologically relevant range. mune dysfunction HLA-mediated, and interferon dysregulation asso- ciated with neutrophil-mediated immune suppression. Since these tumors are targeted by immune checkpoint blockade (ICB) our data P79 highlights the relevance of characterizing the TIME composition in Molecular comparison of tumor microenvironment in primary paired primary and oligometastatic lesions during ICB treatment to lung, melanoma, and kidney tumors versus paired lung metastases optimize treatment approaches. reveals shared perturbations of the immune milieu during oligoprogression Acknowledgements 1 1 Davide Bedognetti, MD, PhD , Jessica Roelands, Master , Angelo Paola Nistico' and Gennaro Ciliberto are co-last authors. 2 2 3 3 Manfredi , Norma Maugeri , Francesca De Nicola , Ludovica Ciuffreda , 3 3 3 Matteo Pallocca , Maurizio Fanciulli , Francesca Di Modugno, PhD , References 3 3 3 Paolo Visca , Barbara Antoniani , Gabriele Alessandrini , Darawan Rinchai, 1. Angelova M, Mlecnik B, Vasaturo A, Bindea G, Fredriksen T, Lafontaine L, 1 1 3 PhD , Wouter Hendrickx, PhD , Paola Nistico', MD , Gennaro Ciliberto, et al. Evolution of Metastases in Space and Time under Immune MD Selection. Cell. 2018 Oct;175(3):751-765.e16. 1 2 Sidra Medicine, Doha, Qatar; IRCCS Ospedale San Raffaele, Milano, Italy; 2. Granton E, Kim JH, Podstawka J, Yipp BG. The Lung Microvasculature Is a Istituto Nazionale Tumori Regina Elena, Rome, Italy Functional Immune Niche. Trends Immunol. 2018 Nov;39(11):890-899. Correspondence: Gennaro Ciliberto (gennaro.ciliberto@ifo.gov.it) 3. Weichselbaum RR. The 46th David A. Karnofsky Memorial Award Lecture: Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P79 Oligometastasis-From Conception to Treatment. J Clin Oncol. 2018 Sep27:JCO1800847. Background 4. Stephens SJ, Moravan MJ, Salama JK. Managing Patients With The importance of tumor-host interactions during cancerogenesis Oligometastatic Non-Small-Cell Lung Cancer. J Oncol Pract. 2018 and metastatic progression has been now widely appreciated. Jan;14(1):23-31. More recently, the impact of the tumor immune microenviron- 5. Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli ment (TIME) to mold tumor evolution was convincingly C, Maiuri L, Maseri A, D'Angelo A, Bianchi ME, Rovere-Querini P, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 44 of 272 Manfredi AA.Activated platelets present high mobility group box 1 to Results neutrophils, inducing autophagy and promoting the extrusion of Overall survival (N=141, median 6.05 months) was not associated neutrophil extracellular traps. J Thromb Haemost. 2014 with PD-L1 positive (>=1% tumor or tumor+immune cells)/nega- Dec;12(12):2074-88. doi: 10.1111/jth.P689. tive, diffuse/non-diffuse, gastric/gastroesophageal, prior therapy Ethics Approval (median=2), tumor IFNgamma signature or CD8+ tumor infiltrate. The study was approved by IFO Institution’s Ethics Board, approval number Differential gene expression analysis identified GRB7, a down- 561/03. stream mediator of HER2 signaling and part of the HER2/ERBB2 amplicon in breast cancer [4], as one of two genes associated with survival >1 year (FDR=0.027). HER2-positivity (medical record) was associated with a 3.5-fold higher median expression of GRB7. Prolonged survival was associated with both HER2-positivity (n= 43/132; HR=0.58, p=0.01) and the top quartile of GRB7 expression (n=25/94; HR=0.48, p=0.007). The median survival for HER2- positive patients was 10.1 months versus 5.95 months for HER2- negative. HER2 status was not associated with PD-L1 status or CD8+ infiltrate. Nearly all HER2-positive (n=40/43) and 2 HER2- negative patients received trastuzumab (median 62 days post- trastuzumab). Prior or best response was not related to 1 year survival and 2 of 3 HER2-positive patients that did not receive trastuzumab had >1 year survival. TMB was also evaluated and significantly associated with HER2-positivity (N=61, p=0.041). In the subset of 61 patients with TMB data, patients with high TMB plus HER2-positivity had the longest median survival of 15.4 months compared to all other patients at 6.7 months (N=14 vs 47; HR=0.47; p=0.04). Conclusions HER2 was associated with improved survival with checkpoint blockade in advanced GEA patients, regardless of response to Fig. 1 (abstract P79). See text for description prior trastuzumab. This study was limited by the lack of pre- treatment biopsies, but consistent with a recent report on Asian GEA patients [5]. The combination of HER2-positivity and relatively higher TMB in a limited dataset led to the greatest observed me- dian survival time, suggesting an interaction between HER2 and P80 TMB that warrants exploration in future GEA studies involving HER2 is associated with prolonged survival in advanced checkpoint inhibition. gastroesophageal adenocarcinoma patients treated with checkpoint blockade Acknowledgements 1 1 2 Carrie Brachmann, PhD , Emon Elboudwarej , Manish Shah, MD , David The authors gratefully acknowledge the patients and their families who 3 4 5 Cunningham , Jean-Philippe Metges , Eric Van Cutsem, MD, PhD , Zev participated in this study. 6 1 1 1 Wainberg, MD , Jingzhu Zhou , Dung Thai , Pankaj Bhargava , Daniel Trial Registration Catenacci, MD Clinicaltrials.gov NCT02862535 1 2 Gilead Sciences, Foster City, CA, United States; Weill Cornell Medicine, NY Presbyterian, New York, NY, United States; Sutton and London References Hospital, London, United Kingdom; Brest University Hospital, Brest, 1. Shah M, Metges J-M, et al. A phase 2, open-label, randomized study to France; University Hospitals Leuven & KU Leuven, Leuven, Belgium; evaluate the efficacy and safety of andecaliximab combined with nivolu- 6 7 UCLA School of Medicine, Santa Monica, CA, United States; University mab versus nivolumab alone in subjects with unresectable or recurrent of Chicago Medical Center, Chicago, IL, United States gastric or gastroesophageal junction adenocarcinoma. ASCO Gastrointes- Correspondence: Carrie Brachmann (carrie.brachmann@gilead.com) tinal Cancers Symposium. 2019. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P80 2. Metges J-M, Elboudwarej E, et al. Exploratory evaluation of baseline tumor biomarkers and their association with response and survival in pa- Background tients with previously treated advanced gastric cancer treated with ande- The benefit of checkpoint blockade in advanced gastric cancer caliximab combined with nivolumab versus nivolumab. ASCO is limited and patient selection biomarkers are needed. In a ran- Gastrointestinal Cancers Symposium. 2019. domizedphase 2study in >=2ndlineadvancedgastroesopha- 3. Brachmann C, Zhang Y, et al. Evaluation of intratumoral T cells in geal adenocarcinoma (GEA) cancer in Europe, US and Australia, biopsies from advanced gastric patients treated with andecaliximab and there was no clinical benefit for the addition of andecaliximab nivolumab. ASCO Gastrointestinal Cancers Symposium. 2019. to nivolumab in the total population or evaluated subgroups 4. Ferrari A, Vincent-Saloman A, et al. A whole-genome sequence and tran- (including PD-L1) [1,2]. Pharmacodynamic analyses demon- scriptome perspective on HER2-positive breast cancers. Nature Commu- strated little to no impact of andecaliximab [3]. This exploratory nications. 2016. biomarker analysis included all patients as a nivolumab-treated 5. Satoh T, Kang Y-K, et al. Exploratory subgroup analysis of patients with prior population. trastuzumab use in the ATTRACTION-2 trial: a randomized phase III clinical Methods trial investigating the efficacy and safety of nivolumab in patients with ad- Evaluation of archival tumor tissue was described [2,3]. Tumor muta- vanced gastric/gastroesophageal junction cancer. Gastric Cancer. 2019. tion burden (TMB) was evaluated by whole exome sequencing with Ethics Approval matched normal. Survival analyses (cox proportional hazards) were This study was approved by the institutional review board or independent adjusted for age and sex. ethics committee appropriate for each site. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 45 of 272 P81 Table 1 (abstract P81). Associations between TMB and Baseline Association of tumor mutational burden with clinical, genomic, Characteristics and treatment characteristics in advanced non-small cell lung cancer 1 1 2 Connor Willis, PharmD , Hillevi Bauer, PharmD , Trang Au, PharmD ,Sudhir 1 1 3 Unni, PhD, MBA , Wallace Akerley, MD , Ashley Sekhon, MD , Firas Badin, 4 5 6 7 MD , John Villano, MD, PhD , Matthew Schabath, PhD , Bing Xia, MD , 8 9 9 Beth Gustafson, PharmD , Komal Gupte-Singh, PhD , Beata Korytowsky , 9 9 John-Michael Thomas, PharmD , Gabriel Krigsfield, PhD , Solomon 9 1 10 Lubinga, PhD , Diana Brixner, PhD , David Stenehjem, PharmD 1 2 University of Utah, Salt Lake City, UT, United States; Long Island University, Brooklyn, NY, United States; MetroHealth Medical Center, Cleveland, OH, United States; Baptist Health, Lexington, KY, United 5 6 States; Markey Cancer Center, Lexington, KY, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; University of Southern California, Los Angeles, CA, United States; Saint Luke's Cancer Institute, Kansas City, MO, United States; Bristol-Myers Squibb, Princeton, NJ, United States; University of Minnesota, Duluth, MN, United States Correspondence: David Stenehjem (stene032@d.umn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P81 Background Tumor mutational burden (TMB) is emerging as a potential predictor of response to immunotherapy in various tumor types. However, the association of TMB data with clinical, demographic, genomic, and treatment characteristics warrants further investigation. Methods Nine U.S. Comprehensive Cancer Centers participated in this observa- tional, cohort study; five centers are members of the Oncology Re- search Information Exchange Network (ORIEN). Adult patients with stage IV non-small cell lung cancer (NSCLC) with tissue-based TMB data from any testing platform were included and their treatment in- formation was abstracted using a standardized case report form. TMB reporting ranged from September 2014 through March 2019. TMB-High and TMB-Low were defined as >10 mutations/megabase (mut/Mb) and <10 mut/Mb, respectively. Clinical, demographic, gen- omic, and treatment characteristics were compared by TMB level. Results There were 426 patients enrolled in the study across seven of the Table 2 (abstract P81). Associations between TMB and Select nine sites. TMB results from comprehensive genomic profiling (CGP) Oncogene Mutations were available for 354 patients. CGP vendors included Foundation Medicine (79.9%), Caris Life Sciences (17.0%), Tempus (2.8%), and NantHealth (0.3%). The median time from diagnosis to CGP testing was 45 days. A comparison of clinical and demographic characteris- tics by TMB is presented in Table 1. TMB-High status was associated with male gender (p<0.01), and positive smoking history (p<0.01). No correlation was found between TMB and PD-L1 (Table 2). TMB-High was positively associated with multiple oncogenes including STK11, LRP1B, TP53, and KDM5C (Table 2). In addition, there were significant negative associations between TMB-High and individual occurrences of altered ALK (p=0.03), EGFR (p<0.01), and ROS1 (p=0.03). The pro- portion of patients receiving first-line immunotherapy increased yearly from 8.5% in 2015, 19% in 2016, 40% in 2017, and 46% in Conclusions These interim results demonstrate the feasibility of conducting multi- site observational electronic health record-based studies with CGP and TMB across a national cohort of comprehensive cancer centers. P82 Immunotherapy utilization has been increasing in the first-line set- High-throughput pairing of single T-cell α and β chains along with ting. Associations between TMB status and driver mutations are indi- phenotypic expression profiling 1 2 3 cative of cancer etiology and informative for treatment decision- Brittany Brown, BS , Miranda Byrne-Steele, PhD , Wenjing Pan, PhD , 2 2 2 4 making. Updated results will be presented with an expanded cohort Song Li , Mary Eisenhower, BS , Daniel Weber , Mollye Depinet, MS , 2 2 2 of patients and future publications with the final cohort (n~1000) will Xiahong Hou, PhD, MD , Alex Moore , Jian Han, MD PhD 1 2 explore treatment, survival and response data. iRepertoire, Inc., Huntsville, AL, United States; iRepertoire, Huntsville, AL, United States; HudsonAlpha Institute for Biotechnology, Huntsville, AL, Acknowledgements United States; iRepertoire.com, Huntsville, AL, United States This study was sponsored by Bristol-Myers Squibb. Recruitment efforts were Correspondence: Jian Han (jhan@irepertoire.com) supported by Mikaela Larson (Huntsman Cancer Institute) and M2Gen® Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P82 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 46 of 272 Background panel was compared to whole-exome sequencing (WES) derived The T-cell receptor (TCR) is responsible for recognizing antigens as TMB. LOH-MHC status reported from PGDx elio tissue complete was peptides bound to a major histocompatibility complex. TCRs typically compared to MHC status determined by WES for accuracy. Addition- contain both an alpha (α) and beta (β) chain that contribute to anti- ally, TMB and MHC status were analyzed together in FFPE samples gen specificity; however, we have seen multiple cases of a single cell from ICI treated patients and correlated to clinical outcomes. containing dual α or dual β chains as well. When analyzing bulk rep- Results ertoires, information about endogenous pairing of α and/or β chains 115 pan-cancer samples were analyzed for microsatellite status and is lost after bulk lysis of T-cell populations. Pairing α and β chains demonstrated an overall agreement of 100.0% with a PCR-based from a single cell while also analyzing the phenotypic expression al- method. TMB was determined in 118 pan-cancer samples and dis- lows us to track TCR specificity and T cell function. This information played a high level of concordance with WES-derived TMB (Pearson can provide direct calculations of clonal frequency in various cell sub- correlation, p=0.903) across a range of TMB scores (0.2-89.7 muts/ sets, allow tracking of specific lymphocytes with treatment, and reveal Mbp). FFPE tissue samples from 98 cancer patients previously treated paired information for both chains of the receptor for downstream Car- with ICIs were then tested for LOH-MHC and demonstrated 88% ac- T development. curacy of detection when compared to WES. Furthermore, analysis of Methods TMB and MHC status in tandem found that patients with both high Here, we developed a method for high-throughput pairing of TCR α TMB and normal MHC status were found to have a significantly and β chains along with expression profiling. We examined, on aver- higher PFS, suggesting greater efficacy of ICI therapy. age, around 15,000 CD4+ cells loaded onto the BD Rhapsody Express Conclusions system. The receptor information is amplified from the same cDNA These data demonstrate the feasibility of measuring MSI, TMB, and using iRepertoire’s proprietary method that incorporates a multiplex evaluating LOH-MHC with high accuracy in a single >500 gene NGS mix of primers associated with both the TCR α and β loci; phenotyp- assay. Additionally, the results presented herein suggest that measur- ing of the cell is obtained using the BD Rhapsody RNA-seq kit. Along- ing TMB and MHC status concurrently can provide added utility in side the high throughput data, we also performed FACS-based single predicting patient response to ICI therapy. cell sequencing on the same individual’s samples through our iPair method (presented previously) and examined the overlapping recep- P84 tor sequences between both methods. Panel-derived tumor mutational burden (TMB) correlates with Results immune checkpoint inhibitors (ICIs) response in gastrointestinal With this mid throughput method, we are able to accurately assess cancers the frequency of single cells containing dual alpha or dual beta TCRs, 1 2 1 San-chi Chen, MD , Kien-Thiam Tan, PHD , Ming-Huang Chen , Yi-Ping which can help to evaluate the high throughput data. 1 2 2 1 Hung, MD , Yi-Lin Hsieh , Yi-Hua Jan , Yee chao Conclusions Taipei Veterans General Hospital, Taipei, Taiwan, Province of China; The described high throughput application should be applicable to ACT Genomics Co., Ltd., Taipei, Taiwan, Province of China any oligo-dT based single cell strategy. Correspondence: Yee chao (ychao@vghtpe.gov.tw) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P84 This study was approved by New England IRB, IRB number 120160202 Background P83 Tumor mutational burden (TMB) has been emerging as a relatively Pan-cancer assessment of composite genomic biomarkers in new biomarker that is independent of PDL1 for the prediction of re- immuno-oncology to predict responses and resistances to immune sponse to the immune checkpoint inhibitor (ICI) treatment. A recent checkpoint inhibitor therapy study has shown that whole exome sequencing (WES)-derived TMB Gustavo Cerqueira, Laurel Keefer, Kelly Gerding, PhD, Kenneth correlates well panel-derived TMB that is estimated using targeted Valkenburg, Christina Oliveras, James White, Leila Ettehadieh, Christopher sequencing. Here, we evaluate the correlation between panel- Gault, James Hernandez, Eric Kong, Isabell Loftin, Samuel Angiuoli, derived TMB with response to ICI treatment in gastrointestinal Abigail McElhinny, John Simmons, PhD cancers. Personal Genome Diagnostics, Baltimore, MD, United States Methods Correspondence: Eric Kong (ekong@pgdx.com) FFPE tumor and normal tissues from 18 patients with gastrointestinal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P83 cancers who had previously received ICI therapy at Taipei Veterans Gen- eral Hospital were retrospectively underwent targeted next-generation Background sequencing (ACTOncoTM) for the identification of somatic variants across Therapeutic response to immune checkpoint inhibitors (ICIs) requires 440 genes and the calculation of TMB. NetMHC and IEDB were used to a prior, suppressed immune response that is released via the inter- predict neopeptide bound to patient-specific HLA class one genotype. action of the checkpoint receptors with their cognate ligands. Micro- RECIST criteria were used to categorize tumor response. satellite instability (MSI) and tumor mutation burden (TMB) have Results emerged as composite genomic metrics that may better predict pa- Patients were grouped into responder (PR or CR, n=10) and non- tient response to ICI treatment, compared to conventional PD-L1 ex- responder (PD or SD, n=7). Among all patients, responders had sig- pression. However, testing for MSI and TMB separately is labor nificantly higher TMB than non-responders (mean 7.37 muts/Mb vs. intensive, increases turnaround time, and consumes valuable tumor 1.24 muts/Mb, p=0.0007). The number of predicted neopeptides tissue samples. Furthermore, recent studies have demonstrated that were significantly higher in responders than in non-responders antigen presentation mechanisms may also play a role in predicting (mean 10.8 vs. 3.6, p=0.0226). Notably, a non-responder harboring outcomes, wherein loss of heterozygosity in MHC class I genes (LOH- EGFR gain-of-function mutation did not respond to the ICI treatment MHC) suggests low likelihood of ICI benefit. despite high TMB. Furthermore, patients harboring MUC16 mutation Methods demonstrated higher TMB than patients without MUC16 mutation Here, we propose that these varied immune-oncology metrics can be (mean 17.38 muts/Mb vs. 3.9 muts/Mb, p=0.0005) (Figure 1). measured together in a single assay, utilizing next-generation se- Conclusions quencing (NGS) technologies. To test this hypothesis, we analyzed Although the cohort size is small, our study showed that panel-based >200 pan-cancer FFPE tumor tissue samples using the PGDx elio™ tis- TMB is predictive of response to ICI. As in lung cancers, EGFR muta- sue complete assay (currently in development; >500 genes panel) to tion is associated with decreased efficacy of ICI in the GI cancers. measure MSI, determine TMB (across 1.3 Mb), and assess MHC status Ethics Approval in a single assay. Detection of MSI was assessed for accuracy against The study was approved by TPEVGH intuition’s Ethics Board, approval a validated PCR approach and TMB determination from our targeted number 2015-07-002BC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 47 of 272 of TMB using publicly available whole-exome cancer sequencing data resulted in high correlation (R2 = 0.902, 0-40 mutations/Mb) and was parallel to the performance of our existing Oncomine Tumor Muta- tion Load Assay (R2 = 0.901, 0-40 mutations/Mb). Empirical analysis and performance of the assay on a common set of cell lines based on a universal reference standard also resulted in a positive correlation. Conclusions A larger tumor only NGS assay was developed to support compre- hensive genomic profiling and routine clinical research in oncology. The assay design and informatics workflow support characterization of mutational signatures and provide normalized TMB estimates. Min- imal input material requirement and rapid sample to report time will have a high impact on clinical research. More detailed information on the assay and an update on performance will be presented. P86 Predictive Immune Modeling enables biomarker discovery in NSCLC patients treated with second line immunotherapy 1 1 2 Natalie LaFranzo, PhD , Steve Daniel, PhD , Walt Carney, PhD , Milan 3 1 Bhagat , Natalie LaFranzo, PhD 1 2 Cofactor Genomics, St. Louis, MO, United States; Walt Carney Biomarkers Consulting, LLC, Boston, MA, United States; TriStar Technology Group, LLC, Washington, DC, United States Fig. 1 (abstract P84). See text for description Correspondence: Natalie LaFranzo (natalie_lafranzo@cofactorgenomics.com) P85 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P86 Development of a pan-cancer NGS assay for detection of tumor mutational burden and targeted biomarkers from FFPE samples Background Dinesh Cyanam, MS, Vinay Mittal, Nickolay Khazanov, Paul Williams, While cancer checkpoint inhibitors have garnered much attention Janice Au-Young, Gary Bee, Sameh El-Difrawy, Aren Ewing, Jennifer due to their ability to generate durable responses and improved sur- Kilzer, Anelia kraltcheva, PhD, Scott Myrand, MS, Yu-Ting Tseng, Cristina vival, the actual number of patients who are eligible for, receive Van Loy, Elaine Wong-Ho, Chenchen Yang, Dinesh Cyanam, MS, treatment with, and respond to these therapies remains modest [1]. Santhoshi Bandla, Warren Tom, PhD, Seth Sadis This is driven by a dependency on legacy diagnostics, built on Thermo Fisher Scientific, Ann Arbor, MI, United States single-analyte biomarkers such as PD-L1, which have failed to cap- Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) ture the complexity of disease [2]. Even in the case of non-small cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P85 lung cancer (NSCLC), an indication where the benefit of IO therapies is considered significant, there is much to be learned about the biol- Background ogy of the patients who respond, or do not respond to these Next-generation sequencing (NGS) is being applied to support routine therapies. clinical research in oncology with a primary focus on evaluating known Methods oncogenic variants. However, the advent of cancer immunotherapies Multidimensional RNA models have emerged to move beyond requires that clinical research solutions must also address biomarkers these legacy methods to reveal the full scope of disease com- such as Tumor Mutational Burden (TMB) and Microsatellite Instability plexity, resulting in increased predictive accuracy. Leveraging a (MSI) for immune checkpoint inhibitors. Therefore, we developed a re- database of gene expression models built using Predictive Im- search use NGS solution for FFPE tissues that expanded upon our mune Modeling, immune context of the tumor microenvironment current Oncomine Tumor Mutation Load Assay by measuring bio- is quantified. In this study, a cohort of NSCLC patients who re- markers for both targeted and immune checkpoint therapies. ceived second-line immunotherapies (checkpoint inhibitors) were Methods evaluated retrospectively. Pre-treatment solid tumor FFPE tissue Gene content was prioritized based on the relevance and variant samples were processed using the ImmunoPrism immune profil- prevalence of biomarkers in solid tumors. Additional genomic regions ing assay to generate comprehensive, individual immune profiles. were added to supplement the coding sequence footprint to support Pathological, demographic, and survival data (including overall TMB. The assay used Ion AmpliSeq™ technology with automated survival and progression-free survival, indicative of therapy re- templating on the Ion Chef™ system and sequencing on the Ion Gen- sponse), was used to group patients for predictive biomarker eStudio™ S5 sequencing platform. An automated tumor-only work- discovery. flow for variant calling, TMB and MSI estimation and sample quality Results reporting was provided within Ion Reporter Software. Decision sup- Individual immune profiles of the patients are compared, both within port tools were used for variant interpretation and evaluation of po- and between relevant cohorts, and statistically-significant biological tential variant relevance. signals are reported. Machine-learning derived multidimensional Results biomarkers were also generated, which are defined by the optimal Over 500 genes with known DNA and RNA alterations were included. combination of all analytes measured in the assay, enabling improve- The panel has broad capability for variant calling, fusion detection, ments in predictive accuracy. This study represents the first data MSI status, in addition to TMB. Specifically, for TMB, DNA repair path- generated using the ImmunoPrism assay with patients receiving ways were comprehensively represented as alterations in these checkpoint inhibitor therapies. genes may lead to high mutation burden. A coding sequence foot- Conclusions print to support TMB was generated. In development studies, the Predictive Immune Modeling enables us to build multidimensional assay displayed high uniformity and consistent read depth to sup- models of disease. When combined with well-curated patient co- port robust variant calling. The automated workflow required min- horts, such as the NSCLC patients described here, predictive bio- imal input of FFPE tumor only DNA and RNA material. Sample to markers may developed which capture more facets of the complex report turnaround time was less than five days. In-silico assessment immune contexture than previously possible. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 48 of 272 References P88 1. Haslam A, Prasad V. Estimation of the Percentage of US Patients Evaluation of a tumor-only pan-cancer targeted semi-conductor With Cancer Who Are Eligible for and Respond to Checkpoint based next-generation sequencing (NGS) test for microsatellite Inhibitor Immunotherapy Drugs. JAMA Netw Open. 2019 May instability in FFPE samples 1 2 2 3;2(5):e192535. Sameh El-Difrawy, Ph D , Anelia kraltcheva, PhD , Vinay Mittal , Elaine 2 2 2 2 2. Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune- Wong-Ho , Dinesh Cyanam, MS , Seth Sadis , Jennifer Kilzer , Cristina 2 2 2 2 checkpoint blockade: response evaluation and biomarker development. Van Loy , Janice Au-Young, PhD , Aren Ewing , Sameh El-Difrawy Nat Rev Clin Oncol. 2017 Nov;14(11):655-668. ThermoFisher Scientific, South San Francisco, CA, United States; Ethics Approval Thermo Fisher Scientific, Carlsbad, CA, United States The human tissue samples utilized for this study were provided by Correspondence: Sameh El-Difrawy (Sameh.El- TriStar Technology Group and have written, informed donor consent Difrawy@thermofisher.com) permitting academic and commercial research for publication, as Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P88 well as approval from a competent ethical committee. Background Comprehensive genomic profiling using next-generation sequencing (NGS) has become an essential tool to support routine clinical research P87 in oncology. Advent of cancer immunotherapies also requires assess- All-in-One, quantitative immune repertoire profiling of PBMC and ment of immune checkpoint inhibitor biomarkers such as microsatellite FFPE for renal cancer treatment evaluation instability (MSI) and tumor mutational burden (TMB). 1 1 2 Mollye Depinet, MS , Wenjing Pan, PhD , Sang-gin Wu, MD, PhD , MSI arises from defects in the mismatch repair (MMR) system and is as- 1 1 1 Xiaohong Hou, MD PhD , Brittany Brown, BS , Mary Eisenhower, BS , sociated with hypermutability of short DNA sequence repeats, micro- 1 1 3 Daniel Weber , Miranda Byrne-Steele, PhD , Michael Lotze , Jian Han, satellite locations, throughout the genome. Such defects are commonly MD PhD observed in colorectal, gastric and endometrial cancers and have been 1 2 iRepertoire, Huntsville, AL, United States; National Taiwan University, shown to be predictive of response to immunotherapy treatment. Trad- Taipei City, Taiwan, Province of China; UPMC Hillman Cancer Center, itionally MSI testing has been done using single biomarker tests such Pittsburgh, PA, United States as PCR/fragment analysis or immunohistochemistry (IHC) that require Correspondence: Jian Han (jhan@irepertoire.com) high sample input and are time consuming. Therefore, we developed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P87 an RUO NGS solution appropriate for FFPE tissues that addresses bio- markers for targeted and immune checkpoint therapies. Background Methods Next generation sequencing of the immune repertoire is a com- The performance of our RUO NGS based MSI approach was tested in prehensive immune profiling methodology that allows detailed, the context of a large Ion AmpliSeq™ panel composed of more than sequence-specific insight into the adaptive immune response. 13,000 amplicons covering 500+ genes. The content includes a di- While immune repertoire analysis of bulk RNA typically focuses verse set of microsatellite markers targeting MSI locations comprised on a single receptor chain, understanding of the variable rear- of mono- and di-nucleotide repeats that range from 7 to 34 bp. Se- rangements of the immune repertoire as a whole provides a quencing was carried out on the Ion 550™ chip and the Ion GeneStu- broader view of the immune landscape with potential prognostic dio™ S5 system. In-sample standards were designed and value. This is accomplished through the study of all seven TCR incorporated as internal references utilized by the analysis pipeline and BCR chains together (i.e., TCR-alpha, TCR-beta, TCR-delta, and a novel algorithm was developed that leverages the unique sig- TCR-gamma, and BCR-IgK and -IgL). One of the key challenges nal processing properties inherent in semi-conductor sequencing. during immune receptor amplification is the formation of dimers, The test provides results for individual microsatellites and generates which can compete with the immune amplicons of interest dur- an MSI score and status for the sample of interest. ing library preparation. Results Methods The performance of the MSI solution was tested using a set of over We therefore developed a novel PCR technique, dimer avoided multi- 400 FFPE and cell-line samples from different tissue types and plex PCR (dam-PCR), that effectively avoids dimer formation during showed excellent concordance with orthogonal tests. We report on PCR and incorporates unique molecular identifiers for direct RNA the sensitivity and specificity of our tumor only approach and quantification and error removal. With one sample, dam-PCR allows propose ideas to utilize generated MSI score in combination with for the amplification of all seven TCR and BCR loci in a single, quanti- other bio markers. tative multiplex reaction. Here, we apply this method to the amplifi- Conclusions cation of both PBMC and FFPE RNA from renal cancer patients An NGS assay was developed to support comprehensive genomic undergoing treatment. profiling and routine clinical research in oncology. The assay design Results and unique informatics workflow support precise characterization of We found that both TCR-alpha and -beta diversity prior to treatment mutational signatures and provides normalized MSI and TMB esti- along with the expression ratio between B cells and T cells are good mates. The performance of the assay was verified over a large cohort predictors of treatment efficacy. of colorectal, gastric and endometrial cancer samples with MSI status Conclusions independently assigned by orthogonal tests. [For Research use Only. Our study suggests that examining multi-chain immune reper- Not for use in diagnostic procedures] toire composition can be valuable for predicting treatment re- sponse and evaluating treatment protocols. Additionally, this method shows promise for future applications in both clinical P89 settings and basic research, as it allows for a cost effective, all- Obesity related changes in AXL-driven inflammatory signaling inclusive, and quantitative immune-profiling analysis of immune impact survival in melanoma repertoires from a range of sample types, including FFPE, where Alicia Gingrich, MD, Kylie Abeson, BS, Alexander Merleev, PhD, Robert sample RNA may be both limited in quantity and degraded in Canter, MD, MAS, FACS, Emanual Maverakis, Amanda Kirane, MD, Alicia quality. Gingrich, MD Ethics Approval University of California, Davis, Sacramento, CA, United States This study was approved by the University of University of Pittsbur- Correspondence: Alicia Gingrich (agingrich@ucdavis.edu) gh's Ethics Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P89 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 49 of 272 Background The TYRO3, AXL and MERTK (TAM) receptor tyrosine kinase (RTK) family have been associated with a number of human cancers, including melanoma.[1-3] Effects attributed to oncogen- esis and metastasis (epithelial-to-mesenchymal transition) of the TAM receptors have been described.[2] Recent evidence correl- ating obesity with a paradoxical improved response to immuno- therapy in melanoma suggests both tumor microenvironment and clinical phenotype play a role in response.[4] Therefore, we sought to build a predictive model of response to therapy from biomarkers, using TAM receptors and conventional markers of checkpoint inhibition such as PD-1. This model was tested in the normal weight, overweight and obese populations. Methods TCGA-SKCM melanoma tumor mRNA expression and clinical data for metastatic melanoma patients were downloaded from the GDC legacy archive (https://portal.gdc.cancer.gov/legacy-archive) (n = 471).[5] Bio- markers were defined as “high” or “low” expression in each patient. Dif- ferences in Kaplan-Meier survival curves based on level of expression were tested using G-rho family tests. Strength of relationships between biomarkers were measured using Pearson’s correlation. All statistical analysis were performed using R package “survival”. Results Normal weight, overweight and obese patients had markedly dif- ferent biomarker profiles associated with survival (Figure 1). In Fig. 1 (abstract P89). KM curves by clinical phenotype the normal weight population, high CD8 (p=0.0093), PD1 (p= and biomarker 0.0093) and CD84 (p=0.022) were associated with improved sur- vival. In the overweight population, high CD8 (p=0.0098), PD1 (p=0.0004) and CD84 (p=0.0081) were associated with improved P90 survival, while high Gas6 (p=0.029) and MERTK (p=0.043) were as- Unique tumor immune microenvironments of potentially PD-L1/ sociated with decreased survival. And in the obese population, TGF-β trap responsive tumors high AXL expression was associated with improved survival (p= Sean Glenn, PhD, Sarabjot Pabla, MSc, PhD, BS, Erik Van Roey, Jonathan 0.004), while CD8 (p=0.91) and PD1 (p=0.89) demonstrated no as- Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, sociation. In correlation analysis, AXL expression was most closely Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, associated with macrophage markers CD163 (r=0.52), CD84(r= Yirong Wang, MS, Carl Morrison, MD, DVM 0.56) and MS4A4A(r=0.53) in the obese but not the normal OmniSeq, Inc., Buffalo, NY, United States weight population. Correspondence: Sean Glenn (sean.glenn@omniseq.com) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P90 Taken together, these data suggest that immunologic response in metastatic melanoma patients is driven by separate immune Background profiles for obese and non-obese populations. AXL appears to Tumors often do not respond to PD-1/PD-L1 axis inhibitors due to im- mediateresponsein theobesepopulation by a macrophage- mune escape mechanisms present in the tumor microenvironment. Bi- driven mechanism as opposed to T cell mediation. Collectively, functional antibody-based immunotherapies that simultaneously target the significant differences in the transcriptomic profiles between immune checkpoints and immunosuppressive cells are being devel- obese and non-obese patients suggest potential clinical implica- oped to slow tumor growth. Anti-PD-L1/ TGF-β trap fusion proteins are tions regarding targets for treatment and application to patients one approach being tested to counter the traditional immune check- based on clinical phenotype. point inhibition via PD-1/PD-L1 axes and simultaneously inhibit the pro-tumor/anti-inflammatory effects of TGF-β.Inthis study, we not only References describe the tumor immune microenvironment of tumors expressing 1. Dransfield I, Farnworth S. Axl and Mer receptor tyrosine kinases: distinct PD-L1 and TGF-β, but also describe potential patient selection strat- and nonoverlapping roles in inflammation and cancer? Adv Exp Med egies based on gene expression measurements of these tumor im- Biol. 2016;930:113-32. mune microenvironments from clinical samples. 2. Verma A, Warner SL, Vankayalapati H, et al. Targeting Axl and Mer kinases Methods in cancer. Mol Cancer Ther. 2011;10:1763-73. RNA-seq was performed for 395 immune transcripts on 1323 FFPE tu- 3. Wu X, Liu X, Koul S, et al. AXL kinase as a novel target for cancer therapy. mors of diverse histologies. To find true TGF-β high expressing tu- Oncotarget. 2014;5:9546-9563. mors, TGF-β gene expression was normalized by a tumor 4. McQuade JL, Daniel CR, Hess KR, et al. Association of body-mass index inflammatory score (average expression rank of 161 inflammation and outcomes in patients with metastatic melanoma treated wtih tar- genes derived from co-expression signature of 1323 tumors spanning geted therapy, immunotherapy, or chemotherapy: a retrospective, multi- 35 tumor histologies). Proportion of PD-L1 IHC positive, tumor muta- cohort analysis. Lancet Oncol. 2018;19:310-322. tional burden (TMB) high and cell proliferation categories was esti- 5. Guan J, Gupta R, Filipp FV. Cancer systems biology of TCGA SKCM: mated for TGF-β high expressing tumors. Inclusion and exclusion efficient detection of genomic drivers in melanoma. Sci Rep. criteria were developed based on PD-L1 and normalized TGF-β 2015;5:7857. expression. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 50 of 272 Results Results Gene expression revealed varying degrees of TGF-β high tumors RNA-seq libraries from control RNAs at both 10 and 100 ng input in all tumor types studied. Sarcoma, pancreatic cancer and breast have less than 10% rRNA reads, replicate correlations greater cancer had the highest proportion of TGF-β high tumors. Within than 99%, and gene-level and transcript-level detection rates that these TGF-β high tumors, 41% were PD-L1 IHC+ (TPS≥1%), and are highly concordant with a conventional library preparation kit. 28% were TMB-high. 11% (n=147/1323) tumors were both TGF-β Additionally, the data confirms comparable dynamic range and high and PD-L1 high making these tumor microenvironments linearity of response of the ERCC spike-in controls. Libraries pre- ideal for a potential PD-L1/TGF-β trap treatment. Interestingly, pared from FACS-sorted T cells show differential expression pro- 47% (n=69/147) of these tumors presented with strong/moderate files consistent with the expected patterns. inflammation, with 53% (n=78/147) being non-inflamed tumors. Conclusions Conversely, there were 11.7% (n=155/1323) tumors that were The Advanta RNA-Seq NGS Library Prep workflow simplifies the high- TGF-β low and PD-L1 low presenting suboptimal tumor micro- throughput generation of RNA-seq libraries, significantly minimizing environment for a potential treatment. Notably, only 26% (n=40/ hands-on time and costly reagent consumption, which will facilitate 155) of these tumors presented with strong/moderate inflamma- the incorporation of RNA sequencing into the immune-oncology tion with clear majority (74%; n=115/155) being strongly or mod- research toolkit. erately inflamed tumors. For Research Use Only. Not for use in diagnostic procedures. Conclusions This large clinically tested tumor cohort suggests an immune phenotype of potentially PD-L1/TGF-β trap responsive tumors ex- ists across multiple histologies. PD-L1/TGF-β high tumors have distinct immune profiles compared to PD-L1/TGF-β low tumors. A P92 clinical immune gene expression assay described in this study Survival benefits of comprehensive genomic profiling and could not only improve patient selection for anti-PD-L1/TGF-β treatment in metastatic non-small cell lung cancer 1 2 trap treatment, but for other bi-specific fusion protein based Alison Sexton Ward, PhD , Jennifer Johnson, MD ,Komal Gupte- 3 3 immunotherapies. Singh, PhD , Mohammad Ashraf Chaudhary, PhD ,Devender 3 1 1 Ethics Approval Dhanda, PhD , Oliver Diaz, PhD , Katherine Batt, MD, MSc , John De-identified specimens and data were analyzed by OmniSeq under Fox 1 2 IRB approved protocol BDR 080316 (Roswell Park Comprehensive Precision Health Economics, Oakland, CA, United States; Jefferson Cancer Center, Buffalo, NY). University Hospital, Philadelphia, PA, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Priority Health, Grand Rapids, MI, United States Correspondence: Komal Gupte-Singh (Komal.Singh@bms.com) P91 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P92 Expression profiling of T cells using nanoscale automation with a full-length RNA sequencing library preparation kit on a Background microfluidic circuit platform 1 1 1 Metastatic non-small cell lung cancer (mNSCLC) patients who receive Thomas Goralski, PhD , Sangpen Chamnongpol , Michael Phelan , 2 1 1 comprehensive genomic profiling (CGP) at diagnosis may be more Jennifer Snyder-Cappione , Julie Alipaz , Joel Brockman ,Brian 1 1 1 1 likely to receive optimal first line (1L) therapies than patients who re- Fowler , Jennifer A. Geis ,Christopher Kubu ,Raphael Kung , 1 1 1 ceive panel testing (PT) with the enhanced ability to identify bio- Benjamin Lacar , Naveen Ramalingam, PhD ,Mandi Wong ,Charles 1 1 markers with associated therapies. The incremental survival benefits Park ,David King 1 2 of receiving optimal treatments following CGP testing at diagnosis Fluidigm Corp, South San Francisco, CA, United States; Boston have yet to be estimated. University School of Medicine, Boston, MA, United States Methods Correspondence: Christopher Kubu (chris.kubu@fluidigm.com) A Markov simulation model of biomarker testing and treatment Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P91 assignment for mNSCLC was built to estimate the survival out- comes associated with CGP versus PT. Biomarkers identified with Background PT were EGFR, ALK, ROS1, BRAF, and PD-L1 (≥50%). All biomarker RNA sequencing (RNA-seq) provides hypothesis-free profiling of tests were tested simultaneously using single gene testing or transcript levels and isoforms. This profiling captures a compre- assay. CGP, which employed Next-Generation Sequencing, identi- hensive view of the peripheral immune system or the tumor fied all the above biomarker changes and estimated tumor muta- microenvironment. The resulting profiles can be used to tional burden (TMB). The model assumed that PD-L1 testing was characterize differential gene expression patterns that can further conducted together with CGP. Biomarker identification, except for the understanding of the immune system. To further enable TMB and PD-L1, was assumed to be mutually exclusive and to these types of studies we have developed a highly cost-effective, occur at published prevalence rates. Incremental false-negative nanoliter-volume microfluidics-based workflow and chemistry rates of each genetic test in PT relative to CGP were applied. compatible with Illumina® sequencing instruments to simultan- Treatment pathways followed NCCN guidelines and current pub- eously generate RNA-seq libraries from up to 48 samples. This lished clinical trial results. Key inputs and assumptions were method fully automates solid-phase capture of polyadenylated tested in sensitivity analyses. RNA, reverse transcription, and index PCR within a compact nano- Results scale integrated fluidic circuit (IFC) on our Juno™ system. The Patient overall survival for each biomarker test within each test- workflow includes reagents necessary to generate full-length, ing strategy are shown in Table 1. Patients receiving CGP had random-primed RNA-seq libraries from as little as 10 ng of total 8.5% (1.4 months) longer survival on average than those who re- RNA, while preserving strandedness information. ceived PT. Patients receiving CGP testing at presentation spent Methods more time on 1L therapies (40% vs. 33%), thereby less time on Multiple replicates of 10 ng and 100 ng of total RNA from con- 2L therapies (23% vs. 26%) compared to patients receiving PT at trol samples spiked with ERCC RNA Spike-In Mixes were used to presentation. prepare RNA-seq library using the Advanta™ RNA-Seq NGS Library Conclusions Prep Kit. The performance was compared to a conventional li- CGP testing among mNSCLC patients at the time of diagnosis re- brary preparation kit. We also used our platform to profile total sulted in survival gains in comparison to PT due to higher proportion RNA purified from FACS-sorted CD3+, CD8+, CD28–,and CD25+/ of patients receiving optimal 1L treatment. hi T suppressor cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 51 of 272 Table 1 (abstract P92). Overall survival (months) by testing strategy & analyzed using Adaptive immunoSEQ in 125 samples. Tumor im- biomarker mune phenotype classification was done using unsupervised consen- sus clustering based on the expression of ICR genes. Results We have built one of the most extensive high-quality datasets for immunogenomic alterations available so far in colon cancer. Our pre- liminary data supports a positive impact of ICR gene expression in colon cancer cohort: patients with a Th-1 polarized microenviron- ment display better survival. Integrative analysis encompassing som- atic mutation, copy number variations, and transcriptome is ongoing and will be presented at the conference (Figure 1). Conclusions This newly generated immune centric NGS dataset, generated in Qatar, will contribute dramatically to elucidating the genetic determi- nants of immune responsiveness in cancer. Acknowledgements This work was supported by Qatar National Research Fund (QNRF) with grant JSREP07-012-3-005 References 1. Wang, E., Worschech, A. & Marincola, F. M. The immunologic constant of rejection. Trends Immunol. 29, 256–262 (2008). 2. Spivey, T. L. et al. Gene expression profiling in acute allograft rejection: challenging the immunologic constant of rejection hypothesis. J. Transl. Med. 9, 174 (2011). 3. Bertucci, F. et al. The immunologic constant of rejection classification P93 refines the prognostic value of conventional prognostic signatures in The advanced immune-centric NGS cohort for colon cancer breast cancer. Br. J. Cancer (2018). doi:10.1038/s41416-018-0309-1 1 1 2 Wouter Hendrickx, PhD , Jessica Roelands, Master , Peter Kuppen , 4. Galon, J., Angell, H. K., Bedognetti, D. & Marincola, F. M. The Continuum 3 1 1 Francesco Marincola, MD , Najeeb Syed , Davide Bedognetti, MD, PhD of Cancer Immunosurveillance: Prognostic, Predictive, and Mechanistic 1 2 Sidra Medicine, Doha, Qatar; Leiden University Medical Center, Leiden, Signatures. Immunity 39, 11–26 (2013). Zuid-Holland, Netherlands; Refuge Biotechnologies, Half Moon Bay, CA, 5. Roelands, J. et al. Genomic landscape of tumor-host interactions with dif- United States ferential prognostic and predictive connotations. bioRxiv 546069 (2019). Correspondence: Davide Bedognetti (dbedognetti@sidra.org) doi:10.1101/546069 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P93 6. Pagès, F. et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. The Background Lancet 391, 2128–2139 (2018). The immune system has a substantial effect on the progression of 7. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with colon cancer. Typically, an immune response defined by a polarized low memory requirements. Nat. Methods 12, 357–360 (2015). Th1 phenotype, characterized by expression of chemokine-receptor 8. Risso, D., Schwartz, K., Sherlock, G. & Dudoit, S. GC-Content Normalization ligands, activation of interferon-stimulated genes, production of cyto- for RNA-Seq Data. BMC Bioinformatics 12, 480 (2011). toxic molecules by effector immune cells, and upregulation of im- Ethics Approval mune regulatory genes, has been associated with immune-mediated Sidra Medicine IRB approval : #1602002725 tumor rejection. We have previously introduced a gene signature, called Immunology Constant of Rejection (ICR), that reflects these im- mune components.[1–4] This signature was able to differentiate quite well the patients with an active immune environment and improved survival vs those who did not[5]. Virtually, all correlative analyses integrating exome and transcrip- tomic data in colon cancer based on publicly available date use the TCGA cohort. Although it is broadly accepted that T-cell infiltration influences prognosis in colon cancer[6], the association between transcriptomic immune signature and patient survival could not be observed in the TCGA colon cancer cohort. This is likely due to the per protocol exclusion of samples with low tumor purity (i.e., higher stromal/immune infiltration), as at that time TCGA consortium fo- cused on defining cancer genetic makeup. To gain more insight into the underlying mechanism of cancer tissue rejection by the immune system in colon cancer, we build an extensive data repository from high quality snap frozen colon cancer samples unbiased for tumor purity. Methods RNA and DNA were isolated from a cohort of 366 colon cancer pa- tients collected over the last decade at the University of Leiden Med- ical Center (LUMC), Netherlands. Tissue sections flanking the corresponding samples were hematoxylin- and eosin-stained. RNA- seq (HiSeq4000) data was obtained using HISAT2 alignment[7] and quantile normalized after GC-correction of the raw counts.[8] Whole Fig. 1 (abstract P93). The advanced immune-centric NGS cohort for Exome Sequencing (WES) (>100X) was performed for normal and colon cancer cancer tissue (366 RNA-seq and 608 WES). T-cell repertoire was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 52 of 272 P94 A potential mechanism of anti-cancer immune response activated by immune-related adverse events (irAEs) in urological cancer patients 1 1 1 Taigo Kato, MD, PhD , Motohide Uemura , Koji Hatano , Atsunari 1 1 1 2 Kawashima , Takeshi Ujike , Kazutoshi Fujita , Kazuma Kioytani , Norio Nonomura 1 2 Osaka University, Osaka, Japan; Japanese Foundation for Cancer Research, Tokyo, Japan Correspondence: Taigo Kato (kato@uro.med.osaka-u.ac.jp) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P94 Background With the spread of usage of Immune checkpoint inhibitors (ICIs), a certain number of patients face discontinuation of ICIs due to severe immune- related adverse events (irAEs). Recently, some reports have shown en- couraging efficacy among patients who discontinued ICIs, leading to the hypothesis that irAEs-experienced patients have strong and long-lasting anti-cancer immune responses. So far, the molecular mechanisms of the immune response, particularly for T cells that play pivotal roles in attack- ing cancer cells, still remain unclear. Thus, characterization of T cell reper- Fig. 2 (abstract P94). Expanded T cells in metastatic site are toire and immune signatures in peripheral blood mononuclear cells detected in systemic (PBMCs) and tumors before and after ICIs treatment should contribute to better understanding of irAEs-related anti-cancer immune responses. Methods P95 In this study, we collected PBMCs from 4 urological cancer patients, Single-cell RNA-sequencing from clinically relevant core needle before ICIs treatment and at the onset of severe irAEs. For 1 kidney biopsies for evaluation of tumor-immune cell interactions in the cancer patient who had long durable response after discontinuation tumor microenvironment of ICIs, we also collected metastatic tissue sample and applied a next Namit Kumar, PhD, Mohan Bolisetty, Peter Szabo, PhD, Xuan Li, Becky generation sequencing approach to characterize T cell receptor (TCR) Penhallow, Ryan Golhar, Alice Walsh, Saumya Pant repertoires using RNAs isolated from tumors and PBMCs. We also Bristol-Myers Squibb, Princeton, NJ, United States measured mRNA expression levels of immune-related genes in the Correspondence: Namit Kumar (Namit.Kumar@bms.com) PBMCs of pre- and post-ICIs treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P95 Results We found that elevated transcriptional levels of CD3, CD4, CD8, GZMA, Background PRF1, andFOXP3 alongwithhighGZMA/CD3and PRF1/CD3 ratiointhe Elucidating biomarkers associated with immunotherapy response peripheral blood at the onset of irAEs. TCR repertoire analysis revealed and resistance will allow for better informed patient selection and drastic expansion of certain T cell clones in metastatic tissue after irAEs treatment decisions as well as enhanced drug development strategy. (Figure 1). Interestingly, some of these abundant TCR clonotypes were Current biomarker strategies are based on cellular markers (eg, im- also increased in peripheral blood at the onset of irAEs (Figure 2). munohistochemistry) or bulk molecular averages (eg, whole-exome Conclusions sequencing). However, there is limited ability to integrate cellular Our findings revealed that a certain number of expanded- and irAEs- and molecular data. Single-cell RNAseq (scRNAseq) is a promising related T cell clones in cancer tissue may also circulate systemically technology allowing for an unbiased analysis of the tumor microen- and then attack tumor cells in distant regions, leading to durable re- viroment (TME) at cellular resolution. Despite the immense potential, sponse in the patients with irAEs. implementation of this technology in clinical trials has been limited due to lack of methodologies applicable to clinically relevant speci- mens such as core-needle biopsies (CNB). Here, we describe the de- velopment of clinically applicable scRNAseq technology and analysis. Methods Treatment-naïve commercially sourced tumor resections were used to gen- erate ex-vivo CNB for scRNAseq analysis with 10X genomics. Post-clustering, unsupervised cell-type identification was performed (SingleR), and down- stream analyses were carried out (Seurat v2, custom R). Cells from multiple patients/tumor types (endometrial, TNBC, NSCLC, ccRCC, gastrointestinal; n= 8), and healthy donors (peripheral blood mononuclear cells; n=3) were com- bined, batch-corrected and aligned using canonical correlation analysis (CCA); and differential gene expression was performed (MAST algorithm). Results CNB scRNAseq was optimized across 5 tumor types, and the resulting data from ~43,000 cells allowed for the unbiased identification of TME cellular components (stromal, epithelial, immune-cell subtypes). The cellular resolution of this dataset allowed us to identify cell pop- ulations with distinct gene signatures. For example, we identified 2 macrophage subclusters—a lung tumor-specific cluster and a tumor- independent cluster. Lung-specific macrophages showed upregula- tion of genes including SPP1, G0S2, RGCC, PHLDA1, and TREM. Differ- ential gene expression analysis evaluated similarities and differences between TME vs healthy PB cells and allowed for surrogate pharma- codynamics marker assessment. In our analysis, 1197 genes were dif- Fig. 1 (abstract P94). Clonal T cell expansion in ferentially expressed; the most enriched genes in tumor-derived pancreatic metastasis monocytes included HSPA1A, IL8, APOE, and SPP1 whereas PB Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 53 of 272 monocytes were enriched for genes including LGALS2, S100A12, P97 S100A9, AHNAK, and CSTA. TCRB repertoire convergence and clonal expansion define the Conclusions NSCLC tumor microenvironment of responders to anti-PD-1 We have demonstrated the feasibility of scRNAseq from single CNB monotherapy 1 2 2 through the development of protocols to enable identification of bio- Timothy Looney, PhD , Katharina Leonards , Ilaria Alborelli , Luca 1 2 markers related to pharmacodynamics, therapeutic response, or disease Quagliatta , Philip Jermann 1 2 progression. Further, we have optimized the bioinformatics workflow to Thermo Fisher Scientific, Austin, TX, United States; University of Basel, derive meaningful biological insights from these scRNAseq datasets, Basel, Switzerland such as mechanisms involved in immune response or resistance that Correspondence: Philip Jermann (philipmartin.jermann@usb.ch) are tumor extrinsic or intrinsic. Our pilot study sets the groundwork to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P97 explore including scRNAseq in future prospective clinical studies. Background Acknowledgements There is an outstanding need to identify predictive biomarkers for re- Bristol-Myers Squibb. sponse to anti-PD-1 monotherapy for NSCLC. Here we investigated TCRB clonal expansion and TCR convergence within the pretreatment tumor microenvironment as predictors of response in a cohort of 37 P96 FFPE-preserved biopsies. For context, we compared the predictive T-cell receptor alpha and beta repertoire profiling using an value of these features with TMB values from the same tumors. augmented transcriptome Methods Eric Levy, PhD, Pamela Milani, Sean Boyle, PhD, Gabor Bartha, Charles Total RNA from FFPE-preserved pretreatment NSCLC biopsies (11 re- Abbott, PhD, Robert Power, Rena McClory, Robin Li, John West, MBA, sponders, 14 non-responders) was extracted for TCRB repertoire se- Richard Chen quencing via the Oncomine TCRB-SR assay (15-265ng RNA input; Personalis, Inc., Menlo Park, CA, United States average 164ng) and the Ion Torrent Gene Studio S5. TMB values Correspondence: Richard Chen (richard.chen@personalis.com) were obtained from FFPE-preserved gDNA from the same biopsies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P96 using the Oncomine Tumor Mutation Burden Assay. TCR conver- gence and clonal expansion were evaluated independently or in a Background combined model as predictors of response. The promise of immunotherapy has revealed the need for compre- Results hensive profiling of the tumor and its immune microenvironment, in- TCRB sequencing revealed increased TCR convergence (p = .02, Wil- cluding analysis of the T-cell receptor (TCR) repertoire. To address coxon) and clonal expansion (p = .06, Wilcoxon) in those who bene- this challenge, we developed ImmunoID NeXT to provide a more fited from anti-PD-1 therapy. A logistic regression classifier comprehensive view of the tumor and tumor microenvironment combining both features was able to discriminate responders from (TME) from limited FFPE tumor biopsies. This includes profiling both non-responders with a sensitivity of .91 and specificity of .71 at the the TCR alpha and beta chains. We show that ImmunoID NeXT accur- optimal cutoff, per the Youden’s J method. The TCR-based classifier ately and reproducibly profiles abundant clones and provides infor- was able to identify responders who otherwise had low to intermedi- mation on the diversity of T-cells in tumor samples. ate (<10muts per Mb) TMB. Methods Conclusions We first analyze the reproducibility of ImmunoID NeXT using repli- TCRB clonal expansion and convergence warrant further evaluation cates of PBMCs. Then, we compare the concordance of clones from as potential predictive biomarkers of response. Importantly, TCRB se- ImmunoID NeXT to the top clones from a standalone TCR sequen- quencing may allow for identification of responders who are other- cing approach. We also analyze the reproducibility of clones in wise missed by TMB-based stratification. patient-derived FFPE samples, and compare to IHC quantification of CD3+ cells to highlight the intra-sample heterogeneity of T-cell abun- dance and diversity. We then analyze the clonal diversity of pre- P98 treatment tumor samples in a cohort of melanoma patients who Automated rarefaction analysis for precision human and mouse B underwent PD-1 blockade. Finally, we use ImmunoID NeXT to profile and T cell receptor repertoire profiling from peripheral blood and the clonal diversity across over 100 solid tumor samples. FFPE-preserved specimens Results Timothy Looney, PhD, Geoffrey Lowman, PhD, Michelle Toro, Jayde Abundances of clones shared between replicates of PBMC samples Chang, Denise Topacio-Hall, BS, MA, Loni Pickle, PhD, Fiona Hyland, have a very high concordance (R2>0.99 with both TRA and TRB). Timothy Looney, PhD Compared to the standalone TCR approach, we identify over 96% of Thermo Fisher Scientific, Austin, TX, United States the top 1000 TRA clones, and over 99% of the top 1000 TRB clones, Correspondence: Timothy Looney (timothy.looney@thermofisher.com) both with highly concordant abundances (R2>0.95 and R2>0.94 in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P98 TRA and TRB, respectively). Subsequent curls of a tumor FFPE sample also have a high concord- Background ance of clonal abundances (R2>0.89 and R2>0.91 in TRA and TRB, re- Identifying the optimal input amount and sequencing depth for B spectively). TCR sequencing also provides a view of the clonal diversity and T cell receptor repertoire profiling is challenging owing to vari- of T-cells in a sample, which is not available with quantification via IHC. ation in material quality and lymphocyte diversity in blood and FFPE Finally, in a melanoma cohort, clonality based on either TRA or TRB is preserved specimens. Rarefaction analysis has emerged as a potential significantly different in responders to checkpoint inhibition. approach for assessing whether immune repertoire libraries have Conclusions been sequenced to saturation. Here we present a novel automated The ImmunoID NeXT platform can provide insight into the diversity method for saturation analysis of IGH and TCRB chain libraries de- of the immune repertoire, highlighting the platform’s ability to pro- rived from sequencing of peripheral blood leukocytes (PBL) and vide comprehensive analysis of both the tumor and tumor micro- FFPE-preserved RNA and DNA. environment. We demonstrate that ImmunoID NeXT is reproducible, Methods sensitive, and accurate at profiling high-abundance TRA and TRB Human TCRB and IGH repertoire libraries were generated using the clones, as well as feasible with FFPE samples. We also highlight how Oncomine TCRB-SR and BCR IGH-SR assays from: (1) 25ng PBL total immune repertoire results from ImmunoID NeXT can be used to gain RNA (2) 500ng PBL gDNA (3) 150ng RNA from FFPE preserved NSCLC understanding about the immunological composition of the TME. Fi- and (4) 200ng gDNA from FFPE preserved brain tissue. Mouse TCRB nally, we show how ImmunoID NeXT can profile the diversity of the and IGH libraries were generated using the Ion Ampliseq TCRB-SR TCR repertoire in tumor samples. and BCR IGH-SR assays and 25ng RNA or 500 ngDNA derived from Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 54 of 272 spleen or lymph node. Libraries were sequenced on the Ion Torrent P100 Gene Studio S5 then analyzed with Ion Reporter to identify clono- Impact of obesity on immunity in gastroesophageal types, quantify clonal expansion and diversity, and for IGH chain li- adenocarcinoma [GEAC] 1 2 1 braries, identify B cell clonal lineages and assess isotype usage. We Sarbajit Mukherjee, MD, MS , Sami Ibrahimi , Yali Zhang , Jianmin 1 1 then repeated clonotyping and analysis of secondary repertoire fea- Wang , Pawel Kalinski, MD, PhD tures using data that had been downsampled to fixed read depths. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Results University of Oklahoma, Oklahoma City, United States We observed an asymptotic relationship between the sequencing depth Correspondence: Sarbajit Mukherjee and the number of B and T cell clones detected, clone Shannon diversity, (sarbajit.mukherjee@roswellpark.org) and B cell clonal lineage richness and diversity, indicating that libraries Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P100 had been sequenced to saturation. By contrast, T and B cell normalized Shannon entropy appeared robust to sequencing depth. Background Conclusions Obesity is associated with an elevated risk of GEAC [1], but the Automated downsampling analysis may serve as a convenient tool molecular mechanism remains unknown. Paradoxically, however, for optimizing sequencing depth and input amount for B and T cell obesity is associated with a superior response to anti-PD-1 treat- repertoire sequencing studies. We expect this approach to become a ment [2,3]. This may be explained by our recent observations routine component of immune repertoire analysis. that obesity enhances PD-1 mediated T-cell dysfunction in a mechanism involving leptin signaling. Prompted by this data, we aimed to identify obesity/leptin-regulated molecular biomarkers in P99 GEAC. TMBler: a bioinformatic tool for measuring and optimizing Tumor Methods Mutational Burden quantification from targeted sequencing panels Based on the body-mass index (BMI), we categorized patients Laura Fancello, Luca Mazzarella, MD PhD, Alessandro Guida, Arnaud into normal (BMI 18-24.9), overweight (BMI 25-29.9) and obese Ceol, Piergiuseppe Pelicci, Luca Mazzarella, MD PhD (BMI ≥30). We then retrospectively analyzed the clinical report of IEO Istituto Europeo di Oncologia IRCCS, Milano, Italy PD-L1 staining by IHC_22C3/Keytruda from metastatic GEAC pa- Correspondence: Luca Mazzarella (luca.mazzarella@ieo.it) tients treated at our institution between 2014-2019. Chi-squared Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P99 test was used to determine the association between categorical variables. Next, we performed RNA-seq analysis of 13 gastric can- Background cer FFPE specimens (8 obese and 5 normal weight) to identify Tumor mutational burden (TMB) is increasingly proposed as a predict- differential gene expression between these two groups. Gene ex- ive biomarker for immunotherapy response in cancer patients. pression was quantified by log-fold changes. Differentially TMB assessed by Whole Exome Sequencing (WES) is considered the gold expressed genes were identified by using DESeq2. Then we standard but remains confined to research settings. Targeted enrichment looked at the association between the expression of leptin and panels of various genomic sizes are emerging as a more sustainable method- immune-related genes from those specimens, using generalized ology for assessing TMB in the clinical setting. However, panel-based TMB linear model implemented in DESeq2. TCGA gastric cancer data- quantification has not been adequately standardized to date, leading to base (TCGA -STAD) was used to validate these associations (using major heterogeneities in TMB measurement and a lack of uniformly accepted Pearson test) independently. A p-value of <0.05. cutoff values, thus limiting the possibility to transfer results across settings. In Results particular, the choice of variants to include in TMB calculation (synonymous, Our analysis of the clinical report of 77 patients with metastatic cancer driver genes or low-allelic frequency mutations, or other features) may GEAC revealed that patients with a BMI =>25 were more likely to strongly affect results and in particular TMB predictive value [1] express PD-L1 than normal-weight individuals (p = 0.03)(Table 1). Methods Our RNA-seq analysis identified the following genes to be up- We developed "TMBler", an R package to calculate TMB from targeted regulated in the obese group: NOS2, FOXP3, IDO1, EOMES, sequencing panels. TMBler allows to select multiple filters on mutation CD160, and CXCR5 (p<0.05). Expression of these genes was posi- counts for TMB quantification. It also includes a set of functions to tively correlated with leptin in our database; however, these asso- simulate custom panels on WES and calculate predictive value based ciations did not reach statistical significance; possibly due to our on available data on immunotherapy response matched with sequen- small sample size. The same analysis within the TCGA-STAD data- cing data [2,3]. Finally, it allows to measure panel-based TMB concord- base identified a strong positive correlation between the expres- ance with WES-based TMB and its predictive value using Receiver sion of all six genes and leptin (p <0.05)(Figure 1). GSEA Operating Characteristic (ROC) curves. identified several up-regulated immune-related pathways (Adap- Results tive Immune System, Antigen Processing Cross Presentation etc.) By simulating custom and commercially available panels, we show that the in the obese group. application of specific filter combinations can significantly influence TMB cal- Conclusions culation and its predictive value, and we identify instances where risk of erro- Our preliminary data suggest that obesity, and specifically lep- neous assignment of patients to responder/nonresponder groups is highest tin, is associated with several immune markers in GEAC. Our Conclusions mechanistic studies will explore how obesity/ leptin regulates TMBler is a useful tool for quantifying TMB from targeted panels. It the immune system and promotes cancer. These studies may can analyze performance of existing panels, optimize analytical pipe- allow us to identify new leptin regulated pathways as thera- line and design novel custom panels through simulations. peutic targets. References References 1. Fancello L, Gandini S, Pelicci PG, Mazzarella L. Tumor mutational burden 1. Garai J, Uddo RB, Mohler MC, Pelligrino N, Scribner R, Sothern MS et al. quantification from targeted gene panels: major advancements and At the crossroad between obesity and gastric cancer. Methods Mol challenges. J Immunother Cancer. 2019 Jul 15;7(1):183 Biol.2015; 1238:689-707. 2. Hellmann MD, Nathanson T, Rizvi , et al. Genomic Features of Response 2. Ibrahimi S, Mukherjee S, Roman D, King C, Machiorlatti M, Aljumaily R. to Combination Immunotherapy in Patients with Advanced Non-Small- Effect of Body Mass Index and Albumin level on Outcomes of Patients Cell Lung Cancer. Cancer Cell. 2018 Receiving Anti PD-1/PD-L1 Therapy. J Clin Oncol 36, 2018 (suppl 5S; abstr May 14;33(5):843-852 213). 3. Samstein RM, Lee CH, Shoushtari AN. Tumor mutational load predicts 3. Wang Z, Aguilar EG, Luna JI, Dunai C, Khuat LT, Le CT et al. Paradoxical survival after immunotherapy across multiple cancer types. Nat Genet. effects of obesity on T cell function during tumor progression and PD-1 2019 Feb;51(2):202-206 checkpoint blockade. Nat Med. 2019 Jan; 25(1):141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 55 of 272 Ethics Approval P101 The study was approved by the Institutional Review Board at Roswell Park Immune-based classification of pleural malignant mesothelioma by Comprehensive Cancer Center, approval number STUDY00000894 / BDR using integrative transcriptome analysis 1 2 1 2 109419. Ernest Nadal, MD, PhD , Ania Alay , David Cordero , Elisabeth Aliagas , 1 1 3 3 José Ruffinelli , Ramón Palmero , Ricard Ramos , Ivan Macía , Anna 3 3 1 Ureña , Fran Rivas , Xavier Solé 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; Bellvitge Biomedical Research Institute, L'Hospitalet, Spain; Bellvitge University Hospital, Table 1 (abstract P100). See text for description L'Hospitalet, Spain Correspondence: Xavier Solé (x.sole@iconcologia.net) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P101 Background Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia. Immune checkpoint inhibitors in MPM demonstrated modest efficacy, partly due to lack of predictive biomarkers of clinical benefit from immunotherapy. The aims of this work were: to identify immune fractions associated with clinical outcome; to stratify MPM patients based on their immune contexture and to characterize the immune-based groups at the genomic and transcriptomic levels. Methods Seven gene-expression datasets of MPM were used to assess theimmunemicroenvironmentof516 samples. The abundance of 20 immune fractions in each sample was inferred using Gene Set Variation Analysis. Identification of clinically-relevant frac- tions was performed with Cox Proportional-Hazards Models ad- justed for age, stage, sex, and tumor histology. Results T-Helper 2 (Th2, HR=2.14, p=1.5x10-4) and cytotoxic T cells (CTC; HR=0.57, p=9.1x10-3) were found to be consistently asso- ciated with overall survival in multiple datasets. Three immune clusters (IG) were subsequently defined based on Th2 and CTC immune infiltration levels: IG1 (54.5% of samples) had high Th2/ low CTC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) had low Th2/high CTC levels. Immune clusters were associated with overall survival independently of tumor histology, with an improving survival from IG1 to IG3 (HR IG2=0.52, 95% CI 0.39–0.69; HR IG3=0.32, 95% CI 0.19–0.53; p= 8.4x10-8; Figure 1). IG3 was significantly enriched in epithelioid tumors (90% IG3 vs. 62% IG1, p=0.001) and patients were youn- ger compared to the other groups(60 yearsIG3 vs. 66years IG1, p=0.021). These groups showed differential molecular pro- files, being IG1 enriched for CDKN2A and IFN-related genes de- letions. No statistically significant differences in the tumor mutational burden was observed, howerver IG3 tumours had fewer mutations than IG1 and IG2 groups. At the transcriptional level, IG1 samples showed upregulation of cell proliferation and DNA repair-related gene-sets, while IG3 samples presented up- regulation of immune checkpoint inhibitors (Figure 2) and inflammation-related pathways. Finally, integration of gene ex- pression with functional signatures of in vitro drug response showed that IG3 patients are more likely to respond to immune checkpoint inhibitors, while IG1 patients might be more sensi- tive to PARP inhibitors. Conclusions Analysis of publicly available gene-expression data of MPM reveals three major immune-based groups, based on Th2 and CTC composition. These clusters are associated with distinct genomic profiles and clinical outcome. Further validation of this classification is warranted in an independent cohort of Fig. 1 (abstract P100). See text for description MPM. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 56 of 272 P102 CT antigens are frequently expressed non-inflamed tumors Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS , Erik Van Roey, Sean Glenn, PhD, Jonathan Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, Yirong Wang, MS, Carl Morrison, MD, DVM OmniSeq, Inc., Buffalo, NY, United States Correspondence: Sarabjot Pabla (sarabjot.pabla@omniseq.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P102 Background Cancer testis (CT) antigens are tumor antigens that have a highly tis- sue restricted expression in germ cells but are often expressed in di- verse malignancies. With their highly immunogenic expression limited to tumor cells, CT antigens have become a prime target for cancer vaccinations and T-cell based therapy with chimeric T-cell re- ceptors. In this study, we investigated the association of two CT anti- gens (NY-ESO-1 and LAGE-1a) with the immune microenvironment of real-world clinical tumors spanning multiple histologies. Furthermore, we describe the association of CT antigens with traditional bio- markers of immunotherapy such as PD-L1 immunohistochemistry (IHC) and tumor mutational burden (TMB), with inflammatory status and cell proliferation status with confirmatory studies performed on a large TCGA pan-cancer cohort of 11,001 tumors. Methods Unsupervised clustering was performed on gene-expression data of 395 immune transcripts of 1323 FFPE tumors to reveal three inflam- matory patient clusters and three distinct gene groups; CT-antigen, inflammatory and neoplastic clusters. Test for proportions was per- formed using Pearson’s chi-squared test to describe association of NY-ESO-1 and LAGE-1a with PD-L1 IHC, TMB, inflammatory cluster and cell-proliferation. A retrospective cohort (n=242) of checkpoint Fig. 1 (abstract P101). Overall survival analysis according to the inhibition (CPI) treated tumors was utilized to perform overall survival immune groups (Kaplan-Meier curves) and response to CPI therapy for CT antigen+ tumors. Survival analysis was confirmed against the Pan-Cancer TCGA cohort (n=11,001). Results Unsupervised clustering showed clear co-expression sub-clustering of CTA genes differentiated from “immune” and from “neoplastic expres- sion”. PD-L1 IHC status was not associated with NY-ESO-1 (p=0.71) or LAGE-1a (p=0.52) status. Interestingly, LAGE-1a positive cases were over-represented in TMB high cases (p=0.016), whereas, NY-ESO-1 sta- tus was not associated with TMB. NY-ESO-1 positive cases were highly over-represented in non-inflamed cluster (p=0.006), whereas, LAGE-1a status was not associated with inflammation status. Both NY-ESO-1 (p= 0.031) and LAGE-1a (p=0.008) were significantly associated with cell- proliferation status. NY-ESO-1 positive tumors have significantly (p= 0.014) higher response rate in retrospective cohort but this was not ob- served for LAGE-1a status. NY-ESO-1 and LAGE-1a status showed trend toward better (p=0.09 and p=0.06 respectively) survival in the retro- spective and TCGA pan-cancer cohort. Conclusions This study presents an in-depth analysis of the immune landscape of CT antigen positive tumors across multiple histologies. CT antigen bearing tumors not only have unique immune profiles but also have significant associations with biologically relevant emerging bio- markers such as inflammatory signature, TMB and cell proliferation. CT antigens are a viable target for non-inflamed tumors for check- point inhibition therapy. Ethics Approval De-identified specimens and data were analyzed by OmniSeq under IRB approved protocol BDR 080316 (Roswell Park Comprehensive Fig. 2 (abstract P101). Expression of immune checkpoint markers Cancer Center, Buffalo, NY). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 57 of 272 P103 Methods Detection of human leukocyte antigen class I loss of Stage I-III subjects (n=24) receiving curative-intent chemo (doxo- heterozygosity in solid tumor types by next-generation DNA rubicin, cyclophosphamide, paclitaxel) were monitored longitudin- sequencing ally (mixed effects linear model) with serial peripheral blood Jason Perera, PhD , Brandon Mapes, PhD, Denise Lau, PhD, Ameen mononuclear cell flow cytometry and quantitative immunose- Salahudeen, Aly Khan, PhD quencing of the T-cell receptor β locus (TCRseq) using the immu- Labs, Chicago, IL, United States noSEQ® assay (Adaptive Biotechnologies, Seattle, WA). To evaluate Correspondence: Jason Perera (jason.perera@tempus.com) for long-term chemo effects, these analyses were repeated in a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P103 cohort of recurrent breast cancer patients who received chemo >12 months preceding analysis (n=9). Wilcoxon rank sum and Background tests of slope were employed to screen for associations with Human leukocyte antigen (HLA) class I proteins are expressed on the chemo response, defined as complete pathologic response (pCR) surface of all nucleated cells and are vital for immune surveillance. at surgical resection. When tumor-specific mutations (neoantigens) are presented on HLA Results molecules to CD8+ T cells, this recognition can drive immune re- By TCRseq, chemo resulted in an acute decline in T-cell fraction (0-8 sponses against the tumor and lead to tumor destruction. One mech- weeks, p12 months following chemo. anism of immune escape for tumors is loss of heterozygosity in HLA Conclusions genes (HLA-LOH), which reduces the total number of neoantigens avail- Curative-intent chemo is associated with T-cell death followed able for presentation to T cells. Due to the highly polymorphic nature by reconstitution, with the resulting T-cell repertoire being more of HLA, the copy number status of HLA genes is extremely challenging clonal and less abundant in naïve T cells. These findings persist to assess by standard bioinformatics approaches. To investigate the at the time of metastatic recurrence, and therefore may contrib- prevalence of HLA-LOH, we developed a specialized pipeline to detect ute to immunotherapy non-response in metastatic disease. Con- HLA-LOH by DNA next-generation sequencing (NGS). versely, we identified T-cell reconstitution as a potential Methods biologic modifier of chemo response. T-cell reconstitution can A cohort of colorectal and non-small cell lung cancer samples be therapeutically targeted with inhibitors of androgen receptor underwent DNA sequencing on the Tempus xT panel using signaling, which in experimental models enables thymic matur- paired, formalin-fixed, paraffin-embedded tumor and normal ation of naïve T-cell clones and an increase in peripheral T-cell (blood or saliva) samples. To detect HLA-LOH from NGS data, we count. This hypothesis is beingevaluatedinanongoingphase used NGS-based HLA typing to resolve the patient’smostprob- II clinical trial of bicalutamide (androgen receptor antagonist) able HLA haplotype. Based on this haplotype, we adaptively rea- plus ipilimumab and nivolumab in metastatic breast cancer ligned reads, extracted a number of features describing the (NCT03650894). relative allele coverage in the tumor and normal samples, and Ethics Approval used these features to make a confident determination of allelic The study was reviewed and approved by Providence Heath and Ser- loss in the patient’s tumor sample. vices Internal review board, approval number 15-162. Results Evidence of HLA-LOH was detected in 16% of non-small cell lung tumor samples and 17% of colorectal tumor samples. We did not ob- P105 serve a significant association between LOH status and tumor muta- PD-L1 isoform as a potential biomarker to predict response for tional burden or neoantigen load. In the colorectal cancer cohort, anti-PD-(L)1 treatment HLA-LOH was observed in tumor samples classified as microsatellite Kunbin Qu (kunbin.qu@beigene.com) instability-high (MSI-H); however, the association between HLA-LOH BeiGene, USA, San Mateo, CA, United States status and MSI status was not statistically significant. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P105 Conclusions We developed a novel method of determining HLA-LOH by DNA Background NGS and demonstrated that HLA-LOH is a readily detectable feature anti-PD-1/anti-PD-L1 (anti-PD-(L)1) therapies have shown clinical ac- in human tumors. These results highlight the complexity of antigen tivity across different cancers. However, predicting patient response presentation, the potential importance of HLA-LOH as a biomarker of remains challenging. Here we explore PD-L1 splicing isoforms as a immunotherapy response and resistance, and lays the groundwork potential predictive biomarker for anti-PD-(L)1 therapy response. Four for future investigations. PD-L1 splicing isoforms exist, including one dominant wildtype tran- script and an alternative isoform which skips the second exon (del- taExon2_PD-L1). P104 Methods Impact of chemotherapy (chemo) on peripheral T-cell diversity and TCGA normalized mRNA transcript counts were downloaded implications for subsequent immunotherapy response in breast from Genomic Data Commons. anti-PD-1 treated melanoma cancer 1 2 3 RNA-Seq data was from Hugo et. al. [1]. Bioinformatics analyses Joanna Pucilowska, PhD , Paul Fields, PhD , Valerie Conrad, BS , David 3 3 3 were performed in statistical package R. Human wildtype and Page, MD , Alison Conlin, MD , Joanna Pucilowska, PhD , Catherine 2 3 3 3 deltaExon2_PD-L1 isoforms were stably transfected into the Sanders, PhD , Raina Tamakawa, MS , Brie Chun, MD , Isaac Kim, MD , mouse cell-line BW5147. A chimeric PD-1 receptor, P3Z, which Mark Schmidt 1 2 fuses the extracellular and transmembrane domains of human Providence Cancer Center, Portland, OR, United States; Adaptive PD-1 to the cytoplasmic domain of human CD3ζ, was stably Biotechnologies, Seattle, WA, United States; EACRI Providence Cancer transfectedintoHuT78 cellsasa reporter assay for PD-1 signal- Center, Portland, OR, United States ing and IL-2 production [2]. Correspondence: David Page (david.page2@providence.org) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P104 By examining protein crystal structures from Protein Data Bank, we found exon2 occupies the physical interface between PD-1 and PD- Background L1. It is also the interface between anti-PD-L1 therapeutics and PD- Immune checkpoint blockade is only modestly effective in metastatic L1. Therefore, anti-PD-L1 molecules may not effectively target PD-L1 breast cancer. One potential contributing factor is chronic lymphode- in patients harboring the deltaExon2_PD-L1 isoform and may lack pletion associated with preceding curative-intent chemo. Here, we clinical activity. The prevalence of the deltaExon2_PD-L1 isoform evaluate the short and long-term effects of chemo on peripheral T- across TCGA tumors is shown in Figure 1. There are 8 cancers where cell counts and clonal diversity in a cohort of breast cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 58 of 272 the isoform is present above 5%, including liver and endometrial cancers. The deltaExon2_PD-L1 isoform was successfully transfected into BW5147 as demonstrated by mRNA expression. In co-cultures of HuT78/P3Z with BW5147/PD-L1 (both the wildtype and deltaEex- on2_PD-L1), IL-2 was secreted from the wildtype but not from the deltaExon2_PD-L1. Incubation with anti-PD-1 reduced IL-2 in a dose- dependent manner with the wildtype only (Figure 2), indicating del- taExon2_PD-L1 does not support PD-1 signaling. Patients expressing only deltaExon2_PD-L1 or a higher ratio of deltaExon2_PD-L1/wildtype may not have optimal PD-(L)1 axis signaling and be less responsive to anti-PD-1 intervention. To test this hypothesis, a ratio metric between deltaExon2_PD-L1 and wildtype was applied to an anti-PD-1 treated melanoma cohort GSE78220. This biomarker ratio stratified responders from non- responders with a p-value of 0.027 (non-responders with no del- taExon2_PD-L1 isoform were excluded, Figure 3), whereas PD-L1 expression did not. Conclusions Patients with deltaExon2_PD-L1 isoform lack the interface be- tween PD-L1 and PD-1, the same interface necessary for anti-PD- L1 therapeutic binding. This may lead to non-optimal signaling through the PD-(L)1 axis. Suboptimal signaling and inability to bind anti-PD-L1 potentially could reduce response to both anti- PD-1 and anti-PD-L1 treatments. This hypothesis needs to be fur- ther validated in additional anti-PD-L1 and anti-PD-1 treated Fig. 2 (abstract P105). See text for description cohorts. Acknowledgements The authors would like to thank Vanitha Ramakrishnan and Jessica Li for scientific discussions. References 1. Hugo W. et. al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell 2016; 165:35- 2. Zhang T. et. al. The binding of an anti-PD-1 antibody to FcγRΙ has a pro- found impact on its biological functions. Cancer Immunol Immunother. 2018; 67:1079-1090. Fig. 3 (abstract P105). See text for description P106 Tumor mutational burden profile (TMB) of oncogenic driver mutations in non small cell lung cancer Paul Walker, MD, Nitika Sharma East Carolina University, Greenville, NC, United States Correspondence: Nitika Sharma (sharman@ecu.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P106 Background Tumor mutational burden has emerged as a potential biomarker pre- dictive of response to Immune checkpoint blockade (ICB) in lung cancer. The utility of this biomarker in oncogenic driver mutations, that account for nearly 20-50% of NSCLC, is still unknown. KRAS mu- tation in lung cancer is a prognostic biomarker whereas EGFR and Fig. 1 (abstract P105). See text for description BRAF pathogenic mutations are predictive of response to tyrosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 59 of 272 kinase inhibitors (TKI). ICB with bevacizumab has demonstrated clin- TMB was higher in current and former smokers (median TMB 10.7 v ical benefit in EGFR mutated lung cancers per IMpower150 clinical 6.4 mutations/megabase(Mb); p < 0.0001), patients without an identi- trial [1]. TMB analysis between actionable/pathogenic EGFR muta- fiable oncogenic driver mutation (median TMB 14.5 v 8.5 mutations/ tions (i.e. exon 19 del, exon 21 L858R, T790M) and EGFR uncommon/ Mb; p = 0.004), and patients with locoregional disease at the time of variants mutations may provide therapeutic implications [2]. To ex- diagnosis (median TMB 10.8 v 8.0 mutations/Mb, p = 0.02). TMB cor- plore the immunological basis for these findings, we evaluated the related closely across all matched tumor pairs (Pearson’s r = 0.85, Fig- immune biomarker profile of NSCLC patients using Caris next- ure 1). Significant increases or decreases in TMB were uncommon in generation sequencing (NGS) platform. paired samples, and we observed no significant change in median Methods TMB with increasing time between specimen collection or with inter- We studied tissue samples on 446 patients with NSCLC from 2016- vening chemotherapy, immunotherapy, radiation therapy, or tar- 18. TMB was measured by counting all non-synonymous somatic mu- geted therapy. tations per megabase of the genome coding area using targeted Conclusions NGS (592 genes). High TMB was defined as ≥ 10 mut/Mb. The ana- In NSCLC, TMB correlated closely across tumor pairs, and increasing lysis was conducted using SAS 9.4. Variables were tested using a Wil- time between sample collections and intervening treatments were coxon signed-rank test. not correlated with significant changes in TMB. Results Ethics Approval KRAS mutations were found in 85 pts (19%), BRAF in 9 pts (2%), EGFR This study was conducted under Dana-Farber/Harvard Cancer Center mutation in 36 pts (8%), EGFR pathogenic mutation in 22 pts (5%), Protocol 02-180. EGFR variants in 14pts (3%). The median TMB of KRAS mutant vs KRAS wt (wild type) was 10 vs 7 mut/Mb (range 0-31, p<0.01). Conclusions This study highlights the unique immune profile of certain oncogenic driver mutations in NSCLC. Our results show that KRAS and BRAF mu- tant subsets have a significantly higher TMB than KRAS and BRAF wild type. In addition, EGFR variants have a higher TMB as compared to actionable pathogenic EGFR mutations. These findings could have therapeutic implications in guiding patient selection for ICB and merit a prospective investigation. References 1. Socinski M, Jotte R,Cappuzzo F. Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC. N Engl J Med. 2018; 378:2288-2301. 2. Offin M, Rizvi H,Tenet M.Tumor Mutation Burden and Efficacy of EGFR- Tyrosine Kinase Inhibitors in Patients with EGFR-Mutant Lung Cancers. Clin Cancer Res. 2018;1102. Ethics Approval The study was approved by ECU Institutional Review Board, approval number UMCIRB 15-001311. P107 Changes in tumor mutational burden in serially biopsied non-small cell lung cancer 1 1 1 James Smithy, MD, MHS , James Smithy, MD, MHS , David Hwang, MD , 2 2 3 1 Yvonne Li , Liam Spurr , Andrew Cherniack, PhD , Lynette Sholl , Mark Awad, MD PhD 1 2 Brigham and Women's Hospital, Boston, MA, United States; Dana- Farber Cancer Institute, Boston, MA, United States; Broad Institute of MIT and Harvard, Boston, MA, United States Fig. 1 (abstract P107). See text for description Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P107 P108 Background Working towards precision medicine of the tumor High tumor mutational burden (TMB) has been associated with re- microenvironment 2 2 2 2 sponse to checkpoint blockade in non-small cell lung cancer (NSCLC) Kyung Kim, PhD , Jeeyun Kim , Seung-Tae Kim , Jung-Yong Hong , 1 1 and other malignancies. However, the degree to which TMB changes Laura Benjamin , Kristen Strand-Tibbitts, PhD 1 2 over time, across anatomical sites, and with intervening treatment re- Oncologie Inc, Waltham, MA, United States; Samsung Medical Center, mains unknown. To evaluate TMB changes across time points, we Seoul, Republic of Korea compared TMB in tissue specimens from patients with serially- Correspondence: Kristen Strand-Tibbitts biopsied NSCLC. (kristen@oncologie.international) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P108 Clinicopathologic characteristics and changes in TMB were analyzed from patients with NSCLC and more than one tissue specimen that Background had undergone targeted next generation sequencing (NGS, OncoPa- Immune therapies for cancer have generated an enhanced focus on nel) at the Dana-Farber Cancer Institute. Those representing distinct controlling cancer through modulation of biologies associated with primary tumors by histologic or genomic analysis were excluded. the tumor microenvironment, rather than the traditional approach of Results targeting cancer cell biology. As more and more targeted therapies 193 NSCLC patients with more than one interpretable NGS result are designed to modulate the tumor microenvironment, we need a were identified; 30 were excluded due to separate primary tumors. better understanding of microenvironmental heterogeneity in human Of the 163 remaining patients included in the analysis, the median cancer. Similar to what has been done to describe patient subsets time between samples was 14 months (range: 0 to 114 months). based on their cancer biology using DNA and RNA signatures, we are Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 60 of 272 working to describe patient subsets based on their microenviron- nivolumab treatment. The CD8 signature may be used alone and/or in ment biology. The ultimate goal is to find effective means of identify- combination with other relevant and potentially independent bio- ing patients for novel therapeutic treatments that target biological markers such as PD-L1 expression or TMB to identify patients likely to pathways that regulate the non-neoplastic cells and drive cancer benefit from anti–PD-1 therapies. progression. Methods Acknowledgements RNA from publicly available sources including microarray and RNA- Bristol-Myers Squibb. Professional medical writing and editorial assistance Seq were analyzed with respect to gene signatures that describe four were provided by Katerina Pipili, PhD, and Jay Rathi, MA, of Spark Medica Inc, different microenvironmental phenotypes. funded by Bristol-Myers Squibb. Results Trial Registration These four microenvironmental subtypes are prognostic, but also NCT02387996 show evidence of being predictive to existing modalities of cancer drugs when analyzed in retrospective analysis. We examined the im- References pact of cancer stage on the distribution of these subtypes and find 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene little variation. signature with CD8 expression by immunohistochemistry (IHC) in Conclusions multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. Future clinical trials to prospectively test these four unique signatures 2. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- as predictive biomarkers for therapy need to be designed. lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. 3. Galsky M, Saci A, Szabo P, et al. Impact of tumor mutation burden on P109 nivolumab efficacy in second-line urothelial carcinoma patients: explora- Predictive performance of a CD8-derived signature by gene tory analysis of the phase II CheckMate 275 study. Ann Oncol. 2017; expression profiling in patients with urothelial carcinoma from 28(Suppl 5): Abstract 848PD. CheckMate 275 Ethics Approval 1 2 1 Peter Szabo, PhD , Padmanee Sharma, MD, PhD , George Lee, PhD , The protocol was approved by site institutional review boards or 1 1 1 1 Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , Robin independent ethics committees and conducted according to Good 1 1 1 1 Edwards, PhD , Saumya Pant , Abdel Saci , Neeraj Adya , Matthew Clinical Practice guidelines, per the International Conference on Galsky, MD Harmonisation. Patients provided written informed consent based on 1 2 Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD Declaration of Helsinki principles. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Institute, New York, NY, United States Correspondence: Peter Szabo (Peter.Szabo@bms.com) P110 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P109 Tumor CD8+ T-cell infiltration assessed by gene expression profiling alone or by immunohistochemistry plus epithelial- Background mesenchymal transition gene expression in urothelial carcinoma in Gene expression profiling (GEP) has been used to identify biomarkers CheckMate 275 1 1 2 of response to immunotherapy. Using a GEP-based inflammation Peter Szabo, PhD , Abdel Saci , Padmanee Sharma, MD, PhD , George 1 1 1 1 1 assay, we derived and analytically validated a CD8 signature to assess Lee, PhD , Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , 1 1 1 T-cell infiltration in the tumor microenvironment (TME) [1]. Here, we Scott Chasalow , Michael Montalto , Robin Edwards, PhD , Saumya 1 1 1 3 retrospectively explore the association of the CD8 signature, alone Pant , Neeraj Adya , Bruce Fischer, MD , Matthew Galsky, MD 1 2 and in relation to established biomarkers PD-L1 and tumor muta- Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD tional burden (TMB), with clinical response to nivolumab treatment. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Methods Institute, New York, NY, United States In the phase 2 CheckMate 275 trial, 270 patients with platinum- Correspondence: Peter Szabo (Peter.Szabo@bms.com) resistant metastatic urothelial carcinoma (UC) and evaluable tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P110 PD-L1 expression received nivolumab treatment. Responses were de- termined by blinded, independent review committee assessments Background [2]. Minimal follow-up time for the current analysis was ~3 years. T- Close proximity of CD8+ T cells to cancer cells has been associated with cell infiltration in the TME was assessed using the CD8 signature and improved outcome with immunotherapy. Using a gene expression pro- by immunohistochemistry (IHC) using an automated commercial pro- filing (GEP)-based inflammation assay, we previously derived gene sig- prietary assay (Dako mouse clone C8/144B; Agilent Technologies Co) natures that defined CD8+ T-cell infiltration (CD8 signature) and [1]. PD-L1 expression on tumor cells was independently assessed by localization to tumor parenchymal and stromal compartments (CD8- IHC using the PD-L1 IHC 28-8 pharmDx assay (Dako). TMB was mea- topology signatures) in multiple tumor types [1,2]. In patients with sured by whole exome sequencing [3]. Cox proportional-hazards re- urothelial carcinoma (UC), high stromal/epithelial-mesenchymal transi- gression assessed the dependence of progression-free survival (PFS) tion (EMT) gene expression has been associated with T-cell exclusion or overall survival (OS), and logistic regression assessed the depend- and poor response to immunotherapy [3]. Here, we assess three CD8- ence of objective response (OR) on biomarker values. The linear ef- derived signatures and compare them with a CD8 immunohistochemis- fects of biomarkers and their multiplicative interaction were included try (IHC)-derived score combined with EMT gene expression (CD8.IH- when multiple biomarkers were evaluated. Likelihood-ratio tests (2- C_EMT) to evaluate associations between these biomarkers and with sided) were used to assess biomarker interaction effects. Associations response to nivolumab in patients with UC in CheckMate 275 [4]. with PFS and OS were investigated using Kaplan–Meier analyses with Methods biomarker scores categorized by tertile. 270 patients with platinum-resistant metastatic UC received nivolumab, Results with response assessed by blinded central review [4]. CD8+ T-cell infil- GEP was evaluable in 205 (76%) and GEP+TMB in 113 (42%) of 270 tration in the TME (assessed using the CD8 signature [1], CD8-topology treated patients. Baseline characteristics, OR, PFS, and OS were similar signatures (parenchymal, stromal) [2], and by IHC using a proprietary between all treated patients and the GEP-evaluable cohort. CD8 signa- commercial assay [Dako mouse clone C8/144B antibody; Agilent Tech- ture scores showed a positive association with OR (P=0.005), PFS (P= nologies Co]) and PD-L1 expression on tumor cells (Dako PD-L1 IHC 28- 0.005), and OS (P 8 pharmDx) were assessed on baseline tumor samples. Predictive per- Conclusions formance of the CD8 signature and CD8-topology signatures individu- These results suggest that the GEP-based CD8 signature may have util- ally, the combined CD8-derived signatures (triple CD8), and ity as a potential biomarker for predicting clinical response to CD8.IHC_EMT, was evaluated using Cox proportional-hazards regression Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 61 of 272 for overall and progression-free survival (OS, PFS) and with logistic re- (NGS). Although the 0.4 cutoff identifies MSI-high status, there is in- gression for objective response (OR). Odds ratios were scaled to reflect sufficient data for this score's repercussion for MSI-stable patients [1]. the difference between the 75th and 25th biomarker percentiles. Two- MSI-high status rarely occurs in lung cancer patients representing sided likelihood-ratio tests were used to assess biomarker and inter- less than 1% of the cases. Therefore, we aim to identify how MANTIS action effects. Associations with PFS and OS were also investigated score correlates with immune profile and clinical outcomes in MSI- using Kaplan–Meier analyses with biomarker scores categorized by stable lung cancer. tertile. Methods Results MANTIS score was calculated for two TCGA (The Cancer Genome GEP was evaluable in 205/270 (76%) patients. Baseline characteristics Atlas) cohorts: squamous cell carcinoma (SqCC, n= 501) and adeno- and clinical outcomes were similar in the overall population and the carcinoma (ADC, n=517). After excluding MSI-high patients (n=3 and GEP-evaluable cohort. Response and survival predictions from the 1, respectively) we stratified each cohort into quartiles. The highest triple CD8 and CD8.IHC_EMT overlapped, and both biomarkers pre- quartile was named MANTIS-high (M-H) and the lowest quartile dicted benefit from nivolumab independent of PD-L1 expression. MANTIS-low (M-L). Immune profile (immune cell infiltration and PD- Odds ratios for OR were 2.59 (95% CI, 1.59–4.21) for triple CD8, 2.12 L1 expression), tumor mutational burden (TMB), neoantigen burden (1.47–3.07) for CD8.IHC_EMT, 2.51 (1.42–4.43) for the CD8 signature, and survival outcomes were compared between M-H and M-L. Tumor and 1.74 (1.22–2.49) for the parenchymal CD8-topology signature. immune landscape was identified using signatures from immune Conclusions metagenes predicting infiltration for 31 immune cells. Combined CD8 and CD8-topology gene signatures (triple CD8) Results showed similar performance to CD8.IHC_EMT for predicting response M-H was associated with higher activated CD4, gamma delta and and survival in nivolumab-treated patients with UC. These data sug- Th17 T cell infiltration when compared with M-L in lung SqCC (all gest potential utility of testing biomarker combinations and support p <0.05). No statistically significant difference in tumor T cell infil- further evaluation of gene signatures associated with parenchymal vs tration was found in ADC (Figure 1,2). M-H patients had a higher stromal CD8+ T-cell localization for predicting response to immuno- TMB when compared with M-L patients in ADC (p<0.05) and the therapy in patients with cancer. same tendency was observed for SqCC (p=0.10) (Figure 3). Add- itionally, M-H correlated with lower PD-L1 (CD274) expression in Acknowledgements both SqCC and ADC (each p<0.05) when compared with M-L. No Bristol-Myers Squibb. Professional medical writing and editorial assistance significant differences in neoantigen burden were demonstrated. were provided by Bernard Kerr, PGDipSci, and Jay Rathi, MA, of Spark Medica M-H patients showed a trend towards lower median overall sur- Inc, funded by Bristol-Myers Squibb. vival in SqCC and ADC (75 vs 63 months p=0.21; 53 vs 50 Trial Registration months, p=0.14, Figure 3C,3D). NCT02387996 Conclusions This is the first report that illustrates the implications of a microsatel- References lite instability score on immune landscape, PD-L1 expression, TMB 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene and clinical outcome from a pool of more than a thousand MSS non- signature with CD8 expression by immunohistochemistry (IHC) in small cell lung cancer patients. multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. 2. Szabo PM, Lee G, Ely S, et al. CD8+ T cells in tumor parenchyma and Reference stroma by image analysis and gene expression profiling: potential 1. Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam biomarkers for immuno-oncology therapy. J Clin Oncol. 2019; 37(Suppl): AM, Waldner MJ, Bindea G, Mlecnik B, Galon J, Trajanoski Z. Abstract 2594. Characterization of the immunophenotypes and antigenomes of 3. Wang L, Saci A, Szabo PM, et al. EMT- and stroma-related gene expres- colorectal cancers reveals distinct tumor escape mechanisms and novel sion and resistance to PD-1 blockade in urothelial cancer. Nat Commun. targets for immunotherapy. Genome Biol. 2015; 16:64. 2018; 9:3503. 4. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. Ethics Approval The protocol was approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation. Patients provided written informed consent based on Declaration of Helsinki principles. P111 Clinical and immunologic implications of a microsatellite instability score in lung cancer 1 1 1 Pedro Viveiros, MD , Misuk Lee , Bhoomika Sukhadia, MD , Kyunghoon 1 2 2 1 Rhee , Victor Wang , Jeffrey Chuang , Young Kwang Chae, MD 1 2 Northwestern University, Chicago, IL, United States; Jackson Laboratory For Genomic Medicine, Farmington, CT, United States Correspondence: Misuk Lee (misuklee55@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P111 Background Microsatellite instability status is currently used to predict susceptibil- Fig. 1 (abstract P111). Immune landscape in squamous ity to immunotherapy. MANTIS score was originally developed to cell carcinoma identify microsatellite instability through next-generation sequencing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 62 of 272 Conclusions This rich and large dataset illustrates the power and scalability of the 10x Genomics Chromium Single Cell Immune Profiling Solution with Feature Barcoding technology and presents an exciting opportunity for researchers to explore and draw further conclusions about the mechanisms of TCR–pMHC interaction. Furthermore, this experiment serves as the next step on the path toward the even larger-scale ex- periments that will be necessary to fully comprehend the rules of antigen recognition in the adaptive immune system in response to cancer and infectious diseases and will be key in the development of successful immunotherapies. Acknowledgements This study was performed in collaboration with our 10x Genomics partners Fig. 2 (abstract P111). Immune landscape in adenocarcinoma Immudex and Biolegend. P112 P113 A new way of immunity exploration by linking highly Dynamic analysis and visualization of the immune infiltration in multiplexed antigen recognition to immune repertoire and human cancer by integrating TCGA data phenotype Mingchao Xie, PhD, Bolan Linghu, PhD, Zhongwu Lai, PhD, Jonathan 1 1 1 Dagmar Walter, PhD , Stephane Boutet , Michael Stubbington, PhD , Dry, Ben Sidders 1 2 1 1 Katherine Pfeiffer , Josephine Lee , Luz Montesclaros , Julia Lau , Daniel AstraZeneca, Waltham, MA, United States 1 1 3 Riordan , Alvaro Martinez Barrio , Liselotte Brix, PhD , Kivin Jacobsen, Correspondence: Jonathan Dry (Jonathan.Dry@astrazeneca.com); Ben 3 4 4 1 PhD , Bertrand Yeung , Xinfang Zhao , Tarjei Mikkelsen Sidders (benjamin.sidders@astrazeneca.com) 1 2 10x Genomics, Pleasanton, CA, United States; 10x Genomics.com, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P113 Pleasanton, CA, United States; Immudex, Copenhagen, Denmark; Biolegend, San Diego,CA, United States Background Correspondence: Tarjei Mikkelsen (tarjei@10xgenomics.com) Understanding of the complex interplay between tumors and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P112 their immunologic microenvironment is critical for immune- oncology (IO) studies, which can facilitate the discovery of Background novel prognostic biomarkers, identification of new drug targets, Recent progress in cancer immunotherapy emphasizes the and determination of drug resistance mechanisms. However, importance of understanding immune-regulatory pathways in due to a lack of proper analysis tools and datasets, systematic- cancer. It has been shown that immune cells play a crucial role ally exploring the tumor–immune interaction is still a big in the tumor microenvironment and can be used for targeted challenge. therapeutics. Therefore, it is important to understand and Methods characterize T cells and their antigen binding specificity and di- Here, we deconvoluted the immune cell compositions and per- versity in order to develop effective targeted immunotherapies. formed IO-related pathway/signature enrichment analysis for 9,721 Recent technological advancements have enabled the integra- primary tumor samples from 33 TCGA cancer types using transcrip- tion of simultaneous cell-surface protein, transcriptome, immune tomic data, and developed a web-based application, IO Browser. repertoire and antigen specificity measurements at single cell Results resolution, providing comprehensive, scalable, high-throughput The browser allows the user to visualize the immune infiltrations of a characterization of immune cells. sample or cohort, and to define disease segments or “immuno-types” Methods based on the presence of single/multiple immune cell types or IO- Using the 10x Genomics Single Cell Immune Profiling Solution related pathway/signatures. Users can then perform survival compari- with Feature Barcoding technology in conjunction with Biolegend sons, explore gene expression of key cancer and IO genes as well as oligo-conjugated antibodies and Immudex DNA barcoded generate oncoprints in the different segments. The browser also pro- peptide-MHC Dextramer® (pMHC), we performed multi-omic vides statistical analysis to identify the gene or mutations enriched in characterization of CD8+ T cell recognition of various virus and the immuno-typed disease segment, and correlate gene expression common cancer antigens in normal patients. Next generation se- or mutations with specific immune cell types in tumor microenviron- quencing libraries were made following the 10x Genomics work- ment (TME). flow, where gene expression and immune repertoire libraries are Conclusions generated alongside libraries from DNA barcodes conjugated to In summary IO Browser enables comprehensive analysis and antibodies or pMHC, allowing quantification of cell surface pro- visualization of the dynamic interactions between tumor and im- teins and identification of T cell receptor (TCR) specificities. Ana- mune landscape, and can aid our understanding of the interplay be- lysis was performed using the latest version of Cell Ranger (v3.0). tween tumor genomics and immune biology to facilitate line of sight The TCR-dist algorithm was used to identify clusters of related and disease segmentation. TCR sequences and enriched CDR3 motifs. Results P114 We performed multi-omic characterization of ~100,000 CD8+ T cells Tissutal immune profile and pathological complete response in from four MHC-matched donors. The multi-omic combination of triple negative breast cancer gene expression, paired alpha/beta T cell receptor (TCR) repertoire, 1 1 1 Andrea Botticelli, MD , Bruna Cerbelli , Simone Scagnoli , Maria Ida cell surface proteins and pMHC binding specificity allowed the identi- 1 1 2 2 Amabile , Alessandro De Luca , Lucio Fortunato , Leopoldo Costarelli , fication of CD8+ T cell subpopulations with specificity for pMHCs 1 1 1 Marianna Nuti, PhD , Giulia D'Amati , Paolo Marchetti within our panel. Within our data, we observed TCRs with cognate 1 2 Sapienza University of Rome, Rome, Italy; San Giovanni Addolorata antigens that had been reported previously, while also identifying Hospital, Rome, Italy entirely new TCR–pMHC interactions. In addition, we observed spe- Correspondence: Bruna Cerbelli (bruna.cerbelli@uniroma1.it) cific expanded non-naïve T cell clones along with more diverse bind- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P114 ing in the naïve compartment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 63 of 272 Background spectrometry to deeply characterize global tumor proteomes to In the neoadjuvant setting, pathological complete response (pCR) is identify proteins and pathways that are associated with pre- more frequently achieved by triple negative breast cancer subtype treatment response to anti-PD-1 immunotherapy. and patients who attain this status show improved survival; However, Methods standard neoadjuvant therapy results in pCR rates slightly over 30% Unbiased, data-independent acquisition (DIA) mass spectrometry of cases. The mechanism underlying the resistance to chemotherapy was used to analyze formalin fixed paraffin imbedded (FFPE) is still unclear and could be related both to the molecular heterogen- tumor tissue samples from subjects with Stage III-IV melanoma eity of cancer cells and to the activation of the treatment-related im- which were resected prior to initiation of first-line anti-PD-1 ICI mune response. For this reason, the search for immune biomarkers therapy. The selected samples represent two distinct clinical sub- able to predict the response to chemotherapy represents a new groups; those who received clinical benefit, with a partial re- promising frontier. Recent reports underscore the role of TILs, PDL-1 sponse or better (PR, SD and CR, n = 13), and those with no and CD73. The aim of our work is to define a novel tissutal immune clinical benefit (PD, n = 9) and no observable response to ther- profile (TIP) able to predict pCR [1,2]. apy. Samples were prepared for mass spectrometry using stand- Methods ard procedures. All samples were analyzed using 2-hour gradients We enrolled 61 pts who received NAC (EC for 4 cycles followed by on a LC-MS/MS setup operated in DIA mode. Data was extracted Paclitaxel q7 for 12 cycles or q21 for 4 cycles) between Jan 2011 and using Spectronaut (Biognosys) with a sample specific spectral li- June 2017 at Policlinico Umberto I and San Giovanni Addolorata Hos- brary which was combined with a large human tissue resource li- pital of Rome. We performed, in basal paraffin-embedded biopsies, brary. Statistical analysis was conducted to identify proteins that stromal TILS evaluation and immunohistochemistry for PD-L1 (Ven- are either up- or down-regulated with respect to benefit group. tana SP142 clone) evaluated both on tumor cells (TC) and tumor- Pathway analysis was also conducted to highlight dysregulated infiltrating immune cells (IC) and CD-73 assessed on TC. We defined biological functions and pathways. “positive tissutal immune profile” (TIP+) the pts with “high TILS” Results (>50%), “PD-L1 positive” ( >1% both on TC and IC ) and “low CD73” 7,590 proteins were quantified across all samples, with 6,627 (<40%), and the others as “negative tissutal immune profile” (TIP- quantified on average per sample. Univariate statistical testing ).Statistical analysis was performed with T di Student test and χ2 test. between groups identified 254 proteins that are dysregulated Results (120 up-regulated and 134 down-regulated) in subjects who re- We enrolled 61 females (median age: 50 y; range 28-75) affected by ceived clinical benefit. Through partial least squares discriminant TNBC. The clinical stage before NAC was as follow: 3 pts cT3 (5%), 3 analysis (PLS-DA) a set of 25 proteins was identified that describe pts cT4 (5%) and 28 pts were cN+ (38%). Twenty-three patients the variance between the two sample groups. When annotated (38%) showed pCR. No significant associations were found between to their sub-cellular location, all up-regulated species are identi- pR and cT, cN, age, and KI-67. Seven patients (11%) were TIP+ and fied as mitochondrial proteins, indicating an enhanced metabolic achieved pCR in 100% of cases; 54 patients were TIP- and pCR were environment, and the down-regulated species are cytosolic, lyso- showed in 16/54 of cases (30%) (p< 0,001). somal or membrane associated. This observation was also Conclusions reflected in pathway analysis which identified up-regulation of ar- TIP+ seems to be associated with higher pCR rate in TNBC patients ginine and citrulline metabolism and down-regulation of adhesion .These preliminary results suggest the possibility of using novel pro- related processes driven by MHC-II and integrins. files combining multiple immune- biomarkers. Conclusions Global profiling of the tumor proteome provides a unique References characterization of melanoma tumor biology. A pathway level ana- 1. Matsumoto H, Koo SL, Dent R, Tan PH, Iqbal J. Role of inflammatory lysis shows increased metabolic processes combined with decreases infiltrates in triple negative breast cancer. J Clin Pathol. 2015;68:506–510. in adhesion related proteins may underly the differences in benefit doi: 10.1136/jclinpath-2015-202944 139. related to ICI therapy. 2. Jiang T, Xu X, Qiao M et al. Comprehensive evaluation of NT5E/CD73 Ethics Approval expression and its prognostic significance in distinct types of cancers. The study was approved by the Istituto Nazionale Tumori IRCCS Fon- BMC Cancer. 2018 Mar 7;18(1):267. doi: 10.1186/s12885-018-4073-7. dazione “G. Pascale” of Napoli Institutution‘s Ethics Board, approval PubMed PMID: 29514610; PubMed Central PMCID: PMC5842577. number 33/17. Ethics Approval Consent CE 4181 Sapienza University of Rome Written informed consent was obtained from the patient for pub- lication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this P115 journal. Deep proteomic characterization of FFPE tumor samples from late- stage melanoma subjects treated with anti-PD-1 immunotherapy 1 1 Nicholas Dupuis, PhD , Jakob Vowinckel, PhD , Domenico Mallardo, P116 2 2 2 2 MD , Mariaelena Capone, MD , Madonna Gabriele , Antonio Sorrentino , Centrifuge-free red blood cell lysis and immunostaining of whole 2 1 2 Vito Vanella , Daniel Heinzmann , Paolo Antonio Ascierto, MD blood for flow cytometry using Laminar Wash™ system 1 2 Biognosys AG, Schlieren, Switzerland; Istituto Nazionale Tumori IRCCS, Ira Kim, Melvin Lye, Chyan Ying Ke, Nadiezda Fernandez Oropeza, Naples, Italy Sigeeta Rajaram, Kong Leong Cheng, Ih Chin Kon, Royce Pek, Namyong Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) Kim, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P115 Curiox Biosystems, Boston, MA, United States Correspondnce: Namyong Kim (namyong@curiox.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P116 Immune checkpoint inhibitors (ICI) have improved the treatment options for patients with advanced stage melanoma, with im- Background proved clinical responses and overall survival compared to stand- Blood cells are prime indicators of immuno-surveillance, and the ease ard systemic therapies. However, a large percentage of melanoma of blood sampling makes blood analysis a key interest for clinical patients do not respond to ICIs, highlighting the need for a and research applications. While current flow cytometry methods are greater understanding of the tumor environment and host im- high-throughput and provide fine resolution in the segregation of mune response. Here, we apply unbiased discovery proteomics, white blood cell (WBC) populations, WBC enrichment involving red based on label-free data independent acquisition (DIA) mass blood cell (RBC) lysis are laborious and typically performed manually, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 64 of 272 contributing to experimental variability especially as blood cells are Clonality assessments ; dualplex and multiplex immunohistochemis- sensitive to physical and chemical stress. try coupled to digital pathology analyses to assess Immune Cells Infil- Methods tration and PD-L1 mediated inhibition (Immunoscore® IC), Immune We describe RBC lysis and leukocyte immunostaining on a Suppression through Regulatory T cells and Myeloid-derives suppres- centrifuge-less platform Laminar Wash™, using a novel wall-less plate sor cells quantification, T-Cell Exhaustion status ; standardized and laminar flow washer. The Laminar Wash™ 24-well plate consists methods for assessment of endothelial activation markers ; flow cy- of an array of hydrophilic spots surrounded by hydrophobic surface, tometry for circulating immune cell subtypes quantification; ICI which functions as a virtual wall that separates each spot. Each well plasma exposure levels. A multimodal integrative Immunogram pres- is capable of staining and lysing 100uL of whole blood. During lysis, entation is proposed for each patients. WBCs settle to the surface of the spot, allowing the spent lysis buffer Conclusions to be removed by a gentle and continuous laminar-flow washing This preliminary study shows that multimodal immune profiling is process on the Laminar Wash™ system, eliminating centrifugation feasible and could be a new tool to understand the biology and and resuspension that may stress cells and disrupt antibody binding. pharmacology of lung cancer resistance to anti-PD1/L1 ICIs and po- Results tentially guide patient management décisions. We observed improved retention of CD45+ lymphocytes while lysing on Laminar Wash™ plates compared to conventional centrifuge Acknowledgements tubes. In studies comparing mouse whole blood lysis and antibody This work is supported by the French National Cancer Agency, Agence staining by conventional tube centrifuge and Laminar Wash, Laminar Nationale du Cancer, through the PIONeeR project financing. Wash achieved dramatically higher staining index and improved Trial Registration resolution of cell cluster by flow cytometry. ClinicalTrials.gov Identifier: NCT03493581 Conclusions Ethics Approval In summary, Laminar Wash system provides gentle, fast and convenient The study was approved by the French Ethic Comitee CPP Ouest II Angers, blood lysis, while improving data quality with superior antibody staining. approval number 2018/08. P117 P118 Immunogram to decipher PD1/L1 ICI resistance: a proof of concept HYDRA platform development to investigate Siglec-engaging in advanced Non-small cell lung cancer patients of the PIONeeR tumor immunosuppressive glyco-codes Project Li Peng, PhD, Adam Petrone, Adam Shoemaker, Jillian Prendergast, PhD, 1 2 3 Florence Monville, PhD , Frederic Vely , Joseph Ciccolini , Florence Zakir Siddiquee, Jenny Che, Lihui Xu, BS, Karl Normington, PhD, MBA, 2 2 1 4 Sabatier , Stephane Garcia , Vanina Leca , Marion Fabre , Christelle James Broderick, Li Peng, PhD 4 1 4 1 Piperoglou , Pernelle Outters , Laurent Arnaud , Laurent Vanhille, PhD , Palleon Pharmaceuticals, Waltham, MA, United States 1 1 1 Caroline Lauge, BA , Anna Martirosyan, Dr , Aurelie Collignon , Marie Correspondence: Li Peng (lpeng@palleonpharma.com) 5 6 7 2 Roumieux , Julien Mazieres , Maurice Perol , Françoise Dignat-George , Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P118 2 2 1 Eric Vivier , Fabrice Barlesi, MD, PhD , Jacques Fieschi, PhD 1 2 3 HalioDx, Marseille, France; AMU, APHM, Marseille, France; AMU, APHM, Background 4 5 IPC, Marseille, France; APHM, Marseille, France; AMU, Marseille, France; The glyco-immune checkpoint (Siglec/sialoglycan axis) has emerged 6 7 Toulouse Universitary Hospital, Toulouse, France; Centre Leon Berard, as a new mechanism of cancer immune escape and offers new thera- Lyon, France peutic interventions to overcome resistance to current immunother- Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) apies. Siglecs (sialic acid-recognizing Ig-superfamily lectins) are type I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P117 transmembrane sialoglycan binding proteins expressed on various immune cells (innate and adaptive). Humans express at least four- Background teen unique Siglecs which have distinct preferred sialoglycan ligands. In the management of advanced Non-Small Cell Lung Carcinoma Tumors upregulate certain sialoglycan patterns to facilitate immune (NSCLC), PD1/L1 immune checkpoint inhibitors (ICIs) have increased cell evasion by engaging these inhibitory Siglec receptors. This tumor overall survival (OS) over standard second-line chemotherapy. While inhibitory “glyco-code” consists of a heterogenous mixture of numer- this long-term increase in OS is driven by about 20% of patients, others ous sialoglycans, binding to Siglecs through low affinity and high display disease progression during the first weeks. PIONeeR workpack- avidity interactions. age 2 aims to understand and eventually predict response and/or re- Methods sistance to those ICIs in stage IV or recurrent NSCLC patients. For that Deciphering the hypersialylation glyco-code of tumors is key to iden- purpose, an Immunogram was designed that integrates a comprehen- tifying cancer patients for glyco-immune checkpoint blockade ther- sive set of biomarkers measured in the tumor microenvironment. apies. However, the heterogeneity and complexity of sialoglycans Methods make characterization of the tumor surface sialoglycome difficult The immune contexture from the PIONeeR trial’s patients is being with current technologies. To overcome this challenge, we developed characterized in a prospective manner and will be confronted to clin- a proprietary sialoglycan-probing reagent, HYDRA, to functionally de- ical data at the end of the study. This multi-modal approach, encom- tect inhibitory tumor sialoglycans engaging Siglecs. HYDRA mimics passing a range of immune scoring assays, is applied to blood and this natural avidity driven Siglec-sialoglycan interaction, consisting of tumor biopsy from each patient, both sampled before and through- multimeric fusions of a Siglec N-terminal extracellular domain con- out anti-PD1/L1 ICI treatment. This work aims at describing pre- taining the carbohydrate recognition domain (CRD), a trimerization treatment samples profiling. motif, and a Fc dimerization domain. Results Results We assessed the feasibility of such a profiling and provide descriptive We have generated several HYDRA constructs with robust expression multi-modal immune profiles for the 10 first PIONeeR-included pa- using a mammalian HEK293 system. Size-exclusion chromatography tients. These profiles combine raw results from more than ten tests, profiles of HYDRA demonstrate high purity and confirmed multimeric corresponding to the following technologies and biomarkers: Gen- assembly. HYDRAs have greater than fifteen-fold increase in binding omic Next Generation Sequencing for Tumor Mutational Burden affinity compared to Siglec-Fc dimers as measured using bio-layer (TMB), DNA mismatch-repair deficiency (MSI/MSS status) and T Cell interferometry Octet. HYDRA also demonstrates sialoglycan-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 65 of 272 binding, as its binding was eliminated when cells were treated with P120 sialidase (which removes terminal sialic-acids of sialoglycan) or using Evaluation of CD8 score by automated quantitative image analysis cells lacking sialoglycans from knocking out UDP-GlcNAc 2- in metastatic melanoma treated with PD1 blockade: preliminary Epimerase. Glycan array binding of HYDRA confirmed similar sialogly- results can preferences of its Siglec counterpart as described in the litera- Anjali Rohatgi, MD PhD, Douglas Hartman, Arivarasan Karunamurthy, ture, suggesting engineering did not alter glyco-recognition Julie Burkette, Yana Najjar, MD, John Kirkwood, MD, Hassane Zarour, MD, properties. These high-affinity and sialoglycan-specific HYDRAs en- Liron Pantanowitz, Diwakar Davar, MD abled us to develop a robust immunohistochemistry (IHC) assay to UPMC, Pittsburgh, PA, United States analyze cancer patient samples. A cohort of tissues (>2,500 patients) Correspondence: Diwakar Davar (davard@upmc.edu) from various indications were analyzed to enable indication Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P120 prioritization for glyco-immune checkpoint therapies. HYDRA IHC on healthy and cancerous human tissues demonstrate unique binding Background patterns with concordance between duplicate primary tumor cores PD1 blockade produces responses in 30-40% of metastatic melan- and primary tumor versus metastatic cores from the same patient in oma (MEL) with durable relapse-free benefit [1,2]. Pre-existing tumor- non-small cell lung, kidney and colon cancer samples. infiltrating CD8+T cell infiltrates (TIL), neoantigen burden and IFN-γ Conclusions gene expression signature (GES) correlate with clinical anti-tumor re- In summary, the HYDRA technology distills the structural hetero- sponse [3-5] to PD1 blockade. However, neoantigen burden and IFN- geneity of tumor surface sialoglycans to a straightforward func- γ GES are cost-prohibitive and time-consuming assays that are not tional readout of immunosuppressive glyco-codes engaging available for clinical use; while CD8 T cell analysis by immunohisto- inhibitory Siglecs, which may allow patient stratification based on chemistry (IHC) is cost-effective and operator-independent. The aim deciphering a tumor-specific surface glycan pattern. of this study is to develop and validate an image analysis algorithm to automatically quantify CD8+ T cells (CD8 score) in patients with metastatic MEL treated with PD1 blockade. P119 Methods Analytical validation of run-to-run and site-to-site performance of Included patients had advanced metastatic MEL treated with PD1 a human immune profiling assay and automated data analysis blockade. Radiographic response assessed using RECIST v1.1. For the solution for CyTOF mass cytometry technology purposes of this analysis, patients were defined as responders (R; Clare Rogers (clare.rogers@fluidigm.com) complete, partial response, stable disease) or non-responders (NR; pro- Fluidigm, South San Francisco, CA, United States gressive disease). Pre-treatment tumor biopsies from 58 patients were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P119 utilized. Brightfield image analysis results were cross-validated with fluorescence-based quantification (AQUA™). A nuclear image algorithm Background designed to run on whole slide images was optimized to manual count. Immune profiling is an essential method for quantifying changes The algorithm was locked down and used on a cohort of whole tissue in immune population numbers and states over time in health sections from MEL patients. All images were reviewed by independent and disease. A cornerstone in translational and clinical research, pathologist blinded to clinical outcomes. Response and outcomes were it is frequently used to investigate chronic inflammation, infec- statistically correlated with image analysis results. tious disease, autoimmune diseases, and cancer. The diversity of Results immune populations demands a high parameter approach to There were 40 R patients and 18 NR patients. Median CD8 score was more fully and efficiently quantify these changes. Mass cytometry, 101 cells/mm3 in R and 48.7 cells/mm3 in NR (p=0.098). Median PFS which utilizes CyTOF® technology, is a single-cell analysis platform were greater in R compared to NR (18 months vs. 2 months, p100 that hasusedasmanyas50 metal-taggedantibodies[1] to re- cells/mm3 (64%). solve discrete cell populations, all in a single tube of sample. It is Conclusions an ideal solution for routine enumeration of immune cell We report the successful technical development and clinical valid- populations. ation of an image algorithm to automate CD8 score for metastatic Methods MEL treated with PD1 blockade. Preliminary results demonstrate We have developed a sample-to-answer solution for human im- CD8 score was directly associated with response and improved mune profiling using mass cytometry: the Maxpar® Direct™ Im- PFS. CD8 score is an assay that could be carried out using exist- mune Profiling System. It includes an optimized 30-marker ing technology in pathology departments. Further analysis will immune profiling panel provided in a dried single-tube format, focus on validating these results in a larger cohort to permit clin- validated SOPs for human whole blood and PBMC staining, an in- ical use. strument data acquisition template, instructions for data acquisi- tion on a Helios™ system, and automated Maxpar Pathsetter™ References software for data analysis. 1. Ribas A, Hamid O, Daud A, Hodi FS, Wolchok JD, Kefford R, Joshua AM, Results Patnaik A, Hwu WJ, Weber JS, Gangadhar TC, Hersey P, Dronca R, Joseph Here we present assay analytical validation data on repeatability, re- RW, Zarour H, Chmielowski B, Lawrence DP, Algazi A, Rizvi NA, Hoffner B, producibility, software precision, software accuracy, and site-to-site Mateus C, Gergich K, Lindia JA, Giannotti M, Li XN, Ebbinghaus S, Kang reproducibility. The repeatability of eight identical donor samples ac- SP, Robert C. Association of Pembrolizumab With Tumor Response and quired on a single Helios instrument resulted in CVs 5% in fre- Survival Among Patients With Advanced Melanoma. JAMA. 2016; quency). Reproducibility of three identical samples acquired on three 315:1600-9. different Helios instruments resulted in CVs 2. Larkin J, Lao CD, Urba WJ, McDermott DF, Horak C, Jiang J, Wolchok JD. Conclusions Efficacy and Safety of Nivolumab in Patients With BRAF V600 Mutant and We conclude that this assay provides a robust solution for broad im- BRAF Wild-Type Advanced Melanoma: A Pooled Analysis of 4 Clinical Tri- mune profiling using mass cytometry, reducing sources of variability als. JAMA Oncol. 2015;4:433-40. and subjectivity in sample preparation and data analysis. 3. Tumeh P, Harview C, Yearley J, Shintaku I, Taylor E, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West A, Carmona M, Kivork C, Seja E, Reference Cherry G, Gutierrez A, Grogan T, Mateus C, Tomasic G, Glaspy J, Emerson 1. Simoni Y, Becht E, Fehlings M et al. Bystander CD8+ T cells are abundant R, Robins H, Pierce R, Elashoff D, Robert C, Ribas A. PD-1 blockade in- and phenotypically distinct in human tumour infiltrates. Nature. 2019; duces responses by inhibiting adaptive immune resistance. Nature. 557:575–579. 2014;515:568-71 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 66 of 272 4. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu to identify patient-specific patterns which might improve the predic- XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber tion of the response to therapy. AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Conclusions Tomassini JE, Loboda A, Kaufman D. Pan-tumor genomic biomarkers for We believe that the Cancer Immunogram has the potential to facili- PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362:197 tate drug development by providing a 360° vision of the tumour im- 5. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee mune contexture and may also help clinicians to personalize W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, advanced cancer patient care. Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN, Chan TA. Mutational References landscape determines sensitivity to PD-1 blockade in non-small cell lung 1. Galon J, Bruni D. Approaches to treat immune hot, altered and cold cancer. Science. 2015;348:124-8. tumours with combination immunotherapies. Nat Rev Drug Discov. Ethics Approval 2019;18:197-218. The study was approved by University of Pittsburgh‘s Institutional Review 2. Pagès F et al. International validation of the consensus Immunoscore for Board, approval number PRO18080253. the classification of colon cancer: a prognostic and accuracy study. Consent Lancet. 2018; 391:2128-2139. Written informed consent was obtained from the patient for publication of 3. Mlecnik B et al. Integrative Analyses of Colorectal Cancer Show this abstract and any accompanying images. A copy of the written Immunoscore Is a Stronger Predictor of Patient Survival Than consent is available for review by the Editor of this journal. Microsatellite Instability. Immunity. 2016; 44:698-711. 4. Blank CU et al. The "cancer immunogram". Science. 2016;; 352:658-60. 5. Sbarrato T et al, Combining multimodal biomarkers as an immunogram P121 to guide immunotherapy use: A proof of concept. Proceedings: AACR Cancer Immunogram: combining multi-parameter approach and Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. machine learning to capture the complexity of tumor immune contexture 1 1 1 Thomas Sbarrato, PhD , Laurent Vanhille, PhD , Mounia Filahi , Anna P122 1 1 1 Martirosyan, PhD , Véronique Frayssinet , Caroline Davin, BA , Caroline Microfluidic-based cell separation method improves workflow for 1 1 1 1 Laugé , Assil Benchaaben , Alboukadel Kassambara , Felipe Guimaraes , evaluation of rare lymphocytes from cancer patient samples 1 2 1 1 1 1 1 1 Régis Perbost , Jérôme Galon , Hélène Girardi , Jacques Fieschi, PhD Jodi Stone, BS , Megan Nichols , Amy Austin , Kala Bradshaw , Jessica E. 1 2 1 1 2 HalioDx, Marseille, France, Centre de Recherche des Cordeliers, Paris, Norris, BS, MT , Jennifer Montague, PhD , Peng Meng Kou , Nitin 2 2 2 2 France Kulkarni , Nirav Sheth , Anya Manning, MBA , Sarah M. Mickool , Kyle 2 2 1 Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) Smith , Ravi Kapur , John Powderly, MD, CPI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P121 Carolina BioOncology Institute, Huntersville, NC, United States; MicroMedicine, Waltham, MA, United States Background Correspondence: John Powderly (jpowderly@carolinabiooncology.org) To tailor clinical care and personalized treatment of cancer patients, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P122 the scientific community together with the practitioners have fo- cused into refining our understanding of cancer biology and resist- Background ance to treatments. In that perspective, the concept of Immunoscore Isolation of rare lymphocyte populations from peripheral blood prod- proposed by Galon et al [1, 2, 3] has highlighted the crucial role of ucts of cancer patients can be challenging due to technician variabil- immune response to the tumor. In parallel, immunotherapies by im- ity and substantial cell loss through standard cell separation mune checkpoint inhibitors (ICI) anti-PD-1/PD-L1 were approved in methods such as Mononuclear Cell Preparation Tubes™ (CPTs). An several cancer indications, such as Non-Small Cell Lung Cancer or automated microfluidic approach was evaluated to determine melanoma, even if only a minority of these patients respond posi- lymphocyte recovery, processing time, and ease of use. Furthermore, tively to the treatment. In addition, ICI are far less effective for other increased yields of rare cells from cancer patients’ peripheral blood high-incidence indications like colorectal cancer (CRC), thus suggest- could potentially substitute for leukapheresis when leukapheresis is ing that multiple factors may be critical for capturing the exact na- not a viable option. ture of the tumour microenvironment (TME). In this context, the Methods comprehensive identification and assessment of these factors could White blood cells (WBCs) or peripheral blood mononuclear cells be key to stratify patients and allow the selection of the optimal (PBMCs) were isolated from human peripheral blood using either treatment. MicroMedicine’s Microfluidic System (MS) or CPTs. Cell viability and Methods lymphocyte recovery were compared using a hematology analyzer In order to support clinical researchers and biopharmaceutical com- and flow cytometry. Further, a rare lymphocyte population was posi- panies in the evaluation of the efficacy of candidate drugs, HalioDx tively immunomagnetically selected from healthy volunteers and has developed the Cancer Immunogram, a solution based on Blank cancer patients. Immunophenotyping was performed pre- and post- CU et al. [4]. Our multi-parameter approach encompassing a unique cell selection, followed by in vitro expansion of the rare lymphocytes. range of immune scoring assays is based on the analysis and the un- Results derstanding of the immune contexture of tumors and offers a per- Using cells collected from healthy volunteers, the automated MS sonalized and dynamic “fingerprint” of tumor-immune system prototype consistently recovered 83.5 ± 10.1% lymphocytes in a total interaction. To address this, the Cancer Immunogram combines dif- of 31 ± 5.8 minutes, including hands-on time, compared to the ferent technologies and biomarkers to assess 1) the tumor character- standard CPT process, which recovered 43.2 ± 7.6% lymphocytes in istics (Tumor foreignness, MSI, PD-L1 expression, common mutation 72.2 ± 4.1 minutes, from 32 – 34 mL blood samples (n = 5). The via- drivers), 2) the immune infiltration (Immunoscore®, CD8/PD-L1 prox- bility was comparable at 97.9 ± 1.6% (MS) and 96.7 ± 3.0% (CPT). In imity, TCR clonality, immune expression signature), 3) the immune further studies with both healthy donors and cancer patients, a rare checkpoint status (T Cell Exhaustion BrightPlex panel) and 4) the im- lymphocyte population was successfully selected from WBCs isolated mune suppression status (Treg, MDSC and M1/M2 macrophage with the MS, enabling immunophenotyping of the rare cell popula- BrightPlex panels). tion and subsequent in vitro expansion. Expansion of this lymphocyte Results population from a colon adenocarcinoma patient was found to be Here, we consolidate our Proof of Concept for the Cancer Immuno- suppressed post-immunotherapy compared to pre-treatment. Cells gram in the context of CRC [5] by leveraging this meta-analysis on a isolated from patients with other malignancies were successfully ex- 20-patients cohort. Using machine learning algorithms to extract the panded. Finally, peripheral blood collected from cancer patients most relevant features, we show that the Cancer Immunogram allows yielded a greater number of rare lymphocytes using the MS for the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 67 of 272 cell expansion study in comparison to the CPT isolation method, NK cells, in mediating antitumor response after immune check- which correlates to having a higher lymphocyte recovery. point inhibition. Conclusions NanoString nCounter is Intended for Research Use Only. Not for Use The MS consistently recovered approximately twice the number in Diagnostic Procedures. of lymphocytes in half the time compared to the traditional CPT Ethics Approval method. The automated cell separation process improves the The study was approved by Yale University Human Investigation consistency of cell isolation while freeing up technician time. Rare Committee, approval number 9505008219. lymphocyte populations could be reliably recovered from periph- eral blood with significantly higher yield compared to the CPT P124 method. While leukapheresis enriches for MNCs and MS isolates A novel cell-mediated immunotherapy for treatment of lung and all WBCs, these results suggest that peripheral blood collection- breast cancer based MS has the potential to complement leukapheresis, espe- Indu Venugopal, PhD, Kathlynn Brown, Michael McGuire, Claire Gormley cially for small- to medium-scale studies. SRI International, Harrisonburg, VA, United States Correspondence: Kathlynn Brown (kathlynn.brown@sri.com) P123 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P124 Identification of mRNA signatures that predict response to immunotherapy in melanoma patients Background 1 2 2 Ioannis Vathiotis, MD , Amy Sullivan , Sarah Warren, PhD , Nicole Cancer Immunotherapies designed to generate a cell-mediated 1 1 1 Gianino , Sandra Martinez-Morilla, PhD , Pok Fai Wong, MD, MPhil , immune response against tumors are emerging as frontline 1 3 1 Harriet Kluger, MD , Konstantinos Syrigos , David Rimm, MD, PhD treatment options for cancer; however, concerns regarding effi- 1 2 Yale University, New Haven, CT, United States; NanoString cacy, safety and cost efficacy have limited the use of these Technologies, Seattle, WA, United States; University of Athens, Athens, treatments. Greece Methods Correspondence: David Rimm (david.rimm@yale.edu) To address these weaknesses, we developed a novel immunother- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P123 apy capable of delivering previously encountered antigenic pep- tides specifically to cancer cells and facilitating their presentation Background through the MHC class I pathway. It utilizes a synthetic nanoparti- Currently, there is no diagnostic test that can accurately predict cle delivery system comprised of three components: a neutral response in melanoma patients treated with immunotherapy. stealth liposome, encapsulated synthetic immunogenic HLA class I NanoString® nCounter® PanCancer IO 360™ panel (Research Use restricted peptides derived from measles virus (MV), and a tumor- Only) measures mRNA from 770 genes related to the tumor and targeting peptide on the external surface of the liposome. The host immune response. Here, we used this panel to assess the targeting peptide results in accumulation of liposomes specifically predictive value of individual genes and weighted gene signa- inside cancer cells, and facilitates presentation of MV-derived im- tures in a cohort of immunotherapy (ITx) treated melanoma munogenic peptides in HLA class I molecules (Figure 1). We refer patients. to this system as TALL (Targeted Antigen Loaded Liposomes). Methods Therefore, TALL can generate a secondary immune response spe- We used pretreatment, formalin-fixed paraffin-embedded (FFPE) cifically against the targeted tumor cells in a patient who has whole tissue sections from 59 melanoma patients that received been previously vaccinated against or infected by MV. In short, single agent or combination immunotherapy (pembrolizumab, we are attempting to trick the immune system into responding nivolumab, or nivolumab plus ipilimumab). Two slides from each as though the cancer cell is infected with MV without the use of patient were macrodissected and RNA was extracted. The mRNA viral particles. transcripts were hybridized and tagged by unique probes for the Results 770-plex PanCancer IO 360 panel and then measured on the We synthesized liposomes encapsulating H250, an immunogenic nCounter platform. RNA counts were correlated with best overall HLA class I restricted peptide identified from measles response (BOR), clinical benefit (CB), progression free survival hemagglutinin protein. These liposomes were targeted to breast (PFS) and overall survival (OS). and lung cancer cells via our targeting peptide, which was identi- Results fied using phage-display methodology. Treatment of lung cancer Indoleamine 2,3-dioxygenase 1 (IDO1) was the best single gene cells with TALL results in functional presentation of H250 in both predictor of BOR (Area under the curve (AUC) = 0.73) and CB MHC and HLA class I molecules. Our in-vitro and in-vivo studies (AUC =0.70).Among othergenes,IDO1 mRNAwas also foundto indicate that presentation of H250 is dependent on the cancer be significantly associated with longer PFS (P < 0.01, False dis- targeting peptide; liposomes that lack the targeting peptide did covery rate (FDR) = 0.18) and OS (P < 0.01, FDR = 0.052). The not accumulate in the cancer cells and presentation of H250 was previously described 18-gene tumor inflammation score (Ayers abrogated. Treatment with TALL substantially reduced growth of TIS) validated for the prediction of BOR (AUC = 0.68), PFS (P < LLC1 and 4T1 tumors in vaccinated C57BL/6 and Balb/c mice 0.05, FDR = 0.18) and OS (P < 0.001, FDR = 0.025). TIS also pre- respectively. dicted CB (AUC = 0.67). Its predictive value remained the same ir- Conclusions respective to immunotherapy agent administered. Nevertheless, it The outcome of our therapy is a robust cytotoxic T lymphocyte re- decreased for patients harboring the BRAF and NRAS mutations sponse directed specifically against the tumor. It's advantages in- (AUC = 0.76 versus 0.51 and 0.44 for patients with BRAF and clude: 1) Bypassing the need to identify tumor-associated antigens or NRAS mutations respectively). The best signatures for this cohort educate the immune system through a primary immune response; 2) were for Cytotoxicity, Immunoproteasome and CD56dim Cells It is anticipated to be effective against tumors with a low mutational which were predictive for BOR (AUC = 0.72, 0.71 and 0.70 re- load, making it efficacious on early-stage and metastatic cancer; 3) It spectively), CB (AUC = 0.69, 0.68 and 0.70 respectively), and OS does not use a live virus or biologically-derived material, allowing for (all FDRs < 0.05). Further work is underway to compare these complete synthetic manufacturing. It also does not require isolation Yale melanoma results with other cohorts. or ex-vivo manipulation of patient’s cells, reducing production time Conclusions and costs. Pretreatment mRNA counts of single genes or weighted signature scores are related to immunotherapy outcomes in melanoma pa- Acknowledgements tients. This work validated the Ayers TIS signature and Research reported in this work was supported by DOD under grant number highlighted the role of the immune microenvironment, especially W81XWH-16-1-0262 and SRI internal research funds Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 68 of 272 receptors. In contrast, ciforadenant inhibited induction of the AdenoSig and AMPSig in all experimental settings at the tran- script and protein level. Conclusions A2AR agonists and AMP induce specific GEPs dominated by im- munosuppressive mediators of MDSC and monocyte/macro- phage biology. These GEPs may be used as biomarkers for patient selection. CD73 antagonists alone may be limited by the induction of compensatory immunosuppressive pathways medi- ated by AMP accumulation. Combination ciforadenant and CPI- 006 treatment may synergize to activate anti-tumor immunity by 1) blocking adenosine production and signaling, 2) directly activating immune cells, and 3) blocking a compensatory induc- tion of AMPSig. This combination strategy is being evaluated in an ongoing Ph1/1b clinical trial in patients with advanced solid tumors. Trial Registration NCT02655822 and NCT03454451 Fig. 1 (abstract P124). TALL mechanism of action P125 Adenosine and AMP gene expression profiles predict response to adenosine pathway therapies and indicate a need for dual blockade of CD73 and A2AR with CD73 inhibitors Stephen Willingham, PhD, Drew Hotson, PhD, Jessica Hsieh, Brian Munneke, Long Kwei, PhD, Joseph Buggy, Richard Miller, MD Corvus Pharmaceuticals, Burlingame, CA, United States Correspondence: Stephen Willingham (swillingham@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P125 Background Extracellular adenosine in the tumor microenvironment gener- ates an immunosuppressive niche that promotes tumor growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. Various agents targeting the adenosine path- way are now in clinical trials as cancer therapies. Ciforadenant is a selective A2AR antagonist and CPI-006 is an anti-CD73 anti- body (Fc-mutant IgG1) that blocksthe enzymaticconversion of AMP to adenosine and directly stimulates immunity. Both agents are now being studied in clinical trials (NCT02655822 and NCT03454451). In this report, we evaluate the role of ad- Fig. 1 (abstract P125). See text for description enosine and AMP-related gene expression profiles (GEPs) that may predict the response of patients receiving adenosine path- way therapies. Ex vivo studies reveal a requirement for dual P126 blockade of CD73 and A2AR for optimal neutralization of AMP Discovery of biomarkers associated with benefit from PD-1 mediated immunosuppression. checkpoint blockade in non-small-cell lung cancer (NSCLC) using Methods high-plex digital spatial profiling Normal human PBMCs were stimulated ex vivo with NECA (stable ad- 1 1 2 Jon Zugazagoitia, MD, PhD , Swati Gupta, PhD , Kit Fuhrman, MS PhD , enosine analog) or AMP. RNA from tumor biopsies and PBMC was an- 1 1 1 Scott Gettinger, MD , Roy Herbst, MD, PhD , Kurt Schalper, MD, PhD , alyzed using NanoString. Renal cell cancer (RCC) tumor biopsies David Rimm, MD, PhD collected from patients treated with ciforadenant (100 mg BID) either Yale University School of Medicine, New Haven, United States; as a single agent (n=18) or in combination with atezolizumab (n=14). Nanostring, Inc., Seattle, WA, United States Results Correspondence: David Rimm (david.rimm@yale.edu) Ex vivo A2AR agonism resulted in dose-dependent increases in CXCR2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P126 ligands (CXCL1,2,3,5,8) and key mediators of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory media- Background tors such as IL-1beta and CCL2,3,7,8, 20 were also observed, as were in- Only a minority of patients with advanced NSCLC truly benefit from creases in SERPINB2, S100A8, PTGS2, THBS1. Preliminary biomarker single-agent PD-1 checkpoint blockade, and more robust predictive analysis suggests ciforadenant anti-tumor activity in RCC was associ- biomarkers are needed to optimally deliver these therapies. The ated with increased expression of select analytes (AdenoSig) in pre- GeoMx Digital Spatial Profiler (DSP) (NanoString, Inc.) allows high- treatment biopsies (Figure 1). plex protein expression analysis in a quantitative and spatially- Ex vivo AMP or AMPalphaS (a non-hydrolyzable AMP analog) resolved manner from single formalin-fixed paraffin embedded tissue stimulation induced a similar GEP (AMPSig), but included specific sections. Here we use this technology as a discovery tool to find pro- decreases in OAS3, BIRC5, CDK1, MX1, IFI27, and IFIT1. CD73 anti- tein markers associated with benefit from single-agent PD-1 check- body and small molecule antagonists amplified the AMPSig by point blockade in NSCLC. preserving AMP, which itself directly stimulates adenosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 69 of 272 Methods biopsies on the Leica Biosystems BOND RX autostainer. The tissues We used the GeoMx DSP in a cohort of 63 immunotherapy-treated were then imaged in five distinct fluorescent channels (DAPI, FITC, NSCLC cases represented in a tissue microarray, 52 of whom had TRITC, Cy5, Cy7) in 3 rounds of image acquisition on the ZEISS Axio pre-treatment samples and received single-agent PD-1 checkpoint Scan.Z1. HALO analysis software was used to identify cell phenotypes blockade. A panel of 40 photocleavable oligonucleotide-labeled pri- and spatial interactions across the whole slide images. UMAP and mary antibodies (NanoString Human IO panel) was used for protein PSDM were also used to characterize cellular phenotypes and similar- detection. Proteins were measured in 4 independent molecularly- ities amongst samples in the cohorts. Downstream H&E staining was defined tissue compartments by fluorescence co-localization (tumor performed on the same slides with a fourth imaging round to pro- [panCK+], leucocytes [CD45+], macrophages [CD68+], and non- duce a fused 12-plex fluorescent and brightfield image. immune stromal cells [CK-/CD45-CD68-/DNA+]). The photocleaved Results oligos were hybridized and digitally counted with the nCounter plat- The multiplex panel was able identify all 12 markers in FFPE samples form. Two cut-points (median and top tertile) were explored for each and immunophenotype single cells through co-expression of several maker. All statistical testing was performed using a two-sided signifi- biomarkers. Of the 4,096 possible phenotypes, the relevant pheno- cance level of α=0.05 without correction for multiple hypothesis types mapped included, but were not limited to: T cells, T-regs, Cyto- testing. toxic T-cells, Exhausted T-cells, B cells, NK cells, M1 and M2 Results macrophages, tumor cells, and expression along the PD-L1 and PD-1 160 protein variables were generated per case (normalized counts immune checkpoint axis. Distance mapping and infiltration indexes within molecularly defined compartments). In univariate analyses were measured in tumor regions, stroma compartments, and along using pre-specified cut-points, 10 markers were associated with clin- invasive margins. ical benefit (CB) or non-CB, 6 markers with PFS, and 13 markers with Conclusions OS. Of these, CD56 (top tertile) and CD4 (median) measured in the In this work, we introduce a tumor and immune cell phenotyping CD45 compartment were the only markers that significantly pre- multiplex immunofluorescence panel for the comprehensive dicted either CB (OR 6.7, p = 0.014 and OR 8.5, p = 0.014, respect- characterization of the tumor microenvironment and its applicability ively) longer PFS (HR 0.38, p = 0.011 and HR 0.33, p = 0.002, across a range of carcinoma and melanoma FFPE tissue samples for respectively) and longer OS (HR 0.44, p = 0.044 and HR 0.31, p = support of deep pathology assessment in drug discovery research. 0.002, respectively). After adjusting for 3 baseline clinical prognostic The ability to colocalize markers in the same compartment for the factors (performance status, liver metastasis, dNLR) in a multivariate identification of thousands of phenotypes when combined with Cox proportional hazard model, both CD56 and CD4 remained pre- brightfield pathological assessment within a single sample has the dictive for PFS (HR 0.39, p = 0.020 and HR 0.37, p = 0.017, respect- potential to accelerate immunotherapy research. ively), while only CD4 was predictive for OS (HR 0.28, p = 0.006). Conclusions P128 This pilot scale, discovery study shows the potential of the DSP tech- Pooled analysis of Programmed Death Factor Ligand 1 (PD-L1) nology in the identification of spatially-informed biomarkers of re- expression as a predictive biomarker using individual data on sponse to PD-1 checkpoint blockade in NSCLC. This works highlights 7,918 randomized study patients a previously undescribed role for CD56+ immune cells and CD4+ T- Andrea Arfe, Geoffrey Fell, Brian Alexander, MD MPH, Mark Awad, MD cells as potential predictors of immunotherapy outcomes in NSCLC. PhD, Scott Rodig, MD, PhD, Lorenzo Trippa, Jonathan Schoenfeld, MD, Ethics Approval MPH All tissue samples were collected and used with specific consent or Dana-Farber Cancer Institute, Boston, MA, United States waiver of consent under the approval from the Yale Human Investi- Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) gation Committee protocol #9505008219. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P128 P127 Background Development of a 12-marker immunofluorescence multiplex panel PD-L1 expression is one of the most studied biomarkers to predict for the in-depth investigation of the tumor immune landscape the efficacy of immune checkpoint inhibitors (ICIs), but its clinical sig- analyzing 4,096 phenotypes nificance is controversial. Several factors have limited the study of Courtney Hauck, Aditi Sharma, Monique Johnson, Wenya Yang, Bonnie PD-L1 expression. Most trials use of hazard ratios (HRs) to measure Phillips, PhD, Mark Burton, HTL ASCP, Douglas Wood, PhD, Stephanie treatment effects on survival outcomes, a questionable practice for Hennek, PhD, Mael Manesse, PhD, J Kent Moore, PhD, Katir Patel, PhD, immunotherapy studies. Additionally, trials use different cut-off Jamie Buell, Sean Downing, PhD values to dichotomize PD-L1 scores, complicating meta-analyses. Ultivue, Cambridge, MA, United States Therefore, we performed a pooled analysis to: i) estimate the distri- Correspondence: Sean Downing (sean.downing@ultivue.com) bution of PD-L1 expression scores in clinical populations, and ii) as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P127 sess the relationship between PD-L1 levels and ICIs’ effects on overall survival (OS). Instead of HRs, we used a more robust metric, i.e. differ- Background ences in restricted mean survival times (ΔRMSTs). Current IHC methods limit the depth of information from a single tis- Methods sue sample to a single target in the case of chromogenic staining, or Following PRISMA guidelines, we analyzed individual-level data re- to sample morphology and general cell identification in the case of constructed from the publications of 14 randomized clinical trials of H&E. Multiplex immunofluorescence (mIF) methods provide insights ICIs. We used an imputation-based approach to estimate i) the distri- into a wide number of markers of interest and their spatial context in bution of PD-L1 scores, ii) the survival distribution in different PD-L1 a single sample but limit the level of marker co-localization detection classes, and iii) pooled ΔRMST estimates. We show the advantage possible because of multiple antigen retrieval or photobleaching provided by meta-analytic estimates such as ours for the design of steps. Here, we demonstrate the utility of a new 12-plex mIF panel future studies in a simulation study. We simulated 10,000 NSCLC tri- using InSituPlex technology that can identify thousands of pheno- als (1:1 randomization; sample size: 500 patients) that compared ICIs types and spatial behavior through the co-localization of markers with standard chemotherapy. Simulated trials followed either i) a de- that was once limited to the domain of flow cytometry. sign that does not use prior information on the distribution of PD-L1 Methods levels and their association with ICIs’ effects, or ii) a design tailored The 12-Plex marker panel was developed including: CD3, CD4, CD8, to our meta-analytic estimates. CD20, Granzyme B, CD56, CD68, CD163, FoxP3, PD-1, PD-L1, and pan- Results Cytokeratin/Sox10 used the InSituPlex and DNA-Exchange technol- We reconstructed data on 7,918 individual patients, 3,496 with ogy to perform mIF staining of FFPE samples from tonsil and tumor NSCLC, 4,529 with other tumors. The estimated distribution of PD-L1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 70 of 272 expression is U-shaped, with most patients presenting a low or high Methods expression: only about 7% had an expression in the 5%-50% range. Serial peripheral blood mononuclear cells were obtained from pa- ΔRMST estimates suggest that i) ICIs provide an OS benefit to all pa- tients with RCC undergoing immunotherapy. Samples were obtained tients, and ii) the magnitude of OS benefits increases along with PD- at baseline (cycle 1) and initiation of each subsequent cycle (up to L1 score, although changes in ΔRMSTs were greater in NSCLC (Figure cycle 6). Flow cytometry identified longitudinal changes in T cell sub- 1). In the simulations, the power to detect a positive treatment effect sets. Additionally, recently activated CD8 T cells, identified by surface increased from 80% to 93% using a design tailored to meta-analytic expression of CD38 and HLA-DR, were sorted at baseline, post-cycle information. 1, and post-cycle 2, and analyzed by RNA seq. Clinical responses Conclusions were determined at the first restaging scans using RECIST v1.1 cri- By highlighting that higher PD-L1 scores predict increasing OS bene- teria, to define those with clinical benefit (complete response, partial fits, our findings extend those of recent meta-analyses that evaluated response, or stable disease) or no clinical benefit (progressive PD-L1 expression scores as predictors of ICIs’ efficacy. They also illus- disease). trate how meta-analytic estimates like ours can improve the power Results of future trials to detect ICIs’ benefits. Our findings also suggest that Of 27 patients analyzed, 10 received nivolumab, 7 nivolumab + the practice of dichotomizing the range of PD-L1 expression scores is NKTR-214, and 10 nivolumab + ipilimumab. Median age was 58 years inadequate for patient stratification. (range 33-78) with a male (70%), Caucasian (89%), and solely clear cell histology (83%) predominance. A burst in circulating activated CD8 T cells as defined by a ≥1.8 fold increase in CD38+HLA-DR+ CD8 T cells from baseline to post-cycle 1 (Figure 1A) was observed in 8/12 patients who had clinical benefit and 6/15 patients with no clinical benefit (Figure 1B). Transcriptional analysis revealed that in patients with the aforementioned immunological response, T-cells had upreg- ulated TCR signaling, CD28 signaling, enhanced glycolysis and iron uptake, and reduced TGF-beta signaling compared to patients with- out an immunologic response. Conclusions Peripheral blood immune monitoring of RCC patients while on im- munotherapy may provide an early predictor of response. One im- portant limitation identified is the treatment specific cycle length defined sample collection timing and therefore may miss transient early immunologic changes. This study advances knowledge regard- ing the newly generated effector CD8 T cells that contain important information about the immunobiology underlying response to immunotherapy. Acknowledgements This work was supported by funding from the NCI grant 1-R00-CA197891 and Nektar Therapeutics. We would like to acknowledge The Yerkes NHP Genomics Core which is supported in part by NIH P51 OD011132, the Emory Flow Cytometry Core (EFCC) supported by the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378, and NIH/NCI under award number, 2P30CA138292-04. Fig. 1 (abstract P128). See text for description References 1. 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Motzer RJ, Tannir NM, McDermott DF, Aren Frontera O, Melichar B, Donald Harvey, PharmD, Bradley Carthon, MD, PhD, Omer Kucuk, MD, Choueiri TK, Plimack ER, Barthelemy P, Porta C, George S, Powles T, Mehmet Bilen, Haydn Kissick Donskov F, Neiman V, Kollmannsberger CK, Salman P, Gurney H, Hawkins Emory University, Atlanta, GA, United States R, Ravaud A, Grimm MO, Bracarda S, Barrios CH, Tomita Y, Castellano D, Correspondence: Jennifer Carlisle (jennifer.w.carlisle@emory.edu) Rini BI, Chen AC, Mekan S, McHenry MB, Wind-Rotolo M, Doan J, Sharma Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P129 P, Hammers HJ, Escudier B, CheckMate I. Nivolumab plus Ipilimumab ver- sus Sunitinib in Advanced Renal-Cell Carcinoma. N Engl J Med. Background 2018;378(14):1277-1290. Although immunotherapy with PD-1 or dual PD-1/CTLA-4 blockade 3. Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, Sica GL, can be a successful treatment for patients with advanced renal cell Yu K, Koenig L, Patel NT, Behera M, Wu H, McCausland M, Chen Z, Zhang carcinoma (RCC), the majority do not respond [1, 2]. Prior peripheral C, Khuri FR, Owonikoko TK, Ahmed R, Ramalingam SS. Proliferation of PD- blood immune profiling studies have shown that a transient rise in 1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung activated CD8 T cells correlates with clinical response to PD1 block- cancer patients. Proc Natl Acad Sci U S A. 2017;114(19):4993-4998. ade in lung cancer [3, 4] and melanoma [5]. The effect of checkpoint 4. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, blockade on circulating CD8 T cells in RCC is unknown, as is the T cell Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, biology underlying clinical response. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 71 of 272 Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, approval of the NYU Institutional Review Board at the NYU Perlmut- Araki K, Ahmed R. Rescue of exhausted CD8 T cells by PD-1-targeted ter Cancer Center with informed consent [11]. therapies is CD28-dependent. Science. 2017;355(6332):1423-1427. Serum samples were run on HuProt Human Proteome Microarrays 5. Huang AC, Postow MA, Orlowski RJ, Mick R, Bengsch B, Manne S, Xu W, containing >19,000 human proteins by CDI Laboratories. Raw serum Harmon S, Giles JR, Wenz B, Adamow M, Kuk D, Panageas KS, Carrera C, IgG signal intensities were processed across staining cohorts via Wong P, Quagliarello F, Wubbenhorst B, D'Andrea K, Pauken KE, Herati interquartile range normalization. RS, Staupe RP, Schenkel JM, McGettigan S, Kothari S, George SM, Pre-existing antibody responses were defined as patient-specific IgG Vonderheide RH, Amaravadi RK, Karakousis GC, Schuchter LM, Xu X, signals >3.5 median absolute deviations above cohort median IgG Nathanson KL, Wolchok JD, Gangadhar TC, Wherry EJ. T-cell invigoration background (modified Z-score). Group statistics were computed to tumour burden ratio associated with anti-PD-1 response. Nature. (GraphPad Prism), and gene ontology enrichment analysis was per- 2017;545(7652):60-65. formed (Enrichr) [12]. Ethics Approval Results Samples are collected under an approved IRB protocol (The Urological Several pre-existing antigen-specific IgG autoantibody targets were Satellite Specimen Bank at Emory University, IRB00055316), and all observed to have associations with good outcomes (SD/PR) or ob- patients provided informed consent. jective clinical responses (PR/CR) versus patients with progressive dis- ease (POD). While final determination of the most predictive subsets is ongoing, many targets represent genes in an axis surrounding im- mune signaling pathways, hereditary neurodegenerative disease, and the ubiquitin proteasome pathway (ie, UBQLN1, UBQLN2). An exemplary example was observed in the autoantibody responses shared by >10% of all patients regardless of clinical outcome. Gene ontology enrichment analysis of these shared melanoma-patient autoantibodies versus KEGG 2019 [12] demonstrates this set of pro- teins is strongly enriched for neurotrophin signaling-associated pro- teins after multi-sample correction (P=0.004) (Table 1). Several other associations were observed cohort-wide for ontologies with tissue- specific enrichment in the brain, neurons, and neuronal processes. Conclusions In this pilot study, we found strong associations across the cohort for autoantibodies against nerve-growth-inducing neurotrophins and genes like UBQLN1 and UBQLN2 which have strong associations with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson’s, and Alzheimer’s – neurodegenerative diseases that are known to have incidences which correlate with melanoma [14–16]; this hints at a potential immunologic connection between the conditions, per- haps related to an antitumor / autoimmune axis involving the targets reported here. Acknowledgements We thank the patients and their families who consented to participate in this study. Funding support for the study was provided by the NYU Cancer Center and NIH/NCI Cancer Center Support Grant P30CA016087, the Marc Jacobs campaign to support melanoma research, Goldberg Charitable Trust, Fig. 1 (abstract P129). See text for description Wings for Things Foundation and Clayman Family Foundation to I. Osman; the American Medical Association foundation, the Melanoma Research Foundation and the American Skin Association grants to M. Gowen. Trial Registration P130 Patient samples included in this study were not part of a randomized Melanoma patients harbor pre-existing IgG autoantibodies controlled clinical trial. targeting neuronal proteins that associate with differential clinical outcomes following checkpoint blockade 1 2 2 2 References Tyler Hulett, PhD , Keith Giles , Michael Gowen, MD , Danny Simpson , 2 2 2 1. Nagele EP, Han M, Acharya NK, DeMarshall C, Kosciuk MC, Nagele RG. Jeremy Tchack , Una Moran , Zarmeena Dawood , Anna Pavlick, MD, 2 1 2 2 Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human MBA , Shaohui Hu , Hua Zhong , Michelle Krogsgaard , Tomas Kirchhoff, 2 2 Sera, and Their Number Is Influenced By Age, Gender, and Disease. PhD , Iman Osman 1 2 Tsokos GC, editor. PLoS ONE. 2013;8:e60726. 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All data was collected with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 72 of 272 7. Anderson KS, Sibani S, Wallstrom G, Qiu J, Mendoza EA, Raphael J, et al. P131 Protein Microarray Signature of Autoantibody Biomarkers for the Early Single cellular interrogation of tumor microenvironment enables Detection of Breast Cancer. Journal of Proteome Research. 2011;10:85–96. diagnosis and prognostication of malignancies 1 1 2 8. Miersch S, Bian X, Wallstrom G, Sibani S, Logvinenko T, Wasserfall CH, Wei Jian Tan , Jian Hang Lam , Mona Meng Wang , Paola Ricciardi- 1 2 3 et al. Serological autoantibody profiling of type 1 diabetes by protein Castagnoli , Anita Sook Yee Chan , Tony Kiat Hon Lim , Joe Poh Sheng 3 1 arrays. Journal of Proteomics. 2013;94:486–96. Yeong , Tong Seng Lim, PhD 1 2 9. Srivastava RM, Lee SC, Andrade Filho PA, Lord CA, Jie H-B, Davidson HC, Menarini Biomarkers Singapore, Singapore, Singapore; Singapopre Eye et al. Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate Research Institute, Singapore, Singapore; Singapore General Hospital, to Trigger Tumor Antigen-Specific T-cell Immunity in Head and Neck Singapore, Singapore Cancer Patients. Clinical Cancer Research. 2013;19:1858–72. Correspondence: Tong Seng Lim (tongseng.lim@mbiomarkers.com) 10. Hulett TW. Coordinated responses to individual tumor antigens by IgG Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P131 antibody and CD8+ T cells following cancer vaccination. 2018;14. 11. Gowen MF, Giles KM, Simpson D, Tchack J, Zhou H, Moran U, et al. Background Baseline antibody profiles predict toxicity in melanoma patients treated Tumor microenvironment contains a diverse array of cell types with immune checkpoint inhibitors. Journal of Translational Medicine with heterogeneous genomic and molecular profiles. Averaging [Internet]. 2018 [cited 2018 Nov 4];16. Available from: https://translational- the characteristics of all cells in a cancerous tissue no doubt ob- medicine.biomedcentral.com/articles/10.1186/s12967-018-1452-4 scures important variations in biomarkers among minority, but 12. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, critical, pathogenic cell populations. High resolution, single-cell Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list analyses are thus needed in the clinic to precisely delineate the enrichment analysis tool. BMC Bioinformatics. 2013;128(14). inherent heterogeneity of tumor microenvironment underlying 13. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, oncogenesis in each patient. Biopsies derived from tumor micro- Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, McDermott MG, environment are routinely used for medical diagnosis or prognos- Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene tications. The number of cells typically available from a biopsy is set enrichment analysis web server 2016 update. Nucleic Acids Research. limited and heterogeneous. The ability to distinguish, select, and 2016; gkw377 . sort rare malignant or pathogenic immune cells from either tissue 14. Olsen JH, Friis S, Frederiksen K. Malignant Melanoma and Other Types of or liquid biopsies poses a unique challenge for single-cell based Cancer Preceding Parkinson Disease: Epidemiology. 2006;17:582–7. diagnosis and prognostication. Heterogeneous cell populations 15. Freedman DM, Curtis RE, Daugherty SE, Goedert JJ, Kuncl RW, Tucker MA. with non-target immunoreactive cells in pausicellular biopsies The association between cancer and amyotrophic lateral sclerosis. Cancer complicate conventional bulk-cell analysis, and could lead to dis- Causes & Control. 2013;24:55–60. ease misdiagnosis or prognostication. 16. Roe CM, Fitzpatrick AL, Xiong C, Sieh W, Kuller L, Miller JP, et al. Cancer Methods linked to Alzheimer disease but not vascular dementia. Neurology. We adopted a state-of-the-art multi-modal strategy including the 2010;74:106–12. real-time imaging-based DEPArray with downstream molecular and Ethics Approval genomic assays [1], and quantitative multiplex immunofluorescent All data collected for this study was collected with approval of the NYU technique [2] in order to predict clinical outcome or direct therapy, Institutional Review Board at the NYU Perlmutter Cancer Center with based on single-cell based diagnostic or prognostic biomarkers de- informed consent. rived from tumor microenvironment. Consent Results No sensitive or patient identifiable information is included in the data We provided proof-of concepts that DEPArray technology enabled presented. automated isolation and recovery of rare malignant or pathogenic immune cells from liquid or tissue biopsies in several malignan- cies including vitreoretinal lymphoma (VRL), hepatocellular carcin- oma (HCC) and colorectal carcinoma (CRC). Rare target B lymphoma cells were distinguished and sorted from pausicellular ocular vitreous biopsies with high resolution and purity required Table 1 (abstract P130). Enrichment of anti-neuronal growth for sensitive single-cell based MYD88 mutational profiling to aid autoantibodies VRL diagnosis [1]. Single cellular imaging revealed the presence of large (>10μm), irregular shaped of a novel population of HCC- infiltrating macrophages in association with improved prognosis after surgery. A unique signature regulatory T-cells (Tregs) popu- lation was identified in both blood circulation and cancerous tis- sues of CRC. These signature Tregs expressed phenotypically distinct surface markers in association with better disease-free and overall survival of CRC patients. Conclusions Using real-time imaging-based, digital sorting DEPArray, we could distinguish, select and sort different types of malignant or target immune cells including B-cells, T-cells and macrophages from het- erogeneous tumor microenvironment or liquid biopsies with low cellularity. Comprehensive genomic and molecular characteriza- tions at single cell resolution revealed crucial biomarkers associ- ated with clinicopathological features that impact clinical outcome of patients. The single cell interrogation using DEPArray technology provides a novel precision medicine tool for diagnos- tics and prognostications of malignancies in future. Acknowledgements This study was supported by research funding from the research collaboration between A. Menarini Biomarkers Singapore Pte Ltd, Singapore Eye Research Institute and Singapore General Hospital. Some data presented here are part of patent filed on 21 August 2018 (#10201807097T). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 73 of 272 References P133 1. Tan, W.J., et al., Single-cell-MYD88 sequencing of isolated B cells High tumor expression of DKK1 is associated with improved from vitreous biopsies aids vitreoretinal lymphoma diagnosis. Blood, clinical benefit and longer progression free survival across 2019. multiple solid tumors when treated with a targeted anti-DKK1 2. Lim, J.C.T., et al., An automated staining protocol for seven-colour im- antibody (DKN-01) munofluorescence of human tissue sections for diagnostic and prognos- Michael Kagey, PhD, Girish Naik, MD, Michael Haas, PhD, Heidi tic use. Pathology, 2018. 50(3): p. 333-341. Heath, Franziska Schurpf-Huber, Walter Newman, PhD, Cynthia Sirard, Ethics Approval MD This study was approved by the SingHealth Institutional Review Board in Leap Therapeutics, Cambridge, MA, United States accordance with the Singapore Guidelines for Good Clinical Practice and Correspondence: Cynthia Sirard (csirard@leaptx.com) the Declaration of Helsinki, approval number #2009/907/B, 2012/104/F Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P133 and #2017/2494 Background Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, con- P132 tributes to an immune suppressive tumor microenvironment Harmony: Integrative tool to analyse and visualise multiplex- and promotes tumor growth, angiogenesis and metastasis. immunofluorescence single-cell data DKN-01, a DKK1 neutralizing antibody, has demonstrated clin- 1 2 3 4 Duoduo Wu , Joe Yeong, MBBS, PhD , Grace Tan , Marion Chevrier , ical activity across multiple solid tumors as both a monother- 5 5 4 Josh Loh , Tony Lim , Jinmiao Chen apy and in combination with checkpoint inhibitors and 1 2 National University of Singapore, Singapore, Singapore; Department of chemotherapies. Nonclinical studies indicate that DKN-01 effi- Anatomical Pathology, Sing, Singapore, Singapore; Nanyang cacy depends on a functioning immune system, notably nat- Technological University, Singapore, Singapore; Agency of Science, ural killer (NK) cells. High tumor expression of DKK1 correlates Technology and Resear, Singapore, Singapore; Singapore General with a worse clinical prognosis in many solid tumors. As such, Hospital, Singapore, Singapore, Singapore we evaluated tumor levels of DKK1 and association with clin- Correspondence: Tony Lim (Lim.Kiat.Hon@singhealth.com.sg); Jinmiao ical outcomes for DKN-01 based therapies. Chen (Chen_Jinmiao@immunol.a-star.edu.sg) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P132 DKK1 mRNA expression in patient tumor biopsies was evalu- ated with a RNAscope in situ hybridization assay. Expression Background levels were semi-quantified with QuPath or manually scored. In the advent of immuno-technology, newer single-cell flow cytome- Data was pooled from three separate clinical trials, DKN-01 as try techniques have greatly increased the capacity for the maximum monotherapy or in combination with paclitaxel or pembrolizu- number of immunological parameters measured. Notably, multiplex- mab in esophagogastric cancer (EGC) (NCT02013154), DKN-01 immunofluorescence (mIF) can perform measurements for 7 markers, as monotherapy or in combination with paclitaxel in epithelial flow cytometry can handle 20, and imaging mass cytometry can endometrial cancer (EEC) or epithelial ovarian cancer (EOC) process up to 37 biomarkers simultaneously. Hence, dimensionality (NCT03395080) and DKN-01 in combination with gemcitabine/ reduction techniques such as t-SNE and UMAP are becoming increas- cisplatin in biliary tract cancer (BTC) (NCT02375880). Survival ingly important for tumour single-cell data analysis. Using human he- analysis was performed by the Kaplan-Meier method and multi- patocellular carcinoma (HCC) tissue samples, we aim to compare and variable Cox proportional-hazards and logistic regression evaluate the use of a new technique, UMAP, as an alternative to t- models were used to study the association of DKK1 H-score SNE in mIF derived single-cell data. cutoffs (tertiles and quartiles) with survival and clinical benefit Methods (CR, PR or SD per RECIST v1.1) outcomes. We adopted an unsupervised clustering approach using FlowSOM to Results identify 8 major cell types present in human HCC tissues by staining Atotal of 120 patients (59EGC,28EEC,20EOC and13BTC) them with 7 markers, including immune-checkpoint molecules and had DKK1 tumor expression with response and survival out- one nuclear counterstain. Following that, UMAP and t-SNE were ran comes. Patients who had an H-score ≥ upper-quartile (≥50) of independently on the dataset to qualitatively compare the distribu- DKK1 expression versus < upper-quartile had a higher-odds of tion of clustered cell types in both dimensionality reduction tools. having clinical benefit/response with an adjusted OR of 4.46 Results (95% CI: 1.78, 11.71) and a longer PFS with an adjusted HR of The key advantage of UMAP is its superior runtime – it takes approxi- 0.49 (95% CI: 0.29, 0.81). Patients who had an H-score ≥ upper- mately one-fifth the time required to run t-SNE. Both techniques pro- tertile (≥35) versus < upper-tertile had a higher-odds of having vide similar arrangements of cell clusters, with the key difference clinical benefit/response with an adjusted OR of 2.82 (95% CI: being UMAP’s extensive characteristic branching. Also, increasing 1.21, 6.67) and a longer PFS with an adjusted HR of 0.53 (95% perplexity values in t-SNE results in a t-SNE visualisation with certain CI: 0.33, 0.85). degrees of branching like that of UMAP’s, albeit limited. When pa- Conclusions rameters such as standard deviation, minimum and maximum inten- Elevated DKK1 tumor expression was associated with a higher sity from the mIF image cytometry data were included, a t-SNE plot clinical benefit/response rate and longer PFS for DKN-01 based with virtually the same morphology as the resulting UMAP plot can treatments across multiple solid cancers. Tumor expression of be visualised. Most interestingly, UMAP’s branching highlighted bio- DKK1 may represent an important patient selection criterion logical lineages, especially in identifying potential hybrid tumour cells for further development of DKN-01 based therapies in solid (HTC). Survival analysis shows patients with higher proportion of HTC cancers. The contribution of high levels of tumor DKK1 expres- have a worse prognosis (p-value = 0.019). sion to an immune suppressive tumor microenvironment and Conclusions the role of NK cells is currently under investigation. We conclude that both techniques are similar in their visualisation capabilities, but UMAP has a clear advantage over t-SNE in runtime, Ethics Approval making it highly plausible to employ UMAP as an alternative to t-SNE Studies were approved by the Institutional Review Boards of each in single-cell data analysis. participating institution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 74 of 272 P134 Background Prognostic value of tumor microenvironment based on PD-L1 Loss of Y chromosome (LOY) is a well know established expression and CD8+ TILs density in locally advanced NSCLC phenomenon associated with cancers and ageing. Recently, LOY treated with concurrent chemoradiotherapy in peripheral blood cells was suggested as a possible biomarker 2 1 1 1 Lukas Käsmann , Kathrin Gennen , Julian Taugner , Chukwuka Eze , for different cancers in males. On the basis of previous findings, 1 1 1 Monika Karin , Olarn Roengvoraphoj , Jens Neumann , Amanda the present case-control study was conducted to evaluate the 3 1 1 1 Tufman , Michael Orth , Simone Reu , Claus Belka association of LOY in peripheral blood cells in prostate (PC) and 1 2 University Hospital, Munich, Germany; University of Munich, Munich, colorectal cancers (CRC) in males [1-4]. Germany; Thoracic Oncology Centre Munich, Munich, Germany Methods Correspondence: Lukas Käsmann (lkaesmann@gmail.com) 30 CRC patients (mean age = 44.03±10.8), 36 PC patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P134 (mean age = 60.8 ± 15.8 yrs) and 36 healthy control male cases (mean age = 54.6± 15.1 years) were recruited. DNA was Background extracted by using a standard phenol-chloroform method. The prognostic role of the tumor immunity microenvironment Multiplex quantitative fluorescent (QF) PCR was used to co- (TIME) in multimodal treatment for locally advanced non-small amplify the homologous sequences present on the Y chromo- cell lung cancer (LA-NSCLC) is unclear. Increasing evidence sug- some and other chromosome followed by their analysis on the gests treatment benefit depending on tumor cell PD-L1 expres- genetic analyzer (ABI 3500) and finally the Y/X ratio was calcu- sion. The purpose of this retrospective single-center study was to lated on the basis of the peak height obtained from the investigate the prognostic value of PD-L1 expression on tumor electropherogram. cells in combination with CD8+ tumor stroma-infiltrating lympho- Results cytes (TILs) density in inoperable LA-NSCLC treated with concur- The mean Y/X ratio was significantly lower in the whole group rent chemoradiotherapy (CRT). of cancer patients (0.709±0.02; p <0.0001) when compared to Methods the controls (0.92±0.044). Also, the Y/X ratio when calculated We collected retrospectively clinical characteristics and initial separately was found to be lower in CRC (0.701±0.078; p < tumor biopsy samples of 31 inoperable LA-NSCLC patients treated 0.0001) and PC (0.717±0.044; p <0.0001) cases, when compared with concurrent CRT. PD-L1 expression on tumor cells (0% versus to controls (0.92±0.044). Multivariate logistic regression was ≥1%), CD8+ TILs density (0-40% vs. 41-100%) and TIME according performed by matching cancer and control subjects with age to classification by Zhang et al. were evaluated for potential and the results suggest that LOY is not influenced by their prognostic value in terms of local control, progression-free (PFS) age. and overall survival (OS) as well as correlations with clinic- Conclusions pathological features investigated. The results support the significant association of LOY in peripheral Results blood cells carcinogenesis in males. LOY can also serve as a non- Median OS was 14 months (range: 3-167 months). The OS rates invasive cancer biomarker to improve the early diagnosis and man- at 1- and 2 years were 68% and 20%. Local control rates for the agement of cancer patients in males. entire cohort at 1 and 2 years were 74% and 61%, respectively. Median PFS and PFS at 1 and 2 years were 13±1.4 months, 58% Acknowledgements and 19%. PD-L1 expression <1% on tumor cells was associated We are highly grateful to Sanjay Gandhi Post Graduate Institute of with improved OS, PFS and local control in patients treated with Medical Sciences, Lucknow, Uttar Pradesh, India for providing the concurrent CRT. Univariate analysis showed a trend for improved infrastructure and lab facilities for research work. The authors also thank OS and local control in patients with low CD8+ TILs density. all the consultant and residents of SGPGIMS, who helped in carrying out Evaluation of TIME appears to be an independent prognostic fac- the study. Dr Ambreen Asim is the first author, who collected data, tor for local control, PFS and OS. The longest and shortest OS carried out all the practical work and drafted this abstract. Prof. Sarita were achieved in patients with type I (PD-L1neg/CD8low) and Agarwal is the corresponding and second author, who helped in type IV (PD-L1pos/CD8low) tumors (median OS: 57±37 vs. 10±5 finalizing, correcting and critical review of the work. Prof.Rakesh Kapoor months, p=0.05), respectively. and Prof. Neeraj Rastogi are the oncologist consultant who has provided Conclusions prostate and colorectal cancer patients blood samples after taking Assessment of the tumor immunity microenvironment (TIME) by informed consent for this study. PD-L1 expression on tumor cells and CD8+ TILs density is a pre- dictive biomarker in patients treated with concurrent CRT for in- References operable LA-NSCLC. 1. Noveski P, Madjunkova S, Stefanovska E. Loss of Y Chromosome in Peripheral Blood of Colorectal and Prostate Cancer Patients. Plos One. Acknowledgements 2016;11(1). The study was funded by the German Center for Lung Research (DZL). 2. Chang Y M, Perumal R, Keat P Y, Rita Y.Y. Yong, Daniel L.C. Kuehn, Ethics Approval Leigh Burgoyne. A distinct Y-STR haplotype for Amelogenin nega- The study was approved by the University Ethics Board, approval number tive males characterized by a large Yp11.2 (DYS458-MSY1-AMEL-Y) 493-16. deletion. Forensic Sci Int. 2007; 166(2-3):115-20. Consent 3. Donaghue C, Mann K, Docherty Z and Ogilvie C M. Detection of Written informed consent was obtained from the patient for publication of mosaicism for primary trisomies in prenatal samples by QF-PCR and this abstract and any accompanying images. A copy of the written consent karyotype analysis.Prenat Diagn .2005; 25: 65–72. is available for review by the Editor of this journal. 4. Plaseski T, Noveski P, Trivodalieva S, Georgi D. Quantitative Fluorescent- PCR Detection of Sex Chromosome Aneuploidies and AZF Deletions /Du- plications. Genetic Teasting, 2008 : 12: 4. P135 Ethics Approval Studying Loss of Y chromosome in colorectal and prostate cancers This study was approved by Sanjay Gandhi Post Graduate Institute of in males for non-invasive cancer biomarkers Medical Sciences Ethics Board; approval number IEC CODE – 2018- 53- Ambreen Asim, PhD, Sarita Agarwal, Rakesh Kapoor, Neeraj Rastogi IMP-103 dated 18th June 2018.” Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Consent India Written informed consent was obtained from the patient for publication of Correspondence: Sarita Agarwal (saritasgpgi@gmail.com) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P135 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 75 of 272 P136 Background Quantitative/spatial analysis of Tregs reveal a prominent PD-L1 protein expression by immunohistochemistry (IHC) meas- biomarker role in human non-small cell lung cancer (NSCLC) urement is the only FDA-approved protein diagnostic biomarker 1 1 1 Richa Gupta , Nicolas Rodriguez-Arriagada , Shruti Desai, PhD , for PD1/PD-L1 immunotherapies [1]. However, the tumor-immune 2 1 Konstantinos Syrigos , Roy Herbst, MD, PhD , Vamsidhar Velcheti, MD interaction is complex: PD-L1 expression alone is not predictive 3 1 1 FACP , David Rimm, MD, PhD , Sarah Goldberg, MD, MPH , Kurt of patient response [2]. This has led to the investigation of other Schalper, MD, PhD PD-1 ligands such as PD-L2 [2]. PD-L2 even in the absence of 1 2 Yale University, Burlington, CT, United States; Athens University, PD-L1 has been associated with clinical response to PD-1 block- Athens, Greece; NYU-Langone Medical Center, Pepper Pike, OH, United ade in multiple tumor types [2]. PD-L2 status has also been inves- States tigated where immunotherapy based on PD-L1 has been less Correspondence: Kurt Schalper (kurt.schalper@yale.edu) successful, such as prostate cancer, where PD-L1 expression is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P136 typically low [3]. Here, significantly higher levels of PD-L2 were associated with multiple survival and response measures [3]. Due Background to the diagnostic and therapeutic potential indicated by the pres- Regulatory T cells (Tregs) mediate potent tolerogenic signals, are involved ence of this key immune checkpoint ligand in patients irrespect- in adaptive anti-tumor immune responses and T-cell reinvigoration using ive of PD-L1 expression [2-4], dependable detection tools for immune checkpoint blockers. Despite their prominent immune suppres- investigating the presence and role of PD-L2 are crucial. To ad- sive role, the tissue distribution and contribution of Tregs to clinical out- dress this need, Abcam have developed and extensively charac- comes in human lung cancer is not well understood. terized and validated a recombinant rabbit monoclonal antibody Methods specific to PD-L2 (CAL28). For checkpoint inhibitors, Abcam The levels and tissue distribution of Tregs and major tumor infiltrating already has research use only versions of three anti-PD-L1 Rab- lymphocyte (TIL) subsets were measured using simultaneous detection MAb® antibodies employed in the clinical setting (73-10, 28-8 and of FOXP3, CD4, CD8, pancytokeratin and DAPI by multiplexed quantita- SP142), co-developed with pharmaceutical and diagnostic tive immunofluorescence in 619 formalin-fixed paraffin embedded companies. (FFPE) NSCLCs from 4 independent cohorts represented in tissue micro- Methods arrays (cohort #1 [Yale, n=210], cohort #2 [Greece, n=192]; cohort #3] A recombinant rabbit monoclonal antibody was generated using [80 immunotherapy-treated NSCLCs]; cohort #4 [Yale, n=137, adenocar- a direct B cell cloning process and characterized for IHC. The cinomas with mutation testing). Markers were measured in different tis- clone was tested using PD-L2-transfected and non-transfected sue compartments and cell phenotypes were used for individual cell HEK293 cells fixed in formaldehyde and processed into paraffin counts and machine-learning-based spatial analysis. We studied the as- wax (FFPE) and further validated alongside In Situ Hybridization sociation between T-cell populations, tissue distribution, clinicopatho- (ISH) for PD-L2 mRNA in FFPE commercial cell lines. Once speci- logic/molecular characteristics and outcomes. ficity was determined, it was tested in positive and negative tis- Results sues and TMAs of Head & Neck Squamous Cell Carcinoma Tregs (DAPI+/CD4+/FOXP3+ cells) were predominantly located in the (HNSCC), Prostate Carcinoma (PC) and Renal Cell Carcinoma stromal compartment and represented 3-10% of the total T-cell popula- (RCC). tion. The level of Tregs was positively associated with higher CD8+ T- Results cell infiltration across the cohorts. There was no consistent association CAL28 demonstrated positive IHC staining on PD-L2- between Treg levels and patient age, gender, smoking status, clinical overexpressed HEK293 cells processed in FFPE with a lack of stage or tumor histology. However, Tregs were significantly higher in staining in the parental line. Additionally, CAL28 demonstrated KRAS mutated lung adenocarcinomas than in EGFR mutant or KRAS/ IHC staining in FFPE cell lines where PD-L2 expression was con- EGFR wild-type cases. As a single marker, the level of Tregs was not sig- firmed with ISH for PD-L2 mRNA. Expression in tumor tissue in nificantly associated with survival. However, the Treg to CD8 signal ratio TMAs from HNSCC, PC and RCC was evaluated with no non- was associated with shorter 5-year overall survival across the cohorts. specific background staining. Reduced survival was also seen in cases with a higher 5-nearest neigh- Conclusions bor (5NN) mean distance between CD4+/Tregs and CD8+/CD4+ cells. We have demonstrated sensitivity, specificity and reproducibility Notably, the survival effect of the Treg-associated metrics was numeric- of a recombinant rabbit monoclonal antibody to PD-L2 in IHC ally higher in patients treated with immune checkpoint blockers. (CAL28). The global, commercial availability of this recombinant Conclusions clone to researchers, pathologists, clinicians and the biopharma- Tregs are prominently less abundant than other TIL subsets in NSCLC ceutical industry will enable further progress to be made in un- microenvironments and they are increased in T-cell inflamed tumors. derstanding the clinical relevance and predictive value that PD-L2 Their positive association with CD8+ cytotoxic TILs suggests their up- promises for cancer immunotherapy. regulation upon adaptive anti-tumor immune pressure and could ex- plain the inconsistent reported relationship between Tregs and References prognosis. Elevated Treg to CD8 signal ratio and reduced spatial clus- 1. Tsao MT, Kerr K, Yatabe Y, et al. PL 03.03 Blueprint 2: PD-L1 Immunohisto- tering between CD4-Tregs and CD8-CD4 are indicative of poor out- chemistry Comparability Study in Real-Life, Clinical Samples. J Thor Oncol. come preferentially in NSCLC patients treated with checkpoint 2017;12(Suppl 2):S1606 blockade suggesting a biomarker role. 2. Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Ethics Approval Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T. All tissues were used after approval from the Yale Human Investiga- Clin Cancer Res. 2017;23:3158-3167 tion committee protocol #9505008219 which approved patient con- 3. Zhao SG, Lehrer J, Chang SL, Das R, Erho N, Liu Y, Sjöström M, Den RB, sent forms or waivers of consent. Freedland SJ, Klein EA, Karnes RJ, Schaeffer EM, Xu M, Speers C, Nguyen PL, Ross AE, Chan JM, Cooperberg MR, Carroll PR, Davicioni E, Fong L, Spratt DE, Feng FY.The Immune Landscape of Prostate Cancer and P137 Nomination of PD-L2 as a Potential Therapeutic Target. J Natl Cancer Inst. Development and high specification validation of two recombinant 2019;111:301-310 rabbit monoclonal antibodies to accurately detect human PD-L2 4. Takamori S, Takada K, Toyokawa G, Azuma K, Shimokawa M, Jogo T, expression in FFPE tissue sections by immunohistochemistry Yamada Y, Hirai F, Tagawa T, Kawahara A, Akiba J, Okamoto I, Nakanishi Simon Renshaw, Will Howat, PhD, Subham Basu, PhD Y, Oda Y, Hoshino T, Maehara Y. PD-L2 Expression as a Potential Predict- Abcam, Cambridge, United Kingdom ive Biomarker for the Response to Anti-PD-1 Drugs in Patients with Non- Correspondence: Subham Basu (Subham.Basu@abcam.com) small Cell Lung Cancer. Anticancer Res. 2018;38:5897-5901 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P137 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 76 of 272 P138 3. Oaknin A, Duska LR, Sullivan RJ, et al. Preliminary safety, efficacy, and Review of evidence for predictive value of microsatellite pharmacokinetic/pharmacodynamic characterization from GARNET, a instability/mismatch repair status in response to non-anti-PD-(L)1 phase I/II clinical trial of the anti–PD-1 monoclonal antibody, TSR-042, in therapies in patients with advanced or recurrent endometrial patients with recurrent or advanced MSI-H and MSS endometrial cancer. cancer Gynecol Oncol. 2019; 154:17. 1 2 2 2 Cara Mathews, MD , Ellie Im , Liliana Alfaya , Karin Travers , Craig 4. Antill YC, Kok PS, Robledo K, et al. Activity of durvalumab in advanced Gibson endometrial cancer (AEC) according to mismatch repair (MMR) status: 1 2 Women and Infants Hospital, Providence, RI, United States; TESARO: A The phase II PHAEDRA trial (ANZGOG1601). J Clin Oncol 37, 2019 (suppl; GSK Company, Waltham, MA, United States abstr 5501). Correspondence: Cara Mathews(cmathews@wihri.org) 5. Djordjevic B, Bruegl A, Fellman B, et al. The prognostic effect of MLH1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P138 loss in endometrial endometrioid adenocarcinoma. Lab Invest. 2014; 94:280A–281A. Background 6. Cohen JG, Goodman MT, Karlan BY, Walsh C. Genomic characterization of Multiple immunotherapies have been evaluated in patients with ad- grade 3 endometrial carcinoma. Gynecol Oncol. 2014; 133:134–135. vanced or recurrent endometrial cancer (EC) using molecular bio- 7. Cosgrove CM, Cohn DE, Hampel H, et al. Epigenetic silencing of MLH1 in markers, including microsatellite instability-high (MSI-H) and stable endometrial cancers is associated with larger tumor volume, increased (MSS) status. Clinical outcomes appear to be different in patients with rate of lymph node positivity and reduced recurrence-free survival. Gyne- MSI-H/mismatch repair (MMR)-deficient status versus MSS/MMR-profi- col Oncol. 2017; 146(3):588–595. doi:10.1016/j.ygyno.2017.07.003. cient status when receiving anti-programmed cell death (ligand) 1 (PD- 8. Kim SR, Pina A, Albert A, et al. Does MMR status in endometrial cancer [L]1) therapies [1,2]. It is unclear if these differences are due to the ther- influence response to adjuvant therapy? Gynecol Oncol. 2018; 151:76–81. apies themselves or to differences inherent to the patient populations. 9. Aghajanian C, Filiaci V, Dizon DS, et al. A phase II study of frontline We sought to evaluate the association between MSI-H/deficient MMR paclitaxel/carboplatin/bevacizumab, paclitaxel/carboplatin/temsirolimus, (dMMR) status and response among patients with advanced or recur- or ixabepilone/carboplatin/bevacizumab in advanced/recurrent rent EC. endometrial cancer. Gynecol Oncol. 2018; 150:274–281. Methods We conducted a systematic review of the Embase, MEDLINE, and P139 Cochrane Central Register of Controlled Trials databases from 2000 Expression of GITR and GITR-L by head and neck squamous cell to present to identify publications (manuscripts and conference pro- cancer ceedings) on studies using chemotherapy, surgery, radiotherapy, hor- 1 2 Rachna Moudgil, MS , Christopher Paustian, PhD , Carmen Ballesteros- monal therapy, or biological therapy (or any combination thereof) in 1 1 1 Merino, PhD , Shawn Jensen, PhD , Hong-Ming Hu, PhD , Walter Urba, adult patients (≥18 years) with stage III or IV advanced or recurrent 1 1 1 MD, PhD , Carlo Bifulco, MD , Marcus Couey, MD, DDS , Traci Hilton, EC, and where MMR or MSI status was identified (by any means). To 2 1 1 1 PhD , Bernard Fox, PhD , Rom Leidner, MD , R. Bryan Bell, DDS, MD better understand the prognostic value of MSI-H/MSS status, we ex- 1 2 Earle A. Chiles Research Institute, Portland, OR, United States; UbiVac, cluded anti-PD-(L)1 therapies from the analysis, as recent evidence Portland, OR, United States suggests that there is a positive predictive value for these agents in Correspondence: Bernard Fox(foxb@foxlab.org) patients with MSI-H/dMMR status [2-4]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P139 Results Our systematic review of MSI/MMR status and recurrence-free survival Background (RFS), progression-free survival (PFS), and overall survival (OS) identified Head and neck squamous cell cancer (HNSCC) ranks as the 6th most a total of 5 studies. One study reported dMMR status was associated common cancer afflicting humans and remains a significant unmet med- with a reduction in RFS (hazard ratio, 2.02) [5], while another study ical need. While interfering with the PD-1/PD-L1 axis improves outcomes, found no significant effect [6]. A third study reported a trend towards a the majority of patients progress and die of their disease. To address this higher rate of recurrence among patients with advanced-stage EC with lack of efficacy our group has explored the immune makeup of HNSCC, dMMR than among patients with MMR proficiency (P value not re- hypothesizing that a better characterization of responders and non- ported) [7]. Two studies reported no significant association between responders will result in improved predictive biomarkers and insights into PFS and dMMR status [8,9]. Three studies found no statistically signifi- strategies to improve outcomes for the majority of patients. cant association between OS and dMMR status [5,6,8]. Methods Conclusions Over the past 7 years we have collected and processed more than This review could not identify a consistent association between 350 HNSCC specimens. When sufficient tumor material was available, dMMR or MSI-H status and recurrence, RFS, PFS, or OS among pa- tumor-infiltrating lymphocytes (TIL) and primary tumor cultures were tients with advanced or recurrent EC receiving therapy other than initiated and characterized for autologous tumor reactivity. Once anti-PD-(L)1. For RFS, where differences were present, they trended established, tumor cell lines were characterized for phenotypic towards worse outcomes for patients with MSI-H/dMMR status. Con- markers by flow cytometry. Flow cytometric analysis and RNASeq has sequently, we have identified no evidence of a prognostic or predict- been performed on some established cell lines and on FFPE tumor ive value of MSI-H or dMMR biomarker status for efficacy outcomes specimens. in patients with advanced or recurrent EC receiving non–anti-PD-(L)1 Results therapy. Further investigation into the prognostic or predictive value Consistent with previous reports increased expression of CD8 T cells of MSI-H/dMMR status is warranted. was associated with improved outcome. In preliminary studies in- creased expression of GITR was also associated with improved out- Acknowledgements come. Initial speculation was that GITR expression was coming from Clinical Trial Registration: N/A immune infiltrates. Subsequently a report suggested that GITR could be expressed by HNSCC. Using flow cytometry we detected low level References GITR expression on 3 HNSCC cell lines and low to high level expres- 1. Segal NH, Wainberg ZA, Overman MJ, et al. Safety and clinical activity of sion of GITR-L on a 8 HNSCC cell lines. Studies are continuing to ex- durvalumab monotherapy in patients with microsatellite instability–high pand on these preliminary observations. (MSI-H) tumors [abstract]. J Clin Oncol. 2019; 37(Suppl 4):670. Conclusions 2. Konstantinopoulos PA, Liu JF, Luo W, et al. Phase 2, two-group, two- Anti-GITR and GITR-L both have the potential to provide positive sig- stage study of avelumab in patients (pts) with microsatellite stable (MSS), nals to immune cells. In addition to APC’s, GITR-L expression by some microsatellite instable (MSI), and polymerase epsilon (POLE) mutated re- HNSCC cells may contribute to the make-up of the immune cells infil- current/persistent endometrial cancer (EC) [abstract]. J Clin Oncol. 2019; trating these cancers. 37(Suppl 15):5502. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 77 of 272 Acknowledgements P141 Funding Support: The Harder Family, Robert and Elsie Franz, Wes and One-year progression-free survival in lung cancer patients treated Nancy Lematta, Lynn and Jack Loacker, the Providence Portland Medical with immune checkpoint inhibitors is significantly associated with Foundation and the Oral and Maxillofacial Surgery Foundation, The Mur- a novel immunomodulatory signature but not PD-L1 staining 1 1 1 2 dock Trust. Harsha Ranganath, MD , Amit Jain , Justin Smith , Julie Ryder , Amina 1 2 2 2 3 Ethics Approval Chaudry , Emily Miller , Felicia Hare , Poojitha Valasareddy , Rob Seitz , 3 3 3 2 The study was approved by the institutional review board of the Providence David Hout , Brock Schweitzer , Tyler Nielsen , Janice Mullins , Gregory Portland Medical Center (12-075A). Vidal University of Tennessee Health Sciences, Indianapolis, IN, United States; 2 3 P140 West Clinic Cancer Center, Memphis, TN, United States; Insight Myeloid cell contexture and IL-8 expression as a candidate Genetics, Nashville, TN, United States immunotherapy target in non-small cell lung cancer (NSCLC) Correspondence: Gregory Vidal (gvidal@westclinic.com) 1 1 Venkata Vamsi Nagineni, MD , Kurt Schalper, MD, PhD , Shruti Desai, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P141 1 2 2 1 PhD , Ignacio Melero, MD , Miguel Sanmamed, MD, PhD , Richa Gupta , 1 1 Roy Herbst, MD, PhD , Venkata Vamsi Nagineni, MD Background 1 2 Yale University, Waukegan, IL, United States; University of Navarra, Immune checkpoint inhibitors (PD-(L)1 inhibitors) have shown Pamplona, Spain promising therapeutic outcomes and have been approved for Correspondence: Kurt Schalper (kurt.schalper@yale.edu) multiple indications. However, widespread use of PD-(L)1 inhibi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P140 tors has been limited by a low response rate and immune- related adverse events. Therefore, an improved method for Background predicting response to the immune checkpoint blockade would Interleukin-8 (IL-8) is a chemokine expressed in multiple cancer types, better identify patients misclassified by conventional testing. We including NSCLC. It exerts various functions in shaping cancer have evaluated a proprietary algorithm which utilizes gene vascularization, cell dedifferentiation and inflammation/immunity. IL-8 expression in solid tumors to assess the presence of an immu- was described as a chemotactic factor for neutrophils and it has been nomodulatory (IM) signature intended to predict immunother- proposed to mediate recruitment of tolerogenic myeloid cells favoring apy response. The purpose of this study was to evaluate the a pro-tumorigenic microenvironment. Although clinical trials targeting performance of the IM signature against progression-free IL-8 are ongoing, its expression and role in NSCLC is unclear. survival (PFS) of patients treated with immune checkpoint Methods inhibitors. We developed a multiplexed quantitative immunofluorescence (QIF) Methods panel for simultaneous and localized measurement of IL-8, myeloperox- In this retrospective study, archival tumor tissue from metastatic idase (MPO), CD15, cytokeratin (CK) and DAPI. We analyzed the expres- lung cancer patients treated with one of three PD-(L)1 inhibitors sion of these markers and their association with PD-L1, CD4 and CD8- (pembrolizumab, nivolumab, and atezolizumab) either as a single positive cells in 3 retrospective NSCLC immunotherapy-naive cohorts agent or in conjunction with standard chemotherapy, from whom represented in tissue microarrays (cohort #1, n=262; #2, n=145; and #3, response data was available, was tested for the IM signature. n=132); 1 cohort of NSCLC patients treated with immune checkpoint Patients were stratified into two groups based on IM signature blockers (#4, n=59) and 1 collection of lung adenocarcinomas (LAC) an- classification as positive or negative, which was compared to im- alyzed for activating mutations in EGFR and KRAS (#5, n=121). We stud- munohistochemistry PD-(L)1 testing with a primary endpoint of ied the level of the targets, their distribution and association with one-year progression-free survival. Additionally, the IM signature immune features, clinicopathological variables and survival. classification was compared with objective response by Spear- Results man’s correlation as a continuous variable. IL-8 protein signal was detected in ~85% of cases with cytoplasmic Results staining pattern and was higher in tumor than in stromal cells. Elevated A total of 71 metastatic lung cancer patients were included in the tumor IL-8 was consistently associated with higher MPO+ neutrophils study with a median follow-up of 29 months. The one-year PFS haz- and CD15+ tumor-associated myeloid cells across the cohorts, but not ard ratio for the IM positive group was 0.31 (95% CI 0.14 to 0.68; with CD4+ and CD8+ T-cells. Increased IL-8 expression was not associ- p=.004 - Figure 1). A total of 62 out of the 71 metastatic lung cancer ated with major clinicopathologic variables. Elevated MPO+ and CD15+ patients had previous PD-L1 staining. Head-to-head analysis of PD-L1 cells was significantly higher in KRAS mutated than in EGFR mutated and IM signature on these patients found the one-year PFS hazard LACs. High MPO and CD15 signal was associated with shorter 5-year ratio for the IM group to be 0.30 (95% CI 0.13 to 0.71; p=0.006) and overall survival in all NSCLC cohorts. The negative prognostic effect of the one-year PFS hazard ratio for PD-L1 positive staining to be 0.76 MPO and CD15 was comparable in both immunotherapy-naïve and (95% CI 0.31 to 1.82; p=0.533). The mean IM correlation value with immunotherapy-treated NSCLC collections. objective response for PD = -0.06; SD = -0.04; PR = 0.14; CR = 0.33; p Conclusions Conclusions IL-8 protein is frequently expressed in NSCLCs associated with increased The IM signature was significantly associated with prolonged one- tumor-associated myeloid cells but independent from intratumor T-cell year progression-free survival among patients treated with PD-L1 in- responses. KRAS mutated LACs have prominent MPO+/CD15+ expres- hibitors while PD-L1 staining failed to be significantly associated. Pa- sion, supporting an immune suppressive role of myeloid cells in these tients classified positive by the IM signature demonstrated a three- malignancies. CD15 and MPO are prognostic markers in NSCLC and IL-8 fold improved hazard ratio compared to those who were negative. blockade could mediate favorable immunomodulatory effects. Funding was provided by Insight Genetics working in cooperation Ethics Approval with West Cancer Center and Research Institute. All tissues were used after approval from Yale Human Investigation Ethics Approval committee protocol #9505008219 which approved the patient con- This study was approved by the West Cancer Clinic Institutional Re- sent forms or waivers of consent view Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 78 of 272 above. The classifier delivered stratifications for response vs nonre- sponse with 84% accuracy, 79% sensitivity, 92% specificity, 75% posi- tive predictive value (PPV) and 95% negative predictive value (NPV). The associations of EpiSwitch™ response calls with overall survival (OS) and progressive free survival (PFS) in the independent cohort were significant (OS and PFS: log-rank p Conclusions The established EpiSwitchTM classifier contains strong binary markers of epigenetic deregulation with features normally attributed to gen- etic markers; the binary status of these classifying markers is statisti- cally significant for survival. Altogether, these findings highlight the potential of the EpiSwitchTM approach for identifying responders and non-responders to immuno-oncology therapies. Acknowledgements The authors would like to thank patients enrolled in the EMR000070-001 JAVELIN Solid Tumor trial for agreeing for their samples to be used for research purposes. This work is funded by Merck KGaA, Darmstadt, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. References 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- cer. Front Genet. 2016; 7:194. Fig. 1 (abstract P141). See text for description 2. Carini C, Hunter E; Scottish Early Rheumatoid Arthritis Inception Cohort investigators, et al. Chromosome conformation signatures define P142 predictive markers of inadequate response to methotrexate in early Development and validation of baseline predictive biomarkers for rheumatoid arthritis. J Transl Med. 2018; 16:18. response to avelumab in second-line (2L) non-small cell lung 3. Jakub JW, Grotz TE, Jordan P, et al. A pilot study of chromosomal cancer (NSCLC) using EpiSwitchTM epigenetic profiling aberrations and epigenetic changes in peripheral blood samples to 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , identify patients with melanoma. Melanoma Res. 2015; 25:406-11. 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan 4. Bastonini E, Jeznach M, Field M, et al. Chromatin barcodes as biomarkers 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem for melanoma. Pigment Cell Melanoma Res. 2014; 27:788-800. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir 5. Yan H, Hunter E, Akoulitchev A, et al. Epigenetic chromatin conformation 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins changes in peripheral blood can detect thyroid cancer. Surgery. 2019; 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, 165:44-49. Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; Ethics Approval Mayo Clinic, Rochester, MN, United States The protocol was approved by the institutional review board or independent Correspondence: Parantu Shah (parantu.shah@emdserono.com); ethics committee at each center. Matthew Salter (matthew.salter@oxfordbiodynamics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P142 P143 Development and validation of baseline predictive biomarkers for Background response to immuno-checkpoint treatments in the context of Development of baseline predictive classifiers for response to treatment multi-line and multi-therapy cohorts using EpiSwitchTM epigenetic can provide advantages for programs of targeted immunotherapies, profiling development of successful combination therapies, and identification of 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , responder populations to active therapies. Chromosome conformations 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan represent strong systemic cellular network deregulations associated 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem with differences in clinical phenotypes and outcomes [1]. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir Methods 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, business of Merck KGaA, Darmstadt, Germany/Pfizer alliance," has ap- Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; plied its proprietary technology EpiSwitchTM to monitor systemic Mayo Clinic, Rochester, MN, United States epigenetic biomarkers for chromosome conformation signatures in Correspondence: Parantu Shah (parantu.shah@emdserono.com) baseline blood samples of patients with multiline anti–PD-L1 (avelu- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P143 mab) treatment of NSCLC. This application was based on the pub- lished methodology for validated predictive biomarkers for response Background to treatment [2], systemic blood-based monitoring of oncological Development of baseline predictive classifiers for response to treatment conditions [3-5], and proprietary programs in collaboration with the can provide advantages for programs of targeted immunotherapies, Mayo Clinic for predictive and response biomarkers in melanoma pa- development of successful combination therapies, and identification of tients treated with anti–PD-1 therapy (pembrolizumab). responder populations to active therapies. Changes in chromosome Results conformations represent strong systemic cellular network deregulations A 14-marker classifier was generated with 12 avelumab-treated pa- associated with differences in clinical phenotypes and outcomes [1]. tients in each response group; in this cohort, responders were de- However, questions remain about the applicability of classifiers across fined as patients with complete or partial response, and non- treatment lines, indications, and drug combinations. responders were defined as patients with progressive disease. Valid- Methods ation of the developed predictive markers was performed on an in- Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a dependent cohort of 75 patients treated with avelumab as either business of Merck KGaA, Darmstadt, Germany/Pfizer alliance, has ap- first-line (1L) or 2L therapy. In the validation cohort, patients with plied its proprietary technology EpiSwitchTM to monitor systemic stable disease were also considered as responders in addition to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 79 of 272 epigenetic biomarkers for chromosome conformation signatures at Methods baseline in patients with multiline anti–PD-L1 (avelumab) treatment Tyramide signal amplification detection was used to inform on the of non-small cell lung cancer (NSCLC). Additionally, epigenetic bio- tumor/stroma/immune contexture (CD8, PanCK, FAP, MHC-I, CD31) and markers to predict outcome and response in patients with melanoma to characterize T-cell functions (PD1, CD3, PanCK, GZMB, and PD-L1). treated with anti–PD-1 (pembrolizumab) and its combination with Whole slide digital pathology scoring algorithms were developed to another agent were identified in collaboration with the Mayo Clinic. identify all phenotypes represented by the markers and their specificity Results and sensitivity was verified against the results by expert observers. Three NSCLC classifiers predicting response to avelumab in first-line Results (1L), second-line (2L), and combined 1L + 2L cohorts were built and ap- Assay performance including accuracy, precision, and sequential markers plied to test sets. Average accuracy, positive predictive value (PPV), and detection was validated on > 200 unique cases of Gastric, Pancreatic, negative predictive value (NPV) for 10-fold cross-validation on data Breast, Lung, Urothelial and Colorectal carcinomas. For an early proof-of- splits were reported. An NSCLC patient set treated with 2L pembrolizu- concept, we analyzed paired pre- vs. post treatment tumor biopsies from mab served as an independent test set. The 2L NSCLC classifier three pancreatic cancer patients treated with a combination of atezolizu- achieved high (defined hereafter as > 0.7) predictive power (PPV, NPV, mab and chemotherapy. Digital pathology algorithms identified biologic- and accuracy) in the 2L test set but not in the 1L test set. A reduced ally and clinically relevant features: MHC-I is highly expressed in tumor version of this classifier achieved a PPV of 0.71 in the 2L pembrolizu- cells of the primary lesions and very rare tumor cells express MHC-I in the mab population. The 1L classifier was not applicable in patients who re- liver met samples. A patient with partial response (PR) showed a signifi- ceived 2L treatment for NSCLC. The 1L + 2L composite classifier had cantly increased tumor MHC-I upon treatment, while two patients with high predictive power in both 1L and 2L cohorts and a high PPV for stable disease (SD) did not show significant changes. The PR patient also identifying responders in the 2L pembrolizumab population. A fourth showed an increased density of CD3+, CD8+, and GZMB+ T cells within classifier starting with preselected NSCLC markers had good predictive the tumor post-treatment, indicating an increased tumoral T cell infiltra- power for classifying responders in patients with melanoma treated tion and activation by the treatment. with pembrolizumab. Finally, a 2L NSCLC classifier trained to classify re- Conclusions sponse groups from pembrolizumab-treated patients also identified The automated 5-plex IHC assays and digital pathology algorithms NSCLC responders with a high PPV from patients treated with pembro- developed in this study provide a robust tool for quantitative and lizumab in combination with an epigenetic drug. spatially resolved whole-slide characterization of the tumor-immune Conclusions contexture. Applying these tools in large-scale clinical investigations Collectively, these results suggest that a set of EpiSwitchTM bio- may provide better understanding of the response/resistance mecha- markers correlates with outcome on anti–PD-1/PD-L1 immunother- nisms to cancer immunotherapies. apies. Classifier signatures could be generated to work across treatment lines, indications, and combinations, and could be helpful Cellular Therapies for baseline patient stratification. P145 Acknowledgements Expanding Iovance’s tumor infiltrating lymphocytes (TIL) from core The authors would like to thank patients enrolled in the EMR000070-001 biopsies for adoptive T cell therapy using a 22-day manufacturing JAVELIN solid tumor trial for agreeing to consent usage of samples for process research purposes. This work was funded by Merck KGaA, Darmstadt, Michelle Abelson, PhD, Kenneth D'Arigo, Florangel Hilton, Maria Fardis, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany PhD, MBA, Cecile Chartier and Pfizer Inc., New York, NY, USA. Iovance Biotherapeutics, Inc., Tampa Bay, FL, United States Correspondence: Cecile Chartier (cecile.chartier@iovance.com) References Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P145 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- Background cer. Front Genet. 2016; 7:194. Iovance’s TIL products Lifileucel and LN-145 have demonstrated re- Ethics Approval markable clinical activity in melanoma and cervical cancer utilizing The protocol was approved by the institutional review board or independent Iovance’s proprietary 22-day manufacturing process and surgically ethics committee at each enrolling center. resected tumor lesions ~ 1.5-cm diameter [1, 2]. Using a core needle biopsy procedure to obtain tumor samples could allow for greater convenience of collecting the tumor from patients [3]. We asked whether a streamlined manufacturing process could be implemented P144 to produce therapeutically relevant TIL from multiple histologies Quantification of tumor-stroma-immune contexture by multiplex starting with a core biopsy. fluorescent immunohistochemistry and whole-slide digital image Methods analysis 1 1 2 Core biopsies obtained from 4 melanoma and 3 pancreatic, 2 breast, Adriana Racolta, PhD , Mehrnoush Khojasteh, PhD , Jennifer Giltnane , 1 1 1 2 ovarian, and 1 lung tumors were processed in vitro, using a 22-day Antony Hubbard, BS , Hongjun Zhang , Miriam Matei , Jessica 1 1 1 expansion method termed ‘Core process’. Core biopsy-derived TIL Baumann , Wenjun Zhang, MD, PhD , Tsu-Shuen Tsao, PhD , Hartmut 2 2 1 1 1 were assessed for expansion, phenotype (lineage, youth/differenti- Koeppen , Lisa Ryner , Xingwei Wang , Jim Martin , Auranuch Lorsakul , 1 1 1 1 ation, activation, and exhaustion markers), function (IFN-gamma and Ilya Ravkin , Smadar Shiffman , Lidija Pestic-Dragovich , Lei Tang, PhD , CD107a mobilization), and TCR repertoire. Yulei Wang, BA PhD 1 2 Results Roche Tissue Diagnostics, Tucson, AZ, United States; Genentech, South Iovance’s Core process successfully generated TIL products from all San Francisco, CA, United States tested samples. One to 2 cores yielded more than 10e9 T cells for 10 Correspondence: Yulei Wang (wang.yulei@gene.com) of the 12 preparations. Phenotypic analyses revealed no significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P144 differences in terms of T cell lineages and memory subsets, or ex- pression of activation, differentiation, and exhaustion markers when Background compared to Iovance’s current products. Core-derived TIL products Understanding response to immunotherapies in relation to tumor- responded to PMA and to anti-CD3 stimulations by inducing levels of immune contexture requires a paradigm shift from a single-marker CD107a mobilization and IFN-gamma secretion like those produced test towards multiplexed immunohistochemistry (IHC). Here we re- by TIL derived from excisional biopsies. Preliminary TCR sequencing port the development, early proof of concept of two fully automated data suggest that high-diversity products can be also be obtained 5-plex fluorescent multiplex IHC assays and accompanying digital from small samples, similar to what is obtained from TIL expansion. pathology algorithms. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 80 of 272 Conclusions observed compared with single transduced CAR T-cells (product A or This work demonstrates that the Iovance 22-day Core manufacturing B). Expression of the IL7R_CCR in both AUTO6NG and product A con- method is highly robust and that it is feasible to expand TIL to thera- ferred exogenous-cytokine-independent viability and homeostatic peutically relevant numbers from as little as 1 to 2 core biopsies from proliferation of modified T-cells, without causing autonomous T-cell multiple histologies with this method. Resulting products were shown growth. Furthermore, AUOTO6NG T-cells and product B but not to be phenotypically comparable to, and as potent as, products gener- product A proved resistant to both TGFb- and PD1/PDL1-mediated ated with Iovance’s process from excisional biopsy. Iovance anticipates immunosuppression in vitro due to the presence of dnTGFbRII and implementing this process in the clinic in the near future. dSHP2 in those genetically engineered CAR T-cells. Finally, intraven- ous delivery of AUTO6NG exhibited potent anti-tumour activity and References extended survival in NSG mice with established tumour burden. 1. Jazaeri AA, Zsiros E, Amaria RN, Artz AS, Edwards RP, Robert Michael Conclusions Wenham RM, et al. Safety and efficacy of adoptive cell transfer using These results demonstrate the feasibility, safety, and efficacy of autologous tumor infiltrating lymphocytes (LN-145) for treatment of AUTO6NG T-cells. The addition of IL7R_CCR, dnTGFbRII and dSHP2 recurrent, metastatic, or persistent cervical carcinoma. Clin Oncol. modules to the AUTO6NG product augment its functions by extend- 2019;37:15:2538 (suppl). ing T-cell persistence and rendering modified T-cells resistant to 2. Sarnaik A, Khushalani NI, Chesney JA, Kluger HM, Curti BD, et al. Safety TGFb- and PD1/PDL1-driven immune inhibition. and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients P147 who progressed on multiple prior therapies including anti-PD-1. J Clin Effect of chemotherapy on cellular kinetics of NKG2D-based CAR T- Oncol. 2019;37:15:2518 (suppl). cells in metastatic colorectal cancer patients 3. Ullenhag GJ, Sadeghi AM, Carlsson B, Ahlström H, Mosavi F, Wagenius G, Erik Marcelo Alcantar Orozco, Eytan Breman, MSc, Marie-Sophie Dheur, Tötterman TH, et al. Adoptive T-cell therapy for malignant melanoma pa- PhD, Fabian Borghese, PhD, Emilie Cerf, PhD, Nathalie Braun, Caroline tients with TILs obtained by ultrasound-guided needle biopsy. Cancer Lonez, PhD, Anne Flament, Frederic Lehmann, MD Immunol Immunother. 2012;61:725–732. Celyad, Mont-Saint-Guibert, Belgium Correspondence: Frederic Lehmann (flehmann@celyad.com) P146 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P147 AUTO6NG: Next generation GD2-targeting CAR T-cell therapy with improved persistence and insensitivity to TGFb and checkpoint Background inhibition for relapsed/refractory neuroblastoma Autologous and allogeneic Chimeric Antigen Receptor (CAR) T-cells are 1 1 1 Daniela Achkova, PhD , Adrian Zarzoso , Yusuf Demir , Fernando under thorough investigation to translate their success in B-cell malig- 1 1 1 1 Gallardo , Maria Stavrou , Marco Della Peruta , Saket Srivastava , Mathew nancies to other types of cancer. Previous studies associated the anti- 1 1 1 1 Robson , Shimobi Onuoha , Simon Thomas , Shaun Cordoba , Martin tumour effect of CAR T-cells to their long-term persistence. Most stud- 1,2 Pule ies use cyclophosphamide and fludarabine (CyFlu) preconditioning 1 2 Autolus Ltd, London, United Kingdom; University College London chemotherapy to facilitate CAR T-cell persistence. However, the effect Cancer Institute, London, United Kingdom of CyFlu preconditioning was rarely compared to other chemotherapies Correspondence: Martin Pule (m.pule@autolus.com) or to CAR T-cells alone. The THINK, SHRINK and ALLOSHRINK trials Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P146 evaluate the safety and clinical activity of NKG2D receptor-based CAR T-cells in metastatic colorectal cancer (mCRC) patients. THINK and Background SHRINK utilize autologous CAR T-cells, whereas ALLOSHRINK utilizes Neuroblastoma is the most common extracranial solid cancer in chil- allogeneic CAR T-cells. In THINK, CAR T-cells are injected without pre- dren with poor long-term survival in those with high-risk disease. A conditioning chemotherapy or after CyFlu. In SHRINK and ALLOSHRINK, currently ongoing phase I clinical study of GD2-targeted CART for re- FOLFOX chemotherapy is given before CAR T-cell injections. Herein we fractory/relapsed neuroblastoma (NCT02761915) shows activity present cellular kinetics results from these three trials. against disseminated disease without inducing on target/off tumor Methods toxicity. However, CART persistence was limited and clinical activity Whole blood samples were drawn at various timepoints from pa- transient and incomplete. tients receiving at least one injection of CAR T-cells. Peripheral blood Building on the GD2 CAR used in this study, we have developed a next mononuclear cells (PBMCs) were isolated by ficoll gradient centrifu- generation T-cell product candidate termed AUTO6NG. The AUTO6NG gation at a central laboratory designated by the Sponsor. Genomic product consists of 3 distinct populations of GD2-targeted CAR T-cells, DNA was isolated using a commercially available kit. Engraftment of produced by dual transduction of T-cells with two separate retroviral CAR T-cells was measured by digital droplet polymerase chain reac- vectors. The first vector directs the expression of a GD2-targeting CAR, tion (ddPCR) using transgene-specific primers and reported as trans- co-expressed with a constitutively signalling IL7 cytokine receptor gene copies per microgram of genomic DNA. Long-term persistence (IL7R_CCR) (product A), while the second vector is a tri-cistronic retro- of CAR T-cells was measured by calculating the area under the curve viral vector encoding the same GD2 CAR, co-expressed with dominant (AUC) using the linear trapezoidal rule. negative TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). Results dSHP2 confers resistance to inhibitory signals such as those from PD1. 35 mCRC patients have been treated in THINK (14), SHRINK (9) and Methods ALLOSHRINK (12). Preliminary results are available for 29 subjects. Cell Human T-cells were either dual transduced with both vectors yield- kinetics for subjects having received one injection of autologous CAR T- ing a mix of product A/B/A+B (AUTO6NG) or single transduced with cells show a seven-fold increase in mean peak levels of T-cell engraft- each vector individually giving raise to product A or B. Both single ment with CyFlu compared to FOLFOX. Mean AUC is four times higher and dual transduced CAR T-cells were extensively evaluated in vitro with CyFlu compared to FOLFOX. Peak levels of engraftment and per- for redirected lysis, cytokine secretion, T-cell proliferation and survival sistence observed with FOLFOX and without previous chemotherapy and resistance to immunosuppressive pathways (including TGFb and are similar. Additionally, allogeneic CAR T-cells exhibit a five-fold in- PD1/PDL1 inhibition) in co-culture assays with GD2-positive and crease in mean AUC and a ten-fold increase in mean peak levels com- negative tumour cell lines. Additionally, anti-tumour activity of pared to autologous cells with the same prior chemotherapy regimen. AUTO6NG was evaluated in vivo by intravenous administration in an Additional analyses will be presented during the congress. established neuroblastoma xenograft model in NSG mice. Conclusions Results Analyses of the initial 29 patients receiving either autologous or allo- AUTO6NG T-cells (product A/B/A+B) were highly potent in cytotox- geneic NKG2D-based CAR T-cells demonstrate that CyFlu enhances icity assays against GD2 positive tumour cell lines with no differences peak levels and persistence of adoptively transferred cells. FOLFOX Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 81 of 272 does not appear to influence engraftment or persistence of CAR T-cells. P149 Allogeneic CAR T-cells show higher peaks and time-averaged persist- Silencing PD-1 using self-delivering RNAi PH-762- to improve ence compared to autologous cells. Analysis of the results is ongoing. Iovance TIL effector function using Gen 2 manufacturing method 1 1 Ethics Approval Inbar Azoulay-Alfaguter, PhD , Michelle Abelson, PhD , Krit Ritthipichai, 1 1 1 2 The studies referred to in this abstract were approved by all relevant DVM, PhD , Kenneth D’Arigo , Florangel Hilton , Marcus Machin, BS , 2 2 1 1 ethical committees and authorities. Dingxue Yan , James Cardia , Maria Fardis, PhD, MBA , Cecile Chartier 1 2 Iovance Biotherapeutics, Inc., Tampa, FL, United States; Phio Pharmaceuticals, Tampa, FL, United States P148 Correspondence: Cecile Chartier (cecile.chartier@iovance.com) High affinity NK cells expressing a PD-L1 chimeric antigen receptor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P149 demonstrate anti-tumor activity in head and neck cancer through multiple distinct mechanisms Background 1 1 1 Yevtte Robbins , Jay Friedman, PhD , Sarah Greene , Kellsye Fabian, Adoptive T-cell transfer with tumor infiltrating lymphocytes (TIL) 1 1 2 2 PhD , Michelle Padget , John Lee, MD , Patrick Soon-Shiong, MD , is an investigational immunotherapy for advanced solid cancers. 3 2 2 1 Kayvan Niazi , Lennie Sender , Laurent Boissel , Jeffrey Schlom, PhD , Ongoing Phase II clinical trials of Iovance’s lifileucel and LN-145 1 1 James Hodge, PhD, MBA , Clint Allen, MD TIL products have demonstrated efficacy with ORRs of 38% and 1 2 NIH, Bethesda, MD, United States; NantKwest, Culver City, CA, United 44% in patients with melanoma and cervical cancer, respectively 3 4 States; Nantworks, Culver City, CA, United States; NIH/NIDCD, Bethesda, [1,2]. Anti-PD-1 therapy has been widely used as a first-line ther- MD, United States apy in several types of cancer. TIL infusion products from the pa- Correspondence: Clint Allen (clint.allen@nih.gov) tients previously treated with anti-PD-1 therapy still sustain PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P148 expression, especially the subset of tumor antigen-specific TIL [3]. Building on the therapeutic efficacy of PD-1 blockade, we rea- Background soned that intrinsic silencing of PD-1 in our TIL products, may A significant portion of head and neck cancers (HNCs) harbor gen- provide similar benefits to systemic administration of anti-PD-1 omic alterations that render them insensitive to T cell detection. For therapy, while decreasing the side effects associated with sys- these patients, natural killer (NK) cellular therapy may be an effective temic anti-PD-1 [3]. Self-delivering small interfering RNA (sd- complementary treatment approach. We studied the anti-tumor ac- rxRNA) is a chemically modified siRNA molecule, which has ability tivity of a novel, off the shelf, NK cellular therapy consisting of high to penetrate cell types with high knockdown efficiency of specific affinity NK cells engineered to express a chimeric antigen receptor target genes [4]. Furthermore, a knockdown approach yields a (CAR) targeting PD-L1 (PD-L1 t-haNKs). transient effect, which may prove a more favorable approach Methods when compared with permanent genetic modification. Here, we Irradiated (15 Gy) PD-L1 t-haNK cells were assessed for direct cytotox- tested the silencing efficiency of a PD-1-targeted sd-rxRNA, icity of five human and two murine HNC cell lines by real-time imped- termed PH-762, in TIL and its effect on TIL phenotype and ance analysis. PD-L1 knockout by CRISPR/Cas9 gene editing was function. performed in select cells to assess PD-L1-specific killing. Co-culture as- Methods says with PD-L1 t-haNKs and murine or human peripheral and tumor TIL from melanoma, breast cancer, lung cancer, H&N cancer, infiltrating leukocytes were performed to determine selective elimin- and sarcoma were expanded ex vivo with Iovance’s proprietary ation of cells. Wild-type C57BL/6 (B6) or NSG mice were engrafted with 22-day process in the presence of PH-762. Resulting TIL parental or PD-L1 knockout murine or human tumors and assessed for products were assessed for PD-1 knockdown, cell expansion tumor growth inhibition (TGI) following PD-L1 t-haNK treatment. and viability, phenotype (T-cell lineage, differentiation, activa- Results tion, and exhaustion), and effector functions (IFN-gamma PD-L1 CAR expression on PD-L1 t-haNKs was verified. PD-L1 t-haNKs induction). killed all human and murine HNC cell lines at low effector:target ra- Results tios. Killing of cells was significantly enhanced with increased PD-L1 Average silencing of the PD-1 levels was 85%. Sixteen of the 19 expression following IFN-γ pre-treatment. Baseline killing was par- tumors tested demonstrated >80% silencing at the surface of tially reversed and IFN-γ -enhanced killing was completely abrogated PH-762-treated TIL relative to control sd-rxRNA-treated TIL. The in PD-L1 knockout cells. Ex vivo co-culture of PD-L1 t-haNKs with per- remaining 3 samples had ~70% silencing efficiency. Expression ipheral and tumor infiltrating leukocytes from tumor bearing mice or of T-cell activation markers including 4-1BB and OX40 was sig- with peripheral leukocytes from HNC patients revealed selective elim- nificantly increased in TIL expanded with PH-762. Importantly, ination of PD-L1 high macrophages and myeloid derived suppressor other inhibitory and exhaustion molecules remained unaffected, cells (MDSC) but not lymphocyte subsets. Treatment of B6 mice bear- suggesting that compensatory mechanisms were not triggered ing murine oral cancers with PD-L1 t-haNKs in vivo resulted in ≥50% by PD-1 silencing. Functionally, PD-1 knockdown TIL displayed reduction in PD-L1 high macrophages and MDSC but no reduction in elevated IFN-gamma secretion when co-cultured with autolo- lymphocytes. Treatment of NSG mice bearing parental human HNC gous tumor cells, indicating improved effector function upon or B6 mice bearing parental murine oral cancer resulted in significant specific T-cell re-stimulation. TGI after PD-L1 t-haNK treatment. TGI was completely abrogated in Conclusions mice bearing PD-L1 knockout tumors. sd-rxRNA-mediated silencing of PD-1 with PH-762 in TIL was Conclusions highly efficient and generated TIL products with elevated PD-L1 t-haNKs mediated potent PD-L1-specific cytotoxicity against effector function, providing a strong rationale for clinical HNC cells and selectively eliminate immunosuppressive macrophages testing. and MDSC expressing high levels of PD-L1 from the periphery and tumor microenvironment. PD-L1 t-haNK monotherapy resulted in PD- Acknowledgements L1-specific TGI in xenograft and syngeneic models. These data pro- PH-762 was kindly provided by Phio Pharmaceuticals. vide the pre-clinical rationale for the clinical study of PD-L1 t-haNKs in solid tumors. Evidence that PD-L1 t-haNKs selectively eliminate im- References munosuppressive macrophages and MDSC support the clinical study 1. Sarnaik A. et al. Safety and efficacy of cryopreserved autologous tumor of PD-L1 t-haNKs as a monotherapy or in combination with treat- infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic ments designed to activate T cell immunity. melanoma patients who progressed on multiple prior therapies Ethics Approval including anti-PD-1. J Clin Oncol. 2019;37:2518-2518. The study was approved by the NIH Animal care and Use Committee, 2. Jazaeri A A, et al. Safety and efficacy of adoptive cell transfer using approval number 1464-18. autologous tumor infiltrating lymphocytes (LN-145) for treatment of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 82 of 272 recurrent, metastatic, or persistent cervical carcinoma. J ClinOncol. 2019;37:2538-2538. 3. Gros A, et al. PD-1 identifies the patient-specific CD8(+) tumor- reactive repertoire infiltrating human tumors. J Clin Invest. 2014;124:2246-2259. 4. Ligtenberg M A, et al. Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malig- nant Melanoma. Mol Ther. 2018;26:1482-1493. P150 1st-in-human CAR T clinical trial for metastatic breast cancers Cynthia Bamdad, PhD , Andrew Stewart, PhD, Pengyu Huang, PhD, Benoit Smagghe, PhD, Scott Moe, PhD, Tyler Swanson, Thomas Jeon, Danica Page, Ketan Mathavan, PhD, Trevor Grant, PhD, Rachel Herrup Minerva Biotechnologies, Waltham, MA, United States Correspondence: Cynthia Bamdad (cbamdad@minervabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P150 Background Minerva will open a 1st-in-human CAR T clinical trial for meta- static breast cancers at the Fred Hutchinson Center September, 2019. huMNC2-CAR44 targets a novel form of MUC1; no thera- peutic that targets this form has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 Fig. 1 (abstract P150). huMNC2-CAR44 T cells kill MUC1* have targeted the tandem repeat domains, which are cleaved positive tumors and shed from the surface of cancer cells. Cleavage and shed- ding of the tandem repeat domain increases as tumor stage in- creases. huMNC2-CAR44 targets the truncated extra cellular P151 domain of MUC1* (muk 1 star), also known as MUC1-C, which is Solid tumor cytotoxicity by natural killer cells expressing a HER2- the transmembrane cleavage product that remains after MUC1 directed chimeric antigen receptor enhanced by MyD88/CD40 (MC) is cleaved and the tandem repeat domain is shed from the can- Xiaomei Wang, PhD, Daniel Jasinski, PhD, Jan Medina, David Spencer, cer cells. The MNC2 antibody, which is the targeting head of PhD, Aaron Foster, PhD, Joseph Bayle, PhD the CAR, cannot bind to full-length MUC1. It binds to an ectopic Bellicum Pharmaceuticals, Houston, TX, United States epitope that is only unmasked by cleavage and release of the Correspondence: Aaron Foster (afoster@bellicum.com); Joseph Bayle MUC1 tandem repeat domain. MUC1* growth factor receptor is (jhbayle@bellicum.com) activated when onco-embryonicgrowth factorNME7ABdimer- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P151 izes its truncated extracellular domain. NME7AB and the huMNC2 antibody both competefor thesamebinding site, Background which is masked in full-length MUC1. The potent, innate anti-tumor cytotoxicity of natural killer (NK) cells Methods combined with their low risk of inducing graft-versus-host disease Monoclonal antibody MNC2 was selected because it recognizes have made NK cells an emerging platform for allogeneic, off-the- a conformational epitope within MUC1* that is created by cleav- shelf CAR-based cell therapies. However, adoptive transfers of NK age by MMP9, which is overexpressed in breast cancers and is cells have shown limited expansion and persistence which may im- an indicatorofpoorprognosis. The luminal edge of some nor- pact their ability to induce durable anti-tumor responses. Here, we mal tissues express a cleaved MUC1*-like form; however, on demonstrate that constitutive expression of a novel chimeric costi- normal tissues, MUC1 is cleaved by a different cleavage enzyme, mulatory protein, comprised of the signaling domains from MyD88 which alters the conformation of the truncated extra cellular and CD40 (MC) and secreted IL-15 dramatically improves the prolifer- domain and it is not recognized by the MNC2 antibody. ation and anti-tumor efficacy of HER2 CAR-redirected NK cells. Results Methods huMNC2-scFv recognizes 95% of breast cancers, across all subtypes, Human CD56-positive cells were enriched from PBMCs derived from wherein the average percent staining for each tissue specimen is healthy donors and activated with irradiated K562 cells in the presence ~80%. Despite this robust staining of cancerous tissues, huMNC2- of IL-15. NK cells were subsequently transduced with retroviral vector en- scFv showed almost no binding to normal tissues and no staining of coding inducible Caspase-9 (iC9), a HER2-specific CAR (HER2.ζ), MyD88/ critical organs. In vitro, huMNC2-CAR44 T cells killed cancer cells, but CD40 (MC) [1] and IL-15. Gene-modified NK cells were evaluated for ex- not non-cancer cells even if they expressed MUC1 or a cleaved pansion, cytotoxicity, cell phenotype and cytokine production, in vitro MUC1. In NSG mice (n>300), huMNC2-CAR44 T cells eliminated and in an HER2 positive OE-19 NSG mouse xenograft model. MUC1* positive tumors from implanted naturally occurring breast Results cancer cells. A single CAR T cell injection eliminated tumors for 100 NK cells were efficiently transduced (>50%) and demonstrated robust days; control animals had to be sacrificed at Day 20. Further, ex vivo expansion (150 fold, 14 days post-activation) in culture rela- huMNC2-CAR44 T cell mediated killing increased as MUC1* density tive to transduced NK cells. In coculture assays with HER2-expressing increased (Figure 1). OE19 and SKOV3 tumor cells, MC-enhanced CAR-NK cells showed po- Conclusions tent cytotoxicity with elevated expression of pro-inflammatory cyto- If successful, huMNC2-CAR44 could treat a wide variety of solid kines and chemokines including MIP1α, IFN-γ, and GM-CSF. In tumors. huMNC2-scFv binds to 95% of breast, 83% ovarian, 78% addition, NK cells expressing the iC9 safety switch could be rapidly pancreatic and 71% of lung cancers. ablated by treatment with 1 nM rimiducid to initiate apoptosis. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 83 of 272 animals engrafted with OE-19 tumor cells, iC9-CAR.ζ-MC-IL15 modi- Conclusions fied NK cells demonstrated significantly improved control of tumor Through the direct cytosolic delivery of antigen, we engineered expansion compared with control NK cells. unfractionated PBMCs to function as potent APCs. This strategy has Conclusions demonstrated significant potential to generate CD8+ T cell responses MyD88/CD40 and IL-15 enhance the proliferation and anti-tumor po- in both mouse and human systems and has been scaled for clinical tency of CAR-modified NK cells. Further, inclusion of the iC9 safety implementation. switch can be used to mitigate potential toxicities. These technolo- Ethics Approval gies have the potential to provide a potent, off-the-shelf allogeneic Human samples were supplied by an approved vendor and animal cell therapy to treat solid tumors. studies were conducted in accordance with SQZ Biotech's Animal Care Program and IACUC which operate according to principles set Reference forth in PHS Policy and the Guide for the Care and Use of Laboratory 1. Collinson-Pautz MR, Chang WC, Lu A, Khalil M, Crisostomo JW, Lin PY, Animals - 8th edition. Mahendravada A, Shinners NP, Brandt ME, Zhang M, Duong M, Bayle JH, Slawin KM, Spencer DM, Foster AE. Constitutively active MyD88CD40 costimulation enhances expansion and efficacy of chimeric antigen re- P153 ceptor T cells targeting hematological malignancies. Leukemia. 2019; Memory CD8+ T cells are more resistant to cancer stem cell (CSC) 33:2195-2207. suppression than effector CD8+ T cells and are more effective at Ethics Approval targeting CSC in a murine melanoma model This study was approved by Bellicum's IACUC and performed in its AAALAC 1 2 2 2 Brooke Bredbeck, MD , Shibin Qu , Alicia Kevelin , Ashley Pepple , Amy approved vivarium. 1 2 1 Felsted , Anutosh Ganguly , Clifford Cho, MD, FACS University of Michigan Medical School, Ann Arbor, MI, United States; P152 Ann Arbor VA Medical Center, Ann Arbor, MI, United States Antigen delivery to PBMCs by microfluidic squeezing primes anti- Correspondence: Clifford Cho (cliffcho@med.umich.edu) tumor immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P153 Matthew Booty, PhD, Kelan Hlavaty, Emrah Ozay, PhD, Carolyne Smith, PhD, Katherine Seidl, PhD, Howard Bernstein, MD, PhD, Armon Sharei, Background Scott Loughhead The ability to suppress immune reactivity is a defining hallmark SQZ Biotechnologies, Watertown, MA, United States of cancer [1-3]. Both the administration and disinhibition of Correspondence: Matthew Booty (matt.booty@sqzbiotech.com) CD8+ T cells, through adoptive immunotherapy and checkpoint Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P152 inhibition respectively, have yielded unprecedented responses in patients with advanced melanoma [4-8]. However, a majority Background of patients remain stubbornly unresponsive to T cell-based The presentation of sufficient antigen on major histocompatibility therapy [9,10]. A better knowledge of cancer-induced T cell sup- complex class I (MHC-I) is a potential barrier to generating potent pression is needed improve efficacy. Memory CD8+ T cells cancer immunizations. We use microfluidics-based squeezing to de- (Tmem) are more effective than effector CD8+ T cells (Teff) at liver antigen directly to the cytosol of target antigen presenting cells controlling melanoma growth after adoptive cell transfer (ACT) (APCs) – resulting in the enhanced presentation of antigen on MHC-I. in a murine melanoma model [11,12]. Melanoma cancer stem In addition to facilitating potent CD8+ T cell priming by professional cells (CSC) are primarily responsible for tumor growth and APCs, this approach can make unfractionated peripheral blood metastasis [13,14]. We hypothesized that Tmem are both more mononuclear cells (PBMCs) effective, unorthodox APCs capable of resistant to CSC suppression and more effective at targeting priming CD8+ T cell responses in mouse and human systems. CSC after ACT. Methods Methods Protein and peptide antigens were delivered to the cytosol of murine The B16F10 melanoma cell line was stably transfected to ex- splenocytes or human PBMCs by microfluidic squeezing. The re- press low levels of lymphocytic choriomeningitisvirus(LCMV) sponse to in vivo immunization was assessed by flow cytometry in a peptide antigen GP33 (B16GP33). Ly5.1+/C57BL/6 mice were in- series of experiments in mice. Tumor experiments were conducted fected with LCMV to isolate Teff and Tmem on post-infection with the TC-1 cell line, which expresses the viral antigens E6 and E7 days 8 or > 30, respectively. Ly5.2+/C57BL/6 mice were inocu- from human papilloma virus type 16 (HPV16). lated with subcutaneous B16GP33 tumors followed by either no Human PBMCs were loaded with synthetic long peptides (SLPs) con- treatment or ACT with Teff or Tmem on days 1 or 7. On day taining MHC-I restricted epitopes from cytomegalovirus (CMV) or 18-20, tumors were harvested for flow cytometric analysis HPV16. These PBMCs were co-cultured with epitope-reactive human (FACS) to characterize tumor-infiltrating lymphocytes (TIL) and responder CD8+ T cells, and interferon gamma production was quan- composition of melanoma CSC versus non-CSC (NCSC) based on tified to assess antigen-specific responses in vitro. expression of the CSC-specific marker aldehyde dehydrogenase Results (ALDH). In mice, we demonstrate that microfluidic squeezing enables delivery to Results all cell subsets within the spleen and that delivered protein antigen is Tumor inhibition was observed after ACT, with greatest treatment rapidly processed and presented on MHC-I. In vivo immunization using effect found after Tmem ACT (Figure 1). FACS analysis of CD8+ splenocytes squeezed with a HPV16-derived E7 SLP primes E7-specific re- TIL showed a predominant exhausted and non-activated pheno- sponses. Prophylactic immunization of mice implanted with TC-1 resulted type after Teff ACT; in contrast, CD8+ TIL exhibited a highly acti- in complete protection and these responses were durable, as mice were vated phenotype as well as superior endogenous CD8+ T cell protected upon TC-1 re-challenge. Therapeutic immunization following recruitment (Figure 2) after Tmem ACT. FACS analysis of tumor TC-1 implantation reduced tumor growth and extended survival com- cells after ACT demonstrated that ALDHhigh CSC fractions were pared to unimmunized mice (25 days vs 50 days). Following therapeutic markedly expanded after Teff ACT, but diminished after Tmem immunization, 85% of tumor infiltrating CD8+ T cells were found to be ACT (Figure 3). E7-specific compared to 3% in unimmunized mice. Conclusions In human cells, we demonstrate that squeezing of primary PBMCs en- Tmem-based ACT resulted in optimal tumor growth suppression, a ables delivery to all cell subsets. Delivery of CMV and HPV16 SLPs leads more activated TIL phenotype with superior CD8+ T cell recruitment, to presentation on MHC-I, as demonstrated by in vitro responses of and substantially stronger clearance of CSC compared to Teff ACT both CD8+ T cell clones and patient-derived memory populations. De- and controls. These observations suggest that use of Tmem may en- livery of CMV antigens at the manufacturing scale (~1 x 10^9 cells) also able cellular therapies to more effectively evade the suppressive ef- results in presentation and activation of CD8+ T cells. fects of melanoma while selectively targeting CSC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 84 of 272 References 1. Marincola F, Wang E, Herlyn M, et al. Tumors as elusive targets of T-cell- based active immunotherapy. Trends Immunol. 2003; 24:335-342. 2. Dunn GP, Old LJ, et al. The three E’s of cancer immunoediting. Ann Rev Immunol. 2004; 22:329-360. 3. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Ann Rev Immunol. 2007; 25:267-295. 4. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell trans- fer immunotherapy. Clin Cancer Res. 2011; 17:4550-4557. 5. Dudley MS, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy folloing non-myeloablative but lymphodepleting chemotehrapy for the treatemnt of patients with refractory metastatic melanoma. J Clin Oncol. 2005; 23:2346-2357. 6. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711-723. 7. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimu- mab or monotherapy in untreated melanoma. N Engl J Med. 2015; 373:23-34. 8. Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013; 369:122-33. 9. Brahmer JR, Tykodi SS, Chow LQM, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366:2455-2465. 10. Royal RE, Levy C, Turner K, et al. Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J Immunother. 2010; 33:828-33. Fig. 2 (abstract P153). CD8+ TIL exhibit a more activated 11. Contreras A, Sen S, Tatar AJ, et al. Enhanced local and systemic anti- phenotype after memory ACT melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice. Cancer Immunol Immunother. 2016; 65:601-611. 12. Contreras A, Beems MV, Tatar AJ, et al. Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive melanoma im- munotherapy in a mouse model. J Immunother Cancer. 2018; 6:41. 13. Maccalli C, DeMaria R. Cancer stem cells: perspectives for therapeutic targeting. Cancer Immunol Immunother. 2015; 64:91-97. 14. Pan Q, Li Q, Liu S, Ning N, Zhang X, Yu Y, Chang AE, Wicha MS. Concise review: targeting cancer stem cells using immunological approaches. Stem Cells. 2015; 33:2085-2092. Ethics Approval This study was approved by University of Michigan’s ethics board (IACUC), approval #1608-004. Fig. 3 (abstract P153). Memory CD8+ T cells target melanoma CSC P154 Development of an antigen-presenting bead kit for activation and expansion of human antigen-specific T cells Yelena Bronevetsky, PhD (yelena.bronevetsky@gmail.com) Berkeley Lights Inc, Alameda, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P154 Background Immunogenicity validation of peptide neoantigens represents a crit- ical bottleneck in the tumor antigen discovery process. Current bio- informatics platforms for antigen prediction are unsatisfactory, forcing researchers to screen many peptides per protein target in order to identify the few bona fide antigens. Standard immunogen- icity assays also cannot discriminate antigen-Human Leukocyte Anti- Fig. 1 (abstract P153). Memory CD8+ T cells vs effector CD8+ T gen (HLA) binding from T cell receptor (TCR) recognition, leading to cells in melanoma unnecessary screening of non-HLA binders in expensive and lengthy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 85 of 272 T cell reactivity assays that require large numbers of expensive pri- products were stimulated ex vivo with a T cell-specific superantigen. mary cells. Further, the use of donor-derived antigen-presenting cells Dogs received ACT of ex vivo-activated T cells followed by five sub- to assay T cell immunogenicity and expand rare, antigen-specific cutaneous IL-2 injections. Dogs were monitored for development of cells from the endogenous repertoire has inherent variability and a metastases via thoracic radiographs every three months. minimal degree of quality control. Berkeley Lights has developed an Results artificial antigen-presenting bead kit that expands antigen-specific T All 14 patients received autologous vaccinations. Due to early metas- cells from peripheral blood. tasis, 11 dogs received ACT. One dog did not receive adjuvant IL-2; Methods ten dogs completed the entire protocol. Toxicity was minimal after Peptide binding to HLA Class I and stability of the peptide-HLA com- pre-medicants (NSAID, antihistamine, and antiemetic) were instituted plex is assayed by loading peptides onto beads and staining with prior to ACT. With premedication, all toxicities were VCOG grade I/II. antibody. Following validation of peptide-HLA binding, primary CD8+ Median disease-free interval for all dogs was 213 days. Median sur- T cells from peripheral blood are stimulated by antigen-presenting vival time (MST) for all dogs was 415 days. Five dogs have survived beads twice over the course of two weeks. Frequencies of antigen- for over 621 days and are disease-free (Figure 1.) In addition, the re- specific T cells in the resulting cells is assayed by tetramer staining. sults included at least one dog with complete regression of distant Antigen-specific T cells can be loaded onto the Berkeley Lights (BLI) macroscopic metastasis. Lightning platform, a novel microfluidic platform that enables thou- Conclusions sands of single cell experiments in parallel. On the BLI Optoselect This immunotherapy protocol is safe and tolerable. Compared to chip, IFNg secretion and CD137 upregulation of antigen-specific T MST for historical amputation alone with or without adjuvant chemo- cells is assayed in response to antigenic stimulation. Following ana- therapy (MST of 307(1) and 134(2) days, respectively), a significant lysis, single cells can be exported for further analysis. survival benefit is noted in this group of patients. Further prospective Results studies are warranted to gain additional immunologic insight to the Berkeley Lights has developed an artificial antigen-presenting bead protocol, further improve disease response and survival, and evaluate that expands antigen-specific T cells from peripheral blood 10 times the translational impact this treatment could have in advancing hu- more effectively than autologous dendritic cells. This system allows man medicine. users to load peptides of choice onto magnetic beads and use them to assay peptide-HLA binding and stability, and to efficiently stimu- References late and expand antigen-specific T cells. Finally, in conjunction with 1. Phillips B, Powers BE, Dernell WS, Straw RC, Khanna C, Hogge GS, Vail the Berkeley Lights Lightning platform and the T cell Phenotype and DM. Use of single-agent carboplatin as adjuvant or neoadjuvant therapy Functional Analytics workflow, multiple functional parameters can be in conjunction with amputation for appendicular osteosarcoma in dogs. assayed from as few as 1000s of T cells, linking peptide-HLA binding J Am Anim Hosp Assoc. 2009; 45:33-8. and recognition to antigen-specific effector function. 2. Spodnick GJ, Berg J, Rand WM, Schelling SH, Couto G, Harvey HJ, Henderson RA, MacEwen G, Mauldin N, McCaw DL, Moore AS, Morrison W, Norris AM, O’Bradovich, J, O’Keefe DA, Page R, Ruslander D, Klausner J, Straw R, Thompson JP, Withrow SJ. Prognosis for dogs with appendicular P155 osteosarcoma treated by amputation alone: 162 cases (1978-1988). J Am Prospective translational study evaluating vaccine-enhanced Vet Med Assoc. 1992; 200(7):995-9. adoptive T cell therapy for treatment of osteosarcoma in Ethics Approval companion dogs The study was approved by University of Missouri’s IACUC, approval number 1 1 Tammie Wahaus, BSBA , Noe Reyes, DVM , Jeffrey Bryan, DVM, MS, PhD, 2 2 DACVIM-Oncology , Jeffrey Bryan, DVM, MS, PhD, DACVIM-Oncology , 3 2 Gary Wood, PhD , Brian Flesner, DVM, MS, DACVIM-Oncology , Lindsay 2 2 Donnelly, DVM, MS, DACVIM-Oncology , Debbie Tate, RVT, VTS 1 2 ELIAS Animal Health, Olathe, KS, United States; University of Missouri, Columbia, MO, United States; TVAX Biomedical, Olathe, KS, United States Correspondence: Tammie Wahaus (twahaus@eliasah.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P155 Background Canine osteosarcoma (OSA) is an aggressively metastatic primary bone malignancy with a 90% mortality rate. Many naturally occurring canine cancers are genetically and biologically similar to their human counterparts. All cancers express neoantigens and therefore are po- tentially susceptible to vaccine-enhanced adoptive T cell therapy. Syngeneic rodent studies demonstrated that metastases could be permanently eliminated with vaccine-enhanced adoptive T cell ther- apy. Canine OSA studies provide an excellent translational bridge be- tween experimental metastatic rodent cancer studies and metastatic human cancer clinical trials. We hypothesized that dogs with OSA could be safely treated at diagnosis with surgery, autologous cancer cell/P. acnes vaccination, adoptive T cell transfer (ACT) of ex vivo-acti- vated T cells, and low dose human interleukin-2 (IL-2) resulting in im- proved survival compared to carboplatin. We further hypothesized that significant efficacy would be achieved by treating dogs with in- tact immune systems and minimal residual disease [1,2]. Methods 14 client-owned cancer bearing dogs were enrolled in a one-arm prospective trial. Dogs were staged with bloodwork, limb and thor- acic radiographs, histopathology, and bone scans prior to amputation to remove the primary bone tumor. Autologous cancer cell/P. acnes Fig. 1 (abstract P155). Survival Analysis of Dogs vaccinations were administered intradermally weekly for three weeks. Completing Protocol Dogs underwent leukapheresis. Mononuclear white blood cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 86 of 272 P156 2. Hodge G, Barnawi J, Jurisevic C, Moffat D, Holmes M, Reynolds PN, Lung cancer sub-types exhibit differential susceptibility to natural Jersmann H, Hodge S. Lung cancer is associated with decreased killer cell cytotoxicity expression of perforin, granzyme B and interferon(IFN)-γ by infiltrating Jason Cahoon, BS, Shilan Dong, MS, Rafet Amoor, Donna Sonntag, MS, lung tissue T cells, natural killer (NK) T-like and NK cells. Clin Exp Immu- Alexander Spurrell, Rachit Ohri, PhD nol. 2014; 178:79–85. Enable Life Sciences, Worcester, MA, United States Correspondence: Rachit Ohri (rachit@enablelifesciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P156 Background Natural Killer (NK) cells hold great promise in immunotherapy, particularly for lung cancer [1]. However, there is a paucity of literature which organizes the susceptibility of various lung can- cer subtypes to NK cells. We evaluated the cytotoxicity (necrosis and apoptosis) of the NK cell line KHYG-1 (Effector) against cell- lines of 3 different subtypes of lung cancer i.e. H1975, H1703, A549 (Target). We also determined the levels of biomarkers rele- vant to NK cell activation and function [2] i.e. the cell-surface biomarker CD107a and 5 soluble biomarkers [Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha]. Methods Three lung cancer cell lines (H1975, H1703, A549) (ATCC, Vir- ginia) were plated at 100% confluency in 96-well plates (1.25 cells/cm^2, 3.2*10^5 cells/mL), while K562 cells (ATCC, Virginia), used as a positive control, were suspended in the plate wells at 3.2*10^5 cells/mL. KHYG-1 cells (JCRB, Japan) were added at a density of 6.4*10^6 cells/mL, at a 20:1 Effector:Target (E:T) ra- tio. Cells incubated for 5 hours at 5% CO2, 37 °C. Subsequently: [i] Cytotoxicity (necrosis and apoptosis) in target cells were quantified using flow cytometry with PerCP-Cy™5.5 Annexin V and Propidium Iodide (PI). [ii] CD107a expression on KHYG-1 cells was evaluated using Fig. 1 (abstract P156). Necrosis flow cytometry with PE-labeled anti-human CD107a Ab. [iii] Expression levels of 5 soluble biomarkers (Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha) were determined in the cell supernatants using Luminex. Results The cytotoxicity data suggests that for necrosis, all target cell- lines were significantly different (all p values < 0.0001) from each other in terms of susceptibility to the effector cells (A549 > H1703 > H1975 > K562) (Figure 1). K562 cells are significantly higher in late apoptosis than all three lung cancer cell lines (Figure 2). Amongst the 3 lung cancer cell-lines, the H1703 cell line is significantly higher in late apoptosis than H1975 and A549 cells (p-value < 0.05) (Figure 3). Although differences were seen in the necrotic and late apoptotic profiles of target cells, the CD107a expression on the KHYG-1 effector cells was similar across all co-cultures (Figure 4). The soluble biomarker data (Luminex) is being collected. Conclusions In conclusion, cell-lines corresponding to different lung cancer subtypes, i.e. A549 (Carcinoma), H1703 (Squamous Cell) and H1975 (Adenocarcinoma) exhibit significant differences in both their necrosis and late apoptosis susceptibility when co-cultured with NK cells. Such insight could be used to better guide NK cell based immunotherapy development. References 1. Aktaş O, Öztürk A, Erman B, Erus S, Tanju S, Dilege S. Role of Natural Killer Fig. 2 (abstract P156). Late Apoptosis Cells in Lung Cancer. J Clin Cancer Res Clin Oncol. 2018; 144:997-1003. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 87 of 272 Background Adoptive cell transfer (ACT) of tumor-targeted T cells has demonstrated encouraging clinical efficacy in some hematological cancers. However, in solid tumors, targeting a single antigen (e.g., CAR-T and TCR therap- ies) can lead to antigen escape and development of resistance. Further- more, although support provided by lymphodepletion or cytokine administration can enhance responses to ACT, these systemic treat- ments are often associated with significant toxicities. Torque’sSlipstream™ T cell manufacturing platform is a high-efficiency process for generating Deep-Primed™ T cells: polyclonal non- genetically engineered T cells that (1) are targeted against multiple tumor-specific antigens and (2) carry immunomodulating cytokine pay- loads to provide prolonged and locally directed immune support with- out systemic toxicities. The Slipstream™ process is designed to resolve the manufacturing challenge of generating high yields of early memory phenotype tumor-reactive T cells, which are associated with clinical benefit. Here, we show that the Slipstream™ process drives robust ex- pansion while preserving favorable memory characteristics of natural tumor-reactive T cells, and we demonstrate that Deep-Priming™ Tcells with Deep IL-15 or Deep IL-12 improves function. Methods Multi-targeted T cells (MTC) were comparatively generated from do- nors via either a first-generation process or the new Slipstream™ process that leverages ex vivo expansion conditions optimized for MTC production. T cell reactivity against tumor-associated antigens, memory, polyfunctionality, cytotoxicity, and response to Deep IL-15 Fig. 3 (abstract P156). Late Apoptosis minus K562 and Deep IL-12 were measured. The modularity of Slipstream™ was tested by training MTC against antigen cassettes including cancer or viral antigens and measuring reactivity against antigen subsets. Results Compared to a first-generation process, MTC generated with Slip- stream™ exhibited >20-fold improvement in antigen-specific reactiv- ity and a substantial improvement in the yield of memory-phenotype antigen-specific T cells including a 10-fold increase in Tcf1-positive cells. Furthermore, the Slipstream™ process yielded MTC with in- creased polyfunctionality and specificity as measured by cytokine production and TCR sequencing, respectively. Notably, T cells ex- panded using the Slipstream™ process showed potent cytotoxicity against human cancer cells as well as responsiveness to Deep IL-15 and Deep IL-12. The Slipstream™ process can also be adapted for simultaneous training of MTC against different antigens including virus-associated tumor antigens. Conclusions The Slipstream™ process is optimized to produce Deep-Primed™ MTC with substantive increases in characteristics associated with clinical efficacy: antigen reactivity, memory phenotype, and polyfunctionality. Modularity of the Slipstream™ process has been demonstrated by simultaneously training T cell clones reactive to cancer and virus- associated antigens, and Deep-Primed™ MTC with cell-associated Deep IL-15 or Deep IL-12 drives enhanced T cell function in vitro. P158 Suboptimal er stress induced autophagy regulates anti-tumor T cell response Fig. 4 (abstract P156). CD107a Expression Shilpak Chatterjee, Danh Tran, Kim Dosung, Satish Nadig, Carl Atkinson, Hongjun Wang, J. Alan Dieh, Shikhar Mehrotra, PhD, Paramita Chakraborty, PhD Medical University of South Carolina, Charleston, SC, United States P157 Correspondence: Shikhar Mehrotra (mehrotr@musc.edu) Optimized process for manufacturing Deep-Primed™ Tcells Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P158 creates product with improved functional characteristics and reactivity against multiple tumor-associated antigens Background Shawn Carey, PhD, Christine McInnis, PhD, Alicia Worthylake, Angela Endoplasmic reticulum (ER) stress induced by external or internal Forte, Elisabeth Brown, Darren Smith, Kate Sackton, PhD, Rosemary stimuli activates a number of well-orchestrated cellular signaling pro- Soucy, Tap Maniar, MD, Karsten Sauer, PhD, Thomas Andresen, PhD, cesses aimed to promote either cell apoptosis or to restore cellular Andy Rakestraw, PhD function and resolve the stress. In tumor microenvironment, induc- Torque Therapeutics, Cambridge, MA, United States tion of ER stress is known to dampen the antitumor activity of T cells Correspondence: Thomas Andresen (tandresen@torquetx.com) by reducing their mitochondrial function. However, if magnitude of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P157 ER stress governs the T cell fate and function is unknown. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 88 of 272 Methods Results We performed our study on B16 murine melanoma model and used Of the 12 peptides with high predicted score, we confirmed 7 (in- standard immunological techniques like flow cytometry, immunoblot cluding NY-ESO-1 antigen SLLMWITQC) strongly activate human pri- analysis, microscopy, real time PCR mary NY-ESO C259-expressing T cells. These off-target peptides Results include peptides with up to 7 amino acid changes (of 9 possible), Using melanoma antigen gp100 reactive T cells, we found that low which could not be predicted using the recognition motif as deter- level of ER stress enhances T cell stemness and promotes mitochondrial mined by alanine scans. biogenesis, whereas high level of ER stress triggers T cell death. More- Conclusions over, upon adoptive transfer, T cells treated with low dose ER stress in- Thus, this replacement scan assay determines the “TCR fingerprint” ducer are able to form long-lived memory in vivo, express reduced and, when coupled with the algorithm applied to the database of level of co-inhibitory molecule, and demonstrate superior anti-tumor human 9-mer peptides binding to HLA-A*02:01, enables identifica- immunity by increasing overall survival of B16 murine melanoma bear- tion of potential off-target antigens and the tissues where they are ing mouse. Mechanistically, we discovered that, upon ER insult at sub- expressed. This platform enables both screening of multiple TCRs to optimal level, a protective autophagy pathway is induced to promote identify the best candidate for clinical development and identifica- cell survival and maintain stemness through the protein kinase R-like tion of TCR-specific cross-reactive peptide recognition and consti- endoplasmic reticulum kinase (PERK)/ activating transcription factor-4 tutes an improved methodology for the identification of potential (ATF4)-dependent manner. Conversely, knockdown of PERK abrogates off-target peptides presented on MHC class I molecules. We used this autophagy activation, hampers mitochondrial biogenesis in response to platform and demonstrate screening of multiple TCRs targeting suboptimal ER stress, which in-turn compromises the antitumor func- tumor antigens. tion of melanoma antigen specific T cells. Furthermore, we demon- strated that blocking autophagy in T cells hampers T cell anti-tumor P160 activity. Lastly, T cells which initiates autophagic process due to sub- Engineered natural killer cells redirected against adenosinergic optimal ER stress show better potential to control tumor compared to immunometabolic suppression for the immunotherapy of lung those, that do not enter into the process carcinoma Conclusions Andrea Chambers, MS, Kyle Lupo, BS, Jiao Wang, PhD, Sandro Matosevic, Overall, these preclinical data highlights that, low level of ER stress PhD response is important for healthy cellular function and therapeutic- Purdue University, Lafayette, IN, United States ally, ER stress pathways can be manipulated in T cells in order to Correspondence: Sandro Matosevic (sandro@purdue.edu) regulate their antitumor potential. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P160 Ethics Approval The study was approved by Medical University of South Carolina‘s Background Ethics Board, approval number 2018-00628. NK cells are powerful effectors in cancer immunotherapy and have po- tential to treat various cancers; however significant challenges remain P159 in the treatment of solid tumors. Energy availability is compromised TCR fingerprinting and off-target peptide identification surrounding solid tumors and NK cell metabolic reprogramming can Armen Karapetyan, Chawaree Chaipan, Katharina Winkelbach, Sandra occur to inhibit NK effector functions [1]. Accumulation of adenosine in Wimberger, Jun Seop Jeong, Bishnu Joshi, Robert Stein, MD PhD, Dennis the tumor microenvironment (TME) from the activity of ectoenzymes Underwood, PhD, Eleni Chantzoura, PhD, Alvaro Yague, Jan Bergmann, CD39 and CD73 on cancer cells is one mechanism that leads to im- John Castle, PhD, Marc Van Dijk, PhD, Volker Seibert paired NK cell function. Our previously published data has established Agenus Inc, Lexington, MA, United States that the effects of TME adenosine on NK cells cause specific Correspondence: John Castle (john.castle@agenusbio.com); Marc Van reorganization of the cells’ metabolism and effector signatures to sup- Dijk (marc.vandijk@agentustherapeutics.com) press NK cell function, and the cytokine combination of IL-12/15 was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P159 hyperresponsive to adenosine [2]. One way to combat immunosuppres- sion induced by cancer-produced adenosine is to engineer NK cells to Background overcome this inhibition. To that end, we engineered NK cells to dir- Adoptive T cell therapy using patient T cells redirected to recognize ectly target CD73 by imparting NK-specific signaling to enhance anti- tumor-specific antigens by expressing genetically engineered high- tumor activity against CD73+ lung carcinoma. affinity T-cell receptors (TCRs) has therapeutic potential for melanoma Methods and other solid tumors. Clinical trials implementing genetically modified Peripheral blood-derived NK cells were isolated from healthy human TCRs in melanoma patients have raised concerns regarding off-target tox- donors and expanded using feeder cells. NK cells were electroporated icities resulting in lethal destruction of healthy tissue, highlighting the ur- using mRNA or transduced with lentivirus expressing the CD73- gency of assessing which off-target peptides can be recognized by a TCR. targeting construct which bears signaling domains derived from Methods FcγRIIIa. Engineered NK cells expressing the construct were tested for As a model system we used the clinically efficacious NY-ESO-1-spe- their killing ability against lung carcinoma A549 cells. The engineered cific TCR C259, which recognizes the peptide epitope SLLMWITQC NK cells were then adoptively transferred into a CD73+ lung cancer presented by HLA-A*02:01. We investigated which amino acids at xenograft into NSG mice. Circulating CD73-CAR NK cells were quanti- each position enable a TCR interaction by sequentially replacing fied for their expression of activating markers NKG2D, DNAM, and every amino acid position outside of anchor positions 2 and 9 with NKp30 and visualized using immunohistochemistry to determine infil- all 19 possible alternative amino acids, resulting in 134 peptides (133 tration into tumors, and mice were assessed for tumor growth. altered peptides plus epitope peptide). Each peptide was individually Results evaluated using three different in vitro assays: binding of the NY-ESO We showed NK cells can be efficiently redirected against CD73 to C259 TCR to the peptide, peptide-dependent activation of TCR- block the generation of immunosuppressive adenosine and rescue expressing cells, and killing of peptide-presenting target cells. To impaired NK cell anti-tumor immunity. Specifically, primary human represent the TCR recognition kernel, we defined Position Weight NK cells were successfully engineered to express the synthetic CD73- Matrices (PWMs) for each assay by assigning normalized measure- FCyRIIIa construct. Retargeted NK cells showed enhanced anti-tumor ments to each of the 20 amino acids in each position. To predict functions in vitro against CD73-expressing A549 cells. Engineered pri- potential off-target peptides, we applied a novel algorithm project- mary NK cells also showed promise in stunting CD73+ lung cancer ing the PWM-defined kernel into the human proteome, scoring NY- tumor growth for up to 3 weeks in vivo. Current and future studies ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 include evaluation of off target effects and local injection to further binding 9-mer peptides. evaluate infiltration of the NK cells in vivo. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 89 of 272 Conclusions Background The microenvironment of solid tumors is highly immunosuppressive Adoptive cancer antigen-specific T cell therapy currently comprised and adenosine has been shown to impair NK cell anti-tumor immun- of chimeric antigen receptor (CAR-) and T cell receptor (TCR) engi- ity. A novel anti-CD73 targeting construct using NK cell signaling neered T cells. Clinical results from CAR-T cells have demonstrated components has been developed and shown to prevent tumor promising results in treating leukemia, while TCR-engineered T cells growth of CD73+ lung carcinoma. which have the advantage of recognizing intracellular tumor anti- gens is still in very early development. References Methods 1. Chambers A *, Lupo K*, Matosevic S. Tumor-microenvironment-induced Here, we report the development of two CD4+ or CD8+ TCRαβ-KO immunometabolic reprogramming of natural killer cells. Front Immunol. reporter T cell lines for the screening and characterization of trans- 2018; 9:2517. genic TCRs. A TCRαβ-KO reporter T cell line was first developed by 2. Chambers A, Wang J, Lupo K, and Matosevic S. Adenosinergic knocking out the endogenous TCR α and β chains in the reporter T signaling alters natural killer cell functional responses. Front Immunol, cell line using CRISPR/Cas9 and the successful knockout is confirmed 2018; 9:2533. by phenotypic assays and TCR v chain locus sequencing. Ethics Approval Results The study was approved by Purdue University Institution's Review Board, We demonstrated that re-introduction of HA peptide-specific HA1.7 approval number 1804020540. TCR α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Furthermore, P161 the select expression of CD4 or CD8 variants in the TCRαβ-KO re- High-efficiency CAR-T cell manufacturing by improved scalable porter T cell line could enable the development of TCRs for both electroporation MHCI- and MHCII-restricted tumor antigen targets. Jian Chen, PhD, George Sun Conclusions Celetrix LLC, Manassas, VA, United States The CD4+ and CD8+ TCRαβ-deficient reporter T cells can serve as Correspondence: Jian Chen (jchen@celetrix.com) valuable tools for screening and characterization of neoantigen- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P161 specific TCRs Background CAR-T cells are currently manufactured for clinical use by infection of P163 human T cells with viral vectors containing the CAR gene. The Effect of common gamma-chain cytokines on myeloid-derived current viral CAR-T manufacturing process is lengthy and costly and suppressor cell and M2 macrophage suppressive function: electroporation has emerged as a promising alternative. However, Implications for cellular immunotherapy 1 2 2 2 clinical use of electroporation technology in CAR-T has been difficult Anna Cole, BA , Charlotte Rivas , Josue Pineda , Corrine Baumgartner , 2 2 and several clinical trials have met significant problems due to the Stephanie Fetzko , Robin Parihar 1 2 low transfection efficiency and/or high cell mortality. Rice University, Houston, TX, United States; Baylor College of Medicine, Methods Houston, TX, United States Our novel understanding of the electroporation mechanism revealed that Correspondence: Robin Parihar (rxpariha@texaschildrens.org) the current widely-used electroporation methods have significant mis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P163 takes in the physical design as well as electroporation buffer design. The first problem is the electroporation sample container design. It is well Background known that electrochemical reaction generates gas bubbles that are Immunotherapy using antigen-redirected lymphocytes such as harmful to the cells and there was no good solution to the problem. Here chimeric antigen receptor (CAR)-T or -NK cells in patients with solid we used a novel pressurization approach to largely eliminate the effect. tumors has shown poor efficacy. Cell therapies are hindered by im- Results munosuppressive cells such as inhibitory macrophages (M2s) and Combined with other improvements including electroporation myeloid-derived suppressor cells (MDSCs) that contribute to a highly buffer design and post-electroporation cell culture strategy, we suppressive tumor microenvironment (TME) [1]. Researchers have have been able to achieve over 80% plasmid transfection effi- armed redirected lymphocytes with the ability to secrete cytokines in ciency in unstimulated T cells and over 90% plasmid transfection hopes of promoting their proliferation and function in suppressive efficiency in stimulated T cells. The viability in survived cells is TMEs [2,3]. However, the effect of these cytokines on other immune over 95% measured by live/dead staining and the true survival cells within the TME, such as MDSCs and M2s, is unknown. rate measured by survived cell number is over 66%. The new Methods electroporation method can achieve over 90% in gene editing To determine how the human common gamma-chain cytokines, and the method is also widely applicable in electroporation of interleukin(IL)-2, IL-7, IL-15, and IL-21 affect human MDSCs and M2s, NK cells, DC cells and monocytes. we exposed ex vivo enriched M2s and MDSCs to each cytokine sep- Conclusions arately and assessed changes in MDSC and M2 phenotype and ability The new method is also scalable as billions of cells can be processed to dampen T-cell activation and proliferation. To further define in the large volume electroporation setting. Our method can poten- cytokine-induced changes in MDSC/M2 function in a more clinically tially eliminate the need for expensive cell expansion and virus pro- relevant system, we tested the ability of cytokine-exposed MDSCs/ duction altogether, therefore cutting the huge economic burden of M2s to impair CAR-T cell proliferation and anti-tumor activity in a CAR-T therapy. TME co-culture. As a clinical correlate, we assessed common gamma- chain cytokine receptor expression on MDSCs and M2s within neuro- blastoma and sarcoma patient tumors and tested the effects of cyto- P162 kine exposure on their suppressive capacity. Development of CD4+ and CD8+ TCRαβ-deficient bioluminescent Results reporter T cells for screening and characterization of neoantigen- Subsets of ex vivo enriched M2s and MDSCs expressed common specific TCRs gamma-chain cytokine receptors. MDSCs expressed receptors for IL-2 Zhi-jie Cheng, PhD, Jamison Grailer, PhD, Michael Slater, Pete Stecha, Jim (22%, avg. MFI=67, n=3), IL-7 (43%, avg. MFI=375, n=3), IL-15 (23%, Hartnett, Frank Fan, PhD, Mei Cong, PhD avg. MFI=310, n=3), and IL-21 (65%, avg. MFI=124, n=4); whereas Promega Corporation, Madison, WI, United States M2s expressed receptors for IL-2 (17%, avg. MFI=59), IL-7 (98%, avg. Correspondence: Zhi-jie Cheng (jey.cheng@promega.com) MFI=543), IL-15 (36%, avg. MFI=619), and IL-21 (91%, avg. MFI=296). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P162 Exposure of human MDSCs or M2s to IL-2, IL-7, IL-15, or IL-21 did not Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 90 of 272 alter their cell-surface phenotype. Exposure of these suppressive median survival of 24 days. Treatment with PM21-NK cells improved myeloid cells to IL-2, IL-7, and IL-15 did not change their ability to survival over untreated (p=0.0003) and PD-L1 alone (p=0.0002) suppress T-cell proliferation. In contrast, exposure of M2s and MDSCs groups having median survival of 40 days. Combination of PM21-NK to IL-21 increased their ability to suppress T-cell proliferation and ac- cells with anti-PD-L1 further improved of survival over the PM21-NK tivation (98% suppression by IL-21 exposed MDSCs vs. 72% suppres- cells alone group (48 days, p=0.042) with 25% of mice still remaining sion by control MDSCs; 98% suppression by IL-21 exposed M2 vs. in good health at day 58. 79% suppression by control M2 at a 2:1 T cell:MDSC/M2 ratio). Conclusions Conclusions These data support the use of anti-PD-L1 in NK cell therapy, regard- These results suggest that IL-21 increases the suppressive capacity of less of initial tumor PD-L1 status. PM21-NK cells can be used for human MDSCs and M2s. Ongoing experiments will define the mech- tumor treatment and to prime tumors to express PD-L1. The PD-L1 anisms by which IL-21 alters MDSC and M2 suppression and further induced upon NK cell treatment can serve as “universal targetable define the effect of IL-21 exposed MDSCs and M2s on tumor growth ligand” if used with humanized anti-PD-L1 antibodies to cause tumor and CAR-T cell therapeutic efficacy in vivo. killing by ADCC. References P165 1. Martinez M, Moon EK. CAR T cells for solid tumors: New strategies for Robust, reproducible and highly scalable manufacturing of P- finding, infiltrating, and surviving in the tumor microenvironment. Front. BCMA-ALLO1, an allogeneic CAR-T stem cell memory product for Immunol. 2019; 10:128. multiple myeloma, from numerous healthy donors 2. Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T Stacey Cranert, PhD, Maximilian Richter, PhD, Min Tong, MS, Leslie Weiss, cells enhance antitumor efficacy and overcome the tumor MS, Yening Tan, MS, Eric Ostertag, MD, PhD, Julia Coronella, PhD, Devon microenvironment. Sci Rep. 2017; 7:10541. Shedlock, PhD 3. Liu D, Song L, Wei J, Courtney AN, Gao X, Marinova E, Guo L, Heczey A, Poseida Therapeutics, San Diego, CA, United States Asgharzadeh S, Kim E, et al. IL-15 protects NKT cells from inhibition by Correspondence: Devon Shedlock (dshedlock@poseida.com) tumor-associated macrophages and enhances antimetastatic activity. J Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P165 Clin Invest. 2012; 122:2221-2233. Ethics Approval Background Tumor tissue use was approved by Baylor College of Medicine IRB study Autologous Chimeric Antigen Receptor (CAR) T cell therapy for re- protocol #26691, and samples were de-identified prior to laboratory lapsed/refractory Multiple Myeloma (MM), such as Poseida’s anti-B evaluation. cell maturation antigen (BCMA) product candidate, P-BCMA-101, have shown significant efficacy in the clinic. P-BCMA-101 is com- P164 prised of a high percentage of stem cell memory T cells (TSCM), Effect of NK cell treatment on PD-L1 expression and anti-PD-L1 resulting in a product that is much safer and potentially more dur- response able than other anti-BCMA autologous product candidates. However, Alicja Copik, PhD, Jeremiah Oyer, Sarah Gitto, Deborah Altomare, PhD individualized products have expensive and time-consuming manu- University of Central Florida, Orlando, FL, United States facturing and significant variability in input patient T cells characteris- Correspondence: Deborah Altomare (deborah.altomare@ucf.edu) tics. We are developing P-BCMA-ALLO1, an off-the-shelf anti-BCMA Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P164 allogeneic (allo) CAR-T product candidate manufactured from serial healthy donor material that circumvents many of the downsides of Background an individualized CAR-T product. PD-1 axis blockade therapies have shown success but responses are Methods limited to ~15% of cancer patients. These responses correlate with P-BCMA-ALLO1 is produced using two key platform technologies: presence of lymphocyte infiltrated, PD-L1 positive tumors. Strategies the nonviral piggyBac® (PB) DNA Modification System and the that increase PD-L1 expression may improve outcomes of PD-1 axis high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. blockade. PD-L1 on tumor cells is induced by IFNγ, secreted by NK The PB transposase mRNA and DNA encoding the PB-based cells. We developed a method for producing therapeutic quantities transgene are electroporated along with the components of the (>1,000 fold expansion within two weeks) of hyper-activated NK cells CC system needed to knockout (KO) the T Cell Receptor (TCR) with high anti-tumor cytotoxicity and enhanced IFNγ secretion. The and beta-2 microglobulin, thereby eliminating expression of Major utilizes particles from Plasma Membrane of K562 cells expressing Histocompatibility Complex (MHC) class I. The T cells are then ex- membrane bound IL21 (PM21-particles). Herein, the ability of PM21- panded using our proprietary "booster molecule.” The resulting particle expanded NK cells to induce PD-L1 expression on various tu- product demonstrates expression of the transgene in nearly all mors was tested in vitro and in vivo in ovarian cancer model. Fur- cells, and after a purification step, have eliminated all TCR expres- thermore, the effect of anti-PD-L1 on NK cell anti-tumor activity was sion and most MHC class I expression. tested in vitro and in vivo. Results Methods We have produced P-BCMA-ALLO1 at both research and near- NK cells were expanded with PM21-particles as described. For in vivo clinical scale from >35 donors with >97% manufacturing success. experiments, NSG mice were implanted with 1x10^6 SKOV-3 cells Efficiencies of TCR-KO ranged from ~50-90%, with final product i.p.. Mice were treated with 10^7 PM21-NK cells (n=6) or with vehicle always demonstrating >99% TCR-KO. T cell expansion varied from control (n=6) on days 8 and 13. Mice were sacrificed on day 20 to ~0.5-20 fold. At clinical production scale, this translates to up to collect tumors. Tumors were perfused and retrieved tumor cells were 250 doses of CAR-T per manufacturing run at a dose of 150x10e6 analyzed for PD-L1 expression while infiltrating immune cells were cells/patient. P-BCMA-ALLO1 demonstrated a high-percentage of phenotyped. TSCM cells (CD45RA+CD62L+CD45RO-). Furthermore, P-BCMA- Results ALLO1 generated from multiple donors demonstrated potent effi- PM21-NK treatment induced PD-L1 on >30% of tumor cells across cacy in the RPMI-8226 xenograft model in NSG mice, thus estab- multiple cell lines. PM21-NK cells are negative for PD-1 and addition lishing the feasibility of using serial individual donors in our of anti-PD-L1 had no effect on their cytotoxicity or cytokine produc- manufacturing process. tion. In in vivo experiment, PM21-NK cell treated mice had increased Conclusions PD-L1+ tumors vs. the untreated group (29.7% vs 14.5%, p<0.0001). In summary, these data demonstrate a robust, reproducible and Despite T-cell depletion, T-cells made up ~22% of hCD45+ events in highly scalable manufacturing process. Moreover, this production perfused tumors, 83% of which were Tregs. PM21-NK cells are PD-1-, process can be expanded for use with additional targets for treat- but are inhibited by Tregs. Untreated and anti-PD-L1 alone mice had ment of other heme or solid tumors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 91 of 272 P166 P167 ET140202 T-cell therapy for the treatment of liver cancer is built Improved efficacy leveraging CAR-T therapeutics that produce upon a novel antibody-T cell receptor (AbTCR) ARTEMIS™ T-cell tightly controlled, tumor proximal, immunomodulatory outputs platform Michon Pinnix, Krista McNally, Jay Danao, BS, Melissa Fardy, Rachel Jun Cui, PhD, Pengbo Zhang, Hongruo Yun, Yiyang Xu, Lucas Horan, Hovde, MS, Charlotte Davis, Nicole Grant, Dianna Lester-Zeiner, David PhD, Shaohua Xu, Sean Xu, Hong Liu Mai, Ben Wang, PhD, Gus Zeiner, PhD Eureka Therapeutics, Inc., Emeryville, CA, United States Chimera Bioengineering, Emeryville, CA, United States Correspondence: Hong Liu (hong.liu@eurekainc.com) Correspondence: Gus ZeinerGus Zeiner (gus@chimera.bio) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P166 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P167 Background Background The use of engineered T cells for the treatment of solid cancers remains Current chimeric antigen receptor (CAR) T cell therapies are clinically challenging. Recently, we developed a novel antibody-T cell receptor efficacious against several B cell malignancies, but are less effective (AbTCR) ARTEMIS™ T-cell platform, which combines antibody-based target at eliminating solid tumors. A key contributor to this observed lack recognition with gamma/delta TCR-based cellular activation [1]. In contrast of efficacy is the tumor microenvironment (TME) that is erected by to chimeric antigen receptors (CARs), the AbTCR forms a natural multi- solid tumors to impose immunosuppressive physical and chemical meric receptor with the endogenous CD3 complex, which feeds into a barriers to T cell function and survival. To break TME-driven immuno- network of signaling pathways that regulate T-cell activation. In addition, suppression, attempts have been made to arm CAR-T cells with the the inclusion of gamma/delta TCR chains within the AbTCR avoids the for- ability to produce immunomodulatory payloads that target features mation of mispaired receptors with unknown cross-reactivity, which is a of the TME. By their nature, these immunomodulators (e.g. Interleu- potential risk associated with current alpha/beta TCR-based therapies. kin 12) are frequently toxic when systemically delivered. Due to a Using the core design of the AbTCR ARTEMIS™ T-cell platform, we de- limited repertoire of existing programmable gene regulators, consti- veloped ET140202 for the treatment of hepatocellular carcinoma (HCC). tutive expression and systemic distribution of immunomodulators by ET140202 features an AbTCR targeting alpha-fetoprotein (AFP)peptide/ armed CAR-T cells is common. MHC complexes (specifically AFP158-166/HLA-A2) expressed on HCC Chimera Bioengineering has characterized a novel, post-transcriptional cancer cells. To optimize T-cell activation and expansion, the AbTCR is gene regulatory node that strictly governs effector outputs in human T co-expressed with a CD28-based co-stimulatory molecule engineered cells. This gene regulatory node, termed Gold, has been used to create to target Glypican 3 (GPC3) expressed on HCC cancer cells. enhanced CAR-T therapeutics that produce tightly controlled immuno- Methods modulatory outputs only upon tumor engagement. To test the specificity and potency of ET140202 T cells in vitro, Methods ET140202 T cells were co-incubated with either target-positive or Methods include: design and characterization of a proprietary bicis- target-negative cells. Lactate Dehydrogenase release was used to tronic GoldCAR lentiviral vector to simultaneously deliver both CAR quantify target cell lysis. CFSE assay was used to measure cell prolif- and IL12 transgenes; production of lentivirus and infection of primary eration. Expression of differentiation and exhaustion markers were human T cells; in vitro characterization of GoldCAR-T cell function determined by flow cytometry. The in vivo anti-tumor activity of utilizing cell based assays, immunoassays and flow cytometry; im- ET140202 T cells was tested in an AFP+/HLA-A2+ Hep G2 liver cancer plantation of subcutaneous xenograft tumors in NSG mice with xenograft model. We also engineered the same anti-AFP158-166/ tumor growth monitored by caliper and bioluminescent imaging HLA-A2 binding moiety onto a CD28-based CAR (AFP-CAR) and com- (BLI) to assess in vivo efficacy; and ex vivo analysis of blood, tumor pared AFP-CAR-T cells to ET140202 T cells in various assays. and lymphatic organs to characterize safety profile. Results Results ET140202 T cells specifically lysed AFP-positive tumor cells. Com- CD19 targeted GoldCAR-T cells delivering IL12 demonstrate im- pared to AFP-CAR-T cells, ET140202 T cells displayed enhanced proved efficacy over standard CD19 CAR-T cells in a subcutaneous in vitro cell killing and proliferation even after repetitive antigen Daudi B cell xenograft mouse model. These GoldCAR-T cells exhibit stimulations. ET140202 T cells also display a less exhausted surface comparable efficacy to CAR-T cells with constitutive IL12 expression, phenotype (e.g. lower PD-1 expression) and a higher percentage of but levels of pro-inflammatory cytokines in the peripheral blood are central memory T cells (CCR7+ CD45RA-) after antigen stimulation. In lower in the mice treated with GoldCAR-T cells. vivo, both intravenous and intratumoral single administration of Conclusions ET140202 T cells led to significant tumor growth inhibition. GoldCAR-T cells with tumor proximal IL12 delivery demonstrate en- Conclusions hanced efficacy over standard CAR-T cells and an improved safety ET140202 is built upon our novel AbTCR ARTEMIS™ T-cell platform, profile compared to CAR-T cells with constitutive IL12 expression. which was designed to harness the natural biology of T cells to fight The implications of this study point to an amplified CAR-T cell re- cancer. Both in vitro cellular assays and in vivo mouse studies sup- sponse in an immunosuppressive tumor microenvironment. port the safety and efficacy of ET140202 T cells. Whether these pre- Trial Registration clinical findings for AbTCR-based ET140202 T-cell therapy translate Not applicable into the clinical setting is currently being tested (clinicaltrial.gov, Ethics Approval NCT0399803). The animal study was conducted under the Institutional Animal Care and Use Committee (IACUC) of LumiGenics, LLC, 750 Alfred Nobel Reference Drive, Suite 103, Hercules CA 94547 1. Xu Y, Yang Z, Horan LH, Zhang P, Liu L, Zimdahl B, Green S, Lu J, Morales JF, Barrett DM et al. A novel antibody-TCR (AbTCR) platform combines P168 Fab-based antigen recognition with gamma/delta-TCR signaling to facili- Role and function of T-cell immunoglobulin– and mucin domain– tate T-cell cytotoxicity with low cytokine release. Cell Discovery 2018, containing (TIM)–3 receptor on natural killer cells in solid tumors 4(1):62. Tram Dao, Sandro Matosevic, PhD Ethics Approval Purdue University College of Pharmacy, West Lafayette, IN, United States All animal experiments were conducted according to protocols approved Correspondence: Sandro Matosevic (smatosev@purdue.edu) by their Institutional Animal Care and Use Committee (IACUC) and in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P168 accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Background Washington, DC, 1996) and the Policy on Humane Care and Use of Natural killer (NK) cells are part of the innate immune system, but are Laboratory Animals (Department of Health and Human Services, capable of participating in both innate and adaptive immune Bethesda, MD). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 92 of 272 responses due to their wide range of cytolytic activities, from de- P169 granulation, secretion of cytokines to antibody-dependent cell- Enriching non-genetically modified antigen specific marrow mediated cytotoxicity. These are possible due to the cells’ ability to infiltrating lymphocytes (MILs) to target HPV+ oropharyngeal recognize self and non-self-entities via the net signal generated from squamous cell carcinoma 1 1 2 their activating and inhibitory receptors upon engagement. One such Danielle Dillard , Vanessa Chan , Lakshmi Rudraraju, MS , Elizabeth 2 1 1 1 receptor is TIM-3, which is expressed on various lymphocytes. In T- DeOliveira , Amy Thomas , Ervin Griffin , Megan Heimann , Luca Biavati, 1 1 1 cells, TIM-3 is an exhaustion marker [1], but on NK cells, results are MD , Elizabeth Zawidzka , Marguerrita El Asmar , Drew Pardoll, MD, 1 1 3 1 conflicting in regards to its function as the receptor exhibits both ac- PhD , Kellie Smith, PhD , Carole Fakhry, MD , Ivan Borrello, MD tivating and inhibitory effects depending on disease type and activa- Johns Hopkins School of Medicine, Baltimore, MD, United States; 2 3 tion status [2-6]. WindMIL therapeutics, Baltimore, MD, United States; Otolaryngology, Methods Head and Neck Surgery, Baltimore, MD, United States NK cells were isolated from peripheral blood of healthy donors. Correspondence: Danielle Dillard (danielle.dillard@jhmi.edu) After expansion, they were co-cultured for 4 hours with glioblast- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P169 oma (U87) at effector:target (E:T) ratios of 2.5:1 and 10:1, and various receptors were screened by flow cytometry, including PD- Background 1, NKG2A, LAG-3, CD158b, CEAMCAM-1 and TIM-3. Then, expres- Human papillomavirus positive (HPV+) oropharyngeal squamous cell sion of TIM-3 was measured when in the presence of patient- carcinoma (HPV-OPSCC) accounts for ~80% of OPSCCs in the United derived primary glioblastoma cells (GBM43) and prostate cancer States. Although HPV-positivity confers improved survival relative to (PC3) for 4 hours. To determine the effect of TIM-3 expression, HPV(-) OPSCC, outcomes for metastatic HPV-OPSCC remain dismal. killing assay are being carried out by blocking TIM-3 on NK cells. High tumor infiltrating lymphocytes (TILs) are associated with better Further investigation is being performed by blocking one of TIM- outcomes in HPV-OPSCC and have the promise of a therapeutic role 3’s primary ligands, Galectin-9, on cancer cells to determine its based upon their intrinsic enhanced tumor specificity. However, not impact on NK cell cytotoxicity. Statistical analyses are completed all tumors possess TILs. To date, therapeutic expansions involve sur- in SAS JMP Pro 14. gical excision of the tumor, expansion of TILs with high-dose IL-2 for Results up to 4 weeks and clinical product success rate between 40-60%. We found that TIM-3 is significantly downregulated on primary hu- Marrow infiltrating lymphocytes (MILs) represent a novel and distinct man NK cells, in both frequency and surface density, when exposed T cell population obtained from the bone marrow (BM) of patients to solid tumor cells such as U87, GBM43 and PC3 at multiple E:T ra- that possess significant tumor-specificity over peripheral blood lym- tios. Unlike other inhibitory NK receptors, this downregulation was phocytes (PBLs). Although the early work was done in myeloma, the unique to TIM-3. However, it is not known why the downregulation unique nature of the BM microenvironment makes it a reservoir of occurs with solid tumors, and whether this change in expression af- antigen-experienced memory T cells in numerous solid tumors. As fects NK killing capacity. Here, we report the role of TIM-3 on NK cell such, we sought to determine whether HPV-specific MILs could be cytotoxicity against solid tumor cell lines and the role of Galectin-9 in identified and expanded ex vivo. mediating NK cell activity. Methods Conclusions We obtained bone marrow and peripheral blood from patients with We found that Tim-3 was significantly downregulated on NK cells in localized, HPV-OPSCC. A modified version of the MANAFEST assay response to solid tumor cells. Understanding the complex roles of was used to evaluate proliferation of peripheral and bone marrow- Tim-3 expression on NK cells allows us to better understand the nu- derived CD8+ T cells in response to HPV early 6 (E6) and early 7 (E7) anced immunomodulatory role of Tim-3 on NK cell anti-tumor re- peptides (HPVFEST) in a HPV-OPSCC patient. sponses, and provide a basis for the development of Results immunotherapies targeting impaired NK cell function in solid tumors T cell receptor sequencing and bioinformatic analysis of each peptide- stimulated culture revealed a markedly increased frequency of HPV- References specific T cells in MILs compared to peripheral blood. HPV-specific MILs 1. Sánchez-Fueyo A, Tian J, Picarella D, Domenig C, Zheng XX, Sabatos CA, showed a higher average TCR clone frequency relative to PBLs (p value Manlongat N, Bender O, Kamradt T, Kuchroo VK, Gutiérrez-Ramos JC, = 0.0037). Additionally, of the 5 highest expanded TCR clones for both Coyle AJ, Strom TB. Tim-3 inhibits T helper type 1-mediated auto- and compartments HPV-specific MILs possessed an increased average rep- alloimmune responses and promotes immunological tolerance. Nat resentative clone frequency (p value = 0.0001). To investigate general Immunol. 2003; 4:1093-101. responsiveness to shared tumor antigens, we incubated autologous BM 2. Ndhlovu LC, Lopez-Vergès S, Barbour JD, Jones RB, Jha AR, Long BR, with lysate from an HPV+ OPSCC and then added ex-vivo activated Schoeffler EC, Fujita T, Nixon DF, Lanier LL. Tim-3 marks human natural MILs and PBLs. Tumor specificity was defined as IFN훾 production in killer cell maturation and suppresses cell-mediated cytotoxicity. Blood. CD3 cells. MILs possessed an average of 50.73% IFN훾 production as 2012; 119:3734-43. compared to 0.11% in PBLs to HPV-OPSCC lysate. 3. Jost S, Moreno-Nieves UY, Garcia-Beltran WF, Rands K, Reardon J, Toth I, Conclusions Piechocka-Trocha A, Altfeld M, Addo MM. Dysregulated Tim-3 expression Collectively, these data indicate that HPV-specific T cells exist in the on natural killer cells is associated with increased Galectin-9 levels in HIV- BM of patients with localized disease and possess a greater clonoyt- 1 infection. Retrovirology. 2013; 10:74. pic frequency and functional tumor recognition compared to PBLs. 4. Gleason MK1, Lenvik TR, McCullar V, Felices M, O'Brien MS, Cooley SA, These data provide the rationale for developing this novel adoptive T Verneris MR, Cichocki F, Holman CJ, Panoskaltsis-Mortari A, Niki T, Hira- cell approach using MILs in this patient population. shima M, Blazar BR, Miller JS. Tim-3 is an inducible human natural killer Ethics Approval cell receptor that enhances interferon gamma production in response to This study was approved by Johns Hopkins University Institution Re- galectin-9. Blood. 2012; 119:3064-72. view Board, approval number IRB00128334 and NA_00028682. 5. Ju Y, Hou N, Meng J, Wang X, Zhang X, Zhao D, Liu Y, Zhu F, Zhang L, Sun W, Liang X, Gao L, Ma C. T cell immunoglobulin- and mucin- P170 domain-containing molecule-3 (Tim-3) mediates natural killer cell sup- Sequential anti-CD19, anti-CD22, and anti-CD20 autologous pression in chronic hepatitis B. J Heptatol. 2010; 52:322-9. chimeric antigen receptor T cell (CAR-T) therapies treating a child 6. da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo with relapsed refractory Burkitt lymphoma VK, Osman I, Bhardwaj N. Reversal of NK-cell exhaustion in advanced mel- Juan Du, MD, Yonghong Zhang anoma by Tim-3 blockade. Cancer Immunol Res. 2014; 2:410-22. Beijing Boren Hospital, Beijing, China Ethics Approval Correspondence: Yonghong Zhang (yhzhang58@126.com) This study was approved by Purdue Intuition’s Ethics Board, approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P170 number 1804020540. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 93 of 272 Background Results Currently, the prognosis of children with relapsed refractory(r/r) Burkitt lym- Using this method, we have retrieved multiple TCRs that confer specific phoma(BL) remains dismal . New therapies are exlored to achieve a higher recognition of a public neoepitope derived from a PIK3CA hotspot mu- remission rate such as immunotherapy for these patients .We have success- tation (H1047L). These TCRs are restricted by HLA-A*03:01, an allele fully treated a case by adopting sequential autologous chimeric antigen re- present in 20.5% of the North American population. Immune- ceptor T cell(CAR-T) therapies, targeting antigen CD19,CD22,and CD20. precipitation/tandem mass spectrometry analysis determined that the Methods endogenously processed and presented public neoepitope is a 9 amino An 8-year-old boy was studied,who presented with a mass on the acid sequence containing a His to Leu substitution at position 2. To right side of the neck and was diagnosed with BL by pathology. The understand the mechanistic basis for the immunogenicity of this public child was treated with standard chemotherapy but suffered from re- neoepitope, we generated x-ray crystallography structures of mutant lapse. Subsequently, anti-CD19, anti-CD22, and anti-CD20 autologous and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These stud- CAR-T cell treatments were sequentially administered. We observed ies revealed significant topologic overlap in the bound peptides. By the clinical manifestations and response to the three cycles of CAR-T contrast, the thermal and kinetic stability of the mutant peptide/HLA- treatments, values of peripheral CAR-T cells were also monitored and A*03:01 complex was significantly enhanced relative to the WT com- side effects were assessed. plex, as measured by differential scanning fluorimetry and fluorescence Results anisotropy assays. Peripheral blood T cells genetically engineered with The patient displayed no response to anti-CD19-CART treatment PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells .After CD-22 directed CART the patient got partial remission (PR),but in a HLA/mutation-specific manner, leaving HLA-mismatched or WT tar- relapse occurred quickly. Finally, after the use of anti-CD20 CAR-T cell get cells unperturbed. therapy, the child achieved complete remission (CR) and has cur- Conclusions rently achieved a 6-month event-free survival (EFS). During the CD19 These findings reveal for the first time the existence of an endogen- and CD20 CAR-T cell treatments, only mild cytokine release syn- ously processed and presented public neoantigen derived from a drome (CRS) were observed in the patient (grade 1) while he devel- PIK3CA hotspot mutation. These results open the possibility of target- oped a grade 3 CRS during CD22 CAR-T therapy, the symptoms ing this common driver oncogene using adoptively transferred and included fever and hypoxemia. genetically redirected T cells. Conclusions Ethics Approval Autologous CAR-T cell therapies targeting multplei tumor antigens The study was approved by Smita Chandran's Institution‘s Ethics could be novel and safe treatments for children with r/r BL. Board, approval number IRB 17-250. Consent Written informed consent was obtained from the patient for publica- P172 tion of this abstract and any accompanying images. A copy of the Shape and material properties increase artificial antigen written consent is available for review by the Editor of this journal. presenting cell effectiveness Savannah Est Witte, BS, Kaitlyn Calebrisi, Jordan Green, PhD P171 Johns Hopkins University, Baltimore, MD, United States T cell receptor gene therapy for a public neoantigen derived from Correspondence: Jordan Green (green@jhu.edu) mutated PIK3CA, a dominant driver oncogene in breast and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P172 endometrial cancers 2 1 3 3 Jiaqi Ma , Martin Klatt, PhD , Friederike Dundar, PhD , Paul Zumbo , Background 1 3 1 Matthew Femia , Doron Betel, PhD , David Scheinberg , Brian Baker, Particulate delivery of artificial antigen presenting cells (aAPCs) is a promis- 2 1 1 PhD , Christopher Klebanoff, MD , Smita Chandran, PhD ing cell-free strategy to initiate selective T cell stimulation for immunother- 1 2 MSKCC, New York, NY, United States; University of Notre Dame, Notre apy in vivo. While 2D aAPC strategies aim to optimize T cell proliferation Dame, IN, United States; Weill Cornell Medicine, New York, NY, United States and selection in vitro for subsequent cell therapy, it would be advanta- Correspondence: Christopher Klebanoff (klebanoc@mskcc.org); Smita geous to deliver “off-the-shelf” biodegradable aAPCs directly as an in vivo Chandran (chandrs1@mskcc.org) therapeutic. 3D aAPC effectiveness has been limited due to inefficiencies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P171 in stimulating T cells as well as rapid clearance of delivered particles. To improve bioavailability as well as increase particulate aAPC effectiveness, Background we have developed a soft, biodegradable, microparticle aAPC (Figure 1). “Public” neoantigens represent immunogenic epitopes encompassing To create a new platform technology for immunoengineering, material hotspot mutations in driver oncogenes that are also restricted by and shape are investigated as parameters for improving T cell stimulation. common HLA alleles. In contrast with patient-specific “private” Methods neoantigens, public neoantigens are conceptually attractive because One micron size particles were synthesized using a poly(ethylene gly- they are tumor-specific, clonally conserved, and shared across pa- col) diacrylate (PEGDA) or using a poly(lactic-co-glycolic acid) emulsion tients. Whether PIK3CA, the most common driver oncogene in breast method [1,2]. Anti-CD3 and anti-CD28 conjugated particles were incu- and endometrial cancer, can yield public neoepitopes that may be bated with primary mouse T cells and proliferation was quantified at 3 exploited for cancer immunotherapy is unknown. and 7 days. For macrophage uptake studies, particles were incubated Methods with macrophages at 37 °C to analyze particle uptake and 4 °C to evalu- We have developed a high-throughput, single-cell functional assay for ate binding of particles to cells. To synthesize soft, ellipsoidal aAPCs, a the discovery and retrieval of TCR alpha/beta gene sequences that con- novel thin-film stretching technique was developed where emulsified fer specific recognition of endogenously processed and presented pub- PEGDA droplets were frozen then cast into films and stretched [3]. lic neoantigens and not the corresponding wild type (WT) sequence. In Results this approach, donor-derived T cells are sensitized with autologous Protein conjugation efficiency and T cell proliferation were 10-fold antigen presenting cells (APCs) electroporated with RNA encoding higher for PEGDA particles than PLGA particles (Figure 2a-b). Uptake PIK3CA hotspot mutations. Expanded T cells are subsequently divided studies indicate a ~20-fold decrease in binding and uptake by the into paired daughter wells for short-term co-culture with APCs electro- PEGDA particles (Figure 2c). Ellipsoidal aAPCs stimulate T cells 3 porated with minigenes containing either mutant or WT PIK3CA se- times more effectively than spherical particles (Figure 3b,d). Uptake quences. Acutely re-stimulated T cells from paired wells are subject to studies indicate a ~10-fold decrease in nonspecific uptake of ellips- single-cell alpha/beta TCR VDJ and RNA sequencing. TCR alpha/beta oidal particles (Figure 3c). gene sequences associated with selective upregulation of TCR signaling Conclusions transcripts to mutant but not WT PIK3CA stimulation are subsequently Particle material and shape are significant factors in designing partic- cloned into retroviral vectors to confirm reactivity. ulates as biomimetic aAPCs for in vitro T cell stimulation. Ongoing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 94 of 272 work on further decoupling these parameters and optimizing in vivo efficacy has the potential to unleash a promising biomimetic plat- form technology for immunoengineering of T cells. References 1. Anselmo AC, Mitragotri S. Impact of Particle Elasticity on Particle-Based Drug Delivery Systems. Adv. Drug Deliv. Rev. 2017; 108:51–67. 2. Meyer RA, Sunshine JC. Biodegradable Nanoellipsoidal Artificial Antigen Presenting Cells for T Cell Activation. Small. 2016; 11:1519–1525. 3. Meyer RA, Meyer RS, Green JJ. An Automated Multidimensional Thin Film Stretching Device for the Generation of Anisotropic Polymeric Micro- and Nanoparticles. J. Biomed. Mater. Res. A. 2015; 103:2747–2757. Fig. 3 (abstract P172). See text for description P173 Case reports: Correlates of response following adoptive transfer of ADP-A2M4, affinity-enhanced T-cells targeting MAGE-A4, in synovial sarcoma 1 1 1 Svetlana Fayngerts, PhD , Zohar Wolchinsky , Shravani Shitole , Joana 1 1 1 Senra , Rebecca Dryer-Minnerly, PhD , Ruoxi Wang , Jean-Marc Navenot, 1 1 1 1 PhD , Olga Ochkur , Gareth Betts, PhD , Natalie Bath, MSc , Erin Van 1 1 1 1 Winkle , Tom Holdich , Malini Iyengar, PhD , Rafael Amado, MD , Marcus 2 3 1 1 Butler, MD , David Hong, MD , Alex Tipping, PhD , Samik Basu, MD , Indu Ramachandran, PhD 1 2 Adaptimmune, Philadelphia, PA, United States; Princess Margaret Cancer Centre, Toronto, Ontario, Canada; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Indu Ramachandran (indu.ramachandran@adaptimmune.com) Fig. 1 (abstract P172). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P173 Background ADP-A2M4 is a genetically engineered autologous affinity-enhanced receptor immunotherapy (SPEAR T-cells) directed towards a MAGE- A4 peptide expressed in the context of HLA-A*02 on tumor cells. Clinical responses with ADP-A2M4 have been reported in patients with advanced MAGE-A4+ synovial sarcoma (SS) tumors. Here, we describe intra-tumoral and peripheral correlates associated with clin- ical response and resistance in two patients with SS. Methods Transduced T-cell persistence was determined by qPCR in PBMCs. Serum cytokines were measured via a multiplexed electrochemiluminescence-based immunoassay (MSD). Immunohisto- chemistry for antigen and immune markers was performed on FFPE tumor biopsies collected from patients prior to and following ADP- A2M4 transfer. A digital PCR-based assay was performed on FFPE tumor biopsies to detect the presence of SPEAR T-cells in the tumor. T-cell cytotoxicity assays were performed in vitro using the IncuCyte® platform. Clinical responses were assessed by RECIST v1.1. Results In the first patient, the best overall response (BOR) following ADP- A2M4 treatment was a partial response. Multiple correlates previously shown to be associated with response were observed. The patient’s pre- and post-infusion tumor biopsies expressed high levels of MAGE-A4 protein. Post-infusion, high levels of persisting transduced cells were observed in peripheral blood. Additionally, the patient had a grade 2 CRS event associated with a high level of serum IFN-g and Fig. 2 (abstract P172). See text for description IL-15 induction. In the post-infusion tumor sample, a notable increase Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 95 of 272 in CD3+ T-cell infiltration, including SPEAR T-cells, was observed and simultaneously any potential TAA. RevTMs were able to effi- along with PD-L1 induction. ciently redirect RevCAR-T-cells specifically against different tumor tar- In the second patient, the BOR was stable disease; then the disease pro- gets. Moreover, we show that combinatorial targeting can be gressed. MAGE-A4 protein expression was lower prior to ADP-A2M4 infu- achieved using our RevCAR system. Here, dual-RevCAR-T-cells were sion, compared to the 1st patient. Minimal peripheral induction of IFN-g efficiently activated only after engagement by two RevTMs targeting and IL-15 was observed post-infusion along with a lower level of trans- the activating or costimulatory RevCAR and different TAAs. duced T-cell persistence, compared with the 1st patient. No CRS was re- Conclusions ported in this patient. Both patients’ manufactured products contained Taken together, we developed a switchable RevCAR platform showing transduced CD8+ T-cells capable of killing antigen-expressing targets high effectiveness, increased specificity, improved safety, easy control- in vitro. In the responding patient, effective target killing was observed in lability, and small size facilitating combinatorial tumor targeting. transduced CD8+ T-cells isolated from the tumor site post-infusion. Eight Ethics Approval additional SS patients have been treated, and we continue to analyze The study was approved by local authorities and the Ethics Board. biomarkers in these patients. Conclusions P175 Based on these two cases, we have identified some factors that may PD-L1: A side-effect of T cell engagement or a main player in MDS contribute to the anti-tumor activity of ADP-A2M4. High antigen ex- tumor immune evasion? pression levels, IL-15 and IFN-g cytokine induction, good engraft- 1 1 1 2 Valentina Ferrari, BA , Alison Tarke , Hannah Fields , Tiffany Tanaka , ment, tumor site trafficking, and cytolytic function of SPEAR T-cells 2 1 1 Rafael Bejar , Thomas Lane, MD , Antonella Vitiello, PhD , Maurizio may be associated with favorable responses in SS patients treated Zanetti, MD with ADP-A2M4. 1 2 PersImmune, San Diego, CA, United States; University Of California San Trial Registration Diego, San Diego, CA, United States NCT03132922 Correspondence: Antonella Vitiello (avitiello@persimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P175 P174 Combinatorial tumor targeting using a novel switchable RevCAR Background system Immune checkpoint inhibitors (ICIs) are being tested in myelodys- 1 1 1 Anja Feldmann, PhD , Anja Hoffmann , Ralf Bergmann , Liliana Loureiro, plastic syndromes (MDS) based on pre-clinical data suggesting that 1 2 1 1 PhD , Enrico Kittel-Boselli , Nicola Mitwasi , Stefanie Koristka , Justyna the relevant targets are expressed on tumor and immune cells. Here 3 1 1 1 Jureczek , Nicole Berndt , Claudia Arndt , Michael Bachmann we study both tumor cells and T cells from patients with higher-risk 1 2 Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; University MDS to assess the role of PD-L1. Hospital Carl Gustav Carus, Dresden, Germany; German Cancer Research Methods Center (DKFZ), Heidelberg, Germany Patients’ CD3+ control cells, CD34+ stem cells, and their autologous Correspondence: Anja Feldmann (a.feldmann@hzdr.de) MDS cell lines (MCLs) were analyzed by DNA and RNA sequencing to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P174 identify somatic variants present in the tumor cells and absent from the control cells. From all somatic variants identified, we generated Background and tested neopeptides in vitro for their ability to induce tumor- Although T-cells genetically modified to express chimeric antigen recep- specific T cell responses. A T cell killing assay was performed to as- tors (CARs) are successfully used to treat hematological malignancies, pa- sess which neopeptide-specific T cells were capable of mediating tients still suffer from several drawbacks of conventional CAR (cCAR) tumor cell lysis (Figure 1A). In parallel, tumor PD-L1 expression levels therapy. CAR-T-cells can cause severe to life-threatening adverse reac- were measured by flow cytometry before and after 24-hour incuba- tions like on-target, off-tumor toxicities which cannot be controlled in pa- tion with tumor-specific autologous T cells. As a control for back- tients. Moreover, cCAR therapy often fails to successfully affect solid ground tumor cell lysis and PD-L1 expression, tumor cells were also tumors and bears the risk to encourage tumor escape variants upon tar- incubated with CEF-specific T cells (CEF: CMV, EBV, and flu peptides). geting of only one single tumor-associated antigen (TAA). In order to Results overcome these problems, we have established a novel on/off-switchable Patients’ tumor cells did not express PD-L1 at baseline. Remarkably, RevCAR system facilitating combinatorial targeting strategies. after co-culture, PD-L1 expression on the tumor cells ranged from Methods 20% to 70% (Figure 1B). Tumor cells, when incubated with CEF- For combinatorial targeting one T-cell has to be modified with two separ- specific T cells, did not upregulate PD-L1, suggesting that PD-L1 ex- ate CARs recognizing different TAAs. The first CAR mediates the activation pression may be linked to target recognition by neoantigen-specific and the second CAR the costimulatory signal. In case of ‘AND’ gate tar- T cells (Figure 2). Interestingly, tumor cell lysis was independent of geting, dual-CAR-T-cells have to recognize both TAAs on the surface of PD-L1 expression on tumor cells (Figure 1C). Additionally, IFNg the target cells to get activated. However, such combinatorial targeting neutralization did not affect PD-L1 expression nor ability to lyse strategies are struggling with several challenges including the adjustment tumor cells (Figure 3). These data show that when tumor cells are in- of signal strength and affinity of both split CARs as well as the CAR size cubated with autologous tumor-specific T cells, the tumor cells up- limiting the number of transduced specificities. In order to overcome regulate PD-L1 expression yet do not escape lysis by T cells. these obstacles, our idea was to construct small RevCARs comprising only Since lysis of tumor cells may occur prior to their upregulating PD-L1, we a small peptide epitope as extracellular domain. By removing the extra- pre-incubated tumor cells with soluble IFNg prior to co-culture with T cells. cellular single-chain variable fragment (scFv) of cCARs, RevCARs avoid Tumor cells were 96% PD-L1+, and were lysed by tumor-specific T cells at tonic signaling induced by scFv dimerization. As RevCARs do not have an the same level of target cells that had not been treated with IFNg (Figure 4). extracellular antigen binding moiety, they cannot bind to any antigen Conclusions per se. Thus, actually they are switched off. Only in the presence of a bis- Collectively, these data lend support to the notion that, in this sys- pecific target module (RevTM), RevCAR-T-cells can be redirected to tumor tem, PD-L1 is not a main player in MDS tumor immune evasion, sug- cells and switched on. Finally, short-living RevTMs allow a repeatedly on/ gesting that tumor immune evasion might function in a PD-L1- off-switch and controllability of RevCAR-T-cells and furthermore a flexible independent way. They also suggest that cognate recognition of redirection of RevCAR-T-cells to any target. tumor cells by neoantigen-specific T cells can cause the upregulation Results PD-L1 on target cells via a yet to be identified mechanism. For proof of concept two small peptide epitopes were selected to Ethics Approval construct the respective RevCARs. Additionally, a series of different The study was approved by the University of California, San Diego's RevTMs was generated recognizing one of the two peptide epitopes Institutional Review Board, HRPP #161345. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 96 of 272 P176 Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) for metastatic uveal melanoma: feasibility of treatment using a product generated from the primary tumor Marie-Andree Forget, PhD, Cara Haymaker, PhD, Orenthial Fulbright, BS, Shawne Thorsen, BS, Esteban Flores, BS, Arely Wahl, MSc, Rene Tavera, BS, Benjamin Tintera, BS, Timothy Woody, Michelle Williams, Yun Shin Chun, Patrick Hwu, MD, Dan Gombos, Sapna Patel, MD, Rodabe Amaria, MD, Chantale Bernatchez MD Anderson Cancer Center, Houston, TX, United States Correspondence: Chantale Bernatchez (CBernatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P176 Background MD Anderson Cancer Center’s tumor-infiltrating lymphocyte (TIL) program has expanded TIL from tumor fragments of cutaneous metastatic melanoma using high dose IL-2 from over 900 patients with a growth success averaging 62% [1]. Surprisingly, this growth success plunges to 45% for metastatic uveal melanoma tumor fragments [2] and less than 20% from primary uveal tu- mors. The reason for this drop is unclear as uveal melanomas have an infiltration of CD8+TIL comparable to cutaneous melan- oma [2], which in theory makes it an attractive candidate for im- Fig. 1 (abstract P175). Ferrari, V SITC 2019 abstract slide 1 munotherapy. However, limited success observed with checkpoint therapy prompted us to explore ex vivo manipulation of the TIL. A previous report demonstrated the feasibility of TIL adoptive cell therapy for metastatic uveal patients using a TIL product ex- panded from metastases [3]. Since the primary and metastatic sites of uveal melanoma dis- play preserved gene mutations, indicating a potential shared antigen landscape, one could propose generating a TIL product from the primary tumor when the patient undergoes enucle- ation and to utilize this product for treatment at time of recurrence. Methods Given the challenge of propagating TIL from a primary uveal tumor in high dose of IL-2 only, we hypothesized that our new TIL3.0 method to propagate TIL from tumor fragments (1st phase of expansion), based on the 3-signals required for optimal activa- tion of a T-cell (TCR engagement, costimulation and cytokine ex- Fig. 2 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 posure) would enable TIL growth from a higher percentage of primary uveal tumors given the success obtained with metastatic sites [1]. This product can be banked and accessed later for treat- ment at recurrence. Results The TIL3.0 expansion platform was shown to be optimal for T-cell propagation allowing for successful expansion of TIL from primary uveal melanoma tumors in >90% of the cases (n=20). This expan- sion was rapid (less than 3 weeks) and consistently composed of CD8+CD3+TIL. This later observation is attributed to the use of the agonistic anti-CD137/4-1BB, Urelumab, as of costimulation sig- nal in our TIL3.0 method. The TIL3.0 method applied to primary tumors could be scaled and adapted for GMP. This process, followed by a rapid expan- sion protocol, was applied to treat the first metastatic uveal pa- tient with a TIL product generated from the primary tumor. The patient was infused with a total of 14.4 billion TIL with a viability of 99%. Conclusions This study demonstrates the feasibility of generating a TIL product from a primary uveal tumor to be used for treatment at recurrence. Trial Registration Fig. 3 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 NCT00338377 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 97 of 272 References Deletion of p50 in patient-derived marrow CD34+ cells and subse- 1. Tavera RJ, Forget MA, et al. Utilizing T-cell Activation Signals 1, 2, and 3 quent production of IMC for adoptive transfer may contribute to the for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over therapy of these and additional cancers, alone or with additional the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma. J immunotherapies. Immunother. 2018; 41:399-405. Ethics Approval 2. Qin Y, de Macedo MP, Reuben A, et al. Parallel profiling of immune The study was approved by the Johns Hopkins University Animal infiltrate subsets in uveal melanoma versus cutaneous melanoma unveils Care and Use Committee, protocol MO19M10. similarities and differences: A pilot study. OncoImmunology. 2017; 6:e1321187. 3. Chandran SS, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two- P178 stage, single-arm, phase 2 study. Lancet Oncol. 2017; 18:792-802. Dual inhibition of PI3Kdelta and PI3Kgamma to enhance Ethics Approval mitochondrial mass and ex vivo expansion of central and stem cell Institutional review board (IRB)-approved protocol# 2004-0069 memory T cells from CLL patients Christopher Funk, BS, , Shuhua Wang, MD, Alexandra Waller, BS, Lauren Fleischer, BS, Aditi Sharma, Harold Spencer, PhD, Vikas Gupta, MD, PhD, P177 Sruthi Ravindranathan, PhD, Mala Shanmugam, PhD, Christopher NF-kB p50-deficient immature myeloid cell (p50-IMC) adoptive Flowers, MD, MS, Edmund Waller, MD, PhD, FACP transfer slows the growth of murine prostate and pancreatic Emory University School of Medicine, Atlanta, GA, United States ductal carcinoma Correspondence: Edmund Waller (ewaller@emory.edu) Rahul Suresh, PhD, David Barakat, PhD, Theresa Barberi, PhD, Lei Zheng, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P178 PhD MD, Elizabeth Jaffee, MD, Kenneth Pienta, MD, Alan Friedman, MD Johns Hopkins University School of Medicine, Baltimore, MD, United Background States For chimeric antigen receptor T-cell (CAR T) therapy to treat Correspondence: Alan Friedman (afriedm2@jhmi.edu) chronic lymphocytic leukemia (CLL), recent work associates remis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P177 sions with infusion of sufficient non-exhausted memory CAR T, capable of oxidative phosphorylation [1]. Our work aims to Background modulate metabolic pathways during ex vivo expansion of T-cells NF-kB p50 binds DNA but, unlike p65, lacks a trans-activation do- for translation of these findings to clinical adoptive therapies. main and recruits co-repressors. Macrophages and dendritic cells Class I catalytic PI3K enzymes, such as PI3Kdelta and PI3Kgamma, lacking NF-kB p50 are skewed towards a pro-inflammatory pheno- regulate T-cell differentiation, regulatory T cell formation, and TCR type, with increased cytokine expression and enhanced T cell acti- signaling [2]. In this study, we hypothesized that pharmacological vation; additionally, murine melanoma, fibrosarcoma, colon inhibition of these pathways during ex vivo culture would in- carcinoma, and glioblastoma grow slower in p50-/- mice. Given crease populations of early memory CAR T with enhanced meta- these data, we evaluated efficacy of p50-deficient immature mye- bolic and survival potential. loid cells (p50-IMC) adoptively transferred into tumor-bearing hosts. Methods Immature cells were utilized to maximize tumor localization, and Healthy- and CLL- donor peripheral blood mononuclear cells pretreatment with 5-fluorouracil (5FU) was examined due to its po- were isolated and cryopreserved. Thawed cells were sorted for tential to impair marrow production of myeloid cells, to target CD3 expression prior to culture (or transduction) and expanded tumor myeloid cells, and to potentially release tumor neoantigens. in G-Rex plates using anti-CD3/CD28 beads, 30 U/mL Methods interleukin-2, and investigational drugs: idelalisib, duvelisib and WT-IMC or p50-IMC were generated by culturing lineage-negative ibrutinib for 9 days. marrow cells from WT or p50-/- mice in media containing TPO, SCF, Results and FL for six days followed by M-CSF for one day on ultra-low at- Class I catalytic enzymes in T-cells, PI3Kdelta and PI3Kgamma, tachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) exhibit domain homology (Figure-1A). Accordingly, maximum cell or K-Ras(G12D) pancreatic ductal carcinoma (PDC)-luciferase cells re- yields for both idelalisib (Figure 1B) and duvelisib (Figure 1C) ceived 5FU followed five days later by three doses of 1E7 IMC every occurred upon inhibition of both isoforms. Comparing doses of three to four days. Some groups also received four doses of anti-PD- duvelisib, idelalisib, and ibrutinib that yielded optimal T-cell 1 antibody twice weekly alone or with p50-IMC. expansion, we showed potent PI3Kdelta/gamma dual antagonism Results maximizes live T-cell yields (Figure 1D) out-performing PCa grew slower in p50-/- mice, and absence of host p50 led to interleukin-2-inducible kinase inhibition. PI3K antagonists in- prolonged survival of mice inoculated orthotopically with PDC. creased frequencies of cells expressing co-stimulatory molecules 5FU followed by p50-IMC slowed PCa and PDC tumor growth ~3- (Figure 2A-B). A dose-dependent increase in cells expressing FAS/ fold in contrast to 5FU followed by WT-IMC, 5FU alone, or p50- FAS-L (Figure 2C) and an increased expression of pro-survival IMC alone. Slowed tumor growth was evident for 93% of PCa tu- BCL-2 (Figure 2D) after anti-CD3/28 stimulation suggests the mors but only 53% of PDC tumors. In PCa, p50-IMC predomin- increase in live cells with a TSCM phenotype is likely due to en- antly generated tumor and draining lymph node F4/80+ hanced cell survival. macrophages, but also CD11b+F4/80-CD11c+ conventional den- Next,weconfirmed thepositiveeffectofdualPI3Kdelta/gamma dritic cells. A subset of tumor and nodal macrophages co- inhibition in expansion of T cells from CLL donors (Figure 3A-B). expressed Ly6C and MHCII and had reduced MR compared to PI3K antagonists increased frequencies of CD8 cells (Figure 3C) host macrophages, collectively indicating a pro-inflammatory and co-stimulatory molecule expressing cells (Figure 3D-E). Inter- phenotype. p50-IMC also produced a 5-fold increase in activated estingly, addition of idelalisib to T cell cultures resulted in a PCa tumor CD8 T cells, and antibody-mediated CD8 T cell deple- dose-dependent decrease in immune checkpoint molecules LAG- tion obviated slower tumor growth induced by 5FU followed by 3, Tim-3, and PD-1 (Figure 3F-J). Given the importance of T cells p50-IMC. Anti-PD-1 markedly slowed PCa growth but had little ef- with the stem cell memory (TSCM) phenotype in adoptive T-cell ficacy against PDC, whereas anti-PD-1 combined with p50-IMC therapy, T-cell differentiation was studied. PI3K antagonists in- slowed PDC tumor growth to prolong survival more effectively crease the frequency of early, T-memory, and effector memory than either alone in an initial experiment. cells (Figure 4A). Notably, the frequency of TSCM doubled (Figure Conclusions 4B). Lastly, PI3K antagonists significantly increased the mitochon- 5FU followed by p50-IMC slows the growth of murine prostate and drial mass (Figure 4C) within total CD3 (Figure 4D) and CD8 (Fig- pancreatic ductal carcinoma and depends upon CD8 T cell activation. ure 4E) subsets. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 98 of 272 Conclusions Dual inhibition of PI3Kdelta and PI3Kgamma modulates aspects of T-cell biology relevant to CAR T remissions. PI3Kdelta/gamma antagonism enhances CD27 expression and mitochondrial mass, decreases immune checkpoint expression, and enriches the TSCM phenotype. Acknowledgements The authors thank the patients and healthy volunteers for their blood donations. CR Funk is supported by a Howard Hughes Medical Research Fellowship. The authors thank Emory’s Clinical Lymphoma Research Team for requesting patient consent and aiding in sample collection. References 1. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 2018; 24:563-571. [2]2. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, and Abraham RT. Leading Edge The PI3K Pathway in Human Disease. Cell. 2017; 170:605-635. Ethics Approval The study was approved by Emory University's Institutional Review Committee, approval number 00057236. Fig. 3 (abstract P178). See text for description Fig. 1 (abstract P178). See text for description Fig. 4 (abstract P178). See text for description P179 Checkpoint Cbl-b siRNA-based APN401 adoptive cell therapy: superior efficacy & immune memory induction in murine hepatocellular carcinoma following APN401 monotherapy and synergism with anti-PD1 1 1 1 Anderson Gaweco, MD, PhD , Kathrin Thell , Maria Urban , Julia 1 1 2 3 Harrauer , Isabella Haslinger , Josef Penninger , Vincent Chung , 4 5 Anthony El-Khoueiry, MD , Carlos Becerra, MD 1 2 Apeiron Biologics, Vienna, Austria; University of British Columbia, 3 4 Vancouver, BC, Canada; City of Hope, Duarte, CA, United States; USC Norris Comprehensive Cancer Center, Los Angeles, CA, United States; Baylor University Medical Center, Dallas, TX, United States Correspondence: Anderson Gaweco (anderson.gaweco@apeiron- biologics.com) Fig. 2 (abstract P178). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P179 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 99 of 272 Background combination of immunomodulatory cytokines: IL12 and IL21. Upon The intracellular master checkpoint Cbl-b, an E3 ubiquitin ligase, administration, SENTI-101 innately homes to peritoneal tumors, se- negatively regulates the innate and adaptive anti-tumor immune re- cretes IL12 and IL21 in a localized and sustained fashion, and induces sponses. Selective cell-based targeting of Cbl-b not only induces a robust anti-tumor immune response. anti-tumor activity in vivo but also overrides immune regulation by Methods the PD-L1/PD-1 pathway in vitro [1]. Human APN401 is an autologous Two syngeneic pre-clinical models of disseminated peritoneal carcin- adoptive cellular therapy of ex vivo Cbl-b-silenced human PBMCs omatosis with distinct immune phenotypes were established by currently in a clinical Phase 1b multiple dose study demonstrating implanting cells in the peritoneal cavities of mice (CT26-fLUC = early clinical safety and tolerability in patients with advanced solid immune-inflamed; B16-F10-fLUC = immune-excluded) [3]. A library of tumors. Herein the preclinical Proof of Concept efficacy of murine over 50 murine MSC lines engineered to express immune effectors APN401 immunotherapy is established in the syngeneic mouse hepa- (cytokines, chemokines, growth factors), either individually or in com- tocellular carcinoma Hepa1-6 tumor rechallenge study. bination, was administered intraperitoneally and evaluated for anti- Methods tumor activity via bioluminescence and tumor weight measurements. Hepa1-6-C57/BL6-tumor bearing mice were treated for 19 days with Immune phenotype was characterized by flow-cytometry and multi- murine APN401, ex vivo silenced immune cells with Cbl-b specific plexed immunohistochemistry. siRNA, as monotherapy or in combination with anti-PD1 (clone Results RMP1-14) versus control siRNA on D7 post-inoculation. Mice were MSCs expressing the combination of IL12 and IL21 (SENTI-101) were se- later rechallenged on D29 with Hepa1-6 s.c. on the contralateral flank lected based on significant tumor-burden reduction and immune pro- in the absence of any further APN401 treatment. file changes in both syngeneic models. Notably, the combination Results outperformed each individual cytokine in extending survival (p=0.02). Significant tumor growth inhibition (TGI) was observed following 19 Intraperitoneal administration of SENTI-101 into tumor-bearing mice days of treatment with APN401 alone or in combination with anti- led to preferential co-localization with tumors (>10-fold higher vs. PD1 starting on D7 after s.c. inoculation (p<0.0001). Following rechal- normal tissues, p=0.001). Local concentrations of IL12 and IL21 were lenge, significant TGI of 86% (p<0.001) was observed in prior ~100-fold greater in the peritoneal space vs. serum (p=0.002). SENTI- APN401-treated mice versus control siRNA-treated mice. Mice that re- 101 treatment reduced tumor-burden more than 200-fold (p50% of ceived APN401 in combination with anti-PD1 prior to rechallenge the mice were tumor-free after 90 days, while control groups and demonstrate profound synergistic anti-tumor efficacy (p<0.001) ver- groups treated with anti-PD1 antibody had a median survival of 21 sus anti-PD1 alone with control siRNA. APN401 was well tolerated to 30 days. Surviving mice were able to reject newly implanted and APN401-treated mice were unremarkable with optimal body tumor cells, demonstrating anti-tumor immune memory. conditions. Anti-tumor effects of SENTI-101 are mediated by a multi-modal im- Conclusions mune response. The frequency of antigen-presenting cells in periton- APN401 monotherapy demonstrates striking anti-tumor efficacy in eal tumor-draining lymph nodes was more than doubled vs. controls the murine hepatocellular carcinoma Hepa1-6 model. The preclinical (p=0.01). This correlated with increased T-cell and B-cell tumor infil- synergistic effects of APN401 with anti-PD1 support its therapeutic trates forming tertiary-lymphoid structures, which are associated with utility as a combination therapy with immune checkpoint anti-PD1 improved prognosis in cancer [4]. T-cell activation markers (CD38, treatment. The significant TGI observed following tumor rechallenge IFNg, GranzymeB) were significantly increased locally. indicate that prior selective cell-based Cbl-b-silencing with APN401 Conclusions alone or in combination with anti-PD1 induced systemic and durable SENTI-101 induces localized immune-modulation, regulates multiple anti-tumor immune memory responses. These findings highlight the steps of the cancer immunity cycle, and results in durable anti-tumor potential promise of a selective adoptive cell-based Cbl-b silencing responses. These data warrant further development of SENTI-101 for by APN401 as a novel immunotherapy for cancer. the loco-regional treatment of advanced solid tumors. Reference References 1. Fujiwara M, Anstadt EJ, Clark RB. Cbl-b Deficiency Mediates Resistance to 1. Desai JP, Moustarah F. Cancer, Peritoneal Metastasis. StatPearls Programmed Death-Ligand 1/Programmed Death-1 Regulation. Front Publishing. 2019 [Updated 2019 Jun 30] Immunol. 2017 Jan 26;8:42. 2. Lengyel E. Ovarian Cancer Development and Metastasis. Am J Pathol. 2010; 177(3): 1053–1064 3. Mosely SI, Prime JE, Sainson RC, et al. Rational Selection of Syngeneic P180 Preclinical Tumor Models for Immunotherapeutic Drug Discovery. Cancer SENTI-101, an allogeneic cell product, induces potent and durable Immunol Res. 2017; 5(1):29-41 anti-tumor immunity in pre-clinical models of peritoneal 4. Sautès-Fridman C, Petitprez F, Calderaro J, Fridman WH. Tertiary carcinomatosis lymphoid structures in the era of cancer immunotherapy. Nat Rev Alba Gonzalez Junca, PhD, Gary Lee, PhD, Archana Nagaraja, PhD, Alyssa Cancer. 2019;19(6):307-325 Mullenix, Russell Gordley, PhD, Daniel Frimannson, PhD, Anissa Benabbas, Chen-Ting Lee, PhD, Tiffany Truong, Allison Quach, Mengxi Tian, Rishi Savur, Rowena Martinez, Alyssa Perry-McNamara, Don-Hong P181 Wang, PhD, Ori Maller, PhD, Dharini Iyer, PhD, Ashita Magal, Christina Multi-phenotype CRISPR-Cas9 screens identify p38 kinase as a Huynh, Carmina Blanco, Jack Lin, PhD, Brian Garrison, PhD, Philip Lee, target for adoptive immunotherapies PhD, Timothy Lu, MD, PhD, Sravani Mangalampalli Devikala Gurusamy, PhD, Suman Vodnala, Amanda Henning, Rigel Senti Biosciences Inc., South San Francisco, CA, United States Kishton, Tori Yamamoto, Arash Eidizadeh, Li Jia, Christine Kariya, Mary Correspondence: Gary Lee (gary.lee@sentibio.com) Black, Robert Eil, Douglas Palmer, Zhiya Yu, Jenny Pan, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P180 Madhusudhanan Sukumar, Shashank J. Patel, Nicholas Restifo, MD National Institutes of Health, Bethesda, MD, United States Background Correspondence: Nicholas Restifo (restifo@nih.gov) More effective therapies for disseminated peritoneal carcinomatosis, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P181 including high-grade serous ovarian cancer, remain a major medical need [1]. Although several treatments offer initial responses to local- Background ized disease, patients with disseminated peritoneal tumors face poor Adoptive T cell transfer immunotherapy (ACT) using tumor-infiltrating overall survival [2]. lymphocytes (TIL) and gene-modified T cells can induce complete and SENTI-101 is a novel therapeutic agent comprising allogeneic mesen- durable regression of metastatic human malignancies that are other- chymal stromal cells (MSCs) genetically modified to express a potent wise refractory to treatment. While successful T cell-based treatments Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 100 of 272 of patients with widely metastatic melanoma, synovial sarcoma, cholan- concentration of 1 x 106 cells/ml with high concentration of hIL-2 giocarcinoma and cancers of the breast, colon, and cervix have been and 5% UltraGRO® for 14 days in our original closed system. Then, reported in recent years, most patients with common epithelial cancers we confirmed the expression of surface markers, CD107a fail to respond to treatment. While several factors can contribute to the mobilization and cell-mediated cytotoxicity against various tumor efficacy of ACT, a major inherent limitation is the induction of termin- cells and normal cells with or without monoclonal antibody drugs ally differentiated phenotype coupled with the loss of proliferative cap- in vitro and antitumor effects against peritoneal dissemination model acity in TIL during current ex vivo expansion protocols. Individual gene using SKOV3 in vivo. knockout approaches for enhancing T cell-based cancer immunother- Results apies are low-throughput and can improve one desired function (T cell [Results and Discussion] Importantly, we’ve found that our GAIA-102 memory) at the expense of another equally important function (expan- exhibited CD3-/CD56bright/CD57- immature phenotype that could sion). Thus, there is significant interest in identifying T-cell intrinsic kill various tumor cells efficiently from various origins, including Raji negative regulatory circuits that limit their ability to expand robustly cells that was highly resistant to NK cell killing. More importantly, ex vivo, while dampening their terminal effector differentiation along massive accumulation, retention, infiltration and sphere destruction with the reduction of oxidative stress and genomic damage. by GAIA-102 were affected neither by myeloid-derived suppressor Methods cells nor regulatory T-lymphocytes. GAIA-102 was also effective To identify the T cell intrinsic negative regulatory circuits, we developed a in vivo to murine model of peritoneal dissemination of human ovar- multi-phenotype genetic screen to systematically target 29 major kinases ian cancer. screen to concurrently measure the impacts of individual gene knockouts Conclusions on T cell expansion, differentiation, oxidative stress and genomic stress. Thus, these findings indicate that GAIA-102 has a potential to be an Using CRISPR-Cas9-based gene perturbation combined with high- ‘upward compatible’ modality over CAR-T strategy, and would be a throughput flow cytometry, we developed and validated a multi-phenotype new and promising candidate for adoptive immunotherapy against screen, which identified Mapk14/p38 kinase as a target that improved all solid tumors. We now just started GMP/GCTP production of this new four phenotypes in CD8+ T cells. We used murine and human ex vivo T cell and powerful NK cells and first-in-human clinical trials in use of expansion models to validate the results from our genetic screen. GAIA-102 will be initiated on 2020. Results Ethics Approval Results from our genetic screen identified p38 kinase as a unique [Ethics Approval] Written informed consent was obtained from all multi-phenotypic regulator of cellular differentiation, oxidative, and healthy volunteers, in accordance with the Declaration of Helsinki. genomic stress while achieving improved cellular expansion. Further- Upon the approval of the institutional ethical committee (ap- more, pharmacological inhibition of p38 kinase in murine and human proval no. 29-315) of Kyushu University, peripheral blood samples ex vivo T cell expansion models validated the results from our gen- were collected from healthy volunteers. The animal experiments etic screen. Cells cultured in the presence of a p38 inhibitor had in- were reviewed and approved by the Institutional Animal Care creased capacity for cytokine production, specifically interferon-γ and and Use Committee of Kyushu University (approval nos. A30-234- demonstrated improved in vivo persistence. Additionally, cells cul- 0 and A30-359-0). tured in the presence of the p38 inhibitor demonstrated enhanced in vivo cell-expansion, tumor infiltration, and anti-tumor efficacy in P183 an immunocompetent tumor mouse model. Development of novel chimeric antigen receptor T cells for Conclusions immunotherapy of hepatocellular carcinoma This study establishes p38 inhibition in T cells as a potentially import- Yukai He, PhD, Leidy Caraballo Galva, Xiaotao Jiang ant strategy for improving ACT immunotherapy for cancer patients. Augusta University, Augusta, GA, United States Ethics Approval Correspondence: Yukai He (yhe@augusta.edu) All human samples were isolated in accordance with approved clin- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P183 ical protocols and in accordance with NIH institutional review board approval and informed consent from patients and healthy donors. Background Immunotherapy has a great potential for hepatocellular carcin- P182 oma (HCC). Several human glypican 3 (hGPC3)-specific chimeric GAIA-102: a new class NK cell-like phenotype manufactured in antigen receptor T cells (CARTs) are being tested for HCC. But, accordance with GMP/GCTP that can eliminate solid tumors most, if not all, are constructed from one monoclonal antibody Yui Harada, PhD, Yoshikazu Yonemitsu, MD, PhD (mAb). It is unknown whether targeting different epitopes of Kyushu University, Fukuoka, Japan hGPC3 will create more effective CARTs. Here, we aim to develop Correspondence: Yui Harada (rkfraile@med.kyushu-u.ac.jp) novel CARTs that target different regions of hGPC3. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P182 Methods BalB/C mice were immunized with hGPC3 protein. Hybridomas Background and mAbs were generated and characterized. Then, CARTs were [Background] Cancer immunotherapy has been established as a new built from the novel mAbs and their antitumor effect was therapeutic category since the recent success of immune checkpoint studied. inhibitors and a type of adoptive immunotherapy, namely chimeric Results antigen receptor-modified T cells (CAR-T). Although CAR-T demon- Twenty-two hGPC3-specific mAbs were identified by ELISA. Out of strated impressive clinical results, serious adverse effects (cytokine them, 14 bound HepG2 cells. Five mAbs were further character- storm and on-target off-tumor toxicity) and undefined efficacy on ized by immunohistochemical staining. Three of them (6G11, 8F8, solid tumors are important issues to be solved. We’ve developed a and 12D7) were found to specifically stain HCC tumor but not cutting-edge, simple, and feeder-free method to generate highly acti- adjacent normal tissues. The 3 mAb’s affinity were in the nano- vated and expanded human NK cells from peripheral blood molar range. 6G11 and 8F8 bound to hGPC3 epitope aa25-39 (US9404083, PCT/JP2018/018236, PCT/JP2019/012744), and have and aa463-496, respectively. No specific epitope was identified been conducting further investigation why our new type of NK cells, for 12D7 though it bound to the N-fragment (25-358aa). CARTs named as GAIA-102, are so effective to kill malignant cells. built from the 3 mAbs underwent expansion in response to Methods HepG2 cell stimulation. However, their effector function was sig- [Materials and Methods] Cryopreserved PBMCs purchased from nificantly different. 8F8 CARTs possessed the strongest effector HemaCare Corporation were mixed and processed by using LOVO function. 6G11 CARTs generated the greatest expansion, but with and CliniMACS® Prodigy (automated/closed systems). CD3+ and slightly weaker function. In contrast, 12D7 CART had the weakest CD34+ cells were depleted, and the cells were cultured at a effector function. Soluble hGPC3 did not activate CARTs, nor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 101 of 272 blocked CART activation by tumor cells. Adoptive transfer of 8F8 P185 and 6G11, but not 12D7, CARTs generated potent antitumor ef- T cell antigen presenting cell (tAPC) is a strategy to induce CAR T fects with complete regression of HCC xenografts, which corre- expansion in vivo in the absence of a tumor for on-target toxicity lated to their expansion in vivo. studies Conclusions Yen Ho, BS, Jon Jones, BA, Patrick Carlson, BS, Cyr de Imus, The three novel CARTs that target different hGPC3 regions pos- Rebekah Turk, Dina Alcorn, Kyle Kolaja, Ruth Salmon, PhD, Thomas sess significantly different effector function and antitumor effects. Long Adoptive transfer of CARTs targeting the hGPC3 N- or C-epitope Celgene Corporation, Seattle, WA, United States results in complete eradication of HCC xenografts. Correspondence: Thomas Long (thomas.long@junotherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P185 P184 Background Activating marrow infiltrating lymphocytes in hypoxia enhances Preclinical safety evaluation of chimeric antigen receptor (CAR) their efficacy in adoptive T-cell therapy T cells presents a number of unique challenges. One of those 1 1 1 1 Megan Heimann , Ervin Griffin , Luca Biavati, MD , Amy Thomas , challenges is the development ofaCynomolgus macaquetoxi- 1 1 2 Danielle Dillard , Elizabeth Zawidzka , Brianna Richardson , Robert cology model as a tool to understand potential on-target CAR T 1 2 3 1 Leone , Gregory Szeto , Kimberly Noonan, PhD , Ivan Borrello, MD cell toxicity. This is important for CAR T cell programs where Johns Hopkins University School of Medicine, Baltimore, MD, United the lead binder is cyno cross-reactive and the target has known States; University of Maryland Baltimore County, Baltimore, MD, United normal tissue expression conserved across species. Unlike many States; WindMIL Therapeutics, Baltimore, MD, United States mouse models, non-human primates lack target-expressing Correspondence: Ivan Borrello (iborrell@jhmi.edu) tumors that can drive CAR T cell activation and expansion; it Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P184 would be advantageous to recapitulate that expansion in the context of a toxicity model. Background Methods Marrow infiltrating lymphocytes (MILs) are a promising candidate for T cell antigen presenting cells (tAPCs) have been reported to adoptive cell therapy (ACT) due to their broader anti-tumor specifi- drive measurable CAR T cell expansion in Rhesus macaques [1]. city and persistence. These characteristics are due to intrinsic proper- The advantages of this strategy include the ease of manufactur- ties of the bone marrow (BM); known to be a reservoir for long-lived ing tAPCs in parallel to CAR T cells and co-engraftment of memory T cells. It has also been established that naïve and memory tAPCs with CAR T cells in hematological niches to ensure anti- T cells are metabolically quiescent, favoring oxidative phosphoryl- gen availability. The optimal tAPC dosing strategy to drive a ation (OXPHOS) over glycolysis, while effector T cells favor glycolysis strong and persistent CAR T cell activity has not been deter- to fuel their rapid proliferation. mined. Here, we generated human anti-CD19 CAR T cells Methods expressing firefly luciferase and human T cells expressing a We examined how activation and expansion of MILs in hypoxia truncated human CD19 (CD19t) lacking the intracellular domain could be used to better understand the inherent properties of as tAPCs. We then dosed mice either with CAR and tAPC con- the BM, to exploit these properties, and enhance the efficacy of currently, or with CAR first then followed by tAPCs three days MILs in ACT, especially when compared to that of peripheral later. blood lymphocytes (PBLs). By activating MILs in hypoxia, we can Results select for and/or alter the cells best suited to mount an effective (Figure 1) shows bioluminescent imaging (BLI) measurements anti-tumor response. that indicate concurrent and delayed tAPC dosing have similar Results CAR T cell expansion kinetics; however, the delayed tAPC Activation under hypoxic conditions alters MILs in several unique dosing exhibits a greater magnitude of CAR T cell expansion. In ways. MILs show greater overall expansion, enhanced tumor- both cases, the CAR T cell expansion occurs in a tAPC dose- specificity, and a unique metabolic profile—upregulating both dependent manner. Imaging shows that concurrent dosing OXPHOS and glycolytic machinery. This metabolic profile suggests leads to CAR T cell proliferation primary in the lungs, whereas that hypoxia-activated MILs possess properties of both effector delayed tAPC dosing leads to more systemic CAR T cell and memory cells. PBLs grown under the same conditions fail to expansion. In addition, flow cytometry data show a significant expand significantly and show no metabolic differences or specifi- depletion of tAPCs in the peripheral blood between day 7 and city. Following activation in hypoxia we have found that MILs day 14. have upregulated metabolism-related genes such as CPT1A and Conclusions GLUT1 and 3, as well as anti-apoptotic factors such as BCL2 and Follow-up studies delivering additional tAPC doses three, six, BCL2L1 at an RNA level. The post-expansion MILs product, using and nine days after CAR T cells show that repeated tAPC ad- intracellular staining and FACs analysis, shows an increase in ministration can significantly increase the CAR T cell exposure mitochondrial proteins, including TOMM20, CPT1a, and SDHa, in- over time compared to a single tAPC dose. Overall, these data creased mTOR signaling, and increased glycolytic machinery—HK2 demonstrate that tAPCs can be used to induce CAR T cell ex- and GLUT1. Additionally, while cell-cycle analysis with Ki67 and PI pansion in vivo in the absence of a tumor and will enable us shows that MILs are more resting at baseline and arrested in G0, to design a tAPC strategy for use in a Cynomolgus macaque upon activation in hypoxia they become more proliferative—mov- model to evaluate the safety of CAR T cell candidates. ing into S and G2/M phase—than normoxic MILs or PBLs and maintain this phenotype over time. This finding is supported by our RNAseq data showing a lack of transcriptional activity at baseline but a significant increase by day 3 of activation. Gene Reference expression analysis has also shown that there is a substantial in- 1. Berger C, Sommermeyer D, Hudecek M, Berger M, Balakrishnan A, crease in expression of IL2 and IL15Ra with a significant decrease Paszkiewicz PJ, Kosasih PL, Rader C, Riddell SR. Safety of targeting in the expression of IL10 transcripts. ROR1 in primates with chimeric antigen receptor-modified T cells. Conclusions Cancer Immunol Res. 2015;3(2):206-16. These findings suggest that hypoxia contributes to the unique Ethics Approval properties of MILs through modification of their metabolic profile All animal studies were conducted in accordance with protocols approved and is being uniquely employed to generate more effective MILs, by the Institutional Animal Care and Use Committee. and not PBLs, for adoptive cell therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 102 of 272 P187 Single-cell RNA sequencing and functional assessment of healthy donor- and cancer patient-derived T and CAR-T cells Zinkal Padalia, MS, Konstantinos Karagiannis, PhD, Brigid Mcewan, MA, Vahan Simonyan, PhD, Jonathan Terrett, PhD, Demetrios Kalaitzidis, PhD CRISPR Therapeutics, Cambridge, MA, United States Correspondence: Demetrios Kalaitzidis (d.kalaitzidis@crisprtx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P187 Background Autologous chimeric antigen receptor T (CAR-T) cell therapies have shown remarkable success in treating relapsed/refractory B-cell ma- lignancies. However, even in indications with high complete re- sponse rates, not all patients respond or have durable responses after CAR-T treatment. Furthermore, autologous CAR-T treatments have not yielded the same impressive outcomes in solid malignan- cies to date. A major limitation of autologous CAR-T therapy may be the dysfunctional state of a patient’s T cell populations used for manufacturing of a drug product. Allogeneic therapeutics can bypass this limitation by enabling the use of healthy donor starting material. Moreover, healthy donor material that exhibits specific T cell attri- butes can be selected for drug product manufacturing. Methods Fig. 1 (abstract P185). See text for description To identify attributes that can be associated with improved perform- ance of CAR-T cells we have characterized T cells from healthy do- nors as well as cancer patients, in particular from chronic P186 lymphocytic leukemia (CLL) patients as these have been described Evaluation of antigen-specific T-cell immunity at the single cell previously to be dysfunctional. level using large panels of DNA barcoded MHC multimers Results 1 2 2 Kivin Jacobsen, PhD , Dagmar Walter , Michael Stubbington , Katherine We show impaired function of cancer patient-derived CAR-T cells 2 1 1 2 Pfeiffer , Charlotte Halgreen , Liselotte Brix, PhD , Stephane Boutet , when compared to healthy donor-derived cells utilizing both in vitro Kivin Jacobsen, PhD and in vivo assays. We have performed single-cell RNA sequencing 1 2 Immudex, Copenhagen, Denmark; 10x genomics, Pleasanton, CA, (scRNA seq) on both starting material T cells and CAR-T cells from United States multiple healthy and CLL donors used in functional assays to uncover Correspondence: Liselotte Brix (lb@immudex.com) both gene expression and population differences associated with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P186 CAR-T cell performance. scRNA seq analysis revealed marked hetero- geneity among starting populations as well as CAR-T lots from the Background cancer patient-derived T cells. Identification of disease-specific T-cell epitopes is key to developing Conclusions novel cancer vaccines and immunotherapies. Profiling disease-specific Our analysis has allowed us to associate distinct cellular subpopula- T cells, emerging during an induced cellular immune response is im- tion and gene expression profiles with preclinical functional outputs. portant for understanding anti-tumor immunity and guide personalized therapy. The MHC dCODE™Dextramer® technology enables simultan- P188 eous screening of high numbers of T-cell specificities in the same sam- Enhanced anti-tumor activity of human placental CD34+ derived ple using MHC multimer-specific DNA barcodes and next generation natural killer cells in combination with ACY-241 for multiple sequencing as readout. Combining this technology with 10x Genomics myeloma immunotherapy Chromium single cell assay further enables the simultaneous analysis of Lin Kang, PhD, Xiaokui Zhang, Shuyang He, Vanessa Voskinarian-Berse, antigen specific T-cells, sequencing of the cognate T-cell receptors and Bhavani Stout, Valentina Rousseva, William van der Touw, James Edinger, single cell gene expression profiling. Robert Hariri Methods Celularity Inc., Warren, NJ, United States Panels of up to 50 dCODE™Dextramer® reagents were used for screening Correspondence: Xiaokui Zhang (xiaokui.zhang@celularity.com) antigen-specific T-cells in human blood samples. Single cell sequencing was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P188 performed on the isolated T-cell subpopulations, and their gene expression profile analyzed in combination with cell phenotype and TCR sequences. Background Results Celularity, Inc. is developing human placental CD34+ derived, off-the- The experiment generated a large dataset and we show one example of shelf, and allogeneic natural killer (PNK) cells for various hematologic how such a dataset can be analyzed to generate useful information. By com- malignancies and solid tumors. ACY-241 is an orally bioavailable and bining the gene expression profile, cellular phenotypes and Dextramer specifi- selective histone deacetylase (HDAC) 6 inhibitor in MM clinical devel- city we identified expanded populations of antigen-specific T cells in the opment. ACY-241 has been shown to sensitize MM cells to endogen- memory T cell compartment and characterized their individual TCR clono- ous NK cell killing [1]. Here, we investigated the potential types based on TCR sequence. pMHC-specific T-cells were also detected in augmentation of PNK mediated anti-MM activity by ACY-241 the naïve T cell compartment, showing a more diverse TCR sequence profile. treatment. Conclusions Methods This experiment demonstrates a novel method of exploring antigen- Placental CD34+ cells were cultivated in the presence of cytokines in- specific T-cell responses. Linking TCR sequences with pMHC specifi- cluding thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 city, cellular phenotypes and gene expression at this scale and reso- days to generate PNK cells. MM cell lines were treated with different lution provide a more comprehensive analysis of the antigen-specific doses (0, 0.1, 0.3, 1, 3, 10 and 30μM) of ACY-241 over 24h, 48h, or T cell response than previously available in a single workflow. Novel 72h. Cytotoxicity of PNK against different doses of ACY-241 pre- biomarkers and improved strategies of T cell based immunothera- treated MM cell lines was assessed by a PKH26/TO-PRO-3 FACS based peutic development will result from T cell analysis at this scale and assay. Ligands to NK activating receptors of ACY-241 treated MM resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 103 of 272 cells were evaluated by flow cytometry. The RPMI8226 subcutaneous- surface, as well as release of damage-associated molecular patterns ly(SubQ) xenografted NOD scid gamma (NSG) mouse model was including HMGB1 and ATP. Moreover, we show that TTFields treated used for in vivo efficacy study. cells promote phagocytosis by DCs, DCs maturation in vitro, and pro- Results mote immune cells recruitment in vivo. We also show that the com- ACY-241 treatment of MM cell lines for >48h resulted in significant bined treatment of TTFields plus anti-PD-1 led to a significant inhibition of cancer cell growth and decreased cell viability at doses decrease in tumor volume and significant increases in CD45+ tumor >10μM. In a dose dependent manner, ACY-241 further enhanced infiltrating cells in both tumor models. In the lung tumors, these infil- cytotoxicity of PNK against MM cells. In a 4h cytotoxicity assay at ef- trating cells, specifically macrophages and DCs, demonstrated upreg- fector to target (E:T) ratio of 10:1, relative to vehicle control, PNK (n= ulation of surface PD-L1 expression following short treatment 3 donors) showed increased cytotoxicity to ACY-241 pretreated MM duration. Correspondingly, cytotoxic T-cells isolated from these cell lines: RPMI8226 (16.3% to 34.9%), MM.1S (15.1% to 26.0%), OPM2 tumors have shown higher levels of IFN-γ production relative to (12.7% to 37.2%), and U266 (0% to 9.1%). Increased expression of li- untreated mice. In the colon cancer tumors, significant increases in gands to activating NK receptors, MIC A/B, CD56, CD54, and CD155 T-cell infiltration was observed following long treatment duration was detected from ACY-241 treated MM cells, suggesting that en- with TTFields plus anti-PD-1. gagement of NKG2D, CD11a or DNAM-1 of NK cells leads to en- Conclusions hancement of the anti-MM effect. Our results demonstrate the potential of TTFields therapy to induce In vivo anti-MM activity of PNK in combination with ACY-241 was ICD. We also demonstrate robust efficacy of concurrent application assessed in a RPMI8226 SubQ xenograft NSG model. Single intraven- of TTFields and anti PD-1 therapy in mouse models of cancer. These ous dosing of 1.0E7 PNK in combination with ACY-241 significantly data suggest that combining TTFields with anti-PD-1 might achieve reduced the tumor growth compared to vehicle control (P<0.001). tumor control by further enhancing antitumor immunity. Conclusions Our data demonstrated that enhanced in vitro anti-MM activity of Acknowledgements PNK in combination with ACY-241. In vivo efficacy of PNK in combin- The authors would like to thank Dr. Kenneth Swanson from Beth Israel ation with ACY-241 was further demonstrated in a RPMI8226 SubQ Deaconess Medical Center, and Dr. Ilan Volovitz from Tel Aviv Sourasky xenograft NSG model. Taken together, our results demonstrate the Medical Center for their helpful and constructive comments. synergistic effects of combining an HDAC inhibitor with an NK cell Ethics Approval therapy for anti-MM enhancement. Further development of a com- This study was approved by Novocure’s Ethics Board and by the Israel binatorial PNK and ACY-241 therapy for MM treatment is warranted. National Ethics Board; approval numbers 160816, 21015, IL-17-3-131 and IL- 19-1-38. Reference 1. Ray A, Das DS, Song Y, Hideshima T, Tai YT, Chauhan D, Anderson KC. P190 Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 anti- Single-day CAR manufacturing platform using mRNA and Flow body enhances anti-tumor immunity and cytotoxicity in multiple mye- Electroporation Technology loma. Leukemia. 2018 Mar;32(3):843-846. 1 1 1 1 Michael Kuo , Robert Keefe, PhD , Linhong Li, PhD , Angelia Viley , Mary 1 2 3 Loveras , Brian Mulhern , Melanie Hartsough , Claudio Dansky Ullmann, 4 1 P189 MD , Dhana Chinnasamy 1 2 Tumor Treating Fields (TTFields) induce immunogenic cell death MaxCyte Inc, Gaithersburg, MD, United States; Scilucent, Washington resulting in enhanced antitumor efficacy when combined with DC, United States; Hartsough Nonclinical Consulting, Gaithersburg, MD, anti-PD-1 therapy United States; MaxCyte, Cambridge, MA, United States 1 1 1 1 Noa Kaynan, PhD , Tali Voloshin , Shiri Davidi , Yaara Porat , Anna Correspondence: Dhana Chinnasamy (dhanac@maxcyte.com) 1 1 1 Shteingauz , Mijal Munster , Rosa Schnaiderman , Catherine Tempel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P190 1 1 1 1 1 Brami , Yaniv Alon , Einav Zeevi , Karnit Gotlib , Roni Blat , Orni Tal 1 1 1 1 Yitzhaki , Shay Cahal , Aviran Itzhaki , Eilon Kirson , Uri Weinberg, MD Background 1 2 1 1 PhD , Adrian Kinzel , Yoram Palti , Moshe Giladi MaxCyte has developed a rapid and potent cell therapy that uti- 1 2 Novocure Ltd., Haifa, Israel; Novocure GmbH, Munich, Germany lizes mRNA in transient Flow Electroporation (FEP) to produce Correspondence: Moshe Giladi (mgiladi@novocure.com) gene-modified cell products, termed CARMA™. This proprietary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P189 CARMA platform modifies peripheral blood mononuclear cells (PBMC) from apheresis to generate a cryopreserved drug product Background in a single-day manufacturing process using the cGMP-compliant, Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic closed MaxCyte GT® Transfection System, dramatically reducing treatment modality delivered via noninvasive application of low-intensity, the labor, facilities investment, and cost of raw materials typically intermediate-frequency, alternating electric fields. In this study we evalu- required for such products. The CARMA one-day manufacturing ated whether TTFields-induced cell death can be immunogenic and process using cGMP grade mRNA has the potential to therefore suitable for combination with anti-PD-1 therapy. revolutionize cell therapy strategies by significantly reducing the Methods wait time for patients receiving treatment. Cancer cells were treated with TTFields using the inovitro(TM) sys- Methods tem. Immunogenic cell death (ICD) was characterized by the expos- We report here the implementation of the CARMA platform to ure of calreticulin on the cell surface, secretion of ATP, and release of manufacture MCY-M11, a PBMC cell therapy product expressing HMGB1. For detection of ER stress, phosphorylation of eIF2α was an anti-mesothelin chimeric antigen receptor (Meso-CAR) de- assessed. TTFields effect on autophagy was evaluated using electron signed to target mesothelin-expressing solid malignancies. MCY- microscopy, and evaluation of LC3. Bone marrow derived dendritic M11 expresses the Meso-CAR in all cells in the PBMC preparation, cells (DCs) were co-incubated with TTFields treated cells and phago- which are processed and cryopreserved without the need for cytosis by DCs and DCs maturation were evaluated. The combination prior activation or selective expansion. MCY-M11 for clinical appli- of TTFields and anti-PD-1 was evaluated in short duration treatment cation is manufactured under the appropriate cGMP quality sys- protocol in orthotopic lung cancer model and long duration treat- tems and controls by MaxCyte at HCATS, a Contract Development ment protocol in subcutaneous colon cancer model. Analysis of infil- Manufacturing Organization (CDMO). Manufacturing release speci- trating cells was performed using flow cytometry. fications are preliminarily assigned, with multiple For Information Results Only (FIO) data points being accumulated during clinical produc- We demonstrate that cancer cells that die during TTFields application tion, while sufficient clinical data is being generated to establish exhibit ER stress leading to calreticulin translocation to the cell meaningful release criteria. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 104 of 272 Results lymph nodes. Given the plasticity of Th17 and Treg cells, we A total of 20 CARMA product development and engineering runs assessed FoxP3 expression within the cell product 10 days post were performed during the technology transfer campaign, with transfer and found that the frequency of FoxP3+ transferred cells analytical test methods and supply chain established. The viable was significantly heightened through IL-6 blockade. cell yield from pre-FEP to post-FEP samples averaged around Conclusions 92%. Meso-CAR expression in T and NK cell subsets in MCY-M11 IL-6 induced by Th17 cell therapy promotes an inflammatory ranged from 42-83% (average 73%) and 28-75% (average 59%), over regulatory phenotype in vivo permitting durable memory respectively. The anti-tumor bioactivity and target specificity of against tumors. The expansion of tumor-specific regulatory cells MCY-M11 was successfully established in vitro by demonstrating from the transferred product is enhanced in the absence of IL-6 antigen-specific cytotoxicity and inflammatory cytokine release in signaling. This work implies that the universal strategy of IL-6 co-culture assays with various mesothelin-expressing human inhibition for cytokine release syndrome may come at the ex- tumor cell lines. Increased survival and efficacy were also demon- pense of long-term efficacy for cell therapy approaches. strated in vivo using a human mesothelin expressing ovarian syn- Ethics Approval geneic mouse tumor model. All animal studies were approved by MUSC's IACUC committee, ap- Conclusions proval number 0488. The CARMA one-day manufacturing process using cGMP grade mRNA has the potential to revolutionize cell therapy strategies by significantly reducing the wait time for patients receiving treat- P192 ment. MCY-M11 is currently being tested in a first-in-human clin- Anti-HLA-G antigen receptor T-cells exhibit potent anti-tumor ical trial for advanced epithelial ovarian cancer and peritoneal effects against human solid tumors mesothelioma (ClinicalTrials.gov Identifier: NCT03608618). Alan Epstein, MD, PhD, Aida Kouhi, Aida Kouhi Trial Registration University of Southern California, Los Angeles, CA, United States NCT03608618 Correspondence: Alan Epstein (aepstein@usc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P192 P191 Background IL-6 fuels durable memory for Th17-mediated responses to tumors 1 1 1 HLA-G is highly expressed on human placenta during pregnancy and has Hannah Knochelmann, BS , Connor Dwyer, PhD , Aubrey Smith, BS , 1 1 1 been found to suppress the NK response to cells that lose their HLA and/ Megan Wyatt, MS , Guillermo Rangel RIvera , Jacob Bowers , Michelle 1 2 3 or beta2-microglobulin expression. [1-2]. In addition, except for pregnancy, Nelson, PhD , Gregory Lesinski, PhD, MPH , Zihai Li, MD, PhD , Mark 1 1 HLA-G is rarely expressed in normal adult tissues. Moreover, roughly 50% Rubinstein, PhD , Chrystal Paulos, PhD of human solid tumors lose their HLA expression to avoid detection by Medical University of South Carolina, Charleston, SC, United States; 2 3 the human immune system. [3] HLA-G is therefore an outstanding target Emory University, Atlanta, GA, United States; Ohio State University, for CAR T-cells since, like the placenta, it is up-regulated in HLA-negative Columbus, OH, United States tumors to suppress NK destruction. [4] We have successfully generated Correspondence: Hannah Knochelmann (knochelm@musc.edu) anti-HLA-G CAR T-cells to treat solid tumors that express HLA-G. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P191 Methods An anti-HLA-G CAR construct was generated by fusing anti HLA-G Background scFv to a second generation CAR containing the CD8α leader se- Accessibility of T cell transfer therapies for most patients is hin- quence, 4-1BB co-stimulation sequence, and CD3ζ signaling domain. dered by cost and time required for product development. Our The CAR vector was then fused with a lentivirus vector in-frame with lab has shown that shortening ex vivo expansion of Th17 cells the CAR backbone, and was used to transduce primary human CD3 licenses a proinflammatory cell product which induces cytokine positive T-cells. After transduction, expanded CAR-T cells were char- stormwithhighlevelsofsystemicIL-6intumor-bearing hosts. acterized for their ability to bind HLA-G antigen and HLA-G positive Despite potential toxicity, briefly expanded Th17 cells eradicate SKOV-3 cells (human ovarian cancer model) using flow cytometry. large established tumors in low doses and generate durable Results memory against tumor rechallenge, suggesting a therapeutic Expanded CAR-T cells were able to bind successfully both the benefit to the inflammatory state. Prior reports show that IL-6 HLA-G antigen and SKOV-3 cells in vitro. Expanded CAR-T cells promotes functional CD4+ T cell memory formation. Given that were then co-cultured with SKOV3-Luc cells and studied for their IL-6 is blocked clinically to manage cytokine release syndrome, epitope-driven cytotoxicity. Anti HLA-G CAR T-cells displayed we addressed the physiologic impact of IL-6 on efficacy and dur- dose-dependent cytotoxicity when co-cultured with tumor cells. ability of Th17 cell therapy. We have recently developed an in vivo model of ovarian cancer Methods that can be used for testing the efficacy of our CAR-T cells. In Th17 cells were expanded ex vivo using the TRP-1 transgenic this model, NSG mice are injected with 2 million SKOV3-Luc cells mouse model in which CD4+ T cells express a TCR that recog- intraperitoneally (ip). Seven-10 days after injection, tumors are nizes tyrosinase-related protein 1 on melanoma. Naïve CD4+ T visible when observed by bioluminescence imaging, at which cells were polarized to the Th17 phenotype and infused into time the treatment group will receive an ip injection of anti HLA- mice with B16F10 melanoma after a nonmyeloablative total G CAR-T cells. Since ovarian cancer rapidly metastasizes to the body irradiation (5 Gy) preparative regimen. Serum cytokine peritoneum, the aforementioned model should provide relevant levels were obtained by multiplex array and IL-6 signaling was clinical data that can be translated to patients, and like inhibited with antibodies targeting the IL-6R and neutralizing IL- hematopoietic cancers, will present antigen quickly after injection 6cytokine. of CAR T-cell to keep them stimulated and functional. Results Conclusions Acute IL-6 blockade post Th17 cell transfer did not impact the We are currently testing the efficacy of anti-HLA-G CAR-T cells primary response against melanoma nor the engraftment of Th17 in vivo using this ip model, and plan to show that HLA-G as a cells. However, blocking IL-6 abrogated long-term responses in- pan tumor target will provide selective and specific cell based creasing the frequency of tumor relapse upon secondary chal- therapy which may in the near future be clinically relevant for lenge and reduced survival. Mechanistically, IL-6 blockade chemotherapy resistant ovarian cancer and other tumors. reduced phosphorylation of STAT3 in transferred T cells associat- ing with diminished Bcl-2 expression. The CD4+ compartment Acknowledgements was reshaped by IL-6 blockade via promoting a greater frequency This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. of FoxP3+ Treg cells in the peripheral blood, tumor and draining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 105 of 272 References Immetacyte is assessing this approach in our current Phase I/II clinical 1. Apps R, Gardner L, Moffett A. A critical look at HLA-G. Trends Immunol. trial of TIL in ovarian cancer patients (EudraCT–2019-000106-30). 2008; 29:313–321. 2. Jurisicova A, Casper RF, MacLusky NJ, Mills GB, Librach CL. HLA-G expres- Acknowledgements sion during preimplantation human embryo development. Proc. Natl. This research was supported by IUK projects 133299 and 104468 Acad. Sci. U. S. A. 1996; 93:161–5. Ethics Approval 3. de Kruijf EM et al. HLA-E and HLA-G expression in classical HLA class I- This study was approved by the South Central Research Ethics Committee : negative tumors is of prognostic value for clinical outcome of early 19/SC/0355 breast cancer patients. J. Immunol. 2010; 185:7452–9. 4. Lin, A. & Yan, W.-H. Human Leukocyte Antigen-G (HLA-G) Expression in P194 Cancers: Roles in Immune Evasion, Metastasis and Target for Therapy. Use of stimulatory cells in conjunction with IL-12 and IL-18 Mol. Med. 2015; 21:782–791. augments NK cell expansion and transduction, drives a Ethics Approval memory phenotype, and improves in vitro and in vivo CAR This study was approved by the IRB of the University of Southern California NK activity protocol #HS-16-00029 on 2-29-16. Anmol Vohra, MS, Katherine Jamboretz, MS, Sasha Lazetic, Denise Gonzalez, Daofeng Liu, PhD, Ivan Chan, PhD, James Trager, PhD P193 Nkarta Inc, South San Francisco, CA, United States CoStAR (Costimulatory Antigen Receptor) enhancement of tumour Correspondence: James Trager (jtrager@nkartatx.com) infiltrating lymphocyte therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P194 Gray Kueberuwa, PhD, John Bridgeman, PhD, Martina Sykorova, Milena Kalaitsidou, Michelle Le Brocq, Robert Hawkins Background Immetacyte, Manchester, Manchester County, United Kingdom NK cells have been expanded on K562 stimulatory cells express- Correspondence: Robert Hawkins (r.hawkins@immetacyte.com) ing membrane-bound (mb) IL-15 and 41BBL for clinical use, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P193 can be genetically modified to express activating chimeric recep- tors [1,2,3]. Engineered NK cells targeting CD19 or ligands of Background NKG2D show in vitro and in vivo cytotoxicity against relevant The efficacy of TIL therapy is limited in some patients due to the tumor targets that can overcome endogenous resistance to NK failure of the cells to respond to tumour sufficiently or persist cells. NK cells activated in the presence of IL-12, IL-15 and IL-18 long enough to have a necessary anti-tumour effect. We have ad- develop cytokine induced memory-like phenotype and function; dressed this issue by developing Co-stimulatory receptors (Co- these cells have shown clinical promise [4]. Here we describe NK StARs) that provide enhanced signaling to tumour-specific T-cells cell function and phenotype achieved by combining the robust upon encountering tumour associated antigens. driven by K562-mbIL15-41BBL with the induction of a cytokine- Since tumour reactivity is determined by natural TCRs that have induced memory phenotype achieved after exposure to IL-12 and undergone thymic selection, this approach does not bear with it the IL-18. risks of other therapies targeting tumour antigens expressed on the Methods cell surface Healthy donor PBMC NK were expanded on K562-mbIL15-41BBL Methods stimulatory cells with IL-2 alone or with IL-2 plus IL-12 and IL-18 In order to identify optimal signalling domain for Co-StAR mole- (12-18). We compared NK cell expansion, cytokine secretion, cyto- cules, several iterations of our prototype receptor were synthe- toxicity against tumor lines at various time points, and persist- sised. The ability of each to enhance T-cell activation, ence in culture over 4 weeks. The expanded NK were transduced proliferation, secretion of cytokines and increase resistance to with CD19 and NKG2D CAR constructs, and the resulting cells apoptosis were assessed. evaluated for CAR expression, cytotoxicity and in vivo efficacy To explore if this approach has the potential of wide applicabil- against relevant cell lines. ity, we went on to assess targeting of two additional ovarian Results cancer tumour associated antigens. To achieve this, the antigen Addition of 12-18 to the K562-mbIL15-41BBL stimulatory cells im- binding moiety was exchanged and signaling domain kept proves NK expansion 2-3 fold [If we have a p value, it’s better to constant. give it:<br>‘…significantly improves NK expansion 2-3 fold (p<x) Results relative to that…’][Will put together later for the poster]relative We show that colorectal cancer specific Co-StAR significantly en- to that achieved using the stimulatory cell line alone, while NK hances the number of T-cells expressing IL2, TNFα,41BB,CD107a cell cytotoxicity is unchanged. IFNγ and TNFα production and and bcl-xL by factors ranging from 2-4 fold in model systems. transduction efficiency are also improved in this setting. Over 3 This shows an increase in activation, effector function and resist- weeks of culture following brief exposure to 12-18, NK cell ance to apoptosis. We also identified an optimal signaling do- phenotype changes, with an increased percentage of CD62L+ main that caused the greatest magnitude of enhancement for and NKG2C+ cells, and increased NKG2C expression per NK cell. the above factors. While the NKG2D-CAR driven cytotoxic activity is unchanged by Observations of Co-StAR enhancement were mirrored in model 12-18 at 14 days post-exposure, cytotoxic activity increases in systems for ovarian cancer, targeting two separate ovarian cancer these cells by day 21. In addition, this improved cytotoxicity at tumour associated antigens. day 21 is reflected by improved CD19-CAR driven in vivo activity In addition, stimulation assays showed that Co-StAR with optimal sig- against the CD19+ tumor target [Raji or Nalm6?]Nalm6 [Oops, naling domain increased T-cell proliferation over 3 weeks in compari- Nalm6, thx.] with an increased presence of circulating NK cells son to prototype Co-StAR, Co-StAR that binds an irrelevant target, or over 4 weeks in the mice. indeed, mock transduced T-cells. Conclusions Conclusions The data demonstrates that the addition of IL-12 and IL-18 to Our optimal Co-StAR provides a means to effectively deliver “signal K562-mbIL15-41BBL stimulatory cells during NK expansion main- 2” to T-cells. Enhancing activation, effector functions and resistance tains in vitro cytotoxicity and improves expansion, transduction, to apoptosis upon contact with target tumour cells. persistence, and in vivo efficacy. Further, IL-12 and IL-18 drive de- Since T-cell activation primarily requires “signal 1”, application to of velopment of a memory phenotype and more sustained NK cell Co-StAR to enhance TIL therapy, which works through natural, thymi- function over the course of several weeks. This activation system cally selected TCRs, provides a means to increase the activity of may allow for development of more robust and potent engi- tumour-reactive TIL without risking severe off-tumour side effects. neered NK cells for clinical use. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 106 of 272 References Conclusions 1. Lapteva N, Durett AG, Sun J, Rollins LA, Huye LL, Fang J, Dandekar V, These promising results infer that adoptive transfer of transgenic T Mei Z, Jackson K, Vera J, Ando J, Ngo MC, Coustan-Smith E, Cam- cells can offer an effective strategy to not only prevent further tumor pana D, Szmania S, Garg T, Moreno-Bost A, Vanrhee F, Gee AP, Roo- growth as rapamycin therapy does, but also to treat and even elimin- ney CM. Large-scale ex vivo expansion and characterization of ate arising tumors. This strategy might offer a cure for patients with natural killer cells for clinical applications. Cytotherapy. LAM, a disease that hits women in the prime of their lives. 2012;14(9):1131-1143 2. Chihaya I, Iwamoto S, Campana D. Genetic modification of primary Acknowledgements natural killer cells overcomes inhibitory signals and induces specific Studies supported by a DoD Tuberous Sclerosis Complex Research Program killing of leukemic cells. Blood. 2005; 106:376-383. Clinical Translational Research Award to CLP. 3. Yang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Ethics Approval Chimeric Receptor with NKG2D Specificity Enhances Natural Killer All animal experiments were approved by the Animal Care and Use Cell Activation and Killing of Tumor Cells. Cancer Res. Committee of Northwestern University and followed the institutional 2013;73(6):1777-1786 guidelines; protocol number IS00008259. 4. Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, Neal CC, Yu L, Oh ST, Lee YS, Mulder A, Claas F, Cooper MA, Fehniger TA. Cytokine-induced P196 memory-like natural killer cells exhibit enhanced responses against mye- The first step toward the universal cell therapy: Simultaneous loid leukemia. Sci Trans Med. 2016;8(357): 357ra123 removal of HLAs (Human leukocyte antigens) using CRISPR- Ethics Approval mediated quadruple genome editing in allogeneic T cells Animal studies were conducted and approved by the Explora IACUC Jeewon Lee, Ph D, Munkyung Kim, Joong Hyuk Sheen, Jihye Ryu, Yu committee. Young Kim, Okjae Lim MOGAM Institute for Biomedical Research, Yongin-si, Gyeonggi-do, Republic of Korea P195 Correspondence: Okjae Lim (blubelle@mogam.re.kr) Loss of function of the TSC1-TSC2 complex renders tumors eligible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P196 for GD3 CART therapy 1 1 1 Ancy Thomas , Saurav Sumughan , Zhussipbek Mukhatayev , Emilia 1 1 2 2 Background Dellacecca , Nicola Lancki , Levi Barse , Jesus Zamora-Pineda , Suhail 2 2 1 2 Chimeric antigen receptor (CAR) T cell therapy is the revolution- Akhtar , Maria Picken , Denise Scholtens , Daniel Dilling , Richard 3 1 ary treatment of choice for hematologic malignancies. Currently Junghans, PhD, MD , Caroline Le Poole 1 2 approved CAR T therapies require patients’ own immune cells, Northwestern University, Chicago, IL, United States; Loyola University, and this autologous T cell manufacturing process involves certain Maywood, IL, United States; Boston University, Boston, MA, United limitations primarily derived from the nature of individualized States therapy. Thus, engineering allogeneic donor cells to evade host Correspondence: Caroline Le Poole immune rejection is required for a broader clinical application of (caroline.lepoole@northwestern.edu) the therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P195 Methods In this study, we attempted to inhibit expression of both HLA I Background and II through the CRISPR/Cas9 gene editing system to reduce Benign tumors can arise from bi-allelic mutations in a single gene. In tu- allo-reactive immune rejection response. First, we screened 60 berous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), gRNAs targeting B2M and 60 gRNAs each targeting alpha chains tumors do not acquire additional mutations, and patients are not eli- of HLA-II molecules (DP, DQ and DR, respectively) to find gRNA gible for therapeutics that rely on neoantigen formation. However, the sequences efficiently ablate expression of HLA molecules on T affected gene is responsible for several predictable phenotypic cell surface. Next, we investigated whether the absence of HLA-I/ changes. As mTOR hyperactivity resulting from mutations in TSC1 or II expression in donor T cells could alleviate immune response TSC2 is associated with overexpression of some melanoma-associated from allogeneic responders using in vitro mixed lymphocyte reac- antigens, de novo expression of ganglioside D3 expression may render tion (MLR) assays. the resulting, benign tumors eligible for immunotherapy. Results Methods We have identified gRNA sequences highly efficient in targeting We probed the expression of GD3 in human TSC lesions of the lungs, B2M and alpha chains of HLA-II molecules without carrying off- kidneys, skin and brain by immunostaining and monitored anti-GD3 target effects. Selected gRNA sequences for HLA-II ablation cov- titers in serum by ELISA. Infiltration by NK cells and NKT was mea- ered the vast majority of each HLA-II alpha chain allele. HLA-I/II sured to look for natural responses to the cell surface antigen. We double negative T cells generated by simultaneous quadruple next isolated tumors cells from TSC2 heterozygote mice and con- genome editing with the selected gRNAs maintained their pheno- firmed loss of heterozygosity by genotyping before challenging types and cytotoxicity upon TCR stimulations compared to the groups of 10 SCID/beige mice in 2 repeat experiments, and subject- control cells treated with non-target gRNA. Furthermore, the MLR ing them to adoptive transfer by GD3-CART cells and measured assays showed that IFN- and TNF-α production in allo-responder tumor sizes over time. Similarly we treated groups of 8 ageing TSC T cells was significantly decreased in the absence of donor HLA-I mice >16 months of age by adoptive GD3 CART-cell transfer, and alone and was further diminished in response to HLA-I/II double measured surface tumor growth on internal organs. negative donor T cells compared with the control cells, implicat- Results ing prolonged survival of the adoptively transferred immune cells. We found consistent overexpression of GD3 in tissues from TSC pa- Conclusions tients compared to healthy controls. GD3 overexpression was not ac- In conclusion, we have identified novel gRNA sequences ablat- companied by an influx of NK(T) cells, and anti-GD3 titers were ing expression of HLA molecules on donor T cell surfaces to reduced rather than elevated in patients, supporting the concept dramatically reduce donor-derived allo-responses, establishing that slow growing tumors in TSC patients are not immunogenic. an essential cornerstone towardsthe universalTcell therapy. However, CART cells responsive to GD3, supplemented by IL-2, medi- Ethics Approval ated prolonged and significant anti-tumor responses in both immu- Human PBMCs were obtained from healthy volunteers by leukapher- nodeficient and immune competent hosts. The majority of TSC2 esis from the Samsung Medical Center (SMC) under IRB approval heterozygote mice treated by CAR T cells displayed no tumors at (SMC IRB no.2018-01-089). end point, versus all mice treated with untransduced T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 107 of 272 P197 we developed a novel modular universal CAR platform termed Uni- The development of an autologous neoantigen specific T cell CAR. UniCAR T-cells are exclusively activated in the presence of a tar- product from peripheral blood, NEO-PTC-01, through the ex-vivo get module (TM), which establishes the cross-link between antigen- induction protocol, NEO-STIM™ specific cancer cells and UniCAR T-cells in an individualized time- 1 1 1 Divya Lenkala, MS , Marit Van Buuren, PhD , Brian McCarthy , Jessica and target-dependent manner. The carbohydrate antigen sialyl-Tn 1 1 1 1 Kohler, PhD , Michael Nelson , Flavian Brown , Yvonne Ware, MS , (STn) is a particularly interesting target due to its expression in sev- 1 1 Yuting Huang, MS , Janani Sridar , Yusuf Nasrullah, MS, United eral types of cancer and absence in normal healthy tissues. Given the 1 1 2 Kingdom , Dewi Harjanto , Joost Van Den Berg, PharmD , Matthew small size of such TMs, they are rapidly eliminated and thus, possible 3 1 Goldstein, MD, PhD , Richard Gaynor, MD side effects and activation of UniCAR T-cells can be easily controlled 1 2 Neon Therapeutics, Cambridge, MA, United States; Netherlands Cancer by TM dosing. In late phases of treatment, TMs with extended half- Institute, Amsterdam, Netherlands; Tango Therapeutics, Cambridge, MA, life may play an important role by improving the eradication of re- United States sidual tumor cells. Correspondence: Marit Van Buuren Methods (mvanbuuren@neontherapeutics.com) In this work, a novel longer-lasting TM against STn was developed, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P197 characterized and compared to the previously developed short-lived anti-STn TM [1]. Short-lived TMs are composed of a tumor-specific Background binding moiety fused to the La peptide epitope (E5B9) which is rec- Neoantigens are tumor-specific antigens that have been shown to be ognized by UniCAR T-cells. In extended half-life TMs, these two com- important in the anti-tumor immune response. These antigens are ponents are fused via an Fc domain derived from the human IgG4 not subject to central immune tolerance and are therefore potentially molecule. Functional and pharmacokinetic properties were assessed more immunogenic than tumor-associated antigens. The goal of our using in vitro and in vivo assays. studies is to generate neoantigen specific T cell responses and per- Results form detailed characterization of the induced T cell responses to- The developed anti-STn IgG4-based TM efficiently activates and redi- wards these neoantigen targets to assess the applicability of the rects UniCAR T-cells to STn-expressing tumors in a highly efficient approach for adoptive cell therapy. target-specific and target-dependent manner, promoting the secre- Methods tion of pro-inflammatory cytokines, tumor cell lysis of breast and Patient-specific neoantigens were predicted using our RECON® bio- bladder cancer cells in vitro and of breast cancer cells in experimen- informatics platform, and the predicted high-quality neoantigens tal mice. A comparable or increased killing efficiency was obtained at were utilized in our proprietary ex-vivo stimulation protocol, NEO- a lower concentration range in comparison to the results obtained STIM to assess immunogenicity. NEO-STIM is used to prime, activate for the anti-STn scFv-based TM. Additionally, PET studies demon- and expand memory and de novo T cell responses from both the strate the specific enrichment of the anti-STn IgG4-based TM at the CD4+ as well as the CD8+ compartment. In-depth analysis was per- tumor site presenting a prolonged serum half-life compared to the formed to characterize the specificity, functionality (cytokine produc- scFv short-lived TM. tion and cytolytic capacity) and diversity of the induced T cell Conclusions responses through high throughput flow cytometric analysis. Taken together, these data demonstrate the effective and potential Results application of this CAR T-cell-derived modular system to target STn Here we present the successful induction of memory and de novo in different types of cancer using different TM formats. The use and CD8+ and CD4+ T cell responses in peripheral blood mononuclear combination of such molecules with different formats and half-lives cells isolated by leukapheresis from five melanoma patients using provides highly promising and customized tools for retargeting of NEO-STIM. We then extensively characterized these T cell responses UniCAR T-cells in a flexible, individualized and safe manner at differ- and show that these responses are functional, specific and have cyto- ent stages of treatment. lytic capacity. Conclusions Reference NEO-STIM is a novel platform to understand in detail the immuno- 1. Loureiro L R, Feldmann A, Bergmann R, Koristka S, Berndt N, Arndt C, genic potential of high-quality neoantigen-targets. Moreover, this Pietzsch J, Novo C, Videira P, Bachmann M. Development of a novel platform can be utilized to generate T cell products from peripheral target module redirecting UniCAR T cells to Sialyl Tn-expressing tumor blood for adoptive cell therapy for patients with a variety of solid cells. Blood Cancer J. 2018; 8(9): 81. tumors. Ethics Approval Ethics Approval All animal activities and procedures were performed in accordance with the The samples for the study were collected under ClinicalTrials.gov: protocols approved by the Institutional Review Board at Semmelweis NCT02897765 and N16NEON protocol University - Budapest, approval number PE/EA/50-2/2019. P198 P199 Short-lived and extended half-life target modules for redirecting Generation of functionally and phenotypically mature, allogeneic UniCAR T-cells against sialyl-Tn expressing cancer cells natural killer cells from human induced pluripotent stem cells 1 1 1 Liliana Loureiro, PhD , Anja Feldmann, PhD , Ralf Bergmann , Stefanie under chemically-defined, feeder- and serum- free culture 1 1 2 2 Koristka , Nicole Berndt , Nikolett Hegedüs , Domokos Máthé , Paula conditions 3 1 1 Videira , Michael Bachmann , Claudia Arndt Kyle Lupo, BS, Andrea Chambers, MS, Sandro Matosevic, PhD Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; Purdue University, Lafayette, IN, United States 2 3 Semmelweis University, Budapest, Hungary; Faculdade de Ciências e Correspondence: Sandro Matosevic (sandro@purdue.edu) Tecnologia/UNL, Caparica, Portugal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P199 Correspondence: Liliana Loureiro (l.loureiro@hzdr.de) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P198 Background While targeted immunotherapy with engineered natural killer (NK) Background cells has emerged as a promising approach for the treatment of solid The development of chimeric antigen receptors (CARs) has rapidly tumors, challenges in sourcing, processing, and genetically modifying emerged as a promising approach in cancer immunotherapy. None- blood-derived NK cells limit the potential for developing life-saving theless, drawbacks associated with CAR T-cell therapies include on- treatments for cancer patients. As an alternative, the use of induced target/off-tumor effects and cytokine release syndrome. Aiming an pluripotent stem cells (iPSCs) offers a promising approach to over- increased clinical safety while preserving the efficacy of such therapy, coming existing challenges faced when engineering NK cell-based Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 108 of 272 immunotherapies. However, approaches for generating NK cells from antigen-specificity, higher CD8:CD4 ratio and ability to persist long- iPSCs described so far have several shortcomings: they utilize sera or term [3]. Based on these differences, we hypothesized that MILsTM feeder layers to adapt iPSCs or culture hematopoietic progenitors, would provide a more robust platform for CAR-T therapy compared take months to complete, and rely on individualized, and thus highly to PBLs. We have previously shown that CAR-modified MILsTM (CAR- variable, iPSC reprogramming protocols, limiting their utility. MILsTM) demonstrate superior killing of tumor target cells in vitro Methods compared to CAR-T cells generated from patient-matched PBLs (CAR- We have generated NK cells from iPS cells using a novel feeder-free PBLs) [4]. In this study, we compared, at the single cell level, func- differentiation protocol starting from either centrally-validated and tionality of patient-matched CAR-MILsTM and CAR-PBLs following banked iPSC lines or iPSCs reprogrammed from donor fibroblasts. antigen-specific in vitro stimulation. Our protocol utilizes a two-step, entirely feeder-free procedure in- Methods volving hematopoietic progenitor generation followed by NK differ- CAR-MILsTM and CAR-PBLs engineered to express a BCMA-specific, 4- entiation. These differentiated NK cells have been characterized for 1BB/CD3z-signaling CAR were produced using cryopreserved lympho- inhibitory and activating marker expression, IFN-γ production, de- cytes from the bone marrow and blood of six patients with multiple mye- granulation, and cytotoxicity against a number of solid tumor targets, loma. CD4 and CD8 T cells isolated from the CAR-MILsTM and CAR-PBLs including primary patient-derived glioblastoma cells. Moreover, these products were stimulated with K562 cells transduced with either BCMA cells were expanded in culture and manipulated to generate a cyto- (K562-BCMA) or nerve growth factor receptor (K562-NGFR) at a ratio of toxic infusible cell therapy product. 1:2 for 20 hrs. After 20 hrs of co-culture, T cells were enriched and loaded Results into IsoCode chips containing ~12,000 microchambers pre-patterned iPS cells were differentiated into hematopoietic progenitor cells, with a 32-plex antibody array. Protein secretion from 1000-2000 single T yielding CD34+/CD45+ and CD34+/CD43+ cell populations at yields cells per product was detected by a fluorescence ELISA-based assay and consistent with results described in literature using feeder-based pro- single cell polyfunctional profiles analyzed using IsoPeak (IsoPlexis). tocols [1]. Following four weeks of NK cell differentiation, cells Results showed high expression of several NK cell maturation markers as CD4 and CD8 T cells from both CAR-MILsTM and CAR-PBLs demon- well as inhibitory and activating receptors (CD56+/CD3-, NKG2D, strated an antigen-specific increase in polyfunctionality (secretion of 2+ NKp30, NKp44, NKp46, DNAM-1, CD16, CD94/NKG2A, and CD158b). cytokines per cell) and polyfunctional strength index (PSI) in response iPSC-NK cells derived using our protocol were also similar to blood- to BCMA stimulation compared to NGFR control. When compared to derived NK cells in morphology, expansion rate, and functionality (in CAR-PBLs, CAR-MILsTM demonstrated increased polyfunctionality and terms of cytotoxicity and degranulation potential). By using centrally- increased PSI in both CD4 and CD8 T cells. The enhanced PSI in CAR- validated iPSC lines, we further demonstrate our ability of avoiding MILsTM was predominated by effector, stimulatory and chemoattrac- donor-specific reprogramming protocols. tive proteins associated with antitumor activity including Granzyme B, Conclusions IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and en- We developed a new protocol for the generation of NK cells from hanced secretion of these same proteins was reported to be associated iPSCs that is entirely feeder and serum-free and can be extended to with improved clinical responses in patients with Non-Hodgkin lymph- the use of validated iPSC lines avoiding donor and reprogramming oma treated with CD19-specific CAR-T therapy [5]. variability. iPSC-derived NK cells using our protocol exhibit character- Conclusions istics of mature blood-derived NK cells and powerful cytotoxicity Based on these data and the inherent antitumor properties of against solid tumor targets. Additionally, these cells offer the advan- MILsTM, we speculate that CAR-MILsTM would be more potent tage of increased expansion rates, improved ease of transfection and effective than currently approved CAR-T products derived while in the iPSC state, and are free of contaminating T-cells associ- from PBLs. ated with GvHD risk, overcoming many limitations of existing NK cell based immunotherapies. References 1. Borrello I and Noonan KA, Marrow-Infiltrating Lymphocytes – Role in Biol- Reference ogy and Cancer Therapy. Front Immunol. 2016 March 30; 7(112) 1. Bock A, Knorr D, and Kaufman D. Development, Expansion, and In Vivo 2. Noonan K.A., Huff C.A., Davis J., Lemas M. V., Fiorino S., Bitzan J., Ferguson Monitoring of Human NK Cells from Human Embryonic Stem Cells A., Emerling A., … Borrello I. Adoptive transfer of activated marrow- (hESCs) and Induced Pluripotent Stem Cells (iPSCs). Journal of Visualized infiltrating lymphocytes induces measurable antitumor immunity in the Experiments : JoVE 74 (2013): 50337. bone marrow in multiple myeloma. Sci. Transl. Med. 2015; 7: 288ra78. 3. Noonan KA, Rudraraju L, Hoyos V, Lutz E and Borrello I. Persistence of Non Gene-Modified Adoptively Transferred Marrow Infiltrating Lympho- P200 cytes (MILs) More Than Five Years Post Transfer. Blood 2016 128:4552. CART-engineered Marrow-infiltrating Lymphocytes (MILsTM) are 4. Lutz ER, Hoyos V, Rudraraju L, DeOliveira E, Jana S, Weiss I, Borrello IM, more polyfunctional than their matched peripheral blood Noonan K. Marrow-infiltrating Lymphocytes (MILs) provide a robust plat- counterparts form for CAR-T cell therapy. Blood 2018 132:3337. 1 1 1 Eric Lutz, PhD , Lakshmi Rudraraju, MS , Elizabeth DeOliveira , Srikanta 5. Rossi J, Paczkowski P, Shen Y, … Bot A. Preinfusion polyfunctional anti- 1 2 2 3 Jana , Jing Zhou, MD, PhD , Sean Mackay, MBA , Ivan Borrello, MD , CD19 chimeric antigen receptor T cells are associated with clinical out- Kimberly Noonan, PhD comes in NHL. Blood 2018 132(8):804-814. 1 2 WindMIL Therapeutics, Baltimore, MD, United States; Isoplexis, Ethics Approval Branford, CT, United States; Johns Hopkins University, Baltimore, MD, The study was approved by the Johns Hopkins University IRB. United States Correspondence: Eric Lutz (lutz@windmiltherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P200 P201 Comparison of phenotype and anti-tumor profile of CD19-CAR-T Background cells generated from either umbilical cord blood- or peripheral WindMIL Therapeutics is developing Marrow-infiltrating Lymphocytes blood-derived T lymphocytes 1 1 1 (MILsTM), a novel form of adoptive T cell therapy composed of bone Cristina Maccalli, PhD , Dhanya Kizhakayil, PhD , Shilpa Ravindran, BSc , 1 1 1 marrow-derived, patient-autologous, polyclonal CD4 and CD8 T cells Saad Rasool , Rebecca Mathew , Valentina Mattei , Monica Casucci, 2 1 1 1 [1]. Genetically unmodified MILsTM have demonstrated antitumor ac- PhD , Sara Deola, MD, PhD , Chiara Cugno, MD , Damien Chaussabel , 1 1 tivity in patients with multiple myeloma [2] and are being developed Sara Tomei, PhD , Christof von Kalle , 1 2 for several other tumor types. Distinguishing features of T cells from Sidra Medicine, Doha, Qatar; San Raffaele Scientific Institute, Milan, Italy bone marrow compared to T cells from peripheral blood lympho- Correspondence: Cristina Maccalli (cmaccalli@sidra.org) cytes (PBLs) include their memory phenotype, inherent tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P201 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 109 of 272 Background Background T lymphocytes expressing antigen-specific chimeric receptors (CARs) Natural killer (NK) cells are innate immune cells with a critical role in im- have been revealed as a powerful therapeutic approach for aggres- mune surveillance against cell transformation and tumor development. NK sive and refectory childhood and adult B cell malignancies. Umbilical cells express an array of unique activating and inhibitory receptors whose cord blood cells (UCB), with their unique capacity of broad leukocyte aggregate signaling determine activation of NK cell effector function. antigen (HLA)-matching, can represent an appealing starting material Adoptive transfer of NK cells has demonstrated the potential to induce an- for the generation of “off-the shelf” CAR-T cells to render this type of titumor responses in the clinic. Celularity has developed a platform for therapy accessible to a large number of cancer patients. generating cytotoxic NK cells from placental CD34+ cells (PNK cells) for Methods adoptive cancer immunotherapy. Although PNK cells demonstrate cytotox- CAR-T cells have been generated from either UCB (N=5, ALLCELLS, icity against diverse cancer cell types, their activating mechanisms are little USA) and peripheral blood lymphocytes (PBLs; N=2) from healthy do- characterized. In this study, we explore the contribution of specific signal- nors. In vitro enriched T cells have been transduced with CD19- ing pathways and upstream NK cell receptors involved in PNK cell cytotox- CD28z-CD3z and CD19-4-1BBz-CD3z encoding lentiviral vectors (LVs). icity against glioblastoma multiforme (GBM) cell targets. Deep phenotype characterization of these CAR-T cells has been per- Methods formed utilizing an in-house designed IF multiparametric (28 PNK cells were transcriptionally profiled using scRNAseq and qRT-PCR markers) panel. Functional assays have been performed to assess to identify candidate pathways regulating cytolytic function. Expression cytokine (IFN-gamma, IL2, IL-5 and IL-17), perforin and granzyme B of major receptors and intracellular signaling molecules were analyzed release (EliSpot or multicolor FluoroSpot) and cytotoxic activity (Del- using flow cytometry and western blot. PNK cell phenotype was com- fia assay) by CAR-T cells following the co-culture with CD19+ or pared to circulating NK cells. PNK cytotoxicity was evaluated against CD19- target cells. In addition, transcriptomic modular repertoire [1- GBM cell lines (LN-18 and U251) in xCELLigence platform and a de- 3] has been applied by parallel quantitative PCR using the high granulation assay using CD107a staining. The role of key signaling path- throughput BioMark HD platform to determine gene expression pro- ways driving PNK effector functions was analyzed in cytolysis assays file of CAR-T cells described above. using small molecule inhibitors of Src kinases, SYK, PLC-γ,PI3Kand Results MAP kinases, including JNK, p38 and ERK. Efficient LV transduction was achieved for UCB-T cells, although requir- Results ing higher MOI as compared to PBL (25 vs. 5; 66-80 vs. 70-88 % of PNK cells highly express genes mediating NK cell effector functions, includ- transduction, respectively). The frequency of CD4+ transduced T cells ing NCR1, NCR2, NCR3, KLRK1 and CD226. Flow cytometry demonstrated (45-59% of positive cells) was superior in UCB as compared to PBL (27- increased expression of NKp44 (99.7±0.2% vs. 69.3±5.2%), NKG2D (68.7± 36% of positive cells) while transduced CD8+ T cells were 18-20 and 7.9% vs. 44.6±5.8%) and GITR 99.7±0.2% vs. 15.0±3.2%) on PNK cells when 40-67%, respectively. CB-CAR-T cells were enriched of compared to circulating NK cells. PNK cells demonstrated strong cytolytic CD45RA+CCR7+CD27+CD62L+ T cells. These cells co-expressed ICOS activity against multiple GBM cell lines. While inhibitors of Src, SYK, PLC-γ, and 4-1BB but these molecules were not detectable on PBL-CAR-T cells. p38 and ERK did not modulate PNK cytotoxicity, inhibitors targeting JNK Markers associated with late differentiation/exhaustion of T cells, such and PI3K pathways significantly suppressed PNK cell cytotoxicity, specific- as LAG-3 and TIM-3, were found only on PBL-CAR-T cells. PD-1 was ally, 64.9±4.7% inhibition by SP600125 (JNK) and 25.2±3.3% inhibition by expressed at higher levels in CB- vs. PBL-derived CAR-T cells (15-40% Ly294002 (PI3K) on LN-18 cells; 100% inhibition by SP600125 and 45.5.5± and 40-60% in CD45RA+ and CD45RO+ T cells, respectively). 8.9% by Ly294002 on U251. JNK and PI3K inhibitors also reduced degranu- Antigen-specific reactivity was shown by CAR-T cells either isolated lation (70.6±3.2% by SP600125 and 58.4±3.6% by Ly294002). Furthermore, from UCB or PBL against acute lymphoblastic leukemia or EBV-B cells PI3K pathway controlled PNK cytokine production upon coculture with overexpressing CD19. U251 cell line, whereas JNK inhibition had minimal effect. Interestingly, differential gene expression profiles were assessed Conclusions through the comparison of UCB- vs. PBL-derived CAR-T cells and Our results demonstrate the importance of PI3K and JNK pathways in their co-culture with antigen-specific target cells. Genes differentially mediating PNK cytotoxicity to GBM cell line targets. These data com- expressed in CD19-CD28z-CD3z vs. CD19-4-1BBz-CD3z CAR-T cells bined with our receptor profiling on PNK cells establish the rationale were also found. for further investigating receptor-ligand interactions that directly Conclusions modulate PI3K and JNK activity. Taken together, these results proved that anti-tumor early differenti- ated/central memory CAR-T cells can be efficiently isolated from UCB P203 with distinctive phenotype as compared to PBL-CAR-T cells. Discovery and characterization of the first fully human Phosphopeptide Tumor Target-specific T cell receptor References 1 1 2 Xavier Michelet, PhD ,Eleni Chantzoura,PhD , Ekaterina Breous-Nystrom, PhD , 1 Chaussabel, D. and N. Baldwin. Democratizing systems immunology with 2 1 1 1 Alessandra Franchino ,RachelSmith , Daniel Pollacksmith ,Jan Bergmann , modular transcriptional repertoire analyses. Nat Rev Immunol, 2014, 1 2 2 2 Alvaro Sebastian Yague , Paisley Myers, PhD , Erin Jeffrey , Benjamin Wolf , 14:271-80; 2 1 1 Dennis Underwood, PhD ,Marc Van Dijk,PhD ,ArthurHurwitz, PhD 2 Altman MC., Rinchai D., Baldwin N., Whalen E., Garand M., Ahamed 1 2 Agentus Therapeutics, Lexington, MA, United States; Agenus, Basel, Kabeer B., et al. A Novel Repertoire of Blood Transcriptome Modules Switzerland Based on Co-expression Patterns Across Sixteen Disease and Physio- Correspondence: Arthur Hurwitz logical States. 2019, bioRxiv; doi: https://doi.org/10.1101/525709. (andy.hurwitz@agentustherapeutics.com) 3 Altman MC., Baldwin N., Whalen E., Al-Shaikhly T., Presnell S., Khaenam P., Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P203 et al. A Transcriptome Fingerprinting Assay for Clinical Immune Monitor- ing. 2019, bioRxiv; doi: https://doi.org/10.1101/587295. Background AgenTus Therapeutics is developing innovative adoptive cell therap- P202 ies to target a novel class of neoantigens called Phosphopeptide Mechanisms underlying human placental CD34+-derived natural Tumor Targets (PTTs). These post-translational modification-based killer cell cytotoxicity against glioblastoma neo-antigens arise in tumor cells through dysregulated kinase and Tanel Mahlakoiv, PhD, Bhavani Stout, Valentina Rousseva, Irene Raitman, phosphatase activities. PTTs represent one of the most promising cell Lin Kang, PhD, Robert Hariri, Xiaokui Zhang, William van der Touw therapy targets, as they are shared within and between cancer indi- Celularity, Warren, NJ, United States cations. Using a mass-spectrometry-based approach that analyzes Correspondence: William van der Touw MHC I-bound peptides, we have analyzed PTTs from several indica- (william.vandertouw@celularity.com) tions. This approach allows us to survey the TCR ligandome of tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P202 cells and healthy tissues. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 110 of 272 Phospho-ligandome analysis identified the phosphopeptide after injection, the alloreactive CD4 T-cells underwent marked expan- EPRpSPSHSM presented by HLA*B07:02+ cancer cells in a patient sion and produced higher levels of interferon-gamma compared to with Acute Myeloid Leukemia (AML). This phosphopeptide results syngeneic CD4 T-cells. This was accompanied by markedly increased from the phosphorylation of the Mixed Lineage Leukemia-1 (MLL1) infiltration of host macrophages within the tumors as early as four protein, a histone lysine methyl transferase that functions as a tran- hours after injection. These tumor-infiltrating macrophages secreted scriptional regulator and has been associated with tumorigenesis. higher levels of interleukin (IL)-1β, IL-12 and IL-23, which are critical for Methods inducing effector T-cell responses. Indeed, 24 hours after injection of + + Using proprietary platforms consisting of primary T cell expansion alloreactive CD4 T-cells, the infiltration of host effector CD8 T-cells from the central compartment and a mammalian display platform into tumors significantly increased, as evidenced by their production of containing TCR α and β chain libraries from the expanded T cells, we high levels of perforin and granzyme B. Furthermore, the melanoma B6 isolated the first fully-human PTT-specific TCR: agenT-04002. mice that survived alloreactive CD4 T-cell therapy developed host Results memory T-cells specific to the B16 melanoma and acquired complete Functional characterization demonstrated that target recognition by resistance to the tumor rechallenge. agenT-04002 is dependent on the phosphoseryl-moiety. Furthermore, Conclusions agenT-04002 shows potent cytotoxic activity against numerous human Results showed that immune reactions triggered by ex vivo-gener- hematologic tumor cell lines in vitro and AML tumor control in vivo in a ated alloreactive CD4 T-cells disrupt immunosuppressive tumor mi- mouse xenograft model. Activated T cells harboring the recombinant croenvironments and establish long-term host antitumor memory T- TCR display a pro-inflammatory phenotype in vitro and in vivo following cell responses. Our findings may help develop new strategies for sig- tumor challenge. Most importantly, when co-cultured with AML cancer nificantly enhancing the efficacy of cancer immunotherapy. cells from patients, agenT-04002 T cells specifically recognize and kill tumor cells while sparing healthy myeloid cells. Acknowledgements Conclusions This work was supported by JSPS KAKENHI Grant Numbers JP15K09659, and AgenTus is developing the next generation of TCRs by targeting a JP19K07754. unique class of neo-antigens with multi-cancer potential. Our data demonstrate feasibility, specificity, and potency of PTT-specific TCRs. References Targeting PTTs across diverse indications will enable us to have 1. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, Bader P, broader applicability of cellular therapies. MR. Verneris MR, Stefanski HE, Myers GD, Qayed M, Moerloose BD, Hiramatsu H, Schlis K, Davis KL, Martin PL, Nemecek ER, Yanik GA, Peters C, Baruchel A, Boissel N, Mechinaud F, Balduzzi A, Krueger J, June CH, Levine P204 BL, Wood P, Taran T, Leung M, Mueller KT, Zhang Y, Sen K, Lebwohl D, Ex vivo-activated allogeneic CD4 T-cells disrupt Pulsipher MA, Grupp SA. Tisagenlecleucel in Children and Young Adults immunosuppressive tumor microenvironment, and induce host with B-Cell Lymphoblastic Leukemia. NEJM. 2018; 378: 439-448. tumor-specific cytotoxic T-cells in mice 2. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, Brudno JN, Stetler- 1 1 1 Kazuhiro Mochizuki, MD, PhD , Shogo Kobayashi , Nobuhisa Takahashi , Stevenson M, Feldman SA, Hansen BG, Fellowes VS, Hakim FT, Gress RE, 1 1 2 3 1 Hideki Sano , Yoshihiro Ohara , Shin Mineishi, MD , Yi Zhang , Atsushi Kikuta and Kochenderfer JN. T cells expressing an anti–B-cell maturation antigen 1 2 Fukushima Medical University, Fukushima City, Japan; Penn State chimeric antigen receptor cause remissions of multiple myeloma. Blood. Cancer Institute, Hershey, PA, United States; Temple University, 2016;128:1688-1700. Philadelphia, PA, United States 3. Majzner RG, Mackall CL. Tumor Antigen Escape from CAR T-cell Therapy. Correspondence: Kazuhiro Mochizuki (mochi-k@fmu.ac.jp) Cancer Discov. 2018; 8: 1219–1226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P204 4. Bezu L, Kepp O, Cerrato G, Pol J, Fucikovag J, Spisekg R, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: Peptide-based vaccines in anticancer Background therapy. Oncoimmunol. 2018; 7: e1511506 (15 pages). Cancer immunotherapies that target tumor-specific or tumor- 5. Negrin RS. Graft-versus-host disease versus graft-versus-leukemia. associated antigens are promising treatments for patients with incur- Hematology Am Soc Hematol Educ Program. 2015; 2015: 225-230 able cancers [1,2]. However, relapses due to the loss of target anti- Ethics Approval gens challenge the success of these therapies [1,3]. Multitargeted Experimental protocols were approved by the Fukushima Medical immunotherapies, such as cancer vaccinations specific to multiple University’s committee on Use and Care of Animals; approval number cancer-associated peptides, are possible approaches. However, clin- 28054, 29039, and 2019048. ical studies have shown that they have limited efficacy with respect to the induction of objective responses [4]. The graft-versus-leukemia effect observed after allogeneic hematopoietic stem cell transplant- P205 ation (allo-HSCT) is another example of strong multitargeted antitu- Automated, closed bioreactors for T cell processing and dendritic mor immunity mediated by donor T-cells that recognize and react to cell-T cell co-culture multiple allo-antigens [5]. In the present study, we demonstrated a Lekhana Bhandary, BS, PhD, Andrew Kozbial, BS, PhD, Shashi Murthy, BS, novel approach for attaining alloreactive CD4 T-cell-induced multi- PhD targeted cancer immunity that does not utilize allo-HSCT. Northeastern University, Boston, MA, United States Methods Correspondence: Shashi Murthy (s.murthy@northeastern.edu) + + Cluster of differentiation (CD)4 and CD8 T-cells isolated form the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P205 spleen of BALB/c mice were separately activated in cultures by den- dritic cells (DCs) generated from the bone marrow of C57BL/6 (B6) Background mice. The resultant host-reactive donor T-cells were injected into B6 Functionally closed and affordable automated cell culture systems mice bearing pre-established B16 melanoma. Host T-cells activated are critical to the success of cell-based immunotherapy. Despite by syngeneic DCs were used as the control. major advances in these therapies, there are few systems available Results that are practical for use at both the pre-clinical and clinical stages. Whereas the intratumoral injection of host-reactive donor CD4 T-cells To address this need, we have designed a system called BATON elicited potent antitumor immunity against established B16 melanoma which is designed for optimal culture of both adherent and suspen- in an alloantigen-dependent manner, intratumoral injection of host- sion cell types (Fig. 1). Cells are cultured via continual perfusion and reactive donor CD8 T-cells or host-type syngeneic T-cells failed to in- the fluid flow loop also enables automated cell loading and harvest. duce antitumor responses. The number of injected donor-type host- This poster will describe two application areas, namely T cell expan- reactive CD4 T-cells diminished after tumor regression and did not in- sion relevant to autologous CAR-T and TCR therapies and dendritic duce graft-versus-host disease-like complications. Interestingly, early cell (DC)-T cell co-culture for neo-antigen-based T cell therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 111 of 272 Methods The T cell expansion capability of the BATON system was evaluated by seeding BATON cartridges each having a surface area of 40 cm2 and volume of 25 mL with 23 million PBMCs along with CD3/28 Dynabeads. Cells were continually perfused with Irvine Scientific Prime XV xeno- free T cell medium with 33 U/mL IL-2 for 9 days. For comparison, a simi- lar culture was performed in a G-Rex 6 well plate. For DC-T cell co- culture experiments enriched monocytes (MOs) were seeded into the BATON system at a seeding density of approximately 600k MOs/cm2 into two cartridges. Monocytes were differentiated into immature DCs by continually perfusing the seeded MOs for 6 days with CellGenix DC Medium supplemented with 350 U/mL IL-4 and GM-CSF (CellGenix). On Day 6, the DCs from one cartridge were harvested for flow cytometry. The other cartridge was drained without removal of the DCs and seeded with approximately 23 million PBMCs. This cartridge was then perfused with Irvine Scientific Prime XV xeno-free T cell medium with 33 U/mL IL-2. Cells were harvested following 7 days of co-culture. In addition to flow cytometry characterization, the cytotoxicity of the T cells was evaluated via co-culture with Jurkat cells. Results BATON achieved high levels of T cell expansion, comparable to G-Rex (Fig. 2-3) and harvested cells showed strong cytotoxic ability (Fig. 4). For DC-T cell co-culture experiments, the BATON system generated DCs from monocytes at high yield (27% of seeded monocytes converted into DCs) (Fig. 5A). Expansion of T cells from the seeded PBMCs was ro- Fig. 2 (abstract P205). See text for description bust, with 26-fold expansion achieved in 7 days (Fig. 5B). Harvested T cells showed strong cytotoxic ability relative to control (Fig. 6). Conclusions The BATON system is an effective platform for reagent- and DC- mediated T cell expansion. Acknowledgements Funding from the NSF via grant 1645205 is gratefully acknowledged. Fig. 1 (abstract P205). See text for description Fig. 3 (abstract P205). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 112 of 272 Fig. 4 (abstract P205). See text for description Fig. 6 (abstract P205). See text for description P206 Transmembrane and linker domain amino acid composition alters chimeric antigen receptor (CAR) membrane residence and may conceal detection of novel functional CAR formats 1 2 1 Dina Schneider, PhD , Virginia Hoglund, MS , Ying Xiong, PhD , Darong 1 1 2 Wu, MS , Boro Dropulic, PhD, MBA , Rimas Orentas, PhD Lentigen Technology, Miltenyi Biotec, Gaithersburg, MD, United States; Seattle Children’s Research Institute, Seattle, WA, United States Correspondence: Rimas Orentas (rimas.orentas@seattlechildrens.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P206 Background The relationship between the structure of the extracellular linker (L) and transmembrane (TM) domains, and CAR-T function has not been fully described. In previous studies we used L and TM domains de- rived from CD8. To better define amino acid sequences governing cell surface expression and anti-tumor activity, we altered the se- quence and length of these domains and tested the impact on CAR T biology. Methods TM domains from glycoproteins expressed on the T cell surface were aligned to CD8 and those with a high degree of similarity were used to create new CARs. In some constructs the extracellular sequence proximal to the membrane of those proteins (L) was also included. CAR function was tested using LV-transduced human T cells. Protein expression was analyzed by flow cytometry and western blot, in vitro function by cytokine release and cell-mediated cytolysis, and in vivo function in xenograft models. CAR protein expression was also ana- lyzed by immunofluorescent microscopy. Results Sequences from T cell-expressed CD antigens, the CD3 complex, acti- vation markers, and members of the tumor necrosis factor receptor superfamily (TNFRSF) were analyzed. Based on sequence conserva- tion we created new CARs expressing combinations of CD4 and CD8 Fig. 5 (abstract P205). See text for description TM domains, as well as TNFRSF9, TNFRSF16, and TNFRSF19 (CD137, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 113 of 272 NGFR, TROY/TAJ). All constructs were detected by western blot. Conclusions Strong T cell surface expression was seen for CD8L/CD4TM, CD8L/ These results demonstrate that 3D-ACT model using ex vivo ex- CD4TM, CD8L/TNFRSF19TM, and TNFRSF16L/TNFRSF16TM. Intermedi- panded TILs and 3D tumoroid models is an effective tool for the ate surface expression was seen for TNFRSF9L/TNFRSF9TM. Con- therapeutic assessment of autologous TILs and indicate that it can structs with TNFRSF19L/ TNFRSF19TM had very poor surface also be used to assess efficacy of other cellular therapy applications. expression. However, these “undetectable” CARs by flow cytometry Furthermore, implementation of this platform in the clinical studies had the highest level of cytotoxicity and cytokine release vs Raji may also allow determining the most effective combinatorial cellular lymphoma. Immunofluorescence studies with transduced T cells on therapy strategies for individual patients. their own, or in the presence of Raji target cells, demonstrated that TNFRSF19 sequence may mediate an intracellular residence profile. Association of CARs with the CD3 complex was also noted. P208 Conclusions Impact of combined blockade of PD1 and activation of CD137 on The production of CARs for clinical use generally requires detection tumor infiltration and tumor cell killing efficacy of TILs in an of the CAR protein on the cell surface. We found that high-activity ex vivo autologous 3D tumoroid model of NSCLC patient samples CAR-T constructs can be created using the linker and TM domains of Jenny Kreahling, PhD, Melba Page, PhD, Mibel Pabon, PhD, Vijayendra TNFRSF19, even though these constructs are expressed on the cell Agrawal, PhD, Soner Altiok, MD, PhD surface at low to undetectable levels. The mechanism by which these Nilogen Oncosystems, Tampa, FL, United States CARs are functionally active, while in a primarily intracellular state, is Correspondence: Soner Altiok (soner@nilogen.com) under investigation. Intracellular residence of CARs may be a novel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P208 mechanism to prevent undesired activation or T cell exhaustion and represents a novel locus of CAR-T activity control. Background Adoptive cell therapy (ACT) with TILs has been of growing interest as anti- cancer treatment in solid tumors. This therapy consists of the outgrowth P207 and expansion of tumor resident T cells from tumor material and their trans- A patient-driven ex vivo 3D tumor organoid model to assess fer back into the same patient to achieve tumor cell killing. However, exist- efficacy of tumor infiltrating T-cell adoptive cell therapy ence of intrinsic immune escape mechanisms may diminish the efficacy of Jenny Kreahling, PhD, Mibel Pabon, PhD, Melba Page, PhD, Vijayendra therapeutic applications of TILs. Here we describe an ex vivo patient derived Agrawal, PhD, Soner Altiok, MD, PhD 3D tumoroid platform utilizing powerful high content confocal imaging mo- Nilogen Oncosystems, Tampa, FL, United States dalities to monitor the impact of PD1 inhibition and CD137 activation on au- Correspondence: Soner Altiok (soner@nilogen.com) tologous TIL infiltration and ACT mediated tumor cell killing. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P207 Methods Human tumor samples were obtained with patient consent and rele- Background vant IRB approval. Fresh patient tumor samples were processed into Adoptive cell transfer (ACT) of ex vivo expanded tumor- tumoroids measuring 100-150 μm in size. For these studies, autolo- infiltrating lymphocytes (TILs) has shown promising therapeutic gous TILs were propagated from each tumor sample. TILs were fluo- efficacy in subsets of patients with several solid tumors including rescently labeled and incubated together with 3D tumoroids in the NSCLC. However, to improve the anti-tumor efficacy of TIL ACT in presence or absence of the PD1 inhibitor nivolumab and/or an agon- solid tumors it is critical to develop rational combination strat- ist anti-CD137 mAb urelumab. TIL infiltration into tumoroids and kill- egies and to identify biomarker(s) predictive of patients who ing of metabolically labeled tumor cells were quantified by advanced would respond favorably to TIL therapy. Here we describe a high confocal microscopy and a custom image analysis algorithm that was content imaging approach using a fresh tumoroid model with in- correlated with flow cytometry and cytokine profiling. tact tumor stroma for quantitative assessment of autologous TIL Results infiltration and target tumor cell killing. We show that nivolumab and urelumab treatments had significant Methods impacts on TIL infiltration in subsets of NSCLC tumoroids. Flow cy- All human tumor samples were obtained with patient consent and tometry analysis demonstrated treatment-mediated activation of TILs relevant IRB approval. For the ex vivo assays 3D tumoroids measuring accompanied by marked changes in the release of pro-and anti- 100-150 micron in size were prepared and cryopreserved during the inflammatory cytokine profiles. Furthermore, we documented the ef- process of ex vivo propagation of autologous TILs. Allogeneic periph- fect of TIL transfer and drug treatment on resident T-cells, Tregs and eral blood mononuclear cells (PBMCs) were used as control. Ex vivo myeloid cell populations within the tumoroids. No correlation was propagated TILs were fluorescently labeled and their growth and found between TIL activity and composition of propagated TILs or functional characteristics in the presence or absence of CD3/CD28 PD-L1 expression on tumor cells. tetramer were assessed via flow cytometry. High content confocal Conclusions analysis was used to quantify TILs infiltration into the tumoroids and This data suggests that combined blockade of PD1 and activation of target tumor cell killing using Nilogen’s 3D-ACT platform. Multiplex CD137 may enhance the therapeutic efficacy of TIL ACT in NSCLC. cytokine assays and flow cytometry analysis were performed to as- Overall, this study also shows that our 3D-ACT tumoroid model al- sess TIL activation upon exposure to tumoroids. lows comprehensive analysis of compensatory mechanisms and se- Results lection of rational combinatorial treatment using adaptive cellular We successfully prepared matched autologous TILs and unpropa- therapy with autologous TILs and likely with other types of cellular gated 3D tumoroids from NSCLC patient tumors. The characteristics therapies. of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved tumor organoids using a cus- tom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of P209 marker-specific cell number, cell viability and apoptosis in addition to Hijacking CAR-CD19 T cells to potently control Her2-positive solid structural and functional analysis of cells in intact 3D tumoroids. High tumors in vitro and in vivo through the use of unique and content confocal imaging analysis demonstrated that CD3/CD28 pre- selective bridging proteins activated TILs with increased activation phenotypes and enhanced Paul Rennert, PhD, Christine Ambrose, PhD, Fay Dufort, PhD, Lihe Dufort, pro-inflammatory cytokine release had marked infiltration into the Alyssa Birt, Thomas Sanford, Lan Wu, Roy Lobb, PhD 3D tumor organoids compared to untreated TILs and PBMCs. The Aleta Biotherapeutics, Natick, MA, United States data was correlated with quantitative tumor cell killing assessment Correspondence: Paul Rennert (paul.rennert@aletabio.com) for tumoroids. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P209 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 114 of 272 Background using antibodies directed against the scFv extracellular region. The Cell therapy success is limited by two critical issues. One is loss of anti- analytic panel included 29 cell surface, activation, exhaustion, and gen expression. This accounts for the ~50% relapse rate seen in CAR- cell-cell adhesion markers to identify/characterize lymphocyte sub- treated B cell malignancies. Solid tumors have highly variable antigen sets; and 9 intracellular markers to characterize functional status and expression and CARs targeting a single antigen fail as antigen-negative activation of signaling pathways. viSNE was used to visualize high- tumor cells escape, driving tumor resistance. dimensional data on a 2D map and quantify CyTOF data. A related issue is that most cell therapeutics fail to persist in the pa- Results tient. This is a particularly true of solid tumor treatment with CAR T Axi-cel products contained a median of 63% (range, 20-86%) trans- cells. The persistence failure may result from unproductive CAR-T inter- duced CAR (CAR+) T cells, and included relatively undifferentiated T cell action with the targeted tumor cell. subsets: a median of 0.4% and 52% of CD4 CAR+ cells were T stem cell We have developed CAR-CD19 T cells (CAR19s) that secrete bridging memory (SCM) and central memory (CM) cells, respectively, and 7% proteins to address these two critical issues. We leverage the ability and 34% of CD8 CAR+ cells were SCM and CM cells, respectively. CAR+ of CAR19s to persist independently of the target tumor cell while T cells in products had significantly higher expression of proliferation, simultaneously endowing these CARs with potent targeting technol- activation, and exhaustion markers (Ki67, CD25, HLA-DR, ICOS, OX-40, ogy. Here we illustrate this technology using the CD19-based bridg- Tim3, LAG3) and higher expression of cell-cell adhesion molecules ing protein that binds both EGFR and Her2. These data demonstrate (CD49d, CD29) compared with CAR-negative (CAR–)Tcells. On day 7, a that CAR19 T cells can be redirected to kill solid tumors in vivo. median of 11% of circulating T cells (range 0.6-58.4%) were CD4 CAR+ Methods and 3% (range 0.6-44.1%) were CD8 CAR+. Both CAR– and CAR+ T cells We cloned a highly stable CD19 extracellular domain (ECD) in frame with showed evidence of activation, but circulating CAR+ T cells expressed an anti-Her2 scFv to create, express and purify CD19-ECD-anti-Her2 bridg- higher levels of Ki67, 4-1BB, Tim3, PD-1, PD-L1, CXCR3, CD29, pZAP70, ing proteins. The sequence was also cloned downstream of a CAR19 do- pSTAT3 and pSTAT5, compared to CAR– Tcells. main and P2A cleavage site in a lentiviral vector. Transduced primary T Conclusions cells expressed the CAR19 and secreted the bridging protein. These CyTOF enables detailed characterization of CAR T cell products com- bridging protein formats were evaluated with in vitro cytotoxicity assays prising heterogeneous T cell subsets. Axi-cel comprises both trans- and Her2+ tumors in vivo. Finally, we added an anti-EGFR scFv sequence, duced and untransduced T cells at various stages of differentiation, creating an extremely potent multi-antigen targeting module. including SCM cells. CAR+ T cells showed higher expression of a broad Results range of proliferation and activation/exhaustion markers, compared to Incorporating a stabilized CD19 ECD in bridging proteins improved pro- CAR– cells, both in axi-cel products and in peripheral blood 7 days after tein expression, including from CAR19 T cells. CAR19 T cells secreting CAR T cell infusion. These data shed light on phenotypic and functional stabilized CD19-anti-Her2 bridging proteins were highly potent in vitro diversity of CAR T cells, pre- and post-infusion, influenced by the manu- and in vivo targeting CD19+ or Her2+ cells. An anti-EGFR scFv was facturing process, conditioning-related homeostatic cytokines and added to the CD19-ECD-anti-Her2 bridging protein. This novel multi- antigen-driven activation. Future studies may explore associations be- antigen targeting bridging protein supports highly potent cytotoxicity tween product composition and clinical outcomes. against single and dual antigen-expressing tumor cells while retaining Trial Registration intrinsic anti-CD19 activity, providing these unique CARs with a tumor- NCT02926833 independent and self-renewing antigen depot in CD19-positive normal B cells. For specific indications a third binding domain is added. P211 Conclusions Combining Deep™ IL-12 Primed and Deep™ IL-15 Primed T cells We have created a robust system for targeting multiple tumor anti- leverages complementary mechanisms to enhance anti-tumor gens simultaneously with a single CAR T cell. Further the use of activity CAR19s supports CAR-T cell persistence independently of tumor anti- Katharine Sackton, PhD, Elena Geretti, PhD, Pengpeng Cao, PhD, Shawn gen expression. This unique technology addresses critical issues in Carey, PhD, Xiaoyan Liang, Jonathan Nardozzi, PhD, Zishu Gui, Alicia cell therapy using a potent technology whose modular nature allows Worthylake, Glenn Leary, Becker Hewes, MD, Tap Maniar, MD, Jonathan for rapid program and pipeline development. Fitzgerald, PhD, Andy Rakestraw, PhD, Karsten Sauer, PhD, Douglas Jones, PhD, Thomas Andresen, PhD P210 Torque Therapeutics, Cambridge, MA, United States Deep phenotypic and functional analysis of transduced anti-CD19 Correspondence: Thomas Andresen (tandresen@torquetx.com) CAR T cells and untransduced T cells in patients treated with axi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P211 cel by single cell mass cytometry 1 1 1 Yohei Arihara, MD, PhD , Caron Jacobson, MD , Philippe Armand, MD , Background 2 2 2 John Rossi, MS , Nathalie Scholler, MD, PhD , Stuart Sievers, PhD , Interleukin-15 (IL-15) and Interleukin-12 (IL-12) play complementary 2 2 2 Edmund Chang, PhD , Mauro Avanzi, MD, PhD , Adrian Bot, MD, PhD , roles as immunomodulators. IL-15 induces T cell memory and supports Jerome Ritz, MD survival, activation and proliferation of CD8+ T and NK cells. IL-12 pro- Dana-Farber Cancer Institute and Harvard Medical School, Boston, motes T cell cytotoxicity and innate immune responses in the tumor MA,United States; Kite, a Gilead Company, Santa Monica, CA, Santa microenvironment. Both cytokines have been explored as cancer im- Monica, CA, United States munotherapies, but clinical success has been limited due to severe side Correspondence: Jerome Ritz (jerome_ritz@dfci.harvard.edu) effects. To limit systemic toxicities, Torque has developed its Deep- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P210 Primed™ T cell therapy technology. Multi-targeted T cells (MTC) specific for multiple tumor antigens are generated from patient apheresis. Cyto- Background kines are tethered to MTCs to support MTC persistence and activity fol- Axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T lowing adoptive transfer into patients, while limiting systemic cytokine cell therapy, has shown high efficacy in relapsed/refractory (R/R) dif- exposure. This study evaluates the combination of cytotoxic T lympho- fuse large B cell lymphoma (DLBCL). Axi-cel contains heterogeneous cytes (CTL) Deep-Primed with Deep™ IL-15 and Deep™ IL-12 to leverage populations of transduced and untransduced T cells. We used single their complementary biology for superior efficacy. cell mass cytometry (CyTOF) to analyze the impact of this heterogen- Methods eity on proliferation and expansion of these cells after infusion. CTLs reactive against MART-1 antigen were generated from healthy Methods donors (MART-1 CTLs). Next, expansion and cytotoxicity of MART-1 CyTOF examined CAR T cell products from 12 patients with R/R CTLs loaded with Deep IL-12, Deep IL-15 or both against MART-1 ex- DLBCL and peripheral blood mononuclear cells obtained 7 days after pressing SKMEL-5 melanoma cells were assessed. In addition, murine axi-cel infusion. We identified anti-CD19 CAR T cells (CAR+ cells) PMEL CD8+ T cells reactive against the B16-F10 melanoma antigen Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 115 of 272 gp100 were loaded with Deep IL-12, Deep IL-15 or both and evalu- Conclusions ated for in vitro expansion, activation and cytotoxicity against B16- Conclusions: These results indicate the viability of TIL ACT for refrac- F10 melanoma cells, as well as for anti-tumor activity in B16-F10 tory ovarian cancer by allowing for the large expansion of anti-tumor tumor-bearing mice. TIL in a short time and consistent manner. A Phase II clinical trial Results based on this work is currently open at UTMDACC to evaluate the Loading with Deep IL-15 promoted MART-1 CTL proliferation and feasibility of TIL ACT in recurrent or refractory OvCa (NCT03610490). preserved antigen reactivity over time. Deep IL-12 loaded MART-1 Ethics Approval CTLs displayed enhanced IFN-γ secretion and cytotoxicity, particularly Ethics approval: Ethical approval and tissue from surgical resections used at low effector:target ratios. Combination of MART-1 CTLs loaded to expand TIL were both obtained under a protocol (PA16-0912 and with Deep IL-12 and Deep IL-15 further enhanced T cell expansion, LAB02-188) approved by the Institutional Review Board of UTMDACC. IFN-γ secretion and cytotoxicity. Similarly, combination of murine PMEL T cells loaded with Deep IL-12 and Deep IL-15 resulted in per- P213 sistent T cell activation, improved memory, and enhanced cytotox- Conversion of peripheral blood mononuclear cells into tumor- icity over individually loaded T cells. Coadministration of Deep IL-12 specific cytolytic cell populations using tumor cells engineered and Deep IL-15 loaded PMEL T cells to B16-F10 melanoma-bearing with multiple immunomodulatory factors mice was well-tolerated, with minimal and reversible body weight 1 1,2 2 Joshua Keegan , James Lederer , Frank Borriello, MD, PhD , Nathan loss, and elicited superior anti-tumor activity. Schomer, MS Conclusions 1 2 BWH Harvard Medical School, Boston, MA, United States; Alloplex Modular tethering of Deep™ IL-12 and Deep™ IL-15 to T cells uniquely Biotherapeutics, Inc., Winchester, MA, United States leverages their complementary functions as immunomodulators to Correspondence: James Lederer (jlederer@alloplexbio.com); Frank maximize anti-tumor activity without notable toxicity in preclinical Borriello (fborriello@alloplexbio.com) models. A Phase I clinical trial of Deep IL-15 Primed MTCs (TRQ15-01) in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P213 solid cancers and lymphoma is enrolling (NCT03815682). Torque is initi- ating clinical evaluation of Deep IL-12 Primed MTCs (TRQ12-01), includ- Background ing a combination arm with TRQ15-01. Numerous studies and two recent clinical approvals have demon- strated the efficacy and safety of cellular immunotherapies to treat P212 diverse cancer subtypes. To date, the only approved cytotoxic cel- Potential clinical application of tumor-infiltrating lymphocyte lular therapies in the U.S. are T-cell based. However, ongoing clin- therapy for ovarian epithelial cancer prior or post-resistance to ical trials using gamma delta T cells and natural killer cells have chemotherapy suggested not only clinical efficacy, but also synergistic beneficial Donastas Sakellariou-Thompson, BS, Marie-Andree Forget, PhD, Emily effects when combined with checkpoint inhibitor antibody therap- Hinchcliff, MD, Joseph Celestino, Patrick Hwu, MD, Amir Jazaeri, MD, Cara ies. Our group developed novel immune stimulatory allogeneic Haymaker, PhD, Chantale Bernatchez tumor cells engineered with multiple immunomodulatory factors as MD Anderson Cancer Center, Houston, TX, United States a novel approach to generate heterologous tumor cell lytic popula- Correspondence: Cara Haymaker (chaymaker@mdanderson.org); tions of human peripheral blood mononuclear cells (PBMCs) for Chantale Bernatchez (cbernatchez@mdanderson.org) cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P212 Methods SK-MEL-2 cells obtained from the NIH were engineered to express a set Background of immunomodulatory proteins with the aim of expanding oncolytic cell Background: Epithelial ovarian cancer (OvCa) is the deadliest populations. Cells were engineered using lentiviral vectors prepared by gynecological cancer, and is estimated to account for almost 14,000 VectorBuilder, and sorted by flow cytometer for high expression of de- deaths in 2018. Traditional management of advanced stage OvCa in- sired immunomodulators. These engineered cells were then mixed with cludes tumor reductive surgery and adjuvant platinum-taxane chemo- freshly thawed human PBMCs and co-incubated for up to 14 days. Cells therapy, which results in high rates of initial complete response. were collected at time points during the incubation period for pheno- However, nearly 90% of patients recur and the 5-year survival rate for typic analysis using mass cytometry (CyTOF). Functional characterization late-stage disease is only 28. Immunotherapy has become a powerful of stimulated PBMCs was conducted using a cytotoxicity assay against treatment option for several solid tumor types. The presence of tumor- targets from several cancer subtypes. infiltrating lymphocytes (TIL) is correlated with better prognosis in ovar- Results ian cancer, pointing at the possibility to benefit from harnessing their CyTOF analysis of stimulated PBMCs revealed expansion of natural anti-tumor activity. The effectiveness of adoptive cell therapy (ACT) killer cells, gamma-delta T cells, and CD8 T cells by 8 days after with TIL has already been shown in metastatic melanoma with object- stimulation (Figure 1). All these populations expressed high levels of ive response rates of 40-50%. This preclinical study explores the feasibil- NKG2D and Granzyme B, suggesting widespread recognition of ity of transposing TIL ACT to OvCa using an improved culture method. tumor antigens and cytotoxic capability. In contrast, PBMCs that were Methods activated and expanded by CD3/CD28 activation beads showed less Methods: High-grade serous ovarian cancer (n=84), pre- or post- heterogenous expansion. PBMCs expanded with immunomodulatory chemotherapy and primary or metastatic, samples were accrued. TIL cell lines demonstrated potent cytotoxic activity towards both the were cultured using either high-dose IL-2 only, high-dose IL-2 with an parental cell line, as well as other melanoma and non-melanoma agonistic antibodies targeting 4-1BB (a41BB), or a combination of IL-2, cancer cell lines (Figure 2). As a control for specific cytotoxicity, both a41BB, and an agonistic anti-CD3 mAb. The cells were phenotyped autologous and allogeneic PBMCs were tested as targets and dis- using flow cytometry in the fresh tissue and after expansion. Tumor re- played no detectable cytotoxicity in our assay. activity was assessed against HLA-matched ovarian cancer cell lines via Conclusions IFN-γ ELISPOT. We developed a novel approach to expand immune cell populations Results from normal human PBMCs demonstrating anti-tumor activity Results: Ovarian cancer is highly infiltrated with CD8+ TIL that are pref- against multiple cancer cell types. This strategy is being developed erentially and robustly expanded with IL-2 and the two agonistic anti- to activate and expand PBMCs from cancer patients that will be used bodies. With a 95% success rate, the TIL are grown to ≥100x10^6 cells for autologous cellular immunotherapy. Taken together, the results in 2-3 weeks without over differentiation. In addition, the CD8+ TIL from this study demonstrate our ability to generate multiple popula- grown with this method showed HLA-restricted tumor recognition. TIL tions of cytotoxic effector cells from PBMCs, which should provide a growth and tumor recognition was independent of surgery site or straight-forward approach to generate clinically-relevant cells for chemotherapy exposure. adoptive cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 116 of 272 replace endogenous TCRs with the native neoTCR sequence in each edited T cell for expression at native TCR levels. Precision genome en- gineering of a colon cancer tumor cell line (SW620) was used to express the patient-specific neoantigen (COX6C-R20Q) at native levels. neoTCR- T cells were co-cultured with SW620 expressing the COX6C-R20Q muta- tion or the COX6C wild-type peptide. Functional readouts were T cell proliferation, cytokine secretion and tumor cell killing. Results Seven neoTCR clonotypes against the mutated COX6C peptide (COX6C-R20Q) presented in the context of HLA-A2 were cloned from imPACT-captured neoE-specific CD8 T cells. Primary human T cells were engineered with the 7 different TCR specificities against the COX6C-R20Q. Each of the seven candidate neoTCR-engineered T cells displayed specific cytotoxicity against tumor cells expressing en- dogenous levels of the COX6C-R20Q neoantigen. At 96 hours, using Effector to Tumor cell ratio (E:T) 1:1, 85-90% tumor elimination was observed (p < 0.000001 for each comparison). Significant tumor cell killing was detected with an E:T ratio as low as 1:5. neoTCR-T cells also proliferated and secreted interferon-gamma in response to co- culture with the relevant tumor target. Importantly, neoTCR-T cell ac- tivity was absent when co-cultured with tumor cells expressing wild type COXC6 protein. Conclusions Fig. 1 (abstract P213). See text for description These results demonstrate that the imPACT Isolation Technology used to capture antigen-experienced, neoE-specific T cells from the blood of patients with cancer authenticates that these neoE-HLA tar- gets are relevant for engineering neoTCR-T cells therapies. Lever- aging this approach, PACT is developing autologous personalized adoptive T cell therapy (NeoTCR-P1 product). A Phase 1 clinical trial to test NeoTCR-P1 T cells in subjects with solid tumors is currently ongoing (NCT03970382). References 1. Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, Urbinati F, Foy S, Hunter T, Tai A, Bethune M, Peng A, Dalmas O, Franzusoff A and Mandl S. Abstract 4783: Highly efficient, non-viral preci- sion genome engineering for the generation of personalized neoepitope- specific adoptive T cell therapies. Cancer Res 2019;79(13 Suppl). 2. Sennino B, Conroy A, Purandare B, Litterman A, Jacoby K, Moot R, Lu W, Nguyen D, Urbinati F, Foy S, Hunter T, Dalmas O, Bethune M, Park T, Peng S, Franzusoff A and Mandl S. Abstract 1433: NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with ‘youn- ger’ phenotypes that rapidly proliferate and kill target cells upon recogni- tion of cognate antigen. Cancer Res 2019;79(13 Suppl). Fig. 2 (abstract P213). See text for description 3. Peng S, Quach B, An D, Sandoval S, Bao R, Pan Z, Bethune M, Dalmas O, Yi M, Meadows C, Heeringa K, Guo L, Yuen B, Sorfleet J, Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, P214 Mandl S and Franzusoff A. Abstract 1435: An ultra-sensitive and high- T cells precision engineered to express neoepitope-specific TCRs throughput technology (imPACT) for the identification and isolation of cloned from a patient with colorectal cancer specifically target and intrinsic and emergent neoepitope-specific T cells from the peripheral kill relevant neoantigen-expressing tumor cells blood and TILs of cancer patients. Cancer Res 2019;79(13 Suppl). Barbara Sennino, PhD, Adam Litterman, John Gagnon, Andrew Conroy, Ethics Approval Bhamini Purandare, Zheng Pan, Dalmas Olivier, Kyle Jacoby, Songming Human samples in this study were procured from a commercial vendor who Peng, Alex Franzusoff, Stefanie Mandl, PhD collected them according to their established ethics policies. PACT Pharma, South San Francisco, CA, United States Correspondence: Alex Franzusoff (afranzusoff@pactpharma.com); Stefanie Mandl (smandl@pactpharma.com) P215 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P214 Depletion of CD45RA-positive cells potentiates the reactivation of EBV-specific T-cells from EBV positive lymphoma patients Background Sandhya Sharma, BSc, Naren Mehta, Kathan Parikh, RA, Cliona Rooney, Neoepitopes (neoE) derived from private tumor-exclusive mutations PhD represent compelling targets for personalized TCR-T cell therapy to Baylor College of Medicine, Houston, TX, United States eradicate tumor cells throughout the body. The imPACT Isolation Tech- Correspondence: Sandhya Sharma (me.sandhya@gmail.com) nology® is an ultra-sensitive and high-throughput process for the cap- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P215 ture of mutation-targeted CD8 T cells from patient blood. NeoTCRs of native sequence, cloned from the captured T cells, were evaluated for Background tumor-targeted functionality by non-viral precision genome engineer- Epstein-Barr virus (EBV)-positive lymphomas express viral type-2 latency ing of fresh human CD8 and CD4 T cells for neoTCR expression [1,2]. proteins (T2-Ags). Autologous EBV-specific T-cells (EBVSTs) directed to Methods T2-Ags have produced complete responses in ~50% lymphoma pa- NeoTCRs were isolated from the blood of a patient with colorectal can- tients[1]. However, in our ongoing clinical trial, we failed to generate cer using the imPACT Isolation Technology® [3]. Subsequently, healthy EBVSTs from 24% of the patients procured; manufacturing failures donor CD8 and CD4 T cells were precision genome engineered to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 117 of 272 associated with lack of T-cell expansion and T2-Ag-specificity. Further, Background in lines successfully expanded, many EBVST lines demonstrated low T2- Administration of Toll-like receptor agonists with adoptively trans- Ag specificity and some contained a high frequency of NK-cells. Our ferred T cells augment tumor immunity. However, systemic or local goal is to improve both the manufacturing success rate and clinical effi- administration of TLR agonists could heighten inflammation and lead cacy of EBVSTs. In EBV-exposed individuals, EBVSTs reside in CD45RA- to toxic side effects when coupled with CAR or TIL-based therapies. CD45RO+ memory compartment, while CD45RA-positive population in- Thus, we hypothesized that TLR agonists could be used ex vivo dur- cludes unwanted naïve T-cells, suppressive regulatory-T cells, and NK- ing T cell expansion and ultimately generate a cell product with en- cells[2,3]. We hypothesized that removal of CD45RA-positive cells from hanced anti-tumor properties for adoptive cell transfer therapy while PBMCs prior to EBV-antigen specific stimulation would improve EBVST reducing potential toxicity to patients. generation and specificity by eliminating competing naïve T-cells, while Methods reducing potentially inhibitory cells capable of inhibiting the outgrowth To test our hypothesis, we employed the Pmel-1 transgenic mouse of antigen-specific T-cells. We, therefore investigated the effects of se- model, in which CD8+ T cells harbor a TCR specific for the gp100 epi- lective depletion of CD45RA-positive cells from PBMCs prior to EBV T2- tope expressed on melanoma and healthy melanocytes. Pmel spleno- Ags stimulation. cytes were stimulated with hgp100 peptide in the presence or absence Methods of TLR9 agonist CpG (ODN-1668) and expanded in IL-2 for one week. T EBVSTs were generated from whole PBMCs and CD45RA depleted (RAD) cell phenotype was analyzed via flow cytometry and anti-tumor activity PBMCs and we measured proliferation by counting, T2-Ag specificity assessed in mice bearing B16F10 melanoma tumors. using IFN-gamma ELIspot assays and cell-phenotype by flow cytometric Results analysis. To compare their in-vivo efficacy, EBVSTs were adoptively trans- T cells expanded with CpG possessed a unique phenotype (IL-2Ralpha- ferred into immunodeficient mice bearing autologous EBV-transformed high, ICOS-high, CD39-low) and mediated more potent responses lymphoblastoid tumor cells and tumor clearance was evaluated. against melanoma in vivo than traditionally expanded T cells. Interest- Results ingly, this phenotype and anti-tumor function was dependent on the RAD-EBVSTs produced greater expansion of EBVSTs from PBMCs and presence of B cells at the start of culture, as their removal resulted in a decreased the frequency of NK cells, which dominated some of our pa- loss of this unique phenotype and anti-tumor efficacy in vivo. Con- tient lines. T2-Ag specificity increased by 3-5 fold as measured by versely, removal of CD4+ T cells, NK cells, dendritic cells, or macro- gamma-IFN release in response to T2-Ags stimulation in both healthy phages from culture did not ablate the phenotype or anti-tumor donors and patients. RAD-EBVSTs maintained antigen specificity over activity of CpG-generated T cells. The CpG-elicited T cell effects were multiple rounds of weekly T2-Ags stimulation. Phenotypic analysis dem- also dependent on the peptide-mediated interaction between the T onstrated decreased expression of exhaustion markers in RAD-EBVSTs. cell and APC in culture as activating with plate-bound or bead-bound Most importantly, this approach restored responsiveness to antigen antibody strategies resulted in a T cell population that was similar to stimulation in most unresponsive patients enabling the successful re- the ineffective vehicle treated cells both phenotypically and therapeut- activation and expansion of EBVSTs from lymphoma patients that failed ically. We further found that CpG-treated B cells expressed heightened previously. This advantage extends to VSTs with specificity for HPV, VZV levels of CD40, suggesting that induction of a CD40-CD40L axis be- and adenoviral antigens, suggesting that we have identified a general tween B and T cells may account for the generation of potent IL- approach for improving the activity of VSTs for immunotherapy. EBVSTs 2Ralpha-high, ICOS-high, CD39-low T cells. generated from RAD-PBMCs demonstrated more rapid tumor clearance Conclusions and superior control of metastatic tumors in our xenograft model. The TLR9 agonist CpG can be used in ex vivo culture to potentiate Conclusions an anti-tumor T cell product for ACT. The CpG-associated T cell This approach to the generation of VSTs is being translated to the phenotype (IL-2Ralpha-high, ICOS-high, CD39-low) and anti-tumor clinic for use in multiple clinical trials. In future, we aim to elucidate ability was dependent on the presence of B cells and a direct inter- the mechanisms underlying the inhibitory effects of the CD45RA action between the anti-tumor T cells and APC via peptide activation. positive population in the reactivation and expansion of EBVSTs. Post CpG stimulation B cells express heightened CD40, which may promote the B cell/T cell interaction and thus the generation of a po- References tent T cell product for ACT. 1. Bollard CM, Gottschalk S, Torrano V, et al. Sustained Complete Responses in Ethics Approval Patients With Lymphoma Receiving Autologous Cytotoxic T Lymphocytes This study was approved by the Institutional Animal Care & Use Com- Targeting Epstein-Barr Virus Latent Membrane Proteins. Journal of Clinical mittee of the Medical University of South Carolina, protocol number Oncology. 2016;32(8):798-808. doi:10.1200/JCO.2013.51.5304. 0488. 2. Krzywinska E, Cornillon A, Allende-Vega N, et al. CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity. Bobé P, ed. P217 PLoS ONE. 2016;11(4):e0150434. doi:10.1371/journal.pone.0150434. An NK cell line (PD-L1 t-haNK) engineered to target PD-L1 3. Krzywinska E, Allende-Vega N, Cornillon A, et al. Identification of Anti- efficiently kills tumor cells and myeloid derived suppressor cells tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With 1 1 1 Kellsye Fabian, PhD , Michelle Padget , Renee Donahue, PhD , Kristen Hematological Cancers. EBioMedicine. 2015;2(10):1364-1376. doi:10.1016/ 1 2 3 Solocinski, PhD , Clint Allen, MD , John Lee, MD , Patrick Soon-Shiong, j.ebiom.2015.08.021. 3 1 1 MD , Jeffrey Schlom, PhD , James Hodge, PhD, MBA Ethics Approval 1 2 NCI/NIH, Bethesda, MD, United States; NIDCD/NIH, Bethesda, MD, The study was approved by Baylor College of Medicine's Ethics Board, United States; NantKwest, Culver City, CA approval number H7634 and H7666 for human subjects. Animal protocol Correspondence: James Hodge (jh241d@nih.gov) AN5551. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P217 P216 Background CD8+ T cells break tolerance to tumors in a B cell-dependent The ability of natural killer (NK) cells to lyse tumor targets without manner via TLR9 signaling prior sensitization and without human leukocyte antigen (HLA)-re- Aubrey Smith, BS, Hannah Knochelmann, BS, Connor Dwyer, PhD, striction make them promising candidates for “off the shelf” cell- Megan Wyatt, MS, Guillermo Rangel Rivera, Jessica Thaxton, PhD, MSCR, based immunotherapy. Here we investigate the anti-tumor efficacy Mark Rubinstein, PhD, Bei Liu, MD MPH, Eric Bartee, PhD, Chrystal Paulos, of a novel NK cell platform, the PD-L1-targeted high-affinity NK (PD- PhD L1 t-haNK), which lacks killer inhibitory receptors (KIRs), carries a high Medical University of South Carolina, Mount Pleasant, SC, United States payload of granzyme and perforin granules, and has been designed Correspondence: Aubrey Smith (butchera@musc.edu) with a chimeric antigen receptor (CAR) to target PD-L1 expressing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P216 cells on cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 118 of 272 Methods Conclusions Frozen, irradiated (15 Gy) PD-L1 t-haNK cells were thawed and charac- The assay provides a sensitive, simple read-out to support discovery terized via flow cytometry and RNAseq. PD-L1 t-haNK lytic activity was research and/or QC lot release applications. assessed in vitro using MDA-MB-231, BT549, T47D, MCF7, SUM149, H460, H441, HCC4006, SW480, SW620, DU145, HTB1, CaSki, and CH22 P220 cell lines as targets in indium-based and flow cytometry-based killing Targeting neoantigens with immunotherapy: Are we limited to assays. The effect of pre-treating tumor targets with IFNγ on PD-L1 t- pre-existing autologous neoantigen-specific T-cells? haNK targeting was also examined. PD-L1 t-haNK cells were co-cultured Aline Bracher, Slavoljub Milosevic, Daniel Sommermeyer with human peripheral blood mononuclear cells (PBMCs) and purified Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany human myeloid derived suppressor cells (MDSCs) to investigate the ef- Correspondence: Daniel Sommermeyer fects of PD-L1 t-haNK on immune subsets. The therapeutic activity of (d.sommermeyer@medigene.com) PD-L1 t-haNK in vivo was studied by adoptive transfer of PD-L1 t-haNK Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P220 cells to NOD scid gamma (NSG) mice engrafted with parental MDA-MB- 231 (PD-L1+) and MDA-MB-231/PD-L1 CRISPR knockout (PD-L1-null). Background Results Several facts shape our considerations regarding the use of neoanti- Here, we show that irradiated PD-L1 t-haNK cells express PD-L1- gens as highly specific targets for immunotherapy of cancer. First, specific CAR, and high levels of perforin and granzyme B. PD-L1 t- adoptive T-cell therapy using TILs seems to be most successful if the haNKs lysed all 14 human tumor cell lines tested in vitro, and in- TILs include T-cells specific for antigens resulting from individual muta- creased cell lysis corresponded with increasing levels of PD-L1 ex- tions. Second, the success of checkpoint inhibitors is often correlated pression on tumor cells. Increasing PD-L1 expression on tumor cells with the mutational load of tumors. However, a mutation must fulfill through IFNγ treatment improved PD-L1 t-haNK-mediated lysis by up several criteria to be effective as a neoantigen that can be recognized to 100%. In vivo, adoptive transfer of PD-L1 t-haNK cells inhibited the by T-cells. Obviously, the mutation must lead to a novel amino acid se- growth of engrafted parental MDA-MB-231 but not PD-L1 null MDA- quence (e.g. single amino acid substitution, fusion- or frameshift- MB-231 tumors. Finally, when co-cultured with human PBMCs and sequence), and be located in a gene expressed in tumor cells. Further- purified human MDSCs expressing PD-L1, PD-L1 t-haNK cells prefer- more, a peptide spanning the new sequence needs to be efficiently entially lysed the MDSC population but not other PBMC subsets. processed and presented by HLA molecules. Finally, a T-cell response Conclusions must be triggered that can specifically recognize the mutated epitope. This study provides a rationale for the potential use of adoptively trans- Targeting neoantigens as patient-individualized epitopes requires ro- ferred irradiated PD-L1 t-haNK cells as a unique immunotherapeutic plat- bust processes for rational and rapid selection and validation of form against a range of human tumor types. In addition to lysing the neoantigens as T-cell targets. Currently, the most challenging step is tumor cells, MDSC killing may be a novel mechanism of action of PD-L1 t- predicting specific T-cell responses. Huge efforts have been made to haNK cells that may potentiate their impact especially in cancers where analyze the reactivity of patients’ T-cells against mutations. However, MDSC’s limit immune approaches. The safety and clinical benefit of PD-L1 this approach is limited to the T-cell repertoire present in patients at t-haNK cells in cancer patients are being assessed in ongoing clinical trials. the time of tumor resection or blood draw and might miss potential Ethics Approval potent T-cell responses that were lacking or no longer present in the The study was approved by the NCI IRB, NIH protocol 99-CC-0168. patient. In our opinion, only screening the T-cell repertoire of several healthy donors can answer the question if a specific mutation can P218 trigger T-cell responses. We present proof-of-concept data how we A novel, bioluminescent assay for the selective detection of target use our high-throughput T-cell receptor (TCR) platform technologies cell killing in mixed cultures and automated processes for fast and efficient screenings of T-cells Richard Somberg, PhD, Brock Binkowski, Aileen Paguio, Peter Stecha, isolated from several partially HLA-matched healthy donors. Chris Eggers, Braeden Butler, Michael Beck, Julia Gilden, PhD, Jey Cheng, Methods Mei Cong, PhD, Frank Fan, PhD Promising neoantigens were predicted and T-cells responses after Promega, Madison, WI, United States stimulation with antigen-presenting cells either transfected with Correspondence: Brock Binkowski (brock.binkowski@promega.com) minigene constructs or loaded with peptides were compared. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P218 Results Peptide stimulation triggered specific T-cells for most tested muta- Background tions, indicating that T-cell repertoires of healthy donors can Efforts to develop and commercialize cellular immunotherapies recognize neoantigens when they are forced to be presented. Also, would benefit from assays that selectively monitor target cell death with the clinically relevant approach using endogenously processed that are sensitive and easy-to-use. To address this, we have devel- antigen encoded by minigenes, specific T-cell responses against oped an approach to selectively quantify target cell death using a neoantigens presented on different HLAs were efficiently triggered, gain-of-signal assay format and bioluminescence read-out. although only against some of the tested epitopes. Methods Conclusions The method relies on the release of a HiBiT-tagged protein from tar- This screening strategy has the aim to develop future TCR-based get cells following cell lysis. HiBiT, an 11 a.a. peptide tag, binds to therapies and can be used for the identification of promising muta- cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconsti- tions for vaccination or as a source for TCRs for adoptive T-cell ther- tute NanoBiT Luciferase. Target cells are engineered to express a apy. Furthermore, generated data can subsequently improve HiBiT-tagged protein using either ectopic expression or CRISPR/Cas9 algorithms predicting the immunogenicity of neoantigens. to tag endogenous lactate dehydrogenase (LDH), and cell lysis is quantified by adding a detection reagent containing LgBiT and furi- P221 mazine substrate (no medium removal). Superior anti-tumor activity of metabolically enhanced bispecific Results antibody armed CAR-less Bionic T cells The signal is proportional to the amount of target cell death, and 1 2 1 Archana Thakur, PhD , John Scholler , Edwin Bliemeister , Carl June, measurements can be made using endpoint or kinetic formats. Cell 2 1 MD , Lawrence Lum, MD, DSc lines have low rates of spontaneous release and fusion proteins that 1 2 University of Virginia, Charlottesville, VA, United States; University of are stable in the extracellular medium, enabling assays up to 24 Pennsylvania, Philadelphia, PA, United States hours or more. The bright signal from NanoBiT Luciferase allows the Correspondence: Archana Thakur (at2fx@virginia.edu) use of low numbers of target cells per well (e.g. 2,500), and the assay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P221 can detect very low levels of target cell death (e.g. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 119 of 272 Background inevitably leads to excessive T cell differentiation ex vivo, and de- The major challenges of T cell-based therapies in solid tumors are creased T cell functionality and persistence in vivo. Importantly, T cell the metabolic insufficiency of T cells to exhibit effector functions and differentiation is tightly controlled by epigenetic mechanisms that persist in an altered metabolic landscape of the tumor microenviron- could be targeted therapeutically. The BET protein BRD4 has been re- ment (TME). To overcome these challenges, we developed the next ported to regulate expression of the transcription factor BATF that generation of chimeric antigen receptors-less (CAR-less) Bionic T cells drives CD8+ T cells towards a more effector-like phenotype, while for the treatment of solid cancers. We hypothesize that metabolically BRD4 inhibition by the drug JQ1 prevented this and thereby en- enhanced Bionic T cells armed with bispecific antibody (BiAb) will hanced in vivo T cell persistence and function in adoptive immuno- show enhanced anti-tumor responses and controlled off-tumor on- therapy models [1]. Ph-29089 is a chemically modified self-delivery target toxicity in solid cancer. RNA inhibitor containing an asymmetric duplex structure (≤ 15 base Methods pair duplex) and a single-strand phosphorothioate tail targeting the We engineered metabolically enhanced “Bionic T cells” (BTC) that are BET protein BRD4. PH-29089 is efficiently delivered to immune cells, devoid of CAR but contain a transmembrane and an intracellular do- including T cells, without the need for specialized formulations or main of a co-stimulatory molecule and TCR signaling domain of mechanical transfection as is observed with current RNAi’s. CD3z. T cells were transduced with CAR-less constructs without co- Methods stimulatory domain-FLAG-ζ (Control) or with co-stimulatory domain- Purified human CD8+ T cells were expanded using the rapid expan- FLAG-4-1BB-ζ, FLAG-CD28-ζ, FLAG-ICOS-ζ and FLAG-OX40-ζ, FLAG-27- sion protocol (REP) developed by the National Cancer Institute. Flow ζ and were tested for their hypoxic tolerance, anti-tumor activity, cytometry was used to study Ph-29089 for its ability to knock down cytokine production and exhaustion phenotype in the presence or BRD4 at the protein level in expanding T cells, and to determine T tumor targets. cell differentiation status during and immediately after ex vivo ex- Results pansion. Release of IFNγ by T cells cocultured with tumor cells was Our data show that hypoxia differentially affected BTC survival de- assessed by ELISA. pending on the co-stimulatory endodomain. Under normoxia Bionics Results with CD28ζ (28z) endodomain show 5% apoptosis versus 61% apop- Ph-29089 elicited a concentration dependent silencing of BRD4 pro- tosis under hypoxic (5% oxygen) condition. On the other hand, Bion- tein with an IC50 of 1-2 μM. The BRD4 silencing persisted at least 5 ics with 4-1BBζ showed only 13% apoptosis under hypoxic condition days post-treatment, whereas media and non-targeting control (NTC) suggesting enhanced hypoxic tolerance. HER2 and EGFR BiAb armed did not significantly affect BRD4 protein levels. Compared to un- BTC were tested against various low-high HER2 and EGFR expressing treated, NTC-treated and JQ1-treated CD8+ T cells, Ph-29089-treated cancer cell lines. Specific cytotoxicity of anti-HER2 BiAb (HER2Bi) and CD8+ T cells contained higher percentages of central memory and anti-EGFR BiAb (EGFRBi) armed BTC against MDA-MB-231, SK-BR-3, stem cell-like memory T cells, as determined by expression of BT-20, MiaPaCa-2 cell lines measured by real time cell analysis using CD45RA, CCR7, CD62L and CD95. Moreover, Ph-29089-treated CD8+ xCELLigence ranged between 75-100% at 2:1 E/T ratio at 72 hours. T cells displayed superior functionality, as indicated by enhanced Sequential killing by HER2Bi armed BTC followed by (f/b) EGFRBi IFNγ production when exposed to the allogeneic melanoma cell lines armed BTC showed efficient killing against target cells (86.2%) or A375 and ROAL. EGFRBi-BTC f/b HER2Bi-BTC (88.2%) compared to the killing by Conclusions HER2Bi-BTC (49.7%) or EGFRBi-BTC (43.5%) alone at low E/T ratio in These findings support the hypothesis that BRD4 silencing by Ph- the presence of 100 IU/ml IL-2 at 96 hours. Cytokine levels of IFN-γ, 29089 is a viable approach for expanding T-cells with superior anti- IL-15, IL-2R, and GM-CSF were significantly higher in culture superna- tumor potential for adoptive cell therapies. tants of tumor cells (SK-BR-3) and HER2Bi-BTC or EGFRBi-BTC co- cultures compared to the control condition with unarmed BTC. Reference Phenotypic data show increased expression of 4-1BB, ICOS and OX40 1. Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, on CD4+ and CD8+ T cells after short antigenic exposure and con- et al. BET bromodomain inhibition enhances T cell persistence and tinuous antigenic exposure. function in adoptive immunotherapy models. J Clin Invest. 2016 Conclusions 126(9):3479–94. Data show that armed BTC: 1) exhibit superior survival in hypoxic Ethics Approval condition; 2) can effectively kill multiple tumor targets in serial killing This study was carried out in accordance with the recommendations of assay; 3) cytokines and chemokines show immune modulating and Karolinska Institutet review board with written informed consent from all tumor killing profile. subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Stockholm Acknowledgements Regional Ethics Committee, with approval number (2011/143-32/1). This study was made possible by start-up funds for LGL from the University of Virginia. AT is a co-founder of NOVA Immune Platform. CJ is a co-founder P223 of T Immunity. LGL is a co-founder of Transtarget, Inc. and sits on the Withdrawn Scientific Advisory Board for Rapa Therapeutics. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P223 P222 Modulating BRD4 in T cells using self-delivery RNAi to improve P224 adoptive cell therapy of cancer 1 1 1 Case studies of sarcoma and MRCLS following treatment with NY- Jeroen Melief, PhD , Laura Van Leeuwe Kirsch, BSc , Esmeralda Hemme , 2 2 2 ESO-1 TCR T Cells (GSK3377794): correlates of predictable John Barrett, PhD , Simon Fricker, PhD , Gerrit Dispersyn, PhD , Rolf response characteristics Kiessling, MD, PhD 1 2 3 1 2 Brian Van Tine, MD, PhD , Sandra D'Angelo, MD , Alexandra Gyurdieva , Karolinska Institute, Stockholm, Sweden; Phio Pharmaceuticals, 3 3 3 3 Laura Johnson , David Turner , Jenna Tress , M. Phillip DeYoung , Yuehui Marlborough, MA, United States 3 3 4 Wu , Aisha Hasan, MBBS MD , Dejka Araujo, MD Correspondence: Rolf Kiessling (rolf.kiessling@ki.se) Washington University in St. Louis, St. Louis, MO, United States; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P222 Memorial Sloan Kettering Cancer Center, New York, NY, United States; 3 4 GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Background Center, Houston, TX, United States Ex vivo expansion of T cells for adoptive cell therapy (ACT) of cancer Correspondence: Brian Van Tine (bvantine@wustl.edu) is commonly done with cytokines and stimuli for efficient activation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P224 and expansion of large numbers of tumor-specific T cells. This Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 120 of 272 Background Background Genetically engineered NY-ESO-1 specific T cells (NY-ESO-1 T Cells; Chimeric Antigen Receptor (CAR) T cell therapy is a revolutionary GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a cancer treatment that genetically alters T cells to redirect and har- self-inactivating lentiviral vector to express an affinity-enhanced NY- ness their cancer killing potential. Currently FDA approved CAR T cell ESO-1-specific T-cell receptor. Phase 1 and 2 trials are evaluating products are autologous-based, requiring individualized blood apher- GSK3377794 in solid tumors and hematologic malignancies. This study esis and manufacture. Deriving patient-specific CAR T cell products is will review biomarker data for eight patients from two ongoing phase expensive, laborious, and time consuming, with logistical and regula- 1/2 pilot studies of GSK3377794 in synovial sarcoma (SS; NCT01343043; tory challenges. The success rate of autologous CAR T cell therapy is N=7) and myxoid/round-cell liposarcoma (MRCLS; NCT02992743; N=1) also limited by the urgency of acute and aggressive cancers, uncer- with prolonged response and stable disease (SD). tainty over T cell number, and intrinsic differences in T cell function- Methods ality. Generating CAR T cells from induced pluripotent stem cells Patients who were progression free ≥4 months following first infu- (iPSC) holds encouraging prospect for generating ‘off-the-shelf’ CAR sion were selected. All received the same lymphodepletion (30 mg/ T cell products and overcoming these challenges. iPSCs can prolifer- m2 x3D fludarabine, 600 mg/m2 x3D cyclophosphamide) followed ate almost infinitely while keeping their pluripotency and lineage by GSK3377794 infusion. Six patients with SS were eligible for second differentiation potential. However, the complexity of T cell develop- infusion and received higher-dose lymphodepletion (30 mg/m2 x4D ment and disturbance of T cell differentiation by CAR expression cre- fludarabine, 1800 mg/m2 x2D cyclophosphamide) before second in- ates a challenge for successful iPSC-derived CAR T cell generation. fusion. Pretreatment biopsies were analyzed for CD3 infiltration by Previously reported iPSC-derived CAR T cells showed innate like phe- RNAScope. Transduced cell persistence was measured by quantitative notypes with weak antigen-specific cytotoxicity and compromised PCR of transgene vectors peripheral blood mononuclear cell (PBMC) cytokine production [1]. DNA. Cytokine expression was measured by Meso Scale Discovery Methods immunoassay. PBMC phenotypes were characterized by flow In our current study, we generated iPSC lines from healthy donor T cells cytometry. by an integration-free method using iPSC reprograming episomal vec- Results tors. The iPSC cells were transduced with clinical grade lentivirus to ex- Five of seven patients with SS had SD for 17.8–105 weeks; two had press CD19-specific CARs (CD19CAR), sorted and colonized to generate partial response/complete response (PR/CR) per RECIST1.1. The pa- a homogeneous CAR+ iPSC cell bank. By using a 3D co-culture system, tient with MRCLS had PR per RECIST1.1 lasting 8.8 months. Six of we successfully generated iPSC-derived CD19CAR T cells. seven patients with SS received second infusion; 2/7 had SD, 3/7 had Results PR, 1/7 had CR. Immunohistochemistry revealed ≥50% of cells with The produced iPSC-derived CD19CAR T cells have a surface marker 2+/3+ NY-ESO-1 expression; this was maintained before second infu- phenotype (CD3+CD5+CD7+TCRalphabeta+CD8alphabeta+ and sion. Baseline tumor samples consistently showed 10 fold over first CD3+CD5+CD7+TCRalphabeta+CD4+) and gene expression signa- infusion; of these, there was one PR and one CR. Cytokine increases tures typical of natural T cells. These iPSC-derived CD19CAR T cells reflecting immune cell activation (eg, IFN gamma, IL-6, and IL-2R expanded robustly within two weeks (~100 fold), and showed potent alpha were observed 4–7 days after both infusions. Transduced T antigen-specific cytotoxicity against CD19+ parental tumor cells such cells within manufactured product showed increased expression of as NALM6 and Raji comparing to their CD19 knockout control cells. It activation markers (eg, CD28, ICOS, and CD40L) versus T cells from is intriguing that the in vitro cytotoxicity potency of iPSC-derived apheresis. In 2 patients, transduced CD8 cells primarily had T effector CD19CAR T cells was superior to conventional PBMC-derived CAR T memory RA+ (CD45RA+CCR7-) and T effector memory (CD45RA- cells generated from the same donor. These iPSC-derived CD19CAR T CCR7-) phenotype. In one, 34.3% transduced CD8 cells had T stem cells also demonstrated efficient degranulation activity and a Th1 cell memory (CD45RA+CCR7+) phenotype. cytokine profile (e.g. IFNgamma and TNFalpha when challenged with Conclusions CD19+ target cells. Furthermore, these cells demonstrated potent SS and MRCLS tumors initially show low immune cell infiltration. anti-tumor activity in vivo in a NSG mouse model using NALM6 as Upon GSK3377794 infusion, increased expression of activated im- target tumor. mune cell cytokines was observed in serum from selected patients. Conclusions Further analysis can provide insights into clinical response character- Our study demonstrates the feasibility of generating naturalistic istics and identification of predictive biomarkers. and functional CAR T cells from iPSCs, which may provide utility in the development of ‘off-the-shelf’ CAR T cell manufacturing Acknowledgements strategies. Medical writing assistance was provided by provided by Fiona Woodward and Chloe Stevenson of Fishawack Indicia Ltd, UK. These studies Acknowledgements (NCT01343043 and NCT02992743) were funded by GlaxoSmithKline (GSK). This research is supported by Mustang Bio. Inc. Trial Registration NCT01343043 and NCT02992743 Reference Ethics Approval 1. Themeli, M., et al., Generation of tumor-targeted human T lymphocytes This study was approved by the appropriate institutional review boards and from induced pluripotent stem cells for cancer therapy. Nat Biotechnol, independent ethics committees. 2013. 31(10): p. 928-33. Ethics Approval The study was approved by the COH Institutional Review Board (IRB) and P225 Office of Human Subjects Protection. Generating iPSC-derived CAR T cells with an endogenous T cell phenotype and conventional CAR T functionality 1 1 Zhiqiang (Daniel) Wang, PhD , Hellen McWilliams-Koeppen, MS , P226 1 1 1 1 Christian Huynh, BS , Hernan Reza , Vibhuti Vyas , Xiuli Wang, PhD , Iovance Gen2 TIL manufacturing process produces drug products 1 1 1 Wen-Chung Chang, MS , Julie Ostberg, PhD , Renate Starr, MS , Jamie that exhibit favorable quality attributes for adoptive cell transfer 1 1 1 1 1 Wagner , Brenda Aguilar, BS , Xiwei Wu , Jinhui Wang , Wei Chen , Chris across 5 solid tumor indications 2 2 1 1 Seet , Gay Crooks , Christine Brown, PhD , Stephen Forman, MD Seth Wardell, Maritza Lienlaf-Moreno, Lavakumar Karyampudi, Anand 1 2 City of Hope, Duarte, CA, United States; UCLA, Los Angeles, CA, United Veerapathran, PhD, Ian Frank, Michelle Blaskovich, BS, Kenneth Onimus, States Arvind Natarajan, Maria Fardis, PhD, MBA, Joe Wypych Correspondence: Zhiqiang (Daniel) Wang (zhwang@coh.org); Christine Iovance Biotherapeutics, Inc., Tampa, FL, United States Brown (cbrown@coh.org); Stephen Forman (sforman@coh.org) Correspondence: Joe Wypych (joe.wypych@iovance.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P225 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P226 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 121 of 272 Background P227 The Iovance Gen2 manufacturing process is a robust T-cell expansion Co-expression of the metabolic enzyme GOT2 with a GPC3- platform that produces a cryopreserved drug product after a 22-day targeted CAR-T overcomes challenges of the solid tumor manufacturing period. Gen2 represents a flexible closed cell produc- microenvironment, substantially improving therapeutic efficacy in tion process that is scalable to meet commercial demand. Drug prod- solid tumor xenografts ucts generated by this process display favorable quality attributes for Kathleen Whiteman, MS, Tapasya Pai, Eugene Choi, Taylor Hickman, Tyler adoptive transfer and the method is reproducible across 5 solid Johnson, Luke Barron, PhD, Taylor Friedman, Madaline Gilbert, Binzhang tumor indications at clinical scale. Shen, Seth Ettenberg, Kathleen McGinness, Greg Motz Methods Unum Therapeutics, Cambridge, MA, United States Methods to assess proliferation, phenotype, and function were ap- Correspondence: Greg Motz (greg.motz@unumrx.com) plied to in-process and final drug products generated with Gen2 at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P227 clinical scale to determine fit within the internally defined target product profile. TIL expansion was assessed through automated enu- Background meration of total and viable nucleated cells. Culture health was The metabolic demands of cancer cells in the solid tumor microenvir- assessed through cellular viability determined by DAPI exclusion. onment (TME) create an unfavorable T cell environment through deple- Immunophenotyping was performed to determine identity and pur- tion of critical nutrients and amino acids and accumulation of waste ity as well as relative levels of activation and differentiation of the products. This drives T cell dysfunction and inhibits the effectiveness of cell product. Cellular function was evaluated as the ability of the cell immunotherapies. To overcome these and other TME challenges, we product to secrete IFN-ɣ in response to CD3, CD28, and 4-1BB recep- developed the BOXR (bolt-on chimeric receptor) platform in which tor engagement. engineered T cells co-express both a chimeric-targeting receptor and a Results “bolt-on” transgene [1]. In a screen of 100+ genes for enhanced T cell Reported herein is the collective experience at Iovance for expansion function when co-expressed with an anti-glypican-3 (GPC3) CAR, we of TIL from five tumor types. The Iovance Gen 2 manufacturing identified the first candidate of our BOXR platform, BOXR1030, which process achieved doses comparable to lifileucel (LN-144, melanoma) co-expresses the transgene glutamic-oxaloacetic transaminase 2 and previously published methods across 5 primary tumor indica- (GOT2), a critical enzyme involved in mitochondrial metabolism. tions (Melanoma: mean 2.83 x 10e10 viable cells, n=82; Cervical: Methods mean 2.31 x 10e10 viable cells, n=53; Head & Neck: mean 5.82 x We compared functional and phenotypic readouts of second- 10e10 viable cells, n=12; Lung: mean 2.09 x 10e10 viable cells, n=3; generation GPC3 CAR-T cells with BOXR1030. Broad transcrip- Sarcoma: mean 1.12 x 10e10 viable cells, n=5). Quality attributes of tome profiling, metabolic characterization, and comprehensive drug products generated with Gen 2 were comparable across all 5 phenotypic assessments were performed; T cell proliferation and primary tumor indications evaluated in terms of T-cell purity, expres- cytokine production under TME-stress conditions (limiting nutri- sion of costimulatory molecules, and memory subsets. Gen 2 drug ents and hypoxia) were evaluated. In vivo, we assessed T cell products across all 5 additional indications continued to exhibit ro- anti-tumor activity, expansion and phenotype using GPC3- bust capacity to produce INF-ɣ upon reactivation, comparable to expressing solid tumor xenograft models in mice. lifileucel. Results Conclusions The addition of GOT2 had pleiotropic effects on BOXR1030 T The Iovance Gen2 manufacturing process allows for the rapid cells, improving multiple T cell functions relative to parent generation of clinical scale doses for patients in urgent need of GPC3 CAR-T cells. BOXR1030 CD4+ T cells had greater polyfunc- therapy. The cryopreserved drug product introduced critical logis- tionality relative to parent CAR-T. BOXR1030 showed improved tical efficiencies and flexibility in distribution that overcame trad- proliferation in vitro, including against TME-challenges. itional barriers to the commercialization of TIL therapy. Gen 2 BOXR1030 CD8+ T cells had a greater proportion of less differ- drug products exhibit favorable quality attributes for adoptive entiated CD27+ cells following production, and CD8+ T cells transfer including high levels of co-stimulatory molecules, and a evaluated ex vivo from xenograft tumors had substantially di- robust capability to secrete cytokine upon reactivation. These minished level of inhibitory receptors (PD-1, Tim-3) suggesting characteristics are reproducible across a broad range of solid resistance to exhaustion in the TME (Figure 1). Further, tumor indications at a high manufacturing success rate opening BOXR1030 was highly efficacious against GPC3-expressing solid the door for many more patients to benefit from this highly tumor models that resisted parental CAR-T therapy (Figure 2), beneficial therapy. [1,2,3] and activity was associated with improved T cell expansion and persistence in peripheral blood. References Conclusions 1. Donia M, Junker N, Ellebaek E, Andersen MH, Straten PT, Svane IM. Co-expression of a metabolic gene to enhance T cell function is Characterization and comparison of ‘Standard’ and ‘Young’ tumor a novel approach to cell therapy for solid tumors. BOXR1030 infiltrating lymphocytes for adoptive cell therapy at a Danish had substantially improved T cell phenotype and function in di- Translational Research Institution. Scand. J. Immunol. 2011;75:157–67. verse ways relative to the parent GPC3 CAR, and GOT2 con- 2. Besser MJ, Shapira-Frommer R, Treves AJ. et al. Clinical responses in a ferred superior activity against numerous TME challenges both phase II study using adoptive transfer of short-term cultured tumor infil- in vitro and in vivo. These results demonstrate that engineering tration lymphocytes in metastatic melanoma patients. Clin Cancer Res. of T cell immunometabolism is an effective and potent strategy 2010;16:2646–55. to overcome the challenges of the solid tumor microenviron- 3. Jin J, Sabatino M, Somerville R, Wilson JR, Dudley ME, Stroncek DF, et al. ment. IND-enabling studies with BOXR1030 are underway with Simplified method of the growth of human tumor infiltrating the expectation that BOXR1030 will be evaluated clinically in lymphocytes in gas-permeable flasks to numbers needed for patient the treatment of GPC3+ malignancies. treatment. J Immunother 2012;35:283–92. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 122 of 272 Reference P228 1. Barron L, Whiteman K, Gilbert M, Pai T, Snyder M, Fray M, Nelson A, Tumor infiltrating lymphocyte recognition of shared neoantigens Johnson T, Lakeman K, Shin J, Boomer R, Ettenberg S, McGinness K, from mutated DNA repair/remodeling proteins in a patient with Motz G. Select metabolic and costimulatory “bolt-on” transgenes metastatic pancreatic adenocarcinoma 1 1 enhance chimeric receptor-bearing T cell activity against solid tu- Ghanshyam Singh Yadav, PhD , Chetana Bhaskarla, PhD , Smriti 1 1 2 2 mors. J Immunother Cancer. 2018;6(Suppl 1):114:110-111, abstract Chaurasia, PhD , Joshua Tobin , Xinming Zhuo , Yinghong Pan , 2 1 P216. Annerose Berndt , Udai Kammula, MD, FACS 1 2 Ethics Approval University of Pittsburgh, Pittsburgh, PA, United States; UPMC Genome This study was approved by Unum Therapeutics’ Institutional Animal Care Center, Pittsburgh, PA, United States and Use Committee (IACUC); approval number 2016-04-004. Correspondence: Udai Kammula (kammulaus@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P228 Background Defective DNA repair, a hallmark of cancer, results in genomic instability and accumulation of genetic abnormalities in many malignancies. Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TIL) rep- resents a personalized cancer immunotherapy capable of targeting shared and private neoantigens resulting from tumor somatic mutations. We sought to interrogate TIL neoantigen reactivity in a tumor harboring a som- atic mutation in the DNA repair gene, ATM (Ataxia-Telangiectasia Mutated). Methods TIL cultures were generated from a surgically resected pancreatic cancer metastasis harboring a somatic ATM mutation. Anti-tumor re- activity of TIL culture was assessed by coculturing TIL with autolo- gous tumor cells and measurement of IFN-gamma release and upregulation of 4-1BB by flow cytometry. Tumor specific mutations were identified by whole genome sequencing (WGS). DNA fragments encoding the altered gene sequences were synthesized and expressed in autologous dendritic cells by RNA electroporation to en- able neoantigen reactivity screening. T cell receptor (TCR) sequencing was performed after single-cell sorting of tumor reactive TIL followed by primer specific PCR for TCR alpha and beta chains. Results Analysis of the pancreatic cancer TIL revealed high level reactivity against autologous tumor. Tumor WGS identified 141 somatic muta- tions (107 SNVs; 19 frameshifts; 15 other). Screening for neoantigen re- activity identified CD8+ T-cell responses against a missense mutation in Fig. 1 (abstract P227). See text for description ATM (23% of TIL) and a frameshift mutation in ARID1A (32% of TIL) but not against respective wild type gene products. TCR sequencing identi- fied a single unique TCR specific for ATM and ARID1A, respectively. Genes encoding the ATM specific TCR were retrovirally transduced into healthy donor T-cells and found to confer strong ATM mutation reactiv- ity without recognition of wild type ATM. Conclusions Over 50% of the TIL expanded from a patient with metastatic pancre- atic cancer were found to recognize neoantigens from either mu- tated ATM or ARID1A, which play a crucial role in DNA repair and chromatin remodeling. ACT using T-cells genetically engineered with these TCRs represents an attractive immunotherapy for patients har- boring these shared tumor mutations. Acknowledgements The study was supported by UPMC Immune Transplant and Therapy Center (ITTC). Ethics Approval This study was reviewed and approved by University of Pittsburgh Institutional Review Board. IRB#18010273. P229 The next generation “off-the-shelf” universal CAR for adoptive immunotherapy 1 1 1 1 Weichih Yang, PhD , Yun Ji , Xiaobing Luo , Huijuan Cui , Michael 1 1 1 1 1 1 Patrick , Yutian Wei , Shigui Zhu , Jiaqi Huang , Xin Yao , Yihong Yao , 2 2 Aibing Liang , Ping Li 1 2 Cellular BioMedicine Group, Gaithersburg, MD, United States; Tongji Hospital of Tongji University, Shanghai, China Correspondence: Yun Ji (yun.ji@cellbiomedgroup.com) Fig. 2 (abstract P227). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P229 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 123 of 272 Background antigen CD4+ and CD8+ responses and cancer-associated antigen- Adoptive immunotherapy using autologous T cells redirected with specific CD8+ T cell responses in vivo. We observe that prophylactic chimeric antigen receptors (CARs) has emerged as a powerful means and therapeutic AAC administration to mice strongly impedes tumor of treating cancer, such as B-cell malignancy. However, this approach growth and extends survival. Following therapeutic immunization, the is limited by the availability of autologous T cells especially for infant anti-tumor responses correlate with an over 10x increase in antigen- patients or patients undergoing multi rounds of chemotherapy. specific CD8+ tumor-infiltrating lymphocytes compared to untreated Methods mice. Finally, in an in vitro human system, we demonstrate that AACs Here we show that employment of the CRISPR-Cas9 system allows can be highly loaded with antigen and adjuvant using CellSqueeze®, highly efficient multiplex gene editing of T Cell Receptor Alpha Con- and that these AACs can be engulfed by human monocyte-derived stant and Beta-2-Microglobulin in primary human T cells, which is dendritic cells. intended to avoid graft-versus-host-disease and minimize the immuno- Conclusions genicity of transferred cells. Furthermore, redirecting the gene- In summary, these results indicate that antigen and adjuvant delivery engineered cells with a B cell maturation antigen (BCMA) CAR led to to APCs in vivo can effectively prime a potent anti-tumor response in their efficient destruction of BCMA+ tumor targets. To further improve mice and support the further study of SQZ AACs as an immunother- the efficacy of these universal BCMA CAR T cells, we use a strategy to apy for cancer treatment. generate the next generation universal CAR T cells by starting from naive precursors and producing the CAR T cells in conditions favoring T P231 memory stem (Tscm) cell expansion. Invariant natural killer T cells as an allogeneic cell therapy Results platform These Tscm enriched gene-engineered BCMA CAR T cells demonstrated Burcu Yigit, PhD, Xavier Michelet, PhD, Simon Yue, Darrian Moskowitz, superior activity compared to the conventional universal CAR T cells Mark Exley, Burcu Yigit, PhD based on their expansion, phenotype, IFN-gamma release, and cytotox- AgenTus Therapeutics, Lexington, MA, United States icity. An early phase clinical trial using this BCMA CAR in an autologous Correspondence: Burcu Yigit (burcu.yigit@agentustherapeutics.com) setting has demonstrated promising clinical readout (NCT03815383). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P231 Conclusions Therefore, we believe this next generation Tscm-enriched universal Background CAR T cells employing the same BCMA vector will provide another al- AgenTus Therapeutics is developing innovative allogeneic and ‘off- ternative choice for multiple myeloma patients in an “off-the-shelf” the shelf’ cell therapies by utilizing invariant natural killer T cells manner similar to other biological drugs. (iNKT) to target solid and liquid tumors. iNKT cells are innate-like lym- phocytes that bridge innate and adaptive immune responses. They P230 can be activated via their invariant T cell receptor recognizing lipid Activating antigen carriers for cancer therapy: preclinical immune antigens (e.g. alpha-Galactosylceramide) presented on CD1d mole- responses drive tumor regression cules, through NKG2D - NKG2D ligand interactions, and by cytokines. Defne Yarar, PhD, Amritha Ramakrishnan, Katarina Blagovic, PhD, Upon activation, large amounts of IFN-gamma production leads to Katherine Seidl, Howard Bernstein, MD, PhD, Armon Sharei recruitment and activation of T cells and NK cells. iNKT cells also SQZ Biotechnologies, Watertown, MA, United States exert potent direct cytolytic activity. While they are found in very low Correspondence: Defne Yarar (defne.yarar@sqzbiotech.com) numbers in human blood (~ 0.01% of T lymphocytes), some of their Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P230 unique properties make them valuable for cell therapy platforms. Due to their invariant antigen receptor, their ability to cause GvHD is Background minimal, and in fact, they have been demonstrated to suppress Productive activation of the immune system by antigen presenting GvHD in BMT settings. This facilitates the use of iNKT cells in an allo- cells (APCs) loaded ex vivo has proven to be challenging. To over- geneic cell therapy setting. In addition, iNKT cells are very efficient in come this, we have developed an approach that harnesses the nat- infiltrating solid tumors to exert their cytotoxic function and activate ural process of red blood cell (RBC) clearance from the body to other anti-tumor immune cells. activate the immune response in vivo. Using the CellSqueeze® micro- Methods fluidics platform, we have generated activating antigen carriers Due to low frequency of circulating iNKT cells, we have developed (AACs), engineered from RBCs, that are highly loaded with antigen and optimized a method to isolate and generate large numbers of and adjuvant and potently activate APCs in vivo. Here, we show that these cells in vitro for use in ‘off-the-shelf’ and allogeneic setting. AAC-mediated antigen and adjuvant targeting to APCs drives antigen Results presentation in vivo and primes potent anti-tumor T cell responses. We can achieve over 40,000-fold expansion of iNKT cells through Methods stimulation of the invariant TCR in less than 30 days. Importantly, To generate AACs, we loaded proteins or synthetic long peptide anti- after such massive expansion, iNKT cells retain their inherent cyto- gens together with adjuvants into murine or human RBCs with CellS- toxic capacity and cytokine production in response to tumor cells. queeze®. Following intravenous AAC injection into mice, we measured Conclusions AAC clearance kinetics from the blood and characterized the site and AgenTus is applying its proprietary Antigen Receptor platforms to cell type of AAC uptake. In addition, we quantified endogenous im- identify novel CARs and TCRs directed against tumor-specific anti- mune responses to AAC administration by flow cytometry. To deter- gens. We believe that through modification with tumor-targeted mine the ability of AACs to control subcutaneously implanted tumors, CARs and TCRs, iNKT cells will serve as potent allogeneic cell therapy we measured tumor growth rates in mice treated either prophylactic- vehicles. This should enable an ‘off-the-shelf’ approach for improving ally or therapeutically with AACs. Finally, to assess if AACs could be patient access to cell therapy. engulfed by antigen-presenting cells in a human system, we quantified in vitro uptake of human AACs by monocyte-derived dendritic cells P232 using flow cytometry and fluorescence microscopy. Characterization of ADCC resistance in multiple cancer types Results David Zahavi, MS, BS, Yongwei ZHANG, Sandra Jablonski, PhD, Louis We demonstrate that CellSqueeze® loads antigen and adjuvant into Weiner, MD AACs effectively. When administered into a mouse, these carriers are Georgetown University, Washington, DC, United States cleared from circulation within one hour and are engulfed by profes- Correspondence: Louis Weiner (weinerl@georgetown.edu) sional phagocytes in both the spleen and liver. Moreover, we find that Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P232 murine SQZ AACs processed with CellSqueeze® stimulate model Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 124 of 272 Background conjugated with H-2Kb OVA, m4-1BBL and mIL-12. Naïve OT1 Antibody-dependent cell-mediated cytotoxicity (ADCC) is an import- cells were transferred into tumor bearing mice followed by ant mechanism of action in targeted monoclonal antibody (mAb) mRBC-321 treatment. Dramatic dose-dependent expansion of cancer immunotherapy. The majority of patients who receive tar- OT1, comprised of functional effector and central memory pheno- geted mAbs develop resistance to therapy and there remains a great types, was observed in peripheral blood (9468-fold Tem; 146-fold need to understand resistance mechanisms. In vitro modeling of Tcm expansion) and secondary lymphoid organs (3323-fold Tem; ADCC provides an experimental system for uncovering tumor cell 725-fold Tcm expansion in spleen) on day 7. mRBC-321 treatment based immune resistance mechanisms. using an EG7.OVA tumor model resulted in tumor regressions in Methods 12/16 mice, 6 of which were cured, and increased survival (p Utilizing our in vitro model system of continuous selection pressure Conclusions with NK92-CD16V effector cells and the mAbs Cetuximab and Trastu- Based on these results, human RTX-321 expressing h4-1BBL, hIL-12, zumab we have generated three ADCC resistant cell lines from par- and HLA-A*02 with an HPV E7 peptide was generated to activate ental A431, SK-OV-3, and FaDu cells. and expand HPV E7-specific T cells. RTX-321 activated TCR signaling Results in an engineered HPV E7-specific TCR Jurkat line and stimulated 4- We show that the induction of ADCC resistance in all three cells lines 1BB and IL-12R signaling in respective reporter cell lines. These re- involves a loss of target cell adhesion properties required for the es- sults support the clinical development of RTX-321, which is currently tablishment of an immune synapse, NK cell activation, and target cell in IND-enabling studies for the treatment of HPV16+ advanced solid cytotoxicity. Remarkably, ADCC-resistant cells possess reduced cell tumors. surface expression of multiple proteins that contribute to intercellular interactions and immune synapse formation. We have termed the P234 loss of a selection of cell surface proteins which contributes to ADCC DAP10 and DAP12 signaling based CAR circumvents ligand- resistance Testudinidosis. This phenomenon is characterized by dys- dependent tonic signaling and mediates potent anti-tumor regulation of protein trafficking and subcellular localization of the cell response in vivo surface molecules. Additionally, ADCC resistant cell lines exhibit aber- Alan Epstein, MD, PhD, Long Zheng, Long Zheng rant IFN/STAT1 signaling. University of Southern California, Los Angeles, CA, United States Conclusions Correspondence: Alan Epstein (aepstein@usc.edu) Using multiple cell lines to model ADCC resistance has led to the dis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P234 covery of a shared mechanism of resistance across cancer types that may reveal potential therapeutic targets for combination Background immunotherapy. The Lym-1 antibody which targets a unique, discontinuous epitope on the HLA-Dr protein expressed in human B-cell lymphomas and leukemias de- P233 veloped in the late 1970’s by co-author ALE has been found to be clinically RTX-321, an allogeneic artificial antigen presenting red cell safe and effective as an I-131 radioimmunoconjugate [1-3]. Based upon therapeutic, expressing MHC I-Peptide, 41BBL and IL12, promotes these early clinical data, we have generated a humanized version (huLym- antigen-specific T cell expansion and anti-tumor activity in HPV16+ 1-B) to construct chimeric antigen receptors (CARs) using the single chain tumors variable fragment (ScFv) of huLym-1-B to treat Lym-1 positive tumors. Xuqing Zhang, PhD, Tiffany Chen Methods Rubius Therapeutics, Cambridge, MA, United States The antibody ScFv was ligated to a lentivirus vector in frame with Correspondence: Tiffany Chen (tiffany.chen@rubiustx.com) CAR backbone and CAR lentiviruses were produced and used to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P233 transduce primary CD3+ human T-cells. After transduction, we char- acterized the expansion, the effector function, and the immune- Background phenotypes of CAR-T cells. The in vivo efficacy of the CAR-T cells was Autologous CAR-T therapy has demonstrated efficacy in a small measured in a metastatic Raji lymphoma xenograft 8 days after the subset of hematological cancers. The wider adoption of antigen- iv injection of 1 million tumor cells in NSG mice. specific therapies has been limited by significant toxicity and a Results lack of effectiveness in solid tumors. Manufacturing is costly, huLym-1-BCAR was successfully transduced at an efficiency between time-consuming and difficult to scale. To address these limita- 50-80%. Surprisingly, the CAR transduced T-cells showed limited expan- tions, Rubius Therapeutics has genetically engineered red cells to sion, reaching approximately 8-fold expansion after 11 days of culture create an allogeneic artificial antigen-presenting cell (aAPC), compared to mock T-cells which had a robust 310 to 380-fold expan- called RTX-321, for the treatment of HPV16+ advanced solid tu- sion. In expanded CAR-T cells, over 28% of cells showed phosphory- mors. RTX-321 presents an HPV E7 peptide on major histocom- lated CD3z and increased PD-1 and LAG3 expression indicative of T-cell patibility complex I (MHC I [HPV]), a costimulatory signal (4-1BBL) exhaustion which was not detected in mock T-cells. Since Lym-1 recog- and a membrane-bound cytokine (IL-12) on the cell’ssurface to nizes a conformational epitope on HLA-Dr which may be weakly mimic the human immunobiology of T cell APC interactions. RTX- expressed by activated T-cells, we speculated that the huLym-1-BCAR 321 is designed to activate and expand tumor-specific T cells may stimulate the transduced T-cells to cause tonic signaling and ex- present within the patient and eliminates the need for manufac- pansion failure. This was confirmed by flow cytometry that detected turing patient-derived T cells. huLym-1-B but not huLym-1-Bmut, a huLym-1-B mutant that lost its Methods binding ability, binding on T cells. Meanwhile, neither huLym-1-Bmut As a proof of principle, red cells engineered to express mouse based CAR or huLym-1-BCAR with inactive CD3z domains showed de- MHC I H-2Kb loaded with OVA 257-264 peptide (H-2Kb OVA), creased expansion or increased CD3z phosphorylation. murine 4-1BBL (m4-1BBL) and murine IL-12 (mIL-12) were used to Conclusions stimulate OT1 cells. Although the proliferation issue did not compromise CAR-T cells’ ef- Results fector function, this would impose a potential challenge for large-scale Compared to cells expressing H-2Kb OVA or m4-1BBL alone, manufacture. We discovered that replacing the BB3z in huLym-1-BCAR these cells induced up to a 14-fold expansion of OVA antigen- with signaling domains from DAP10 and DAP12 addressed this prolifer- specific OT1 cells in vivo. These expanded cells displayed a mem- ation issue but enabled potent anti-tumor efficacy in vivo. In addition, ory phenotype and enhanced antigen-specific tumor killing of the expanded huLym-1-B DAPCAR-T cells enabled a lower dose of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate, injected CAR T-cells to induce durable control of metastatic lymphoma mRBC-321, was created using murine red cells chemically in mice with large tumor burdens. Further testing of DAP signaling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 125 of 272 sequences in other CAR T-cells may show that this change also im- suggesting myeloid cell proliferation. Finally, stable or partial re- proves the clinical efficacy of CAR T-cell therapy directed against other sponse to therapy tracked with either a dense T cell- or myeloid-cell antigens targeted in both hematopoietic and solid tumors. infiltrate while patients who progressed displayed low infiltrate levels. Acknowledgements Conclusions This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. In conclusion, immunotherapy associated skin rash contains a com- plex immune infiltrate, with increased cellular proliferation among References sites of dense infiltration. The highest levels of T cell or myeloid cell 1. Epstein AL, et al. Two new monoclonal antibodies, Lym-1 and Lym-2, re- infiltrate were seen in stable or responding patients, suggesting a active with human B-lymphocytes and derived tumors, with immunodi- link between the immune profile of skin rash and therapy response. agnostic and immunotherapeutic potential, Cancer Res. 1987; 47:830-840. Ethics Approval 2. DeNardo GL, et al. Low-dose, fractionated radioimmunotherapy for B-cell The study was approved by The Northwestern University Ethics Board maligancies using 131I-Lym-1 antibody. Cancer Biother Radio. 1998; 13:239-254. 3. Hu E, et al. A phase 1a clinical trial of LYM-1 monoclonal antibody ser- otherapy in patients with refractory B cell malignancies. Hematol Oncol. P236 1989; 7:155-166. Nivolumab related side effects based on patient-reported Ethics Approval outcomes: A multicenter study from real-life setting 1 1 This study was approved by the IRB of the University of Southern California, Canan Karadas , Zehra Gok Metin, Assoc Prof, PhD, RN , Nur Izgu, PhD, 1 2 2 protocol number HS-16-00029 approved 2-29-16, and by IACUC protocol RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, Checkpoint Blockade Therapy Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Correspondence: Canan Karadas (karadas.canan@gmail.com) P235 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P236 A dense, proliferative myeloid and T cell-rich, immune infiltrate characterizes immunotherapy-induced skin rash Background Cormac Cosgrove, PhD, Cory Kosche, Suyeon Hong, Caroline Le Poole, Immune checkpoint inhibitors provide an effective treatment option Jennifer Choi for patients with melanoma and other cancer types. Nivolumab, as Northwestern University, Chicago, IL, United States an immune checkpoint inhibitor, acts via blockade of the PD-1 recep- Correspondence: Cormac Cosgrove tor, and limits immune responses against tumors. As reported for (cormac.cosgrove@northwestern.edu) nivolumab, the anti-PD-1 antibodies can induce immune-related ad- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P235 verse events [1, 2]. Nivolumab related adverse effects compose of general, pulmonary, gastrointestinal, neurologic, skin, and infusion re- Background actions. These adverse effects may be life threatening, so following Checkpoint inhibitor immunotherapy is associated with a unique tox- of patients receiving nivolumab is essential for early diagnosis and icity profile, collectively known as immune related adverse events management. Therefore, this study aimed to examine nivolumab re- (irAEs). One of the earliest and most common irAEs is skin rash. Inter- lated adverse effects based on patient- reported outcomes (PROs) estingly, development of rash has been associated with improved from a real-life setting. survival, and is likely an early indicator of a successfully activated im- Methods mune response. More severe toxicities also occur, can affect nearly In total, 40 patients receiving first cycle of nivolumab were included every organ and are clinical justification for dose reduction or termin- from three hospitals located in Turkey. Patient Information Form and ation of immunotherapy. However, the mechanism underpinning Patient Monitoring Checklist-Nivolumab were used for data collection. rash development and its link to disease outcome or toxicities is not Patient Monitoring Checklist-Nivolumab included totally 46 questions: known. Thus, we examined the makeup of immunotherapy- (21-general adverse effects, 3-pulmonary, 8-gastrointestinal, 5- associated skin rash infiltrates with the goal of uncovering mechanis- neurological, 2-skin, and 7-infusion reactions) and two options in each tic insights behind rash development. item that evaluating presence of related adverse effect, as “yes” or “no”. Methods PROs were analyzed by frequency and percentages. Immunohistochemistry was used to describe the immune infiltrate in Results skin biopsies from healthy subjects and from a lesional site of patients The mean age of patients was 55.47±16.48 years, and the majority of who developed rash secondary to immunotherapy. Rash samples were them (82.5%) had diagnosed with cancer longer than one year. The obtained from 7 patients receiving α-PD1, α-CTLA4/α-PD1 combination cancer diagnosis composed of melanoma (52.5%), renal cell carcin- or α-PDL1/α-NKG2A combination. Acetone-fixed sections from frozen oma (17.5%), nasopharyngeal carcinoma (7.5%) and other cancer biopsies were stained for CD3, CD4, CD8, CD68, CD11c, CD1a, CD207, types (23.5%). Considering differences in terms of gender, only two or Ki67. Cell abundance in the dermis was compared among groups. items including 8th item related general adverse effects, quiring loss Results of hair, and 37th item evaluating changes in mental status, percep- : Rash samples showed significant enrichment of T cells (CD3 p= tion, judgement, and memory had a significant difference. Female 0.01), CD8 T cells (p=0.03) and dendritic cells (CD11c p=0.03) in the patients reported higher adverse effects than male regarding afore- dermis vs controls. More moderate enrichment of macrophages mentioned items (p (CD68) was observed in rash versus control samples while the abun- Conclusions dance of dermal Langerhans cells (CD1a or CD207) was comparable PROs related nivolumab in this study included fatigue, rash and itch- among groups. We observed more proliferating cells in the dermis of ing, and diarrhea as consisted with previous reports. A comprehen- rash vs control samples (Ki67 p=0.024), associated with areas of sive approach is needed to reduce and manage nivolumab related dense immune infiltration. Ki67 expression was highly correlated with adverse effects. both CD68 (r=0.89; p=0.012) and CD11c (r=0.786; p=0.048), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 126 of 272 References aware of the rare, debilitating, and possibly previously undescribed 1. Naidoo J, Page DB, Li BT, Connell LC, Schindler K, Lacouture ME, Wolchok paraneoplastic and autoimmune toxicities that may occur. JD. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint anti- bodies. Ann Oncol. 2015; 26:2375–2391. References 2. Spain L, Diem S, Larkin J. Management of toxicities of immune 1. Forster MD, Devlin MJ. Immune Checkpoint Inhibition in Head and Neck checkpoint inhibitors. Cancer Treat Rev. 2016; 44:51–60. Cancer. Front Oncol. 2018; 8:310. Ethics Approval 2. Pollack MH, Betof A, Dearden H. Safety of resuming anti-PD-1 in pa- The study was approved by clinical trials ethics committee of the University tients with immune-related adverse events (irAEs) during combined of Health Sciences Ankara Oncology Training and Research Hospital anti-CTLA-4 and anti-PD1 in metastatic melanoma. Ann Oncol. 2018; (decision number: 2018–06/52) and performed in accordance with the 29:250-255. Helsinki Declaration. 3. Bataller L, Wade DF, Graus F. Antibodies to Zic4 in paraneoplastic neurologic disorders and small-cell lung cancer. Neurology. 2004; 62. 4. Schwab KS, Kristiansen G, Schild HH. Successful Treatment of Refractory P237 Squamous Cell Cancer of the Head and Neck with Nivolumab and A case of anti-Zic4 antibody-mediated cerebellar toxicity induced Ipilimumab. Case Rep Oncol 2018;11:17–20. by dual checkpoint inhibition in HNSCC Consent Sunil Iyer, MD, Nidah Khakoo, MD, Gabriella Aitcheson, MD, Marina Consent was obtained from the patient for publication of this abstract. Kushnirsky, MD, Cesar Perez, MD University of Miami, Miami Beach, FL, United States Correspondence: Sunil Iyer (sunil.iyer@jhsmiami.org) P238 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P237 Longitudinal immune and genomic monitoring reveals signatures of response and Immune-related adverse events in cancer patients Background receiving checkpoint inhibitor therapy Combined checkpoint inhibition therapy targeting the PD-L1 and Shaheen Khan, PhD, David Gerber CTLA4 pathways has been a successful approach in the treatment of UT Southwestern Medical Center, Dallas, TX, United States metastatic melanoma, leading to its investigation in the treatment of Correspondence: David Gerber (david.gerber@utsouthwestern.edu) head and neck squamous cell carcinoma (HNSCC) with PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P238 sion [1]. Despite the potential for excellent responses, an increased rate of autoimmune neurological toxicity and paraneoplastic conditions has Background been observed [2]. We present the case of a patient with metastatic Despite the remarkable success of immune checkpoint inhibitor (ICI) HPV-positive HNSCC treated with ipilimumab/nivolumab who experi- therapy, a significant number of patients develop severe and unpredict- enced severe cerebellar ataxia, with a positive screen for the anti-Zic4 able immune-related adverse events (irAEs) affecting a wide variety of antibody, which has been associated with cerebellar degeneration in organs. Concerns over irAE have led to the exclusion of patients with small cell lung cancer (SCLC) and has not been reported in HNSCC [3]. autoimmune disease from ICI clinical trials. Role of host genetic and im- Results mune factors in mediating irAEs remain unclear and it is not clear if the A 40-year-old man diagnosed with HPV-positive HNSCC with meta- manifestations of irAEs is associated with response to therapy. static recurrence after radiation treatment of the initial tumor was Methods started on a clinical trial of a DNA-PK inhibitor. His disease pro- We used multi-faceted approach to identify blood-based biomarkers gressed, and given his PD-L1 tumor proportion score of 70% he was predictive of irAEs and response to ICI therapy. We characterized initiated on ipilimumab/nivolumab. After his second cycle, he pre- changes in host immune system in 200 patients receiving ICI therapy sented with sudden blurred vision and mild ataxia, which rapidly pro- at baseline and post-immunotherapy (100 with irAEs and 100 without gressed to severe ataxia and dysarthria. Autoimmune toxicity was irAEs). We assessed genetic predisposition to autoimmunity in these suspected; initial brain imaging and serum testing were unremark- patients using the Illumina GSA SNP array and via targeted resequen- able. While awaiting the results of complex autoimmune and para- cing of over 150 immunoregulatory loci including the HLA region. We neoplastic CSF testing, he was treated with multiple modalities in an evaluated serum levels of cytokines/chemokines, Antinuclear auto- escalating fashion with minimal improvement, including pulse-dose antibodies (ANA) and 124 autoantibodies and performed RNA se- corticosteroids, IVIG, and plasmapheresis. The paraneoplastic panel quencing and flow cytometry on peripheral blood mononuclear cells returned negative for common autoimmune culprits in cerebellar en- (PBMCs) at baseline and post immunotherapy in patients with and cephalopathy including anti-Hu and anti-Yo; however, anti-Zic4 was without irAEs. detected at borderline levels. Repeat MRI showed an enhancing le- Results sion in the cerebellum. Finally, rituximab was initiated, and the pa- Our preliminary data analysis identified signatures of autoantibodies tient is slowly improving. Notably, restaging scans show a mixed and cytokines correlating with response and toxicity. We also identi- response with resolution of previously extensive metastatic disease fied HLA haplotypes linked with autoimmunity in selected patients in the thorax, however with worsening osseous lesions. that developed immune-related adverse events. In this meeting, we Conclusions will present immune and genetic correlates of irAEs and response to We present a case of anti-Zic4-mediated cerebellar toxicity in the set- therapy in our patient cohort. ting of dual PD-L1/CTLA4 inhibition in the treatment of metastatic Conclusions HNSCC. Anti-Zic4 has been historically associated with cerebellar- In-depth analysis of immune and genetic datasets is currently under- predominant paraneoplastic neurological disorders in SCLC [3], and way. We hope that our studies can help identify blood-based bio- to our knowledge, has not been described in HNSCC. Although the marker signatures predictive of irAEs and/or response and reveal patient experienced an impressive partial response, he suffered novel insights into the mechanisms underlying irAE. Our current gen- grade 4 cerebellar neurotoxicity. Cases have demonstrated excellent etic data suggests further expanding the genetic studies in larger pa- clinical responses utilizing dual PD-L1/CTLA4 inhibition in HNSCC [4], tient population to identify the role of underlying genetic however high-grade adverse events have also been reported in this predisposition to autoimmunity in mediating irAEs. We hope our regimen’s more established use in metastatic melanoma [2]. Despite findings may ultimately help identify customize therapy, expand use the exciting advances in cancer immunotherapy, clinicians must be of immunotherapy and prevent toxicities. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 127 of 272 P239 Table 1 (abstract P239). ICI-arthritis patient characteristics (total n=372) Immune checkpoint inhibitor-associated arthritis: a systematic literature review of case series and case reports 1 2 3 Michael Tiongson, BA , Nilasha Ghosh, MD , Carolyn Stewart, BA , 2 2 2 Karmela Chan, MD , Bridget Gatto, MLIS , Anne Bass, MD 1 2 Albany Medical College, Nanuet, NY, United States; Hospital for Special Surgery, New York, NY, United States; Weill Cornell Medicine, New York, NY, United States Correspondence: Nilasha Ghosh (GhoshN@HSS.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P239 Background As immune checkpoint inhibitors (ICI) continue to revolutionize can- cer treatment, immune-related adverse events (irAE) are becoming more prevalent. Inflammatory arthritis occurs in approximately 4% of ICI-treated patients [1] but remains poorly characterized. We per- formed a systematic literature review to identify all reports of ICI- associated inflammatory arthritis in order to describe it phenotypic- ally and serologically. Methods PubMed, Embase and Cochrane databases were searched for pub- lications reporting musculoskeletal irAEs secondary to ICI treat- ment through the search date, May 31, 2019. Publications were included if they provided individual patient-level data regarding the pattern of joint involvement. Two reviewers screened all ab- stracts and full texts to extract demographics, clinical features, se- rologies, treatment data and outcomes. Descriptive statistics were used to summarize results. Results 4339 articles were screened, of which 67 were included (42 case reports, 15 case series, 10 retrospective chart reviews) encom- passing 372 patients (Table 1). Mean age was 63 +/- 11 years; 61% patients were male. The majority of patients had metastatic melanoma (57%) and were treated with anti-PD1 or anti-PDL1 P240 therapy (78%). Median time to onset of arthritis was 4 months Quantitative cell-based bioassays to advance immunotherapy (range: 1 day-53 months). 49% had polyarticular arthritis, 17% oli- programs targeting immune checkpoint receptors goarthritis, 3% monoarthritis, 10% arthralgia and 21% polymyalgia Vanessa Ott, PhD, Jamison Grailer, PhD, Jun Wang, Julia Gilden, PhD, rheumatica (PMR). 9% tested positive for rheumatoid factor (RF) Pete Stecha, Denise Garvin, Michael Beck, Jim Hartnett, Frank Fan, PhD, or cyclic citrullinated peptide (CCP) antibodies. 74% required cor- Mei Cong, PhD, Zhi-jie Jey Cheng ticosteroids and 45% required additional therapies, including 5% Promega, Madison, WI, United States requiring a TNF inhibitor. 63% of patients achieved control of Correspondence: Zhi-jie Jey Cheng (jey.cheng@promega.com) their musculoskeletal symptoms with treatment, and 32% were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P240 ultimately able to discontinue anti-rheumatic treatments. ICI were continued in 49%, transiently withheld in 11%, and permanently Background discontinued due to musculoskeletal irAEs in 13%. At last follow- The human immune system is comprised of a complex network of im- up, 27% had progression of their cancer. mune checkpoint receptors that are promising new immunotherapy tar- Conclusions gets for the treatment of a variety of cancers and autoimmune-mediated Half of reported ICI-associated arthritis cases have a polyarthritis disorders. Immunotherapies designed to block co-inhibitory receptors (e.g. (often in an RA distribution) but only 9% are seropositive. PMR is also PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of can- commonly seen. The vast majority of ICI-arthritis cases are in melan- cer. However, not all patients and tumor types respond to this approach. oma patients treated with anti-PD1/PDL1 therapy. Most patients re- This has resulted in broadening of immunotherapy research programs to spond to steroids alone but about half require additional anti- target additional co-inhibitory (e.g. LAG-3, TIM-3) and co-stimulatory (e.g. rheumatic agents. Further studies are needed to determine long- 4-1BB, GITR, OX40, ICOS) receptors individually and in combination. term musculoskeletal outcomes in these patients and the impact of Methods arthritis treatment on cancer survival. A major challenge in the development of biologics is access to quantita- tive and reproducible functional bioassays. Existing methods rely on pri- Reference mary cells and measurement of complex functional endpoints. These 1. Kostine M, Rouxel L, Barnetche T, Veillon R, Martin F, Dutriaux C, assays are cumbersome, highly variable and fail to yield data quality re- Dousset L, Pham-Ledard A, Prey S, Beylot-Barry M, Daste A. Rheum- quired for drug development in a quality-controlled environment. To ad- atic disorders associated with immune checkpoint inhibitors in pa- dress this need, we have developed a suite of cell-based functional tients with cancer—clinical aspects and relationship with tumour bioassays to interrogate modulation of immune checkpoint receptors indi- response: a single-centre prospective cohort study. Ann Rheum Dis. vidually (e.g.PD-1,CTLA-4,LAG-3, TIM-3, GITR, 4-1BB) and in combination 2018; 77:393-8. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 128 of 272 (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) that express luciferase reporters driven by response elements under the and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) precise control of mechanistically relevant intracellular signals. could serve as subpopulations relevant for patient stratification (Figure Results 2). We further validated these results using CyTOF wherein these same The bioassays reflect mechanisms of action for the drug candidates de- subpopulations were differentially abundant between responders and signed for each immune checkpoint receptor and demonstrate high spe- non-responders at the pre-therapy time-point. cificity, sensitivity and reproducibility. For example, using the PD-1 Conclusions blockade bioassay, TCR-mediated luciferase activity is recovered with Collectively, our results revealed that (1) PD-1 blockade-based anti-PD-1 and PD-L1 blocking Abs but not with unrelated control anti- treatment-induced gene expression profiling changes (increase cell bodies. Similarly, TCR and CD28-mediated luciferase activity is recovered proliferation, metabolism and activation) of CD8 T cells are detect- in the CTLA-4 blockade bioassay with an anti-CTLA-4 blocking antibody able as early as EOC1 in the PB; (2) specific subpopulations of plastic (ipilimumab), but not with unrelated control antibodies. In a PD-1+CTLA- CD8 T cells identified have the potential to serve as an actionable 4 combination bioassay, anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimu- biomarker to select AML patients most likely to benefit from such im- mab) blocking antibodies individually induced luciferase activity (3.0- and mune checkpoint therapies. These findings need to be confirmed in 5.6-fold, respectively), the combination of both antibodies resulted in a larger studies with αPD-1 based therapies in AML. synergistic 18-fold increase in luciferase activity. Similar antibody-induced Acknowledgements luciferase activity is observed in a panel of bioassays specific for agonist NIH (R01CA174385), CPRIT (RP180466), MRA Established Investigator Award antibodies, including GITR, 4-1BB, OX40 and CD40. This response can be to NV (509800), Welch Foundation (E1774), NSF (1705464), CDMRP enhanced following Fc receptor cross-linking, reflecting in vivo activity. (CA160591), and Owens foundation. We would like to acknowledge the Conclusions MDACC Flow Cytometry and Cellular Imaging Core facility for the FACS Cell-based reporter bioassays overcome the limitations of primary sorting (NCI P30CA16672), UH Seq-N-Edit core for RNA-sequencing service, cell-based assays for functional characterization of antibody and and Intel for the loan of computing cluster. other biologics drugs targeting individual or combination immune Trial Registration checkpoint receptors. Here we show a portfolio of mechanism of NCT02397720 action-based bioassays for co-inhibitory and co-stimulatory immune References checkpoint receptors that can be used for antibody screening, 1. Daver, N. et al. Efficacy, Safety, and Biomarkers of Response to Azacitidine characterization, potency and stability studies. and Nivolumab in Relapsed/Refractory Acute Myeloid Leukemia: A Nonrandomized, Open-Label, Phase II Study. Cancer Discov. 2019; 9: 370–383. P241 2. Corces, M. R. et al. Lineage-specific and single-cell chromatin accessibility Single-cell deconvolution identifies T-cell correlates of response to charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016; PD-1 blockade treatment in AML 48: 1193–1203. 1 1 2 2 Xingyue An, BS , Jay R Adolacion , Mansour Alfayez , Jairo Matthews , 3. Linsley, P. S., Speake, C., Whalen, E. & Chaussabel, D. Copy number loss of 2 2 1 2 Wilmer Flores , Steven Kornblau, MD , Arash Saeedi , Sreyashi Basu , the interferon gene cluster in melanomas is linked to reduced T cell 2 1 Naval Daver, MD , Navin Varadarajan, PhD infiltrate and poor patient prognosis. PLoS One. 2014; 9. 1 2 University of Houston, Houston, TX, United States; Univ. of Texas MD 4. Szczepanski, M. J. et al. Increased frequency and suppression by Anderson Cancer Center, Houston, TX, United States regulatory T cells in patients with acute myelogenous leukemia. Clin. Correspondence: Navin Varadarajan (nvaradarajan@uh.edu) Cancer Res. 2009; 15: 3325–3332.[5. Knaus, H. A. et al. Signatures of CD8+ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P241 T cell dysfunction in AML patients and their reversibility with response to chemotherapy. JCI Insight 2018; 3. Background Ethics Approval The combination of the αPD-1 (nivolumab) and hypomethylating agent All patients signed an informed consent form approved by the Institutional azacytidine demonstrated encouraging response in R/R acute myeloid Review Board (IRB) from The University of Texas MD Anderson Cancer leukemia (AML) patients, but the percentage of patients who achieved Center. The study was conducted in accordance with the Declaration of IWG 2016 responses was limited[1]. Early predictive biomarkers to facili- Helsinki. The study was approved by the IRB from University of Houston. tate future trials patient selection are desirable. A better understanding of T cells in AML pre-therapy and on-therapy should yield valuable in- Table 1 (abstract P241). See text for description sights on the treatment-induced anti-tumor response. Methods We performed RNA-sequencing on T cells from a cohort of AML pa- tients who were treated with azacytidine and nivolumab (Table 1). By leveraging subset definitions based on single-cell RNA-sequencing results from T cells of cancer patients, we implemented deconvolu- tion of our bulk T-cell RNA-sequencing data to obtain the relative abundance of different T-cell subsets (in-silico dissection). Results For validation purpose, we compared the gene expression of periph- eral blood (PB) T cells from AML patients and healthy donors (HD)[2,3]. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients[4,5] (Figure 1). Compared with HD T cell, circulating AML CD4 T cells consisted of a higher frequency of Treg[4]. PB CD8 T cells from AML patients were with a significantly lower frequency of naïve, and higher frequencies of effector and exhausted phenotypes[5]. Independent of the clinical responses, comparison of the pre-treatment CD8 T cells from bone marrow (BM) and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PB CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression changes in the AML circulating CD8 T cells, characterized by increased cell me- tabolism and cell proliferation. Deconvolution identified that pre- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 129 of 272 contribute to immune suppression in the tumor microenvironment, inhibiting angiogenic pathways may normalize the tumor vasculature and relieve immunosuppression to augment antitumor immunity, es- pecially with concurrent immune checkpoint inhibition. Therefore, we characterized lucitanib’s selectivity and investigated the antitu- mor efficacy and mechanisms of action of lucitanib combined with anti-PD-1 in nonclinical models. Methods The kinase inhibition profile of lucitanib was evaluated using func- tional biochemical assays. Kinase phosphorylation in cancer cells or mouse tissues was assessed by western blot. In vivo efficacy studies were conducted in syngeneic mouse models with monotherapy or combinations of lucitanib (10 mg/kg daily), DC101 (mouse VEGFR2 monoclonal antibody; 40 mg/kg every 2 days), or anti-PD-1 (5−10 mg/kg biweekly). Treated tumors were analyzed for gene and protein expression and immune composition. Results In vitro, lucitanib demonstrated selective and potent inhibition of the tyrosine kinases VEGFR1-3, PDGFRalpha/beta, FGFR1-3, CSF1R, DDR1, and RET. Lucitanib caused dose-dependent inhibition of VEGFR2 phosphorylation in vivo; one 10 mg/kg dose sustained inhibition for 12 hours. Compared with DC101, lucitanib significantly enhanced tumor growth inhibition and survival in MC38 colon tumor-bearing mice. Lucitanib combined with anti-PD-1 significantly increased anti- tumor activity relative to single agents and to DC101 plus anti-PD-1. Fig. 1 (abstract P214). See text for description Lucitanib-treated MC38 tumors exhibited gene expression changes beyond those observed with DC101 treatment. Across multiple syn- geneic mouse models, tumor growth was significantly inhibited 81%−98% by lucitanib combined with anti-PD-1 and 57%−88% by lucitanib alone. The combination significantly extended survival by 90% to >186% and 15% to >35% compared with vehicle or the best mono- therapy, respectively. Lucitanib combined with anti-PD-1 increased gene expression associated with T-cells, cytotoxic cells, and T-cell sig- naling in BR5FVB1-Akt ovarian tumors, relative to each monotherapy. However, lucitanib alone appeared sufficient to modulate innate and adaptive immunity-related gene expression in MC38 tumors. Additional tumor profiling and mechanism of action studies are ongoing. Conclusions Lucitanib, a potent and selective angiogenesis inhibitor, is differenti- ated from DC101 and displays enhanced antitumor activity in com- bination with PD-1 inhibition in multiple syngeneic models. Gene expression changes associated with tumor immune infiltration and increased antitumor immunity were observed in the combination- treated tumors and may contribute to the increased antitumor activ- ity. Results from these studies support the clinical development of the combination of lucitanib and immune checkpoint blockade as a potential treatment for patients with solid tumors. Ethics Approval The studies were conducted in accordance with the Shanghai Medi- cilon Inc.Guidelines for Use and Care of Animals or an approved IACUC protocol at Crown Biosciences. P243 Fig. 2 (abstract P214). See text for description Modulating the immunogenicity of low-mutation soft tissues sarcomas with epigenetic targeted therapy Himavanth Gatla, PhD, Maggie Phillips, Brian Ladle Johns Hopkins Medicine, Owings Mills, MD, United States P242 Correspondence: Brian Ladle (bladle@jhmi.edu) Combination of the angiogenesis inhibitor lucitanib with immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P243 checkpoint blockade augments anti-tumor activity in syngeneic models Background Rachel Dusek, PhD, Liliane Robillard, PhD, Thomas Harding, PhD, Andrew Sarcomas account for 13% of all cancers in young adults under the Simmons, PhD, Minh Nguyen age of 20. For recurrent and metastatic sarcomas, the very poor sur- Clovis Oncology, Inc., San Francisco, CA, United States vival rate despite surgery and chemotherapy warrants better sys- Correspondence: Rachel Dusek (rdusek@clovisoncology.com) temic therapies. As opposed to cancers that show good responses to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P242 immune checkpoint blockade which have high mutation burden, pediatric sarcomas present with low mutation burden, and a Background complete ineffectiveness of immune checkpoint blockade as a mono- Lucitanib is an anti-angiogenic, small molecule multi-tyrosine kinase therapy, suggesting poor immune system activation, and immune inhibitor that has demonstrated potent tumor growth inhibition in infiltration. multiple cancer xenograft models. Because angiogenic factors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 130 of 272 Methods types of DNA damage repair deficiencies and immune evasion have We have used a mutated Kras-driven murine sarcoma syngeneic been lacking. Hence, we have modeled the biology of ovarian cancer tumor model (KP Sarc) and GM-CSF-secreting whole cell tumor cell using patient-relevant mutational landscapes in an immune- vaccine approach (GVAX) to investigate if epigenetic targeted ther- proficient, syngeneic mouse model in order to help us identify the apy induced tumor associated antigens are capable of generating contribution of common driver mutations to the immune repertoire sustained anti-tumor immunity in the tumor microenvironment, and thus to responses of HGSOC tu- Results mors to immunotherapy. We show that sequential combination of epigenetic modifying drugs Methods - DNA methyl transferase inhibitor (decitabine) and HDAC inhibitor We hypothesize that the immune composition and gene expression (entinostat) significantly increases the expression of cancer testis an- signatures of the resulting tumors will vary based on the combin- tigens (CTAs), compared to either drugs alone. In addition, these ation of genetic alterations and the DNA repair proficiency of the drugs modify chemokine expression including increased expression transformed cells. To this end, we have engineered novel syngeneic of CXCL10. Significantly improved immune responses can be gener- mouse models from murine-fallopian-tube epithelium using CRISPR/ ated to decitabine and entinostat pre-treated KP sarc tumor cells as Cas9 technology. These tumors capture the most common combina- assessed by slowed tumor growth, increased T cell infiltrates, and in- tions of co-occurring mutations observed in HR-deficient and -profi- creased cytokine production. Furthermore, immune checkpoint cient patient samples. blockade therapy potentiates the tumor regression mediated by the Results combination of decitabine and entinostat more effectively than the To validate the DNA repair proficiency of the transformed cells, we regression mediated by either drugs alone. This suggests that com- measured Rad51 nuclear focus formation after ionizing radiation (IR) bination therapy induced antigenicity generates better anti-tumor and PARP inhibitor and DNA-damaging agent sensitivity. The HR- immune responses. Rechallenging the mice which rejected epigeneti- deficient cell lines had significantly fewer Rad51 nuclear foci and cally modified KP Sarc tumor formation with similarly treated KP Sarc were more sensitive to PARP inhibition in comparison to HR- cells did not result in tumor formation, whereas untreated KP Sarc proficient cells. Initial immune /stromal analysis using flow cytometry, cells grew uninhibited, suggesting that epigenetic therapy induced scRNA seq transcriptomic and immunofluorescence analysis re- tumor associated antigens are capable of generating a sustained vealed substantial differences in the myeloid and T-cell regulatory memory immune response. By depleting CD4 and CD8 T lympho- compartments between HR-proficient and -deficient primary and cytes, we show that epigenetic targeted therapy induced anti-tumor metastatic tumors and within the ascitic fluid. Preliminary results responses are mediated by both CD4 and CD8 T lymphocytes. Diffi- also suggest that inhibition of the DNA damage response (DDR), culty with in vivo treatment includes the myelosuppressive side ef- checkpoint kinase 1 (Chk1) in combination with immune check- fects of decitabine and entinostat which can inhibit T cell responses point inhibitors, potentiates antitumor effects and augments cyto- immediately after treatment. Proper sequencing of the drugs when toxic T-cell infiltration. given in vivo will be crucial to generate successful adaptive T cell re- Conclusions sponses to newly expressed antigens. Understanding the genetic basis of these complex cellular interac- Conclusions tions will be critical to better tailor combinations of existing targeted Epigenetic targeted therapy induced tumor associated antigens are treatments and immunotherapies in ovarian cancer to fight this dev- capable of generating sustained anti-tumor immune responses. astating disease. P244 P245 The genomic architecture of serous carcinomas shapes the tumor Durvalumab after concurrent chemoradiotherapy in inoperable microenvironment and modulates responses to targeted and stage III non-small cell lung cancer (NSCLC) – a German radiation immunotherapies oncology survey 1 2 3 4 Sonia Iyer, PhD , Shuang Zhang , Anniina Farkkila , Sean Smith , David Lukas Kaesmann, MD, Chukwuka Eze, Julian Taugner, Olarn 5 5 1 1 1 Pepin , Raghav Mohan , Tian Xia , Ferenc Reinhardt , Tony Chavarria , Roengvoraphoj, Claus Belka, Farkhad Manapov 1 6 3 7 Esmee Hoefsmit , Shailja Pathania , Yunlan Zhou , Kevin Elias , Benjamin University Hospital, Munich, Germany 8 1 Neel , Robert Weinberg, PhD Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) Whitehead Institute for Biomedical Research, Cambridge, MA, United Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P245 2 3 States; NYU Langone Health, New York, NY, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Institute of Background Technology, Cambridge, MA, United States; Massachusetts General Consolidation PD-L1 inhibition with durvalumab after platin-based Hospital, Boston, MA, United States; University of Massachusetts, Boston, concurrent chemoradiotherapy (CRT) has become the standard of MA, United States; Brigham and Women's Hospital, Boston, MA, United care in inoperable stage III non-small cell lung cancer (NSCLC) States; NYU-Langone Medical Center, New York, NY, United States based on the excellent PACIFIC trial results. Treatment recommen- Correspondence: Sonia Iyer (iyers@wi.mit.edu); Robert Weinberg dations need time for implementation in nationwide settings and (weinberg@wi.mit.edu) require the close interaction of different medical specialities. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P244 this nationwide survey, we questioned the distribution and clin- ical settings of durvalumab treatment after concurrent CRT, ob- Background served side effects of this treatment and summarize follow-up High-grade serous ovarian cancer (HGSOC) is the most frequent and management. most aggressive histologic subtype of ovarian cancer. The corner- Methods stone of the existing treatment of HGSOC is DNA-damaging chemo- We surveyed radiation oncology institutions in Germany via an an- therapy; however, practically all patients eventually develop the onymous online questionnaire sent by e-mail to all members of the progressive disease and the 5-year survival is only 40%. Immunother- German Radiation Oncology Society. apy would seem to be an attractive alternative treatment to chemo- Results therapy, yet existing immunotherapies perform poorly in ovarian We received a total of 255 responses (response rate: 18%). Of cancer, with only ~10% of patients responding to checkpoint block- which 203 (80%) were completed and returned and thus eligible ade. Why this is the case remains poorly understood and there is a for further evaluation. The respondents work in 87 different cities pressing need to understand the underlying biology of immune eva- and 44% in a private medical practice, 29% in university and sion in ovarian cancer. One critical area of interest is the role of hom- 22% in a general hospital. Responses of the same department ology dependent DNA repair (HR) in immune evasion. Unfortunately, were analysed for congruence. Durvalumab was implemented in the preclinical tools required to explore the relationship between the clinical routine by 143 (70%) respondents. Reasons for failed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 131 of 272 implementation in clinical practice were patient ineligibility, deci- months. All patients were alive at the time of evaluation. Four (25%) sion of medical oncologists or absence of updated German evi- patients have developed oligoprogression. Metastastic sites were dence review (S3-guidelines) regarding this treatment approach. bone, brain, adrenal gland and distant lymph nodes. Two patients re- Durvalumab was generally administered according to the re- ceived second-line chemotherapy after distant failure. Another two spondents by private oncological practices in 32%, general or received stereotactic body radiotherapy for all metastatic sites and university hospital in 57% and in the radiation oncology de- continued on durvalumab. Common toxicity during durvalumab was partment, which delivered the CRT in 11% of cases. Importantly, dermatitis (I-II° CTCAE) which occurred earliest after 2 cycles in 10 according to 36% of all respondents initial PD-L1 status was (65%) patients and pneumonitis II° CTCAE in 2 (13%) and III° CTCAE present in ≤30% of all patients. 82% of respondents have treated in 2 (13%) patient between 2-7 months after completion of CRT. In 1-15 patients with durvalumab and 14% of respondents >15 pa- total, 3 (19%) patients discontinued durvalumab treatment after a tients. Furthermore, no respondent had applied durvalumab in median of 4 months due to distant progression or unacceptable less than 14 days after the completion of CRT. 65 (46%)and 49 toxicity. (34%) respondents started durvalumab 14-28 days and later than Conclusions 28 days after CRT, respectively. The majority of respondents Durvalumab was well tolerated with reversible acute toxicity. 25% of (>80%) re-staged the patients with CT (thorax/upper abdomen) patients develop oligoprogression after a mean time of 5.5 months prior to durvalumab. Severe side effects requiring hospital admis- after the end of CRT. sion in more than 10% of all patients were reported by only 12% Ethics Approval of all respondents. The study was approved by the University Ethics Board, approval Conclusions number 17-230. Durvalumab was implemented in the multimodal treatment of inop- Consent erable stage III NSCLC and administered by the absolute majority of Written informed consent was obtained from the patient for publica- respondents. Low testing rates of PD-L1 at initial diagnosis were ob- tion of this abstract and any accompanying images. A copy of the served and should be considered a major barrier to universal adop- written consent is available for review by the Editor of this journal. tion and integration in the clinical work-flow. Durvalumab appears to be well tolerated. However, treatment-related side effects need to be P247 considered during and after multimodal therapy. TLR3-targeting combinatorial chemokine modulation sensitizes “Cold” tumors for the therapeutic effectiveness of immune Acknowledgements checkpoint inhibition We would like to thank the Board of the German Society for Radiation 1 2 Kathleen Kokolus, MS, PhD , Natasa Obermajer, PhD , Per Basse, MD, Oncology (DEGRO) for their approval and their office team for providing the 1 1 PhD , Pawel Kalinski, MD, PhD mailing list. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Ethics Approval UPMC Hillman Cancer Center, Pittsburgh, PA, United States The Board of the German Society for Radiation Oncology (DEGRO) approved Correspondence: Pawel Kalinski (Pawel.Kalinski@roswellpark.org) the survey. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P247 P246 Background Prospective evaluation of outcome and toxicity of durvalumab Immune checkpoint inhibition (ICI) has emerged as life-prolonging treatment after chemoradiotherapy in inoperable stage III non- and occasionally curative treatment for many cancer patients, but small cell lung cancer (NSCLC) patients their activity remains disappointing in many common tumors. ICI Lukas Kaesmann, MD, Julian Taugner, Chukwuka Eze, Olarn therapies are effective against “hot” tumors infiltrated with cytotoxic Roengvoraphoj, Claus Belka, Farkhad Manapov T lymphocytes (CTLs) but inefficient against “cold” tumors lacking University hospital, Munich, Germany CTLs. The importance of CTLs, availability of CTL targets and local ex- Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) pression of PD-L1 and PD-L2 (induced by CTL-produced IFNγ) in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P246 overall effectiveness of ICI remain controversial. We have observed that chemokine-modulatory (CKM) regimens combining TLR3 ligands Background with type-1 IFNs are up to 100-fold more effectiveness in inducing Consolidation PD-L1 inhibition with durvalumab after concurrent CTL attractants (CXCL9, CXCL10, CCL5) compared to either factor chemoradiotherapy (CRT) has become the standard of care in inoper- alone. Moreover, CKM suppresses local Treg attractants, and targets able stage III non-small cell lung cancer (NSCLC) based on the excel- tumor microenvironment (TME) rather than healthy tissues [1-3]. lent PACIFIC trial results. The aim of this prospective single center Thus, we tested whether local or systemic CKM treatments enhance study was to evaluate the outcome and toxicity of durvalumab treat- CTL infiltration in “cold” tumors and determined the feasibility of ment after CRT in a tertiary cancer center. short-term CKM to sensitize poorly immunogenic, αPD-1 resistant, tu- Methods mors to PD-1 blockade. We prospectively collected clinical characteristics, toxicity and out- Methods come of all patients with inoperable stage III NSCLC treated with dur- C57BL/6 mice inoculated with MC38 (colorectal) or ID8 (ovarian) can- valumab after CRT/RT since 9th November 2018. Toxicity was cer cells were treated starting on day three (low-stage disease) or collected using the Common Terminology Criteria for Adverse Events eight (late-stage disease). A two dose course of CKM (IFNα and rinta- version 5 before and during treatment. Re-staging after CRT and be- tolimod [2]) followed by three doses of αPD-1 in two distinct regi- fore the start of durvalumab consisted of a CT scan (thorax/upper ab- mens: 1) Sequentially (following CKM) or 2) Concurrent with CKM. domen). 18F-FDG-PET-CT was performed 3 months and CT 6 months Mice were monitored for intratumoral CTL, tumor growth and after start of consolidation treatment. survival. Results Results Data of 16 patients treated with durvalumab after CRT/RT were eval- We observed strong effectiveness of CKM in promoting intratumoral uated. Three patients (19%) were female and 13 (68%) male, median increases in CTL and PD-L1 expression in the TME. CKM aids in the age at treatment start was 64 years. 10 (53%) patients had T4 or T3 sensitization of the largely αPD1-resistant tumors to PD1 blockade. tumors, four (25%) patients had N3 and 9 (56%) N2 disease. 15 Pa- Both sequential and concurrent CKM allowed antitumor effectiveness tients had CRT with a medium radiation dose of 63.20 Gy and were of αPD-1, resulting in overall prolongation of survival and 30-100% treated with two concurrent cycles of platin-based chemotherapy. cures, depending on treatment initiation. Sensitizing tumors to αPD- One patient was treated with moderate hypofractionated radiother- 1 did not require intratumoral CKM administration and was observed apy without chemotherapy. Median follow-up was 7 (range:2-16) with systemic application at distant sites, consistent with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 132 of 272 preferential activation of tumor tissues by CKM observed in tumor 95% CrI: 0.42-2.73) and avelumab+axitinib (HR=1.49, 95% CrI: 0.76- explant model [3]. Although strong antitumor effects were seen in 2.96) over P+A but were not statistically significant. the absence of any vaccination component, a stronger effect could In the intermediate + poor IMDC risk group, P+A showed a significant OS be observed by vaccination with tumor-loaded dendritic cells. benefit over sunitinib (HR=0.52, 95% CrI: 0.37-0.74) and was favored over Conclusions the other two interventions evaluated, but not statistically significant We demonstrate that local or systemic CKM sensitizes mice with [cabozantinib (HR=0.65, 95% CrI: 0.38-1.11) and N+I (HR=0.79, 95% CrI: poorly immunogenic tumors for subsequent effectiveness of PD-1 0.53-1.18)]. P+A showed a significant PFS benefit over sunitinib (HR=0.67, blockade. Thus, promoting intratumoral CTL accumulation may be 95% CrI: 0.53-0.85) and was favored over N+I (HR=0.87, 95% CrI: 0.65- sufficient for therapeutic effectiveness of ICI against “cold” tumors 1.17), but not statistically significant. PFS benefit favored cabozantinib with low mutational load. Our data provides rationale for clinical test- (HR=1.40, 95% CrI: 0.85-2.31) over P+A, but was not statistically significant. ing of sequential regimens where short-term CKM is followed by rou- Conclusions tine ICI, limiting the inconvenience for patients and facilitating the The results of this analysis suggest that pembrolizumab+axitinib may inclusion of CKM into routine immunotherapy plans. have PFS and OS advantages over most alternative first-line treat- ment options for mRCC, irrespective of IMDC risk groups. Acknowledgements Funded by 1P01CA132714, Rustum Family Foundation and Institutional P249 Support. Time-dependent blood transcriptomic perturbations differentially associated with mono and combination checkpoint inhibitor References therapy 1. Muthuswamy R, Berk E, Junecko BF et al. Cancer Res. 2012; 72:3735-3743. 1 2 1 Darawan Rinchai, PhD , Emily Hinchcliff, MD , Wouter Hendrickx, PhD , 2. Theodoraki MN, Yernei S, Sarkar et al. Cancer Res. 2018; 78:4292-4302. 1 1 Jessica Roelands, Master , Damien Chaussabel , Davide Bedognetti, MD, 3. Obermajer H, Urban J, Wieckowski E et al. Nat Protoc. 2018; 13:335-357. 1 2 PhD , Amir Jazaeri, MD Ethics Approval 1 2 Sidra Medicine, Doha, Qatar; The University of Texas MD Anderson This study was approved by Roswell Park Comprehensive Cancer Center's Cancer Center, Houston, TX, United States IACUC (protocol 1398M). Correspondence: Amir Jazaeri (aajazaeri@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P249 P248 Pembrolizumab plus axitinib (P+A) versus other first-line (1L) Background systemic therapies for advanced/metastatic clear-cell renal cell The rationale for combination immunotherapy is based on presumed carcinoma (ccmRCC) by IMDC Risk Status – a network meta- additive or synergistic properties of each individual drug [1-2]. analysis (NMA) Understanding the molecular mechanisms modulated by a given 1 1 1 Ian McGovern, MPH , Rohan Shirali, MS , Andrew Simon, ScM , Yichen drug is critical to implement more efficient therapeutic approaches. 2 2 1 Zhong, PhD , Rodolfo Perini, MD , Maria Lorenzi, MSc , Oluwakayode We conducted a study to determine 1) whether response to anti- Adejoro, MD, MPH anti-CTLA4 monotherapy (Tremelimumab) or combination of anti- 1 2 Precision Xtract, Boston, MA, United States; Merck & Co. Inc., PDL1(Durvalumab) and anti-CTLA4 (Tremelimumab) therapy could be Kenilworth, NJ, United States measured via blood transcriptome profiling; and 2) whether differ- Correspondence: Oluwakayode Adejoro ences between both treatment groups could be observed. In blood (oluwakayode.adejoro@merck.com) transcriptomic correlative studies performed so far, samples have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P248 been collected at limited time points (i.e., before and after treat- ments), preventing the description of the kinetic and dynamic Background changes associated with specific treatment modalities [3]. The International Metastatic Renal Cell Carcinoma Database Consortium Methods (IMDC) risk group classification is an important prognostic factor for ef- An adaptively randomized phase II trial of sequential versus combination ficacy outcomes of first-line systemic treatment of advanced/metastatic administration of Tremelimumab and Durvalumab (MEDI4736) in patients mRCC. IMDC risk is predictive of outcomes including overall survival with recurrent platinum resistant ovarian, peritoneal or fallopian tube (OS), progression-free survival (PFS) and overall response rate (ORR). cancers at the M.D. Anderson Cancer Center. Peripheral blood samples Pembrolizumab in combination with axitinib showed superior and clin- were collected serially before treatment and at 6 time points post- ically meaningful improvements in OS, PFS and ORR versus sunitinib in treatment from patients receiving Tremelimumab, alone or in combin- subjects with untreated ccmRCC in the KEYNOTE-426 trial. This NMA ation with Durvalumab, administered every 28 days. A total of 91 samples synthesized evidence from randomized clinical trials (RCTs) to indirectly were analyzed. Time points include C1D01 (baseline), C1H12 (12 hours compare the relative treatment effects of P+A vs other therapies in sub- after treatment), C1D08 (cycle one day 8), C1D15 (cycle one day 15), jects with favorable and intermediate + poor IMDC risk groups. C2D01 (cycle 2 day one) and C3D01 (cycle 3 day one). Blood transcrip- Methods tome profiles were generated by RNA-seq (Illumina HiSeq4000) at Sidra Fixed-effect Bayesian NMA was conducted to determine the relative ef- medicine. A set of 382 transcriptional modules was used for the analysis ficacy of treatments. Hazard ratios (HRs) for PFS and OS were estimated of this dataset using a pre-defined framework [4-5]. A module is consid- with 95% credible intervals (CrIs). Analyses were conducted among ered to be “responsive” to the treatment when significant changes in subjects with favorable risk, and intermediate + poor risk disease. abundance are observed for a proportion of its constitutive transcripts Results that is greaterthatwhatcouldbeexpectedbychance[4-5]. Among subjects with favorable IMDC risk, the estimated HRs for OS Results favored P+A over the other 3 interventions evaluated [nivolumab+i- We identified changes in blood transcript abundance in both treatment pilimumab (N+I) (HR=0.53, 95% CrI: 0.18-1.60), sunitinib (HR=0.64, groups, the modular perturbation peaking at cycle 1 day 15 post- 95% CrI: 0.24-1.70) and pazopanib (HR=0.73, 95% CrI: 0.26-2.03)] but treatment (Figure 1). Perturbations of Cell cycle, Protein synthesis and none were statistically significant. For PFS, P+A had statistically sig- Gene transcription modules were observed in both groups. But import- nificant benefit over 2 out of 9 interventions evaluated [interferon- ant qualitative differences were observed as well. Most notably, abun- alpha (IFN) (HR=0.30, 95% CrI 0.15-0.60) and bevacizumab (B)+ tem- dance of gene sets associated with Interferon, Tumor necrotic factor, sirolimus (HR=0.41, 95% CrI 0.18-0.98)]. The results numerically fa- cytotoxic lymphocyte and erythroid signatures was specifically in- vored P+A over 5 out of 9 interventions evaluated [ranging from creased in patients receiving combination immunotherapy B+IFN (HR=0.50, 95% CrI 0.23-1.10) through, atezolizumab, pazopa- Conclusions nib, N+I, to sunitinib (HR=0.81, 95% CrI: 0.53-1.23)], but were not sta- We characterized differential, time-dependent, systemic perturbations tistically significant. PFS benefits favored B+atezolizumab (HR=1.07, associated with mono vs combination immune checkpoint blockade. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 133 of 272 The peak of this immune modulation is observed at day 15 after P250 treatment. The mechanistic and clinical relevance of these findings Combination treatment of the oral CHK1 inhibitor, SRA737 and remains to be explored in a larger group of patients low dose gemcitabine, enhances the effect of PD-L1 blockade by Trial Registration modulating the immune microenvironment in small cell lung NCT03026062 cancer Triparna Sen, PhD (triparnasen@gmail.com) References Memorial Sloan Kettering Cancer Center, New York, NY, United States 1. Naumann RW, Coleman RL. Management strategies for recurrent Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P250 platinum-resistant ovarian cancer. Drugs 2011; 71: 1397-412. 2. Davis A, Tinker AV, Friedlander M. "Platinum resistant" ovarian cancer: what is Background it, who to treat and how to measure benefit? Gynecologic oncology 2014; Small cell lung cancer (SCLC), the most aggressive form of lung cancer, 133: 624-31. shows poor response rates to immunotherapy targeting the pro- 3. Friedlander P, Wassmann K, Christenfeld AM, Fisher D, Kyi C, Kirkwood JM, grammed cell death protein 1 pathway (PD-(L)1). Our group previously Bhardwaj N, Oh WK. Whole-blood RNA transcript-based models can predict discovered that SCLC exhibits high expression of checkpoint kinase 1 clinical response in two large independent clinical studies of patients with (CHK1) and that the CHK1 inhibitor SRA737 activates the innate im- advanced melanoma treated with the checkpoint inhibitor, tremelimumab. mune STING pathway, demonstrating robust anti-tumor activity and J Immunother Cancer 2017 Aug 15;5(1):67. doi: 10.1186/s40425-017-0272-z. synergy in combination with anti-PD-L1 in an SCLC model. 4. Chaussabel D, Baldwin N. Democratizing Systems Immunology with Modular Methods Transcriptional Repertoires Analyses. Nat Rev Immunol. 2014 Apr;14(4):271–80. As SRA737 is being tested in SCLC patients in combination with low 5. Altman MC, Rinchai D, et al. A Novel Repertoire of Blood Transcriptome dose gemcitabine (LDG), we evaluated the efficacy and immune cor- Modules Based on Co-expression Patterns Across Sixteen Disease and relates (including macrophages associated with resistance to immune Physiological States. bioRxiv 525709; doi: https://doi.org/10.1101/525709 checkpoint blockade) of the SRA737+LDG regimen in combination Ethics Approval with anti-PD-L1 in an SCLC model. The study was approved by the MD Anderson Cancer Center Ethics Board, Results approval number IRB 5 IRB00006023 and Sidra Medicine's IRB, approval Trp53, Rb1 and p130 (RPP) triple knockout SCLC cells were implanted 1804022877 into the flank of B6129F1 immunocompetent mice. After the mice developed tumors, they were treated with single agents or various drug combinations. Anti-PD-L1 and LDG demonstrated minimal ef- fect on tumor growth as single agents and only a modest effect as a combination. Moderate to strong anti- tumor activity was however observed with SRA737 monotherapy which directly correlated with dosing intensity. The most profound and synergistic anti-tumor activity was observed when anti-PD-L1 was com- bined with the SRA737+LDG regimen, with all animals showing durable regressions. Analysis of tumor infiltrating immune cells at the end of this treatment regimen showed a dramatic induction of cytotoxic T- cells and a reduction of exhausted and regulatory T cells. Similarly, pro- inflammatory M1 type macrophages and dendritic cells were increased while immunosuppressive M2 type macrophages and MDSC cells were dramatically decreased. As monotherapy, the more dose intensive SRA737 schedule resulted in similar effects on lymphocytes when com- bined with anti-PD-L1. These effects are consistent with our previous data showing that SRA737 treatment leads to an induction of STING and type I interferon signaling in tumors, which is associated with the establishment of an anti-tumor immune microenvironment. Conclusions Our findings suggest that the combination of anti-PD-L1 with the SRA737+LDG regimen may represent the optimal implementation of these agents, leading to a dramatic anti-tumor activity accom- panied by the establishment of a strong anti-tumor immune microenvironment. Given that anti-PD-(L)1 drugs are approved but show limited efficacy in SCLC, our preclinical data provide a strong rationale for combining these agents with the SRA737+LDG regimen to enhance clinical response rates. P251 CTX-8371, a novel bispecific targeting both PD-1 and PD-L1, is more potent than combination anti-PD-1 and PD-L1 therapy and provides enhanced protection from tumors in vivo Diana Albu, PhD, Pearl Bakhru, Wilson Guzman, BS, Michael Ophir, Rachel McCrory, BS, William Carson, Jason Kong, Beata Bobrowicz, Pia Muyot, Amanda Oliphant, Dalton Markrush, Rachel Rennard, Cheuk Lun Leung, Sara Haserlat, Michael Schmidt, Jose Gonzalo, Bing Gong, Robert Tighe, BS, Diana Albu, PhD, Benjamin Wolf Compass Therapeutics, Cambridge, MA, United States Correspondence: Benjamin Wolf (benjamin.wolf@compasstherapeutics.com) Fig. 1 (abstract P249). Modular repertoire analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P251 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 134 of 272 Background Results Monoclonal antibody immunotherapies targeting immune check- From 10/2017 to 2/2019, 83 patients received I+N for mRCC and point receptors have shown great promise for a subset of cancer were included. Demographics are shown in Table 1. By International patients. However, novel combination therapies are still needed Metastatic RCC Database Consortium (IMDC) risk criteria [2], 20.5% to increase the benefit of cancer immunotherapy and bring it to were favorable, 61.4% intermediate, and 18.1% poor risk. 65% were broader patient populations. Here we describe the preclinical stage IV at diagnosis, 63.9% were untreated, and 16.9% patients had evaluation of CTX-8371, which combines PD-1 and PD-L1 target- prior nivolumab exposure. 77.1% of patients had clear cell pathology. ing in one bispecific, tetravalent molecule. 12/83 (14.4%) have sarcomatoid differentiation: 2 have an ongoing Methods response and 7 have died. At the data cutoff date, 44/83 (53%) pa- Our proprietary Stitchmabs®™ bispecific screening platform was used tients have progressed or died. Median PFS was 5.3 months (95% CI to conduct an unbiased screen of bispecifics comprised of various 3-8.5) (Figure 1). OS rates at 6, 12, and 18 months were 76.2%, 63.8%, checkpoint blocking antibodies. This screen yielded the surprising and 51.5%, respectively (Figure 2). Rates of best radiographic re- discovery that a bispecific containing both PD-1 and PD-L1 binding sponse were CR 4.8%, PR 22.9%, SD 18.1%, PD 32.5%, and unknown arms was more potent than the combination of parent monoclonal 21.7%. 44/83 (53%) patients experienced no adverse event (AE). 18/ antibodies. We then generated common light chain bispecifics con- 83 (21.7%) patients experienced a grade 3/4 AE (most commonly taining compatible anti-PD-1 and PD-L1 antibodies and used multiple diarrhea, n=7), 20/83 (24%) patients experiencing a grade 1-2 AE at in vitro assays to identify our lead, CTX-8371. Additional in vitro and worst (most commonly hypothyroidism, n=14), and one grade 5 AE oc- in vivo experiments confirmed CTX-8371’s reactivity across species, curred. 23/83 (27.7%) patients have died, with 10/83 (12.0%) patients in vivo anti-tumor effects, and the underlining mechanisms driving dying within 90 days of receiving the first dose of I+N. 4/83 patients its distinctive activity. have achieved a complete response. Two of these patients discontin- Results ued treatment at 11 and 12 months with a sustained response at 1 and We found that CTX-8371 binds to human and cynomolgus monkey 5 months, respectively. Three other patients remain off therapy for AEs PD-1 and PD-L1 targets with sub-nanomolar affinities and is cross- and have not progressed after 11, 5, and 3 months. reactive to mouse PD-1 and PD-L1. Compared to Keytruda®, CTX- Conclusions 8371 increased T cell activation and tumor cell killing in vitro, signifi- In our real-world cohort of mRCC patients, I+N has similar clinical effi- cantly delayed tumor growth, and prolonged survival in human cell cacy as previously described; however, the cohort is more frail, with transfer tumor models. Additionally, CTX-8371 demonstrated efficacy 16.9% of patients treated in the nivolumab refractory setting. Five in transplantable mouse syngeneic models. Investigation into the patients remain off therapy. This forms the basis for larger prospect- mechanisms responsible for the enhanced efficacy of CTX-8371 unex- ive treatment discontinuation trials (ie. Alliance A031704 phase 3 pectedly found that the bispecific causes a massive loss of PD-1 from trial) with prospective treatment discontinuation for complete re- the T cell surface, which was not observed in response to monoclo- sponse patients at 1-year. nal antibodies alone or combined. This robust PD-1 downregulation, potentially mediated through bridging together the T cell and tumor References cell, may explain the ability of CTX-8371 to reverse PD-1 suppression 1. Motzer RJ, Tannir NM, McDermott DF, et al. Nivolumab plus Ipilimumab more potently than standard blocking antibodies. versus Sunitinib in Advanced Renal-Cell Carcinoma. N Engl J Med. Conclusions 2018;378(14):1277-1290. Taken together, the results demonstrate that the bispecific, tetrava- 2. Heng DY, Xie W, Regan MM, et al. Prognostic factors for overall survival lent antibody CTX-8371 has increased potency in vitro and in vivo as in patients with metastatic renal cell carcinoma treated with vascular compared to clinical checkpoint blockade agents. Some of its effects endothelial growth factor-targeted agents: results from a large, multicen- are likely attributable to its unique mechanism of action, driving ro- ter study. J Clin Oncol. 2009;27(34):5794-5799. bust downregulation of cell-surface PD-1. Thus, CTX-8371 has the po- Ethics Approval tential to increase the number of patients that benefit from PD-1/PD- This study was approved by the Duke University IRB (#Pro00101984) L1 checkpoint blockade. P252 A retrospective study to evaluate real-world clinical outcomes in patients with metastatic renal cell carcinoma (mRCC) treated with Ipilimumab and Nivolumab Landon Brown, MD, Emily Kinsey, MD, Chester Kao, MD, Patrick Healy, Andrew Armstrong, MD, Megan McNamara, MD, Sundhar Ramalingam, MD, Michael Harrision, MD, Daniel George, MD, Tian Zhang, MD Duke University, Durham, NC, United States Correspondence: Landon Brown (landon.brown@duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P252 Background The combination of nivolumab at 3mg/kg plus ipilimumab at 1mg/ kg (I+N) followed by maintenance nivolumab has greatly improved outcomes in patients with intermediate or poor-risk untreated mRCC [1]. Real-world series of patients treated with this combination are scarce. In this retrospective analysis, we present a real-world experi- ence with this combination immunotherapy. Methods A search was performed to identify all mRCC patients treated in the Duke Cancer Institute network with I+N. An extensive chart review was conducted. Patient characteristics are summarized with descrip- tive statistics; Kaplan Meier analysis was performed for progression Fig. 1 (abstract P252). See text for description free survival (PFS) and overall survival (OS). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 135 of 272 Methods We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. Results We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young [3]. αPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibit- ing this property. Efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC dif- fered by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM-3 expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. Conclusions Treatment differences in aged versus young could depend on im- mune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. PD-L2 expression differences could be a mechanism for treatment differences. We are now identi- fying mechanisms for increased PD-L2 and contributions to αPD- L2 efficacy in aged, and testing TCSC effects on treatments (Fig- ure 1-3). Our work can improve cancer immunotherapy in aged Fig. 2 (abstract P252). See text for description hosts and further provide important insights even in young hosts. Table 1 (abstract P252). See text for description Acknowledgements South Texas MSTP training grant (NIH T32GM113896), TL1TR002647, R01 CA231325. References 1. Schildberg FA,Klein SR,Freeman GJ,SharpeAH. Coinhibitory Pathways in the B7-CD28 Ligand-Receptor Family. Immunity. 2016;44(5):955-72. 2. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 ther- apy. Nature. 2016;537(7620):417-21. 3. Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol. 2018;105:146-54. Ethics Approval All animal work was done under UTHSA Institutional Animal Care and Use Committee approved studies in compliance with the Guide for the Care and Use of Laboratory Animal Resources (published by National Research Council of the National Academies), Animal Welfare Act (AWA) (published by USDA), Public Health Service Policy on Humane Care and Use of Laboratory Animals (published by NIH) and US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Approval number: 20180021AR. P253 Distinct clinical and immunological responses to αPD-1, ⍺PD-L1 and ⍺PD-L2 immunotherapy in B16 melanoma in aged versus young hosts includes T-cell stem cell effects and PD-L2 expression differences Myrna Garcia, BS, Alvaro Padron, Yilun Deng, MD, PhD, Harshita Gupta, PhD, Aravind Kancharla, Tyler Curiel, MD University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Correspondence: Tyler Curiel (curielt@uthscsa.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P253 Background Aging is the biggest risk factor for cancer, yet little is known about can- cer immunotherapy effects. αPD-1 can block PD-L1 and PD-L2 while ⍺PD-L1 blocks PD-1 and CD80 [1]. A recent key finding in young hosts Fig. 1 (abstract P253). ⍺PD-L2 treats B16 melanoma in aged mice including humans is that melanoma response to αPD-1/αPD-L1 corre- but not young mice lates with CD8+TCF-1+ T cell stem cell (TCSC) generation [2]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 136 of 272 functional Fc as monotherapies or in a combination with anti-PD-1 was carried out in order to better understand the effects of anti-TIGIT anti- body with functional Fc at molecular level at different time points. Results Here we demonstrate using mouse tumor models that anti-mTIGIT antibodies require interactions with Fc gamma receptors on myeloid cells in the tumor microenvironment for effective anti-tumor re- sponse. Our observations reveal that the anti-mTIGIT therapeutic ef- fect is not achieved by depletion of intratumoral regulatory T cells, but instead is mediated by “reverse activating signals” through Fc gamma receptors on myeloid cells, inducing expression of various mediators such as cytokines, including TNF-alpha and IL-23, and che- Fig. 2 (abstract P253). PD-L2 expression is significantly higher in mokines, such as CXCL10 and CXCL11, thus generating the condi- aged mice when compared to young tions for potentially promoting immune infiltrates into the tumor microenvironment. In addition, up-regulation of co-stimulatory mole- cules, such as CD80, CD86, and CD40, has been observed, consistent with the heightened anti-tumor activity of Fc gamma receptor binding com- petent anti-mTIGIT antibodies. Furthermore, we discovered induction of a robust and persistent granzyme B and perforin response from the in vivo treatment of anti-mTIGIT antibody with a functional Fc, distinct from a predominantly interferon-gamma-driven anti-PD-1 blockade. Conclusions Our observations for the first time provide mechanistic insights into the requirement for Fc engagement of anti-mTIGIT monoclonal anti- bodies for effective anti-tumor activity in vivo which has implications for the various human antibodies of various isotypes are currently under intense clinical investigations. P255 Preclinical characterization and efficacy of MG1124, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy 1 1 1 Jae-Chul Lee, Master degree , Minkyu Hur , Hye-Young Park , Mi-Young 1 1 1 2 3 Oh , Hye-mi Nam , Hye In Yum , HyungSuk Choi , Jaehwan Kim , 3 1 Byoung Chul Cho, MDphD , Yangmi Lim MOGAM Institute for Biomedical Research, Yong-in, Korea, Republic of; 2 3 GC Pharma, Yongin-si, Korea, Republic of; Yonsei Cancer Center, Seoul, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P255 Fig. 3 (abstract P253). αPD-1, αPD-L2 and αPD-L1 elicit distinct Background TCSC that also differ by age CEACAM1 is one of the several immune checkpoint receptors expressed on T cells and NK cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction in- P254 duces downregulation of ZAP70 phosphorylation in response to T cell re- Requirement of Fc gamma receptor-mediated myeloid-cell ceptor (TCR) stimulation. CEACAM1 is also highly expressed on non-small activation for effective cancer immunotherapy with an anti-TIGIT cell lung cancer (NSCLC) and its expression is correlated with cancer pro- antibody gression and poor prognosis. We developed a fully human monoclonal Jin-hwan Han, PhD, Mingmei Cai, Jeffery Grein, Samanthi Perera, PhD, antibody MG1124, targeting human CEACAM1. Hongmei Wang, Mike Bigler, Roenna Ueda, Thomas Rosahl, Elaine Methods Pinheiro, PhD, Drake LaFace, Wolfgang Seghezzi, Sybil Williams, PhD T cell activation of MG1124 was determined by an NFAT-luciferase Merck Research Laboratories, South San Francisco, CA, United States reporter assay with CEACAM1 overexpressing Jurkat stable cells. Correspondence: Sybil Williams (sybil_williams@merck.com) Evaluation of the homophilic interaction of CEACAM1 or interaction Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P254 of CEACAM1 with CEACAM6 was performed by protein ELISA. In vitro efficacy of MG1124 was examined using an NK-mediated tumor cell Background killing assay. The anti-tumor efficacy of MG1124 alone or in combin- The molecule “T cell immunoreceptor with immunoglobulin and ITIM ation was studied in vivo in a humanized mouse model engrafted domain”, or TIGIT, has recently received much attention as a promis- with NSCLC patient-derived tumor xenografts. ing target in the treatment of various malignancies. In spite of the Results quick progression of anti-TIGIT antibodies into clinical testing both as Anti-CEACAM1 antibody MG1124 bound to CEACAM1 but not to other monotherapy and in combination with programmed death-1 (PD-1)– CEA family members. MG1124 blocked CEACAM1-CEACAM1 homophi- directed immune checkpoint blockade, the molecular mechanism be- lic interaction and CEACAM1-CEACAM6 heteropilic interation by bind- hind the observed therapeutic benefits remains poorly understood. ing to the N domain of CEACAM1. Especially CEACAM1-CEACAM1 Methods homophilic interaction induced downregulation of ZAP70 phosphoryl- Anti-Mouse TIGIT (mTIGIT) blocking antibodies of two distinct isotypes ation in response to TCR stimulation in a CEACAM1 overexpressing Jur- (mouse IgG1 with D265A mutation and mouse IgG2a) and TIGIT- kat stable cell line, which was rescued by MG1124 resulting in deficient mice were generated and used to demonstrate the require- augmentation of NFAT activity and IL-2 expression. NK cell-mediated ment of IgG-Fc gamma receptor interaction for effective anti-tumor re- tumor lysis was increased by MG1124 in a CEACAM1 expression- sponse in vivo studies. Gene expression profiling of whole tumors after dependent manner. In an NSCLC PDX-huNSG mouse model, MG1124 in vivo treatment of anti-mTIGIT antibody with functional or non- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 137 of 272 suppressed tumor progression as a monotherapy and combination Conclusions with pembrolizumab in a CEACAM1 high expressing model. In single Our data validate the therapeutic potential of providing IL-7 signals to mouse trial analysis, MG1124 suppressed tumor progression more than overcome PD-1 resistance. The bifunctional anti-PD1/IL-7 favors the T- 30% as monotherapy (53%, 10/19) as well as in combination (73%, 16/ cell effector over T-regulatory immune balance by stimulating effector 22) with pembrolizumab (5 mpk, 2qW). Moreover, PDXs of adenocarcin- and exhausted T-cells while disarming Tregs suppressive functions. oma origin with more than 50% of CEACAM1 expression were more ef- ficiently prohibited for progression with MG1124, suggesting the P257 potential therapeutic use of MG1124 in patients with NSCLC. Obesity is associated with diminished anti-PD-1-based Conclusions immunotherapy response rates in renal cancer MG1124, an anti-CEACAM1 antibody, blocked CEACAM1-mediated 1 1 1 Rachael Orlandella, BS , Shannon Boi , Justin Gibson, BS , William negative regulation and restored T/NK cell activities. MG1124 showed 1 2 2 1 Turbitt , Gal Wald , Lewis Thomas , Katlyn Norris , Lakshminarayanan effective anti-tumor activity in in vivo mouse models and its combin- 1 1 1 Nandagopal, MD , Peng Li, PhD , Eddy Yang, MD, PhD , Tatiana ation with PD-1 blockade further enhanced treatment efficacy. 1 1 Marquez-Lago, PhD , Lyse Norian, PhD MG1124 is a potential therapeutic candidate for immune checkpoint 1 2 University of Alabama, Birmingham, AL, United States; University of blockade in cancer therapy. Iowa, Iowa City, IA, United States Correspondence: Lyse Norian (lnorian@uab.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P257 P256 A novel bifunctional anti-PD-1 / IL-7 fusion protein potentiates Background effector function of exhausted T cell and disarms Treg suppressive Obesity is a regarded as a major risk factor for developing renal cell car- activity cinoma (RCC). Despite the success of anti-PD-1 checkpoint blockade in 1 1 1 Aurore Morello, PhD , Justine Durand , Caroline Mary , Virginie RCC, response rates remain low (20-30%). Recent studies have observed 1 1 1 2 Thepenier , Margaux Seite , Géraldine Teppaz , Nicolas Poirier that obesity is associated with heightened frequencies of PD-1+ CD8 T 1 2 OSE immunotherapeutics, Nantes, France; Poirier Household, Nantes, France cells [1] and favorable outcomes and responses to immunotherapy in Correspondence: Nicolas Poirier (nicolas.poirier@ose-immuno.com) melanoma [1, 2]. However, the effects of obesity on anti-tumor immun- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P256 ity and immunotherapeutic efficacy in renal cancer remain unknown. Methods Background PD-1 expression on tumor-infiltrating CD8 T cells from treatment-naive Despite the clinical success of anti-PD(L)1 therapies, most patients re- RCC subjects with (BMI >30 kg/m2) or without (BMI < 30 kg/m2) obes- main unresponsive or fail to develop a durable response. We explored ity (n = 18) was determined via flow cytometry. In a separate retro- a second generation of PD-1 antibody by fusing IL-7 cytokine to the Fc spective study, outcome data were queried for RCC patients with (BMI portion. IL-7 is an optimal target for immunotherapy to preferentially >30 kg/m2) or without (BMI < 30 kg/m2) obesity that were treated with stimulate effector T-cell (Teff) functions over regulatory T-cells (Treg), anti-PD-1 as standard of care and had at least 6 months of follow-up due to the differential expression of IL-7R. Moreover, It has been pub- (n=58). Overall survival (OS) was analyzed using Kaplan-Meier methods lished that PD-1 blockades increase IL-7R expression and improve IL-7 and Cox proportional hazards regression after controlling for patients’ signaling in exhausted T-cells rationalizing our combinatorial approach. age, sex, and number of prior treatments. For murine studies, BALB/c Methods mice were randomized to and maintained on either standard chow or Proliferation (H3 thymidine), IFN-γ, IL-7R signaling (pSTAT5) and NFAT (PD- high-fat diet for 20 weeks to generate age-matched lean or diet- 1 bioassay, Promega) assays were tested to determine anti PD-1/IL-7 effi- induced obese (DIO) mice. Mice were then given an orthotopic renal cacy on naïve and/or exhausted-like T-cells. For the suppressive assay, CD4 tumor challenge with syngeneic Renca cells and treated with an anti- Treg and autologous CD8 Teff were co-cultured (1:1) and proliferation was PD-1-based combination immunotherapy or saline. assessed on Day 5. Tumor infiltrating T cells (TILs) were isolated from orthotopic tumor-bearing mice (Hepa1.6, LLC-1,AK7) and subjected to IL-7 Results ex vivo, IL-7R signaling pSTAT5 was determined by flow cytometry. Obesity was associated with reduced frequencies of intratumoral PD- Results 1highCD8+ T cells in treatment-naive murine and human renal tumors. Our anti PD-1/IL-7 bispecific antibody efficiently blocks the PD-1/PD- Although the majority (73%) of lean mice responded to immunotherapy, L1 and PD-L2 interactions and the PD-1-mediated inhibitory signal DIO mice exhibited a reduced response rate (44%). Lean and DIO re- (pSHP1). Importantly, we observed that the IL-7 portion synergizes sponders exhibited favorable ratios of activated CD8+ T cells to myeloid- with the anti-PD-1 to enhance TCR mediated signaling (NFAT). Al- derived suppressor cells (MDSC), reduced PD-1 expression on CD8+ T though IL-7R expression on T cells decrease over repeated antigen cells, and elevated concentrations of CCL5 in renal tumors. Neutralization stimulation, we demonstrated that IL-7 still efficiently activate par- of CCL5 in lean immunotherapy-treated mice yielded a reduced response tially and fully-exhausted human T-cells (pSTAT5) and maintain their rate (43%), unfavorable ratios of activated CD8+ T cells to MDSCs, and di- proliferation capacity. We next characterized sensitivity of TILs to IL-7 minished IFNg secretion from intratumoral CD8+ T cells. The translational in multiple orthotopic mouse models. In PD-1 sensitive tumor (Meso- relevance of our murine findings was reflected in metastatic RCC patients, thelioma), only 10% of TILs express IL-7R whereas in PD-1 resistant as patients with obesity had a trending reduction in OS following stand- model (Hepatocarcinoma and Lung carcinoma), 40-60% of TILs (CD4 ard of care nivolumab (p= 0.06) and a 10.2 month reduction in OS. and CD8) express IL-7R and respond to IL-7 stimulation ex vivo as Conclusions measured by pSTAT5 signaling. These data suggest that the anti PD- Our data suggest that obesity is associated with reduced responses to 1/IL-7 bispecific can reactivate TILs that are resistant to PD-1 therapy. anti-PD-1 based immunotherapies in the context of renal cancer. Contin- Knowing that Tregs have a key suppressive function, we also ex- ued study of this critical issue is needed to better inform patient care. plored the possibility that the anti-PD-1/IL-7 fusion protein affect Treg functions. In a human Treg/Teff coculture assay, we observed Acknowledgements that the anti PD-1/IL-7 molecule abrogate the Treg capacity to inhibit Financial support was provided by NIH grant R01CA181088 to LAN; proliferation and IFN-gamma secretion of CD8+ Teff. Moreover, IL-7 CPCTP T32 fellowship #T32CA047888 to RMO; CPCTP R25 fellowship and the anti-PD-1/IL-7 does not stimulate Treg proliferation, in con- #R25CA047888 to SKB; CMDB T32 fellowship #T32GM008111 to JTG; and trast to IL-2 and IL-15 cytokines. Oncology T32 fellowship #T32CA183926 to WJT. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 138 of 272 References 1. McQuade, J.L., et al., Association of body-mass index and outcomes in patients with metastatic melanoma treated with targeted therapy, immunotherapy, or chemotherapy: a retrospective, multicohort ana- lysis. Lancet Oncol, 2018. 19(3): p. 310-322. 2. Wang, Z., et al., Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): p. 141-151. Ethics Approval Human subject studies were approved by the UAB IRB (protocol # X151013003); murine studies were approved by UAB IACUC (protocol #20233). P258 Combination of NK Cells and anti-PD-L1 Ab with ADCC Fig. 1 (abstract P258). See text for description enhances the anti-tumor effects in PD-L1 high cancer cells 1 2 1 Ji-Eun Park, BS , Bhumsuk Keam, MD, PhD , Ha-ram Park , Soyeon Kim, 3 2 2 2 PhD , Chan-Young Ock, MD, PhD , Miso Kim , Tae Min Kim, MD, PhD , P259 2 2 Dong-Wan Kim, MD PhD , Dae Seog Heo Hydrogel-enabled intratumoral co-delivery of anti-PD-1 antibody Seoul National University Cancer Research Institute, Seoul, Korea, and adenosine deaminase in a mouse model of renal cell Republic of; Seoul National University Hospital, Seoul, Korea, Republic carcinoma of; Biomedical Research Institute, SNUH, Seoul, Korea, Republic of Ketki Velankar, MS, Ngoc Pham, BS, Wilson Meng, PhD, Ellen Gawalt, Correspondence: Bhumsuk Keam (bhumsuk@snu.ac.kr) Nathan Schueller Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P258 Duquesne University, Pittsburgh, PA, United States Correspondence: Wilson Meng (meng@duq.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P259 Although Programmed cell death-1 (PD-1)/ Programmed death-ligand 1 (PD-L1) inhibitors showed remarkable antitumor activity, a large por- Background tion of cancer patients do not response to PD-1/PD-L1 inhibitors even It is hypothesized that tumor resistance to anti-PD-1 monoclo- in the PD-L1 high tumor. Most of PD-L1 inhibitors were modified in FcR nal antibodies is due in part to the accumulation of adenosine binding site to prevent antibody-dependent cellular cytotoxicity (ADCC) (ADO) generated in the tumor microenvironment (TME). ADO against PD-L1 expressing non-tumor cells. IMC-001, developed by impairs the activation and proliferation of effector T cells while ImmuneOncia, is a fully human PD-L1 recombinant monoclonal anti- expanding regulatory T cell (Treg) population, which is in- body that did not modify FcR binding and preserved ADCC. Therefore, versely related to the overall survival of cancer patients, includ- IMC-001 would be synergistic with NK compared to other PD-L1 mono- ing those with renal cell carcinoma (RCC). We propose to clonal antibodies (mAbs). We evaluate anti-tumor efficacy of IMC-001 develop an injectable system by which ADO are degraded in and NK cells against several PD-L1 high cancer cell lines through ADCC. the TME in order to enhance the efficacy of anti-PD1 treat- Methods ment. To this end, we have developed a hydrogel to co-deliver PD-L1 expression was measured by flow cytometry. Standard 51Cr-release anti-PD-1 antibody with adenosine deaminase (ADA), which ca- and CD107a degranulation assays were performed to evaluate the in vitro tabolizes ADO. The hydrogel contains a bioaffinity module ADCC efficacy of 3 groups: control, anti-PD-L1 Ab without ADCC (atezolizu- (named “Z15_EAK”) to retain the anti-PD-1 antibody in tumors mab), anti-PD-L1 Abs with ADCC (IMC-001, Anti-hPD-L1-hIgG1 [atezolizu- while limit the diffusion of ADA in TME for extended durations. mab with wild type FcR binding site, hPD-L1mab]). Various cancer cell We have previously shown that Z15_EAK hydrogel can retain lines were used as target cells, including head and neck squamous carcin- IgG at subcutaneous injection site for at least two weeks [1]. oma (HNSCC), lung cancer, stomach cancer, ovarian cancer, bladder cancer The expectation is that persistent co-localization of anti-PD-1 and lymphoma cell lines. 51Cr-release assay was performed using NK-92- and ADA in the TME will expand Th1 T cells and reduce Treg CD16 as an effector cell with effector to target ratio (E:T) of 30:1. CD107a in draining lymph nodes (DLN) and systemic lymphoid tissues. degranulation assay was performed using peripheral blood mononuclear This postulation was tested in an immunocompetent mouse cells (PBMC) from healthy donors with E:T ratio of 1:1. PBMC was activated model of RCC. by IL-15 and grouped by CD16 V158F genotyping individually. Methods Results A mouse RCC cell line (RENCA) was cultivated for in vitro assays and The expression of PD-L1 is high in several cell lines including SNU- in vivo inoculation into BALB/c mice. Beginning three days after 1076 (8.8±1.3), FaDu (15±0.1), HN31 (21.8±1.1) and H1975 (11.3±1.7). tumor inoculation, the hydrogel loaded with an anti-PD-1 IgG anti- NK cell cytotoxicity in PD-L1 high cell lines was more potent in IMC- body and ADA was injected subcutaneously in the peri-tumoral re- 001 or anti-hPD-L1-hIgG1 compared to control treatment or atezoli- gion for three doses three days apart. DLN, spleen, and tumors were zumab. The PD-L1 high or PD-L1 low tumor cell specific lysis was de- collected for flow cytometric analysis and ELISA measurements. tected by 51Cr-release assay in control group (isotype and Results atezolizumab) vs. and anti-PD-L1Ab with ADCC groups (IMC-001 vs. After three doses, DLN in mice received the hydrogel loaded with Anti-hPD-L1-hIgG1) (Figure 1). Besides, in CD107a degranulation anti-PD-1 antibody and ADA were five times larger than those in assay, activated PBMC cytotoxicity was increased when target cells mice received saline control (Figure 1). In addition, the lymph nodes are opsonized by anti-PD-L1 Abs with ADCC. NK cells that character- in treated mice contained fewer CD4+CD25+FoxP3+ Treg cells com- ized with CD16 high affinity genotype (V/F) are more enhancing pared to controls (Figure 2). After ex vivo re-stimulation with RENCA ADCC than (F/F) low affinity genotype. Two genotyped (V/F vs. FF) for expansion, lymphocytes in treated mice exhibited higher NK cell lysis induced by IMC-001 in FaDu cells was 39.3% vs. 12.8%. interferon-gamma levels than controls, indicating elevated Th1 Conclusions phenotype in the DLN. Anti-PD-L1 Abs with ADCC, such as IMC-001, enhanced the cytotoxic Conclusions activity of NK cells on various PD-L1 high cell lines. This study provides The preliminary data indicate that the local delivery of anti-PD- rationale that NK92-CD16 or NK cell immunotherapy for PD-L1 high 1 and ADA with the hydrogel shifted the local T cell popula- tumor through combination with ADCC preserved anti-PD-L1 Ab. tion toward an effector phenotype (Th1) while limiting the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 139 of 272 Treg expansion. An extended co-localization of anti-PD-1 and P260 ADA in the TME not only modulates immune events in the Characterization of AB154, a humanized, non-depleting α-TIGIT local lymphoid tissues but can also enhance the anti-tumor re- antibody undergoing clinical evaluation in subjects with advanced sponse systemically. Furthermore, the localized delivery reduces solid tumors off-target toxicities of anti-PD-1 antibody. Alejandra Lopez, BSc, Joanne Tan, PhD, Amy Anderson, PhD, Akshata Udyavar, PhD, Nell Narasappa, MSC, Susan Lee, PhD, Daniel DiRenzo, Reference PhD, Kristen Zhang, BS, Hema Singh, Sharon Zhao, Kimberline Gerrick, 1. Pham, N.B., et al. Toward reducing biomaterial antigenic potential: a Adam Park, Lisa Seitz, MA, Nigel Walker, PhD, Matthew Walters, PhD miniaturized Fc-binding domain for local deposition of antibodies. Biomaterials Arcus Biosciences, Inc., Hayward, CA, United States Science. 2019: 7(3): 760-772. Correspondence: Joanne Tan (jtan@arcusbio.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P260 The animal study was approved by Duquesne University's Ethics Board, approval number 180604. Background TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). CD226 is an activating receptor found on NK cells, monocytes and a subset of T cells. TIGIT and CD226 are paired receptors that compete for shared ligands CD155 and CD112, which are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression, whereas binding of the same ligand to CD226 promotes immune activation. AB154, designed to lack FcɣR binding, blocks human TIGIT with minimal risk of depleting intra-tumoral antigen-experienced CD8+ T cells. Methods Translational studies quantifying TIGIT, CD226 and CD155 expression in vari- ous tumor types and normal tissues were performed using flow cytometry, immunohistochemistry (IHC) and by mining publicly available RNASeq data- sets. TIGIT occupancy (RO) and Ki-67 levels from Ph1 dose escalation cohorts were quantified by flow cytometry. Downstream transcriptional effects of TIGIT/CD155 interaction in CD8+ T cells and Treg were assessed using Nano- string®. AB154, AB154 modified to restore wild-type (wt) IgG1 effector func- tion or to display enhance FcɣR binding via Fc mutations, were used in functional assays and antibody-dependent cell cytotoxicity (ADCC) studies. Results AB154, regardless of IgG1 variant, effectively abrogated the TIGIT- mediated inhibitory effects on activated T cells. In contrast, only non- depleting AB154 lacked ADCC activity in mixed cultures containing NK cells and activated T cells. Data assembled from TCGA, confirmed by flow cytometry as well as IHC, identified multiple tumor types bearing high TIGIT and CD155 expression. In particular, antigen-experienced T cells isolated from late stage head and neck squamous cell carcinoma tumors express higher levels of TIGIT and PD-1 than of CD226. Levels of Fig. 1 (abstract P259). See text for description TIGIT expression in this subset was equivalent, if not higher, than intra- tumoral Treg. Preliminary results from our Phase-1 dose escalation study demonstrated near complete target engagement by AB154 in T cells, NK cells, and NKT cells, coupled with concomitant increases in Ki- 67 expression within the aforementioned subsets. Conclusions Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented here provide: 1) rationale for clinical development of a non-depleting a- TIGIT blocking antibody (AB154), 2) evidence of AB154-related immune activation in subjects with advanced solid tumors, 3) evidence supporting AB154 as a rational combination partner with a-PD-1 (AB122). Trial Registration NCT03628677 P261 Recruitment of CD103+ DCs via tumor stroma-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy John-Michael Williford, PhD, Jun Ishihara, PhD, Ako Ishihara, Aslan Mansurov, BChen, Tiffany Marchell, Melody Swartz, PhD, Jeffrey Hubbell University of Chicago, Chicago, IL, United States Correspondence: Jeffrey Hubbell (jhubbell@uchicago.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P261 Background Checkpoint inhibitor antibody (CPI) therapy has demonstrated signifi- cant clinical benefit in a number of tumor types. Unfortunately, certain Fig. 2 (abstract P259). See text for description tumor characteristics, such as the lack of immune cell infiltration, often Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 140 of 272 correlate with poor responses to CPI therapy. Studies have identified C- P262 C Motif Chemokine Ligand 4 (CCL4) as a key molecule necessary for Schweinfurthins Cause Rapid Induction of Ecto-calreticulin Expression the recruitment of cross-presenting, CD103+ dendritic cells (DCs) to the Raymond Hohl, MD, Jeffrey Neighbors, PhD, Ruoheng Zhang, MBBS tumor; tumors lacking CCL4 expression exhibit a “cold tumor” pheno- Penn State College of Medicine, Hershey, PA, United States type and respond poorly to immunotherapy [1]. Based on these results, Correspondence: Raymond Hohl (rhohl@pennstatehealth.psu.edu) we hypothesized that tumor-targeted CCL4 could enhance immune cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P262 infiltration into the tumor and synergize with CPI therapy. Methods Background We generated a fusion protein comprised of CCL4 and a collagen bind- Our previous study demonstrated that schweinfurthin analogs im- ing domain (CBD) derived from von Willebrand factor, a tumor-stroma prove anti-PD-1 immunotherapy in a murine melanoma model by in- targeting strategy developed in our lab [2]. Anti-tumor efficacy studies ducing sustainable in vivo anti-tumor immunity.[1] We speculate that were performed in mouse syngeneic models, including B16F10 melan- the induction of immunogenic cell death (ICD) contributes to these oma, EMT6 breast cancer, and PyMT breast cancer. Flow cytometry was effects. The release of immunogenic danger-associated molecular employed to evaluate the tumor immune infiltrate. patterns (DAMPs) from tumor cells during ICD directly activate anti- Results cancer immunity.[2] Cell surface exposure of calreticulin (ecto-CRT) is Utilizing exposure of collagen in leaky tumor vasculature due to its disor- the major determinative DAMP because it stimulates cancer cell dered structure, we observed that intravenous (i.v.) infusion of CBD-CCL4 phagocytosis by dendritic cells and further activates anti-cancer im- fusion proteins, but not native CCL4, can enhance infiltration of CD103+ munity.[3] Phosphorylation of eukaryotic translation inhibition factor DCs, CD8+ T cells, and natural killer cells and slow B16F10 tumor growth eIF2α, an indicator of ER stress, is highly correlated to CRT exposure when combined with CPI therapy consisting of anti-cytotoxic T- on the cell surface in some forms of ICD.[4] Previous studies of the lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand schweinfurthin family of compounds have shown that they increase 1 antibody (PD-L1) (Figure 1, A-B) Further analysis showed strong correla- the phosphorylation of eIF2α and have complex effects on lipid tions between the presence of CD103+ DCs and CD8+ T cells and tumor homeostasis.[5] We hypothesize that schweinfurthin analogs enhance regression. Similarly, in the EMT6 breast cancer model, tumor-targeted CRT exposure during induction of ICD via eIF2α phosphorylation to CCL4 in combination with CPI, but not native form CCL4, enhanced recruit- improve anti-cancer immunity. ment of CD103+ DCs, CD8+ T cells, and led to a reduction in tumor Methods growth. To confirm the importance of CD103+ DCs in mediating anti- B16.F10 murine metastatic melanoma cells were cultured with increas- tumor responses, we utilized Batf3 knockout mice bearing B16F10 tumors; ing concentrations of the schweinfurthin analog, TTI-3114 or vehicle for in this instance, anti-tumor efficacy of CPI + CBD-CCL4 was completely lost. 24 hours, followed by measurement of ecto-CRT expression by flow cy- Efficacy studies in PyMT breast cancer models highlighted the therapeutic tometry. Kinetics of ecto-CRT exposure was also assessed. To determine benefit of CBD-CCL4 delivery (Figure 1C); CPI therapy alone led to the importance of lipids in this process, studies were carried out with complete tumor remission in only 10% of mice, whereas combination normal or charcoal-stripped lipid-free media. In addition, the role of therapy of CPI + CBD-CCL4 cured 50% of the treated mice (Figure 1D). eIF2α phosphorylation in TTI-3114-induced CRT exposure was evalu- Conclusions ated using western blots for total and phosphorylated eIF2α with thap- These results highlight the utility of recruiting CD103+ DCs to the sigargin acting as a positive control for the induction of ER stress. tumor to improve the efficacy of CPI therapy. This engineered chemo- Results kine delivery strategy demonstrates significant translational potential by targeting the tumor stroma following systemic administration. TTI-3114 induces rapid surface calreticulin exposure on B16.F10 murine melanoma cells in a concentration-dependent manner, References starting at 30nM. 1. Spranger S, Gajewski T. Impact of oncogenic pathways on evasion of Lipid depletion sensitizes melanoma cells to TTI-3114-induced antitumor immune responses. Nat Rev Cancer. 2018; 18:139-147. calreticulin exposure. 2. Ishihara J et al. Targeted antibody and cytokine cancer immunotherapies TTI-3114 causes eIF2α phosphorylation before CRT exposure in through collagen affinity. Sci Transl Med. 2019; 11:eaau3259. melanoma cells, indicating potential ER stress response. Conclusions Based on the above results, we hypothesize that schweinfurthins in- duce CRT exposure on the cell surface likely by promoting a form of ER-stress, and this effect is augmented by lipid depletion. Future ex- periments will investigate the function of various lipids in schweinfurthin-induced CRT exposure and subsequent immunogenic cell death. We will also explore the exact mechanisms which are re- sponsible for the CRT cell surface exposure phenomena. References 1. Kokolus, K. M. et al. Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity. Oncoimmunology 8, 1–13 (2019). 2. Zhou, J. et al. Immunogenic cell death in cancer therapy: Present and emerging inducers. J. Cell. Mol. Med. 4854–4865 (2019). doi:10.1111/ jcmm.14356 3. Obeid, M. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61 (2007). 4. Bezu, L. et al. eIF2α phosphorylation: A hallmark of immunogenic cell death. Oncoimmunology 7, 1–3 (2018). 5. Kuder, C. H. et al. Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification. Mol. Fig. 1 (abstract P261). See text for description Pharmacol. 82, 9–16 (2012). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 141 of 272 P263 either alone or in combination with other immune checkpoint therap- Targeting EZH2 enhances antigen presentation, antitumor ies. We hypothesized that an antibody with a unique binding mode immunity and circumvents anti-PD-1 resistance in head and neck could activate T cells in an Fc effector-less format. Hence, we developed cancer 7A5, a CD137 agonist monoclonal antibody which potentially has a 1 2 1 Liye Zhou, PhD , Ravindra Uppaluri, MD, PhD , Tenny Mudianto, BS , ligand-like structural binding mode and demonstrated that it effectively 1 1 Xiaojing Ma, PhD , Rachel Riley, BS engages the CD137 receptor in preclinical studies. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Methods Women's Hospital/DFCI, Boston, MA, United States 7A5 was identified from a human Fab phage display library screen Correspondence: Ravindra Uppaluri and engineered to an IgG1 Fc effector null antibody. Solid phase (ravindra_uppaluri@dfci.harvard.edu) binding assays with recombinant CD137 protein and cell-based as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P263 says in CD137 expressing cells were used to evaluate binding and functional activity in vitro. To assess agonist activity, 7A5 was tested Background in NF-kB luciferase reporter, PBMC co-stimulation and Treg suppres- Anti-programmed death-1 (PD-1) receptor-based therapeutics im- sion assays. To determine antitumor activity in vivo, human tumor prove survival in recurrent head and neck squamous cell carcinoma xenograft mouse models (NSG mice harboring human NCI-H292 or (HNSCC) patients but many do not benefit due to a low response HCC827 NSCLC tumors) reconstituted with human PBMCs or T cells, rate. Multiple mechanisms of immunoevasion have been identified in were used. Treatments included 7A5 monotherapy and the combin- HNSCCs including in the antigen presentation machinery. Herein, we ation with anti-PD-L1 antibody in these models. identified enhancer of zeste homolog 2 (EZH2) as a therapeutic tar- Results get in HNSCCs that enhanced tumor cell antigen presentation and In this study, we characterized 7A5, a fully human IgG1 Fc effector subsequently sensitized resistant tumors to anti-PD-1 therapy. null monoclonal antibody. We showed 7A5 binds CD137 and the Methods binding epitope overlaps with the CD137 ligand binding site. 7A5 en- EZH2 regulation of antigen presentation was defined using EZH2 in- gages the CD137 receptor and activates signaling independent of hibitors (GSK126 and EPZ6438) in human and mouse HNSCC cell cross-linking or Fc effector function. It binds to activated primary T lines. Mechanistic dissection of EZH2 in regulation of antigen presen- cells and leads to T cell stimulation in cell-based assays. Monother- tation was investigated using flow cytometry, qRT-PCR, ELISA and apy with 7A5 inhibits tumor growth in humanized mouse models chromatin-immunoprecipitation assays. EZH2 deficient cell lines were and this activity is enhanced when combined with a PD-L1 antagon- generated using CRISPR-CAS9. GSK126 and anti-PD-1 blocking anti- ist antibody. Furthermore, changes to the intra-tumoral immune body were used in testing combinatorial therapy in vivo. gene expression signature in response to 7A5 is highly suggestive for Results a mechanism of enhanced T cell infiltration and activation. EZH2 expression was negatively correlated with antigen processing Conclusions machinery (APM) pathway components in HNSCC TCGA datasets. In summary, CD137 antibody 7A5 represents a differentiated agonist EZH2 inhibition resulted in significant upregulation of MHC class I ex- with preclinical biological properties that support its further develop- pression in both human and mouse HNSCC lines and increased anti- ment as an anti-cancer immunotherapy. gen presentation in mouse models. This increased antigen presentation on the tumor cell by EZH2 inhibitors or CRISPR medi- P265 ated EZH2 deficiency, increased antigen specific CD8+ T cell prolifer- Immune checkpoint inhibitors induce response in a dose- ation, IFNγ production and tumor cell cytotoxicity. Mechanistically, dependent manner while their immune related adverse events are EZH2 inhibition reduced the histone H2K27me3 modification on the dose-independent, a meta-analysis β-2-microglobulin promoter to regulate antigen presentation. Finally, 1 2 1 Osama Rahma, MD , Joshua Reuss, MD , Anita Giobbie-Hurder, MS , in an anti-PD-1 resistant model of HNSCC, combination EZH2 inhib- 3 4 5 Ghazaleh Razavi, MD , Pooja Mehra, MD , Seema Gupta , Rawad Elias, ition with anti-PD-1 suppressed tumor growth at least partially due 6 5 MD , Samir Khleif, MD to the upregulation of antigen presentation capacity of tumor cells. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; John Hopkins, Conclusions Baltimore, MD, United States; Georgia Cancer Center, Augusta, GA, Our results demonstrated that targeting EZH2 enhanced antigen presen- United States; University of Virginia, Charlottesville, VA, United States; tation and circumvented anti-PD-1 resistance. Thus, combining EZH2 tar- 5 6 Georgetown University, Washington, DC, United States; Hartford geting with anti-PD-1 may increase therapeutic susceptibility in HNSCC. Healthcare, Hartford, CT, United States Correspondence: Samir Khleif (snk48@georgetown.edu) P264 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P265 Characterization of a human CD137 (4-1BB) receptor binding monoclonal antibody with differential agonist properties that Background promotes antitumor immunity Despite the expansion of Immune Checkpoint Inhibitor (ICI) indi- Helen Kotanides, Rose Marie Sattler, Maria Lebron, Carmine Carpenito, cations, the relationship between ICI dose-escalation and toxicity PhD, Juqun Shen, Jingxing Li, David Surguladze, Jaafar Haidar, Colleen or response has not been established. To understand this correl- Burns, Leyi Shen, Ivan Inigo, BS, Anthony Pennello, Amelie Forest, MSc, ation, we performed a meta-analysis of all available clinical trials Xinlei Chen, Darin Chin, Andreas Sonyi, Michael Topper, Lauren Boucher, investigating ICIs. Prachi Sharma, Yiwei Zhang, Douglas Burtrum, Ruslan Novosiadly, Dale Methods Ludwig, Gregory Plowman, Michael Kalos We searched PubMed and abstracts presented at (inter)national Eli Lilly and Company, Indianapolis, IN, United States meetings for trials (T) using FDA-approved ICIs including ipilimumab, Correspondence: Helen Kotanides (helen.kotanides@lilly.com) atezolizumab, nivolumab, and pembrolizumab. The reported rates of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P264 treatment-related grade 3-5 adverse events (G3-5AEs), immune- related adverse events (irAEs), and response were collected. For each Background ICI, comparisons of incidence rates between doses or diseases were CD137 (4-1BB) is a member of the TNFR receptor superfamily that plays based on marginal, exact generalized linear models. a key role in mediating immune response through costimulatory sig- Results nals that promote T cell proliferation, survival and memory. CD137 A total of 74T (7469 patients (pts)) published between 1/2010 – 1/2017 agonism has the potential to reinvigorate potent antitumor immunity were included (15T-ipilimumab (1058 pts), 30T-nivolumab (2281 pts), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 142 of 272 29T-pembrolizumab (4130 pts)) (Figure 1). For ipilimumab, the overall incidence of G3-5AEs was 34%. A significant 27% reduced risk of G3- 5AEs was seen with 3 mg/kg compared to 10 mg/kg (p=0.002) (Figure 2). However, there was no relationship observed between dose of ipili- mumab and incidence of irAEs or response to therapy (Figure 3). With nivolumab, the overall incidence of G3-5AEs was 20.1%. Incidence of G3-5AEs was significantly lower in NSCLC, with risk reductions of 24- 38% when compared to RCC or melanoma (p≤0.05). No dose-toxicity relationship was seen for G3-5AEs or irAEs (Figure 4). In both melanoma (6T) and NSCLC (7T), a dose-response association was observed, with significantly decreased odds of response of 17% and 64% for 1mg/kg compared to 3mg/kg in melanoma and NSCLC, respectively (Figure 5,6) with no further increase in response for doses above 3 mg/kg. This as- sociation was not observed in RCC (Figure 7). For pembrolizumab, the overall incidence of G3-5 AEs was 13.3%. Risk of G3-5AEs was 17% lower in melanoma than in NSCLC (p=0.03). No dose-toxicity relation- ship was seen for G3-5AEs or irAEs (Figure 8). In melanoma (7T), 2mg/ kg every 3 weeks (q3w) had 22% decreased odds of response com- pared to 10mg/kg (q2w) (p=0.01) (Figure 9). For NSCLC (5T), no dose- response relationship was noted (Figure 10). Conclusions We found no correlation between dose of ipilimumab and odds of G3-5iAEs or response. For pembrolizumab and nivolumab, no dose-toxicity correlation was seen but a dose-response correlation was observed suggesting that, for the PD-1 inhibitors, efficacy ap- Fig. 2 (abstract P265). Bootstrap analysis for G3-4 AEs pears to be dose-dependent while toxicity does not. Accordingly, of ipilimumab future clinical trial design of ICIs should use a dose escalation method with a primary objective of identifying an effective dose rather than a maximum tolerated dose. Acknowledgements Merck and BMS for providing input Fig. 1 (abstract P265). Consort Diagram of Literature Search Fig. 3 (abstract P265). Bootstrap analysis for ORR for ipilimumab Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 143 of 272 Fig. 6 (abstract P265). Bootstrap analysis for ORR for nivolumab Fig. 4 (abstract P265). Bootstrap analysis for G3-4 AEs in NSCLC for nivolumab Fig. 5 (abstract P265). Bootstrap analysis for ORR for nivo Fig. 7 (abstract P265). Bootstrap analysis for ORR for nivolumab in melanoma in RCC Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 144 of 272 Fig. 10 (abstract P265). Bootstrap for ORR for pembrolizumab in NSCLC Fig. 8 (abstract P265). Bootstrap for incidence of G3-4 AE P266 in pembrolizumab Evaluation of immunomodulatory receptor/ligand expression on matched human biospecimens Shawn Fahl (shawn.fahl@dls.com) Discovery Life Sciences, Huntsville, AL, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P266 Background The integration of immunomodulatory receptor signaling is crucial for the activation status of responding T cells, and modulation of these re- ceptors, and their ligands, may be of therapeutic benefit. Indeed, recent breakthroughs in checkpoint inhibitor therapies, and in particular those that target the PDL1/PD1 interaction, have demonstrated success in nu- merous oncological indications. Understanding the expression of these receptors and their cognate ligands within the complex cellular archi- tecture of solid tumors will be fundamentally important to the design on the next-generation of immunotherapies. Methods Bulk RNASeq analysis of primary human tumor tissue revealed the expression of numerous co-stimulatory (LIGHT/HVEM, 41BB/41BBL, OX40/OX40L, GITR/GITRL) and co-inhibitory (Lag3, VISTA, PVR/PVRL2/ TIGIT, Tim3/Galectin-9) receptors and ligands within the tumor micro- environment. Using multiparametric flow cytometry, we have profiled the expression of these immunomodulatory receptors and their re- spective ligands on the major cellular components of the tumor microenvironment and correlated it with expression on cellular sub- sets within matched peripheral blood. P267 Phenotyping of TIGIT pathway members may be used for cancer selection in the clinical application of anti-TIGIT antibody EOS884448 Noemie Wald, PhD, Julia Cuende, PhD, Marjorie Mercier, Florence Nyawouame, MSc, Margreet Brouwer, MSc, Erica Houthuys, PhD, Gregory Driessens, PhD, Veronique Bodo, PhD, Catherine Hoofd iTeos Therapeutics, Gosselies, Belgium Correspondence: Gregory Driessens Fig. 9 (abstract P265). Bootstrap for ORR for pembrolizumab (gregory.driessens@iteostherapeutics.com) in melanoma Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P267 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 145 of 272 Background The complement system consists of a network of more than 50 dif- TIGIT is a T cell co-inhibitory receptor that drives tumor cell mediated ferent plasma and membrane associated proteins. It is a part of the immunosuppression. Predominantly expressed on CD4+ Tregs, CD8+ T innate immune system and plays a key role in host defense against and NK cells in healthy individuals, TIGIT is further upregulated in these pathogens as well as in tissue homeostasis. The anaphylatoxin C5a is cells in cancer patients. In patients, it is frequently co-expressed with formed upon cleavage of C5 during the process of complement acti- exhaustion markers such as PD-1. DNAM-1/CD226, a co-stimulatory re- vation. C5a is the most potent chemoattractant and induces recruit- ceptor, is expressed on NK and T cells and competes with TIGIT for ment and activation of different immune cells to inflamed tissue, PVR/CD155 binding, but with a lower affinity. Since cancer cells express among which are neutrophils, eosinophils, monocytes, basophils, and high level of CD155 and because TIGIT expression is increased on TILs, mast cells. C5a binds to the seven transmembrane-spanning recep- the TIGIT pathway represents a major mechanism for immunosuppres- tors C5aR1 (CD88) and C5aR2 (C5L2). Blocking C5aR1 thus appears as sion within the tumor. We developed EOS884448, an antagonist anti- a potent mean to control the myeloid suppressive cells in the TME. TIGIT antibody, to prevent TIGIT-mediated immunosuppression in can- In this context we developed IPH5401, a fully human blocking anti- cer patients. C5aR1 monoclonal antibody that prevents binding to C5a. Methods Results To support selection of indications for clinical application of We first explore further the expression profile of C5aR1 in the TME EOS884448, we used flow cytometry and immunohistochemistry both at mRNA and protein levels in several solid cancer indications (IHC) to characterize peripheral and tumoral expression of TIGIT, showing various levels of infiltration by C5aR1 positive immune cells. CD155 and CD226 in healthy or cancer donors. Then, we demonstrated that IPH5401 can block activation and migra- Results tion of Human neutrophils and macrophages in vitro. TIGIT is expressed on multiple immune subsets in healthy donors. Simi- Conclusions lar analysis on matched PBMCs and TILs from 15 cancer donors Altogether, these results support our ongoing multi-center, open highlighted the overexpression of TIGIT on cells from those samples. label, dose-escalation and dose expansion Phase I/II clinical trial Interestingly, ex vivo polyfunctional analysis of cytokine production (STELLAR-001) evaluating the safety and efficacy of IPH5401 in com- demonstrated immunosuppression of TIGIT+ TILs versus their TIGIT- bination with durvalumab, an anti-PD-L1 immune checkpoint inhibi- counterparts. Among PBMCs and TILs assessed by flow cytometry, tor, as a treatment for patients with advanced solid tumors. tumor-infiltrating Tregs exhibit the highest TIGIT expression (frequency of positive cells and receptor density). This finding was confirmed by P269 IHC on tumor samples, supporting the potential value of an ADCC- IL-15 together with TIGIT blockade reverses PVR-mediated NK cell competent antibody targeting preferentially tumor-infiltrating Tregs. dysfunction in melanoma Finally, intrinsic expression of TIGIT on tumor cells was detected on 1 1 1 Joe-Marc Chauvin, PhD , Mignane Ka , Ornella Pagliano , Carmine several haematological malignancies, opening the potential for 1 1 1 2 Menna , Quanquan Ding , Cindy Sander , Jiajie Hou , Soldano Ferrone, EOS8844488 to directly kill tumor cells in addition to its activities to re- 1 1 1 MD, PhD , Diwakar Davar, MD , John Kirkwood, MD , Robert Johnston, invigorate immunity. The expression of the TIGIT ligands CD155 and 3 3 2 1 PhD , Alan Korman, PhD , Mark Smyth , Hassane Zarour, MD CD226 co-receptor were also analysed by IHC in tissues (n=284-307) 1 2 University of Pittsburgh, Pittsburgh, PA, United States; QIMR Berghofer from 9 cancer indications. CD155 is mostly expressed by tumor cells, Medical Research Institute, Queensland, Australia; Bristol-Myers Squibb, ranging from a median of 2% of CD155high tumor cells for cervix to Redwood City, CA, United States 50% for pancreatic cancer. CD155 expression is highest in pancreatic, Correspondence: Joe-Marc Chauvin (chauvinj@upmc.edu) prostate, kidney, gastric and colon cancers. CD226 is detected on im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P269 mune cells infiltrating tumors. The median percentage of tissue area positive for CD226 ranged from 0.07% in head and neck to 0.98% in Background gastric cancer, with gastric, lung and renal cancer showing the highest Natural Killer cells (NKs) play a critical role in tumor immunosurveil- CD226 expression. lance. Multiple activating and inhibitory receptors regulate NK cell- Conclusions mediated tumor cytotoxicity. The inhibitory receptor TIGIT and its Together, these findings strongly support the relevance of targeting counter-receptor CD226 exert opposite effects on NK cell function, TIGIT with an ADCC-competent antibody and provide a method to with TIGIT blockade reinvigorating NK cell-mediated tumor reactivity. select cancer types that may benefit from treatment with Whether and how the manipulation of the TIGIT/CD226/PVR axis may EOS884448. reactivate NK-cell mediated antitumor activity in melanoma patients Ethics Approval (MPs) has not yet been thoroughly evaluated. The study was approved by UCL‘s Ethics Board, approval number Bio- Methods bank2019/09MAI/005 Flow cytometry was used to evaluate the phenotype and function of NKs in the periphery (cNKs) and tumor sites (TiNKs) in MPs. CD226 P268 mRNA was evaluated by RT-PCR. CD226 and TIGIT internalization was IPH5401 anti-human C5aR antibody targets suppressive myeloid evaluated on isolated cNKs by Imagestream analysis. Melanoma lung cells in the TME metastasis tumor models in WT and TIGIT-/- mice were used to Joanna Fares, Léa Simon, Caroline Soulas, Marion Loivet, Elodie Bonnet, evaluate in vivo effects of TIGIT and CD226 blockades on NK- Luciana Batista, Romain Remark, PhD, Cécile Bonnafous, Robert Zerbib, mediated tumor control with or without IL-15 treatment. MSc, Mathieu Bléry Results Innate Pharma, Marseille, France In sharp contrast with CD8+ T cells, TiNKs downregulated both TIGIT Correspondence: Robert Zerbib (robert.zerbib@innate-pharma.fr) and CD226 expression as compared to cNKs. TiNKs exhibited de- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P268 creased expression of activation markers, lytic potential and melan- oma killing capacity as compared to cNKs in MPs. Membrane-bound Background PVR, but not soluble PVR, triggered CD226 internalization and deg- A hallmark mechanism of synergy in immunotherapy is the elimin- radation, leading to NK dysfunction. IL-15 stimulation increased ation of immunosuppressive cells, such as myeloid cells and neutro- CD226 and TIGIT expression levels and NK cell function, and TIGIT phils, to allow for the reactivation of effector cells. These blockade further increased TiNK proliferation and function against immunosuppressive cells are indeed associated with poor prognosis MHC class-I deficient tumors as compared to IL-15 or TIGIT blockade in many cancer types as well as resistance to checkpoint blockade. alone. TIGIT blockade promotes tumor antigen-specific CD8+ T cells From a therapy perspective, we aimed to specifically target the re- proliferation independently of NK cells. TIGIT blockade impeded me- cruitment of these major mediators of pro-tumoral inflammation into tastasis in mice in a CD226-dependent manner and only in presence the tumor microenvironment (TME). of IL-15. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 146 of 272 Conclusions P271 Membrane-bound PVR plays a critical role in the tumor microenviron- Anti-SIRPalpha antibodies exert anti-tumor activity in both CD47- ment by modulating TIGIT/CD226 expression and the function of dependent and CD47-independent manners TiNKs. IL-15 together with TIGIT blockade, counteracts PVR-mediated Hongtao Lu, PhD, Xiaofeng Niu, PhD, Qinglin Du, PhD, Jingfeng Yu, TiNK dysfunction in melanoma, and prevent metastasis occurrence in Roumei Xing, Yanfen Hu, Jinfeng Zhao, Fengli Wang, Zhihao Wu, PhD, mice in a CD226 dependent manner. Our findings support the devel- Yangsheng Qiu, Hongtao Lu, PhD opment of novel combinatorial immunotherapy with IL-15 and TIGIT Elpiscience Biopharma, Shanghai, China blockade to promote NK cell-mediated destruction of MHC class I- Correspondence: Hongtao Lu (hongtaolu@elpiscience.com) deficient melanoma, which are refractory to CD8+ T cell-mediated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P271 immunity and PD-1 blockade. Background Signal-regulatory protein alpha (SIRPα), is an inhibitory receptor P270 expressed on myeloid cells and dendritic cells. Ligation of CD47 Expression and clinical significance of the CD47/SIRPα pathway as to SIRPα delivers a “don’teat me” signal to suppress phagocyt- a candidate immunotherapy target in non-small cell lung cancer osis. Tumor cells frequently overexpress CD47 to evade (NSCLC) macrophage-mediated destruction. Currently, agents targeting 1 1 1 Shruti Desai, PhD , Franz Villarroel-Espindola , Patricia Gaule, PhD , Adam CD47 have proceeded to clinical trials and demonstrated promis- 1 2 2 Ducler , Marisa Peluso, MS , Benjamin Lee, MD PhD , Kurt Schalper, MD, ing anti-tumor results. However, these agents have been associ- 1 2 PhD , Pamela Holland, PhD ated with hemolytic anemia and thrombocytopenia. In addition, School of Medicine, Yale University, New Haven, CT, United States; universal expression of CD47 causes antigen sink, which leads to Surface Oncology, Cambridge, United States reduced efficacy. We therefore consider targeting SIRPα to Correspondence: Kurt Schalper (kurt.schalper@yale.edu) achieve an improved efficacy with a better safety profile. We Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P270 have developed 2 classes of anti-SIRPα antibodies: CD47-SIRPα interaction “blocker” and “non-blocker”. Both groups of antibody Background functionally stimulate phagocytosis of multiple cancer cell types Immunostimulatory therapies have revolutionized the treatment of by macrophages. NSCLC. Multiple studies show that activation of the CD47/SIRPα path- Methods way can mediate cancer immune evasion by blocking phagocytic ac- Using SIRPα extracellular domain (ECD), SIRPα overexpression tivity of macrophages. Although early stage clinical trials blocking stable cell line and plasmid encoding SIRPα as immunogens, this pathway are ongoing, the expression, tissue distribution and clin- anti-SIRPα antibodies were generated by traditional hybridoma ical significance of the CD47 axis in NSCLC remains unknown. technology. Pan-allele/SIRP family homologue binding proper- Methods ties, and species cross-reactivity profile were evaluated by ELISA Using control tissue samples/cell line transfectants, we validated anti- and FACS. In vitro function activity was determined by phago- bodies to reliably detect CD47 and SIRPα protein in FFPE tissue and cytosis assay. In vivo safety profile was assessed in hCD47/ standardized a multiplexed quantitative immunofluorescence panel for hSIRPα double knock-in mice. Lead clone was humanized via simultaneous measurement of DAPI, pan cytokeratin, CD8/CD47/SIRPα. CDR grafting and back mutation screening. Stress tests were We used this panel to interrogate two retrospective NSCLC Yale cohorts carried out to evaluate the developability of candidate represented in tissue microarrays (#1: n=297 and #2: n=175). Cohort #3, antibody. n=139 adenocarcinomas with mutation testing were also analyzed. We Results studied the levels of the targets, tissue distribution, association with A panel of anti-SIRPα antibodies that stimulate phagocytosis of clinicopathologic/molecular variables and survival. multiple cancer cells were developed. One lead clone (B4) was se- Results lected from the “Blocker” group based on ranking of in vitro bind- Predominant tumor CD47 expression (cytoplasmic/membranous) was ing/function properties. Subsequently, ES004-B4 was identified as recognized in 82 -88% of cases in the NSCLC cohorts. SIRPα protein the candidate antibody after humanization, affinity maturation, was detected in 94- 98% of cases located in the stroma. Elevated ex- and property characterization. It recognized pan-allele human pression of CD47/SIRPα was significantly associated with high CD8+ SIRPα with high affinity (KD hSIRPα V1/V2, 0.86nM/1.43nM). ES004- tumor infiltrating lymphocytes in the cohorts. The targets showed no B4 was shown to stimulate phagocytosis of multiple cancer cell consistent associations between major clinicopathologic variables. types. Another lead antibody (N4) was selected from the CD47/ Lung adenocarcinomas with KRAS mutation showed significantly SIRPα interaction “non-blocker” group. ES004-N4 also induced lower levels of CD47 than EGFR mutated or EGFR/KRAS WT tumors. strong phagocytosis of multiple cancer cell types by macrophages, Although individual CD47/SIRPα levels were not prominently associ- albeit it did not disrupt CD47/SIRPα interaction, suggesting an ated with outcome, their co-localization in stroma was positively as- unique mode of action. ES004-B4 and ES004-N4 didn’t trigger sociated with longer 5-year overall survival. hemagglutination and had no negative impact on T cell prolifera- Conclusions tion in vitro. No severe hemolytic anemia and thrombocytopenia CD47 and SIRPα are frequently expressed in NSCLCs with higher were observed in hCD47/hSIRPα double knock-in mice treated levels in CD8+/T-cell inflamed tumors, suggesting adaptive upregula- with 10mg/kg of each antibody, suggesting a low safety risk tion of this pathway associated with anti-tumor immune pressure. in vivo. CD47 levels are associated with major oncogenic signaling events in Conclusions lung adenocarcinomas. Localized measurement of stromal CD47/ In summary, we have developed 2 anti-SIRPα antibodies with “Best- SIRPα co-expression in intact tumor specimens could provide valu- in-Class” potential: 1) CD47/SIRPα interaction “Blocker” ES004-B4, 2) able information about the pathway activation. CD47/SIRPα interaction “Non-Blocker” ES004-N4. Both antibodies Ethics Approval greatly enhance macrophage-mediated tumor cell destruction, pos- All tissues were used after approval from the Yale Human Investiga- sibly through different mechanisms of action. We are currently ad- tion committee protocol #9505008219 which approved the patient vancing the development of ES004-B4 and ES004-N4 into clinical consent forms or waiver of consent. candidates. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 147 of 272 P272 P273 SRF231, a fully human high-affinity anti-CD47 antibody, exerts Blockade of glyco-immune checkpoint using EAGLE to potentiate potent preclinical antitumor activity through engagement of the anticancer immunity Fc receptor (FcR), CD32a Lizhi Cao, Jenny Che, Abhishek Das, PhD, Sujata Nerle, Wayne Gatlin, Marisa Peluso, MS, Kshama Doshi, PhD, Caroline Armet, BS, Li Zhang, Robert Leblanc, Zakir Siddiquee, Hui Xu, Karl Normington, PhD, MBA, Rachel O'Connor, BS, Matthew Rausch, PhD, Jonathan Hill, PhD, Weiguo Yao, James Broderick, Li Peng, PhD Benjamin Lee, MD PhD, Pamela Holland, PhD, Vito Palombella, PhD, Palleon Pharmaceuticals, Waltham, MA, United States Alison Paterson, PhD Correspondence: Li Peng (lpeng@palleonpharma.com) Surface Oncology, Inc., Cambridge, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P273 Correspondence: Marisa Peluso (mpeluso@surfaceoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P272 Background The glyco-immune checkpoint (Siglec/sialogylcan) axis has recently Background emerged as a new mechanism of cancer immune evasion. We have CD47 is a transmembrane protein that acts as a “Don’t Eat Me” signal previously described a bifunctional antibody-sialidase fusion platform to evade immune recognition. It is overexpressed in multiple cancer named EAGLE (Enzyme-Antibody Glyco-Ligand Editing) to inhibit this subtypes and is associated with poor prognosis. Several anti-CD47 axis by selectively removing the terminal sialic acids of sialoglycans molecules designed to antagonize the CD47 axis are being tested in on tumor cells. Using a bacterial sialidase for proof-of-concept stud- the clinic. Preclinical characteristics and antitumor mechanisms of ies, we have shown that EAGLE leads to enzyme-dependent robust the investigational agent SRF231, a fully human antibody targeting monotherapy efficacy with complete regressions and immune mem- CD47, are described. ory in syngeneic mouse tumor models. Methods Methods SRF231 monovalent affinity and binding properties were evaluated Since the bacterial sialidase poses immunogenicity concerns, we by Surface Plasmon Resonance (SPR) technology and in vitro agglu- engineered and optimized a human sialidase for EAGLE overcoming tination assays. Directly labeled SRF231 was used to profile tumor vs the low expression yield and poor stability of human sialidases. We normal cell binding. SRF231-mediated antitumor activity was confirmed the antitumor activity of human sialidase-containing assessed in tumor using macrophage coculture systems designed to EAGLE in vitro and in vivo using coculture of cancer cells and primary evaluate the impact of SRF231 on tumor cell phagocytosis, cell death, human immune cells and immunocompetent tumor models. Further- and cell depletion. Receptor occupancy (RO)/activity relationships more, we explored and identified predictive and correlative pharma- and dependency on FcR were also assessed. A xenograft tumor codynamic (PD) biomarkers to EAGLE treatment in preclinical tumor model was used to characterize the pharmacokinetic (PK)/pharmaco- models. dynamic (PD)/tumor exposure/efficacy relationship of SRF231 follow- Results ing single dose administration. The expression yield of human sialidase was improved by ~400 fold Results compared to the wild type through protein engineering, which en- SRF231 is a fully human, high-affinity anti-CD47 antibody with a slow ables the production of human sialidase-based EAGLE. We con- off-rate and binding mode that does not lead to agglutination of structed EAGLE-408, consisting of the engineered human sialidase patient-derived red blood cells or tumor cells. Increased binding to and anti-HER2 antibody trastuzumab, and confirmed its robust desia- several tumor vs normal cells is observed with SRF231. Analyses lylation efficiency using various HER2-expressing tumor cells in vitro. probing the relationship between FcR and SRF231 activity revealed EAGLE-408 enhanced macrophage-mediated phagocytosis of tumor that SRF231 leads to antitumor activity through both phagocytosis cells in vitro. EAGLE-408 also demonstrated enzyme-dependent and cell death mechanisms in a manner largely dependent on the monotherapy efficacy with complete regressions and immune mem- activating receptor FcγRIIa (CD32a), predominantly expressed by ory in syngeneic EMT6-HER2 mouse tumor models. Furthermore, in myeloid cells. Additionally, SRF231-mediated antitumor activity is the PD study, we observed EAGLE-408 treatment enhanced CD8+ T retained in longer-term assay conditions and in washout conditions. cell infiltration into tumors, increased CD8+ T cells in the draining Moreover, submaximal SRF231 RO is sufficient for maximal phagocyt- lymph nodes, and augmented NK cells and myeloid cells in osis induction in vitro. In a B-cell lymphoma xenograft model, a sin- circulation. gle dose of SRF231/mouse yields tumor stasis out to 21 days with Conclusions submaximal tumor exposure. Antitumor activity is associated with an In summary, EAGLE with engineered human sialidase offers a increase of host macrophage infiltration and cytokine induction sug- new immunomodulatory approach to overcome resistance to gestive of an innate immune response. current immunotherapies by effectively inhibiting Siglec/sialogly- Conclusions can axis in the tumor microenvironment. SRF231 is a high affinity, CD47-targeting antibody that delivers an ac- tivating signal to myeloid cells via CD32a and displays favorable pre- P274 clinical characteristics regarding its RO/tumor exposure/efficacy Antibody targeting of tumor associated macrophages in relationship. SRF231 is currently being evaluated in a Phase 1 clinical pancreatic cancer and melanoma remodel the tumor trial [NCT03512340] in advanced solid tumors and lymphomas. Un- microenvironment and revives immune targeting of tumor cells derstanding these PK/PD/tumor exposure/efficacy relationships, as Dhifaf Sarhan, PhD (dhifaf.sarhan@ki.se) well as the role of target vs FcR affinity, offers guidance on the devel- Karolinska Institute, Stockholm, Sweden opment of CD47 antagonists for patients with cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P274 Ethics Approval Mice were used in compliance with protocols approved by the IACUC Background of Mispro Biotech Services, Cambridge, MA (#2017-03-21SUR-1), Immunotherapy for cancer has revolutionized clinical practice and Charles River Accelerator and Development Lab, Cambridge, MA enabled cures for previously lethal cancers. However, the clinical re- (#CR-008), Charles River Laboratories, Worcester, MA (#I023), or MI sponses are variable and highly influenced by immune regulatory Bioresearch, Ann Arbor, MI (VUF #26). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 148 of 272 compartments in the tumor microenvironment (TME). This is espe- tumor types. Immunotherapeutic agents have a mechanism of action cially true for pancreatic cancer (PC) where clinical trials aiming to re- distinct from chemotherapy and are being used across a broad array cover T cell anti-tumor activity have been disappointing. Thus, in PC of tumor types. A standardized, universal scoring system for patho- and other cancers there is a clinical need for alternative treatments logic response that encompasses features characteristic for immuno- We have previously shown that antibodies targeting the scavenger therapy and spans tumor types is needed. receptor MARCO expressed on tumor-associated macrophages Methods (TAMs), reduces tumor growth and impair metastasis in murine can- Hematoxylin and eosin-stained slides from neoadjuvant surgical re- cer models. Here we investigated targeting of the scavenger receptor sections and on-treatment biopsies were assessed for features of MARCO on human TAMs in pancreatic ductal adenocarcinoma immune-related pathologic response (irPR). 258 specimens from pa- (PDAC) and hypothesized that targeting this receptor will remodel tients with 11 tumor types as part of ongoing clinical trials for anti- the suppressive environment and relive the anti-tumor responses to PD-1 were evaluated. An additional 98 specimens from patients re- increase the efficacy of immunotherapy. ceiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types. Methods Results To test our hypothesis, analysis of MARCO gene expression data Common irPR features (immune activation, cell death, tissue repair, from the Human Protein Atlas (HPA) project was performed in- regression bed) were present in all tumor types reviewed, including vestigating pancreatic tumors (n=176), as these consist of up to melanoma, non-small cell lung, head and neck squamous cell, Merkel 80% stroma, compared with healthy pancreatic tissues (n=171). In cell, and renal cell carcinoma, amongst others. Features were consist- vitro, cytokine differentiated macrophages alternatively cultured ent across primary tumors, lymph nodes, and distant metastases. with PC cell lines under hypoxia and normoxia conditions were Specimens from patients treated with anti-PD-(L)1 in combination co-cultured with cytotoxic cells to mimic their interaction in the with another agent also exhibited irPR features. TME. Later, macrophages were treated with anti-MARCO Abs and Conclusions their phenotype and function were examined prior and following irPR features are consistent across tumor types and treatment set- interaction with immune effector cells. Subsequently, anti-MARCO tings. Standardized, pan-tumor immune-mediated pathologic re- ab anti-tumor efficacy was tested in vitro and in vivo in PC and sponse criteria (irPRC) are defined and associated specimen-handling melanoma models. considerations are described. Future, prospective studies are merited Results to validate irPRC in larger datasets and to associate pathologic fea- We found a 30-fold increase in MARCO expression in pancreatic tu- tures with long-term patient outcomes. mors compared to healthy tissues. Also, a significant (p=0.03) correl- Ethics Approval ation between high expression and decreased survival was noted in The study was approved by the Johns Hopkins University Institu- pancreatic cancer patients. Furthermore, pancreatic cancer cell lines tional Review Board. induced MARCO expression on macrophages and dedifferentiated them towards myeloid-derived suppressor cells (MDSC). This ef- fect was amplified by hypoxic condition. Notably, MARCO+ MDSC P276 in contrast to control monocytes and macrophages suppressed T- Efficacy of PD-1/PDL-1 Immune Checkpoint Inhibitors in and Natural Killer (NK) cell anti-tumor activities, which was re- Elderly Patients with Non-Small-Cell Lung Cancer; A Subgroup versed by treatment with anti-human MARCO Abs. In addition, Meta-analysis of Randomized Controlled Trials targeting TAMs with anti-MARCO Abs, abolished their anti- 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD inflammatory phenotype in vitro and in vivo and normalized their 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of metabolism towards pro-inflammatory. Moreover, in B16 melan- Medicine at Mount Sinai, New York, NY, United States; King Edward oma tumor model the anti-MARCO Ab mediated an anti-tumor Medical University, Lahore, Pakistan; The University of Texas, effect that was dependent on NK cells and their TRAIL-mediated Houston, TX, United States killing mechanism. Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P276 Thus, our study reveals a novel interaction between TAMs and cyto- toxic cells as a result of TAM targeting with monoclonal Ab demon- Background strating a promising approach as immunotherapy that remodel the PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as immune TME. an efficacious drug class for the treatment of non-small-cell lung can- Ethics Approval cer (NSCLC). The efficacy of PD-1/PD-L1 ICPI therapy in the elderly The study was approved by Institutional Ethics Board, approval num- (patients age ≥65 and ≥75) has not been thoroughly investigated. ber Dnr 2013.977-31.1. The aim of this study was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy in elderly patients with NSCLC. Methods P275 A systematic review of the literature to identify randomized con- Pan-tumor pathologic scoring of response to PD-(L)1 blockade trolled trials (RCTs) which reported overall survival (OS) and progres- Julie Stein, Evan Lipson, MD, Tricia Cottrell, MD, PhD, Patrick Forde, sion free survival (PFS) of elderly patients with NSCLC who were MD, Robert Anders, MD, PhD, Ashley Cimino-Mathews, Elizabeth randomized to receive PD-1/PDL-1 ICPIs or docetaxel/investigator’s Thompson, MD PhD, Mohamad Allaf, MD, Mark Yarchoan, Josephine choice chemotherapy. The hazard ratios (HR) of OS and PFS in elderly Feliciano, MD, Elizabeth Jaffee, MD, Drew Pardoll, MD, PhD, Suzanne patients (along with their 95% confidence intervals; CI) were ex- Topalian, MD, Janis Taube, MD, MSC tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Johns Hopkins University School of Medicine, Baltimore, MD, United 1 versus chemotherapy stratified by patient age (≥65 and ≥75). A States random effects model was employed only when there was significant Correspondence: Janis Taube (jtaube1@jhmi.edu) heterogeneity among studies (>40%, as assessed by I-squared). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P275 Results Screening of 15,092 studies yielded four RCTs (two with patients age Background ≥75) which enrolled a total of 2,429 patients. No significant difference Pathologic response assessment of tumor specimens from patients in PFS with PD-1/PDL-1 treatment versus chemotherapy was found receiving systemic treatment provide an early indication of thera- among patients aged ≥65 (HR 0.8, 95% CI 0.53-1.06, I2 78.30%) or pa- peutic efficacy and predict long-term survival. Grading systems for tients aged ≥75 (HR 1.1, 95% CI 0.43-1.77,I2 0.00%). Patients aged ≥65 pathologic response were first developed for chemotherapy in select had an improved OS with PD-1/PD-L1 ICPI treatment versus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 149 of 272 chemotherapy (HR 0.67, 95% CI 0.51-0.84, I2 62.50%). Patients aged 1 versus chemotherapy stratified by the degree of PD-L1 expression. ≥75 did not show any significant difference in OS when treated with A random effects model was employed only when there was signifi- PD-1/PD-L1 versus chemotherapy (HR 1.02, 95% CI 0.35-1.69,I2 0.00%). cant heterogeneity among studies (>40%, as assessed by I-squared Results Conclusions Screening of 15,092 studies yielded four RCTs (two reporting only Patients aged ≥65 show an improved OS with PD-1/PD-L1 therapy. How- PD-L1 expression ≥50%) which enrolled a total of 2,429 patients. An ever, NSCLC patients ≥75 do not show a significant difference in OS or improved PFS with PD-1/PDL-1 treatment versus chemotherapy was PFS when treated with PD-1/PD-L1 ICPI versus chemotherapy (Figure 1). found among patients with PD-L1 expression ≤1% (HR 0.78, 95% CI This may be a consequence of aging and its impact on individuals’ cap- 0.57-0.98, I2 25.30%) ≥1% (HR 0.62, 95% CI 0.46-0.78, I2 0.00%), ≥5% ability to mount an anti-tumor response. Further studies assessing the ef- (HR 0.46, 95% CI 0.31-0.6, I2 0.00%), and ≥10% (HR 0.44, 95% CI ficacy of PD-1/PD-L1 ICPI use in patients ≥75 are warranted. 0.29-0.58, I2 0.00%). Similarly, an improved OS was observed with PD-1/PDL-1 treatment versus chemotherapy was found among patients with PD-L1 expression ≥1% (HR 0.7, 95% CI 0.52-0.87, I2 0.00%), ≥5% (HR 0.54, 95% CI 0.38-0.69, I2 0.00%), and ≥10% (HR 0.51, 95% CI 0.35-0.68, I2 0.00%), but not with PD-L1 expression of ≤1%, ≤5%, and ≤10% (figure 1). No significant difference in OS and PFS was observed with PD-1/PD-L1 treatment versus chemotherapy among patients with ≥50% PD-L1 expression. Of the two RCTs which reported ≥50% PD-L1 expression, one used Nivolumab (and reported a negative outcome in OS and PFS) whereas the other used Pembrolizu- mab (and reported a favorable OS and PFS). Conclusions NSCLC patients who express PD-L1 have an improved OS and PFS with PD-1/PD-L1 ICPI use versus chemotherapy. However, high PD-L1 expression (≥50%) may not translate into a better response to all PD- 1/PD-L1 ICPIs (Figure 1). Further studies are needed to assess the intra-class efficacy of different PD-1/PD-L1 ICPIs in NSCLC patients with a high PD-L1 expression. Fig. 1 (abstract P276). OS and PFS in Elderly Patients with NSCLC P277 Utility of PD-L1 Expression in Non-Small-Cell Lung Cancer Patients Treated with PD-1/PDL-1 Immune Checkpoint Inhibitors; A Subgroup Meta-analysis of Randomized Controlled Trials 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States; King Edward Medical University, Lahore, Pakistan; The University of Texas, Houston, TX, United States Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P277 Background Fig. 1 (abstract P277). OS and PFS Stratified by PD-L1 Expression PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an efficacious drug class for the treatment of non-small-cell lung can- cer (NSCLC) evident by findings of multiple randomized controlled P278 trials (RCTs). The utility of PD-L1 expression analysis in treatment PD-1/PDL-1 immune checkpoint inhibitors in smokers with non- planning and prognosis remains questionable. The aim of this study small-cell lung cancer; a subgroup meta-analysis of randomized was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemo- controlled trials therapy in patients with NSCLC stratified by PD-L1 expression 1 2 3 4 Faisal Ali , Bibek Pannu , Maryam Hussain , Arafat Farooqui , Taha Methods 1 1 5 Alrifai , Phyo Myint , Syed Jafri, MD A systematic review of the literature to identify RCTs which reported 1 2 Saint Joseph Hospital, Chicago, IL, United States; John H Stroger Jr overall survival (OS) and progression free survival (PFS) of patients Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount with NSCLC who were randomized to receive PD-1/PDL-1 ICPIs or do- Sinai, New York, NY, United States; King Edward Medical University, cetaxel/investigator’s choice chemotherapy and underwent PD-L1 ex- Lahore, Pakistan; The University of Texas, Houston, TX, United States pression analysis. The hazard ratios (HR) of various degrees of PD-L1 Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) expression (along with their 95% confidence intervals; CI) were ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P278 tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 150 of 272 Background P279 PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an effi- PDL-1 is highly expressed in ovarian germ cell tumor and cacious drug class for the treatment of non-small-cell lung cancer (NSCLC) associated with cancer stem cells population expressing CD44 evident by findings of multiple randomized controlled trials (RCTs). How- Salmah Alamri, Miral Mashhour, Kholoud Alwosaibai, PhD ever, the efficacy of PD-1/PDL-1 ICPIs in patients with NSCLC who are King Fahad Specialist Hospital, Dammam, Saudi Arabia current or former smokers remains to be debated. The aim of this study Correspondence: Kholoud Alwosaibai (kh20978@gmail.com) was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P279 in patients with NSCLC who were active or former smokers Methods Background A systematic review of the literature to identify RCTs which reported Immunotherapy using checkpoint inhibitors have proposed beneficial overall survival (OS) and progression free survival (PFS) among effects for some types of cancer such as lung cancer and melanoma. former or active smokers with NSCLC who were randomized to re- However, using PD-1/PDL-1 inhibitors to treat ovarian cancer was lim- ceive PD-1/PDL-1 ICPIs or docetaxel or investigator’s choice chemo- ited and further studies are needed to identify the patients that will therapy. The hazard ratios (HR) of these subgroups (along with their benefit from this treatment. In this study we have explored predictive 95% confidence intervals; CI) were extracted to compute a pooled biomarkers in ovarian cancer that might associate with checkpoint (HR) to report the efficacy of PD-1/PDL-1 versus chemotherapy in treatment outcome. For the first time, we have investigated the role of former or active smokers with NSCLC. A random effects model was PDL-1 expression in the tumor microenvironment cells that includes employed only when there was significant heterogeneity among immune cells and cancer stem cells in different types of ovarian cancer. studies (>40%, as assessed by I-squared) Methods Results 66 surgical samples of different types of ovarian cancer have been Screening of 15,092 studies yielded six RCTs which enrolled a total of collected from pathology department. IHC staining has been per- 3,584 patients. PFS did not differ significantly with PD-1/PDL-1 treat- formed using PD-L1 IHC 22C3 pharmDx to detect PDL-1, CD8 and ment or chemotherapy among non-smokers (HR 1.33, 95% 0.80-1.80; I2 CD4 to detect tumor infiltrating lymphocyte (TIL), and CD44, CD117 20.6%) and former or active smokers (HR 0.83, 95% CI 0.63-1.04; I2 and OCT3/4 to detect stem cell markers. 67.5%). Survival analysis revealed that former or active smokers tended Results to have an improved OS when treated with PD-1/PDL-1 inhibitors ver- We found that 47% of ovarian cancer patients express PDL-1. The expres- sus chemotherapy (HR 0.72, 95% CI 0.54-0.90; I2 70.5%), whereas OS sion of PDL-1 have been detected in different types of ovarian cancer in- did not differ significantly among non-smokers when treated with PD- cluding, serous carcinoma, germ cell tumor, endometrioid. The majority 1/PDL-1 inhibitors versus chemotherapy (HR 0.76, 95% CI 0.48-1.04; I2 (73%) of germ cell tumor tissues express PDL-1 whereas serous cancer 0.0%) and endomitrioid express PDL-1 in 46% and 50% of the cancer tissue, re- Conclusions spectively. However, PDL-1 protein was undetectable in some histological NSCLC patients who are former or active smokers have an improved type of ovarian cancer such as granulosa tumor and mucinous tumor. OS when treated with PD-1/PDL-1 ICPIs, though the PFS does not dif- Also we determined the expression levels of TIL in the ovarian cancer fer significantly (Figure 1). This may be a consequence of a higher tissue that either PDL-1 positive or negative. We found that 81% of mutation burden among smokers. Further research into the inter- ovarian cancer samples have TIL that express CD8 and 92% of these action between carcinogens, toxins and proinflammatory substances ovarian cancer samples are associated with PDL-1 expression.TIL that of smoking and ICPIs are warranted. express CD4 have been found in 66% of all ovarian cancer samples and 77% of these samples are associated with PDL-1 expression. Further, we have studied the association between PDL-1 expression and cancer stem cell markers such as CD44, CD117, OCT 3/4. We found that all PDL-1 positive samples are expressing CD44 and also, we found strong association between CD117 expression and PDL-1 expression. Conclusions Immunotherapy treatment using PDL-1 inhibitor could be considered for ovarian cancer patients that expressing PDL-1 particularly, germ cell ovarian cancer. In addition, PDL-1 expression is strongly associated with CD44. Inhibiting PDL-1 using immunotherapy might downregulate stem cell populations and decrease the chemotherapy resistance and recurrence that derived by stem cell residual in the cancer tissue. Ethics Approval The study was approved by the IRB at King Fahd Specialist Hospital- Dammam. approval number ONC0340 P280 Impact of tumor mutational burden on overall survival in patients with non-small cell lung cancer treated with immunotherapy 1 2 1 Mark Awad, MD PhD , Navin Mahadevan , Andrew Polio , Natalie 1 1 1 1 Vokes , Elizabeth Aguilar , Biagio Ricciuti, MD , Giuseppe Lamberti, MD , 1 1 1 Gonzalo Recondo, MD , Giulia Leonardi , Anika Adeni , Pasi Janne, MD 1 1 1 1 PhD , Eliezer Van Allen, MD , Adem Albayrak, MS , Renato Umeton , Lynette Sholl 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Women’s Hospital, Boston, MA, United States Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P280 Background In non-small cell lung cancer (NSCLC), tumor mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint in- hibitors, but the optimal TMB cutpoint associated with a survival benefit Fig. 1 (abstract P278). OS and PFS in Smokers and Non-Smokers remains unclear. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 151 of 272 Methods We collected clinicopathologic data from patients with NSCLC se- quenced by the OncoPanel NGS platform at the Dana-Farber Cancer Institute. The relationship between TMB and clinical outcomes after treatment with immune checkpoint inhibitors was investigated in the subset of patients treated with immunotherapy. Results Among 2690 patients identified, median TMB was significantly higher Fig. 3 (abstract P280). See text for description among current smokers compared to former (P<0.0001) and never smokers (P<0.0001) and among squamous tumors compared to smoking-related nonsquamous tumors (P=0.01) (Figure 1). Patients without oncogenic drivers and those harboring BRAF or KRAS mutation had the highest median TMB (10.6, 11.1 and 9.8 mutations/megabase [mut/Mb], respectively), while tumors with ROS1, MET, RET and ALK al- terations had the lowest median TMB (6.7, 6.1, 5.3, 5.3 mut/Mb, respect- ively) (Figure 2). Among patients treated with immunotherapy (N=489), a recursive partitioning algorithm identified an optimal TMB cut-off for PFS and OS of 18.5 mut/Mb, which represents the 88th percentile for TMB in this cohort. Baseline clinicopathologic characteristics were well- balanced between patients with a TMB of ≥18.5 and <18.5 mut/Mb (Table 1). Patients with a TMB of ≥18.5 mut/Mb had a significantly Table 1 (abstract P280). See text for description higher response rate (43.3% vs. 17.5%, P<0.0001), a longer median progression-free survival (8.2 vs. 2.7 months, HR:0.52 [95%CI:0.38-0.72], P<0.0001), and a longer median overall survival (20.7 vs. 10.2 months, HR:0.55 [95%CI:0.38-0.79], P=0.001) compared to those with a TMB < 18.5 mut/Mb (Figure 3). After adjusting for performance status, smoking history, and PD-L1 expression, a TMB of ≥18.5 mut/Mb was associated with a significantly longer PFS (HR:0.56 [95%CI: 0.41-0.78], P =0.001) and OS (HR: 0.57 [95%CI: 0.40-0.82], P =0.003) in multivariate analysis. Conclusions Tumor-only NGS identifies clinical and genomic correlates of high TMB in NSCLC. Patients with a TMB ≥88th percentile are most likely to experi- ence a survival benefit when treated with immune checkpoint inhibitors. Fig. 1 (abstract P280). See text for description Fig. 2 (abstract P280). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 152 of 272 P281 L1+ tumors, do not benefit. The magnitude of immune activation Durable response after immunotherapy discontinuation: a promoted by PD-1/PD-L1 axis blockade can be further enhanced multicenter real-life experience through concomitant T-cell co-stimulation such as that achieved 1 2 3 Maria Bassanelli, MD, PhD , Diana Giannarelli, MD , Marco Russano , through CD137 agonism; however, clinical applications of such an 2 4 5 Fabiana Letizia Cecere , Maria Rita Migliorino , Silvana Giacinti , Viola approach may be limited by toxicity associated with the systemic ef- 4 6 5 4 Barucca , Emilio Bria , Enzo Maria Ruggeri , Fabio Calabrò , Alain fects of CD137 agonists. Here we characterize PD-L1 and CD137 7 3 1 Gelibter , Daniele Santini , Anna Ceribelli tumor expression supporting the development of a PD-L1xCD137 1 2 San Camillo de Lellis Hospital, Rieti, Italy; Regina Elena National Cancer bispecific molecule that provides PD-1 axis blockade coupled with 3 4 Institute, Rome, Italy; University Campus Bio-Medico, Rome, Italy; San tumor-targeted CD137 co-stimulation. Camillo Forlanini Hospital, Rome, Italy; Belcolle Hospital, Viterbo, Italy; Methods 6 7 Fondazione Pol. Universitario A.Gemelli, Rome, Italy; Policlinico In situ hybridization (ISH) and multicolor flow cytometry was per- Umberto I, Rome, Italy formed to characterize PD-L1 and CD137 expression in tumor biop- Correspondence: Maria Bassanelli (maria.bassanelli@yahoo.it) sies. A PD-L1xCD137 bispecific molecule (PD-L1xCD137) was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P281 constructed based on PD-L1 blocking mAbs and CD137-engaging mAbs and was evaluated for binding to respective antigens. Its func- Background tional activity was evaluated in CD3 or SEB-driven T-cell activation Immune checkpoint inhibitors (ICIs) have significantly improved over- systems, MLR assays and tumor microenvironment models. Anti- all survival (OS) in several cancer types. Unlike chemotherapy, the tumor activity in vivo was evaluated in combination with tumor tar- optimum treatment duration with ICIs is not clearly established [1,2] geted anti-CD3 based bispecific DART® molecules. Methods Results We conducted an observational, retrospective analysis of 46 consecu- ISH revealed expression of PD-L1 in significant proportion of surgi- tive patients (pts) with advanced solid tumors who discontinued cally resected carcinomas; noteworthy, many such tumors contained immune-based therapies for any reason except progressive disease. CD137+ immune infiltrate adjacent to PD-L1+ cells. Moreover, The aim of this study was to assess the outcome and the antitumor ex vivo co-incubation of tumor and immune cells in the presence of activity of ICIs after treatment discontinuation. Treatment-free sur- CD3-based bispecifics or Fc-enhanced antibodies further induces PD- vival (TFS) was defined as the time from interruption of immunother- L1 and CD137 expression. PD-L1xCD137 binds and blocks PD-L1, re- apy for any reason except progressive disease to start of subsequent versing PD-1-mediated T-cell inhibition equipotently to the effect of anticancer therapy or best supportive care or death. Median OS, pro- approved PD-L1 benchmark mAbs; it also binds CD137, but absent gression free survival (PFS), TFS and the 95% confidence interval (CI) clustering supported by PD-L1+ cells, fails to induce CD137 signaling. were estimated with the Kaplan -Meier method In the presence of PD-L1-expressing cells, however, PD-L1xCD137 Results drives CD137 activation and immune cell co-stimulation. Robust T- 46 pts (median age 68 years [range 41-86]; male: 65.2%) with advanced cell activation and cytokine secretion was induced by PD-L1xCD137, cancer (n.39 non-small-cell lung cancer, n.15 renal cell carcinoma and with significantly greater activity than that observed with the com- n.2 melanoma) were treated with ICIs, as clinically indicated, at eight bination of PD-L1 blocking and CD137 agonistic mAbs. Notably, Italian institutions: 44 pts received programmed death 1 (PD-1) inhibi- when combined with tumor targeted immunotherapies, PD- tors (n.31 nivolumab, n.13 pembrolizumab) and 2 pts programmed L1xCD137 supports enhanced activation of effector cells and anti- death ligand 1 (PD-L1) (n.1 durvalumab, n.1 atezolizumab). A median of tumor activity. 8 cycles were administered [range 1 to 52]. 36 pts discontinued ICIs Conclusions due to toxicities (diarrhoea, pneumonitis, hepatotoxicity)and 10 pts for These data show that an investigational PD-L1xCD137 bispecific can reasons non immune-related. The median PFS from the beginning of switch on CD137 co-stimulation in a PD-L1-dependent fashion. While ICIs was 12.4 months (mo) [95% CI: 8.2-16.6] and the median OS was tumor adaptive resistance via PD-L1 induction promotes tumor im- 20.0 mo (95% CI: 11.8-28.2). Median PFS from ICIs completion was 5.0 mune escape, PD-L1xCD137 can exploit the checkpoint ligand up- mo [95% CI: 2.7-7.3] and median OS was 16.1 mo (95% CI: 5.4-26.8). Me- regulation by contributing a co-stimulatory signal in addition to dian TFS was 7.4 (95% CI: 5.8-8.9) mo checkpoint blockade. PD-L1xCD137 provides a potential therapeutic Conclusions approach to overcome limitations of existing PD-1/PD-L1-targeting This study shows the durable cancer-specific immune response in pts strategies either as monotherapy or in combination with comple- with advanced cancer even after stopping the ICIs for any reason ex- mentary immune based therapeutic modalities, such as CD3 based cept progressive disease bispecifics or Fc-enhanced mAbs. References P283 1. Kim PS, Ahmed R. Features of responding T cells in cancer and chronic Generation and characterization of a PD-1 resistant mouse tumor infection. Curr Opin Immunol 2010; 22: 223–230. model 2. Schadendorf D, Hodi FS, Robert C et al. Pooled analysis of long-term sur- Marie Bernardo, PhD, Tatiana Tolstykh, Yu-An Zhang, Dinesh Bangari, Hui vival data from phase II and phase III trials of ipilimumab in unresectable Cao, PhD, Joon Sang Lee, PhD, Natalia Malkova, Jack Pollard, PhD, or metastatic melanoma. J Clin Oncol 2015; 33: 1889–1894. Fangxian Sun, Dmitri Wiederschain, Timothy Wagenaar Sanofi Oncology Research, Cambridge, MA, United States P282 Correspondence: Timothy Wagenaar (timothy.wagenaar@sanofi.com) Tumor-targeted T-cell activation via an investigational PD-L1 x Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P283 CD137 bispecific molecule Alexey Berezhnoy, PhD, Ling Huang, Daorong Liu, Jennifer DiChiara, Background Jonathan Li, PhD, Douglas Smith, PhD, Jill Rillema, BS, Valentina Immune checkpoint blockade elicits durable anti-cancer responses Ciccarone, PhD, James Tamura, PhD, Ralph Alderson, PhD, Gundo in the clinic, however a large proportion of patients do not bene- Diedrich, Ezio Bonvini, PhD, Paul Moore, PhD fit from treatment. Several mechanisms of innate and acquired MacroGenics, Inc, Rockville, MD, United States resistance to checkpoint blockade have been defined and include Correspondence: Paul Moore (moorep@macrogenics.com) mutations of MHC I and IFNg signaling pathways. However, such Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P282 mutations occur in a low frequency of patients and additional mechanisms have yet to be defined. In an effort to better under- Background stand acquired resistance to checkpoint blockade, we generated Blockade of the PD-1/PD-L1 axis can improve outcome in a variety of a mouse tumor model exhibiting in vivo resistance to anti-PD-1 cancers; yet, many patients, including subsets of patients with PD- antibody treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 153 of 272 Methods CD8+ T cells in the blood of E2 supplemented huNOG mice. Tumor A PD-1 resistant mouse tumor model was generated by serially pas- immunohistochemistry showed low number of TILs and very low ex- saging MC38 tumors in mice treated with anti-PD-1. The resistant pression of programmed death-ligand 1 (PD-L1). No clear anti-tumor tumor line was characterized using a range of molecular techniques. effects were observed with pembrolizumab treatment compared to Results the isotype control group. MC38 tumors acquired resistance to PD-1 blockade following serial Conclusions in vivo passaging. Lack of sensitivity to PD-1 blockade could not be In conclusion, estrogen supplementation was needed to support attributed to dysregulation of PD-L1 or B2M expression, as both were orthotopic ER+ breast cancer growth. However, estrogen decreased expressed at similar levels in parental and resistant cells. Similarly, survival of female huNOG mice. Estrogen had immunomodulatory ef- IFNg signaling and antigen processing and presentation pathways fects and induced adverse effects including anemia. Due to these were functional in both parental and resistant cell lines. Unbiased deleterious effects and decreased number of immune cells, no direct gene expression analysis was used to further characterize potential conclusions can be drawn for the possible anti-tumor effects of pem- resistance mechanisms. RNA-sequencing revealed substantial differ- brolizumab in this orthotopic ER+ breast cancer model. Caution ences in global gene expression with PD-1 resistant tumors display- should be taken when evaluating efficacy of immunotherapies in ing a marked reduction in expression of immune-related genes hormone-dependent preclinical cancer models, and using ER+ breast relative to parental MC38 tumors. Transcriptomic data revealed that cancer models where tumor growth is supported by local microenvir- PD-1 resistant tumors exhibit reduced immune infiltration across onment, such as in bone metastasis models, should be considered. multiple cell types, including T and NK cells, while pathway analysis Ethics Approval identified activation of two major tumor promoting signaling path- This study was approved by the National Animal Experiment Board ways in PD-1 resistant tumors. Pharmacological inhibition of these in Finland; license number ESAVI-2331-04 10 07-2017. pathways in combination with PD-1 blockade inhibited tumor growth and extended the survival of mice bearing resistant tumors. P285 Conclusions Integrated molecular characterization of primary resistance This study describes a novel PD-1 resistant mouse tumor model and mechanisms to immune checkpoint blockade in advanced non- underscores the importance of two well defined signaling pathways small cell lung carcinoma (a-NSCLC) to response to immune checkpoint blockade. 1 2 2 Benjamin Besse, MD PhD , Caroline Fraslon, PhD , Hui Cao, PhD , Joon 2 2 2 Sang Lee, PhD , Stephanie Malyszka, MS , Marielle Chiron, PhD , Anne 2 2 1 P284 Caron, PhD , Souâd Naimi, PhD , Laura Mezquita, MD , David Planchard, 1 1 1 Pitfalls in preclinical development of immunotherapies for ER+ MD, PhD , Jean-Yves Scoazec, MD , Ludovic Lacroix, PharmD , Etienne 1 1 breast cancer: estrogen as an immunomodulator potentially Rouleau, MD , Naima Imam-Sghiouar, PhD , Aurélien Marabelle, MD 1 1 1 2 influencing pembrolizumab efficacy in a breast cancer model in PhD , Eric Angevin, MD , Christophe Massard, MD , Jack Pollard, PhD 1 2 humanized mice Gustave Roussy Cancer Center, Villejuif, France; Sanofi, Vitry sur Seine, 1 1 1 2 Tiina Kähkönen , Mari Suominen , Jenni Mäki-Jouppila , Emrah Yatkin , France 3 3 1 1 Philip Dube , Azusa Tanaka , Jussi Halleen , Jenni Bernoulli, PhD Correspondence: Caroline Fraslon (caroline.fraslon@sanofi.com) 1 2 Pharmatest Services, Turku, Finland; University of Turku, Turku, Finland; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P285 Taconic Biosciences, Rensselaer, NY, United States Correspondence: Jenni Bernoulli (jenni.bernoulli@pharmatest.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P284 Reinvigoration of anti-tumor immunity via immune checkpoint block- ade (ICB) has transformed outcomes in a-NSCLC. However, a majority Background of patients are innately resistant to ICB, and a better understanding Immunotherapies have the potential to improve outcomes in triple- of the resistance mechanisms may guide the development of new negative breast cancer patients but evidence is less consistent in es- treatment strategies and patients therapies. trogen receptor -positive (ER+) patients. To advance preclinical devel- Methods opment and understand the effects of immunotherapies against ER+ Biopsies performed immediately before treatment with single agent breast cancer, we aimed to establish a novel orthotopic ER+ breast ICB in patients with a-NSCLC (MATCH-R trial [NCT02517892]) were an- cancer model in humanized mice and to study efficacy of pembroli- alyzed. The stromal microenvironment and immune context was zumab in the model. characterized via an integrated analysis of whole transcriptome Methods (RNA-seq), whole exome sequencing (WES), and immunohistochemis- Female CIEA NOG® (NOG) mice and NOG mice engrafted with human try (IHC) of CD3, CD8, FOXP3 and PDL1. Specifically, the immune con- CD34+ hematopoietic stem cells (huNOG, Taconic Biosciences) were text and the relative abundance of 10 immune and stromal cell types implanted with 5 μg/day estradiol (E2)-releasing implants, and one were assessed with integrated IHC and Cell Populations-counter week later inoculated with ER+ MCF-7 human breast cancer cells into (MCP-counter) [1] analysis of the RNA-seq. Somatic mutations and the mammary fat pad. One group of huNOG mice did not receive E2 Tumor Mutation Burden (TMB) were evaluated. The transcriptional implants. Orthotopic tumor growth was followed by caliper mea- state of the tumor and its microenvironment was assessed by GSVA surements. At study week 2, the E2 supplemented mice were strati- analysis [2] of the MSigDB collection [3]. Patient’s outcome was asso- fied to receive human IgG4 isotype control or pembrolizumab (5 ciated to molecular data. Primary resistance to ICB was defined as PD mg/kg, i.p., Q5D) until the end of the study. The study was termi- in the first radiological examination, or a median PFS inferior to 3 nated at study week 7 and tumors were processed for histological months. and immunohistochemical analysis of tumor infiltrating lympho- Results cytes (TILs). Changes in blood cell counts were assessed by flow cy- Thirty-one patients with adenocarcinoma were enrolled: Median age tometry and hematology. was 60 (34-80), 13 were female, 28 were smokers, 10 were re- Results sponders, and 21 were non-responders. Median tumor cellularity was ER+ orthotopic breast tumors grew only in the presence of E2 sup- 60% (30%-90%). plement. However, general condition of huNOG mice started to de- Responders had higher TMB and immune infiltration compared to crease after 3 weeks on E2 supplementation and their survival was non-responders. Non-responders could be divided into two classes: decreased compared to huNOG mice without E2 supplement. those with equal infiltration to responders “hot tumors” and those Hematological analysis indicated that estrogen decreased the levels with less infiltration “cold tumors”. Neutrophil infiltration was associ- of white and red blood cells, hemoglobin and hematocrit that were ated with resistance to ICB in both “hot” and “cold” resistant tumors. further decreased by concomitant pembrolizumab treatment. Flow Mutations in the IFN-γ and/or KRAS/STK11/KEAP pathways were asso- cytometry analysis confirmed lower numbers of CD3+, CD4+ and ciated to ICB resistance. Increased activation of hypoxia-response, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 154 of 272 transforming growth factor (TGF-β) and MYC pathways from the partial responders and non-responders (high expression), thereby GSVA analysis were also associated to ICB resistance. TGF-β pathway suggesting that the identified target is relevant to the clinical activation was associated to the “cold tumor” ICB-resistant tumor situation. class. Conclusions Conclusions The mAb is currently under preclinical development as a novel im- ICB sensitivity was associated to TMB, IFN-γ pathway mutation, and munotherapeutic antibody to overcome resistance to anti-PD-1 im- immune infiltration. We have further refined our understanding of mune checkpoint blockade. the primary ICB resistance mechanisms in that there are both “hot” and “cold” non-responsive tumors, which suggests that different References therapeutic approaches be may be required in these two subtypes, 1. Perrot I, Michaud HA, Giraudon-Paoli M, et al. Blocking Antibodies i.e. targeting the TGF-β pathway in the “cold” non-responding tumor Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Im- class. We are continuing to increase our baseline cohort size and will mune Responses in Combination Cancer Therapies. Cell Rep. 2019 include post-treatment biopsies collected with this protocol. May 21;27(8):2411-2425.e9. doi: 10.1016/j.celrep.2019.04.091 2. Hugo W, Zaretsky JM, Sun L et al. Genomic and Transcriptomic References Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. 1. Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, Selves J, Cell. 2016 Mar 24;165(1):35-44. doi: 10.1016/j.cell.2016.02.065. Epub Laurent-Puig P, Sautès-Fridman C, Fridman WH, de Reyniès A. Estimating 2016 Mar 17. the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol. 2016; 17(1):218-238. P287 2. Hänzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for A novel bispecific checkpoint inhibitor antibody to preferentially microarray and RNA-seq data. BMC Bioinformatics. 2013;14:7-22. block PD-1 and LAG-3 on dysfunctional TILs whilst sparing Treg 3. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, activation Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 1 1 2 Laura Codarri Deak , Patrick Weber, Mr , Stefan Seeber, Dr , Mario Perro, 2011; 27(12):1739-40. 1 1 1 1 Dr , Xavier Miot, Mr , Heidi Poulet, Mrs , Iryna Dekhtiarenko, Dr , Ethics Approval 3 3 4 Matthias Füth , Henry Kao, Dr , Christine McIntyre, Dr , Francesca This study was approved by Gustave Roussy Scientific Board, the French 3 1 1 Michielin, Dr , Christian Klein, Dr rer nat , Pablo Umana, PhD , Lin-Chi National Health Agency and Ethical Committee (ID-RCB : 2014-A01147- 5 2 3 Chen, Dr , Christoph Markert, Dr , Merlind Mücke, Dr 40) 1 2 Roche Innovation Center Zurich, Zurich, Switzerland; Roche Innovation Center Munich, Munich, Germany; Roche Innovation Center Basel, Basel, P286 Switzerland; Roche Innovation Center Welwyn, Welwyn, United In vivo genetic screens in PD-1 resistant mouse models identified Kingdom; Roche Innovation Center New York, New York, NY, United modulators of anti-PD1 response with relevance to States pembrolizumab-treated human metastatic melanoma Correspondence: Laura Codarri Deak (laura.codarri_deak@roche.com) Aurélie Docquier, PhD, Cécile Déjou, Gilles Alberici, Armand Bensussan, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P287 PhD, Jeremy Bastid, Nathalie Bonnefoy, PhD OREGA Biotech, Ecully, France Background Correspondence: Jeremy Bastid (jeremy.bastid@orega-biotech.com) Check point inhibitors targeting PD-1 have shown unprecedented Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P286 clinical efficacy in several cancer indications and therefore have, revolutionized the standard of care. However, despite this ad- Background vancement in cancer immunotherapy, only ~20-30% of the pa- We previously reported the identification of CD39 as a regulator of tients derive durable benefit from such a treatment. One of the anti-PD-1 response in combination with oxaliplatin in resistant suggested reasons for this limited success is the expression/acti- models using our screening approach [1]. vation of compensatory inhibitory pathways such as LAG-3 on Methods tumor-reactive T cells. These pathways compensate for the loss Similar approach was used to identify novel targets that modulate of function of PD-1 upon its blockade. Therefore, it is envisioned anti-PD-1 response. Here we report the discovery of a novel potent that simultaneous antagonism of PD-1 and LAG-3 receptors regulator of anti-PD-1 therapy in MCA205 fibrosarcoma and B16K1 would overcome this adaptive resistance mechanism and allow a melanoma syngeneic mouse models harboring no or low response more profound reinvigoration of dysfunctional tumor-reactive T rates to PD-1 immune checkpoint inhibitor. cells. Conversely, a recent report has highlighted that blockade of Results LAG-3 on regulatory T cells (Tregs) increases their suppressive Remarkably, the Knock-Out (KO) of the identified target had limited, function and, therefore may off-set its benefit on the reinvigor- if any, impact on tumor growth in various mouse models. Whereas ation of dysfunctional tumor-reactive T cells. the mouse models used are broadly resistant to anti-PD1 therapy, Methods treatment in the KO background markedly improved the efficacy of We therefore developed a 1+1 PD1-LAG3 bispecific antibody (BsAb) anti-PD-1 mAb by increasing the response rates and by increasing with a 10-20 fold higher affinity for PD-1 than for LAG-3, allowing an the rate of complete vs partial responses, which translated into im- avidity driven selectivity gain to PD-1 and LAG-3 double positive T proved mouse survivals. We generated various human-mouse cross- cells. reactive blocking antibodies to the target, including a humanized Results mAb. The neutralizing antibodies mimicked the KO phenotype and Hence, PD1-LAG3 BsAb is assumed to have the following advantages markedly improved the response to anti-PD1 therapy in preclinical over monospecific and other bispecific aPD/L1 and aLAG-3 anti- mouse models. The mechanism of action is being investigated. We bodies: 1) improved targeting to dysfunctional T cells rather than found that the expression of target within the tumor induces an im- Tregs due to the selectivity gain and different expression patterns of munosuppressive tumor immune microenvironment by upregulating PD-1 and LAG-3 on these two T cell types, 2) reduced internalization, several immunoregulatory cytokines and chemokines. Interestingly, 3) Fc silent-mediated resistance to drug-shaving by macrophages. we re-analyzed transcriptomic data from 28 metastatic melanoma tu- Conclusions mors prior to anti-PD-1 pembrolizumab therapy [2] to validate our These characteristics translated in a significant increase in 1) in vitro target and related signaling pathways. We found a stepwise in- T cell effector functions even in the presence of Tregs, and 2) in vivo creased expression of our target, its receptor and the identified tar- tumor control/eradication in mouse models compared to combin- gets of the pathway from complete responders (low expression) to ation of monospecific anti-PD1 and anti-LAG3 antibodies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 155 of 272 P288 P289 CCR5+CTLA4+ Treg cell subset characterizes renal tumors (RCC) Macrophages modulate patient response to immune checkpoint immunosuppressed by PD1 blockade inhibition in a novel lung tumour explant model 1 1 1 1 1 Agathe Dubuisson , Agathe Dubuisson , Charles Bayard , Sebastien Lauren Evans, BSc, MSc , Kate Milward, DPhil , Richard Attanoos, BSc, MB 1 1 1 1 2 1 1 Lofek , Nicolas Voisin , Séverine Mouraud , Delphine Bredel , Sandrine BS, FRCPath , Aled Clayton, PhD , Rachel Errington, PhD , Zsuzsanna 1 1 1 2 1 Susini , Mathieu Rouanne, MD , Hervé Beaumert , Bastien Parier , Tabi, PhD 1 1 1 2 Aurélien Marabelle, MD PhD , Laurence Zitvogel, MD, PhD , Mélodie Cardiff University, Cardiff, United Kingdom; University Hospital Wales, 1 1 Bonvalet , Mélodie Bonvalet Cardiff, United Kingdom 1 2 Gustave Rousy, Villejuif, France; APHP, Le Kremlin Bicetre, France Correspondence: Lauren Evans (Evansl57@cardiff.ac.uk) Correspondence: Mélodie Bonvalet (bonvaletmelodie@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P289 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P288 Background Background The tumour microenvironment (TME) consists of a dynamic interplay A small subset of patients undergoing PD1 blockade therapy develop between the tumour and stroma. Various stroma-resident and a hyper-progressive disease (HPD), corresponding to an acceleration tumour-infiltrating immune cells are associated with pro-tumour ac- of tumor growth rate [1]. However, the mechanisms underlying HPD tivity. Whilst immunotherapies may trigger anti-tumour immune re- are unknown, and the identification of predictive biomarkers remains sponses, anti-inflammatory M2-like tumour-associated macrophages an unmet clinical need. We aimed at better understanding the mech- (TAMs) within the TME present an obstacle for effective treatment anism of hyper-progression following nivolumab treatment by exam- [1]. Pre-clinical research has predominantly used mouse models to ining the tumor immune infiltrate of fresh RCC using an ex vivo represent the in vivo TME. Unfortunately, such models do not faith- model system. fully replicate the human immune system therefore, providing an in- Methods adequate measure of immunotherapy response. Human tumour- Fifteen fresh primary RCC were processed according to methods pre- derived explants maintain the original 3D tumour architecture and viously reported [2] and stimulated for 3 days with nivolumab. At combination of multiple cell types. Therefore, we established an baseline, we monitored the phenotype of the immune infiltrate by ex vivo tumour explant model of non-small cell lung cancer (NSCLC) flow cytometry. After 3 days of culture, we measured 26 soluble fac- incorporating TAMs, to determine their role in immunotherapeutic tors which were released into the supernatant. We then examined response. the relationship between baseline phenotype and functional immune Methods reactivity to nivolumab, considering variations of at least 2 independ- Tumour explants (ca. 1mm³) were generated from fresh tumour tis- ent soluble factors in the range of sue. Autologous CD14 peripheral blood mononuclear cells (PBMCs) Results were added to explants for 48h followed by flow cytometry pheno- Nivolumab induced a strong inhibition of soluble factor release in 5 out typing using a 7-marker macrophage panel (CD14; CD64; PDL-1; of 15 RCC. The decreased soluble factors were IL1RA (3 out of 5 tumors CD163; CD206; CD23; CD200R). The functional contribution of macro- (3/5)), IFNg, CXCL10, IL10, G-CSF, GM-CSF (2/5), IL-4, IL-5, IL-6, IL-8, IL-9, phages to explant-mediated immunosuppression was assessed + + CCL4, CCL5, PDGFbb (1/5). In these hypo-sensitive tumors (HS), nivolu- through measuring IFNγ/TNFα production from CD4 /CD8 T cells by mab induced a significant decrease of IL-4, IL-8, G-CSF (p<0.05). flow cytometry. T cells, in the presence of explants, were stimulated Conclusions with a viral peptide pool and incubated for 6 days ±250μg/mL Atezo- The TME of HS renal tumors that are immunosuppressed by nivolu- lizumab, prior to intracellular cytokine staining. Culture supernatants mab display high levels of the CCR5 ligands and CCR5+ CTLA4+ Treg were collected and the Th1/Th2 cytokine profile determined using a cells, compared to NHS tumors. As reported by Kamada et al. [3], dis- LEGENDplex bead-based immunoassay (BioLegend). Transcriptomic tinct Treg subset might be involved in the deleterious effects of nivo- analysis was performed on patient tumour tissue and peripheral lumab observed in HPD. Prospective clinical studies should blood using the NanoString PanCancer IO360 gene expression panel. determine if nivolumab-associated HPD is caused by a pre-existing Results pool of highly immunosuppressive CCR5+ CTLA4+ Treg cells. Tumour explants significantly promoted M2-like macrophage differ- entiation and suppressed T cell activity ex vivo. The PDL-1 inhibitor, Acknowledgements Atezolizumab significantly improved T cell function in some patients This work was supported by Bristol-Myers Squibb. and reduced explant-mediated immunosuppression, particularly in the presence of macrophages. This suggests that response or resist- References ance to anti-PDL-1 therapies may be partially TAM dependent. The 1. Champiat S, Dercle L, Ammari S, Massard C, Hollebecque A, Postel-Vinay differential effect of Atezolizumab observed in the presence of S, Chaput N, Eggermont A, Marabelle A, Soria JC, Ferté C. Hyperprogres- patient-derived tumour explants indicates the potential of this model sive Disease Is a New Pattern of Progression in Cancer Patients Treated in predicting immunotherapy responses. Ongoing transcriptomic by Anti-PD-1/PD-L1. Clin Cancer Res. 2017;23(8):1920-1928. analysis of lung cancer tissue aims to reveal associations between 2. Jacquelot N, Roberti MP, Enot DP, Rusakiewicz S, Ternès N, Jegou S, the TME and T cell function. Variations in the cytokine secretion pro- Woods DM, Sodré AL, Hansen M, Meirow Y, Sade-Feldman M, Burra A, file of tumour explant co-cultures are also being studied under differ- Kwek SS, Flament C, Messaoudene M, Duong CPM, Chen L, Kwon BS, An- ent experimental conditions. derson AC, Kuchroo VK, Weide B, Aubin F, Borg C, Dalle S, Beatrix O, Conclusions Ayyoub M, Balme B, Tomasic G, Di Giacomo AM, Maio M, Schadendorf D, Using the tumour explant model, we found that alleviation of Melero I, Dréno B, Khammari A, Dummer R, Levesque M, Koguchi Y, Fong explant-mediated immunosuppression by Atezolizumab may be L, Lotem M, Baniyash M, Schmidt H, Svane IM, Kroemer G, Marabelle A, macrophage dependent. This model could be used to predict patient Michiels S, Cavalcanti A, Smyth MJ, Weber JS, Eggermont AM, Zitvogel L. response to anti-PDL-1 immunotherapy, and explore combination Predictors of responses to immune checkpoint blockade in advanced therapies. Ongoing research aims to improve immunotherapy re- melanoma. Nat Commun. 2017 ;8(1):592. sponses through reprogramming TAMs using immune-modifying 3. Kamada T, Togashi Y, Tay C, Ha D, Sasaki A, Nakamura Y, Sato E, Fukuoka drugs. S, Tada Y, Tanaka A, Morikawa H, Kawazoe A, Kinoshita T, Shitara K, Sakaguchi S, Nishikawa H. PD-1+ regulatory T cells amplified by PD-1 Acknowledgements blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A. Study supported by research funds from Cardiff University, School of 2019 May 14;116(20):9999-10008. Medicine. We would like to thank the Wales Cancer Bank and Lung Ethics Approval Multidisciplinary Research Group for their ongoing involvement in patient The study was approved by the Comité de Protection des Personnes Ile de recruitment and sample acquisition, and to the patients who donated their France III Ethics Board, approval number 2016-A00732-49. samples for our research. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 156 of 272 Reference P291 1. Cassetta L, Kitamura T. Targeting Tumor-Associated Macrophages as a Po- Driving T cell dysfunction in vitro for rational immunotherapy tential Strategy to Enhance the Response to Immune Checkpoint Inhibi- design tors. 2018;6. Matthew Hancock, Cailin Joyce, PhD, Thomas Horn, Simarjot Pabla, Ethics Approval Benjamin Duckless, Andrew Basinski, Dhan Chand, PhD, Jeremy Waight, Ethical approval for this project was provided through the Wales Cancer PhD, Mariana Manrique, PhD, Nicholas Wilson, Alex Duncan, PhD, Bank (WCB). The WCB has ethics approval as a Research Tissue Bank from Jennifer Buell, PhD, David Savitsky, PhD, Lukasz Swiech, John Castle, PhD the Wales Research Ethics Committee 3, reference 16/WA/0256 (previous Agenus Inc, Lexington, MA, United States approval references – 06/MRE09/26 and 11/WA/0279). This approval Correspondence: John Castle (john.castle@agenusbio.com) covers the collection of samples (including consent), processing and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P291 storing samples across multiple collection and storage sites. The approval also allows release of anonymised samples to researchers carrying out Background cancer related activity, following successful application approval from the Immune checkpoint blockade (ICB) elicits durable responses in some WCB External Review panel (project 17/016). Sample collection of blood cancer patients, but novel targets and combination approaches are and tissue, from non-small cell lung cancer patients undergoing surgical needed to address resistance and broaden clinical benefit. Here, we resection, was performed at the University Hospital of Wales, Cardiff. present a system for characterizing mechanisms of T cell dysfunction in the tumor microenvironment (TME) and apply the system to un- cover novel approaches to ICB resistance. P290 Methods Interferon gamma production by regulatory T cells is necessary for We developed a long-term human co-culture system comprised of response to cancer immunotherapy primary T cells and cancer cells that enables controlled differentiation Angela Gocher, PhD, Dario Vignali, PhD, Creg Workman, PhD, Sanah of naïve T cells to effector, memory and dysfunctional states. We lon- Handu gitudinally monitored T cell effector functions, protein and RNA ex- University of Pittsburgh, Pittsburgh, PA, United States pression across states and single cells. Finally, we challenged the Correspondence: Dario Vignali (dvignali@pitt.edu) system with PD1 antibody to uncover biomarkers and mechanisms Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P290 of therapeutic resistance. Results Background T cells in our system become activated and then gradually progress Our lab has shown that murine regulatory T cells (Tregs) produce the to a terminally dysfunctional state driven by multiple cancer antigen cytokine interferon gamma (IFNγ) during anti-PD1 therapy and that exposures. T cell cytotoxicity is maintained over several antigen ex- Treg response to IFNγ is necessary for tumor eradication by anti-PD1 posures before sharply decreasing whereas cytokine secretion begins therapy [1]. However, the role of this cellular source of IFNγ in the to decrease with only one prior antigen exposure. The expression of tumor microenvironment (TME) and how Treg derived IFNγ dictates known T cell regulators and novel factors is altered over the time response to other cancer immunotherapies has yet to be studied. In course, with known factors reflecting previous observations in vivo. addition, it has been shown that IL-12-induced production of IFNγ is Anti-PD1 prolongs cytotoxic capacity but T cells eventually fail to re- necessary for response to anti-PD1 therapy [2]. However, it has yet to spond. Single cell mapping in the presence of anti-PD1 reveals an ex- be determined whether Tregs are a key responder to IL-12 during panded population of T cells that co-express PD1, TIGIT and cancer immunotherapy. Thus, elucidating the interplay of IL12, IFNγ activation markers. Consistent with this, the combination of PD1 and and Tregs in the TME is essential for maximizing efficacy and minim- TIGIT blockade enhances cytotoxicity relative to monotherapies. izing the clinical resistance to current cancer immunotherapies. Conclusions Methods These findings demonstrate the utility of our system to deeply inter- Our lab has generated two novel murine models that allow for Treg- rogate therapeutic response and resistance. Moreover, its scalability restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/ and modularity enable genome-scale screening to discover novel tar- LFoxp3Cre-YFP). These murine models were validated and used to gets that could enhance antitumor activity of both natural and adop- assess the contribution of Treg-derived IFNγ on the growth of syn- tively transferred T cells. geneic MC38 (colon adenocarcinoma) and B16 (melanoma) tumors and response to a various cancer immunotherapies (checkpoint blockade, vaccination and tumor-specific antibodies). Flow cytometry P292 was conducted on tumor and control non-draining lymph nodes to Tissue site of tumor growth dictates anti-tumor immunity and phenotype the immune infiltrate in response to immunotherapy. response to checkpoint blockade Results Brendan Horton, PhD, Stefani Spranger, PhD Mice with Tregs either unable to generate IFNγ (IfngL/LFoxp3Cre- Massachusetts Institute of Technology,, Cambridge, MA, United States YFP) or respond to the IFNγ-inducing cytokine IL-12 (Il12rb2L/ Correspondence: Stefani Spranger (spranger@mit.edu) LFoxp3Cre-YFP) were unable to eradicate tumors in response to im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P292 munotherapy. In addition, preliminary results suggest that Il12rb2- deficient Tregs may be more suppressive as indicated by an increase Background in tumor growth compared to control. An emerging area of clinical importance is differential responsiveness Conclusions to checkpoint blockade immunotherapy across different tissues sites These data suggest that IFNγ producing Tregs are necessary to shift of tumor growth, leading to partial responses and ultimately cancer- the balance from an immunosuppressive TME to one that favors the related deaths [1, 2]. However, tissue-specific anti-tumor immune re- reinvigoration of the anti-tumor response generated by current can- sponses are not well understood. We used mouse models to deter- cer immunotherapeutic agents. Future studies will determine mine how anti-tumor immune responses differ across tissue sites and whether the capacity of Tregs to produce IFNγ can predict response how this relates to immunotherapy efficacy. to immunotherapy. Methods Mice were inoculated subcutaneously or intravenously with syngen- References eic KP lung cancer cells, then treated with anti-CTLA-4 and anti-PD- 1. Overacre-Delgoffe A, Chikia M. Interferon-γ Drives Treg Fragility to Pro- L1 antibodies, and analyzed for tumor burden. Response to check- mote Anti-tumor Immunity. Cell. 2017; 169: 1130-1141. point blockade was correlated with tumor-infiltrating T cells (TIL) and 2. Garris C, Arlauckas S. Successful Anti-PD1 Cancer Immunotherapy Re- systemic immune responses using flow cytometry and immuno- quires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL- histochemistry. Mice were also tested for their ability to generate sys- 12. Immunity. 2018; 49: 1148-1161. temic and protective immunity against a second tumor challenge. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 157 of 272 Results ~500 cancer causing genes) as the number of nonsynonymous coding Comparing lung and subcutaneous tumors, we observed striking dif- mutations per megabase and the optimal cut-off for TMBhigh was de- ferences within the TIL compartment. Lung tumors contained more termined using the 98th percentile of newly-diagnosed tumors and TIL with higher expression of PD-1. Despite this, lung tumors did not segmented linear regression analysis. Patients were stratified by histo- respond to anti-CTLA-4 + anti-PD-L1, while subcutaneous tumors did. molecular subtype, IDH mutation, 1p/19q co-deletion, TMB, and ICB TIL expanded in subcutaneous tumors after immunotherapy, while treatment under clinical trials and expanded access. Overall (OS) and lung tumors had no change in the number of TIL. We further tested progression-free (PFS) survival were estimated by the Kaplan-Meier for concomitant immunity and found that subcutaneous KP tumors method and evaluated using multivariable Cox regression. generated an immune response that could protect against a second Results tumor. However, KP lung tumors failed to generate a protective sys- We identified 1,223 HGG patients with genomics, including 64 temic immune response. These results were reproduced using a pan- hypermutated tumors. Overall the cohort consisted of 79% newly- creatic cancer cell line, indicating that tumors growing in the lung diagnosed and 21% recurrent gliomas and subclass distribution generated weaker systemic immune responses than subcutaneous was 75% IDH-wildtype glioblastomas/anaplastic astrocytomas,16% tumors. Protective concomitant immunity required CD8+ T cells, and IDH-mutant glioblastomas/anaplastic astrocytomas, and 6% 1p/19- to a lesser extent CD4+ T cells, but not NK cells. Therefore, we inves- codeleted anaplastic oligodendrogliomas. Hypermutated HGG tigated differences in the generation of systemic, antigen-specific were predominantly seen in the setting of recurrence (18% of re- CD8+ T cell responses between subcutaneous and lung KP tumors. current HGGs vs 2% of newly diagnosed, 5% overall incidence). Using KP.SIY tumors, elispot assays found that lung tumors gener- Comparisons of biomarkers and genomics in hypermutated versus ated fewer antigen-specific T cells in the spleen. Transferred 2C T non-hypermutated HGGs showed distinct characteristics in glioma cells proliferated less in the spleens of lung tumor-bearing hosts than subtypes with implications for differential mechanisms of in hosts with subcutaneous tumors, even though proliferation in TMBhigh acquisition in HGGs with the most important biomarkers draining lymph nodes was similar. Consistently, KP.SIY lung tumors of differential hypermutation risk being MGMT promoter methyla- generated weaker concomitant immunity than subcutaneous KP.SIY tion (22% of methylated vs. 6% of unmethylated cases, p=0.02) tumors. and IDH1/2 mutation (25% of IDH-mutant vs. 12% of IDH- Conclusions wildtype HGGs, p=0.007). The tissue site of tumor growth determines the number and pheno- 129 (11%) of HGGs (mostly IDH-wildtype GBMs/AAs) received PD-1/ type of TIL, the generation of systemic immunity, and the response PD-L1 ICB therapy, including 13% (n=8) of hypermutated HGGs. to checkpoint blockade. Response to immunotherapy correlates not Immunohistopathologic assessment of tumor responses and immune with the number of TIL, or their phenotype, but with the ability of infiltrates was conducted and correlated with the genomic profiling. the host to mount a systemic anti-tumor CD8+ T cell response. Because the majority of ICB-treated cases were IDH-wildtype HGGs, this subset was further evaluated in analyses which were risk- References adjusted by age, sex, histomolecular subgroup, MGMT promoter 1. Khoja, L., et al., Patterns of response to anti-PD-1 treatment: an explora- methylation, and prior therapy (i.e. RT/TMZ/CCNU/bevacizumab). Cor- tory comparison of four radiological response criteria and associations relations between TMB, molecular genotypes, and clinical response with overall survival in metastatic melanoma patients. Br J Cancer, 2016. to ICB in HGGs will be presented. 115(10): p. 1186-1192. Conclusions 2. Lee, J.H.J., et al., Metastasis-specific patterns of response and progression Our study represents the largest set of genomically characterized gli- with anti-PD-1 treatment in metastatic melanoma. Pigment Cell Melan- omas and gliomas with hypermutation with data related to re- oma Res, 2018. 31(3): p. 404-410. sponses to PD-1/PD-L1 ICB therapy. Our data support systematic TMB Ethics Approval characterization and clinical molecular genotyping to assist therapy- The animal work in these experiments was reviewed and approved by the decision making in gliomas and provide foundational data essential MIT Committee on Animal Care. The approved animal protocol number to future clinical trial designs of immunotherapeutics. for this work is 0117-012-20. Ethics Approval This study was approved by the Partners HealthCare (#2015P002352) and Dana-Farber Cancer Institute (10-417) institutional review boards. P293 Tumor mutational burden (TMB) and genomic predictors of clinical outcomes to PD-1/PD-L1 checkpoint blockade in high-grade P294 gliomas Long survival associated with receipt of anti-CTLA-4 in patients 1 1 1 1 Bryan Iorgulescu, MD , Mehdi Touat , Yvonne Li , Liam Spurr , Gareth with metastatic melanoma from acral lentiginous, mucosal and 1 1 1 1 Grant , William Pisano , Mary Jane Lim-Fat , Eudocia Lee , Lakshmi uveal primary tumors 1 2 2 3 1 Nayak , E Chiocca , Raymond Huang , Andrew Cherniack , Patrick Wen , Nicholas Klemen, MD, Melinda Wang, BS, Kelly Olino, MD, James Clune, 1 4 1 1 Ahmed Idbaih , Franck Bielle , David Reardon, MD , Keith Ligon MD, Stephan Ariyan, MD, Charles Cha, MD, Sarah Weiss, MD, Harriet 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Kluger, MD, Mario Sznol, MD Women's Hospital, Boston, MA, United States; Broad Institute, Yale University School of Medicine, New York, NY, United States Cambridge, MA, United States; Sorbonne Université, Paris (UPMC), Paris, Correspondence: Mario Sznol (mario.sznol@yale.edu) France Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P294 Correspondence: David Reardon (david_reardon@dfci.harvard.edu); Keith Ligon (Keith_Ligon@dfci.harvard.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P293 Metastatic melanoma from acral lentiginous, mucosal and uveal pri- mary tumors responds poorly to checkpoint inhibitors (CPI), poten- Background tially due to a low burden of immunogenic neoantigens [1-7]. Long- High tumor mutational burden (TMB) is an emerging biomarker for term outcomes of these patients after treatment with CPI have not predicting response to PD-1/PD-L1 immune checkpoint blockade been established. (ICB) in a spectrum of cancer patients, however, its clinical value and Methods therapeutic implications in high-grade gliomas (HGG) is not yet We performed a retrospective review of a single institutional experi- established. ence using antibodies against CTLA-4, PD-1 and PD-L1 for patients Methods with stage IV melanoma. Primary tumor histology was categorized as We retrospectively reviewed all HGGs at our institutions from 2013- cutaneous, acral, mucosal or uveal. Patients with unknown primary 2018 that underwent genomic characterization. TMB was determined were excluded. We measured overall survival (OS) after the first dose from clinical targeted exome next-generation sequencing (DFCI-Profile, of CPI using the Kaplan-Meier method. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 158 of 272 Results We treated 428 patients with metastatic melanoma from 2007-2019. Pri- mary tumors were cutaneous in 283 (66%) patients, unknown in 55 (13%), acralin22(5%), mucosalin38(9%)and uvealin30(7%)(Table 1).Mucosal patients had a slight female preponderance. The proportion staged M1c was higher in mucosal and uveal patients. Patients with cutaneous primary tumors had median OS after CPI of 45 months, compared with 17 months for acral (P = 0.047), 18 months for mucosal (P = 0.003) and 12 months for uveal (P < 0.001)(Figure 1). Five-year survival for cutaneous, acral, mucosal and uveal patients was 46%, 34%, 21% and 22% respectively. Next we combined the patients with acral, mucosal and uveal melan- oma (n = 90) and performed survival analysis stratified by the first type of CPI treatment. Median OS after anti-PD-1 or anti-PD-L1 was 9 months, compared with 18 months after anti-CTLA-4 (P = 0.010) and 20 months after combination therapy with anti-CTLA-4 plus anti-PD-1 (P = 0.003)(Figure 2). While 21 of 31 (68%) patients treated with anti- CTLA-4 later were treated with anti-PD-1, only 5 of 18 (28%) patients Fig. 1 (abstract P294). Overall survival stratified by histology treated with anti-PD-1 later received anti-CTLA-4 (P = 0.02). There were 21 patients who survived at least three years after CPI, all of whom were treated with anti-CTLA-4 with or without anti-PD-1. Of the 10 patients with actual five-year survival, 3 had complete re- sponses while the other 7 all required local and/or regional therapies to control progressive disease (Table 2). Conclusions Long survival in patients with metastatic melanoma from acral, mu- cosal and uveal primary tumors was uniformly associated with re- ceipt of anti-CTLA-4. Our experience shows that while acral, mucosal and uveal melanomas have worse outcomes than cutaneous melan- oma, with an aggressive multidiscliplinary approach five-year survival is still possible for 25-32% of these patients. References 1. Luke JJ, Callahan MK, Postow MA, Romano E, Ramaiya N, Bluth M, et al. Clinical activity of ipilimumab for metastatic uveal melanoma. Cancer. 2013;119:3687–95. 2. Algazi AP, Tsai KK, Shoushtari AN, Munhoz RR, Eroglu Z, Piulats JM, et al. Clinical outcomes in metastatic uveal melanoma treated with PD-1 and PD-L1 antibodies. Cancer. 2016;122:3344–53. 3. D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy Fig. 2 (abstract P294). Overall survival stratified by treatment and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis. JCO. 2017;35:226–35. 4. Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for Patients With Advanced Mucosal Melanoma. The Table 2 (abstract P294). Characteristics of five-year survivors Oncologist. 2013;18:726–32. 5. Bello DM, Chou JF, Panageas KS, Brady MS, Coit DG, Carvajal RD, et al. Prognosis of acral melanoma: a series of 281 patients. Ann Surg Oncol. Springer US; 2013;20:3618–25. 6. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. Nature Publishing Group; 2012;44:1006–14. 7. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al. Mutational heterogeneity in cancer and the search for new cancer- associated genes. Nature. 2013;499:214–8. Ethics Approval The study was approved by the Yale-New Haven Hospital Institutional Review Board, approval number 2000021595. Table 1 (abstract P294). Demographics and first treatment P295 The impact of metastatic sites on checkpoint inhibitor outcomes in patients with cutaneous and unknown primary melanoma Penina Krieger, MPhil, Francisco Sanchez-Vega, Nikolaus Schultz, Alexander Shoushtari, MD Memorial Sloan Kettering Cancer Center, Bronx, NY, United States Correspondence: Penina Krieger (peninakrieger@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P295 Background Clinical biomarkers of response to Programmed Death-1 (PD-1) based therapies are sorely needed in melanoma. The American Joint Committee on Cancer (AJCC) 8th Edition Staging system is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 159 of 272 derived from the pre-checkpoint inhibitor era and divides patients into locally advanced or soft tissue disease (M0/M1a), lung metastases (M1b), other visceral metastases (M1c), and brain metastases (M1d). It is unclear whether this prognostic classification remains valid in the mod- ern therapeutic era. The number of metastatic sites influences out- comes with BRAF-MEK therapy [1], but its prognostic importance with PD-1-based therapy is unknown. Methods All patients with melanoma who had prospective tumor molecu- lar profiling at a single center (MSK-IMPACT) and subsequently received frontline PD-1 blockade as single agent (Nivolumab or Pembrolizumab) or combination therapy with Ipilimumab (combo) were included. Demographic and clinical data were collected, in- cluding metastatic sites present at time of PD-1 therapy. Overall survival (OS) and time to treatment failure (TTF) were calculated from the onset of PD-1 therapy using Kaplan-Meier methodology. The impact of specific metastatic sites on TTF and OS was determined using Cox Proportional Hazards Regression Models. Tumor mutational burden (TMB) was calculated as previously described [2]. Results Fig. 1 (abstract P295). See text for description 309 patients received frontline PD-1 monotherapy (n=179) or PD-1 combo (n=130). Overall survival varied by AJCC stage (p<0.0001, Fig- ure 1); M1b and M0/M1a groups had similar OS (median=NR, p = 0.27) followed by M1c (median=39 mo) and M1d (median=28 mo). Patients with 3+ metastatic sites had worse median OS than those with 0-2 metastatic sites (45 vs 39 mo, p<0.0001, Figure 2). Among patients with M1c disease, those with bone or liver me- tastases had worse median OS than those without them (39 vs NR mo, Liver HR=2.4, p=0.036, Bone HR=2.6, p=0.021, Figure 3). Among patients with M1d disease, those with liver metastases had shorter TTF than those without when treated with PD-1 com- bination therapy (HR=2.4, p=0.044). There was no significant dif- ference in median TMB by AJCC M stage at treatment or by site of metastasis. Conclusions AJCC 8th edition M1b disease has a similar prognosis to M0/M1a dis- ease in the era of PD-1-based therapy. The presence of liver or bone metastases at time of PD-1 therapy portends worse OS and TTF even within M1c and M1d disease. Patients with 0-2 metastatic sites live longer than those with 3+ sites. These clinical observations are not explained by differences in TMB. Future trials of PD-1 blockade in ad- vanced melanoma should account for these differences. Fig. 2 (abstract P295). See text for description Acknowledgements Research reported in this abstract was supported by the National Cancer Institute of the National Institutes of Health under Award Number R25CA020449 and by National Cancer Institute Cancer Center Core Grant P30CA008748, Kravis Center for Molecular Oncology. The content is the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1. Long G, Grob J, Nathan P, Ribas A, Robert C, Schadendorf D, Lane S, Mak C, Legenne P, Flaherty K, Davies M. Factors predictive of response, disease progression, and overall survival after dabrafenib and trametinib combination treatment: a pooled analysis of individual patient data from randomised trials. Lancet Oncol. 2016; 17:1743–54. 2. Samstein RM, Lee CH, Shoushtari AN, Hellmann MD, Shen R, Janjigian YY, Barron DA, Zehir A, Jordan EJ, Omuro A, Kaley TJ, Kendall SM, Motzer RJ, Hakimi AA, Voss MH, Russo P, Rosenberg J, Iyer G, Bochner BH, Bajorin DF, Al-Ahmadie HA, Chaft JE, Rudin CM, Riely GJ, Baxi S, Ho AL, Wong RJ, Pfister DG, Wolchok JD, Barker CA, Gutin PH, Brennan CW, Tabar V, Mel- linghoff IK, DeAngelis LM, Ariyan CE, Lee N, Tap WD, Gounder MM, D’An- gelo SP, Saltz L, Stadler ZK, Scher HI, Baselga J, Razavi P, Klebanoff CA, Yaeger R, Segal NH, Ku GY, DeMatteo RP, Ladanyi M, Rizvi NA, Berger MF, Riaz N, Solit DB, Chan TA, Morris LGT. Tumor mutational load predicts sur- vival after immunotherapy across multiple cancer types. Nat Genet. 2019; 51:202–206 Ethics Approval The study was approved by Memorial Sloan Kettering's Ethics Board, Fig. 3 (abstract P295). See text for description approval number 18-244. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 160 of 272 P296 BMI, irAE and gene expression signatures predict resistance and survival to immune-checkpoint inhibition in renal cell carcinoma 1 1 1 2 Brian Labadie, MD , Ping Liu, PhD , Riyue Bao, PhD , Michael Crist, MD , 3 4 5 Ricardo Fernandes, MD , Laura Ferreira Freire , Scott Graupner , Andrew 5 4 3 6 Poklepovic, MD , Ignacio Duran , Saman Maleki Vareki , Arjun Balar, MD , Jason Luke, MD, FACP 1 2 University of Chicago, Chicago, IL, United States; NYU School of Medicine, New York, NY, United States; Western University, London, 4 5 Ontario, Canada; HUMV, Santander, Spain; Virginia Commonwealth University, Richmond, VA, United States; New York University, New York, NY, United States; University of Pittsburgh, Pittsburgh, PA, United States Correspondence: Jason Luke (lukejj@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P296 Background Treatment with immune-checkpoint inhibition (ICI) has changed the treatment paradigm in ccRCC however many do not respond to these treatments and no reliable molecular biomarker exists to pre- dict response to ICI in individual patients. Clinical variables may cor- relate with lack of response to treatment (primary resistance) or clinical benefit. Methods Via an international multi-institution collaboration, clinical charac- Fig. 1 (abstract P296). Kaplan-Meier curves depicting teristics from patients with ccRCC treated with anti-PD-1/L1 ther- survival outcomes apy were collected. Patients with primary resistance (defined as progression on initial computed tomography scan) were com- pared to patients with clinical benefit. Multivariable analysis was performed to identify factors associated with improved time to progression or death. The Cancer Genome Atlas Kidney Renal P297 Clear Cell Carcinoma cohort (TCGA-ccRCC) was examined for the A semi-mechanistic platform model to capture individual animal correlation between gene expression patterns, clinical factors, and responses to checkpoint inhibitors in a syngeneic mouse model 1 2 1 2 2 survival outcomes. Lin Lin, PhD , Alison Betts , Carissa Young , Wendy Qiao , Jatin Narula , 2 2 2 2 Results Peter O’Brien , Derek Bartlett , Andrea Hooper , Jason Williams , John 1 1 1 1 Of 90 patients, 38 (42.2%) had primary resistance and 52 (57.8%) Burke , Joshua Apgar , Lore Gruenbaum, PhD , Fei Hua, PhD 1 2 had clinical benefit. Compared with the cohort of patients with Applied BioMath, LLC, Concord, MA, United States; Pfizer Worldwide initial benefit, primary resistance was more likely to occur in pa- R&D, Cambridge, MA, United States tients with worse ECOG performance status (p=0.03), earlier stage Correspondence: Fei Hua (fei.hua@appliedbiomath.com) at diagnosis (p=0.04), no prior nephrectomy (p=0.04) and no Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P297 immune-related adverse events (irAE) (p=0.02). In the entire co- hort, improved overall survival was significantly correlated with Background lower International Metastatic RCC Database Consortium risk Syngeneic mouse models have been widely employed in preclinical score (p=0.02) and lower neutrophil:lymphocyte ratio (p=0.04). In discovery of checkpoint inhibitors as they enable study of drug im- patients with clinical benefit, improved progression free survival pact on the intact immune system. However, the interpretation of was significantly associated with increased BMI (p=0.007) and irAE such studies remains challenging partly due to the large variability in occurrence (p=0.02) while improved overall survival was signifi- individual animal responses to drug treatment. cantly correlated with overweight BMI (BMI 25-30)(p=0.03) and no Methods brain metastasis (p=0.005). In the TCGA-ccRCC analysis, higher ex- In this work, we describe the generation of a model platform that pression of angiogenesis gene signature was found to be corre- captures essential aspects of the pharmacokinetics, cellular and lated with lower neoplasm histologic grade and better survival (p tumor growth effects of murine surrogates of two checkpoint thera- < 0.05). Angiogenesis and T cell-inflamed gene signatures were peutic antibodies, anti-PD1 and anti-CTLA4, in the CT26 syngeneic inversely correlated in tumors of high T cell-inflamed gene ex- tumor model. The model describes individual animal responses with pression (p=0.008), a pattern not observed in non-T cell-inflamed regard to drug exposure, key intra-tumoral cell kinetics and tumor tumors. (Figure 1) volume changes and provides biologically plausible explanations for Conclusions the observed differences between good and poor responders to Identification of BMI, performance status and prior nephrectomy as treatment with anti-PD1 or anti-CTLA4. predictors of response to PD1/L1 in ccRCC may help inform treat- Results ment selection. The inverse association of angiogenesis gene signa- We used the model to predict the antibody dose-response relation- tures with ccRCC histologic grade highlight opportunities for ships for individual animals and to identify dose thresholds above adjuvant combination VEGFR2 TKI and ICI. which complete tumor elimination can be achieved in good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 161 of 272 responders. In contrast, our models predict that poor responders demonstrated higher expression of angiogenesis-related profiles and would not achieve complete response even with much higher drug increased CD31 expression. Furthermore, Pbrm1 deficient Renca sub- doses. The parameters in our model that impact the response in cutaneous tumors in mice demonstrated longer latency but more re- poor responders are not drug-related. This finding suggests that sistance to programmed death-1 (PD-1) blockade. Analysis of the immune-cell related barriers have to be crossed in order to achieve a IMmotion150 cohort revealed that ccRCC patients with PBRM1 muta- therapeutic response in these animals - possibly via combination tions were associated with decreased immune infiltrates and a re- therapy. duced response rate to atezolizumab monotherapy or combination In addition, we identified the net tumor cell doubling rate, one po- therapy with bevacizumab. tential parameter that contributes to individual variability in response Conclusions to treatment, as the most sensitive biological parameter determining Pbrm1 and PBRM1 loss reduced IFN gamma-STAT1 signaling. Pbrm1 tumor volume changes upon treatment with anti-PD1 or anti-CTLA4. inactivation was associated with a less immunogenic tumor micro- Measuring individual animal tumor cell growth characteristics may environment in animal and human tissue samples. Response to PD- help with the experimental design and qualification of animals for L1 blockade is reduced in patients with PBRM1 mutations in the studies (in addition to absolute tumor volume), and thereby reduce IMmotion 150 study. This study forms a framework for future mech- inter-animal variability and enhance the interpretability of study re- anistic and clinical studies on the interaction between genomic fea- sults, especially in combination with a model such as the one pre- tures in RCC and response to immunotherapy. sented here. Conclusions Acknowledgements This model platform can be adapted to capture and compare check- We acknowledge the TCGA Research network. This work was supported by point drug effects in different syngeneic tumor models. Moreover, it funding from DOD grant W81XWH-17.1.0307, DOD grant CA160728P1, UT can be expanded to add additional drug mechanisms and can serve MD Anderson Cancer Center CCSG grant 5 P30 CA016672 (Biostatistics as a tool to inform the experimental design of mouse studies. shared resource group) and the Adopt-a-Scientist Foundation. Ethics Approval The animal protocols (2018-0376) were approved by Institutional Animal P298 Care and Use Committee (IACUC) of The Health Science Center, Texas A&M PBRM1 loss defines distinct tumor phenotype associated with University. Human subject protocol (2007-0511) was approved by immunotherapy resistance in renal cell carcinoma Institutional Research Board at M.D. Anderson Cancer Center. 1 1 1 1 Xiande Liu, PhD , Wen Kong , Christine Peterson , Daniel McGrail , Anh 1 1 1 1 Hoang , Xuesong Zhang , Truong Lam , Patrick Pilie, MD , Haifeng Zhu, 1 2 2 3 PhD , Kathryn Beckermann , Scott Haake , Sevinj Isgandrova , Margarita P299 3 1 2 Martinez-Moczygemba , Nidhi Sahni , W. Kimryn Rathmell , Eric Jonasch, Neuropilin-1 is a T cell memory checkpoint limiting long-term MD tumor immunity 1 2 1 2 3 MD Anderson Cancer Center, Houston, TX, United States; Vanderbilt Chang Liu, PhD , Ashwin Somasundaram, MD , Sasikanth Manne , 3 1 1 1 University Medical Center, Nashville, TN, United States; Texas A&M Angela Gocher, PhD , Andrea Szymczak-workman , Kate Vignali , Daniel 2 1 1 Health Science Center, Houston, TX, United States Normolle, PhD , Robert Ferris, MD, PhD , Tullia Bruno, PhD , E. John 3 1 1 Correspondence: Eric Jonasch (ejonasch@mdanderson.org) Wherry, PhD , Creg Workman, PhD , Dario Vignali, PhD 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P298 University of Pittsburgh, Pittsburgh, PA, United States; UPMC Hillman Cancer Center, Pittsburgh, PA, United States; University of Pennsylvania, Background Philadelphia, PA, United States Polybromo-1 (PBRM1), encoding a mammalian specific subunit of the Correspondence: Dario Vignali (dvignali@pitt.edu) switch/sucrose non fermenting (SWI/SNF) chromatin remodeling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P299 complex, is the second most frequently mutated gene in clear cell renal cell carcinoma (ccRCC). Data thus far on the effect of PBRM1 Background loss on immune responsiveness are inconsistent. The impact of Robust CD8+ T cell memory is essential for long-term protective im- PBRM1 mutation on response to immunotherapy in patients with munity but is often impaired in cancer due to T cell exhaustion, which renal cell carcinoma (RCC) has become a topic of intense debate. causes a loss of memory precursors. Immunotherapy via checkpoint RCC-specific mechanistic and large-scale clinical data are needed to blockade does not effectively reverse this defect in the majority of pa- precisely further characterize the influence of PBRM1 loss on re- tients, potentially underlying disease relapse. Resistance mechanisms sponse to immunotherapy. that underlie poor CD8+ memory development remain unknown. Methods Methods An immunocompetent murine RCC model was applied to investigate The development of post-surgical tumor immunity [1] was interrogated in the response to anti-PD-1 therapy. Multiple human RCC datasets the CD8+ T cell-restricted Neuropilin-1 (Nrp1)-deficient mice (E8ICreNrp1L/ (TCGA, IMmotion150 and ICGC), a murine pre-malignant dataset and L), by surgically removing the primary B16F10 tumor followed by re- a Renca tumor dataset were used to perform gene signature enrich- challenge 30- or 60-days post resection. The synergy between CD8-specific ment analysis (GSEA). Immunohistochemistry and Multiplex Opal Im- Nrp1 deficiency and anti-PD1 blockade was investigated with the MC38 munofluorescence were performed to assess immune cell infiltration. tumor model. In a competitive setting, the Nrp1–/– and Nrp1+/+ pMel-T Real-time PCR, western blot, ELISA and chromatin immunoprecipita- cells [2] were co-transferred into the same host (CD45.1) followed by tion (ChIP) were used to study the activity of the interferon gamma gp100-B16 tumor inoculation, where the in vivo long-term persistence of signaling pathway. the donor cells was assessed. The transcriptomic modulation by Nrp1 defi- Results ciency was studied using bulk population RNA sequencing (bpRNAseq) on Pbrm1 knockout in murine RCC Renca cells impaired the binding of the pMel-T cells (Nrp1–/– vs. Nrp1+/+) recovered from various phase of brahma-related gene 1 (BRG1), the adenosine-triphosphate- in vivo activation (effector, memory and recall). Lastly, the physiological rele- dependent enzyme subunit of the SWI/SNF complex, to the promoter vance of NRP1 expression on CD8+ T cells in cancer patients was studied of IFN gamma receptor 2 (Ifngr2) and reduced Ifngr2 expression. in a cohort of peripheral blood leukocyte (PBL) samples from treatment- PBRM1/Pbrm1 deficiency impaired IFN gamma-induced phosphoryl- naïve patients with head and neck squamous cell carcinoma (HNSCC). ation of Janus kinase 2 (JAK2) and STAT1, and the subsequent ex- Results pression of downstream target genes involved in tumor The E8ICreNrp1L/L mice exhibited substantially enhanced protection from microenvironment (TME) modulation, such as Cxcl9, Icam1, Irf1, and tumor re-challenge, despite unchanged primary tumor growth. Enhanced Stat1 itself. In both human and murine RCC tumors, PBRM1/Pbrm1 responsiveness to anti-PD1 immunotherapy was also observed. NRP1 was loss was associated with lower expression of immune-related profiles co-expressed with multiple inhibitory receptors (IRs) on CD8+ T cells and and reduced T cell infiltration. PBRM1/Pbrm1 loss tumors also restrained memory differentiation and development by repressing an Id3- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 162 of 272 dependent transcriptional program. NRP1 was also highly expressed on medications received before recruitment to INT-NA had significant the exhausted CD8+ T cells found in the HNSCC patients and negatively relevance in the outcome. Naïve patients or previously challenged associated with the size of memory T cell pool and disease prognosis. by Immunotherapy had the best clinical benefit compared to Conclusions those receiving first line BRAF/MEK inhibitors (RR 40% vs 10%; p= These data reveal NRP1 as a unique “immune memory checkpoint” 0.001). Again, after disease progression treatment with target ther- with a mode of action that is distinct from other immune check- apy in naïve and immunotherapy previously treated patients, points. NRP1 blockade may promote the establishment of long-term showed a different beneficial pattern compared to patients re- T cell memory that is essential for durable anti-tumor immunity. ceived BRAF/MEK inhibitors before immunotherapy. The clinical parameters correlated with an increase of clinical benefit were References genus (female vs male), age (older [+60] vs younger), LDH score 1. Zhang, P., et al., Induction of postsurgical tumor immunity and T-cell mem- (normal vs high and very high), neutrophil/lymphocyte ratio (ele- ory by a poorly immunogenic tumor. Cancer Res, 2007. 67(13): p. 6468-76. vated vs normal). The introduction of an algorithm including the 2. Overwijk, W.W., et al., Tumor regression and autoimmunity after reversal of a clinical variables above mentioned could define four predictable functionally tolerant state of self-reactive CD8+ T cells. J Exp Med, 2003. 198(4): cohorts of benefit with a 95% of accuracy. p. 569-80. Conclusions Ethics Approval From the real-life analysis, we generated a simple algorithm that All animal experiments were performed in the American Association for the might drive clinical decision. Our finding clearly showed how previ- Accreditation of Laboratory Animal Care-accredited, specific-pathogen-free ous treatment impacted outcome: patients treated with iBRAF/iMEK facilities in Division of Laboratory Animal Resources, University of Pittsburgh after treatment with ICI showed better clinical outcome respect pa- School of Medicine (UPSOM). Animal protocols were approved by the tients treated with iBRAF/iMEK before treatment with ICI. Institutional Animal Care and Use Committees of University of Pittsburgh. Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) Acknowledgements electing to undergo treatment were offered the option to participate in the The study was supported by the Institutional Project "Ricerca Corrente" of University of Pittsburgh Cancer Institute (UPCI) protocol for research. All Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy patients signed an informed consent that was approved by the Institutional Review Board (IRB) of the University of Pittsburgh. References 1. Balar AV, Weber JS. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother 2017; 66(5):551–564.2. P300 2. Larkin J, Minor D, D’Angelo S et al. Overall survival in patients with Real World data analysis related to metastatic melanoma patients advanced melanoma who received nivolumab versus investigator’s treated with immunotherapy from 2012 to 2018 at Istituto choice chemotherapy in CheckMate 037: a randomized, controlled, Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy open-label phase III trial. J Clin Oncol 2018; 36(4): 383–390. Gabriele Madonna, Medical Biotechnology (LS) , Mariaelena Capone, 3. Schachter J, Ribas A, Long GV et al. Pembrolizumab versus ipilimumab 1 1 1 1 MD , Marilena Tuffanelli , Marcello Curvietto, PhD , Miriam Paone, PhD , for advanced melanoma: final overall survival results of a multicentre, 1 1 1 Assunta Esposito, PhD , Antonio Sorrentino , Marco Palla , Luigi randomised, open-label phase 3 study (KEYNOTE-006). Lancet 2017;390: 1 1 1 Scarpato , Domenico Mallardo, MD , Ester Simeone, MD , Antonio 1853–1862.4. 1 2 2 2 Grimaldi, MD , Kristina Viktorsson , Lisa Villabona , Rolf Lewensohn , 4. Larkin J, Chiarion-Sileni V, Gonzalez R et al. Combined nivolumab and ipi- 2 1 Giuseppe Masucci, MD, PhD , Paolo Antonio Ascierto, MD limumab or monotherapy in untreated melanoma. N Engl J Med2015; Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, Naples, Italy; 373: 23–34.5. Karolinska Institutet, Stockholm, Sweden 5. Robert C, Long GV, Brady B et al. Nivolumab in previously untreated Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) melanoma without BRAF mutation. N Engl J Med 2015; 372(4):320–330. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P300 6. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbé C, et al: Guidelines for the evaluation of immune therapy activity in solid tumors: Background immune-related response criteria. Clin Cancer Res 2009, 15:7412–20. Immuno checkpoint inhibitors (ICI) have improved the prognosis for Ethics Approval patients with advanced malignancy [1-5]. Their real-life application The study was approved by the internal ethics board of the Istituto may give different outcome compared to the benefit presented by Nazionale Tumori IRCCS Fondazione “G. Pascale” in Napoli Italy, approval clinical trials as the inclusion and exclusion criteria might be selective number of registry 33/17 and give overoptimistic survival rates. Here we present the analysis of cutaneous metastatic melanoma patients treated with check-point inhibitors at Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” P301 of Napoli Italy (INT-NA). Expression of tumor matrix metalloproteases ADAM10 and Methods ADAM17 correlates with low PD-L1 protein-to-mRNA ratio in We investigated retrospectively, from 2012 to 2018, 578 stage IV multiple tumors, predicting poor outcomes melanoma patients received ipilimumab, pembrolizumab or nivo- Aaron Mansfield, MD, Jacob Orme, MD PhD, Roxana Dronca, MD, lumab as monotherapy at the INT-NA. Ipilimumab was adminis- Haidong Dong, MD, PhD tered intravenously at the dosage of 3 mg/kg every 3 weeks for Mayo Clinic, Rochester, MN, United States four doses, pembrolizumab at the dosage of 200 mg every 3 Correspondence: Jacob Orme (orme.jacob@mayo.edu) weeks and nivolumab at the dosage of 3 mg/kg every 2 weeks Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P301 until disease progression or unacceptable toxicity appeared. Dis- ease evaluation was performed at baselineand then every 12 Background weeks until progression or the discontinuation of treatment ac- ADAM10 and ADAM17 portend poor prognosis in many malig- cording to the Response Evaluation Criteria in Solid Tumors nancies [1]–[6]. We previously showed these proteases cleave Pro- (RECIST 1.1) [6]. Survival analysis was performed using the Kaplan grammed death-ligand 1 (PD-L1) from tumors in soluble form Meier method and with the log-rank test. Cox regression was (sPD-L1) [7]. sPD-L1 engages immune cell Programmed death 1 used in the univariate and multivariate analysis. The results were (PD-1) to inhibit tumor immunity. It is unknown how broadly this considered significant if p mechanism occurs in solid malignancies. We hypothesized (1) Results ADAM10 and/or ADAM17 may be elevated in tumors with low Patients treated at INT-NA with nivolumab and pembrolizumab PD-L1 protein despite high PD-L1 (CD274) mRNA and (2) this low showed comparable better clinical benefit toward patients treated tumor PD-L1 protein-to-mRNA ratio may predict lower overall with ipilimumab (RR 44.5% vs 20.7%; p=0.01). The anti-tumoural survival. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 163 of 272 Methods immunity – a resistance mechanism to PD-1 checkpoint blockade We queried the Cancer Genome Atlas (TCGA) for all solid tumors in melanoma,” in CRI-CIMT-EATI-AACR International Cancer Confer- with Level 3 reverse phase protein array (RPPA) PD-L1 protein ence, 2018. levels and RNA-seq sequence per million mapped fragments Ethics Approval (FPKM) PD-L1 (CD274), ADAM10, and ADAM17 mRNA levels. We Lab studies involving human subjects are approved by Mayo Clinic‘s calculated a PD-L1 protein-to-mRNA ratio for each sample. Groups Institutional Review Board (IRB), approval number 15-000934. of high and low PD-L1 protein-to-mRNA ratios were evaluated for (1) ADAM10 and ADAM17 expression and (2) overall survival by Cox proportional hazards modeling, adjusting for age and stage at diagnosis. Results Tumor samples demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly more ADAM10 and/or ADAM17 in 23 of 25 cancer types (Table 1). Cox proportional hazards ratios for Table 1 (abstract P301). See text for description death in each group were calculated and reported as a forest plot including hazard ratios and 95% confidence intervals for overall survival for each cancer subtype adjusted for patient age and tumor stage (Figure 1). Patients with tumors demonstrating low PD-L1 protein-to-mRNA ratio experienced significantly worse outcomes in 8 of 25 tumor types and improved outcomes in 2 tumor types (Table 2). Conclusions In this work we report that reduced human PD-L1 protein-to-mRNA ratios are associated with (1) high ADAM10 and/or ADAM17 expres- sion and (2) poor outcomes in multiple cancers. We previously showed in multiple cell lines that ADAM10 and ADAM17 cleave PD- L1 from the surface of tumor cells [7]. Our results suggest that ADAM10 and/or ADAM17 may cleave PD-L1 to cause a low PD-L1 protein-to-mRNA ratio in these tumors. This process may explain poorer survival of patients with low PD-L1 protein-to-mRNA ratios in some cancers. Our findings may explain why some tumors that do not have detect- able PD-L1 expression on immunohistochemistry respond to PD-(L)1 inhibitor therapy given the solubilization of PD-L1 and its subsequent systemic negative regulatory effects on T cells. Further, ADAM10/ ADAM17 inhibition may prevent PD-L1 shedding and sensitize tu- mors to therapy. While this work is correlative in nature, studies ex- ploring this mechanism of tumor immune system evasion are ongoing. Table 2 (abstract P301). See text for description Acknowledgements The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. References 1. M. Uhlen et al., “A pathology atlas of the human cancer transcriptome.,” Science, vol. 357, no. 6352, p. eaan2507, Aug. 2017. 2. P. C. Buchanan et al., “Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17.,” J. Biol. Chem., vol. 292, no. 15, pp. 6339–6351, Apr. 2017. 3. M. E. Powers, H. K. Kim, Y. Wang, and J. Bubeck Wardenburg, “ADAM10 Mediates Vascular Injury Induced by Staphylococcus aureus α- Hemolysin,” J. Infect. Dis., vol. 206, no. 3, pp. 352–356, 2012. 4. S.-S. Ni, J. Zhang, W.-L. Zhao, X.-C. Dong, and J.-L. Wang, “ADAM17 is overexpressed in non-small cell lung cancer and its expression correlates with poor patient survival,” Tumor Biol., vol. 34, no. 3, pp. 1813–1818, Jun. 5. Y.-Y. Wang, Z.-Y. Ye, L. Li, Z.-S. Zhao, Q.-S. Shao, and H.-Q. Tao, “ADAM 10 is associated with gastric cancer progression and prognosis of patients,” J. Surg. Oncol., vol. 103, no. 2, pp. 116–123, Feb. 2011. 6. B. You, Y. Shan, S. Shi, X. Li, and Y. You, “Effects of ADAM10 upregulation on progression, migration, and prognosis of nasopharyngeal carcinoma,” Cancer Sci., vol. 106, no. 11, pp. 1506–1514, Nov. 2015. 7. J. J. Orme et al., “Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 164 of 272 “responders” and “non-responders” to ICI drugs and is also being used to screen co-stimulatory agonists, peptide biologics and other drug classes. Conclusions Taken together, our data indicate that the recall antigen potency assay we established is highly potential to screen the drug candidates for im- mune checkpoint inhibitor and enhancer drug candidates. P303 PD-1 checkpoint blockade in advanced melanoma patients: Neutrophils, NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates Yago Pico de Coaña, Maria Wolodarski, MD, Irene van der Haar Àvila, Takahiro Nakajima, Stamatina Rentouli, Andreas Lundqvist, PhD, Giuseppe Masucci, MD, PhD, Johan Hansson, Rolf Kiessling, MD, PhD Karolinska Institute, Stockholm, Sweden Correspondence: Yago Pico de Coaña (yago.pico.de.coana@ki.se) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P303 Background Blockade of the PD-1 receptor has revolutionized the treatment of meta- static melanoma, with significant increases in overall survival and a dra- matic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of benefitting patients is limited and there is a need for predictive biomarkers and a deeper mechanistic ana- lysis of the cellular populations involved in a clinical response. Methods Fig. 1 (abstract P301). See text for description With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients undergoing treatment with pem- P302 brolizumab (n=7) or nivolumab (n=30) treatment at Karolinska Uni- A reversible T cell exhaustion-like in vitro assay to screen versity Hospital. Blood samples were collected from patients at the candidate drugs following time points: Before treatment and at the time of the sec- 1 2 2 Wushouer Ouerkaxi , Eden Kleiman , Pirouz Daftarian ond and fourth doses. Peripheral blood mononuclear cells (PBMCs) 1 2 MBL International, Woburn, MA, United States; JSR Lifesciences, were isolated by density gradient centrifugation and stained for flow Sunnyvale, CA, United States cytometric analysis within two hours of sample collection. Correspondence: Pirouz Daftarian (pdaftarian@jsrlifesciences.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P302 Two distinct cellular populations were inversely correlated with survival: Neutrophils, and Monocytic myeloid derived suppressor cells (MDSCs). Background Furthermore, overall survival and progression free survival were also T cells undergo different layers of suppression, with a spectrum of events found to be inversely correlated with the activation status of NK cells. that may overlap such as dysfunction, anergy, unresponsiveness, toler- Finally, PD-L1 expression in different monocytic subsets was signifi- ance, and exhaustion. Many factors have been attributed to this including cantly increased in patients with shorter progression free survival and multiple co-inhibitory receptor surface expression, altered transcription was correspondingly correlated inversely with overall survival. factor expression, epigenetic rewiring and dysregulated metabolism. Anti- Conclusions gen persistence is necessary for driving TEX maintenance in both the Our results suggest that cellular populations other than T cells can chronic viral infection setting and cancer. In addition to persistent antigen be critical in the outcome of PD-1 blockade treatment. Specifically, exposure, tumor-infiltrating lymphocytes (TILs) within the tumor micro- the frequencies of activated NK cells and monocytic MDSCs are in- environment (TME) encounter numerous tumor-mediated immunosup- versely correlated with survival and clinical benefit and their role as pressive metabolic byproducts, suppressive cytokines, hypoxia and predictive biomarkers should be further evaluated. cellular debris which converge to suppress T cell function and uniquely Ethics Approval alter is transcription factor profile. These suppressed or dysfunctional T The protocol was approved by the local Ethics Committee and the In- cells are incapable of mounting an optimal anti-tumor response in part stitutional Review Board at Karolinska Institute (approval number due to lack of fitness in competing for glucose and oxygen. Our team 2015/1862-32) and all patients provided written informed consent in has utilized the recall antigen potency assay as a tool for function-based accordance with the Declaration of Helsinki. screening of immune checkpoint inhibitor (ICI) drug candidates. Methods P304 A recall antigen potency assay has been used as a tool for function- DNA damage response gene alterations are associated with high based screening of immune checkpoint inhibitor (ICI) drug candidates. In tumor mutational burden and clinical benefit from programmed this assay, healthy human PBMCs are stimulated with peptide(s) derived death 1 axis inhibition in non-small cell lung cancer from either viruses or tumor proteins and grown in culture for one week. 1 1 1 Biagio Ricciuti, MD , Gonzalo Recondo, MD , Renato Umeton , Giuseppe Day 4 supernatants are functionally assayed by ELISA for IFN-γ secretion 1 2 2 1 Lamberti, MD , Mizuki Nishino , Lynette Sholl , Michael Cheng , Mark and cells are assayed on day 7 by flow cytometry for CD8+ or CD4+ T cell Awad, MD PhD expansion by using a single or cocktail of pMHC tetramers. 1 2 Dana Farber Cancer Institute, Boston, MA, United States; Brigham and Results Women’s Hospital, Boston, MA, United States We have shown that in roughly 30% of donor PBMCs, ICI drugs such as Correspondence: Biagio Ricciuti (biagio_ricciuti@dfci.harvard.edu) pembrolizumab are able to boost both IFN-γ secretion and antigen-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P304 recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory re- Background ceptor downstream signaling is ameliorated with ICI drugs releasing these DNA damage response (DDR) gene alterations are associated with in- T cells from the repressive effects of these co-inhibitory receptors. Further, creased tumor infiltrating lymphocytes, higher genomic instability, this assay has the potential to screen for donors who are in vitro Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 165 of 272 and higher tumor mutational burden (TMB) in cancer. Whether DDR Table 1 (abstract P304). See text for description alterations are associated with benefit from immune-checkpoint in- hibitors (ICIs) in non-small cell lung cancer (NSCLC) is unknown. Methods Clinicopathologic and genomic data were collected from patients (pts) with advanced NSCLC at the Dana-Farber Cancer Institute (DFCI) treated with PD-(L)1 inhibitors. Targeted next-generation sequencing (NGS) by OncoPa- nel was used to determine DDR gene mutation status and TMB. Patients were categorized based on the presence or absence of deleterious DDR gene alterations in a panel of 53 DDR genes. All loss-of-function alterations in DDR genes (including nonsense, frameshift, or splice site) were classified as pathogenic. Missense mutations were evaluated using the Catalogue of Somatic Mutations in Cancer (COSMIC) [1], and ClinVar databases [2], as well as the PolyPhen-2 (Polymorphism Phenotyping v2) functional predic- tion tool [3]. Missense mutations were classified as pathogenic if annotated as pathogenic by either COSMIC or ClinVar and damaging by Polyphen-2. Because only tumor tissue was sequenced, common single nucleotide poly- morphisms (SNPs) were filtered if present at ≥0.1% in Genome Aggregation Database (gnomAD) version 2.1.1 [4]. Clinical outcomes to immunotherapy were evaluated according to DDR mutation status. Results Among 256 pts with successful NGS who received ICIs, 134 (52.3%) were identified as having deleterious DDR mutations (DDR-positive). DDR-positive and DDR-negative groups were well balanced in terms of baseline clinico- pathological characteristics (Table 1). The median TMB was significantly higher in the DDR-positive group compared to the DDR-negative group (12.17 vs 8.36 mutations/megabase, P< 0.0001), as well as among never smokers (9.40 versus 5.70 mut/Mb, P = 0.035, Figure 1B). Compared to DDR-negative pts (N=122), DDR-positive pts had a significantly higher ob- jective response rate (28.6% vs 16.4%, P=0.025, Figure 2A), longer median progression-free survival (4.2 vs 2.2 months, HR: 0.64 [95%CI: 0.49-0.84], P= 0.001, Figure 2B) and overall survival (17.5 vs 9.9 months, HR: 0.60 [95%CI: 0.43-0.82], P=0.002, Figure 2C) with PD-(L)1 therapy. DDR-positive status was associated with significantly longer PFS (HR: 0.70 [0.51-0.95], P=0.024) and OS (HR: 0.61 [95%CI: 0.43-0.85], P=0.004) in multivariate analysis (Table 2). Conclusions Deleterious DDR alterations are frequent in NSCLC and are associated with higher TMB and improved clinical outcomes in NSCLC pts treated with PD-1 axis inhibition. References 1. Forbes SA, Beare D, Boutselakis H, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic acids res. 2017; 45:D777-D783. 2. Landrum MJ, Lee JM, Benson M, et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic acids res. 2016; 44:D862-D868 3. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat. Methods. 2010; 7:248 4. https://gnomad.broadinstitute.org Fig. 1 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 166 of 272 Table 2 (abstract P304). See text for description Response to Pembrolizumab and tumor microenvironment composition is associated with IL8 expression in a head and neck squamous-cell carcinoma cohort 1 2 2 1 Arun Khattri, PhD , Jason Reeves , SuFey Ong , Riyue Bao, PhD , Arya 2 1 2 Bahrami, PhD , Yi-Hung Carol Tan, PhD , Andrew White, BSc , Michael 2 2 2 Bailey , Heather Brauer, PhD , Sarah Warren, PhD , Joseph Beechem, 2 3 PhD , Tanguy Seiwert, MD 1 2 University of Chicago, Chicago, IL, United States; NanoString Technologies, Seattle, WA, United States; Johns Hopkins University, Baltimore, MD, United States Correspondence: Tanguy Seiwert (tseiwert@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P305 Background A subset of head and neck squamous-cell carcinomas are known to re- spond to immune checkpoint inhibitors. To better understand response to therapy in these tumors, a cohort of samples treated with Pembrolizumab was examined to determine if specific cell types are associated with re- sponse to intervention by combined profiling of standard bulk expression assays, ISH staining, and spatially resolved multiplexed protein analysis. Methods RNA was extracted from archival FFPE samples (n = 107) col- lected prior to therapeutic intervention and profiled using the NanoString® nCounter® PanCancer IO 360™ panel (Research Use Only). Gene expression signatures were calculated for immune cell subsets, as well as the Tumor Inflammation Signature (TIS; Ayers 2017 JCI). Individual gene expression and signatures were compared to patient outcome. Subsequently, expression of IL8 was validated by RNAScope in a subset of responders and non- responders (n = 9). To determine whether the IL8 staining pat- tern observed was consistent with specific cell types, a set of six tumor samples (three IL8+ and three IL8-) were further character- ized by multiplexed protein expression analysis on the GeoMx™ digital spatial profiling (DSP) platform to quantitate expression of 40 antibodies. IL8 staining was used to guide DSP selection of re- gions of interest (ROI) within the tumors that were either IL8+ or IL8-. Protein expression was specifically measured from tumor or stromal areas based on Pan-cytokeratin immunofluorescence. Results Initial analysis of the head and neck cohort found that previously reported signatures of response, including TIS, were not associ- ated with patient outcome in this cohort. In contrast, IL8 expres- sion was observed to be highest in patients with progressive disease. Further investigation demonstrated that IL8 expression was most specifically associated with neutrophil markers/expres- sion signatures. DSP profiling confirmed that tumor and stromal segments from IL8+ regions were associated with high expression of CD66B and ARG1 and lower expression HLA-DR consistent with neutrophil/granulocytic MDSCs presence. Furthermore, these re- gions were shown to have lower expression of T-cell markers in- cluding CD3, CD8 and CD4. Conclusions These results demonstrate that, in addition to previously reported biomarkers, IL8 expression and neutrophil presence may be related to response to checkpoint therapy in head and neck cancers. De- creased T-cell marker expression in IL8+ regions may reflect de- creased response in the larger cohort. Ethics Approval The study was approved by the University of Chicago‘s Ethics Board, approval number 8980 and 16-1269 P306 Innate immune cells play a role in therapy resistance to anti-PD1 in Hu-mice melanoma model Raj Somasundaram, PhD, Meenhard Herlyn, DVM PhD P305 The Wistar Institute, Philadelphia, PA, United States Correspondence: Raj Somasundaram (shyam@wistar.org) Fig. 2 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P306 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 167 of 272 Background constituted the majority of cases in both groups. The overall re- Immune checkpoint inhibitor therapy is rapidly emerging as a front- sponse rates of the CPI+M group were significantly higher than those line treatment option for many solid tumors. However, only a third of treated with CPI only (75% vs 53.3%, p = 0.05). melanoma patients respond to immune checkpoint blockade. Cur- Conclusions rently available mouse models have many short comings and are un- The data from this chart review shows an apparent benefit with respect able to address the basis of therapy resistance and immune non- to overall response rates in patients treated with M and CPIs. The cohorts responsiveness that are observed in patients. remain small in this study, but the data is significant. These results align Methods well with data from mouse studies and two small retrospective human Our laboratory has developed a novel humanized mouse melanoma studies in melanoma [2] and lung cancer [3], hence, demonstrating an in- model. Immuno-deficient NSG mice were reconstituted with human creased immune response and higher response rates. Currently, we are CD34+ cells and after 8-12 weeks, mice are fully reconstituted with hu- expanding our cohort number and data-set as we gained access to a big- man innate and adaptive immune cells. Humanized mice were then ger data warehouse. More prospective data are awaited from an ongoing challenged with HLA-matched melanoma cells and the functional abil- phase Ib (UMIN000028405), and phase II (NCT03800602, NCT03048500) ity of human immune cells to restrict tumor growth was monitored. trials that should shed more light on the effects of metformin on Results immuno-oncologic therapies. Restricted tumor growth was observed in humanized mice indicating in vivo sensitization of human immune cells to melanoma. References In therapy studies, tumor-bearing humanized mice treated with anti- 1. Scharping N, Menk A, Whetstone R, Zeng X, Delgoffe G. Efficacy of PD-1 PD-1 showed restricted tumor growth. Anti-PD-1 therapy resulted in blockade is potentiated by metformin-induced reduction of tumor hyp- enhanced infiltration of T-cells that correlated with tumor response. oxia. Cancer Immunol Res. 2017;5(1):9-16. MassCyTOF studies was performed using a panel of immune markers 2. Afzal M, Mercado R, Shirai K. Efficacy of metformin in combination with to understand the mechanism of therapy non-responsiveness in immune checkpoint inhibitors (anti-PD-1/anti-CTLA-4) in metastatic some tumors. Results indicated downmodulation of HLA-class I mole- malignant melanoma. J Immunother Cancer. 2018;6(1):64. cules and increased presence of mast cells cells in the tumor region. 3. Afzal M, Dragnev K, Sarwar T, Shirai K. Clinical outcomes in non-small-cell In tumor-bearing mice, combination of therapy drugs targeting c- lung cancer patients receiving concurrent metformin and immune kit+ mast cells and anti-PD1 caused complete regression of tumor le- checkpoint inhibitors. Lung Cancer Manag. 2019:1-12 (online publication). sions. Tumor free mice were able to reject freshly challenged melan- Ethics Approval oma cells indicating the presence of memory T-cell responses. The study was approved by Louisiana State University Health Science Center Conclusions of Shreveport’s Institutional Review Board separately for each site, IRB Our results suggest that humanized mouse melanoma model can be numbers are STUDY00000891, and STUDY00001017. explored further to understand the therapy resistance mechanisms to immune-based treatments. Further, model will be useful for devel- P308 oping new therapeutic strategies for treating melanoma patients. The impact of obesity on the response rates of checkpoint inhibitor (CPI) cancer immunotherapy 1 2 P307 Philip Haddad, MD, MPH , David Sommerhalder, MD The impact of metformin (M) on the response rates of checkpoint Overton Brooks VA Medical Center/LSUHSC-S, Shreveport, LA, United inhibitors (CPI) States; Louisiana State University Health Science, Bossier City, LA, United 1 2 Philip Haddad, MD, MPH , David Sommerhalder, MD States 1 2 Overton Brooks VA/ LSUHSC, Shreveport, LA, United States; Louisiana Correspondence: Philip Haddad (haddad8838@msn.com) State University Health Science, Bossier City, LA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P308 Correspondence: Philip Haddad (haddad8838@msn.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P307 Background With durable responses, improved clinical benefit, and relatively fewer Background toxicities, CPIs targeting cytotoxic T-lymphocyte-associated protein 4 Immunotherapies in oncology have brought significant change and (CTLA4), programmed death-1 (PD-1), and its ligand (PD-L1) have estab- hope to the field over the last several years. Responses, however, re- lished themselves as essential components of cancer immunotherapy main unpredictable and low in most cases. One of the aspects thought across multiple cancer types. Obesity is a known risk factor for several to be limiting immunotherapies is the tumor microenvironment which cancer types. It is associated with increased progression and cancer- favors tumor growth and immunosuppression. It has been hypothe- related death. This is thought to be the result of inflammaging and PD- sized that dysregulated tumor metabolism creates a hypoxic tumor 1 mediated immune suppression. Recently, two large retrospective microenvironment which acts as a barrier to antitumor immunity. Met- studies found that obesity conferred a survival advantage for cancer formin has been shown to reduce oxygen consumption and subse- patients treated with CPIs which may be independent of sex [1,2]. How- quently reduce the microenvironment hypoxia, leading to improved ever, the mechanistic explanation of this observed obesity paradox, as- response rates of checkpoint inhibitors in murine in vitro and in vivo suming it is real, has been the subject of many scientific conjectures. models [1]. We performed a retrospective review to evaluate response These studies focused on the impact of obesity on CPI overall survival rates in our patients who were treated with CPI+M. which can be affected by many confounding variables. Instead, we ex- Methods plored the effect of obesity on CPI response rates. We reviewed all adult cancer patients who were treated with a CPIs. Pa- Methods tients treated with M and CPIs were compared to those who were We retrospectively reviewed every cancer patient that received CPIs at treated with CPIs only. All tumor types and all CPI drugs were included. Overton Brooks VA Medical Center (OBVAMC) between 2015 and 2019. The primary endpoint was overall response rate, which included stable Patient’s BMI scores at the beginning of CPI therapies were calculated. disease, partial response, and complete response. Additional data was Based on the WHO definition, the patients were grouped according to captured for subgroup analysis. Patients were excluded if they had never their BMIs into overweight and obese (Group A) versus normal and received the treatment, or if they were never assessed for response. underweight (Group B). Our primary outcome of interest was defined as Results the presence or absence of CPI response. Patients who attained stable As of this date, 144 patients had been included in the study data-set. disease, partial response, and complete response were categorized as re- Of those, 24 patients were treated with M and CPIs and 120 patients sponders. Those who progressed on CPI were labeled as non-responders. received CPIs only. Both groups were comparable with respect to Thesignificanceofthe associationbetween the grouped BMI categories sex. However, the M+CPI group was slightly older. Lung cancer and the occurrence of any response was analyzed statistically. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 168 of 272 Results Results Between 2015 and 2019, 65 patients were treated with CPIs and had In the neoadjuvant ICB cohort, increasing age correlated with better documented responses. Both groups were comparable with respect to response to immunotherapy (OR= 0.91, P=0.039) and remained a sig- age, sex, race, and types of CPIs. Lung cancer constituted the majority nificant predictor of response in the multivariable model (OR=0.88, of cases in both groups. Head and neck cancers were more prevalent P=0.03) after adjusting for sex, tumor site, stage, prior therapies, and in Group B while renal and bladder cancer and melanoma were more toxicity; similar results were observed in the OpACIN neoadjuvant prevalent in Group A. Group B had a significantly higher response rate trial study (NCT02977052). Tumor mutational load was positively cor- (80% vs 50%, p=0.01). Furthermore, a higher response rate was ob- related with age (r=0.4) but did not reach statistical significance (P= served between normal BMI and overweight patients (p=0.001) and 0.1). Differential gene expression analysis revealed genes associated normal BMI and obese patients (p=0.06). No difference in response with MHC class II antigen expression (HLA-DQB1, HLA-DRB1) as sig- rates was observed between underweight and obese patients. nificantly overexpressed in younger patients upon treatment with Conclusions ICB. Furthermore, MHC class II regulator interferon-gamma signaling This is the first report to show a detrimental effect of overweight was also observed upregulated in younger patients (P=0.03). PD-1 ex- and obesity on CPI response rates in a retrospective cohort of pression (by IHC) was lower in older patients upon treatment with non-selected consecutive cancer patients in a real-world clinical ICB (r=-0.47, P=0.035). In keeping with human cohort, mice injected setting. with Yumm1.7 melanoma cells showed significant increase in im- mune cell populations expressing MHC class II antigen in the young References mice versus aged mice (P=0.0048). 1. McQuade J, et al. Association of body-mass index and outcomes in pa- Conclusions tients with metastatic melanoma treated with targeted therapy, immuno- Increased age was associated with improved outcomes in melanoma therapy, or chemotherapy: a retrospective, multicohort analysis. Lancet patients receiving neoadjuvant ICB, similar to results in stage IV. The Oncol. 2018;19(3):310-322. mechanisms behind this association are likely multifactorial, and may 2. Xu H, Cao D, He A, Ge W. The prognostic role of obesity is independent relate in part to MHC class II antigen expression mediated immune of sex in cancer patients treated with immune checkpoint inhibitors: A evasion in younger patients, though further studies are needed to pooled analysis of 4090 cancer patients. Int Immunopharmacol. delineate contributing factors. 2019;74:1-10. Trial Registration Ethics Approval NCT02519322 The study was approved by the Louisiana State University Health Science Ethics Approval Center of Shreveport Institutional Review Board, IRB number This trial was approved by the MD Anderson Cancer Center Institu- STUDY00001017. tional Review Board. The trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and with adherence to the Good Clinical Practice guidelines, as defined by the Inter- P309 national Conference on Harmonization. This protocol was conducted Older age predicts better outcome to neoadjuvant immune with compliance with all relevant ethical regulations. checkpoint blockade in metastatic melanoma Consent 1 2 1 Rohit Thakur , Stephen Douglass, PhD , Beth Helmink, MD PhD , Rodabe Written informed consent was obtained from all participants. The MD 1 1 1 Amaria, MD , Hussein Tawbi, MD, PhD , Jennifer McQuade , Eliza Anderson Data Safety Monitoring Board reviewed the data at 12- 3 1 4 Rozeman , Elizabeth Burton , Sangeetha Reddy, MD, MSci , John month increments. 5 3 6 Wherry , Christian Blank, MD PhD , Georgina Long , Jeffrey Gershenwald, 1 1 1 MD , Michael Davies, MD, PhD , Michael Tetzlaff, MD PhD , Ashani 7 1 Weeraratna , Jennifer Wargo, MD, MMSc P310 1 2 MD Anderson Cancer Center, Houston, TX, United States; The Wistar Optimal priming prevents the induction of dysfunctional CD8 T- Institute, Philadelphia, PA, United States; The Netherlands Cancer cells in subprimed conditions, reversing resistance to anti-PD-1 Institute, Amsterdam, Netherlands; UT Southwestern Medical Center, Vivek Verma, PhD, Rahul Nandre, PhD, Jose Lopez, Seema Gupta, PhD, Houston, TX, United States; University of Pennsylvania, Philadelphia, PA, Samir Khleif, MD 6 7 United States; Melanoma Institute Australia, Sydney, Australia; Johns Georgetown University Medical Center, Washington, DC, United States Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Samir Khleif (snk48@georgetown.edu) Correspondence: Jennifer Wargo (JWargo@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P310 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P309 Background Background Suboptimal-priming of lymphocytes by low-affinity antigens is a mechan- There is a growing appreciation for studying the impact of host and ism for prevention of generation of strong immune responses against environmental factors on response to immune checkpoint blockade self-antigens. However, we recently found that the suboptimally-primed (ICB). Recently it was reported that older age correlated with better CD8 cells have a pre-disposition to develop a dysfunctional phenotype outcome in stage IV melanoma patients treated with ICB. We exam- that is marked by expression of CD38 on PD1+CD8+T-cells. Interestingly, ined the association of age with response to ICB and anti-tumor im- the number of these dysfunctional cells were significantly increased upon munity in melanoma patients treated neoadjuvantly (NCT02519322) PD-1 blockade in these suboptimally-primed CD8 cells that served as a and interrogated underlying mechanisms in a murine model. reason for resistance to anti-PD-1 therapy. On the other hand, anti-PD-1 Methods blockade of optimally-primed CD8 cells did not generate these dysfunc- Tumor samples were obtained from neoadjuvant ICB trial patients tional cells and led to cell activation and generation of effector functions pre-treatment, on-treatment and on-surgery. Transcriptome profil- [1]. Hence, here we investigated the effect of optimal-priming on revers- ing was performed using Illumina NextSeq platform and whole ing the resistance to anti-PD-1 therapy. exome sequencing using Illumina HiSeq 2500 platform. Univari- Methods able and multivariable analyses were performed using logistic re- Mice were inoculated with TC-1 cells (a mouse lung epithelial cell- gression modeling. Immune profiling was performed by line, expressing human papillomavirus-specific E7-peptide) in the Immunohistochemistry (IHC). The effects of age on anti-tumor im- presence or absence of concomitant priming with gp100 peptide, a munity were examined by implanting 2x105 Yumm1.7 cells sub- non-cognate tumor vaccine. Seven days later mice were treated with dermally in young (8wks) and aged (>12months) male mice. anti-PD-1 either alone or followed by combination of tumor-specific Tumors were harvested after 30 days of growth and immune sur- E7-peptide vaccine+anti-PD-1. Tumor growth rates, mice survival, face markers were analyzed by flow cytometry. and immune responses in the TME were estimated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 169 of 272 Results results in anti-tumor efficacy. TPST-1495 is a first-in-class, orally avail- We found that in suboptimally-primed TC-1 tumor-bearing mice, able, small molecule, selective dual antagonist of the human PGE2 anti-PD-1 treatment did not show any anti-tumor effects. Therefore, receptors EP2 and EP4, currently under development by Tempest. to check if the optimal priming of CD8 T-cells could reverse this re- Methods sistance, we vaccinated mice with gp100 at the time of tumor im- The effects of TPST-1495 as monotherapy or in combination with plantation with TC-1 cells. We found that compared to suboptimally- anti-PD1 were evaluated in the syngeneic mouse colon models CT26 primed mice, anti-PD-1 treatment of primed-mice resulted in signifi- and Apcmin/+. The mechanism of anti-tumor immunity of TPST-1495 cant retardation of tumor growth and a prolonged mice survival. was evaluated using in vitro primary dendritic cell (DC) differentiation Interestingly, tumor-specific vaccination (E7-peptide) of these and activation assays. Characterization of in vitro differentiated im- primed-mice at the time of anti-PD-1 treatment further enhanced the mune cells or tumor infiltrating lymphocytes were performed using therapeutic efficacy of PD-1 blockade. Moreover, in gp100-primed- flow cytometry. ELISA was used for measurement of cytokine mice we found a significant reduction in the number of PD-1+CD38hi production. dysfunctional cells compared to suboptimally-primed mice. The num- Results ber of these dysfunctional cells was further reduced upon anti-PD-1 Treatment with TPST-1495 reversed PGE2 immune suppression treatment of primed-mice. In addition, the priming state also affected in vitro and in vivo compared to antagonism of EP4 alone or all 4 EP the functionality of CD8 T-cells. Although gp100 alone prevented the receptors. TPST-1495 prevented PGE2 inhibition in vitro of DC differ- induction of these dysfunctional cells, the functionality of CD8 T-cells entiation and activation from human donor monocytes; single EP2 or was only increased when anti-PD-1 was given subsequent to priming EP4 antagonists were sub-optimal in this assay. Significantly, combin- with gp100 peptide. ation with EP1 and/or EP3 antagonists reversed the effect of dual Conclusions EP2 and EP4 blockade on PGE2 immune suppression, suggesting that Here we demonstrate that optimal priming of CD8 cells reverses re- COX-2 inhibition is not optimal for blocking the effects of PGE2. TPST- sistance to anti-PD-1 therapy. The suboptimal-priming of the CD8+ T- 1495 induced potent anti-tumor immune responses and significant cells induces higher numbers of dysfunctional PD-1+CD38hi CD8+ T- tumor regression as a monotherapy in two different murine tumor cells and their frequency further increases upon anti-PD-1 therapy, treatment models of colon cancer, CT26 and Apcmin/+. CT26 tumors leading to therapeutic failure. Since in most tumors, T-cells are analyzed from mice treated with TPST-1495 alone revealed a significant suboptimally-primed [2,3], our mouse data demonstrate the import- increase of infiltrating effector T cells. TPST-1495 combination with anti- ance of appropriately primed T-cells in responding to anti-PD-1 PD1 synergistically inhibited CT26 tumor progression. treatment. Conclusions TPST-1495 is a differentiated highly potent selective dual antagonist References of EP2 and EP4 that overcomes prostaglandin-mediated immune 1 Verma, V. et al. PD-1 blockade in subprimed CD8 cells induces dysfunc- suppression and promotes anti-tumor efficacy. tional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat Immunol, doi:10.1038/s41590-019-0441-y (2019). P312 2 Vonderheide, R. H. The Immune Revolution: A Case for Priming, Not Ipilimumab treatment immunophenotypic changes are associated Checkpoint. Cancer Cell 33, 563-569, doi:10.1016/j.ccell.2018.03.008 (2018). with progression of disease with sequential nivolumab therapy in 3 Vonderheide, R. H., Domchek, S. M. & Clark, A. S. Immunotherapy for metastatic melanoma Breast Cancer: What Are We Missing? Clin Cancer Res 23, 2640-2646, 1 1 2 David Woods, PhD , Andressa Sodre Laino, PhD , Aidan Winters , Jason doi:10.1158/1078-0432.CCR-16-2569 (2017). 1 1 1 Alexandre , Jeffrey Weber, MD, PhD , Pratip Chattopadhyay 1 2 NYU Langone Health, New York, NY, United States; UCSF, San P311 Francisco, CA, United States Dual antagonism of prostaglandin receptors EP2 and EP4 by TPST- Correspondence: Pratip Chattopadhyay 1495 suppresses tumor growth and stimulates anti-tumor (Pratip.Chattopadhyay@nyulangone.org) immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P312 1 2 2 Chan Whiting, PhD , Kim Fischer, PhD , Bryan Laffitte, PhD , Lisa 2 2 2 Rahbaek, PhD , Nick Stock, PhD , Davorka Messmer, PhD , Austin Chen, Background 2 2 2 1 PhD , Traci Olafson , Natalie Nguyen , Amanda Enstrom, PhD , Derek Nivolumab (nivo) and ipilimumab (ipi) combination immunotherapy has 1 1 3 Metzger , Brian Francica , Dingzhi Wang, PhD , Raymond Dubois, PhD, a ~60% response rate in metastatic melanoma patients. However, the im- 3 1 2 1 MD , Ginna Laport, MD , Peppi Prasit, PhD , Thomas Dubensky, PhD pact of these therapies on immune cell phenotypes and the relationship 1 2 Tempest Therapeutics, San Francisco, CA, United States; Inception of those changes to patient outcomes remains under-investigated. Sciences, San Diego, CA, United States; Medical University of South Methods Carolina, Charleston, SC, United States High dimension flow cytometry on baseline and at week 13 (e.g. Correspondence: Brian Francica (bfrancica@tempesttx.com) after initial nivo or ipi therapy) was performed for peripheral blood Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P311 samples from 33 metastatic melanoma patients receiving sequential nivo-ipi or the reverse sequence. We used a novel computational ap- Background proach to analyze the data through semi-comprehensive Boolean Progression of diverse malignancies is promoted by elevated levels gating in which immune cell lineages (e.g. CD3+CD4+) were evalu- of Prostaglandin E2 (PGE2). High PGE2 levels results from dysregula- ated for all possible combinations for up to 15 markers. tion of Cyclooxygenase-2 (COX-2), the enzyme that produces this Results lipid. PGE2 stimulates tumor cell proliferation, survival, evasion and 3,844 measured immunophenotypes were significantly altered post-nivo, metastasis along with host angiogenesis. PGE2 suppresses anti-tumor and 7,133 immunophenotypes were altered post-ipi. The frequency of immunity through inhibiting the function of critical immune effectors 584 immunophenotypes were significantly changed in both treatments, such as NK and T cells, and M1 macrophages, while promoting the with 59 of those changing in opposing directions. In the nivo-ipi cohort, activity of suppressive immune cells including myeloid derived sup- 260 baseline and 662 post-nivo immunophenotypes were significantly as- pressor cells, M2 macrophages, and regulatory T cells. PGE2 signals sociated with response and survival (outcomes). In the ipi-nivo cohort, 432 through a family of four homologous E-prostanoid (EP) G-coupled re- baseline and 668 post-ipi immunophenotypes were associated with out- ceptors, known as EP1, EP2, EP3 and EP4; which,are activated via dis- comes. Two highly similar immunophenotypes associated with outcomes tinct signal transduction pathways. Published literature and overlapped between the cohorts, CD14+CD11C+CD33+CD15-CD19-PDL1- experimental results presented here demonstrate that selective an- PDL2+CD163+GAL9-CD80-CD86-41BBL+CD40+OX40L+ cells. While lower tagonism of both EP2 and EP4 receptor signaling, but not EP1 and levels of these cells were associated with response and improved survival EP3, effectively overcomes PGE2-mediated immune suppression and in nivo-ipi treated patients, lower levels were associated with better Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 170 of 272 outcomes in the ipi-nivo treated patients. Of the 3,844 immunopheno- Specifically, these data show that changes in secretome production types altered post-nivolumab, 100 were also associated with ipi-nivo re- potential of T-cells after treatment with the PD1 blocking antibody sponse. Of these 100, 97% were altered in a manner positively associated nivolumab are associated with metastatic melanoma patient out- with response (e.g. upregulated by nivolumab and higher in responders). comes. We identified a signature of secreted molecules that were as- For example, nivo upregulated CD4+CD45RO-CCR7+ frequencies, which sociated with patient outcomes, providing rationale for targeting were associated with response and longer survival in ipi-nivo treated pa- these molecules to increase the efficacy of nivolumab. Work is under- tients. Of the 7,133 immunophenotypes altered post-ipi, 110 were also as- way to validate the observed associations in an independent set of sociated with nivo-ipi response. Of these, 95% were altered in a manner patient samples. negatively associated with response. This includes ipi associated upregula- Ethics Approval tion of a population of CD4+CD38+CD39+CD127-GARP- cells that are The study was approved by NYU Langone Health's IRB, approval negatively associated with coutcomes in nivo-ipi treated patients as well number S16-00035. as downregulation of a CD4+CD127+CD45RO+CD95+CCR7+ population of cells positively associated with outcomes. P314 Conclusions Alterations of DNA damage response signaling in the development These results demonstrate that nivo and ipi altered the peripheral im- of antibody-dependent cellular cytotoxicity (ADCC) resistance mune landscape in distinct ways. While immunophenotypic changes Louis Weiner, MD, Yongwei Zhang, Dalal Aldeghaither, David Zahavi, MS, BS post-nivo favored response in ipi-nivo treated patients, changes associ- Lombardi Cancer Center, Washington, DC, United States ated with ipi treatment favored progression with ipi-nivo. These data Correspondence: Louis Weiner (weinerl@georgetown.edu) suggest that the immunophenotypic impact of ipi alters the immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P314 landscape in a manner that may impair a subsequent response to nivo. These data also highlight several novel immune cell populations that Background are associated with both treatment effects and patient outcomes. Accumulating evidence has shown that DNA damage response Ethics Approval (DDR) is closely associated with immune response. Innate im- The study was approved by NYU Langone Health's IRB, approval mune responses, such as Natural killer (NK) cell-mediated killing, number S16-00035. are dependent on the DDR essential kinases Ataxia telangiectasia mutated (ATM) or ATM- and RAD3-related (ATR) [1]. However, P313 DNA damage inducing agents activate the NF-kappaB-regulated Single-cell secretome assessment of metastatic melanoma patient interferon immune response pathway [2]. Moreover, DDR inhib- peripheral T-cells reveals a pharmacokinetic signature of patient ition resulting from DNA repair deficiency or cell cycle check- response to nivolumab therapy point inhibition can potentiate efficacy of antibody-based David Woods, PhD, Andressa Sodre de Castro Laino, Daniel Freeman, immunotherapy, such as immune checkpoint blockade and ADCC Jeffrey Weber, MD, PhD, Pratip Chattopadhyay [3-5]. However, the mechanistic role of DNA damage response NYU Langone Health, New York, NY, United States signalinginthe developmentofimmunotherapy resistance re- Correspondence: David Woods (David.Woods@nyumc.org) mains unclear. A NK cell-mediated cetuximab-dependent killing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P313 in vitroADCCmodel wasusedtostudy this involvement. Our previous studies have suggested a loss of cell surface adhesion Background molecules in ADCC resistant cells [6]. Here we study the alter- Therapies targeting T-cell co-inhibitory molecules (e.g. PD1) have dem- ations of DNA damage response signaling in the process of onstrated unprecedented efficacy in the treatment of metastatic melan- ADCC resistance development. oma. However, not all patients respond to checkpoint inhibition, so Methods there is an unmet need to identify mechanisms of resistance/response. A431, a human epidermoid carcinoma cell line, develops resist- Methods ance to cetuximab and NK92-CD16v cell-mediated killing after Using IsoLight, a platform for assessing the secretion of 32 analytes 35 challenges of continuous exposure. Surviving cells following at single-cell resolution, we evaluated previously frozen peripheral each ADCC challenge were collected and studied for protein ex- blood T-cells from six responding and six progressing patients (ac- pression by western blot, immunofluorescence and flow cytome- cording to RECIST 1.1 criteria) treated with nivolumab. Baseline and try. Neutral comet assay was used to measure the level of DNA week 13, post-treatment CD4+ and CD8+ T-cell samples were double strand breaks. Fluorescence-based cytotoxicity methods assessed for each patient. T-cells were stimulated with CD3 and were used to determine the activity of ADCC. Apoptosis was CD28 activating antibodies overnight and subsequently placed on measured by Annexin-V-PI staining. Small interference RNA capture chips for 20 hours. Approximately 400 single-cell events were (siRNA) were used to knockdown gene expression. assessed for each sample. Results Results Levels of gammaH2AX, a marker of DNA damage signaling, CD4+ T-cells from progressing patients, compared to those from were significantly increased with exposure to ADCC, but no in- responding patients, had significantly (p<0.05) higher mean pro- creased DNA breaks were detected in ADCC resistant cells. p53, duction of IL-17F post-nivolumab and an increased proportion of phosphorylated-p53 and signal transducer and activator of tran- cells secreting IL-13, RANTES, IL-6, soluble CD137, TNF, MIP1a and scription 1 (STAT1) were also enhanced during the process of MIP1b relative to baseline. Using an elastic net machine learning ADCC resistance development. Interestingly, phosphorylated- algorithm with cross validation, we assessed the ability of a STAT1 reached a peak prior to the emergence of ADCC resist- manually curated list of analytes to predict patient outcomes. ance and then decreased until cells became entirely resistant. Delta values (post-treatment minus baseline values) were used for There was less apoptosis induction, no caspase activation, less 15 parameters. A receiver operating characteristic with an area induction of gammaH2AX and no activation of p53 in response under the curve of 0.898 was achieved. The most important fea- to ADCC in resistant cells. Inhibition of p53 or STAT1 by siRNA, tures in these models were the percentage of CD4+ T-cells ex- and ATM/ATR inhibitors enhanced ADCC activity in A431 cells pressing IL-6, MIP1a and soluble CD137 along with the but not in ADCC resistant cells.TP53knockdown activated percentage of CD8+ T-cells expressing IL-13. STAT1 in A431 cells, but reduced activation of STAT1 in ADCC Conclusions resistant cells. These results demonstrate the ability of single-cell, high-dimension Conclusions technologies coupled with machine learning to reveal complex asso- DNA damage response signaling was altered during the develop- ciations between immune cell function and clinical outcomes. ment of ADCC resistance, which is involved in ADCC activity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 171 of 272 regulation and might become be a signature of cell sensitivity in PD1/PD-L1 therapies. PD-L2 expression is observed in multiple tumor response to immunotherapy. Further DDR-related multiplex mech- types, and animal studies suggest PD-L2 may be involved in T-cell anisms are being investigated. suppression. However, the role of PD-L2 expression in the TME and the role as a predictive biomarker in immunotherapy is not as well References understood as PD-L1. Here, we investigated the expression of PD-L2 1. Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathway in multiple cellular components of the TME and its potential impact regulates innate immune system ligands of the NKG2D receptor. Nature. on the efficacy of PD-L1 blockade in cancer patients. 2005;436(7054):1186-90. Methods 2. Brzostek-Racine S, Gordon C, Van Scoy S, Reich NC. The DNA damage re- Single-cell RNA sequencing (scRNAseq) was performed using 10X sponse induces IFN. J Immunol. 2011;187(10):5336-5345. genomics for two commercial NSCLC samples and ~4,500 cells were 3. SenT,Rodriguez BL,ChenL,Corte CMD, Morikawa N, Fujimoto J, clustered by their expression pattern using shared nearest neighbor. Cristea S,Nguyen T, Diao L, Li L, Fan Y, Yang Y, Wang J, Glisson BS, Gene expression data for lung adenocarcinoma (LUAD) and squa- Wistuba II, Sage J, Heymach JV, Gibbons DL, Byers LA. Targeting mous carcinoma (LUSC) in TCGA were analyzed. Baseline tumor tran- DNA Damage Response Promotes Antitumor Immunity through scriptomes were profiled for 97 1L+ NSCLC patients treated with PD- STING-Mediated T-cell Activation in Small Cell Lung Cancer. Cancer L1 inhibitor durvalumab (NCT01693562). PD-L1 and PD-L2 immuno- Discov. 2019,9(5):646-661. staining was performed on baseline samples in CP1108. Gene expres- 4. Fenerty KE, Padget M, Wolfson B, Gameiro SR, Su Z, Lee JH, sion signatures for macrophage, fibroblast, dendritic cells, cancer Rabizadeh S, Soon-Shiong P, Hodge JW. Immunotherapy utilizing associated fibroblast (CAF) and interferon gamma were curated in- the combination of natural killer- and antibody dependent cellu- house or adopted from previous studies. lar cytotoxicity (ADCC)-mediating agents withpoly (ADP-ribose) Results polymerase (PARP) inhibition. J Immunother Cancer. 2018,6(1):133- In scRNAseq data, PD-L2 mRNA was found in over 10% of macro- 136. phage and fibroblast cells, and 5. Germano G, Lamba S, Rospo G, Barault L, Magrì A, Maione F, Russo M, Conclusions CrisafulliG, Bartolini A, Lerda G, Siravegna G, Mussolin B, Frapolli R, PD-L2 mRNA expression is mainly in immunosuppressive cellular Montone M, MoranoF, de Braud F, Amirouchene-Angelozzi N, Marsoni S, components of the TME in NSCLC, including macrophage and cancer D'Incalci M, Orlandi A,Giraudo E, Sartore-Bianchi A, Siena S, Pietrantonio associated fibroblast cells and low PD-L2 expression in PD-L1 high F, Di Nicolantonio F,Bardelli A. Inactivation of DNA repair triggers neoan- NSCLC patients may associate with improved OS. tigen generation and impairstumour growth. Nature. 2017,552(7683):116- Trial Registration 120. NCT01693562 6. Aldeghaither DS, Zahavi DJ, Murray JC, Fertig EJ, Graham GT, Zhang Ethics Approval YW,O'Connell A, Ma J, Jablonski SA, Weiner LM. A Mechanism of This study was conducted according to the Declaration of Helsinki Resistance to Antibody-Targeted Immune Attack. Cancer Immunol Res. and approved by the independent ethics committee/institutional re- 2019;7(2):230-243. view board at each participating center, with informed consent ob- tained from all patients. P316 MG1131, a novel TIGIT-targeted monoclonal antibody, induces T cell activation and anti-tumor immune response and suppresses Treg cell activity 1 1 2 1 Hyemi Nam, MS , Hye-Young Park , Eun Jung Song , Eunhee Lee , Hye 1 1 1 1 1 In Yum , Munkyung Kim , Jeewon Lee, Ph D , So Jung Lim , Okjae Lim , Yangmi Lim MOGAM Institute for Biomedical Research, Yongin-si, Korea, Republic of; GC Pharma, Yongin-si, Gyeonggi-do, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P316 Background Fig. 1 (abstract P314). Enhancement of DDR signaling T cell immunoreceptor with Ig and ITIM domain (TIGIT) is a co- pathway molecules inhibitory receptor expressed on CD8+ T cells, CD4+ T cells, NK cells, and regulatory T cells (Treg). TIGIT binds two ligands PVR (CD155) and PVRL2 (CD112) and these ligands are expressed by P315 T cells, APCs, and tumor cells. As malignancies progress, PVR The expression of programmed death ligand 2 (PD-L2) in over-expressed in tumor cells interacts with TIGIT expressed on immunosuppressive tumor microenvironment of non-small cell tumor infiltrating lymphocytes (TIL) and suppresses TIL activity lung cancer (NSCLC) and its potential association with by sending an inhibitory signal to immune cells, which is an im- immunotherapy 1 2 2 2 mune escape mechanism in cancer. In cancer, TIGIT blockade Qu Zhang, PhD , Stefan Bentink , Vinay Pawar , Farzad Sekhavati, PhD , 1 1 1 results in improved effector CD8+ T cell and NK cell function as Keith Steele, DVM, PhD , Jason Hipp , Song Wu, PhD 1 2 well as decreased Treg-cell-mediated suppression. Therefore, we AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, developed MG1131, a novel anti-TIGIT antibody, to modulate Germany the tumor microenvironment towards a more effective anti- Correspondence: Qu Zhang (zhangq@medimmune.com) cancer response. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P315 Methods TIGIT-targeting antibody candidates were screened out of a Background phage display library. The TIGIT antigen binding affinity and PVR In the tumor microenvironment (TME), programmed death-1 receptor blocking of anti-TIGIT antibodies were evaluated through both (PD-1), combined with its ligands PD-L1 and PD-L2, play an important protein-based and cell-based assays. Functional consequences of suppressive role in the immune response to cancer. PD-L1 expression MG1131 were determined using a cell-based reporter assay for T has been shown to be a key predictive biomarker of response to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 172 of 272 cell activation, a Treg cell functional assay and an NK-mediated group were hypertension (32%), anemia (29%), colitis/enterocolitis tumor killing assay. Treg cells and NK cells were isolated from (26%), and nausea/vomiting (25%). Median survival was signifi- healthy donor PBMC. cantly longer in the IO group relative to the CT group (18.6 Results months and 15.1 months, respectively; adjusted HR 0.73, 95% CI We screened out a few of the clones which stood out showing 0.66-0.81, Figure 1). high affinity binding to TIGIT and significant blocking of the Conclusions TIGIT-PVR interaction. Among the tested antibodies, MG1131 was Among the veteran population in real-world settings, patient identified to have the strongest affinity for human TIGIT and sig- characteristics were similar for 2L IO or CT therapy, with the ex- nificant blocking activity, resulting in competition with PVR in a ception of age and geographic region. Rates of common AEs dose dependent manner. Furthermore, MG1131 was cross-reactive were as expected. Our findings indicate improved survival among with cynomolgus monkey TIGIT, but not with mouse TIGIT. Our patients receiving IO versus CT in the 2L setting. More in vitro efficacy data demonstrated that MG1131 significantly en- population-based studies are needed to confirm these findings in hances T cell activation and NK-mediated tumor killing activities other healthcare settings. in a PVR-dependent manner and MG1131 induces IFN-γ secretion and proliferation of CD8+ T cells by inhibiting Treg suppressive Acknowledgements function. The team would like to thank Daniel Lane, PharmD, PhD, MBA for his Conclusions support of this study. We developed an anti-TIGIT antibody, MG1131, with pronounced in- Ethics Approval hibitory activity on the TIGIT-PVR signaling axis. In this study, This study was approved by the Durham VA Institutional Review Board (IRB MG1131 significantly enhanced T cell activation and NK-mediated #02009). tumor killing activity, and efficiently suppressed Treg cell function. Therefore, MG1131 is a potential candidate for cancer immunotherapy. P317 Utilization of second-line immuno-oncology agents and associated health outcomes among united states veterans with advanced non-small cell lung cancer 1 2 3 Mina Allo, PharmD, MPH , Lin Gu, MS , Vishal Vashistha, MD , Ashlyn 2 2 2 Press, MPH , Michael Kelley, MD , Christina Williams, PHD, MPH 1 2 Bristol-Myers Squibb, Princeton, NJ, United States; Durham Veterans Affair, Duke Cancer Ins, Durham, NC, United States; Dept of Medicine, Duke University, Durham, NC, United States Correspondence: Mina Allo (mina.allo@bms.com); Christina Williams (christina.williams4@va.gov) Fig. 1 (abstract P317). KM curve of overall survival of patients in 2L Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P317 Background Studies describing treatment safety, effectiveness and patterns of use are needed to evaluate the real-world impact of immuno-oncology P318 (IO) in advanced non-small cell lung cancer (NSCLC) relative to Large-scale evaluation of concordance of genomic scores in whole chemotherapy (CT). This retrospective cohort analysis assessed exome sequencing and Foundation Medicine comprehensive utilization of IO and CT agents and associated outcomes in second- genomic platform across cancer types 1 1 1 1 line (2L) treatment among stage IV NSCLC patients receiving care in Deepti Aurora-Garg , Andrew Albright, PhD , Ping Qiu, PhD , Yongjin Li , 1 2 1 1 the Veterans Affairs (VA). Xiaoqiao Liu , David Fabrizio, PhD , Lixin Lang , Jared Lunceford, PhD , Methods Razvan Cristescu, PhD 1 2 The VA Corporate Data Warehouse (CDW) oncology database was Merck & Co., Inc., Kenilworth, NJ, United States; Foundation Medicine, used to determine survival and 14 common adverse events (AE) Cambridge, MA, USA, Cambridge, MA, United States of adult patients with stage IV NSCLC diagnosed from 2012 to Correspondence: Deepti Aurora-Garg (deepti.aurora-garg@merck.com) 2017 who received systemic non-targeted (ALK, EGFR) therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P318 within 120 days of diagnosis and were followed until death or end of the study period in June 2019. Descriptive statistics were Background used to summarize treatment and AE occurrence. Kaplan-Meier Whole exome sequencing (WES) is a comprehensive method to methodology and multivariate Cox regression were used to evalu- evaluate the clinical relevance of DNA molecular characteristics, in- ate survival. cluding pan-exomic genomic scores (eg, tumor mutational burden Results [TMB] and homologous recombination deficiency–loss of heterozy- We identified 1655 patients who received 2L therapy, with 42% gosity [HRD-LOH]) and individual alterations (eg, BRCA1/2). Although (n=695) receiving IO monotherapy (nivolumab, pembrolizumab, comprehensive targeted genomic panels are available to measure atezolizumab, and durvalumab), 56.5% (n=935) receiving CT only, TMB and HRD-LOH, including FoundationOne® CDx (F1CDx), imple- and 1.5% (n=25) receiving IO+CT (not included in the current mentation of WES as a diagnostic approach in clinical practice can analysis due to limited sample). Greater than 99% of 2L IO users be challenging. To assess the feasibility of translating findings using used CT only in 1L setting, and >96% of 2L CT only group used WES as an exploratory tool into a practical diagnostic device such as CT in 1L (~ 3% used IO monotherapy, 0.6% IO+CT). Median age F1CDx, we evaluated the concordance of genomic scores (TMB and was 67 vs. 65 years in the IO and CT groups, respectively (p= HRD-LOH) and single-gene alterations between WES and F1CDx in a 0.006). No statistically significant differences between the IO and large pan-tumor data set. CT groups were observed by sex (~97% male), race (~77% White), Methods ethnicity (~98% non-Hispanic), smoking history (~95% current/ This analysis used solid tumor samples from patients with advanced former smoker), or histology (~58% adenocarcinoma). The most disease who received pembrolizumab monotherapy in the second common AEs in the IO group were dyspnea (50%), colitis/entero- line or later during single-arm clinical trials. WES and F1CDx (Dx1 colitis (40%), and anemia (28%); most common AEs in the CT baitset) were used to analyze samples from 436 patients across 22 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 173 of 272 tumor types. Spearman rank-order correlation and linear regression June 2019. Patient Information Form, Brief Fatigue Inventory (BFI), were used to determine concordance and cutoff equivalence for Functional Living Index-Cancer (FLIC), and Dermatology Life Quality TMB and HRD-LOH, each calculated by both WES [1] and F1CDx Index (DLQI) were used for data collection at 1st, 2nd, 3rd, and 4th (Foundation Medicine proprietary pipeline QSR_F1Dx_v1.0.3). cycles of nivolumab. Descriptive statistics, Mann Whitney U and Results Friedman tests were utilized for data analysis. Using WES and F1CDx, high concordance was observed in the pan- Results tumor assessment of TMB (Spearman correlation, 0.7; n=413) and The majority of the patients was male (60%), and the mean age of HRD-LOH (Spearman correlation, 0.5; n=364). When individual indica- patients was 54.10±18.88 years. The mean time of diagnosis for pa- tions were considered, the concordance was further improved for in- tients with melanoma was 40.50±47.84 months (range 2-168). There dications with higher distribution medians. TMB concordance was were no significant differences between patients’ BFI, FLIC, and DLQI higher when restricted to non–small cell lung cancer (Spearman cor- total scores in terms of age and gender (p>.05). The mean scores of relation, 0.8; n=38). HRD-LOH concordance was higher when re- BFI scores were 4.15±2.90 at the 1stcycle, and 3.75±2.98 at the 4th stricted to ovarian cancer (Spearman correlation, 0.7; n=54) and cycle; FLIC scores were 88.15±9.69 and 95.26±12.07; and DLQI scores breast cancer (Spearman correlation, 0.6; n=80). Regression analysis were 2.60±6.30 at 1.90±3.11, respectively. Considering the changes of TMB using both platforms identified F1CDx (Foundation Medicine within time in terms of all scale scores, no significant differences proprietary pipeline QSR_F1Dx_v1.0.3) TMB cutoffs of 10 and 13 mu- were found in BFI (p=.29), and DLQI (p=.49). With regard to FLIC tations/megabase to correspond to WES TMB of ~150 and ~175 mu- scores a significant difference was found from the 1st to the 4th tations/exome, respectively. Assessment of BRCA1/2 deleterious cycle of nivolumab (p=.05). mutations also demonstrated agreement between WES and F1CDx, Conclusions with 305 of 309 (98.7%) samples showing agreement; 282 samples The present study may be the first effort to evaluate changes in BFI, showed wild-type status by both methods and 23 samples showed FLIC and DLQI scores during nivolumab treatment in patients with mel- mutant status by both methods. anoma from the 1st to the 4th cycle. The study findings revealed that Conclusions improvements in BFI, and DLQI scores following nivolumab, even not The high level of concordance between WES and F1CDx suggests statistically significant. Lastly supporting literature [3,4], significant in- that molecular biomarker discoveries, including clinically relevant cut- crease has been found in FLIC scores with nivolumab treatment. offs and molecular epidemiology findings evaluated on the transla- tional WES platform, may be translated successfully in the diagnostic References setting. To our knowledge, this is the first evaluation of concordance 1. Dine J, Gordon R, Shames Y, Kasler MK, Barton-Burke M. Immune checkpoint of genomic scores performed in the context of clinical trial data inhibitors: an innovation in immunotherapy for the treatment and manage- across many indications and in a large data set. ment of patients with cancer. Asia Pac J Oncol Nurs. 2017; 4(2):127-135. 2. Shepherd FA, Douillard JY, Blumenschein GR. Immunotherapy for non- Reference small cell lung cancer: Novel approaches to improve patient outcome. J 1. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for Thorac Oncol. 2011; 6(10): 1763-1773. PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362. pii: 3. Ramirez RA, Lu J, Thomas KE. Quality of life for non-small cell lung cancer eaar3593. patients in the age of immunotherapy. Transl Lung Cancer Res. 2018; Ethics Approval 7(Suppl 2):149-152. The studies in which patient samples were collected were approved by an 4. Schadendorf D, Larkin J, Wolchok J, Hodi FS. Chiarion-Sileni V, Gonzalez independent ethics committee before being initiated at each site. R, Wagstaff J. Health-related quality of life results from the phase III Check Mate 067 study. Eur J Cancer. 2017; 82: 80-91. Ethics Approval P319 The study was approved by clinical trials ethics committee of the University Changes in fatigue severity, health related, and dermatology of Health Sciences Ankara Oncology Training and Research Hospital related quality of life in melanoma patients receiving nivolumab: (decision number: 2018–04/52) and performed in accordance with the Preliminary results of a prospective study from real-life experience Helsinki Declaration. 1 1 Canan Karadas , Nur Izgu, PhD, RN , Zehra Gok Metin, Assoc Prof, PhD, 1 2 2 RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD P320 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Novel in vivo preclinical humanized models for the evaluation of Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, human specific immune checkpoint inhibitors 4 1 2 1 1 Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Anya Avrutskaya , Fabienne Sonego , Jacob Hauser , Emily O’Koren , 1 2 2 1 1 Correspondence: Canan Karadas (karadas.canan@gmail.com) Robin Ball , Gaëlle Martin , Julie Chaix , Thi Bui , Ian Belle , Elizabeth 1 1 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P319 Reap , Patrick Fadden, PhD , Chassidy Hall , Kader Thiam , Paula Miliani de Marval 1 2 Background Charles River Discovery, Wilmington, MA, United States; Genoway, lyon, Previous studies have reported that nivolumab, as an immune check- France point inhibitor, may enhance survival, reduce therapy related toxic- Correspondence: Paula Miliani de Marval ities and improve quality of life (QoL) among melanoma patients (paula.milianidemarval@crl.com) compared with traditional cytotoxic chemotherapy [1,2]. However, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P320 nivolumab is frequently associated with fatigue, increased risk of skin toxicity and autoimmune-related adverse events. Thus, patients may Background experience important changes in daily living activities, health related In the last few years, there has been an increasing demand for suitable pre- QoL, and skin integrity during nivolumab treatment. In view of this clinical mouse models for evaluating the efficacy of checkpoint inhibition- issue, studies are needed evaluating these important changes related based cancer immunotherapies. During tumor progression, immune cells to nivolumab, concurrently. Therefore, this study aimed to investigate can become unresponsive and evade immune surveillance upon chronic fatigue severity, health related QoL, and dermatology related QoL in activation and expression of the programmed cell death protein-1 (PD-1) melanoma patients receiving nivolumab. the ligand PD-L1 on tumor cells or expression of the T lymphocyte associ- Methods ated antigen 4 (CTLA4) in T-cells cells resulting in tumor immune-tolerance. A total of 20 patients, scheduled to receive first dose of nivolumab, We have previously demonstrated that murine anti-PD-1, anti-PD-L1 and in three leading hospitals located in Ankara, was included in this de- CTLA-4 blockade can effectively enhance immune normalization and re- scriptive, prospective and multicenter study. All the patients received activate the antitumor response against multiple syngeneic tumor models. at least four cycle of nivolumab infusion between October 2018 and While these models proved instrumental for evaluating murine immune- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 174 of 272 checkpoint inhibitors (ICI), there is a clear need for additional mouse models Conclusions to evaluate the efficacy of ICI specific for human targets. While heterogeneous patient characteristics may have influenced Methods the results of this study, the trends observed suggest favorable To address this need, we describe the development of humanized PD- outcomes in patients with aNSCLC treated with IO monotherapy 1 and CTLA-4 knock-in (KI) mouse models. The main advantage of in the 1L setting. Further research should explore whether this is these models is that human PD-1 or CTLA-4 proteins are expressed in related to a predominance of patients with high PD-L1 expression the context of a fully functional immune system. To validate these among those who received IO monotherapies. TTD for IO-based models we evaluated the response to pembrolizumab or ipilimumab in therapies in this real-world setting appears to be shorter than a colorectal carcinoma and a glioblastoma preclinical tumor models. PFS reported in previous trials, indicating an unmet need may re- Results main and needs to be explored. However, these results could be We observed significant tumor growth inhibition and growth delay in the influenced by effects of informative censoring or other underlying MC38 tumor model with either monotherapy, but not when treated with clinical factors. Additionally, future studies should investigate dif- the murine counterparts: anti-PD-1 (clone RPM1-14) or CTLA-4 (clone 9H10). ferences in the tolerability profiles of 1L regimens, as well as To extend our validation studies to other tumor models, we implanted how treatment sequences contribute to outcomes. GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembro- Acknowledgements lizumab compared to both the control group and the group treated with This study was funded by Merck KGaA, Darmstadt, Germany, as part of an alliance murine anti-PD-1 antibody. Furthermore, we found that pembrolizumab between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. and ipilimumab therapy results in enhanced effector functions of CD4+and CD8+ T cells associated with increased expression of Granzyme B. Reference Conclusions 1. Blumenthal GM, Gong Y, Kehl K, et al. Analysis of time-to-treatment dis- In summary, the results shown here underscore the value of resour- continuation of targeted therapy, immunotherapy, and chemotherapy in cing to humanized knock-in (KI) mouse models as tools to evaluate clinical trials of patients with non-small-cell lung cancer. Ann Oncol. human specific immune-checkpoint based therapeutics alone and in 2019;30(5):830-8. combination with other agents. Ethics Approval The study was reviewed and granted exception and waiver of consent by the US Oncology, Inc. Institutional Review Board. P321 Real-world clinical outcomes among patients with advanced non- small cell lung cancer who initiated first-line regimens 1 2 3 3 Eric Nadler, MD , Bhakti Arondekar, PhD , Kathleen Aguilar , Jie Zhou , Table 1 (abstract P321). See text for description 2 4 4 Jane Chang , Xinke Zhang , Vivek Pawar 1 3 Texas Oncology, Medical Oncology, Dallas, TX, United States; Pfizer Inc., New York, NY, United States; McKesson Life Sciences, The Woodlands, TX, United States; EMD Serono, Inc., Billerica, MA, United States Correspondence: Eric Nadler (eric.nadler@USONCOLOGY.COM) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P321 Background While clinical trials have demonstrated the clinical benefit of immuno-oncology (IO) regimens for the treatment of advanced non- small cell lung cancer (aNSCLC), either in combination or as mono- therapies, limited research has evaluated clinical outcomes with these therapies in a real-world setting. This retrospective observa- tional study evaluated time to treatment discontinuation (TTD) and overall survival (OS) in patients with aNSCLC receiving care in US community oncology clinics. Previous research suggests that TTD is a pragmatic real-world efficacy endpoint, as TTD and progression-free survival (PFS) are associated across different types of therapy in NSCLC clinical trials [1], therefore TTD was explored in this study. Methods Patients with aNSCLC who initiated first-line (1L) treatment with sys- temic chemotherapies, targeted therapies, or IO regimens in the US Oncology Network between 3/1/15 and 8/1/18 were included in the study population. Electronic health record data for these patients was captured through 2/1/19. Descriptive analyses were performed to assess baseline characteristics and treatment patterns, and the Kaplan-Meier method was used to evaluate TTD and OS from the start of 1L treatment. Results In total, 7,746 patients were included in this analysis (Table 1): 5,859 (75.6%) initiated 1L systemic chemotherapies, 656 (8.5%) targeted therapies, 907 (11.7%) IO monotherapies, and 324 (4.2%) IO combin- ation regimens (with chemotherapies or targeted therapies). Of these, 51.8%, 50.3%, 21.7%, and 17.6%, respectively, proceeded to a subsequent treatment following 1L discontinuation. Median TTD ranged from 2.0 months (95% CI 1.9-2.1) in patients who received systemic chemotherapies to 3.5 months (95%CI: 2.8-4.2) in patients who received IO monotherapies (Figure 1). Similarly, median OS was longest in patients who received IO monotherapies (19.9 months Fig. 1 (abstract P321). See text for description [95%CI: 16.6-24.1]; Figure 2). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 175 of 272 Conclusions We describe the development of a very high affinity antagonistic TIGIT antibody, COM902, that is currently in preclinical development. Co-expression of TIGIT with PVRIG in TILs and their non-redundant in- hibitory effects on T cell activation suggest a potential therapeutic advantage in clinical combinations targeting both pathways. Towards this end we are planning a trial that will eventually incorporate com- binations of COM902 with the anti-PVRIG antibody, COM701. P323 IPH5301, a CD73 blocking antibody targeting the adenosine immunosuppressive pathway for cancer immunotherapy Ivan Perrot, Caroline Denis, PhD, Marc Giraudon-Paoli, Severine Augier, Rachel Courtois, Diana Jecko, Violette Breso, Thomas Arnoux, Nicolas Gourdin, PhD, Romain Remark, PhD, Cecile Bonnafous, Ariane Morel, PhD, Eric Vivier, Yannis Morel, PhD, Pascale Andre, Carine Paturel, PhD Innate Pharma, Marseille, France Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P323 Fig. 2 (abstract P321). See text for description Background CD73 is an extracellular ectonucleotidase highly expressed by tumoral P322 or stromal cells in the tumor microenvironment. By inducing tumor cell COM902, a novel therapeutic antibody targeting TIGIT augments T death, conventional anti-cancer therapies induce extracellular release cell function and the activity of PVRIG pathway blockade in vitro of adenosine triphosphate (ATP), which is degraded by CD39 into ad- and in vivo enosine monophosphate (AMP) and then by CD73 into adenosine, an Maya Kotturi, BS, PhD, Eran Ophir, PhD, Sarah Whelan, PhD, Spencer inhibitor of immune response. Blockade of CD73-mediated degradation Liang, Kathryn Logronio, BS PhD, Kyle Hansen, BS, Zoya Alteber, PhD, of AMP may therefore stimulate anti-tumor immunity across a wide Mark White, BS, PhD range of tumors through preventing the production of adenosine. Compugen, South San Francisco, CA,United States IPH5301 is a humanized effector-silent IgG1 monoclonal antibody that Correspondence: Eran Ophir (erano@cgen.com) selectively binds to and inhibits the activity of both membrane-bound Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P322 and soluble human CD73. IPH5301 is designed to enhance anti-tumor immune responses by inhibiting the enzymatic activity of CD73 in the Background tumor microenvironment, thus releasing tumor-infiltrating lymphocytes TIGIT is a coinhibitory receptor that is highly expressed on tumor in- from adenosine-mediated suppression. Here, we described the expres- filtrating lymphocytes (TILs), including effector and regulatory (Treg) sion of CD73 in several human solid tumors, characterized IPH5301 CD4+ T cells, effector CD8+ T cells, and NK cells. Engagement of antibody properties and its efficacy in vitro. TIGIT with its cognate ligand PVR directly suppresses lymphocyte ac- Methods tivation. TIGIT and PVR are broadly expressed in different types of CD73 expression was assessed by immunochemistry on cohorts of solid tumors, suggesting that TIGIT-PVR signaling may be a dominant solid tumors i.e breast, ovarian, lung, melanoma, pancreatic and head immune escape mechanism for cancer. Utilizing COM902, a thera- and neck cancer. In vitro efficacy of IPH5301 was evaluated (1) in hu- peutic antibody targeting TIGIT, we demonstrate that co-blockade of man T cell proliferation assays; and (2) in enzymatic assays with lym- TIGIT and a new checkpoint inhibitor, PVRIG, augments T cell re- phocytes and serum from healthy donors and human CD73-knock-in sponses in vitro and in vivo. (huCD73KI) mice. To get more insight into the mechanism of action Methods of IPH5301, CD73-IPH5301 complex was analyzed using electron mi- Multi-color flow cytometry analysis of dissociated tumors was used croscopy and the crystal structure of IPH5301 Fab in complex with to quantify TIGIT and PVRIG expression on TILs. Membranous PVR CD73 ectodomain was determined. and PVRL2 expression was characterized by immunohistochemistry. Results The ability of COM902 to promote T cell responses in vitro, alone Whereas inter-patient variability was observed in all tested indications, and in combination with an anti-PVRIG antibody, COM701, was eval- CD73 expression was always detected mainly on tumor cells and did not uated in a primary TIL assay. To examine the in vivo effects of TIGIT correlate with the expression of CD39 or PD-L1. In vitro IPH5301 effi- blockade with COM902 a chimeric antibody with the constant region ciently restored T cell proliferation and blocked adenosine-mediated sup- of mouse IgG1 was generated. The anti-tumor activity of the chimeric pression of Tcellproliferationinamixedlymphocytereactioninadose- COM902 antibody in combination with an anti-mouse PVRIG anti- dependent manner. IPH5301 did not induce CD73 down-modulation and body was assessed in the mouse CT26 colon carcinoma model. did not directly activate B cells. Furthermore, IPH5301 efficiently blocked Results CD73 enzymatic activity in human serum and whole blood as well as in COM902 is a fully human antibody that binds TIGIT with high affinity serum and splenocytes from huCD73KI mice. Finally, we showed that and specificity and disrupts the binding of TIGIT to PVR. This anti- IPH5301 contrains CD73 in an intermediate inactive form. body binds to TIGIT on human CD8+ T cells with higher affinity than Conclusions tested benchmark antibodies. In dissociated tumor samples, TIGIT ex- These results indicate that IPH5301 blocks CD73 with a differentiated pression was highest on TILs in endometrial, head and neck, kidney mechanism of action compared to benchmarked anti-CD73 clinical and lung tumors, and directly correlated with PVRIG expression. Ex- candidates and support the clinical development of IPH5301 for can- cept for breast tumors, PVR was moderately to highly expressed in cer immunotherapy, potentially in combination with chemotherapy all tumor types examined, while PVRL2 expression was highest in or immune checkpoint inhibitors. prostate, ovarian, liver and endometrial tumors. Combination of COM902 and COM701 resulted in enhanced CD3+ TIL activity Acknowledgements in vitro. Furthermore, the combination of chimeric COM902 and anti- The research leading to CD73 results were obtained within the TumAdoR PVRIG resulted in significant CT26 tumor growth inhibition and en- collaborative consortium that received funding from the European hanced overall survival, which was comparable to the combination Community's Seventh Framework Program (FP7/2007-2013) under grant of chimeric COM902 and anti-PD-L1. agreement n°602200. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 176 of 272 Clinical Trial Completed outcomes in other cancer types and in randomized settings will pro- vide additional insight into their prognostic or predictive character. P324 Pan-tumor analysis of the association of cancer and immune Acknowledgements biology-related gene expression signatures with response to Joanne E Tomassini for writing support and Sheila Erespe for editorial pembrolizumab monotherapy support, both employees of Merck & Co., Inc. 1 1 Razvan Cristescu, PhD , Michael Nebozhyn, PhD , Chunsheng Zhang, Trial Registration 1 1 1 1 PhD , Andrew Albright, PhD , Julie Kobie, PhD , Lingkang Huang , Qing NCT01295827, NCT01866319, NCT02335411; NCT02335424; NCT01848834; 1 1 1 1 1 Zhao, MD PhD , Anran Wang , Hua Ma , Andrea Webber , Petar Jelinic , NCT02255097; NCT02447003; NCT02674061; NCT02853344 1 1 1 1 Mohini Rajasagi , Sandra Souza , Raluca Predoiu , Z. Alexander Cao , 1 1 1 1 Junshui Ma , Michael Morrissey , Clemens Krepler, MD , Stephen Keefe , References 1 1 1 Jonathan Cheng, MD , Vassiliki Karantza , Sukrut Shah , Rodolfo Perini, 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-gamma-related mRNA pro- 1 2 3 4 MD , Antoni Ribas, MD, PhD , Petros Grivas, MD, PhD , David Cescon , file predicts clinical response to PD-1 blockade. J Clin Invest 1 1 1 Terrill McClanahan, PhD , Alexandra Snyder, MD , Mark Ayers, PhD , 2017;127:2930-2940. 1 1 Jared Lunceford, PhD , Andrey Loboda, PhD 2. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for 1 2 Merck Inc., Boston, MA, United States; University of California, Los PD-1 checkpoint blockade-based immunotherapy. Science Angeles, Los Angeles, CA, United States; University of Washington, 2018;362:eaar3593. Seattle, WA, United States; Princess Margaret Cancer Centre, Toronto, 3. Ayers M, Nebozhyn M, Cristescu R, et al. Molecular Profiling of Cohorts of Canada Tumor Samples to Guide Clinical Development of Pembrolizumab as Correspondence: Razvan Cristescu (razvan_cristescu@merck.com) Monotherapy. Clin Cancer Res 2019;25:1564-1573. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P324 Ethics Approval The clinical trials included in this analysis were approved by the appropriate Background ethics committees at each participating study center. RNA sequencing (RNASeq) data on baseline tumor biopsies from pa- tients in pembrolizumab monotherapy studies were used to explore Table 1 (abstract P324). See text for description potential relationships between key biological gene expression signa- tures and objective response rate (ORR) in the trials. Methods A canonical set of 10 consensus signatures representative of key tumor biology and tumor microenvironment (TME) elements beyond the 18- gene T-cell inflamed gene expression profile (GEP [1]) was defined using the independent Merck-Moffitt and TCGA databases [2,3], exter- nal to any pembrolizumab trial and prior to relating RNASeq data to clinical outcomes from studies evaluated. These signatures (Angiogen- esis, Hypoxia, Glycolysis, Proliferation, MYC, RAS, gMDSC, mMDSC, Stroma/EMT/TGFβ, WNT) were evaluated in the trial dataset blinded to clinical outcome, to test the association with ORR (RECIST 1.1; where re- sponse=PR or CR). Studies with available RNASeq data (N=1188) in- cluded: KN001/KN006-Melanoma (N=476; pembrolizumab-treated and ipilimumab-naïve), KN052-urothelial (N=186), KN012/KN055-HNSCC (N= 147; HPV-negative by whole exome sequencing), KN086-TNBC (N=132), KN059-Gastric (N=92), and KN427-RCC (N=78), KN100-Ovarian (N=77). Pan-cancer logistic regression analysis of ORR for consensus signatures included terms adjusting for cancer type, ECOG performance status, and the T-cell inflamed GEP, an approach equivalent to evaluating asso- ciation between ORR and the residuals of consensus signatures after detrending them for their relationship with the T-cell inflamed GEP and cancer type. Testing of the 10 pre-specified consensus signatures for P325 negative association (except Proliferation with a hypothesized positive- Tumor Infiltrating Lymphocytes (TILs) in triple-negative breast association) with ORR was adjusted for multiplicity. cancer: High Immunoscore is associated with pathological CR in Results patients receiving neoadjuvant chemotherapy. 1 2 3 4 Covariance patterns of the 11 signatures (including GEP) in Merck- Bernardo Rapoport, MD , Simon Nayler , Jerome Galon, Dr , T Mlecnik , 1 1 4 5 Moffitt and TCGA showed highly concordant co-expression patterns Teresa Smit , Jacqui Barnard-Tidy , Aurelie Fugon , Marine Martel , 6 2 in the RNASeq data from pembrolizumab trials. As anticipated, the T- Ronald Anderson, Professor , Carol Benn cell inflamed GEP demonstrated the strongest association with ORR The Medical Oncology Centre of Rosebank, Johannesburg, South Africa; 2 3 to pembrolizumab. Beyond the positive association seen with T-cell Prof., Johannesburg, South Africa; LABORATORY OF INTEGRATIVE inflamed GEP, three other RNA signatures, Angiogenesis, mMDSC CANCER IMMUNOL, France, France; Luminy Biotech enterprises 163 Ave and Stroma/EMT/TGFβ, exhibited negative associations at the 0.05 de Lu, Marseille, France; HalioDx, Marseille, France; level after adjusting for multiple testing (Table 1). Immunology,University of Pretoria, Pretoria, South Africa Conclusions Correspondence: Teresa Smit (pharmacist@rapoport.co.za) Pan-cancer testing of exploratory gene expression signatures using Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P325 the RNASeq platform in 1188 patients from single-arm pembrolizu- mab trials suggests that features beyond interferon gamma-related Background T-cell inflammation may be relevant to response to anti-PD1 mono- The presence of high levels of tumor infiltrating lymphocytes (TILs) has therapy and may define other axes of tumor biology as rational can- been associated with better prognosis in early triple-negative breast didates for pembrolizumab combinations. These features cancer (TNBC). Immunoscore is a prognostic tool, which categorizes the (Angiogenesis, mMDSC and Stroma/EMT/TGFβ) have been previously densities of spatially positioned CD3 and CD8 cells in both invasive hypothesized to represent immune-suppressive axes with potential margins (IM) and the center of the tumor (CT) yielding a five-tiered clas- negative impact on immunotherapy efficacy. Future evaluation of sification (0–4). High immunoscores have been reported to be associ- the association of these signatures with response and survival ated with improved outcomes in patients with colorectal cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 177 of 272 Methods low initial immunity, characterized as 0.05. Furthermore, patients with We performed the Immunoscore in a cohort of 103 breast cancer low initial immunity who received the DNA-based vaccine showed (BC) patients previously receiving neo-adjuvant chemotherapy. There improved immunogenicity towards the unvaccinated extracellular were triple-negative (TNBC)=53, Luminal=32, Her2+=18 who received domain of HER2 at 1 month and 6 months, p treatment with anthracycline and/or taxane- and/or trastuzumab- Conclusions based neo-adjuvant chemotherapy. Pre-treatment tumor samples A comparison of DNA-based and peptide-based vaccines targeting were immune-stained for CD3 and CD8 T-cell markers. Quantitative HER2 intracellular domain epitopes revealed a more robust and long- analysis of the immune cells was carried out using a computer- lasting immunogenic response with the DNA-based vaccine while assisted image analysis in different tumor locations. maintaining an excellent safety profile. Additionally, immune re- Results sponses to extracellular domain regions of HER2 demonstrate the The pathological complete response (pCR) rate of the entire cohort intra-epitope spreading potential in the DNA-based vaccine. was 44%. On univariate analysis factors associated with higher pCR included primary tumor size (T1=43.48% vs. T2=52.31% vs. T3+T4 Acknowledgements 6.67%, Chi2=10.3201, p40=56.86% vs.15-39=40.54% vs. A special thanks to the Boyd Scholarship for financial assistance during my T-cell density subsets (CD3, CD8) and Immunoscore were significantly research. higher in TNBC compared to non-TNBC patients. Receiver-operating Trial Registration characteristic (ROC) curve analysis was used to determine the opti- Clinicaltrials.gov: NCT00436254 and NCT0034310 mal cut-off points for CD3 and CD8. A high density of CD3 (> than 800mm2) and CD8 (> than 400mm2) positive T-cells in the CT was References associated with higher pCR (CD3 CT:60% vs.25%, p=0.00035 and CD8 1. Datta J., et. al. Progressive loss of anti-HER2 CD4+ T-helper type 1 re- CT: 64% vs.27%, p=0.00016). Analysis of CD3 (> than 1400mm2) (CD3 sponse in breast tumorigenesis and the potential for immune restoration. IM:63% vs.19%, p=0.0001) and CD8 densities in the IM (> than Oncoimmunology. 2015;4(10):e1022301. 500mm2) was also significantly associated with pCR (CD8 IM:63% vs. 2. Mittendorf EA, et. al. Clinical trial results of the HER-2/neu (E75) vaccine 15%, p=0.00003). High immunoscore (24/38 pts (63%)) vs. intermedi- to prevent breast cancer recurrence in high-risk patients: from US Military ate (17/48 pts (35%)) vs. low (4/17 pts (24%)) was significantly associ- Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer. ated with pCR (p=0.00674). In a logistic regression model, Ki-67 (p 2012;118(10):2594-602. Conclusions 3. Disis, M.L., et al., Generation of T cell immunity of the HER-2/neu protein The results of this study show a significant prognostic and potentially after active immunization with HER-2/neu peptide based vaccine. J. Clin predictive role for the Immunoscore and Ki-67 in BC patients, particu- Onc, 2002;20(11): 26424-2632 larly in the TNBC subset. 4. Disis, M.L., et al., Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based Acknowledgements vaccine. Clin Cancer Res, 1999;5(6):1289-97. Dr Ronwyn van Eeden 5. Disis ML, et. al. A phase I trial of the safety and immunogenicity of a Ethics Approval DNA- based vaccine encoding the HER-2/neu (HER2) intracellular domain Ethics Approval was obtained from Pharmaethics SA and University of PTA, in subjects with HER2+ breast cancer. ASCOAnnual Meeting. 2014; approval no 517/2017 6. Disis ML, et. al. Phase II study of a HER-2/neu (HER2) intracellular domain Consent (ICD) vaccine given concurrently with trastuzumab in patients with newly Written informed consent was obtained from the patient for publication of diagnosed advanced stage breast cancer. SABCC. 2009; this abstract and any accompanying images. A copy of the written consent 7. Aurisicchio L., Ciliberto G. Genetic cancer vaccines: Current status and is available for review by the Editor of this journal. perspectives. Expert Opin. Biol. Ther. 2012;12:1043–1058. P326 P327 A retrospective analysis of DNA plasmid and peptide-based Evaluation of PD-L1 and cutoff selection to define a predictive vaccine therapy in treatment of HER-2/neu+ breast cancer biomarker for pembrolizumab monotherapy in esophageal cancer 1 1 1 Aaron Stewart, BS , William Gwin, MD , Mary Disis, MD, FACP , Jennifer using KEYNOTE-180 1 2 1 1 Childs , James Dai , Doreen Higgins, RN, BSN, OCN , Angela Kask Mary Savage, Serafino Pantano, Qi Liu, Jared Lunceford, PhD, Peter Kang, 1 2 University of Washington, Seattle, WA, United States; Fred Hutchinson Pooja Bhagia, MBBS, MD, Kenneth Emancipator, MD Cancer Research Center, Seattle, WA, United States Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United States Correspondence: William Gwin (wrgwin@medicine.uw.edu) Correspondence: Kenneth Emancipator Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P326 (kenneth.emancipator@merck.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P327 Background Patients with HER-2/neu+ overexpressing breast cancer often lose Background immunity toward the HER2 antigen [1]. Vaccines are capable of indu- Interim analysis of the KEYNOTE-180 study (NCT02559687) was used cing a cytotoxic T lymphocyte immune response toward overexpress- to establish a relationship between PD-L1 expression levels and ob- ing antigens, leading to targeted tumor destruction [2-4]. Clinical jective response rate (ORR) in patients with esophageal cancer whose trials have explored peptide-based and DNA-based vaccines as pos- disease progressed after ≥2 lines of therapy and to select a PD-L1 sible vehicles for vaccine delivery, though a direct comparison of cutoff for further validation in the randomized setting (ie, KEYNOTE- safety and immunogenicity has not been previously studied[5-6]. We 181). hypothesize that the DNA-based vaccine will produce superior im- Methods munogenicity due to stable plasmid persistence within the tissue KEYNOTE-180 was a single-arm, open-label, phase 2 study of pem- leading to prolonged HER2 immune response[7]. brolizumab in patients with previously treated, advanced esophageal Methods cancer. Pembrolizumab 200 mg was given intravenously every 3 We retrospectively analyzed adverse events and ELISpot data from weeks. The primary objective was ORR. Patients were required to 104 patients treated with vaccines targeting the intracellular domain provide a tumor sample for retrospective analysis of biomarkers, of HER2/neu using either DNA or peptide fragments. which may predict response to pembrolizumab. PD-L1 expression Results was measured using the PD-L1 IHC 22C3 PharmDx assay and evalu- Adverse event profiles of the 104 patients analyzed were similar with ated using a combined positive score (CPS). CPS is the ratio of PD- no reported grade 3, 4, or 5 events. There was no significant effect L1–expressing cells (tumor cells, lymphocytes, macrophages) to vi- on left ventricular ejection fraction (p=0.88 and p=0.59). Patients with able tumor cells. Testing for a relationship between CPS and ORR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 178 of 272 (per RECIST v1.1 by central review) was conducted using logistic re- Background gression. Cutoff selection proceeded by joint evaluation of the ORR BL-8040 (BL), a CXCR4 antagonist, increases T cell entry into the enrichment profile, the sensitivity and specificity profile (receiver op- bloodstream and thereafter into tumor in humans. We hypothesized erating characteristic analysis), the prevalence of patients with late- that BL in combination with pembrolizumab (P) may thereby pro- line esophageal cancer selected by the cutoff, and trends in overall mote efficacy in metastatic pancreatic cancer (mPC). survival (OS) by Kaplan-Meier (KM) curves. Methods Results Methods: This phase IIb open label study enrolled patients with There were 8 responders among 105 patients with available PD-L1 progression after at least one prior chemotherapy for mPC. Two results at the time of interim analysis (March 1, 2017). CPS was statis- weeks of single agent BL (1.25 mg/kg) was followed by 3-week tically significantly associated (P=0.03) with probability of response. A cycles of P (200 mg IV d1) plus BL (d1,4,8,11). Biopsies for tumor cutoff of CPS 1 did not show enrichment of ORR, whereas higher cut- biology were performed before treatment, after BL monotherapy offs did (Table). CPS 10 showed >3-fold enrichment in ORR above (optional), and after BL/P combination versus below the cutoff. Although higher cutoffs indicated further Results enrichment for ORR, attendant drops in sensitivity and prevalence As of July 2019, 20 pts enrolled; 15 were evaluable for the pri- occurred. Separation between KM OS curves for CPS ≥10 versus CPS mary endpoint of radiologic response. Baseline characteristics: Conclusions median age 66, 10M/10F, median 2 prior lines of therapy (range CPS was useful for identifying patients who responded to pembroli- 1-3). Best overall response includes 1 PR, 2 SD, 12 PD yielding zumab monotherapy. Through the evaluation of several clinical utility 21.4% disease control (1PR+2SD). Median TTP was 2 months over- dimensions, CPS 10 was chosen for further validation in the random- all and 7 months for the PR/SD pts. Median OS was 7 months ized setting based on its ability to enrich for ORR and simultaneously overall and 12 months in PR/SD pts. The combination was well to preserve sensitivity. The preservation of sensitivity, along with tolerated with most AEs being injection site discomfort. Five pa- prevalence, was considered particularly important because of the tients experienced grade 3/4 toxicities. Grade 3 toxicities included safety profile of pembrolizumab and the paucity of treatment op- HTN (n = 1), Alk Phos (n = 1), N/V (n = 2), ascites (n = 1), dys- tions in this patient population. pnea (n = 1), and abd pain (n = 2). One pt had grade 4 dyspnea. Trial Registration Paired biopsies have been analyzed for six patients (1 PR 2 SD 3 ClinicalTrials.gov, NCT02559687 PD). Patients with PR/SD had, at baseline, trends towards greater Ethics Approval T cell, especially cytotoxic CD8+ T cell counts within the tumor The study and the protocol were approved by the Institutional Re- niche than patients with PD (T cells: 188-627 cells/mm2 vs 7-41 view Board or ethics committee at each site. cells/mm2, Cytotoxic CD8+ T cells: 18-137 cells/mm2 vs 0-2 cells/ Consent mm2). The PR patient demonstrated an increase in cytotoxic All patients provided written informed consent to participate in the CD8+ T cell number in the tumor niche and reduction in the clinical trial. stroma following treatment. Additional molecular profiling data from multiplex IF will be available at the meeting. Conclusions Table 1 (abstract P327). See text for description This combination of immunotherapy with pembrolizumab plus BL-8040, without cytotoxic chemotherapy, shows clinical activity in patients with pancreatic cancer even in this heavily pretreated population. The combination was well tolerated. We noted a trend towards greater CD8+ T cell infiltrate at baseline within the tumor cell niche among patients who demonstrated clinical bene- fit. OS for all comers was longer than expected in this heavily pretreated patient group suggesting that P + BL might have a salutary effect on survival time if used earlier in the disease course. Trial Registration NCT02907099 Ethics Approval This study was approved by the M.D. Anderson Institutional Review Board, approval number 2016-0410. P329 PolyPEPI1018 off-the shelf vaccine as add-on to maintenance P328 therapy achieved durable treatment responses in patients with A phase IIB study of Pembrolizumab plus BL-8040 in metastatic microsatellite-stable metastatic colorectal cancer patients (MSS pancreatic cancer: Clinical outcomes and biological correlates 1 1 1 mCRC) David Fogelman, MD , Michael Overman, MD , Robert Wolff , Milind 1 1 1 2 1 2 1 Joleen Hubbard, MD , Chiara Cremolini, MD , Rondell Graham, MD , Javle, MD , Shubham Pant, MBBS , Gauri Varadhachary , Rachna Shroff , 1 1 2 1 1 Roberto Moretto, MD , Jessica Mitchell, CNP , Jaclynn Wessling , Eniko Ignacio Wistuba, MD , Carmelia Barreto, PhD , Renganayaki 1 3 3 3 3 3 3 Toke , Zsolt Csiszovszki, PhD , Orsolya Lőrincz , Levente Molnár , Eszter Pandurengan, MS , Sandesh Subramanya, PhD , Debora Ledesma, PhD , 4 4 4 3 3 3 3 3 Somogyi , Mónika Megyesi , Kata Pántya , József Tóth , Péter Páles , Abi Vainstein-Haras, MD , Ella Sorani, PhD , Tzipora Lustig , Osnat 4 4 5 6 3 2 1 István Miklós , Alfredo Falcone, MD , Joleen Hubbard, MD Kashtan , Yosi Gozlan , Steven Townson, PhD , Jeanne Fahey, PhD , 1 1 2 Mayo Clinic, Rochester, MN, United States; Azienda Ospedaliera James Yao, MD 1 2 3 Universitaria Pisana, Pisa, Italy; Treos Bio, Budapest, Hungary M.D. Anderson Cancer Center, Houston, TX, United States; University of Correspondence: Joleen Hubbard (joleenhubbard@gmail.com) Arizona, Tuscon, AZ, United States; MD Anderson Cancer Center, 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P329 Houston, TX, United States; Bioline Rx, Modi'in, Israel; Merck & Co., Inc., Shoreline, WA, United States; Merck & Co., Inc, Boston, MA, United Background States PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine against CRC, Correspondence: David Fogelman (dfogelman@mdanderson.org) containing 12 immunogenic epitopes derived from 7 conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P328 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 179 of 272 cancer antigens frequently expressed in mCRC based on the analysis tologous cell products. Whilst feasible in such relatively small of 2,931 biopsies. Here we report the results of the phase I study of patient populations, delivering an autologous product to large PolyPEPI1018 vaccine as an add-on to maintenance therapy in MSS cohorts of patients is likely beyond the current logistical cap- mCRC patients. abilities. In the phase 1 alloSHRINK study, we tested the first-in- Methods class non-gene edited allogeneic CAR T-cell therapy, CYAD-101, 11 patients with MSS mCRC in the first-line setting were vaccinated administered concurrently with chemotherapy, for the treatment with PolyPEPI1018 just after the transition to maintenance therapy of metastatic colorectal cancer (mCRC). The NKG2D-based CAR with a fluoropyrimidine and a targeted agent (bevacizumab). (Part A: of CYAD-101 targets eight ligands present at high frequencies n= 5, single dose, 12 weeks follow-up; Part B: n= 6, 3 doses, Q12W). in mCRC, not only on tumor cells but also cells from the tumor Primary endpoints were safety and immunogenicity. Multiple analysis microenvironment, and co-express a T-cell receptor (TCR) inhi- of vaccine-induced immune responses in blood and tumor were per- biting molecule (TIM) that interferes with TCR signaling in an at- formed. Both immune response and clinical benefit were predicted tempt to avoid the main issue of allogeneic T-cell therapy, the using the autologous HLA-genotype determined from patient’s saliva graftversushostdisease (GvHD). sample. Methods Results The alloSHRINK study (NCT03692429) evaluates the safety and The vaccine was well tolerated; most common side effects were tran- clinical activity of multiple infusions of CYAD-101, administered sient skin reactions and flu-like syndrome. No vaccine-related SAE oc- concurrently with standard of care FOLFOX chemotherapy, in pa- curred. 90% of patients had vaccine-specific CD8+ T-cell responses of tients with non-resectable mCRC who received prior chemother- memory-effector type against at least 2 of the 7 vaccine antigens, 5 apy lines (i.e. rechallenge population). Three dose-levels (DL; on average. Vaccine specific CD4+ T-cell responses were detected in 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated all patients. Ex vivo CD8+ T cell responses of effector type were de- through a 3+3 design. tected in 71% of patients, as well as increased fractions of CRC- Results reactive, polyfunctional, circulating CD8+ and CD4+ T cells in pa- In total 12 patients have been enrolled in the dose escalation tient’s PBMC after vaccination. Among the 11 patients 3 patients had segment, now completed (3 at DL1, 3 at DL2 and 6 at DL3). At objective tumor response according to RECIST v1.1, one of them re- the time of submission, only data from the first two DLs were ceived a single dose and 2 of them received 3 doses. For the Part B available. At DL1 and DL2, there was no report of dose-limiting of the study, the Objective Response Rate (ORR) was 33% (2/6) and toxicity (DLT) and no patient experienced Grade ≥ 3 related ad- the Disease Control Rate (DCR) was 67% (4/6). Notably, one patient verse events (uncleaned database). No clinical evidence of GvHD experienced complete tumor shrinkage on 2 of 3 target lesions and has been recorded. Best overall response ≥ 3monthsinclude 1 partial response on 1 lesion after 25 weeks of treatment, qualifying partial response and 3 stable disease over the first 6 patients for curative surgery. Median duration of disease control was 9 (DL1 and 2). At DL1 and 2, preliminary data show a dose- months (95%CI 6.3-11.5) (mPFS not reached during the study). The dependent effect on the cell kinetics and control of the host- 10 month PFS was 50% (3/6). Predicted vaccine antigen-specific versus-graft response against CYAD-101 cells as evidenced by the CD8+ T cell responses were confirmed in vitro with a PPV of 79% (p= similar levels of CYAD-101 engraftment after 2nd and 3rd 0.01). Predicted multiantigenic immune responses tend to correlate infusions. with both PFS and tumor volume reduction. Conclusions Conclusions As of August 2019, no GvHD has been observed following infu- Treatment with PolyPEPI1018 vaccine and maintenance therapy was sions of non-gene edited allogeneic CAR T-cells to mCRC pa- safe, well-tolerated, and demonstrated evidence of immunological tients at the first two DLs, with preliminary signals of clinical and clinical activity in MSS mCRC tumors. In addition predicted multi- activity. The study will have reached protocol-specified end- antigenic immune responses indicated treatment benefit, which sup- points for analysis at the time of presentation and safety, clin- ports further development of a companion diagnostic together with ical and cell engraftment will be presented. The results from the vaccine. this study, in comparison with a study evaluating the autolo- Trial Registration gous analog of CYAD-101 in mCRC will provide critical informa- NCT03391232 tion to support the development of CAR-T therapy in solid Ethics Approval tumors. This study was approved by Mayo Clinic Institutional Review Board Trial Registration and by Central Ethics Committee, Italy (Protocol number: OBERTO- NCT03692429 101). Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics P330 Boards and authorities. Results from the completed dose-escalation of the alloSHRINK phase I study evaluating the allogeneic NKG2D-based CAR T- cell therapy CYAD-101 in metastatic colorectal cancer patients P331 1 1 2 Hans Prenen, MD , Marika Rasschaert, MD , Alain Hendlisz, MD , Leila Results from the completed dose-escalation phase I SHRINK study 2 3 3 Shaza, MD , Erik Alcantar-Orozco, MD, PhD , Emilie Cerf, PhD , Florence evaluating the autologous NKG2D-based CAR T-cell therapy CYAD- 3 3 3 4 Renard , Caroline Lonez, PhD , Anne Flament , Jeroen Dekervel, MD , 01 in metastatic colorectal cancer patients 4 1 1 1 Eric Van Cutsem, MD, PhD Leila Shaza, MD , Alain Hendlisz, MD , Ahmad Awada, MD, PhD , Jean- 1 2 2 3 University Hospital Antwerp (UZ Antwerp), Edegem, Belgium; Luc Canon, MD , Javier Carrasco , Eric Van Cutsem, MD, PhD , Jeroen 2 3 3 4 4 Institut Jules Bordet, BRUSSELS, Belgium; Celyad, Mont-Saint- Dekervel, MD , Erik Alcantar-Orozco, MD, PhD , Florence Renard , Emilie 4 4 4 4 Guibert, Belgium; University Hospital Leuven (UZ Leuven), Leuven, Cerf, PhD , Caroline Lonez, PhD , Anne Flament , Marc Van den Eynde, 5 5 Belgium MD , Jean-Pascal Machiels, MD, PhD 1 2 Correspondence: Caroline Lonez (clonez@celyad.com) Institut Jules Bordet, Brussels, Belgium; Grand Hôpital de Charleroi Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P330 (GHdC), Charleroi, Belgium; University Hospital Leuven (UZ Leuven), 4 5 Leuven, Belgium; Celyad, Mont-Saint-Guibert, Belgium; Cliniques Background Universitaires Saint Luc, Brussels, Brussels, Belgium Current success of chimeric antigen receptor T-cell (CAR-T) ther- Correspondence: Caroline Lonez (clonez@celyad.com) apy in hematological malignancies has been achieved using au- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P331 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 180 of 272 Background Methods Chimeric antigen receptor T-cell (CAR-Ts) therapies have yet to demon- The antigen-specific T cell response in the patients who received a strate positive results in the context of solid tumors largely due to the vaccine targeting the prostate cancer antigen, TARP (TCRγ alternate lack of suitable target antigens. NKG2D-based CARs target 8 stress ligands reading frame protein)[1] for biochemically recurrent (D0) prostate notably expressed to a very high frequency across the metastatic colorec- cancer on NCT00972309 was assessed. Patients with HLA-A0201 re- tal cancer (mCRC) patient population. The autologous NKG2D-based ceived vaccination at weeks 3, 6, 9, 12 and 15 following 1:1 CAR-T therapy CYAD-01 achieved stable disease in several patients with randomization between a vaccine consisting of TARP peptides, mon- mCRC when given as a monotherapy in a multiple injection setting with- tanide ISA 51 VG and GM-CSF versus autologous dendritic cell (DC) out any other supportive therapy (THINK study). In the SHRINK phase 1 pulsed with TARP peptides. Both vaccines used two types of pep- study, CYAD-01 was given concurrently with FOLFOX chemotherapy. tides; wild type (WT) TARP 27-35 (TARP2735) and epitope-enhanced Methods TARP 29-37 peptides (TARP2937-9V). The peripheral blood mono- The SHRINK phase 1 study (NCT03310008) evaluated the safety and clin- nuclear cells were collected at baseline and study weeks following ical activity of multiple infusions of CYAD-01, administered concurrently vaccination to be stored in liquid nitrogen until analysis. Cells were with FOLFOX chemotherapy in mCRC patients. Three dose-levels (DL; thawed and stimulated in vitro with TARP peptides with cytokine 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated through support for multicolor flow cytometry. a 3+3 design in two different mCRC patient populations: (i) resectable Results liver dominant mCRC with FOLFOX chemotherapy as 1st line treatment CD8+ T cell subsets were analyzed for association with the dis- (i.e. neoadjuvant population), and (ii) non-resectable mCRC with prior ease response in 5 patients each who were responders and non- chemotherapy lines for mCRC including FOLFOX and/or FOLFIRI (i.e. re- responders whereas response was defined as slowing of PSA challenge population). slope log value as previously published by Wood et al [3]. The Results proportion of PD1-expressing CD8+ cells showed statistically im- The three DL have been completed with 9 patients in total (3 at each portant differences from baseline with an increase in responders DL), without any report of dose-limiting toxicity (DLT). Only 1 patient and a decrease in non-responders at week 12 after stimulation experienced Grade 3 related adverse event (AE) and no patient expe- with TARP 2735 (p= 0.016), TARP 2937 (p=0.032) and TARP 2937- rienced Grade 4 related AE (uncleaned database as of August 2019). 9V (p= 0.016) peptides. Other CD8+ subsets with granzyme A, Best overall response ≥ 3 months includes 1 partial response and 6 IFN-γ, IL-2, TNFα and perforin positive cells were investigated. stable disease out of 9 patients. Preliminary data show a dose- CD8+TNFα+ cells at week 12 (p=0.032) and CD8+perforin+ cells dependent effect on the cell kinetics. The study will have reached at week 18 (p=0.032) stimulated with TARP 2937 also showed protocol-specified endpoints for analysis at the time of presentation. statistically large differences with an increase in responders and a Conclusions decrease in non-responders. Early data show preliminary signs of clinical activity with the concurrent Conclusions administration of CYAD-01 and FOLFOX chemotherapy in the present Antigen-specific CD8+ T cells from responders as defined by reduced SHRINK study. Safety, clinical and translational research data (cell engraft- PSA slope log showed statistically greater activation as assessed by ment) will be presented. The results from this study, in comparison with expression of activation marker PD-1 than those from non- the results from a similar Phase I study evaluating the allogeneic analog responders after the vaccination in a first-in-human trial targeting of CYAD-01 in mCRC patients (i.e. CYAD-101), will provide critical informa- TARP. The antigen-specific T cell response to the vaccine peptides is tion to support the development of CAR T-cell therapy in solid tumors. an immune correlate of vaccine-induced protection and may be used Trial Registration to guide the ongoing study NCT02362451 that does not have HLA NCT03310008 restrictions. Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics Acknowledgements Boards and authorities. This work was supported by the Center for Cancer Research, National Cancer Institute, National Institue of Health. Trial Registration P332 NCT00972309 Cancer vaccine against prostate cancer antigen TARP induces antigen-specific CD8+ T cells with upregulation of activation marker References PD1 in patients with decreased PSA velocity in D0 prostate cancer 1 1 2 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein Hoyoung Maeng, MD , Brittni Moore , Lauren Wood, MD , Seth 1 1 1 expressed in prostate and breast cancer cells derived from an alternate Steinberg , Katherine McKinnon, MS , Masaki Terabe, PhD , Purevdorj 1 1 1 reading frame of the T cell receptor gamma chain locus. Proc Natl Acad Olkhanud , Ira Pastan, MD , Jay Berzofsky, MD, PhD 1 2 Sci U S A. 2000;97(17):9437–9442. National Cancer Institute, Bethesda, MD, United States; PDS 2. Oh S, Terabe M, Pendleton CD et al. Human CTLs to wild-type and en- Biotechnology, Berkeley Heights, NJ, United States hanced epitopes of a novel prostate and breast tumor-associated pro- Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) tein, TARP, lyse human breast cancer cells. Cancer Res 2004;64(7):2610- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P332 3. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated Background with slowing in PSA velocity and decreasing tumor growth rates in With the success of immune checkpoint inhibitors, anti-tumor im- patients with StageD0prostatecancer. Oncoimmunology. munity is at the focus of cancer therapy. The pursuit of the mechan- 2016;5(8):e1197459. ism to boost anti-tumor T-cell responses is critical to improve the Ethics Approval suboptimal response rate to checkpoint inhibitors. Cancer vaccines The study was approved by the National Cancer Institute Ethics Board can be used to induce such tumor-specific T-cell responses as one of assigned a local number 09C0139, approval number P08397.” the solutions to low responses to checkpoint inhibitors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 181 of 272 P333 Ethics Approval Timed anti-tumor vaccination during chemotherapy induces This study was approved by the Central Committee of Human Investigations strong T-cell immunity and prolonged survival of late stage and by the ethical board of the Leiden University Medical Center (LUMC): cervical cancer patients EudraCT 2013-1804-12. 1 1 Marij Schoenmaekers-Welters, PhD , Marij Welters, PhD , Cornelis Melief, 2 3 1 MD, PhD , Ignace Vergrote , Judith Kroep, MD, PhD , Gemma Kenter, 4 5 6 7 P334 MD,PhD , Nelleke Ottevanger , Wiebren Tjalma , Hannelore Denys , 1 8 8 Concurrent cetuximab (CTX) and nivolumab (NIVO) in patients Mariette van Poelgeest, MD, PhD , Hans Nijman , Anna Reyners , Thierry 9 9 10 1 1 with recurrent and/or metastatic (R/M) head and neck Velu , Frederic Goffin , Roy Lalisang , Nikki Loof , Sanne Boekestijn , 2 2 2 squamous cell carcinoma (HNSCC): Safety results of a phase I/II Willem Jan Krebber , Leon Hooftman , Sonja Visscher , Brent 11 2 5 study Blumenstein, PhD , Richard Stead , Winald Gerritsen , Sjoerd van der 1 1 2 3 Christine Chung, MD , Marcelo Bonomi, MD , Conor Steuer, MD , Burg, PhD 1 2 1 1 1 1 Michael Schell , Jiannong Li , Matthew Johnson , Caitlin McMullen ,J. Leiden University Medical Center, Leiden, ZA, Netherlands; ISA 3 1 1 1 1 Trad Wadsworth , Krupal Patel , Julie Kish, MD , Jameel Muzaffar, MD , Pharmaceuticals, Leiden, Netherlands; University Hospital Leuven, 4 1 4 3 Kedar Kirtane , James Rocco , Nabil Saba, MD Leuven, Belgium; Center for Gynecological Oncology, Amsterdam, 5 1 2 Moffitt Cancer Center, Tampa, FL, United States; Ohio State University Netherlands; Nijmegen University Medical Center, Nijmegen, 6 7 3 Medical Center, Columbus, OH, United States; Emory University, Atlanta, Netherlands; University Hospital Antwerp, Antwerp, Belgium; University 8 4 GA, United States; Ohio State University, Columbus, OH, United States Hospital Gent, Gent, Belgium; University Medical Center Groningen, Correspondence: Christine Chung (christine.chung@moffitt.org) Groningen, Netherlands; Chirec Cancer Institute, Maastricht, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P334 Netherlands; University Medical Center Maastricht, Maastricht, Netherlands; Trial Architecture Consulting, Washington, DC, United Background States Use of anti-Programmed Death-1 (anti-PD-1) inhibitors is a Correspondence: Marij Schoenmaekers-Welters (M.J.P.Schoenmaekers- standard of care for patients (pts) with R/M HNSCC, but only Welters@lumc.nl) limited numbers of pts achieve long term clinical benefits. Im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P333 proving its efficacy and maintaining low toxicity profile are critical in combination strategies. We report the safety results Background of a phase I/II trial of CTX and NIVO in pts with R/M HNSCC. High-risk human papilloma virus type 16 (HPV16) is the major cause Methods of inducing cervical cancer. The oncoproteins E6 and E7 are respon- Pts were treated with CTX 500 mg/m2 IV on Day (D) -14 as a lead-in sible for the cancer development and therefore targeted by the followed by CTX 500 mg/m2 IV and NIVO 240 mg/m2 IV on D1 and therapeutic synthetic long peptide (SLP) vaccine ISA101. Monother- D15 every 28-D cycle (C). Pts with CTX infusion reaction or who did apy induced HPV16 E6/E7-specific T cells and was clinically effective not receive C1D1 for any reason were considered to be non- in half of the HPV16-SLP vaccinated patients with HPV16+ high- evaluable and were replaced. The toxicities with possible, probable, grade premalignant lesions of the vulva. However, in HPV16+ cervical and definite attribution were included in treatment-related adverse cancer patients additional measures need to be taken as the vaccine- events (TRAEs) and immune-related adverse events (IRAEs) analyses. induced T cells encounter an immunosuppressive milieu in the tumor NIVO dose reduction was not allowed. microenvironment. Therefore, in the current study the ISA101 vaccin- Results ation is combined with standard-of-care chemotherapy. For the phase I cohort, 3 pts were enrolled. No dose limiting toxic- Methods ities were observed during 4 weeks of observation period after C1D1, Late stage cervical cancer patients (n=77) were treated 3 times with and no dose reduction was required. An additional 44 pts were en- ISA101 with a 3-week interval in a single arm dose escalation study rolled, and 2 pts were non-evaluable. A total of 45 pts were analyzed. testing 4 different doses of ISA101 and with the addition or not of The median age was 64 (range 24-77), with 37 males and 8 females. pegylated IFN alpha (PegIntron). The start of ISA101 vaccination was The ECOG performance status at baseline was 0 (9 pts, 20%), 1 (33 at day 15 after the second cycle of standard-of-care chemotherapy, pts, 73.3%), and 2 (3 pts, 6.7%). The primary sites were oral cavity 10 which consisted of carboplatin (AUC6)/paclitaxel (175mg/m2). Blood (22%), oropharynx 24 (53%), hypopharynx 3 (7%), larynx 6 (13%), and samples taken during the study were subjected to a set of comple- unknown primary 2 (4%). The p16 status was positive 22 (49%), mentary immune assays to determine the vaccine-induced T-cell negative 11 (24%), and unknown 12 (27%). The p16 status of the responses. subsite, oropharynx, was positive 20 (83%) and negative 4 (17%). The Results smoking status was current 6 (13%); former 27 (60%) and never 12 In 43% of the 72 evaluated patients an objective clinical response (27%) with median pack years of 20 (range 0-185). Prior chemother- was observed. Carboplatin/paclitaxel depleted myeloid suppressive apy was given in 44 (98%). Prior radiotherapy was given in 36 (80%). cells (p The most common grade 3 TRAEs occurring >2% were fatigue 5 Conclusions (11%) and rash-acneiform 2 (4.4%). The only grade 4 TRAE was CTX Our study demonstrates that chemotherapy combined with immuno- infusion reaction in 1 (2.2%). The most common grade 3 and 4 IRAEs therapy, in this case HPV16-SLP vaccination, can be exploited to ef- occurring >2% were fatigue 2 (4.4%). No grade 5 TRAEs or IRAEs oc- fectively treat HPV16+ cervical cancer patients and warrants curred. TRAEs led to CTX dose reduction in 4 (9%) of pts: infusion re- confirmation in a randomized controlled trial. action, diarrhea, hypomagnesemia, fatigue (one each). Conclusions Acknowledgements The combination of CTX and NIVO is well tolerated and remains to This work was financially supported by the Dutch Cancer Society grant 2009-4400 be an option for future studies. (to C.J.M. Melief and S.H. van der Burg). ISA Pharmaceuticals sponsored the trial. Ethics Approval Trial Registration The study was approved by Advarra CIRBI, approval number 00000971 ClinicalTrials.gov NCT02128126. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 182 of 272 P335 Surprisingly, FOXP3+, and CD68+ staining cells were positively as- The tumor immune microenvironment and its association with pCR sociated with pCR. Additional analyses currently being conducted in NRG Oncology/NSABP B-52: Quantification of PD-1, PD-L1, CD8, to assess during treatment biopsies and the spatial relationships FOXP3, and CD68 by multiplex fluorescent-immunohistochemistry between different immune markers may provide further mechan- 1 1 1 1 1 Marion Joy, PhD , Ying Wang , Rim Kim , Nan Song , Ashok Srinivasan , istic insights. 1 1 2 1 Huichen Feng , Corey Lipchik , Reena Cecchini , Samuel Jacobs , Joseph 2 3 4 5 Costantino , Sandra Swain , Eleftherios Mamounas , Priya Rastogi , Acknowledgements 6 7 2 8 Soonmyung Paik , C. Kent Osborne , Norman Wolmark , Peter Lucas , U10CA180868; U24CA196067; UG1CA189867; Genentech, BCRF 9 1 Mothaffar Rimawi , Katherine Pogue-Geile Trial Registration 1 2 NRG Oncology/NSABP, Pittsburgh, PA, United States; NRG Oncology/ NCT02003209 NSABP and the University of Pittsburgh, Pittsburgh, PA, United States; Ethics Approval NRG Oncology/NSABP and Georgetown Lombardi Comprehensive Chesapeake IRB: Samples are exempt based on the Determination for NSABP Cancer Center, Georgetown University Medical Center, Washington, Foundation, Inc. Protocol TB-2 “NSABP TB-2: Comprehensive Survey of DC, United States; NRG Oncology/NSABP and Orlando Health, UF Prognostic and Predictive Markers for Breast and Colon Cancer” Health Cancer Center, Orlando, FL, United States; NRG Oncology/ (Pro00005069). All patients provided written informed consent to the NSABP NSABP and the University of Pittsburgh Cancer Institute, Pittsburgh, B-52 Clinical Study, which was reviewed and approved by the NCI CIRB. No PA, United States; NRG Oncology/NSABP and Yonsei University personal identifiable information is included. College of Medicine, Pittsburgh, PA, United States; NRG Oncology/ NSABP and The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States; NRG Table 1 (abstract P335). See text for description Oncology/NSABP and the University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; NRG Oncology/NSABP The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States Correspondence: Katherine Pogue-Geile (katherine.pogue- geile@nsabp.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P335 Background The NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test whether addition of estrogen deprivation (ED) would improve pCR rate in HER2+/ER+ breast cancer patients treated with docetaxel, carboplatin, trastuzumab, and pertuzu- mab (TCHP). A numerical increase in pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. B-52 provided the opportunity to explore potential predictive markers of pCR to possibly guide new treatment strategies. We examined the tumor immune microenvironment (TME) of B-52 baseline biopsy tumors to determine its associ- ation with pCR. Methods Pretreatment (N=238) biopsies were assessed for CD8, FOXP3, P336 CD68, PD-L1, and PD-1, with multiplex fluorescent immunohisto- Impact of cytokine release syndrome on cardiac function following chemistry (mf-IHC) utilizing the Vectra® Quantitative Pathology CD19 CAR-T cell therapy in children and young adults with acute Imaging System and inForm® Advanced Image Analysis software. lymphoblastic leukemia 1 1 2 Tumor and stromal regions were defined with a panCK antibody Amita Kulshrestha , Haneen Shalabi, Do , Vandana Sachdev , Douglas 2 2 3 4 (included in the same multiplex) and annotated by a patholo- Rosing , Stanislav Sidenko , Crystal Mackall, MD , Brandon Wiley , Daniel 5 6 gist. Digital quantitation of all markers was assessed in the Lee , Nirali Shah 1 2 tumor and stromal regions (defined by panCK). In our pre-specified, National Institutes of Health, Bethesda, MD, United States; National CTEP-approved analysis, we tested the association of PD-L1 in tumor+- Heart, Lung and Blood Institute, Bethesda, MD, United States; Stanford, stroma with a cut-point optimized by ROC curves (Table1). In explora- Stanford, CA, United States; Mayo Clinic, Rochester, MN, United States; 5 6 tory analyses, we also tested a clinically meaningful PD-L1 cut-point of University of Virginia, Charlottesville, VA, United States; National Cancer >1%; other markers were tested for associations with pCR using chi- Institute, Kensington, MD, United States square tests and a median cut-off (Table 1). Correspondence: Nirali Shah (nirali.shah@nih.gov) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P336 Based on our pre-specified analysis, total PD-L1 (assessed in both stroma+tumor) was positively associated with pCR across Background trial arms (45% v 30%, p=0.038). PD-L1 was not significantly as- Cytokine release syndrome (CRS) is the main toxicity of CAR-T cell sociated with pCR with a clinically utilized cut-off of >1% within therapy, which may require hemodynamic support. The impact of the stromal-immune cell compartment (CD8+FOXP3+CD68). Sur- CRS on cardiac function has not been well described. prisingly, in both stromal and tumor cell compartments, CD68 Methods was positively associated with pCR when the two arms are eval- We report on cardiac toxicity seen in children and young adults with uated together. When the treatment arms are examined separ- ALL treated on our phase I trial of CD19 CAR-T cell therapy (clinical- ately, CD68 in the stromal compartment correlates positively trials.gov NCT01593696). All patients had a baseline echocardiogram. with pCR. FOXP3 was also positively correlated with pCR in the Cumulative anthracycline exposure was calculated from prior expos- stromal cell compartment across arms and in the TCHP+ED arm. ure. Additional studies included increased frequency of echocardio- PD-1 and CD8 were not significantly associated with pCR. grams upon ICU transfer, and serial troponin and proBNP. Conclusions Results B-52 showed a positive association of PD-L1 expression with pCR From July 2012 to March 2016, 52 patients, with a median age of 13.4 based on our pre-specified analysis but the extremely low cut-off years (range, 4.2-30.3) were treated on-study; 23 underwent at least (0.05%) makes the clinical utility of this observation doubtful. one prior allogeneic stem cell transplantation. CRS was seen in 37/52 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 183 of 272 (71%), which was grade 3-4 CRS in 8 subjects (21.6%). The median prior anthracycline exposure was 205 mg/m2 (range, 70-620 mg/m2) in doxorubicin equivalents. The median baseline LV ejection fraction (LVEF) was 62% (range 52%-71%). The median LV global longitudinal strain (GLS), at baseline was abnormal: -17 (range, -14 to -24, n=35). ICU transfers occurred in 20 patients, 11 of whom required vasoactive hemodynamic support, with 5 necessitating more than 1 pressor. Seven patients received tocilizumab and 4 patients received steroids. Six (16%) patients developed cardiac dysfunction, amongst whom 4 had grades 3-4 CRS. (Figure 1) Severe cardiac dysfunction, (LVEF < 30%) was seen in 3, with one patient developing cardiac arrest with subse- quent full recovery following placement of an intra-aortic balloon pump, steroids, and tocilizumab. In 2 of these patients, anthracycline exposures exceeded > 360 mg/m2. All but 2 patients had full resolution of cardiac dysfunction by day 28 post CAR. Troponin elevations were seen in 4 of 6 patients with low LVEF. In a limited cohort of patients with pre/post pro-BNP, pro-BNP was higher during CRS, with the high- est levels correlating with more severe cardiac dysfunction. (Figure 2) Conclusions Patients with higher-grade CRS are more likely to experience significant cardiac side effects from CAR T-cell therapy. In most cases, resolution to near baseline was seen coinciding with resolution of CRS, with most having near complete resolution by day 28 post infusion. Implementation of more frequent echocardiogram monitoring and incorporation of BNP and troponin into daily laboratory panel may help to identify those at highest risk of severe cardiac Fig. 2 (abstract P336). Changes in BNP pre and post-CAR infusion dysfunction at an earlier time point, allowing for earlier interven- tion in CRS to potentially limit acute cardiac toxicity. P337 Acknowledgements Survival prolongation by dendritic cell vaccination in combination This research was supported by the Intramural Research Programs of the Center with OK-432, gemcitabine and/or S-1 in patients with advanced of Cancer Research, National Cancer Institute, NIH and the Clinical Center. pancreatic cancer Trial Registration Masahiro Ogasawara, MD, PhD, Shuichi Ota, MD PhD The clinical trial is registered at clinicaltrials.gov NCT01593696 Sapporo Hokuyu Hospital, Sapporo, Japan Ethics Approval Correspondence: Masahiro Ogasawara (ogasawara@hokuyu-aoth.org) This study was approved by the National Cancer Institute Institutional Review Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P337 Board. Background Pancreatic cancer is the most fatal human cancer, with a 5-year overall survival rate of less than 5%. In the current study, we have evaluated the clinical and the immunological responses in patients with advanced pancreatic cancer who received dendritic cell (DC) vaccination in combination with a toll like receptor (TLR) 4 agonist, OK-432 and chemotherapeutic agents, gemcitabine (GEM) and/or S-1. Methods Twenty three patients (13 males, 10 females; aged 37-83 years, me- dian 64 year old) with advanced pancreatic cancer refractory to standard treatment were treated with DC vaccination in combination with OK-432, GEM and/or S-1 from 2012 to 2013 at Sapporo Hokuyu Hospital. Autologous DCs were generated by culturing adherent mononuclear cells with interleukin-4 and granulocyte-macrophage colony stimulating factor. DCs were then loaded with synthetic pep- tides derived from cancer antigens such as Wilms’ tumor 1 (WT1) and MUC1 following maturation by prostaglandin E2 and OK-432. Peptide-loaded mature DCs and OK-432 were administered intrader- mally every 2 weeks, 7 times. The induction of vaccine-induced T cell responses was monitored by using HLA-tetramer and ELISPOT assays. Results The treatment was well tolerated and none of the patients experi- enced more than grade 3 adverse events during the treatment period. Of 23 patients, 1 had partial response (PR), 8 had stable dis- ease (SD) and 14 had progressive disease after one course of vaccin- ation. The median overall survival (OS) was 9.6 months. Survival of patients achieving PR or SD (responders) was longer than those who did not respond to the treatment (non-responders) (median OS; 18.0 Fig. 1 (abstract P336). Change in Ejection Fraction Post CRS Onset vs 5.8 months). An HLA-tetramer assay showed an increase in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 184 of 272 positivity of WT1-specific CD8+ T cells in both responders and non- Results responders after vaccination. However, the increment in the positivity 792 patients were enrolled (intention-to-treat [ITT] population), includ- was remarkable in responders in comparison with non-responders; ing 529 with PD-L1+ tumors (primary analysis population) and 263 with 46.3 and 10.7 fold in responders and non-responders, respectively. PD-L1− tumors. (n=263). At data cut-off (March 4, 2019) in the PD-L1+ Similarly, an ELISPOT assay showed marked increase in spot-positive population, median duration of follow-up for OS was 35.4 months in cells in responders. The median OS in patients showing the positivity the avelumab arm (n=264) and 34.7 months in the docetaxel arm (n= in both assays was longer than those who were positive in either 265); study treatment was ongoing in 25 (9.5%) vs 0 patients, and 17 assay or who were negative in both assays; a median OS was 18.4 (6.4%) vs 74 (27.9%) had received a posttreatment checkpoint inhibitor months, 9.7 months and 4.7 months, respectively, suggesting a cor- (CPI), respectively. 2-year OS rates with avelumab vs docetaxel in differ- relation between an immune response and a clinical outcome. ent PD-L1+ subgroups are shown (Table 1). Of patients with PD-L1+ tu- Conclusions mors alive at 2 years, 67% had received a posttreatment CPI in the DC vaccination combined with a conventional chemotherapy in pa- docetaxel arm compared with 13% in the avelumab arm. In patients tients with advanced pancreatic cancer was demonstrated to be safe with PD-L1+ tumors who had an objective response with avelumab (50 and can elicit immune responses against tumor antigens, which was [18.9%]) or docetaxel (28 [10.6%]), median duration of response (DOR; correlated with clinical effects. investigator assessed) was 19.1 months (95% CI: 10.8-34.8) vs 5.7 Ethics Approval months (95% CI: 4.1-8.3), and proportions with a response lasting ≥6 This study was approved by the Ethics and Internal Review Board at months were 86.0% (95% CI: 72.9%-93.1%) vs 48.1% (95% CI: 28.7%- Sapporo Hokuyu Hospital, approval number 131111.08 65.2%), respectively. Safety profiles of avelumab and docetaxel were similar to those in previous analyses. Conclusions P338 Updated data from JAVELIN Lung 200 showed that although avelu- 2-year follow-up from JAVELIN Lung 200, an open-label, mab did not significantly prolong OS vs docetaxel in the primary randomized, phase 3 study of avelumab vs docetaxel in patients confirmatory analysis, 2-year OS rates were doubled with avelumab with platinum-treated advanced non-small cell lung cancer vs docetaxel in higher PD-L1+ subgroups, and median DOR was >12 (NSCLC) months longer with avelumab vs docetaxel. 1 2 3 Fabrice Barlesi, MD, PhD , Mustafa Özgüroğlu , Johan Vansteenkiste , 4 5 6 7 David Spigel , James Yang , Hidenobu Ishii , Marina Garassino , Filippo Acknowledgements 8 9 10 11 de Marinis , Aleksandra Szczesna , Andreas Polychronis , Ruchan Uslu , This study was funded by Merck KGaA, Darmstadt, Germany, as part of an 12 13 14 Maciej Krzakowski , Jong-Seok Lee , Luana Calabro, MD , Osvaldo alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, 15 16 17 17 Aren Frontera , Barbara Ellers-Lenz , Marcis Bajars , Mary Ruisi , NY, USA. Keunchil Park Trial Registration Aix-Marseille University, Assistance Publique - Hôpitaux de Marseille, NCT02395172 Livon, France; Cerrahpaşa Medical Faculty, Istanbul University, Leuven, Ethics Approval 3 4 Belgium; University Hospital KU Leuven, Leuven, Belgium; Sarah The study protocol was approved by institutional review boards and ethics Cannon Research Institute, Nashville, TN, United States; National Taiwan committees at each institution. The study was done in accordance with the University Hospital, Taipei, Taiwan, Province of China; Kurume University trial protocol, Good Clinical Practice guidelines, and the Declaration of School of Medicine, Kurume, Japan; Fondazione IRCCS Istituto Helsinki. All patients provided written informed consent. Nazionale dei Tumori, Milan, Italy; Istituto Europeo di Oncologia, Milan, 9 10 Italy; Regional Lung Disease Hospital, Otwock, Poland; Mount Vernon Cancer Centre, Northwood, Middlesex, United Kingdom; Ege University 12 Table 1 (abstract P338). See text for description Hospital, Izmir, Turkey; Centrum Onkologii-Instytut Im. M. Skłodowskiej- Curie w Warszawie, Warszawa, Poland; Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea, Republic of; University Hospital of Siena, Siena, Italy; 15 16 Instituto Nacional del Cáncer, Santiago, Chile; Merck KGaA, Darmstadt, Germany; EMD Serono Inc, Billerica, MA, United States; Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence: Fabrice Barlesi (Fabrice.BARLESI@ap-hm.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P338 Background P339 Avelumab, a human IgG1 anti–PD-L1 monoclonal antibody, is ap- Survival is improved by antigen-specific cytotoxic T lymphocytes proved as monotherapy for metastatic Merkel cell carcinoma and (CTL) responses after treatment with the vaccine Tedopi in HLA-A2 platinum-treated urothelial carcinoma in various countries, and in positive advanced non-small cell lung cancer (NSCLC) patients 1 2 combination with axitinib to treat advanced renal cell carcinoma in Benjamin Besse, MD PhD , Enriqueta Felip, MD PhD , Giuseppe 3 4 5 6 the United States. In the JAVELIN Lung 200 study, avelumab did not Giaccone , Rafal Dziadziuszko , Elisabeth Quoix, MD , Werner Hilgers , 7 8 9 10 significantly prolong overall survival (OS) vs docetaxel in patients Federico Cappuzzo , Christophe Borg , Jordi Remon , Nicolas Poirier , 10 10 11 with platinum-treated PD-L1+ NSCLC (primary objective); however, Dominique Costantini , Bérangère Vasseur , Santiago Viteri 1 2 prespecified exploratory analyses showed longer OS with avelumab Gustave Roussy, Villejuif, France; Vall d'Hebron University Hospital, vs docetaxel in patients with higher PD-L1+ tumors. We report up- Barcelona, Spain; Georgetown University, Washington, DC, WA, United 4 5 dated data from JAVELIN Lung 200. States; Medical University of Gdańsk, Gdańsk, Poland; Nouvel Hôpital Methods Civil, Strasbourg, France; Institut Sainte Catherine, Avignon, France; 7 8 Patients with stage IIIB/IV or recurrent NSCLC and disease progres- AUSL Romagna, Ravenna, Italy; Centre Hospitalier Universitaire, 9 10 sion following platinum-doublet chemotherapy were randomized 1:1 Besançon, France; CIOCC- Barcelona, Barcelona, Spain; OSE to avelumab 10 mg/kg Q2W or docetaxel 75 mg/m2 Q3W. The pri- Immunotherapeutics, Nantes, France; University Hospital Dexeus, mary endpoint was OS; the primary analysis population was patients Barcelona, Spain with PD-L1+ tumors (≥1% tumor cell expression; PD-L1 IHC 73-10 Correspondence: Benjamin Besse (benjamin.besse@gustaveroussy.fr) pharmDx assay). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P339 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 185 of 272 Background Tedopi (OSE2101) is a multiple epitope vaccine restricted to HLA-A2 positive patients (45%), targeting five tumor-associated antigens (TAA) frequently expressed in solid tumors: carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER-2/neu), melanoma-associated antigen type 2 and 3 (MAGE2 and MAGE3), and p53. Tedopi is composed by 2 wild type and 7 chemically modi- fied peptides to increase HLA-A2 or T cell receptor (TCR) affinity. A pan-DR epitope (PADRE) of helper T-lymphocyte (HTL) has been added to increase the Cytotoxic T Lymphocyte (CTL) responses. In previously treated advanced NSCLC patients, Tedopi showed a strong CTL immune response, which correlated with overall survival (OS) [1]. The aim of the current translational study was to explore the predict- ive effect of the epitope type and number of epitopes on OS. Methods Fig. 1 (abstract P339). Overall survival in patients with 3-6 vs 0-2 Out of 64 previously treated HLA2+ advanced NSCLC patients en- CTL responses rolled in a phase II trial testing the efficacy of Tedopi (1mL subcuta- neously Q3W for 6 cycles, then Q8W for the reminder year 1 and Q12W up to year 2), 33 patients were assessed for epitope-specific cytotoxic response and HTL responses using an interferon gamma P340 enzyme-linked immunosorbent assay. Leukapheresis was performed Region-focused deep survival learning on PD-L1 stained tissue at baseline, at week 9, 18 and 30 for immunogenicity assays. Predict- samples for data-driven stratification of durvalumab-treated ive analyses of OS were performed using Cox regression. NSCLC patients Results 1 1 1 Nicolas Brieu, PhD , Ansh Kapil , Armin Meier, PhD , Keith Steele, DVM, Patients were stage IV (64%), or locally advanced stage IIIb (36%). 2 2 1 PhD , Marlon Rebelatto, DVM, PhD, DACVP , Guenter Schmidt, PhD Eleven patients were assessed for all 10 epitopes, and 33 for 6 se- 1 2 Definiens AG, Munich, Germany; AstraZeneca, Gaithersburg, MD, lected epitopes (2 CEA, 1 HER-2, MAGE2, MAGE3, PADRE). Median United States survival was 30 months. Correspondence: Nicolas Brieu (nbrieu@definiens.com) There was at least one CTL response to one vaccine epitope in >90% Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P340 of patients. Eight epitopes were highly immunogenic (from 55% to 91%), while HER-2 wild type and one p53 analogue shown a lower Background response (respectively 36% and 9%). The selection of metastatic non-small cell lung cancer (NSCLC) pa- In patients evaluated for 6 selected epitopes, the best cut-off of num- tients that are likely to respond to an anti-PD-L1 checkpoint mono- ber of CTL responses to discriminate OS were 1-6 versus 0, 2-6 vs 0-1 therapy can be guided by the visual assessment by pathologists of or 3-6 vs 0-2. All of three were statistically significant. As an example, the Tumor Cell (TC) score on PD-L1 stained tissue samples [1]. Deep patients with CTL responses to 3-6 epitopes (n=23) had a median OS learning approaches have recently enabled the computer-based rep- of 38 months compared to 15 months in patients (n=10) with CTL re- lication of this visual TC score [2,3] and of its ability to predict overall sponses to 0-2 epitopes (HR=0.39; p=0.04) (Figure 1). CTL response survival (OS) [4]. Because these methods try to reproduce as close as to HER-2 analogue, MAGE3, PADRE and one p53 analogue were pre- possible the visual scoring methodology, they are built on extensive dictive of better OS. prior hypotheses (e.g. definition of cell positivity, score and cut-off) Conclusions and do not enable the data-driven discovery of novel stratification In NSCLC patients, survival was significantly prolonged in patients immu- rules. We present here a novel region-focused end-to-end deep- nized to epitope specific Tedopi vaccine. HER-2, MAGE3, PADRE and p53 learning approach that enables the data-driven generation of survival were identified as vaccine predictive epitopes for prolonged survival. risk heatmaps and the stratification of patients into two risk groups. Methods Acknowledgements On a subset (N=151) of core needle biopsies and tissue resections from We thank François Montestruc and Constant Josse (eXYSTAT, Malakoff, the NCT01693562 clinical trial (NSCLC), epithelium regions are automat- France) for the statistical analysis ically segmented within the manually delineated tumor area [3]. A patch-based convolutional neural network (CNN) is trained on selected Reference patches in a two-fold pre-validation procedure to maximize a log partial 1. Barve M, Bender J, Senzer N, Cunningham C, Greco FA, McCune D, Steis likelihood derived from the Cox proportional hazards model [5,6]. To R, Khong H, Richards D, Stephenson J, Ganesa P, Nemunaitis J, Ishioka G, avoid a disproportionately large number of patches from tissue resec- Pappen B, Nemunaitis M, Morse M, Mills B, Maples PB, Sherman J and tions, a random subset of up to 10K patches is selected for each patient Nemunaitis JJ. Induction of Immune Responses and Clinical Efficacy in a within the segmented regions. The overall survival risk is predicted and Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, aggregated by mean on the detected epithelium regions only. Patients in Metastatic Non-Small-Cell Lung Cancer. J Clin Oncol. 2008;26(27):4418– are finally stratified based on the cohort median of the resulting aggre- gated risk scores. For baseline comparison, the same steps are repeated Ethics Approval considering the complete delineated tumor area instead of the sole The study protocol and its related documents (including the patient segmented epithelium regions. information and informed consent form) received approval from the Institutional Review Board (IRB), and the Competent Authority prior to Results study initiation. The proposed epithelium-focused and data-driven survival CNN Consent yields similar patient stratification (HR=0.525, p=0.003) as obtained Each patient gave his/her written informed consent prior to study with 25% cut-off on visual (HR=0.574, p=0.01) or automated (HR = enrolment. 0.539, p=0.004) TC score (Figure 1), while releasing prior hypotheses Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 186 of 272 on PD-L1 region positivity, score methodology, and cut-off value. As P341 expected on durvalumab-treated patients, high and low risks are as- Activation of toll-like receptors via PS-targeting monoclonal sociated with low and high PD-L1 staining respectively. No relevant antibodies risk groups are identified if the analysis is performed on the full Rolf Brekken, PhD (rolf.brekken@utsouthwestern.edu) tumor area instead. UT Southwestern Medical Center, Dallas, TX, United States Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P341 Our results suggest, for the first time on core needle biopsies and tis- sue resections, (i) the ability of end-to-end deep survival learning to Background directly learn relevant patient stratification as well as (ii) the neces- Multifocal immune suppression in the tumor microenvironment is a sity, in case of small patient cohorts, to restrict the analysis to auto- major underlying cause for the limited efficacy of immune check- matically detected meaningful regions. point blockade. Persistent immune suppression prevents the devel- opment of a robust T cell response to tumor specific antigens that is References required for effective downstream immune checkpoint blockade. An 1. Rebelatto et al., Development of a programmed cell death ligand-1 im- underappreciated but significant contributor to immune suppression munohistochemical assay validated for analysis of non-small cell lung in tumors is the exposure of the membrane phospholipid phosphati- cancer and head and neck squamous cell carcinoma, Diagnostic Path- dylserine (PS) on the surface of tumor cells and tumor-derived micro- ology 2016 vesicles. PS is recognized by receptors on immune cells where it 2. A. Kapil et al., Deep Semi Supervised Generative Learning for Automated triggers the secretion of immune suppressive cytokines, prevents the Tumor Proportion Scoring on NSCLC Tissue Needle Biopsies, Scientific differentiation of myeloid-derived suppressor cells (MDSCs) and in- reports, 2018 hibits dendritic cell (DC) maturation; events that prevent a productive 3. A. Kapil et al., DASGAN - Joint Domain Adaptation and Segmentation for anti-tumor T cell response. Bavituximab, a chimeric monoclonal the Analysis of Epithelial Regions in Histopathology PD-L1 Images, arXiv antibody (mAb) that targets PS and inhibits PS-mediated im- preprint arXiv:1906.11118, 2019 munosuppressive signaling, drives immune activation by reducing 4. N. Brieu et al., Deep learning-based PD-L1 tumor cell (TC) scoring im- the levels of MDSCs, by polarizing tumor-associated macrophages proves survival prediction compared to pathologists on durvalumab- towards an immune stimulatory phenotype and by promoting treated NSCLC patients, SITC 2018. the maturation of dendritic cells (DCs). Bavituximab and other PS- 5. P. Mobadersany et al., Predicting cancer outcomes from histology and targeting mAbs (2aG4 and 1N11) bind to PS via beta-2 glycopro- genomics using convolutional networks, PNAS 2018. tein 1 (β2GP1). β2GP1, an abundant serum glycoprotein, was re- 6. A. Meier et al., End-to-end learning to predict survival in patients with cently identified as a novel component of innate immunity via gastric cancer using convolutional neural networks, Annals of Oncology activation of Toll-like receptors (TLRs). (ESMO), 2018. Methods Ethics Approval Human β2GP1. Monoclonal-antibodies: Bavituximab and 1N11. qPCR, For the Phase 1/2 durvalumab trial (NCT01693562), the study protocol was WB, IP, ICC/IHC, CD, TEM, MST and ELISA reviewed and approved by the Institutional Review Board of the Results participating centers and informed consent was obtained from all We investigated whether the innate immune changes induced by patients. PS-targeting mAbs is mediated in part by β2GP1-induced activation of TLRs in myeloid cells. β2GP1 has a closed conformation that pre- vents its interaction with PS and cell surface receptors. However, interaction of β2GP1 with LPS can induce an open conformation of the protein that allows interaction of β2GP1 with PS and cell surface receptors, including the TLRs. We found through circular dichroism and transmission electron microscopy that the PS-targeting anti- bodies also induce conformational changes in β2GP1, shifting it to an open conformation. In addition, we demonstrate that PS-targeting mAb-mediated dimerization of β2GP1 stimulated pro-inflammatory polarization of bone marrow-derived macrophages (BMDMs) and in- duced TLR2 signaling. Finally, we investigated the expression of a newly characterized TLR-induced transcription factor, Spi-C in bone marrow progenitor cells after stimulation with TLR specific agonists or PS-targeting mAbs +/- β2GP1. Conclusions These studies demonstrate that the PS-targeting mAbs stimulate Spi- C expression in a β2GP1-dependent manner. Overall these data sup- port that one mechanism of innate immune activation induced by PS-targeting mAbs is through TLR2 stimulation on myeloid cells. Fu- ture studies are focused on validating these results in vivo using Tlr- deficient and β2gp1-deficient mice. Trial Registration NCT01999673, NCT03139916, NCT03519997 Ethics Approval All animal experiments were done according to the Animal Research Center (ARC) at UT Southwestern Medical Center. All clinical trials Fig. 1 (abstract P340). See text for description were conducted in accordance with all Federal and State Laws. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 187 of 272 P342 P343 Long term outcomes of a phase I study with UV1, a second Initial results from a Phase II study (TACTI-002) in non-small cell generation telomerase based vaccine, in patients with advanced lung cancer, or head and neck cancer patients receiving non-small cell lung cancer eftilagimod alpha (LAG-3 fusion protein) and pembrolizumab 1 2 2 1 2 3 Wenche Rasch, PhD , Paal Brunsvig, MD PhD , Martha Nyakas, MD , Clau Julio Peguero, MD , Enriqueta Felip, MD PhD , Bernard Doger , Margarita 2 2 2 4 5 6 7 Reisse, MD , Jon Amund Kyte , Hedvig Vidarsdotter Juul , Steinar Majem , Enric Carcereny , Tim Clay , Pawan Bajaj , Matthew Krebs, MD 1 3 2 8 9 Aamdal , Gustav Gaudernack, PhD , Else Marit Inderberg PhD , Frederic Triebel, MD, PhD 1 2 1 2 Ultimovacs ASA, Oslo, Norway; Oslo University Hospital, Oslo, Norway; Oncology Consultants, Houston, TX, United States; Vall d’ Hebron 3 3 Ultimovacs ASA, Prof. Emeritus, Oslo University Hospital, Oslo, Norway Institute of Oncology, Barcelona, Spain; Fundación Jimenez Díaz, Correspondence: Wenche Rasch (wenche.rasch@ultimovacs.com) Madrid, Spain; Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P342 Institut Català d'Oncologia Badalona, Barcelona, Spain; St John of God Subiaco Hospital, Perth, Australia; Tasman Health Care, Queensland, Background Australia; The University of Manchester and The Christie NHS A first generation hTERT vaccine (GV1001) showed evidence of clin- Foundation Trust, Manchester, United Kingdom; Immutep, Orsay, France ical efficacy in patients with advanced non-small cell lung cancer Correspondence: Frederic Triebel (ftriebel@immutep.com) (NSCLC). We have now tested a second generation hTERT vaccine, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P343 UV1. This vaccine is designed to give high population coverage and is composed of three synthetic long peptides containing multiple Background epitopes identified by epitope spreading data from long-term survi- Eftilagimod alpha (efti; previously IMP321) is a recombinant LAG-3 Ig vors who participated in previous hTERT vaccination trials. fusion protein that binds to MHC class II molecules to mediate anti- Methods gen presenting cell (APC) and CD8 T-cell activation. The stimulation Eighteen non-HLA-typed patients with stage III/IV NSCLC with no evi- of the dendritic cell network and subsequent T cell recruitment at dence of progression after prior treatments, were enrolled in a phase the tumor site with efti may lead to stronger anti-tumor CD8 T cell I dose-escalation study of UV1 vaccination with GM-CSF as adjuvant, responses than observed with pembrolizumab alone. Combining an evaluating safety, immune response, and long term clinical outcome. APC activator with an immune checkpoint inhibitor (ICI) aims to in- The present study also aimed to provide a rationale for combining crease efficacy without additional toxicity. We hereby report initial re- UV1 vaccine with PD-1/PD-L1 blockade. sults of stage 1 of this phase II trial (NCT03625323). Results Methods Treatment with GM-CSF and UV1 was well tolerated with no serious The study is based on a Simon's optimal two-stage design, with ob- adverse events observed. All patients experienced one or more ad- jective response rate (ORR) as primary endpoint. Secondary end- verse events, the majority grade 1, such as injection site reactions points include progression free survival and overall survival. Blood and fatigue. Seventeen patients were evaluable for tumor response; for PK/PD assessments and anti-drug antibody evaluation is col- 15 patients had stable disease as best response, while 2 patients had lected. During the first stage of the study, patients (pts) are recruited progressive disease. The median progression free survival (PFS) was into each of three indications: A: 1st line, PD-X naïve NSCLC; B: 2nd 12.3 months and the median overall survival (OS) was 28.2 months. line, PD-X refractory NSCLC; C: 2nd line PD-X naïve HNSCC. Additional The OS at 3 years was 44%. None of the 7 long-term surviving pa- patients (N2) will be recruited for each part if the pre-specified tients (median survival 4.96 years, range 4.04-5.51) have received threshold for ORR is met. In total 109 patients are planned to be en- checkpoint blockade therapy after UV1 vaccination. UV1-vaccination rolled. Eftilagimod alpha is administered as 30 mg subcutaneous in- induced specific T helper 1 (Th1) immune responses in the majority jection every 2 weeks for the first 8 cycles and every 3 weeks for the (67%) of patients. Both immune responses and OS were dose related. 9 following cycles. Pembrolizumab is administered at a standard Conclusions dose of 200 mg intravenous infusion every 3 weeks for maximum 2 The highest dose of UV1 (700 μg) resulted in the highest proportion years. The study was approved by all relevant ethics committees and of immune responses. These responses occurred more rapidly and institutional review boards. were stronger compared to lower doses and the patients in this Results group had a 3-year OS of 83%. This, together with the safety and Between 05 March and 24 July 2019, 27 pts were enrolled and clinical outcome data, favours 700 μg as the preferred UV1 dose in treated in the study. The mean age was 67 (range 53-84) and 74% this patient population. These results provide a rationale for further were male. The ECOG PS was 0 in 59% of the pts and 1 in 41% of clinical studies in NSCLC with UV1 vaccination in combination with the pts. The treatment has been well tolerated with the most com- immune checkpoint blockade. mon AEs being cough (9%), dyspnea (9%), diarrhea (6%) and asthe- nia (5%). Eleven treatment related SAEs were reported in ten pts. Acknowledgements Thirteen (13) pts of part A are evaluable (data cut-off 24th July 2019) We thank all the patients for their participation in the study for efficacy. The vast majority had only one post-baseline tumor as- Trial Registration sessment. Four pts of 13 (31%) had a partial response and six (46%) The clinical trial UV1/hTERT-L was performed with NoMA approval and is pts had stable disease according to iRECIST at data cut-off. registered with Clinicaltrials.gov on February 11, 2013 (NCT01789099). Conclusions Patients provided written informed consent to participate. Enrollment started Thirty (30) mg efti s.c. every 2 weeks in combination with standard on April 8, 2013. dose of pembrolizumab is safe and shows encouraging antitumor Ethics Approval activity. The study was approved by the institutional protocol board, the regional Trial Registration Ethical Committee (REC 2012/1114, EudraCT 2012- 001852-20) and the EudraCT: 2018-001994-25 Norwegian Medicines Agency (NOMA) and the study was registered at NCT: 03625323 clinicaltrials.gov (NCT01789099). Ethics Approval Consent The study was approved by Advarra IRB (US), approval number N/A, Individual patient consent was not applicable as no information in this approval date: 13/08/2018; London - Harrow Research Ethics Com- abstract/poster can be categorized as identifiable. mittee (UK), approval number 18/LO/1889; Instituto de Investigación Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 188 of 272 Hospital 12 de Octubre (Spain), approval number 18/376; Belberry 3.3mos), or overall survival (p=0.40, overall mOS 11.4 mos); therefore, HREC (Australia), approval number 2018-08-636; St John of God the study closed to enrollment at interim analysis. PD-L1 expression Health Care (Australia), approval number 1450. was not associated with response (p=0.52). IF demonstrated an asso- ciation between intratumoral CD4+/PD1+/Ki67+ cells and response (p=0.02). P344 Conclusions A randomized multi-center phase 2 study of combined PD-L1/ We did not observe a benefit adding targeted radiotherapy concur- CTLA-4 inhibition with or without radiation in non-small cell lung rently with combined PD-L1/CTLA-4 therapy in a PD-(L)1 inhibitor re- cancer patients who progressed on PD-(L)1 directed therapy: fractory NSCLC population. However, across cohorts PD-L1/CTLA-4 ETCTN 10021 was generally tolerable and led to response/disease control in some 1 2 3 Arta Monjazeb, MD, PhD , Anita Giobbie-Hurder, MS , Ana Lako , Mark patients, including responses >6 months. Multiplex IF suggests tumor 3 4 5 6 Awad, MD PhD , Ryan Gentzler, MD , Carrie Lee , Joleen Hubbard , infiltration by Ki-67+/PD-1+ CD4 T cells is associated with response 7 8 9 James Abbruzzese, MD , Salma Jabbour, MD , Nataliya Uboha , Kevin and worthy of further investigation. Additional correlative genomic 10 11 12 13 Stephans , Jennifer Johnson, MD , Haeseong Park , Liza Villaruz, MD , and immune analyses are planned. 3 14 15 Katrina Kao , Elad Sharon, MD, MPH , David Raben, MD , Raymond Trial Registration 3 14 14 Mak , Howard Streicher, MD , Helen Chen, MD , Mansoor Ahmed, ClinicalTrials.gov Identifier: NCT02888743 14 16 3 PhD , Scott Rodig, MD, PhD , F. Stephen Hodi, MD , Jonathan Ethics Approval Schoenfeld, MD, MPH This study was approved by the NCI Central IRB. 1 2 UC Davis Cancer Center, Sacramento, CA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; University of Virginia, Charlottesville, Table 1 (abstract P344). See text for description VA, United States; University of North Carolina, Chapel Hill, United 6 7 States; Mayo Clinic, Rochester, United States; Duke, Durham, United States; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States; University of Wisconsin, Madison, WI, United States; 10 11 Cleveland Clinic, Cleveland, United States; Jefferson Medical Center, Philadelphia, PA, United States; Washington University in St. Louis, St. Louis, United States; University of Pittsburgh Medical Center, Pittsburgh, PA, United States; National Institutes of Health, Bethesda, MD, United States; University of Colorado, Greenwood Village, CO, United States; Brigham and Women's Hospital, Boston, MA, United States Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P344 Background Preclinical data support combined PD-L1/CTLA-4 inhibition and sug- gest synergy between PD-L1/CTLA-4 inhibition and targeted radi- ation via enhanced systemic anti-tumor immune responses. We aimed to evaluate combined PD-L1/CTLA-4 inhibition in NSCLC pa- tients who progressed on prior PD-(L)1 inhibitors and determine whether high- or low-dose radiation could increase objective re- sponses outside the radiation field. Methods ETCTN 10021 is a multicenter randomized phase 2 study evaluating the addition of repeated low-dose fractionated radiotherapy (0.5 Gy BID x 2 days) or hypofractionated radiation (8 Gy x 3) concurrently P345 with PD-L1/CTLA-4 inhibition (durvalumab 1500mg/tremelimumab PARP inhibition when combined with PD-L1 inhibition has a 75mg q4w for 4 cycles followed by durvalumab monotherapy) in suppressive effect on T cells in patients with relapsed or recurrent NSCLC patients progressive on prior PD-(L)1 inhibitors (intervening small cell lung cancer therapy allowed). Patients were randomized 1:1:1 to durvalumab/tre- Nobuyuki Takahashi, MD, PhD, Vinodh Rajapakse, Min-Jung Lee, Akira melimumab alone or with low-dose or hypofractionated radiother- Yuno, Sunmin Lee, Sehyun Kim, Rasa Vilimas, Samantha Nichols, Jane apy. The primary endpoint was objective response per RECIST v1.1 Trepel, Anish Thomas excluding irradiated lesions with planned interim analysis. Correlative National Cancer Institute, Bethesda, MD, United States analyses were performed on tissue obtained following progression Correspondence: Anish Thomas (anish.thomas@nih.gov) on prior PD-(L)1 inhibitors using PD-L1 immunohistochemistry and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P345 multiplex immunofluorescence (IF) evaluating CD8, CD4, PD1, Ki67, and cytokeratin in tandem. Background Results Poly ADP-ribose polymerase (PARP) inhibition increased PD-L1 ex- We randomized 78 patients (26 per each of 3 arms) who received >= pression, augmented cytotoxic T-cell infiltration and potentiated the 1 cycle of study therapy between August 2017 and March 2019 anti-tumor effect of PD-L1 blockade in small cell lung cancer (SCLC) across 18 sites. Patients received PD-(L)1 inhibitors for a median of 1 in vivo [1]. Yet in clinical studies, PARP inhibitor plus PD-L1 inhibitor cycles (range 1-5) prior to enrollment; 68% had prior radiation. Treat- did not improve responses in relapsed or recurrent SCLC patients ment related adverse events (TRAE) of any grade were observed in compared to historical controls of PD-L1 inhibitor alone [2, 3]. Given 53 subjects (68%), and grade ≥3 events in 18 subjects (23%), includ- the role of PARPs in activating inflammatory gene expression [4], we ing 1 grade 5 respiratory failure. Response rate across all cohorts investigated the effects of PARP inhibition plus PD-L1 blockade on were 10% (n=8, 95% exact CI: 5%-19%), and disease control 19% (n= the adaptive immune system in SCLC patients. 15, 95% exact CI: 11-30%). Median duration of response was 10.3 Methods months (95% CI: 1.4 months – not reached). Response and disease NCT02484404 SCLC cohort is an open label phase 2 study evaluating control weren’t significantly different between arms (Table 1, p=0.99/ the combination of durvalumab (1500 mg iv, Q4W) and olaparib (300 0.52, respectively), nor was time to progression (p=0.88, overall mTTP Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 189 of 272 mg BID) in patients with relapsed or recurrent SCLC [2]. Peripheral Preliminary efficacy from a cohort of NSCLC patients who re- blood lineages (pretreatment [C1D1], 2 weeks [C1D15] and 6 weeks ceived prior immune checkpoint therapy was previously pre- [C3D1] after treatment) were serially assessed by flow cytometry. sented (SITC-2017); baseline tumor samples were collected to Results assess mechanisms of resistance to prior therapy. Here we de- 20 patients were evaluated. Activated Ki67+ HLA-DR+ T cells sig- scribe the baseline tumor microenvironments (TMEs) of 2 co- nificantly decreased post-treatment (median [interquartile range] horts of patients, melanoma and NSCLC, who experienced on C1D1 and C3D1: 3.5% [2.2–5.9] vs. 2.1% [1.8–3.3], p=0.033 progressive disease on prior anti–PD-(L)1 treatment before among CD4+ T cells; 2.6% [1.8–5.1] vs. 1.4% [1.0–2.3], p=0.002 starting bintrafusp alfa administration. among CD8+ T cells). Activated Ki67+ PD-1- T cells also signifi- Methods cantly decreased post-treatment (1.9% [1.2–3.9] vs. 1.3% [1.1–2.4], Fresh tumor biopsies from melanoma (n=29) and NSCLC (n=64) pa- p=0.020 among CD4+ T cells; 3.2% [2.0–5.0] vs. 2.4% [2.0–2.7], p= tients refractory or resistant to prior anti–PD-(L)1 therapy were proc- 0.025 among CD8+ T cells). By contrast, exhausted Ki67- TIM-3+ essed for FFPE and subjected to RNA sequencing. Based on the CD8+ T cells significantly increased post-treatment (0.8% [0.5–1.3] mechanism of action of bintrafusp alfa, genes and gene signatures vs. 1.2% [0.8–2.0], p=0.002). PD-1 expression on regulatory related to immune and TGF-β pathways were evaluated and com- Foxp3+ CD25+ T cells (Treg) and effector regulatory CD45RA- pared between tumor types. Foxp3hi T cells (eTreg) significantly increased post-treatment (me- Results dian [IQR] of mean fluorescence intensity [MFI] ratio on C1D1 Melanoma had elevated CD8+ T-cell gene signatures but low levels and C3D1: 2.2 [1.6–2.8] vs. 3.1 [2.2–3.4], p=0.002 among Treg; 2.4 of predicted tumor neo-antigens. NSCLC had elevated gene signa- [1.7–2.8] vs. 3.4 [2.3–4.2], p=0.002 among eTreg). CTLA-4 expres- tures suggesting increased infiltration of inhibitory immune cells and sion on non-regulatory Foxp3- CD4+ T cells (non-Treg) also in- gene signatures related to the TGF-β pathway. Lastly, certain mesen- creased post-treatment (0.18 [0.16–0.20] vs. 0.21 [0.18–0.23], p= chymal markers were expressed at a higher level in melanoma com- 0.007). pared with NSCLC—in particular, the gene encoding vimentin, which Conclusions is associated with metastasis and poor prognosis, was 4-fold higher The combination of olaparib and durvalumab resulted in significant in melanoma. decrease in peripheral blood activated T cells, whereas exhausted T Conclusions cells and inhibitory markers on Treg, eTreg and non-Treg cells signifi- The results suggest that this cohort of melanoma patients may cantly increased. These findings contrast with the expected changes have resisted prior immune therapy due to a low neo-antigen under PD-L1 inhibitor treatment alone. These paradoxical changes of count and/or a mesenchymal phenotype. In contrast, the mecha- immune subsets likely reflect the anti-inflammatory effect of olaparib, nisms of resistance in NSCLC patients in this study may have which may have attenuated the antitumor immune efficacy of been related to high levels of inhibitory immune and TGF-β path- durvalumab. way genes. Collectively, these results provide novel insights into Trial Registration TMEs of melanoma and NSCLC in patients refractory or resistant NCT02484404 to anti–PD-(L)1 agents. Ethics Approval References This study was performed with IRB (#15C0179) and FDA approval 1. Sen T, Rodriguez BL, Chen L, Corte CMD, Morikawa N, Fujimoto J, et al. and is registered with Clinicaltrials.gov on August 7, 2015 Targeting DNA Damage Response Promotes Antitumor Immunity (NCT02517398). Patients provided written informed consent to through STING-Mediated T-cell Activation in Small Cell Lung Cancer. Can- participate. cer Discov. 2019;9(5):646-61. 2. Thomas A, Vilimas R, Trindade C, Erwin-Cohen R, Roper N, Xi L, et al. Dur- P347 valumab in Combination with Olaparib in Patients with Relapsed SCLC: Immunomodulation in tumor and peripheral blood following Toca Results from a Phase II Study. J Thorac Oncol. 2019. 511 & Toca FC treatment in patients with solid tumors 3. Krebs M, Ross K, Kim S, De Jonge M, Barlesi F, Postel-Vinay S, et al. P1.15- 1 2 3 4 Gerald Falchook, MD , Jordi Rodon , Shree Venkat , Arthur Donahue , 004 An Open-Label, Multitumor Phase II Basket Study of Olaparib and 4 3 5 Peder Horner , Amber Thomassen , William Accomando , Maria Durvalumab (MEDIOLA): Results in Patients with Relapsed SCLC. J Thorac 5 5 5 5 Rodriquez-Aguirre , Cornelia Bentley , Daniel Hogan , Derek Ostertag , Oncol. 2017;12(11):S2044-S5. 5 5 5 5 Sharon Yavrom , Thian Khoeh , Douglas Jolly, PhD , Harry Gruber, MD , 4. Rosado MM, Bennici E, Novelli F, Pioli C. Beyond DNA repair, the 5 3 Jolene Shorr , Jaime Merchan immunological role of PARP-1 and its siblings. Immunology. 1 2 Sarah Cannon Research Institute, Denver, CO, United States; MD 2013;139(4):428-37. Anderson Cancer Center, Barcelona, Spain; University of Miami, Miami, Ethics Approval FL, United States; Diversified Radiology of Colorado, Denver, CO, United The trial was conducted under a National Cancer Institute Center for States; Tocagen Inc., San Diego, CA, United States Cancer Research–sponsored investigational new drug application Correspondence: Gerald Falchook (gerald.falchook@sarahcannon.com) with institutional review board approval; approval number 15-c- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P347 Background P346 Toca 511 (vocimagene amiretrorepvec) is a cancer-selective, gamma- Comparison of TMEs from melanoma and NSCLC patients retroviral replicating vector encoding yeast cytosine deaminase, an refractory or resistant to anti–PD-(L)1 therapies enzyme that converts 5 fluorocytosine (5-FC) into 5-fluorouracil in George Locke, Cherie Taglienti, PhD, Laureen S. Ojalvo, Christoph the tumor microenvironment. Preclinical models indicated that Toca Helwig, MSc, Alex Rolfe, Olaf Christensen, Isabelle Dussault, PhD 511 and 5-FC treatment kills dividing cancer and nearby immunosup- EMD Serono Research & Development, Billerica, MA, United States pressive cells, leading to T-cell priming and antitumor immune activ- Correspondence: Isabelle Dussault (isabelle.dussault@emdserono.com) ity [1]. A Phase 3 trial of Toca 511 & Toca FC (extended-release 5-FC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P346 for treatment of recurrent high grade glioma is ongoing, following Phase 1 observations of prolonged survival and durable complete re- Background sponses in some patients [2]. Bintrafusp alfa (M7824), an innovative first-in-class bifunctional Methods fusion protein composed of the extracellular domain of the This Phase 1b, single-arm, multicenter study (Toca 6) was designed TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb to investigate immunological changes following Toca 511 & Toca FC blocking PD-L1, has shown evidence of clinical activity in treatment in patients with advanced solid tumors. Patients received phase 1 studies of patients with advanced solid tumors. intravenous (IV) Toca 511 for 3 days (Week 1), underwent biopsy of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 190 of 272 metastatic tumor (Week 2), were dosed with oral Toca FC (Weeks 5 Background and 6), underwent follow-up biopsy (~Week 9), and then repeated The anti-tumor responses to immune checkpoint inhibitors (ICI) is oral Toca FC every 4-6 weeks. Longitudinal peripheral blood mono- limited in malignancies such as pancreatic adenocarcinoma (PA), nuclear cell (PBMC) samples were immunophenotyped by flow cy- head and neck squamous cell carcinoma (HNSCC) and castration re- tometry, and tumor biopsies were analyzed by sistant prostate cancer (CRPC) through establishment of inaccessible immunohistochemistry (IHC). hypoxic regions [1,2]. Under hypoxic conditions, evofosfamide (EVO) Results releases the alkylating agent Br-IPM, which decreases hypoxia, re- A total of 21 patients with a median 4 lines of prior chemotherapy duces density of MDSC, restores T cell infiltration and increases were enrolled (17 colorectal cancer, 2 sarcoma, 1 each non-small cell tumor antigen presentation [3]. Across syngeneic models, EVO dem- lung and pancreas cancer). PBMC results suggest T-cell shifts from onstrates strong therapeutic cooperativity with ICI [4]. naïve to effector phenotypes, CD4+ memory T-cell expansion, and/or Methods B-cell increases after Toca FC in 41% of patients with pre- and post- A phase 1, dose-escalation trial using 3+3 design was conducted to Toca FC blood samples. Following treatment with Toca FC, some pa- determine the safety, tolerability and activity of EVO in combination tients showed marked changes in tumor infiltrating immune popula- with ipilimumab (IPI) for the treatment of 4 tumor types: metastatic tions assessed by IHC, including decreases in CD11b+ myeloid cells, or locally advanced PA, HPV negative HNSCC, ICI-refractory melan- Tregs, and exhausted T-cells, and increases in CD8+ T-cells. In oma and CRPC (NCT03098160). The study drugs (EVO, IPI) were given addition, IV Toca 511 led to viral expression in tumor, which was de- at the following doses respectively: level 1 (400mg/m2, 3mg/kg), creased post-Toca FC. Treatment has been generally well tolerated, level 2 (480mg/m2, 3mg/kg), level 3 (560mg/m2, 3mg/kg), level 4 with no related Grade 4 adverse events. At data cut-off, 9 patients (640mg/m2, 3mg/kg). EVO was administered on days 1 and 8 in cy- were alive (median follow-up 10.4 months); median overall survival cles 1 and 2. IPI was administered on day 8 of each 3 week cycle for was 9.6 months (95% CI 6.3, 16.4). A patient receiving concomitant a maximum of 4 doses after which retreatment was allowed in those panitumumab had a partial response. with irCR/ irPR/ irSD or irPD anytime after study initiation. Tumor re- Conclusions sponse was assessed using irRECIST. Change from baseline in periph- Results suggest Toca 511 infects metastatic tumor following IV eral blood and tumor tissue immune and hypoxia parameters were administration, and subsequent Toca FC induces tumor and im- evaluated as potential biomarkers of activity for this combination. munosuppressive cell killing. Preliminary analyses indicate Toca Results 511 & Toca FC treatment may be associated with T-cell mediated Twenty-one patients with a median age of 67 years were enrolled in immune activity in peripheral blood and metastatic tumor, con- the study, of whom a majority had CRPC (n=11) followed by PA (n= sistent with the immunologic mechanism of action observed in 7), melanoma (n=2) and HNSCC (n=1). Three patients were enrolled preclinical models. Preliminary clinical data suggest a signal of ac- at level 1 and six in level 2, 3 and 4 each. The most common any tivity in these heavily pretreated patients warranting further grade adverse events were rash (n=17), anemia (n=16) and investigation. leukopenia (n=12). The most common grade 3 adverse events were Trial Registration transaminitis (n=4), lymphopenia (n=3) and anemia (n=3). One pa- NCT02576665 tient required treatment discontinuation and 3 required EVO dose re- duction for toxicities. Out of 18 patients with measurable disease at References baseline, three (16.7%) had PR (2 with CRPC and 1 with HNSCC) and 1. Mitchell LA, Lopez Espinoza FL, Mendoza D, et al. Toca 511 gene transfer twelve (66.7%) had SD. The best responses were observed at dose and treatment with the prodrug, 5-fluorocytosine, promotes durable anti- level 3 (Figure 1). Reduced hypoxic exposure of myeloid stroma, cor- tumor immunity in a mouse glioma model. Neuro Oncol. 2017;19:930- relating with reduced suppressive polarization was observed. 939. Conclusions 2. Cloughesy TF, Landolfi J, Vogelbaum, et al. Durable complete responses No new or unexpected safety signals were observed with combined in some recurrent high-grade glioma patients treated with Toca 511 + EVO and IPI. The combination showed evidence of activity in heavily Toca FC. Neuro Oncol. 2018;20:1383-1392. pretreated refractory solid tumors. Dose expansion is planned at EVO Ethics Approval 560mg/m2 and IPI 3mg/kg. This study was approved by the institutional review boards of University of Miami Hospitals and Clinics, The University of Texas MD Anderson References Cancer Center, and Sarah Cannon Research Institute at HealthONE. 1. Blank CU, Haanen JB, Ribas A, et al: CANCER IMMUNOLOGY. The "cancer immunogram". Science 352:658-60, 2016 2. Chouaib S, Noman MZ, Kosmatopoulos K, et al: Hypoxic stress: obstacles P348 and opportunities for innovative immunotherapy of cancer. Oncogene A phase 1 dose escalation study to evaluate the safety and 36:439-445, 2017 tolerability of evofosfamide in combination with ipilimumab in 3. Duan JX, Jiao H, Kaizerman J, et al: Potent and highly selective hypoxia- advanced solid malignancies activated achiral phosphoramidate mustards as anticancer drugs. J Med 1 1 1 Aparna Hegde, MD , Priyamvada Jayaprakash, PhD , Elizabeth Sumner , Chem 51:2412-20, 2008 1 1 1 1 Di Nguyen , Hira Zain , Sarina Piha-Paul, MD , Daniel Karp , Jordi 4. Ai M, Budhani P, Sheng J, et al. Tumor hypoxia drives immune 1 1 1 Rodon , Shubham Pant, MBBS , Siqing Fu, MD, PhD , Ecaterina suppression and immunotherapy resistance. J Immunotherapy of Cancer 1 1 1 Dumbrava, MD , Timothy Yap, MD PhD , Vivek Subbiah, MD , Priya 2015;3(Suppl 2):P392. 1 2 2 2 Bhosale, MD , Jack Higgins, PhD , Eric T.Williams , Thomas F. Wilson , Ethics Approval 1 1 1 Funda Meric-Bernstam, MD , Michael Curran, PhD , David Hong, MD The study was approved by University of Texas MD Anderson Cancer The University of Texas MD Anderson Cancer Center, Bee Cave, TX, Center's Ethics Board, approval number IRB00000121. United States; Molecular Templates, Austin, TX, United States Consent Correspondence: Michael Curran (MCurran@mdanderson.org); David Written informed consent was obtained from the patient for publication of Hong (dshong@mdanderson.org) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P348 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 191 of 272 to reduce α-SMA expression vs RT, suggesting that bintrafusp alfa can reduce RT-induced fibrosis, presumably via TGF-β blockade. Conclusions Collectively, these preclinical findings support the clinical develop- ment of bintrafusp alfa and RT combination therapy and support the rationale for a clinical trial investigating bintrafusp alfa in combin- ation with chemoradiation (CRT) in stage III non-small cell lung can- cer (NSCLC; NCT03840902). In addition, the enhanced efficacy seen in multiple murine models supports the broad application of this combination for treatment of additional cancer indications. Ethics Approval This study was approved by the Institutional Animal Care and Use Committee at EMD Serono, Inc.; approval number [17-008]. P350 Clinical signal/profile in a phase I study of T cCell receptor (TCR) affinity-enhanced specific T cells (TAEST) in advanced cancer patients 1 1 2 3 Yi Li, PhD , Zhaosheng Han, PhD , Xing Zhang, MD , Jian Zhang , 4 1 2 Chengzhi Zhou , Haiping Gong , Desheng Weng, MD , Jianchuan Xia, 2 5 4 3 PhD MD , Johnson Lau , Shiyue Li , Weiliang Zhu Fig. 1 (abstract P348). See text for description 1 2 Guangdong Xiangxue Life Sciences, Ltd., Guangzhou, China; Sun Yat- sen University Cancer Center, Guangzhou City, Guangdong Provi, Peoples Republic of China; Zhujiang Hospital, Guangzhou, China; 4 5 Guangzhou Institute of Respiratory Healt, Guangzhou, China; Axis Therapeutics Ltd., Hongkong, Hong Kong P349 Correspondence: Shiyue Li (lishiyue@188.com) Effects of bintrafusp alfa (M7824) and radiation combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P350 therapy on antitumor activity, immune response, and radiation- induced fibrosis in multiple cancer models Background Yan Lan, MD, Chunxiao Xu, PhD, Huakui Yu, Guozhong Qin, Bo Marelli, T-cell triggering thresholds can be improved by engineered TCR with Jin Qi, Rachel E. Fontana, Amit Deshpande, George Locke, Alex Rolfe, enhanced binding affinity. TAEST for NY-ESO-1 was designed for po- Molly H. Jenkins, Joern-Peter Halle, Kin-Ming Lo tentially better efficacy and good safety profile. EMD Serono Research & Development, Billerica, MA, United States Methods Correspondence: Yan Lan (yan.lan@emdserono.com) Preclinical: Determined the TCR affinities. Expression of CD3, CD4, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P349 CD8, and TCR were traced by antibodies/tetramer. Specificity/efficacy in vitro/in vivo and TAEST infiltration in tumor/lymph node were Background evaluated. Clinical: Phase I study – 14 advanced cancer patients were We recently reported the enhanced preclinical antitumor activity of bin- treated with TAEST. trafusp alfa (M7824), an innovative first-in-class bifunctional fusion pro- Results tein composed of the extracellular domain of the TGF-βII receptor (a Preclinical: TAEST had higher affinity to its antigen vs wild type T- TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In phase 1 cells and with great expression (80-90% positive engineered TCR-T and 1b expansion studies in patients with advanced solid tumors, bin- cells), with ~5-6X more CD8+ over CD4+ cells. There was great trafusp alfa showed early evidence of clinical activity. Bintrafusp alfa is in vitro and in vivo efficacy with strong evidence of tumor specific a particularly rational combination partner for radiation therapy (RT) be- TAEST infiltration. Clinical: Stage I - TAEST alone was dosed in 3 cause RT induces expression of TGF-β, which can promote epithelial-to- NSCLC patients with demonstrated safety and stable disease (SD) ob- mesenchymal transition (EMT), fibrosis, and metastasis, and the expres- served for 28-165 days (OS: 77-308 days). Stage II - lymphodepletion sion of PD-L1. Furthermore, the induction of abscopal effects requires was added to TAEST in 11 patients (NSCLC 4, thyroid CA 1, liver the combination of RT with immunotherapy in mouse models, and CA 1, breast CA 1, colon CA 1, melanoma CA 1, synovial sarcoma abscopal responses have been reported in patients receiving RT in 1,and fibrotic sarcoma 1,) and treated with 0.8-2.15x1010 TAEST combination with an immune checkpoint inhibitor. cells: the synovial sarcoma patient had PR (> 70% tumor size re- Methods duction) with > 12 months duration; the breast CA patient had a The combination of bintrafusp alfa and RT was compared with bin- > 40% tumor shrinkage with healing of skin metastatic ulcers trafusp alfa monotherapy or RT alone in MC38 colorectal carcinoma, during Rx; Two other patients (liver, thyroid CA) showed SD but GL261-luc2 glioma, and 4T1 breast cancer murine models. Antitumor significant tumor necrosis (>50%) with symptomatic relief of local activity was evaluated via tumor growth, survival, and lung metasta- pain. Three NSCLC patients had SD with 59-188 days (Survival ses. Enzyme-linked immune absorbent spot (ELISpot) and immuno- 129-392 days). The fibrotic patient had SD for 87 days (Survival: histochemistry were used to measure the function and infiltration of 273 days); the melanoma patient had SD with 105 days (Survival: CD8+ T cells and the quantity of α-SMA, a marker of cancer- 176 days); last 2 patients ( NSCLC 1, colon CA 1) had PD at 14- associated fibroblasts. Gene expression signature scores of different 16 days post infusion ( OS: 92-129 days) . pathways were calculated from targeted RNAseq analysis. The treatment was tolerated well with fever (12/14), chills (4/14), neu- Results tropenia (5/14), thrombocytopenia(1/14), diarrhea(2/14), chest pain The combination therapy enhanced antitumor activity in all three mur- (1/14), and skin rash (3/14) observed. Expected cytokine response, ine models, increased tumor-specific and tumor-infiltrating CD8+ T cells TCR-gene detection/persistence (>60 days), were also observed in in the MC38 and 4T1 models, respectively, and potentiated an abscopal the patients above (particularly, >362 day for synovial sarcoma effect in secondary MC38 tumors. In the 4T1 model, combination ther- patient). apy decreased lung metastases vs either monotherapy and decreased the expression of EMT and VEGF pathway gene signatures vs RT. Ex- Conclusions pression of α-SMA significantly decreased with bintrafusp alfa mono- (1) TAEST, with its enhanced TCR binding affinity, is safe and toler- therapy in this model, whereas it significantly increased with RT able in a clinical phase I study; (2) TAEST exhibits encouraging effi- monotherapy. However, the combination with bintrafusp alfa was able cacy (DCR: 85.7%, 12/14) with a near CR for synovial sarcoma patient Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 192 of 272 (duration >12 months), and marked tumor necrosis with two more Conclusions patients (liver CA, thyroid CA); (3) Lymphodepletion pretreatment ap- The discordance between disease control per irRECIST and RECIST peared to be critical for efficacy/cytokine response/persistence of suggests that for approximately one in 12 patients, irRECIST is a bet- TAEST cells. ter indicator of clinical benefit from ICI treatment than RECIST. How- ever, overall no stronger association was observed between OS and irPFS compared with between OS and PFS. Thus, neither RECIST nor Acknowledgements irRECIST showed a clear advantage for predicting OS for clinical deci- The National key R&D Program of China, 2016YFC1303404; The Sciences and sions or regulatory purposes. Technology Program of Guangzhou, No. 201704020220. Trial Registration Acknowledgements ClinicalTrials.gov Identifier: NCT03159585; NCT03029273; NCT03462316 This study was funded by Merck KGaA, Darmstadt, Germany, as part of an Ethics Approval alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, The study of bone sarcoma and soft tissue sarcoma was approved by Sun NY, USA. Yat-sen University Cancer Center, approval number B2017-023-01. Trial Registration The study of NSCLC was approved by The First Affiliated Hospital of All trials were registered at clinicaltrials.gov, trial numbers NCT01772004 and Guangzhou Medical University, approval number 2016 No.63. NCT02155647. The study of multiple solid tumor was approved by Zhujiang Hospital of Ethics Approval Southern Mediacl University, approval number 2017-ZLZX-001. The trials were approved by the institutional review board or independent Consent ethics committee at each participating center. Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. P352 Workflow for Immune Monitoring during Clinical Trials by using unsupervised high dimensional augmented intelligence assisted P351 analysis Association between response assessment using RECIST and Alessandra Metelli, PhD, Carsten Krieg, PhD, Luis Cardenas, BS irRECIST in 1765 patients with advanced solid tumors treated with Medical University of South Carolina, Charleston, SC, United States avelumab monotherapy 1 2 1 Correspondence: Carsten Krieg (KriegC@musc.edu) Juliane Manitz , Peter Eggleton , Marcis Bajars , Oliver Bohnsack, MD, 3 4 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P352 PhD, MBA , James Gulley, MD, PhD, FACP EMD Serono Research and Development Institute, Inc, Billerica, Background Massachusetts, United States; Merck KGaA, Darmstadt, Germany, Following checkpoint inhibitors, which significantly improved cancer Darmstadt, Germany; PAREXEL Informatics, Berlin, Germany, Berlin, treatment, an increasing number of combination therapeutics is be- GERMANY; Center for Cancer Research, National Cancer Institute, ing tested in clinical trials. To find possible clinical or biological corre- National Cancer Institutes of Health, Bethesda, MD, United States lates of response or intervention, high throughput multi-omic Correspondence: Juliane Manitz (juliane.manitz@emdserono.com) approaches are necessary to catch all features of possible immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P351 responses during treatment. Methods Background To this aim, we present a portfolio of multi-omic approaches includ- A subset of patients receiving immune checkpoint inhibitor (ICI) ing high-dimensional mass cytometry (CyTOF) and single cell se- treatment may have unconventional response patterns, such as pseu- quencing in combination with unsupervised machine-learning doprogression, which can be classified as best overall response (BOR) bioinformatics to perform in depth characterization of immune re- of progressive disease (PD) by Response Evaluation Criteria in Solid sponses during clinical (immuno)therapy. The analysis is data driven, Tumors (RECIST) v1.1; therefore, immune-related (ir) response criteria, can be adapted to high throughput approaches and can model arbi- irRECIST, have been proposed. This analysis reports the differences in trary trial designs. response assessment by RECIST v1.1 and irRECIST and their associ- Results ation with overall survival (OS) in patients with advanced solid tu- We here show three proof of concept projects using biobanked per- mors treated with avelumab monotherapy (anti–PD-L1). ipheral blood mononuclear cells (PBMCs). In the first study, 51 pa- Methods tients with stage IV melanoma before and after 12 weeks of anti-PD- Data from patients with metastatic or locally advanced solid tumors 1 therapy were analyzed. We observed a clear T cell response on (n=1677, data cutoff, February 15, 2017) enrolled in the phase 1, therapy. The most evident difference in responders before therapy open-label JAVELIN Solid Tumor trial (NCT01772004), and data from was an enhanced frequency of CD14+ CD16+HLA-DRhi classical patients with metastatic Merkel cell carcinoma with disease progres- monocytes. We validated our results using conventional flow and sion after prior chemotherapy (n=88, data cutoff, March 24, 2017) en- found a clear correlation of enhanced monocyte frequencies before rolled in part A of the phase 2 open-label JAVELIN Merkel 200 trial therapy initiation with clinical response such as lower hazard and ex- (NCT02155647) were pooled. Patients with castration-resistant pros- tended progression-free and overall survival. In a second study, we tate cancer from the JAVELIN Solid Tumor study were excluded. All used CyTOF to monitor immune response in 21 non-small cell lung patients received avelumab 10 mg/kg every 2 weeks by intravenous cancer (NSCLC) patients that initially responded and then progressed infusion. BOR, disease control rate, and progression-free survival under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 (PFS) were evaluated. Concordance of disease control rates, Kaplan- plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a Meier, landmark OS, and correlation analyses were performed. response in the CD8+ T cell compartment was observed. Unexpect- Results edly, our high dimensional unbiased analysis was able to detect and A total of 1765 patients were included. All patients had ≥3 months characterize a strong expansion of innate tumor-reactive effector NK of follow-up. The pooled data set included 12 tumor types. The dis- cells starting around day 4 of therapy. In our third unpublished study cordance between the tumor assessment criteria for disease control we were able to identify neutrophils as predictors of outcome in lung rate was 8.3% (n=147), i.e. complete response + partial response + cancer patients. stable disease (SD) per irRECIST and PD + not evaluable per RECIST; Conclusions most patients (n=135) had a BOR of PD by RECIST and irBOR of SD Taken together, our unbiased artificial intelligence-driven immune by irRECIST. The Kaplan-Meier analysis according to Wolchok et al ex- workflow is an extraordinary instrument to monitor immune re- hibited clear separation of the respective (dis)concordant subgroups. sponses during (immuno)therapy and serves as a novel approach for The rank correlations between OS and PFS and OS and irPFS were therapeutic target identification. 0.73 (95% CI, 0.70-0.75) and 0.75 (95% CI, 0.72-0.78), respectively. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 193 of 272 Trial Registration P354 ClinicalTrials.gov Identifier: NCT02523469 Exploring correlates of clinical and immune response to cancer Ethics Approval immunotherapy using FAUST, a novel unbiased cell population This study was approved by the MUSC and Zurich institutional review discovery method, in whole blood flow cytometry board. Steven Fling, PhD, Nirasha Ramchurren, PhD, Leonard D'Amico, Martin Cheever, MD, Evan Greene, Greg Finak, Raphael Gottardo, PhD Fred Hutchinson Cancer Research Center, Seattle, WA, United States P353 Correspondence: Steven Fling (sfling@fredhutch.org) Phase 1 pilot study of RRx-001 + nivolumab in advanced Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P354 metastatic cancer (PRIMETIME) 1 2 1 3 Corey Carter, MD , Bryan Oronsky, MD PhD , Mary Quinn , Jane Trepel , Background 4 5 Nacer Abrouk, PhD , Jeff Skinner, MD The purpose of this study is to describe an immune monitoring ap- 1 2 EpicentRx, Inc., La Jolla, CA, United States; EpicentRx Inc, La Jolla, CA, proach that combines multi-parameter whole blood flow cytometry 3 4 United States; NIH, Bethesda, MD, United States; Clinical Trials with an automated, unbiased, cell population evaluation method to Innovations, Mountain View, CA, United States; Walter Reed National Mil facilitate discovery of informative correlative biomarkers. The Cancer Medical Center, Bethesda, MD, United States Immunotherapy Trials Network (CITN) coordinates multi-center can- Correspondence: Mary Quinn (mquinn@epicentrx.com) cer immunotherapy trials, wherein multiparameter flow cytometry is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P353 performed in real time on longitudinally collected whole blood samples. Background Methods RRx-001 is a minimally toxic small molecule that downregulates We recently reported results from two CITN multi-center clinical trials. CD47 and repolarizes tumor associated macrophages (TAMs) as well We also reported a non-parametric method for unbiased cell popula- as normalizes aberrant tumor perfusion. On the premise that the tion discovery that annotates cell populations with biologically inter- interaction between a CD-47 downregulator like RRx-001 and an pretable phenotypes through a new procedure called Full anti-PD-1 inhibitor like nivolumab may serve to activate both arms of Annotation Using Shape-constrained Trees (FAUST). We used FAUST the immune system, a phase 1 pilot study was undertaken to deter- to analyze extensive flow cytometry data in these two CITN clinical mine the safety and feasibility of RRx-001 and nivolumab in patients trials and demonstrate that candidate biomarkers can be associated with advanced cancer and no standard options. with clinical outcome. Here we compare flow cytometry data ana- Methods lyzed both by conventional manual gating strategies as well as by This single arm, open-label pilot study (NCT02518958) called PRIME- the FAUST method. TIME was designed to evaluate the safety profile of RRx-001 and Results nivolumab in patients with advanced malignancies and no other We highlight the value of FAUST in identifying predictive biomarkers of standard therapeutic options. A 3+3 trial design was used to estab- clinical responses to immunotherapy within fresh whole blood. By com- lish safety of the combination at each dose level and guide the deci- bining whole blood flow staining with FAUST, our results demonstrate sion to escalate dose. RRx-001 is infused once weekly while the ability to capture important minor cell subpopulations, including nivolumab is given at 3mg/kg once every 2 weeks. The RRx-001 start- within the CD8 T cell compartment, that otherwise are missed by man- ing dose was 2 mg IV weekly with 4 dose level escalations up to 16 ual gating. Manual gating can be biased and limited to characterizing mg IV weekly. From January 2015 to November 2015, twelve patients cell populations considered a-priori to be significant. received treatment for only 4 cycles (total 12 weeks) with the com- Conclusions bination due to unavailability of nivolumab, which was not supplied Our results emphasize the unique value of performing flow cytome- to the Sponsor. Treatment-emergent (all cause, TEAEs) and try in multi-center trials using fresh whole blood which preserves the treatment-related (TRAEs) adverse events that occurred within 16 minor cell populations identified by FAUST which may be lost or weeks of the first dose of RRx-001 and nivolumab were characterized compromised by standard cryopreservation methods. according to CTCAE v4.03. Results Twelve patients received >1 dose of RRx-001 and nivolumab. One P355 discontinuation occurred due to pneumonitis and one to volun- Multicenter, open-label, phase 1 study of DSP-7888 Dosing tary withdrawal after a post-procedural infection. There were no Emulsion (DSP-7888) in patients with advanced malignancies 2 3 4 5 DLTs. The main adverse event related to RRx-001 was infusion re- Morris Groves , Aaron Hansen , Wael Harb , Kelly Curtis, MD , Erina 6 7 7 8 action (33.3%). The main adverse event related to the combin- Koga-Yamakawa , Makoto Origuchi , Zhonggai Li , Jose Iglesias , Walid 9 1 ation was pseudoprogression manifested by larger tumors in Shaib, MD , Alexander Spira, MD, PhD, FACP 1 2 patients that were symptomatically improved (25%). The most Virginia Cancer Specialists, Fairfax, VA, United States; Texas Oncology- common immune-related treatment-emergent AEs were pneu- Austin Midtown, Austin, TX, United States; UHN Princess Margaret monitis (8.3%), and hypothyroidism (8.3%). The objective response Cancer Centre, Toronto, Canada; Horizon Oncology Research, LLC, rate at 12 weeks was 25% and the disease control rate (DCR) Lafayette, IN, United States; Syneos Health, Phoenix, AZ, United States; consisting of > SD was 67% by Response Evaluation Criteria in Sumitomo Dainippon Pharma Co., Ltd, Cambridge, MA, United States; 7 8 Solid Tumors (RECIST) version 1.1 25% of the patients progressed Boston Biomedical, Inc., Cambridge, MA, United States; Former on the combination. employee, Boston Biomedical, Inc., Cambridge, MA, United States; Conclusions Emory University, Atlanta, GA, United States The combination of RRx-001 and nivolumab was safe and well- Correspondence: Alexander Spira (Alexander.Spira@USOncology.com) tolerated with preliminary evidence of anti-cancer activity. Further Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P355 analyses with a larger sample size will be required to confirm the ac- tivity of the combination and to determine the optimum schedule Background for RRx-001 and nivolumab. DSP-7888, a cancer vaccine composed of 2 synthetic peptides de- Ethics Approval rived from Wilms’ tumor 1 (WT1) protein, may induce WT1-specific The study was approved by all the revelant Institution‘s Ethics cytotoxic T-lymphocytes (CTLs) and helper T-lymphocytes–mediated Boards. immune responses against WT1-expressing tumors. This dose- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 194 of 272 escalation study (NCT02498665) evaluated DSP-7888 in patients with infiltrating leukocyte (TIL) analysis. CTL, IgG, and TIL were mea- recurrent or progressive advanced malignancies, despite receiving sured by ELISPOT assay, Luminex assay, and IHC (CD8 positive), standard therapy, or in patients intolerant to standard therapy or for respectively. whom no standard of therapy existed for their malignancy. The pri- Results mary objectives were safety, tolerability, and identification of the rec- Seventeen patients were enrolled in 9mg (n=10) and 27mg (n= ommended phase 2 dose (RP2D). Secondary and exploratory 7) groups; the median age was 65 years, and 53% of the pa- objectives included overall survival and WT1-specific CTLs induction. tients had ECOG PS 1. There was no serious adverse drug reac- Methods tion (ADR) in any patient. All ADRs were of grade 1 or 2, with Patients who failed or were intolerant to prior lines of treatment and the most frequent being dermatological injection site reaction, tested positive for HLA-A*02:01, HLA-A*02:06, or HLA-A*24:02 re- in 7/10 (70%) and 6/7 (86%) patients and pyrexia, in 1/10 (10%) ceived escalating doses of intradermal (ID) or subcutaneous (SC) and 2/7 (29%) for the 9mg and 27mg groups, respectively. The DSP-7888 in a rolling study design: 3.5, 10.5, or 17.5 (ID only) mg best overall response was stable disease, in 2/10 (20%) and 2/7 every week for 4 weeks, then every 1–2 weeks for 6 weeks, and every (28%) patients. One patient with cancer of unknown origin re- 2–4 weeks thereafter until progression or other discontinuation event ceived prolonged administration (over 10 months) of the 9mg was met. Dose-limiting toxicities (DLTs) were evaluated over days 1– dose.Inthe 9mgand 27mggroups, antigenspecific IgG was 29. The dose at which ≤1 of 6 patients had a DLT was eligible to be augmented in 9/10 (90%) and 7/7 patients (100%), antigen spe- the RP2D. WT1-specific CTL inductions were assessed by HLA Tetra- cific CTL was detected in 2/10 (20%) and 3/7 patients (43%), mer with peripheral blood. and TIL counts were increased in 2/3 (67%) and 3/4 patients Results (75%), respectively. Twenty-four patients received ID (3.5 mg, n=4; 10.5 mg, n=3; 17.5 Conclusions mg, n=3) or SC DSP-7888 (3.5 mg, n=9; 10.5 mg, n=5). The most fre- TAS0313 demonstrated safety, tolerability, and immunological re- quent adverse event (AE) was injection site reaction (ISR; n=15; sponses in patients with advanced solid tumors in the 9mg and 62.5% [ID: 100% of patients, SC: 36%]); all were grade 1 or 2. No DLT 27mg groups. A phase II part, evaluating the efficacy of combin- was observed. ID DSP-7888 10.5 mg was determined to be the dose ation therapy with pembrolizumab in patients with urothelial car- level for further study based on the RP2D identified in a phase 1/2 cinoma and monotherapy in glioblastoma patients, is currently study of DSP-7888 in patients with myelodysplastic syndrome underway. (NCT02436252). Four patients (ID 17.5 mg, n=1; SC 3.5 mg, n=1; SC Trial Registration 10.5 mg, n=2) had stable disease, 16 had progressive disease, and 4 JapicCTI-183824 were not evaluable. Twenty-one patients were evaluable for WT1- Ethics Approval specific CTL detection. In evaluable patients, WT1-specific CTL induc- The study was approved by National Cancer Research Center Central tion was observed in 6 of 9 ID patients (66.7%) and 5 of 12 SC pa- Hospital’s Ethics Board, approval number T4499. tients (41.7%). Consent Conclusions Written informed consent was obtained from the patients for publi- DSP-7888 was well tolerated, with no DLTs, in patients with ad- cation of this abstract. A copy of the written consent is available for vanced malignancies, supporting further evaluation of DSP-7888. The review by the Editor of this journal. 10.5 mg ID dose was identified as a dose level and route of adminis- tration for further evaluation. P357 Phase I/II clinical and immune responses for locally advanced or P356 metastatic pancreatic cancer using anti-CD3 x anti-EGFR bispecific First-in-human study of the cancer peptide vaccine, TAS0313, in antibody armed T cells (BATs) 1 1 2 1 patients with advanced solid tumors: phase I dose finding part Lawrence Lum, MD, DSc , Tri Le , Minsig Choi , Archana Thakur, PhD , 1 1 3 1 results Matthew Reilly , Paul Kunk, MD , Abhinav Deol, MD , Karen Ballen, MD , 1 1 1 Noboru Yamamoto, MD, PhD, Jun Sato, MD, PhD, Satoru Iwasa, MD, Tamila Kindwall-Keller, DO , Dana Schalk , Ewa Kubicka , Manley Huang, 1 3 3 3 4 PhD, Kan Yonemori, MD, PhD, Takafumi Koyama, MD, Kenji Tamura, MD, PhD , Philip Philip, MD , Hussein Aoun , Gregory Dyson, PhD , Qin Liu , PhD, Toshio Shimizu, MD, PhD, Syunsuke Kondo, Shigehisa Kitano Anthony Shields, MD PhD 1 2 National Cancer Center Hospital, Tokyo, Japan University of Virginia, Charlottesville, VA, United States; Stony Brook Correspondence: Toshio Shimizu (tosshimi@ncc.go.jp) University, Stony Brook, NY, United States; Karmanos Cancer Institute, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P356 Wayne State U, Detroit, MI, United States; Wistar Institute, Philadelphia, PA, United States Background Correspondence: Lawrence Lum (lawrenceglum@cs.com) TAS0313 is a cancer vaccine cocktail containing three long peptides, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P357 with a total of 12 cytotoxic T lymphocyte (CTL) epitope peptides. These peptides were derived from eight cancer-associated antigens Background that are highly expressed in various cancers. We report the results of Chemotherapy for locally advanced pancreatic cancer (LAPC) and a phase I part examining the tolerability, safety, potential efficacy, metastatic pancreatic cancer (MPC) has poor responses and survival and immunological responses of 9 mg and 27 mg TAS0313 in pa- rates. Retargeting anti-CD3 activated T cells (ATC) by arming them tients with advanced solid tumors. with anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) makes ATC Methods into specific cytotoxic T lymphocytes (EGFR BATs). Targeting pancre- The enrolled patients had ECOG PS 0-1 and at least one of the atic cancer cell lines induces cytokine secretion, proliferation, cyto- following HLA types: HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, toxicity, and inhibits tumor growth. We present 5 phase I and 13 -A*24:02, -A*31:01, or -A*33:03. Emulsified TAS0313 solution with phase II patients for a total of 18 evaluable patients out of 21 who an immunological adjuvant (Montanide ISA-51) was subcutane- underwent apheresis. ously administered on Days 1, 8, and 15 of Cycles 1 and 2 and Methods on Day 1 of Cycle 3 or later in 21-day cycles until disease pro- In the phase I, LAPC or MPC patients at Karmanos Cancer Institute gression, or unacceptable toxicity.. Tolerability was assessed in at (KCI) on Protocol #2011-025, in a dose escalation, were given 10, 20, least six patients during the first cycle. Tumor response was eval- and 40 x 10^9 BATs/infusion weekly for 3 weeks, followed by a uated using RECIST v1.1. Blood samples were collected pre- and booster infusion 3 months later if patients were stable or better. post-treatment for the analysis of antigen specific CTL and IgG. There were no dose limiting toxicities, and all infusions were given in Optional serial tumor biopsies were performed for tumor the outpatient setting. In the phase II portion, 13 PC patients at KCI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 195 of 272 (NCT02620865) and University of Virginia (NCT03269526) received Methods twice weekly infusions of 10 x 10^9 BATs/infusion over 4 weeks for a Patients with advanced SS were enrolled to cohorts based on NY-ESO-1 total of 80 x 10^9 EGFR BATs. expression (Cohort 2, low; Cohort 4, high) determined by immunohisto- Results chemistry. Treatment response (RECIST v1.1), safety (CTCAE v4.0), and Eighteen patients were evaluable. Four patients were stable at 6.1, GSK3377794 persistence in transduced PBMCs (transgene vector copies 6.5, 5.3, and 39 months. Two patients developed complete responses measured by qPCR) were assessed. Progression-free survival (PFS) was (CR) when chemotherapy was restarted after their BATs infusions. Pa- defined as the interval between first infusion and first documented dis- tient IT20104 was stable for 1 year on capcitabine, developed “pseu- ease progression or death. Safety was monitored throughout. The study doprogression,” achieved a CR after restarting capcitabine, and was was not designed/powered for cohort comparison. off therapy until 54 months after enrollment when relapse occurred. Results The median overall survival is 14.8 months with a time to progres- As of April 2019, 50 patients were enrolled (N=13 Cohort 2; N=15 Co- sion of 6.6 months. Specific cytotoxicity mediated by peripheral hort 4). Table 2 summarizes response outcomes by Cohort. Median PFS blood mononuclear cells (PBMC) peaked at 31% two weeks after the (95% CI) was 13.1 weeks (7.9, 13.9; Cohort 2) and 22.4 weeks (11.3, 26.6; third infusion, and IFN-γ EliSpots rose from Cohort 4). Median peak (range) persistence of ~64,712 DNA copies/μg Conclusions (13,364–197,546) occurred in Cohort 2 first week post-infusion versus EGFR BATs infusions were safe and induced specific adaptive anti- ~16,468 DNA copies/μg(163–131,175) in Cohort 4. No significant cor- tumor responses. This phase I/II study strongly suggests that multiple relation was observed between peak persistence and best overall re- infusions of EGFR BATs may provide a survival benefit in patients sponse in either cohort (p>0.05). Grade 3/4 adverse events occurring in with pancreatic cancer, and that BATs therapy may increase the ef- ≥40% of patients in both cohorts were leukopenia, neutropenia, fectiveness of subsequent chemotherapy, which will drive the design anemia, thrombocytopenia, lymphopenia, and hypophosphatemia. of future combination trials of BATs and other modalities of therapy. Conclusions Cohorts 2 and 4 showed similar ORRs; more durable responses were Acknowledgements observed in Cohort 4, with prolonged DoR, duration of stable dis- These studies were made possible thanks to philanthropy from Karmanos Cancer ease, and PFS. Peak persistence of GSK3377794 was higher in Cohort Institute and start-up funds for LGL from the University of Virginia. LGL and MH 2, likely due to higher lymphodepletion, but this did not correlate are co-founders of Transtarget, Inc. LGL is a member of the Scientific Advisory with response, unlike data previously reported in other cohorts. Fur- Board for Rapa Therapeutics. AT is a co-founder of NOVA Immune Platform. ther development in SS will be based on previously reported data Trial Registration from Cohort 1. Protocol #2011-025; NCT02620865; NCT03269526 Ethics Approval Acknowledgements These studies were approved by the Karmanos Cancer Institute / Wayne Medical writing assistance was provided by provided by Fiona Woodward State University IRB, approval numbers 2011-25 and 2015-100, and the and Victoria Hunter of Fishawack Indicia Ltd. This study (NCT01343043) was University of Virginia IRB, approval number HSR 19236. funded by GlaxoSmithKline. Trial Registration NCT01343043 P358 Ethics Approval Phase 1 trial of NY-ESO-1-specific adoptive T-cell therapy with This study was approved by the appropriate institutional review boards and GSK3377794 in patients with advanced synovial sarcoma independent ethics committees. 1 2 3 Sandra D'Angelo, MD , George Demetri , Brian Van Tine, MD, PhD , 4 5 6 7 Mihaela Druta , John Glod, MD , Warren Chow , Jenna Tress , M. Phillip 7 7 7 7 7 DeYoung , Aisha Hasan, MBBS MD , Yuehui Wu , David Turner , Ran Ji , 7 8 Alexandra Gyurdieva , Dejka Araujo, MD Table 1 (abstract P358). See text for description Memorial Sloan Kettering Cancer Center, New York, NY, United States; 2 3 Dana-Farber Cancer Institute, New York, NY, United States; Washington University in St. Louis, St. Louis, MO, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; National Cancer Institute, Bethesda, MD, United States; City of Hope Comprehensive Cancer Center, Duarte, 7 8 CA, United States; GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P358 Background Genetically-engineered NY-ESO-1 specific T-cells (NY-ESO-1 T-Cells; GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a self-inactivating lentiviral vector to express affinity-enhanced NY-ESO-1- specific T-cell receptors (TCRs). Ongoing phase 1 and 2 trials are evalu- ating GSK3377794 in solid tumors and multiple myeloma. Study NCT01343043 (208466) is a phase 1 clinical trial assessing GSK3377794 in patients with previously treated, advanced metastatic synovial sar- coma (SS), stratified into 4 cohorts (Table 1). Of 12 patients receiving GSK3377794 infusion in Cohort 1, responses were observed in 6 pa- tients (1 complete response [CR]/5 partial responses [PR]), with an over- all response rate (ORR) of 50% (95% confidence interval [CI]: 0.21–0.79). Median progression-free survival (PFS) was 15.2 weeks (95% CI: 7.6– 37.9); median duration of response (DoR) was 30.9 weeks (95% CI: 14– 72). As of October 15, 2018, median overall survival (OS) was 105 weeks (95% CI: 37–NA). This abstract reports data from Cohorts 2 and 4. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 196 of 272 Table 2 (abstract P358). See text for description manageable adverse events. Our study shows feasibility of tracking phenotypic profiles of adoptively transferred tumor-antigen-specific T cells in patients and derive association between adoptive T cell sta- tus and clinical read-outs. Ethics Approval The study was approved by Mie University Ethics Board, approval number H2018-092. P360 The influence of Durvalumab/Tremelimumab Combination Therapy on Sarcomas Immune Microenvironment profile in a phase II clinical trial (NCT02815995) 1 1 1 Edwin Parra, MD, PhD , Carmelia Barreto, PhD , Ruth Salazar, MD , Cara 1 1 1 1 Haymaker, PhD , Heather Lin , Carmen Behrens, MD , Mei Jiang , Luisa 1 1 1 Solis, MD , Krishna Pandurenga, MS , Sandesh Subramanya, PhD , Young 2 1 1 1 Kim, PhD , Chantale Bernatchez , Jack Lee, PhD , Taylor Tate , Teresa 1 1 1 Simmons , Alexander Lazar, MD, PhD , Wei-Lien Wang , Zachary Cooper, 3 3 3 PhD , Jaime Rodriguez-Canales, MD , Jean Soria, MD , Anthony Conley, 1 1 1 MD , Ignacio Wistuba, MD , Neeta Somaiah, MD, MBBS 1 2 MD Anderson Cancer Center, Houston, TX, United States; Translational Molecular Pathology, Houston, TX, United States; AstraZeneca, Gaithesburg, MD, United States Correspondence: Edwin Parra (erparra@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P360 Background To determine the tumor microenvironment (TME) changes after the combination of durvalumab/tremelimumab treatment, longitudinal P359 sarcoma tissue collections were obtained and analyzed for in-depth Tracking and profiling of NY-ESO-1 TCR-transgenic T cells upon immunoprofiling. adoptive transfer in patients with NY-ESO-1-expressing solid Methods tumors: in vivo differentiation associated with response 1 1 1 Sixty-two patients were enrolled and 36 paired samples (Liposarco- Michael Fehlings , Alessandra Nardin, DVM , Faris Kairi , Evan Newell, 2 3 3 3 ma,LS=6; Angiosarcoma,AS=2; Leiomyosarcoma,LMS=2; Osteo/Chon- PHD , Yoshihiro Mihayara , Shinichi Kageyama , Hiroshi Shiku, MD 1 2 drosarcoma,OS/CS=3/1; Undifferentiated Pleomorphic Sarcoma,UPS= immunoSCAPE, Singapore, Singapore; Fred Hutchinson Cancer 3; Alveolar Soft Part Sarcoma,ASPS=8; Synovial Sarcoma,SS=3; Chor- Research Center, Singapore, Singapore; Mie University School of doma,C =2; and other types,OT=6), were evaluable for TME changes Medicine, Tsu, Japan and correlated with clinical benefit (PR or SD). All patients were Correspondence: Hiroshi Shiku (shiku@clin.medic.mie-u.ac.jp) treated with durvalumab/tremelimumab every 4 weeks for four cy- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P359 cles and then continued durvalumab every 4 weeks for up to 1 year. Biopsies were collected prior to-and- during treatment (Wk6). Malig- Background nant cells (MCs) PD-L1+ was studied by immunohistochemistry. NY-ESO-1 is highly expressed in the majority of synovial sarcomas as Tumor-infiltrating-lymphocytes (TILs), and macrophages were interro- well as other solid tumors and may be an effective target for T cell- gated by multiplex immunofluorescence, Figure-1. The combination based therapies. We conducted a clinical study of adoptive transfer of three T-cell phenotypes (CD3+,CD3+CD8+ and CD3+CD8+GZB+) of lymphocytes transduced with NY-ESO-1-specific TCR in refractory greater than the median density as higher TILs (TILs+) was used to cancer patients with preconditioning (TBI-1301). define “inflamed” tumors and ≤ than the median of 1 or 2 of these Methods as lowest TILs (TILs–) to define “non-inflamed” tumors, through base- High-dose of 5 billion autologous transduced and expanded lympho- line to Wk6. To characterize patterns of the TME and changes be- cytes, consisting of >96% T cells, was transferred into 6 patients, three of tween baseline and Wk6 we stratified the tumors in four groups whom with synovial sarcoma. Longitudinal PBMC samples were obtained using an approach similar as Teng's criteria (1). for immunomonitoring. We used high-dimensional mass cytometry and Results combined a 36-antibody panel with a multiplexed combinatorial Overall, all the phenotype median densities increased from baseline to peptide-MHC tetramer staining approach to longitudinally track and Wk6, Table-1. PR or SD was observed in 17/36 with paired samples, phenotypically characterize adoptively transferred HLA-A*02:01-NY-ESO-1 47% (3 LS, 1 LMS, 1 OS, 7 ASPS, 1 SS, 2 C, and 2 OT). Five ASPS showed transgenic TCR T cells 14, 28, and 56 days after treatment. PR and 2 SD out of 8 cases. We categorized as inflamed tumor 1 LPS, 1 Results AS, 1 LMS, 1 OS, and 5 ASPS at baseline. Interestingly 1 AS, 1 OS, 1 CS, Three out of 6 patients with tumors expressing >75% NY-ESO-1 experi- 1ASPS, 1 SS and 2 OT defined as non-inflamed tumors at baseline chan- enced an objective clinical response (PR) and had cytokine-release syn- ged to inflamed tumors at Wk6 (Figure-2) and from those, OS and ASPS drome (CRS) with high-levels of IL-6 and MCP-1 that could be managed showed SD and PR, respectively. Finally, 4/17 inflamed tumors showed with tocilizumab. The infusion products had variable percentages of SD and 3/17 PR. Furthermore, CD3+CD8+CD45Ro+ increase in the in- naive, TEMRA and EM CD8+ cells, with the three clinical responders flamed tumors at Wk6 than non-inflamed tumors (P=0.005). The most having the highest proportion of EM T cells. NY-ESO-1 TCR transgenic T frequent TME pattern detected at baseline and Wk6 was the immuno- cells could be detected in the circulation of 5 out of 6 treated patients, logical ignorance (TILs-PD-L1-, 61% and 47%, respectively). Interestingly, with frequencies peaking at day 14 and day 28; specific T cells were un- adaptive immune resistance pattern changed from baseline to Wk6 detectable in all patients by day 56. In responders, a substantial num- (TILs+PD-L1+, 14% and 22%, respectively), Figure-3. ber of circulating NY-ESO-1-specific CD8+ T cells showed a phenotypic Conclusions profile consistent with antigen-experience, activation and differenti- Combination of durvalumab/tremelimumab influenced the TME in ation into an effector phenotype 28 days post transfusion. the selected sarcomas cohorts. The immunologic score assessment in Conclusions this longitudinal collection demonstrates the capability to distinguish Adoptive transfer of NY-ESO-1 TCR-transgenic T cells has shown signs non-inflamed vs inflamed tumors and relate it with a clinical benefit, of efficacy in patients with high NY-ESO-1 tumor expression, with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 197 of 272 showing the value of use these markers as possible immune prog- nostic markers in sarcomas. Trial Registration This trial is registered with ClinicalTrials.gov (NCT02815995) References 1. Teng MW, Ngiow SF, Ribas A, Smyth MJ. Classifying Cancers Based on T- cell Infiltration and PD-L1. Cancer Res. 2015;75(11):2139-45. Ethics Approval The study was approved by MD Anderson Institution Ethics Board, Clinical Trail number NCT02815995 Fig. 3 (abstract P360). See text for description Table 1 (abstract P360). See text for description P361 Molecular and immunologic profiling of CD8+ T cell responses in patients receiving a multiple antigen-engineered dendritic cell vaccine 1 2 2 Juraj Adamik, PhD , Patricia Santos, PhD , Samuel Du, BS , Lazar 2 1 3 Vujanovic, PhD , Timothy Howes , Sarah Warren, PhD , Andrea 2 2 1 Gambotto, MD , John Kirkwood, MD , Lisa Butterfield, PhD Parker Institute for Cancer Immunotherapy, San Francisco, CA, United States; University of Pittsburgh, Pittsburgh, PA, United States; NanoString Technologies, Seattle, WA, United States Correspondence: Lisa Butterfield (lbutterfield@parkerici.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P361 Background Despite the immunogenicity and safety profile of dendritic cell (DC) vaccines, the importance of vaccine-induced antigen-specific T cell responses is unclear across clinical trials, and therapeutic efficacy re- mains low with limited clinical responses. Our comprehensive characterization of T cell responses, cell-intrinsic and soluble immune checkpoint molecules and immune-related gene expression profiles reveal novel insights into CD8+ T cells specific for melanoma- associated antigens (MAA) from patients who received autologous DC engineered to express three full length melanoma antigens: tyro- sinase, MART-1 and MAGE-A6 [1]. Methods MAA-specific T cell responses were examined by standardized IFNγ Fig. 1 (abstract P360). See text for description ELISPOT assays at baseline, day 43 (post DC vaccines) and d89 (post observation or IFNα). Luminex was used to detect serum checkpoint and costimulatory molecules, and whole blood flow cytometry was used to quantify PBMC subsets. Targeted mRNA and protein expres- sion analyses in circulating lymphocytes and melanoma tumor sam- ples were performed using NanoString nCounter platform (RUO). Results The majority of the 35 patients were successfully vaccinated, and the total vaccine-induced T cell responses were higher among those exhibiting a favorable clinical outcome. Patients who received check- point blockade treatment prior to DC vaccination had higher base- line MAA-specific CD8+ T cell responses, yet they did not respond more strongly to the vaccine. Two patients who received checkpoint blockade post-DC vaccine showed very strong amplification of their MAA-specific T cells. Molecular profiling in circulating lymphocytes and tumor biopsies showed that elevated PD-1 and CTLA-4 protein levels and gene expression signatures representing checkpoint sig- naling, interferon response and T-cell exhaustion were associated with unfavorable clinical outcome. Gene signatures showing positive correlation with PD-1 protein expression included CD28-dependent PI3K-AKT signaling, the IL12/STAT4 pathway and pan-semaphorin re- ceptor interactions. CTLA-4 protein levels correlated with type I inter- feron response and NOTCH signaling genes. Interestingly, B cell receptor pathways negatively correlated with PD-1 expression, while gene signatures downstream of T cell receptor activation and IL-2 signaling were negatively correlated with CTLA-4 expression. Serum levels of PD-1 and PD-L2 were inversely correlated and post-vaccine Fig. 2 (abstract P360). See text for description serum levels PD-L2 correlated with decreased circulating Treg and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 198 of 272 favorable outcome in patients, suggesting that it may serve as a bio- (22.4%). Median follow-up was 21.2 months (range, 14.9-36.6). marker of clinical response. The ORR was 39.7% (95% CI: 30.7%-49.2%), including 19 patients Conclusions (16.4%) with a CR and 27 (23.3%) with a PR. In patients with PD- Collectively, our study shows that specific checkpoint molecular path- L1+ (n=21 [18.1%]) or PD-L1− (n=87 [75.0%]) tumors, ORRs were ways are critical for vaccine outcomes and for the activation of anti- 61.9% (95% CI: 38.4%-81.9%) and 33.3% (95% CI: 23.6%-44.3%), tumor responses in melanoma patients. Comprehensive profiling of respectively. Median DOR was 18.2 months (95% CI: 11.3 months- MAA-specific T cell responses suggests that DC-vaccine immunization not estimable). 35 patients had a response lasting ≥6months followed by immune checkpoint blockade may be an optimal se- (durable response rate, 30.2% [95% CI: 22.0%-39.4%]). 6- and 12- quential therapy to improve antitumor immunity in melanoma. month PFS rates were 41% (95% CI: 32%-50%) and 31% (95% CI: Trial Registration 23%-40%), respectively. Median OS was 20.3 months (95% CI: FDA IND #15044 and NCT01622933. 12.4 months-not evaluable), and the 12-month OS rate was 60% (95% CI: 50%-68%). In PD-L1+ and PD-L1− subgroups, 12-month Reference OS rates were 71% (95% CI: 47%-86%) and 56% (95% CI: 45%- 1. Butterfield LH, Vujanovic L, Santos PM, Maurer DM, Gambotto A, Lohr J, 66%), respectively. Treatment-related adverse events (TRAEs) of Li C, Waldman J, Chandran U, Lin Y, Lin H, Tawbi HA, Tarhini AA, any grade occurred in 94 patients (81.0%), including grade ≥3 Kirkwood JM. Multiple antigen-engineered DC vaccines with or without TRAEs in 21 (18.1%). No treatment-related deaths occurred. IFNa to promote antitumor immunity in melanoma. JITC. 2019; 7:113. Conclusions Ethics Approval Updated results from JAVELIN Merkel 200 confirm that first-line ave- The clinical trial was fully approved by the Univ. Pittsburgh PRC and IRB lumab treatment was associated with durable responses, a clinically (PRO12010416, #09–021). meaningful OS benefit, and an acceptable safety profile in patients with mMCC. P362 Acknowledgements First-line avelumab treatment in patients with metastatic Merkel This study was funded by Merck KGaA, Darmstadt, Germany, as part of an cell carcinoma: primary analysis after ≥15 months of follow-up alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, from JAVELIN Merkel 200, a registrational phase 2 trial NY, USA. 1 2 3 Sandra D'Angelo, MD , Celeste Lebbé , Laurent Mortier , Andrew Brohl, Trial Registration 4 5 6 7 8 MD , Nicola Fazio , Jean-Jacques Grob , Natalie Prinzi , Glenn Hanna , Registered at www.clinicaltrials.gov, NCT02155647 9 10 11 12 Jessica Hassel, MD , Felix Kiecker , Barbara Ellers-Lenz , Marcis Bajars , Ethics Approval 12 13 Meliessa Hennessy, MPH , Paul Nghiem, MD, PhD The trial was conducted in accordance with international good clinical Memorial Sloan Kettering Cancer Center, New York, NY, United States; practice standards and approved by the independent ethics committee at 2 3 Saint Louis Hospital, Paris, France; Lille Hospital–Claude Huriez Hospital, each participating center Lille Cedex, France; H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States; European Institute of Oncology (IEO), IRCCS, Milan, Italy; Aix-Marseille University, AP-HM Hospital, Marseille, P364 France; Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; A gp100 targeting TCR-based soluble T cell engaging bispecific 8 9 Dana-Farber Cancer Institute, Boston, MA, United States; Heidelberg induces mobilisation and activation of peripheral T cells in patients University Hospital, Heidelberg, Germany; Charité Universitätsmedizin with metastatic melanoma Berlin, Campus Charité Mitte, Berlin, Germany; Merck KGaA, Darmstadt, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc Germany; EMD Serono Research and Development Institut Inc, PhD, Mariantonella Vardeu, Jacob Hurst, PhD, Cheryl McAlpine, MSN Billerica, MA, United States; University of Washington Medical Center at Immunocore Ltd, Abingdon, United Kingdom South Lake Union, Seattle, WA, United States Correspondence: Sion Lewis (Sion.Lewis@immunocore.com) Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P364 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P362 Background Background ImmTAC® (immune-mobilizing monoclonal TCRs Against Cancer) Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine car- molecules are a new class of bispecific therapeutics, consisting of cinoma with a poor prognosis. In the pivotal phase 2 JAVELIN Merkel a high affinity TCR fused to an anti-CD3 single-chain variable 200 trial (NCT02155647), avelumab, a human anti–PD-L1 monoclonal fragment (scFv) T cell-activating moiety [1]. Tebentafusp (gp100 antibody, yielded durable responses in patients with metastatic MCC antigen specific ImmTAC) can elicit a polyfunctional T cell re- (mMCC) who had received prior chemotherapy (part A), and a high sponse and has demonstrated monotherapy activity in advanced objective response rate (ORR) in an initial subgroup treated in the metastatic melanoma [2-5]. Clinical benefit was associated with a first-line metastatic setting (part B), leading to regulatory approval reduction in peripheral CXCR3+ T cells and concurrent increase in worldwide. Here we report the primary analysis of JAVELIN Merkel serum CXCL-10 [6, 7]. The chemokine receptor CCR5 is under- 200 part B after ≥15 months of follow-up in the full patient stood to drive T cell extravasation and potentialy synsergise with population. CXCR3 to promote tumour infiltration [8, 9]. Therefore the aim of Methods this study was to investigate the effect of tebentafusp on the dy- Eligible patients had no prior systemic therapy for mMCC and were namics of T cell mobilisation and activation in metastatic melan- enrolled irrespective of biomarker status; PD-L1+ status was defined oma patients. as ≥1% expression in tumor cells (PD-L1 IHC 73-10 pharmDx assay). Methods All patients received avelumab 10 mg/kg IV every 2 weeks. The pri- HLA-A2+ patients with metastatic melanoma were enrolled on a mary endpoint was durable response, defined as objective response first-in-human, multicentre, Phase I/II, open-label, dose-finding (complete response [CR] or partial response [PR] per RECIST v1.1, ad- study (NCT01211262). Immunophenotypic analysis was under- judicated by independent endpoint review committee) lasting ≥6 taken to assess baseline levels and pharmacodynamic changes months. Secondary endpoints included best overall response, dur- in peripheral immune subsets. PBMC samples were analysed by ation of response (DOR), progression-free survival (PFS), overall sur- flow cytometry from patients at baseline (n=38) and on- vival (OS), and safety. treatment (n=22) over the first dosing cycle, and from age/sex- Results matched healthy control subjects (n=18). Data is reported on T At data cut-off on May 2, 2019, 116 patients had been treated cell populations, markers of activation (CD25) and function with avelumab. Median treatment duration was 5.5 months (CCR5, Ki67) and represented as mean ± standard deviation of (range, 0.5-35.4), and treatment was ongoing in 26 patients subset frequency or percentage change from baseline, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 199 of 272 relationships with overall survival (OS) assessed by univariate vaccine adjuvants to support T cell responses to peptide vaccines. Cox proportional hazards model. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC and/ Results or low-dose metronomic cyclophosphamide (mCy) would be safe Tebentafusp induced T cell extravasation within 24hrs (p and would support strong and durable CD4+ T cell responses in T cells from patients on-treatment exhibited an increase in activation combination with an incomplete Freund’s adjuvant (IFA). marker expression and an expansion of memory and effector T cell Methods subsets (p<0.05). An adaptive design based upon toxicity and durable immune re- Conclusions sponse (dRsp) was used to assign participants with resected stage Tebentafusp administratation induces the rapid extravasation of che- IIA-IV melanoma to one of four study regimens, including a vaccine mokine receptor expressing T cells and the expansion and activation comprising 6 melanoma peptides restricted by Class II MHC (6MHP), of memory T cells. The association between clinical benefit and base- administered in an emulsion with IFA (Montanide ISA-51), with or line levels of peripheral immune subsets may aid our mechanistic un- without the TLR3 agonist polyICLC and with or without systemic derstanding of its anti-tumour activity in metastatic melanoma mCy. Toxicities were recorded (CTCAE v4). T cell responses were mea- patients. sured in peripheral blood lymphocytes (PBL) and in vaccine-site Trial Registration draining lymph node (sentinel immunized node, SIN) with IFNγ ELI- NCT01211262 spot assay ex vivo. Serum antibody responses to 6MHP were mea- sured by ELISA, and changes in circulating regulatory T cells were References assessed by flow cytometry. 1. Lowe KL, et al. Cancer Treat Rev. 2019. Results 2. Boudousquie C, et al. Immunology 2017;152:425–38. Forty-eight eligible patients were enrolled and treated. Following an 3. Middleton M, et al. Presented at ASCO 2016. Abstract 3016. adaptive design, early safety data and T cell response data favored en- 4. Carvajal R, et al. Presented at SITC 2017. Abstract P208. rollment on arm D. At study conclusion, total enrollment was 3, 7, 6, 5. Middleton M, et al. Presented at ASCO 2019. Abstract 9523. and 32 individuals for arms A-D, respectively. Treatment-related dose- 6. Middleton M, et al. Presented at ASCO 2019. Abstract 9530 limiting toxicities (DLTs) were observed in 1/7 (14%) patients on arm B 7. Mullins et al, Cancer Res 2004; 64, 7697-7701 and 2/32 (6%) on arm D, with no treatment arm exceeding the DLT 8. Hong M et al, Cancer Res 2011; 71, 22:6997-7009 25% threshold for early stopping. Strong and durable T cell responses 9. Harlin, Cancer Res 2009;69(7):3077–85: to 6MHP were detected ex vivo in 0%, 29%, 50%, and 50% of patients Ethics Approval enrolled on arms A-D, respectively (Table 1). IgG antibody responses This study was approved by following institutions’ Ethics Boards: were also induced and were greatest for arms C and D (Figure 1). Circu- lating regulatory T cell frequencies were not altered by use of mCy. Oxfordshire Research Ethics Committee; 10/H0604/47, Approved Conclusions June 4, 2010. Combination vaccine adjuvants with IFA, polyICLC, and mCy were Mary Crowley Cancer Research Center; MCMRC IRB # 12-06, Ap- well-tolerated. The dRsp rate for arm D (IFA + polyICLC + mCy) of proved March 16, 2012. 50% (90% CI: [34,66]) exceeded the 18% dRsp rate (90% CI: [11,26]) Human Investigation Committee, Yale University; HIC Protocol # from prior experience with 6MHP in IFA alone. The regimen with IFA 1302011504, Approved March 22, 2012. + pICLC alone also showed promise for enhancing T cell and anti- IntegReview; Protocol No IMCgp100/01, Approved November 13, body responses. Addition of mCy does not alter circulating T reg fre- 2013. quencies but shows some promise as a systemic vaccine adjuvant. Western Sydney Local Health District; HREC2012/7/4.1 (3552) AU RED HREC/12/WMEAD/237, Approved on October 24, Acknowledgements 2012. We thank the Cancer Research Institute/ Ludwig Institute for Cancer Western Institutional Review Board; Panel 1, Study Num 1147687, Research for providing the polyICLC used in the vaccines. Funding was WIRB Pro Num 20141184, Approved July 15, 2014. provided by NCI R01 CA178846 (CLS); 5K25CA181638 (NW); P30 CA044579 Memorial Sloan Kettering Cancer Center, Institutional Review (Biorepository and Tissue Research Facility, Office of Clinical Research, and Board; Protocol # 14-152, August 28, 2014. Biostatistics Shared Resource). Trial Registration The clinical trial Mel63 is registered with Clinicaltrials.gov (NCT02425306). P365 Ethics Approval A trial to evaluate the immunogenicity and safety of a melanoma The clinical trial Mel63 was performed with IRB (#17860) and FDA approval helper peptide vaccine plus incomplete Freund’s adjuvant, (IND #10825). cyclophosphamide, and polyICLC (Mel63) 1 1 1 Craig Slingluff, MD , Gina Petroni, PhD , Kimberly Chianese-Bullock, PhD , 1 1 1 1 Nolan Wages, PhD , Walter Olson, PhD , Kelly Smith , Lynn Dengel, MD , 1 2 1 Anna Dickinson , Caroline Reed , Elizabeth Gaughan, MD , William 1 1 1 1 Grosh, MD , Varinder Kaur, MD , Nikole Varhegyi , Mark Smolkin , 1 1 Table 1 (abstract P365). T cell responses to 6MHP Nadejda Galeassi , Donna Deacon, BS 1 2 University of Virginia, Charlottesville, VA, United States; Emory University School of Medicine, Atlanta, GA, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P365 Background Cancer vaccines require adjuvants to induce effective and durable protective immunity. However, there is no consensus on optimal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 200 of 272 assay. Circulating antibody (Ab) responses to overlapping NY-ESO-1 peptides were detected by ELISA. Tumor biopsies obtained pre- treatment and day 85 were evaluated for immune infiltrates by multi- spectral immunofluorescence histology. Results Target enrollment was 27; study closed early for slow enrollment. Eight patients enrolled and were treated (Table 1). All had ≥ 1 treat- ment emergent adverse event (TEAE); most common (≥50%): rash, fatigue, injection site reaction, pruritus, and diarrhea. Two patients had Gr3 TEAEs related to IPI but not to vaccine. There were no DLTs. Best responses: SD (n=4); PD (n=4). T cell responses to NY-ESO-1 were detected in 6 of 8 (75%) patients.[1] Both patients without T cell response had PD as best response. Ab responses were detected in 7/8 (88%) patients (Table 1). The patient without Ab response had PD as best response. The breadth of Ab responses to NY-ESO-1 was greater for patients with SD than those with PD (p = 0.02). Evaluation of TME of 5 patients revealed increases in proliferating (Ki67+) CD8 T cells, decreases in RORγt+ CD4+ T cells (Figure 1). Interestingly, there were increases in density of CD8+ and CD4+ cells for those with SD (n=3), but decreases for those with PD (n=2, not shown). Conclusions T cell responses and Ab responses to NY-ESO-1 were induced in most patients and were evident ex vivo, suggesting that IPI may have en- hanced the T cell responses to NY-ESO-1 protein and OLP4. Inte- grated T cell and antibody responses were associated with tumor control. Preliminary data of the TME suggests increased activating and proliferating T cells after vaccination plus IPI, especially in pa- tients with tumor control. Acknowledgements The trial was supported by the Ludwig Institute for Cancer Research, the Fig. 1 (abstract P365). Antibody responses to 6MHP Cancer Research Institute, and by the National Institutes of Health, including support from the University of Virginia Cancer Center Support Grant (NIH/NCI P30 CA44579:, Clinical Trials Office, Biorepository and Tissue Procurement Facility, Flow Cytometry Core, and Biomolecular Core Facility). P366 Earlier presentation of results of this clinical trial [1] has been expanded in A phase 1 study of NY-ESO-1 vaccine + ipilimumab (ipi) in patients the present abstract with additional biologic correlates. with unresectable or metastatic melanoma 1 2 3 Trial Registration Craig Slingluff, MD , Hassane Zarour, MD , Michael Postow, MD , Philip 4 5 1 This trial was registered at ClinicalTrials.gov (NCT01810016). Friedlander, MD PhD , Craig Devoe, MD , Ileana Mauldin, PhD , Kelly 1 6 Smith , Mary Macri, BSc 1 2 Reference University of Virginia, Charlottesville, VA, United States; University of 1. Slingluff CL Jr, Zarour HM, Postow MA, Friedlander P, Devoe CE, Macri M, Pittsburgh Cancer Center, Pittsburgh, PA, United States; Memorial Sloan Ryan A, Venhaus R, Wolchok J. J Clin Oncol. 2018; 36(suppl; abstr e15175). Kettering Cancer Center, New York, NY, United States; Mount Sinai Ethics Approval Medical Center, New York, NY, United States; Northwell Health Cancer The study was approved by each institution's Ethics Board, with approval Institute, Lake Success, NY, United States; Ludwig Institute for Cancer numbers: IRB#12-253 (Memorial Sloan Kettering), HS#13-00471 (Mount Research, New York, NY, United States Sinai), IRB#14-133B (Northwell Health), MOD13030240-02/ Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) PRO13030240(University of Pittsburgh), and HRS#16347(University of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P366 Virginia), and to the FDA with IND 10369. Background Ipilimumab (IPI) is an approved immunotherapy for advanced melan- oma. It can enhance immunity to cancer-testis antigen NY-ESO-1. Vac- Table 1 (abstract P366). Enrollment, immune and clinical responses cines with NY-ESO-1 protein or NY-ESO-1 overlapping long peptides (OLP4) have enhanced immunity when administered with Montanide ISA-51 (Montanide) and/or Poly-ICLC (pICLC) adjuvants. This trial assessed safety, immunogenicity, clinical responses (irRC), and effects of IPI + NY-ESO-1 vaccines on the tumor microenvironment (TME). Methods This Phase 1, open-label study enrolled patients among 3 arms: IPI (3 mg/kg i.v. q3 wks x 4) + NY-ESO-1 protein + pICLC + Montanide (Arm A); IPI + NY-ESO-1 OLP4 + pICLC + Montanide (Arm B); and IPI + NY-ESO-1 OLP4 + pICLC (Arm C). Patients had measurable NY-ESO- 1+ tumors. Treatments were administered days 1, 22, 43, 64. Circulat- ing T cell responses were assessed by ex vivo IFN-gamma ELIspot Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 201 of 272 Conclusions Seviprotimut-L is very well tolerated. Subgroup efficacy analyses identi- fied two populations who may benefit from Seviprotimut-L: those with AJCC stage IIB/IIC melanoma and those under age 60. These data support proceeding to the definitive final part of the MAVIS phase III trial testing seviprotimut-L for stage IIB/C patients, in particular those under age 60. Acknowledgements We acknowledge the support of all investigators and clinical coordinators responsible for enrolling patients to this trial. Trial Registration This trial was registered at ClinicalTrials.gov: NCT01546571. References 1. Bystryn JC, Zeleniuch-Jacquotte A, Oratz R et al. Double-blind trial of a Fig. 1 (abstract P366). See text for description polyvalent, shed-antigen, melanoma vaccine.[see comment]. Clinical Can- cer Research 2001; 7: 1882-1887. P367 2. Dorshkind K, Swain S. Age-associated declines in immune system devel- A multicenter, double-blind, placebo-controlled trial of opment and function: causes, consequences, and reversal. Curr Opin seviprotimut-L polyvalent melanoma vaccine in post-resection Immunol 2009; 21: 404-407. melanoma patients at high risk of recurrence 1 2 3 Ethics Approval Craig Slingluff, MD , Brent Blumenstein, PhD , Karl Lewis, MD , Robert 4 5 The study was approved by the Ethics Board at each participating institution (IRB#), as Andtbacka, MD, CM, FACS, FRCSC , John Hyngstrom, MD , Mohammed 6 7 8 follows: University of Virginia Hospital (16223); Anschutz Cancer Pavilion, UC Milhem, MBBS , Svetomir Markovic, MD, PhD , Omid Hamid, MD , Leonel 9 10 11 Denver(1134601); Huntsman Cancer Institute, / Univ of Utah Health Care (55911); Hernandez-Aya, MD PhD , Tawnya Bowles, MD , Prejesh Philips, MD , 12 13 14 University of Iowa Hospitals and Clinics (1133782); Mayo Clinic Cancer Center / Joel Claveau, MD , Sekwon Jang, MD , Jose Lutzky, MD, FACP , Anna 15 16 Mayo Clinic Rochester (12-002308); The Angeles Clinic and Research Institute Bar, MD , Peter Beitsch, MD 1 2 (1196134); Washington University School of Medicine (201205056); Intermountain University of Virginia, Charlottesville, VA, United States; Tri Arc Medical Center (1024288); University of Louisville (15.0039); CHU de Quebec, Consulting, Washington, DC, United States; University of Colorado, L'Hotel Dieu de Quebec (MP-20-2015-2318); Inova Melanoma and Skin Cancer Aurora, CO, United States; Seven and Eight Biopharmaceuticals, Salt Center (1152774); Mount Sinai Medical Center (12-16-H-03); Oregon Health and Lake City, UT, United States; Huntsman Cancer Institute/ Univ of Utah, Science University (IRB00011848); Cancer Solutions (1197259). Salt Lake City, UT, United States; University of Iowa Hospitals and Clinics, Iowa City, IA, United States; Mayo Clinic Rochester, Rochester, MN, United States; The Angeles Clinic & Research Institute, Los Angeles, Table 1 (abstract P367). Enrollment and adverse events CA, United States; Washington University School of Medicine, Saint Louis, MO, United States; Intermountain Medical Center, Murray, UT, United States; University of Louisville, Louisville, KY, United States; 12 13 CHU de Quebec, L'Hotel Dieu de Quebec, Quebec, Canada; Inova Melanoma and Skin Center, Fairfax, VA, United States; Mount Sinai Medical Center, Miami Beach, FL, United States; Oregon Health and Science University, Portland, OR, United States; Cancer Solutions, Dallas, TX, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P367 Background Seviprotimut-L is a vaccine prepared from antigens shed by 3 human mel- anoma cell lines, administered with alum. Prior formulations showed prom- ising immunogenicity for T cell and antibody responses and improved survival in a small phase II clinical trial[1]. Part B1 of MAVIS (Melanoma Anti- gen Vaccine Immunotherapy Study, a three part, Phase III clinical program), was a multicenter, double-blind, placebo-controlled trial to assess the effi- cacy of seviprotimut-L, with the primary endpoint of relapse-free survival (RFS) in patients at high risk of recurrence after definitive surgical resection. Methods For MAVIS Part B1, patients with AJCC v7 stage IIB-III cutaneous melan- oma, after surgical resection, age 18-75, ECOG PS 0-1, were randomized 2:1 to seviprotimut-L 40 mcg or placebo, administered subcutaneously every 2 weeks x 5, then monthly x 4, then every 3 months to month 24. Patients were stratified by stage (IIB/C, IIIA, IIIB/C). Target enrollment was 325. The study was powered for assessment of RFS, with target hazard ratio (HR) of 0.625, one-sided alpha of 0.10, and power 80%. Results 347 patients were randomized, and arms were well-balanced. Treatment- emergent adverse events (AEs) were similar for seviprotimut-L and placebo patients (Table 1). By intent-to-treat (ITT) analysis, RFS was not significantly enhanced for seviprotimut-L in the full study population, but trended slightly higher (Figure 1A). Analysis of subgroups based on pre-planned stratification suggested enhanced RFS for seviprotimut-L among Stage IIB/IIC patients (HR 0.59, 95% CI[0.33,1.07], Figure 1B). Age has been identified as a cause of de- creased immune competence[2]; thus, outcomes were assessed as a function of age as an effect modifier. Figures 1C and 1D show all randomized patients Fig. 1 (abstract P367). Clinical outcome (Figure 1C) and Stage IIB/IIC subset (Figure 1D) by arm and age split at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 202 of 272 Clinical Trial In Progress 3. Bernhardt SL, Gjertsen MK, Trachsel S, et al. Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose es- P368 calating phase I/II study. Br J Cancer. 2006;95:1474-1482 ZI-H04 - A novel MHC class II restricted TCR based cellular therapy targeting hTERT to treat solid tumours Jens-Peter Marschner, MD, Mona Welschof, PhD, Miguel Forte, Eva P369 Kristine Klemsdal, Sylvie Pollmann, Namir Hassan Feasibility of a phase I personalized adoptive T-cell therapy in Zelluna, Seeheim-Jugenheim, Germany patients with relapsed/refractory solid tumors 1 3 Correspondence: Jens-Peter Marschner Apostolia Tsimberidou, MD, PhD , Ali Mohamed , Stephen Eck, MD, 4 4 1 1 (jenspeter.marschner@zelluna.com) PhD , Harpreet Singh , Patrick Hwu, MD , Cassian Yee, MD , Borje Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P368 Andersson, MD, PhD 1 2 MD Anderson Cancer Center, Houston, TX, United States; MD Background Anderson, Houston, TX, United States; Immatics Biotechnology, Chimeric Antigen Receptor T-cells (CAR-T) are highly effective in the Tubingen, Germany; Immatics, Houston, TX, United States treatment of some hematological malignancies but solid tumors re- Correspondence: Apostolia Tsimberidou (atsimber@mdanderson.org) main a challenge for cellular therapies. A few T-cell Receptor T- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P369 cells (TCR-T) have been investigated in solid tumors. To our knowledge, there is only one published study and one case re- Background port using MHC Class II restricted TCRs targeting MAGE-A3 and Adoptive cellular therapy (ACT) is limited in solid tumors due to lack of NY-ESO-1, respectively [1,2]. suitable immunotherapy targets with high specificity and frequent re- lapse following immunotherapy to single targets often associated with Methods loss of target expression in the tumor. ACTolog® is a personalized, ZI-H04 represents a novel approach of TCR based therapies. multi-targeted ACT approach in which autologous T-cell products are Autologous T-cells from patients are genetically modified by manufactured against the most relevant tumor target peptides for indi- lentiviral transduction to express the TCR targeting hTERT in vidual patients whose tumors are positive against predefined targets. the context of the MHC Class II allele, HLA-DPB1*04:01. The Methods TCR was isolated from a pancreatic cancer patient who experi- Patients with advanced metastatic cancers and HLA-A*02:01 enced clinical benefit following a peptide-based cancer vaccin- phenotype, undergo a tumor biopsy. Patients whose tumors ex- ation against hTERT [3]. The TCR clone responded to press >1 of 8 cancer targets undergo leukapheresis. Autologous T autologous tumor and preclinical data demonstrate that ZI-H04 cells are primed against the expressed ACTolog targets in the exhibits high sensitivity to hTERT peptide as well as recogni- presence of IL-21 followed by HLA tetramer-guided cell sorting tion of processed antigen. Furthermore, specificity analysis sup- and rapid expansion. Patients who meet criteria for treatment re- ports the safety of the TCR-T. The restricted combined ceive lymphodepletion with Fludarabine 40 mg/m2 i.v. and Cyclo- expression of hTERT plus HLA class II on normal cells limits the phosphamide 500 mg/m2 i.v. (Days, -6 to -3). T-cells are infused potential for on-target off tumor toxicity. A first-in-human on Day 0, followed by low-dose of IL-2 for 14 days (www.clinical- study is designed to treat patients with relapsed/refractory trials.gov NCT02876510). solid tumors lacking an option of further treatments. Patients Results must be tested positive for HLA-DPB1*04:01 and the tumors From 7/2017 to 7/2019, 203 patients signed an informed consent to must express hTERT. Adequate organ function and lab parame- participate in the study, 91 had HLA-A*02:01 phenotype, 52 had a ters are required. CNS involvement, autoimmune diseases, in- tumor biopsy and 34 patients underwent leukapheresis. To date, 9 fections and immunosuppressive medication are main exclusion patients have received treatment (median age, 38 yrs; range, 25-58 criteria. Primary objectives are safety and tolerability. Part 1 of yrs; 2 men and 7 women; breast cancer, 3; sarcoma, 3; ovarian can- the study, starting in 2020, will be a dose finding part, Part 2 cer, 1; nasopharyngeal, 1; anal carcinoma, 1; median time from diag- a dose extension part with 5 cohorts. Prior to adoptive cell in- nosis 4 years, range, 2-18 years; median number of prior therapies 6, fusion patients will receive a low dose conditioning regimen range 3-12). Very high ACTolog cell doses could be administered. Pa- consisting of 2 x 600 mg/m² cyclophosphamide followed by 3 tients received a median of 2 target-specific ACTolog products (range x 25 mg/m² fludarabine. Patients will be observed for safety, 1-3). Treatment was overall well tolerated. The most common ad- efficacy and exploratory biomarkers. verse events were cytopenias and cytokine release syndrome. All pa- Conclusions tients are alive to date. At 6 weeks, restaging imaging studies ZI-H04 is a novel TCR-T with potentially favourable characteristics. demonstrated stable disease in all patients. One patient with squa- The TCR was isolated from an hTERT vaccinated pancreatic cancer mous cell carcinoma of the anus treated with T cells directed to patient that experienced clinical benefit. A lower probability of COL6A3, exon 6, and PRAME had 26% decrease in tumor measure- off-target activity is expected since no engineering was done to ments at week 6 associated with high T-cell frequencies at 2 weeks the TCR. The MHC Class II restriction provides the possibility to but her disease subsequently progressed. Another patient with naso- induce a multi-pronged immune response including antigen pharyngeal cancer treated with COL6A3 tumor stroma-specific T cells spreading as demonstrated in a case report using an MHC Class had resolution of tumor associated pain and has not required further II TCR-T in melanoma [2]. More than 50% of patients are HLA- treatment for 11 months. A recent tumor biopsy demonstrated nec- DPB1*04:01 positive and the expression rate of hTERT is >80% in rotic cells and no tumor cells could be identified. many tumors. Therefore, a substantial population may benefit Conclusions from ZI-H04 treatment. ACTolog IMA101 is well-tolerated and no safety issues have been noted to date. The study is ongoing. References Trial Registration 1. Lu Y-C, Parker LL, Lu T, et al. Treatment of patients with metastatic cancer www.clinicaltrials.gov NCT02876510 using a major histocompatibility complex class II-restricted T-cell receptor Ethics Approval targeting the cancer germline antigen MAGE-A3. J Clin Oncol. The study was approved by MD Anderson's IRB. 2017;35:3322-3329 Consent 2. Hunder NN, Wallen H, Cao J, et al. Treatment of metastatic melanoma Written informed consent was obtained from the patient for publica- with autologous CD4+ T cells against NY-ESO-1. N Engl J Med. tion of this abstract and any accompanying images. A copy of the 2008;358:2698-703 written consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 203 of 272 P370 Trial Registration The positive correlation between baseline absolute eosinophil NCT02791334 count (AEC) in blood and clinical benefit to PD-(L)1 inhibition monotherapy Reference 1 1 1 Anna Szpurka, PhD , Danni Yu, PhD , Michelle Carlsen , Antoine Tanizaki, Junko, Koji Haratani, Hidetoshi Hayashi, Yasutaka Chiba, Yasushi 2 3 4 Hollebecque, MD , Hyun Cheol Chung, MD, PhD , Amita Patnaik , Nakamura, Kimio Yonesaka, Keita Kudo, et al. "Peripheral Blood 5 6 7 Johanna Bendell, MD , Antoine Italiano, MD , Yung-Jue Bang, MD PhD , Biomarkers Associated with Clinical outcome in Non–Small Cell Lung 8 9 10 Chia-Chi Lin, MD, PhD , Marcus Butler, MD , Timothy Yap, MD PhD , Cancer Patients Treated with Nivolumab." Journal of Thoracic Oncology 11 11 María José de Miguel, MD , María José de Miguel, MD , Jean-Pascal 13, no. 1 (2018/01/01/ 2018): 97-105. 12 13 14 Machiels, MD, PhD , Marc Peeters, MD, PhD , Wu-Chou Su, MD , Ethics Approval 15 1 1 Victor Moreno, MD , Yumin Zhao, PhD , Erik Rasmussen, PhD , Xiaojian The study included multiple investigator sites, and some of these had Xu, MD approval dates instead of approval numbers. The following are listed 1 2 Eli Lilly and Company, Indianapolis, IN, United States; University of Paris showing approving committee followed by approval date or approval # Sud, Villejuif, France; Yonsei University College of Medicine, Seoul, (if available): IntegReview, 15Jun2016; IntegReview, 21June2016; MD Korea, Republic of; South Texas Accelerated Research Therape, San Anderson Office of Protocol Approval IRB, 26May2016; Princess Margaret Antonio, TX, United States; Sarah Cannon Research Institute, Nashville, Cancer Centre, University Health Network Research Ethics Board, 16-5824 6 7 TN, United States; Institut Bergonié, Bordeaux, France; Seoul National (initial approval date 01Feb2017); Comite de protection des Personnes University Hospital, Seoul, Korea; National Taiwan University Hospital, (CPP), EudraCT number 2016-000440-33. Taipei, Taiwan, Province of China; Princess Margaret Cancer Center, Toronto, Canada; The University of Texas, Houston, TX, United States; 11 12 START-HM Sanchinarro, Madrid, Spain; jean- pascal.machiels@uclouvain.be, Brussels, Belgium; Antwerp University Hospital,, Antwerp, Belgium; National Cheng Kung University Hospital, Tainan, Taiwan, Province of China; START Madrid-FJD, Madrid, Spain Correspondence: Danni Yu (yu_danni@lilly.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P370 Background Anti-PD-(L)1 immunotherapies have increased the response rate in certain cancer subtypes however, some patients who may have clin- ical benefit are not identifiable with existing predictive biomarkers. Research is ongoing to identify routinely available blood and clinical markers to predict response to PD-(L)1 therapies. In this study, we ex- Fig. 1 (abstract P370). See text for description plored absolute eosinophil count (AEC) as a biomarker in patient’s re- sponse to PD-(L)1 treatment. Methods P371 This is a phase 1a/1b study of an anti-PD-L1 antibody (LY3300054) Cytokine Microdialysis for real-time immune monitoring in administered alone or in combination with other agents in patients Glioblastoma patients undergoing Checkpoint Blockade 1 2 2 with advanced refractory solid tumors. Eligible patients were ≥18 John Lynes, MD , Victoria Sanchez , Anthony Nwankwo , Gifty 2 2 2 2 years old, had ECOG status ≤1 and had at least 1 measurable lesion Dominah , Xiang Wang, MS , Isac Kunnath , Samantha Dill , Gretchen 2 2 2 2 2 per RECIST v1.1. We assessed the association of AEC with confirmed Scott , Christi Hayes , Tianxia Wu , Marta Penas-Prado , Jing Wu , Eric 2 2 2 2 best overall response (BORc). The AEC cutoff 0.155 (10^9/L) maxi- Burton , John Heiss , Christopher Hourigan, MD, PhD , Mark Gilbert , mized the difference in ORR, similar to previous reports (Tanizaki etal, Edjah Nduom, MD 1 2 2018, JTO [13] e85-e86) . The impact of AEC status was demonstrated Georgetown University, Bethesda, MD, United States; National by a 3-dimensional waterfall plot depicting the best change in tumor Institutes of Health, Bethesda, MD, United States size and overall survival (OS). Correspondence: Edjah Nduom (edjah.nduom@nih.gov) We also tested the correlation between AEC and OS/proxy-progres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P371 sion-free survival (PFS; time to next treatment, TTNT) in NSCLC pa- tients (n=455) who received anti-PD-1 therapy with same cutoff from Background an independent Flatiron database. Glioblastoma is the most common primary malignancy of the brain, Results with a dismal prognosis. Immunomodulation via checkpoint inhibition As of 8 December 2017, 30 patients (MSI-H: n=22, M: n=8) were has provided encouraging results in non-CNS malignancies, but predic- treated. There were no deaths due to adverse events. Two patients tion of responders has proven to be challenging in glioblastoma pa- in MSI-H cohort experienced grade 3 treatment-related AEs (TRAEs): tients. OBJECTIVES: To determine the proportion of patients who have diarrhea (n=1, 4.5%), blood creatinine phosphokinase increased (n=1, a measurable increase of interferon gamma levels in brain tumor tissue 4.5%), and hyponatraemia (n=1, 4.5%). No grade 3 events were re- after their first dose of nivolumab; to evaluate the safety of using brain ported in M cohort, and no grade 4/5 TRAEs were reported in either tumor microdialysis to monitor for immune response; to evaluate the cohorts. There were no TRAEs leading to discontinuation of study safety of the combination of anti-programmed death 1 (PD-1) and anti- treatment. Preliminary efficacy data in MSI-H cohort showed ORR of lymphocyte activation gene 3 (LAG-3) checkpoint inhibition in recurrent 36% [CR in 1 pt (5%)(ovarian), PR in 7 pts (32%)(small intestine glioblastoma patients. adenocarcinoma [1 pt], endometrial [3 pts], colon [3 pts])], DCR in Methods 64% [SD in 6 pts (27%)]; mPFS was 7.39 months (95% CI 1.7, NR). In The study design is a single-center, nonrandomized phase 1 clinical the M cohort, DCR was 63% [PR in 1 pt (13%), SD in 4 pts (50%)]. As trial. Up to 20 adult patients with recurrent glioblastoma will be en- of data cut-off, 16 pts (53%) remain on treatment. Preliminary bio- rolled with the goal of 10 patients completing the trial over an antici- marker analysis, including but not limited to, PD-L1 and CD8 expres- pated 18 months. Patients will undergo biopsy; placement of sion and circulating markers will be presented. microdialysis catheters and lumbar drains; treatment with anti-PD-1 Conclusions checkpoint inhibition; comprehensive immune biomarker collection; LY3300054 was well-tolerated and demonstrated antitumor activity tumor resection; and then treatment with anti-PD-1 and anti-LAG-3 in patients with MSI-H solid tumors; combination expansions are checkpoint inhibition until progression (Figure 1). Three patients ongoing. have undergone all study procedures (Figures 2 and 3). There have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 204 of 272 been no serious adverse events related to the research surgical pro- cedure, nor during the microdialysis portion of the trial. Enrollment is ongoing. EXPECTED OUTCOMES: We expect interferon gamma levels to in- crease in the brain as measured via microdialysis in treated patients. Based on published reports, microdialysis in this patient population is expected to be safe, and anti-LAG-3 and anti-PD-1 combined will likely have a similar side effect profile to other checkpoint inhibitor combinations. The failure of recent trials of immune therapies in glioblastoma un- derscores the need to appropriately measure response in the treated tissue. This trial may provide insight on indicators of which patients will respond to immune therapy. Acknowledgements Funding and support came from: Intramural Research Program of the Na- tional Institute of Neurological Disorders and Stroke Trial Registration Clinicaltrials.gov: NCT03493932 (Registration Date: April 11, 2018) Fig. 3 (abstract P371). See text for description Ethics Approval Institutional Approvals: National Institutes of Health Combined Neurosciences Internal Review Board number - 18-N-0077 P372 Circulating immune cell biomarkers predict response to immune checkpoint inhibitor therapy in metastatic breast cancer Jin Sun Bitar, MD, Colt Egelston, PhD, Susan Yost, Wanqiu Hou, Padam Simran, Paul Frankel, PhD, Mina Sedrak, Jana Portnow, MD, Joanne Mortimer, MD, Christina Yeon, MD, Arti Hurria, MD, Aileen Tang, Norma Martinez, Peter Lee, MD, Yuan Yuan City of Hope, Duarte, CA, United States Correspondence: Yuan Yuan (yuyuan@coh.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P372 Background The role of immune checkpoint PD-1/PD-L1 inhibitor (ICI) in breast cancer (BC) is being investigated in clinical trials. Preclinical evidence Fig. 1 (abstract P371). See text for description strongly supports the synergistic effects of CDK4/6 inhibitor and ICI [1]. A phase II trial is testing the safety and efficacy of the combin- ation of letrozole, palbociclib and pembrolizumab in patients with hormone receptor positive (HR+) BC (NCT02778685). Currently, there is no well-defined circulating biomarker to predict response to ICI. Methods Peripheral blood mononuclear cells (PBMC) were collected at day 1 of cycles 1 (pre-treatment), 2, 4, 6 and 8. The comprehensive characterization of circulating immune cell composition was per- formed using 15-color flow cytometry. Results Preliminary analysis included 9 patients with the following responses by RECIST 1.1: 1 complete response, 4 partial response, 2 stable disease, and 2 progressive disease. Higher baseline frequencies of CD4+ effector memory (p=0.01) and CD8+ CD45RA+ effector memory cells (p=0.01) were observed in patient responders. Additionally, patient responders demonstrated higher fre- quencies of T cells expressing KLRG1, a marker of effector T cell differenti- ation, on both CD4+ (p=0.001) and CD8+ T cells (p=0.004) at baseline. An increase in the frequency of circulating CXCR5+ CD8+ T cells (p=0.01) at cycle 2 was identified in all treated patients and an increase in CCR10+ CD8+ T cells (p=0.03) was detected at cycle 2 in patient responders indi- cating changes in T cell trafficking. Finally, a shift in myeloid cell compos- ition from predominantly classical to non-classical monocytes was observed in patient responders between baseline and cycle 2 (p=0.007). Conclusions High baseline levels of both CD4+ and CD8+ effector T cells indicate the necessity for a pre-existing favorable T cell composition in check- point blockade responders. Temporal changes in T cell trafficking mole- Fig. 2 (abstract P371). See text for description cules and shifts in myeloid cell composition over the course of therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 205 of 272 indicate potential changes in T cell priming and regulation. Further ana- 2. Bernstein V, Ellard SL, Dent SF, et al. A randomized phase II study of lysis is currently ongoing to understand correlates of systemic immune weekly paclitaxel with or without pelareorep in patients with metastatic changes and changes in the tumor microenvironment. breast cancer: final analysis of Canadian Cancer Trials Group IND.213. Breast Cancer Res Treat 2018;167:485-93. Trial Registration 3. Nuciforo, P., Pascual, T., Cortés, J., Llombart-Cussac, A., Fasani, R., Paré, L., NCT02778685 … Holgado, E. (2018). A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2- Reference positive breast cancer treated with chemo-free dual HER2 blockade. An- 1. Goel S, DeCristo MJ, Watt AC, et al. CDK4/6 inhibition triggers anti- nals of Oncology. https://doi.org/10.1093/annonc/mdx647 tumour immunity. Nature. 2017;548(7668):471–475. Ethics Approval Ethics Approval This study was approved by the Spanish Health Authority, protocol number The study was approved by City of Hope National Cancer Center‘s Ethics 2018-003345-42. Board, approval number 16058 P373 A window-of-opportunity study of pelareorep in early breast cancer (AWARE-1) 1 2 3 4 Luis Manso , Patricia Villagrasa , Nuria Chic , Juan Cejalvo , Yann 5 5 6 3 Izarzugaza , Blanca Cantos , Salvador Blanch , Manel Juan , Blanca 3 7 8 7 Gonzalez , Rita Laeufle , Gerard Nuovo , Grey Wilkinson, PhD , Matt 7 3 3 2 2 Coffey , Azucena Gonzalez , Patricia Galvan , Laia Paré , Jordi Canes , 9 3 6 Xavier Gonzalez , Aleix Prat, MD PhD , Joaquín Gavilá 1 2 Hospital Universitario 12 de Octubre, Madrid, Spain; SOLTI Breast Cancer Research Group, Barcelona, Spain; Hospital Clinic, Barcelona, 4 5 Spain; Hospital Clínico, Valencia, Spain; Hospital Universitario, Madrid, Spain; Instituto Valenciano de Oncología (IVO), Valencia, Spain; 7 8 Oncolytics Biotech Inc., San Diego, CA, United States; Ohio State University, Columbus, OH, United States; Hospital Universitari, Sant Cugat del Vallés, Spain Fig. 1 (abstract P373). See text for description Correspondence: Aleix Prat (ALPRAT@clinic.cat) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P373 Background P374 Pelareorep is an intravenously delivered (IV) unmodified oncolytic reo- TCR repertoires from peripheral blood correlate with prognostic virus. Clinical studies have demonstrated that IV delivered pelareorep can response in TNBC cancer vaccine immunotherapy replicate in tumor tissue and promote an inflamed tumor phenotype Sadanand Vodala, PhD, Andrew Nguyen, PhD, Noe Rodriguez, Peter characterized the recruitment of CD8+ T cells and upregulation of PD-L1 Sieling, Charles Vaske, Jon Van Lew, Kayvan Niazi, John Lee, MD, Patrick [1]. Consistent with pelareorep’s role in promoting adaptive anti-tumor Soon-Shiong, MD, FRCS, FACS, Shahrooz Rabizadeh immunity, a randomized phase 2 study in metastatic breast cancer dem- ImmunityBio, Inc, Culver City, CA, United States onstrated a statistically significant improvement in overall survival when Correspondence: Kayvan Niazi (kayvan.niazi@immunitybio.com) pelareorep was combined with paclitaxel [2]. We hypothesize that pelar- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P374 eorep mediated anti-tumor immune responses, such as those mediated by T cells, represent a novel strategy for the control or elimination of Background tumor cells in breast cancer. Specifically, in the preoperative setting for TNBC is an aggressive, heterogeneous, and high-grade subtype that rep- early breast cancer, we examined if pelareorep in combination with anti- resents 10-20% of breast carcinomas. Recently, TECENTRIQ and Abraxane PD-L1 therapy, atezolizumab, and other breast cancer therapies offers were approved for the treatment of PD-L1+ unresectable locally ad- clinical benefit in terms of CeLTIL score, a metric for quantifying tumor vanced or mTNBC suggesting a role for immunotherapy in the treatment cellularity (Cel) and tumor-infiltrating lymphocytes (TIL) [3]. of this disease. Growing evidence suggests that chemotherapeutic agents Methods and immunotherapies synergize in patients. Cancer vaccines are also a This exploratory, non-randomized, window of opportunity study, will promising option for TNBC due to the discovery of neo-antigens and evaluate the safety and effect of pelareorep ± atezolizumab on the tumor tumor associated antigens that mobilize anti-tumor T cells. microenvironment in 38 women with early breast cancer. Patients will re- Methods ceive study treatment for ~21 days prior to definitive surgery or neoadju- Here, we characterize T cell receptor repertoires in patients enrolled in a vant chemotherapy. Five cohorts will be examined (Figure 1): Cohort 1: Phase Ib/2 clinical trial (NCT03387085) following treatment by a regimen HR+/HER2-neg (10 patients), pelareorep + letrozole. Cohort 2: HR+/HER2- intended to induce a synchronized, multi-compartment, anti-tumor im- neg (10 patients): pelareorep + letrozole + atezolizumab. Cohort 3: TNBC mune response by mitigating the immune-suppressive effects of the (6 patients): pelareorep + atezolizumab. Cohort 4: HER2+/HR+ (6 pa- tumor microenvironment and activating immunogenic tumor cell death. tients): pelareorep + trastuzumab + atezolizumab. Cohort 5: HER2+/HR- The trial combined metronomic low-dose chemotherapy, SBRT, an allo- (6 patients): pelareorep + trastuzumab + atezolizumab. CelTIL, viral repli- geneic NK cell line expressing high affinity CD16, yeast and adenoviral cation, and other immune-based biomarkers will be used to examine tumor-associated antigen vaccines, an IL15RαFc super-agonist, check- treatment-related changes within the tumor microenvironment. Blood point inhibition, and an anti-angiogenic agent in a manner predicted to and tumor tissue biopsies will be collected at screening, Day 3 (after maximize cytotoxic T-cell mediated immunological recognition of tumor pelareorep but before atezolizumab), and at surgery (Day ~21). cells. Tumor associated antigens included adenoviral vector-based CEA, Trial Registration MUC1, brachyury, and yeast-based brachyury and CEA vaccines. Blood Spanish clinical studies registry: 2018-003345-42 was collected pre- and post-treatment and target lesion analysis was per- formed using irRC and Recist1.1. Total RNA from PBMCs was used to gen- References erate sequencing libraries from each longitudinal sample. Using NGS, 1. Samson A, Scott KJ, Taggart D, et al. Intravenous delivery of oncolytic TCR-α and -β CDR3s were clonotyped and tracked over serial blood reovirus to brain tumor patients immunologically primes for subsequent draws. Additionally the Shannon-Wiener Diversity Index (SWDI) was calcu- checkpoint blockade. Sci Transl Med 2018;10. lated for each time point. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 206 of 272 Results (T+CTX) versus 1 experimental arm (M+CTX) + either MGA012 or Patient samples showing consistent positive responses by irRC/Recist1.1 MGD013, depending on the interim analysis from part 1; with 250 pa- showed emergence and persistence of new TCR clones post-induction. tients each. The primary efficacy endpoint for cohort A (both parts) is This was further reflected in acute surges in SWDI. Furthermore, a high ORR per RECIST 1.1; for cohort B part 2 it is overall survival. SWDI at baseline and post-treatment indicated clinical benefit suggest- ing an inverse correlation between disease severity and peripheral rep- Acknowledgements ertoire diversity. A TNBC super responder showed dramatic increases in The authors thank all the patients, their families, and the entire staff who are mean SWDI index from 74 prior treatment to 1177 at first biopsy post participating in this trial. Professional medical writing support was provided treatment (34% decrease by irRC and 26% by Recist1.1 analysis) and by Meredith Rogers, MS, CMPP, of The Lockwood Group (Stamford, achieved an index as high as 3516 in a subsequent biopsy (83% and Connecticut, USA), in accordance with Good Publication Practice (GPP3) 64% decrease by irRC and Recist 1.1 respectively). guidelines, with funding by MacroGenics, Inc. (Rockville, MD, USA). Trial Registration Conclusions NCT number to come Our findings strongly suggest that peripheral blood TCR repertoires are prognostic indicators/biomarker for TNBC cancer vaccine immunotherapy References and for T cell-based immunotherapy in general. Taken together, these re- 1. Nordstrom JL, Gorlatov S, Zhang W, et al. Anti-tumor activity and toxico- sults strongly indicate activation and expansion of anti-tumor T cell clones kinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with en- following combination therapy. Further functional studies will expand our hanced Fcγ receptor binding properties. Breast Cancer Res. 2011;13:R123. understanding of T cell based cancer vaccine immunotherapy in TNBC. 2. Nordstrom JL, Muth J, Erskine CL, et al. High frequency of HER2-specific immunity observed in patients (pts) with HER2+ cancers treated with margetuximab (M), an Fc-enhanced anti-HER2 monoclonal antibody P375 (mAb). J Clin Oncol. 2019;37(suppl; abstr 1030). Margetuximab combined with anti-PD-1 (MGA012) or anti-PD-1/LAG-3 3. Catenacci DVT, Limet KH, Uronis HE, al. Antitumor activity of (MGD013) +/- chemotherapy in first-line therapy of advanced/metastatic margetuximab (M) plus pembrolizumab (P) in patients (pts) with HER2+ gastroesophageal junction (GEJ) or gastric cancer (GC) 1 2 2 advanced HER2+ (IHC3+) gastric carcinoma (GC). J Clin Oncol. Daniel Catenacci, MD , Minori Rosales , Jon Wigginton, MD ,HyunCheol 3 4 5 6 7 2019;37(suppl 4; abstr 65). Chung, MD, PhD , Harry Yoon ,Lin Shen , Yoon-Koo Kang ,MarkusMoehler 1 2 4. Fuchs CS, Doi T, Jang RW, et al. Safety and efficacy of pembrolizumab University of Chicago, Chicago, IL, United States; MacroGenics, Inc. monotherapy in patients with previously treated advanced gastric and Rockville, MD, United States; University College of Medicine, Seoul, gastroesophageal junction cancer: phase 2 clinical KEYNOTE-059 trial. Korea, Republic of; Mayo Clinic Cancer Center, Rochester, MN, United 5 6 JAMA Oncol. 2018;4:e180013. States; Beijing Cancer Hospital, Beijing, China; Asan Medical, Seoul, 5. Kang YK, Boku N, Satoh T, et al. Nivolumab in patients with advanced Korea, Republic of; University Medical Center Mainz, Mainz, Germany gastric or gastro-oesophageal junction cancer refractory to, or intolerant Correspondence: Daniel Catenacci (dcatenac@bsd.uchicago.edu) of, at least two previous chemotherapy regimens (ONO-4538-12, ATTR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P375 ACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;390:2461-2471. Background Ethics Approval Trastuzumab (T), a monoclonal antibody (mAb) targeting the human Each investigator’s institutional review/ethics board approved the study. epidermal growth factor receptor 2 (HER2) is the standard of care pal- liative first-line therapy for advanced HER2+ GEJ and GC patients. Mar- getuximab (M) is an Fc-engineered anti-HER2 mAb targeting the same P376 HER2 epitope, but with higher affinity for both 158V (high binding) and NOUS-209: A phase I, first-in-human, multicenter, open-label study 158F (low binding) alleles of the activating Fc receptor CD16A. Even of Nous-209 genetic vaccine for the treatment of microsatellite more, M coordinately enhanced both innate and adaptive immunity, in- unstable solid tumors cluding antigen-specific T-cell responses to HER2 [1,2]. Programmed cell Anna Gorrasi, PhD, Elisa Scarelli, Maria Teresa Catanese, Cinzia Traboni, death receptor 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) are Paola Antonini, Denis Brkic both T-cell checkpoint molecules that suppress T-cell function. MGA012 Nouscom Srl, Rome, Italy (INCMGA00012) is a humanized, hinge-stabilized, IgG4κanti-PD-1 mAb Correspondence: Elisa Scarelli (booking@nouscom.com) blocking binding of PD-L1 or PD-L2 to PD-1. MGD013 is a humanized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P376 Fc-bearing bispecific tetravalent protein that concomitantly binds to PD-1 and LAG-3, inhibiting their respective ligand-binding. We previ- Background ously reported that a chemotherapy (CTX)-free regimen consisting of MSI-H tumors are caused by a defective DNA mismatch repair M+PD-1 blockade was well tolerated in GEJ/GC patients, and induced a (dMMR) system that leads to the accumulation of mutations within 30% objective response rate (ORR) [3]. This was 2- to 3-fold greater than microsatellite regions. Insertions or deletions (indels) in microsatel- in historical controls with checkpoint inhibitors alone [4,5]. This lites of coding regions can result in the synthesis of tumor-specific registration-directed trial investigates the efficacy, safety, and tolerabil- frameshift peptides (FSPs). FSPs are considered safe and potent ity of M+checkpoint inhibition ± CTX in metastatic/locally advanced, neoantigens because they are not expressed in the normal human treatment-naïve, HER2+ GEJ/GC patients. proteome. We selected shared FSPs among patients with MSI cancers Methods with the aim of developing an off-the-shelf vaccine for the cure of This adaptive open-label phase 2/3 study includes 2 cohorts. In the first MSI tumors. 209 FSPs were assembled into 4 artificial genes and single arm, CTX-free cohort A, M+MGA012 is evaluated in HER2+ (im- cloned into 4 Great Apes Adenoviral (GAd) and 4 Modified Vaccinia munohistochemistry [IHC] 3+) and PD-L1+ (excluding microsatellite in- Ankara (MVA) vectors to generate a viral vectored vaccine called stability high) patients. After 40 patients are evaluated for response/ Nous-209. We showed that treatment of tumor-bearing mice with safety, 60 more patients will be enrolled if the threshold for study con- GAd/MVA-based neoantigen vaccines synergizes with Checkpoint In- tinuation is met. In the randomized cohort B, HER2+ (IHC 3+ or IHC 2+/ hibitors (CPI), resulting in a 3-fold-increase of cured animals over CPI fluorescent in situ hybridization+) patients, are enrolled irrespective of monotherapy. PD-L1 status. Part 1 randomizes patients to 1 of 4 arms (50 patients Methods each): control arm (T+CTX) or 1 experimental arm (M+CTX; A Phase-I, FIH study was designed to evaluate the safety, tolerability, M+CTX+MGA012; M+CTX+MGD013). CTX is investigator’s choice of and immunogenicity of Nous-209 genetic polyvalent vaccine in XELOX or mFOLFOX-6. Part 2 (pick-the-winner) consists of the control Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 207 of 272 combination with the licensed programmed death receptor-1 (PD-1)- Conclusions blocking antibody pembrolizumab and to detect any preliminary evi- The combination of vactosertib plus pembrolizumab was tolerable dence of anti-tumor activity. Nous-209 is administered intramuscu- with no additional safety concern. The activity of this combination in larly, with a heterologous prime/boost regimen composed of 1 prime CRC and GC patients will be further evaluated in the Dose Expansion with the mixture of 4 GAd vectors (GAd20-209-FSP) and 3 boosts part of the study. Clinical trial information: NCT03724851 with 4 MVA vectors (MVA-209-FSP). The target population includes Trial Registration adult patients with unresectable or metastatic dMMR or MSI-H colo- NCT03724851 rectal cancer (CRC), gastric, and gastroesophageal (G-E) junction Ethics Approval tumors. The study was approved by Ethics Board from Asan Medical Center, The study is composed of two sequential cohorts. In the first cohort Samsung Seoul Hospital, Severance Hospital, Seoul National Univer- (dose escalation), the Recommended Phase 2 Dose (RP2D) will be sity Bundang Hospital, and National Cancer Center, with approval established. In the second part (dose expansion), additional patients number 2018-1215, SMC 2018-07-146-006, 4-2018-0728, B-1808/487- will be evaluated to consolidate the safety of the RP2D and establish 003 and NCC2019-0042, respectively. the immunogenicity of the vaccination. NOUS-209 IND has been cleared by the US Food and Drug Adminis- P378 tration (FDA). The trial will be enrolling up to 30 patients at US clin- Phase II study of combination ipilimumab, nivolumab, and ical sites. Preliminary results from the study are expected in early panitumumab in patients with KRAS, NRAS, and BRAF wild-type (WT) microsatellite stable (MSS) metastatic colorectal cancer (mCRC) 1 2 3 P377 Michael Lee, MD , Patrick Loehrer, MD , Iman Imanirad, MD , Stacey 4 5 1 Safety and anti-tumor activity of the transforming growth factor β Cohen, MD , Kristen Ciombor, MD , Cheryl Carlson, MD,PhD , Hanna 1 1 receptor I kinase inhibitor, vactosertib, in combination with Sanoff, MD , Autumn McRee, MD pembrolizumab in patients with metastatic colorectal or gastric Univ of North Carolina at Chapel Hill, Chapel Hill, NC, United States; 2 3 cancer Indiana University, Indianapolis, IN, United States; Moffitt Cancer 1 2 3 4 Keun-Wook Lee, MD , Young Suk Park, MD, PhD , Joong Bae Ahn , Sun Center, Tampa, FL, United States; University of Washington, Seattle, WA, 3 4 4 5 5 Young Rha , Hark Kyun Kim , Park Young Lee , Min-Hee Ryu , Jeeyun United States; Vanderbilt University, Nashville, TN, United States 2 6 6 6 Lee, MD, PhD , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kiim , Tae Correspondence: Michael Lee (michael_s_lee@med.unc.edu) Won Kim, MD, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P378 Seoul National University Bundang Hospit, Seongnam, Korea, Republic 2 3 of; Samsung Medical Center, Seoul, Korea, Republic of; Severance Background Hospital, Seoul, Korea, Republic of; National Cancer Center, Kyunggi-do, Panitumumab is an IgG2 monoclonal antibody (mAb) targeting the Korea, Republic of; Asan Medical Center, Seoul, Korea, Republic of; epidermal growth factor receptor (EGFR) and is a standard therapy Medpacto, Inc, Seoul, Korea, Republic of for patients with KRAS, NRAS, and BRAF WT mCRC. Preclinical data Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) shows that anti-EGFR mAbs require functional innate and adaptive Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P377 immunity to mediate efficacy. Anti-EGFR therapy causes a tumor- specific adaptive immune response and immunogenic apoptosis Background [1,2], and anti-EGFR antibodies require functional T cells for in vivo Vactosertib is a highly selective and potent inhibitor of transforming efficacy [3]. However, resistance to anti-EGFR therapy inevitably de- growth factor beta (TGF-β) receptor type 1. Recent studies have re- velops and is associated with increased regulatory T cells expressing vealed that inhibition of TGF-β signaling reverses immunosuppres- CTLA-4 [4] and activated immunosuppressive macrophages with up- sive tumor microenvironment and poor responses to cancer regulation of PD-L1 [5]. Thus, resistance to anti-EGFR antibody ther- immunotherapy. To date, antitumor efficacy by immune check point apy is associated with increased expression of both CTLA-4 and PD- inhibitors in colorectal or gastric/gastroesophageal cancer as mono- L1. We hypothesized that treatment with ipilimumab (anti-CTLA-4) therapy is known to be limited. A combination of TGF-β and PD-1 in- and nivolumab (anti-PD-1) synergizes with panitumumab to signifi- hibition may induce immune restoration and improve antitumor cantly improve the response rate in patients with KRAS, NRAS, and responses. We are reporting Dose Finding part of Phase 1b/2a study BRAF WT MSS mCRC. evaluating the combination of vactosertib plus pembrolizumab in Methods metastatic colorectal cancer (CRC) or diffuse gastric cancer (GC). LCCC1632 is a multicenter, single-arm, phase II clinical trial with a Methods pre-specified safety run-in of panitumumab, ipilimumab, and nivolu- Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and have mab in KRAS/NRAS/BRAF WT, MSS mCRC (NCT03442569). Eligible pa- no prior exposure to immunotherapy including anti-CTLA-4, anti-PD-1, tients must have received 1-2 prior lines of therapy and no prior anti-PD-L1, and TGFβR1 kinase inhibitors. The primary objective is to as- anti-EGFR or immune checkpoint inhibitor therapy. A 6-subject safety sess the safety and the recommended dose of vactosertib given 5 days run-in was treated with ipilimumab 1 mg/kg IV q6wk, nivolumab 240 on/2 days off in combination with pembrolizumab 200 mg every 3 mg IV q2wk, and panitumumab 6 mg/kg IV q2wk and observed for weeks. The Dose Finding part starts with vactosertib 200 mg BID plus 12 weeks for dose-limiting toxicities (DLTs), followed by expansion pembrolizumab. Secondary objectives include characterization of vac- into a Simon’s two stage phase II trial, with 26 more subjects enrolled tosertib pharmacokinetics and anti-tumor activity by response rate. in the first phase and 56 total subjects planned. The primary end- Results point is response rate defined by RECIST 1.1. Secondary endpoints in- As of July 8, 2019, among 10 patients enrolled to 200 mg BID cohort, 6 clude response rate by irRECIST, progression-free survival (PFS), were with CRC and 4 diffuse type GC. Median age was 51 (range 31-71), overall survival (OS), and duration of response. Correlative studies in- 50% were male, median number of previous lines of chemotherapy was 4 clude Consensus Molecular Subtype analysis by archival tissue and (range 2-6). All patients were immune checkpoint inhibitor naïve. No dose assessment of peripheral immune cell activation. limiting toxicity was reported. Common adverse events (AE) were anorexia Results (33%), fatigue (33%), abdominal pain (33%), and fever (33%). There were 3 Within the 12-week DLT period, only one grade 3-4 toxicity (grade 3 serious adverse events (SAE) reported; bilirubin elevations (1), pleural effu- increased lipase) and no DLTs were observed. The most common sion (1), and ileus (1). All SAEs were not related to the study drugs. One pa- grade 1-2 treatment-related adverse events within the DLT period in- tient (1/6) with microsatellite stable (MSS) metastatic CRC achieved partial cluded acneiform rash, hypomagnesemia, decreased lymphocyte response. Biomarker data will be presented at the meeting. count, anemia, nausea, vomiting, hypothyroidism, fatigue, cough, oral Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 208 of 272 mucositis, and elevated AST. No dose modifications were required. CD73 levels will derive greater benefit from CD73 inhibition. Immu- Five of the 6 subjects (83%) had disease control (1 unconfirmed par- nohistochemistry analyses were performed on serial sections of tial response, 4 stable disease) at 12 weeks. tumor tissue to correlate protein and gene expression. Conclusions Results The combination of panitumumab, ipilimumab, and nivolumab was The HV study enrolled more than 50 participants, randomized 3:1 (ac- well-tolerated, without unexpected toxicities encountered in the tive: placebo). AB680 exhibited a good safety profile and displayed a safety run-in cohort. The response rate and disease control rate dem- long half-life following a 30-60 minute intravenous (IV) infusion, con- onstrate early signs of clinical activity. Enrollment in the phase II ex- sistent with the intended Q2W dosing schedule in cancer patients. pansion is ongoing. Doses were identified that provided maximal inhibition of peripheral Trial Registration AMP-ase activity. Our bioinformatics analyses identified tumors that ClinicalTrials.gov Identifier: NCT03442569 have high CD73 expression relative to TNAP and identified pan-RAS mutations that correlate with upregulated CD73 and poor prognosis. References Conclusions 1. Garrido G, Rabasa A, Sanchez B, et al. Induction of immunogenic AB680 is the first potent and selective small-molecule CD73 inhibitor apoptosis by blockade of epidermal growth factor receptor activation to be tested in humans. This first-in-human study demonstrates that with a specific antibody. J Immunol 2011;187:4954-66. AB680 is well tolerated and has optimal PK/PD to support its contin- 2. Pozzi C, Cuomo A, Spadoni I, et al. The EGFR-specific antibody cetuximab ued evaluation in cancer patients. combined with chemotherapy triggers immunogenic cell death. Nat Trial Registration Med 2016;22:624-31. ClinicalTrials.gov NCT03677973 3. Garrido G, Lorenzano P, Sanchez B, et al. T cells are crucial for the anti- Ethics Approval metastatic effect of anti-epidermal growth factor receptor antibodies. The study was approved by Bellberry Limited Ethics Board, approval Cancer Immunol Immunother 2007;56:1701-10. number 2018-08-673 4. Jie HB, Schuler PJ, Lee SC, et al. CTLA-4(+) Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cyto- toxicity and Correlate with Poor Prognosis. Cancer Res 2015;75:2200-10. P380 5. Pander J, Heusinkveld M, van der Straaten T, et al. Activation of tumor- Phase 1b/2 study of BXCL701, a small molecule inhibitor of promoting type 2 macrophages by EGFR-targeting antibody cetuximab. dipeptidyl peptidases, with bempegaldesleukin (bempeg, NKTR- Clin Cancer Res 2011;17:5668-73. 214) and avelumab (anti-PD-L1) in unresectable or metastatic Ethics Approval pancreatic cancer 1 1 2 3 The study was approved by the Institutional Review Board of the University of North Louis Weiner, MD , Benjamin Weinberg, MD , Stina Singel , Cedric Burg , 3 2 3 Carolina at Chapel Hill (IRB number 17-1832) and by the IRB of each subsite. Diane Healey , Jonathan Zalevsky, PhD , Chetan Lathia , Willem 2 4 2 Overwijk, PhD , Cristian Massacesi , Joyce Acbay , John MacDougall, 3 3 PhD , Vincent O'Neill P379 Georgetown Lombardi Comprehensive Cancer Center, Washington, DC, Phase 1 safety study in healthy volunteers of AB680, a small- United States; Nektar Therapeutics, San Francisco, CA, United States; 3 4 molecule inhibitor of CD73 and rationale for combination therapy BioXcel Therapeutics, New Haven, CT, United States; Pfizer, New York, in patients with gastrointestinal malignancies NY, United States Devika Ashok, PhD, Irene Luu, Akshata Udyavar, PhD, Lixia Jin, Lijuan Fu, Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) Elaine Ginn, Ken Lawson, Jenna Jeffrey, PhD, Manmohan Leleti, PhD, Jay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P380 Powers, PhD, Eric Connor, Andy Pennell, Daniel DiRenzo, PhD, Dana Piovesan, MSc, Joanne Tan, PhD, Amanda Garofalo, Wade Berry, BA, Background Matthew Walters, PhD, Steve Young, PhD, Fangfang Yin, PhD, Dominic Treatment of pancreatic cancer continues to have poor outcomes with Lai, Lisa Seitz, MA currently available therapies including checkpoint inhibitors. BXCL701 Arcus Biosciences, Hayward, CA, United States (talabostat, previously PT100) is an orally administered, small molecule Correspondence: Dominic Lai (dlai@arcusbio.com) inhibitor of dipeptidyl peptidases (DPP) specifically DPP4, DPP8 and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P379 DPP9. Inhibition of DPP8 and DPP9 triggers a process in macrophages called pyroptosis leading to innate immune proinflammatory stimula- Background tion of the tumor microenvironment[1,2,3]. BXCL701 also inhibits fibro- Extracellular adenosine, present at high concentrations in the tumor blast activation protein (FAP) releasing the FAP-mediated block of T-cell microenvironment (TME), suppresses immune function. The enzymes migration into the tumor[4]. In syngeneic animal models, significant ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phos- tumor responses were observed when BXCL701 was used with check- phatase (TNAP) catalyze extracellular conversion of adenosine mono- point inhibition[2]. Bempegaldesleukin (bempeg, NKTR-214) is a CD122- phosphate (AMP) into adenosine. Inhibition of CD73 eliminates a preferential interleukin-2 (IL-2) pathway agonist being investigated for major pathway of adenosine production in the TME and can reverse its potential to leverage the clinically validated IL-2 pathway and select- adenosine‐mediated immune suppression. Here we present the first ively stimulate an immune response, without overacting the immune results from a Phase 1 healthy volunteer (HV) study of AB680, a po- system. Bempeg has demonstrated robust anti-cancer activity when tent, reversible and selective small-molecule CD73 inhibitor. This used with checkpoint inhibition in multiple murine tumor models and placebo-controlled HV study assessed the safety, tolerability, pharma- recently in multiple human cancers[5,6]. Avelumab is a checkpoint in- cokinetic (PK) and pharmacodynamic (PD) profile of AB680. hibitor that binds PD-L1 resulting in the release of immune inhibitory Methods effects of this pathway thereby restoring immune responses, including Male or female healthy volunteers aged 18-55 with a body mass anti-cancer immune responses. In a syngeneic mouse model of pancre- index of 18-30 kg/m2 were eligible for enrollment in the atic cancer (Pan02), the triple combination demonstrated potent anti- AB680CSP0001 study (NCT03677973). Escalating doses of AB680 were cancer activity, including long-lasting anti-cancer immunity[7]. These re- evaluated in a single ascending dose (SAD) and repeat dosing study. sults provide therapeutic rationale for testing of this combination in pa- Post dosing, participants were admitted for evaluation and serially tients with pancreatic cancer. assessed for adverse events. Blood samples were collected at various Methods timepoints to elucidate the PK and PD profiles of AB680. AB680 This is an open-label, multicenter study to determine the safety and effi- plasma concentrations were determined using LC-MS/MS and PD ef- cacy of the triple combination therapy of BXCL701, bempeg and avelu- fects were evaluated by monitoring AMP-ase activity in serum. Linear mab. Patients with pancreatic cancer should have received at least 1 line models were used to predict tumor up-regulation of CD73 in mul- of gemcitabine-based therapy and no more than 2 lines of chemother- tiple tumor types with the assumption that tumors expressing higher apy for unresectable or metastatic disease, received no prior anti-PD-1/ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 209 of 272 PD-L1, IL-2 based or other T-cell directed anti-cancer therapy, and have observed when BXCL701 was used in combination with checkpoint in- ECOG 0-1. Patients must agree to biopsy of metastatic disease. Part 1 hibition[2]. Therefore, it is believed BXCL701 mediated activation of the (Phase 1b), is the 3+3 dose escalation phase designed to evaluate the innate immune system via macrophage pyroptosis inflames the cancer safety of escalating doses of BXCL701 with bempeg and avelumab. Part microenvironment and t-SCNC might become responsive to checkpoint 2 (Phase 2) will begin once the recommended combination dose is de- inhibition combined with BXCL701. termined. A Simon two-stage design will be used in Phase 2, initially en- Methods rolling 13 patients. If 2 or more responses are observed, the cohort will This is an open-label, multicenter study in patients with progressive, expand to 34 patients. The primary efficacy parameter is objective re- metastatic castration resistant prostate cancer (CRPC) as defined by sponse by RECIST 1.1. The study will also assess other parameters meas- PCWG3. Patients should have received at least 1 line of systemic uring clinical benefit and mechanistic effects on the immune system therapy and no more than 2 lines of cytotoxic chemotherapy for and tumor microenvironment. The study is not yet recruiting in the US. CRPC, received no prior anti-PD-1/PD-L1 or other T-cell directed anti- Trial Registration cancer therapy, and have ECOG 0-2. Patients in Phase 2 must also Pending have evidence of SCNC,NEPC by central pathology and agree to bi- opsy of metastatic disease. Phase 1b, is the 3+3 dose escalation References phase designed to evaluate the safety of 0.4 mg and 0.6mg BXCL701 1. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP QD on days 1 to 14 of 21-day cycle plus fixed dose pembrolizumab inhibitor, and immune checkpoint inhibitors discovered using AI and Big 200 mg administered IV on day 1 every 21 days to determine the Data analytics. Cancer Research 2017;77(13 Suppl):Abstract nr 2629. recommended dose for phase 2. A Simon’s two-stage design will be 2. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- used in Phase 2 and initially 15 patients with SCNC/NEPC will be en- caspase-1-dependent monocyte and macrophage pyroptosis. Nature rolled. If more than 2 responses are observed, then the cohort will Chemical Biology. 2017;13(1):46-53 expand to 28 patients. Primary efficacy parameter is the composite 3. Okondo M, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the response defined as achieving 1 or more of the following: • Objective Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 response by RECIST 1.1 • CTC conversion from > 5/7.5 mL to < 5/7.5 4. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- mL per Veridex assay by Week 12 • Greater than 50% PSA decline rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. from baseline by Week 12. The study is open in the US with expan- 2015;75(14):2800-2810. sion to the UK underway. 5. Charych D, Khalili S, Dixit V et al. Modeling the receptor pharmacology, Trial Registration pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically- NCT03910660 controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. EUDRACT:2018-003734-32 PLOS ONE 2017; 12(7): e0179431. 6. Diab A, Tannir N, Bernatchez C, et al. A phase ½ study of a novel IL-2 References cytokine, NKTR-214, and nivolumab in patients with select locally ad- 1. Aggarwal R, Huang J, Alumkal JJ, et al. Clinical and Genomic vanced or metastatic solid tumors. J Clin Oncol. 2017;35(suppl):e14040. Characterization of Treatment-Emergent Small-Cell Neuroendocrine Pros- 7. Rastelli L, Gupta S, Jagga Z, et al. Efficacy and immune modulation by tate Cancer: A Multi-institutional Prospective Study. J Clin Oncol. 2018; BXCL701 and dipeptidyl peptidase inhibitor, NKTR-214 a CD122-biased 36(24): 2492-2505 immune agonist with PD1 blockage in murine pancreatic tumors [ab- 2. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP stract]. J Clin Oncol. 2018;36(suppl):3085 inhibitor, and immune checkpoint inhibitors discovered using AI and Big Ethics Approval Data analytics [abstract]. In: Proceedings of the American Association for This study was approved by Institution Review Boards or Ethics Committees Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. affiliated with participating institutions Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2629. 3. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- caspase-1-dependent monocyte and macrophage pyroptosis. Nature P381 Chemical Biology. 2017;13(1):46-53 Phase 1b/2 study of BXCL701, a small molecule inhibitor of 4. OkondoM, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the dipeptidyl peptidases (DPP), with pembrolizumab, (anti-PD-1) Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 monoclonal antibody, in small cell neuroendocrine prostate cancer 5. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- (SCNC, NEPC) rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. 1 2 2 Diane Healey , Rahul Aggarwal, MD , Rahul Aggarwal, MD , Vincent 2015;75(14):2800-2810. 1 1 1 3 O'Neill , Cedric Burg , Diane Healey , Jiaoti Huang , Johann De Bono, 6. cBioPortal version 1.4.3 (dataset accessed on 9th March, 2017) 4 2 MD , Eric Small Ethics Approval 1 2 BioXcel Therapeutics, New Haven, CT, United States; UCSF Helen Diller The study was approved by Institution Review Boards or Ethics Committees Family Comprehensive Cancer Center, San Francisco, CA, United States; affiliated with participating institutions. Duke University School of Medicine, Durham, NC, United States; Institute for Cancer Research Royal Marsden NHS Foundation Trust, London, United Kingdom P382 Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) KEYNOTE-365 cohort D: phase 1b/2 study of pembrolizumab plus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P381 abiraterone acetate and prednisone in metastatic castration- resistant prostate cancer 1 2 3 Background Leonard Appleman, MD, PhD , Josep Piulats , Nataliya Mar , José 4 5 6 7 Treatment emergent Small Cell Neuroendocrine Prostate Cancer (t-SCNC) Arranz , Anthony Joshua, MD , Tina Mayer , Neal Shore, MD , Haiyan Wu, 8 8 9 is an aggressive with poor survival outcomes on standard therapies given PhD , Charles Schloss , Evan Yu 1 2 for metastatic castration-resistant disease[1 ]. BXCL701 (talabostat previ- UPMC Hillman Cancer Center, Pittsburgh, PA, United States; Catalan ously PT100) is an orally administered, small molecule inhibitor of dipepti- Cancer Institute, Barcelona, Spain; UC Irvine Medical Center, Orange, CA, dyl peptidases (DPP) specifically DPP4, DPP8 and DPP9 triggering Uunited States; Hospital General Universitario Gregorio Marañon, macrophage cell death via pyroptosis resulting in proinflammatory stimu- Madrid, Spain; St. Vincent’s Hospital Sydney, Sydney, NSW, Australia; lation of the innate immunity pathway[2,3,4]. BXCL701 also inhibits fibro- Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United blast activation protein (FAP) releasing the FAP-mediated block of T-cell States; Carolina Urologic Research Center, Myrtle Beach, SC,United 8 9 migration into the tumor[5]. FAP, DPP8 and DPP9 are expressed and acti- States; Merck & Co. Inc., Kenilworth, NJ, United States; University of vated in neuroendocrine CRPC[6]. Correlation is robust between the ex- Washington, Seattle, WA, United States pression of PD-L1 and the targets of BXCL701, particularly FAP, DPP8 and Correspondence: Leonard Appleman (applemanlj@upmc.edu) DPP9[2]. In syngeneic animal models, significant tumor responses were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P382 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 210 of 272 Background Background For patients with metastatic castration-resistant prostate cancer HPN424 is a PSMA-targeting T cell engager derived from the TriTAC (mCRPC), additional therapeutic options are needed to improve over- platform (Tri-specific T Cell-Activating Construct). PSMA is a well- all outcomes and to delay the use of chemotherapy. In early-phase validated antigen specific to prostate epithelial cells upregulated trials, pembrolizumab, an anti–PD-1 antibody, showed some activity upon malignant prostate cancer with limited expression in normal as monotherapy in heavily pretreated patients with mCRPC. Cohort D tissues. HPN424 is a recombinant polypeptide of ~50kDa containing of the nonrandomized, multicohort, open-label, phase 1b/2 three humanized antibody-derived binding domains, targeting PSMA KEYNOTE-365 (NCT02861573) study has been designed to evaluate (for tumor binding), albumin (for half-life extension) and CD3 (for T the safety and efficacy of pembrolizumab combined with abiraterone cell engagement). It has been engineered to be a small, globular pro- acetate and prednisone in patients with mCRPC who have not re- tein to enable efficient exposure in solid tumor tissue with prolonged ceived chemotherapy for mCRPC. half-life and excellent stability under physiological conditions. Methods HPN424 binds monomerically to CD3 and PSMA, minimizing non- Adults (≥18 years) with histologically or cytologically confirmed pros- specific T-cell activation. These features are designed to increase the tate cancer, without small cell histology, and who experience pro- therapeutic index compared to earlier generations of T cell engagers gression ≤6 months before screening and have an ECOG PS score of by minimizing off-target toxicities. HPN424 mediates potent target 0 or 1 are eligible. Patients must be chemotherapy naïve for mCRPC tumor cell killing in a PSMA-specific manner in vitro and in xenograft and must not have received second-generation hormonal therapy for models in the presence of T cells, demonstrated at very low antigen mCRPC or must not have experienced failed treatment with enzaluta- densities. Consistent with its mechanism of action (MOA), tumor cell mide or become intolerant to enzalutamide for mCRPC. Patients will killing is accompanied by T cell activation, cytokine induction, and T receive pembrolizumab 200 mg IV every 3 weeks, abiraterone acet- cell expansion. ate 1000 mg once daily, and prednisone 5 mg twice daily. Responses Methods will be radiographically assessed every 9 weeks during year 1 and This is a Phase 1, open-label, multicenter, dose escalation and dose every 12 weeks thereafter. Pembrolizumab treatment will continue expansion study to evaluate the safety, tolerability, clinical activity, for up to 35 cycles (approximately 2 years) or until disease progres- and pharmacokinetics of HPN424 in adult patients with meta- sion, unacceptable toxicity, or patient/physician decision to withdraw. static castrate-resistant prostate cancer (mCRPC) who have pro- Patients who discontinue 1 of the 2 drugs in the combination be- gressed on the prior regimen (per PCWG3 criteria) and received cause of drug-related adverse events can continue with the other at least 2 prior systemic therapies approved for mCRPC. HPN424 combination partner. All patients who discontinue treatment will be is administered once weekly as one-hour IV infusion by single- monitored until trial completion. Primary end points are prostate- patient cohorts until either a Grade ≥2 adverse event (AE) that is specific antigen (PSA) response rate, defined as a PSA decrease of possibly related to HPN424 is observed or an estimated thera- ≥50% from baseline measured on 2 occasions at least 3 weeks apart peutic dose level has been reached. Then a conventional 3+3 de- for confirmation, objective response rate (ORR) per RECIST v1.1 by sign is implemented. Dose escalation will continue until a blinded independent central review (BICR), and safety. Secondary recommended phase 2 dose (RP2D) is determined. In dose ex- end points include time to PSA progression, ORR based on Prostate pansion, up to 18 patients receive HPN424 at the established Cancer Working Group 3 (PCWG3)–modified RECIST v1.1 assessed by RP2D. Additional expansion cohorts may be added. Patients may BICR, duration of response based on RECIST 1.1 and PCWG3-modified continue weekly HPN424 treatment as long as they are receiving RECIST 1.1 assessed by BICR, radiographic progression-free survival clinical benefit. Primary endpoints are number and severity of based on PCWG3-modified RECIST 1.1 assessed by BICR, and overall DLTs following treatment with escalating doses of HPN424 during survival. Recruitment began in December 2018 and will continue escalation, and overall response rate (per PCWG3 criteria) in dose until ~100 patients are enrolled. expansion. Secondary endpoints include AEs, preliminary anti- Ethics Approval tumor activity, pharmacokinetic and pharmacodynamic parame- The study and the protocol were approved by the Institutional Re- ters based on the proposed MOA of HPN424. view Board or ethics committee at each site. Trial Registration Consent NCT03577028 All patients provided written informed consent to participate in the Ethics Approval clinical trial. This study was approved by each participating institution's Institu- tional Review Board. P383 Withdrawn P385 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P383 Pembrolizumab plus enzalutamide versus placebo plus enzalutamide for metastatic castration-resistant prostate cancer: phase 3 KEYNOTE-641 study 1 2 3 4 Julie Nicole Graff , Joseph Burgents , Li Wen Liang , Arnulf Stenzl P384 Knight Cancer Institute, Oregon Health & Science University, Portland, A phase I dose escalation and expansion study of HPN424, a OR, United States; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, 3 4 PSMA-targeting T cell engager, in patients with advanced prostate United States; MSD, China, Beijing, China, Beijing, China; University of cancer refractory to androgen therapy Tuebingen Medical School, Tuebingen, Germany, Tübingen, Germany 1 2 3 Johanna Bendell, MD , Mark Stein, MD , Johann de Bono, MD , Richard Correspondence: Julie Nicole Graff (graffj@ohsu.edu) 4 4 4 Austin, PhD , Sue Hirabayashi , Che-Leung Law, PhD , Bryan Lemon, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P385 4 4 5 PhD , Holger Wesche, PhD , Aaron Weitzman, MD FACP , Lawrence Fong, MD Background Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, Treatment options for patients with metastatic castration-resistant 2 3 United States; Columbia University, New York, NY, United States; Royal prostate cancer (mCRPC) are noncurative, and life expectancy is only Marsden Hospital and The Institute of Cancer Research, Sutton, United about 3 years. Enzalutamide is an androgen receptor inhibitor used Kingdom; Harpoon Therapeutics, South San Francisco, CA, United for the treatment of patients with mCRPC. Pembrolizumab is a pro- States; Weitzman Consulting Group, Los Altos Hills, CA, United States; grammed death 1 (PD-1) inhibitor with antitumor activity as mono- University of California, San Francisco, San Francisco, CA, United States therapy in mCRPC. Results of clinical studies have shown that the Correspondence: Johanna Bendell (jlee@samornbiosciences.com) mechanisms of action of pembrolizumab and enzalutamide may be Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P384 synergistic. In the phase 1b/2 KEYNOTE-365 (NCT02861573) study, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 211 of 272 antitumor activity of pembrolizumab plus enzalutamide was ob- [1]. To further assess the economic impact of NIVO+IPI associated served in mCRPC patients pretreated with abiraterone acetate. Also, with TFS, this study compared healthcare costs among untreated in a single-arm, phase 2 study (NCT02312557) of patients who pro- intermediate/poor-risk aRCC patients with different lengths of TFS. gressed on enzalutamide, some patients had profound anticancer re- Methods sponse when pembrolizumab was added to enzalutamide that lasted This study used individual patient data from the NIVO+IPI arm in Check- years. KEYNOTE-641 (NCT03834493) is a randomized, double-blind, Mate 214 (database lock, August 6, 2018; minimum follow-up, 30 phase 3 trial to evaluate efficacy and safety of pembrolizumab plus months). TFS is defined as the time from last dose of NIVO+IPI to the start enzalutamide versus placebo plus enzalutamide for patients with of subsequent systemic therapy or death, whichever occurs first. All inter- mCRPC. mediate/poor-risk aRCC patients who received NIVO+IPI and provided Methods consent were classified into 3 cohorts based on the length of TFS: cohort Approximately 1200 patients will be randomly assigned 1:1 to receive 1 remained on NIVO monotherapy maintenance; cohort 2 had TFS ≤6 enzalutamide 160 mg/day plus pembrolizumab 200 mg Q3W or months; and cohort 3 had TFS >6 months. Patient characteristics and enzalutamide 160 mg/day plus placebo. Treatment will be stratified overall survival from randomization were described for the 3 cohorts. per prior abiraterone acetate treatment (yes/no), metastases (bone Monthly costs from randomization to last known date alive, including only/liver/other), and prior docetaxel treatment for metastatic study treatment costs, all-cause grade 3/4 adverse event costs, terminal hormone-sensitive prostate cancer (yes/no). Adults (≥18 years) with care costs, and subsequent treatment costs were compared between co- histologically or cytologically confirmed prostate cancer and mCRPC hort 3 versus cohort 1 and cohort 3 versus cohort 2 using Wilcoxon rank- who experienced biochemical or radiographic progression are eli- sum tests. All costs were adjusted to 2019 United States dollars. gible. Patients who received chemotherapy for mCRPC, checkpoint Results inhibition, or any treatment with a second-generation androgen re- Of the 420 eligible patients, 16.4% (N=69) remained on NIVO mono- ceptor inhibitor (eg, enzalutamide, apalutamide, or darolutamide) are therapy maintenance, 60.2% (N=253) had TFS ≤6 months, and 23.3% excluded. Patients intolerant of or experiencing progression with (N=98) had TFS >6 months by the end of patient follow-up. Patient prior abiraterone acetate therapy are included. Patients must have characteristics were mostly similar between cohort 3 versus cohort 1 or ECOG PS 0/1, adequate organ function, and tissue for biomarker ana- 2. By definition, all patients (100%) in cohort 1 were alive at the end of lysis. Responses will be assessed by CT/MRI and radionuclide bone follow-up. The survival probabilities were 94% for cohort 3 and 60% for imaging per PCWG-modified RECIST v1.1 every 9 weeks during the cohort 2 by 18 months and 83% for cohort 3 and 39% for cohort 2 by first year and every 12 weeks thereafter. Treatment will continue with 30 months. Patients with TFS >6 months had significantly lower enzalutamide plus pembrolizumab/placebo until radiographic disease monthly costs ($8,318) compared with those who never discontinued progression, unacceptable toxicity, or consent withdrawal, with a NIVO+IPI ($16,374) and those with TFS ≤6 months ($22,811) (Figure 1). maximum of 2 years of treatment for the pembrolizumab/placebo Conclusions component of the combination. Dual primary end points are overall This retrospective healthcare cost assessment of 3 patterns of patient survival and radiographic progression-free survival by blinded inde- outcomes during and after treatment with NIVO+IPI suggests an eco- pendent central review. The key secondary efficacy end point is time nomic value of achieving prolonged TFS (>6 months). Thus, manage- to subsequent anticancer therapy or death. Additional secondary ment strategies that would lead to prolonged TFS could be end points include objective response rate, duration of response, beneficial both clinically and economically. prostate specific antigen (PSA) response rate, PSA-undetectable rate, time to PSA progression, time to pain progression, and time to radio- Acknowledgements graphic soft tissue progression. Safety and tolerability will also be Writing support was provided by Analysis Group, Inc. and editorial support reported. was provided by Parexel, funded by Bristol-Myers Squibb. Trial Registration Trial Registration ClinicalTrials.gov; NCT03834493 NCT02231749. Ethics Approval The study and the protocol were approved by the Institutional Re- Reference view Board or ethics committee at each site. 1. McDermott DF, Rini BI, Motzer RJ, Tannir NM, Escudier B, Consent Kollmannsberger CK, Hammers HJ, Porta C, George S, Donskov F, Gurney All patients provided written informed consent to participate in the HP. 874P Treatment-free interval (TFI) following discontinuation of first- clinical trial. line nivolumab plus ipilimumab (N+ I) or sunitinib (S) in patients (Pts) with advanced renal cell carcinoma (aRCC): CheckMate 214 analysis. Ann Oncol. 2018; 29(suppl 8):mdy283.083. P386 Ethics Approval Economic benefits associated with treatment-free survival of This trial was approved by the institutional review board or ethics committee immuno-oncology agents among untreated patients with at each site. intermediate/poor-risk advanced or metastatic renal cell carcinoma 1 2 3 Michael Harrison, MD , Meredith Regan, PhD , Michael Atkins, MD , 4 4 4 Sumati Rao, PhD , Shuo Yang, PhD , Jennifer Johansen, PharmD, BCPS , 5 5 5 5 Ella Du, MESc , Chenyang Gu , Ela Fadli , Keith Betts, PhD , David McDermott, MD 1 2 Duke Cancer Institute, Chapel Hill, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Georgetown Lombardi Comprehensive Cancer, Washington, DC, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Analysis Group, Inc, Los Angeles, CA, United States; Beth Israel Deaconess Medical Center, Milton, MA, United States Correspondence: Michael Harrison (Michael.Harrison@Duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P386 Background Nivolumab plus ipilimumab (NIVO+IPI) was associated with signifi- cantly longer treatment-free survival (TFS) compared with sunitinib in intermediate/poor-risk patients with previously untreated advanced Fig. 1 (abstract P386). See text for description or metastatic renal cell carcinoma (aRCC) in the CheckMate 214 trial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 212 of 272 P387 Trial Registration A multicenter, open-label, exploratory platform study to evaluate https://clinicaltrials.gov/ct2/show/NCT03835533 biomarkers and immunotherapy combinations for the treatment of patients with metastatic castration-resistant prostate cancer References (PORTER) 1. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin 1 2 3 Leo Nissola, MD , Karen Autio, MS, MD , Nina Bhardwaj, MD, PhD , AV, et al. Signatures of mutational processes in human cancer. Nature 3 4 5 Matthew Galsky, MD , Kristopher Wentzel, MD , Vanessa Lucey , Cheryl 2013;500(7463):415–21. Beer TM, Kwon ED, Drake CG, Fizazi K, Logothetis 1 1 1 Selinsky, PhD , Christopher Perry , Christopher Cabanski, PhD , Ari C, Gravis G, et al. 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Intratumoral T but not B lymphocytes are related to clinical out- form of prostate cancer, has shown limited benefit from immune come in prostate cancer. APMIS 2012;120(11):901–8. checkpoint inhibition as monotherapy, with two randomized phase 3 6. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. trials with ipilimumab failing to show a survival benefit, and a large VISTA is an inhibitory immune checkpoint that is increased after phase 2 trial with pembrolizumab demonstrating an overall response ipilimumab therapy in patients with prostate cancer. Nat Med rate (ORR) of 3-5%. Clearly novel combinations are needed and a 2017;23(5):551–5. deeper understanding of immune resistance. 7. Graff JN, Alumkal JJ, Drake CG, Thomas GV, Redmond WL, Farhad M, Using a multi-arm, multi-stage platform design, the PORTER study et al. Early evidence of anti-PD-1 activity in enzalutamide-resistant pros- will adaptively test multiple immunotherapeutic combinations to ac- tate cancer. Oncotarget 2016;7(33):52810–7. Kantoff PW, Higano CS, tivate the innate and adaptive immune systems. Coupled with deep Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel- immune biomarker profiling, this design will enable rapid insights Timmunotherapy for castration-resistant prostate cancer. N Engl J Med into the immune responses for each combination, providing data for 2010;363(5):411–22. potential larger definitive trials, while generating hypotheses for new 8. Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den Eertwegh AJM, cohorts. et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after Methods docetaxel chemotherapy (CA184-043): a multicentre, randomised, PORTER is an open-label, non-randomized, exploratory platform double-blind, phase 3 trial. Lancet Oncol 2014;15(7):700–12. study designed to assess the safety and antitumor activity of multiple 9. Lee P, Gujar S. Potentiating prostate cancer immunotherapy with immunotherapy combinations in participants with mCRPC who have oncolytic viruses. Nat Rev Urol 2018;15(4):235–50. received prior secondary androgen inhibition therapy. Each cohort 10. Lopez-Bujanda Z, Drake CG. Myeloid-derived cells in prostate cancer pro- has a two-stage design (initial n = 15, expansion n = 15) with a deci- gression: phenotype and prospective therapies. J Leukoc Biol sion to expand based on the safety, clinical activity, and biomarker 2017;102(2):393–406. results observed in the initial stage. 11. Martin AM, Nirschl TR, Nirschl CJ, Francica BJ, Kochel CM, van Cohort A is open and recruiting, testing the combination of bempe- Bokhoven A, et al. Paucity of PD-L1 expression in prostate cancer: In- galdesleukin (“BEMPEG”, NKTR-214; a CD-122 preferential IL-2 path- nate and adaptive immune resistance. Prostate Cancer Prostatic Dis way agonist) with nivolumab (PD-1 inhibitor), postulating that this 2015;18(4):325–32. will increase PD-L1 expression, intratumoral T and NK cells, and in- Ethics Approval duce an IFN gamma signature. The study was approved by WIRB‘s Ethics Board, IRB Tracking Number: Cohort B will combine CDX-301 (Flt3L), poly-ICLC (PAMP-adjuvant), nivolumab and stereotactic body radiation therapy, in 1-5 metastatic sites, inducing immunogenic cell death, mobilizing and activating dendritic cells increasing tumor antigen presentation, and overcom- P388 ing adaptive immune resistance in mCRPC. Pembrolizumab plus docetaxel and prednisone for enzalutamide- Cohort C will evaluate INO-5151, a DNA vaccine encoding PSA, PSMA, or abiraterone acetate–pretreated patients with metastatic and IL-12 delivered via intramuscular electroporation, in addition to castration-resistant prostate cancer: phase 3 KEYNOTE-921 study 1 2 3 CDX-301 and nivolumab. This is a multi-pronged approach to Neal Shore, MD , Daniel Petrylak, MD , Mostefa Bennamoun , Raffaele 4 5 6 6 7 mobilize and activate dendritic cells, stimulate anti-tumor CD8 T cells, Ratta , Josep Piulats , Ben Li , Charles Schloss , Karim Fizazi and circumvent adaptive immune resistance. Carolina Urologic Research Center, Myrtle Beach, SC, USA, Myrtle Beach, Inclusion criteria include: histologically-confirmed mCRPC that is SC, United States; Smilow Cancer Hospital at Yale University, New measurable or non-measurable by Prostate Cancer Clinical Trials Haven, CT, USA, New Haven, CT, United States; Institut Mutualiste Working Group 3 and progressing despite secondary androgen re- Montsouris, Paris, France, Paris, France; Hopital Foch, Suresnes, France, ceptor signaling inhibitor therapy. Suresnes, France; Catalan Cancer Institute, Barcelona, Spain, Barcelona, The primary endpoint: safety, as assessed by the incidence and se- Spain; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United verity of adverse events. Secondary endpoints: Composite ORR (PSA States; Gustave Roussy, Villejuif, France, Villejuif, France reduction >50%, confirmed CR or PR per RECIST 1.1, or change in cir- Correspondence: Neal Shore (nshore@gsuro.com) culating tumor cell (CTC) from >5 cells/7.5 ml to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P388 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 213 of 272 Background (HD) IL-2 regimen has demonstrated superior overall response rate Docetaxel is an established treatment for patients with metastatic (ORR), depth and durability of response versus lower dose alterna- castration-resistant prostate cancer (mCRPC). Pembrolizumab is a tives. Resistance mechanisms exploited by tumors may play a domin- programmed death 1 inhibitor that was found to have antitumor ac- ant role in limiting the effectiveness of T-cell mediated cancer tivity as monotherapy in mCRPC. In the phase 1b/2 KEYNOTE-365 therapies. The PD-1/PD-L1 interaction is a major pathway hijacked by study (NCT02861573), docetaxel plus pembrolizumab and prednisone RCC tumors to suppress immune control. Antibody-mediated PD1 had activity in patients treated with abiraterone acetate or enzaluta- blockade with pembro results in spontaneous and durable regres- mide for mCRPC, warranting further evaluation of this treatment sions for a subset of RCC tumors (SS Tykodi et al., ASCO 2019, ab- combination. KEYNOTE-921 (NCT03834506) is a randomized phase 3 stract #4570). PD1 blockade has entered clinical practice for trial to evaluate the efficacy and safety of pembrolizumab plus doce- advanced RCC as both a front-line and salvage therapy option. A fa- taxel and prednisone in chemotherapy-naïve patients who were pre- vorable safety profile for PD1 blockade has encouraged exploration viously treated with enzalutamide or abiraterone acetate for mCRPC of novel immuno-oncology combinations. and experienced progression while on therapy. Methods Methods Methods: This is an investigator-initiated, phase I trial of IL-2 plus Approximately 1000 patients will be randomly assigned 1:1 to receive pembro in patients with advanced, clear cell RCC. The study will use docetaxel 75 mg/m2 every 3 weeks (Q3W) plus prednisone/prednis- a 3 + 3 trial design to test three IL-2 dose levels in combination with olone 5 mg twice daily (BID) and pembrolizumab 200 mg Q3W or do- pembro given every 3-weeks at 200 mg flat dosing. Cohorts will re- cetaxel 75 mg/m2 Q3W plus prednisone/prednisolone 5 mg BID plus ceive subcutaneous IL-2 given once daily, 5 days per week for 6 placebo Q3W. Treatment will be stratified per previous treatment with weeks (250,000 U/kg week 1, 125,000 U/kg weeks 2-6); or IV bolus a next-generation hormonal agent (abiraterone acetate or enzaluta- dosing at 72,000 U/kg or 600,000 U/kg (HD IL-2) every 8 hours to a mide) and metastases (bone only, liver, other). Adult (≥18 years) maximum of 14 doses on week 1 and 4 of a 12-week treatment patients with chemotherapy-naïve histologically or cytologically con- course. Patients with stable or responding disease and without firmed mCRPC who experienced progression while receiving androgen treatment-limiting toxicity can receive up to 3 courses of therapy. deprivation therapy (or postbilateral orchiectomy) within 6 months be- The HD IL-2 cohort will enroll an additional 9 patients to gain further fore screening were eligible. Patients must have experienced progres- insight into anti-tumor efficacy. The primary objective is to evaluate sion after ≥8weeks (≥14 weeks for those with bone progression) or safety and tolerability for IL-2 plus pembro. The secondary objective become intolerant after ≥4 weeks of abiraterone acetate or enzaluta- is to assess antitumor activity by RECIST 1.1 for ORR, disease control mide treatment (but not both) with androgen-deprivation therapy in rate, and progression free survival. Exploratory endpoints will include the chemotherapy-naïve mCRPC state. Patients must have ECOG PS 0 pretreatment tumor analysis for PD-L1 expression, and quantitation or 1, adequate organ function, and tissue for biomarker analysis. Re- of regulatory T cell frequency in peripheral blood and tumor sponses will be assessed by CT or MRI and radionuclide bone imaging microenvironment. perProstateCancer Working Group–modified RECIST v1.1 by blinded Trial Registration independent central review (BICR) Q9W during the first year and Q12W Trial Registration: ClinicalTrials.gov, NCT03260504 thereafter. Treatment will continue with docetaxel and prednisone for Ethics Approval up to 10 cycles and with pembrolizumab for up to 35 cycles or until Ethics Approval: This study was approved by the Fred Hutchinson radiographic disease progression, unacceptable toxicity, or consent Cancer Research Center Institutional Review Board, approval number withdrawal. Primary end points are radiographic progression-free sur- 9611. vival by BICR and overall survival. The key secondary efficacy end point is time to initiation of subsequent anticancer therapy or death. Add- P390 itional secondary end points include prostate-specific antigen response Pembrolizumab plus olaparib vs enzalutamide or abiraterone in rate (decline of ≥50% from baseline, measured on 2 occasions at least patients with metastatic castration-resistant prostate cancer who 3 weeks apart), time to PSA progression, and objective response rate experienced progression on chemotherapy: phase 3 KEYLYNK-010 and duration of response per PCWG-modified RECIST 1.1 as assessed study by BICR. Safety and tolerability will also be reported. 1 2 3 4 5 Evan Yu , Se Hoon Park , Yi-Hsiu Huang , Mostefa Bennamoun ,Lu Xu , Trial Registration 5 6 Jeri Kim , Emmanuel Antonarakis, MD ClinicalTrials.gov, NCT03834506 1 2 University of Washington, Seattle, WA, United STates; Samsung Medical Ethics Approval Center, Seoul, South Korea, Seoul, Korea, Republic of; Taipei Veterans The study and the protocol were approved by the Institutional Re- General Hospital, Taipei, Taiwan, Taipei, Taiwan, Province of China; view Board or ethics committee at each site. 4 5 Institut Mutualiste Montsouris, Paris, France, Paris, France; Merck & Co., Consent Inc., Kenilworth, NJ, USA, Kenilworth, United States; Johns Hopkins All patients provided written informed consent to participate in the University, Baltimore, MD, United States clinical trial. Correspondence: Evan Yu (evanyu@u.washington.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P390 P389 A phase I trial of Interleukin-2 (IL-2) and Pembrolizumab (Pembro) Background Combination Therapy for patients with advanced renal cell The docetaxel-pretreated, metastatic castrate-resistant prostate can- carcinoma cer (mCRPC) disease state remains an unmet need for new therapeu- Scott Tykodi, MD, PhD, Johanna Whitney, Sumia Dakhil, Eleanor Bergren, tics. The programmed death 1 (PD-1) inhibitor pembrolizumab and Vivian Nguyen, Samantha Kiriluk, Shailender Bhatia, MD, John Thompson, the polyadenosine diphosphate ribose polymerase (PARP) inhibitor ola- MD parib have some independent antitumor monotherapy activity for University of Washington, Seattle, WA, United States mCRPC. In patients with mCRPC who were unselected for homologous Correspondence: Scott Tykodi (stykodi@fredhutch.org) recombination deficiency (HRD), promising activity was seen with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P389 combination of pembrolizumab and olaparib in the phase 1b/2 KEYNOTE-365 study (NCT02861573), warranting further investigation in Background this population. KEYLYNK-010 (NCT03834519) is a randomized, open- Background: Cellular immune responses play a key role modulating label, phase 3 trial to evaluate the efficacy and safety of pembrolizu- renal cell carcinoma (RCC) progression. IL-2 (aldesleukin) is a potent mab plus olaparib in molecularly unselected enzalutamide-pretreated growth and differentiation factor for T and NK cells with anti-tumor or abiraterone acetate–pretreated patients with mCRPC whose disease activity for advanced RCC across a broad dose range. A high dose progressed on or after taxane chemotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 214 of 272 Methods adjunctive therapy after completion of standard optimal therapy. Key Approximately 780 patients will be randomly assigned 2:1 to receive eligibility criteria are a diagnosis of stage III or IV primary epithelial pembrolizumab 200 mg intravenously Q3W plus olaparib 300 mg or- ovarian cancer, successful establishment of a short-term cancer cell ally twice daily or abiraterone acetate 1000 mg orally once daily plus line, a successful leukapheresis collection of monocytes, and a Kar- prednisone/prednisolone 5 mg orally twice daily (for enzalutamide- nofsky Performance Status of 70 or greater at the time of pretreated patients) or enzalutamide 160 mg/day orally (for abirater- randomization, which takes place shortly after completion of primary one acetate–pretreated patients). Arms will be stratified per prior therapy. Tumor is collected at the time of surgery from which a treatment (abiraterone acetate/enzalutamide) and presence of meas- short-term cell line is derived. Dendritic cells are produced by incu- urable disease (yes/no). Eligible patients (≥18 years) must have histo- bating peripheral blood monocytes in the presence of GM-CSF and logically confirmed mCRPC, experienced progression while receiving IL-4. The antigen source is a lysate of irradiated tumor cells from the androgen deprivation therapy within 6 months before screening, cell culture. Six to seven months after tumor collection, after comple- previously received treatment with abiraterone acetate or enzaluta- tion of concurrent surgery and primary systemic therapy, patients are mide (but not both), and previously received treatment with chemo- stratified by whether they have detectable residual disease, and then therapy (1 prior docetaxel-based regimen). Patients must have an randomized 2:1 to receive the dendritic cell vaccine or autologous ECOG PS of 0 or 1, adequate organ function, and tumor tissue suit- monocytes. Both are admixed with GM-CSF and injected subcutane- able for biomarker analysis. Responses will be assessed by CT/MRI ously at weeks 1, 2, 3, 8, 12, 16, 20, and 24 for up to eight doses. The and radionuclide bone imaging per Prostate Cancer Working Group objective is to achieve a 50% reduction in the risk of death in the (PCWG)–modified RECIST v1.1 by blinded independent central review vaccine arm. (BICR) Q9W during the first year and Q12W thereafter. Treatment will Results continue with up to 2 years of pembrolizumab (35 cycles) and ola- Cell line success rate for submitted tumor samples is 22/22 with 1 in parib or abiraterone acetate/enzalutamide until radiographic disease progress. A satisfactory leukapheresis product has been obtained for progression, unacceptable toxicity, or consent withdrawal. Primary 14/14 patients, but was repeated for 2. 12 of a planned 99 patients end points are overall survival and radiographic progression-free sur- have been randomized. 11 have started treatment; 7 have completed vival. The key secondary efficacy end point is time to initiation of all 8 doses, 1 discontinued early for disease progression, 3 are cur- subsequent anticancer therapy. Other secondary end points are ob- rently in treatment. A total of 77 doses have been administered. No jective response rate and duration of response per PCWG-modified significant toxicity directly attributed to the vaccine has been RECIST v1.1 by BICR, time to prostate-specific antigen progression, reported. time to first symptomatic skeletal event, and safety and tolerability. Conclusions Prognostic or predictive molecular biomarkers (eg, genomic HRD sta- Although logistically complex, this patient-specific vaccine approach tus, microsatellite instability) and patient-reported outcomes will be is feasible, and has been well-tolerated. [NCT02033616] explored. Trial Registration Trial Registration ClinicalTrials.gov NCT02033616 ClinicalTrials.gov, NCT03834519 Ethics Approval Ethics Approval This study was approved by the Western Institutional Review Board The study and the protocol were approved by the Institutional Re- 20171661 view Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the P392 clinical trial. Phase 1 combination study of the CHK1 inhibitor prexasertib (LY2606368) and anti-PD-L1 antibody LY3300054, in patients with high-grade serous ovarian cancer and other advanced solid tumors P391 1 1 1 1 Khanh Do , Claire Manuszak , Sarah Kelland , Allison Powers , Adrienne Randomized phase II trial of autologous dendritic cells loaded with 1 2 1 Anderson , Alona Muzikansky , Andrew Wolanski , Mariano Severgnini, autologous tumor cell antigens from self-renewing cancer cells in 1 1 1 MSc , Geoffrey Shapiro, MD, PhD , Khanh Do, MD patients with newly diagnosed stage III or IV ovarian cancer 1 2 1 2 1 Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Lisa Abaid, MD , Richard Friedman, MD, PhD , John Brown, MD , Alberto 1 1 3 4 General Hospital, Boston, MA, United States Mendivil, MD , Tiffany Beck, MD , Bradley Corr , Leslie Randall , James 5 6 6 6 Correspondence: Khanh Do (Khanh_Do@dfci.harvard.edu) Mason , Candace Hsieh , Gabriel Nistor, MD , Robert Dillman, MD, FACP 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P392 Hoag Hospital, Newport Beach, CA, United States; Disney Family Cancer Center, Burbank, CA, United States; University of Colorado, Background Aurora, CO, United States; University of California Irvine, Orange, CA, Ovarian cancers are characterized by defects in DNA damage repair United States; Scripps Green & Memorial Hospitals, La Jolla, CA, United and high levels of replication stress, creating susceptibility to inhibition States; AiVita Biomedical, Inc., Irvine, CA, United States of checkpoint kinase 1 (CHK1). CHK1 inhibition results in intratumoral Correspondence: Robert Dillman (bob@aivitabiomedical.com) DNA damage that can drive T cell infiltration, as well as PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P391 sion. Combined CHK1 inhibition and immune checkpoint blockade therefore has the potential to enhance T cell activation against tumors. Background Methods Despite recent advances, the 5-year survival rate for patients who We conducted an open-label phase 1 study of prexasertib-mediated present with stage III or IV ovarian cancer remains less than 40%. CHK1 inhibition combined with LY3300054-mediated PD-L1 blockade Standard therapy includes surgical debulking and neoadjuvant and/ following a 3+3 design evaluating 3 administration schedules: lead-in or adjuvant combination chemotherapy, with or without bevacizu- of LY3300054 alone (Arm A), lead-in of prexasertib alone (Arm B), mab, with or without intraperitoneal therapy in certain stage III pa- and combined LY3300054 and prexasertib at outset (Arm C). Both tients, and increasingly the use of PARP inhibitors. So far advanced agents were administered on days 1 and 15 of a 28-day cycle. The ovarian cancer has been relatively refractory to anti-checkpoint ther- MTD was defined as the highest dose level at which less than one- apy, presumably because of limited host anti-tumor immune re- third of at least 6 patients experienced a DLT during C0+C1. Flow cy- sponses. Adjunctive treatment with an effective vaccine could tometry of peripheral blood mononuclear cells was performed for increase immune responses and improve survival. analysis of T cell subsets. Plasma cytokine and chemokine analyses Methods were conducted using the Luminex platform. Patients enrolled to the This randomized phase II trial was approved by Western IRB. AV- currently ongoing expansion phase of the study undergo mandatory OVA-1, patient-specific dendritic cell vaccines loaded with autologous tumor biopsies during C0 and on C1D16 after the combination. tumor antigens from self-renewing cancer cells, is administered as an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 215 of 272 Results 41-77) with ECOG 0-1 and a median of 4 prior lines of systemic treat- Fifteen patients have been treated in the dose escalation phase. The ment (ranging from 1 to 8). All 12 patients received platinum-based combination of both agents is tolerable at the RP2D with prexasertib treatment prior to study entry. No DLTs were observed. The most fre- at 105mg/m2 IV on days 1 and 15 in combination with LY3300054 at quent AEs regardless of relationship to study drug were fatigue (7 700mg flat dosing. Two DLTs occurred, including febrile neutropenia patients), nausea (7 patients) and UTI (5 patients). Overall, 7 patients (Arm C) and prolonged grade 4 neutropenia lasting > 5 days (Arm B). had AEs of > grade 3, 4 of which were considered related to study Most common drug-related adverse events included leukopenia, drug by the investigator. Pharmacodynamic assessments included neutropenia, thrombocytopenia, and anemia. Confirmed partial re- immune phenotyping at the periphery where immune activation was sponses have been observed in 2 patients with CCNE1-amplified observed following AGEN2034 treatment. In this subset of recurrent HGSOC ongoing for 9 and 10 months, respectively. One additional ovarian cancer patients, 1 of 12 patients developed a durable partial CCNE-1 amplified HGSOC patient has had prolonged SD for 11 response (42 wks) at the lowest dose level (1 mg/kg), 8 patients months. Preliminary data on T-cell subset analysis and cytokine pro- demonstrated at least stable disease lasting 8.7 -65.7 weeks, with 5 file show immune modulatory effect of prexasertib, confirming of them meeting the DCR criteria of at least 12 weeks of duration. proof-of-mechanism. Four patients demonstrated progressive disease at the first on treat- Conclusions ment tumor evaluation. Full-dose prexasertib in combination with immune checkpoint block- Conclusions ade is tolerable and has preliminary clinical activity in patients with AGEN2034, a PD-1 inhibitor, is well tolerated with no DLTs observed HGSOC with durable responses. An expansion cohort in this popula- at all dose levels evaluated. The clinical activity and safety observed tion is currently being enrolled utilizing schedule B. Comprehensive in the recurrent ovarian cancer subset were consistent with the over- characterization of the immune microenvironment will be performed all Phase 1 study population. Biomarker evaluations (including PD-L1 in paired tumor biopsies, with attention to pharmacodynamic proof- status) are ongoing. of-mechanism endpoints, including T cell infiltration and PD-L1 ex- Ethics Approval pression and their correlation with the induction of DNA damage. 20170314 - IRB tracking for Copernicus Additionally, immune signatures will be correlated with genomic pro- Consent file and response duration. Written informed consent was obtained from the patient for publica- Ethics Approval tion of this abstract and any accompanying images. A copy of the This study was approved by Dana-Farber Cancer Institute's Ethics written consent is available for review by the Editor of this journal. Board. Consent P394 Written informed consent was obtained from the patient for publica- A Phase 1 study of INCMGA00012, a PD-1 inhibitor, in patients tion of this abstract and any accompanying images. A copy of the with advanced solid tumors: Preliminary results for patients with written consent is available for review by the Editor of this journal. advanced cervical cancer (POD1UM-101) 1 2 3 Janice Mehnert, MD , Luis Paz Ares, MD, PhD , Joanna Pikiel, Dr n med , 4 5 6 P393 Udai Banerji, PhD , Anna Kryzhanivska, MD , Nehal Lakhani, MD, PhD , 7 8 Single agent activity of a novel PD-1 inhibitor AGEN2034 in Sebastian Ochsenreiter, Dr med , Tobias Arkenau, MD, PhD , Nawel 9 9 9 recurrent ovarian cancer:Subset analysis of phase I dose escalation Bourayou, MD , Deanna Kornacki, PhD , Chuan Tian, PhD , Thomas 9 10 NCT03104699 study Condamine, PhD , Itziar Gardeazabal González 1 1 2 3 1 David O'Malley , John Hays , Charles Drescher , Jasgit Sachdev , Wilberto Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United 4 5 6 7 2 3 Nieves-Neira , Breelyn Wilky , Marylin Huang , Kathleen Moore , Waldo States; Hospital Universitario, Madrid, Spain; Szpitale Pomorskie Sp. z 8 8 8 8 4 Ortuzar , Anna Wijatyk , Hagop Youssoufian , Remigiusz Kaleta, MD , o.o., Gdansk, Poland; Royal Marsden NHS Foundation Trust, Sutton, 8 8 8 5 Inbal Sapir , Christopher Dupont, PhD , Irina Shapiro , Debra Richardson, United Kingdom; Regional Clinical Oncology Center, Ivano-Frankivsk, 7 6 7 MD Ukraine; START Midwest, Grand Rapids, MI, United States; Charité 1 8 The James Cancer Center Hospital, Columbus, OH, United States; Comprehensive Cancer Center, Berlin, Germany; Sarah Cannon Institute, 2 3 9 Swedish Cancer Institute, Seattle, WA, United States; HonorHealth London, United Kingdom; Incyte Corporation, Wilmington, DE, United 4 10 Research Institute, Scottsdale, AZ, United States; Northwestern States; Hospital Vall d’Hebron, Barcelona, Spain University, Stroger Hospital, Chicago, IL, United States; University of Correspondence: Janice Mehnert (mehnerja@cinj.rutgers.edu) Colorado Anschultz Medica, Aurora, CO, United States; Sylvester Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P394 Comprehensive Cancer Center, Miami, FL, United States; Stephenson Cancer Center, Oklahoma City, OK, United States; Agenus Inc, Background Lexington, MA, United States Background Correspondence: Christopher Dupont INCMGA00012 is an investigational humanized, hinge-stabilized IgG4 (Christopher.Dupont@Agenusbio.com) monoclonal antibody that binds to PD-1. At all doses tested, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P393 INCMGA00012 has an acceptable tolerability profile with no dose- limiting toxicities or maximum tolerated dose. A dose of 3 mg/kg Background Q2W was initially selected for the tumor specific cohorts. Multiple AGEN2034 is a novel, fully human monoclonal immunoglobulin G4 fixed doses were also examined in this study. The 500 mg Q4W and (IgG4) antibody, designed to block PD-1 from interacting with its li- 375 mg Q3W doses are selected for further development based on gands PD-L1 and PD-L2 with high affinity. The overall objective of favorable pharmacokinetics and safety. the study was to assess safety, MTD, and pharmacokinetic (PK) and Methods pharmacodynamic (PD) characteristics of AGEN2034 monotherapy in Methods patients with advanced, refractory malignancies. The initial expansion phase contained 4 tumor-specific cohorts Methods (endometrial [unselected], cervical, soft tissue sarcoma, and non- Between April 2017 - April 2019, 50 patients with advanced solid tu- small cell lung) treated for up to 2 years. Eligible patients presented mors were enrolled in a phase 1 dose escalation study treated with with a histologically proven, unresectable locally advanced or meta- infusion of AGEN2034 every 2 weeks at the dose range of 1-10 mg/ static tumor, ECOG performance status (PS) ≤1, disease progression kg. Within the study population a subset of patients with heavily pre- during or following ≤5 prior treatments, measurable disease per treated recurrent epithelial ovarian cancer was identified. RECIST v1.1, and no prior treatment with immune checkpoint inhibi- Results tors. The primary endpoint is safety (using CTCAE v4.03 grading). Twelve patients with recurrent epithelial ovarian cancer were en- Confirmed best overall response rate and duration of response were rolled in the Phase I dose escalation. Median age was 58 years (range evaluated by RECIST v1.1 (investigator's assessment). Treatment past Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 216 of 272 progression was allowed for patients experiencing clinical benefit. A recent study of enoblituzumab combined with pembrolizumab Preliminary safety and efficacy results for patients with unresectable showed this combination is feasible and well tolerated with minimal locally advanced or metastatic cervical cancer are presented. additive toxicity [5]. While studies of monotherapy pembrolizumab in Results this population report responses below 17% [6], the overall response Results rate of PD-1/PD-L1 inhibitor-naïve patients (post platinum) receiving As of 24 APR 2019, 35 patients with cervical cancer were treated with 3 enoblituzumab plus pembrolizumab was 33% (6/18) including 1 con- mg/kg INCMGA00012. Median age was 51 (29-81) years, 88.6% were firmed CR and 5 confirmed PRs [5]. This suggests a cooperative white, 48.6% had an ECOG PS of 1. All patients were pretreated with at mechanism and provides a rationale for further development of this least 1 prior platinum-based chemotherapy for recurrent or advanced combination in patients with recurrent/metastatic SCCHN. disease, 91.4% were treated with radiotherapy, and 62.9% underwent MGA012 (also known as INCMGA00012) is an investigational anti-PD-1 surgery. Median drug exposure was 4.4 (0.03-16.0) months. Fourteen monoclonal antibody with a tolerable safety profile and efficacy signal patients (40.0%) experienced Grade (G) 3/4 AEs regardless of causality. consistent with other agents in its class [7] demonstrated in early studies. Seven patients (20.0%) had immune-related AEs (colitis [G2, n=1; G3, Methods n=2], infusion-related reaction [G1, n=1; G3, n=1], diarrhea [G1], hyper- This is a Phase 2/3, randomized, open label study in first-line treatment of thyroidism [G2], and maculopapular rash [G3]). Three patients with col- patients with R/M SCCHN not curable by local therapy (Figure 1). We itis discontinued study treatment. No treatment-related deaths hypothesize that combining enoblituzumab and PD-1 inhibition (with or occurred. Confirmed responses per RECIST v1.1 were observed in 6/31 without chemotherapy) will improve objective response rates and OS (19.4%) response evaluable patients, with 1 patient having a confirmed compared to pembrolizumab/chemotherapy in patients in R/M SCCHN. CR. Median duration of response was not reached as 5/6 patients re- Approximately 200 patients will be randomized in a 1:1:1:1 ratio to main on treatment (10.3, NE months). An additional 12 patients had one of four treatment arms to select the preferred enoblituzumab stable disease for an overall disease control rate of 58.1% (18/31). combination treatment for further evaluation based primarily on Conclusions ORR. In subsequent Phase 3 portion, the selected enoblituzumab/ Conclusions MGA012 regimen (with or without chemotherapy) will be compared INCMGA00012 has been generally well tolerated with evidence of to pembrolizumab and chemotherapy with an endpoint of OS. significant and durable antitumor activity in platinum-refractory cer- Trial Registration vical cancer. These data support further development of To be registered on clinicaltrials.gov INCMGA00012 in cervical cancer. Trial Registration References NCT03059823, 2017-000865-63 1. Siegel R, Naishadham D, and Jemal A, Cancer statistics, 2013. CA Cancer Ethics Approval J. Clin, 2013. 63(1): p. 11-30. The study was approved by institutional review boards or independ- 2. Price KA and Cohen EE, Current treatment options for metastatic head ent ethics committees of participating institutions. and neck cancer. Curr Treat Options Oncol, 2012. 13(1): p. 35-46. 3. Keynote 048. 1200/JCO.2019.37.15_suppl.6000 Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019) 6000-6000. P395 4. Collins M, Ling V, and Carreno BM, The B7 family of immune-regulatory li- Phase 2/3 open-label trial of enoblituzumab in combination with gands. Genome Biol, 2005. 6(6): p. 223. MGA012, with and without chemotherapy, in the treatment of 5. Aggarwal C et al, Open-Label, Dose Escalation Study of Enoblituzumab in patients with recurrent or metastatic head and neck squamous cell Combination with Pembrolizumab in Patients with Select Solid Tumors. carcinoma 1 2 1 33rd Annual Meeting of The Society for Immunotherapy of Cancer Wash- Fernanda Arnaldez , Charu Aggarwal, MD MPH , Scott Currence , Jan 1 1 3 ington, DC, USA November 7–11, 2018 Baughman, MPH , Paul Moore, PhD , George Blumenschein, MD , Jon 1 4 6. Cohen EE, Harrington KJ, Tourneau C, Dinis J, Licitra L, Ahn M, et al., Wigginton, MD , Robert Ferris, MD, PhD 1 2 Head and Neck Cancer, Excluding Thyroid. ESMO, 2017. 28. MacroGenics, Inc., Rockville, MD, United States; Abramson Cancer 3 7. Mehnert J et. At. 33rd Annual Meeting of The Society for Center, Philadelphia, PA, United States; MD Anderson Cancer Center, 4 Immunotherapy of Cancer Washington, DC, USA November 7–11, 2018 Houston, TX, United States; UPMC Hillman Cancer Center, Pittsburgh, Ethics Approval PA, United States Each institution will obtain Ethics Board approval prior to enrollment Correspondence: Fernanda Arnaldez (farnaldez@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P395 Background Squamous cell carcinoma of the head and neck (SCCHN) accounts for >500,000 new cases and nearly 300,000 deaths annually worldwide as of 2012 [1]. Patients with recurrent/metastatic (R/M) SCCHN have a poor prognosis with median overall survival (OS) of Enoblituzumab is an investigational Fc-modified monoclonal antibody that binds B7-H3, which is over-expressed in a wide range of cancers including SCCHN [4], but not in most normal tissues. It has increased affinity for the acti- vating FcγR IIIA (CD16A) and decreased affinity for the inhibitory FcγRIIB (CD32B). The engineered Fc domain confers enoblituzumab with target-specific antibody-dependent cellular cytotoxicity in vitro and anti-tumor activity in preclinical studies, and in vivo and clinical data suggest that Fc-optimized antibodies such as enoblituzumab can en- gage both innate and adaptive immunity as mediators of anti-tumor activity [6]. Enoblituzumab was well tolerated in a Phase 1 monother- Fig. 1 (abstract P395). Study Schema apy trial with no maximum tolerated dose defined up to 15 mg/kg. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 217 of 272 P396 Key secondary endpoints include occurrence and severity of treatment- A phase 2 efficacy and safety trial of ADU-S100 and emergent adverse events and changes from baseline in safety assess- pembrolizumab in adults with head and neck cancer ments. This trial is currently in the recruitment phase. 1 2 3 Ezra Cohen, MD , Robert Ferris, MD, PhD , Douglas Adkins, MD , Dan 2 4 5 Zandberg, MD , Arkadiusz Dudek, MD, PhD , Matthen Mathew , Lara References 6 7 8 Dunn, MD , Juneko Grilley-Olson, MD , Ammar Sukari, MD , Rebecca 1. Chen PL, Roh W, Reuben A, et al. Analysis of Immune Signatures in 9 10 11 Redman, MD , Julie Bauman, MD, MPH , John Kaczmar, MD , Lisle Longitudinal Tumor Samples Yields Insight into Biomarkers of Response 12 13 14 15 Nabell, MD , Nabil Saba, MD , Eric Nadler, MD , Young Kim, MD , and Mechanisms of Resistance to Immune Checkpoint Blockade. Cancer 16 17 17 Ranee Mehra, MD , Nitya Nair, PhD , Somayeh Honarmand, MS , Discov 2016;6:827-37. 17 18 Richard Cutler Jr. , Barbara Burtness, MD 2. Gajewski TF, Fuertes MB, Woo SR. Innate immune sensing of cancer: University of California at San Diego, La Jolla, CA, United States; clues from an identified role for type I IFNs. Cancer Immunol University of Pittsburgh Medical Center, Pittsburgh, PA, United States; Immunother 2012;61:1343-7. Washington University of St. Louis, St. Louis, United States; 3. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the HealthPartners Regions Cancer Care Center, St. Paul, MN, United States; tumor microenvironment. Nat Immunol 2013;14:1014-22. Columbia University Medical Center, New York, United States; 4. Gajewski TF, Woo SR, Zha Y, et al. Cancer immunotherapy strategies Memorial Sloan-Kettering Cancer Center, New York, NY, United States; based on overcoming barriers within the tumor microenvironment. Curr University of North Carolina at Chapel Hill, Chapel Hill, NC, United Opin Immunol 2013;25:268-76. States; Wayne State University School of Medicine, Detroit, MI, United 5. Woo SR, Corrales L, Gajewski TF. The STING pathway and the T cell- 9 10 States; University of Louisville, Louisville, United States; The University inflamed tumor microenvironment. Trends Immunol 2015;36:250-6. of Arizona Cancer Center, Tucson, United States; MUSC Hollings Ethics Approval Cancer Center, Charleston, SC, United States; University of Alabama at This study was approved or is currently under review by an institutional Birmingham, Birmingham, United States; Emory University, Atlanta, GA, review board at each site. United States; Baylor Charles A. Sammons Cancer Center, Dallas, United States; Vanderbilt University School of Medicine, Nashville, P397 United States; University of Maryland, Greenebaum Comprehensive Interim analysis of the combination of durvalumab and cetuximab Cancer Center, Baltimore, United States; Aduro Biotech, Berkeley, CA, in a phase II trial of patients with recurrent and metastatic head United States; Yale University School of Medicine, New Haven, CT, and neck squamous cell carcinoma United States 2 1 1 1 Shuchi Gulati, MD , Sarah Palackdharry , Layne Weatherford , Sarah Wilson , Correspondence: Richard Cutler Jr. (rcutler@aduro.com) 1 1 1 3 1 Shireen Desai , Aubrey Steele ,KashifRiaz , Vinita Takiar ,Trisha Draper Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P396 1 2 University of Cincinnati, Cincinnati, OH, United States; University of Cincinnati Cancer Institute, Cincinnati, OH, United States; University of Background Cincinnati/Barrett Cancer, Cincinnati, OH, United States Immune checkpoint inhibitors such as the PD-1 blocking antibody Correspondence: Shuchi Gulati (gulatisi@ucmail.uc.edu) pembrolizumab have demonstrated marked improvements in duration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P397 of response and long-term survival over standards of care (SOC) in head and neck squamous cell carcinoma (HNSCC) and other cancers. Background However, the significant percentage of patients who are nonresponsive Cetuximab ( IgG1 isotype monoclonal antibody) monotherapy is con- to these immunotherapies (primary resistance) or experience disease sidered standard of care therapy for recurrent and metastatic head relapse following an acquired immune resistance mechanism (second- and neck squamous cell carcinoma (HNSCC).[1] Cetuximab results in ary resistance) [1] highlights the need for new therapies. As tumor re- NK cell mediated ADCC and inhibition of the EGFR signaling path- sponsiveness to immunotherapy may depend, in part, on the way.[1] NK cell activation increases secretion of plasma transforming immunophenotype of the tumor microenvironment (TME) [2-5], one ex- growth factor β (TGFβ) and interleukin 10 (IL-10), resulting in in- ploratory approach to establish, re-establish, or enhance active immune creased expression of PD-1 on T cells and PD-L1 expression on tumor surveillance conditions within the TME is to inject innate immune mod- cells. Blocking the PD-1/PD-L1 check-point receptor pathway in- ulators directly into the tumor to promote an adaptive tumor-specific creases the cytotoxic response of NK cells in mice.[2] Therefore, it immune response. ADU-S100 (MIW815) is a novel synthetic cyclic di- was hypothesized that cetuximab and PD-1/PD-L1 blockade would nucleotide that activates the stimulator of interferon genes (STING) be synergistic. Here we report our findings on the combination of pathway within the TME leading to activation of tumor-resident APCs cetuximab with a PD-L1 inhibitor, durvalumab on T cells, NK cells and priming of tumor antigen specific CD8+ T cells. Direct activation of and cytokines from a phase II open-label single site clinical trial in STING via intratumoral injection of ADU-S100 (MIW815) has been HNSCC patients with recurrent or metastatic disease. (NCT03691714.) shown to overcome active tolerance mechanisms through stimulation Methods of resident leukocyte populations. Preclinical models indicate that sur- Interim analysis includes a total of 15 enrolled patients. Using vival and local tumor shrinkage were significantly enhanced when flow cytometry and Luminex, we evaluated the immune cell phe- ADU-S100 (MIW815) was administered with an anti-PD-1 antibody, sug- notypes and cytokine profiles of peripheral blood in patients be- gesting the PD-1 blockade may act synergistically with concomitant fore and after treatment with the combination of cetuximab and STING activation. In phase 1 trials, tumor shrinkage and durable re- durvalumab with respect to overall response rate (ORR). sponses have been observed after treatment with S100 alone or in Results combination with a PD-1 inhibitor. The primary objective of this trial is Fourteen patients who received at least 2 cycles of treatment were to evaluate the clinical efficacy of intratumoral ADU-S100 (MIW815) included in the interim analysis. Median age was 66 years (range 47- when administered in combination with pembrolizumab. 75), majority of patients were male (79%). Eight patients (57%) had Methods received 1 line of prior chemotherapy, while 3 (21%) had received 2 This open-label, multicenter phase 2 clinical trial (NCT03937141) aims to prior chemotherapies. Seven patients (50%) had received prior im- enroll 33 adults with PD-L1 positive, recurrent or metastatic HNSCC for munotherapy. Of the 7 patients who had next generation sequen- which pembrolizumab is indicated as SOC in the first-line setting. Patients cing completed, 1 was PDL1 positive (14%), all had MSI-high tumors with at least one lesion that is accessible for repeat intratumoral injection (100%) and all had TP53 mutations (100%). One patient achieved a and can provide tumor tissue for eligibility determination and biomarker partial response, and 3 were noted to have stable disease; overall re- analyses will receive intravenous infusions of pembrolizumab (200 mg) at sponse rate (ORR) was noted as 27%. No grade 3/ 4 adverse events Day 1 and intratumoral injections of ADU-S100 (MIW815) (800 mcg/le- attributable to study drugs were reported. Results from peripheral sion) at Day 1 and 8 in 21-day dosing cycles up to 35 cycles, or until cri- blood flow cytometry analyses showed an increase in cytokine pro- teria for treatment discontinuation are met. The primary endpoint is the ducing NK cells and CD3+ T cells in all responders. Luminex assay objective response per Response Evaluation Criteria in Solid Tumors v1.1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 218 of 272 revealed that all responders had a drop in their GM-CSF levels, and an increase in their TNF-alpha and CXCL-10 levels. Conclusions The combination of cetuximab and durvalumab results in a pro- tumorigenic profile with a modest ORR. References 1. Ferris, R. L. et al. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation. Cancer Treat. Rev. 63, 48–60 (2018). 2. Hsu, J. et al. Contribution of NK cells to immunotherapy mediated by PD- 1/PD-L1 blockade. J. Clin. Invest. 128, 4654–4668 Ethics Approval The study was approved by University of Cincinnati's Ethics Board, approval no. FWA #: 000003152 P398 miRNA-A and programmed death ligand 1 (PD-L1) expression in oral squamous cell carcinoma 1 2 2 2 Hong Hyung, MD PhD , Yoon Ho Ko , Lee Hee JIn , Sang Hoon Jeon 1 2 Catholi, Seoul, Korea, Republic of; Catholic Universtiy, Uijeongbu-si, Korea, Republic of Correspondence: Yoon Ho Ko (koyoonho@catholic.ac.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P398 Background Overexpression of PD-L1 in cancer cells is involved not only in the im- Fig. 1 (abstract P398). A and B. See text for description mune evasion but also in cancer progression. Increasing evidence indi- cates that dysregulation of miRNA(miR)s contributes to the pathogenesis of oral squamous cell carcinoma (OSCC). Here, we identified miR-C that regulate the expression of PD-L1 and elucidated whether miR-C affects chemotherapy responsiveness via regulating PD-L1 expression in OSCC. Methods To further verify the role of miRNAs on PD-L1 in OSCC, we carried out the functional study in human head and neck cancer cell line CAL27 and YD8. After transfection with scrambled miRNA-A, B, C (Scr), miRNAs-A, B, C for 48 h, PD-L1 mRNA and PD-L1 protein levels were assessed by RT-qPCR and western blot analysis. To perform EGFP reporter assay, cells were transfected with miRNA-C and re- porter constructs, containing the putative PD-L1 3’-UTR target sites, along with a control vector, EGFP levels were assessed by western blotting. Cell viability was assessed after treatment with 5-FU for 72 h using by MTT solution. GAPDH mRNA and its protein level were used for normalization and as a loading control. Results We investigated various miRs that were negatively correlated with PD-L1 in The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset and could recognize PD-L1 3'-UTR by analyzing TargetScan. Three miRs (miR-A, miR-B, miR-C) were identi- fied which had not been reported to be as associated with PD-L1 be- fore. EGFP reporter assay of only miR-C out of three miRs showed a decrease in the relative PD-L1 expression. This would indicate that only miR-C can recognize target sites in the 3’-UTR of PD-L1 mRNA in OSCC cells. Overexpression of miR-C induced the decrease of PD-L1 mRNA and protein (Figure 1A,1B). The sensitivity of CAL27 and YD8 cells to 5-FU was increased when miR-C was overexpressed (Figure 2). Also, the level of cleaved PARP, one of the apoptotic markers, was increased according to miR-A overexpression (Figure 3). Conclusions Our data suggest that miR-C can regulate PD-L1 expression by tar- geting PD-L1 mRNA, and our present findings shed new light on the complex regulation of PD-L1 in human tumors, and on miR-C in can- Fig. 2 (abstract P398). See text for description cer immuno-based therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 219 of 272 Trial Registration NCT03246958, NCT03341936, NCT03425331, NCT02971956, NCT03075527, NCT02635061 Ethics Approval The present studies were reviewed and approved by the Dana- Farber/Harvard Cancer Center (DF/HCC) institutional review board (Boston, Massachusetts, USA) and all were performed in accordance with relevant guidelines and regulations. Consent Written informed consent was obtained from all subjects prior to par- ticipation in these studies. Informed consent by patients to DF/HCC protocol 02-180 enabled collection of clinical and demographic data, and genomic characterization. P400 Sitravatinib and Nivolumab for resectable Oral cavity squamous cell carcinoma Window of opportunity study (SNOW) 1 1 2 Marc Oliva Bernal, MD , Douglas Chepeha, MD , Amy Prawira, MD , Fig. 3 (abstract P398). See text for description 1 1 3 Anna Spreafico, MD PhD , Scott Bratman, MD , Tina Shek, MD , John De 1 3 1 1 Almeida, MD , Ivan Yeung, MD , Aaron Hansen , Andrew Hope, MD , 1 1 P399 David Goldstein, MD , Ralph Gilbert, MD , Doug Vines, BSc, MRT(N), 3 1 1 1 Instructive conclusions from performing immune correlatives on IO CNMT , Patrick Gullane , Dale Brown, MD , Ilan Weinreb, MD , Bayardo 1 4 4 trials: a meta-analysis of patient tumor and blood samples Perez-Ordoñez, MD , Trevor Pugh, PhD , Pamela Ohashi, PhD , Ben 4 1 5 Patrick Lizotte, PhD, Megan Cavanaugh, Melissa Jean, Cloud Paweletz, Wang, PhD , Jonathan Irish, MD , Hirak Der-Torossianh, MD , Isan Chen, 5 1 PhD MD , Lillian Siu, MD Dana-Farber Cancer Institute, Boston, MA, United States Princess Margaret Cancer Centre, University of Toronto, Toronto, Correspondence: Cloud Paweletz Canada; The Kinghorn Cancer Centre, St Vincent’s Hospital, Darlinghurst, (CloudP_Paweletz@DFCI.HARVARD.EDU) Australia; Princess Margaret Cancer Centre, University of Toronto; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P399 Quantitative Imaging for Personalized Cancer Medicine, TECHNA Institute, University Health Network., Toronto, Canada; University of Background Toronto, Toronto, Canada; Mirati Therapeutics, San Diego, CA, United Blood-based immune phenotyping provides a cost-effective, States minimally invasive, longitudinal, and logistically convenient Correspondence: Lillian Siu (lillian.siu@uhn.ca) assay that may provide two critical pieces of information for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P400 cancer patients receiving immunotherapy: 1.) if the therapeutic is working as intended, and 2.) if there will be any benefit to Background the patient. Sitravatinib is a receptor tyrosine kinase inhibitor that blocks TAM Methods and VEGF family of receptors. Based on non-clinical findings, it is pre- Our group has performed multi-parameter flow cytometric immune- dicted to increase M1 macrophage response and decrease immuno- profiling on hundreds of blood and tumor samples from patients suppressive Tregs and MDSCs in the tumor microenvironment. treated with immunotherapy. We have compiled this dataset of im- Sitravatinib combined with nivolumab showed a safe toxicity profile mune correlatives from patients receiving immune checkpoint block- and promising antitumor activity in non-small cell lung cancer pa- ade enrolled on Dana-Farber clinical trials in the following cancers: tients (pts) progressing on anti-PD-1 agents [1]. The CheckMate-358 thyroid cancer (tumor n = 24; blood n = 180) , head & neck squa- study revealed that preoperative nivolumab was safe and active in mous cell carcinoma (tumor n =18; blood n = 118) , mesothelioma oral cavity squamous cell carcinoma (OCSCC) [2]. We hypothesize (tumor n = 41) , non-small cell lung cancer (tumor n = 34) , and that preoperative sitravatinib and nivolumab have synergistic im- gastric-esophageal cancer (tumor n= 55). munogenic and antitumor effects in OCSCC. Results Methods Our meta-analysis of blood and tumor flow-based immune profil- SNOW is an investigator-initiated, single-center, non- ing confirms that tumor tissue remains the benchmark for deter- randomized, window-of-opportunity study evaluating pre- mining therapeutic efficacy to immune checkpoint blockade. operative sitravatinib and nivolumab in pts with resectable, However, our profiling of blood has produced several previously untreated OCSCC. Pts with T2-4a, N0-2 or T1 generalizable findings: 1.) IO-relevant markers are generally (>1cm)-N2 tumors as per AJCC 8th edition, ECOG >/=1, ad- expressed at very low levels by circulating T cells and only subtly equate organ function and no autoimmune disorders are eli- change after treatment, 2.) the abundance of different leukocyte gible. Figure 1 summarizes study design and treatment. lineages is also largely static, 3.) serial blood profiling at time- Primary objective is to evaluate the immune and pharmaco- points later than three weeks after initiation of treatment are dynamic effects of the treatment combination. Secondary ob- minimally informative, 4.) some immune parameters significantly jectives are: (a) safety, including rate of treatmen-related correlated with therapeutic efficacy are simply indicative of adverse events (TRAEs), surgery completion within the immune-related adverse events, which are historically associated planned window and postoperative complications; (b) antitu- with improved therapeutic efficacy and also easy to diagnose mor activity, including clinical and pathologic responses; rate without immune correlatives, and 5.) it is impossible to delineate of pathological extranodal extension (ENE) and positive mar- between reinvigorated or activated tumor-specific circulating T gins; (c) pharmacokinetics/pharmacodynamics of sitravatinib cells and global reinvigoration or activation of, for instance, by- alone and combined with nivolumab. Correlative studies in- stander T cells without more sophisticated and costly TCR clude: immune biomarkers by multiplex immunohistochemis- deconvolution. try, tumor and blood immunophenotyping; tumor genome Conclusions and transcriptome analyses; intratumoral hypoxia changes We conclude that blood-based immune phenotyping can be, in using 18FAZA-PET. Preliminary results as of June 30th, 2019 some contexts, highly informative, but invites over-analysis. are reported. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 220 of 272 Results P401Neoadjuvant Seven out of the 12 planned evaluable pts have been en- and adjuvant pembrolizumab plus standard of care (SOC) in rolled: 1 pt is currently undergoing study treatment and thus patients with resectable, locally advanced head and neck excluded from this analysis. Median follow-up: 19.5 weeks. All squamous cell carcinoma (HNSCC): the phase 3 KEYNOTE-689 pts completed study treatment and had surgery within the study 1 2 3 planned window. None required sitravatinib dose reduction/ Ravindra Uppaluri, MD, PhD , Nancy Lee, MD , William Westra , Ezra 4 5 6 hold or nivolumab delay. No G3/G4 TRAEs occurred pre- Cohen, MD , Robert Haddad , Stephane Temam , Christophe Le 7 8 9 10 surgery. One pt had G3 neck infection and G3 bleeding from Tourneau , Rebecca Chernock , Sufia Safina , Arkadiy Klochikhin , 11 12 13 the tracheostomy site 11 days post-surgery, both resolved Amichay Meirovitz , Irene Brana, MD , Joy Yang Ge , Ramona Swaby, 13 13 8 and deemed possibly related to study drugs. Tumor reduction MD , Cecilia Pinheiro , Douglas Adkins, MD as per investigator’s assessment was observed in all pts. Five Dana-Farber Cancer Institute and Brigham and Women’s Hospital, pts had pathological downstaging, including 1 complete Boston, MA, United States; Memorial Sloan Kettering, New York, NY, pathological response (Table 1); all pts had clear margins and USA, Sylmar, CA, United States; Icahn School of Medicine, New York, no ENE. All pts received standard of care postoperative radio- NY, USA, New York, United States; University of California San Diego, La therapy based on clinical stage. None required postoperative Jolla, CA, USA, La Jolla, CA, United States; Dana-Farber Cancer Institute chemotherapy. All pts are alive with no recurrence to date. and Brigham and Women's Hospital, Boston, MA, United States; 6 7 Conclusions Gustave Roussy, Villejuif, France, Villejuif, France; Institut Curie, Paris, These preliminary results suggest that preoperative sitravatinib France, Paris & Saint-cloud, France; Washington University School of and nivolumab is a safe and active combination in OCSCC. On- Medicine, St. Louis, MO, United States; Republican Dispensary of going biomarker and tumor immunophenotyping analyses will Tatarstan MoH, Kazan, Russia, Kazan, Russian Federation; Yaroslavl be presented Regional Clinical Oncology, Ulitsa Chkalov, Yaroslavl, Russia, Yaroslavl, Russian Federation; Hadassah-Hebrew University Medical Center, Acknowledgements Jerusalem, Israel, Jerusalem, Israel; Hospital Vall d’Hebron, Barcelona, The authors would like to thank patients and their families for their Spain, Barcelona, Spain; Merck & Co., Inc., Kenilworth, NJ, USA, participation and Mirati Therapeutics for drug supply and their support of Kenilworth, NJ, United States this study. Correspondence: Ravindra Uppaluri Trial Registration (ravindra_uppaluri@dfci.harvard.edu) NCT03575598 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P401Neoadjuvant References Background 1. Leal et al. ESMO Meeting 2018, Abstract 1129O. Neoadjuvant and adjuvant pembrolizumabshowedevidenceof 2. Ferris et al. ESMO Meeting 2017, Abstract LBA46. pathological response (PR) and acceptable safety in patients Ethics Approval with high-risk, resectable, locally advanced (LA) HNSCC in phase This study was approved by the University Health Network Research Ethics 2 studies (NCT02296684 and NCT02641093). KEYNOTE-689 Board (Study number: 18-5537) on July 12th 2018. (NCT03765918), a randomized, open-label, phase 3 trial, will as- sess efficacy and safety of neoadjuvant pembrolizumab and ad- juvant pembrolizumab plus SOC in patients with previously untreated, resectable LA HNSCC. Methods Eligible patients are adults with newly diagnosed, resectable HNSCC (stage III oropharyngeal p16-positive disease [T4 (N0-N2), M0]; stage III/IVA oropharyngeal p16 negative; or stage III/IVA larynx or hypo- pharynx or oral cavity, independent of p16 status) [1] and ECOG per- Fig. 1 (abstract P400). SNOW study design and treatment plan formance status 0 or 1. Patients will be randomly assigned 1:1 to arms A and B, with randomization stratified by primary tumor site (oropharynx/oral cavity vs larynx vs hypopharynx), tumor stage (III vs Table 1 (abstract P400). Tumor downstaging following study IVA), and PD-L1 status defined by tumor proportion score 50% treatment (TPS≥50% vs TPS Trial Registration ClinicalTrials.gov, NCT03765918 Reference 1. American Joint Committee on Cancer. AJCC Cancer Staging Manual, Eight Edition. Amin MB, ed. Chicago, IL: American College of Surgeons; Ethics Approval The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 221 of 272 P402 BELINDA : A phase 3 study evaluating the safety and efficacy of tisagenlecleucel versus standard of care in adult patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma 1 3 4 5 Michael Bishop, MD , Ian Flinn, MD , Peter Borchmann , Ulrich Jaeger , 6 7 8 9 Jason Westin, MD , Nada Hamad , Duncan Purtill , Richard Greil , 10 11 12 13 Simone Thomas , Takanori Teshima , Hideo Harigae , Carlos Garcia , 14 15 16 17 Pere Barba , Abhinav Deol, MD , Paul Shaughnessy , Jessie Gu , 18 17 17 Giovanna Andreola , Marcela Martinez Prieto , Lida Pacaud , Stephen Schuster, MD 1 2 University of Chicago, Chicago, IL, United States; Univeristy of Chicago, Chicago, IL, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University Hospital of Cologne, Cologne, Germany; 5 6 Medical University of Vienna, Vienna, Austria; University of Texas, MD Anderson Cancer Center, Houston, TX, United States; St Vincent’s Hospital, Sydney, Australia; Fiona Stanley Hospital, Murdoch, Australia; 9 10 Paracelsus Medical University, Salzburg, Austria; University Hospital Fig. 1 (abstract P402). See text for description Regensburg, Regensburg, Germany; Hokkaido University, Hokkaido, Japan; Tohoku University Graduate School of Med, Miyagi, Japan; 13 14 University Hospital "12 de Octubre", Madrid, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain; Karmanos Cancer Institute, P403 Wayne State University, Detroit, MI, United States; Texas Transplant Serum soluble CD25 may predict the early therapeutic response in Institute, San Antonio, TX, United States; Novartis Pharmaceuticals pediatric patients with B-cell non-Hodgkin's lymphoma(B- Corporation, East Hanover, NJ, United States; Novartis Pharma AG, Basel, NHL)during autologous chimeric antigen receptor T cell(CAR-T) Switzerland: Abramson Cancer Center, University of Pennsylvania, therapy Philadelphia, PA, United States Jing Guo, Yonghong Zhang, Professor, Juan Du, MD Correspondence: Michael Bishop Beijing Boren Hospital, Beijing, China (mbishop@medicine.bsd.uchicago.edu) Correspondence: Yonghong Zhang (yhzhang58@126.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P402 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P403 Background Background Around one-third of aggressive B-cell non-Hodgkin lymphoma (NHL) CAR-T therapies have been widely employed in B-NHL.Immune acti- patients will not respond to, or will relapse or progress after frontline vation induced by CAR-T therapies includes significant changes of treatment; >50% of treatment failures occur within one year. Progno- some inflammatory cytokines. In this study we analyzed the changes sis is particularly poor in these patients, regardless of salvage chemo- of serum cytokine levels in 12 pediatric patients with B-NHL during therapy and autologous hematopoietic stem cell transplant (auto- CAR-T therapy, so as to explore the relationship between serum cyto- HSCT). Novel therapies are therefore needed for refractory or early- kine levels and early treatment response. The association between relapsed patients with NHL. cytokine levels and cytokine-release syndrome(CRS) grade was also Methods investigated. BELINDA (NCT03570892) is a randomized, open-label, multicenter, Methods phase 3 study to compare the safety and efficacy of two treat- 12 B-NHL pediatric patients aging from 4 to 14 in stage II to IV ac- ment strategies: tisagenlecleucel with standard of care (SOC) cording to st.jude stage were enrolled .Each patient received 1 to 3 immunochemotherapy followed by auto-HSCT in adult patients rounds sequential CAR-T treatment, consisting of CD19, CD20, CD22 with aggressive B-cell NHL whose disease relapsed or progressed CAR-T treatment. A total of 18 rounds CAR-T were performed, includ- after frontline immunochemotherapy. Eligible patients are aged ing 12 CD19, 3 CD20 and 3 CD22. Early tumor response were evalu- ≥18 years, have histologically confirmed aggressive B-cell NHL re- ated on day 15, 30 and 60 of each round of CAR-T and early side lapsed/refractory to frontline therapy containing rituximab and effect known as CRS were observed and graded. Serum samples anthracycline within one year of last dose, and are eligible for were collected at baseline and on day 3,7,11,15,20,30,60 of each auto-HSCT. Patients are apheresed prior to enrollment and ran- round of CAR-T. Levels of Interleukin-6 (IL-6), soluble CD25 (sCD25) domized 1:1 to receive tisagenlecleucel (Arm A) or SOC (Arm B) and interferon-γ (IFN-γ) interleukin-10 (IL-10), tumor necrosis factor- (Figure 1). Randomization is stratified by remission duration (re- α(TNF-α)were measured by ELISA(enzyme-linked immunosorbent fractory or relapse assay). We analyzed the cytokine changes in different treatment out- Trial Registration comes, explored the predictive value of different cytokines using NCT03570892 ROC curve .The relationship between cytokine level and CRS grade Ethics Approval was also analyzed. Tumor response was assessed per RECIST 1.1. The The study is done in accordance with the principles of Good Clin- ROC curve take” response “and “no response” as the outcome indica- ical Practice, the Declaration of Helsinki, and all local regulations. tors ,stable disease (SD) and progressive disease (PD)were defined as The study protocol and all amendments were reviewed and ap- “no response”, complete response(CR) and partial response(PR )were proved by independent ethics committees or institutional review defined as “response”. Adverse Event (AE) grade categorization is ac- boards for each center. All patients provided written informed cording to CTCAE 4.0. consent. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 222 of 272 Results with B cell malignancies for which there are no effective therap- There were statistically significant differences in sCD25 values among ies that induce durable remissions. different treatment responses (P<0.01), with an average decrease in SD patients and an average increase in PR patients ,it is speculated P405 that sCD25 may play a predictive role in treatment response. The CASSIOPEIA: A phase 2 study evaluating efficacy and safety of ROC curve analysis shows that sCD25 had a predictive effect on the tisagenlecleucel in first-line therapy for high-risk pediatric and response to treatment, and the AUC was 0.719,95%ci =(0.516, 0.922) young adult patients with B-ALL who are MRD positive at the EOC ,excluding 0.5, indicating that the difference is statistically significant. 1 1 Shannon Maude, MD, PhD , Hunger Stephen, MD , Jochen Buechner, In this study, other cytokines were not found to be predictive 2 1 3 4 5 MD , Stephen Grupp, MD , Susana Rives , Andre Baruchel , John Levine , markers of therapeutic response. Besides,Spearman rank correlation 6 7 8 9 Joerg Krueger , Theodore Laetsch , Marianne Ifversen , Aiesha Zia , coefficient showed that there was a positive correlation between 10 10 11 Jaclyn Davis , Eric Bleickardt , Mignon Loh sCD25 and CRS grade (r=0.693,P=0.001). University of Pennsylvania; Children’s Hospital of Philadelphia, Conclusions Philadelphia, PA, United States; Oslo University Hospital, Oslo, Norway; sCD25 may be a useful predictive marker for early response of CAR-T 3 4 Sant Joan de Deu Hospital, Barcelona, Spain; Hôpital Robert and level of sCD25 are correlated with CRS grade. Clinical trial Debré&Université de Paris, Paris, France; Mount Sinai School of information:ChiCTR18000144 Medicine, New York, NY, United States; The Hospital for Sick Children, Toranto, Canada; UT Southwestern Medical Center and Child, Dallas, TX, P404 United States; Copenhagen University Hospital Rigshospi, Copenhagen, 9 10 Developing canine CART-19 to fully leverage comparative Denmark; Novartis Pharma AG, Basel, Switzerland; Novartis oncology and inform human clinical trials Pharmaceuticals Corporation, East Hanover, NJ, United States; Kumudhini Haran, MS, Ailian Xiong, Enrico Radaelli, Patrick Savickas, University of California, San Francisco, CA, United States Avery Posey, Donald Siegel, Nicola Mason, BVet Med PhD Correspondence: Shannon Maude (maude@email.chop.edu) University of Pennsylvania, Springfield, PA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P405 Correspondence: Nicola Mason (nmason@vet.upenn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P404 Background Survival is compromised for patients with high-risk (HR) B-cell acute Background lymphoblastic leukemia (B-ALL) who have a poor response to first- CD19 specific chimeric antigen receptor T cell (CART-19) therapy has line chemotherapy. A Children’s Oncology Group (COG) phase-3 resulted in unprecedented durable clinical responses in adult and study for HR B-ALL, AALL0232, showed poor 5-year disease free sur- pediatric patients with B-cell malignancies. However, poor quality of vival (DFS) of 39% in patients with minimal residual disease (MRD) patient T cells, failed persistence, reduced effectiveness within an im- ≥0.1% at end of induction (EOI) and MRD ≥0.01% at the end of con- munosuppressive microenvironment and target antigen loss, repre- solidation (EOC) [1]. The objective of this trial is to determine the effi- sent some of the challenges to improving CART19 efficacy. cacy and safety of tisagenlecleucel in pediatric and young adult Furthermore, correlative biomarkers that predict CART-19 response patients with de novo HR B-ALL who received first-line treatment remain elusive. and remain MRD-positive after the EOC therapy. Pet dogs spontaneously develop B-NHL and B cell leukemias that Methods share oncogenic pathways and similar immunosuppressive features CASSIOPEIA (NCT03876769) is a phase-2, single-arm, global, multicen- to human B cell malignancies. Therefore, they provide an immuno- ter, open-label study being conducted in collaboration with COG. Pa- logically intact, parallel patient population in which to evaluate next tients aged 1-25 years with de novo National Cancer Institute generation CAR T cell strategies and combination approaches that defined HR B-ALL (presenting white blood count >50,000/μL or over address current CART19 challenges. Previously, we have demon- the age of 10 years) who are in first complete remission (CR1) but re- strated the ability to generate functional CD20-targeting canine CAR main MRD-positive (≥0.01% by flow cytometry determined at a cen- T cells. Their use in client owned animals with B-NHL can lead to the tral reference laboratory) at EOC are eligible. Prior to screening, development of canine anti-mouse antibody (CAMA) formation and patients will complete a standard of care first-line 4-drug induction, target antigen escape. To address these issues and provide a parallel MRD assessment at EOI, a Berlin-Frankfurt-Münster phase-1b consoli- reagent that can inform human CAR T cell strategies, we have devel- dation, and MRD assessment at EOC. Eligible patients undergo leuka- oped a fully canine CD19 targeting CAR and confirmed its function pheresis either at the EOI or EOC. Prior to tisagenlecleucel infusion, in vitro against CD19 expressing targets. patients will receive interim maintenance including high-dose Methods methotrexate. Following lymphodepleting chemotherapy, patients We employed a canine scFv phage display library to isolate canine receive a single infusion of tisagenlecleucel based on body weight; CD19-specific scFvs following 3-4 rounds of panning against the sol- 0.2-5.0x10^6 chimeric antigen receptor (CAR)-positive viable T-cells uble extracellular domain of canine CD19. Twelve unique scFvs were per kg in patients ≤50 kg or 0.1-2.5x10^8 CAR-positive viable T-cells isolated and their binding to soluble canine CD19 and cell surface in patients >50 kg. Patients may receive a second infusion based on expressed CD19 was confirmed by ELISA and flow cytometry respect- B-cell recovery and MRD status. Efficacy will be assessed at day 29, ively. One of the highest binding candidates was cloned into a fully then every 3 months for the first year, every 6 months for the second canine CD28ζ CAR in a pMX retroviral plasmid. Retroviruses pseudo- year, then yearly until the end of study. The primary outcome is 5- typed with both RD114 and VSV-G envelope proteins were generated year DFS rate by local investigator assessment, defined as the time using standard protocols and used to transduce primary canine T from tisagenlecleucel infusion to morphologic relapse, occurrence of cells activated using anti-canine CD3/CD28 beads in the presence of secondary malignancy or death from any cause, whichever occurs RetroNectin®. Successful transductions of canine T cells were ob- first. Secondary outcomes include percentage of patients in remis- tained with 45% of T cells expressing CAR on their cell surface by sion without allogeneic transplantation at 1 year, MRD negativity at flow cytometry. Canine CART-19 cells demonstrated antigen-specific month 3, overall survival, cellular kinetics, and safety. The primary proliferation and cytokine production in vitro. We now aim to per- analysis of DFS will be undertaken when 40 DFS events are observed form a pilot study to evaluate the safety and efficacy of this ap- or 6 years after first-patient-first-treatment, whichever occurs later. proach in canine patients with relapsed, refractory B-NHL. This work The estimated enrollment for this study is 160 patients (with 140 in- will serve the dual purpose of enabling pet dogs with spontan- fused). The study is currently enrolling patients in the U.S., Europe eous B cell malignancies to accelerate application of next gener- and Canada. ation CART cell therapies into the human clinics as well as Trial Registration provide much needed immunotherapeutics for canine patients NCT03876769 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 223 of 272 Reference 2. Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, 1. Borowitz MJ, Wood BL, Devidas M et al. Prognostic significance of Sadelain M. CAR T cell-induced cytokine release syndrome is mediated minimal residual disease in high risk B-ALL: a report from Children's On- by macrophages and abated by IL-1 blockade. Nat Med. 2018 cology Group study AALL0232. Blood. 2015;126(8):964-971. Jun;24(6):731-738. Ethics Approval Ethics Approval The study is done in accordance with the principles of Good Clinical UCLA IRB #19-000604 Practice, the Declaration of Helsinki, and all local regulations. The study protocol and all amendments were reviewed and approved by P407 independent ethics committees or institutional review boards for each Phase 3 KEYNOTE-937: adjuvant pembrolizumab versus placebo in center. All patients provided written informed consent. patients with hepatocellular carcinoma and complete radiologic response after surgical resection or local ablation 1 2 3 4 P406 Masatoshi Kudo, MD, PhD , Andrew Zhu , Arndt Vogel , Thomas Yau , 5 6 6 6 Interleukin-1 blockade to prevent severe immune effector cell- Jian Zhou , Erluo Chen , Usha Malhotra , Abby Siegel , Ann-Lii Cheng, associated neurotoxicity syndrome; Trial in progress MD PhD Caspian Oliai, MD, Anna Crosetti, John Timmerman, MD Kindai University School of Medicine, Osaka, Japan, Osaka-Sayama, UCLA, Los Angeles, CA, United States Japan; Massachusetts General Hospital Cancer Center, Harvard Medical Correspondence: John Timmerman (jtimmerman@mednet.ucla.edu) School, Boston, MA, United States; Medizinische Hochschule, Hannover, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P406 Germany, Hannover, Germany; University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong PRC; Zhongshan Hospital, Fudan Background University, Shanghai, China, Shanghai, China; Merck & Co., Inc., CAR T-cell therapy targeting CD19 is a promising new treatment for Kenilworth, NJ, USA, Kenilworth, NJ, United States; National Taiwan relapsed/refractory B-cell lymphomas and leukemias. However, se- University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan vere grade 3 neurotoxicity (immune effector cell-associated neuro- Correspondence: Masatoshi Kudo (m-kudo@med.kindai.ac.jp) toxicity syndrome, or ICANS) is seen in up to one-third of patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P407 Recently, preclinical animal studies of human CD19 CAR T-cell therap- ies have shown that while IL-6 and IL-1 receptor antagonists could Background prevent cytokine release syndrome (without impairing anti-tumor ef- For patients with hepatocellular carcinoma (HCC) who are undergo- ficacy), only IL-1 blockade could prevent neurotoxicity [1,2]. The re- ing potentially curative surgical resection or local ablation, 5-year re- combinant IL-1 receptor antagonist Anakinra was used successfully currence rates are up to 50%-80%; there is no standard of care for to avert lethal neurotoxicity in mice. Anakinra crosses the blood brain adjuvant treatment. The programmed death 1 inhibitor pembrolizu- barrier and has been shown to be safe and efficacious in rheumatologic mab is approved for the treatment of patients with HCC previously conditions driven by high levels of monocyte lineage-associated IL-1 in- treated with sorafenib. There is no direct evidence of benefit with cluding rheumatoid arthritis and neonatal onset multisystem inflamma- pembrolizumab in the HCC adjuvant setting, but a favorable benefit/ tory disease, for which it is FDA approved. We are conducting the first risk profile is anticipated based on data from other indications. human trial of Anakinra to treat ICANS in B-cell lymphoma patients KEYNOTE-937 (NCT03867084) is a randomized, double-blind, phase 3 treated with anti-CD19 CAR T-cells. The trial has been approved by the trial to examine the safety and efficacy of adjuvant pembrolizumab U.S. FDA under an investigator-sponsored IND, and Anakinra is supplied versus placebo in patients with complete radiologic response after by the agent’s manufacturer (Sobi Pharmaceuticals). surgical resection or local ablation of HCC. Methods Methods Patients with diffuse large B-cell lymphoma receiving standard of Eligible patients are aged ≥18 years and have confirmed HCC, complete care CAR T-cells are eligible for enrollment. The primary objectives radiologic response after complete resection or local ablation, Eastern are to: 1) Evaluate the effectiveness of IL-1 blockade in reducing the Cooperative Oncology Group performance status of 0, and class A incidence and duration of severe ICANS in participants receiving anti- Child-Pugh score. Patients with past or ongoing HCV or controlled HBV CD19 CAR T-cells, 2) Assess the safety of Anakinra in CAR T-cell pa- are eligible if they meet certain criteria. Patients (N=~950) will be ran- tients, 3) Measure cytokines (including IL-1, IL-6, IL-15, TNF-alpha, domly assigned 1:1 to receive pembrolizumab 200 mg or placebo every interferon-gamma) and nitric oxide in the serum and CSF of treated 3 weeks and stratified by geographic region, prior local therapy (resec- patients prior to and during CAR T-cell therapy for correlation with tion vs ablation), recurrence risk, and alpha-fetoprotein level at diagno- ICANS events, and 4) Determine the tumor response rate in compari- sis. Treatment will continue for up to 17 cycles (~1 year) or until son to historical controls. Upon development of grade 1 ICANS, or documented disease recurrence, unacceptable toxicity, or investigator/ grade 3 CRS (which is often followed by ICANS), participants will re- patient decision to withdraw. Dual primary end points are recurrence- ceive Anakinra 100 mg subcutaneously every 6 hours for at least 12 free survival (RFS) and overall survival. Secondary end points are safety, doses, or until ICANS returns to grade 1 in participants who develop tolerability, and quality of life. Exploratory end points include distant grade 2 neurotoxicity. Patients will be continuously evaluated for tox- metastases–free survival (DMFS); time to recurrence (TTR); and gen- icity, and assessed for overall tumor response by day 120 with PET/ omic, metabolic, and/or proteomic biomarkers. RFS, DMFS, and TTR will CT scanning. Thirty-six participants will be treated in this multi- be assessed radiographically by the investigator and/or by subsequent center trial, at four centers within the University of California biopsy and confirmed by blinded independent central review. Adverse Hematologic Malignancies Consortium (UC Los Angeles, UC San events (AEs), graded per National Cancer Institute Common Termin- Francisco, UC San Diego, and UC Davis). The trial is powered to ology Criteria for Adverse Events version 4.0, will be recorded up to 30 detect a 50% reduction in the rate of severe ICANS compared to days after last dose (90 days for serious AEs). historical rates. Trial Registration ClinicalTrials.gov, NCT03867084 References Ethics Approval 1. Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito The study and the protocol were approved by the Institutional Re- F, Ponzoni M, Doglioni C, Cristofori P, Traversari C, Bordignon C, Ciceri F, view Board or ethics committee at each site. Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and Consent IL-6 are differentially required for cytokine-release syndrome and neuro- All patients provided written informed consent to participate in the toxicity due to CAR T cells. Nat Med. 2018 Jun;24(6):739-748. clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 224 of 272 P408 P409 LEAP-002: phase 3 study of first-line lenvatinib plus A case report of personalized neoantigen peptide vaccine in pembrolizumab for patients with advanced hepatocellular treating patients with biliary tract cancer 1 2 1 3 3 carcinoma Fang Yong , Fan Mo , Jiawei Shou , Huimin Wang , Lin Chen , Shanshan 1 2 3 4 1 1 1 4 Josep Llovet , Masatoshi Kudo, MD, PhD , Ann-Lii Cheng, MD PhD , Zhang , Hongsen Li , Weidong Han , Hongming Pan , Shuqing Chen 4 5 6 7 1 2 Richard Finn , Peter Galle , Shuichi Kaneko, MD PhD , Tim Meyer , Sir Run Run Shaw Hospital, Hangzhou, China; Vancouver Prostate 8 9 10 10 3 Shukui Qin , Corina Dutcus , Erluo Chen , Leonid Dubrovsky , Abby Centre, UBC, Hangzhou, China; Hangzhou Neoantigen Therapeutics Co., 10 11 4 Siegel , Andrew Zhu Hangzhou, China; Zhejiang University, Hangzhou, China 1 2 Icahn School of Medicine at Mount Sinai, New York, NY, USA; Kindai Correspondence: Shuqing Chen (chenshuqing@zju.edu.cn) University School of Medicine, Higashiosaka, Japan, Osaka-Sayama, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P409 Japan; National Taiwan University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan; David Geffen School of Medicine at UCLA, Los Background Angeles, CA, USA, Los Angeles, CA, United States; University of Mainz Despite recent advance in immune checkpoint blockade therapies in Medical Center, Mainz, Germany, Mainz, Germany; Kanazawa University cancer, the overall response rate is still low in malignancy treatment. Hospital, Kanazawa, Japan, Kanazawa, Japan; University College London Arisen from tumor somatic mutations, neoantigens provide tumor Cancer Institute, London, United Kingdom, London, United Kingdom; specific targets for developing personalized cancer vaccines, further 8 9 Jinling Hospital, Nanjing, China, Nanjing, China; Eisai Inc., Woodcliff eliciting strong T cell-mediated immune response. A single-arm, Lake, NJ, USA, Woodcliff Lake, NJ, United States; Merck & Co., Inc., open-labelled, investigator-initiated clinical study was carried out to Kenilworth, NJ, USA, Kenilworth, NJ, United States; Massachusetts examine the safety and efficacy of personalized peptide vaccine General Hospital Cancer Center, Harvard Medical School, Boston, MA, (iNeo-Vac-P01). Total of 22 patients with solid tumors had been en- USA, Boston, MA, United States rolled in the trial from Feb 7th, 2018 to May 31st, 2019. A biliary tract Correspondence: Josep Llovet (Josep.Llovet@mountsinai.org) cancer patient achieved unique neoplastic changes. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P408 Methods The 63-year-old male, initially diagnosed with intrahepatic biliary Background tract cancer in 2013, was treated with surgical excision in Jun. 2013 Lenvatinib, a multikinase inhibitor, is approved for first-line treatment and postoperative chemotherapy in Apr. 2014 respectively. Both of unresectable hepatocellular carcinoma (HCC). Pembrolizumab, a tumor recurrence and metastases were confirmed with CT scan in programmed death 1 inhibitor, is approved for second-line treatment Nov. 2017. And then, he was treated with 6 cycles of PD-1 antibody of advanced HCC in patients previously treated with sorafenib. The in a clinical trial and dropped out due to disease progression. Under phase 1b KEYNOTE-524 trial showed that lenvatinib plus pembrolizu- his consent, his biopsy and blood samples were obtained for whole mab was well tolerated and demonstrated promising antitumor ac- exome sequencing (WES), RNA sequencing (RNA-seq), and neoanti- tivity in patients with unresectable HCC. LEAP-002 (NCT03713593) is gen identification [1-4]. Finally the total of 7 peptides were synthe- a phase 3 study to evaluate the safety and clinical benefit of lenvati- sized and pooled into 2 groups. nib plus pembrolizumab in patients with previously untreated ad- On Mar 22th, 2018, he started to receive iNeo-Vac-P01 subcutane- vanced HCC. ously (s.c.). The injection sites were the two upper arms. He was Methods scheduled to receive vaccinations with GM-CSF as adjuvant on Eligible patients are aged ≥18 years and have confirmed HCC, East- day 1, 4, 8,15 and 22 (i.e.priming phase),aswellas 6 subse- ern Cooperative Oncology Group performance status (ECOG PS) 0 or quent boosters [5-8]. 1, Barcelona Clinic Liver Cancer stage C or stage B disease not amen- Results able to locoregional or curative therapy, class A Child-Pugh score ≤7 After the last booster vaccination, a grade 3~4 allergic reaction days before study day 1, and ≥1 measurable lesion (per RECIST v1.1 (under NCI-CTCAE 4.03) happened, while clinical manifestations were by blinded independent central review [BICR]). Past or ongoing HCV nausea, vomiting and rash. The treatment-relating allergic reaction infection and controlled HBV are allowed. Patients will be randomly maybe result from peptide-specific antibody accumulation, however, assigned 1:1 to receive oral lenvatinib 12 mg (body weight [BW] ≥60 this hypothesis needs experimental validation by enzyme-linked im- kg) or 8 mg (BW 400 ng/mL); and ECOG PS (0 vs 1). Tumor imaging munosorbent assay. The CT scans indicated an evident increase of will be performed every 9 weeks. Dual primary end points are tumor size at 5th month, and a surprisingly decrease of tumor size at progression-free survival (PFS), assessed per modified RECIST v1.1 by 8th month, implying a pseudo-progression. The duration of stable BICR, and overall survival. Secondary end points are objective re- disease was 14.5+ months, and he had been keeping progression- sponse rate (ORR), duration of response (DOR), disease control rate free since then. The results of IFN-γ ELISPOT assay shows that the (DCR), and time to progression (TTP) per RECIST v1.1 by BICR, efficacy neoantigen peptides stimulated highest number of IFN-γ spots and outcomes (PFS, ORR, DOR, DCR, and TTP) per modified RECIST v1.1 induced significant peptide-specific T-cell response. TCR sequencing by BICR, pharmacokinetics, and safety. Exploratory end points are ef- demonstrated the evident increase of peripheral T cells with three ficacy outcomes evaluated per RECIST v1.1 and iRECIST assessed by TCRs (data unshown). the investigator. Adverse events (AEs), graded per National Cancer In- Conclusions stitute Common Terminology Criteria for Adverse Events version 4.0, The preliminary results demonstrated that iNeo-Vac-P01 treatment will be monitored throughout the treatment period and for 90 days was feasible and safe, and can prolong progression-free survival and after the last dose (120 days for serious AEs). overall survival. Trial Registration ClinicalTrials.gov, NCT03713593 Acknowledgements Ethics Approval The authors would like to gratitude all the patients who participated in the The study and the protocol were approved by the Institutional Re- trial and their families, as well as the Sir Run Shaw clinical site. This study was view Board or ethics committee at each site. funded by Hangzhou Neoantigen Therapeutics Co. Consent Trial Registration All patients provided written informed consent to participate in the This trial had been registration on ClinicalTrials.gov, the identifier number clinical trial. was NCT03662815. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 225 of 272 References dose finding part. Secondary objectives include characterization of 1. Chen F, Zou Z, Du J, Su S, Shao J, Meng F, Yang J, Xu Q, Ding N, Yang Y vactosertib pharmacokinetics and anti-tumor activity by response et al. Neoantigen identification strategies enable personalized rate (RECIST v1.1). immunotherapy in refractory solid tumors. J Clin Invest. 2019; 130. Results 2. Hundal J, Carreno BM, Petti AA, Linette GP, Griffith OL, Mardis ER, Griffith As of July 18, 2019, 13 patients were enrolled to the study (7 in 100 M. pVAC-Seq: A genome-guided in silico approach to identifying tumor mg BID cohort and 6 in 200 mg BID cohort). Median age was 66 neoantigens. Genome Med. 2016; 8(1):11. (range 45-76), 62% were male, median number of previous lines of 3. Ng AWR, Tan PJ, Hoo WPY, Liew DS, Teo MYM, Siak PY, Ng SM, Tan EW, chemotherapy was 4 (range 2-8). All patients were PD-L1 less than Abdul Rahim R, Lim RLH et al. In silico-guided sequence modifications of 25% by SP263 antibody assay. At 100 mg BID cohort, no dose limit- K-ras epitopes improve immunological outcome against G12V and G13D ing toxicity was observed. The most frequently reported adverse mutant KRAS antigens. PeerJ. 2018; 6:e5056. events (AE) were skin rash (30.8%), nausea (23.1%), and pruritis 4. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, Zhang W, Luoma A, (23.1%). There were 3 serious adverse events (SAE) reported; pleural Giobbie-Hurder A, Peter L et al. An immunogenic personal neoantigen effusion (1), skin eruption (1), and empyema (1), and no patients with vaccine for patients with melanoma. Nature. 2017; 547(7662):217-221. reported cardiotoxicity. Among 7 tumor response evaluable patients, 5. Gjertsen MK, Buanes T, Rosseland AR, Bakka A, Gladhaug I, Soreide O, best responses to treatment were SD in 3 patients; 5.9%, 10.4%, and Eriksen JA, Moller M, Baksaas I, Lothe RA et al. Intradermal ras peptide 26.4% decreases from baseline. Biomarker data will be presented at vaccination with granulocyte-macrophage colony-stimulating factor as the meeting. adjuvant: Clinical and immunological responses in patients with pancre- Conclusions atic adenocarcinoma. Int J Cancer. 2001; 92(3):441-450. The combination of vactosertib plus durvalumab has been tolerated 6. Keskin DB, Anandappa AJ, Sun J, Tirosh I, Mathewson ND, Li S, Oliveira G, thus far with no safety concerns; the study is ongoing. The anti- Giobbie-Hurder A, Felt K, Gjini E et al. Neoantigen vaccine generates tumor activity of this combination in patients with advanced NSCLC intratumoral T cell responses in phase Ib glioblastoma trial. Nature. 2019; will be further explored. Clinical trial information: NCT03732274 565(7738):234-239. Trial Registration 7. Weden S, Klemp M, Gladhaug IP, Moller M, Eriksen JA, Gaudernack G, NCT 03732274 Buanes T. Long-term follow-up of patients with resected pancreatic can- Ethics Approval cer following vaccination against mutant K-ras. Int J Cancer. 2011; The study was approved by Ethics Board of Severance Hospital (ap- 128(5):1120-1128. proval number 4-2018-0892) and National Cancer Center (approval 8. Kirner A, Mayer-Mokler A, Reinhardt C. IMA901: a multi-peptide cancer number NCC2019-0057) vaccine for treatment of renal cell cancer. Hum Vaccin Immunother. 2014; 10(11):3179-3189. P411 Ethics Approval Treating advanced non-small lung cancer (NSCLC) patients after This study was approved by the institutional review board and independent checkpoint inhibitor treatment failure with a novel combination of ethics committee of Sir Run Run Shaw Hospital, Zhejiang University Viagenpumatucel-L (HS-110) plus nivolumab School of Medicine; approval number 20180109-9. 1 1 Daniel Morgensztern, MD , Saiama Waqar, MD , Lyudmila Bazhenova, Consent 2 3 4 MD , Rachel Sanborn, MD , Lori Mcdermott, RN, MSc , Jeff Hutchins, Written informed consent was obtained from the patient for publication of 4 5 6 PhD , Luis Raez, MD, FACP, FCCP , Corey Langer, MD , Roger Cohen, this abstract and any accompanying images. A copy of the written MD consent is available for review by the Editor of this journal. Washington University School of Medicine, St. Louis, MO, United States; 2 3 Moores Cancer Center, La Jolla, CA, United States; Earle A. Chiles P410 Research Institute, Portland, OR, United States; Heat Biologics, Tampa, Safety and anti-tumor activity of the transforming growth factor β FL, United States; Memorial Cancer Institute, Pembroke Pines, FL, United receptor I kinase inhibitor, vactosertib, in combination with States; Perelman School of Medicine, Philadelphia, PA, United States durvalumab in patients with advanced non-small cell lung cancer Correspondence: Lori Mcdermott (lmcdermott74@yahoo.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P411 1 2 2 Ji-Youn Han, MD, PhD , Kyoung-Ho Pyo , Jea Hwan Kim , Chun-Feng 2 3 3 3 Xin , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kim , Byoung Chul Cho, Background 2 2 MDphD , Byoung Chul Cho, MDphD Viagenpumatucel-L (HS-110) is an allogeneic cellular vaccine derived 1 2 National Cancer Center, Goyang-si, Korea; Severance Hospital, Seoul, from a human lung adenocarcinoma cell line transfected with the Korea, Republic of; Medpacto, Inc, Seoul, Korea, Republic of gp96-Ig fusion protein that functions as an antigen chaperone for Correspondence: Byoung Chul Cho (cbc1971@yuhs.ac) cross presentation and dendritic cell activation. DURGA is a multi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P410 cohort study evaluating the combination of HS-110 and anti-PD-1 monoclonal antibodies in patients with advanced NSCLC. We report Background on Cohort B, which enrolled patients with progressive disease (PD) TGF-β signaling is known to be associated with poor response to single- after receiving a minimum of 4 months of treatment with a check- agent immune checkpoint inhibitors by immunosuppressive microenviron- point inhibitor (CPI) at any time prior to study entry. ment through strong epithelial-mesenchymal transition (EMT) induction. Methods Combined inhibition of immune checkpoint and TGF-β signal is anticipated Patients with previously treated NSCLC received weekly HS-110 (1 X as a promising therapeutic strategy because these two key pathways have 107 cells) intradermally for 18 consecutive weeks and nivolumab IV independent and complementary immunosuppressive functions. We are 240 mg every 2 weeks, followed by nivolumab maintenance until reporting the Dose Finding part of a Phase 1b/2a study evaluating the tumor progression or intolerable toxicity. Tissue was tested at base- combination of vactosertib, a highly selective and potent TGF-β inhibitor, line for PD-L1 expression (≥ 1% or < 1%) and tumor infiltrating lym- with durvalumab in patients with advanced non-small cell lung cancer phocytes (TILs). TIL high was defined as >10% CD8+ lymphocytes in (NSCLC) who progressed following platinum-based chemotherapy. the tumor stroma. The primary endpoint was objective response rate Methods (ORR) by RECIST 1.1. Secondary endpoints included ORR and clinical Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and benefit rate using iRECIST, progression-free survival (PFS), overall sur- have no prior exposure to immune checkpoint inhibitors, or TGFβ R1 vival (OS) and adverse events (AEs). kinase inhibitors. The primary objective is to assess the safety and Results the recommended dose of vactosertib given 5 days on/2 days off in As of March 2019, 56 patients were enrolled and evaluated for efficacy. combination with durvalumab 1500 mg every 4 weeks. Two dose The median number of prior treatment lines was 2 [range 1 to 6]. Seven levels of vactosertib (100 mg BID and 200 mg BID) were tested in the patients (13%) achieved partial response and 26 patients (46%) had Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 226 of 272 stable disease. Median PFS and median OS were 3.2 months and 11.8 Hossein Borghaei et al., Nivolumab versus Docetaxel in Advanced months, respectively. Immune ORR and clinical benefit rate by iRECIST Nonsquamous Non–Small-Cell Lung Cancer. The New England Journal of were 14% and 61%, respectively. Patients experiencing injection site re- Medicine. 2015; 373:1627-1639 actions (ISR) had improved PFS (3.7 vs 1.8 months; HR 0.21, p =0.0021) Achim Rittmeyer et al., Atezolizumab versus docetaxel in patients with and improved OS (12 vs 5 months; HR 0.16, p=0.0005) compared to previously treated non-small-cell lung cancer (OAK): a phase 3, open- those without ISR. 96% of patients experienced at least one adverse label, multicentre randomised controlled trial. The Lancet. 2017; 389 event, and 92% of all AEs were grade 1 or 2. The most common AEs (10066): 255-265 were fatigue (34%), hypocalcemia (18%), cough (16%) and diarrhea and Ethics Approval dyspnea (14% each). There were four grade 4 events: QTc prolongation, The last amendment was approved in June 2019 by WIRB and Copernicus stroke, pericardial tamponade, and hyponatremia, none of which were IRB, approval number 420160463 deemed related to treatment. There were no grade 5 AEs. Conclusions P413 The combination of HS-110 and nivolumab is well tolerated, and A Phase 1b/2 study of galunisertib in combination with nivolumab does not appear to increase the incidence of immune-related AEs as in solid tumors and NSCLC compared to CPI monotherapy. Patients continue to be enrolled into 1 2 3 Ernest Nadal, MD, PhD , Mansoor Saleh, MD , Santiago Ponce Aix, MD , this cohort. Data suggest that re-challenging the immune system 4 5 6 Maria Ochoa de Olza , Sandip Patel, MD , Scott Antonia, MD, PhD , with nivolumab and HS-110 after CPI treatment failure restores re- 7 7 7 Yumin Zhao, PhD , Ivelina Gueorguieva, PhD , Michael Man, PhD , sponsiveness and clinical benefit for some patients. 7 7 8 7 Shawn Estrem, PhD , Emin Avsar , Wen Hong Lin , Karim Benhadji , 7 9 1 Susan Guba , Inmaculada Ales Diaz , Ernest Nadal, MD, PhD Acknowledgements 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; University of Thank you to the Investigators, their staff, and the patients and family Alabama, Birmingham, AL, United States; Hospital 12 de Octubre, members that made this research possible. Madrid, Spain; Hospital Universitario Vall d'Hebron, Barcelona, Spain; Trial Registration 5 6 University of California, La Jolla, CA, United States; H. Lee Moffitt NCT 02439450 Cancer Center and, Tampa, FL, United States; Eli Lilly and Company, Ethics Approval Indianapolis, IN, United States; Bristol-Myers Squibb, New York, NY, This study was approved by Advarra IRB, Western IRB, Washington University United States; Hospital Universitario Regional de Mala, Malaga, Spain IRB, Cleveland Clinic IRB, UCSD IRB, Providence Portland IRB, NYU Winthrop Correspondence: Ernest Nadal (esnadal@iconcologia.net) IRB, Baptist Health Louisville IRB, Lifespan IRB, and US Oncology IRB. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P413 P412 Background Validation of a single-blinded (patients only) study design for the TGF-β promotes immune suppression. In this study, both TGF-β and prevention of premature patient consent withdrawal in the PD-1 were targeted in patients with advanced refractory solid tumors immuno-oncology trial DUBLIN-3 and recurrent/refractory NSCLC using galunisertib, an oral small mol- Ramon Mohanlal, MD, PhD, MBA, Huang Lan, PhD ecule inhibitor of TGF-β receptor I, in combination with nivolumab, a BeyondSpring Pharmaceuticals, Inc., New York, NY, United States monoclonal antibody that binds PD-1. Correspondence: Ramon Mohanlal Methods (rmohanlal@beyondspringpharma.com) This is a Phase 1b/2 open-label study. Eligible patients were ≥18 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P412 years old, had ECOG status ≤1, and were treatment-naive for anti-PD- 1/PD-L1, or TGFβ R1 kinase inhibitor. Patients had advanced solid tu- Background mors that were refractory to standard systemic therapy (Phase 1b). Patients (pts) generally prefer immunotherapy (IO) over chemother- NSCLC patients (Phase 2) were required to have received prior apy (Chemo) in clinical trials and may prematurely withdraw consent platinum-based treatment. Phase 2 portion of the trial evaluated the if allocated to Chemo. This may impact study outcome (Barlesi Lan- safety of 150 mg BID galunisertib administered on a 14 days on, 14 cet Onc 2018). Due to pts awareness of their treatment allocation in days off dosing schedule in combination with nivolumab given at 3 unblinded IO trials, ‘premature’ consent withdrawal (thus before re- mg/kg Q2W. Efficacy, pharmacokinetics (PK) and pharmacodynamic ceiving first dose of study drug) is consistently and significantly (p data were also evaluated. Methods Results ‘Premature’ pts consent withdrawal rate was calculated for the Plin/ 15 patients were enrolled in Phase 1b and 25 in Phase 2. No dose- Doc (n=174) and Doc (n=181) arms in DUBLIN-3 (NCT02504489) limiting toxicities were observed in the Phase 1 portion of the study. around the time of the first pre-planned Interim Analysis (IA). In the Phase 2 NSCLC cohort, the most frequent treatment-related Results grade 3 AEs included immune-related encephalitis, diarrhea, fatigue, ‘Premature’ consent withdrawal rate in DUBLIN-3 was 1.1 % for Doc ALT/ AST/GGT increase, blood alkaline phosphatase increase, abdom- and 2.3 % for Plin/Doc (p=0.53; NS). Premature consent withdrawal inal distension, cutaneous rash (n=1 each), and cholestasis (n=2) that rate of the Doc arm was significantly (p resolved or were resolving at the time of data cutoff. Two deaths on Conclusions treatment (multi-organ failure and myocardial infarction), both unre- A single-blinded design (for pts only) is effective in preventing pre- lated to study treatment, were observed. 6 (24%) patients had con- mature and imbalanced patient consent withdrawal. This finding firmed partial response (PR) and 4 (16%) had stable disease; 1 may have relevance for the design of future IO trials. A second pre- patient had confirmed PR after initial pseudo-progression. Among planned IA for DUBLIN-3 to evaluate OS is projected for end 2019. the 6 responders, 5 had low or negative PD-L1 expression (≤50%). Trial Registration Median PFS was 5.26 months (95% CI: 1.77, 9.20) and median OS was NCT02504489 11.99 months (95% CI: 8.15, NR). Phase 1b PK data showed rapid ab- sorption (1-3h) and elimination of galunisertib within 48h. Additional References biomarker data including tumor mutational burden and gene- Fabrice Barlesi et al., Avelumab versus docetaxel in patients platinum-treated expression data will be presented. advanced non-small-cell lung cancer (JAVELIN Lung 200): an open-label, Conclusions randomised, phase 3 study. The Lancet. 2018; 19 (11): 1468-1479 Combination treatment of galunisertib at the RP2D of 150 mg BID Roy S Herbst et al., Pembrolizumab versus docetaxel for previously treated, for 14 days on 14 days off schedule with nivolumab 3 mg/kg Q2W PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a was well tolerated. Preliminary efficacy was observed in a subset of randomised controlled trial. The Lancet. 2016; 387 (10027): 1540-1550 patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 227 of 272 Trial Registration Conclusions NCT02423343 Preliminary data suggest the combination of pepinemab plus avelu- Ethics Approval mab is well tolerated and shows initial signals of antitumor activity in The study was performed in accordance with the Declaration of patients with IO failure. We will present updated clinical response Helsinki and was approved by ethics committees in multiple investi- data, as well as additional immunophenotyping of tissue biopsies, in- gator sites. cluding but not limited to activated T cells, regulatory T cells, DCs, monocytes, macrophages, and importantly myeloid-derived suppres- sor cells (MDSCs). P414 Interim results from CLASSICAL-Lung, a phase 1b/2 study of Acknowledgements pepinemab (VX15/2503) in combination with avelumab in All of the CLASSICAL-Lung investigators, site staff, and patients advanced non-small cell lung cancer patients Trial Registration 1 2 2 Michael Shafique, MD , Terrence Fisher, PhD , Elizabeth Evans, PhD , NCT03268057 2 2 2 John Leonard, MD PhD , Desa Rae Pastore , Crystal Mallow, BS , Ernest 2 3 4 5 Smith, PhD , Andreas Schroeder, MD, PhD , Kevin Chin , Joseph Beck , References 6 7 Megan Baumgart, MD , Ramaswany Govindan, MD , Nashat Gabrail, 1. Evans EE et al. Antibody blockade of semaphorin 4D promotes immune 8 9 10 MD , Jonathan Goldman, MD , Rachel Sanborn, MD , Alexander Spira, infiltration into tumor and enhances response to other 11 12 13 MD, PhD, FACP , Nagashree Seetharamu , Yanyan Lou, MD , Aaron immunomodulatory therapies. Cancer Immunol Res. 2015; 3: 689-701 13 2 2 Mansfield, MD , Maurice Zauderer, PhD , Terrence Fisher, PhD 2. Clavijo PE et al. Semaphorin4D inhibition improves response to immune 1 2 Moffitt Cancer Center, Tampa, FL, United States; Vaccinex, Inc, checkpoint blockade via attenuation of MDSC recruitment and function. 3 4 Rochester, NY, United States; Merck KGaA, Darmstadt, Germany; EMD Cancer Immunol Res. 2019; 7(2):282-291. Serono, Rockland, MA, United States; Highlands Oncology Group, Ethics Approval Fayetteville, AZ, United States; University of Rochester, Rochester, NY, This protocol and its amendments were approved by the appropriate IRBs at United States; Washington University School of Medicine, St. Louis, MO, each site. 8 9 United States; Gabrail Cancer Center, Canton, OH, United States; UCLA Medical Center, Paramus, NJ, United States; Earle A. Chiles Research Institute, Portland, OR, United States; Virginia Cancer Specialists, Fairfax, P415 VA, United States; Northwell Health, New York, NY, United States; Tumor Treating Fields (TTFields, 150 kHz) concurrent with Mayo Clinic, Jacksonville, FL, United States standard of care treatment for stage 4 non-small cell lung cancer Correspondence: Terrence Fisher (tfisher@vaccinex.com) (NSCLC) in Phase 3 LUNAR Study Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P414 Ori Farber, Moshe Giladi, Ze'ev Bomzon, Eilon Kirson, Uri Weinberg, MD PhD Background Novocure Ltd., Haifa, Israel Despite progress of immune checkpoint blockade therapies, many Correspondence: Uri Weinberg (weinberg@novocure.com) patients with non-small cell lung cancer (NSCLC) do not receive dur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P415 able clinical benefit from these agents, and even in those who do re- spond initially, acquired resistance and tumor recurrence can Background develop. Pepinemab is an IgG4 humanized monoclonal antibody tar- Tumor Treating Fields (TTFields) are a non-invasive, anti-mitotic treat- geting semaphorin 4D (SEMA4D, CD100). In vivo preclinical studies ment that disrupts the formation of the mitotic spindle and disloca- demonstrated antibody blockade of SEMA4D promoted immune infil- tion of intracellular constituents. TTFields plus temozolomide tration and reduced function and recruitment of immunosuppressive significantly extended survival in newly diagnosed glioblastoma. Effi- myeloid cells within the tumor [1,2]. Importantly, preclinical combina- cacy of TTFields in NSCLC has been shown in preclinical models, and tions of anti-SEMA4D with various immunotherapies enhanced T cell safety in combination with pemetrexed in a pilot study. In the Phase infiltration and activity, as well as durable tumor regression. 3 LUNAR study [NCT02973789], we investigated if the addition of Methods TTFields to immune checkpoint inhibitors or docetaxel increases The CLASSICAL-Lung clinical trial evaluates the combination of overall survival (OS). pepinemab with anti-PD-L1 antibody avelumab to couple benefi- Methods cial modifications of the immune microenvironment via pepine- Trial Design: mab with immune activation via checkpoint inhibition. This Patients (N=534), with squamous or non-squamous NSCLC, are strati- ongoing phase 1b/2, open label, single arm, first-in-human com- fied by their selected standard therapy (immune checkpoint inhibitors bination study is designed to evaluate the safety, tolerability and or docetaxel), histology and geographical region. Key inclusion criteria efficacy of the combination in patients with advanced (stage IIIB/ are disease progression, ECOG 0-2, no electronic medical devices in the IV) NSCLC, including a dose escalation cohort and expansion co- upper torso, and absence of brain metastasis. TTFields (150 kHz) are ap- horts consisting of 1) 17 immunotherapy-naïve patients and 2) 33 plied to the upper torso for at >18 hours/day until progression in the patients whose tumors progressed during or following immuno- thorax and/or liver. The primary endpoint is superiority in OS between therapy (IO failure). patients treated with TTFields in combination with the standard of care Results treatments versus standard of care treatments alone. Key secondary The combination was well tolerated with no concerning safety sig- endpoints compare the OS in patients treated with TTFields and doce- nals identified to date. No patient experienced a treatment-related taxel versus docetaxel alone, and patients treated with TTFields and im- adverse event leading to permanent treatment discontinuation or mune checkpoint inhibitors vs those treated with immune checkpoint death and the most frequent related AEs were grades 1 or 2 fatigue, inhibitors alone. An exploratory analysis will test non-inferiority of pyrexia, or chills. Interim analysis focused on the IO failure cohort TTFields with docetaxel compared to checkpoint inhibitors alone. Sec- which included 22 evaluable patients. Two patients experienced a ondary endpoints include progression-free survival, radiological re- partial response (PR) with 49% and 37% tumor reduction on study sponse rate, quality of life based on the EORTC QLQ C30 questionnaire. following acquired resistance to prior treatment with pembrolizu- The sample size is powered to detect a HR of 0.75 in TTFields-treated mab. In addition, stable disease of at least 8 weeks was observed in patients versus control group. In January 2019, an independent Data 11 patients and 4 patients have remained on study for ≥20 weeks. Monitoring Committee (DMC) performed a review of the LUNAR trial Analysis of pre- and on-treatment lung biopsies demonstrated no or data collected to that point. The DMC concluded that no unexpected low tumor burden detected in 2 patients with PR, and interestingly safety issues could be found in patients treated with the combination no detectable tumor was observed in the biopsies from 3 of 4 pa- of immune checkpoint inhibitors and TTFields, and recommended to tients with stable disease. continue the LUNAR study as planned. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 228 of 272 Trial Registration P417 NCT02973789 ATLAS™ identifies relevant neoantigens for therapeutic anti-tumor vaccination and may serve as a biomarker for efficacy of immunotherapy of solid tumors P416 Parul Agnihotri, Tulin Dadali, Parul Agnihotri, PhD A phase 1 dose escalation with expansion study to evaluate the Genocea Biosciences Inc, Cambridge, MA, United States safety, tolerability, pharmacokinetics, and efficacy of AMV564 in Correspondence: Tulin Dadali (tulin.dadali@genocea.com) subjects with advanced solid tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P417 1 1 2 Raghad Abdul Kairm, MD , Anthony Tolcher , Victoria Smith , Sterling 2 2 2 Eckard, PhD , Jeanmarie Guenot , Patrick Chun Background NEXT Oncology and Texas Oncology, San Antonio, TX, United States; Mutation-derived neoantigen cancer vaccines are promising as Amphivena Therapeutics, Inc., South San Francisco, CA, United States next generation cancer therapies. However, the success of vaccin- Correspondence: Patrick Chun (pchun@amphivena.com) ation is dependent on the ability to identify the right neoanti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P416 gens for vaccine inclusion, which remains a critical challenge. Computationally-identified neoantigens do not necessarily gener- Background ate immunogenic responses. Recently, we reported interim im- Overcoming the suppressive tumor microenvironment is a major munogenicity results from the ongoing GEN-009 personalized challenge in immune therapy. The critical cellular effectors of immunotherapy Phase 1/2a clinical trial (NCT03633110). For GEN- the suppressive tumor microenvironment are the myeloid- 009, ATLAS, an ex vivo, cell-based assay selects neoantigens for derived suppressor cells (MDSC). MDSC are elevated in both the vaccine inclusion based on a patient’s own pre-existing T cell re- tumor microenvironment and periphery in cancer patients and sponses. The interim results revealed that vaccination elicited T are associated with immune dysfunction, repression of anti- cell responses to over 98% of administered peptides. Here, we tumor immunity and poor response to immunotherapy. MDSC explore the relationship between ATLAS readouts and immuno- secrete a variety of immunosuppressive factors that directly in- genicity outcomes in the same subjects. hibit both the cytolytic activity and proliferative capacity of Methods anti-tumor T cells. AMV564 is a bivalent, bispecific antibody that Antigens were profiled by expressing each mutation, identified by engages both CD3 and CD33. Preferential binding of AMV564 to whole exome sequencing, as individual clones in E. coli which regions of high CD33 density enables the selective targeting of are subsequently processed by each subject’sown antigenpre- MDSC. Data from both ex vivo studies [1] and an ongoing clin- senting cells and presented to autologous CD4+ or CD8+ T cells. ical trial in acute myeloid leukemia (AML) support the ability of Antigen-specific responses were determined based on cytokine AMV564 to selectively deplete monocytic and granulocytic secretion in the supernatant after overnight incubation. GEN-009, MDSC while sparing monocytes and neutrophils. composed of 4 pools of 1-5 unique ATLAS-identified neoantigen- Methods specific peptides combined with Hiltonol® was administered to AMV564-301 is an open label, phase 1, multicenter, dose- each subject. Both ex vivo and ten day in vitro stimulated Fluoro- escalation with expansion trial of AMV564 in patients with ad- Spot assays were performed on unsorted PBMC and CD4- and vanced solid tumors for which no recognized standard curative CD8-sorted T cells at baseline and 50 days post vaccination to therapy options are available. The key objectives of the dose- identify peptides to which T cells from the vaccinated patients escalation stage of the study are to characterize the safety and responded. tolerability of AMV564 and identify a maximum tolerated dose Results (MTD) or a recommended phase 2 dose (RP2D) for further study. In the first cohort of six patients, ATLAS identified neoantigens In the dose expansion stage of the study, the safety and toler- by recalling both stimulatory and inhibitory neoantigen-specific T ability of AMV564 will be further characterized in addition to cell responses. One subject, who had a greater proportion of in- evaluating the preliminary efficacy of AMV564. Other objectives hibitory to stimulatory responses detected, progressed prior to include characterization of AMV564 pharmacokinetics, pharmaco- vaccination while no vaccinated patients have experienced pro- dynamics, and immunogenic potential. gressive disease. Compared to NetMHCPan results, more than half Approximately 90 patients with locally advanced or metastatic of the ATLAS-identified neoantigens were not predicted. More- solid tumors will be enrolled. The Dose Escalation Stage will in- over, the predicted epitopes did not result in better immunogen- clude up to approximately 40 patients, depending on the dose icity outcomes post-vaccination than the non-predicted ATLAS- at which the MTD/RP2D is determined, and approximately 50 identified neoantigens. Comprehensively profiling T cell responses additional patients will be enrolled in the Expansion Stage. over time shows consistency of results in patients with no evi- AMV564 will be administered once daily as a subcutaneous in- dence of disease. jection for 14 days in each 21-day cycle. Patients will be treated Conclusions until disease progression, unacceptable toxicity, or withdrawal Neoantigens selected by immune response data from ATLAS and of consent. included in the GEN-009 vaccine were immunogenic and many Trial Registration were not algorithm-predicted, confirming that ATLAS identifies NCT pending relevant neoantigens. ATLAS will be useful for profiling epitope spread in tumor-bearing subjects post-vaccination. The proportion Reference of inhibitory to stimulatory neoantigen-specific responses may be 1. Cheng P, Eksioglu E, Chen X, Wei M, Guenot J, Fox J, List A, Wei S. a biomarker of immunotherapy success. Combination of GEN-009 Immunodepletion of MDSC by AMV564, a novel tetravalent bispecific with standard-of-care checkpoint blockade therapy is currently CD33/CD3 T cell engager restores immune homeostasis in MDS in vitro. ongoing. Blood. 2017;130:51. Trial Registration Ethics Approval ClinicalTrials.gov NCT03633110 This study will be approved by the Institutional Review Board (IRB) or Ethics Approval Independent Ethics Committee (IEC) at each participating institution The study was approved by Western Institutional Review Board, ap- prior to patient enrollment. proval number 1-1078861-1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 229 of 272 P418 paired peripheral blood analysis revealed a treatment-related in- Intratumoral IL-12 plus pembrolizumab combination therapy in crease of KLRG1+/CD127- SLECs as well as a treatment-related reduc- treatment refractory solid tumors: a safety and biomarker analysis tion of MDSCs in the periphery predominantly in responding Pablo Fernandez-Penas, MD, PhD , Matteo Carlino, MBBS, PhD, BMedSC, patients. 1 2 3 4 5 F , Victoria Atkinson, MD , Melinda Telli , Rohit Joshi , Sajev Thomas , Conclusions 6 4 7 Katy Tsai, MD , Rachel Roberts-Thomson , Andrew Haydon, MBBS PhD , TAVO + pembrolizumab continues to be well tolerated in patients 8 9 10 11 Andrew Mant , Tom Van Hagen , Katharine Cuff , Bianca Devitt , Igor with advanced solid tumors and peripheral blood analyses demon- 12 13 14 Puzanov, MD, MSCI, FACP , Marcus Butler, MD , Catalin Mihalcioiu , strates both local and most importantly, systemic signals of IL-12 me- 15 16 17 Hatem Soliman, MD , John Hyngstrom, MD , Mecker Moller , Gregory diated anti-tumor immunity in the absence of clinical signs of 18 19 20 Daniels, MD, PhD , Eric Whitman, MD, FACS , Erica Browning, BS , systemic IL-12 exposure. Thus, TAVO acts as an in situ vaccine to po- 20 20 20 20 Reneta Hermiz , Lauren Svenson , Jack Lee , Donna Bannavong , tentiate the anti-tumor activity of pembrolizumab with a favorable 20 20 20 20 Jendy Sell , Kellie Malloy , David Canton, PhD , Christopher Twitty , toxicity profile. 6 6 Adil Daud, MBBS MD , Alain Algazi, MD Trial Registration 1 2 Westmead Hospital, University of Sydney, Westmead, Australia; Princess NCT03132675; NCT03567720 Alexandra Hospital, University of Queensland, Woolloongabba, Australia; Ethics Approval Stanford University Medical School, Stanford, CA, United States; These studies were approved by the appropriate ethics committees. 4 5 Adelaide Oncology and Haematology, Adelaide SA, Australia; UF Consent Health Cancer Center, Orlando Health, Orlando, FL, United States; Written informed consent was obtained from the patient for publica- University of California San Francisco, San Francisco, CA, United States; tion of this abstract and any accompanying images. A copy of the 7 8 The Alfred Hospital, Melbourne, Australia; Box Hill Hospital, Box Hill, written consent is available for review. 9 10 Victoria, Australia; St. John of God Hospital, Subiaco, Australia; Princess Alexandra Hospital, Woolloongabba QLD, Australia; Eastern Health P419 Clinical School, Box Hill, VIC, Australia; Roswell Park Cancer Institute, A phase 1/2 study of GB1275, a novel CD11b modulator, as Buffalo, NY, United States; Princess Margaret Cancer Centre, Toronto, monotherapy and with an anti-PD-1 antibody in specified Canada; McGill University Health Centre, Montreal, QC, Canada; 15 16 advanced solid tumors or with chemotherapy in metastatic Moffitt Cancer Center, Tampa, FL, United States; Huntsman Cancer pancreatic cancer (mPDAC) Institute and Hospital, Salt Lake City, UT, United States; Sylvester 1 2 3 Johanna Bendell , Drew Rasco, MD , Wungki Park, MD , Lei Zhou, MD, Comprehensive Cancer Center, University of Miami, Miami, FL, United 18 4 4 4 4 MS , Anna Galkin, PhD , Debbie Slee, PhD , Laura Carter, PhD , David States; University of California San Diego, La Jolla, CA, United States; 19 20 4 4 4 4 Nickle, PhD , Rebecca Tran, MS , Jack Li, PhD , Beatrice Ferguson, MS , Atlantic Health System, Morristown, NJ, United States; OncoSec 4 5 3 Jakob Dupont, MD , Vineet Gupta, PhD , Eileen O'Reilly Medical Inc., San Diego, CA, United States 1 2 Sarah Cannon Research Institute, Nashville, TN, United States; The Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) START Center for Cancer Care, San Antonio, TX, United States; Memorial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P418 Sloan Kettering Cancer Center, New York, NY, United States; Gossamer Bio, San Diego, CA, United States; Rush University Medical Center, Background Chicago, IL, United States Intratumoral inflammation, including IL-12 expression and intratu- Correspondence: Johanna Bendell (ttobore@samornbiosciences.com) moral T cell infiltration, is a prerequisite for response to anti-PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P419 therapies. Previously, we demonstrated that enhanced intratumoral IL-12 expression via injection of plasmid IL-12 (tavokinogene telse- Background plasmid; TAVO) followed by electroporation (IT-tavo-EP) can increase Tumor influx of CD11b-expressing Myeloid-Derived Suppressor Cells TIL infiltration, ratios of CD8+ T cell:suppressive immune subsets, and (MDSCs) and M2 Tumor-Associated Macrophages (TAMs) creates an IFN-gamma gene signatures, converting weakly immunogenic tumors immunosuppressive tumor microenvironment that is associated with into highly inflamed, immunologically active lesions that regress with resistance to anti-PD-1 antibody therapy [1, 2, 3]. GB1275 is a novel, anti-PD-1 antibody therapy. Here, we present further support for our first-in-class, CD11b modulator that in vivo led to reduced MDSCs hypothesis that local IT-tavo-EP induces local and systemic immune and TAMs at the tumor site, repolarized M2 immuno-suppressive modulation with minimal systemic toxicity. TAMs towards an M1 phenotype, and subsequently increased tumor Methods infiltration of activated CD8+ T cells [4]. In combination settings with Melanoma (KEYNOTE-695) and mTNBC (KEYNOTE-890) patients were an anti-PD-1 antibody or chemotherapy, these immunomodulatory treated every three weeks with IT-tavo-EP on days 1, 5, and 8 of effects translated into potent anti-tumor effects and prolonged sur- every odd numbered cycle. Clinical toxicity was assessed at 3-week vival in orthotopic PDAC models [4]. We hypothesize that GB1275 ad- intervals and graded by CTCAE v4. In addition, pre- and post- ministration can alleviate myeloid cell-mediated immunosuppressive treatment tumor biopsies and peripheral blood samples were interro- effects and improve cancer treatment outcomes. gated for treatment-related changes in the frequency of CD8+ TIL Methods and other key IL-12-driven peripheral immune cell populations. In This is an open-label, first-in-human study consisting of a Phase 1 particular, we examined circulating short-lived effector T cells (SLECs, Dose Escalation phase with Regimen A using GB1275 monotherapy KLRG1+/CD127-), which are induced by IL-12 exposure, and granulo- and Regimen B using GB1275 with an anti-PD-1 antibody in pts with cytic myeloid derived suppressor cells (gMDSCs or PMN-MDSCs), pancreatic, esophageal, gastric/GEJ, triple negative breast, castration which serve a regulatory function, inhibiting effective anti-tumor im- resistant prostate, or Microsatellite Stable Colorectal Cancer (MSS mune responses. CRC) and Regimen C (GB1275 with Nab-paclitaxel + Gemcitabine Results (Nab-P+Gem)) in mPDAC, followed by a Phase 2 Expansion phase 62 patients were assessed including 46 patients with anti-PD-1 with three cohorts planned: newly diagnosed stage IV mPDAC, MSS antibody-refractory melanoma, and 16 patients with chemotherapy- CRC and PD-L1+ gastric/GEJ cancer. The study starts with Regimen A refractory mTNBC. TAVO in combination with pembrolizumab was with Regimen B starting after the completion of the first few cohorts well tolerated with only 2 of 46 (4.3%) patients from KEYNOTE-695 of Regimen A. Regimen C will start when Regimen A is completed. (cellulitis and presyncope) and 1 of 16 (6.3%) from KEYNOTE-890 Key Inclusion Criteria: Age ≥18 years, histologically confirmed locally (acute renal failure) experiencing grade 3 treatment-related adverse advanced/metastatic tumor specified, ECOG 0-1, prior immunother- events (TRAEs). Paired biopsies were available from both advanced apy is permissible in Dose Escalation phase for Regimen A and B, but melanoma patients and mTNBC patients. Flow cytometry on fresh bi- not for the expansion or Regimen C. Key Exclusion Criteria: untreated opsies from the KEYNOTE-695 revealed significant increases in CD8+ or symptomatic CNS metastasis, received prior myeloid targeting T cells after 1 cycle of treatment. Despite previous data demonstrat- agent or other prohibited medications, history of clinical significant ing non-detectable circulating IL-12 levels after treatment with TAVO, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 230 of 272 cardiovascular disease. Pts with active autoimmune disease requiring consistent with those expected from the poly-ICLC adjuvant alone, systemic therapy will be excluded from Regimen B. Primary objec- and no DLTs. ATLAS results show high interpatient variability as de- tives for the Dose Escalation phase are to determine the MTD/RP2D scribed previously. In an interim analysis of patients, vaccination has and PK profile of GB1275 monotherapy and in combination with an generated both CD8 and CD4 T cell responses measured by ex vivo anti-PD-1 antibody, and safety in combination with Nab-P+Gem. The fluorospot (Table 1). Ten-day in vitro stimulation (IVS) fluorospot as- primary objective for the Basket Expansion phase is to assess says confirm even broader immune responses. Overall, T cell re- efficacy. sponses were measured to 98% of administered peptides. Statistical Considerations: 3+3 design for the Dose Escalation Phase Conclusions and Simon’s 2-stage design for Expansion Phase. AEs graded per GEN-009 is a neoantigen vaccine that targets tumor specific immune CTCAE v5.0, responses per RECIST v1.1. The study is open for recruit- antigens recognized by the individual patient’s lymphocytes and ment and clinical trial registration on clinicaltrials.gov is pending likely expressed by tumor cells. Immunogenicity data show that (NCTxxxxx). ATLAS can, with very high frequency, identify relevant neoantigens and exclude putatively deleterious (immune inhibitory) antigens. References Clinical vaccination together with Standard of Care PD-1 blockade- 1. Fleming, V., Hu, X., Weber, R., Nagibin, V., Groth, C., Altevogt, P., et al. based regimens is in progress. Targeting myeloid-derived suppressor cells to bypass tumor-Induced im- Trial Registration munosuppression. Front Immunol. 2018; 9: 398. ClinicalTrials.gov NCT03633110 2. Kumar, V., Patel, S., Tcyganov, E. and Gabrilovich, D. I. The nature of Ethics Approval myeloid-derived suppressor cells in the tumor microenvironment. Trends The study was approved by Western Institututional Review Board, ap- Immunol. 2016; 37(3): 208-220. proval number 1-1078861-1. 3. Mantovani, A., Sozzani, S., Locati, M., Allavena, P. and Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized Table 1 (abstract P420). See text for description M2 mononuclear phagocytes. Trends Immunol. 2002; 23(11): 549-555. 4. Panni R., Herndon J., Zuo C., et al. Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. Sci Transl Med. 2019; 11: eaau9240. Ethics Approval The study was approved by the local IRB at each participating study site. P420 Broad immunogenicity from GEN-009, a neoantigen vaccine using ATLAS™, an autologous immune assay, to identify immunogenic and inhibitory tumor neoantigens 1 2 3 Roger Cohen, MD , Melissa Johnson, MD , Przemyslaw Twardowski, MD , P421 4 5 6 Mark Stein, MD , Ulka Vaishampayan, MD , Maura Gillison, MD, PhD , Lisa Phase 1 study of the safety, tolerability and preliminary anti-tumor 7 7 7 McNeil, PhD , Louisa Dowal, PhD , James Foti, PhD , Parul Agnihotri, activity of COM701 monotherapy in patients with advanced solid 7 7 7 7 PhD , Daniel DeOliveira, PhD , Manish Jain, MS , Jessica Price , Richard tumors 7 7 7 1 2 3 Hernandez , Arthur DeCillis, MD , Narinderjeet Singh, MS, MBA , Thomas Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD , Ryan 7 7 4 5 5 Davis, MD , Jessica Flechtner, PhD Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 1 2 6 7 7 University of Pennsylvania, Philadelphia, PA, United States; Sarah Adam ElNaggar, MD , John Hunter, PhD , Judy Olweny , Adeboye 3 7 8 9 Cannon Research Institute, Nashville, TN, United States; John Wayne Adewoye, MD , Bartosz Chmielowski, MD, PhD , Dale Shepard, MD PhD , 4 10 11 6 Cancer Institute, Duarte, CA, United States; Columbia University Medical Manish Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD , Drew 5 5 Center, New York, NY, United States; Karmanos Cancer Institute, Detroit, Rasco, MD 6 1 2 MI, United States; MD Anderson Cancer Center, Houston, TX, United The MD Anderson Cancer Center., Houston, TX, United States; The 7 3 States; Genocea, Cambridge, MA, United States University of Chicago, Chicago, IL, United States; Sarah Cannon Correspondence: Thomas Davis (tom.davis@genocea.com) Research Institute/TN Oncology, Nashville, TN, United States; 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P420 Massachusetts General Hospital, Needham, MA, United States; The START Center for Cancer Care, San Antonio, TX, United States; West Background Cancer Center and Research Institute, Memphis, TN, United States; 7 8 Tumor-specific neoantigens provide personalized targets for im- Compugen USA Inc, South San Francisco, CA, United States; University munotherapy. Vaccines against epitopes predicted by in silico ap- of California Los Angeles, Los Angeles, CA, United States; Cleveland proaches very rarely induce CD4+ and CD8+ ex vivo T cell responses Clinic, Cleveland, OH, United States; START - Midwest Cancer Center, regardless of formulation. ATLAS selects neoantigens for vaccine in- Chicago, IL, United States; Columbia University Medical Center, New clusion using ex vivo screening of all patient-specific mutations to York City, NY, United States identify pre-existing CD4+ or CD8+ T cell responses and to exclude Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) inhibitory peptides that may suppress immunity and potentially ac- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P421 celerate tumor progression. Preliminary data suggest that the inhibi- tory peptide profile may predict tumor response to immunotherapy. Background Methods COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of GEN-009-101 is a phase 1/2a study testing safety, immunogenicity poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It in- and clinical activity in immune responsive tumors (NCT03633110). hibits the binding of PVRIG with its ligand, PVRL2. PVRIG is a member After next-generation tumor sequencing and ATLAS testing of au- of the DNAM/TIGIT signaling axis regulating the activity of T/NK-cells. tologous leukocytes, each personalized vaccine is created using up In preclinical experiments we have demonstrated that PVRIG inhib- to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC. ition alone and in combination with anti-PD-1 and/or TIGIT blockers The immunogenicity pilot includes 8 patients in remission (NED), leads to activation of T cells in the tumor microenvironment generat- who received a course of GEN-009 monotherapy. ing an anti-tumor immune response and tumor growth inhibition [1]. Results Although ICI revolutionized cancer treatment, there is an urgent Eight patients have participated and reached the primary immuno- need to develop treatments for patients who are refractory or relapse genicity readout at day 50 (some data pending). The 24 doses given after treatment with ICI. We hypothesized that COM701 will be safe, across all patients have induced only grade 1/2 adverse events tolerable and demonstrate preliminary anti-tumor activity. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 231 of 272 Methods inhibits the binding of PVRIG with its ligand, PVRL2. Nivolumab is an A phase 1a, dose-escalation of COM701 monotherapy utilizing a hybrid anti-PD-1 antibody approved in patients with several malignancies accelerated and 3+3 study design was conducted to determine safety, [2]. PVRIG is a member of the DNAM/TIGIT signaling axis regulating tolerability, to assess the pharmacokinetics (PK), pharmacodynamics, to the activity of T/NK-cells. PD-1 inhibitors play an important role in determine the recommended phase 2 dose and to evaluate preliminary this axis by modulating DNAM activation [3]. In preclinical experi- anti-tumor activity of COM701. Patients with performance status ECOG 0- ments we have demonstrated that PVRIG inhibition alone and in 1 and advanced solid tumors who failed standard of care treatments combination with anti-PD-1 leads to activation of T cells in the tumor were eligible for inclusion. Prior ICIs were permissible. COM701 0.01, 0.03, microenvironment generating an anti-tumor immune response and 0.1, 0.3, 1, 3 and 10 mg/kg IV every 3 weeks were administered until pro- tumor growth inhibition [1]. Although ICI revolutionized cancer treat- gression, intolerable toxicity or investigator or patient discretion. Adverse ment there is an urgent need to develop treatments for patients events were reported per CTCAE v4.03 and anti-tumor activity was evalu- who are refractory or relapse after treatment with ICI. We hypothe- ated using RECIST v1.1. Dose-limiting toxicities (DLTs) were evaluated sized that COM701 will be safe and tolerable and demonstrate pre- within a 21-day window. Data cutoff date was August 09, 2019. liminary antitumor activity as monotherapy and in combination with Results nivolumab in patients with advanced solid tumors. A total of 13 patients were enrolled and treated during dose escalation Methods of COM701, including 6 patients with metastatic colorectal cancer (CRC), This is a phase 1 study with single patient cohorts and 3+3 study design 5 with microsatellite stable status (MSS) and 1 unknown. Patients were of COM701 in escalating doses as monotherapy IV Q3 weeks and in com- heavily pretreated with a median of 7 prior anticancer therapies (range 2- bination with nivolumab 360mg IV Q3 weeks. Key Inclusion Criteria: Age 15). No DLTs have been reported up to 10 mg/kg COM701 dose level. ≥18 years, histologically confirmed advanced solid tumor, performance The most frequent toxicities were fatigue (8%), abdominal pain (6%). status ECOG 0-1, prior anti-PD-1, anti-PD-L1, anti-CTLA-4, OX-40, CD137 Likely immune-related adverse events: elevated TSH and rash were ob- treatments are permissible. Key Exclusion Criteria: Active autoimmune dis- served in 2 patients. Overall 7/13 patients (54%) maintained best re- ease requiring systemic therapy in the last 2 years, symptomatic intersti- sponse of stable disease (SD) ≥12 weeks (13.6 – 43 weeks), including 5/6 tial or inflammatory lung disease, untreated or symptomatic central (83%) of patients with CRC. Five patients continue on study treatment. nervous system metastases. Primary objectives: to evaluate the safety Peripheral PVRIG receptor occupancy (≥90%) was demonstrated at and tolerability of COM701 monotherapy and in combination with nivo- ≥1mg/kg dose of COM701 and PK profile supports Q3 weekly dosing. lumab measured by the incidence of adverse events and dose-limiting Conclusions toxicities (21-day window), to evaluate the pharmacokinetics of COM701, COM701 monotherapy demonstrates an acceptable safety and toler- and to identify the maximum tolerated dose and/or the recommended ability profile with preliminary anti-tumor activity in a patient popula- dose for expansion as monotherapy and in combination with nivolumab. tion that had received multiple prior anti-cancer therapies. Updated Secondary objectives: to characterize the immunogenicity and prelimin- data will be presented at the conference. ary antitumor activity of COM701 in combination with nivolumab. Statis- Trial Registration tical Considerations: AEs will be reported as per CTCAE v4.03 and tumor Clinical trial identification: NCT03667716. responses will be evaluated per RECIST v1.1. Analyses of objectives are descriptive and hypothesis generating. Reference Results 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody At the time of submission no DLTs have been observed up to dose targeting the novel immune checkpoint PVRIG, for the treatment of level 7 of COM701 monotherapy and dose level 1 of COM701 in cancer. J Clin Oncol. 2017; (suppl; abstr 3074) combination with nivolumab 360mg IV Q3 weeks. Ethics Approval Conclusions The study was approved by each site's ethics board. Assessment of safety and tolerability is ongoing for all patients. Up- Consent dated results will be presented at the congress. Written informed consent was obtained from the patient for publication of Trial Registration this abstract and any accompanying images. A copy of the written Clinical trial identification: NCT03667716. consent is available for review by the Editor of this journal. References 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody P422 targeting the novel immune checkpoint PVRIG, for the treatment of Phase 1 study of COM701 monotherapy and in combination with cancer. J Clin Oncol. 2017; (suppl; abstr 3074) nivolumab in patients with advanced solid tumors 2. Nivolumab package insert. http://packageinserts.bms.com/pi/pi_opdivo.pdf. 1 2 3 Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD ,Ryan Accessed 07/22/2019. 4 5 5 Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 3. Wang B, Zhang W et al., Combination cancer immunotherapy targeting 6 7 7 Adam ElNaggar, MD ,JohnHunter, PhD ,JudyOlweny , Adewoye Adewoye, PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory 7 8 9 MD , Bartosz Chmielowski, MD, PhD ,DaleShepard,MDPhD ,Manish phenotype. Sci. Immunol. 2018; Nov 2:3(29). 10 11 6 5 Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD ,DrewRasco,MD Ethics Approval The University of Texas MD Anderson Cancer Center, Houston, TX, The study was approved by the Investigational Review Board/Ethics United States; The University of Chicago, Chicago, IL, United States; Committee of the participating sites. Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United States; Massachusetts General Hospital, Harvard Medical School, Needham, MA, United States; The START Center for Cancer Care, San P423 Antonio, TX, United States; West Cancer Center and Research Institute, SURPASS trial design: A phase 1 dose escalation trial to assess Memphis, TN, United States; Compugen USA Inc, South San Francisco, safety and efficacy of ADP-A2M4CD8 in HLA-A2+ patients with CA, United States; University of California Los Angeles, Los Angeles, CA, MAGE-A4+ tumors 9 10 2 3 4 United States; Cleveland Clinic, Cleveland, OH, United States; The David Hong, MD , Marcus Butler , Melissa Johnson, MD , Tanner 5 1 1 START-Midwest Center for Cancer Care, Chicago, IL, United States; Johanns , Francine Brophy , Rebecca Dryer-Minnerly, PhD , Trupti 11 1 1 1 Columbia University Medical Center, New York City, NY, United States Trivedi, MS , Rafael Amado, MD , Paula Fracasso, MD, PhD 1 2 Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) Adaptimmune, Philadelphia, PA, United States; MD Anderson Cancer Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P422 Center, Houston, TX, United States; Princess Margaret Cancer Centre, Toronto, Canada; Sarah Cannon, Nashville, TN, United States; Background Washington University in St. Louis, St. Louis, MO, United States COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of Correspondence: Paula Fracasso (paula.fracasso@adaptimmune.com) poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P423 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 232 of 272 Background tumors are presented. The study (NCT03548467) was approved by ADP-A2M4CD8 specific peptide enhanced affinity receptor (SPEAR) T- Central Ethics Committee in Heidelberg, Germany. cells are genetically engineered to target MAGE-A4+ tumors in the Methods context of HLA-A*02. ADP-A2M4CD8 are autologous CD4+ and CD8+ Patients with melanoma, NSCLC, clear RCC, urothelial cancer or SCCHN T-cells that express a high affinity MAGE-A4-specific T-cell receptor who did not reach complete responses after >12 weeks of immune (TCR) and an additional CD8α co-receptor. The ADP-A2M4 TCR is be- checkpoint inhibitor (CPI) therapy as standard of care were eligible. ing explored in a pilot study (NCT03132922), where clinical responses After patient-specific vaccine production, patients receive up to 14 vac- have been observed and the TCR has been well tolerated in doses cinations as intramuscular jet injections over a one-year period. CPI up to 10 × 10^9 transduced cells. Because CD4+ T-cells have a weak treatment continued. CT/MRI scans were performed according to hospi- effector function in response to class I antigens, a CD8α co-receptor tals’ routine. Immune responses were assessed by IFN-γ ELISpot. was genetically engineered alongside the TCR in ADP-A2M4CD8, to Results increase TCR binding avidity and enhance the polyfunctional re- At July 22, 2019 data cut-off, 15 patients (9 RCC, 4 SCCHN, 1 Melan- sponse of engineered CD4+ T-cells against MAGE-A4+ tumors. This oma, 1 NSCLC) had received ≤ 11 VB10.NEO vaccinations. Most com- approach is intended to widen the immune response to the tumor mon AEs were injection site reactions which all subsided within days. and improve depth and durability of clinical responses. 6 patients reported ≥ Grade 3 AEs, the most frequent ones were Methods injection-related hypertensive episodes normalizing within hours. In This phase 1, dose-escalation, open-label trial (SURPASS Trial) will the 4 patients assessed with low TMB (2 RCC, 2 SCCHN) strong T-cell characterize safety, tolerability, and antitumor activity across multiple responses towards 63% of selected neoepitopes were observed after tumor types. Patients who are HLA-A*02+ with MAGE-A4+ advanced 3-6 vaccinations. An amplification of existing neoepitope-specific T- esophageal, esophagogastric junction, gastric, head and neck cancers, cells (average of 250-fold increase) as well as de novo responses non-small cell lung, ovarian and urothelial carcinoma, melanoma, myx- were observed suggesting that VB10.NEO increases both the breadth oid/round cell liposarcoma, or synovial sarcoma and who meet all other and strength of the immune responses. inclusion criteria are eligible. Up to 30 patients will be enrolled. 10 patients were evaluable with >1 scan after VB10.NEO start (after Following apheresis, T-cells are isolated, transduced with CD8α_- being on CPI for 9-32 months). 4 patients (3 low TMB, 1 medium MAGE-A4c1032TCR, and expanded. Prior to ADP-A2M4CD8 infusion, TMB) started VB10.NEO with progressive disease (PD) development, patients will receive lymphodepletion consisting of fludarabine (30 of which 3 showed as stable disease (SD) in target lesions after vac- mg/m2/day x 4 days) and cyclophosphamide (600 mg/m2/day x 3 cination (followed up to 7 months), one developed PD after 5 days). During dose escalation, patients will be treated in one of three months. New lesions were detected in 2 patients. 6 patients (5 low ADP-A2M4CD8 dose groups. The initial dose of ADP-A2M4CD8 will TMB, 1 medium TMB) had SD at VB10.NEO start, 5 remained SD be 0.8 × 10^9 - 1.2 × 10^9 to be escalated to 1.2 × 10^9 - 3 × 10^9 (followed up to 9 months), while one had a best target lesion reduc- and then to 3.0 × 10^9 - 6.0 × 10^9 transduced cells in a modified 3 tion of 40%. Updated data will be presented. + 3 dose escalation scheme. Patients will be monitored for dose- Conclusions limiting toxicities (DLTs). Once the tolerability and safety of the lym- Vaccinations with VB10.NEO in addition to CPI were well tolerated. phodepletion regimen and cell dose has been demonstrated, the VB10.NEO induces strong T cell responses towards personalized dose range will increase to a maximum of 10 × 10^9 transduced neoepitopes; both novel T cell specificities and amplification of pre- cells in the expansion phase. Disease will be assessed per RECIST existing T cell responses were observed. Clinical signs of effect on v1.1 by CT/MRI at weeks 4, 8, 16, and 24, and every 3 months for 2 tumor size will continuously be monitored in the trial and early signs years, then every 6 months up to 15 years or until progression. Blood are promising. and tumor biopsy samples will be obtained pre- and post-infusion to Trial Registration evaluate safety, monitor persistence of transduced T-cells, and iden- NCT03548467 tify tumor-intrinsic correlates of response or resistance to therapy. Ethics Approval Trial Registration The study was approved by Central Ethics Committee in Heidelberg. NCT04044859 Ethics Approval P425 This trial is under review by the local institutional review boards for Phase 1b study of GX-I7, a long-acting interleukin-7, evaluating the each proposed study center. safety, pharmacokinetics and pharmacodynamics profiles in patients with advanced solid cancers 1 1 2 P424 Minkyu Heo , Minkyu Heo , Tae Won Kim, MD, PhD 1 2 Preliminary safety, efficacy and immunogenicity results from a Genexine, Inc, New York, NY, United States; Asan Medical Center, phase 1/2a study (DIRECT-01) of cancer neoantigen DNA vaccine Seoul, Korea VB10.NEO in patients with locally advanced or metastatic solid Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P425 1 2 3 1 Jürgen Krauss , Angela Krackhardt , Elke Jaeger , Anja Williams , Reza 3 4 4 4 Rafiyan , HEDDA WOLD, MSc , LIsa Gerner , Monika Sekelja , Agnete Background 4 4 4 Fredriksen, PhD , Karoline Schjetne , Mads Axelsen , Agnete Fredriksen, Cancer and treatment-related lymphopenia is associated with 4 4 PhD , Hedda Wold, MSc higher mortality in patients with various oncologic malignancies. 1 2 NCT, University Hospital Heidelberg, Heidelberg, Germany; Klinicum Interleukin-7(IL-7), a homeostatic cytokine of T lymphocytes, plays rechts der Isar, TUM, Munich, Germany; Krankenhaus Nordwest, a critical and non-redundant role in T cell development and Frankfurt, Germany; Vaccibody AS, OSLO, Norway homeostasis of mature T lymphocytes. IL-7 is a potent amplifier Correspondence: Hedda Wold (hwold@vaccibody.com) of naïve and memory T cells, thereby correcting T cell deficiency Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P424 and contributing to immune reconstitution. This may result in sig- nificant clinical benefit when combined with lymphopenia- Background inducing radiation/chemotherapy or immunotherapy where anti- Generation of potent neoantigen-specific T-cell responses has shown tumor effects are mediated by T cells. promising preclinical efficacy as well as clinical responses, especially Methods in patients with high tumor mutational burden (TMB). VB10.NEO is a A phase 1b study was conducted to assess the safety, pharmacokin- DNA vaccine with intrinsic adjuvant designed for delivery of 20 per- etics and pharmacodynamics of single-agent GX-I7(human IL-7 fused sonalized neoepitopes to antigen presenting cells. Preliminary results to the half-life extension hyFcTM) administered intramuscularly q3w from the ongoing phase 1/2a study treating patients with solid to advanced solid cancer patients who have no available effective Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 233 of 272 treatments (n=21). The dose escalation phase followed the 3+3 de- (NCT03416335). Primary objectives are to evaluate DSP-0509 safety/ sign of GX-I7 doses ranged from 60 to 1,200 μg/kg. Adverse events, tolerability, determine the maximum tolerated dose (Part A mono- PK, and subset analysis of peripheral blood monocytes(PBMCs) were therapy), and identify DSP-0509 monotherapy recommended phase evaluated. 2 dose (RP2D) and the RP2D of DSP-0509 in combination with pem- Results brolizumab for future studies. Secondary objectives include pharma- GX-I7 was well tolerated without DLT and cytokine release syn- cokinetics and antitumor activity. drome. Injection site reactions were the most common Methods treatment-emergent adverse events, which were Grade1 or 2 and Eligible patients are aged ≥18 years with advanced solid tumors (Part resolved. GX-I7 was slowly but steadily absorbed with a Tmax A and C) or melanoma or head and neck squamous cell carcinoma range of 12-48 hours with delay in higher doses. Following GX-I7 with acquired immune checkpoint inhibitor resistance (grouped high administration, up to 4-fold increase in absolute lymphocyte or low CD8+ cell density in tumor tissue; Part B). In Part A, approxi- count(ALC) were demonstrated. Importantly, the number of vari- mately 21–30 patients will be enrolled in each of the monotherapy ous subsets(naïve, TEM, TCM and TEMRA) of both CD4+ and and combination arms. DSP-0509 will be given as a constant rate IV CD8+ T cells was in a greater magnitude than that of ALC, infusion over 3 minutes at a fixed dose. Five provisional dose levels coupled with enhanced expression of Ki-67 peaked at day 7. of DSP-0509 may be tested, with approximately 3–6 patients at each Among T cell subsets, increase in naïve CD4+ and CD8+ T cells level (3 escalation levels at target doses of 0.3, 1, and 3 mg; 2 de- was most prominent. IL-7 receptor alpha(CD127) expression was escalation levels at target doses of 0.6 and 1.8 mg). During induction reduced during the first week, and recovered to baseline after treatment, patients will receive 5 doses of DSP-0509 every week for 4 2~3 weeks post GX-I7 administration. CCR5 expression in both weeks on days 1, 8, 15, 22, and 29 followed by every 2-weeks until CD4+ and CD8+ T cells increased transiently in a dose dependent discontinuation. In the combination arm, pembrolizumab will be ad- manner, suggesting GX-I7 promotes migration of T cell to tumor ministered IV at 200 mg every 3 weeks (Q3W). Dose limiting toxicities environment. No apparent increases in the number of other im- will be monitored within the first 6 weeks of dosing. The mono- and mune cells (NK cell, monocytes, B cells) were observed. combination DSP-0509 RP2Ds will be determined using a Bayesian Conclusions logistic regression model. In Part B, approximately 20–40 patients will A 3-week interval repeated IM administration of GX-I7 appears to receive DSP-0509 at the RP2D using the same dosing schedule as be well tolerated in dose range of 60 – 1,200 μg/kg in advanced Part A. In Part C, approximately 3–6 patients will be treated using the solid cancer patients. Following GX-I7 administration, dose- RP2D with the same induction treatment schedule as Part A, but dependent increase of ALC and T cell subsets(not Treg) were ob- followed by Q3W maintenance dosing. This study is currently recruit- served. These findings suggest that GX-I7 can be an excellent ing patients. combination partner for chemo-radiation, cancer vaccines and im- mune checkpoint inhibitors such as anti-PD-1/PD-L1 antibodies, P427 by increasing T lymphocytes and thereby contributing to en- A phase 1 study of IMC-001, novel anti-PD-L1 antibody, in patients hanced anti-tumor effects. with advanced solid tumors Trial Registration 1 1 Bhumsuk Keam, MD, PhD , Tae Min Kim, MD, PhD , Do-Youn Oh, MD, ClinicalTrials.gov Identifier: NCT03478995 1 1 2 PhD , Chan-Young Ock, MD, PhD , Won Ki Kang, MD, PhD , Yeon Hee Ethics Approval 2 2 3 Park, MD, PhD , Jeeyun Lee, MD, PhD , Ji Hye Lee, MD , Yun Jeong The study was approved by the Severance Hospital, Asan Medical 3 2 Song, MD , Young Suk Park, MD, PhD center, and Catholic Medical Center Institutional Review Board, proto- 1 2 Seoul National University Hospital, Seoul, Korea, Republic of; Samsung col number GX-I7-CA-003. Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, Republic of; ImmuneOncia Therapeutics Inc., Gyeonggi, South P426 Korea A first-in-human phase 1, multicenter trial of toll-like receptor Correspondence: Young Suk Park (pys27hmo@skku.edu) (TLR) 7 agonist DSP-0509 as monotherapy and in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P427 with pembrolizumab in adult patients with advanced solid tumors 2 3 4 Jared Weiss, MD , Anthony Olszanski, MD, RPh , Jordan Berlin, MD , Background 5 5 5 5 Makoto Origuchi , Zhonggai Li , Bella Ertik , Hongliang Cai , Daniel IMC-001 is a fully human IgG1 monoclonal antibody that binds to hu- 5 6 7 1 Clancy , Jose Iglesias , Vivek Subbiah, MD , Shadia Jalal, MD man PD-L1 and mediate the antibody-dependent cell-mediated cyto- Indiana University School of Medicine, Indianapolis, IN, United States; toxicity. The main objectives of this study were to evaluate the 2 3 UNC School of Medicine, Chapel Hill, NC, United States; Fox Chase safety, pharmacokinetics, and pharmacodynamics of IMC-001 in pa- Cancer Center, Phildelphia, PA, United States; Vanderbilt University tients with advanced solid tumors. Additional objectives were to ex- Medical Center, Nashville, TN, United States; Boston Biomedical Inc, plore the anti-tumor activity and identify the maximum tolerated Cambridge, MA, United States; Former employee, Boston Biomedical dose (MTD) of IMC-001. Inc, Cambridge, MA, United States; University of Texas, Houston, TX, Methods United States This is a phase 1, open-label study of IMC-001 in patients with meta- Correspondence: Shadia Jalal (sjalal@iu.edu) static or advanced solid tumors. IMC-001 was administered intraven- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P426 ously every two weeks with a standard 3+3 dose-escalation design until disease progression or unacceptable toxicity. Dose limiting tox- Background icity (DLT) window was defined as 21 days from the first dose. Ad- DSP-0509 is a TLR7 agonist designed to have high water solubility verse events (AEs) were assessed using CTCAE v4.03, and tumor allowing for intravenous (IV) administration and has rapid elimin- response was assessed by the Response Evaluation Criteria In Solid ation, partially due to excretion via organic anion transporting pep- Tumors, version v1.1. tide transporters. In preclinical models, DSP-0509 suppressed tumor Results volume and lung metastasis versus vehicle control. Furthermore, Fifteen patients (8 Male, 7 Female; Median age: 58 [range 39-69]) DSP-0509 in combination with anti–programmed cell death protein 1 were included in 5 dose escalation cohorts, dose ranging from 2 (PD-1) antibody suppressed tumor growth versus vehicle, DSP-0509 to 20 mg. Of the 15 subjects, 5 colorectal cancers, 3 biliary tract alone, or anti–PD-1 antibody alone. This 3-part dose escalation (Part cancers and 2 thymic cancers were included. No DLT was ob- A), dose expansion (Part B), and maintenance dose schedule evalu- served and the maximum tolerated dose was not reached. Most ation (Part C) study will investigate DSP-0509 alone or in combin- common AEs were decreased appetite, pyrexia, and cough. No ation with PD-1 inhibitor pembrolizumab (Part A only) Grade 4 or 5 treatment emergent AEs were reported during the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 234 of 272 study and no TEAE or serious AE led to treatment discontinuation Acknowledgements or death. There were no infusion-related reactions during this This study was funded by OSE Immunotherapeutics (the sponsor of the study. Two grade 2 serious AEs suspected to be related to IMC- study) in collaboration with Boehringer Ingelheim. 001 were seen in one subject at 2mg/kg cohort. Over the dose Trial Registration range 2 to 20 mg/kg IMC-001, AUC ,AUC ,and C gen- This study is registered under ClinicalTrials.gov Identifier: NCT03990233 0-14d 0—∞ max erally appeared to increase in a dose proportional manner for each step of dose escalation. Efficacy evaluation is ongoing and Reference will be presented in the future. 1. Gauttier V, Pengam S, Durand J, Morello A, Conchon S, Vanhove B and Conclusions Poirier N. Selective SIRPa blockade potentiates dendritic cell antigen IMC-001 demonstrated a favorable safety profile up to 20mg/kg cross-presentation and triggers memory T-cell antitumor responses. Can- given IV every 2 weeks in patients with advanced solid tumors. cer Res 2018;78(13 Supplement): abstr 1684. doi:10.1158/1538- Trial Registration 7445.AM2018-1684. Clinical trial identification : NCT03644056 Ethics Approval Ethics Approval The study protocol and its related documents (including the patient This study was approved by Institutional Review Board; approval information and informed consent form) received approval from the Ethics number SMC 2018-01-007-001 and H-1801-042-913. Committees, and the Competent Authority prior to study initiation. Each patient gave his/her written informed consent prior to study enrolment. P428 A phase 1 study evaluating BI 765063, a first in class selective P429 myeloid SIRPa inhibitor, as stand-alone and in combination with BI A phase 1/2a dose escalation and expansion study of HPN536, a 754091, a PD-1 inhibitor, in patients with advanced solid tumours mesothelin-targeting T cell engager, in patients with advanced 1 2 3 Nuria Kotecki, MD , Philippe Cassier , Jean-Pierre Delord, MD , Stéphane cancers expressing mesothelin who have failed standard therapy 4 1 2 3 1 2 2 Champiat , Christiane Jungels , Armelle Vinceneux , Iphigenie Korakis , Erika Hamilton, MD , Richard Austin, PhD , Sue Hirabayashi , Che-Leung 5 6 6 2 2 2 Richard Huhn , Nicolas Poirier , Dominique Costantini , Bérangère Law, PhD , Bryan Lemon, PhD , Holger Wesche, PhD , Debra Richardson, 6 4 3 Vasseur , Aurélien Marabelle MD 1 2 1 Institut Jules Bordet, Brussels, Belgium; Centre Léon Bérard, Lyon, Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United 3 4 2 3 France; IUCT, Oncopole, Toulouse, France; Gustave Roussy, Villejuif, States; Harpoon Therapeutics, South San Francisco, CA, United States; Sarah 5 6 France; Boehringer Ingelheim, Ridgefield, CT, United States; OSE Cannon Research Institute/Stephenson Cancer Center at the University of Immunotherapeutics, Nantes, France Oklahoma Health Sciences Center, Oklahoma City, OK, United States Correspondence: Nuria Kotecki (nuria.kotecki@bordet.be) Correspondence: Erika Hamilton (ehamilton@tnonc.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P428 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P429 Background Background Signal Regulatory Protein α [SIRPα] is a polymorphic protein, strongly HPN536 is a mesothelin-targeting T cell engager derived from the TriTAC expressed on myeloid suppressive cells. BI 765063 (OSE172), a hu- platform (Tri-specific T Cell-Activating Construct). Mesothelin (MSLN) is a manized IgG4 monoclonal antibody (mAb), is a selective antagonist tumor antigen overexpressed in malignant mesothelioma, ovarian carcin- of SIRPα/CD47 interaction, it does not bind to SIRPɣ, known to assist oma, pancreatic carcinoma, lung cancer, and triple negative breast cancer T cell co-stimulation and migration. BI 765063 strongly binds V1 al- with limited expression in normal tissues. HPN536 is a recombinant poly- lele, one of the 2 major functional allele of SIRPα expressed in more peptide of ~50kDa containing three humanized antibody-derived binding than 80% of general population and Asian (in 60%). domains, targeting mesothelin (for tumor binding), albumin (for half-life ex- Anti-tumor effect was shown in various in vivo cancer models using tension) and CD3 (for T cell engagement). It has been engineered to be a the validated anti-mouse SIRPα mAbs surrogate, as single agent. The small, globular protein to enable efficient exposure in solid tumor tissue effect was more pronounced in combination with T checkpoint inhib- with prolonged half-life and excellent stability under physiological condi- itors [1]. BI 765063 mechanism of action includes promotion of tions. HPN536 binds monomerically to CD3 and MSLN, minimizing non- tumor-antigen-presentation while preserving T-cell activation and in- specific T-cell activation. These features are designed to widen the thera- crease tumor phagocytosis. peutic index compared to earlier generations of T cell engagers by minimiz- The trial plans to assess the safety profile and preliminary efficacy of ing off target toxicities. HPN536 mediates potent target tumor cell killing in BI 765063, a first in class myeloid check point inhibitor antagonist of a MSLN-specific manner in vitro and in xenograft models in the presence of SIRPα on myeloid cells. T cells. Consistent with its mechanism of action (MOA), tumor cell killing is Methods accompanied by T cell activation, cytokine induction, and T cell expansion. This study comprises a dose escalation (step 1) to determine the Methods Dose-Limiting Toxicities, Maximum Tolerated Dose (MTD), and Rec- This is a Phase 1/2a, open-label, multicenter, dose escalation and ex- ommended Phase 2 Dose (RP2Ds) of BI 765063 monotherapy and pansion study to evaluate the safety, tolerability, clinical activity, and with BI 754091, and dose-confirmation expansion cohorts (step 2). pharmacokinetics of HPN536 in adult patients with advanced cancers In Step 1, ascending dose of BI 763063 once every 3 weeks intraven- expressing mesothelin who have failed standard available therapy. This ously (iv) using a Bayesian approach with overdose control are tested. study will be divided into 2 parts: Dose Escalation (Part 1) and Expan- When MTD determined, BI 763063 will be tested with BI 754091, a PD- sion (Part 2). Eligible patients with ovarian cancer will be enrolled in 1 mAb inhibitor. In step 2, 2 parallel randomized, non-comparative Dose Escalation. Dose expansion will include patients with ovarian can- mono and combination cohorts will further confirm the RP2Ds and as- cer, pancreatic carcinoma and mesothelioma. HPN536 is administered sess the safety and preliminary efficacy (RECIST 1.1 and iRECIST). once weekly as one-hour IV infusion by single-patient cohorts until ei- Patients ≥ 18 years, PS:0-1, with advanced solid tumor who failed or ther a Grade ≥2 adverse event (AE) that is possibly related to HPN536 is are not eligible to standard therapy will be included. V1/V1 and V1/ observed or an estimated therapeutic dose level has been reached. V2 patients (central testing) are evaluated in separate cohorts in step Then a conventional 3+3 design will be implemented. Dose escalation 1. In step 2, a selected population of V1/V1 patients with advanced- will continue until a recommended phase 2 dose (RP2D) is determined. stage cancers (e.g. non-small cell lung cancer, triple negative breast In dose expansion, up to 20 patients per group receive HPN536 at the cancer, or gastro-intestinal cancers) will be included. established RP2D based on a Simon 2-stage design. Patients may con- Pharmacokinetics (PK), SIRPα receptor occupancy (RO) and a compre- tinue weekly HPN536 treatment cycles until disease progression. Pri- hensive translational program (in blood and tumour) will assess PK/ mary endpoints are number and severity of DLTs following treatment PD profile and biomarkers of activity. with escalating doses of HPN536 during escalation, and overall re- A total of 116 (56 instep1 and60 in step2) patients willbe enrolled. sponse rate (by RECIST v1.1 for ovarian and pancreatic, mRECIST v1.1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 235 of 272 for mesothelioma) in dose expansion. Secondary endpoints include with a PD-1 inhibitor. After defining the MTD and/or RP2D, additional AEs, preliminary anti-tumor activity, pharmacokinetic and pharmacody- subjects may be enrolled at the respective dose schedules to further namic parameters based on the proposed MOA of HPN536. evaluate safety, pharmacodynamic effects, and anti-tumor activity Trial Registration Trial Registration NCT03872206 NCT04009681 Ethics Approval This study was approved by each participating institution's Institu- Table 1 (abstract P430). See text for description tional Review Board. P430 Open-label, multicenter phase 1/2 dose escalation and expansion study of THOR-707 as a single agent and in combination with a PD-1 inhibitor in adult subjects with advanced or metastatic solid tumors 1 2 3 David Luo , Raghad Abdul-Karim, MD , Arun Azad, MD , Joanna Bendell, 4 5 6 7 MD , Hui Gan, MBBS PhD , Filip Janku, MD, PhD , Shiraj Sen, MD, PhD , 8 9 1 1 Tira Tan, MBBS , Judy Wang, MD , Lisa Schechet , Lauren Baker, PhD , 1 10 Joseph Leveque, MD , Tarek Meniawy, MBBS FRACP 1 2 Synthorx Inc, La Jolla, CA, United States; NEXT Oncology, Texas Oncology, San Antonio, TX, United States; Peter MacCallum Cancer Centre, Melbourne, Austrailia; Sarah Cannon Research Institute, Nashville, TN, United States; Austin Hospital, Melbourne, Victoria, Australia; University of TX, MD Anderson Cancer Center, Houston, TX, United States; Sarah Cannon Research Institute at HealthONE, Houston, TX, United States; National Cancer Centre Singapore, Toronto, Canada; 9 10 Florida Cancer Specialists, Sarasota, FL, United States; Linear Clinical Research, Nedlands, WA, Australia Correspondence: Lauren Baker (lbaker@synthorx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P430 Background Recombinant interleukin-2 (aldesleukin), an approved immunotherapy in metastatic melanoma and renal cell carcinoma, can induce complete durable responses in some patients. The anti-neoplastic properties of IL-2 are mediated by activation of effector memory T cells and newly recruited naïve CD8+ T cells against the tumor. The widespread use of P431 IL-2 has been limited due to its high affinity bias for the IL-2 receptor Semi-mechanistic PK and target-occupancy modeling to support alpha chain (IL-2R⍺) on regulatory CD4+ T cells, leading to immunosup- dose justification for anti-PD-L1 clinical candidate CK-301 (TG- pression and, eosinophilic recruitment and activation on innate lymph- 1501) in oncology patients oid cells in the vascular endothelium causing vascular leak syndrome 1 1 2 3 Lin Lin, PhD , James Hilbert , Leonid Gorelik , Jian-Ping Tang , James (VLS). THOR-707 is a recombinant human IL-2 variant that is site- 4 1 1 1 Oliviero , Joshua Apgar , Lore Gruenbaum, PhD , John Burke specifically pegylated, providing a “not alpha” pharmacologic profile 1 2 Applied BioMath, LLC, Concord, MA, United States; Fortress Biotech, designed to prevent engagement of IL-2R⍺, thereby providing an im- Waltham, MA, United States; TG Therapeutics, New York, NY, United proved safety profile while still promoting newly recruited and effector States; Checkpoint Therapeutics, New York, NY, United States memory T cell anti-tumor activity In preclinical studies, eosinophilia was Correspondence: Lore Gruenbaum (lgruenbaum@gothamtx.com) not observed at a doses 10-fold higher than the dose responsible for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P431 eliciting maximal expansion of peripheral CD8+ T cells. Based on these findings, a first-in-human study of THOR-707 was started in June 2019. Background Methods Mathematical modeling was used in conjunction with in vitro, pre- This open-label, multicenter, dose escalation and expansion study in clinical and clinical data to facilitate dose selection of CK-301 (also adult subjects with advanced or metastatic solid tumors will evaluate known as TG-1501), an anti-PD-L1 monoclonal antibody (mAb), for THOR-707 as a single agent and in combination with a PD-1 inhibitor. ongoing and future clinical trials in oncology patients. Study objectives are to define the maximum tolerated dose (MTD) and/ Methods or recommended phase 2 dose (RP2D) of THOR-707 as single agent and A semi-mechanistic pharmacokinetic/target-occupancy (PKTO) model in combination with a PD-1 inhibitor; and to evaluate the overall safety was developed to predict pharmacokinetics (PK) of CK-301 at steady state and tolerability as well as, pharmacokinetics, pharmacodynamics, and and its tumor target occupancy (TO) under various dosing regimens. The preliminary anti-tumor activity. The study will be conducted in 3 parts. model captures the interactions between CK-301, PD-L1, soluble PD-L1 and PD-1 in 3 compartments: tumor, circulation (central) and other tissues (per- Part 1 will evaluate THOR-707 as a single agent across different dos- ipheral). The model was calibrated with CK-301 PK data from the first 5 pa- ing schedules (e.g., dosing every 2 weeks [Q2W] or 3 weeks [Q3W]). tients in a clinical study, CK-301-101, and PK data from published Phase 1 Part 2 will evaluate THOR-707 (Q3W) in combination with a PD- studies of 3 marketed anti-PD-L1 mAbs: atezolizumab, avelumab and durva- 1 inhibitor. lumab. Additionally, the model incorporated experimentally determined Part 3: Dose expansion will begin after the RP2D for THOR-707 as binding affinities for the 3 marketed anti-PD-L1 mAbs and CK-301. a single agent or in combination with a checkpoint inhibitor has Results been determined and will enroll selected populations (e.g., spe- Using the PKTO model, plasma Ctrough values and tumor TO of CK-301 at cific tumor types, treatment history, and/or biomarker profile). steady-state with 800 and 1200 mg q2w or q3w were projected. The TO of CK-301 were compared with predicted steady-state Ctrough TOs of atezoli- Between 50-100 subjects may be enrolled in the dose escalation phase zumab, avelumab and durvalumab at their marketed doses. The steady- to determine the MTD and/or RP2D as a single agent and in combination state Ctrough values of CK-301 are predicted to give >99% tumor TO for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 236 of 272 patients with a nominal or a 10-fold greater than nominal PD-L1 tumor bur- P433 den. This is similar to predicted TO for atezolizumab and durvalumab. The Initial results of the phase 1 portion of an ongoing phase 1/2 PKTO model was used to simulate PK and TO of CK-301 in 1000 virtual pa- study of RP1 as a single agent and in combination with nivolumab tients. The simulations predicted that, at 800 and 1200 mg q2w or q3w, in patients with solid tumors 1 2 3 ≥93.0% of patients with a nominal PD-L1 tumor burden or ≥80.1% of pa- M Middleton, MD PhD , Joseph Sacco , Jaime Merchan , Amber 3 4 5 tients with 10-fold higher than nominal PD-L1 tumor burden would have a Thomassen , Brendan Curti, MD , Ari VanderWalde, MD, MPH, MBioeth , 2 1 >99% tumorTOatsteady-stateCtrough. Anna Olsson-Brown, MBChB (Hons), BSc (Hons) , Francesca Aroldi , Nicos 6 5 7 Conclusions Fotiadis , Scott Baum , Howard Kaufman, MD, FACS , Kevin Harrington, At the proposed CK-301 dosing regimens of 800 and 1200 mg q2w or MD 1 2 q3w, a >99% TO is expected throughout the dosing interval. Relative to University of Oxford, Childrey, United Kingdom; University of Liverpool, atezolizumab and durvalumab treatments, similar percentages of pa- Liverpool, United Kingdom; University of Miami, Miami, United States; 4 5 tients would possibly benefit from CK-301 treatment. Providence Medical Center, Portland, OR, United States; West Cancer Center, Germantown, TN, United States; The Institute of Cancer Research, London, United Kingdom; Replimune, Woburn, MA, United States P432 Correspondence: Howard Kaufman (Howard.Kaufman@replimune.com) A phase 1 dose-escalation study of safety, tolerability, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P433 pharmacokinetics (PK) of ABBV-368 monotherapy and combination in patients (pts) with locally advanced or metastatic solid tumors Background 1 2 3 Christophe Le Tourneau , Wu-Chou Su, MD , Ki Chung, MD , Patricia Background: RP1 is an enhanced-potency oncolytic HSV-1 expressing 4 5 6 LoRusso, DO , Chia-Chi Lin, MD, PhD , Fabrice Barlesi, MD, PhD , Her- a fusogenic glycoprotein (GALV-GP R-) and GM-CSF which is being 7 8 9 Shyong Shiah , Eric Angevin, MD , Alexander Spira, MD, PhD, FACP , tested in a Phase 1/2 clinical trial in ~150 patients with a range of 10 11 Amita Patnaik, MD FRCP(C) , John Powderly, MD, CPI , Dimitrios solid tumors (NCT03767348). 12 13 14 Colevas , Helen Chew, MD , Maulik Patel, PharmD, PhD , Stacie Methods 14 14 14 14 Lambert , Yan Li , Daniel Da Costa , Martha Blaney, PharmD , Methods: The objectivesweretodefinethesafety of RP1 aloneand with 14 15 Michael McDevitt, MD, PhD , Philippe Cassier nivolumab, determine the recommended phase 2 dose (RP2D), and in 1 2 Institut Curie, Paris & Saint-cloud, France; National Cheng Kung University 30 patient phase 2 cohorts, assess efficacy in melanoma, non-melanoma Hospital, Tainan, Taiwan, Province of China; GHS Cancer Institute, skin cancer, urothelial carcinoma and MSI-H tumors. Initial phase 1 re- Spartanburg, SC, United States; Yale Cancer Center, New Haven, CT, United sults will be reported where patients were treated by intra-patient dose States; National Taiwan University Hospital, Taipei, Taiwan, Province of China; escalation of RP1 (up to 10mL of 104-108PFU/mL) by intratumoral injec- 6 7 Aix Marseille Univ Hôpitaux de Marseille, Livon, France; Taipei Medical tion into a single tumor Q2W up to 5 times followed by 12 patients University, Taipei, Taiwan, Province of China; Gustave Roussy, Villejuif, France; dosed 8 times at the RP2D combined with nivolumab (240mg Q2W for 9 10 Virginia Cancer Specialists Research Ins, Fairfax, VA, United States; South 4 months from the second RP1 dose, then 480 mg Q4W for 20 months). Texas Accelerated Research Thera, San Antonio, TX, United States; Carolina Clinically accessible lesions were directly injected, with imaging guidance BioOncology Institute, Huntersville, NC, United States; Stanford University, for deep/visceral lesions. Pre- and on-treatment tumor biopsies were ob- 13 14 Stanford, United States; UC Davis, Sacramento, United States; Abbvie Inc, tained for biomarker analysis. Viral shedding and anti-HSV antibody titers North Chicago, IL, United States; Léon Bérard Cancer Center, Lyon, France were also monitored. Correspondence: Michael McDevitt (michael.mcdevitt@abbvie.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P432 Results: 22 heavily pretreated patients with advanced tumors were enrolled into the dose-rising phase with largely low-grade adverse Background events, including febrile and other constitutional symptoms, local in- ABBV-368 is a humanized anti-OX40 monoclonal antibody. OX40 flammation and erythema. No clear differences were seen between is a member of the TNF receptor superfamily, which exerts its ac- superficial and visceral dosing. RP1 was detected at the injection site tion via activating T effector cells and inhibiting the suppressive and in blood for up to 14 days (next injection), suggesting virus repli- capacity of T reg cells. Preclinical data have shown ABBV-368 an- cation. All HSV seronegative patients seroconverted after three injec- titumor activity in animals. tions. Biological activity was demonstrated including tumor necrosis Methods and shrinkage, with extended clinical benefit and delayed (post-treat- This is a multicenter, phase 1, dose-escalation study (NCT03071757) ment termination and initial PD) systemic reduction in multiple tu- in pts (≥18 years; Eastern Cooperative Oncology Group performance mors in two patients (ipilimumab/nivolumab-refractory melanoma status 0–2) with locally advanced or metastatic solid tumors. The and chemotherapy-refractory cholangiocarcinoma) without interven- study consisted of 3 parts: 1) dose escalation (DE1); 2) cohort expan- ing treatment. The RP2D was selected as up to 10mL of 106PFU/mL sion (CE2); and 3) imaging substudy (IA3). Specific inclusion criteria followed Q2W by multiple doses of 107PFU/mL. Twelve evaluable pa- and dosing schedules for each cohort are shown in the table (Table tients (6 direct injection, 6 image-guided) were then enrolled into 1). In DE1, the primary endpoints are safety, tolerability, and PK of the phase 1 expansion combined with nivolumab. This demonstrated ABBV-368 monotherapy to establish the maximum tolerated dose or tolerability and clinical activity, including complete and partial re- reach the maximally administered dose; the secondary endpoint is sponses in patients with chemotherapy-refractory cutaneous squa- preliminary antitumor activity. Preliminary results for DE1 have been mous carcinoma, and ipilimumab/nivolumab-refractory melanoma. reported (Powderly et al. ESMO 2018). For CE2, the primary end- Treatment remains ongoing, and current data will be presented, in- points are safety, tolerability, and PK of ABBV-368 monotherapy and cluding biomarker data (CD8, PD-L1 staining and Nanostring analysis in combination with ABBV-181 (a humanized anti-programmed cell from tumor biopsies). death 1 monoclonal antibody), and to establish the recommended Conclusions phase 2 dose; the secondary endpoint is preliminary antitumor activ- Conclusions: The Phase 1 clinical data supports the safety and effi- ity of ABBV-368 monotherapy and combination therapy. The primary cacy of RP1 alone and when combined with nivolumab, including endpoints of the IA3 part are safety and tolerability. For all cohorts, demonstration of abscopal anti-tumor effects in patients refractory to AEs will be assessed according to the NCI CTCAE v4.03; response will prior checkpoint inhibitors. The Phase 2 portion of this clinical trial is be assessed Q2 months (mo) for 12 mo, and Q3 mo thereafter, as open in the US and the UK. per the immunotherapy Response Evaluation Criteria in Solid Tumors Trial Registration (iRECIST), and RECIST v1.1. As of 12 Jul 2018, enrollment into DE1 NCT03767348 was completed and CE2 has started. Ethics Approval Trial Registration The study was approved by applicable institutional review or ethics NCT03071757 boards. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 237 of 272 P434 2. Piccione et al. Preclinical and initial phase I clinical characterization of A phase 1/1b study to evaluate the humanized anti-CD73 CPI-006: an anti-CD73 monoclonal antibody with unique immunostimula- antibody, CPI-006, as a single agent, in combination with CPI-444, tory activity. Presented at Society for Immunotherapy of Cancer Meeting; and in combination with pembrolizumab in adult patients with November 7-11, 2018; Washington, DC, USA: Abstract P205. advanced cancers Ethics Approval 1 1 1 1 Mehrdad Mobasher , Richard Miller , Brian Munneke , Deborah Strahs , The study was approved by Western IRB, approval number 1-1066703-1. 1 1 1 Gabriel Luciano , Emily Piccione, PhD , Suresh Mahabhashyam , Jaime 2 3 4 Merchan , John Powderly, MD, CPI , Lauren Harshman, MD , Minal 5 6 7 8 Barve , Walter Stadler, MD , Patricia LoRusso, DO , Melissa Johnson , 9 10 Abhishek Tripathi, MD , Sumanta Pal, MD , Ben Markman, MBBS 11 12 13 FRACP , Jason Luke, MD, FACP , Thomas Marron, MD PhD 1 2 Corvus Pharmaceuticals Inc, Burlingame, CA, United States; University of Miami, Miami, United States; Carolina BioOncology Institute, Huntersville, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Mary Crowley Cancer Research Center, Dallas, TX, United States; University of Chicago Comprensive Cancer Center, Chicago, IL, United States; Yale University School of Medicine, New Haven, CT, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University of Oklahoma, Stephenson Cancer Center, Oklahoma City, OK, United States; City of Hope, Duarte, CA, United 11 12 States; Monash Health, Melbourne, Australia; UPMC, Pittsburgh, PA, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States Correspondence: Mehrdad Mobasher (mmobasher@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P434 Background CD73 expression is elevated in tumors and contributes to increas- ing levels of immunosuppressive adenosine in the tumor micro- environment. CD73 knockout mice exhibit reduced tumor growth and resistance to experimental metastasis. Inhibition of CD73 ac- tivity with an anti-CD73 antibody blocks adenosine production, Fig. 1 (abstract P434). See text for description shown to inhibit tumor growth in syngeneic models. Dual inhib- ition of CD73 and A2aR improves anti-tumor immune responses in mouse tumor models[1]. CPI-006 is a humanized IgG1 Fc gamma receptor binding-deficient anti-CD73 antibody that has a P435 dual mechanism of action. It blocks CD73 catalytic activity and A window of opportunity trial using intratumoral injection of adenosine production. In addition, it has immunomodulatory ac- glatiramer as an immune modulator in patients with resectable tivity on CD73 positive immune cells including B cells, T cells and head and neck and cutaneous squamous cell cancer antigen presenting cells. CPI-006 relieves adenosine-mediated im- Ghulam Rehman Mohyuddin, MD, Joaquina Baranda, MD, Andres Bur, munosuppression in vitro as a single agent and in combination Lisa Shnayder, Kiran Kakarala, Terry Tsue, Prakash Neupane, Gregory Gan, with ciforadenant[2]. CPI-006 is now being investigated in this Joshua Mammen, Daniel Aires, Sufi Thomas, Stephen Williamson, Nelli Phase 1/1b multicenter, open label trial as single agent (SA), in Lakis, Rashna Madan, Prabhakar Chalise, Scott Weir, Andrew Godwin, combination with ciforadenant, an oral, small molecule, selective Greg Reed, Cory Berkland A2aR antagonist and in combination with pembrolizumab, an Kansas University Medical Center, Kansas City, KS, United States anti-PD1 indicated for the treatment of patients across a number Correspondence: Joaquina Baranda (gioncol@gmail.com) of malignancies (NCT03454451). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P435 Methods Up to 462 subjects will be enrolled at approximately 35 sites in the Background US, Canada and Australia. Eligible patients with: non-small cell lung Immunotherapy using checkpoint inhibition improves outcome of cancer (NSCLC), renal cell carcinoma cancer (RCC), urothelial bladder patients with melanoma, lung, bladder, microsatellite instability-high cancer, cervical cancer, colorectal cancer, ovarian cancer, pancreatic and other tumors[1]. However, systemic administration of immuno- cancer, prostate cancer, head and neck cancer, triple-negative breast therapy may have limited activity in some tumors partly due to fail- cancer, endometrial cancer, select sarcomas and non-Hodgkin lymph- ure of activated T cells to migrate to tumor[1]. Intratumoral injections oma (NHL) who are relapsed, refractory or intolerant to 1 to 5 stand- (ITI) may allow high concentration of immunostimulatory products lo- ard therapies; aged ≥ 18 yo; with adequate organ function and cally while using small amounts of drugs. This may also facilitate mul- measurable disease. Study details is presented in Figure 1. tiple combination therapies and avoid systemic off-target toxicities. The primary objective of the dose escalation is to assess safety/ tolerabil- By capitalizing on existing data and experience, repurposing ap- ity, MTD or MDL of CPI-006 SA, in combination with ciforadenant and proved drugs for cancer represents an opportunity to rapidly ad- with pembrolizumab in ascending dose levels. Secondary objectives are vance promising therapies. to evaluate the PK of CPI-006 as SA or in combinations and analyze po- Glatiramer acetate is an agent commonly used for multiple scler- tential predictive biomarkers. In dose escalation, the primary objective is osis[2]. It acts as an immunomodulator and has essentially no sys- to assess the safety and tolerability of CPI-006 SA and in combinations in temic bioavailability but exhibits a high prevalence of injection site patients with selected advanced cancers. Secondary endpoints include reactions[2]. It upregulates the activity of natural killer cells in efficacy; PK of CPI-006 as SA and in combinations and to evaluate the re- leukemia cell lines [3], suggesting potential for immunostimulatory lationship between biomarkers and clinical activity. effect for ITI. For percutaneously accessible tumors for which the standard of care is surgical resection without any neoadjuvant ther- References apy, there exists a window of opportunity where ITI of glatiramer can 1. Young et al. Co-inhibition of CD73 and A2AR adenosine signaling im- be performed before surgery. proves anti-tumor immune responses. Cancer Cell. 2016; 30(3):391-403. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 238 of 272 Methods blood pressure, and must have provided a tumor sample evaluable for This is a proof-of-concept, investigator-initiated, window of opportunity PD-L1; and must not have immunodeficiency, active central nervous trial in subjects with percutaneously accessible head and neck or cuta- system metastases (except in GBM cohort), or be on steroid therapy neous squamous cell cancer who are to undergo surgery. Subjects will (patients with GBM may be on dexamethasone ≤2 mg/day orally or receive glatiramer 40 mg by ITI 3 times a week prior to surgery. Sub- equivalent and stable for 5 days at the time of enrollment). Patients will jects will receive at least one dose and up to 3 doses of glatiramer. receive lenvatinib 20 mg daily and pembrolizumab 200 mg every 3 About 10 eligible patients will be included in this trial. Safety data will weeks (Q3W) for ≤2 years, or until confirmed disease progression (may be collected. Tumor tissue at the time of diagnosis and at the time of continue lenvatinib if receiving clinical benefit), unacceptable toxicity, surgery will be collected and compared for biomarkers. Primary end- or study withdrawal. Patients with confirmed complete response may point is safety. Secondary endpoint is effect of ITI of glatiramer on bio- discontinue after ≥24 weeks combination therapy and ≥2pembrolizu- marker levels. Pre- and post-treatment tumor samples will be tested mab doses after initial complete response date. Tumor imaging will using an immunology panel that profiles immunology genes and pro- occur at baseline, Q9W (or for GBM patients: Q6W until 18 weeks, then teins including major classes of cytokines, interferons, KIR family, and Q9W) for the first 54 weeks, Q12W until week 102 (~2 years), and TNF-receptor. Tumors samples will also be evaluated for the Ki-67 pro- Q24W thereafter using RECIST v1.1/RANO by investigator assessment in liferative index and for caspase-3 and cleaved caspase-3 immunoex- the initial cohorts and by BICR after cohort expansion. Primary end- pression. Paired T-test or the Wilcoxon signed rank test will be used to points are objective response and safety (adverse events graded using assess the changes in the variables. We hypothesize that this approach NCI CTCAE v4.0, and discontinuation due to adverse events). Secondary will break the immunosuppressive tumor microenvironment as evi- endpoints include disease control, duration of response, progression- denced by an increase in inflammatory cytokines, chemokines, and im- free survival, and overall survival. Initially, approximately 180 patients mune cell infiltration. Decline in Ki-67 and increase in caspase-3 may be will be enrolled (30/cohort; each cohort may be expanded to 100 after a signal of anti-tumor activity. planned interim analysis). Enrollment is ongoing at 44 sites in 10 coun- Trial Registration tries across North America, South America, Europe, Asia, and Australia. NCT03982212 Acknowledgements References Writing support was provided by Shilpa Aggarwal, PhD, of C4 MedSolutions, 1. Whiteside, T.L., et al., Emerging Opportunities and Challenges in Cancer LLC (Yardley, PA, USA), a CHC Group company, funded by Eisai Inc. and Merck Immunotherapy. Clin Cancer Res, 2016. 22(8): p. 1845-55. Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. 2. Wingerchuk, D.M. and J.L. Carter, Multiple sclerosis: current and emerging Legal Entity Responsible for the Study: Eisai Inc. and Merck Sharp & Dohme disease-modifying therapies and treatment strategies. Mayo Clin Proc, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA 2014. 89(2): p. 225-40. 3. Maghazachi, A.A., K.L. Sand, and Z. Al-Jaderi, Glatiramer Acetate, Dimethyl Funding Source Fumarate, and Monomethyl Fumarate Upregulate the Expression of Funding for this research was provided by Eisai Inc. and Merck Sharp & CCR10 on the Surface of Natural Killer Cells and Enhance Their Chemo- Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. taxis and Cytotoxicity. Front Immunol, 2016. 7: p. 437. Trial Registration Ethics Approval NCT03797326 This study was approved by Kansas University Medical Center Institutional Ethics Approval Review Board, approval number: HSC00144030 An independent institutional review board or ethics committee approved the protocol at each study site, and the trial is being conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. P436 Phase 2 study of lenvatinib plus pembrolizumab in previously treated patients with solid tumors: LEAP-005 P437 1 2 3 4 5 Ravit Geva , Seock-Ah Im , Zarnie Lwin , Susan Weil , Lei Xu , Anne Disease-related biomarkers are associated with extended 5 5 6 Morosky , Kevin Norwood, MD , Hyun Cheol Chung, MD, PhD progression free survival after treatment with NEO-PV-01 in 1 2 Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; Seoul National combination with anti-PD1 in patients with metastatic cancers 3 1 2 University Hospital, Seoul, Korea, Republic of; University of Queensland, Patrick Ott, MD, PhD , Ramaswamy Govindan, MD , Aung Naing, MD, 4 3 4 5 6 Queensland, Australia; Eisai Inc., Woodcliff Lake, NJ, United States; FACP , Terence Friedlander, MD , Kim Margolin, MD , Jessica Lin, MD , 5 6 7 8 Merck & Co., Inc., Kenilworth, NJ, United States; Yonsei University Nina Bhardwaj, MD, PhD , Matthew Hellmann, MD , Mark Awad, MD 1 9 9 9 College of Medicine, Seoul, Korea, Republic of PhD , Amy Wanamaker , Lisa Cleary , Michael Rooney , Julian Scherer, 9 9 9 9 Correspondence: Ravit Geva (ravitg@tlvmc.gov.il) PhD , Meghan Bushway , Melissa Moles , Zakaria Khondker , Richard 9 9 9 9 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P436 Gaynor, MD , Lakshmi Srinivasan, PhD , Andrew Chi , Joel Greshock , Siwen Hu-Lieskovan, MD, PhD 1 2 Background Dana Farber Cancer Institute, Boston, MA, United States; Washington Lenvatinib (multiple receptor tyrosine kinase inhibitor of vascular endo- University, Saint Louis, MO, United States; MD Anderson Cancer Center, thelial growth factor receptors 1–3, fibroblast growth factor receptors 1– Houston, TX, United States; University of California San Francisco, San 4, platelet-derived growth factor receptor α,RET,and KIT) andanti–PD-1 Francisco, CA, United States; City Of Hope, Duarte, CA, United States; 6 7 inhibitor pembrolizumab have shown clinical benefit as monotherapies Massachusetts General Hospital, Boston, MA, United States; Mt. Sinai across multiple cancers. In preclinical studies, lenvatinib plus PD-1 block- Medical Center, New York, NY, United States; Memorial Sloan Kettering ade improved antitumor activity vs either agent alone. LEAP-005 Cancer Center, New York, NY, United States; Neon Therapeutics, (NCT03797326) evaluates the efficacy and safety of lenvatinib plus pem- Cambridge, MA, United States; Huntsman Cancer Institute, Los brolizumab in patients with previously treated selected advanced tumors. Angeles, CA, United States Methods Correspondence: Joel Greshock (jgreshock@neontherapeutics.com) This global, open-label, phase 2 study enrolls patients ≥18 years with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P437 the following previously treated histologically/cytologically confirmed advanced tumors: triple negative breast, ovarian, gastric, colorectal Background (non-MSI-H/pMMR), glioblastoma multiforme (GBM), or biliary tract (ex- Neoantigens arise from mutations in cancer cell DNA and are import- cluding ampulla of Vater). Patients must have progressed on or since ant targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a last treatment; have measurable disease per RECIST v1.1 (modified to personal neoantigen vaccine of up to 20 peptides designed by the follow ≤5 target lesions/organ [10 total]) or the RANO criteria (GBM RECON® bioinformatics platform using patient neoantigen and HLA only), assessed locally and confirmed by blinded independent central profiles. Here we report biomarker correlates of clinical benefit for review (BICR); ECOG performance score 0–1, adequately controlled NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 239 of 272 with nivolumab in anti-PD1 naïve metastatic melanoma, NSCLC and antigen recall studies (Hanson 2018) showed that soluble vopra- bladder cancer patients (NCT02897765). telimab stimulated a polyfunctional cytokine response only in Methods CD4 T cells that were ICOS hi, further supporting the hypothesis Patients received 12 weeks of nivolumab monotherapy (240 mgs that vopratelimab induces activation and proliferation of CD4 T Q2W), then NEO-PV-01 in a prime-boost format spanning 12 weeks, effector cells only after an initial priming event induces an ICOS nivolumab continued for up to 2 years. The primary objective was hi CD4 T cell phenotype. Furthermore, in melanoma patients safety, secondary objectives were overall response rate (ORR), treated with ipilimumab, a sustained increase in the frequency of progression-free survival (PFS), and overall survival. Comprehensive ICOS-positive CD4 T cells correlated with clinical benefit and sur- comparisons of baseline and serial molecular and immunological vival (Carthon 2010). In contrast, emergence of these ICOS hi cells characteristics between patients with vs. without durable PFS were has not been noted with PD-1/PD-L1 inhibitors (Hanson 2018). performed for all tumor cohorts. We hypothesized that the combination of vopratelimab with ipili- Results mumab will enhance the presence and functionality of ICOS hi A total of 34 melanoma, 27 NSCLC and 21 bladder cancer patients re- CD4 T effector cells, thereby potentially increasing the likelihood ceived nivolumab therapy, of which 27, 18 and 15 initiated vaccine of clinical benefit. respectively. The median follow up time was 13.4, 12.0 and 14.7 Methods months for melanoma, NSCLC and bladder cancer respectively. No This open label, multi-center, phase 2 study will evaluate efficacy, treatment-related serious adverse events were noted. The median safety, PK, and exploratory pharmacodynamics of vopratelimab in PFS for the melanoma cohort was not reached (95% CI: 3.3, NE), and combination with ipilimumab in adult patients with non-small cell the ORR was 47%. The median PFS in both the NSCLC and bladder lung cancer or urothelial cancer who have been previously cohort was 5.6 months (95% CI’s: 2.3, 8.7; 2.0, 8.1 respectively) with treated with PD-1/PD-L1 inhibitors. We expect to enroll approxi- ORR’s of 22% and 24% respectively. RECON tumor neoantigen abun- mately 200 evaluable subjects in total. Primary endpoint is ORR. dance was predictive of durable PFS in melanoma patients. Analyses Secondary endpoints include safety and tolerability, PFS, OS as of pre-treatment peripheral TCR repertoires reveal a more clonal T well as PK/PD. cell population in melanoma patients with extended PFS. Other fac- Trial Registration tors that associated with durable PFS included the abundance of B ClinicalTrials.gov NCT03989362 cells and CD8+ T cells in the tumor microenvironment. Finally, across Ethics Approval cohorts, longitudinal tumor biopsies from patients with extended Study was approved by the applicable Institution Ethics Boards PFS showed higher rates of initial pathologic responses after vaccin- ation vs. biopsies from patients with shorter PFS, suggesting vaccine- P439 related anti-tumor responses in this subset. Phase 1 first in human study of programmed cell death receptor- Conclusions 1(PD-1) inhibitor monoclonal antibody (mAb) JTX-4014 in adult NEO-PV-01 in combination with nivolumab is safe and leads to post- subjects with advanced refractory solid rumor malignancies vaccine immune and pathologic responses, indicating further clinical 1 2 Kyriakos Papadopoulos, MD , Gerald Falchook, MD , Nehal Lakhani, MD, evaluation is warranted. The association of baseline disease charac- 3 4 4 4 4 PhD , Gosia Riley , Jian Xu, PhD , Johan Baeck , Gilad Gordon , Elizabeth teristics with prolonged PFS suggests future patient enrichment 4 5 Trehu, MD , Judy Wang, MD strategies. 1 2 START, San Antonio, TX, United States; Sarah Cannon Research Inst. at Trial Registration Healthone, Denver, CO, United States; START-Midwest, Grand Rapids, NCT02897765 MI, United States; Jounce Therapeutics, Cambridge, MA, United States; Ethics Approval Florida Cancer Specialists - SCRI, Sarasota, FL, United States This trial has been approved by all institutional Review Boards of Correspondence: Kyriakos Papadopoulos every clinical trial site involved with this study. (Kyri.Papadopoulos@startsa.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P439 P438 Phase 2 Multicenter Trial of ICOS agonist vopratelimab and a Background CTLA-4 inhibitor in PD-1/PD-L1 inhibitor experienced adult JTX-4014 is a fully human mAb consisting of 2 identical hinge- subjects with Non-small Cell Lung Cancer or Urothelial Cancer stabilized immunoglobulin gamma 4 (IgG4, S228P) heavy and two (EMERGE) identical kappa (Igκ) light chains, that specifically binds to PD-1. The 1 1 2 Russell Pachynski, MD , Ramaswamy Govindan, MD , Ellen Hooper, MD , mechanism of action of JTX-4014 is to block the interaction of PD-1 2 2 2 Christopher Harvey, PhD , Amanda Hanson , Sean Lacey, MA , Rachel with its ligands, PD-L1 and PD-L2, and augment anti-tumor T-Cell ac- 2 2 2 2 McComb , Courtney Hart , Haley Laken , Johan Baeck , Elizabeth Trehu, tivity. This Phase 1 trial objectives were to evaluate the safety and MD tolerability of the drug along with its maximum tolerated dose (MTD) Washington University School of Medicine, St. Louis, MO, United States; and recommended Phase 2 dose. Jounce Therapeutics, Cambridge, MA, United States Methods Correspondence: Russell Pachynski (rkpachynski@wustl.edu) Key inclusion criteria included age ≥18 yrs, histologically or cytologic- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P438 ally confirmed extracranial solid tumor refractory to at least one prior line of therapy, no concurrent anticancer treatment, no prior anti-PD- Background 1 or anti-PD-L1 therapy, no requirement for selection based on PD- ICOS is a costimulatory molecule upregulated on activated T cells. L1 expression, no history of immune-mediated conditions, and ad- Vopratelimab (JTX-2011) is an IgG1 ICOS agonist monoclonal anti- equate renal, hepatic, and bone marrow function. The trial was a body known to activate and proliferate primed CD4 T effector standard 3+3 design with 5 fixed dose levels ranging from 80 mg cells in vitro, with established preclinical efficacy in multiple Q3wk to 1200 mg Q3wk given by IV infusion. In addition, there was tumor models. In the Phase 1/2 ICONIC trial (NCT02904226), one arm of 800 mg Q6wk. vopratelimab in patients with advanced solid tumors (Yap 2019) Results has shown to be safe and well tolerated as monotherapy and in 18 patients were enrolled in the trial (10 males, 8 females) with an combination with nivolumab. The ICONIC study showed no correl- average age of 66.3 yrs. Tumor types included ovarian (n=4), salivary ation between tumor reductions and ICOS and PD-L1 levels in gland, sarcoma, prostate and mesothelioma (n=2 each). The max- pre-treatment tumor samples by IHC. However, emergence of imum administered dose was 1200mg; MTD was not reached. There peripheral blood ICOS High (hi) CD4 T effector cells following were no deaths, no dose limiting toxicities. One treatment-related treatment with vopratelimab +/- nivolumab was associated with serious adverse event of pneumonitis occurred after the second dose tumor reductions and improved PFS and OS. In addition, ex vivo at 1200 mg Q3wk. Adverse events occurring in > 15% of patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 240 of 272 included fatigue, anemia, AST increased, dizziness, and tumor pain. 1 SA arm and 3 combination arms in which TPST-1120 is combined Only fatigue was noted as related in more than one patient (all with nivolumab, docetaxel or cetuximab. The RP2D of TPST-1120 to Grade 1 and 2). Grade 3 related AEs included increase alkaline phos- proceed to DEx will be determined by safety and biomarkers includ- phatase and pneumonitis. At time of data cutoff, median number of ing analysis of FAO/PPARα gene expression in the peripheral blood doses administered was 3 (range 1-11). Preliminary investigator and in tumor biopsies. The DEx arms will follow a 2-stage expansion assessed antitumor activity included: confirmed partial response (PR) design. This trial began accrual in May 2018 at U.S sites and is cur- in 1 patient with salivary gland carcinoma, unconfirmed PR in 1 pa- rently enrolling into the Monotherapy/Dose Escalation cohort. Expan- tient with ovarian cancer (both PD-L1+ by IHC) and best response of sion cohorts are projected to open in early 2019. The total sample stable disease in 6 patients. Systemic exposure of JTX-4014 increased size is up to 338 pts. dose proportionally; mean terminal half-life ranged from 11 to 17 Trial Registration days. JTX-4014 pharmacokinetics was comparable to other approved NCT03829436 anti-PD-1 mAbs. No anti-drug antibodies were observed. Ethics Approval Conclusions This study is being conducted in accordance with Good Clinical Prac- JTX-4014 is well-tolerated and appears to have similar qualities to tice and the Helsinki Declaration and has been approved by the known anti-PD-1 inhibitors in terms of pre-clinical and clinical charac- Western IRB/Copernicus Group, tracking # 20190182. teristics. Antitumor activity was observed in the difficult to treat population enrolled. Phase 2 testing JTX-4014 is planned. P441 Trial Registration ARTISTRY-2: a phase 1/2 study of subcutaneously administrated NCT03790488 ALKS 4230 as monotherapy and in combination with Ethics Approval pembrolizumab in patients with advanced solid tumors The study was approved by the relevant Institutions' Ethics Board 1 2 3 John Powderly, MD, CPI , Bradley Carthon, MD, PhD , Marc Ernstoff, MD , 4 5 6 Anthony Olszanski, MD, RPh , Stephen Liu, MD , Kelly Curtis, MD , 7 7 7 7 P440 Yangchun Du, PhD , Lei Sun, PhD , Emily Putiri, PhD , Yan Wang, PhD , 7 7 8 Phase 1/1b multicenter trial of TPST-1120, a peroxisome Heather Losey, PhD , Bruce Dezube, MD , Ulka Vaishampayan, MD 1 2 proliferator-activated receptor alpha (PPARα) antagonist as a Carolina BioOncology, Huntersville, NC, United States; Emory University, single agent (SA) or in combination in subjects with advanced Atlanta, GA, United States; Roswell Park Comprehensive Cancer Center, cancers Buffalo, NY, United States; Fox Chase Cancer Center, Phildelphia, PA, 1 2 2 5 John Powderly, MD, CPI , Saurin Chokshi, MD , Johanna Bendell, MD , United States; Georgetown University, Washington, DC, United States; 3 3 4 6 7 Leisha Emens, MD, PhD , Jason Luke, MD, FACP , Brian Francica , Chan Syneos Health, Phoenix, AZ, United States; Alkermes, Inc., Waltham, 4 4 4 8 Whiting, PhD , Thomas Dubensky, PhD , Ginna Laport, MD MA, United States; Barbara Ann Karmanos Cancer Institute, Detroit, MI, 1 2 Carolina BioOncology, Huntersville, NC, United States; Sarah Cannon United States Research Institute, Nashville, TN, United States; University of Pittsburgh, Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Pittsburgh, PA, United States; Tempest Therapeutics, South San Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P441 Francisco, CA, United States Correspondence: Thomas Dubensky (tdubensky@tempesttx.com); Background Ginna Laport (glaport@tempesttx.com) ALKS 4230 is an engineered fusion protein of circularly permuted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P440 interleukin-2 (IL-2) and IL-2 receptor α (IL-2Rα) designed to selectively bind the intermediate-affinity IL-2R for selective expansion of natural Background killer (NK) and CD8+ T cells (Figures 1 and 2). Compared with recom- Tumor cells initially rely on glucose consumption via aerobic glyco- binant human IL-2, ALKS 4230 exhibited enhanced pharmacokinetic lytic pathways. However, as tumor cells proliferate and metastasize in and selective pharmacodynamic properties in mice, resulting in im- an increasingly hypoxic tumor microenvironment (TME), tumors in- proved antitumor efficacy [1]. Intravenous dosing of ALKS 4230 is be- creasingly utilize fatty acid oxidation (FAO) as glucose stores are de- ing studied in the ARTISTRY-1 trial of patients with advanced solid pleted. FAO supports both tumor growth and suppressive immune tumors (NCT02799095), which has more than 50 patients enrolled cells in the TME, facilitating tumor progression. PPARα is a ligand- to date [2]. Here, we present a study investigating ALKS 4230 ad- activated nuclear transcription factor which regulates lipid metabol- ministered subcutaneously. Potential advantages of subcutaneous ism, FAO and inflammation. TPST-1120 is a first in class, oral, selective dosing over intravenous include: (i) lower peak serum drug con- PPARα antagonist that blocks transcription of PPARα target genes centrations with a prolonged exposure profile, which may result leading to a metabolic shift from FAO to glycolysis. Antagonism of in a milder safety profile and improved tolerability; (ii) lymphatic FAO in the TME leads to direct killing of tumor cells dependent on absorption, which may facilitate direct immunologic effects; and FAO and facilitates the cytotoxicity of effector cells. Preclinical studies (iii) a more convenient dosing schedule than daily inpatient intra- with various tumor models demonstrate efficacy of TPST-1120 as venous dosing. monotherapy and in combination with anti-PD1 antibodies and Methods chemotherapy. TPST-1120 has an IC50 of 0.04 nM with a >35 fold se- ARTISTRY-2 (NCT03861793) is a phase 1/2 study of ALKS 4230 ad- lectivity over other PPAR isoforms. ministered subcutaneously as monotherapy and in combination Methods with pembrolizumab in patients with advanced solid tumors. The We have initiated a phase 1/1b multicenter, open label trial to evalu- study will be conducted in 2 parts. In the first part (dose escal- ate TPST-1120 as a SA and in combination (combo) with other sys- ation; phase 1), multiple ascending doses of ALKS 4230 will be temic therapies including nivolumab, an anti-PD1 monoclonal administered subcutaneously every 7 days (q7d) or every 21 days antibody; docetaxel, a cytotoxic chemotherapeutic agent and cetuxi- (q21d) during a 6-week lead-in period. Injection site locations will mab, an anti-EGFR monoclonal antibody. The objectives are to 1) include the back of the arm, the thigh, or the abdomen. If the evaluate safety and tolerability of continuous dosing of TPST-1120 2) patient has tolerated ALKS 4230 monotherapy treatment, combin- identify a recommended phase 2 dose (RP2D) 3) evaluate efficacy, ation therapy with pembrolizumab (200 mg) administered as an and 4) evaluate PK/PD parameters. Eligibility criteria: 1) patients with intravenous infusion over 30 minutes q21d will be added to the advanced non-small cell lung, hepatocellular, renal cell, triple- ongoing ALKS 4230 regimen. In the second part (dose expansion; negative breast, urothelial, pancreatic, gastro-esophageal, castration- phase 2), ALKS 4230 will be administered subcutaneously at the resistant prostate, head and neck, or MSS colorectal cancer, or chol- selected recommended phase 2 dose (RP2D) and dosing schedule angiocarcinoma, or sarcoma; and 2) 1-5 prior therapies for metastatic from phase 1 in combination with pembrolizumab in 5 tumor- disease. This phase 1/1b adaptive design is composed of Dose Escal- specific cohorts of patients with non-small-cell lung cancer, small- ation (DEs) and Dose Expansion (DEx) cohorts. DEs consist of 4 arms, cell lung cancer, hepatocellular carcinoma, squamous cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 241 of 272 carcinoma of the head and neck, and squamous cell carcinoma Background of any tissue origin. Additional eligibility criteria for the study in- T-cell activation requires effective co-stimulation along with T-cell re- clude Eastern Cooperative Oncology Group performance status of ceptor (TCR) engagement. CD80 provides a well-characterized costi- 0 to 1 and adequate bone marrow, liver, and kidney function. mulatory signal by binding to CD28 on the surface of T cells. Outcomes include RP2D, safety, pharmacokinetics/pharmaco- Following activation, T cells upregulate CTLA4 on the cell surface dynamics, immunogenicity, and antitumor activity. Efficacy end- which binds to CD80 with higher affinity than CD28, disrupts effect- points include overall response rate, disease control rate, duration ive CD80-CD28 signaling, and inhibits T-cell activation. FPT155 is a of response, time to response, and progression-free survival and novel CD80 extracellular domain-Fc fusion protein that directly in- overall survival at 6- and 12-month milestones. duces T-cell activation and cytokine production by binding to CD28 and de-represses endogenous CD80-CD28 activity in the tumor Acknowledgements microenvironment by binding to CTLA4. FPT155 has potent efficacy The study is sponsored by Alkermes, Inc. Medical writing and editorial in syngeneic preclinical tumor models, including some that are not support was provided by Parexel and funded by Alkermes, Inc. responsive to agents targeting PD-1. FPT155 is not a superagonist as Trial Registration it requires separate, concurrent TCR engagement. ClinicalTrials.gov NCT03861793 Methods FPT155 is being investigated in a multi-center, open-label, first- References in-human phase 1 trial. The dose escalation portion of the trial 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of is currently enrolling patients with advanced solid tumors that ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic have progressed after treatment with available therapies. A tumor models alone and in combination with immune checkpoint minimum anticipated biological effect level (MABEL) based ap- inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. proach was used to select the initial dose in humans. Patients 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, receive a fixed dose of FPT155 every three weeks with single- McDermott DF, et al. Intravenous administration of ALKS 4230 as patient accelerated titration cohorts through the first four dose monotherapy and in combination with pembrolizumab in a phase I levels of 0.07, 0.21, 0.7 and 2.1 mg and a standard 3+3 dose- study of patients with advanced solid tumors. J Clin Oncol. escalation design for the subsequent 7, 21, 42, and 70mg dose 2019:37(Suppl). Abstract TPS2649. levels. The primary objective of the phase 1a portion of the trial Ethics Approval is to determine the recommended dose and evaluate the safety This study was approved by Ethics and Institutional Review Boards (IRBs) at and tolerability of FPT155. all study sites; IRB reference numbers 20182543 (Western IRB), 00006731 Results (Roswell Park Comprehensive Cancer Center), STUDY00000056 As of June 17, 2019, 7 patients have been treated on study with (Georgetown University, MedStar Health Research Institute). FPT155 doses ranging from 0.07-7mg; median age was 58 years, 57% had ECOG PS 1 and median number of prior therapies was 4 (range: 2-8). To date, no dose-limiting toxicities or ≥Grade 3 treatment- emergent adverse events (TEAEs) from causes other than disease progression have been reported. There have been no serious adverse events or ≥grade 3 TEAEs attributed to FPT155 and the only TEAE at- tributed to FPT155 in more than one patient has been fatigue (Gr1, Gr2; 1 pt each). 2/7 patients continue on treatment. Conclusions FPT155 as monotherapy has been well tolerated to date. Enrollment Fig. 1 (abstract P441). ALKS 4230 is a fusion of IL-2 and IL-2Rα of accelerated titration cohorts is complete with dose-escalation in 3+3 cohorts ongoing. Trial Registration ACTRN12618001955202 Ethics Approval The study was approved by IRBs at all participating study sites. P443 Expansion cohorts of non-small cell lung cancer (NSCLC) and castration resistant prostate cancer (CRPC) in COSMIC-021, a phase 1b study of cabozantinib plus atezolizumab 1 1 2 Nick Salgia, PhD , Sumanta Pal, MD , Santiago Ponce Aix, MD , Yohan 3 4 5 6 Loriot , Robert Dreicer , Ulka Vaishampayan, MD , Toni Choueiri , Patrick Fig. 2 (abstract P441). Cell activation by IL-2 and ALKS 4230 7 8 8 9 10 Schöffski , Giri Ramsingh , Amy Liu , Farah Lim , Joel Neal, MD, PhD , Neeraj Agarwal, MD 1 2 City of Hope, Duarte, CA, United States; University Hospital 12 de Octubre, Madrid, Spain; Institut de Cancérologie Gustave Roussy, Villejui, P442 France; University of Virginia School of Medicin, Charlottesville, VA, A phase 1 study of FPT155, a first-in-class CD80 extracellular United States; Karmanos Cancer Institute, Detroit, MI, United States; domain-Fc fusion protein, in patients with advanced solid tumors 2 3 4 5 6 7 Dana-Farber Cancer Institute, Boston, MA, United States; Leuven Jermaine Coward , Hui Gan, MBBS PhD , James Kuo , Michael Millward , 6 7 7 7 8 Cancer Institute, Leuven, France; Exelixis, Alameda, CA, United States; Gary Richardson , Wei Deng , Siddhartha Mitra , Maike Schmidt , Hong 7 8 1 9 10 Barts Cancer Institute, London, United Kingdom; Stanford University Xiang, PhD , Lisa Horvath , Amy Prawira, MD 1 2 11 Medical Center, Standford, CA, United States; Huntsman Cancer St. Vincent’s Hospital Sydney, Darlinghurst, Australia; Icon Cancer Institute, Salt Lake City, UT, United States Centre, Brisbane, Australia; Olivia Newton-John Cancer Center, Correspondence: Nick Salgia (jennifer.humbert@fishawack.com) Melbourne, Victoria, Australia; Scientia Clinical Research, Randwick, 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P443 Australia; Linear Clinical Research, Nedlands, Australia; Cabrini Hospital, Malvern, Australia; Five Prime Therapeutics, Inc, South San Francisco, Background CA, United States; Chris O’Brien Lifehouse, Camperdown, Australia Cabozantinib inhibits tyrosine kinases involved in tumor growth, Correspondence: Amy Prawira (amy.prawira@svha.org.au) angiogenesis, and immune regulation, including MET, VEGFR, RET, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P442 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 242 of 272 ROS1, and TAM family kinases (TYRO3, AXL, MER). Encouraged by with minimal toxicities at the dose levels studied (1×108 HPVSTs/m2) preclinical and clinical studies that suggested that cabozantinib pro- so far. However, most patients remained with disease after infusion. motes an immune-permissive environment, the safety and efficacy of A great challenge for HPVST therapy is to generate more specific and cabozantinib or cabozantinib in combination with atezolizumab are potent HPVSTs as ~30% of our HPVST products failed the potency being evaluated in the COSMIC 021 phase 1b study (NCT03170960) criterion, evaluated by γ-IFN ELISpot assay. The aim of this work was in solid tumors including NSCLC and CRPC. Cabozantinib has demon- to increase the potency and success rate of HPVST manufacturing. strated clinical activity as monotherapy in advanced NSCLC and in Methods previously treated CRPC [1,2]. Here we provide updated trial details The current manufacturing strategy uses peripheral blood mono- for expansion cohorts of NSCLC and CRPC patients. nuclear cells (PBMCs) as starting material for the enrichment and ex- Methods pansion of HPVSTs, in the presence of dendritic cells and cytokines. The dose-escalation stage of this global, open-label trial is com- Either low frequency or anergy of HPVSTs, even in HPV-exposed do- pleted; in the expansion stage, 20 combination cohorts are being en- nors, impede growth, and manufacturing failure is largely attributed rolled at the recommended dose of cabozantinib 40 mg QD PO + to non-specific T cell outgrowth. To overcome this problem, we eval- atezolizumab 1200 mg Q3W IV. uated CD45RA depletion of PBMCs to remove the bulk of non- NSCLC cohorts include patients with: (1) nonsquamous (nsq)NSCLC specific cells (naïve T cells and natural killer (NK) cells). The CD45RA with prior immune checkpoint inhibitor (ICI) therapy (anti–PD-1 or fraction also contains B-cells and T regulatory cells that may inhibit anti–PD-L1); (2) nsqNSCLC without prior systemic anticancer therapy specific outgrowth. We also evaluated the use of an HLA-negative for metastatic disease; (3) EGFR-mutant nsqNSCLC with prior EGFR- universal lymphoblastoid cell line (uLCL), developed in our center, as targeting therapy. An additional exploratory cohort will assess cabo- a co-stimulatory cell line to rapidly expand the cells while maintain- zantinib monotherapy (60 mg) in nsqNSCLC patients with prior ICI ing HPV specificity. therapy. Results CRPC cohorts include patients with: (1) metastatic CRPC adenocarcin- HPVSTs manufactured using CD45RA negative PBMC populations as oma with measurable disease and prior enzalutamide and/or abira- starting material consistently displayed overarchingly higher specifi- terone therapy; (2) high-risk (measurable visceral metastasis or city than HPVSTs manufactured from PBMCs. Interestingly, uLCLs not prostate-specific antigen doubling time of only supported exponential growth of HPVSTs, but increased their The study allows an initial enrollment of 30 patients in each cohort HPV specificity, opening the possibility of producing sufficient with potential for expansion per recommendation by the Study Over- HPVSTs for higher dose levels. We reported successful production sight Committee. Based on preliminary efficacy per RECIST v1.1 and using PBMC from HPV-exposed healthy donors and cancer patients, safety, the original cohorts of nsqNSCLC with prior ICI therapy and and greatly improved HPVST specificity in all. metastatic CRPC adenocarcinoma with measurable disease and prior Conclusions enzalutamide and/or abiraterone therapy are being expanded to 80 These changes will be incorporated in our HPVST manufacturing patients each. protocol with the goal of improving the anti-tumor activity of our The primary endpoint of the expansion stage is the objective re- product. sponse rate for each cohort. Exploratory objectives include correl- ation of tumor and plasma biomarkers and immune cell profiles with References clinical outcome. 1. Centers for Disease Control and Prevention https://www.cdc.gov/cancer/ Trial Registration hpv/index.htm NCT03170960 2. Forastiere AA, Ang KK, Brizel D, et al. Head and neck cancers. J Natl Compr Canc Netw. 2008; 6:646–695. References 3. 11. Greer BE, Koh WJ, Abu-Rustum N, et al. Cervical cancer. J Natl Compr 1. Smith DC, Smith MR, Sweeney C, Elfiky AA, Logothetis C, Corn PG, Canc Netw. 2008; 6:14–36 Vogelzang NJ, Small EJ, Harzstark AL, Gordon MS, Vaishampayan UN. J Ethics Approval Clin Oncol. 2013; 31:412-419. This study was approved by Baylor College of Medicine Institutional Review 2. Drilon A, Rekhtman N, Arcila M, Wang L, Ni A, Albano M, Van Board; approval number H7634, H7666 and HESTIA and HESTIA IND. Voorthuysen M, Somwar R, Smith RS, Montecalvo J, Plodkowski A. Lancet Oncol. 2016; 17:1653-60. P445 Preliminary results of a Phase 1 trial with a personalized P444 neoantigen vaccine (ADXS-NEO) in advanced and refractory cancer The quest for highly potent human papillomavirus-specific T patients 1 2 3 Lymphocytes for Adoptive Immunotherapy of HPV-associated Frank Tsai, MD , Jonathan Goldman, MD , Marc Matrana , Sumitra 4 4 4 4 malignancies Sheeri , John Heyburn , Megan Parsi , Andres Gutierrez, MD PhD , Joel 1 2 2 2 2 Pei Yun Teo, PhD , Sandhya Sharma, BSc , Alex Salyer , Dimitrios Hecht , Joel Hecht 2 2 2 3 1 Wagner , Benjamin Shin , Sachin Thakar , Li-Chun Huang , Shian Jiun Honor Health Virginia Piper Cancer Care, Scottsdale, AZ, United States; 3 2 2 2 Shih , Carlos Ramos , Cliona Rooney, PhD UCLA Jonsson Comprehensive Cancer Center, Paramus, NJ, United 1 3 4 Tessa Therapeutics/ Baylor College of Medicine, Houston, TX, United States; Ochsner Cancer Center, New Orleans, LA, United States; Advaxis 2 3 States; Baylor College of Medicine, Houston, TX, United States; Tessa Inc, Princeton, NJ, United States Therapeutics, Singapore, Singapore Correspondence: Joel Hecht (JRHecht@mednet.ucla.edu) Correspondence: Cliona Rooney (crooney@bcm.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P445 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P444 Background Background ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based im- The human papillomavirus is linked to 42,700 new cases of cancers munotherapy. This vaccine is a bioengineered Lm vector that se- each year [1]. While many HPV-associated cancers can be eradicated cretes an antigen-adjuvant fusion protein consisting of up to 40 by multimodal therapies, recurrent diseases have dismal prognosis unique (personal) neoantigens and a truncated fragment of listerioly- [2,3]. HPV-positive tumors express viral antigens (E6 and E7) that are sin O (tLLO), which has adjuvant properties. Preliminary clinical and recognized by HPV-specific T cells (HPVST). We are evaluating the immunogenicity results from two dose-levels of ADXS-NEO mono- adoptive transfer of ex vivo expanded autologous HPVSTs for the therapy evaluated in the ongoing Phase 1 trial are herein reported. treatment of HPV-positive cancers in a phase I clinical trial (HESTIA). Methods To date, 12 patients have been treated and promising outcomes ADXS-NEO-02 is a phase 1 dose-escalation study of ADXS-NEO mono- have been attained- 1 complete response and 1 partial response, therapy in subjects with advanced and refractory metastatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 243 of 272 microsatellite stable-colon cancer (MSS-CRC), metastatic squamous was also examined. Furthermore, the possibility of combination treatment histology head and neck cancer, and metastatic non-small cell lung of TAS-116 and anti-PD-1 mAb were investigated in animal models cancer (NSCLC). Manufacturing of ADXS-NEO starts with whole ex- Results ome sequencing of each pt-matched normal and tumor samples to TAS-116 significantly reduced Treg cells, particularly effector Treg cells in detect genetic alterations in the coding regions of the genome both peripheral blood and the TME, resulting in augmentation of tumor followed by its production under GMP specifications. ADXS-NEO is in- antigen-specific CD8+ T cells. STAT5, one of the HSP90 client proteins fused intravenously every 3 weeks until disease progression or limit- that is important for Treg cell development, maintenance and function ing toxicity. Main endpoints include safety, tolerability, preliminary was degraded by TAS-116, thereby reducing FoxP3 expression in effector efficacy and immune-correlative data. Treg cells. TAS-116 augmented tumor antigen-specific CD8+ T cells in Results animal models with reduction of Treg cells in the TME. Additionally, com- The turnaround time for manufacturing ADXS-NEO has consistently bination treatment with PD-1 blockade exhibited a far stronger antitumor been 7-8 weeks from biopsy to first dose. Two pts treated at 1X109 effect than either treatment alone. Moreover, in a phase I trial, the com- CFU (dose level 1) experienced dose limiting toxicities (i.e., Gr 3 hyp- bination of an anti-PD-1 mAb and TAS-116 exhibited a notable clinical ef- oxia ± Gr 3 hypotension) within 4 hours of completing the infusion ficacy in patients with microsatellite-stable (MSS) colorectal cancer of the second dose. These acute adverse events were manageable accompanied by effector Treg cell reduction in the TME. and reversible with tocilizumab and/or steroids. A de-escalated dose Conclusions of 1X108 CFU, has been found to be safe, tolerable and immuno- We propose a novel concept to control eTreg cells by targeting a genic in a cohort of 3 pts. ADXS-NEO at both doses induced: 1) acti- Treg cell-critical signaling pathway and the potential as a combin- vation and proliferation of CD4+ / CD8+ T cells; 2) neoantigen- ation therapy with PD-1 blockade. specific T cell responses -including hotspot mutations- after 1 week Trial Registration of the initial priming dose in pooled ELISPot analysis and 3) T cell re- UMIN000032801 sponses to neoantigens found in the pts’ tumor, but not included in Ethics Approval the construct (i.e., antigen spreading). Deconvolution ELISPot data This study was approved by the institutional review boards of the Na- from the first MSS-CRC pts. analyzed, showed T cell responses to tional Cancer Center and was conducted in accordance with ethical 90% of the targets in the ADXS-NEO construct. Two out of 4 initial guidelines, including the Declaration of Helsinki.All mouse experiments pts treated had stable disease. were approved by the Animals Committee for Animal Experimentation Conclusions of the National Cancer Center, Japan, and Taiho Pharmaceutical Co. Ltd. A safe and tolerable dose of ADXS-NEO monotherapy has been established (1X108 CFU) which elicited fast and broad antitumor im- P447 munity, including T cell responses to neoantigens and antigen ALKS 4230, an engineered IL-2 fusion protein, in monotherapy spreading. Enrollment in a combination therapy arm with pembroli- dose-escalation and combination therapy with pembrolizumab in zumab is due to start in 4Q2019. patients with solid tumors: ARTISTRY-1 trial Trial Registration 1 2 Ulka Vaishampayan, MD , Jameel Muzaffar, MD , Vamsidhar Velcheti, MD, NCT03265080 3 4 5 FACP , Christopher Hoimes, DO , Lucy Gilbert, MD , David McDermott, Ethics Approval 6 7 8 9 MD , Anna Spreafico, MD PhD , Quincy Chu, MD , Kelly Curtis, MD , This clinical tria has been performed in accordance with the Declar- 10 10 10 Yangchun Du, PhD , Harald Mackenzie, MB , Lei Sun, PhD , Emily ation of Helsinki and has been approved by appropriate ethics com- 10 10 10 Putiri, PhD , Heather Losey, PhD , Bruce Dezube, MD , Marc Ernstoff, mittee at UCLA LA, Ochsner Cancer Center LA and Honor Health MD Virginia G Piper Cancer Care AX. Barbara Ann Karmanos Cancer Institute, Detroit, MI, United States; 2 3 Moffitt Cancer Center, Tampa, FL, United States; Perlmutter Cancer P446 Center, NYU Langone Health, New York, NY, United States; UH A novel regulatory T Cell-Targeted Immunotherapy by targeting Cleveland Medical Center, Cleveland, OH, United States; Cedars Cancer their crucial signal by HSP90 inhibitors Center, Montreal, QC, Canada; Beth Israel Deaconess Medical Center, Ayaka Tsuge, MD, Yosuke Togashi, MD, PhD, Kohei Shitara, MD, Hiroyoshi Milton, MA, United States; Princess Margaret Cancer Centre, Toronto, Nishikawa, MD, PhD ON, Canada; Cross Cancer Institute, University of Alberta/Alberta Health National Cancer Center, Kashiwa, Japan Services, Edmonton, AB, Canada; Syneos Health, Phoenix, AZ, United 10 11 Correspondence: Hiroyoshi Nishikawa (hnishika@east.ncc.go.jp) States; Alkermes, Inc., Waltham, MA, United States; Roswell Park Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P446 Comprehensive Cancer Center, Buffalo, NY, United States Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P447 Cancer immunotherapy, particularly immune checkpoint inhibitors opened a new era of cancer therapy. Yet, the clinical efficacy is limited Background due to the complexed immune suppressive mechanisms in the tumor ALKS 4230 is an engineered fusion of IL-2 and IL-2Rα designed to se- microenvironment (TME). Regulatory T (Treg) cells, an immune suppres- lectively expand NK and CD8+ T cells (Figures 1 and 2). In preclinical sive subset of CD4+ T cells, are abundant in tumor tissues and play a key studies, ALKS 4230 exhibited enhanced pharmacokinetic and select- role as an immune suppressive mechanism in the TME via inhibiting ef- ive pharmacodynamic properties with improved antitumor efficacy fective antitumor immunity. While various Treg cell-targeted reagents is relative to IL-2 [1]. under development, none of them have not been translated into the Methods clinic due to the difficulty of specific Treg cell depletion in the TME. The ARTISTRY-1 (NCT02799095) is a phase 1/2 study investigating major obstacle to develop effective Treg cell-targeted reagents was the ALKS 4230 as monotherapy and in combination with pembrolizu- lack of molecules specifically expressed by Treg cells in the TME. We mab in adults with advanced solid tumors [2]. For monotherapy therefore focused on the specific signal(s) used in Treg cells in the TME. dose escalation, ALKS 4230 is administered intravenously over 30 Methods minutes once daily for 5 days every 14 or 21 days. For combin- We focused on HSP90 inhibitor, TAS-116 as a Treg cell regulator, especially ation therapy, the same regimen of ALKS 4230 is administered terminally-differentiated effector Treg cells. Peripheral blood mononuclear with pembrolizumab every 21 days in cohorts based on tumor cells (PBMCs) were treated with TAS-116, and the changes in T cell popula- type, prior anti-PD-1 therapy, and rollover from monotherapy. tions including Treg cells were analyzed. In addition, we explored the Outcomes include the monotherapy recommended phase 2 dose mechanism(s) of Treg cell reduction using PBMCs and FoxP3+ T cell lines. (RP2D), safety, pharmacodynamics, and antitumor activity (RECIST The effect of TAS-116 on tumor antigen (NY-ESO-1)-specific CD8+ T cells 1.1). Results of the completely enrolled cohorts of dose-escalation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 244 of 272 phase and of combination therapy in anti-PD-1-unapproved tu- mors as of June 21, 2019, are presented. Results For dose escalation, 36 patients received ALKS 4230 monotherapy ≤6 μg/kg/d. Maximum tolerated dose has not been reached. Most fre- quent adverse events (AEs), regardless of relationship, were pyrexia (75%) and chills (72%); the majority were grades 1 or 2. Grade ≥3 AEs related to ALKS 4230 occurred in 11 patients (31%) and were mainly transient leukopenia. One death from aspiration pneumonia was considered unrelated to ALKS 4230 by the investigator. ALKS 4230 induced dose-dependent increases in circulating NK and CD8+ T cells with minimal, non-dose-dependent effects on regulatory T Fig. 2 (abstract P447). ALKS 4230 structure and activity cells (Tregs). At 3 and 6 μg/kg/d, 8 of 14 patients with evaluable scans had stable disease. One patient with heavily pretreated pancre- atic adenocarcinoma had prolonged stable disease with 6+ months of monotherapy; CA19-9 decreased from 2571 U/mL (pretherapy) to P448 673 U/mL (nadir). Data from 20 patients enrolled in the combination Phase I study of Veliparib and Nivolumab in adults with refractory therapy cohort of PD-1-unapproved tumors indicate no new toxic- advanced solid tumor and lymphoma ities; 7 of 11 patients with evaluable scans had stable disease or bet- Young Kwang Chae, MD, Pedro Viveiros, MD, Sheetal Kircher, Valerie ter. One patient (ovarian cancer) had confirmed partial response; CA- Nelson, Aparna Kalyan, Devalingam Mahalingam 125 normalized from a peak of 282 to 24.5 U/mL after 2 months of Northwestern University, Chicago, IL, United States therapy. Correspondence: Young Kwang Chae (ychae@nm.org) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P448 ALKS 4230 is a promising agent with acceptable tolerability and pre- liminary clinical benefit. It selectively expanded CD8+ T cells and NK Background cells with minimal Treg expansion. The intravenous monotherapy Tumors with high mutation burden often respond to immunother- RP2D was established as 6 μg/kg/d. Safety and pharmacodynamic apy. Veliparib, a Poly (ADP-ribose) polymerase (PARP) inhibitor carries data enabled selection of the 3 μg/kg dose for initial evaluation in anti-neoplastic activity by accumulating DNA damage, possibly en- combination with pembrolizumab. hancing the effect of checkpoint inhibitors. Here we report safety and preliminary efficacy of veliparib and nivolumab combination Acknowledgements from the dose escalation phase of [NCT03061188] clinical trial. The authors would like to thank all the patients who are participating in this Methods study. The study is sponsored by Alkermes, Inc. Medical writing and editorial We conducted a phase I study of veliparib in combination with nivo- support was provided by Parexel and funded by Alkermes, Inc. lumab in chemo-refractory stage IV/ unresectable solid cancer pa- Trial Registration tients. Nivolumab 240mg IV day 1 and 15 q28 days for 4 cycles; ClinicalTrials.gov NCT02799095 480mg IV q28 days Cycle 5 onwards, combined with veliparib start- ing 300mg PO bid 3+3 dose escalation until maximum tolerated References dose (MTD) was established. Treatment continued until disease pro- 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of gression or limiting toxicity. Primary objective was establishing MTD ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic for veliparib, defined as the highest dose causing dose-limiting tox- tumor models alone and in combination with immune checkpoint icity (DLT) in < 2 of 6 patients. Secondary objective was to assess inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. safety, tolerability and early efficacy of combination. 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, Results McDermott DF, et al. Intravenous administration of ALKS 4230 as Nine patients with adequate end-organ function and performance monotherapy and in combination with pembrolizumab in a phase I status were enrolled. Tumor types included colon cancer (n = 3) and study of patients with advanced solid tumors. J Clin Oncol. pancreatic cancer (n = 2). Four patients (44%) had BRCA-related som- 2019:37(Suppl). Abstract TPS2649. atic mutations (BRCA1, BRCA2, ATM and BRIP1). Most common ad- Ethics Approval verse events categorized as possibly or probably related to treatment This study was approved by Ethics and Institutional Review Boards (IRBs) at were fatigue (n = 6, 67%), anemia (n = 5, 56%), nausea (n = 4, 44%) all study sites; IRB reference numbers 16-229 (Dana-Farber Cancer and diarrhea (n = 3, 33%). Grade 3 and 4 events were anemia (n = 3, Institute), MOD00003422/PH285316 (Roswell Park Comprehensive Cancer 33%), fatigue and alkaline phosphatase elevations (n = 2, 22% each), Center), 20160175 (Western IRB), i15-01394_MOD23 (New York University AST, ALT elevations, low platelet count, hypokalemia and hyperten- School of Medicine), STUDY20190090 (Cleveland Clinic), 0000097 sion (n = 1, 11% each). One patient experienced DLT, grade 3 fatigue, (ADVARRA). at dose level 2 (400 mg BiD) and the MTD was established as 400mg bid. Disease control rate after 24 week follow up was 11%. Five pa- tients presented disease progression (56%). One patient withdrew consent at Cycle 3 and another developed limiting fatigue at Cycle 3, both had stable disease (SD). A patient succumbed due to complica- tions of disease before first assessment. One patient with refractory metastatic pancreatic carcinoma harboring a BRIP1 L680fs*9 muta- tion had SD after a 35-week follow-up. Median progression-free sur- vival and overall survival were 9 and 25 weeks, respectively. Conclusions The recommend phase 2 dose of Veliparib is 400mg bid when com- bined with Nivolumab. The side effect profile is on par with the ones Fig. 1 (abstract P447). ALKS 4230 structure and activity previously described for veliparib and nivolumab in monotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 245 of 272 We are expanding the cohort to now include tumors harboring DNA Effects on tumor infiltrating immune cells and molecular signatures repair defects. in correlative biomarker analyses provide insights to ALX148’s mech- Trial Registration anism as a myeloid checkpoint inhibitor. NCT03061188 Ethics Approval Acknowledgements The study was approved by Northwestern University Ethics Board. We would like to thank all of the participating patients and their families as STU00204250. well as site research staff. Trial Registration ClinicalTrials.gov identifier NCT03013218. P449 Pharmacodynamic biomarker characterization of ALX148, a CD47 References blocker, in combination with established anticancer antibodies in 1. Kauder et al., ALX148 blocks CD47 and enhances innate and adaptive patients with advanced malignancy antitumor immunity with a favorable safety profile. PLOS ONE. 2018 1 2 3 Hong Wan, PhD , Laura Chow, MD , Justin Gainor, MD , Nehal Lakhani, 13(8): e0201832 4 5 6 MD, PhD , Hyun Chung, MD, PhD , Keun-Wook Lee, MD , Jeeyun Lee, 2. Lahkani et al., A phase 1 study of ALX148, a CD47 blocker, alone and in 7 8 9 MD, PhD , Patricia LoRusso, DO , Yung-Jue Bang, MD PhD , Stephen combination with established anticancer antibodies in patients with 10 11 1 12 Hodi , Wells Messersmith, MD , Philip Fanning, PhD , Pierre Squifflet , advanced malignancy and non-Hodgkin lymphoma. Journal of Clinical 1 1 1 1 Feng Jin , Tracy Kuo , Sangeetha Bollini , Jaume Pons, PhD , Sophia Oncology 2018 36:15_suppl, 3068-3068 Randolph, MD, PhD 3. Lahkani et al., A phase 1 study of ALX148: CD47 blockade in combination 1 2 ALX Oncology, Burlingame, CA, United States; University of with anticancer antibodies to bridge innate and adaptive immune Washington, Seattle, WA, United States; MGH Cancer Center, Boston, responses for advanced malignancy. Journal for ImmunoTherapy of MA, United States; START Midwest, Grand Rapids, MI, United States; Cancer 2018 6 (Suppl 1):114. Abstract 335. 5 6 Yosei Cancer Center, Seoul, Korea, Republic of; Seoul University 4. Chow et al., A phase I study of ALX148, a CD47 blocker, in combination Bundang Hospital, Seongnam, Korea, Republic of; Samsung Medical with established anticancer antibodies in patients with advanced Center, Seoul, Korea; Yale Cancer Center, New Haven, CT, United States; malignancy. Journal of Clinical Oncology 2019 37:15_suppl, 2514-2514. 9 10 Seoul National University Hospital, Seoul, Korea; Dana Farber Cancer Ethics Approval Center, Boston, MA, United States; University of Colorado Cancer The study was approved by institutional review boards or independent Center, Aurora, CO, United States; International Drug Development ethics committees of participating institutions (approval numbers on file Institute, Brussels, Belgium at ALX Oncology). Correspondence: Hong Wan (Hong@alxoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P449 P450 Background A phase I/IIa, open-label, dose-escalation and expansion study to CD47 is a myeloid checkpoint upregulated by tumor cells to investigate the safety, tolerability, pharmacokinetics and evade immune destruction. ALX148 (A) is a fusion protein com- pharmacodynamics of TJ107 in Chinese patients with advanced prised of a high affinity CD47 blocker linked to an inactive hu- solid tumors 1 1 1 1 1 1 man immunoglobulin Fc region [1]. We have previously shown in Jin Li , Ye Guo , Wei Peng , Junli Xue , Wei Zhao , Xiaoxiao Ge , Liqiong 1 1 1 1 2 the first-in-human clinical trial, that ALX148 is well tolerated in Xue , Wenbo Tang , Li Zhou , Min Zhang , Bingshi Guo , Liping Wang, 2 2 2 2 combination with trastuzumab (T) or pembrolizumab (P) with no MD , Jiyuan Guo , Feifei Cui, PhD , Haiyun Suo 1 2 maximum tolerated dose (MTD) identified [2, 3]. Antitumor activ- Shanghai East Hospital, Shanghai, China; I-Mab Biopharma, Shanghai, ity of ALX148 in combination with T or P was observed in pa- China tients with advanced gastric/ gastroesophageal junction (G/GEJ), Correspondence: Jin Li (lijin@csco.org.cn) head and neck squamous cell carcinoma (HNSCC) and non-small Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P450 cell lung cancer (NSCLC) [4]. The objective of this exploratory analysis was to characterize tumor infiltrating immune cells and Background molecular signatures from tumor biopsies and peripheral blood TJ107, an immuno-oncology agent also known as Hyleukin, is a samples obtained from this trial. T cell amplifier comprising a homodimer of engineered human Methods interleukin-7 (IL-7) fused with Genexine’s proprietary long-acting Patients with HER2-positive malignancy (including G/GEJ cancers pro- platform hybrid Fc. IL-7 is a critical homeostatic factor for T gressed on T + fluoropyrimidine and platinum-based therapy) re- cells, acting on T cells to increase their number, diversity and ceived A+T. Patients with advanced malignancy including NSCLC functionality. TJ107 could play a pivotal role in reconstitution [checkpoint inhibitor (CPI)-resistant/refractory or PD-L1 tumor propor- and reinvigoration of T cell immunity in cancer patients, provid- tion score (TPS) ing unique opportunities for immuno-oncology combination Results strategies. The aim of this study (NCT04001075) is to determine Eighty-two patients received A+T (n=30) or A+P (n=52) as of the safety, tolerability and PKPD profile of TJ107 in Chinese can- April 18, 2019. In dose expansion cohorts (N=60), anticancer ac- cer patients. tivity was observed in response-evaluable patients [G/GEJ (n= Methods 18) 4PR, 5SD; HNSCC (n=19) 3PR, 6SD and NSCLC (n=18) 3SD]. This ongoing study is to evaluate the safety, tolerability, PK profile, Near complete CD47 TO was maintained throughout the dosing and anti-tumor activity of TJ107 in patients with advanced solid tu- interval. No dose-dependent changes were apparent in mors who failed standard therapy. Patients receive TJ107 every 4 peripheral lymphocyte populations. Preliminary results from weeks by intramuscular (IM) injection. Dose escalation is aided by a paired biopsies (n=15) demonstrated increased tumor-associated 3+3 scheme from 240μg/kg to 1200μg/kg. A dose expansion cohort macrophages and lymphocytes in both intra-tumoral and peri- is being planned after the RP2D is determined. Safety is assessed by tumoral regions after treatment with ALX148 combinations. monitoring AEs and the associated grades per NCI CTCAE v5.0. Gene expression signatures of tumor inflammation and immune Tumor response will be assessed per RECIST v1.1. Samples will be cell subsets are being investigated. Results will be updated at collected for PK, PD, ADA, immunophenotyping and TCR repertoire presentation. analysis. Conclusions Results ALX148 demonstrates excellent tolerability with objective responses Three patients with colorectal cancer were enrolled in the first observed in patients with advanced G/GEJ cancer and HNSCC that cohort (240μg/kg).TJ107 was well tolerated and no DLTs were re- have progressed on prior systemic and HER2-targeted therapies. ported during the first cycle at this dose level. The preliminary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 246 of 272 PK results shows that TJ107 was rapidly absorbed and reached limiting toxicities (injection-site reaction [ISR], lymph node pain; serum peak concentration around 24 hours post-dose. TJ107 was ISR). Among all treated patients, the most common adverse slowly cleared from the body and remained detectable in serum events (AEs) were ISR (89.5%), fatigue (39.5%), nausea (28.9%), until Day 14 post-dose. A substantial increase in absolute constipation, and decreased appetite (23.7% each). Nine (23.7%) lymphocyte count (ALC) from baseline was observed and peaked patients had MEDI5083-related ≥G3 events, most commonly ISR. around 3 to 4 weeks post first dose. FACS analysis revealed in- Six (15.8%) patients discontinued due to a MEDI5083-related AE. creases in CD3+, CD4+ and CD8+ T cells. The numeric increase in There were 4 (10.5%) deaths due to AEs (sequential cohort, T cells is consistent with increased Ki67 expression on Day 8 post MEDI5083 5mg, n=2 and MEDI5083 7.5mg, n=1; concurrent co- first dose. There were no notable changes in B cells, monocytes, hort, MEDI5083 4mg, n=1), 3 unrelated to treatment and 1 pos- NK cells, neutrophils, nor Tregs, as expected. sibly related to MEDI5083. The maximum tolerated dose for Conclusions MEDI5083 was 5mg. In the response evaluable population (N=36), Preliminary results from this trial suggest that TJ107 activated a PR was observed (head and neck squamous cell carcinoma; se- IL-7 pathway and expanded T cells in cancer patients in a simi- quential cohort, MEDI5083 3mg; time to response 5.7 months) lar way to data previously reported in healthy subjects. TJ107 and 11 (30.6%) patients had SD (sequential cohort, n=7; concur- exhibits a promising safety and tolerability profile in cancer pa- rent cohort, n=4). Six patients had SD ≥24 weeks. The ORR (95% tients under current dose. These findings support further clinical CI) was 2.8% (0.1–14.5%). MEDI5083 showed dose-dependent investigation. pharmacological activity in the mobilization of peripheral blood B Trial Registration cells, and induced measurable increases in activated proliferative Investigate the Safety, Tolerability, Pharmacokinetics and Pharmaco- Ki-67+ CD8+ T cells in peripheral blood. dynamics of TJ107 in Chinese Patients With Advanced Solid Tumors. Conclusions ClinicalTrials.gov Identifier: NCT04001075 Subcutaneous administration of MEDI5083 caused high rates of injec- Ethics Approval tion site reactions. The toxicity profile does not support further devel- The study was approved by the Ethics Committee Shanghai East Hos- opment of the subcutaneous formulation of this drug. pital's, approval number 2018 (058). Trial Registration ClinicalTrials.gov NCT03089645 Ethics Approval P451 This study was approved by the Institutional Review Board/Independ- First-in-human study of CD40 agonist MEDI5083 in advanced solid ent Ethics Committee at each investigational site participating in the tumors with durvalumab administered sequentially or concurrently study 1 2 3 Ben Tran, MBBS FRACP , Mark Voskoboynik , Johanna Bendell, MD , 4 5 Martin Gutierrez, MD , Charlotte Lemech, MBBS BSc(med) MD(res) , 6 6 7 Daphne Day , Sophia Frentzas , Ignacio Garrido-Laguna , Chris P452 8 8 8 8 DelNagro , Fujun Wang , Charles Ferte, MD, PhD , Mayukh Das , SPEARHEAD-1 trial design: A phase 2, single arm, open-label Benedito Carneiro, MD clinical trial of ADP-A2M4 SPEAR T-cells in patients with advanced 1 2 Peter MacCallum Cancer Center, Melbourne, Australia; Nucleus synovial sarcoma or myxoid/round cell liposarcoma 3 1 2 3 4 Network, Melbourne, Australia; Sarah Cannon Research Institute, Dejka Araujo, MD , Jean-Yves Blay , Sandra Strauss , Claudia Valverde , 4 5 5 5 Nashville, TN, United States; Hackensack University Medical Center, Erin Van Winkle , Malini Iyengar, PhD , Rafael Amado, MD 5 1 2 Hackensack, NJ, United States; Scientia Clinical Research, Sydney, MD Anderson Cancer Center, Houston, TX, United States; Leon Berard, 6 7 3 Australia; Monash Medical Centre, Clayton, Australia; Huntsman Cancer Lyon, France; University College London Hospitals, London, United 8 4 Institute, Salt Lake City, UT, United States; AstraZeneca, San Francisco, Kingdom; Vall D'Hebron University Hospital, Barcelona, Spain; 9 5 CA, United States; The Warren Alpert Medical School, Providence, RI, Adaptimmune, Philadelphia, United States United States Correspondence: Erin Van Winkle (erin.vanwinkle@adaptimmune.com) Correspondence: Mayukh Das (mayukh.das@medimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P452 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P451 Background Background ADP-A2M4 specific peptide enhanced affinity receptor (SPEAR) T-cells MEDI5083 is a homodimeric fusion protein of three single-chain are genetically engineered to target MAGE-A4+ tumors in the con- CD40L domains linked to an immunoglobulin G fragment text of HLA-A*02. MAGE-A4 has been described as having high ex- crystallizable domain, and activates the CD40 pathway to promote pression in synovial sarcoma (SS) and myxoid/round cell liposarcoma immune responses. This first-in-human study evaluated the safety (MRCLS). In recent studies [1, 2], immunohistochemistry (IHC) ana- and clinical activity of MEDI5083 given sequentially or concurrently lyses showed that 82% of SS samples and 68% of MRCLS samples with the PD-L1 antibody durvalumab in patients with advanced solid expressed MAGE-A4. A pilot study (NCT03132922) of ADP-A2M4 in- tumors. duced clinical responses in patients with SS. Methods Methods Eligible patients with metastatic or recurrent tumor types progressing This phase 2, open-label trial (SPEARHEAD-1 Trial) will evaluate the on or refractory to prior therapy were enrolled in multiple cohorts of efficacy, safety and tolerability of ADP-A2M4 SPEAR T-cells. Patients MEDI5083 (3mg, 4mg, 5mg, 6mg, and 7.5mg) administered subcuta- who are HLA-A*02+ (excluding A*02:05, and A*02:07 and A*02 null neously (SC) Q2W for 4 doses. In the sequential-treatment cohort, as sole A*02 alleles), who have advanced/metastatic SS or MRCLS MEDI5083 was followed by a 4-week wash-out, then durvalumab who have received prior chemotherapy, and have MAGE-A4 expres- 1500 mg intravenously (IV) Q4W. In the concurrent-treatment cohort, sion assessed by IHC at ≥2+ in ≥ 30% of tumor cells, and who meet MEDI5083 was administered with concurrent durvalumab 1500 mg IV all other inclusion criteria are eligible for treatment. Up to 60 patients Q4W for 2 doses, followed by durvalumab 1500 mg IV Q4W. The pri- will be treated. mary endpoint was safety. Secondary endpoints included pharmaco- Following apheresis, T-cells are isolated, transduced with MAGE- kinetics, immunogenicity, and efficacy based on investigator- A4c1032TCR, and expanded. Prior to infusion, patients will receive assessed RECIST V1.1. lymphodepletion consisting of fludarabine (30 mg/m2/day x 4 days) Results and cyclophosphamide (600 mg/m2/day x 3 days). Patients will re- As of June 30, 2019, 38 patients were treated; 29 received se- ceive 1 – 10 × 10^9 transduced T-cells. Futility analysis will be con- quential treatment (MEDI5083 3mg, n=4; 4mg, n=4; 5mg, n=18; ducted after 15 patients are dosed and have been followed for at 7.5mg, n=3) and 9 patients received concurrent treatment least 4 months from the time of T-cell infusion. An independent Data (MEDI5083 3mg, n=3; 4mg, n=6). Two patients (sequential cohort, Safety Monitoring Board will review ongoing safety and benefit:risk MEDI5083 5mg and 7.5mg) had MEDI5083-related G3/4 dose- during the interventional phase of the study. Disease will be assessed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 247 of 272 by independent review per RECIST v1.1 by CT/MRI at weeks 4, 8, 12, Results 16, 24, and every 2 months thereafter until confirmed disease pro- The primary objective of sub-study 2 is to evaluate GSK3377794 gression. Once disease progression is established, patients will enter efficacy by overall response rate per RECIST v1.1 (central inde- the long-term follow-up phase of the study, with visits every 6 pendent review). Secondary objectives include: time to and dur- months through Year 5, and annually thereafter for Years 6-15. ation of response; disease control rate; progression-free survival; Trial Registration overall survival; potential immune response to GSK3377794; safety NCT04044768 and tolerability. Exploratory objectives include: correlation of T- cell persistence with safety, clinical responses, and phenotype of References infused T cells. Impact on quality of life and daily functioning will 1. Iura K, et al. Cancer-testis antigen expression in synovial sarcoma: NY- also be assessed. ESO-1, PRAME, MAGEA4, and MAGEA1. Human Pathology 2017a; 61:130- Conclusions 139. Based on the encouraging clinical activity of GSK3377794 observed 2. Iura K, et al. MAGEA4 expression in bone and soft tissue tumors: its utility in earlier trials, this larger clinical trial is being initiated to establish as a target for immunotherapy and diagnostic marker combined with and further discern the efficacy and safety of GSK3377794 in this NY-ESO-1. Virchow Archiv 2017b;471:383–392. biomarker-selected metastatic SS patient population. This innovative Ethics Approval Master Protocol study design permits evaluation of GSK3377794 This trial is under review by the institutional review board of the trial sites treatment in other NY-ESO-1+ tumor types in HLA A*02+ patients within separate sub-studies. P453 Acknowledgements Autologous T cells with NY-ESO-1-specific T-cell receptor Medical writing assistance was provided by Fiona Woodward of Fishawack (GSK3377794) in HLA-A*02+ previously-treated and -untreated Indicia Ltd, UK, funded by GlaxoSmithKline (GSK). This study (NCT03967223) advanced metastatic/unresectable synovial sarcoma: A master was funded by GSK. protocol study design Trial Registration 1 2 3 4 Sandra D'Angelo, MD , Jean-Yves Blay , Warren Chow , George Demetri , NCT03967223 5 6 7 Fiona Thistlethwaite, MD, PhD , Michael Wagner , David Loeb , Steven 8 9 10 Attia , Albiruni Razak , John Haanen, MD PhD , Aisha Hasan, MBBS References 11 11 11 11 11 MD , Julia Billiard , Laura Pearce , Yuehui Wu , Ran Ji , Laura 1. Riedel RF, Jones RL, Italiano A, et al. Systemic anti-cancer therapy in syn- 11 11 11 12 Johnson , Chandra Srinath , Aiman Shalabi , Sandra Strauss , ovial sarcoma: A systematic review. Cancers 2018; 10:E417. 4 13 1 Katherine Thornton , Crystal Mackall, MD , William Tap , Brian Van Tine, 2. Vlenterie M, Litière S, Rizzo E, et al. Outcome of chemotherapy in MD, PhD advanced synovial sarcoma patients: Review of 15 clinical trials from the Memorial Sloan Kettering Cancer Center, New York, NY, United States; European Organisation for Research and Treatment of Cancer Soft Tissue 2 3 Centre Léon Bérard, Lyon, France; City of Hope Comprehensive Cancer and Bone Sarcoma Group; setting a new landmark for studies in this Center, Duarte, CA, United States; Dana-Farber Cancer Institute, Boston, entity. Eur J Cancer 2016; 58:62–72. MA, United States; The Christie NHS Foundation Trust, Manchester, Ethics Approval United Kingdom; Fred Hutchinson Cancer Research Center, Seattle, WA, This Master Protocol will be conducted under approval by the appropriate United States; Montefiore Medical Center, New York, NY, United States; institutional review boards and independent ethics committees. 8 9 Mayo Clinic in Florida, Jacksonville, FL, United States; Princess Margaret Cancer Centre, Toronto, Canada; Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, Netherlands; GlaxoSmithKline, Collegeville, PA, United States; University College London Hospitals, London, United Kingdom; 13 14 Stanford University, Stanford, CA, United States; Washington University in St. Louis, St. Louis, MO, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P453 Background Synovial sarcoma (SS) comprises ~5%–10% of soft-tissue sarcomas [1]. Anthracycline-based chemotherapy is a 1st-line treatment in advanced metastatic/unresectable disease, but response rates are low Fig. 1 (abstract P453). Study Design Methods A clinical trial is underway utilizing a Master Protocol design allowing investigation of GSK3377794 in multiple tumor types (NCT03967223). The first 2 sub-studies are single-arm trials evaluating treatment in previously-untreated (sub-study 1) and previously-treated (sub-study P454 2) HLA A*02+ patients with NY-ESO-1+ metastatic SS. Sub-study 1 is Induction of serum CXCL10 by tebentafusp, a gp100-CD3 a pilot study evaluating efficacy as 1st-line treatment (N=10). Sub- bispecific fusion protein, was associated with survival in uveal study 2 plans to enroll 55 patients with metastatic/locally advanced melanoma in a Phase I/II Study unresectable SS who have progressed following anthracycline-based 1 2 2 Marcus Butler, MD , Brandon Higgs , Cheryl McAlpine, MSN , Joseph chemotherapy. Inclusion criteria include: ≥10 years of age; measur- 3 4 5 Sacco , Jessica Hassel, MD , Shaad Abdullah, MD , Koustubh Ranade, able disease; adequate organ function; ECOG performance status 0– 5 6 PhD , Richard Carvajal, MD 1. Exclusion criteria include: CNS metastases; clinically significant sys- 1 2 Princess Margaret Cancer Centre, Toronto, Canada; Immunocore, Ltd, temic illness; prior gene therapy with integrating vector or NY-ESO-1- Oxfordshire, United Kingdom; Clatterbridge Cancer Centre, Liverpool, specific T cells, vaccine or targeting antibody; prior autoimmune dis- United Kingdom; University Hospital Heidelberg, Heidelberg, Germany; ease or allogeneic hematopoietic stem-cell transplant. Patients will 5 6 Immnuocore, Ltd, Rockville, MD, United States; Columbia University undergo eligibility screening; leukapheresis and manufacture of Medical Center, New York, NY, United States GSK3377794; lymphodepletion and infusion of GSK3377794 followed Correspondence: Brandon Higgs (brandon.higgs@immunocore.com) by safety follow-up and disease assessments; and a 15-year follow-up Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P454 under a separate protocol (Figure 1). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 248 of 272 Background P455 Tebentafusp (formerly IMCgp100) is a TCR–anti-CD3 bispecific fusion A randomized phase 2 study of neoadjuvant talimogene protein targeting melanocyte-expressed gp100 antigen; Phase I/II laherparepvec (T-VEC) plus surgery vs surgery for resectable stage clinical studies showed monotherapy activity in metastatic melanoma IIIB-IVM1a melanoma: 2-year primary analysis of recurrence-free including uveal (UM) [1]. Historical 1-year survival (OS) rate for 2L survival (RFS) 1 2 3 metastatic UM is ~35% [2]. In a phase I clinical study of tebentafusp Reinhard Dummer, MD , David Gyorki , John Hyngstrom, MD , Adam 4 5 6 7 in cutaneous and 19 UM patients, serum IFNg-induced chemokines, Berger, FACS, MD , Robert Conry, MD , Lev Demidov , Anjali Sharma , 7 7 7 8 particularly CXCL10, were induced by tebentafusp and high induced Sheryl Treichel , Kevin Gorski , Abraham Anderson, PhD , Mark Faries , levels correlated with OS and tumor reduction. In a subsequent Merrick Ross, MD 1 2 phase I/II trial (NCT02570308) of 2L UM, we sought to confirm this University Hospital of Zurich, Zurich, Switzerland; Olivia Newton-John and increase mechanistic understanding with blood mRNA analysis. Cancer Centre, Melbourne, Australia; University of Utah Huntsman Methods Cancer Inst, Salt Lake City, UT, United States; Rutgers Cancer Institute of NCT02570308 was conducted in HLA-A*0201+ patients with ad- New Jersey, New Brunswick, United States; University of Alabama vanced 2L UM; this exploratory analysis focused on further investiga- School of Medicine, Birmingham, AL, United States; N.N. Blokhin Russian tion of an initial 40 patient cohort [3]. Intra-patient escalation dosing Cancer Research Ce, Moscow, United States; Amgen Inc, Thousand regimen used low initial dosing at Cycle1, Day1 (C1D1, 20 mcg) and Oaks, United States; John Wayne Cancer Institute, Santa Monica, United C1D8 (30 mcg). From C1D15, 19 patients received between 54-73 States; University of Texas MD Anderson Cancer, Houston, TX, United mcg , 22 patients received 68 mcg (expansion phase). Sera from 18 States and 22 patients in escalation and expansion phases, respectively Correspondence: Reinhard Dummer (Reinhard.Dummer@usz.ch) were profiled pre-treatment and post first and third dose with 11 im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P455 mune markers (Luminex); whole blood from 19 escalation phase pa- tients was analysed for gene expression (NanoString). Low/High Background groups were defined at the median for OS and Mann-Whitney tests Risk of recurrence and death after resection of stage IIIB-IVM1a mel- were used for time contrasts. anoma is high. In the previous 1-year interim analysis, T-VEC plus sur- Results gery demonstrated a pathological complete response rate of 22.8% In an updated analysis of rash (on-target, off-tumor toxicity), 31 of 40 and improved RFS compared to upfront surgery (Dummer et al, patients with Grade 2+ rash had 1-year OS rate ~77%; 1-year OS in ASCO 2019). Here, we present results from the primary 2-year RFS remaining patients was ~35%. Tebentafusp induced a transient re- and biomarker analyses (CT.gov identifier: NCT02211131). sponse in cytokines, reaching maximal changes at 8-24 hours post Methods first and third doses. High levels of CXCL10 induced at first dose cor- Patients with resectable stage IIIB-IVM1a melanoma, ≥ 1 injectable cuta- related with improved OS (HR=0.37 95%CI=[0.15, 0.89]); high induced neous, subcutaneous, or nodal lesions ≥10 mm, and no systemic treat- CXCL9 levels also trended with OS (HR=0.52 95%CI=[0.21,1.3]). ment 3 months prior were randomized 1:1 to 6 doses/12 wks of CXCL9/CXCL10 transcripts increased at the same time points, as did neoadjuvant T-VEC followed by surgery during weeks 13-18 (Arm 1) signatures for neutrophils, type I IFN, and eosinophils (folds>1.5, p<- versus surgery during weeks 1-6 (Arm 2). T-VEC was given at standard 2, p<-1.5, p dosing until surgery, no remaining injectable tumors, or intolerance. Conclusions The primary analysis estimated a between-group difference in 2-yr RFS In this exploratory analysis, high CXCL10 levels induced by tebenta- on the intent-to-treat set. RFS event was defined as the first local, re- fusp associated with improved OS in UM. Tebentafusp reduced CD8+ gional, or distant recurrence or death due to any cause after surgery. and CD4+ gene signatures in the blood and induced systemic cyto- Per protocol, patients who withdrew prior to surgery or had an R1 or kine and gene expression responses, consistent with T cell redirec- R2 resection were counted as an RFS event at randomization. An add- tion and immune activation. Patients who develop tebentafusp- itional analysis calculated RFS from randomization to the date of first induced Grade 2+ rash appear to have better survival than those post-surgery event regardless of surgical margin status. who do not. Results Trial Registration 150 pts were randomized (76 arm 1, 74 arm 2). Median (range) NCT02570308 follow-up time was 31.2 (0.1–49.9) months. 75% in Arm 1 and 93% in Arm 2 had surgery as planned. In the per protocol analysis, 29.5% of References patients in Arm 1 and 16.5% of patients in Arm 2 remained recur- 1. Middleton MR. J Clin Oncol 37, 2019 (suppl; abstr 9523) rence free (HR: 0.75, P=0.07). In the additional analysis, 50.5% of pts 2. Rantala ES, Hernberg M, Kivelä TT. Overall survival after treatment for in Arm 1 and 30.2 % in Arm 2 remained recurrence free (HR: 0.66, P= metastatic uveal melanoma: a systematic review and meta-analysis. Mel- 0.038). 2-year overall survival rates were 88.9% in Arm 1 and 77.4% anoma Res. 2019 Jan 16. doi: 10.1097/CMR.0000000000000575. in Arm 2 (HR: 0.49, P=0.050). In arm 1, T-VEC treatment resulted in a 3. Sato T DOI: 10.1200/JCO.2017.35.15_suppl.9531 Journal of Clinical 3-fold increase (P Oncology 35, no. 15_suppl (May 20 2017) 9531-9531. Conclusions Ethics Approval Neoadjuvant T-VEC improved 2-year RFS and OS in resectable stage This study was in accordance with the Declaration of Helsinki and was IIIB-IVM1a melanoma. T-cell influx and PD-L1 upregulation after T- approved by all IRBs/ethics committees from each clinical sites VEC treatment support a role for the adaptive immune system con- participating in the study. Specific approval numbers can be provided sistent with the mechanisms of action. Additional biomarker results upon request. including clinical correlations will be presented at the congress. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 249 of 272 Trial Registration Ethics Approval CT.gov identifier: NCT02211131 The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Reference Consent 1. Dummer R, et al. Presented at The American Society of Clinical All patients provided written informed consent to participate in the clinical Oncology; May 31–June 3, 2019; Chicago IL, USA. J Clin Oncol 37, 2019 trial. (suppl; abstr 9520) Ethics Approval P457 The study was approved by participating institutions' Ethics Board. Randomized phase II neoadjuvant study: PD-1 inhibitor TSR-042 vs. combination PD-1 inhibitor TSR-042 and Tim-3 inhibitor TSR- P456 022 in borderline resectable stage III or oligometastatic stage IV KEYNOTE-630: phase 3 study of adjuvant pembrolizumab versus melanoma 1 2 2 placebo in patients with high-risk, locally advanced cutaneous Zahra Kelly, DO , Yana Najjar, MD , Hassane Zarour, MD , John Kirkwood, 2 3 2 4 squamous cell carcinoma MD , Suthee Rapisuwon, MD , Hong Wang , Marc Ernstoff, MD , Joseph 1 2 2 5 2 6 Jessica Geiger , Gregory Daniels, MD, PhD , Ezra Cohen, MD , Joy Yang Drabick, MD, FACP, FIDSA , Diwakar Davar, MD , Mohan Bala 3 3 3 1 Ge , Burak Gumuscu, MD PhD , Ramona Swaby, MD , Anne Lynn University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 4 2 3 Chang University of Pittsburgh, Pittsburgh, PA, United States; Georgetown 1 2 4 Cleveland Clinic, Cleveland, OH, United States; University of California, University, Washington, DC, United States; Roswell Park Comprehensive 3 5 San Diego, La Jolla, CA, United States; Merck & Co., Inc., Kenilworth, NJ, Cancer Center, Buffalo, NY, United States; Penn State Cancer Institute, 4 6 United States; Stanford University Medical Center, Stanford, CA, United Palmyra, PA, United States; Tesaro, Inc, Wltham, MA, United States States Correspondence: Diwakar Davar (davard@upmc.edu) Correspondence: Jessica Geiger (GEIGERJ@ccf.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P457 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P456 Background Background Neoadjuvant PD-1 blockade produces pathological responses in Despite undergoing current standard-of-care surgical resection and ~30% of patients (pts) with high-risk resectable melanoma (MEL) adjuvant radiotherapy, ~20% of patients with high-risk, locally ad- with durable relapse-free benefit, and increased circulating acti- vanced cutaneous squamous cell carcinoma develop local recurrence vated CD8+ T cells (1,2). TIM-3 is an inhibitory immune check- within 5 years [1]. Recent data show effective antitumor activity and point and mediates immune escape; blockade of which produces acceptable safety of programmed death 1 inhibitors in patients with anti-tumor immune responses and synergizes with anti-PD-1 (3– locally advanced or metastatic cutaneous squamous cell carcinoma. 5). TSR-042/dostarlimab is an IgG4 humanized monoclonal anti- KEYNOTE-630 (NCT03833167), a randomized, double-blind, placebo- body that binds with high affinity to PD-1, inhibiting binding to controlled phase 3 trial, will evaluate the efficacy and safety of adju- PD-L1 and PD-L2. TSR-042 has been studied in patients with ad- vant pembrolizumab in patients with high-risk locally advanced or vanced non-small cell lung (NSCLC) and endometrial cancers with metastatic cutaneous squamous cell carcinoma. promising results (6). TSR-022 is an IgG4-k isotype humanized Methods monoclonal antibody that binds with high affinity to TIM-3, thus Patients with high-risk locally advanced cutaneous squamous cell enhancing T cell activity. TSR-042/TSR-022 combination has been carcinoma who have undergone surgical resection and radiother- studied in a phase I/II study that demonstrated promising efficacy apy will be randomly assigned 1:1 to intravenous pembrolizumab of combination in PD-1 refractory melanoma and NSCLC (7). We (400 mg every 6 weeks) or placebo for up to 9 cycles (~1 year) hypothesized that neoadjuvant therapy with TSR-042/TSR-022 or until disease recurrence, unacceptable toxicity, or investigator combination may improve pathologic response rates compared to or patient decision to withdraw. Randomization will be stratified TSR-042 monotherapy in high-risk resectable MEL. by extracapsular extension (yes vs no), cortical bone invasion (yes Methods vs no), and prior systemic therapy (yes vs no). Eligible patients Pts with regionally advanced (stage IIIB-D) or oligometastatic are adults with histologically confirmed locally advanced cutane- (stage IVA-B) melanoma who have yet to undergo definitive sur- ous squamous cell carcinoma with ≥1 high-risk features at the gery are eligible. Primary endpoints are rate of major pathologic primary site of malignancy and macroscopic resection with or response (MPR), safety, and incidence of dose-limiting toxicities without microscopic positive margins who completed adjuvant (DLT). Secondary endpoints are radiographic response, relapse- radiotherapy, were disease free ≤28 days from randomization, free survival (RFS) and overall survival (OS). Pathological response and have Eastern Cooperative Oncology Group performance sta- will be assessed depending on residual volume of tumor (RVT) tus 0 or 1. The primary efficacy end points are investigator- using prior cutoffs: 0% (complete response, pCR); 0%<RVT< assessed and biopsy-confirmed recurrence-free survival. Secondary RVT50% (non-response, pNR) (8–10). Sample size is 28 patients end points are overall survival, health-related quality of life, and per arm. Patients will receive neoadjuvant therapy for 6 weeks safety. To assess treatment response, radiographic imaging will prior to planned surgery. Surgery will occur 1-3 weeks after com- be performed at least every 12 weeks in year 1, then every 6 pletion of pre-operative therapy. After surgery, subjects will re- months until the end of year 5. All patients meeting crossover or ceive additional maintenance TSR-042 for approximately 48 weeks retreatment criteria at first disease recurrence may receive pem- (Figure 1). Sample size provides 80% power to detect an im- brolizumab 400 mg every 6 weeks for up to 18 cycles. Adverse provement of 30% upon MPR rate of 30% with anti-PD-1 events will be recorded until 30 days (90 days for serious adverse monotherapy. events) after study end and will be graded per NCI CTCAE v4.0. Enrollment of ~570 patients is planned, and recruitment is on- References going in 18 countries. 1. Amaria, R. N. et al. Neoadjuvant immune checkpoint blockade in high- Trial Registration risk resectable melanoma. Nature Medicine 24, 1649–1654 (2018). ClinicalTrials.gov, NCT03833167 2. Huang, A. C. et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nature Medicine 25, 454–461 Reference (2019). 1. Porceddu SV, Bressel M, Poulsen MG, et al. Postoperative concurrent 3. Fourcade, J. et al. Upregulation of Tim-3 and PD-1 expression is asso- chemoradiotherapy versus postoperative radiotherapy in high-risk cuta- ciated with tumor antigen–specific CD8+ T cell dysfunction in melan- neous squamous cell carcinoma of the head and neck: the randomized oma patients. The Journal of Experimental Medicine 207, 2175–2186 phase III TROG 05.01 trial. J Clin Oncol. 2018;36:1275-1283. (2010). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 250 of 272 4. Chauvin, J.-M. et al. TIGIT and PD-1 impair tumor antigen–specific CD8+ signals and tumor antigens that can be captured and processed by T cells in melanoma patients. Journal of Clinical Investigation 125, 2046– IT co-administered CD1c (BDCA-1)+ myDC, reinvigorating the cancer 2058 (2015). immunity cycle. 5. Woo, S.-R. et al. Immune Inhibitory Molecules LAG-3 and PD-1 Synergis- Methods tically Regulate T-cell Function to Promote Tumoral Immune Escape. Patients with advanced melanoma who failed standard therapy were Cancer Research 72, 917–927 (2012). eligible for IT injections of ≥1 non-visceral metastasis with T-VEC 6. Moreno, V. et al. Abstract CT053: Preliminary safety, efficacy, and PK/PD (10^6 PFU/mL; max total volume of 4 mL) on day 1 followed by IT in- characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 jection of autologous, non-substantially manipulated CD1c (BDCA-1)+ monoclonal antibody, TSR-042, in patients with recurrent or advanced myDC on day 2. Injection of T-VEC (10^8 PFU/mL; max total volume NSCLC and MSI-H endometrial cancer. Cancer Research 78, CT053–CT053 of 4 mL) was repeated on day 21 and every 14 days thereafter. Pa- (2018). tients were treated with 0.5x10^6, 1x10^6, or 10x10^6 CD1c (BDCA- 7. Davar, D. A phase 1 study of TSR-022, an anti-TIM-3 monoclonal anti- 1)+ myDC in cohort-1, -2, and -3, respectively. Primary objectives body, in combination with TSR-042 (anti-PD-1) in patients with colorectal were safety and feasibility. Repetitive biopsies of treated lesions were cancer and post-PD-1 NSCLC and melanoma. (2018). performed. 8. Cottrell, T. et al. Pathologic Features of Response to Neoadjuvant Anti- Results PD-1 in Resected Non-Small Cell Lung Carcinoma: A Proposal for Quanti- In this ongoing trial, 2 patients were treated in cohort-1, 2 patients in tative Immune-Related Pathologic Response Criteria (irPRC). Annals of on- cohort-2, and 3 patients in cohort-3. Patients received a median of 6 cology : official journal of the European Society for Medical Oncology (range 3-10) injections of T-VEC. All patients are evaluable for re- (2018). doi:10.1093/annonc/mdy218 sponse. The best overall tumor response (according to iRECIST) was a 9. Stein, J. et al. Major pathologic response on biopsy (MPRbx) in CR (pathologic CR) and one PR (confirmation pending; pathologic CR patients with advanced melanoma treated with anti-PD-1: evidence of treated lesions). Both patients were treated in cohort-3 and had for an early, on-therapy biomarker of response. Annals of oncology : previously progressed on anti-PD-1 checkpoint inhibition, and one official journal of the European Society for Medical Oncology 30, patient also on anti-CTLA-4 therapy. Adverse events include G1 fever 589–596 (2019). in 4 patients, G1-2 flu-like symptoms in 5 patients, transient G1-2 10. Tetzlaff, M. et al. Pathological assessment of resection specimens after local pain and redness at the injection-site in 3 patients, and G1 neoadjuvant therapy for metastatic melanoma. Annals of oncology : gastrointestinal symptoms in 4 patients. The patient with CR devel- official journal of the European Society for Medical Oncology 29, 1861– oped an asymptomatic G3 eosinophilia during treatment; the patient 1868 (2018). with PR developed a transient purpuric rash at the site of skin metas- tases after the first treatment. Multiplexed immune-profiling (Ultivue) of baseline and on-treatment tumor biopsies is ongoing. Conclusions IT co-injection of autologous CD1c (BDCA-1)+ myDC with T-VEC is feasible and tolerable and resulted in encouraging early signs of anti- tumor activity in patients with immune checkpoint inhibitor refrac- tory melanoma who received high dose CD1c (BDCA-1)+ myDC. Trial Registration ClinicalTrials.gov: NCT03747744 Ethics Approval This study was approved by the Ethics Board of Universitair Zieken- huis Brussel. Consent Written informed consent was obtained from the patient for publica- tion of this abstract and any accompanying images. A copy of the written consent (NL,FR) is available for review by the Editor of this journal. P459 New generation chimeric antigen receptor T-Cell Therapy Fig. 1 (abstract P457). See text for description (CoupledCAR) induces high rate remissions in solid tumor Chengfei Pu, Lei Xiao Innovative Cellular Therapeutics, Shanghai, China Correspondence: Lei Xiao (xiaolei@ictbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P459 P458 Background A phase I clinical trial on intratumoral administration of Conventional CAR T cell therapy showed weak CAR T expansion in autologous CD1c (BDCA-1)+ myeloid dendritic cells plus patients, thus achieved no or little response for treating solid tu- talimogene laherparepvec (T-VEC) in patients with advanced mors. Here, we generated "CoupledCAR" T cells including an anti- melanoma TSHR CAR molecule. Compared with conventional CART cells, "Cou- Julia Katharina Schwarze, MD, MSc, Gil Awada, Louise Cras, Inès Dufait, pledCAR" T cells successfully improved the expansion of CART cells Ramses Forsyth, Ivan Van Riet, Bart Neyns more than 100 times and enhanced CAR T cells’ migration ability, UZ Brussel, Brussels, Belgium allowing the CAR T cells to resist and infiltrate the tumor microenvir- Correspondence: Julia Katharina Schwarze onment and killed tumor cells. (juliakatharina.schwarze@uzbrussel.be) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P458 We designed a “CoupledCAR” lenti-vector containing a scFv targeting hTSHR. Patient‘s CD3 T cells were isolated and transduced with the Background lentivirus. Then,transduction efficiency was evaluated. After infusion, Intratumoral (IT) myeloid dendritic cells (myDC) play a pivotal role in peripheral blood samples were collected to analyze expansion and initiating antitumor immune responses and re-licensing of anti-tumor cytokine release. The evaluation of response level for patients were cytotoxic T-lymphocytes within the tumor microenvironment. IT in- performed at month 1,month 3,and month 6 by PET/CT. jection of the oncolytic virus T-VEC leads to the release of maturation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 251 of 272 Results and documented D0 prostate cancer whose PSADT is between 3 and To verify the effect of “CoupledCAR” T cells on solid tumors, we have 15 months are eligible. The participant should have normal organ func- completed several clinical trials for different solid tumors, including tions, ECOG 0-1. Patient should not be on any other cancer treatment two patients with thyroid cancer. Immunohistochemistry (IHC) results at enrollment. The primary objective is to assess the PSA slope log showed thyroid stimulating hormone receptors (TSHR) were highly change at week 24 and 48 compared to baseline. Secondary objectives expressed in thyroid cancer cells. In vitro co-culture experiments are 1) to assess the safety of ME-TARP DC vaccines 2) To characterize showed TSHR CAR T cells specifically recognized and killed TSHR- cellular and humoral immune responses associated with vaccination. positive tumor cells. Animal experiments showed TSHR CAR T cells Results inhibited the proliferation of TSHR-positive tumor cells. Therefore, we The lead-in safety cohort (N=6) completed the treatment without designed "CoupledCAR" T cells expressing a binding domain against DLT and we are currently enrolling the randomized arms (N=66). TSHR. Further,we did clinical trials of two group patients that were Conclusions successfully treated using conventional TSHR CAR T cells and "Cou- Multi-Epitope TARP Peptide Autologous Dendritic Cell Vaccination in pledCAR" T cells, respectively. In the group using conventional Men with Stage D0 Prostate is safe and open for further accrual in TSHR CAR T cells, patients showed weak cell expansion and less mi- randomized arms to compare vaccine versus placebo. gration ability. In the group using TSHR "CoupledCAR" T cells, pa- tients showed rapid expansion of CAR T cells and killing of Acknowledgements tumor cells. One month after infusion (M1), the patient was evalu- This study is supported by the Center for Cancer Research, National Cancer ated as PR(Partial Response): the lymph node metastasis disappeared, Institute, National Institute of Health. and thoracic paratracheal tumors decreased significantly. Three Trial Registration months after infusion (M3), the patient was evaluated as a durable NCT02362451 response, and the tumor tissue was substantially smaller than M1. Further, two patients with colorectal cancer were enrolled in this References trial and infused "CoupledCAR" T cells. One patient achieved PR and 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein the other one achieved SD. expressed in prostate and breast cancer cells derived from an alternate Conclusions reading frame of the T cell receptor gamma chain locus. Proc Natl Acad “CoupledCAR” T cells can effectively promote expansion, migration Sci U S A. 2000;97(17):9437–9442. and killing ability of CAR T cells in patients with thyroid cancer. “Cou- 2. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated pledCAR” T cell technology is a technological platform, which may with slowing in PSA velocity and decreasing tumor growth rates in be used to treat other cancer types. Next, we are recruiting more pa- patients with StageD0prostatecancer. Oncoimmunology. tients for clinical trials using “CoupledCAR” T cells. 2016;5(8):e1197459. Ethics Approval The study was approved by National Cancer Institute Ethics Board, approval P460 number P121083 and assigned a local number 15C0075. A randomized, placebo-controlled phase II study of multi-epitope TARP peptide autologous dendritic cell vaccination in men with stage D0 prostate cancer P461 1 3 1 Hoyoung Maeng, MD , Lauren Wood, MD , Seth Steinberg , David Phase 1b study of INCMGA00012, a programmed cell death-1 (PD- 2 1 1 Stroncek, MD , Masaki Terabe, PhD , Jay Berzofsky, MD, PhD 1) inhibitor, in combination with chemotherapy in patients with 1 2 National Cancer Institute, Bethesda, MD, United States; National advanced solid tumors (POD1UM-105) 3 1 2 2 Institute of Health, Bethesda, MD, United States; PDS Biotechnology, David Planchard, MD, PhD , Jill Bowman , Nawel Bourayou, MD 1 2 Berkeley Heights, NJ, United States Gustave Roussy Institute, Villejuif, France; Incyte Corporation, Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) Wilmington, DE, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P460 Correspondence: David Planchard (david.planchard@gustaveroussy.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P461 Background TARP is a 58 amino acid protein expressed in normal and malignant Background prostate tissue [1]. TARP is highly expressed in 95% of prostate cancers Background: The combination of PD 1/programmed death-ligand 1 including all Gleason types and in both castration sensitive (CSPC) and (PD-L1) checkpoint inhibitors with chemotherapy has proven clinic- castration resistant prostate cancer (CRPC). In the pilot study of 1st gen- ally meaningful efficacy and manageable safety profile based on sev- eration TARP peptide-pulsed autologous dendritic cell vaccination eral randomized trials in treatment-naive advanced non-small cell (TARP DC vaccine, NCT00972309, N= 41) utilizing TARP WT 27-35 and lung cancer (NSCLC) [1,2,3] and encouraging data are emerging in epitope enhanced EE29-37-9V in HLA-A*0201 positive men with stage unresectable advanced malignant pleural mesothelioma (MPM) [4]. D0 prostate cancer (PSA biochemical recurrence) published by Wood et INCMGA00012 is an investigational humanized immunoglobulin G4 al, the vaccine was found to be immunogenic and safe [2]. TARP vaccin- (IgG4) monoclonal antibody against human PD-1. In the phase 1 ation was also associated with a decreased slope log PSA in more than POD1UM-101 study, INCMGA00012 has demonstrated acceptable tol- 70 % of the patients at 24 and 48 weeks and a decrease in calculated erability with clinical activity observed in multiple tumor types, in- tumor growth rate constant. Standard of care in D0 prostate cancer cluding a confirmed objective response rate (by Response Evaluation ranges from watchful waiting, salvage radiation and anti-androgen Criteria in Solid Tumors [RECIST] version1.1) of 19% in an interim ana- therapy without strong evidence to support one or the other. In the lysis of a cohort of patients with platinum-refractory NSCLC [5]. The current study of multi-epitope (ME)-TARP DC vaccines, 5 overlapping POD1UM-105 trial aims to investigate INCMGA00012 in combination 18-20-mer peptides encompassing the full sequence of TARP are added with standard-of-care chemotherapy regimens in patients with ad- to 1st generation TARP peptides to pulse the autologous DCs. The use vanced NSCLC or MPM. of synthetic long peptides can increase the chance of a durable multi- Methods valent anti-TARP response. Methods: POD1UM-105 is a phase 1b, global, multicenter study, in pa- Methods tients with histologically or cytologically confirmed advanced/meta- This is a single-blinded, randomized, placebo-controlled phase II study static NSCLC or unresectable MPM and regardless of PD-L1 expression, in men with Stage D0 prostate cancer. Men with a PSADT between 3 not previously treated with systemic therapy. Key eligibility criteria in- and 15 months will be randomized 2:1 to receive a ME TARP DC vac- clude no prior systemic treatment (except for patients [with known sen- cine or a monocyte placebo. HLA restriction is not required. Patients sitizing mutations] who have disease progression on or following an will receive a total of 6 doses of vaccine or a placebo, 20e6 cells/dose approved targeted tyrosine kinase inhibitor, or chemotherapy com- intradermally every 3 weeks. Males older than 18 with adenocarcinoma pleted > 6 months before enrollment), no prior checkpoint inhibitor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 252 of 272 therapy, measurable or nonmeasurable disease by RECIST version 1.1, Combination Immunotherapies and Eastern Cooperative Oncology Group performance status ≤1. P462 Patients will be assigned to 1 of 4 treatment groups (12-24 patients ImmTAC®-chemotherapy combination: A preclinical evaluation each), and will receive INCMGA00012 every 3 weeks for up to 2 years shows potential benefits in combination with standard doses of chemotherapy agents (4 to 6 Filipa Bravo-Lopes, Nora Rippaus, Kristina Petrovic, Francesca Amicarella, cycles) (Treatment Group A: gemcitabine/cisplatin; Group B: peme- Adam Taylor, Laure Humbert, Rupert Kenefeck, Adel Benlahrech, BSc trexed/cisplatin; Group C: pemetrexed/carboplatin; Group D: pacli- PhD taxel/carboplatin) (Figure 1). Immunocore Ltd, Abingdon, United Kingdom The primary study objective is to evaluate safety, tolerability (DLTs), and Correspondence: Rupert Kenefeck (rupert.kenefeck@immunocore.com); determine a recommended phase 2 dose of INCMGA00012 in combin- Adel Benlahrech (adel.benlahrech@immunocore.com) ation with chemotherapy. Secondary objectives include determining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P462 preliminary clinical activity (measured by objective response rate, dur- ation of response, and disease control rate) and pharmacokinetics. Ex- Background ploratory objectives include assessment of additional efficacy measures ImmTAC molecules are bispecific T cell redirectors comprised of an (progression-free survival and overall survival), and relevant biomarkers. affinity‐enhanced TCR recognizing tumour antigen in the context of HLA molecules and a T cell engaging anti‐CD3 domain. Paclitaxel is a Acknowledgements chemotherapy drug which stabilises microtubules and it is widely This study is sponsored by Incyte Corporation (Wilmington, DE). used in combination with platinum-based chemotherapies against Trial Registration multiple indications. Building on the recent evidence that chemo- NCT03920839 therapy positively combines with immunotherapy [1], this study was designed to evaluate the potential of combining Paclitaxel with References ImmTAC-mediated T cell activation. 1. Gandhi L, Rodriguez-Abreu D, Gadgeel S, et al. Pembrolizumab plus Methods chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. In vitro assays to assess T cell-mediated lysis, T cell expansion, 2018;378:2078-2092. and cytokine release in response to ImmTAC-mediated T cell re- 2. Langer CJ, Gadgeel SM, Borghaei H, et al. Carboplatin and pemetrexed direction were performed with a range of ImmTAC concentrations with or without pembrolizumab for advanced, non-squamous non-small- and/or Paclitaxel. T cell-mediated lysis of antigen positive cells cell lung cancer: a randomised, phase 2 cohort of the open-label was monitored in real time while Granzyme A and B, IL-2, IL-8, KEYNOTE-021 study. Lancet. 2016;17:1497-1508. IL-10, IFN-γ, TNF-α,MIP-1α, IP-10, and MIG were measured in cell 3. Socinski MA et al. Atezolizumab for first-line treatment of metastatic non- culture supernatants. T cell expansion was measured by flow squamous NSCLC. N Engl J Med. 2018;378:2288-2301 cytometry. 4. Nowak AK, Lesterhuis WJ, Hughes BGM, et al. DREAM: a phase II study of Results durvalumab with first line chemotherapy in mesothelioma─first results. J ImmTAC molecules were highly effective at redirecting T cells to Clin Oncol. 2018;36(suppl):8503 proliferate, produce pro-inflammatory cytokines, chemokines and 5. Mehnert JM, Joshua AM, Lakhani N, et al. First-in-human phase 1 study of granzymes, and to lyse antigen positive targets. ImmTAC- INCMGA00012 in patients with advanced solid tumors: interim results of the co- Paclitaxel combination showed substantially enhanced lysis of tar- hort expansion phase. J Immunother Cancer. 2018;6(suppl 1):115. Abstract P669. get cells compared to either agent alone (in both magnitude and Ethics Approval kinetics). With 100 pM ImmTAC and 25 nM Paclitaxel, the median The study was approved by institutional review boards or independent cytolysis area under the curve using the compounds in combin- ethics committees of participating institutions. ation was 6269 compared to 3782 for Paclitaxel alone or 3196 for ImmTAC alone. Additionally, the time it took to lyse 80% of target cells decreased from 84 hours for ImmTAC alone to 51 hours when in combination with Paclitaxel. Despite enhanced kill- ing, some reduction of cytokine release and T cell proliferation were observed when ImmTAC and Paclitaxel were combined. Not- ably, cytokine secretion and T cell proliferation were restored, and improved killing maintained by pre-treating target cells with Paclitaxel. Similarly, pre-treatment of effector T cells with Pacli- taxel did not impact their ability to kill and proliferate in re- sponse to ImmTAC. Conclusions These in vitro studies show enhanced ImmTAC-mediated tumour cell lysis with Paclitaxel when administered either in combination with ImmTAC or sequentially. These data support the premise that Pacli- taxel positively combines with ImmTAC and provide strong rationale for further clinical investigation. Reference 1. Liu SV, Camidge DR, Gettinger SN, Giaccone G, Heist RS, Hodi FS, Ready NE, Zhang W, Wallin J, Funke R, Waterkamp D, Foster P, Iizuka K, Powderly J. Long-term survival follow-up of atezolizumab in combination with platinum-based doublet chemotherapy in pa- tients with advanced non-small-cell lung cancer. Eur J Cancer. 2018;101:114-122. Fig. 1 (abstract P461). See text for description Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 253 of 272 P463 melanoma efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine, correlating Immunotherapy combinations for betel-nuts related HNSCC: one with increases of T-cells and CD8α+ DCs institutional experiences in Taiwan Methods Jo-Pai Chen, MD, Ruey-Long Hong, MD, PhD, Wei-Chen Lu, MD Utilizing the B16F10 syngeneic mouse melanoma model, vaccina- National Taiwan University Hospital, Taipei, Taiwan, Province of China tions are administered by intramuscular electroporation with CpG ad- Correspondence: Ruey-Long Hong (rlhong@ntu.edu.gov.tw) juvant three times at one-week intervals beginning five days post Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P463 lethal tumor implantation. Aza is given intraperitoneally at 1mg/kg, and IFNα therapy is given in a series of one high followed by three Background low doses, as noted. Tumor sizes, growth, and survival were all Betel-nuts chewing might contribute to (1) strong inflammation, in- assessed. Tumor-infiltrating lymphocytes (TILs) were assessed by vasion, and angiogenesis; (2) poor response to traditional therapeis. stimulating the purified lymphocyte fraction of tumors with vaccine In the 1st line setting of R/M HNSCC, EPF offers survival benefit and antigens followed by intracellular cytokine staining flow cytometry. immune checkpoint inhibitor(anti-PD1 monoclonal antibody), like Dendritic Cells were assessed by flow cytometry. nivolumab or pembrolizumab, has already brought survival benefit in Results the 2nd line treatment. We demonstrate that the addition of IFNα and Aza treatments to Methods mice vaccinated with the MIP-3α-Gp100-Trp2 vaccine led to signifi- From 2016 to early 2019, 30 R/M HNSCC patients receiving cantly reduced tumor burden and overall increases in mouse survival, immunotherapy-containing regimens in Yun-lin Branch of National increasing median survival by 39% over vaccine. Importantly, this in- Taiwan University Hospital were reviewed. crease in efficacy was dependent on the presence of all three com- Results ponents, as vaccine plus IFNα or vaccine plus Aza did not differ These patients consisted of 1 HPV and 29 non-HPV; 18 pembrolizu- significantly from vaccine alone. The addition of Aza and IFNα to the mab and 12 nivolumab; 10 with afatinib(6 pembrolizumab & 4 nivo- vaccine increased T-cell tumor infiltration, altered the proportion of lumab); 5 with bevacizumab; 6 with chemotherapy. The objective CD8+T-cells, and increased CD8α+ DC infiltration. response rate was 47%(14/30) and clinical benefit was 80%(24/30). Conclusions 16 patients were still under use(4 afatinib with pembrolizumab; 4 afa- Efficient targeting of antigen to immature dendritic cells with a tinib with nivolumab). 1 patient under afatinib and pembrolizumab chemokine-fusion vaccine offers a potential alternative approach presented hyperprogression but then got pCR after bevacizumab to classic and dendritic cell-based vaccines currently undergoing combined with strong chemotherapy. 1 patient under low dose nivo- clinical investigation. Combining this approach with IFNα and Aza lumab in 20 mg biweekly with oral metronomic cyclophosphamide treatments significantly improved vaccine efficacy, with efficacy had mild tumor response. 1 patient under nivolumab with high dose correlating with changes in TILs and tumor infiltrating DCs. Fur- ifosfamide developed nephritis. 1 patient had rapid skin metastasis ther potential therapy optimization currently undergoing investi- over previous radiation fields after pembrolizumab, bevacizumab, gation offers promise for this line of investigation to become a and chemotherapy. 3 patients under afatinib & pembrolizumab de- novel melanoma therapy. veloped autoimmune cholestasis(2 also with pneumonitis). Afatinib & Ethics Approval nivolumab had similar efficacy but less toxicity. 10 pateints receiving All procedures performed in studies involving animals were in ac- afatinib combined with anti-PD1(8 faliling EPF, 7 with pleural/pericar- cordance with the ethical standards of the IACUC of the Johns Hop- dial/skin metastasis. 5 rapid progression within 3 months after defin- kins University under Protocols #MO16H147 and MO19H319. ite CCRT) had 70% response rate(7/10) and 90% clincial benefit(9/10). Post-progression use of anti-PD1 with other treatments were seen in P465 4 patients(esp. 1 with nivolumab & ipilimumab for sarcomatous Immune-mediated mechanisms involved in the synergistic anti- change). 3 patients got benefits and had longer survival. tumor efficacy of NHS-IL12 combined with the class I HDAC Conclusions inhibitor entinostat Immunotherapy-containing combinations are of clinical significance Kristin Hicks, PhD, Yohei Ozawa, MD, PhD, Karin Knudson, PhD, Jeffrey in refractory betel-nuts related HNSCC in Taiwan. Afatinib has several Schlom, Sofia Gameiro, PharmD, PhD immuno-modulatory effects. In high risk patients(pleural/pericardial/ National Cancer Institute, NIH, Bethesda, MD skin metastasis failing EPF and rapid progression within 3 months Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) after definite CCRT) in Taiwan, afatinib with anti-PD1 may be a good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P465 option to avoid hyperprogression. Earlier use, well biomarkers, best combinations, and optimal sequencing will be future goals. Background Combining epigenetic agents with immunotherapy has shown P464 clinical promise. Preclinically, we and others have shown that the IFN-α and 5-Aza-2’-deoxycytidine enhance the anti-tumor efficacy class I histone deacetylase (HDAC) inhibitor entinostat can po- of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by tentiate the anti-tumor efficacy of immunotherapies. Mechanistic- affecting T-cell and Dendritic Cell recruitment into tumor ally, this is partially mediated by tumor MHC Class I upregulation, James Gordy, PhD, Richard Markham, BS MD impairing regulatory CD4+ T cells (Tregs) and monocyte derived Johns Hopkins Bloomberg School of Public, Baltimore, MD, United States suppressive cells (MDSCs) and/or enhancing CD8+ T cells and Correspondence: Richard Markham (rmarkha1@jhu.edu) granzyme B levels in the tumor microenvironment (TME). In these Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P464 studies, we observed that entinostat induced murine tumor necrosis. Background Methods The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic Therefore, we investigated if this treatment-induced necrosis cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated anti- could be targeted by NHS-IL12, an IL-12 NHS76 conjugate that gens have shown improved efficacy and immunogenicity, compared binds free DNA in regions of tumor death/necrosis. NHS-IL12 has to vaccines lacking the chemokine. To optimize the therapy, our la- been shown to have significant anti-tumor efficacy in preclinical boratory has added agents designed to further enhance the T cell ac- murine models and has been demonstrated to be safe in pa- tivating function of DCs and overcome immunoregulatory tients. We hypothesize that increasing the pro-inflammatory, im- mechanisms of the tumor microenvironment. Here, we report that mune stimulatory cytokine IL-12 in the TME will synergize with the combination of type-I interferon therapy (IFNα) with 5-Aza-2’- the entinostat-mediated immune effects to induce significant deoxycitidine (Aza) profoundly enhanced the therapeutic anti- tumor control. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 254 of 272 Results paclitaxel was 54.5% (6/11), the median PFS was 6.4 months (95% CI In the EMT6 breast cancer murine model, administration of NHS- 3.7 to 9.2 months), and the median OS was 14.1 months (95% CI 3.2 IL12 at the onset of entinostat-induced necrosis synergized to to 25.0 months). produce significant anti-tumor efficacy, including resolving well- Conclusions established tumors and enhancing survival. These studies are Our preliminary data suggest that carboplatin and paclitaxel often in- now being extended to additional murine models of solid carcin- duce objective responses in patients with prior treatment with anti- omas. Examination of immune subsets in the TME by flow cytom- PD1/PD-L1 antibodies and that the clinical outcomes compare favor- etry revealed that the combination increased infiltration of ably to those seen in checkpoint inhibitor naïve patients. If con- granzyme B+ CD8+ T cells and M1-like CD38 expressing tumor firmed, these data suggest that there is no “opportunity cost” for macrophages. Currently, we are using depletion studies to assess administering immunotherapy in the first line. the relative contribution of these immune subsets to the anti- Ethics Approval tumor efficacy. Further, we are examining the effect of this com- Approval by the UCSF IRB has been requested and will be obtained bination on CD8+ T cell function and macrophage polarization, before this work is presented. phenotype, and function. Conclusions Overall, the combination of NHS-IL12 and entinostat is showing en- P467 couraging results in a preclinical solid tumor model, providing a ra- ALPN-202, a conditional CD28 costimulator and dual checkpoint tionale to examine this combination clinically. inhibitor, enhances the activity of multiple standard of care modalities Katherine Lewis, PhD, Mark Maurer, BS, Sherri Mudri, BS, Kayla Susmilch, Acknowledgements MS, Fariha Ahmed-Qadri, MS, Chelsea Gudgeon, BS, Steven Levin, PhD, The authors thank Curtis Randolph for excellent technical assistance. Martin Wolfson, BS, Stacey Dillon, PhD, Kristine Swiderek, PhD, Stanford Peng, MD, PhD Alpine Immune Sciences, Seattle, WA, United States P466 Correspondence: Katherine Lewis Carboplatin and paclitaxel after anti-PD1 or anti-PDL1 therapy: A (katherine.lewis@alpineimmunesciences.com) retrospective study in patients with squamous cell carcinoma of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P467 the head and neck Audrey Humphries, BS, Madeleine Welsh, BA, Alain Algazi, MD Background UCSF, San Francisco, CA, United States Checkpoint inhibitors targeting the PD-1 axis have transformed Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) cancer treatment. However, objective response rates remain low, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P466 suggesting that novel therapeutics and/or combination treat- ments are needed. At the same time, non-immuno-oncology Background therapeutic approaches such as chemotherapy remain standard Historically, cytotoxic chemotherapy has been the first line stand- of care for many malignancies. ALPN-202 is a variant CD80 ard of care for patients with RM-SCCHN. More recently, a ran- vIgD™-Fc fusion that mediates PD-L1-dependent CD28 costimula- domized phase 3 clinical trial demonstrated improved overall tion and inhibits the PD-L1 and CTLA-4 checkpoints. This novel survival in RM-SCCHN patients receiving pembrolizumab in the mechanism of action provides potent single agent immunomodu- first line versus those receiving the prior standard carboplatin, latory activity in mouse tumor models, and thus has the potential 5FU, cetuximab. Additional studies have suggested that sequen- to complement other therapeutic modalities, such as checkpoint cing of therapies impacts outcomes. Several small case series inhibitors or chemotherapies. have described clinical outcomes in patients treated with chemo- Methods therapy after immunotherapy, but these have often included pa- Mice were implanted subcutaneously with human (hu) PD-L1- tients treated with a variety of cytotoxic regimens such that transduced MC38 colon carcinoma and B16-F10 melanoma cell lines. comparison to historical controls is difficult. Here we present clin- Once measurable tumors were established, mice were treated with ical outcomes in patients treated with carboplatin and paclitaxel anti-mouse checkpoint (i.e. PD-1 or CTLA-4) blocking monoclonal immediately following disease progression on anti-PD1 or anti- antibodies (mAbs) or oxaliplatin, a platinum-based chemotherapeutic PDL1 therapy. agent, alone or in combination with ALPN-202, to evaluate compati- Methods bility of the novel ALPN-202 protein with existing cancer therapies. A chart review was performed to identify all patients with RM- Anti-tumor responses were evaluated by serial tumor volume measure- SCCHN, including tumors originating in the oral cavity, oropharynx, ments and RNA-Seq analysis of tumors isolated from treated mice. hypopharynx, larynx, and sinuses treated with an anti-PD1 or anti- Results PDL1 antibody immediately followed by carboplatin and paclitaxel Anti-PD-1, anti-CTLA-4, or oxaliplatin alone were only modestly ef- through December 2018. Patients were required to have both pre- fective as monotherapy in huPD-L1+ MC38 tumor-bearing mice, treatment and post-treatment imaging associated with treatment while ALPN-202 has potent anti-tumor activity in this model. with both immunotherapy and chemotherapy or documented death When the checkpoint inhibitors or chemotherapy were adminis- from disease after administration of at least 1 cycle of chemotherapy. tered in combination with ALPN-202, significantly greater reduc- Anti-PD1 / PDL1 treatment history, p16 IHC (for OPC), and baseline tions in tumor growth over time were observed than with any of PD-L1 IHC (where available) were assessed. The best overall response these agents alone (Figure 1 and Figure 2,respectively).Further- was assessed by RECIST v1.1 and PFS and OS were determined using more, ALPN-202 was extremely effective (92% tumor growth in- the Kaplan-Meier method. hibition) in improving the anti-tumor activity of anti-PD-1 mAb in Results mice bearing huPD-L1+ B16-F10 tumors, a tumor that is known A total of 11 patients meeting the inclusion criteria were identified to be poorly immunogenic and treatment-recalcitrant (Figure 3). including 5 patients with p16+ SCC of the oropharynx, and 3 with RNA-Seq analysis of tumors from the MC38 studies was per- SCC of the oral cavity. 5 patient has detectable staining for PD-L1 at formed to explore in-depth the mechanisms, including enhance- baseline, 2 did not, and 3 patients had no PD-L1 IHC available. 6 pa- ment of T cell effector transcript expression, that play a role in tients receive pembrolizumab alone or in combination, 4 received the ability of ALPN-202 to provide anti-tumor immunity and to durvalumab, and 1 patient was treated with nivolumab. The median enhance the activity of checkpoint inhibitors and the chemother- duration of prior PD1 / PDL1 exposure prior to chemotherapy was apeutic oxaliplatin. 2.7 months (range 1.4 to 18.2 months). The BORR to carboplatin and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 255 of 272 Conclusions P468 ALPN-202 demonstrates potent anti-tumor efficacy as monother- Targeting the ICOS pathway in combination with apy and significantly improves the anti-tumor activity of other chemotherapy to enhance T-cell mediated anti-tumor immune only modestly effective treatment modalities, such as checkpoint- responses only blockade mAbs and chemotherapy. ALPN-202 has the poten- Tamer Mahmoud, PhD, Jeffrey Riggs, Madhu Ramaswamy, PhD, Leigh tial to be significantly effective as a monotherapy, and its com- Hostetler, Brian Naiman, Ilyssa Ramos, Sean Turman, Fernanda Pilataxi, patibility with checkpoint inhibitors and chemotherapeutics Christopher Morehouse, MD, Alex Alfaro, Norman Peterson, Ryan suggest versatility in its potential to improve outcomes in the Fleming, Nazzareno Dimasi, Kyle Kuszpit, Ronald Herbst, Gianluca frontline setting alone and/or in combination with standard of Carlesso, PhD care of multiple cancer types. A first-in-human clinical study with AstraZeneca, Gaithersburg, MD, United States ALPN-202 is in preparation. Correspondence: Gianluca Carlesso (carlessog@medimmune.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P468 The study was approved by the vivarium's International Animal Care and Use Committee (IACUC), IR# 17-01. Background ICOS (Inducible T-cell Costimulator) is a member of the CD28 superfamily that is detected on activated and memory T lym- phocytes. Treatment with Immune checkpoint inhibitors (ICI) in clinical studies has shown that expansion of ICOS-expressing T cells is associated with positive patient outcome. Preclinical studies have validated the rationale for targeting ICOS and the clinical investigation of ICOS agonist antibodies (mAbs) com- bined with ICI is under way. To enhance tumor immunogenicity and increase the response rate to immunotherapy, we examined the efficacy of combination therapy of ICOS mAbs with stand- ard of care chemotherapy using a colorectal tumor syngeneic animal model. Methods For the microarray analysis, freshly isolated normal primary hu- man T cells were stimulated with sub-optimal concentration of Fig. 1 (abstract P467). See text for description anti-CD3 mAb with either ICOS, OX40 mAbs, or GITRL-FP for 4 hours. ¬In-vitro proliferation and cytokine secretion assays were performed using primary human T cells stimulated with anti- CD3 mAb and ICOS mAb in the presence of different inhibitors. For in-vivo studies, CT26 tumor cells were subcutaneously injected into BALB/c mice and ICOS or control mAbs were ad- ministered intraperitoneally (ip) or in combination with an intra- venous injection of 5-Fluorouracil (5-FU) at day 10 post tumor implantation. For the biodistribution study, CT26 tumor-bearing mice were dosed ip with 89Zr-labeled anti-ICOS or control mAb and imaged using positron emission tomography (PET)/SCAN. For the depletion study, CT26 tumor-bearing mice were injected ip with either CD4 or CD8 mAb twice a week starting one day before tumor implantation, whereas sphingosine-1 phosphate re- ceptor (S1PR) modulator (FTY720) was orally administered daily starting at day 9 post-implantation. Tumor growth was assessed three times a week. Results Fig. 2 (abstract P467). See text for description Compared to other T-cell agonists, in-vitro co-stimulation of human T cells with ICOS mAb lead to enhanced metabolic T cell reprogramming, activation of the PI3K/mTOR pathways, and secretion of IFN-gamma and IL-10. Immuno-PET scan imaging analysis revealed ICOS expression across tumor and secondary lymphoid tissues. ICOS mAb and 5-FU combination therapy on CT26 tumor-bearing mice resulted in a significant increase in anti-tumor responses compared to single-arm treatment or con- trol groups. Moreover, the efficacy of either anti-ICOS or 5-FU monotherapy or combination required cytotoxic T cell activity and was dependent on the S1P-mediated egress of immune cells from peripheral lymph nodes. Conclusions Collectively, the results of these studies show that ICOS agonism in combination with chemotherapy may provide an effective therapeutic option for the activation of T cells in solid tumor Fig. 3 (abstract P467). See text for description indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 256 of 272 P469 4. Cappello P, et al. Vaccination with ENO1 DNA prolongs survival of Chemotherapy enhances effector T cell responses to tumor genetically engineered mice with pancreatic cancer. Gastroenterology. associated antigens in human and mouse pancreatic cancer 2013;144:1098–106. 1 1 1 Giorgia Mandili, PhD , Claudia Curcio, PhD ,SaraBulfamante, MS , Ethics Approval 1 1 2 Laura Follia, MS , Daniele Giordano, MD , Rossella Spadi, MD ,Maria The study was approved by the local research ethical committee 2 1 Antonietta Satolli, MD , Paola Cappello, PhD MS , Francesco Novelli, (Azienda Ospedaliera Città della Salute e della Scienza di Torino, PhD Turin) and investigations were performed according to the Helsinki 1 2 University of Turin, Turin, Piedmont, Italy; Azienda Ospedaliera Declaration principles. Universitaria Città della Salute e della Scienza di Torino, Turin, Piedmont, Italy Correspondence: Francesco Novelli (franco.novelli@unito.it) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P469 P470 Impact of treatment-induced necrosis in the anti-tumor Background efficacy of Entinostat combined with the immunocytokine Pancreatic Ductal Adenocarcinoma (PDA) is one of the most NHS-IL12 lethal cancer, both for lack of effective screening method and Yohei Ozawa, MD, PhD, Kristin Hicks, PhD, Karin Knudson, PhD, Jeffrey resistance to chemotherapy (CT). Immunotherapy (IT) trials with Schlom, Sofia Gameiro, PharmD, PhD immune check-point inhibitors did not achieve significant gain National Cancer Institute, NIH, Bethesda, MD, United States of survival yet [1]. However, some CT agents, such as gemcita- Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) bine (GEM), have several immune modulating effects [2] and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P470 starting from the hypothesis that more immunogenic antigens can be induced by CT treatment, its ability to increase the sus- Background ceptibility of PDA to IT was evaluated. Tumor necrosis resulting from hypoxia, inflammation, or ab- Methods normal angiogenesis is associated with poor outcome in sev- Sera from 28 PDA patients before and after CT (BCT and ACT eral solid malignancies. However, theroleoftreatment- respectively) were profiled by serological proteome analysis induced necrosis is still controversial. The class I HDAC inhibi- (SERPA) [3]; the recognized TAAs were identified by mass spec- tor entinostat (Syndax) has been shown to promote significant trometry and confirmed by ELISA. The proliferation, phenotype tumor control in combination with immunotherapies through and cytokine production of T cells were evaluated on patients’ immune-mediated mechanisms. In addition, we observed that PBMCs from the same cohort after in vitro stimulation with entinostat induces tumor necrosis in murine solid tumors. We four selected TAAs (ENO1, G3P, K2C8 and FUBP1). Mice that hypothesize that entinostat-induced necrosis could be tar- spontaneously develop PDA (KC) were treated with GEM prior geted by NHS-IL12 (M9241, Merck KGaA), an IL-12 NHS76 con- of DNA vaccination against ENO1 [4]. Tumor lesions, immune jugate designed to bind free DNA in regions of tumor death/ infiltration and the titer of TAAs-specific antibody were evalu- necrosis, therefore promoting a synergistic anti-tumor effect. ated. TAAs-specific IFNγ production from splenocytes was NHS-IL12 has demonstrated significant anti-tumor efficacy in analyzed. preclinical solid tumor models and has been safely adminis- Results tered to cancer patients. The number of TAAs recognized by IgG in PDA patients’ sera, Methods as well as their ability to induce a complement dependent cyto- The amount of necrosis entinostat induced over time was exam- toxicity against PDA cells, was increased in ACT sera. Some ined in three distinct murine solid tumor models: MC38 (colon), identified TAAs showed a positive correlation between the in- 4T1 (triple-negative breast) and EMT6 (breast). Necrosis was crease of ACT antibody titer and longer patients’ survival. An in- evaluated by histological analysis of H&E staining and a ter- creased T cell TAAs-specific proliferative response after CT and minal deoxynucleotidyl transferase dUTP nick end labeling the evaluation of IFNγ/IL10 ratio was detected, revealing that (TUNEL) assay. To assess if NHS-IL12 could bind to necrotic ATC T cells shifted TAAs-specific responses from regulatory to tumor regions, tumor sections from mice treated with PBS or effector one. After stimulation with TAAs, in mostly patients the entinostat were incubated in vitro with NHS-IL12. The binding ratio between CD8 and CD4 Treg cells was increased in ATC T and distribution of NHS-IL12 in the tumor was then examined cells. The role of CT to enhance the TAAs-specific adaptive re- by immunofluorescence staining usingananti-humansecondary sponse prompt us to exploit its effect in combination with the antibody. DNA vaccination. Of clinical relevance, KC mice treated with Results GEM prior of ENO1 DNA vaccination displayed smaller tumor le- Entinostat promoted tumor control in all three tumor models, sions together with an increase of tumor-infiltrating CD4 and albeit to different degrees. The onset of entinostat-induced ne- CD8, in comparison to mice vaccinated or GEM-treated only. crosis was observed after two weeks of continuous dosing. In Furthermore, CT increased specific antibodies and IFNγ- EMT6 tumors, areas of tumor necrosis identified via H&E stain- producing T cells in vaccinated mice not only against ENO1, the ing also displayed DNA fragmentation measured by the TUNEL target TAA of vaccination, but also against G3P, suggesting an assay. Of note, in the entinostat-treated tumors there were antigen spreading effect of the combinatory treatment. areas of tumor death/necrosis with increased immune cell infil- Conclusions tration identified by H&E staining. Furthermore, initial immuno- Overall these data indicated that in pancreatic cancer CT effect- fluorescence studies indicate that NHS-IL12 binds to these areas ively ameliorates T cell responses against TAA and it might be in the entinostat-treated tumors in vitro. These preliminary find- reconsidered to render them suitable targets for IT. ings are being confirmed utilizing specimens from mice treated with all the agents. Additionally, in the EMT6 model, entinostat and NHS-IL12 syner- References gized to produce significant anti-tumor efficacy, including 1. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients resolving well-established tumors and enhancing survival. The with advanced cancer. N Engl J Med. 2012;366:2455–65. combination therapy promoted tumor infiltration of CD8 T cells 2. Cappello P, et al. Next generation immunotherapy for pancreatic and M1-like macrophages. Ongoing correlative studies in tumor cancer: DNA vaccination is seeking new combo partners. Cancers. specimens are examining the spatial distribution between 2018;10(2):51 entinostat-induced tumor necrosis, NHS-IL12 binding, and CD8 3. Tomaino B, et al. Autoantibody signature in human ductal pancreatic and M1 infiltration. adenocarcinoma. J Proteome Res. 2007;6:4025–31. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 257 of 272 Conclusions Journal of Clinical Oncology, ASCO, 2017 35, e14544. Overall, our results suggest that entinostat-induced necrosis may 2. Pankaj Gaur et al. CXCR4 antagonist (BL-8040) to enhance antitumor promote a tumor microenvironment conducive to NHS-IL12 bind- effects by increasing tumor infiltration of antigen-specific effector T-cells. ing, resulting in significant anti-tumor efficacy. These results in- Journal of Clinical Oncology, ASCO, 2018, 36, 73. form a rationale to examine this combination in the clinic for 3. Manuel M. Hidalgo et al. Evaluation of pharmacodynamic (PD) patients with solid carcinomas. biomarkers in patients with metastatic pancreatic cancer treated with BL- 8040, a novel CXCR4 antagonist. Acknowledgements Journal of Clinical Oncology, ASCO, 2018 36, 88-88. The authors thank Curtis Randolph for his excellent technical assistance. Ethics Approval The study was approved by the Hebrew University Ethics Board, approval number 18-15644-4. P471 Combination of BL-8040, anti PD-1 and chemotherapy significantly reduced pancreatic tumor growth and changed the balance between CD4+/FOXP3+ cells and CD8+ cells in the tumor Amnon Peled, PhD (peled@hadassah.org.il) Hadassah University Hospital, Jerusalem, Israel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P471 Background Cancer cells shape the tumor microenvironment (TME) to support their growth by recruiting immune suppressing cells such as T regulatory cells, as well as inhibiting the recruitment and activa- tion of effector CD8+ T cells. In this study, we investigated the effect of combining the CXCR4 antagonist BL-8040 [1, 2], anti PD- 1 immune checkpoint inhibitor and chemotherapy of Irinotecan, Fluorouracil and Leucovorin) IFL (on pancreatic tumor immune cell composition and growth. Methods The effect of BL-8040, anti PD-1 and IFL on tumor growth and im- mune cell constitution was assessed using the syngeneic Panc02 tumor mouse model. Tumors were established by s.c. injection . The Fig. 1 (abstract P471). Tumor growth accumulation of immune cells in the TME was assessed by immuno- histochemical staining for CD8, CD4, Foxp3, and CD69. Results Treatment of tumors with anti PD-1 or BL-8040 alone, had no effect on tumor growth, whereas, IFL treatment had significant effect on tumor growth (67% inhibition). Combination of anti PD-1 + IFL, had no significant better effect on tumor growth compared to IFL (p<0.09), whereas, BL-8040 + IFL, had a signifi- cantly better effect on tumor growth compared to IFL (p<0.04). Moreover, IFL + BL-8040 + anti PD-1, had a highly significantly better effect on tumor growth, compared to IFL alone (p<0.004) (Figure 1). The triple combination treatment (TCT), further re- duced tumor growth, compared to chemotherapy alone, by 58%.Inthe TCT, 3out of 8micedid notdevelop tumoratall, compared to 1 mouse that did not develop tumor in the IFL alone group. The TCT did not significantly change the number of CD8+ T cells accumulating in the tumor but increased their activation status (Figure 2A,B). Only in the TCT, the CD8+ T cells Fig. 2 (abstract P471). A, B, C. See text for description were larger in size inside the tumor parenchyma (p<0.01, Fig. 2C) and expressed CD69. Interestingly, we found that tumors treated with the TCT, had significantly reduced numbers of CD4+ and CD4+, Foxp3+ cells (Figure3). Conclusions TCT reduced significantly the number CD4+ and CD4+FOXP3+ cells and increased the numbers of activated CD8+, CD69+ cells in the TME. We hypothesize, that the ability of BL-8040 to modulate the TME may allow better activation of immune effector cells, contributing to the effect of chemotherapy and immunotherapy on tumor growth .A Phase IIa, multicenter, open label trial in patients with metastatic pancreatic cancer (the COMBAT study cohort II, [3]) is currently ongoing to as- sess the effect of the triple combination with BL-8040, +Pem- brolizumab and ILF on disease progression. References 1. Michal Abraham et al. Effect of BL-8040, high-affinity CXCR4 antagonist, on T-cell infiltration, tumor growth, and synergy with immunomodula- Fig. 3 (abstract P471). See text for description tory agents. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 258 of 272 P472 Background STAT3 ASO and Cisplatin combination sensitizes protected tumor While immune checkpoint blockade has revolutionized cancer care, microenvironments to checkpoint-mediated therapy many patients remain refractory to checkpoint inhibition. Converting Theresa Proia, PhD, Maneesh Singh, PhD, Nanhua Deng, Minwei Ye, checkpoint-refractory tumors into checkpoint-responsive tumors is a Frank McGrath, Douglas Ferguson, Simon Barry major challenge for cancer immunotherapy. Interleukin-12 (IL-12) is a AstraZeneca, Waltham, MA, United States potent cytokine that holds potential to reshape the immune environ- Correspondence: Simon Barry (simon.t.barry@astrazenea.com) ment in solid tumors. Its clinical utility, however, has been limited by Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P472 severe toxicities both from systemic administration and from expres- sion by adoptively transferred gene engineered T cells. We report Background here that adoptive cell therapy (ACT) with T cells carrying surface- Chemotherapy-immunotherapy (chemo-IO) combinations are being tethered DeepTM IL-12 overcomes these challenges, enhances T cell explored in the clinic. Immune responses mediated by PD-1 or PD-L1 therapeutic efficacy, activates immune cells in the tumor and over- inhibition may be enhanced by the immunogenic effects of cytotoxic comes resistance to checkpoint blockade. agents, which can increase tumor antigens. Chemo-IO combination Methods strategy relies on drug dose and schedule optimization, to minimize Activity of Deep IL-12 Primed T cells were evaluated in B16-F10 melan- direct T cell killing with chemotherapy, enhance antigen presenta- oma tumors, a cell line resistant to checkpoint inhibition. Mouse PMEL tion, and promote T cell activation. CD8 T cells, which are reactive against the B16-F10 melanoma antigen Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that se- gp100, were surface-tethered with Deep IL-12 and evaluated for anti- lectively targets STAT3, a master regulator of immune suppression, tumor activity in mice bearing B16-F10 melanoma. We additionally and is currently in Ph 1/2 clinical trials in combination with an anti- evaluated effects in the tumor of Deep IL-12 Primed T cells and PMEL T PD-L1 antibody, durvalumab. In preclinical tumor models, we demon- cells alone or co-administered with IL-12. Further studies evaluated strated that mouse surrogate STAT3 ASO remodels the suppressive combinations of Deep IL-12 Primed T cells with checkpoint inhibition. tumor microenvironment to enhance cytotoxic T cell activity in com- Results bination with anti-PDL1 (Singh et al. SITC 2019). ACT of Deep IL-12 Primed PMEL T cells significantly improved tumor Methods growth inhibition and overall survival of B16-F10 tumor bearing mice We sought to maximize the therapeutic benefits of chemo-IO by compared to PMEL T cells alone or combined with systemic co- dose and schedule optimization of adding cisplatin to STAT3 ASO administration of IL-12. In the tumor, Deep IL-12 repolarized im- and anti-PDL1. In patients, cisplatin dose ranges from 50mg/m2- munosuppressive monocytic myeloid-derived suppressor cells (M- 100mg/m2. We modeled relevant preclinical doses in the range of 5- MDSC) into an immune-activating antigen-presenting cell (APC) 10 mg/kg based on mouse plasma exposure, and proceeded with 5 phenotype. Consistent with an anti-tumor role for the repolarized M- mg/kg to represent a low clinical therapeutic (~60mg/m2) dose. MDSC, administration of an antibody to deplete these cells reduced Using the immunogenic MC38 model, various schedules of chemo-IO the efficacy of Deep IL-12 Primed T cells. Further evaluation revealed were explored; (1) Cisplatin priming on day 3 (to increase antigens), high expression of the checkpoint ligand PD-L1 on the repolarized followed by STAT3 ASO/anti-PDL1 on day 7. (2) STAT3 ASO pre- M-MDSC. To test the hypothesis that this limits efficacy of Deep IL-12 treatment on day 3 (to remodel the suppressive tumor microenviron- Primed T cell ACT, Deep IL-12 Primed T cells were co-administered ment), followed by cisplatin/anti-PDL1 on day 7. (3) All 3 agents with PD-L1 blockade. This further improved anti-tumor efficacy, dosed simultaneously on day 7 post implant. resulting in durable long-term responders. Efficacy was further im- Results proved by repeat dosing of Deep IL-12 Primed PMEL T cells. Cisplatin treatment in MC38-tumor bearing mice resulted in variable Conclusions tumor growth inhibition, with one tumor regression. Flow cytometry Our data demonstrates that ACT with tumor-specific T cells carrying analysis confirmed that cisplatin treatment had no detrimental effect surface-tethered Deep IL-12 repolarizes suppressive M-MDSC in the on T cell number or functionality, and showed a trend of increased tumor, enhances anti-tumor efficacy, and synergizes with checkpoint CD11b+/Ly6C+ dendritic cells, providing confidence to explore effi- inhibition in a checkpoint refractory cancer model. Torque is apply- cacy of the triplet combination. ing this approach to develop a novel adoptive cell therapy for can- Regardless of schedule, we observed 20% complete response rate in cer, Deep IL-12 Primed multi-targeted T cells (TRQ12-01), which is the triplet combinations, compared with 0 complete responses in expected to start clinical evaluation in 2019. any other treatment group; but interestingly, schedule 1 required dose holidays. Flow cytometry analysis of the triplet compared with P474 vehicle revealed enhanced CD4 T cell functionality (1.6x increase Efficacy and toxicity evaluation of anti-human CD47 and SIRPa IFNγ, p<0.001, and 1.2x increase IL-2, p=0.001), and enhanced NK antibodies in genetically humanized B-hSIRPa/hCD47 mice functionality (1.3x increase Granzyme B+, p<0.01; 4.3x increase TNFα, Frank An, Yanan Guo, Jie Xiang, Chaoshe Guo p<0.001). Biocytogen, Boston, MA, United States Conclusions Correspondence: Jie Xiang (info@biocytogen.com); Chaoshe Guo Collectively, we have generated data to support the combination of (info@Biocytogen.com) low-dose chemotherapy and immunotherapy to enhance the anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P474 tumor immune response. As chemo-IO combinations are being ex- plored in the clinic, it will be important to optimize dose and sched- Background ule to minimize toxicity and maximize therapeutic benefit. CD47 is a transmembrane protein expressed ubiquitously on the sur- face of human cells, including. SIRPa, one of the binding partners of P473 CD47, is a member of the signal-regulatory-protein (SIRP) family which Adoptive transfer of Deep IL-12 Primed T cells increases sensitivity is expressed on many myeloid cells including phagocytic cells. Engage- to PD-L1 blockade for superior efficacy in checkpoint refractory ment of SIRPa by CD47 elicits the “do not eat me” signal to prevent tumors phagocytosis of “self” cells by macrophages. Anti-CD47 and anti-SIRPa 1 1 2 Gulzar Ahmad, PhD , Jonathan Nardozzi, PhD , Lars Petersen, PhD , antibodies that interfere with the CD47-SIRPa interaction have demon- 2 2 1 Esben Christensen, MSc , Ditte Jaegher , James Suchy, PhD , Karsten strated promise in clinical trials as new class of therapeutics. Despite 1 1 1 Sauer, PhD , Douglas Jones, PhD , Thomas Andresen, PhD such exciting potential, side effects associated with CD47/SIRPa block- 1 2 Torque Therapeutics, Cambridge, MA, United States; Denmark ade such as anemia may represent a significant toxicity concern. There- Technical University, Kgs. Lyngby, Denmark fore, evaluation of both efficacy and toxicity of human CD47/SIRPa Correspondence: Thomas Andresen (tandresen@torquetx.com) antibody candidates emerges as one of most actively investigated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P473 areas in immuno-oncology in recent years. However, lack of animal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 259 of 272 models that enable expedient testing of anti-human CD47 or anti- further increased with loss of both PD1 and LAG3, as a result of en- human SIRPa antibodies in vivo has been a limiting factor for CD47/ hanced proliferation (Ki67/BrdU) – a phenotype demonstrated to be SIRPa antibody development. intrinsically regulated in the co-adoptive transfer system. Although Methods expression of TIM3, TIGIT and 2B4 IRs that normally co-express with To accelerate direct efficacy and toxicity testing of anti-human CD47 PD1 are maintained, CD8+ TIL isolated from Pdcd1L/L E8ICre.GFP and SIRPa antibodies, Biocytogen has generated the double human- and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP show increased functionality ized mice, B-hSIRPa/hCD47, where the human extracellular domains (IFNg, TNFa and GzmB release) by flow cytometry. Moreover, CD8+ of SIRPa and CD47 replace their respective murine counterparts. TIL polyfunctionality is evident with PD1/LAG3 loss that was largely Homozygous B-hSIRPa/hCD47 mice express humanized but not the driven by effector and chemoattractive secretions by analysis with a wild type mouse SIRPa and CD47. Further, we also created two triple 28-plex single-cell cytokine response panel (Isoplexis). humanized mouse strains, B-hPD1/hSIRPa/hCD47 and B-hPD-L1/ Conclusions hSIRPa/hCD47, where humanized PD1 and PD-L1 extracellular do- Overall, these data suggest that PD1 and LAG3 synergize to have a mains replace their mouse counterparts, respectively, in the B- dominant effect on CD8+ TIL and limit antitumor immune effects, as hSIRPa/hCD47 background. intrinsic removal of both IRs results in reduced B16-F10 tumor Results growth which has a substantive impact on the development of sys- We present here that B-hSIRPa/hCD47 mice were successfully used temic anti-tumor immunity. These results are encouraging for the for screening anti-human CD47 and anti-human SIRPa antibodies for continued development of LAG3 targeting agents in the clinic, which efficacy and toxicity in tumor models of the engineered MC38- would hopefully yield improved clinical responses in combination hCD47 cell line that expresses human CD47 in MC38 cells. Anti- with anti-PD1. human CD47 and anti-human SIRPa antibodies were efficacious in controlling MC38-hCD47 tumor growth in B-hSIRPa/hCD47 mice. Var- P476 ied toxicity profiles were observed in terms of body weight loss, The combination of a STING agonist with cytokines results in blood cell counts, and blood liver enzyme levels in anti-human CD47 robust anti-tumor effects in autochthonous tumor models antibody treatments. Anti-human PD-1 and anti-human CD47 anti- 1 1 1 2 Cristina Blaj, PhD , Yingjoy Li , Allen Chen , Anthony Descien, PhD , bodies showed single agent and combination anti-tumor effect in B- 2 2 3 Brian Francica, PhD , Sarah McWhirter , Lora Picton , K. Christopher hPD1/hSIRPa/hCD47 mice. So did anti-human PD-L1 and anti-human 3 1 Garcia , David Raulet, PhD CD47 antibodies in B-hPD-L1/hSIRPa/hCD47 mice. 1 2 University of California Berkeley, Berkeley, CA, United States; Aduro Conclusions Biotech Inc, Berkeley, CA, United States; Stanford University School of Taken together, we have validated three double and triple human- Medicine, Stanford, CA, United States ized CD47/SIRPa mouse models and demonstrate that these human- Correspondence: Cristina Blaj (cristina.blaj@berkeley.edu) ized mice are useful tools in facilitating development of therapeutics Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P476 targeting human CD47/SIRPa. Background P475 Cancer immunotherapies based on immune checkpoint blockade are PD1 and LAG3 converge to limit polyfunctionality and systemic highly effective, but only in a limited number of tumor types. Even in immunity transplanted preclinical models, monotherapy rarely results in cures. 1 2 2 Lawrence Andrews, PhD , Sasikanth Manne , E. John Wherry, PhD , Creg Moreover, preclinical subcutaneous models appear to be more respon- 1 1 Workman, PhD , Dario Vignali, PhD sive to therapies than preclinical carcinogen or genetically engineered 1 2 University of Pittsburgh, Pittsburgh, PA, United States; University of mouse (GEM) autochthonous models of cancer, or human tumors. To Pennsylvania, Philadelphia, PA, United States extend immunotherapy to a broader range of tumor types, our strategy Correspondence: Dario Vignali (dvignali@pitt.edu) is to rationally design combination immunotherapies with the potential Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P475 to boost innate and adaptive immune responses and overcome im- munosuppressive environments characteristic of human tumors. Our Background regimen includes a stimulator of interferon genes (STING) agonist, cyto- Targeting PD1 has yielded clinical success across a variety of tumor kines and checkpoint inhibitors. types, yet a significant proportion of patients remain unresponsive to Methods treatment. Thus, overcoming inhibitory receptor (IR)-mediated toler- Autochthonous tumors induced by a carcinogen or in GEM models, ance is essential to improve immunotherapeutic responses. Co- as well as subcutaneous models derived from the GEM models, were expression of PD1 and LAG3 on CD8+ tumor-infiltrating T cells (TIL) is treated with combination immunotherapies. We used flow cytometry, associated with an exhausted phenotype, exemplified by a severe de- immunofluorescence, Luminex assays and qPCR to characterize the fect in cytokine production, cytolytic activity and inability to proliferate. immune cell infiltration, activation status, receptor expression and se- In a number of murine tumor models, dual PD1/LAG3 blockade syner- cretion of cytokines in response to therapy. gistically limits tumor growth greater than targeting PD1 alone, yet the Results relative and synergistic contributions of PD1 and LAG3 on CD8+ T cells Intratumoral injection of cyclic dinucleotides (CDNs, specifically ADU- in preventing effective anti-tumor immunity is unknown. S100, a STING agonist) resulted in ~20% stable regressions of estab- Methods lished transplanted tumors and immunity to re-challenge in a sub- To understand the cellular and mechanistic basis for PD1/LAG3 syn- cutaneous sarcoma model. The same protocol resulted in significant ergy, conditional knockin mice “surgically dissect” Pdcd1 and/or Lag3 tumor growth delay in autochthonous GEM models and a carcinogen floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. To allow model. Antibody-mediated depletion studies revealed that natural for intrinsic analysis of PD1 and/or LAG3 on antigen-specific CD8+ T killer (NK) cells as well as CD4 and CD8 T-cells played an important cells, these mice have been generated as a pmel-1 transgenic back- role in mediating the anti-tumor effects. The combination of CDNs ground, with each mutant strain uniquely congenically marked for use and an IL-2 superkine resulted in synergistic anti-tumor efficiency in in a co-adoptive transfer system allowing analysis of PD1 and/or LAG3- all models tested. deficient CD8+ T cells, and controls, in the same host. Autochthonous tumors are clinically more relevant, as they resemble Results the tumor resistance signature observed in human cancers. Our sar- Mice with CD8+ T cells deficient in PD1 or PD1 and LAG3 (Pdcd1L/L coma models represent excellent platforms to dissect the differences E8ICre.GFP and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP, respectively) show between the refractory GEM models and the more responsive trans- attenuation of B16-F10 tumor growth with improved survival, com- planted tumors. Flow cytometry and immunofluorescence analysis pared to LAG3-deficient mice (Lag3L/L-yfp E8ICre.GFP) and controls revealed the prevalence of tumor associated macrophages in the (E8ICre.GFP). CD8+ TIL frequency is increased with loss of PD1, and GEM models, consistent with the established role of macrophages in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 260 of 272 promoting an immunosuppressive environment. Additionally, we dis- played by TGFb1 in this model. Based on these promising data, we covered a number of differentially secreted cytokines that might play have generated novel humanized 13A1 variants with in-vitro charac- a role in the anti-tumor response. The roles of macrophages and cy- teristics similar to the parental antibody. tokines are being tested. Conclusions Conclusions Isoform-specific blockade of active TGFb1 with antibody 13A1 is as The combination of cyclic dinucleotides with an IL-2 superkine efficacious or better than pan-TGFb blockade in enhancing the anti- produced strong antitumor effects in transplanted and autoch- tumor efficacy of PD-L1 checkpoint therapy in mice with established thonous tumor models and may translate into an efficacious ap- EMT6 tumors. Humanized 13A1 antibodies that maintain the in-vitro proach to treat human cancers. Our studies provide indications features of the biologically active parental murine 13A1 antibody are for additional targets that might modulate the tumor microenvir- therefore attractive clinical candidates for combination therapy with onment to enhance antitumor responses and synergize to en- checkpoint antibodies, especially in patients with low response rates hance immunotherapy effects. to current anti-checkpoint mono-therapies. Ethics Approval The study was approved by the University of California Berkeley Insti- References tutional Animal Care And Use Committee, approval number AUP- 1. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, de 2015-1-8058-1. Gramont A. Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther. 2015;147:22-31; Colak S. and Ten Dijke P. Targeting TGFb signaling in cancer. Trends Cancer 2017; 3(1):56-71.; Tauriello, D.V.D., Palomo-Ponce P477 S., et al. TGFb drives immune evasion in genetically reconstituted colon Isoform-specific blockade of active TGFb1 with mAb 13A1 cancer metastasis. Nature, 2018; 554:538-543 enhances the efficacy of PD-L1 checkpoint therapy in a EMT6 2. Poniatowski LA, Wojdasiewicz P, Gasik R, Szukiewicz D. 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J Leukoc Biol. 2011 Jun; 89(6):1001-1007 AND Brioschi M., States Cheou P., van Snick, J., Uyttenhove C., Coukos, G., Ritter, G., Dunn, S. Jour- Correspondence: Steven Dunn (steven.dunn@chuv.ch) nal for ImmunoTherapy of Cancer 2018, 6(Suppl 1):115 Abstr. Nr. 482 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P477 4. Gupta A, Budhu S, Giese R, van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Isoform specific TGF-β inhibition in combin- Background ation with radiation therapy as a novel immune therapeutic ap- TGFb is a highly pleiotropic cytokine implicated in tumor escape proach to cancer therapy. Journal for ImmunoTherapy of Cancer and progression. Targeting TGFb has recently emerged as an ex- 2017; 5 (suppl 2):87 Abstract 326 AND Gupta A, Budhu S, Giese R, citing new approach to overcome TGFb-mediated resistance to van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Target- checkpoint cancer immunotherapy [1]. In humans, three isoforms ing specific TGF-β isoforms in combination with radiation therapy of TGFb (TGFb1, -2 and -3) have been shown to individually drive leads to differential antitumor effects in mouse models of cancer. context-dependent physiological and phenotypic responses [2]. Cancer Res. 2018; 78(13 suppl): Abstract 4716 Most current therapeutic TGFb reagents do not adequately distin- 5. Mariathasan S., Turley S.J. et al. TGFb attenuates tumor response to PD-L1 guish among the three TGFb isoforms and could give rise to on- blockade by contributing to exclusion of T cells. Nature, 2018; 554:544- target off-tumor toxicity, undesirable inflammatory adverse events or lack of activity. To address this unmet need for more selective therapeutic reagents, we generated a panel of murine antibodies with mono-isoform specificity for TGFb1 (13A1) or TGFb3 (1901) P478 and successfully humanized these antibodies, carefully maintain- SEMA4D antibody blockade overcomes mechanisms of immune ing their selective specificity and neutralization potency [3]. The suppression and combination immunotherapy including TGFβ murine TGFb isoform-specific antibodies enhanced anti-tumor effi- blockade promotes efficient tumor regression 1 1 1 cacy in-vivo in B16 melanoma and 4T1 breast cancer models [4]. Terrence Fisher, PhD , Crystal Mallow, BS , Holm Bussler, PhD , Sebold 1 1 1 2 We now expanded our in-vivo studies into the immune-exclusion- Torno, BS , Desa Rae Pastore , Alan Howell, MS , Luis Ruffolo, MD , 2 2 1 1 type tumor model EMT6, in which a pan-specific TGFb antibody Nicholas Ullman , Brian Belt, JD , Joe Bucukovski , Christine Reilly, BS , 2 1 2 was shown to overcome TGFb mediated resistance to PD-L1 Benjamin Dale , Ernest Smith, PhD , David Linehan, MD , Maurice 1 1 checkpoint therapy [5]. Zauderer, PhD , Elizabeth Evans, PhD 1 2 Methods Vaccinex, Rochester, NY, United States; University of Rochester, Efficacy of the murine TGFb1 antibody 13A1 and a pan-TGFb anti- Rochester, NY, United States body (1D11) in combination with anti-mPD-L1 were evaluated in Correspondence: Elizabeth Evans (eevans@vaccinex.com) established orthotopic EMT6 murine breast carcinomas. Antibody Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P478 humanization was performed using molecular engineering, combin- ing framework grafting, competitive screening and selective back Background mutations guided by assaying for TGFb neutralization potency in Despite progress of immune checkpoint blockade therapies, resist- TMLEC reporter cells. Further humanized 13A1 variants with distinct ance mechanisms including myeloid suppression and upregulation kinetic properties were generated by error-prone mutagenesis and of TGFβ signaling prevent durable clinical benefit in many cancer pa- selective library screening. tients. Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody pro- Results motes immune infiltration, reduces immunosuppression, and We found that isoform-specific neutralization of active TGFb1 with enhances T cell activity in the tumor microenvironment (TME), result- mAb 13A1 was highly efficacious in overcoming the low efficacy of ing in increased tumor control in preclinical models when combined PD-L1 checkpoint mono-therapy, enhancing control of tumor growth with various immunotherapies [1,2]. Clinical trials of immune check- and increasing survival of mice with established EMT6 tumors. Fur- point inhibitors (ICI) in combination with pepinemab (VX15/2503), a ther, 13A1 appeared at least as potent as the pan-TGFb antibody humanized anti-SEMA4D antibody, are currently underway in several when combined with anti-PD-L1, highlighting the dominant role cancer indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 261 of 272 Methods Background Activity of anti-SEMA4D antibody in combination with immune Tumor necrosis factor receptor 2 (TNFR2) is central to immune bal- checkpoint inhibitors and TGFβ blockade was evaluated in preclinical ance control in humans and mice. We created human-directed anti- mouse tumor models. Ongoing clinical trials of immune checkpoint TNFR2 antibodies as a therapeutic approach in cancer, with positive inhibitors (ICI) in combination with pepinemab include: (i) a Phase findings [1]. We have now identified a murine-directed surrogate an- 1b/2a combination trial of pepinemab with avelumab in ICI naïve or tagonistic antibody to TNFR2. This antibody shares traits identified in ICI refractory or relapsed NSCLC (CLASSICAL-Lung) (NCT03268057, N= our human-directed antibodies as critical to limiting regulatory T cell 65); (ii) neoadjuvant integrated biomarker trials in patients with (Treg) expansion and activating T effector (Teff) cells. Here we metastatic melanoma (NCT03769155, n=36), metastatic colorectal, present data on this surrogate anti-TNFR2 antibody in two syngeneic pancreatic (NCT03373188, n=32) and head and neck (NCT03690986, mouse models of colon cancer. n=36) cancers treated with pepinemab in combination with nivolu- Methods mab or ipilimumab. Gene expression and immunohistochemical ana- We studied the therapeutic effects of solo and combined immuno- lysis are employed to evaluate changes in immunophenotype as well therapy using the murine-directed anti-TNFR2 antibody in CT26 and as TGFβ-induced effects on TME and tumor progression. MC38 colon tumor models. We compared the new mouse surrogate Results anti-TNFR2 antibody therapy, a commercially available anti-PD1 ther- Anti-SEMA4D antibody enhanced tumor regression when combined apy, and anti-TNFR2/anti-PD1 combination immunotherapy. Mice with antibodies targeting CTLA-4, PD-1, PD-L1, LAG3, and TGFβ in sev- were dosed bi-weekly (100ug/mouse antibody). Antigen-specific CD8 eral preclinical models. For example, anti-SEMA4D plus anti-TGFβ treat- and Treg infiltrates were also studied. ment resulted in maximal tumor growth delay (TGD) of 239% (p<0.01) Results and 10/15 complete tumor regressions (CR) (p<0.05), compared to 10% In the CT26 model, anti-TNFR2 antagonism alone or co-treatment TGD and 0/13 CR with single agent anti-TGFβ or 29% TGD and 1/10 CR with anti-PD1 and anti-TNFR2 was highly efficacious (55-62% of mice with anti-SEMA4D alone in MC38 colon carcinoma model. SEMA4D cured). Anti-PD1 alone was less efficacious (25% cured). In the MC38 blockade reversed expression of genes related to EMT. Additionally, the model, therapy with anti-TNFR2 alone showed some efficacy (20% combination of anti-SEMA4D, folfirinox, and ICI improved survival in cured) and anti-PD1 alone had the least efficacy (10% cured), but KP2-tumor bearing mice, a KPC-derived pancreatic adenocarcinoma anti-PD1 in combination with anti-TNFR2 yielded the best overall sur- model of immune exclusion, myeloid suppression and active TGFb sig- vival (70% cured). Sequential antibody dosing with anti-PD1 followed naling. In clinical trials, pepinemab was well-tolerated and analysis of by anti-TNFR2 yielded no synergy. In contrast, sequential treatment pre- and on-treatment biopsies revealed increased CD8:FoxP3 ratios with anti-TNFR2 first followed by anti-PD1 or the combination of and reduced presence of myeloid derived suppressor cells within TME. anti-TNFR2 plus anti-TNFRF2 showed synergy and highest efficacy. Conclusions Anti-TNFR2 therapy was distinct from anti-PD1 therapy in showing SEMA4D antibody blockade modulates the TME to enhance anti- pronounced regulatory Treg depletion and enhanced Teff infiltration tumor immunity and combination therapies further enhance anti- in the tumor microenvironment, demonstrating in vivo specificity for tumor activity and overcome important resistance mechanisms. Pre- disease-causing cells only in the tumor. liminary data suggest the combination of pepinemab plus immune Conclusions checkpoint therapy is well tolerated and shows initial signals of anti- Anti-TNFR2 immunotherapy provides benefits in two colon cancer tumor activity in patients. Ongoing analysis of various therapeutic models, both as a single agent and when administered in combin- combinations and immunophenotyping of tissue biopsies will shed ation with anti-PD1. Anti-PD1 before anti-TNFR2 was associated with light on mechanism of action of SEMA4D antibody blockade in sev- poor outcomes for survival, histology and lack of long-term cure, eral combination therapies. suggesting that non-specific unleashing of the immune system with anti-PD1 destroys the tumor microenvironment specificity of anti- Acknowledgements TNFR2. These results highlight the value of anti-TNFR2 antagonism We would like to thank the clinical and research teams at Emory University, in vivo in mouse tumor models as solo therapy or as a combination including Doctors Greg Lesinsky, Christina Wu, Conor Steuer, Nabil Saba, therapy, administered first or concurrently with anti-PD1. This study Michael Lowe, Ragini Kudchadkar, and Brian Olson. We also extend gratitude of new immunotherapy combinations highlights the need to test to the Avelumab team at EMD Serono, as well as clinical investigators and both single agent therapy as well as the sequencing of combination their teams related to the CLASSICAL-Lung trial. therapy as new agents are brought forward to the clinic. Trial Registration NCT03268057 References NCT03769155 1. Torrey H, Butterworth J, Mera T, et al.Targeting TNFR2 with antagonistic NCT03373188 antibodies inhibits proliferation of ovarian cancer cells and tumor- NCT03690986 associated Tregs. Sci Signal. 2017;10(462). pii: eaaf8608. Ethics Approval References Mice were tested and monitored for tumor growth by either Champions 1. Evans EE, et al. Antibody Blockade of Semaphorin 4D Promotes Immune Oncology (Hackensack, NJ) or a third-party pharmaceutical company in Infiltration into Tumor and Enhances Response to Other accordance with their animal welfare guidelines. Immunomodulatory Therapies. Cancer Immunol Res. 2015;3(6):689-701. 2. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith ES, Zauderer M, Evans P480 EE, Allen CT. Semaphorin4D inhibition improves response to immune Heterologous prime-boost vaccination safely enhances antitumor checkpoint blockade via attenuation of MDSC recruitment and function. immunity to the colorectal antigen GUCY2C Cancer Immunol Res. 2019;7(2):282-291 John Flickinger, BS, Robert Carlson, Jagmohan Singh, Trevor Baybutt, BS, Elinor Leong, Alicja Zalewski, Amanda Pattison, Jeffrey Rappaport, Joshua P479 Barton, Scott Waldman, Adam Snook, PhD Evaluation of a TNFR2 antibody with and without anti-PD-1 Thomas Jefferson University, Philadelphia, PA, United States therapy in two murine colon cancer models Correspondence: Adam Snook (adam.snook@jefferson.edu) Katie Case, Lisa Tran, Hui Zheng, Michael Yang, Denise Faustman, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P480 PhD Massachusetts General Hospital/Harvard Medical School, Charlestown,, Background MA, United States The transmembrane receptor guanylyl cyclase C (GUCY2C) is an Correspondence: Denise Faustman (faustman@helix.mgh.harvard.edu) emerging target for colorectal cancer immunotherapy. Recently, an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P479 adenovirus-based vaccine against GUCY2C was tested in a phase I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 262 of 272 clinical trial where it was found to safely induce GUCY2C- Although paradoxically, obesity has been found to improve re- specific immune responses [1]. However, GUCY2C immune re- sponse to immunotherapy in a subgroup of patients with mel- sponses following immunization wane over time and optimal anoma [4,5]. GUCY2C immunity may require multiple GUCY2C vaccinations. Methods Moreover, repeated vaccination utilizing adenovirus-based Wildtype C57BL/6femalemicewererandomizedto ahigh-fat vectors is hindered by the production of adenovirus-specific (60%) or low-fat standard chow (14%) diet for 16 weeks to gen- antibodies following first vaccination. For this reason, we have erate diet-induced obese (DIO) or age-matched lean controls, generated a recombinant strain of Listeria monocytogenes respectively. Animals were then challenged with the syngeneic secreting GUCY2C (Lm-GUCY2C) to boost GUCY2C immune E0771 mammary carcinoma cell line. Tumor outgrowth was responses. These studies assess the immunogenicity, thera- quantified by caliper measurements, bioluminescent imaging peutic efficacy, and safety of a heterologous prime-boost (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, immunization utilizing adenovirus and Listeria monocytogenes and endpoint tumor weights. Once tumors were palpable, ani- vectors. mals were randomized to receive no therapy or immunotherapy Methods consisting of intratumoral CpG co-administered with non- T-cell responses following vaccination were assessed by IFNy ELISpot. replicative adenovirus (Ad) encoding murine TNF-related apop- Tumor protection following vaccination was assessed by challenging tosis inducing ligand (TRAIL; AdT). Whole tumor immunogenetic mice with a luciferase-expressing CT26 colorectal cancer cell line. Lu- gene expression profiles were evaluated using nanoString and minescence following luciferin injection and overall survival were immune populations were assessed via multi-parameter flow cy- quantified. Safety was assessed by histopathologic evaluation of tometry. T cell cytokine production was evaluated via flow cy- known GUCY2C-expressing tissues following vaccinations. tometry following ex vivo CD3/CD28 stimulation. Results Results Construction of Lm-GUCY2C was validated by GUCY2C western DIO mice had significantly increased body weights at tumor blot on J774A.1 macrophage cells infected with Lm-GUCY2C. challenge versus lean controls (45 versus 25 grams, p <0.0001) Optimal GUCY2C immunogenicity was achieved utilizing All methodologies demonstrated that obesity significantly in- adenovirus-GUCY2C to ‘prime’ GUCY2C immune responses with creases primary mammary tumor outgrowth and alters cellular Lm-GUCY2C to ‘boost’ and was found to be superior to homolo- and immunogenetic profiles within the tumor microenviron- gous administration using either adenovirus-GUCY2C or Lm- ment. Notable alterations include significant reductions in the GUCY2C vectors. Similarly, anti-tumor studies found heterol- frequency of CD4+ T cells, CD8+ T cells, and CD19+ B cells; ogous administration of adenovirus-GUCY2C and Lm-GUCY2C to with a simultaneous increase in the frequency of myeloid- be superior to homologous administrations. Importantly, histo- derived suppressor cells (MDSCs). Following immunotherapy ad- pathologic evaluation of mice following heterologous prime- ministration, lean animals controlled tumor growth whereas DIO boost revealed no toxicity. animals experienced progressive tumor growth. Despite these Conclusions differential tumor outcomes, both lean and DIO animals dis- Heterologous prime-boost vaccination utilizing adenovirus and played robust intratumoral effector CD8+ T cell accumulation Listeria vectors expressing the tumor antigen GUCY2C demon- and ex vivo function. In contrast, immunotherapy reduced the strate superior immunogenicity and antitumor efficacy over intratumoral accumulation of monocytic and granulocytic MDSCs homologous immunization with either vector, a strategy that only in lean animals. Both MDSC populations persisted in the can be translated to colorectal cancer patients. tumors of animals with DIO, resulting in less favorable effector CD8+ T cell to MDSC ratios. Acknowledgements Conclusions The authors thank the Center for Cell and Gene Therapy, Baylor College of Our data implicate obesity as a causal factor in impairing im- Medicine for assistance in adenovirus vaccine manufacturing. munotherapeutic efficacy in a pre-clinical model of breast can- cer, potentially via accumulation of MDSCs. Our data suggest Reference that clinical investigation and consideration is needed for fac- 1. Snook AE, BaybuttTR, XiangB,Abraham TS,Flickinger JC,HyslopT, tors such as body composition and body mass index when Zhan T, Kraft WK, Sato T, and Waldman SA. Split tolerance permits treating breast cancer patients with immunotherapy. safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J. Immunother Cancer. 2019;7, 104. Acknowledgements Ethics Approval This study was supported in part by NIH-NIGMS training grant T32GM008111 Studies were approved by the Thomas Jefferson University IACUC (Protocol # to JTG. 01956). References P481 1. Incio, J., et al., Obesity promotes resistance to anti-VEGF therapy in breast Obesity impairs immunotherapeutic efficacy in pre-clinical breast cancer by up-regulating IL-6 and potentially FGF-2. Sci Transl Med, 2018. cancer 10(432). Justin Gibson, BS, Rachael Orlandella, BS, William Turbitt, Robert Sorge, 2. Sheng, X., et al., Adipocytes Sequester and Metabolize the PhD, Lyse Norian, PhD Chemotherapeutic Daunorubicin. Mol Cancer Res, 2017. 15(12): p. 1704- University of AlabamaBirmingham, AL, United States 1713. Correspondence: Lyse Norian (lnorian@uab.edu) 3. Lehuede, C., et al., Adipocytes promote breast cancer resistance to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P481 chemotherapy, a process amplified by obesity: role of the major vault protein (MVP). Breast Cancer Res, 2019. 21(1): p. 7. Background 4. McQuade, J.L., et al., Association of body-mass index and outcomes in Obesity has long been known to worsen prognosis and survival patients with metastatic melanoma treated with targeted therapy, im- for breast cancer patients. Recent reports further indicate that munotherapy, or chemotherapy: a retrospective, multicohort analysis. obesity negatively impacts response to targeted anti-VEGF ther- Lancet Oncol, 2018. 19(3): p. 310-322. apy [1] and efficacy of chemotherapeutics [2,3]. However, no 5. Wang, Z., et al., Paradoxical effects of obesity on T cell function during studies have yet investigated the impact of obesity on re- tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): sponse to immunotherapy in the context of breast cancer. p. 141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 263 of 272 P482 P483 STACT: A novel therapeutic platform that delivers TLR enhanced GVAX elicits tumor-specific tissue resident memory immunomodulatory payloads to tumor-resident myeloid cells After T cells independent of T cell priming IV dosing and demonstrates potent anti-tumor efficacy in Michael Korrer, Young Kim, MD, PhD, David Taylor preclinical studies Vanderbilt University Medical Center, Nashville, TN, United States Laura Glickman, PhD, Christopher Rae, PhD, Alexandre Iannello, PhD, Correspondence: Young Kim (young.j.kim@vanderbilt.edu) Anastasia Makarova, PhD, Haixing Kehoe, MS, John Faulhaber, Bill Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P483 Hanson, Christopher Thanos, PhD Actym Therapeutics, Inc, Berkeley, CA, United States Background Correspondence: Christopher Thanos (cthanos@actymthera.com) GVAX, a genetically modified whole cell vaccine, is proposed to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P482 work by recruiting and activating antigen-presenting cells which then traffic to the draining lymph node and elicit tumor-specific Background Tcells.Inmouse models GVAX haslittle therapeutic benefit as Many experimental therapies developed to promote proper T- a monotherapy, but when combined with a Toll-like receptor cell infiltration in immune-excluded tumors are too toxic for sys- (TLR) 4 adjuvant, TLR Enhanced GVAX (TEGVAX) significantly re- temic administration, which will be required in a metastatic dis- duces tumor burden. Paradoxically, the increased therapeutic ease setting. These include innate targets such as STING and benefit of TEGVAX corresponds with a decrease in delivery of TLR agonists, co-stimulatory receptor agonists, and type I/II tumor antigen to the draining lymph node. In order to improve cytokine receptor combinations. To address these limitations, the efficacy of the TEGVAX platform, it is critical to understand we have engineered a highly attenuated, microbial-based im- the mechanism by which it induces anti-tumor immune re- munotherapy platform called STACT (S. Typhimurium Attenuated sponses. Since TEGVAX requires T cells to work, yet significantly Cancer Therapy). Upon IV administration, the microbe traffics to reduces antigen delivery to the draining lymph node, we and enriches in the tumor microenvironment. There, it is specif- hypothesize that TEGVAX functions independent of lymph node ically phagocytosed and lysed by tumor-resident myeloid cells, priming. enabling efficient delivery of plasmids encoding immunomodu- Methods latory payloads. Using our proprietary platform, we have gener- B16-mOVA cells injected s.c. into B6 mice. TEGVAX (1e6 B16- ated multiple systemically-administered therapies that target mOVA + 1e5 B78H1-GM Irradiated cells + 20ug MPLA) injected several well-characterized, yet intractable immune pathways. s.c. into opposite flank 5 days after tumor injection. 10ug daily Characterization of STACT microbes encoding constitutively ac- FTY720 i.p. on day 4. tive STING variants (STACT-STING) and IL-2 (STACT-IL2) are pro- Results vided as examples. To determine if TEGVAX alters the priming of CD8 T cells, we Methods performed longitudinal studies of OT-1 CD8 T cell proliferation The STACT platform strain has been engineered using precision in vivo comparing TEGVAX to GVAX. We found that GVAX in- genome modifications for enhanced tolerability, reduced im- duced rapid proliferation of OT-1 cells, whereas TEGVAX failed to munosuppressive inflammation, and tumor specificity. STACT- induce OT-1 cell proliferation as shown by FACS and in vivo im- mediated delivery of immunomodulatory proteins in primary aging. We then determined if TEGVAX required myeloid or NK mouse and human cells was confirmed by in vitro functional as- cells to reduce tumor burden. We found that TEGVAX reduced says. STACT strains were evaluated in vivo for tumor-specific en- tumor burden in NK depleted, but not myeloid cell depleted richment, payload delivery, tolerability, and therapeutic efficacy mice. These results raised the question if priming in the draining following IV administration in several subcutaneous syngeneic lymph node was required for therapeutic efficacy of TEGVAX. To tumor studies. determine this, we performed tumor growth studies with mice Results administered FTY720, which sequesters circulating T cells in STACT was found to be 100,000-fold enriched in tumors, relative lymph nodes. We demonstrated that TEGVAX significantly re- to spleen, after tail vein injections in mice. Flow cytometry duced B16-mOVA tumor growth even in the presence of FTY720 staining revealed that STACT does not infect stromal or tumor treatment, suggesting T cell priming was not required. Immune cells and is specifically targeted by tumor-resident myeloid cells phenotyping of TEGVAX treated mice, showed a significant in- (TAMs, DCs, and monocytes). STACT is rapidly phagocytosed crease in tumor infiltrating tissue-resident memory (Trm) T cells. and then destroyed by these cells, delivering its plasmid DNA Conclusions contents encoding immunomodulatory protein expression cas- Our results demonstrate that combining TLR4 agonist with GVAX settes. We have measured highly efficient heterologous gene (TEGVAX) completely alters the immune response to vaccination. transfer and protein expression within primary mouse and hu- TEGVAX does not prime naïve T cells nor require trafficking of T cells man M2 macrophages treated with STACT, at levels comparable from LN to tumor to function. We observed an increased number of to DNA transfection. Therapeutically relevant levels of IL-2 were Trm CD8 T cells infiltrating the tumor leading us to conclude that measured in the tumor microenvironment of STACT-IL2 treated TEGVAX is functioning by eliciting a tumor-specific Trm T cell re- mice several weeks after dosing. For STACT-STING, significant sponse independent of lymph node priming. tumor growth inhibition, including complete tumor regressions Ethics Approval were observed, and the therapy was well tolerated. Immune The study was approved by Vanderbilt University Animal care and correlates were consistent with on-target expression in the use board tumor microenvironment, and the anti-tumor effect was adap- tive immune mediated. P484 Conclusions CB-708, an orally bioavailable small molecule inhibitor of CD73 STACT is a highly attenuated, microbial-based therapeutic platform with immunostimulatory and anti-tumor activity engineered to deliver immunomodulatory payloads, alone or in com- Clarissa Lee, PhD, Deepthi Bhupathi, Roland Billedeau, Jason Chen, Lijing bination, to phagocytic cells of the solid tumor microenvironment Chen, Rosalyn Dang, Matthew Gross, Tony Huang, Weiqun Li, PhD, Yong after systemic administration. The goal of STACT therapy is to pro- Ma, Andrew MacKinnon, Gisele Marguier, MS, Silinda Neou, MS, mote immune-mediated tumor clearance of T-cell excluded solid tu- Francesco Parlati, PhD, Natalija Sotirovska, MS, Sandra Spurlock, Timothy mors and elicit durable anti-tumor immunity. Stanton, Susanne Steggerda, PhD, Jing Zhang, Winter Zhang, Jim Li Ethics Approval Calithera Biosciences, South San Francisco, CA, United States All animals were used according to protocols approved by an Institu- Correspondence: Jim Li (jli@calithera.com) tional Animal Care and Use Committee and maintained in specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P484 pathogen-free conditions in a barrier facility. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 264 of 272 Background Methods High adenosine (ADO) in the tumor microenvironment suppresses CT26 tumor measurement seven days after treatment was used to the immune response against cancer cells by inhibiting immune ef- define tumors as KD033-surrogate responders (decreasing tumor vol- fector functions and promoting the development of immunosuppres- umes), non-responders (no change or increasing tumor volumes) and sive cells. Extracellular ADO can be generated from ATP released by non-targeted IL-15 best responders. RNA was isolated from these tu- cells undergoing stress or death through the combined actions of mors and analyzed using the Nanostring PanCancer IO 360 Gene Ex- the ectonucleotidases CD39 (ATP to AMP) and CD73 (AMP to ADO). pression Panel for immune cell responses. Combination therapy with Inhibition of ADO production via CD73 is a promising therapeutic ap- genes identified through Nanostring analysis was evaluated in a proach for the treatment of cancer. tumor model where KD033-surrogate monotherapy showed minimal Methods efficacy such as 4T1, an aggressive breast carcinoma murine model We developed CB-708, a potent and selective small molecule inhibi- involving spontaneous metastases to other organs. 4T1 cells were tor of CD73. The potency of CB-708 was evaluated against recombin- injected into the mammary gland of Balb/c mice and grown to 100 ant CD73 and CD73-expressing cells using a malachite green assay. mm3 prior to treatments. Tumors and metastasis nodules in the lung Selectivity against related ectonucleotidases was also assessed. Inhib- were evaluated. ition of CD73 in plasma was measured using LC/MS to assess conver- Results sion of 15N5-AMP into 15N5-ADO. Reversal of AMP-mediated Transcriptional analysis showed that CTLA-4 was one of the top immune suppression of human CD8+ T cells was determined by genes that was differentially upregulated after KD033-surrogate treat- measuring T cell activation in the presence of exogenous AMP. T cell ment in comparison to non-targeting IL-15. In the 4T1 tumor model, proliferation was assayed by flow cytometry and cytokine levels were monotherapies of both KD033-surrogate or anti-CTLA-4 did not have measured by ELISA. The EG7 and CT26 syngeneic tumor models were any effect on 4T1 tumor growths; however, the combination therapy used to assess the therapeutic effect of CB-708. with single dose of KD033-surrogate and repeat dose of anti-CTLA-4 Results showed a decrease in the average number of lung metastases and a CB-708 potently and completely inhibited soluble human CD73 (IC50 significant tumor growth inhibition compared to vehicle-treated = 170 pM) and cell-bound human CD73 (IC50 = 210 pM), but did not animals. inhibit human CD39, ENTPD2, or ENTPD3. CB-708 retained high po- Conclusions tency in the presence of whole human plasma (IC50 = 380 pM) and Analysis of murine tumors treated with KD033 surrogate in vivo re- reversed AMP-mediated suppression of human CD8+ T cell prolifera- sulted in combination strategies, including KD033 in combination tion and production of IFNγ and granzyme B in vitro. Oral administra- with CTLA-4, that can be exploited in targeting resistant and refrac- tion of CB-708 was well-tolerated in tumor-bearing mice, resulted in tory cancers. Based on the therapeutic activity and improved safety sustained exposure above mouse plasma IC50, and exhibited single- of the fusion protein, Kadmon plans to initiate clinical studies of agent tumor growth inhibition in syngeneic tumor models including KD033 in 2019. established EG7 tumors. Efficacy in the EG7 model was dependent Ethics Approval on CD8+ T cells and was correlated with pharmacodynamic inhib- Animal studies were conducted for Kadmon by Crown Bioscience Inc. ition of CD73. Enhanced tumor growth inhibition was observed when with approved SOP and IACUC protocol. CB-708 was combined with checkpoint inhibition (anti-PD-L1) or with chemotherapy (oxaliplatin, doxorubicin, docetaxel) in the EG7 model. P486 Conclusions Releasing the break on T cell activation through novel small CB-708 is an orally bioavailable and highly potent small molecule in- molecule inhibition of HPK1 hibitor of CD73. CB-708 reverses the immunosuppressive effects of Minhui Shen, Gayathri Bommakanti, PhD, Deanna Mele, PhD, Neil AMP-derived ADO in vitro and in vivo and has anti-tumor activity. Grimster, PhD CB-708 is expected to enter clinical development in 2019. AstraZeneca, Waltham, MA, United States Correspondence: Deanna Mele (Deanna.Mele@astrazeneca.com) P485 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P486 Synergistic efficacy of anti-PD-L1/IL-15 fusion protein in combination with anti-CTLA-4 antibody in a murine orthotopic 4T1 Background breast carcinoma model Loss of immune surveillance is required for cancer cell growth and 1 2 2 2 Stella Martomo, PhD , Dan Lu, MA , Jeegar Patel , Zhanna Polonskaya , metastasis, tumors co-opt suppressive mechanisms to evade detec- 2 2 Xenia Luna , Kevin McCracken tion by the immune system. The immune system is equipped with 1 2 Kadmon Corporation, New York, NY, United States; Kadmon, New York, multiple feedback mechanisms to limit inflammation in order to pre- NY, United States vent autoimmunity, tumors activate these pathways to escape im- Correspondence: Stella Martomo (stella.martomo@kadmon.com) munity. HPK1 (hematopoietic progenitor kinase 1) is a negative Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P485 regulator of T cell activation, the kinase activity limits T cell signaling and tumoricidal cytokine production. Recent literature has shown in- Background activation of the kinase function of HPK1 prevents tumor progression Administration of immune checkpoint inhibitors anti-PD1/PD-L1 have in murine tumor models [1]. led to durable objective responses in select cancers. However, a sub- Methods stantial number of patients fail to respond or become resistant to these We used lentiviral delivered shRNAs and CRISPR/Cas9 technology to therapies. We have generated a therapeutic fusion protein (KD033) by delete HPK1 in Jurkat cells and primary human T cells, respectively. combining a proprietary high affinity anti-human-PD-L1 (or anti- Jurkat T cells (wt/KO) were activated with anti-CD3 antibody and cell murine-PD-L1, (KD033-surrogate)) antibody with human IL-15. Initial as- lysates were assessed by western blot to examine signaling events sessment of this fusion antibody showed enhanced tolerability relative downstream of the T cell receptor (TCR). Primary human T cells or to a non-targeted IL-15 fusion protein in addition to its potent anti- CRISPR/Cas9 KO T cells were activated in vitro using anti-CD3/anti- tumor activity. In the CT26 murine colorectal tumor model, a single CD28 antibodies in the presence or absence of HPK-1 inhibitors +/- dose of KD033-surrogate consistently resulted in antitumor response prostaglandin E2 (PGE2). Cell viability and numbers were assessed by that included tumor clearance and long-term tumor-free survival. Initial flow cytometry. Cytokines were quantified by ELISA. analysis of KD033-surrogate treatment showed robust adaptive and Results cytotoxic immune gene signatures in tumors leading to tumor inhib- We present novel findings demonstrating that the role of HPK1 is ition and memory responses. We further analyzed tumors from KD033- conserved in human Jurkat cells as well as in primary human T cells. surrogate responders and non-responders to evaluate possible thera- Loss of HPK1 enhanced T cell receptor signaling in Jurkat cells. In peutic combinations to broaden the response of KD033. CRISPR/Cas9 KO HPK1 primary human T cells, TCR activation resulted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 265 of 272 in enhanced cytokine secretion and proliferation concomitant to a 100% lethality by 60 days, but 3 doses of AgN2a 4P leads to 100% decrease in pSLP76, the target molecule phosphorylated by HPK1. survival at 50 days after both allogeneic and syngeneic BMT. Consistent with the KO phenotype, our HPK1 inhibitors resulted in Conclusions enhanced T cell activation, cytokine secretion and proliferation. In Co-culture of NK cells with an engineered costimulatory vaccine is an addition, the inhibitors were able to rescue T cells from PGE2 medi- effective strategy to induce apoptosis of neuroblastoma tumor cells ated suppression. In vivo studies are currently underway to examine by increasing NK-mediated cytokine production and cytotoxicity, and anti-tumor activity of these compounds in various syngeneic models. enhances anti-tumor effects after BMT. Usage of cell-based vaccines Conclusions after BMT could be an effective strategy to augment NK cell activity In summary our small molecule inhibitors of HPK1 could enhance against neuroblastoma. anti-tumor immunity through increased T cell function overcoming suppressive signals in the tumor microenvironment and thus Acknowledgements broaden the response to check point inhibitors for cancer AgN2a 4P was a gift from Dr. Bryon Johnson at Medical College of immunotherapy. Wisconsin. This work was supported by grants from the St. Baldrick’s – Stand up to Cancer Pediatric Dream Team Translational Research Grant SU2C- Acknowledgements AACR-DT-27-17, NCI/NIH R01 CA215461, American Cancer Society Research Minhui Shen1, Gayathri Bommakanti1, Kevin Xu1, Kun Song1, Rob Ziegler1, Scholar grant RSG-18-104-01-LIB, Hyundai Hope on Wheels and the MACC Jason Kettle2, Adelphe Mfuh1 Jason Sheilds2, Neil Grimster1, Lisa Drew1, Fund (C.M.C). We would like to thank the UWCCC core facilities, who are Stephen Fawell1, Deanna A. Mele1 supported in part through NCI/NIH P30 CA014520. Stand Up to Cancer is a 1AZ Discovery Early Oncology, Boston, MA, 2AZ Discovery Early Oncology, division of the Entertainment Industry Foundation. Research Grants are Cambridge UK, MA administered by the American Association for Cancer Research, the Scientific Partner of SU2C. Reference Ethics Approval 1. Hernandez S, Qing J et al. The Kinase Activity of Hematopoietic The study was approved by University of Wisconsin-Madison Animal Care Progenitor Kinase 1 is Essential for the Regulation of T cell Function and Use Committee, approval number M005915. Cancer. Cell. 2018; 25: 80-94. P488 P487 IPH5201, a blocking antibody targeting the CD39 Combining an engineered costimulatory vaccine with NK cells immunosuppressive pathway, unleashes immune responses in induces an anti-tumor effect against murine neuroblastoma combination with cancer therapies 1 1 1 1 in vitro and after bone marrow transplant in vivo Pascale Andre , Ivan Perrot , Caroline Denis, PhD , Marc Giraudon-Paoli , 1 1 1 1 Nicholas Mohrdieck, BS, Paul Bates, Sean Rinella, Katharine Tippins, Severine Augier , Rachel Courtois , Diana Jecko , Thomas Arnoux , 1 1 2 Christian Capitini, MD Violette Breso , Nicolas Gourdin, PhD , Nadia Luheshi, PhD , Ariane 1 1 1 1 University of Wisconsin-Madison, Madison, WI, United States Morel, PhD , Yannis Morel, PhD , Eric Vivier , Carine Paturel, PhD 1 2 Correspondence: Christian Capitini (ccapitini@pediatrics.wisc.edu) Innate Pharma, Marseille, France; AstraZeneca, Milton, Cambridge, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P487 United Kingdom Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P488 High risk neuroblastoma remains a challenge to cure with only 50% survival, despite multi-modality treatment. Natural killer (NK) cells Background have been previously shown to have activity versus neuroblastoma CD39 is an extracellular ectonucleotidase highly expressed in the but have not been consistently successful in clinical trials. NK cell ac- tumor microenvironment, by stromal cells and some immune in- tivation via co-culture with a vaccine engineered to express CD54, filtrating cells. CD39 contributes to the production of adenosine, CD80, CD86, and CD137L, called AgN2a 4P, was studied to investi- an inhibitor of immune response, via sequential hydrolysis of ad- gate NK cells’ ability to induce cytotoxicity of murine neuroblastoma enosine triphosphate (ATP) and adenosine diphosphate into ad- tumor cells in vitro and in vivo. enosine monophosphate, which then is degraded into adenosine Methods by CD73 enzyme. In contrast, ATP has immune-stimulatory activ- NKs and irradiated AgN2a 4P were co-cultured in ratios of 1 (NKs):0.5 ity through promoting dendritic cell (DC) maturation. Blockade of (AgN2a 4P) and 1:1, and compared to NK only and AgN2a 4P only CD39-mediated degradation of ATP may therefore stimulate anti- controls, with all groups receiving IL-15/IL-15Ralpha, and then ana- tumor immunity across a wide range of tumors by preventing lyzed by flow cytometry, multiplex cytokine analysis, and cytotoxicity production of immunosuppressive adenosine and by promoting in vitro after 1, 3, 5, 7, and 9 days. To study the efficacy of in vivo accumulation of immunostimulatory ATP in the tumor microenvir- vaccination with AgN2a 4P after bone marrow transplant (BMT), onment. IPH5201 is a humanized monoclonal antibody that se- C57BL/6 or B6AJ recipients were lethally irradiated, followed by trans- lectively binds to and inhibits the activity of both membrane- plantation of T-cell depleted C57BL/6 donor bone marrow on day +0. bound and soluble human CD39. Here, we explored the efficacy BMT recipients were then treated with the AgN2a 4P vaccine for 2 of IPH5201 in vitro and in vivo in immunocompetent human versus 3 weekly doses, and with or without adoptive transfer of CD39 knockin (huCD39KI) mouse model in combination with im- donor NK cells to accelerate immune reconstitution. All recipients mune checkpoint inhibitor. were then challenged with NXS2 neuroblastoma tumor, and followed Methods for tumor growth and survival. In vitro, efficacy of IPH5201 was evaluated (1) on the phenotypic Results changes and stimulatory potential of monocyte-derived DC, (2) on The NK:AgN2a 4P co-culture at 1:0.5 and 1:1 increases Ly49A+ NKs the inflammasome pathway by assessing interleukin-1b secretion from 6% to 21%, and Ly49D+ NKs from 3% to 30% from day +0 to from in vitro-derived M1 macrophages, and (3) on T cell proliferation. day +9. pSTAT1 activation remains consistently high between 80%- HuCD39KI mice were characterized for the expression and function 98%, and pSTAT3 activation remains 20%-50%, across the co-culture of human CD39. To assess CD39 blockade in vivo, a mouse IgG1 ver- period. NK cells release increased levels of IFN-gamma and IL-6 at sion of IPH5201 was produced (moIPH5201), which contained key the co-cultured ratios of 1:0.5 and 1:1, and CXCL1 at the 1:1 ratio, as point mutations in the Fc region to abrogate Fc receptor interactions. compared to the NK with IL-15/IL-15Ralpha controls. The NK:AgN2a Antitumor efficacy of CD39 blockade was assessed in huCD39KI mice 4P ratios of 1:0.5 and 1:1 induce significantly increased apoptosis of grafted with mouse tumor cells not expressing mouse CD39. Neuro2a neuroblastoma cells than NK cells with IL-15/IL-15Ralpha HuCD39KI mice were treated with blocking anti-human CD39 Ab, ei- alone. In vivo, injection of 2 doses of AgN2a 4P vaccine leads to ther alone or in combination with a blocking anti-mouse PD-L1 Ab. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 266 of 272 Results the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and As hypothesized, in vitro IPH5201 enhanced the phenotypic 5 mg/kg induced significant tumor growth inhibition (55% and 57%, maturation and the activation of DCs and macrophages by inhi- respectively) as compared to vehicle control or Fc-silenced huIgG1 biting ATP hydrolysis. IPH5201 also efficiently restored T cell controls. Additionally, 2 out of 9 animals in the 5 mg/kg MCLA-145– proliferation in a dose-dependent manner to the levels ob- treated group had complete tumor regression. MCLA-145 increased served in the absence of ATP addition. Thus, IPH5201 preserved the number of infiltrating CD8+ T cells, as well as the percentage of extracellular ATP, thereby promoting the activation of DCs and central memory CD8+ T cells. macrophages and limiting adenosine accumulation and its im- Conclusions munosuppressive effect on T cells. Furthermore, moIPH5201 MCLA-145 is currently undergoing clinical development mice treatment efficiently inhibits membrane and soluble hu- (NCT03922204). man CD39, from huCD39KI mice ex vivo without mediating CD39-expressing cell depletion in vivo. Finally, blockade of P490 CD39 potentiates the anti-tumor efficacy of anti-PD-L1 Ab Antibody derived from an elite responder to checkpoint inhibitor monotherapy. therapy relieves immunosuppression by tumor associated Conclusions macrophages Together these data indicate that blocking CD39 in conjunction with Randi Simmons, Siddarth Chandrasekaran, Melissa Conerly, Tyrel Smith, PD-1/PD-L1 checkpoint inhibitors provides increased anti-tumor effi- Sam Lam, Jacqueline Pham, Ray Fox, Darbie Whitman, Meghan Zuck, cacy and support the rational for assessing this combination in clin- Sara Carbonetti, Kamal Puri, PhD ical trials. Oncoresponse Inc, Seattle, WA, United States Correspondence: Kamal Puri (kpuri@oncoresponseinc.com) P489 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P490 Identification and characterization of MCLA-145 (CD137 x PD-L1): a bispecific antibody that requires PD-L1 binding to activate CD137 Background 1 1 1 Simon Plyte , Cecile Geuijen , John de Kruif , Pieter Fokko van Loo, Tumor-associated macrophages (TAM)s in the tumor microenviron- 1 1 1 1 PhD , Paul Tacken , vanessa Zondag-vander Zande , Rinse klooster , ment (TME) contribute to tumor immune evasion by suppressing 1 1 1 1 hans van Maaden , Erik Rovers , steef engels , floris franzen , abdul anti-tumor immune responses and by promoting a tumorigenic mi- 1 1 1 2 basmeleh , willem bartelink , Mark Throsby , Patrick Mayes , Horacio lieu. High infiltration of immunosuppressive myeloid cells generally 2 2 2 2 2 Nastri , shaun stewart , jing zhou , steve wang , Chen-yen Huang , predicts unfavorable prognosis. Reduction or repolarization of sup- 2 2 2 2 thomas codamine , ashwini kularni , yao bin lui , arpita mondal , leslie pressive myeloid cells is an attractive strategy to enhance clinical re- 2 2 2 2 2 hall , soeon kim , marina martinez , shaun o'brien , edmund moon , sponses to immune checkpoint inhibitor (CPI) therapy. Cancer steven albelda patients who achieved durable response to CPI therapy (elite re- 1 2 Merus, Utrecht, Netherlands; Incyte Research Institute, Wilmington, DE, sponders) may harbor antibodies that contribute to clinical response United States by promoting an anti-tumor TME. Correspondence: Simon Plyte (simon.plyte@merus.nl) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P489 B cells derived from elite responders were cloned and screened for IgG antibodies binding to myeloid derived suppressor cells. Background Hits were prioritized based on myeloid binding profiles and their CD137 (4-1BB) is costimulatory receptor on T and NK cells that re- variable-regions sequenced, cloned, and expressed as recombin- quires clustering to elicit its effects on target cells and enhance adap- ant IgG1. Cloned antibodies underwent further characterization to tive immune responses against tumors. The development of CD137 evaluate their ability to reverse the immunosuppressive effects of targeted agents for cancer therapy has been hampered by on-target myeloid cells in assays modelling the TME. Primary human mono- off-tumor toxicity in the case of agonistic monospecific antibodies, or cytes and T cells were used to interrogate antibody-dependent limited antitumor activity in the case of Fcγ-mediated crosslinking of immunomodulatory responses in vitro. A humanized mouse mAbs. model was used to evaluate the anti-tumor activity of the lead Methods antibody, OR2805. To address the issues of toxicity and efficacy, we have identified a Results highly selective and potent CD137xPD-L1 bispecific antibody, MCLA- The target of OR2805 is highly expressed on TAMs and M2-like 145. Collections of common light chain Fabs recognizing CD137 and macrophages. OR2805 does not bind to other hematopoietic PD-L1 were produced based on antibody panels from immunized cells nor a panel of human primary non-immune cells. The anti- MeMo® mice. Unbiased, combinatorial, functional screening was then body stains positively on M2-like TAMs from primary human performed on a large and diverse panel of CD137xPD-L1 bAbs to lung tumor samples. OR2805 treatment reduces expression of identify those for which CD137 mediated activation is dependent on cell-surface markers associated with tumor-promoting M2c-like the presence of PD-L1 on a neighboring cell macrophages. In co-culture assays, OR2805 relieves the suppres- Results sive effect of M2 macrophages and resulted in increased T cell Both the CD137 and PD-L1 Fab arms block the interaction with their activation and proliferation, upregulation of T cell activation respective ligands as demonstrated in competition flow cytometry or markers, and enhanced T cell-mediated tumor cell killing. Ad- ELISA assays, respectively. MCLA-145 drives transactivation of CD137 ministration of OR2805 in humanized NSG-SGM3 mouse tumor in the vicinity of cells expressing PD-L1 and the degree of CD137 ag- models resulted in approximately 50% reduction in A549 tumor onistic activity in T cells correlated with the expression level of PD-L1 growth and a 60% reduction in H1975 tumor growth. In this on neighboring cells. CD137 signaling was induced by MCLA-145 in model, OR2805 treatment significantly increased the proportions multiple primary human immune cell assays and reversed T cell sup- of human CD8+ T cells and human CD11b+ myeloid cells in the pression mediated by M2 macrophages or Tregs, in vitro. In one hu- spleen as well as significantly enhanced expression of activation manized mouse tumor model, human T cells expressing NY-ESO markers (ICOS, OX-40) by human CD8+ T cells. specific TCR were adoptively transferred to mice bearing A549 tu- OR2805 reduces TAM-mediated immunosuppression and enhances mors, which expressed NY-ESO antigen and human PD-L1. MCLA-145 anti-tumor immune responses. OR2805 treatment induces robust treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) anti-tumor activity in lung cancer xenograft models in humanized as compared to T cell only–treated mice. In the tumors of MCLA- mice. This data justifies further development of OR2805 as anti- 145–treated mice, the percentage of NY-ESO specific CD8+ T cells cancer therapy in combination with other CPI treatments. OR2805 were significantly increased compared with controls. In a second has the potential to increase the number of patients who may bene- model, mice engrafted with human CD34+ cells were implanted with fit from current CPI therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 267 of 272 P491 References Preclinical development of a novel TNFRSF25 agonist antibody, 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. PTX-35, for cancer immunotherapy combinations Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 Matthew Seavey, PhD, Jayalakshmi Miriyala, MS, Jason Rose, MS, Vikas 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg Tahiliani, PhD, Patrick Dillon, PhD, Elena Gorovits, PhD, Jeff Hutchins, expansion in mice by TNFRSF25 prevents allergic lung inflammation. J PhD, Rahul Jasuja, PhD Clin Invest.; 120(10):3629-3640 Heat Biologics, Inc., Durham, NC, United States 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed Correspondence: Matthew Seavey (mseavey@heatbio.com) M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P491 That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell Background responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Tumor Necrosis Factor Receptor Super Family 25 (TNFRSF25), 5. Schreiber T.H., et al.; (2014). Comparative combination cancer also known as Death Receptor 3 (DR3), is preferentially immunotherapy with vaccination and TNFRSF stimulation. Society of expressed by activated and antigen-experienced T-cells. Immunotherapy of Cancer (SITC) Conference, Poster: https:// TNFRSF25 is a potent costimulatory molecule, similar to OX40, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ 4-1BB and GITR. PTX-35 was developed as a humanized, affinity heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf matured, IgG2 mAb against TNFRSF25 for use with current can- 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ cer immunotherapy options, including cancer vaccines. All regulatory T cells and the severity of acute graft-versus-host disease. pharmacology and IND-enabling PK and toxicology has been Blood 128 (24), 2846-2858 completed for PTX-35 [1-6]. Methods Previous proof-of-concept studies were completed elsewhere. P492 Pharmacology studies described here were completed with a sur- A new generation anti-TGFβ antibody, SAR439459, relieves rogate antibody, mouse-IgG1-PTX-35 (mPTX-35). Regulatory T-cell immunosuppression and improves anti-tumor efficacy of PD1 expansion studies were completed with Foxp3-RFP+ transgenic blockade mice (FIR). CD8+ T-cell expansion studies were conducted with Rita Greco, Hongjing Qu, Joachim Theilhaber, Gary Shapiro, Richard adoptively transferred OVA-specific, TCR-transgenic, CD8+ T-cells Gregory, PhD, Christopher Winter, Natalia Malkova, Lily Pao, Mikhail Levit, (OT-1). Human PTX-35 was tested in 28-day mouse, and 2-week Alexei Protopopov, Jack Pollard, PhD, Tun Tun Lin, MD, Dmitri and 8-week non-human primate, toxicology studies. Human, Wiederschain, Sharad Sharma, PhD mouse, and monkey, in vitro, tissue-cross reactivity tests were Sanofi, Cambridge, MA, United States also performed to check species cross-reactivity. Human PTX-35 Correspondence: Sharad Sharma (sharad.sharma@sanofi.com) was also tested in a human PBMC stimulation assay, in vitro, to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P492 check for impact on proliferation and cytokine release. Background Results TGFβ is a potent immunosuppressive cytokine that acts on TNFRSF25-engagement in mice expanded antigen specific CD8+ multiple cell types of the innate and adaptive arms of the im- T-cells when given in the context of vaccination (6 and 19-fold mune system within the tumor microenvironment. Emerging increase in CD8+ T-cells in blood over vaccination alone at peak data implicates role of TGFβ in tumor immune evasion, resist- and boost, respectively), and Tregs were expanded in vaccine ance to cancer therapy and poor prognosis. We report preclin- absence in FIR animals (2-fold increase in CD4+ Foxp3+ T-cells ical data on SAR439459, a new pan anti-TGFβ antibody, which in blood).The MABELand NOEL,inFIR mice,using mPTX-35, is capable of neutralizing all active isoforms of human and was determined to be 0.01 mg/kg and 0.001 mg/kg, respect- murine TGFβ. ively. The 2-week PTX-35 treatment of cynomolgus monkeys confirmed findings in mice, that treatment results in the expan- Methods sion of Teff and Treg cell subsets. Intravenous bolus injection Cancer patient databases were analyzed for various gene signa- once every 2-weeks over an 8-week period was well tolerated tures. In vitro experiments were performed to examine the effi- giving a NOAEL of 100 mg/kg. Species cross-reactivity was ob- cacy of SAR439459 in preventing TGFβ-mediated suppression of served for mouse, human and monkey tissues. No adverse primary human T and NK cells. To evaluate anti-tumor efficacy, events were recorded for the 28-day mouse toxicology study. MC38 and EMT6 mouse tumor models were treated with Testing PTX-35 in a human PBMC, anti-CD3 proliferation assay, SAR439459, anti-PD1, or combination. MC38 model was used to in vitro, showed that TNFRSF25-engagement provided the ne- examine immune cell functions ex vivo. cessary costimulation to drive cellular division at sub-optimal Results concentrations of anti-CD3 (2-fold increase), providing the ne- TGFβ was found to be upregulated in cancer patients and its cessary in vitro, human proof-of-concept data, which was con- increased activation correlated with reduced overall survival (OS) sistent across four different human blood donors. in PD-1 refractory cancer patients. SAR439459 blocked TGFβ- Conclusions mediated suppression of human T and NK cells. TGFβ impaired PTX-35 is a potent costimulatory agonist targeting a novel pathway the activity of anti-PD1 mediated T cell response, while that can work in concert with cancer vaccines. Due to the ability of SAR439459 restored this activity. In MC38 and EMT6 tumor PTX-35 to possibly stimulate both pro-inflammatory and anti- models, treatment with SAR439459 resulted in anti-tumor efficacy; inflammatory pathways, depending on treatment context, thera- the combination of SAR439459 with anti-PD1 enhanced this activ- peutic modulation can provide numerous opportunities for cancer ity and resulted in complete tumor regression and elicited a pro- and inflammatory diseases. longed anti-tumor response. Conclusions Acknowledgements This data demonstrates that combination of SAR439459 with anti- Pelican Therapeutics would like to thank the Cancer Prevention and PD1 generates a strong anti-tumor immune response and im- Research Institute of Texas (CPRIT) that helped fund these studies. We would proves anti-tumor efficacy in several tumor models. The preclin- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for ical data presented here formed the basis for the ongoing clinical mouse TL1A-Ig and advice, both located at the University of Miami. investigation of SAR439459 in cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 268 of 272 P493 Background High-dimensional analysis delineates modulation of myeloid and Heat (Heat) Biologics has developed a next generation cellular vac- lymphoid compartments with STAT3 ASO and PDL-1 combination cine platform that incorporates a tumor antigen chaperone (gp96-Ig) therapy in a tumor cell line and a host of over-expressed cancer associated Theresa Proia, PhD, Maneesh Singh, PhD, Larissa Carnevalli, PhD, Gayathri neoantigens. Viagenpumatucel-L (HS110), a human lung adenocar- Bommakanti, PhD, Nanhua Deng, Matthew Griffin, Lukasz Magiera, Adina cinoma cell line, stably transfected to express gp96-Ig, is being tested Hughes, Laura Prickett, Patricia McCoon, PhD, Corinne Reimer, Simon in a phase 1/2 clinical trial (NCT#02439450) for NSCLC. Heat has re- Barry, PhD cently developed HS130, an allogeneic cell-based vaccine, designed AstraZeneca, Waltham, MA, United States to secrete tumor-associated antigens along with a costimulatory mol- Correspondence: Theresa Proia (theresa.proia@astrazeneca.com); ecule, OX40L. Preclinical results of mouse HS130 (mHS130) in com- Gayathri Bommakanti (gayathri.bommakanti@astrazeneca.com) bination with mouse HS110 (mHS110) has shown a potent anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P493 tumor effector and memory CD8+ T cell response, followed by tumor regression. In our current study, we further characterized the role of Background mHS110 and mHS130 in combination with an agonist TNFRSF25 STAT3 is a ubiquitously expressed transcription factor and master monoclonal antibody, PTX35. PTX35 is a potent stimulator of effector regulator of immune suppression in the tumor microenvironment and memory CD8+ T cell responses, which taken-together with (TME). Danvatirsen, a therapeutic antisense oligonucleotide (ASO) HS110 and HS130 has the potential of treating human cancers [1-7]. that selectively targets STAT3, has shown clinical benefit alone and in Methods combination with durvalumab (anti-PDL1) and is currently in Phase To study expansion, contraction, and maintenance of tumor-specific 1/2 clinical studies. CD8+ T cell responses, mHS110 and/or mHS130, in combination with Methods different doses of mouse-IgG1-PTX35 (mPTX35) was administered to To gain mechanistic insight into the therapeutic response induced C57BL/6 mice that were adoptively transferred with syngeneic OVA- by mouse surrogate STAT3 ASO in the CT26 syngeneic mouse tumor specific T cells (OT-I). Mice were then challenged with murine melan- model, we have used two complementary forms of high-dimensional oma tumors (B16F10-OVA) to characterize the tumor-specific immune profiling; mass cytometry (CyTOF) and flow cytometry. We supported cells in the periphery, spleen, and tumor-microenvironment that the in vivo mouse findings with in vitro studies in human macro- were involved in tumor regression. phages treated with danvatirsen. Results Results Combination of mPTX-35 with mHS-110 and mHS130 increased the Multidimensional immune profiling studies provided key mechan- expansion of tumor-specific CD8+ T-cells, in a mPTX-35 dose- istic observations: (1) Robust reduction of total STAT3 protein in dependent manner. This cellular expansion was significantly higher myeloid lineage cells, including an 80% reduction in macro- in the 1 mg/kg dose of mPTX-35 and far exceeded the additive value phages and 50% reduction in dendritic cells, but not in CD8+ T of mPTX-35, mHS130, and mHS110 treatment alone. Systemic admin- cells. (2) In the combination treatment arm, STAT3 ASO treatment istration of mPTX35, in combination with mHS110 and mHS130, led promoted a two-fold reduction of intratumoral immunosuppres- to a significant increase in the expansion of activated CD8+ T cells in sive macrophages, doubling of iNOS positive activated macro- the blood and stimulated activation of KLRGhi IL7Rlo short-lived ef- phages and enhanced proliferation and IFNγ production from fector cells (SLECs). Importantly, this combination resulted in higher tumor antigen specific T cells. (3) The tumor-associated mono- frequencies of tumor infiltrating lymphocytes (TILs), which enhanced cyte/macrophage compartment is highly complex and dynamic regression of established B16F10-OVA tumors and increased overall and displays a spectrum of activation states ranging from a pre- survival. dominantly anti-inflammatory phenotype (F4/80+ CD206+ IL4r+ Conclusions MerTK+; six fold higher) in progressively growing control tumors These results strongly suggest that mPTX35 synergizes with mHS110 to a predominantly proinflammatory phenotype (F4/80+ iNOS+ and mHS130 to amplify activated tumor-specific CD8+ T cells, pro- CCR2+; three fold higher) in responding tumors from combination gram a strong memory response, and allow for tumor regression. treated groups. The combinations of these three treatments in the clinic may trans- In vitro, human macrophages were highly sensitive to danvatirsen late into an efficacious approach to treating human cancers. treatment, with an IC50 of 60nM for total STAT3. Human ‘M2-like’ macrophages were generated in the presence of IL10 and M-CSF and Acknowledgements treated with danvatirsen, which promoted an increase in IFNγ, IL-12, Pelican Therapeutics would like to thank the Cancer Prevention and and TNFα, as well as increased CD80/CD86 expression, consistent Research Institute of Texas (CPRIT) that helped fund these studies. We would with polarization from a suppressive phenotype to a pro- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for inflammatory phenotype. mouse TL1A-Ig and advice, both located at the University of Miami. Conclusions Our data support the hypothesis that STAT3 reduction in the myeloid References lineage results in activation of macrophages entering the TME and 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. enhanced effector T cell responses in combination with checkpoint Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 inhibition. Our ongoing work is focused on exploring the effects of 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg STAT3 reduction in other key immune cells in which we have ob- expansion in mice by TNFRSF25 prevents allergic lung inflammation. J served robust knockdown including Tregs, endothelial cells, and Clin Invest.; 120(10):3629-3640 CAFs. 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 P494 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell A novel TNFRSF25 agonist, PTX35, synergizes with Gp96-Ig/OX40L- responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Ig to enhance effector and memory anti-tumor CD8+ T cell 5. Schreiber T.H., et al.; (2014). Comparative combination cancer responses and delay tumor growth immunotherapy with vaccination and TNFRSF stimulation. Society of Vikas Tahiliani, Patrick Dillon, PhD, Jayalakshmi Miriyala, MS, Jason Rose, Immunotherapy of Cancer (SITC) Conference, Poster: https:// MS, Anh Trinh, Rahul Jasuja, PhD, Jeff Hutchins, PhD, Matthew Seavey, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ PhD heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf Heat Biologics, Inc., Durham, NC, United States 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ Correspondence: Matthew Seavey (mseavey@heatbio.com) regulatory T cells and the severity of acute graft-versus-host disease. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P494 Blood 128 (24), 2846-2858 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 269 of 272 7. FrommG,deSilva S, GiffinL,Xu X,Rose J,Schreiber TH.Gp96-Ig/ P496 Costimulator (OX40L, ICOSL, or 4-1BBL) Combination Vaccine Im- Inhibition of autophagy enhances multifunctional genetically- proves T-cell Priming and Enhances Immunity, Memory, and Tumor engineered NK cell-based immunotherapy of glioblastoma Elimination. Cancer Immunol Res. 2016;4(9):766-778. doi:10.1158/2326- Jiao Wang, PhD, Sandro Matosevic, PhD 6066.CIR-15-0228 Purdue University, West Lafayette, IN, United States Correspondence: Sandro Matosevic (sandro@purdue.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P496 P495 Secondary immune resistance mechanisms induced by therapeutic Background cancer vaccines which prevent tumor regression and foster Despite aggressive treatments, the median life expectancy for GBM recurrences patients is only around 15 months, highlighting the need for new Sjoerd Van der Burg, PhD, Elham Beyranvand Nejad, Camilla Labrie, therapeutic approaches. NK cells are showing potential for immuno- Suzanne van Duikeren, Ing, Ramon Arens, PhD, Thorbald van Hall, PhD, therapy of GBM. However, NK cells struggle to cross the blood brain Sjoerd van der Burg, PhD barrier (BBB) and infiltrate into GBM [1]. Moreover, the immunosup- Leiden University Medical Center, Leiden, Netherlands pressive tumor microenvironment (TME) impairs NK cell activity, for Correspondence: Sjoerd van der Burg (shvdburg@lumc.nl) instance due to adenosine-mediated downregulation of NKG2D ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P495 pression [2]. Methods Background We developed an innovative NK cell-based immunotherapy for Immunotherapy may induce complete tumor regressions but often GBM that targets multiple “checkpoints” simultaneously, by tumors partially regress followed by tumor recurrence. Here, we fo- combining 1) multifunctional NK cells which consist of a cleav- cused on the underlying mechanisms. able scFv targeting CD73 alongside dual chimeric antigen recep- Methods tors directed against GD2 and NKG2D ligands and 2) inhibition The TC-1 mouse tumor model in which different formulation and ap- of autophagy in GBM cells to sensitize them to NK cell lysis and plication of an HPV16 SLP vaccine results in full cure or tumor recur- promote NK cell infiltration into GBM via the secretion of GBM- rence and therapy resistance after initial full tumor regression. specific chemoattractants. Tumors, spleens and lymph nodes were analyzed by mass- and flow- Results cytometry. Mice were treated with antibodies to PD-1, PD-L1, OX-40, We have designed and synthesized a multifunctional CAR con- 4-1BB, NKG2A and TGFβ. Cell-sorted tumor cells were RNA se- struct that expresses an anti-CD73 scFv which is cleavable by quenced. Immune parameters were assessed in 5-10 mice, survival GBM-associated proteases, and a dual CAR that enables NK cells analyses were performed on at least 10 mice per group. All experi- to avoid antigen escape common to GBM (Figure 1A). We have ments were performed 2-3 times. generated engineered NK-92 or primary human NK cells that effi- Results ciently express the construct, from which the anti-CD73 scFv Optimal vaccination resulted in about 7% circulating tumor- could be functionally released via uPA treatment (Figure 1B and specific CD8+ T cells and complete cure of all mice, whereas sub- C). Engineered NK cells showed a significantly higher in vitro abil- optimal vaccination led on average to 1.7% tumor-specific T cells ity to kill patient-derived GBM43 targets (Figure 1D and E). To and tumor regression followed by recurrence in all mice. Neither target autophagy, BECN1- GBM43 cells were generated, and their booster vaccinations, which increased the numbers of circulating in vivo subcutaneous growth in RAG-1-/- mice validated the crit- tumor-specific type 1 cytokine-producing CD4+ and CD8+ T cells ical role of autophagy in GBM onset and progression (Figure 1F). (p<0.01), nor the co-administration of (combinations of) anti- We further showed that targeting autophagy inhibited the bodies against PD-1, PD-L1, 4-1BB, or OX-40 prevented tumor re- in vitro proliferation of GBM43 itself (Figure 1G), sensitized GBM currence or improved survival after vaccination. Immune escape to NK cell lysis (Fig. 1H), and induced elevated chemokine secre- was intrinsic to the tumor cells as the direct reinjection of ex- tion (CCL5), which in turn increased NK cell migration across the vivo cell-sorted recurrent tumor cells into groups of 10 naïve BBB using an in vitro BBB model (Figure 1I). hosts did not result in any response to vaccination while in all Conclusions cases the reinjected ex-vivo cell-sorted nontreated tumor cells We have generated multifunctional NK cells that can target mul- displayed vaccine-induced tumor regression followed by relapse tiple “checkpoints” at once showing improved cytotoxicity against (p<0.01). Ex-vivo analyses of escaped tumor cells showed no al- GBM through increased resistance to the immunosuppressive terations in the expression of MHC-I or the E7 tumor-antigen or TME via adenosinergic CD73 blockade and the ability to avoid their sensitivity to tumor-specific CTL mediated killing. RNA se- antigen escape by GBM via dual CARs. We have also found that quencing on bulk sorted (CD45-) tumor cells from non-treated blocking the autophagy pathway in GBM displayed potent syn- (n=4) and escaped (n=4) tumors, revealed a specific vaccine- ergy with NK cell-mediated immunotherapy. Based on these re- induced downregulation of the TNF- and P53-signaling pathways sults, to achieve combined therapeutic effects in vivo, we are and upregulation of TGFβ signaling. Indeed, more TGFβ positive currently evaluating this immunotherapy in an orthotopic GBM fibroblasts surrounded the escaped tumors (p<0.05). Recurrent tumors mouse model. Taken together, this approach provides a promis- displayed strongly reduced numbers of infiltrated CD8+ T cells (p< ing platform for the combination treatment of GBM with engi- 0.001), whereas this was not the case for escaped tumor cells- neered NK cells. reinjected tumors. However, both types of escaped tumors displayed lower numbers of tumor-infiltrating Ly6C+MHCI-II+ inflammatory mac- rophages (p<0.01). TGFβ-blockade delayed but did not prevent relapses References to recur (p=0.07). The use of inflammation inducing chemotherapy rein- 1. Kmiecik J, Zimmer J, Chekenya M. Natural killer cells in intracranial stalled the infiltration of tumors with inflammatory myeloid cells after neoplasms: presence and therapeutic efficacy against brain tumours. J vaccination, prevented relapse and reinstalled sensitivity of escaped tu- Neurooncol. 2014 ;116(1):1-9. mors to therapeutic vaccination (p=0.01). 2. Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting Conclusions enhances immunotherapy of CD73+ solid tumors with piggyBac- The sequential clinical phases during non-curative immunotherapy engineered chimeric antigen receptor natural killer cells. J Immunother may involve several distinct secondary escape mechanisms. Cancer. 2018;6(1):136. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 270 of 272 not show tumor growth inhibition in this model, DRP-104 significantly inhibited tumor growth and the combination further enhanced efficacy, illustrated by extended survival for both DRP-104 alone (50 days) and combination (96 days) treatment groups compared to vehicle (33days) and anti-PD-L1 alone (33days). Combination treatment also resulted in long term durable cures in 50% of the mice. Conclusions DRP-104 treatment results in dramatic remodeling of the tumor micro environment, leading to enhanced function of multiple immune cells distinct from activities obtained by anti-PD-1 Ab. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti- tumor efficacy including long-term durable cures even in checkpoint in- hibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in com- bination with PD-1/PD-L-1 checkpoint inhibitors. P498 Blockade of PD-1 and LAG-3 on CD8+ T cells, induced by vaccination, elicits superior anti-tumor efficacy Christopher Zahm, PhD, Douglas McNeel, MD, PhD, Laurne Delmastro UW Carbone Cancer Center, Madison, WI, United States Correspondence: Douglas McNeel (dm3@medicine.wisc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P498 Fig. 1 (abstract P496). See text for description Background T cell immune checkpoint receptors (ICR) and their ligands have emerged as a major mechanism by which tumors avoid immune de- tection. ICR blockade targeting PD-1/PD-L1 and/or CTLA-4 have revo- P497 lutionized the treatment of many cancer types. However, not all DRP-104, a novel broad acting glutamine antagonist, induces cancers respond to ICR blockade, in large part mediated by the pres- distinctive immune modulation mechanisms and synergistic ence or absence of tumor infiltrating CD8+ T cells. We have focused efficacy in combination with immune checkpoint blockade on tumor vaccines as a means to increase the number of tumor- Yumi Yokoyama, PhD, Michael Nedelcovych, PhD, Robert Wild, PhD specific CD8+ T cells. We have previously demonstrated that activa- Dracen Pharmaceutical, New York,, NY, United States tion of CD8+ T cells by vaccination leads to increased expression of Correspondence: Robert Wild (rwild@dracenpharma.com) specific ICR, and that blockade of these ICR with vaccination leads to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P497 better anti-tumor response than either alone. Differences in expres- sion of specific ICR, notably PD-1 and LAG-3, appeared dependent on Background presentation of antigen by professional versus non-professional APC, Glutamine is an essential amino acid for rapidly proliferating cancer hence we hypothesized that blockade of both of these ICR with vac- cells, thus depriving the same fuel from immune cells and contributing cination should be superior to either alone. to tumor immune evasion. DRP-104 was designed as a novel prodrug Methods of the broad acting glutamine antagonist 6-Diazo-5-oxo-L-norleucine In these studies we directly assessed the expression of PD-1, LAG-3, (DON). DRP-104 is inert in its prodrug form, affords high levels of CTLA-4, and TIM-3 on CD8+ T cells following activation in the pres- plasma and gastro-intestinal (GI) tissue stability; has high tumor cell ence or absence of professional APC. Next, we transferred these cells permeability and preferential tumor versus plasma/GI tissue distribution into tumor bearing mice, alone or in combination ICR blocking anti- for DON. Here we sought to (1) compare immunological modulation of bodies, to directly evaluate their anti-tumor efficacy. Finally, we im- DRP-104 to anti-PD-1Ab, and (2) evaluate the combination effect of munized tumor-bearing HLA-A2-transgenic mice with different anti- DRP-104 with PD-1/PD-L1 checkpoint inhibitors. tumor DNA vaccines that have previously been shown to elicit CD8+ Methods T cells preferentially expressing either PD-1 or LAG-3, and used each Immunomodulatory effects of DRP-104 as a single agent and com- vaccine alone or in combination with ICR blockade. bination with anti-PD-1Ab was evaluated in the CT26 mouse colon Results carcinoma model by flow cytometry and Luminex assay. In vivo anti- We found that PD-1, LAG-3, CTLA-4 and TIM-3 are all increased on tumor efficacy of combination with anti-PD-1/PD-L1Ab was evaluated CD8+ T cells after activation by professional APC, however LAG-3 in CT26 and H22 hepatocellular carcinoma models. alone was increased on CD8+ T cells activated in the absence of pro- Results fessional APC (Figure 1). When these cells were adoptively trans- DRP-104 treatment showed broad immune cell modulation effects in- ferred into tumor bearing mice, LAG-3 blockade improved the anti- cluding increased T, NK, and macrophages; while anti-PD-1Ab affected tumor efficacy of CD8+ T cells activated without APC, and PD-1 mainly CD8+T cells. Cytokine modulation in tumor and plasma revealed blockade improved the anti-tumor efficacy of CD8+ T cells activated that DRP-104 decreased pro-tumorigenic cytokines such as VEGF and by APC (Figure 2). Immunization with different DNA constructs [1-3] KC(IL-8) while anti-PD-1Ab showed either no change or slight increase in combination with ICR blockade led to improved anti-tumor re- in these cytokines. CT26 bearing mice treated with anti-PD-1Ab alone, sponses, however combining LAG-3 blockade with PD-1 blockade DRP-104, and the combination showed tumor growth inhibition at day showed no benefit over PD-1 blockade alone (Figure 3). 12 of 48%, 90%, and 94%, respectively. Median survival days were 31.5, Conclusions 36, and 56 days, respectively (vehicle; 17.5 days). Notably 9 mice These data support our previous finding that PD-1 blockade improves treated with combination of anti-PD-1 with DRP-104 were tumor free at the efficacy of CD8+ T cells activated by vaccination. In this model, we end of the experiment (day 77) and 100% of these mice rejected a detected no additional benefit to concurrent LAG-3 blockade. The role of CT26 tumor re-challenge. In the H22 model, mice were treated with ei- other ICR in limiting anti-tumor immunity, and strategic blockade of these ther anti-PD-L1 Ab, DRP-104, or combination. While anti-PD-L1Ab did receptors following T-cell activation, is an area of active investigation. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 271 of 272 References 1 Smith, H. A., Rekoske, B. T. & McNeel, D. G. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses. Vaccine 32, 1707-1715, doi:10.1016/j.vac- cine.2014.01.048 (2014). 2 Rekoske, B. T., Smith, H. A., Olson, B. M., Maricque, B. B. & McNeel, D. G. PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization. Cancer Immunol Res 3, 946-955, doi:10.1158/2326-6066.CIR-14-0206 (2015). 3 Colluru,V. T., Zahm,C.D.&McNeel,D.G.Mini-intronicplasmid vac- cination elicits tolerant LAG3+ CD8 T cells and inferior anti-tumor re- sponses. OncoImmunology, 0-0, doi:10.1080/2162402X.2016.1223002 (2016). Fig 3 (abstract P498). Some vaccines only effective when combined with ICR blockade P499 Nano-Pulse Stimulation in combination with the TLR 7/8 agonist, resiquimod, synergizes to eliminate murine melanoma through innate and adaptive immune responses Joel Benjamin, PhD, Amanda McDaniel, BA, Kristin von Rothstein, Sasha Farina, Bruce Freimark, PhD, Richard Nuccitelli, MS, PhD Pulse Biosciences, Inc, Hayward, CA, United States Correspondence: Richard Nuccitelli (rnuccitelli@pulsebiosciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P499 Fig. 1 (abstract P498). Priming with professional APCs leads to Background ICR expression Nano-Pulse Stimulation (NPS) is a non-thermal treatment modality that provides high amplitude electrical energy pulses in the nanosec- ond range that is focal and directly acts on cellular structures and membranes to initiate regulated cell death. Previous work has shown that NPS induces release of tumor antigen and stimulates an in situ anti-tumor immune response [1, 2, 3]. The TLR 7/8 agonist resiqui- mod (RES) has been used as an immune adjuvant in previous cancer vaccine treatments in murine models to aid in antigen processing and presentation [4]. We have evaluated the combination of NPS and RES treatment to inhibit tumor growth and induce innate and adaptive immune responses. Methods The B16-F10 melanoma in C57BL/6j mice was used to investigate the potential combined effects of NPS and RES. B16-F10 tumor cells (2x10e5) were injected intradermally on the left flank and treated with NPS 5 days after inoculation. RES (50 μg) was then dosed in multiple combinations and timing to assess optimal tumor cell elimination and immune stimulation from the combin- ation treatment. Tumor efficacy (volume) was measured twice per week. Immune biomarkers included flow cytometry and IHC of T cell and myeloid immune cells from tumors, draining lymph nodes and spleens. Results Low energy NPS and up to 3 doses of RES as monotherapies partially inhibit tumor growth. However, combination of NPS with 24-hour, post treatment of RES resulted in complete regression in a large frac- tion of treated animals. This efficacy is concomitant with increased antigen-specific and memory CD8 populations in the spleen and lymph node, as well as an increase in certain innate immune cell Fig. 2 (abstract P498). APC-induced ICR signaling compromised populations. Tumor regressions showed no sign of regrowth 90 days anti-tumor response after treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 272 of 272 Conclusions Methods Compared to monotherapy treatments, the combination of NPS and Three models of colon tumorigenesis were used to conduct the RES treatments induce stronger tumor growth inhibition which per- present study (IACUC#3408), which included a colitis (AOM/DSS –in- sists as long-term tumor regression. Persistent tumor regression by duced), a spontaneous (APCMin-driven), and a syngeneic (engrafted combination treatment is associated with increases in B16F10 with MC-38 or CT-26 cells) model. Several mutant mice were used in antigen-specific and memory CD8 populations, as well as increases in these models including PARP-1-/-, PARP-1-/+ APCMin/+, APCMin/ innate immune populations with potential for antigen presentation. +PARP-1-/-+/−, APCMin/+PARP-1-/+ as well as WT mice. Mice were These data support a mechanism by which NPS combined with randomized and assigned to the different experimental groups. TLR7/8 agonists activates an enhanced immune response. Some groups of mice were administered olaparib, anti-mouse PD-1 antibodies, or a combination of the two agents. Mice were scarified References according to the requirements of each model and tumor and tissues 1. Nuccitelli R, Berridge JC, Mallon Z, Kreis M, Athos B, Nuccitelli P. were collected for the analysis. MDSCs were generated by incubating Nanoelectroablation of murine tumors triggers a CD8-dependent inhib- bone marrow cells with GM-CSF, G-CSF, and IL-6. Tumor MDSCs were ition of secondary tumor growth. PLoS ONE. 2015; 10(7): e0134364 1-17. generated by enzymatic digestion of MC-38-engrafted tumors 2. Nuccitelli R, McDaniel A, Anand S, Mallon Z, Berridge JC, Uecker D. Nano- followed by positive selection. The suppression assay was performed Pulse Stimulation is a physical modality that can trigger immunogenic by co-cultured with CD3/CD28-stimulated CFSE-labeled T cells and tumor cell death. J Immunotherapy Cancer. 2017; 5:32 DOI 10.1186/ proliferation was assessed by FACS. s40425-017-0234-5. Results 3. Skeate JG, DaSilva DM, Chavez-Juan E, Anand S, Nuccitelli R, Kast W.M. Here, we show that partial PARP-1 inhibition via gene heterozygosity or Nano-Pulse Stimulation induces immunogenic cell death in human a moderate olaparib dose was sufficient to protect against colitis- or papillomavirus-transformed tumors and initiates an adaptive immune re- APCMin-mediated intestinal tumorigenesis, while extensive inhibition sponse. 2018; PLoS ONE. 13(1): e0191311 via gene knockout or a high olaparib dose was ineffective or aggra- 4. Caisova V, Vieru A, Kumzakova Z, Glaserova S, Husnikova H, Vacova N, vated the burden despite anti-inflammatory effects and promotion of a Krejcova G, Padoukova L, Jochmanova I, Wolf KI, Chmelar J, Kopecky J, tumor-suppressive microenvironment. A sub-IC50 metronomic dose of Zenka J. Innate immunity-based cancer immunotherapy: B16-F10 murine olaparib or PARP-1 heterozygosity was also sufficient to block tumori- melanoma model. 2016; BMC Cancer.16(1); 940; DOI:10.1186/s12885-016- genesis in syngeneic colon cancer models by modulating the suppres- 2982-x. sive function, but not differentiation or intratumoral migration, of myeloid-derived suppressor cells (MDSCs). These effects occurred through a reduction of arginase-1, iNOS, and COX-2 expression but in- P500 dependently of PARP-1-trapping on chromatin. Interestingly, the metro- Targeting PARP-1 with metronomic therapy as a new approach to nomic olaparib dose increased the intratumoral numbers, but not modulate MDSC function and enhance anti-PD1 immunotherapy in percentages, of CD8+ T cells. Adoptive transfer of WT bone marrow- colon cancer derived MDSCs abrogated the protective effects of PARP-1 heterozy- 1 1 1 Salome Valentina Ibba , Mohamed Ghonim , Abdelmetalab Tarhuni , gosity against the tumor burden. A metronomic olaparib dose was 1 1 1 Matthew Dean , Hamid Boulares, PhD , Augusto Ochoa, MD , Youssef highly synergistic with anti-PD1-based immunotherapy leading to al- 1 1 1 1 Errami , Ali Elbahraway , Ilyes Benslimane , Dorota Wyczechowska , Luis most complete eradication of tumors on mice. 1 2 1 Del Valle, MD , Amir Al-Khami , Hanh Luu Conclusions Louisiana State University-Health Science Center, New Orleans, LA, Our results support a paradigm-shifting concept that expands the United States; Tanta University, Tanta, Egypt utility of PARPi and encourage testing metronomic dosing of PARPi Correspondence: Hamid Boulares (hboulr@lsuhsc.edu) to enhance efficacy of check-point inhibitor-based immunotherapies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P500 not only in cancer of the colon but also that of other tissues ultim- ately benefiting a large proportion of cancer patients. Background Ethics Approval PARP inhibitors (PARPi) have important anti-tumor effects in BRCA- IACUC#3408 defective cancers but efficacy requires their use at/near maximum- tolerated-doses to achieve trapping of the enzyme on damaged Publisher’s Note chromatin. However, the benefits of targeting non-DNA repair as- Springer Nature remains neutral with regard to jurisdictional claims pects of PARP with low metronomic doses have not been explored. in published maps and institutional affiliations.
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Springer Journals
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Copyright © 2019 by The Author(s).
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Medicine & Public Health; Oncology; Immunology
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10.1186/s40425-019-0763-1
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Abstract

Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 https://doi.org/10.1186/s40425-019-0763-1 MEETING ABSTRACTS Open Access 34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 1 National Harbor, MD, USA. 6-10 November 2019 Published: 6 November 2019 About this supplement These abstracts have been published as part of Journal for ImmunoTherapy of Cancer Volume 7 Supplement 1, 2019. The full contents of the supplement are available online at https://jitc.biomedcentral.com/articles/supplements/volume-7-supplement-1. Please note that this is part 1 of 2. Results Poster Presentations Preliminary cohort of 10 patients includes 4 with recurrence at a median Biomarkers, Immune Monitoring, and Novel of 2.4 years and 6 with no recurrence at a median of 12 years post-LT. Technologies We found that patients with recurrence post-LT have significantly higher densities of MPO+ PMNs compared to those with no recurrence. This dif- P1 ference is primarily driven by PMNs located within the peritumoral Peritumoral neutrophil infiltration predicts recurrence of stroma (Median [interquartile range [IQR] 2.46 [1.99 - 2.92] vs 1.23 [0.723 hepatocellular carcinoma following liver transplantation -1.78], p=0.019). Intratumoral PMN infiltration was not associated with re- 1 1 1 1 Marc Najjar, MD , Michael Ross , Ayush Srivastava , Robyn Gartrell, MD , currence (Median [IQR] 0.91 [0.59 - 1.20] vs 1.33 [0.56 – 1.90], p=0.308). 1 1 1 Emanuelle Rizk, BA , Olivia Perez , Evan Lieberman , Charles Drake, MD, Moreover, density of CD3, both intratumoral and peritumoral, did not cor- 1 1 1 1 PhD , Ladan Fazlollahi , Helen Remotti , Elizabeth Verna , Karim relate with recurrence, nor did the tissue-derived NLR. Further, we found 2 1 1 Halazun , Jean Emond , Yvonne Saenger, MD that the tissue-derived NLR did not correlate with NLR in blood. 1 2 Columbia University Medical Center, New York, NY, United States; Weill Conclusions Cornell Medicine, New York, NY, United States Higher densities of peritumoral PMNs are associated with post-LT Correspondence: Marc Najjar (mn2594@cumc.columbia.edu) HCC recurrence. Evaluation of TME using qmIF can be used to predict Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P1 recurrence in post-LT HCC. Further, tissue based analysis of PMNs does not correlate with serum NLR allowing potential for composite Background biomarkers. As this is preliminary, further analysis is underway and Hepatocellular carcinoma (HCC) is the most common liver malig- will be validated on the larger cohort of patients. nancy and the 5th cause of cancer-related mortality worldwide. Though previous studies have found that serum neutrophil-to- Reference lymphocyte ratio (NLR) is predictive of survival post liver transplant 1. Gartrell RD, Marks DK, Hart TD, et al. Quantitative Analysis of Immune (LT), peritumoral neutrophil (PMN) infiltration in the tumor micro- Infiltrates in Primary Melanoma. Cancer Immunol Res 2018;6:481-93. environment (TME) of HCC has not been thoroughly investigated yet. In this study we sought to evaluate tissue based PMN infiltration in HCC post LT using quantitative multiplex immunofluorescence (qmIF), previously used to study the TME of several other tumor types[1]. Methods A database of 634 patients was created at Columbia University Irving Medical Center (CUIMC) including adult patients with available clin- ical follow up who underwent liver transplantation (LT) for HCC be- tween 1998 and 2018. We evaluated a preliminary cohort of 10 patients using qmIF, excluding patients with viral hepatitis. FFPE tumor sections were pre-selected by a GI pathologist. Slides were stained using qmIF for MPO (PMNs), CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), HLA-DR (immune activation), and Hep- Par1 (hepatocytes/tumor). Multiplex images were visualized using Vectra (Akoya) and processed using inForm (Akoya). Data was ana- lyzed using R Studio for concatenation, density, nearest neighbor Fig. 1 (abstract P1). Quantitative multiplex immunofluorescence and statistical analysis. Serum NLR was calculated using complete images of HCC blood counts collected prior to LT(Figure 1). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 2 of 272 P2 determined via flow cytometry. Analyte secretion was determined Single-cell RNAseq analysis of the effects of cryopreservation on from supernatant using Milliplex MAP Human CD8+ T-cell Panel. primary tumor tissue Results Shawn Fahl (shawn.fahl@dls.com) We detected pembrolizumab binding to T-cells in a dose dependent Discovery Life Sciences, Huntsville, AL, United States manner and an increase in the activation marker CD69 on T-cells fol- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P2 lowing tumor cell and pembrolizumab treatment in three of four pa- tients tested. We devised an initial E:T optimization screen to identify Background a patient-specific ratio which renders our subsequent therapy re- The tumor microenvironment is a complex mixture of multiple cell sponse profiling highly personalized. CD3+CD8+ T-cell mediated types, and numerous therapeutic interventions have been developed tumor cell death and enhanced killing was detected in the presence targeting distinct aspects of this environment. Tumor tissue samples of pembrolizumab. Immune cell infiltration as well as therapy related are an integral part of identifying and understanding potential thera- cell death was observed in our 3D microtumors. Altered patient spe- peutic targets within the tumor microenvironment of multiple cancer cific cytokine secretion was measured when the cultures were indications. As early biomarker discovery is often hindered by the lo- treated with pembrolizumab and significantly correlated with pem- gistical demands of sourcing fresh human tumor tissue, cryopre- brolizumab induced reduction of microtumor growth rates. served dissociated tumor cell suspensions provide a viable Conclusions alternative for accessing multiple, highly-annotated tumor samples The data generated from these two complex 3D in vitro models al- for complex studies. Previous evaluations of cryopreservation on vi- lows us to better understand immune responses to autologous able tumor tissue have relied on flow or mass cytometry which, while tumor cells and checkpoint blockade. Our models are therefore ideal powerful, are limited in the number of targets that can be analyzed. and complimentary for preclinical testing of new I/O agents as well Single cell gene expression can analyze the expression of signifi- as patient response predictions to I/O based therapies. cantly more targets and provide a clearer picture on the effects of Ethics Approval cryopreservation on the cellular composition of the tumor. Tissue was acquired with approval from Prisma Health's Institutional Methods Review Board, PRO# 00069834. Multiple unique primary tumor samples were dissociated to the single-cell level and profiled by flow cytometry. These single cell sus- P4 pensions were subsequently subjected to single cell RNASeq using Novel immune competent murine glioblastoma models derived the 10X Genomics platform prior to, and immediately following, cryo- T2 L/L L/L L/L from Nestin-CreER ; Quaking ; P53 ; PTEN mice preservation. Data was subsequently analyzed to determine how 1 1 2 Chao-Hsien Chen, MD , Renee Chin, MS , Genevieve Hartley, PhD , cryopreservation impacted the cellular composition of the tumor 1 2 2 Cheng-En Hsieh, MD , Rishika Prasad, MS , Takashi Shingu, PhD , David microenvironment. 2 2 2 Hong, MD , Jian Hu, PhD , Michael Curran, PhD The University of Texas MD Anderson Cancer Center UTHealth P3 Graduate School of Biomedical Sciences, Houston, TX, United States; Predicting patient response to checkpoint blockade therapy using The University of Texas MD Anderson Cancer Center, Houston, TX, in vitro 3D cultures United States Kathryn Appleton, PhD, Ashley Elrod, Qi Jin Guo, Dennis Ruder, Tessa Correspondence: Michael Curran (MCurran@mdanderson.org) DesRochers, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P4 KIYATEC, Inc., Greenville, SC, United States Correspondence: Tessa DesRochers (tessa.desrochers@kiyatec.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P3 The widely used glioblastoma multiforme (GBM) model GL261 is highly immunogenic and readily cured by checkpoint blockade limit- Background ing its use for pre-clinical modeling of immunotherapy for human Knowledge of immune responses that correlate with clinical outcome GBM [1,2]. We developed four novel murine immunocompetent glio- T2 is essential for the development of strategies to harness a patient’s blastoma stem cell (QPP) lines derived from Nestin-CreER Quaking L/L L/L L/L immune system to eradicate cancer. Pre-clinical platforms that recap- (QKI) ; P53 ; PTEN mice, reflecting a common set of alterations itulate the immune response in the context of cancer are necessary in patients [3-5]. The four QPP cell lines are syngeneic to C57BL/6J for adequate understanding and detection of clinical efficacy, how- mice and exhibit distinct responses to T-cell checkpoint blockade. ever, the technology to accurately test immuno-oncology (I/O) ther- Methods apy response is lacking. Despite the value animal models provide in The differential responsiveness of each QPP line was assessed a pre-clinical setting, they lack matched patient tumor and immune through analysis of tumor growth in the brain versus the flank in un- cell interactions. To address this shortcoming, we developed in vitro treated, αPD-1, or αCTLA-4 treated mice. The impact of tumor gen- 3D tissue models that maintain autologous patient tumor cells and omic landscape on responsiveness at each site was measured immune cells for the testing and prediction of immune cell re- through whole exome sequencing. To understand cellular factors sponses. We hypothesize that these 3D tissue models will recapitu- modulating responsiveness of these GBM lines to checkpoint block- late the patient tumor microenvironment and detect response to I/O ade, the immune microenvironments of sensitive (QPP7) versus re- agents. sistant (QPP8) lines were compared in the brain using high Methods parameter flow cytometry. Drivers of flank sensitivity versus brain re- Tumor cells and T-cells were obtained from seven melanoma patient sistance were also measured for QPP8. biopsies and screened for PD-L1 and lymphocyte populations prior Results to incorporation into 3D culture. Effector cell to Tumor cell (E:T) QPP GBM lines demonstrate a range of sensitivities to CTLA-4 and optimization assays were conducted with expanded T-cells at differ- PD-1 blockade when implanted on the flank ranging from complete ent densities and co-cultured at different time points with tumor sensitivity (QPP7) to complete resistance (QPP4). In the brain, QPP7 cells. Viability was measured using CellTiter-Glo® 3D. T-cell response remains sensitive to both antibodies, but QPP4 and QPP8 fail to re- was determined using flow cytometry following 24-hour co-culture spond to blockade of either checkpoint (Figure 1). Analysis of the with tumor cells. Microtumors were established using a biologically QPP8 immune infiltrate in skin reveals enhanced ratios of CD8s to inert scaffold and extracellular matrix components. Microtumor via- Treg and myeloid suppressors in response to checkpoint blockade; bility was determined using PrestoBlue and T-cell infiltration was however, none of these benefits manifest in the brain QPP8 except a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 3 of 272 very specific increase in CD8s relative to granulocytic suppressors (Figure 2). Brain-implanted QPP8 reacts adaptively to checkpoint blockade by upregulating PD-L1 expression across its myeloid stroma. In contrast, immune-responsive QPP7 does not induce PD-L1 and shows markers of enhanced CD8 T cell fitness. Consistent with these observations, genomic analysis reveals a higher mutation dens- ity in QPP7 versus the other QPP lines. Using checkpoint-insensitive QPP4/8, we have now identified agonists of the Stimulator of Inter- feron Genes (STING) pathway as highly promising therapeutics for treating these tumors in the brain. Conclusions We have developed novel syngeneic models of GBM with relevant genetics and immune sensitivities relative to human disease. Through comparing T cell checkpoint blockade sensitive versus in- sensitive variants of these QPP lines, and through comparing variant sensitivity dictated by site of implantation, we have begun to identify the genetic and cellular components that govern immunotherapeutic sensitivity of GBM. References 1. Reardon DA, Omuro A, Brandes AA, Rieger J, Wick A, Sepulveda J, Phuphanich S, de Souza P, Ahluwalia MS, Lim M, Vlahovic G, Sampson J (2017) OS10.3 Randomized Phase 3 Study Evaluating the Efficacy and Safety of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: CheckMate 143. Neuro-Oncology 19: iii21-iii21. 2. Reardon DA, Gokhale PC, Klein SR, et al. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model. Cancer Immunol Res. 2016;4(2):124-135. 3. Hu J, Ho AL, Yuan L, et al. From the Cover: Neutralization of terminal differentiation in gliomagenesis. Proc Natl Acad Sci U S A. 2013;110(36):14520-14527. Fig. 2 (abstract P4). Immune landscape of QPP8 TME in 4. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic different niches landscape of glioblastoma. Cell. 2013;155(2):462-477. 5. Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating P5 endolysosomal degradation. Nat Genet. 2017;49(1):75-86. Laminar Wash™ AUTO system: a reliable walk-away sample Ethics Approval preparation solution for better TIL recovery without centrifugation All experiments were conducted according to protocols approved by the 1 1 2 2 Ira Kim , Melvin Lye , Roberta Zappasodi, PhD , Isabell Schulze , University of Texas MD Anderson Cancer Center Institutional Animal Care 3 1 1 Christoph Eberle, PhD , Chyan Ying Ke , Kong Leong Cheng , Ih Chin and Use Committee. 1 1 2 1 Kon , Royce Pek , Taha Merghoub, PhD , Namyong Kim, PhD 1 2 Curiox Biosystems, Boston, MA, United States; MSKCC, New York, NY, United States; Charles River Laboratories, Worcester, MA, United States Correspondence: Namyong Kim (namyong@curiox.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P5 Background The naturally occurring tumor infiltrating lymphocytes (TILs) exist in a complex microenvironment containing the extracellular matrix, blood vessels, and stromal and endothelial components in addition to vari- ous immune cells. While a growing number of preclinical mouse models is under development, the heterogeneity of the cell compos- ition in a solid tumor poses considerable technical challenges in iso- lating and characterizing the TILs for the downstream analysis. One common problem with TILs preparation occurs during solid tumor dissociation, whereby the TILs are left in a mixture with tissue debris and dead cells in suspension. Consequently, a preparation of autolo- gous TILs often requires further costly and laborious processing such as density gradient centrifugation, immune cell sorting and enrich- ment, and dead cell and debris removal in combination with multiple centrifugation steps. We introduce a novel Laminar Wash™ technol- ogy, which can help overcome these technical challenges. Methods We performed pilot studies on syngeneic (MC38, CT26, Cloud- manS91, 4T1) and humanized mouse tumor models as well as with human PBMCs and tumor biopsies using the Laminar Wash™ technol- ogy. Briefly, we evaluated various functional parameters of TILs such as polyfunctional CD8+ T cell responses and glucose update effi- ciency (2-NBDG) as well as conducting a side-by-side comparison of the TIL recovery rate and immunophenotypic characteristics of lymphoid and myeloid subsets on the Laminar Wash™ and the Fig. 1 (abstract P4). Orthotopic QPP survival and immune sensitivity centrifugation-based systems. In addition, the cell retention rate, cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 4 of 272 viability, debris removal, epitope preservation and the overall pro- detectable in the periphery starting at 2 weeks with stable levels (up cessing time were assessed and compared. Furthermore, we intro- to 40% of live cells to 8 weeks) with increasing T effector memory duce a complete walk-away approach to sample preparation that cells over the course of study. All tumors evaluated had high levels eliminates operator-based variability while significantly enhancing re- of TIL as measured by flow cytometry and immunohistochemistry. producibility and consistency of downstream analysis. Costimulatory and inhibitory molecules evaluated on CD4 and CD8 Results included 4-1BB, TIM-3, LAG-3, OX-40, as well as PD-1, which was Our data demonstrate that the Laminar Wash™ method resulted in expressed on both peripheral blood cells and in TIL. Tumor growth higher cell retention and viability, more clearly defined immune sub- kinetics was unaltered by PBMC humanization through a 5-week sets, a lowered background signal, and an enhanced yield of the TILs study window. from freshly dissociated tumor samples compared to the Conclusions centrifugation-based counterparts. The Laminar Wash™ system can PBMC-humanized NSG-B2M mice may represent a model for evaluat- effectively remove the floating debris in suspension while keeping ing of IO therapeutics with a long study window due to the lack of the live cells unperturbed, allowing the cell surface architecture and xGVHD. While PBMC engraftment kinetics are donor dependent, simi- epitopes better retained for improved downstream analysis with flow lar phenotypes are observed and T cell subsets expressing several cytometer. Additionally, the Laminar Wash™ AUTO system offers a relevant therapeutic targets, including PD-1 are present. This model completely automated sample processing solution for dissociated may permit a rapid in vivo method to study checkpoint blockade tumor samples, simplifying and expediting cell preparation with en- and other T-cell-directed IO therapeutics. hanced consistency and reproducibility. Ethics Approval Conclusions The study was approved by Champions Oncology's Institutional Ani- Laminar Wash™ results in healthy, viable, and well defined popula- mal Care and Use Committee (IACUC). tion of TILs, while improving the overall quality of data. The AUTO station provides an automated, centrifuge-free, and walk-away work- P7 flow for dissociated tumor samples for cytometry-based assays. Development of a natural killer (NK) ImmunoGraft platform for the evaluation of the pharmacodynamics of immuno-oncology Acknowledgements therapeutics Laminar Wash™ results in healthy, viable, and well defined population of TILs, 1 1 2 Bhavana Verma, PhD , Bruce Ruggeri , Jon Weidanz, PhD , Amy Wesa, while improving the overall quality of data. The AUTO station provides an PhD automated, centrifuge-free, and walk-away workflow for dissociated tumor 1 2 Champions Oncology, Rockville, MD, United States; Abexxa Biologics, samples for cytometry-based assays. Arlington, TX, United States Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); P6 Amy Wesa (awesa@championsoncology.com) Development of a peripheral blood mononuclear cells (PBMC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P7 ImmunoGraft platform to evaluate the pharmacodynamics of Immuno-oncology therapeutics Background Bhavana Verma, PhD, Bruce Ruggeri, Amy Wesa, PhD Harnessing NK cell anti-cancer cytotoxicity has gained interest as a Champions Oncology, Rockville, MD, United States therapeutic strategy, and consequently improved preclinical models Correspondence: Bruce Ruggeri (bruggeri@championsoncology.com); supporting the translation of NK cell–mediated therapies to the clinic Amy Wesa (awesa@championsoncology.com) are desired. Reproducible models with human NK engraftment into Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P6 immunodeficient mice co-engrafted with cell line-derived xenograft or patient-derived xenograft tumor models have been lacking due to Background an inability to support NK cell engraftment and persistence. Here we Humanized immune system (HIS) mouse models enable in vivo stud- evaluated IL-15-NOG mice for the engraftment and sustained survival ies in the context of the human immune cells with a human tumor of both ex vivo expanded and primary human NK cell isolates for es- and are critical for the development of next generation immune- tablishing models that engraft effectively with both human NK cells oncology (IO) agents. Humanization of immunodeficient mice and a PDX or CDX tumor. through the adoptive transfer of normal adult PBMC leads to rapid Methods engraftment of human T cells to study immune-modulatory agents NK cells from normal adult peripheral blood mononuclear cells in the context of human tumor xenografts, but is limited by the de- (PBMC) donors (N=3) were expanded using two different commer- velopment of xenogeneic graft-versus host disease (xGVHD). In this cially available kits and evaluated for NK phenotype, expansion rates study, we evaluated the engraftment of PBMC in β2microglobulin and yields. Titrated doses of ex vivo expanded NK cells were adop- null super-immunodeficient mice NSG-B2M mice, that lack MHC Class tively transferred into IL-15-NOG mice for human chimerism, and the I on host tissues. A cell line-derived xenograft model (CDX) co- persistence and survival of NK cells and their immunophenotype engrafted with PBMC (PBMC-ImmunoGraft) was characterized for were assessed. In separate studies, naïve NK cells enriched from humanization, tumor infiltrating leukocytes (TIL) phenotype and PBMC were also evaluated for NK cell persistence and expansion tumor response to checkpoint inhibitors. in vivo. To establish an NK ImmunoGraft, NK cells were engrafted in Methods xenograft tumor bearing mice and tumor growth kinetics were PBMC from healthy donors (N=7) were implanted and engraftment characterized. in peripheral blood was assessed by flow cytometry. T cell memory Results phenotypes were assessed over time in a small cohort, and costimu- Donor dependent NK expansion was observed ex vivo, with 28 to latory and inhibitory T cell subsets were evaluated at the terminal 50-fold expansion by two weeks. NK cells expanded ex vivo were time point in blood and secondary lymphoid organs. Next, NSG-B2M CD3-CD16+CD56± and varied based on the expansion kit utilized. mice were co-implanted with MDA-MB-231 breast cancer cell line s.c, Nearly all CD45+ cells in circulation were NK cells, and these peaked humanized with PBMC and tumor growth kinetics were monitored. by week 2, and were maintained for up to 10 weeks in IL-15-NOG Efficacy studies evaluating check point inhibitors are currently mice. Primary NK cells engrafted with slower kinetics, with peak ongoing. abundance at 3-4 weeks. NK cells expressed granzyme B, and further Results functional studies are in progress. For all NK cell populations, cell Successful PBMC engraftment without xGVHD was observed in NSG- density-dependent engraftment was observed with a largely stable B2M mice up to 8 weeks, in contrast with MHC Class I expressing im- NK phenotype observed across the study. In the absence of any munodeficient mice that developed xGVHD within 4-5 weeks. Dose therapeutic treatment, NK cell persistence and expansion in vivo did and donor- dependent chimerism was observed. T cells were not inhibit tumor xenograft growth kinetics in IL-15-NOG mice Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 5 of 272 Conclusions sensitivity and as such could be utilized to assess efficacy of the IL-15-NOG mice support the survival and persistence of human NK monoclonal antibody administered. There are also potential applica- cells from both ex vivo expanded and naïve NK cells, suggesting the tions related to rapid drug screening using the patient-derived xeno- universality of this platform for human NK engraftment. Our prelimin- graft model. Our future plans are focused on adapting these ary studies support IL-15 NOG mouse model as a suitable system for solutions to the characterization of immune checkpoint inhibitor evaluation of NK cellular therapies or NK cell-modulating therapies in therapeutics in standard-of-care FFPE tissues obtained from patients the context of patient-derived or cell-line derived xenograft (PDX or undergoing immunotherapy. CDX) mouse models Ethics Approval P9 The study was approved by Champions Oncology's Institutional Ani- Immune checkpoint biomarkers in hepatocellular carcinoma (HCC): mal Care and Use Committee (IACUC). Assessment of PD-L1 and tumour mutation burden in tumour samples from clinical patients 1 1 1 1 P8 Hisani Horne, PhD, MPH , Young Lee , Todd Creasy , Rebecca Fish , 1 1 2 1 Monoclonal antibody detection from formalin-fixed paraffin- Jonathan Cairns , Paul Scorer , Janine Feng , Marietta Scott, PhD , Mark 1 1 1 embedded tumor tissues using Fab-selective proteolysis nSMOL Gustavson , Aleksandra Dudek-Madej , Craig Barker , Nicholas 1 1 2 2 coupled with liquid chromatography and triple quadrupole mass Holoweckyi , Rebecca Halpin , Peiyi Wang , Quinea Lassiter , Xiaoling 2 2 1 1 spectrometry Xia , Mohammed Abdelwahab , Weimin Li , Alejandra Negro , Jill 1 1 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Noriko Iwamoto, PhD , Walker 2 2 2 1 2 Yoshinobu Koguchi, MD, PhD , John Cha , Brian Piening, PhD , Eric Tran, AstraZeneca, Gaithersburg, MD, United States; Roche Tissue 2 2 2 2 PhD , Hong-Ming Hu, PhD , Bernard Fox, PhD , William Redmond, PhD Diagnostics, Oro Valley, United States 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Correspondence: Hisani Horne (hisani.madison@astrazeneca.com) Cancer Center, Portland, OR, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P9 Correspondence: Takashi Shimada (tashimada@shimadzu.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P8 Background Programmed cell death ligand-1 (PD-L1) expression and tumour Background mutation burden (TMB) have been shown to be predictive of re- With the development of immune checkpoint inhibitors, the focus of sponse to anti-PD-1/PD-L1 immunotherapies in various cancers. cancer therapy is shifting to immunotherapy. Our purpose is to de- The prevalence and distribution of PD-L1 expression and/or velop the drug efficacy index by rapid analysis of antibodies accumu- tumour mutations in HCC and correlation with clinical characteris- lating in cancer tissue using liquid chromatography and mass tics are poorly understood. A better understanding of these bio- spectrometry (LC-MS/MS) and by characterization of the antibody markers may help inform appropriate patient selection strategies distribution in the tumor microenvironment. Using a novel proteoly- in HCC. sis method in which antibody molecules are collected on a 100 nm Methods resin pore and trypsin is immobilized on a 200 nm nanoparticle sur- PD-L1 expression was evaluated on tumour cells (TC), immune cells face, we have developed a method for physicochemically limiting (IC), or combined TC and IC using the VENTANA PD-L1 (SP263) Assay trypsin access to antibody and identifying the structural specificity of in three independent HCC sample sets: 2 from commercial tissue complementarity-determining regions while minimizing extra pep- banks (n = 500 and n = 2417) and 1 from patients enrolled in tides and protease without depending on the type of antibody. NCT02519348, encompassing a wide range of stage and grade of dis- Using this method to detect antibodies from formalin-fixed paraffin- ease. NCT02519348 is a phase 2 study evaluating safety, efficacy out- embedded (FFPE) tumor tissues, we aim to develop novel diagnostics comes of durvalumab with or without tremelimumab in advanced that can aid in therapeutic dosing and predicting responses to HCC. TMB was assessed in tissue by whole exome sequencing in a antibody-based therapies. subset of 70 patients from NCT02519348. Methods Results To demonstrate the feasibility of these approaches, the human At a cut-off date of Feb 28, 2019, 282/335 (84.2%) patients en- breast and epidermoid carcinoma cell lines SKBR3 and A431 were in- rolled in NCT02519348 were successfully evaluated for PD-L1 cubated with either trastuzumab and cetuximab, which bind to (Table 1). Significant expression was seen in ICs relative to TCs. erbB2 and EGFR, respectively. FFPE cell blocks were then prepared Patients in NCT02519348 showed higher PD-L1 expression in TCs and proteins were extracted from 8 μm sections after deparaffiniza- than commercial cohorts. In a univariate analysis using 1% cut tion and decrosslinking. The extracted proteins were subjected to offs, higher TC (but not IC or combined TC and IC) PD-L1 expres- the Fab-selective proteolysis nSMOL, and the signature peptides of sion was associated with patients with HCV infection (p=0.003). each antibody, IYPTNGYTR for trastuzumab and SQVFFK for cetuxi- Of 70 study patients tested, 55 were evaluable for TMB (median mab, were detected via triple-quadrupole LC-MS/MS. SCID mice were 2.59, range 0.46 - 5.61 Mut/Mb). Among patients with available subcutaneously implanted with BT474 cells and 5 days later were in- TMB data there was no observed correlation between TMB and fused with 10 mg/kg or 20 mg/kg trastuzumab. 24 h after administra- PD-L1 expression. tion, tumor and other tissues were harvested and FFPE block were Conclusions prepared for trastuzumab quantitation in FFPE tissues. PD-L1 expression was observed in both TC and ICs in HCC, with the Results latter being more prevalent. Viral status and disease stage may im- As a result of the pretreatment protocol using the cell block, the con- pact PD-L1 expression in this setting, but further work is needed to ditions of deparaffinization, decrosslinking, and protein extraction confirm this. TMB and PD-L1 appear to identify distinct patient sub- were optimized. Mass spectra of the signature peptides from trastu- sets in HCC. zumab and cetuximab could be detected using 20,000 cells. This Trial Registration condition was also applied to xenograft tissue and the degree of NCT02519348 trastuzumab accumulation was detected in FFPE tumor tissue in a Ethics Approval dose-dependent manner. The study (NCT02519348) is performed in accordance with eth- Conclusions ical principles that have their origin in the Declaration of We show that these approaches can be utilized to quantify antibody Helsinki and are consistent with ICH/GCP, and applicable regula- concentrations in typically-challenging FFPE specimens with good tory requirements. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 6 of 272 Table 1 (abstract P9). See text for description different subsets, compared to DGM, potentially allowing more as- says to be executed from the same blood sample. Overall, this tech- nology has the potential to transform cell separation by automating a variable and labor-intensive processes, and therefore has utility in applications that require consistent cell quality and functionality. Table 1 (abstract P10). See text for description P10 Table 2 (abstract P10). See text for description Inertial microfluidics enables highly consistent separation and concentration of leukocytes from human peripheral blood for downstream B-cell and T-cell functional assays Sarah Mickool, Eric Smith, Aleksander Jonca, Gustavo Arnal, Mary Vincent Larcom, Melanie Scully, Peng Megn Kou, PhD, Nitin Kulkarni, Kyle Smith MicroMedicine, Inc., Waltham, MA, United States Correspondence: Kyle Smith (kyle@micromedicine.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P10 Background Cell separation plays a vital role in research and clinical settings for the development and monitoring of cutting-edge therapies. Despite its labor-intensiveness and variability, density gradient P11 centrifugation-based method (DGM) has remained the primary Early detection of breast cancer (BCa) through MDSC and method of upstream cell isolation for decades due to a lack of viable lymphocyte immunophenotyping: from manual gating to pattern alternatives. This is problematic as DGM is a non-scalable, manual recognition neural networks 1 1 1 process. To address this lack of innovation, we have developed an George Dominguez, PhD , John Roop , Alexander Polo, BS , Anthony 1 2 1 automated Microfluidic System based on inertial focusing that en- Campisi, BS , Dmitry Gabrilovich, MD/PhD , Amit Kumar, PhD 1 2 ables label-free white blood cell (WBC) separation and concentration Anixa Biosciences, San Jose, CA, United States; The Wistar Institute, from 3-75mL of whole blood in short timescale with high Philadelphia, PA, United States consistency, providing reliable sample preparation for downstream Correspondence: George Dominguez (george@anixa.com) functional assays. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P11 Methods WBCs were isolated from 15% ACD-A anticoagulated peripheral hu- Background man blood using the Microfluidic System or DGM. Cell number, via- Myeloid-derived suppressor cells (MDSCs) are contributors in sup- bility, and immune phenotype were evaluated by hematology porting tumor progression and escape [1,2]. Studies have quantified analyzer and flow cytometry. To assess B-cell function, cells were MDSCs to detect tumor development, monitor progression, and/or cryopreserved post separation, thawed, and stimulated with IL-2 and predict therapeutic responses [3, 4]. Here, we compared several ma- R848, followed by Human IgG and IgM ELISPOT. To assess T-cell func- chine learning (ML) approaches to analyze flow cytometry data to tion, thawed cells underwent bead-based granulocyte depletion and detect breast cancer (stage I/II) through manual gating and hyper- stimulation with CEF peptide pool, followed by Human IFNγ ELISPOT. voxelation of cell events. Results Methods The prototype Microfluidic System consistently processed 40mL of We used standard multiparametric flow cytometry techniques to anticoagulated blood in approximately 20 minutes with minimal measure myeloid-derived suppressor cell (MDSC), myeloid, and hands-on time as opposed to 60–90 minutes for DGM with signifi- lymphocyte cell populations found in the peripheral blood of 99 cant hands-on time. While DGM collects only peripheral mononuclear biopsy-confirmed early stage BCa patients and 88 healthy donor fe- cells (PBMCs), the System isolates the total WBC population and may male (HDF) controls. Manual gating was performed to generate be beneficial for immunophenotyping. As shown in Table 1, the gated values, and raw flow cytometry data were transformed using Microfluidic System consistently provided improved WBC or PBMC HyperVOX to generate hypervoxelated cytometry event counts. The recovery, viability, purity, RBC depletion, and platelet depletion as ML algorithms used were: support vector machine (SVM), Bayes SVM, compared to DGM. Immune phenotyping shows that the Microfluidic Ensemble SVM, k-nearest neighbor (kNN), and pattern recognition System also consistently resulted in improved recovery of lympho- neural network (PRNN). All algorithms were trained using data from cyte subsets, including CD19+, CD3+, CD4+, and CD8+ cells (Table 2). 64 BCa patients and 69 HDF controls. Predictions were evaluated B-cell and T-cell functionality were found to be equivalent between using the performance of each trained ML algorithm on 35 early the two cell isolation methods based on IgM/IgG and IFNγ secretion, stage BCa patients and 19 HDF that were not used for training (hold- respectively. With the improved cell recovery using the Microfluidic out test set). System, more target cells from the same blood sample may be col- Results lected for downstream assays. Using manually gated counts, the resulting accuracies were: SVM = Conclusions 75.4%, Bayes SVM = 71.3%, Ensemble SVM = 65.6%, and kNN = The Microfluidic System offers a faster, more reliable method than 69.7%. Using hypervoxelated event counts, the resulting accuracies DGM for upstream cell separation from whole blood. The System were: SVM = 78.7%, Bayes SVM = 77.1%, Ensemble SVM = 57.4%, consistently recovers more cells, including functional lymphocytes of kNN = 67.2%, and PRNN = 92.6%. Hypervoxelated data analyzed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 7 of 272 using PRNN resulted in the highest accuracy with a sensitivity of regulated; q-value > 0.05 and log2 fold change > 0.58). In addition to the 91.4% and a specificity of 94.7%; the resulting AUC = 0.9098 (95%CI acute phase proteins (e.g. CRP and SAA1) which were previously verified = 0.8031 to 1.000). Additionally, we tested 26 samples collected from to be elevated in subjects with NSCLC, partial least squares discriminant patients with confirmed ductal carcinoma in situ (DCIS) using hyper- analysis helped identify additional proteins that are differentially voxelated counts with a PRNN. Even though they are clinically expressed between the sample groups. Most relevant to immune func- deemed as pre-cancerous (stage 0), 18 out of 26 (AUC = 0.8421; tion was CLC (Galectin-10), which was elevated in NSCLC samples and 95%CI = 0.7163 to 0.9679) were classified as BCa suggesting utility has been identified as key component supporting the suppressive func- for detecting the existence of even a non-invasive cancerous lesion. tion of Tregs.[1] Furthermore, F13A1 was suppressed in the NSCLC sam- Conclusions ples which is known to be associated with macrophage activation. Although further study is needed, we believe that using PRNN with Conclusions MDSC immunophenotyping, in conjunction with other known clinical 162 proteins were identified as candidate biomarkers and reflect the risk factors, would allow for clinicians to make a more informed diag- host immune response via acute phase response signaling, innate im- nosis and treatment recommendation when screening and for mune response, and other proinflammatory stimuli. Several of these recommending subsequent interventions for early stage breast markers have been linked to patient outcomes and poor prognosis. cancer. Reference References 1. Kubach, J., et. al.; Blood 2007 110:1550-1558 1. Kumar V, Patel S, Tcyganov E, Gabrilovich D. The nature of myeloid- derived suppressor cells in the tumor microenvironment. Trends Immu- P13 nol. 2016; 37:208-220. Immunomodulatory effects of Interleukin 2 in the circulation of 2. Marvel D, Gabrilovich D. Myeloid-derived suppressor cells in the tumor melanoma patients and the added impact of VEGF inhibition with microenvironment: expect the unexpected. J Clin Invest. 2015; 125:3356- Ziv-aflibercept 1 2 3 Arjun Khunger, MD , Ghanashyam Sarikonda , Paul Frankel, PhD , Jenn 3. Elliott L, Doherty G, Sheahan K, Ryan E. Human tumor-infiltrating myeloid 2 2 2 2 Tsau, PhD , Zeni Alfonso, PhD , Jane Gao, MS , Anil Pahuja, BSc , cells: phenotypic and functional diversity. Front Immunol. 2017; 8:86. 2 2 2 Christine Vaupel, PhD , Naveen Dakappagari , Shabnam Tangri, PhD , 4. Okla K, Wertel I, Wawruszak A, Bobinski M, Kotarski J. Blood-based ana- Ahmad Tarhini, MD, PhD lyses of cancer: circulating myeloid-derived suppressor cells – is a new 1 2 Memorial Hospital West, Pembroke Pines, FL, United States; Navigate era coming? Crit Rev Clin Lab Sci. 2018. BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA, United Ethics Approval 3 4 States; City of Hope, Duarte, CA, United States; Emory University and The study was approved by the Virtua Oncology (#20161), University of Winship Comprehensive Cancer center, Atlanta, GA, United States Pennsylvania (#826544), and Cooper Health (#17-174) IRBs. Correspondence: Ahmad Tarhini (tarhiniaa@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P13 P12 Deep characterization of the depleted plasma proteome in Background subjects with NSCLC using data independent acquisition mass Interleukin 2 (IL-2) plays a key role in antitumor immunity by en- spectrometry reveal host immune response mechanisms hancing survival of antitumor cytotoxic T lymphocytes and nat- Nicholas Dupuis, PhD, Linda Sensbach, Sebastian Müller, Lukas Reiter ural killer (NK) cells and promoting proinflammatory cytokines, Biognosys AG, Schlieren, Switzerland that can lead to durable responses in patients with melanoma. Correspondence: Nicholas Dupuis (nicholas.dupuis@biognosys.com) High levels of vascular endothelial growth factor (VEGF) are asso- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P12 ciated with non-response to IL-2 and combination biotherapy with Ziv-aflibercept (inhibitor of the VEGF pathway) and high- Background dose IL-2 may lead to improved antitumor efficacy. Mechanistic Measurement of circulating biomarkers in cancer has proven utility studies utilizing peripheral blood of melanoma patients treated for early detection, differential diagnosis, and predicting pre- with this biotherapy may illuminate the underlying mechanisms treatment response to therapy. More recently, circulating proteomic of immune susceptibility and resistance [1]. biomarkers for pre-treatment prediction of therapeutic response Methods have received additional attention due to the heterogeneous re- Patients with stage III or stage IV inoperable melanoma were sponses to immunotherapies. To develop a greater understanding of treated with high-dose IL-2 alone or in combination with Ziv- the circulating plasma proteome in subjects with cancer we have op- aflibercept in a phase 2 clinical trial [1] (NCI8628; Tarhini et al. timized a depleted plasma proteomic workflow, based on label-free Cancer. 2018). Peripheral blood mononuclear cells (PBMC) from data independent acquisition (DIA) mass spectrometry, and applied it treated patients (N=89) on this trial were tested at baseline (be- to plasma from subjects with late stage NSCLC. This approach pro- fore initiating systemic immunotherapy), and 6-weeks (following vides a deep and unbiased description of the plasma proteome and immunotherapy initiation). High complexity (14-color) flow cytom- the dysregulated biological pathways associated with lung cancer. etry designed to detect key immunological biomarkers such as Methods myeloid-derived suppressor cells (MDSCs), regulatory T cells Plasma samples from subjects with Stage III-IV non-small cell lung (Tregs), proliferating T-cells, PD-1 and TIM3 expression on T-cells, cancer (NSCLC, n = 15) and age matched healthy donors (n = 15) and differentiation of T-cells into Th1, Th2 or Th17 phenotype were depleted of 14 high abundance proteins using MARS Hu-14 were used to evaluate the correlation between immunological spin columns (Agilent). All samples were prepared for mass spectro- biomarker expression and efficacy. Statistical significance was de- metric acquisition using two-hour gradients on a C18 column termined using ANOVA or paired student’st-test. coupled online to a Thermo Scientific Q Exactive HF-X operated in Results DIA mode. Targeted data extraction was performed using Spectro- Treatment with high dose IL-2 resulted in significant immune activa- naut (Biognosys) with a hybrid library approach. Statistical analysis tion as detected by significant increases in both proliferating CD4+ was conducted to identify disease associated biomarker candidates (p<0.0001) and CD8+ (p<0.0001) T-cells at 6-weeks post-treatment in and pathway analysis highlights dysregulated biological functions. both treatment arms in addition to increase in Tregs (CD4+ CD25+ Results Foxp3+ T-cells; p<0.0001). Addition of VEGF inhibition showed a gen- A comprehensive protein spectral library was created containing 1,827 eral trend towards decrease in classical monocytes (CD14+ CD16-; p= unique proteins. In DIA acquisition, in total 1,304 proteins were quantified 0.0769) as well as Th17 cells (defined as CD45RA- CCR6+ CXCR3- across all samples (1,105 average per sample). Univariate statistical testing CCR4+; p=0.0597). In patients receiving combination therapy, a identified 162 dysregulated proteins (125 up-regulated and 37 down- higher proportion of subjects experienced CBR (Clinically Beneficial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 8 of 272 Response = CR+PR+SD) compared to monotherapy and this CBR cor- P15 related with a decrease in CD4+ ICOS+ (p=0.0219), classical mono- Microsatellite instability detection with cell-free DNA next- cytes (CD14+ CD16-; p=0.0141), Th17 cells (CD45RA- CCR6+ CXCR3- generation sequencing CCR4+; p=0.0445) as well activated CD4+ T-cells (CD4+ CD38+ HLA- Ariane Lozac’hmeur, MS, Jason Perera, PhD, Denise Lau, PhD, Aly Khan, DR+; p=0.0285). PhD, Ariane Lozac’hmeur, MS Conclusions Tempus Labs, Chicago, IL, United States VEGF inhibition with Ziv-aflibercept adds significant immunomod- Correspondence: Ariane Lozac’hmeur ulatory effects when combined with IL-2. Further correlative ana- (ariane.lozachmeur@tempus.com) lyses determining the effect of combination therapy on Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P15 progression-free survival and identifying predictive biomarkers of therapeutic efficacy are ongoing and will be presented at the Background meeting. Microsatellite instability is a clinically actionable genomic indication for cancer immunotherapy. In microsatellite instability-high (MSI-H) tumors, Acknowledgements defects in DNA mismatch repair (MMR) can cause a hypermutated This United States (U.S.) National Cancer Institute (NCI)-sponsored study was phenotype where alterations accumulate in the repetitive microsatellite initiated by the California Cancer Consortium under N01 contract NO1-CM- regions of DNA. MSI detection is typically performed by subjecting 2011-00038. Laboratory correlatives were supported by Navigate BioPharma. tumor tissue (“solid biopsy”) to clinical next-generation sequencing or Trial Registration specific assays, such as MMR IHC or MSI PCR. Circulating cell-free tumor https://clinicaltrials.gov/ct2/show/NCT01258855 DNA (cfDNA) testing (“liquid biopsy”) is rapidly emerging as a less inva- sive method for cancer detection and monitoring disease progression. Reference Here, we explore the possibility of detecting MSI in cfDNA and develop 1. Tarhini AA, Frankel P, Ruel C, Ernstoff MS, Kuzel TM, Logan TF, et al. NCI a novel cfDNA MSI detection assay with high specificity. 8628: A randomized phase 2 study of ziv‐aflibercept and high‐dose Methods interleukin 2 or high‐dose interleukin 2 alone for inoperable stage III or The Tempus cfDNA targeted panel contains 39 highly informative IV melanoma. Cancer. 2018;124(22):4332-41. microsatellite loci previously used by the clinically validated Tempus Ethics Approval xT 595-gene panel. For each microsatellite locus, we identified all se- The study was initiated after approval by the ethics committee at the quencing reads that mapped to the corresponding microsatellite re- participating sites and was conducted in accordance with the gion and quantified the number of repeat units contained within the Declaration of Helsinki. sequencing read. Next, three distinct summary statistics were calcu- lated to characterize the distribution of the number of repeat units for each locus. Finally, using 54 labeled patient samples (17 MSI-H, P14 37 microsatellite stable) sequenced with the Tempus cfDNA panel, a Validation of dendritic cell and natural killer cell signatures for k-Nearest Neighbor (k-NN) classifier was trained to classify each locus clinical biomarker development for a new sample. Patient samples with more than 50% unstable loci Bolan Linghu, PHD, Pei Zhang, PhD, Marylens Hernandez, Mingchao Xie, were classified as MSI-H. PhD, Christine Barbon, Srimathi Srinivasan, Deanna Russell, MS, Anna Results Coenen-Stass, Deanna Mele, PhD, Patricia McCoon, PhD, Jonathan Dry, We validated the ability of our model to detect MSI on a new inde- Ben Sidders, Kris Sachsenmeier, PhD pendent validation dataset. MSI-H status was detected in 6 patient AstraZeneca, Waltham, MA, United States samples. In 3 of these patients (2 colorectal, 1 skin cancer), abnormal Correspondence: Ben Sidders (benjamin.sidders@astrazeneca.com); Kris MMR IHC confirmed the detected MSI-H status. In the other 3 pa- Sachsenmeier (kris.sachsenmeier@astrazeneca.com) tients (1 colorectal, 1 non-small cell lung cancer, and 1 endometrial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P14 cancer), MSI-H status was confirmed by our clinically validated solid tumor MSI assay. Furthermore, the reliability of the model was vali- Background dated in 10 technical replicates from 2 MSI-H patients in our training Quantification of immune cell abundance using gene signatures from dataset. The results were 100% concordant with all 10 replicates clas- mRNA profiling has the potential to inform clinical studies of cancer sified as MSI-H. immunotherapy. However, few of the signatures reported in previous Conclusions studies have been validated therefore the concordance of signature These results demonstrate the ability of our assay to detect MSI in scores with corresponding immune cell abundance is unknown. cfDNA with high specificity, providing a transformative opportunity Methods to report a clinically actionable insight alongside other somatic To tackle this challenge we designed a two-stage validation strategy. changes detected from cfDNA. Firstly we validate signatures computationally using previously pub- lished datasets. Secondly we generate expression profiling data from an immune cell spike-in experiment with human PBMCs. As a proof P16 of concept experiment, we implemented the method to validate two Circulating immunological biomarkers for predicting response to gene signatures for CD141+ dendritic cells (DC) and CD56+ natural neoadjuvant chemotherapy in TNBC patients killer (NK) cells. Charlotte Milton, PhD, Thanussuyah Alaguthurai, Atousa khiabany, Mres, Results Sheeba Irshad, MD PhD We demonstrate gene signatures for both CD56+ NK and CD141+ Kings College London, London, United Kingdom DC cell types show high and significant agreement to the corre- Correspondence: Sheeba Irshad (sheeba.irshad@kcl.ac.uk) sponding immune cell abundance. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P16 Conclusions This work establishes a starting point for validating gene signatures Background through an approach that is tractable yet recapitulates real-world Triple negative breast cancer (TNBC) accounts for 10-20% of breast variability we might expect in clinical use. cancer and is associated with particularly poor prognosis. Patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 9 of 272 are commonly treated with neoadjuvant chemotherapy (NAC) and decisions. In this work, we perform whole plasma and plasma- response to treatment is a strong predictor of overall survival. Re- derived exosome proteomic profiling to construct a predictive model cently, the ability of chemotherapeutics to stimulate an anti-tumour of immunotherapy response and toxicity, and to glean further bio- immune response has been appreciated as an important mechanism logic insight into the mechanisms underlying resistance to ICB. of action; possibly contributing to the elimination of distant micro- Methods metastatic disease by resetting of the attenuated functional immun- Whole plasma was analyzed in a cohort of 55metastatic melanoma ity. In TNBCs, higher levels of tumour-infiltrating lymphocytes correl- patients receiving anti-PD1 antibodies (MGH IRB #11-181) at baseline, ate with response to NAC and high intra-tumoral levels of immune- and on-treatment at 6 week and 6 month time-points. Exosomes related genes, including those associated with type I interferon re- were analyzed in 15 of these patients for all time-points. Proteomic sponses, and the presence of CD8+ cytotoxic T lymphocytes, correl- analysis was performed using an innovative multiplex proximity ex- ate with improved disease outcome. tension assay that enabled detection of more than 1000 proteins Methods simultaneously. A linear mixed model with maximum likelihood esti- The underlying hypothesis of this study is that phenotypic profiling mation for model parameters was used to analyze differences be- of peripheral blood cells have the potential to inform clinical deci- tween patient groups, and significant differences were determined sions and help predict therapeutic response, with lower costs and after Benjamini and Hochberg multiple hypothesis correction. higher compliance than serial tumour biopsies, due to their minimal Results invasiveness. Whilst significant research efforts have been made to Between plasma baseline and on-treatment time-points, 67 differen- assess circulating markers such as circulating tumour cells and circu- tially expressed proteins were identified including markers of inflam- lating tumour DNA as potential biomarkers; understanding the evolv- mation such as PD1, CXCL9, CXCL10, CXCL11, IL10, CCL3 and TNFR2. ing peripheral “immunological status” of TNBC patients on NAC is Exosome samples had a distinct protein signature over the treatment warranted. period compared to plasma, including differential expression of We therefore set out to analyse serial blood samples from TNBC pa- CXCL16, CCL18, CCL20, and IL6, among others. 41 proteins were dif- tients receiving NAC to monitor the changes in the peripheral im- ferentially expressed in plasma between ICB responders and non- mune response through deep analysis of functional and phenotypic responders including several inflammatory proteins such as CD28, immune markers. We investigated (1) whether chemotherapy affects TNFb, MCSFRa and IL8, and others implicated in melanoma resist- the immune phenotype; and (2) whether a defined peripheral blood ance, such as MIA and ERBB2. Similarly, exosome revealed a distinct immune phenotypic profile relates to treatment response. protein signature between responders and non-responders com- Results pared to plasma consisting of CXCL9, CXCL13, CXCL16, CCL19, CD8a, Here we present preliminary results from 10 TNBC patients receiving GZMA and CD5 expression. Whereas plasma proteins reflected a NAC. Analysis of 39 PBMC populations using mass cytometry by myeloid signature, exosome proteins reflected a lymphoid signature, time-of-flight (CyTOF), highlighted phenotypic changes in B cell pop- suggesting that the two compartments may capture elements of dif- ulations in response to treatment, in particular a dramatic increase in ferent immune processes. Integrating data from both plasma and circulating regulatory B cells (CD19+CD24+CD38+) post- exosome proteomics, we applied machine learning tools to build a chemotherapy (5.4% and 46.2% of B cells pre- and post- predictor of response. Further analysis to look for predictors of tox- chemotherapy, respectively, p=0.0004). We also detected an increase icity is currently underway. in expression of exhaustion markers (CD38+CD39+) on CD8+ T cells Conclusions which was associated with poor response to chemotherapy (0.8 and Overall, our work suggests that plasma and exosome protein signa- 2.7 fold increase from baseline in exhausted CD8+ T cells in patients tures are distinct and may reflect unique immunological processes. with pathological complete response and residual disease, respect- Proteomic analysis of these compartments may be an effective way ively, p=0.008). for non-invasive liquid biopsy to predict ICB response. Conclusions We now plan to integrate these data with Luminex profiling of 36 P18 serum cytokines, mass spectrometry analysis of circulating exosomes Liquid biopsy protein biomarkers to predict responses and and clinicopathological and standard of care blood monitoring. elucidate resistance to cancer immunotherapy Taken together, this study aims to provide a comprehensive analysis 1 2 3 Arnav Mehta, MD PhD , Marijana Rucevic, PhD , Gyulnara Kasumova , of the utility of immune monitoring to understand TNBC patient re- 2 2 3 Emmett Sprecher , Lina Hultin Rosenberg , Dennie Frederick , Ryan sponse to NAC. 3 3 3 3 Sullivan, MD , Nir Hacohen , Keith Flaherty , Genevieve Boland Ethics Approval 1 2 MGH and Broad Institute, Boston, MA, United States; Olink Proteomics, The study was approved by NRES Committee London - Chelsea, ap- Watertown, MA, United States; MGH, Hanover, MA, United States proval number 13/LO/1248. Correspondence: Marijana Rucevic (m.rucevic@olink.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P18 P17 Role of plasma-derived exosome in monitoring immunotherapy Background response and toxicity The response of metastatic melanoma to anti-PD1 is heterogeneous. 1 2 3 Arnav Mehta, MD PhD , Gyulnara Kasumova , Alvin Shi , Lina Hultin We performed proteomic profiling of patient plasma samples to 4 4 2 2 Rosenberg , Emmett Sprecher , Dennie Frederick , Ryan Sullivan, MD , build a predictor of immunotherapy response and uncover biological 2 1 2 Keith Flaherty , Nir Hacohen , Genevieve Boland , Marijana Rucevic, insights underlying primary resistance. PhD Methods 1 2 MGH and Broad Institute, Boston, MA, United States; MGH, Hanover, An initial cohort comprised 55 metastatic melanoma patients re- 3 4 MA, United States; MIT, Cambridge, MA, United States; Olink ceiving anti-PD1 (Pembrolizumab or Nivolumab) at Massachu- Proteomics, Uppsala, MA, United States setts General Hospital (MGH), and 116 additional patients Correspondence: Arnav Mehta (nawi214@gmail.com) comprised a validation cohort. Plasma samples were collected Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P17 baseline and on-treatment, at 6 weeks and 6 months’ time- points, and profiled for 1000 proteins by a multiplex Proximity Background Extension Assay (PEA, by Olink Proteomics). A subset of patients Immune checkpoint blockade (ICB) has revolutionized the treatment had single-cell RNA-seq (Smart-Seq2 protocol) performed on of many solid tumors, including metastatic melanoma. Despite recent tumor tissue. Group differences and treatment effects were eval- successes, many patients fail to respond or are overcome by severe uated using linear mixed models with maximum likelihood esti- toxicities that limit further treatment. To date, there are no non- mation for model parameters, and Benjamini and Hochberg invasive predictors of response and toxicity that can guide treatment multiple hypothesis correction. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 10 of 272 Results immunohistochemical staining of Sema4D of the tumor associated At the baseline, 6 differentially expressed proteins were identified inflammatory cells (TAIs). between responders (R) and non-responders (NR) whereas im- Results mune suppression marker ST2 and IL-6 were found significantly sSema4D levels in plasma of HNSCC and the autoimmune individuals higher among NR. Kaplan-Meier survival curves stratified by the (p=0.18, independent-samples Mann-Whitney test), were not statisti- baseline differentially expressed proteins were highly predictive cally significantly different, but sSema4D levels were significantly of overall survival (OS) and progression-free survival (PFS). At 6- higher in the HNSCC and the autoimmune groups compared to weeks on-treatment time point, 80 proteins were found differen- healthy donors (p<0.001 for both comparisons). Three histological tially expressed between R and NR including several proteins im- patterns of tumor inflammation were defined according to the extent plicated in primary or acquired resistance (IL8, MIA, TNFR1 of stromal inflammation and TAIs infiltrate into the tumor islands. among others). Several 6-weeks differentially expressed proteins First; the inflamed type (TAIs infiltrated the tumor cells), second the were highly predictive of survival (ICOSL, IL8, MIA). Furthermore, TAIs excluded type (inflamed stroma but TAIs did not infiltrate the 160 significantly differentially expressed (DE) proteins were identi- tumor islands and/or were excluded by a thin peri-tumoral fibromyx- fied across the treatment period majority of which are reflective oid zone) and third as deserted ( minimal to no TAIs in the peri- of immune activation under the pressure of the immunotherapy. tumroal stroma or the tumor islands). The paired tumor tissue and Analysis of single-cell RNA-seq data of tumor tissue from a subset blood samples collected at the same time point, showed that high of these patients revealed that gene expression of most proteins levels of sSema4D in plasma, correlated directly with TAIs excluded predictive of response were enriched among tumor myeloid cells, histological pattern of tumor inflammation (p= 0.04). with the remainder of proteins being reflective of exhausted T Conclusions cell states. Our data presents a novel role of Sema4D as a soluble immune bio- Conclusions marker that can read in real time the histological pattern of tumor in- These results unveil a putative role of myeloid cells within the flammation. This opens new avenues for personalized immunotherapy tumor microenvironment in anti-PD1 response or primary resist- and HNSCC patient stratification. ance. Whole plasma proteomic profiling of anti-PD1 treated pa- tients revealed DE proteins between R and NR that may enable a References liquid biopsy to predict anti-PD1 response. Importantly, we dem- 1. Derakhshandeh R, Sanadhya S, Lee Han K, Chen H, Goloubeva O, Webb onstrate the relationship of serum biomarkers to OS and PFS and TJ, Younis RH. Semaphorin 4D in human head and neck cancer tissue are currently attempting to build machine learning classifiers as and peripheral blood: A dense fibrotic peri-tumoral stromal phenotype. predictors of response to checkpoint therapy leveraging early and Oncotarget. 2018; 9:11126-11144. late on-treatment time points. 2. Younis RH, Han KL, Webb TJ. Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid- Derived Suppressor Cells. J Immunol. 2016; 196:1419-29. P20 3. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith E, Zauderer M, Evans Semaphorin 4D in peripheral blood of head and neck squamous EE, Allen CT. Semaphorin4D Inhibition Improves Response to Immune- cell carcinoma reads the histological pattern of tumor Checkpoint Blockade via Attenuation of MDSC Recruitment and Function. inflammation in real time Cancer Immunol Res. 2019; 7:282-291. Ioana Ghita, Manar Elnaggar, Risa Chaisuparat, John Papadimitriou, 4. Ayers M, et al. IFN-gamma-related mRNA profile predicts clinical response Joshua Lubek, Rania Younis, BDS, MDS, PhD, Soren Bentzen, PhD to PD-1 blockade. J Clin Invest. 2017; 127:2930–40 University of Maryland, Baltimore, MD, United States Ethics Approval Correspondence: Rania Younis (ryounis@umaryland.edu) The study was approved by University of Maryland institutional review board, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P20 Institutution‘s Ethics Board, approval number (HCR-HP-00073603) Background There is an urgent need for immune biomarkers that can monitor P21 the status of inflammation of cancer patients. Soluble biomarkers Activation Profiling of tumor infiltrating CD8+ T cells reveals CTLA- represent a convenient prognostic and diagnostic method. Sema- 4 mean fluorescence intensity correlates with response in phorin 4D (Sema4D) is a glycoprotein that can function as a trans- treatment naïve melanoma membrane protein or a cleaved soluble form (sSema4D), that we Lauren Levine, MD, Katy Tsai, MD, James Lee, MD, Clinton Wu, BS, Kelly previously detected in peripheral blood [1]. The role of Sema4D as Mahuron, MD, Alain Algazi, MD, Michael Rosenblum, MD PhD, Adil an inflammatory mediator in several pathological aspects and its role Daud, MD in tumor immune suppression [2,3], highlights its significance as a University of California, San Francisco, San Francisco, CA, United States molecule to be further investigated for translational potential. The Correspondence: Adil Daud (daudai@gmail.com) objective of this work was to investigate the level of sSema4D in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P21 plasma in relation to the histological pattern of tumor inflammation of head and neck squamous cell carcinoma (HNSCC) patients in real Background time. Background: Activation markers such as PD-1 and PDL-1 as well as Methods tumor mutation burden and IFN-gamma gene expression profiling Under University of Maryland institutional review board approval and have been explored as markers for response in melanoma and in upon patient consent, we obtained paired peripheral blood and other cancers. PD-1 inhibition activates checkpoint positive cytotoxic tumor tissue of thirty-nine HNSCC patients, collected at the same T lymphocytes (cpCTLs) inducing tumor regression. We have previ- time point to allow for real time correlative analysis. Thirty eight pa- ously demonstrated that baseline peCTL frequency predicts response tients of classic autoimmune conditions, thirteen allergy patients, to anti–PD-1 monotherapy and combination CTLA4/PD-1 blockade in seven osteoarthritis patients and thirty-one healthy donors were in- metastatic melanoma. We evaluated the frequency of this CD8+ T cell cluded as controls. The level of Sema4D in plasma was detected subset at baseline and after immunotherapy treatment and evaluated using tailored direct ELISA assay. The histological pattern of tumor in- the utility of the intensity of expression activation marker expression flammation [4] was analyzed by three pathologists using the as a surrogate for tumor response as assessed by flow cytometry. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 11 of 272 Methods proportional hazard modeling to associate gene expression and We identified 490 patients with melanoma biopsied pre and post signature scores with the clinical annotations. PD-1 therapy and available for analysis. Of these 148 patients Results had unresectable stage III or stage IV melanoma and were treat- In the PanCancer IO 360 analysis, genes and signatures are compared ment naïve and started PD-1 therapy following biopsy. An add- to clinical annotations through heat maps, volcano plots, forest plots, itional 61 patients were identified with PD-1 resistant melanoma. box plots, waterfall plots, swim lane plots, Kaplan Myer plots, scatter Approximately 2 × 106 cells were stained with anti-hCD3, anti- plots, and the IO 360 wheel plot. The report is delivered in an HTML hCD8, anti-hCD45, anti-CD4 , anti-Foxp3, anti–hCTLA-4 (14D3), format that provides interactive visualizations, quality control, and anti–PD-1 , anti–HLA-DR, anti–PD-L1, and LIVE/DEAD Fix- able downloadable results. Data are analyzed individually and as part of Aqua Dead Cell Stain (Life Technologies). Data were acquired by larger treatment groups. an LSRFortessa (BD Biosciences) and analyzed using FlowJo soft- Conclusions ware (Tree Star, Inc.). Objective Responses were evaluated by The PanCancer IO 360 assay is a tool for characterizing transcriptional RECIST 1.1, CR/PR were classified as “responders” and SD/PD as patterns associated with tumor-immune interactions that can be ap- “non-responders.” plied across a wide range of cancer types. Gene signatures enable ro- Results bust characterization of immune activity from small sample cohorts, : cpCTL percentage correlated with response. The mean cpCTL was and the report simplifies the interpretation of results. This combin- 27.1% for treatment naïve responders (R), 16.52% for treatment naïve ation enables researchers to have insight into clinically relevant biol- non-responders (NR) and 8.59% for PD-1 resistant patients post treat- ogy that will ultimately lead to help drive the immune-oncology ment (ANOVA p=0.0003 for R/NR, 801 (ANOVA p=0.0002). field. Conclusions PD-1 progressive patients are significantly depleted in cpCTL even References compared to treatment naïve non-responders, suggesting that add- 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-γ-related mRNA profile pre- itional T cell influx may be needed for effective checkpoint blockade dicts clinical response to PD-1 blockade. J Clin Invest. 2017;127(8):2930- in these patients. In treatment naïve melanoma, CD8+ activation as shown by CTLA-4 MFI has an optimal range along the activation- 2. Danaher P, Warren S, Dennis L, et al. Gene expression markers of Tumor dysfunction spectrum, and strongly correlates with response to PD-1 Infiltrating Leukocytes. J Immunother Cancer. 2017;5:18. checkpoint therapy. 3. Danaher P, Warren S, Lu R, et al. Pan-cancer adaptive immune resist- ance as defined by the Tumor Inflammation Signature (TIS): results Acknowledgements from The Cancer Genome Atlas (TCGA). J Immunother Cancer. We gratefully acknowledge the patients who participated in this study 2018;6(1):63. Ethics Approval The study was approved by UCSF's Ethics Board approval number 138510 P23 High dimensional immune monitoring of peripheral blood samples P22 from breast cancer patients using mass cytometry (CyTOF) 1 1 2 Transcriptomic characterization of immune response within Jose Villasboas, MD , Kaitlyn McGrath, MS , El-ad David Amir, PhD , 1 1 1 diverse tumor environments using the NanoString® nCounter® Roberto Leon-Ferre, MD , Matthew Goetz, MD , Judy Boughey, MD , 1 1 1 PanCancer IO 360™ assay Jody Carter, MD, PhD , Krishna Kalari, PhD , Liewei Wang, MD, PhD , 1 1 Jessica Perez, PhD, Lei Yang, David Henderson, PhD, Heather Brauer, Vera Suman, PhD , Richard Weinshilboum, MD , Stephen Ansell, MD, PhD, Sarah Warren, PhD PhD 1 2 NanoString, Seattle, WA, United States Mayo Clinic, Rochester, MN, United States; Astrolabe Diagnostics, Fort Correspondence: Sarah Warren (swarren@nanostring.com) Lee, NJ, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P22 Correspondence: Jose Villasboas (Villasboas@mayo.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P23 Background The efficacy of immune response in solid tumor settings is driven Background by many factors including the biology of the tumor, the immune CyTOF produces high dimensional single cell data allowing simultan- system, and the microenvironment. The Tumor Inflammation Sig- eous monitoring of multiple immune cell subsets. This enables nature (TIS) is an 18-gene Research Use Only (RUO) signature that characterization of the immune system in normal and disease states. measures the presence of a preexisting immune response on the We developed a standardized pipeline to study human peripheral nCounter platform and enriches for response to pembrolizumab blood mononuclear cells (PBMCs) of cancer patients. Here we detail [1]. We have incorporated TIS into the PanCancer IO 360 panel, a our process and present early findings on a cohort of 40 patients 770-gene RUO expression assay containing 48 additional signa- with early-stage triple-negative breast cancer (TNBC) treated with tures of tumor-immune biology. To accompany this panel, we neoadjuvant chemotherapy. have created analysis software that associates the gene expres- Methods sion and signature scores with annotations of the samples to Thirty commercially-available metal-tagged antibodies were opti- characterize the immune system, tumor, and stroma within the mized to identify major cell subsets using a 4-point titration scheme. tumor microenvironment to give insight into underlying biology Replicates of cryopreserved PBMCs from a pool of 4 healthy donors of response to treatment, disease progression, survival, and other were created for panel titration and used as longitudinal references. sample characteristics. We studied 40 cryopreserved PBMCs from patients with TNBC. We Methods stained samples individually using standard protocol, barcoded over- The PanCancer IO 360 assay relies on gene signatures to describe night during DNA intercalation, and pooled for acquisition. Debar- biological processes, measure the presence of 14 different im- coded output data was normalized on a per-batch basis to the mune cell populations, or report the expression of key thera- median intensity of EQbeads. We uploaded files to an automated peutic targets. Data from The Cancer Genome Atlas (TCGA) was platform for unbiased processing. Patient-level meta-data was added used for signature training and development. Signatures are ei- to experiment matrix to determine differential abundance of immune ther single genes, weighted linear sums of multiple genes with subsets across clinical and pathological groups. coregulated expression, or algorithms to determine under- Results expression of genes in a coregulated pathway [2,3]. The analysis We required 7 rounds of titration to optimize antibody concentra- software leverages differential expression analysis and Cox tions. Data was collected on over 23 million live single-cell events Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 12 of 272 (Table 1) assigned to 31 canonical populations (Figure 1A). The me- Table 1 (abstract P23). See text for description dian frequencies of main populations were: B cells (11.9%), T-CD4+ cells (34.3%), T-CD8+ cells (11.7%), NK cells (8.6%), and monocytes (11.3%). At the profiling level, 76 subsets were agnostically identified, with B, T, NK, and monocytes broken down into 10, 32, 8, and 13 subsets respectively. Activated (CD38+CD161+) CD16+NK cells (Fig- ure 1B) were more prevalent in TNBC samples (median 5.2%, range 0.5%-11.9%) compared to normal blood (median 0.76%, range 0.1%- 2.4%). A population with phenotype suggestive of myeloid derived suppressor cells (LineagenegHLA-DRLowCD66b+CD24+CD16+; Figure 1C) was also more prevalent in TNBC samples (median 1.2%, range P24 0.1%-17.3%) compared to normal blood (median 0.6%, range 0.2%- Molecularly guided digital spatial profiling for highly multiplexed 1.0%). These populations demonstrated opposite association trends analysis of gene expression with spatial and single cell resolution when patients were stratified by clinical outcomes. Activated NK cells 1 2 2 Anushka Dikshit, PhD , Chris Merritt, PhD , Jamie Rose Kuhar , Karen were more frequent in patients achieving pathological complete re- 2 2 1 1 Nyugen , Kristina Sorg , Bingqing Zhang , Courtney Anderson, PhD , sponse while MDSC-like cells were more frequent in those with re- Xiao-Jun Ma sidual disease (Figures 1D-1E). 1 2 Advanced Cell Diagnostics, Newark, CA, United States; NanoString Conclusions Technologies, Seattle, WA, United States We demonstrated the feasibility of a complete pipeline for deep phe- Correspondence: Xiao-Jun Ma (xiao-jun.ma@bio-techne.com) notyping of cryopreserved PBMCs in cancer patients. Our approach Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P24 identified rare cell subsets using an unbiased analysis tool, linking specific populations to opposite clinical outcomes. High dimensional Background immune monitoring is feasible and should be applied to study the The tumor microenvironment (TME) is a network of complex inter- immune system of cancer patients at large. actions between the tumor and surrounding immune cells. Im- munotherapies including immune checkpoint blockade have Acknowledgements demonstrated therapeutic efficacy and durable responses for sev- This work was part of the Mayo Clinic Cancer Immunome Project which is eral tumor types, however most patients are nonresponsive or de- supported by the Wohlers Family Foundation. Samples were obtained in velop resistance to such immunotherapies. To identify new collaboration with investigators from the Mayo Clinic Breast Cancer Genome predictive biomarkers to better stratify patients, it is essential to Guided Therapy (BEAUTY) study. The BEAUTY study is funded in part by the comprehensively characterize the immune cells within the TME at Mayo Clinic Center for Individualized Medicine; Nadia’s Gift Foundation; John the molecular level. Traditional methods to assess gene expression P. Guider; the Eveleigh Family; George M. Eisenberg Foundation for Charities; in tissues lack either spatial information or sensitivity/specificity. To generous support from Afaf Al-Bahar; and the Pharmacogenomics Research address this, we have developed a novel workflow combining the Network (PGRN). Other contributing groups include the Mayo Clinic Cancer single molecule and single cell visualization capabilities of the RNA- Center and the Mayo Clinic Breast Specialized Program of Research scope in situ hybridization (ISH) assay with the highly multiplexed Excellence (SPORE). spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler Ethics Approval (DSP) RNA assays (Research Use Only). The study was reviewed approved by the Mayo Clinic Institutional review Methods board (IRB). The fully automated RNAscope Multiplex Fluorescent assay was used to visually identify CD3E (T-cell)-enriched regions and CD19 and CD20 (B-cell)-enriched regions within FFPE human lung cancer tis- sues. Using the GeoMx DSP, 10 CD3E-enriched regions of interest (ROI) and 10 CD19-enriched ROI were spatially profiled for 78 genes related to immune-oncology research. The RNAscope Multiplex Fluor- escence assay was used again to visually confirm the differentially expressed genes between the T and B-cell-enriched regions with sin- gle cell resolution. Results To show a workflow combining RNAscope molecularly guided visualization and GeoMx DSP profiling is feasible, we confirmed that both assay protocols are compatible. We then examined con- cordance between GeoMx DSP and RNAscope ISH data, demon- strating that RNAscope and GeoMx DSP data can be obtained on the same section. To test the full automated workflow, we com- pared the differentially expressed genes within the T cell and B cell-enriched ROI. The RNAscope assay confirmed that, while the expression of the immunoregulatory molecules CTLA4, PD-L1, PD- 1, and ICOSLG were detected in both ROI, the CD3E (T-cell)- enriched ROI demonstrated significantly higher expression of these checkpoint markers. Compared to the CD19-enriched ROI, the CD3-enriched ROI also showed increased inflammatory signa- ture, demonstrated by elevated levels of cytokines and chemo- kines such as CCL5, CXCL9 and IFNG. Conclusions We present a robust workflow that overcomes the historical limita- tions of ISH and IHC by combining high resolution imaging with high plex profiling. With this workflow, the RNAscope ISH technology can molecularly guide the GeoMx DSP to precisely profile ROI while retaining the morphological context of heterogenous tumors. Fur- Fig. 1 (abstract P23). High dimensional immune monitoring of thermore, RNAscope assays can be used to confirm GeoMx DSP- breast cancer PBMCs identified gene expression signatures at single cell resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 13 of 272 P25 4. Borbulevych OY, Santhanagopolan SM, Hossain M, Baker BM. TCRs used A conserved MART-1 T cell receptor motif is predictive of in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens responses to checkpoint blockade via distinct mechanisms. J Immunol. 2011;187(5):2453-2463. 1 2 2 Ariel Isser, BS , Tatsuya Yoshida , Junya Ichikawa , Jeffrey Weber, MD, Ethics Approval 2 3 PhD , Jonathan Schneck, MD, PhD The protocol was approved by the NYU Institutional Review Board, i16-01975 1 2 Johns Hopkins University, Baltimore, MD, United States; New York School of Medicine, New York, NY, United States; Johns Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Jeffrey Weber (jeffrey.weber@nyulangone.org); Jonathan Schneck (jschnec1@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P25 Background Since the introduction of checkpoint blockade inhibitors for cancer immunotherapy, numerous studies have sought to identify bio- markers predictive of patient response [1]. However, the relevance of antigen-driven responses to the tumor has yet to be investigated. To address this question, we examined T cell responses to MART-1, an antigen overexpressed in melanoma cells and a target for melanoma clinical trials that have had variable degrees of success. We hypothe- sized that features of patients’ MART-1 CD8+ T cell repertoires could predict their response to checkpoint blockade. Methods To understand the MART-1 T cell repertoire, MART-1 CD8+ T cells were expanded from HLA-A2+ melanoma patients and healthy do- nors using artificial antigen presenting cells (aAPC) or peptide-pulsed dendritic cells. Tetramer positive cells were sorted after 14-22 days and CDR3β sequenced. Motif analysis based on sequence homology was performed using the Immunomap algorithm by clustering 11,252 unique MART-1 CDR3β sequences from 33 samples and 20 donors, including five nivolumab responders and five non- responders [2]. Fig. 1 (abstract P25). See text for description Results No significant difference in the frequency of MART-1 expanded T cells was seen between healthy donors and melanoma patients with or P26 without checkpoint therapy. There was no immunodominant Vβ gene Murine T cell phenotype and function in a single-well format: a usage and limited clonotype overlap between donors. However, se- novel, multiplexed and high-throughput assay workflow using the quence homology showed extensive overlap between donors, driven iQue platform by two clusters present in 60% and 80% of samples at average frequen- Veronica Bruce, PhD, Caroline Weldon, John O'Rourke, Veronica Bruce, cies of 10% and 14%, respectively. These clusters were homologous to PhD each other as well as the DMF4 T cell receptor (TCR), one of the first Sartorius, Albuquerque, NM, United States clinically used genetically engineered T cells, with a known crystal struc- Correspondence: John O'Rourke (John.ORourke@Sartorius.com) ture [3,4]. The core region of these clusters contained a conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P26 amino acid motif that was identical to contact residues between the DMF4 TCR and MART-1 peptide bound to HLA-A2. The motif identified Background from the core region of these clusters was highly conserved across Immunotherapy is an actively growing arena in oncotherapeutics re- samples, present almost exclusively in the junctional region between search and development. In this context, whether testing CAR-T cells, the D and J genes of the CDR3β, and encoded by a diverse range of checkpoint inhibitors, or novel bispecific antibodies, the ultimate nucleotides, all evidence of selective pressure. Despite its conservation, goal is to modulate the immune system to harness its tumor killing the frequency of this motif was nearly six times lower in pre-therapy power. T cells play a critical role in immune-regulated clearance of samples expanded from non-responders compared to responders (40% both liquid and solid tumors. Upon antigenic stimulation and activa- vs. 7%, p=0.0045, Figure 1). tion, T cells rapidly expand, secrete cytokines, and differentiate to Conclusions various functional subsets (e.g. effector T cells, memory T cells). On Since the frequency of the identified MART-1 TCR motif is significantly the other hand, suppression of T cells (i.e. exhaustion) leads to im- lower in non-responders compared to responders, it could potentially mune escape and the spread of tumor cells. Mouse models remain be used as a biomarker to predict response of HLA-A2+ melanoma pa- the most commonly used animal system for in vivo and in vitro can- tients to checkpoint blockade prior to the onset of therapy. cer biology research and drug discovery. As researchers move for- ward to either better understand the role of T cells in cancer biology References or to develop novel immunotherapies, there is a need for improved 1. Zappasodi R, Wolchok JD, Merghoub T. Strategies for Predicting methods to quickly gather comprehensive data on T cell biology in Response to Checkpoint Inhibitors. Curr Hematol Malig Rep. this model. To address this, we demonstrate a multiplexed, high- 2018;13(5):383-395. throughput, robust assay workflow capable of measuring multiple 2. Sidhom J-W, Bessell CA, Havel JJ, Kosmides A, Chan TA, Schneck JP. murine T cell biology endpoints quickly and reproducibly in a single- ImmunoMap: A Bioinformatics Tool for T-Cell Repertoire Analysis. Cancer well format. Immunol Res. January 2017; 6(2):151-162. Methods 3. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of Tumor-Infiltrating In our workflow, stimulated mouse T cells were assayed in a 96-well Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with plate using fluorescent antibodies against CD3, CD4, CD8, CD69, Metastatic Melanoma. N Engl J Med. 1988;319(25):1676-1680. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 14 of 272 CD44, CD62L, and PD-1, QBeads for cytokine detection, and markers the tumor tissue upon drug treatment. The impact of different for cell viability and proliferation. Data were acquired on the iQue3 immuno-oncology drug treatments ex vivo on TME will be discussed. technology (VBR configuration) and analyzed on a plate-based level Application of this platform in the clinical studies may also allow de- using the integrated ForeCyt software. termining the most effective combinatorial therapeutic strategies for Results individual patients. Our assay workflow enabled simultaneous evaluation of viability, in- terrogation of helper and cytotoxic T cells for markers of activation P28 and exhaustion, and identification of key memory subsets. Prolifera- Mass spectroscopy-based highly multiplexed super-resolution tion and secreted cytokines (IFN-gamma and IL-2) were also quanti- imaging method for fine details of tumor microenvironment fied. Data analysis and visualization of multiple endpoints was monitoring and tumor-immune cell interactions streamlined and performed in real time using the ForeCyt software. 1 1 1 2 Yunhao Bai, BS , Bokai Zhu , Michael Angelo, MD, PhD , Yongxin Zhao , Conclusions 1 1 1 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Garry Nolan, PhD The assay was completed in four hours, including data analysis. This 1 2 Stanford University, Stanford, CA, United States; Carnegie Mellon workflow saves the end user’s time and resources by combining mul- University, Pittsburgh, PA, United States tiple experiments into a single, multiplexed workflow, and helps Correspondence: Sizun Jiang (sizunj@stanford.edu); Xavier Rovira Clave minimize subject-to-subject variability. Altogether, our workflow al- (xrovira@stanford.edu); Garry Nolan (gnolan@stanford.edu) lows for easy phenotype and functional profiling of murine T cells in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P28 a single-well format while generating actionable results in a matter of hours. Background In tumor microenvironment, tumor-immune interactions are indi- P27 cated by cell surface proteins such as T cell receptor (TCR) and PD- Functional 3D-plEX quantitative multiplex immunofluorescence L1. The key workhorse for studying these cellular interactions is via platform to assess IO drug impact on tumor microenvironment in imaging; conventional imaging methods are limited by the number ex vivo treated intact 3D-tumor organoids of fresh patient tumor of channels and the spatial resolving capabilities. tissue A new modality of imaging, Multiplexed Ion Beam Imaging (MIBI) Jenny Kreahling, PhD, Vijayendra Agrawal, PhD, Melba Page, PhD, Mibel [1,2], can resolve >40 parameters simultaneously in biological sam- Pabon, PhD, Soner Altiok, MD, PhD ples. MIBI can current attain single cell resolutions but has difficulties Nilogen Oncosystems, Tampa, FL, United States in resolving fine subcellular features. Here, we present Expansion Correspondence: Soner Altiok (soner@nilogen.com) MIBI (ExMIBI), which combines a physical expansion of a biological Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P27 sample with the MIBI imaging method. ExMIBI will be critical for the scientific community to obtain previously inaccessible insights into Background the fine details of tumor microenvironment and cancer-immune cell The tumor stroma consists of various components of the tumor interactions, and promises to unravel fundamental insights in patient microenvironment including tumor cells, fibroblasts, immune cells immunotherapy responses. and the extracellular matrix. Spatial organization and dynamic inter- Methods play of the complex cell-to-cell interactions play an important role Expansion microscopy (ExM) [3,4] is a technique that can physical ex- in cellular phenotypes that can result in permanent alterations in pansion of biological specimens 4 to 10 folds through polymer cellular functions and response to oncology as well as immuno- chemistry, three-color fluorescent imaging of cellular features with oncology drug treatments. While informative, conventional 2D an apparent lateral resolution of 70 nm in diffraction-limited confocal tumor dissociated models do not maintain the stromal- microscopes has been achieved. However, the expanded gel is fragile stoichiometry of the tumor microenvironment, lacking vital support and contains up to 99.9% water, which limits its usage in imaging mechanisms necessary to accurately assess ex-vivo tumor cell via- method that requires high vacuum condition. We explored a way to bility and immune-cell activation after drug treatment. Here, we de- collapse the tissue-containing gel on a complementary charged sub- scribe a functional quantitative multiplex immunofluorescence strate to achieve a vacuum-compatible gel that can be imaged by platform, 3D-plEX, to quantify drug-mediated changes in tumor im- the MIBIscope, with lateral resolution <100 nm. Various methods for mune microenvironment and tumor cell viability in intact 3D tumor sample charging removal are systematically tested for imaging a organoids of patient tumor samples. non-conductive gel in MIBI. Methods Results All patient tumor samples were obtained with patient consent We have established a robust method, ExMIBI, that allows ExM and relevant IRB approval. Unpropagated live 3D tumoroids hydrogels to be compatible with the high vacuum imaging condi- measuring 100-150 micron in size were prepared from fresh pa- tions of the MIBI. This method can achieve 40 parameters. A vali- tient tumors using a proprietary technology, pooled together to dated panel of MIBI compatible antibodies, focusing on the immune represent tumor heterogeneity and equally distributed to differ- system, is being tested and established for ExMIBI in FFPE tissues ent treatment groups including nivolumab, ipilimumab, atezolizu- (Figure 1). mab and urelumab singly or in different combinations. Cell Conclusions media was collected for multiplex cytokine release assay. Tumor- The combination of ExM and MIBI, termed ExMIBI, permits highly oids were fixed and embedded for multiplex immunofluorescence multiplexed super resolution imaging of tissue samples. We will now studies. In addition to tumor cell killing, treatment-mediated be able to map previously inaccessible, finer details of the tumor changes in TME was analyzed in each treatment group side-by- microenvironment. The application of ExMIBI to dissect cellular im- side using multiplex immunofluorescence markers including CD4, mune interactions, in their spatial biological context, will allow a bet- CD8, FoxP3, CD68, Pan-CK, PD-L1 and Ki67. ter understanding into the basic principles of our immune system in Results healthy and disease states. Our results demonstrated that 3D-plEX platform using clinically rele- vant intact, uniformly sized tumoroids of fresh patient tumor tissue is Acknowledgements highly versatile and reliable approach to quantify drug-mediated We thank Matt Newgren for tireless technical support on the MIBI changes in cellular composition and spatial organization of the tumor instrument. This research has received advice and help from Prof. Michael immune microenvironment. Angelo, Prof. Sean Bendall and their research group. B.Z. is supported by the Conclusions Stanford Graduate Student fellowship. S.J is supported by a Stanford Dean’s Combination of this approach with multiplex cytokine release assay Fellowship and the Leukemia & Lymphoma Society Career Development allows a comprehensive understanding of dynamic changes within Program. X.R.-C. is supported by a long-term EMBO fellowship. This work was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 15 of 272 supported by grants from the FDA, NIH, Parker Institute for Cancer separate tumor and stromal compartments, detect tumor/stroma Immunotherapy, the Bill and Melinda Gates Foundation, as well as the margins, and identify leukocytes in immunostained tissues were im- Rachford and Carlota A. Harris Endowed Professorship to G.P.N. plemented in each tissue analyzed. Resulting image markups of cell detection and biomarker expression measured by image analysis References were reviewed by an MD pathologist for acceptance. Tissues not 1. Angelo M, Bendall SC, Finck R, et al. Multiplexed ion beam imaging of meeting acceptance criteria were re-analyzed until acceptable to the human breast tumors. Nature Medicine. 2014;20(4):436–42. reviewing pathologist. 2. Keren L, Bosse M, Marquez D, et al. A Structured Tumor-Immune Micro- Results environment in Triple Negative Breast Cancer Revealed by Multiplexed We demonstrate the synergistic value of layered image analysis algo- Ion Beam Imaging. Cell. 2018;174(6):1373-87.E19. rithms which provide context to biomarker expression in NSCLC tis- 3. Chen F, Tillberg PW, Boyden ES. Expansion Microscopy, Science. sues. Samples were grouped in to immune desert, excluded, and 2015;347(6221):543-8. inflamed phenotypes based on total leukocyte and CD8 expression 4. Tillberg PW, Chen F, Piatkevich KD, et al. Protein-retention expansion mi- patterns in the tumor, stroma, and margin. PD-L1 expression was croscopy of cells and tissues labeled using standard fluorescent proteins scored based on percentages of tumor and stromal expression, as and antibodies. Nature Biotechnology. 2016;34(9):987–92. well as digital representations of common PD-L1 scoring paradigms. Additionally, samples were stratified by PD-L1 patterns of constitu- tive, induced, immune, or ignorant expression. Conclusions Digital image analysis of IHC stained tissues creates comprehensive tissue biomarker profiles that are useful in assessment of tumor and immune interactions in IO drug development and patient stratifica- tion. Complex algorithms that utilize AI and machine learning can be overseen by MD pathologists to create clinically acceptable digital analysis solutions. References 1. Rimm DL, Han G, Taube JM, et al. A Prospective, Multi-institutional, Pathologist-Based Assessment of 4 Immunohistochemistry Assays for PD- L1 Expression in Non–Small Cell Lung Cancer. JAMA Oncol. 2017;3(8):1051–1058. 2. Hendry S, Salgado R, Gevaert T, et al. Assessing Tumor-infiltrating Lym- phocytes in Solid Tumors: A Practical Review for Pathologists and Pro- Fig. 1 (abstract P28). The workflow and sample images of ExM-MIBI posal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcin- oma In Situ, Metastatic Tumor Deposits and Areas for Further Research. P29 Adv Anat Pathol. 2017;24(5):235–251. Comprehensive image analysis of immunostained NSCLC tissues 3. Taube JM, Galon J, Sholl LM, et al. Implications of the tumor immune provides necessary context for immune oncology biomarker microenvironment for staging and therapeutics. Mod Pathol. profiling 2018;31(2):214–234. Charles Caldwell, PhD, Jenifer Caldara, BS, Will Paces, BS, Kelsey Weigel, 4. Silva MA, Ryall KA, Wilm C, Caldara J, Grote HJ, et al. PD-L1 immunostain- PhD, Roberto Gianani, MD ing scoring for non-small cell lung cancer based on immunosurveillance Flagship Biosciences, Westminster, CO, United States parameters. PLOS ONE 2018; 13(6): e0196464 Correspondence: Charles Caldwell (ccaldwell@flagshipbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P29 P30 Background Deep spatial profiling of the immune landscape of MSI and MSS Manual pathology assessments of Immunohistochemistry (IHC) colorectal tumors markers in immune oncology (IO) is often challenging and results Sarah Church, PhD, Jason Reeves, Daniel Zollinger, Jill McKay-Fleisch, can be highly variable[1,2]. Measuring biomarker presence in IO must Andrew White, BSc, Michael Bailey, Arya Bahrami, PhD, Chris Merritt, take in to account both immune and tumor environments and pro- PhD, Margaret Hoang, Sarah Warren, PhD, Joseph Beechem, PhD vide contextual information on the interaction between tumor and NanoString Technologies, Everett, WA, United States immune biomarker landscapes [3]. Due to the complex nature sur- Correspondence: Sarah Church (schurch@nanostring.com) rounding tissue biomarker interpretation in IO, digital image analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P30 (IA) solutions have been developed that layer complex artificial intelligence (AI) and machine learning algorithms to obtain full tissue Background biomarker profiles necessary for drug development and patient In colorectal cancer (CRC) there have been many recent advances in stratification[4]. immune-related biomarkers that are both prognostic and predictive Here, a comprehensive tissue analysis solution is presented in mono- of response to immunotherapy. Microsatellite instability (MSI)/mis- plex PD-L1 and CD8 stained slides that includes precise digital bio- match repair deficiency (dMMR) is present in ~15-20% of CRCs and marker scoring in tumor and stromal compartments, recapitulation of corelates with increased immunogenic mutations that often augment common scoring paradigms, analysis of biomarker expression at the lymphocyte infiltration into the tumor microenvironment (TME). Add- tumor/stroma interface (margin), and quantification, scoring, and itionally, location of tumor infiltrating T-cells in two areas of the TME, spatial localization of leukocytes in the tumor and stroma. Aggrega- the tumor center (CT) and invasive margin (IM) has also been shown tion of all cellular and biomarker data generates tissue phenotypes to be prognostic and predictive of response to immunotherapy. Here that characterize the IO landscape of each tissue. we use multiplexed protein and RNA digital spatial profiling to elicit Methods the immune landscape of MSI-MSS characterized CRC tumors. Serial sections of 20 NSCLC samples were IHC stained for PD-L1 and Methods CD8 expression. Stained slides were scanned at 20x magnification Forty-eight CRC tumors were analyzed for gene expression (GX) and analyzed using Flagship Biosciences’ image analysis solutions. using the NanoString® nCounter® PanCancer IO 360™ Research Use Image analysis algorithms which quantify biomarker expression, Only (RUO) Gene Expression Panel and assessed for 48 cell typing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 16 of 272 and biological signatures, including MMR loss/MSI predictor and the Polaris® with pre-defined acquisition parameters. Scans were un- Tumor Inflammation Signature (TIS). A subset of 18 CRC tumors (6 mixed and analyzed with inForm® software using a pre-configured al- MSI-TIS-hi, 6 MSS-TIS-hi, 6 MSS-TIS-lo) was selected for analysis with gorithm tailored to the PD1/PD-L1 Lung Cancer Panel Kit. Spatial the RUO GeoMx™ Digital Spatial Profiler (DSP) using 40 antibodies, analyses and visualizations were performed using the phenoptr and 84 or 1,600+ in situ probes. Selection of regions of interest (ROIs) in phenoptrReports R-based packages and custom scripts. two locations, CT and IM were guided by staining with fluorescent Results markers (CD45, CD3, pan-CK, DNA). 300-600 μM diameter circle ROIs The pre-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer Panel were selected, and in some cases segmented by pan-CK+/pan-CK-. Kit was able to visualize the panel targets (PD-L1, PD-1, CD8, CD68, Results FoxP3, and Cytokeratin) across the variety of lung cancer samples in Using whole tissue GX, we first confirmed MSI/dMMR characterization the TMA. Cell phenotyping and spatial analyses revealed core-to-core and TIS status of 48 CRC tumors using PanCancer IO 360 signatures. variations in cell densities and proximities among different markers. We selected 18 tumors within this cohort based on TIS status to fur- Measurement of the dynamic range of PD-L1 expression across dif- ther dissect the location-dependent immune contexture of the TME, ferent cores also revealed the improved sensitivity in PD-L1 detection with a particular emphasis on differentiating MSI-TIS-hi and MSS-TIS- provided by unmixing. hi CRCs. DSP confirmed loss of dMMR markers (MSH2/MLH1) and Conclusions identified an increased amount of potentially suppressive macro- The end-to-end Phenoptics staining, imaging, unmixing, and spatial phages (CD163+PD-L1+) in MSI-TIS-hi versus MSS-TIS-hi tumors. Seg- analysis workflow described here provides a robust and sensitive mentation of ROIs based on tumor versus stroma (pan-CK+/-) platform for exploring the immune landscape within the tumor identified samples with high proportions of tumor-invading TILs. microenvironment. These samples were then further profiled using probes against 1600+ mRNA targets revealing distinct pathways related immune cell Reference orientation within the TME. 1. Lu S, Stein JE, Rimm DL, et al. Comparison of Biomarker Modalities for Conclusions Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Here we show the use of novel high-plex spatial profiling to profile Review and Meta-analysis. JAMA Oncol. Published online July 18, 2019. location and pathways in the TME of MSI and MSS CRC tumors. doi:10.1001/jamaoncol.2019.1549 These findings elicit unique biology related to the location and sig- naling of immune cells, which have the potential to unveil targets for P32 therapeutic combinations. Differential immune contexture of human colorectal carcinomas with mismatch repair deficiency (MSI-H) and increased DNA P31 damage responses (DDR) 2 2 2 Applying multispectral unmixing and spatial analyses to explore Shruti Desai, PhD , Venkata Nagineni , Micaela Morgada , Aravind 2 2 1 2 tumor heterogeneity with a pre-optimized 7-color immuno- Kalathil , Ila Datar , Charles Fuchs, MD, MPH , Patricia LoRusso, DO , 2 2 oncology workflow Ranjit Bindra , Kurt Schalper, MD, PhD 1 2 Carla Coltharp, PhD, Bethany Remeniuk, PhD, Chichung Wang, Rachel Yale University, New Haven, CT, United States; Yale University, School Schaefer, Linying Liu, Glenn Milton, Victoria Duckworth, Michael McLane, of Medicine, New Haven, CT, United States Peter Miller, Yi Zheng, Carla Coltharp, PhD Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Akoya Biosciences, Hopkinton, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P32 Correspondence: Yi Zheng (YZheng@akoyabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P31 Background Tumor cells accumulate deleterious genomic alterations through sus- Background tained mutagenic exposure and defective DNA repair. Approximately The tumor microenvironment hosts a myriad of cellular interactions 15% of human colorectal carcinomas (CRCs) display mismatch repair that influence tumor biology and patient outcomes. Multiplex im- deficiency (MSI-H) associated with increased somatic mutations and munofluorescence (mIF) provides the ability to investigate a large sensitivity to immune checkpoint blockers. Advanced tumors can number of these interactions in a single tissue section, and has been harbor additional DNA-repair alterations with functional/therapeutic shown to outperform other testing modalities for predicting re- implications. Increased double strand DNA breaks have been re- sponse to immunotherapies [1]. ported across solid tumors and can be detected by changes in Multispectral imaging (MSI) improves the capabilities of mIF by pro- Serine139-phosphorylated histone H2AX (γH2AX). We studied the im- viding the ability to spectrally unmix fluorescence signals. This in- mune composition of human CRCs with MSI-H and elevated DDR. creases the number of markers that can be probed in the same scan Methods and allows for separation of true immunofluorescence signals from Using multiplexed quantitative immunofluorescence (QIF), we stud- tissue autofluorescence background. ied the level of major adaptive and innate immune markers in a Here, we apply MSI to explore spatial interactions observed in lung retrospective collection of 265 stage I-IV CRCs from Yale represented cancer samples using an end-to-end translational workflow based on in tissuemicroarrays. We used previously validated QIF panels includ- the PhenopticsTM platform. The workflow includes a pre-optimized ing the markers DAPI, cytokeratin, γH2AX, CD3, CD4, CD8, CD20, PD- 7-color staining panel kit along with a pre-configured analysis algo- L1, CD15, myeloperoxidase (MPO), IL-8, Ki-67, granzyme-B (GZB), rithm for cell phenotyping. Beta-2 microglobulin (B2M), HLA-class I and HLA-class II. The MSI sta- Using tissue microarrays (TMA), we demonstrate the heterogeneity of tus was determined using clinical-grade immunohistochemistry de- spatial interactions observed among different lung cancer samples tection of MLH1, MSH2, MSH6 and PMS2. We analyzed the and the improved sensitivity of detection afforded by unmixing association between localized measurement of markers and with multispectral scans. major clinicopathologic variables/survival. Methods Results A lung cancer TMA was created using the 3DHistech TMA Master II From 252 evaluable cases, 12.1% were classified as MSI-H. Relative to from five formalin-fixed paraffin-embedded lung cancer tissue blocks. MSS tumors, MSI-H CRCs showed significantly higher levels of PD-L1, The TMAs were stained using the Opal Polaris 7-Color PD1/PD-L1 lower CD20 and non-significant increases in CD3, CD4, CD8, T-cell Ki- Lung Cancer Panel Kit on the Leica BOND RXTM automated stainer 67 and T-cell GZB. MSI-H cases displayed lower tumor-cell B2M and using the associated preloaded Opal 7-Color Panel Kit protocol. increased stromal HLA-class II expression. MSI-H tumors also showed Whole slide MOTiFTM multispectral scans were acquired on Vectra significantly higher levels of IL-8 and MPO+ cells than MSS Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 17 of 272 counterpart. The level of γH2AX was comparable between MSI-H and clinicopathologic associations were found. γH2Ax was not prognostic MSS malignancies. Cases with increased tumor-cell γH2AX (> cohort as single marker. However, elevated simultaneous expression of median) showed significantly higher levels of PD-L1 and all studied γH2Ax and CD3 was associated with longer 5-year overall survival in lymphocyte markers than cases with lower γH2AX. In addition, these the immunotherapy-naïve cohorts. In patients treated with PD-1 axis tumors displayed significantly higher T-cell proliferation, mild in- blockers, elevated baseline γH2Ax/CD3 was associated with a clear creases in T-cell GZB and higher levels of HLA-class I/class II proteins. trend toward longer survival but did not reach statistical significance. The levels of IL-8 and MPO+ cells were comparable across the γH2AX Conclusions groups. The DNA repair and immune markers were variably associ- Active DDR as measured by tumor-cell γH2Ax expression occurs in a ated with 5-year overall survival in the cohort. high proportion of human NSCLCs and is associated with T-cell in- Conclusions flamed tumors. Despite their association with smoking, lung adeno- DNA repair deficiency defines human CRCs with distinct innate and carcinomas harboring activating mutations in KRAS display lower adaptive immune contexture. While mismatch repair deficiency is as- DDR markers than EGFR mutant or EGFR/KRAS wild type malignan- sociated with mild/moderate intratumor T-cell responses and prom- cies. Collectively, our results support the use of combination therapy inent myeloid cell features; elevated DDR display prominent adaptive targeting DDR and immunostimulatory therapies in a fraction of immunity and unaltered myeloid-cell changes. Our data indicate that NSCLC. MSI-H and DDR phenotypes are independent features in human CRC Ethics Approval and this could be used to design optimal therapeutic strategies. All tissues were used after approval from the Yale Human Investiga- Ethics Approval tion committee protocol #9505008219 which approved the patient All tissues were used after approval from the Yale Human Investiga- consent forms or waiver of consent. tion committee protocol #9505008219 which approved the patient consent forms or waiver of consent. P34 A fully optimized end-to-end solution for I/O multiplex P33 immunofluorescence staining using Opal Polaris 7-Color PD1/PD- DNA damage response (DDR) is associated with increased adaptive L1 Panel Kits for lung cancer and melanoma anti-tumor responses and PD-L1 expression in human non-small Yi Zheng, Rachel Schaefer, Linying Liu, Glenn Milton, Carla Coltharp, cell lung cancer 2 2 1 PhD, Victoria Duckworth, MS, Michael McLane, Peter Miller, MS Shruti Desai, PhD , Aravind Kalathil , Roy Herbst, MD, PhD , Ranjit 2 1 2 Akoya Biosciences, Hopkinton, MA, United States Bindra , Patricia LoRusso, DO , Kurt Schalper, MD, PhD 1 2 Correspondence: Peter Miller (pmiller@akoyabio.com) Yale University, New Haven, CT, United States; Yale University, School Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P34 of Medicine, New Haven, CT, United States Correspondence: Kurt Schalper (kurt.schalper@yale.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P33 Understanding cellular heterogeneity and spatial relationships be- tween biomarkers within the tumor microenvironment (TME) is a key Background component to translational research in immuno-oncology. Multiplex Tumor cells accumulate genomic alterations as a consequence of immunofluorescence (mIF) on formalin-fixed, paraffin-embedded sustained mutagenic events and defective DNA repair mechanisms, (FFPE) tissue is the multiparameter assay most frequently chosen collectively called DNA damage response (DDR). Targeting DDR path- across all current I/O clinical trials, as it allows for quantitative assess- ways can induce synthetic lethality and prominent anti-tumor re- ment of these relationships in situ. Running medium to large scale sponses in neoplasms with DNA repair deficiency. In addition, translational studies on FFPE tissue demands an assay that is repro- increased DNA damage could favor anti-tumor immune responses by ducible, quantitative, easy-to-use, and standardized, yet still allows increasing the neo-antigenic load and T-cell recognition. Despite its for flexibility when detecting differentially expressing biomarkers therapeutic implications, the frequency and significance of DDR alter- across samples. In this study, we demonstrate a fully developed, flex- ations in human non-small cell lung cancer (NSCLC) remains poorly ible, end-to-end workflow solution for tissue biomarker discovery by understood. applying miF in lung cancer and melanoma. This newly developed Methods Phenoptics™ solution provides an integrated MOTiF™ workflow in- Using irradiated cell line preparations and expression controls, we cluding primary antibodies and image analysis algorithms enabling a standardized a multiplexed quantitative immunofluorescence more comprehensive and specific TME analysis with minimal user (mQIF) panel for simultaneous and localized measurement of optimization. DAPI (all cells), cytokeratin for tumor epithelial cells (AE1/AE3, Methods DAKO), γH2AX to map active DNA damage/repair responses FFPE samples from human lung cancer and melanoma were stained (JBW301, Millipore), CD3 for T-lymphocytes (Rabbit polyclonal, using Opal Polaris 7-Color PD1/PD-L1 Lung Cancer and Melanoma DAKO) and PD-L1 (E1L3N, CST) in formalin-fixed paraffin- Panel Kits. Staining was performed on the Leica BOND RX™ auto- embedded (FFPE) tissue samples. We used this panel to interro- mated stainer with the pre-loaded MOTiF protocol. Multispectral gate 4 retrospective NSCLC cohorts from Yale represented in tis- scans were acquired on Vectra Polaris® with pre-optimized acquisi- sue microarray format including immunotherapy-naïve cases tion parameters and analyzed with a pre-configured phenotyping al- (Cohort#1: n=297 and #2:n=175); lung adenocarcinomas tested gorithm in inForm®. Spatial analyses and visualizations were for major oncogenic mutations (Cohort #3, n=139); and baseline performed in R using phenoptr and phenoptrReports. NSCLC samples from patients treated with immune checkpoint Results blockers (Cohort #4, n=84). We analyzed the levels of the markers This simplified end-to-end solution results in better quantification of in different tumor tissue compartments and their association with cancer-immune interactions by providing: major clinicopathological variables. Results Detectable nuclear tumor-cell γH2Ax was recognized in 37-58% of  Well-optimized Opal Polaris 7-Color PD1/PD-L1 Lung Cancer NSCLCs. Elevated tumor-cell γH2Ax expression was consistently asso- and Melanoma Panel Kits. Along with the pre-loaded Leica ciated with smoking history, increased intratumor CD3+ T-cells and BOND RX automation protocol, we provide a staining workflow PD-L1 protein expression across the cohorts. The level of γH2Ax was with pre-defined primary antibody concentration, fixed staining significantly lower in KRAS mutant lung adenocarcinomas than in order, and Opal™ dye-antibody pairs, leaving Opal concentra- EGFR mutant or EGFR/KRAS wild type tumors. No additional tions as a flexible dial. Recommended image acquisition Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 18 of 272 parameters on the Vectra Polaris® that significantly simplify lymphoid aggregates. Micro-regions were picked and sequenced. visualization of multiple markers via multispectral isolation. Pre- Hierarchical clustering and differential expression analysis differ- configured image analysis algorithms that make quantitative entiated the three micro-region types and revealed tumor- and T analysis at a per-cell and per-slide level streamlined and cell-specific expression signatures. CIBERSORT demonstrated the standardized. presence of T cell-associated transcriptomic profiles in lymphoid aggregates and in TIL-containing micro-regions that were propor- tional to the number of T cells retrieved. Aligned RNA-seq reads Conclusions were further analyzed via TraCeR to identify TCR α and β chain The 7-Color PD1/PD-L1 panel kits utilizing MOTiF whole slide scan- sequences from retrieved TILs. ning enable visualization of multiple biomarkers at the whole slide Conclusions level, revealing distribution patterns and their spatial context across These data establish the potential of combining multi-parameter IF the entire tissue section. With these new assays, we have demon- microscopy with highly focused RNA sequencing as a powerful tool strated an easy-to-use yet comprehensive end-to-end Phenoptics re- for investigation and biomarker discovery for immuno-oncology. search workflow. We have radically simplified the Opal method and facilitated the development and optimization of translational multi- plex fluorescent assays by providing pre-defined staining conditions P36 while still giving researchers the flexibility to balance signals based The complexity of myeloid-derived suppressor cells in non-small on their tissue samples. Complementary pre-configured phenotyping cell lung cancer: A combinatorial multiplex IHC and flow cytometry provides researchers faster access to quantitative data across study approach samples. 1 2 1 1 Amanda Finan, PhD , Muriel Smet , Maroua Tliba , Manon Motte , Jean- 1 1 1 Philippe Coton , Domenico Lazzaro , Renaud Burrer 1 2 Histalim, Montpellier, France; Barc Lab, Ghent, Belgium P35 Correspondence: Renaud Burrer (rburrer@histalim.com) Pick-Seq®: a spatial analysis tool for immuno-oncology biomarker Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P36 discovery utilizing multi-parameter imaging and RNA sequencing of tissue micro-regions Background 1 1 1 Nolan Ericson , Rebecca Podyminogin , Jennifer Chow, PhD , Yu-An Lung cancer is the most common cause of cancer-related deaths 2 2 2 2 1 Chen , Jia-Ren Lin , Zoltan Maliga , Peter Sorger , Kyla Teplitz , Melinda worldwide with non-small cell lung cancer (NSCLC) representing the 1 1 1 Duplessis, PhD , Eric Kaldjian, MD , Tad George, PhD gross majority of the cases. The immune microenvironment of NSCLC 1 2 RareCyte, Seattle, WA, United States; Harvard Medical School, Boston, is diverse with many players that can impact tumor development MA, United States and clinical outcomes. In particular, myeloid-derived suppressor cells Correspondence: Tad George (tgeorge@rarecyte.com) (MDSC) are important components of the immunosuppressive net- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P35 work that can hinder the activity of T cells, natural killer cells, and dendritic cells. MDSC in the blood may represent prognostic markers Background for NSCLC patients and for monitoring a patient’s response to im- Pick-Seq is a novel workflow uniquely enabled by the RareCyte Cyte- munotherapies. There is a gap in the relevance of MDSC within the Finder® Instrument that combines visualization of multiple protein tissue context due to limitations with conventional immunohisto- markers with investigation of gene expression from selected micro- chemistry. Multiplex immunofluorescence offers a technical advan- regions on tissue slides, providing spatial and contextual investiga- tage by allowing the detection of co-expression and spatial tion of tumors and their microenvironment. organization of multiple targets within a preserved tissue architecture Methods on a single slide. Frozen breast carcinoma and formalin-fixed, paraffin-embedded ton- Methods sil sections were stained by multi-parameter immunofluorescence (IF) We have developed the multiplex immunofluorescence for markers of T cells, B cells, and cytokeratin. Slides were imaged Histoprofile-MDSC panel to identify monocytic MDSC (M-MDSC) with the CyteFinder® Instrument and 40 μm micro-regions were re- and polymorphonuclear MDSC (PMN-MDSC) in situ. Five human trieved with the integrated CytePicker® Retrieval Module. RNA was NSCLC tissue samples were investigated by multiplex immunofluor- isolated and whole transcriptome amplified (SMART-seq v4), followed escence and H&E staining. After multispectral acquisition, the MDSC by Nextera XT library preparation, sequencing on Illumina MiSeq, and populations were evaluated with the imaging software HALO. gene expression analysis. Differentially expressed genes were se- Paired peripheral blood was analyzed for circulating M-MDSC by lected to create a Pick-Seq-informed IF staining panel to confirm flow cytometry. RNA expression results. Cell compositions of each micro-region were Results deconvolved with CIBERSORT. The development and verification of the multiplex panel are pre- Results sented. The NSCLC subtype of the samples was determined by a Tonsil micro-regions from one T cell zone and two adjacent follicles pathologist from the H&E sections. Monocytes, neutrophils, M-MDSC, were retrieved for RNA sequencing. Transcriptomic analysis con- and PMN-MDSC were evaluated in the five tissue samples. The neu- firmed increased expression of B cell markers in follicles and T cell trophils, monocytes, and M-MDSC in the peripheral blood could be markers in the T cell zone. CIBERSORT analysis revealed distinct cellu- assessed by flow cytometry. A varying distribution of the cell popula- lar compositions between T cell zones and the B cell follicles. tions in the lung tissue and the peripheral blood of the different Principle component analysis of gene expression found that micro- NSCLC subtypes can be appreciated. The two approaches are regions retrieved from the two follicles clustered independently from compared. each other, and from the T cell zone micro-regions. Differential ex- Conclusions pression analysis between the adjacent follicles revealed distinct pat- We present an in-depth combined approach for MDSC investigation terns of CD21 expression, a marker which was not present in the in lung tissue and the peripheral blood of NSCLC patients. The ap- original IF staining panel. Subsequent staining confirmed differential proaches presented here demonstrate the power of multiplex immu- protein expression of CD21, indicating that only one follicle con- nohistochemistry and flow cytometry in the identification and tained a germinal center. In breast carcinoma, ROI were identified for quantification of multiple immune cell populations with a limited micro-region retrieval that included tumor cells, tumor cells with quantity of patient sample and the potential application of this interspersed tumor infiltrating lymphocytes (TIL), or adjacent method in both preclinical and clinical studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 19 of 272 P37 8. Otvos B, Silver DJ, Mulkearns-Hubert EE, et al. Cancer Stem Cell-Secreted ImmunoPET imaging of glioma-infiltrating myeloid cells using Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Sup- Zirconium-89-labeled anti-CD11b antibody pressor Cell Function and Facilitates Glioblastoma Immune Evasion. Stem Alexandra Foster, BS, Rajeev Kumar, Shubhanchi Nigam, Lauren McCarl, Cells. 2016; 34:2026–2039. Robert Edinger, Ian Pollack, Carolyn Anderson, Wilson Edwards, Gary 9. Meyer C, Cagnon L, Costa-Nunes CM, et al. Frequencies of circulating Kohanbash, Alexandra Foster, BS MDSC correlate with clinical outcome of melanoma patients treated with University of Pittsburgh, Pittsburgh, PA, United States ipilimumab. Cancer Immunol Immunother. 2014; 63:247–257. Correspondence: Gary Kohanbash (gary.kohanbash2@chp.edu) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P37 The study was approved by University of Pittsburgh's Institutional Animal Care and Use Committee (IACUC). Background Gliomas are the most common primary central nervous system tumor, P38 with malignant gliomas causing significant morbidity and mortality. Sensitive methodologies for tracking T cell immunotherapy by MRI Thirty percent of a glioma’s cellular mass may be attributed to immuno- 1 2 1 2 Brooke Helfer, PhD , Deanne Lister , Charles O'Hanlon III , Eric Ahrens , suppressive and pro-tumoral tumor-associated myeloid cells (TAMCs), Brooke Helfer, PhD primarily myeloid-derived suppressor cells (MDSCs) and tumor- 1 2 Celsense, Inc, Pittsburgh, PA, United States; UCSD, La Jolla, CA, United associated macrophages (TAMs) [1-4]. Multiple preclinical studies and States clinical trials have attempted to target these cells; however, monitoring Correspondence: Brooke Helfer (brooke@celsense.com) responses to these therapies remains a challenge. Quantifying TAMCs Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P38 within gliomas using an antibody-based tracer for non-invasive posi- tron emission tomography (immunoPET) may allow for better patient Background stratification, monitoring of treatment efficacy, and ultimately improve Cancer immunotherapies have made a great progress and hold much survival rates [5-9]. Integrin CD11b is a cellular marker expressed on the promise in the treatment of cancer. Specifically, in the case of B-cell surface of TAMCs frequently used to identify macrophages and micro- malignancies (such as Acute Lymphoblastic Leukemia, or ALL), CAR glia. We therefore hypothesized that radiolabeled anti-CD11b antibody (chimeric antigen receptor) and TCR (T-cell receptor) therapies have (Ab) could be used for immunoPET imaging of TAMCs in a preclinical demonstrated encouraging clinical results. As we begin to target solid orthotopic syngeneic glioma model. tumors with TCR and CAR T-cells, the hurdle of being able to select a Methods suitable target and achieve successful cellular delivery/homing to the The human/mouse cross-reactive anti-CD11b Ab (clone M1/70) was site of disease remains. With this in mind, being able to visualize a rap- conjugated with p-NCS-Bz-DFO chelator and radiolabeled with 89Zr idly dividing cellular population is another obstacle to consider. for PET imaging with specific activity of 2 μCi/μg. PET/CT imaging, Methods with or without a blocking dose of anti-CD11b Ab, was performed in Here we demonstrate the application of two clinically applicable per- mice bearing established orthotopic syngeneic GL261 gliomas. Flow fluorocarbon (PFC) tracers, one commercially available and a next- cytometry and histology in tissues collected from post-imaging bio- generation magnetic resonance imaging (MRI) probe called FETRIS. distribution validated targeting of CD11b+ TAMCs. Both of these agents enable the migration and persistence of cellular Results therapies to be noninvasively imaged by 19F MRI, while the FETRIS Standard uptake values (SUV) indicated significant 89Zr-anti-CD11b reagent adds additional detection sensitivity. Ab uptake in the tumor ipsilateral right brain (SUVmean = 2.6 ± 0.24) Results compared to contralateral left brain (SUVmean = 0.6 ± 0.11). Blocking Using a general T-cell expansion protocol, we show that adding a cellular with 10-fold lower specific activity 89Zr-anti-CD11b Ab reduced the label does not alter the viability or release characteristics of T cells. By SUV in right brain with (SUVmean = 0.11 ± 0.06). Spleen and lymph pairing the PFC signal with conventional proton MRI from the same im- nodes also showed high uptake, while bone and muscle showed low aging session, the images are able to be overlaid, allowing cells to be uptake. Biodistribution analysis confirmed these results. Additionally, traced to their anatomical location. With nominal exogenous fluorine nat- no uptake was observed in the brain of non-tumor bearing mice that urally present in tissue, labeled cells appear with little background. received 89Zr-ant- CD11b. Flow cytometry with QuantiBRITE Fluores- Conclusions cence Quantitation Kit demonstrated that the majority of tumor- Images of both reagents show the detection and sensitivity of the infiltrating immune cells expressed CD11b at an average of 54,076 method and how they can be applied to monitor the distribution of CD11b molecules per cell in GL261. cells over time. Conclusions Imaging TAMCs with 89Zr-labeled anti-CD11b Ab may be feasibility for preclinical studies, patient stratification, and monitoring of immunotherapy. P39 References Looking beyond the assay: Comparison of multiplex chromogenic 1. Gabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate and fluorescent immunohistochemistry for standardized immune immune cells resemble M0 macrophage phenotype. JCI insight. 2016; oncology profiling in non-small cell lung carcinoma patients 1 2 2 1(2). Ana Hidalgo Sastre, PhD , Lorenz Rognoni, PhD , Monika Baehner , 2 2 2 2 2. Kennedy BC, Showers CR, Anderson DE, et al. Tumor-associated macro- Marco Testori , Jessica Chan , Andreas Spitzmüller , Nicolas Brieu, PhD , 3 3 3 phages in glioma: friend or foe? J oncol; 2013. 2013. Bonnie Phillips, PhD , Katir Patel, PhD , Sean Downing, PhD , Alex 4 4 2 3. Kohanbash G, Okada H. Myeloid-derived suppressor cells (MDSCs) in gli- Haragan , John Field , Florian Leiss, PhD 1 2 3 omas and glioma-development. Immunolo invest. 2012; 41:658–679. Definiens, Munich, Germany; Definiens AG, Munich, Germany; Ultivue, 4. Lapa C, Linsenmann T, Lückerath K, et al. Tumor-associated macrophages Cambridge, MA, United States; Liverpool University Hospital, Liverpool, in glioblastoma multiforme-a suitable target for somatostatin receptor- United Kingdom based imaging and therapy? PloS one. 2015; 10. Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) 5. Kohanbash G, McKaveney K, Sakaki M, et al. GM-CSF Promotes the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P39 Immunosuppressive Activity of Glioma-Infiltrating Myeloid Cells through Interleukin-4 Receptor-α. Cancer Res. 2013; 73:6413–6423. Background 6. Raychaudhuri B, Rayman P, Huang P, et al. Myeloid derived suppressor cell Given the heterogeneity of tumors and the variety of potential bio- infiltration of murine and human gliomas is associated with reduction of markers in immune oncology, there is a need for quantitative standard- tumor infiltrating lymphocytes. J Neurooncol. 2015; 122:293–301. ized assays to reliably assess the immune status of a patient’stumor to 7. Okada H, Kohanbash G, Zhu X, et al. Immunotherapeutic approaches for be able to extract the true biological information across cohorts. Here, glioma. Crit rev immunol. 2009; 29:1–42. two different tissue-based approaches have been compared: multiplex Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 20 of 272 immunofluorescence (mIF) and multiplex chromogenic immunohisto- P40 chemistry (mIHC). Independently of the technique used, assay reproduci- Tumour immunity signatures to expand current diagnostic bility and standardized quantification of staining intensity are a approaches in mismatch repair deficient cancers in the context of prerequisite for obtaining consistent results. Using a cohort of non-small Lynch Syndrome through InSituPlex technology and Tissue cell lung carcinoma (NSCLC) patients, we identified patterns of immune Phenomics integration 1 2 3 cell infiltration that were comparable, independent of the assay applied. Ryan Hutchinson, Fellow , Armin Meier, PhD , Bonnie Philips ,Katir 3 3 3 1 Methods Patel, PhD , Sean Downing, PhD , Karan Sharma , Julia Como ,Simin 1 2 4 1 Formalin-fixed paraffin-embedded (FFPE) true consecutive slides from 7 Daneshvar , Gillian Livock , Ingrid Winship , Christophe Rosty ,Mark 5 2 1 NSCLC resections were stained with a multiplex chromogenic panel (in- Jenkins ,GunterSchmidt , Daniel Buchanan ,RyanHutchinson, cluding CD3, PD-L1, CD68, CD8, PD-1) at Mosaic Laboratories [1] and with Fellow the UltiMapper kits (I/O PD-L1 and I/O PD-1) from Ultivue. mIHC scans Victorian Comprehensive Cancer Centre, Melbourne, Australia; 2 3 were acquired with an Aperio AT Turbo scanner (Leica), while mIF scans Definiens AG, Munich, Germany; Ultivue, Cambridge, MA, USA, Boston, 4 5 were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss) both as whole MA, United States; Royal Melbourne Hospital, Melbourne, Australia; The slide images. mIHC and mIF images were co-registered, and Definiens University of Melbourne, Melbourne, Australia custom algorithms for digital image analysis were applied [2,3]. Correspondence: Daniel Buchanan (daniel.buchanan@unimelb.edu.au) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P40 Densities of immune cell populations and their locations in different com- partments (invasive margin vs tumor center and tumor epithelium vs Background tumor stroma) were measured (Figure 1). For instance, CD3 cell density Deficiency in the mismatch repair (dMMR) can result from had a Pearson correlation of 0,91 and a Spearman correlation of 0,89 be- inherited mechanisms (Lynch Syndrome (LS)) or from somatic tween both assays (mIHC vs mIF). Differentiation between tumor epithe- inactivation caused by hypermethylation of the MLH1 gene lium and tumor stroma was based on a histology-driven deep learning promoter (MLH1 methylated). A third subtype of dMMR colo- approach for mIHC and on pan Cytokeratin for mIF (Figure 1). rectal cancer (CRC) and endometrial cancer (EC) have neither Conclusions LS nor MLH1 promoter methylation and are referred to as sus- By applying mIHC and mIF in true consecutive tissue slides we re- pected Lynch syndrome (SLS). There remains a knowledge gap trieved the information of tumor immune cell infiltrates that was as to whether the tumour microenvironment (TiME) is different consistent across the different assays and distinguished it from infor- between LS, MLH1-methylated and SLS dMMR CRC and EC. The mation that is specific to either of the assays. We believe that being aimofthisstudy wastocharacterise and identify immune pat- able to relate across staining techniques could help pathologists and terns within the TiME that may enhance the current clinical research centers draw conclusions across cohorts that were stained triaging of LS, SLS and MLH1-methylated subtypes of dMMR with the same markers but with different assays. CRC and EC. Methods References Ten FFPE samples from seven individuals were studied: CRC (N=5; 1. Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of 1xLS, 2xMLH1 methylated, 1xSLS and 1x proficient MMR (pMMR)) inflammatory cell patterns and densities using multiplex and EC (N=2; 1xLS and 1xpMMR) and where available adjacent nor- immunohistochemistry immuno-oncology assays [abstract]. In: Proceed- mal tissue (N=5: 3xcolon and 2xendometrium) were characterized ings of the AACR-NCI-EORTC International Conference: Molecular Targets using the Ultivue UltiMapper I/O portfolio (InSituPlex) on a Leica and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia Bond autostainer and digitally acquired using the Zeiss Axio Scan Z1. (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl): Abstract nr B069. We evaluated CD3, CD8, CD11c, CD20, CD45RO, CD68, CD163, Gran- 2. Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated zyme B, Ki-67, MHC II, PD-1, PD-L1, and pan-cytokeratin. Tissue phe- quantification of whole-slide multispectral immunofluorescence images to nomic approaches were developed to spatially characterize, quantify identify spatial expression patterns in the lung cancer microenvironment. immune cell patterns and visualize heterogeneity within the TiME SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. (Figure 1). 3. Brieu, Nicolas & Meier, Armin & Kapil, Ansh & Schönmeyer, Ralf & Gavriel, Results Christos & Caie, Peter & Schmidt, Günter. (2019). Domain Adaptation- InSituPlex technology enabled the visualization of the based Augmentation for Weakly Supervised Nuclei Detection. heterogenous infiltration and co-localization patterns across LS Ethics Approval dMMR CRC (Figures 2&3). Tissue phenomic approaches demon- Ethical approval was granted by the Liverpool Research Ethics Committee, strated the following; within the SLS category the colon cancer reference number 97/141. had higher mean areas of intraepithelial (IE) PD-L1 (6% vs. 2%), CD8 (18% vs. 8%) and CD68 (28% vs. 12%) compared to the pMMR EC. The MLH1 methylated tumour with a high tumour mu- tation burden (33.84 mutations/MB) had a higher mean area of IE PD-L1 (8% vs. 2%) and CD8 (30% vs. 5%) while the tumour with low TMB had a higher mean area of IE CD68 (15% vs. 8%). Within LS, the EC had a higher mean area of IE PD-L1 compared to the CRC (14% vs. 0%), in contrast the CRC had a higher IE CD8 area (27% vs. 10%) (Figures 4&5). Conclusions This study evaluated the immune contexture within inherited and sporadic subtypes of dMMR CRCs and ECs, highlighting dif- fering immune infiltration patterns and phenomic densities. In- tegration of multiplex technologies and Tissue Phenomics can enhance the understanding of the dMMR TiME and with poten- tial utility in clinical triaging and to inform immune-oncology clinical trials. Acknowledgements We thank the investigators and participants of the ACCFR and ANGELS Fig. 1 (abstract P39). Consecutive slides from NSCLC resection studies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 21 of 272 Fig. 2 (abstract P40). TiME regions of immune infiltration Fig. 1 (abstract P40). Phenotypes Fig. 3 (abstract P40). TiME regions of immune infiltration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 22 of 272 P41 Multiplexed Imaging for the simultaneous detection of nucleic acids and proteins to dissect the tissue immune landscape and microenvironment of viral diseases 1 1 2 Sizun Jiang, PhD , Xavier Rovira Clave, PhD , Chi Ngai Chan, PhD , Bokai 1 1 1 1 Zhu , Yunhao Bai, BS , Marc Bosse, PhD , David McIlwain, PhD , Sean 1 1 2 Bendall, PhD , Michael Angelo, MD, PhD , Jacob Estes, PhD , Garry Nolan, PhD 1 2 Stanford University, Stanford, CA, United States; Oregon Health and Sciences University, Stanford, CA, United States Correspondence: Jacob Estes (estesja@ohsu.edu); Garry Nolan (gnolan@stanford.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P41 Background Multiplexed Ion Beam Imaging (MIBI) is a novel imaging modal- ity capable of resolving >40 parameters simultaneously in bio- logical samples. Here, we developed viralMIBI, a highly sensitive method capable of detecting down to single copies of nucleic acids, in addition to protein epitopes. ViralMIBI en- ables the functional dissection of the immune landscape in viral driven diseases, such as that of tumor viruses (HBV, EBV, LCV) and others (HIV, SIV, Zika, Ebola). The combination of vir- alMIBI and cutting-edge cell neighborhood analytical methods will be paramount to better understand the immunological host-pathogen interactions for viral diseases, revealing insights into virus-induced immunodeficiency as well as virus-driven cancers. Methods To allow for the sensitive detection of nucleic acids, we took advantage of a customized branched DNA amplification method that can be easily adapted to a variety of multiplexed Fig. 4 (abstract P40). TiME regions of immune infiltration imaging platforms. Formalin-Fixed and Paraffin-Embedded (FFPE) tissue samples from Rhesus macaque animal models for a number of viral diseases were processed for viralMIBI nucleic acid and protein marker detection. Imaging was performed with the MIBIscope, a secondary ion mass spectrometry based device. Results We have established a robust method for highly multiplexed nucleic acid and protein epitope detection in FFPE tissue samples. As a proof of concept, we were able to detect down to single integrated copies of SIV. The establishment and validation of a Rhesus macaque spe- cific antibody panel allowed for the in-depth characterization of cel- lular identities at the single-cell level, while maintaining their tissue geopositions. Conclusions ViralMIBI enables the MIBI to achieve highly sensitive nucleic acid detection, in addition to its multiplexed protein capabil- ities. Here, we leveage this method for the detection of various viral pathogens. ViralMIBI is also applicable to other targets, such as genomic amplifications frequently seen in cancers, or gene expression studies. The ability to image >40 parameters in tissue samples will vital for a better understanding of im- mune regulation of diseases, such as the establishment of viral related cancers as well as latent tissue reservoirs of pathogens. These discoveries can then be translated to better immuno- therapy treatments against viral driven diseases. Acknowledgements We thank Matt Newgren for tireless technical support on the MIBI instrument, Rachel Finck, Xiao-Jun Ma and Bingqing Zhang for helpful discussions. S.J was supported by a Stanford Dean’s Fellowship and the Leukemia & Lymphoma Society Career Development Program. X.R.-C. was supported by a long-term EMBO fellowship. This work was supported by grants from the FDA, NIH, Parker Institute for Cancer Immunotherapy, the Bill and Melinda Gates Foundation, as well as Fig. 5 (abstract P40). TiME regions of immune infiltration the Rachford and Carlota A. Harris Endowed Professorship to G.P.N. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 23 of 272 P42 An integrated multiplexing approach for the immunoprofiling of the tumor microenvironment of ovarian granulosa cell tumors 1 2 1 1 Juncker-Jensen, PhD , Tyvette Hilliard , Nicholas Stavrou , Erinn Parnell , 1 1 1 1 Judy Kuo , Eric Leones , Flora Sahafi , Josette William, PhD, MD , Sharon 2 1 Stack , Anna Juncker-Jensen 1 2 NeoGenomics, Aliso Viejo, CA, United States; University of Notre Dame, South Bend, IN, United States Correspondence: Anna Juncker-Jensen (anna.juncker- jensen@neogenomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P42 Background Ovarian granulosa cell tumors (GCTs) are rare tumor accounting for 2-5% of all ovarian cancers. The main current treatment for GCT is surgery, however a subset require chemotherapy for residual and re- current disease. GCT malignancies are often low-grade, however a clinical characteristic of these tumors is a tendency for late recur- rence which is the most critical factor for GCT death. As the onset of recurrence is unpredictable, future research should focus on identify- ing both biomarkers for prognosis prediction, as well as targets that could help guide clinical trials in the development of targeted ther- apies for this rare indication. As GCTs are rare tumors making tissue Fig. 1 (abstract P41). Validation of viralMIBI: Detection of SIV in availability very limited, we used a dual multiplexing approach in infected tissue order to maximize the data output from a total of 14 FFPE tumor samples (6 primary tumors, and 8 recurrent tumors). Methods For protein multiplexing we have used MultiOmyx™, an immunofluores- cence (IF) multiplexing assay utilizing a pair of directly conjugated Cya- nine dye-labeled (Cy3, Cy5) antibodies per round of staining (Figure 1). Each round of staining is imaged and followed by dye inactivation en- abling repeated rounds of staining and deactivation, while deep learn- ing based cell classification algorithms identify positive cells for each biomarker. We generated a 15-marker panel consisting of CD3, CD4, CD8, FoxP3, CD68, CD163, HLA-DR, CD34, CTLA-4, PD-1, PD-L1, Ki67, vimentin, S100, and Pan Cytokeratin. For the gene expression analysis RNA was extracted from the adjacent 10 μm section and then analyzed using the Nanostring nCounter assay, specifically the 770 gene PanCan- cer Immune Panel. Hybridization, purification and immobilization and counts were based on manufacturer’sprotocol. Results On protein level we confirmed previous findings that ovarian GCTs are so-called “cold” tumors, with a very low density of T cell infiltra- tion. When we analyzed the presence of macrophages in the tumor microenvironment however, we found a 113% increase in TAM dens- ity in recurrent tumors compared to primary tumors. When searching for markers differentially expressed between primary and recurrent tumors we detected 4 genes in our PanCancer immune panel that were either significantly down-regulated (CCND3 or TOLLIP), or up- regulated (MAP3K and TNFSF4) in recurrent tumors. TNFSF4 encodes the protein OX40L, and interestingly a high expression of its receptor OX40 has previously been shown to be indicative for response to chemotherapy in recurrent ovarian cancer [1]. Conclusions We have used a dual multiplexing approach on both gene and pro- tein level in order to immunoprofile the tumor microenvironment of ovarian rare granulosa tumors. Reference 1. Ramser M, Eichelberger S, Däster S, Weixler B, Kraljević M, Mechera R, Tampakis A, Delko T, Güth U, Stadlmann S, Terracciano L, Droeser RA, Singer G. High OX40 expression in recurrent ovarian carcinoma is Fig. 2 (abstract P41). Detection of single integration events of SIV indicative for response to repeated chemotherapy. BMC Cancer. with viralMIBI 2018;18:425-433. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 24 of 272 custom panel of more than 25 markers on the PBMC samples and acquired the images using a Keyence benchtop microscope. Results The CODEX system was used to generate highly multiplexed im- mune profiles of human PBMC samples using an optimized cus- tom panel of CODEX antibodies. The images were processed using the CODEX Software Suite and cell phenotypes were clus- tered and annotated using the Multiplexed Analysis Viewer (MAV). Antibody specificity and panel performance were evalu- ated by assessing co-expression and mutually exclusive expres- sion of relevant immune markers with the CODEX analysis pipeline. Conclusions Simultaneous analysis of tens of markers in blood or plasma sam- ples can have several applications in the discovery of cellular bio- markers, immune monitoring and drug discovery and development. This preliminary study shows the compatibility of the CODEX system with cell suspensions for highly multiplexed, single-cell analysis and offers a more cost-effective method for immune profiling of blood samples. P44 Solar-IHC: Cell-to-cell distances in the tumour immune microenvironment of Hepatocellular Carcinoma has the potential Fig. 1 (abstract P42). Immunofluorescent overlay image of GCT to prognosticate survival 1 2 2 recurrent tumor Matthew Leong, NA , Toh Han Chong , Choo Su Pin , Kiat Hon Lim 3 2 2 4 Tony , Joycelyn Lee , David Wai , Poh Sheng Joe Yeong , Jin Miao Chen, PhD Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore, Singapore; National Cancer Centre Singapore, Singapore, Singapore; Singapore General Hospital, Singapore, P43 Singapore, Singapore; Department of Anatomical Pathology, Singapore A novel platform for highly multiplexed, single-cell imaging of cell General hospital, Singapore, Singapore, Singapore; Singapore suspensions Immunology Network, Agency of Science, Technology and Research, 1 2 1 1 Anum Khan , Won-Mean Lee , Jon Mulholland , Dhananjay Wagh , John Singapore, Singapore, Singapore 1 2 Coller , Gabriel Mercado Correspondence: Jin Miao Chen (Chen_Jinmiao@immunol.a- 1 2 Stanford University, Palo Alto, CA, United States; Akoya Biosciences, star.edu.sg) Menlo Park, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P44 Correspondence: Won-Mean Lee (wmlee@akoyabio.com); Gabriel Mercado (gmercado@akoyabio.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P43 Hepatocellular carcinoma (HCC) is a lethal cancer, being the fourth leading cause of cancer-associated mortality worldwide Background due to its low five-year survival and high reoccurrence rates [1], Analyzing populations at the single cell level has become increas- and identifying indicators of prognosis is key in developing novel ingly important in the study of cancer and autoimmune disorders treatments and improving survival of HCC patients. With the ad- due to high levels of population heterogeneity and rare cell phe- vent of digital pathology, the immune-architecture of solid tu- notypes that can drive disease pathogenesis and progression. mours has become a central interest of cancer research and has Until recently, characterizing protein markers on single cells was been studied for the development of predictive and diagnostic limited to a handful of markers due to the technical and logis- applications. Here, we have assessed if intercellular Euclidean dis- tical challenges of flow cytometry platforms. New advancements tances in the tumour immune microenvironment can possibly be in single cell analysis technologies have enabled researchers to used to predict patient prognosis. study more than 30 parameters per cell. But these platforms are Methods expensive and require significant panel design, thereby limiting In this study, biopsies were taken from 110 HCC patients who access and usability. Here we demonstrate the use of the recently underwent surgical resection. The solar-IHC pipeline involves ar- launched CODEX System to generate an in-depth immune profile ranging the liver biopsies into tissue arrays and subsequently of human PBMC samples. studying them using automated multiplex immunohistochemistry/ Methods immunofluorescence (mIHC/IF) protocol developed in Singapore The CODEX® System is an affordable, benchtop instrument that General Hospital, with biomarkers Ecadherin, CD3, CD8, CD103 integrates with existing fluorescence microscopes and enables and PD1 [2], followed by image analysis software inForm version highly multiplexed imaging of over 40 markers in fresh frozen 2.4.2. and FFPE tissue samples. The CODEX technology uses a DNA- Results based barcode library to label antibodies and iterative cycles of Ecadherin was adopted as the tumour cell marker, and 26 im- adding and removing cognate dye-labeled oligonucleotides to mune cell phenotypes are defined by variable levels of immune reveal the staining of three markers per cycle. Data acquisition markers CD8, CD103, and PD-1 (as shown in Table 1) Dimension- is fully automated by the CODEX instrument. We tested a ality reduction and unsupervised clustering of the distances Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 25 of 272 between tumour and immune cell phenotypes showed distinct P45 clusters of patients with significant differences in clinical out- Multiplex immunofluorescence staining, whole slide imaging, and comes. Long cell-cell distances between immune cell phenotypes spatial phenotyping of T-cell exhaustion, regulatory T cells, and and tumor cells was associated with an improved overall survival myeloid-derived suppressor cells in tumor FFPE samples 2 3 1 1 (p-value = 0.02) and disease-free survival (p-value = 0.01), while Kyla Teplitz , Katir Patel, PhD , Michael Tomac, MS , Kate Lillard, PhD , 3 1 the opposite was true for short cell-cell distances between im- Mael Manesse, PhD , Anne Hellebust, PhD 1 2 mune cell phenotypes and tumor cells. This was observed in the Indica Labs, Inc., Alcester, United Kingdom; Rarecyte, Inc, Seattle, WA, analysis of all CD8+, CD8-, CD103+, CD103-, PD1+ and PD1- cells, United States; Ultivue, Inc, Cambridge, MA, United States possibly due to the suppressive immunomodulatory effect Ecad- Correspondence: Kate Lillard (kate@indicalab.com) herin+ tumour cells has on neighbouring immune cells [3]. Fur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P45 thermore, machine learning enabled the prediction of clusters with considerable accuracy, with a K-fold cross-validation of 91%. Background This indicates a strong association of cell-cell distances with pa- The immune cell milieu that comprises the tumor microenviron- tient survival, and a robust reproducibility of distance pattern- ment (TME) is highly heterogeneous and complex. Depending based predictors. on biological interactions and functional state, immune cell Conclusions populations can either promote or suppress tumor progression. In this study, our data suggests that the analysis of intercellular CD8+ T cells, for example, are the primary mediators of anti- distances has the potential to be used as a prognostic indicator tumor immunity; however, they are often ineffective either be- in HCC. Coupled with next generation machine learning tech- cause they are unable to infiltrate the tumor or because they niques, this novel approach to cell-to-cell distance analysis has become functionally exhausted [1,2], Pathologically activated the potential to be an easily implementable algorithm to predict myeloid-derived suppressor cells (MDSCs) which infiltrate the patient prognosis in HCC. This bioinformatics approach can also tumor are also associated with tumor progression [3,4]. Multiple be utilized in the analysis of other biomarkers and cancer types, biomarkers are required to accurately identify these individual and this brings exciting prospects for the future of cancer immune cell types and their functional states. In this work, we research. employ advanced multiplexing techniques to observe biologic- ally and functionally distinct T cell and MDSC populations and References to quantify their density and distribution within the TME of sev- 1. McGlynn KA, Petrick JL, London WT. Global epidemiology of eral tumor types. hepatocellular carcinoma: an emphasis on demographic and regional Methods variability. Clinics in liver disease. 2015;19(2):223-38. UltiMapper assays were used to perform multiplex immunofluor- 2. LimJCT,Yeong JPS, LimCJ, OngCCH,WongSC, Chew VSP, escence on multiple tumor FFPE samples (lung, colorectal, breast). Ahmed SS, Tan PH, Iqbal J: An automated staining protocol for Three multilpex panels were run in this study: UltiMapper PD-1 seven-colour immunofluorescence of human tissue sections [CD3, CD45RO, PD-1, CK/Sox1], UltiMapper T-reg[CD4, CD8, FoxP3, for diagnostic and prognostic use. Pathology. 2018 CK, Sox10], and UltiMapper MDSC[CD11b, CD14, CD15, HLA- Apr;50(3):333-341 DR].FFPE slides were stained using the BOND RX autostainer from 3. Nagl S, Haas M, Lahmer G, Büttner-Herold M, Grabenbauer GG, Fietkau R, Leica Biosystems and scanned on the CyteFinder® II HT Instru- et al. Cell-to-cell distances between tumor-infiltrating inflammatory cells ment from RareCyte, Inc. This instrument performs high-speed, have the potential to distinguish functionally active from suppressed in- whole-slide scanning in the 5 channels used in the UltiMapper flammatory cells. Oncoimmunology. 2016;5(5):e1127494. Kits and outputs an open source, stitched, pyramidal TIFF. Image Ethics Approval analysis was conducted using HALO 3.0 software to perform cell This study was approved by the Institutional Review Board (IRB), approval phenotyping, proximity analysis, image registration, and density number 2014/590/B. mapping. Results Cell counts for relevant phenotypes were obtained for each panel to identify exhausted T cells, T-regs, cytotoxic T cells, M-MDSCs, and Table 1 (abstract P44). See text for description PMN-MDSCs. Spatial analysis was employed to map the degree of T- cell infiltration and exhaustion correlating to T-reg and MDSC expres- sion in the tumor microenvironment. Conclusions Here we present a workflow for tackling the complexity of the tumor immune microenvironment by leveraging high-quality multiplex panels, high-speed whole-slide imaging, and quanti- tative spatial analysis. UltiMapper assays used in this study (PD-1, T-reg, and MDSC) were able to identify single-cell phe- notypes through co-localization and negative selection of markers. Using HALO image analysis, cell populations were enumerated and quantified to measure the level of T-cell ex- haustion caused by T-cell regulation and myeloid-derived im- mune cell suppression. References 1. Fridman WH, Zitvogel L, Sautes-Fridman C, Kroemer G. The immune con- texture in cancer prognosis and treatment. Nat. Rev. Clin. Oncol. 2017; 14:717–734. 2. Thommen DS, Schumacher TN. T Cell Dysfunction in Cancer. Cancer Cell. 2018; 33:547-562. 3. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 2012; 12:253–268. 4. Kumar V, Patel S, Tcyganov E, Gabrilovich DI. The Nature of Myeloid- Derived Suppressor Cells in the Tumor Microenvironment. Trends Immu- nol. 2016; 37:208–220. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 26 of 272 P46 medicine but without the complex workflow, safety, and half-life limi- Same-slide multiplex immunofluorescence and brightfield tations. In this study, we compared the behavior of monocytes histological staining as a new research tool for fast and loaded with nanoparticles in vitro and nanoparticles injected intra- comprehensive pathology assessment of the tumor venously and subsequently taken up by phagocytic cells (in situ load- microenvironment ing) and imaged using MPI the differences in biodistribution and Mael Manesse, PhD, Douglas Wood, PhD, Heike Boisvert, PhD, Sean migration of monocytes in in naïve and tumor-bearing and naïve Downing, PhD, Mael Manesse, PhD (control) mice. Ultivue, Cambridge, MA, United States Methods Correspondence: Mael Manesse (mael.manesse@ultivue.com) Twenty mice were implanted with 300,000 4T1 tumour cells in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P46 4th mammary fat pad. CD11b+ mouse monocytes were harvested using EasySep® Mouse CD11b positive selection kit II (StemCell Tech- Background nologies). The isolated monocytes were prelabeled with Vivotrax® Innovative and efficient translational research tools enabling a better (100 μg/mL). On day 7 post-implantation, either 5 million prelabeled understanding of the tumor and its microenvironment are a keystone cells or free Vivotrax (6 mg/kg) were intravenously injected into nor- of the development of digital pathology. Current immunohistochem- mal or tumor-bearing mice for in vitro or in situ targeting experi- istry (IHC) methods limit the depth of information from a single tis- ments (N=5 mice for all four groups). 3D MPI images using a sue sample to a single target in the case of chromogenic staining, or MOMENTUM MPI system (Magnetic Insight) were acquired 1, 4, 7 to sample morphology and general cell identification in the case of and 10 days after injection. MicroCT images (CT120, Trifoil Imaging) hematoxylin and eosin staining (H&E). True phenotyping requires the were acquired and co-registered using VivoQuant (Invicro). Tumors, use of a single section, as serial sections may not contain the same liver, spleen and draining lymph nodes were then harvested, imaged, cells, especially small immune cells such as T-cells. Multiplex im- fixed, and stained with Prussian blue and analyzed for iron contents. munofluorescence (mIF) methods have been established to provide Results insights into a wide number of markers of interest and their spatial Tumor-bearing mice showed a significant accumulation of nanoparti- context in a single sample. Here, we demonstrate a new research ap- cles for both the in situ and in vitro targeting methods, although the proach combining multiplexed detection of protein markers with time and amount of accumulation was different. For both experi- standard H&E pathology review in tumor samples, in a streamlined, ments, nanoparticles were predominately detected in the expanding single-day sample-to-answer workflow. margins of the tumor. For the in vitro labeled monocytes, accumula- Methods tion was rapid, with the maximum accumulation being at 24 hours InSituPlex technology was used to perform multiplex immunofluores- post-injection, while for the in situ labeled cells, accumulation was cence staining of formalin-fixed, paraffin-embedded (FFPE) samples slower. from human tonsil and primary tumor biopsies on the Leica Biosys- Conclusions tems BOND RX autostainer. The tissues were then imaged in five dis- By combining the sensitivity, specificity as well as accurate quantita- tinct fluorescent channels (DAPI, FITC, TRITC, Cy5, Cy7) before being tion potentials of MPI, information can be obtained on labeled stained using standard H&E protocols and imaged again. Fluorescent monocytes and their biodistribution in tumour models. Other cells and brightfield whole-slide images were acquired on a ZEISS AxioS- can also be labeled (dendritic cells, MDSCs, NKs, and T cells) and this can.Z1 slide scanner. Images of the same tissue section were co- information can be utilized to better understand the factors influen- registered and fused into a single image for analysis using Indica cing immune cell migration in and around tumors. Labs HALO software. Results P48 The InSituPlex technology enables deep phenotyping of immune Turning ‘cold’ tumours ‘hot’: Guided magnetic hyperthermia for cells through colocalization and co-expression of multiple protein tumour immune stimulation markers in tumor samples. Phenotypic information was then overlaid 1 1 2 1 Patrick Goodwill , Daniel Hensley , Zhi Wei Tay , Elaine Yu , James with the H&E image of the same section to facilitate identification 1 1 1 1 Mansfield, Msc , Blayne Kettlewell , Ryan Orendorff , Kyle Fields , Steve and immuno-profiling of specific cells in the tumor and its environ- Conolly ment. The fused images were also analyzed to provide cell counts, 1 2 Magnetic Insight, Alameda, CA, United States; University of California distance mapping, and expression levels of each of the markers. Berkeley, Berkeley, CA, United States Conclusions Correspondence: James Mansfield (jim@jmansfield.com) In this work, we present a new modality for pathology research with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P48 a convenient workflow that enables fast tissue review and deep immuno-profiling and phenotyping of the tumor via fusion of H&E Background and mIHC staining of the same tissue section. Cancer immunotherapy is now the “fifth pillar” of cancer thera- peutics [1]. Although hugely successful, there are limitations. In P47 many studies, less than half the patients are responsive to ther- Imaging cancer immunology: Systemic tracking of immune cells apy. One hypothesis is that refractory tumours are immunologic- in vivo with magnetic particle imaging ally ‘cold’– i.e., there are insufficient immune cells in the tumour 1 1 2 James Mansfield, Msc, Gang Ren , Jeff Gaudet , Yanrong Zhang , Sara for the therapy to be efficacious [2]. Thus, methods to stimulate 2 2 1 Ghobadi , Max Wintermark , Patrick Goodwill an immunogenic response in solid tumours to improve immuno- 1 2 Magnetic Insight, Alameda, CA, United States; Stanford University, Palo therapy efficacy are desirable. Alto, CA, United States Hyperthermia is known to induce a local immunogenic response, Correspondence: James Mansfield (jim@jmansfield.com) making it a potential adjunct to radiation and immune therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P47 One hyperthermia method is Magnetic Fluid Hyperthermia (MFH), which is based on electromagnetic heating of magnetic nanoparti- Background cles (MNPs) [3,4]. , However, poor control of heating localization and The rapid growth of research into immuno-oncology research has magnitude have prevented MFH’s widespread clinical adoption. fueled a need to track be able to determine the location of a variety Magnetic Particle Imaging (MPI) is an emerging tracer imaging tech- of immune cells systemically and in solid tumors. However, existing nique that directly detects and quantitates superparamagnetic iron- methods for cell tracking that have generally been insufficient. Mag- oxide nanoparticles with exceptional contrast and high sensitivity at netic Particle Imaging (MPI) is a novel tomographic molecular im- millimeter-scale resolutions [5]. MPI’s contrast is similar to nuclear aging technique that can be used to non-invasively track iron-oxide medicine, but without the complex workflow, safety, and half-life lim- tagged immune cells in 3D in vivo, with contrast similar to nuclear itations of a radioactive tracer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 27 of 272 Methods conventional Treg and CTL populations in GIT tissue sections Here we describe how MPI and MFH can be combined to pro- from IBD patients versus normal individuals by multiplex duce spatially localized heating and accurate control of heating immunofluorescence. magnitude. Spatial localization is achieved using a unique Methods mechanism, magnetic localization. Localization is effected by Conventional Treg are typically defined as lymphocytes with a CD3+/ using a strong magnetic field gradient to produce a “field-free CD4+/CD25+/FoxP3+ immuno-phenotype. This complex antigenic region” (FFR) where nanoparticles are heated, while nanoparti- signature has made it difficult to definitively label Treg populations cles outside the FFR are quenched and do not heat. The use of in tissue sections by immunohistochemistry. We combined a 5-plex an FFR thus enables millimeter-scale control over which MNPs (CD3, CD4, CD8α, CD25, FoxP3) immunofluorescence assay using Ulti- are heated [6-8]. vue InSituPlex® multiplex technology with image analysis using Results Indica Labs HaloTM software to identify, localize and enumerate: 1) MPI is first used to quantitate the MNPs prior to heating, to enable total CD3+ T cells, 2) CD8α+ cytotoxic T lymphocytes (CTL) and 3) treatment planning and prediction of the heating dose. MFH can CD3+/CD4+/CD25+/FoxP3+ conventional Treg in formalin-fixed then be induced in target regions of interest located anywhere in paraffin-embedded (FFPE) sections of GIT from patients with UC and the body while avoiding regions containing MNPs that should not be CD versus controls. Using this approach, we were able to definitively heated, such as the liver or lymph nodes. identify and enumerate these immune cell populations on single Conclusions FFPE tissue sections from each specimen. Combined MPI-MFH enables new treatment workflows that ex- Results ploit spatially localized MFH and accurate control of heating We found greater Treg and CTL cell densities (cells/mm2) in colon magnitude. These workflows may resemble image-guided radi- from CD and UC patients versus controls and higher densities of Treg ation therapy or image-guided high-intensity focused ultra- and lower densities of CTL in small intestine from patients with CD sound. Combined MPI-MFH also prevents damage to nearby versus controls. healthy tissue while enabling new applications such as targeted Conclusions immunogenic stimulation. MPI-MFH also enables new heat- The Ultivue InSituPlex© assay was capable of discretely localizing actuation applications involving systemic injection of MNPs conventional Tregs and CTL in human tissues. This multiplex platform followed by local targeting such as local release of a drug [9] could be used to simultaneously localize Tregs and CTL in FFPE surgi- (break thermally labile bonds/nanocarriers) without requiring ac- cal resections and biopsies of neoplastic tissue as well. tive chemical targeting. While currently only available for small animal use, its underlying physics does not prevent its transla- References tion to human sizes 1. Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, Panaccione R, Ghosh S, Wu JCY, Chan FKL, Sung JJY, Kaplan GG. References Worldwide incidence and prevalence of inflammatory bowel disease in 1. Zaidi, N; Jaffee, E. J Clin Invest (2018). the 21st century: a systematic review of population-based studies. Lancet. 2. Sharma, P. et al. Curr Opinion Immunol (2016). 2018; 390:2769-78. 3. Jordan, A. et al. Journal of Magnetism and Magnetic materials, (1999). 2. Corridoni D, Arseneau KO, Cominelli F. Inflammatory bowel disease. 4. Latorre, M. Puerto Rico health sciences journal, 28(3) (2009). Immunol Lett. 2014; 161:231-5. 5. Gleich, B. & Weizenecker, J. Nature 435,1214–1217 (2005). 3. van Herk EH, Te Velde AA. Treg subsets in inflammatory bowel disease 6. Murase, K. Physica Medica, 29(6), 624-630 (2013). and colorectal carcinoma: Characteristics, role, and therapeutic targets. J 7. Hensley, D. Physics in Medicine & Biology, 62(9), 3483 (2017). Gastroenterol Hepatol. 2016; 31:1393-404. 8. Tay, Z. ACS nano, 12(4), 3699-3713 (2018). 4. Yamada A, Arakaki R, Saito M, Tsunematsu T, Kudo Y, Ishimaru N. Role of 9. Liu, J. F., Small, 14(44), 1802563 (2018). regulatory T cell in the pathogenesis of inflammatory bowel disease. World J Gastroenterol. 2016; 22:2195-205. Ethics Approval P49 Human tissues were obtained from the National Disease Research Use of Ultivue InSituPlex® multiplex immunofluorescence to Interchange (NDRI) with support from NIH grant U42OD11158. Tissues localize and quantify regulatory T lymphocytes in formalin-fixed were collected for research purposes under IRB-approved informed paraffin-embedded human tissue sections consent and collection procedures and provided to Pfizer in accordance 1 1 2 Shawn O'Neil, DVM, PhD , Renee Huynh , Courtney Hebert , Jamie with applicable government regulations and guidelines. 2 2 1 1 Buell , Sean Downing, PhD , John Jakubczak, PhD , Yutian Zhan, MS 1 2 Pfizer, Cambridge, MA, United States; Ultivue, Inc., Cambridge, MA, United States P50 Correspondence: Shawn O’Neil (llospo@gmail.com) Rapid high-plex staining and simultaneous imaging for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P49 immunophenotyping of tissue sections 1 2 1 Benjamin Pelz, PhD , Daniel Migliozzi , Diego Dupouy , Anne-Laure 3 3 2 Background Leblond , Alex Soltermann , Martin Gijs 1 2 The inflammatory bowel diseases ulcerative colitis (UC) and Lunaphore Technologies, Lausanne, Switzerland; École Polytechnique Crohn’s disease (CD) are chronic, relapsing inflammatory disorders Fédérale de Lausanne, Lausanne, Switzerland; Universitätsspital Zürich, of the gastrointestinal tract (GIT) that affect millions of individuals Zurich, Switzerland worldwide [1]. The pathogenesis of these disorders is thought to Correspondence: Benjamin Pelz (benjamin.pelz@lunaphore.com) involve dysregulation of mucosal immune homeostasis in the GIT Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P50 in response to environmental factors in genetically susceptible in- dividuals [2]. Regulatory T cells (Treg) are CD4+ T lymphocytes Background that play a central role in peripheral immune tolerance, actively The tumor microenvironment plays a vital role in cancer development. inhibiting inflammation upon antigenic stimulation. There are two Multiplex immunostainings allow studying the interaction of different cell major populations of Treg: conventional Treg and TR1 cells [3]. types in the tumor microenvironment using a single tissue slide. Though Conventional Treg arise from the thymus (tTreg) or can be in- several techniques are available to perform high-plex stainings, they re- duced in the periphery (pTreg); both tTreg and pTreg constitu- quire intensive manual handling, are highly time consuming or not com- tively express FoxP3 and CD25 (IL-2Rα). An imbalance in patible with tissue sections on standard microscope slides. Here we conventional Treg and effector T cells in the GIT microenviron- present a fully automated microscope integrated method for rapid high- ment is thought to play a part in the pathogenesis of inflamma- plex sequential fluorescent immunostaining and imaging of tissue tory bowel disease (IBD) [4]. Thus, we sought to quantify sections. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 28 of 272 Methods P51 Formalin-fixed, paraffin-embedded tissue sections underwent manual dewax- Phenotypic and spatial analysis of inter- and intra-tumor ing and antigen retrieval step. All subsequent steps of staining, antibody elu- heterogeneity using multiplexed ion beam imaging tion and imaging were automated on the microscope integrated (MIBI) 1 2 2 microfluidic device. A single tissue section was stained sequentially for 24 dif- Jason Ptacek, PhD , Robert Johnson, PHD , Joann Palma, PHD , Jay 1 1 1 1 1 ferent immunophenotyping and tissue structural markers. Each staining cycle Tarolli , Rachel Finck , Murat Aksoy , Yi Zhang , Jessica Finn , Jason consisted of incubation of the tissue section with a pair of mouse and rabbit Ptacek, PhD 1 2 primary antibodies, followed by the corresponding fluorescently labelled sec- Ionpath, Inc, Menlo Park, CA, United States; AbbVie, North Chicago, IL, ondary antibodies and DAPI. The section was imaged after each staining United States cycle and subsequently eluted before staining the next pair of markers. Correspondence: Jessica Finn (jessica.finn@ionpath.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P51 Our microscope integrated microfluidic system allowed automated 24-plex staining with conventional primary and fluorescently labelled secondary Background antibodies in less than five hours, including image acquisition steps. The Elucidating both the cell types present in the tumor microenvir- microfluidic tissue processor enabled fast fluidic exchange and thereby re- onment and the spatial relationship between immune and sulted in reduced staining time down to 10-12min per marker. Integration cancerous cells is at the forefront of immunotherapy research. of a window into the microfluidic chip allowed direct tissue imaging under To address this, MIBI has been developed to image up to 40 the microscope avoiding the removal and mounting of the slide. Protocol markers at single cell resolution. optimization resulted in a high signal to background noise ratio for each Methods marker and complete elution of antibodies from the previous staining step. Staining of 10 NSCLC formalin-fixed paraffin embedded (FFPE) A comparison of a 10-plex staining with standard chromogenic stainings tissue sections was performed similarly to traditional IHC ex- on sequential sections showed high concordance for the stained area on cept that a panel of 20 metal labeled antibodies were stained tonsil as well as lung cancer tissue sections (Figure 1). simultaneously. The tissue was imaged at subcellular resolution Conclusions using an ion beam and time-of-flight secondary ion mass spec- With the microscope integrated microfluidic system, it is possible to trometry (ToF-SIMS). The masses of detected species were then perform fast multiplex stainings including image acquisition without assigned to target biomolecules given the unique label of each the need to handle the tissue slide. Moreover, due to the sequential na- antibody and multi-step processing and segmentation were ture of the system it would be easily possible to further increase the performed to create images of the TME and enable quantitative number of markers in the multiplex staining. We foresee this technique metrics of different cell subsets. to greatly facilitates the execution of high-plex stainings and thereby Results the discovery of novel tumor-microenvironment interactions. Control samples imaged at study start and end showed con- sistent marker quantification (inter-run R2>0.99), indicating MIBI staining and acquisition is reproducible and robust. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. Highly expressed nuclear, membrane, and cytoplasmic markers were utilized in conjunc- tion to accurately determine cell boundaries in tissue images. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relation- ships between cells of different types. At the highest level, cells were classified as positive for markers that are indicative of immune and tumor cells based on measured intensities of marker expression, such as CD45+ and keratin+ cells in epi- thelial cancers, respectively. Co-expression of markers were used to classify immune cells into subsets, including T cells and macrophages (Figure 1). Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. Conclusions MIBI offers high-parameter capability, at sensitivity and reso- lution uniquely suited to understanding the complex tumor im- mune landscape, including the spatial relationship of immune and tumor cells and the expression of immunoregulatory proteins. Fig. 1 (abstract P50). Automated microfluidics-assisted multiplexing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 29 of 272 Conclusions Anti-ILT3 mAb treatment induced a conversion from intra- tumoral immune suppression to activation in a SK-MEL-5 hu- NSG tumor model. CyTOF in anti-ILT3 drug discovery holds promise to effect a paradigm-shift in our ability to under- stand MOA and evaluate the impact of therapeutic interven- tions that can accelerate biomarker discovery and drug development. The anti-ILT3 mAb activity in human immune cells in vitro and tumor efficacy in vivo is presented in a companion poster. Ethics Approval The study was approved by Merck Institutional Animal Care and Use Committee, approval number 2022-200518-FEB. P53 Pixelwise H-score: a novel digital image analysis-based metric to quantify membrane biomarker expression from IHC images Amy-Jackson Fisher, Pamela Whalen, Cory Painter, Pamela Vizcarra, Eric Powell, MD, Sripad Ram, PhD Pfizer, Inc., San Diego, CA, United States Correspondence: Sripad Ram (sripad.ram@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P53 Fig. 1 (abstract P51). Cell segmentation and classification Background Immunohistochemistry (IHC) assays play a central role in evaluat- ing biomarker expression in tissue sections for diagnostic and re- P52 search applications. Manual scoring of IHC images, which is the CyTOF in anti-ILT3 mAb drug discovery - humanized tumor model current standard of practice, is based on qualitative criteria and selection and in vivo PD/biomarker exploration are known to have several shortcomings in terms of reproduci- Yujie Qu, MD, Alan Byford, Caniga Michael, Ying Huo, Barbara Joyce- bility and scalability to large scale studies. While digital image Shaikh, BS, Laurence Fayadat-Dilman, Veronica Juan, Carl Mieczkowski, analysis (DIA) based approaches hold significant promise to over- Laura Bald, Jeanne Baker, Michael Meehl, Scott Pruitt, MD, PhD, Stephen come these limitations, current DIA methods pose several chal- Alves, Lily Moy, Philip Brandish, PhD, Jie Zhang-Hoover, Jie Zhang- lenges that have limited their widespread use in analyzing Hoover clinical samples. Merck, Boston, MA, United States Methods Correspondence: Jie Zhang-Hoover (jie.zhang-hoover@merck.com) We introduce a novel DIA metric, the pixelwise H-score (pix H- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P52 score), that quantifies biomarker expression from whole-slide scanned IHC images. Pix H-score is unique in that it does not rely Background on the detection of individual cells or the delineation of subcellu- ILT3 on human monocytic myeloid cells is linked to immune tol- lar compartments (e.g. nucleus and cell membrane) which are ne- erance in transplantation and immune suppression in cancer. cessary for traditional scoring algorithms such as the H-score. All Anti-ILT3 mAb is being developed as a cancer immunotherapy to DIA metrics are calculated using either commercially available reverse the suppression and increase T cell activation. To evaluate (HALO, Visiopharm) or open-source (QuPath) digital pathology the effect of an anti-ILT3 mAb in vivo, we sought to select an ap- software packages. propriate humanized tumor model and identify immune activa- Results tion signatures in the model that are associated with the We compute the pix H-score, the ATM score [1] and the trad- treatment efficacy. itional H-score [2] from IHC images for several biomarkers includ- Methods ing PD-L1. Our results show that the pix H-score exhibit tight Humanized tumor models were generated by subcutaneously concordance to multiple orthogonal measurements such as mRNA implanting Panc 08.13 or SK-MEL-5 tumor cells in NSG mice levels and pathologist score, and provide consistently better per- engrafted with human cord-blood CD34+ hematopoietic stem cells formance over other DIA metrics. (hu-NSG). Tumor-bearing mice were treated with a human-mouse Conclusions chimeric anti-ILT3 mAb. Single cell mass cytometry (CyTOF) that sim- We anticipate that the new metric introduced here will be ultaneously quantifies over 40 cell surface and intracellular markers broadly applicable to quantify biomarker expression from a was used to phenotype tumor infiltrating cells (TILs) in these tumor wide variety of IHC images. Although not shown here, the new models. metric can also be applied to immunofluorescence images. Results Moreover, these results underscore the benefit of digital image The CyTOF phenotyping of untreated mice showed an overall im- analysis-based approaches which offer an objective, reprodu- mune suppressive environment in the tumor in both Panc 08.13 and cible and highly scalable strategy to quantitatively analyze IHC SK-MEL-5 hu-NSG models. ILT3 expression was detected in both images. models. However, the levels of ILT3 expression on CD14+ myeloid cells and percentage of CD14+ myeloid cells among TILs were higher in SK-MEL-5 compared to Panc 08.13 tumors, which led to the selec- References tion of the SK-MEL-5 model for further exploration. Anti-ILT3 mAb 1. Choudhury KR, Yagle KJ, Swanson PE, Krohn KA, Rajendran JG, A Robust treatment in the SK-MEL-5 hu-NSG model increased activation of Automated Measure of Average Antibody Staining in CD14+ myeloid sub-populations in TILs by viSNE CyTOF clustering Immunohistochemistry Images. J Histochem Cytochem. 2010; 58: 96-107. analysis. Furthermore, the treatment increased levels of CD69 and 2. Hatanaka Y, Hashizume K, Nitta K, Kato T, Itoh I, Tani Y, Cytometrical HLA-DR expression on CD4+ T cells, while reducing the percentage image analysis for immunohistochemical hormone receptor status in of naïve CD4+ T suppressor cells in CD45+ TILs. breast carcinomas. Pathol Int. 2003; 53: 693-699. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 30 of 272 P54 P55 Development of a 9-color immunofluorescence assay using Combining the best of two worlds: Transfer of multiplex tyramide signal amplification and multispectral imaging for immunofluorescence images from non-small cell lung carcinoma patients high-throughput studies on FFPE tissue sections into pseudo multiplex chromogenic immunohistochemistry images 1 2 1 1 Bethany Remeniuk, PhD, Carla Coltharp, PhD, Kristin Roman, MS, Lorenz Rognoni, PhD ,Ana HidalgoSastre, PhD ,Linda Brützel , Philipp Wortmann , 1 1 1 3 Chichung Wang, Clifford Hoyt, MS Monika Baehner ,Marco Testori ,Jessica Chan , Bonnie Phillips, PhD , Katir Patel, 3 3 4 4 1 Akoya Biosciences, Hopkinton, MA, United States PhD , Sean Downing, PhD , Alex Haragan ,JohnField , Florian Leiss, PhD 1 2 3 Correspondence: Clifford Hoyt (choyt@akoyabio.com) Definiens AG, Munich, Germany; Definiens, Munich, Germany; Ultivue, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P54 Cambridge, MA, United States; Liverpool University Hospital, Liverpool, United Kingdom Background Correspondence: Ana Hidalgo Sastre (ahidalgo@definiens.com) In cancer research, advancing our understanding of the under- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P55 lying mechanisms driving disease progression is key to devel- oping new therapeutic regimens and improving patient Background outcomes. Over the past several years, multiplex immunofluor- One of the biggest challenges in multiplex chromogenic IHC (mIHC) is to escence (mIF) has played a vital role in elucidating novel accurately identify and quantify double positive cells. Multiplex immuno- immune-tumor interactions and identifying targets of interest fluorescence (mIF) instead, allows for visualization of plenty of biomarkers for drug discovery and development. at once with true co-localization. However, visualizing tissue morphology Emerging studies utilizing mIF have revealed complex cell-to- in mIF images can be challenging and the vast color combinations over- cell interactions within the tumor microenvironment (TME), whelming. Pathologists are key to retrieve biological information from however, greater interrogation of the biology comprising multiplex assays and provide annotations for assay validation. To support these interactions, including cellular composition and pathologist analysis, resections of non-smallcelllungcarcinoma (NSCLC) functional status, require higher levels of multiplexing. With patients were stained with mIF and displayed as pseudo mIHC images. the rapidly increasing number of available multiplexing ap- Additionally, consecutive slides were stained with a mIHC panel. PD-L1 proaches, there is an inherent tradeoff between capability positive macrophages from the pseudo mIHC images were quantified and throughput. and compared to the readouts identified in the real chromogenic IHC. In this study, we demonstrate a streamlined workflow to Methods develop and optimize a 9-color assay on the Leica BOND RX™ 7 formalin-fixed paraffin-embedded (FFPE) resections from NSCLC patients autostainer. This methodology offers an optimal balance be- were stained using Ultivue’s UltiMapper I/O PD-L1 kit and I/O PD-1 kit and tween multiplexing and sample throughput to facilitate research whole image scans were acquired with a Zeiss Axio Scan.Z1 scanner (Zeiss). and support translational studies on whole formalin-fixed Consecutive slides were stained with a multiplex chromogenic panel (in- paraffin-embedded (FFPE) tissue. cluding CD68, CD8, PD-1) at Mosaic Laboratories (1) and scanned with an Aperio AT Turbo scanner (Leica). Images were analyzed using an automated Methods workflow for quantitative multiplex image analysis developed at Definiens Forthe9-colorassay,Opal™ fluorophores were used on serial (2). Afterwards, mIF images were converted into pseudo mIHC images. Pa- sections of lung cancer FFPE tissue. The panel was designed on thologists annotated double positive macrophages for CD68 and PD-L1 on Akoya’s Mantra 2 semi-automated multispectral microscope, both images. Results were compared with automatically detected double which allows for rapid analysis of staining performance. Once positive cells and across assays. In addition, pathologists qualitatively optimized, multispectral images were acquired on both the assessed visual similarity of real and artificial chromogenic images. Mantra 2 and Vectra Polaris of the same tissue regions and an- Results alyzed to show equivalence between the platforms. Cell counts, Pathologists annotated double positive macrophages for CD68 and densities, and spatial parameters were generated using Akoya’s PD-L1 markers on both images (mIF and pseudo mIHC). Results were inForm imageanalysissoftwareand theRscript package compared with those obtained using artificial intelligence to auto- phenoptrReports, which produces quick, summarized outputs of matically detect double positive cells and across assays. In addition, the image analysis data. These same analyses were also used pathologists qualitatively assessed visual similarity of real and artifi- to evaluate reproducibility of all markers when run in a high- cial chromogenic images (Figure 1). throughput process. Conclusions Results Transferring mIF into pseudo mIHC images helps to combine the advan- Dynamic range of measured per cell signals for all markers had tages from both approaches: true colocalization of biomarkers whilst main- a median of 200:1. Agreement between the Mantra 2 and taining tissue morphology, facilitating visual evaluation of digital images Vectra Polaris-based measurements was generally >95% when by pathologists. This technology could be used to complement research, comparing cellular expression signals and cell counts based on clinical routine diagnostic, drug development and biomarker discovery. cell phenotyping classifiers. Cross talk was undetectable after spectral unmixing despite significant spectral overlap inherent References in a 9-color assay. Reproducibility across three batches of five (1) Lisa M. Dauffenbach, Christopher A. Kerfoot, et al. Characterization of serial sections of lung cancer tissue was generally <10% coeffi- inflammatory cell patterns and densities using multiplex immunohistochemistry cient of variation for all markers in the assay, supporting a immuno-oncology assays [abstract]. In: Proceedings of the AACR-NCI-EORTC high-through process of approximately 20 resection samples per International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct day. 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Conclusions Suppl): Abstract nr B069. We have successfully established a standardized process for 9- (2) Lorenz Rognoni, PhD; Vinay Pawar, PhD; Tze Heng Tanet, et al. Automated color multiplexing that offers a balance between elucidating the quantification of whole-slide multispectral immunofluorescence images to intricate cellular biology driving disease progression and thera- identify spatial expression patterns in the lung cancer microenvironment. peutic responsiveness within the TME while simultaneously SITC Annual Meeting; 2018 Nov 7-11; Washington, DC. Poster nr P442. providing a practical and reliable assay that can be imple- Ethics Approval mented to support translational, high-throughput studies in clin- Ethical approval was granted by the Liverpool Research Ethics Committee, ical research. reference number 97/141. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 31 of 272 using matched 3,3′-Diaminobenzidine chromogenic and single bio- marker fluorescent controls tonsil tissue, which were validated using tumor samples from patients with high grade glioma. We characterized the spatial arrangement of myeloid subpopulations, ex vivo and corre- lated the changes in spatial orientation, quantity and localization of cells to the tumor. We determined that dynamic re-arrangement of myeloid cells can be observed under pressure of immunotherapy within the tumor, and confirm both a time-dependent and dose- dependent effect of oHSV-1 on this immune cell modulation. Conclusions These data suggest a unique, multiplexed approach to study spatial ar- rangement of myeloid and T-cell populations and their spatial distribu- tion within tumors under basal growth conditions or in the presence of anticancer immunotherapies, which may implicate the activity of mye- loid cells with treatment responses. These findings could impact per- sonalized cancer immunotherapy for patients receiving care. Ethics Approval The samples were collected under IRB approval. Consent The samples were collected under written patient consent for publi- cation of this abstract. P57 An ex-vivo human system elucidates a role for natural killer cells in the anticancer effect of drug combinations in triple negative breast cancer Fig. 1 (abstract P55). mIF and pseudo mIHC 1 2 2 Aaron Goldman , Douglas Best , Saravanan Thiyagarajan , Misti Jain, 2 2 1 2 PhD , Basavaraja Shanthappa , Munisha Smalley, PhD , Hans Gertje, BS , Aaron Goldman P56 1 2 Brigham and Women's Hospital; Mitra Biotech, Woburn, MA, United States Interrogating the effect of oncolytic Herpes simplex virus-1 on Correspondence: Aaron Goldman (goldman1@mit.edu) spatial arrangement of myeloid cells in glioblastoma multiforme Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P57 using an ex vivo human system and multiplex immunohistochemistry 1 2 2 Background Munisha Smalley, PhD , Misti Jain, PhD , Saravanan Thyiagarajan , Emily 3 3 4 2 Response and resistance to cancer therapy relies on the presence of ac- Alonzo , Katherine Crosby , Douglas Best , Hans Gertje, BS , Basavaraja 2 5 5 1 tive immune cells in the tumor microenvironment, which recalibrate Shanthappa , Ralph Pulchalski , Charles Cobbs , E. Antonio Chiocca , 1 1 1 the body’s own defense largely by modulating exhaustion of cytotoxic Sean Lawler, PhD , Aaron Goldman , Munisha Smalley 1 2 lymphocytes including T cells and natural killer (NK) cells. However, Brigham and Women's Hospital, Woburn, MA, United States; Mitra there is a critical gap in our understanding for the role of immune cells Biotech RxDx, Bangalore, India; Cell Signaling Technologies, Danvers, to drive response or resistance to drugs and immunotherapies at the MA, United States; University of Birmingham, Birmingham, United individual patient level. This is primarily due to limitations in complex Kingdom; Swedish Neuroscience Institute, Seattle, WA, United States tumor-immune interfaces that exist in many current tumor models. Correspondence: Aaron Goldman (goldman1@mit.edu) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P56 Here, we deployed an ex-vivo human system that uses an explant of native, patient-derived solid tumors including autologous immune cells. Background Utilizing biopsied tumor tissue from patients diagnosed with triple- The role of myeloid cell populations within a tumor and their contribu- negative (ER- PR- HER2-) breast cancers (TNBC, N=7), we studied drug- tion to effective cancer immunotherapy is emerging with considerable induced cell death (cleaved caspase-3) and spatial heterogeneity of NK interest. However, assessing the role of intratumoral myeloid cells cells (CD3-CD56+PanCK-) using multiplex immunohistochemistry under therapy pressure has it’s challenges. Here, we implemented a (mIHC). Spatial orientation of cells in the microenvironment, including multiplex immunohistochemistry (mIHC) panel and a human ex vivo proximity of NK to tumor and NK cell density within regions of the system to interrogate key myeloid subsets as they affiliate with infiltra- tumor vs. stroma were performed using HALO-based quantitative ana- tion and activation of an emerging immunotherapy for glioblastoma lyses. Finally, we deployed in-vitro co-culture studies using 3-D TNBC multiforme (GBM) – oncolytic Herpes simplex virus-1 (oHSV-1). mIHC organoids and human-derived NK cells (NK-92MI). combined with advancements in digital pathology and machine learn- Results ing algorithms have enabled identification, quantification and spatial First, we report the ability of the ex-vivo human system to retain the orientation of multiple cell types in a single field of view (FOV). spatial orientation and total population of natural killer cells and T- Methods cells over the course of a 72h explant culture. Next, using Spearman Cell Signaling Technology antibodies (CD3e,D7A6E™, ID: 85061), (CD68, correlation analyses and principal component analysis (PCA), we de- D4B9C, ID: 76437), (CD11c, D3V1E, 49420), (MHC Class II (HLA-DRB) LGII- termined that drug response to both immunotherapy and conven- 612.14), (Pan-Keratin, C11, 4545) were optimized for mIHC staining, tional cancer drugs, indicated by high incidence of cleaved caspase-3 using a tyramide signal amplification approach to pin-point, in a single after drug pressure, is directly associated with changes to the tumor- FOV: intratumoral T-cells, defined by CD3e+; macrophages, defined by NK cell proximity and density of NK cells within the tumor bed vs. CD68 and conventional dendritic cells defined by CD11c+MHCII +, in the stroma. Finally, using the 3-D tumor organoid cultures with NK relation to the surrounding tissue architecture defined by pan cytokera- cells, we determined that activity and tumor cytolysis by NK cells is tin. These biomarkers were integrated with incidences of oHSV-1 infil- hampered through cancer cell-activated cytokines, which diminish tration and replication (via expression of green fluorescent protein). expression of activating biomarkers including NKG2D/C. Results Conclusions First, we confirmed an optimized protocol for treating GBM ex vivo Taken together, these results provide a method to study the spatial with oHSV-1 such that tissue viability, infiltration and replication of arrangement of immune cells in an entirely human system, which the virus are optimal. The staining of the mIHC panel was optimized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 32 of 272 can be perturbed with anticancer drugs to reliably influence the ex- P59 pression and growth patterns of immune cells. We further demon- Highly consistent automated multiplex immunofluorescence for strate that this strategy can help to guide in-vitro studies to further immunoprofiling of solid tumors in clinical trials: assay validation elucidate mechanisms of action of drugs, which influence response study using multispectral imaging and digital analysis 1 2 2 vs. resistance via immune cell activity. Michael Surace, PhD , Lorenz Rognoni, PhD , Farzad Sekhavati , Andrew 2 2 2 1 Ethics Approval Fisher, PhD , Andreas Spitzmueller , Sara Batelli, PhD , Karma Dacosta , 2 3 4 Anonymous breast cancer tissue samples were collected under IRB Vinay Pawar , Clifford Hoyt , Edwin Parra, MD, PhD , Jaime Rodriguez- approval with due written consent from each patient. Canales, MD 1 2 AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, 3 4 Germany; Akoya Biosciences, Hopkinton, MA, United States; UT - MD P58 Anderson Cancer Center, Houston, TX, United States Brain MRI performed within 4 weeks of PD-1 inhibitors as a Correspondence: Jaime Rodriguez-Canales potential prognostic marker for non-small cell lung cancer (rodriguezcanalesj@medimmune.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P59 Ammar Sukari, MD, Misako Nagasaka, MD, Seongho Kim, PhD, Tahmida Chowdhury, Natasha Robinette, MD Background Karmanos Cancer Institute, Detroit, MI, United States Novel multiplex immunofluorescent (mIF) platforms have been devel- Correspondence: Ammar Sukari (sukaria@karmanos.org) oped for immunoprofiling of solid tumors to understand the tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P58 microenvironment and to identify biomarkers for immunotherapy. One of these methods employ IF and tyramide signal amplification (TSA) to Background generate between 4 to 9-marker multiplex panels analyzed with multi- PD-1 inhibitors aim to re-instate the natural anti-cancer immune- spectral imaging. Although this method can provide reliable data, they mediated cytotoxicity. Although PD-1 inhibitors are now consid- can show variability in consistency depending on the markers [1], ered part of standard of care treatment in advanced metastatic which affects the reliability of mIF for use in clinical trials. The goal of NSCLC [1], little is known about the effects of PD-1 inhibitors on this study was to develop and validate a highly consistent mIF method asymptomatic central nervous system (CNS) metastases. We hy- for its use in clinical trials in the pharma and academic environments. pothesized that early MRI brain imaging due to the development Methods of neurological signs and symptoms following the initiation of A mIF panel for the analysis of carcinomas was optimized using an PD-1 inhibitor may help delineate a subset of NSCLC patients automated stainer (Leica) and automated multispectral scanner (Po- with asymptomatic and undiagnosed CNS metastases prior to ini- laris, Akoya Biosciences). The markers included keratins (AE1/AE3), tiation of therapy and may predict for worse outcomes. CD68, PD-L1, PD1, CD8 and Ki67. Each primary antibody was first per- Methods formed on standard chromogenic IHC according to previously vali- Data from NSCLC patients who received at least one dose of PD- dated protocols. The mIF panel was developed with a secondary 1 inhibitors between September 2013 through the data cut-off of antibody detection and TSA, using specific fluorophores for each May 2017 were captured from our institution’s pharmacy data- marker. Once optimized, the miF panel was tested on serial sections base. The primary objective was to describe the characteristics of of formalin-fixed human tonsil controls and six non-small cell lung patients with MRI brain being performed within 4 weeks of the carcinomas, including replicates, together with standard IHC staining first dose of PD-1 inhibitors and the secondary objectives were of each individual marker for comparison. A set of three repeats on estimation of progression free survival (PFS) and overall survival different days for each mIF with all cases was performed to test (OS) for the same population. consistency and reproducibility of the mIF method. All slides were Results digitally scanned and analyzed using Automated Definiens Insights 140 NSCLC patients received at least one dose of PD-1 inhibitors Platform with custom algorithms (Definiens AG, Munich, Germany), prior to data cut-off. Median age was 64 (range: 24-86). 83 (59%) comparing the cell populations in the serial section slides between were male. 64 (46%) were treated on a clinical trial. There were standard IHC and mIF, and between mIF repeated rounds. The data 92 (66%) adenocarcinoma, 41 (29%) squamous cell carcinoma was statistically analyzed using Pearson’s correlations. (SCC) and 7 (5%) poorly differentiated NSCLC. 84 (40%) had a Results pre-PD1 inhibitor MRI brain performed and 25 (18%) had been Using an automated workflow, the mIF data compared with standard diagnosed with baseline CNS metastases. 128 (91%; Group 1) did IHC showed correlations between 0.83 to 0.99. The data from the not have an MRI brain performed within 4 weeks of starting PD-1 three rounds of mIF performed on different days showed correlations inhibitors, while 12 (9%; Group 2) patients did. 9 out of 12 pa- between 0.89 and 0.99. The marker that showed the lowest correl- tients had new or worsening CNS metastases. Of the 9, 1 had ation was CD68 (r=0.83), the possible cause was the difficulties on WBRT, 1 had gamma knife, 1 went onto hospice while a decision cell segmentation due to morphologic irregularity shown by macro- was made to monitor imaging and symptoms in 6 patients. The phages. Overall our present data showed a much higher consistency median PFS was 5.28 months (95% CI, 3.90 to 8.03) and 1.75 than our previously published results using a non-automated mIF months (95% CI, 1.08 to NE) for Group 1 and Group 2, respect- protocol, which correlations ranged from 0.17 to 0.87[1]. ively. The median OS was not reached (95% CI, 15.38 to NE) and Conclusions 5.77 months (95% CI, 2.85 to NE) for Group 1 and Group 2, Our data demonstrates that using an automated workflow including respectively. automated staining, scanning and analysis, and with properly vali- Conclusions dated IHC markers, mIF becomes a highly consistent methodology In this retrospective analysis, patients who had MRI brain within and it is compatible for its use with clinical trial tissue specimens. 4 weeks of starting PD-1 inhibitors had worse outcomes. Trial Registration Not applicable Reference 1. NCCN Clinical Practice Guidelines in Oncology. Non-Small Cell Lung Can- Reference cer. Version 5. 2019- June 7, 2019. https://www.nccn.org/professionals/ 1. Parra ER, Uraoka N, Jiang M, Cook P, Gibbons D, Forget MA, Bernatchez physician_gls/default.aspx#site, last accessed 7/30/2019. C, Haymaker C, Wistuba II, Rodriguez-Canales J. Validation of multiplex Ethics Approval immunofluorescence panels using multispectral microscopy for immune- The study was approved by the Wayne State University Institution's Ethics profiling of formalin-fixed and paraffin-embedded human tumor tissues. Board, approval number 062616M1E. Sci Rep. 2017; 7:13380-13391. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 33 of 272 P60 P61 Combining transcriptomic immune population inference with Segmentation and classification of single cells using multiplexed automated digital masking of H&E images finds immune effectors ion beam imaging preferentially distribute within stroma regions Jay Tarolli, Rachel Finck, Murat Aksoy, Yari Sigal, Noah Newgren, Jessica 1 2 2 Christopher Szeto, PhD , Mustafa Jaber, PhD , Liudmila Beziaeva , Kevin Finn, Jason Ptacek, PhD 1 1 1 Kazmierczak , Steve Benz , Shahrooz Rabizadeh IONpath, Menlo Park, CA, United States 1 2 ImmunityBio, Santa Cruz, CA, United States; NantOmics, Culver City, Correspondence: Jay Tarolli (jay@ionpath.com) CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P61 Correspondence: Steve Benz (Steve.Benz@nantomics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P60 Background When studying the tumor microenvironment, knowing not only Background the types of immune cells present but also the spatial distribu- Multiple methods to characterize immune-cell populations in tion and relationship of these immune cells to other immune and tumor microenvironment (TME) are being assessed as potential tumor cells provides crucial information. In the past, techniques biomarkers of immunotherapy response. These include manual used to analyze these spatial relationships have been limited by pathological assessment of lymphocyte infiltration, immunohisto- the number of biomarkers that could be simultaneously mea- chemical (IHC) staining for specific adaptive response markers sured. Recently, with the development of multiplexed ion beam such as CD8, and more recently transcriptomic-based deconvolu- imaging (MIBI), 40+ biomarkers can be simultaneously measured tions of immune populations such as xCell and TIMER. Here we in a single scan [1]. By probing with an ion beam, tissue sections combined digital masking using deep-neural nets with transcrip- can be imaged at a spatial resolution on the same order of mag- tomic deconvolution to infer where immune-subpopulations may nitude as light based techniques, providing subcellular resolution. reside in the TME. This combination of multiplexed biomarker measurements and Methods subcellular spatial resolution enables segmentation of the image An unselected set of 187 clinical samples from the ImmunityBio data- into individual cells, making possible subsequent cell type classifi- base were analyzed. Each had H&E stained diagnostic slides with cation and quantification. pathologist-annotated tumor regions, as well as deep whole- Methods transcriptomic sequencing (>200M reads). Deep neural networks pre- Samples of placenta, lung, tonsil, lymph node, thymus, and liver viously trained on TCGA slide images were used to generate digital were imaged with MIBI. Segmentation of these images was per- spatial masks for 3 characteristics: tumor-content, lymphocytes, and formed in two steps. First, a MaskRCNN [2] model was trained stroma. Patients were scored based on the presence of intratumoral to utilize multiplexed MIBI data to predict the location of cell lymphocytes (iTIL) and stromal lymphocytes (sTILs). Immune subpop- instances in a MIBI image for a single class of objects by learn- ulations were inferred from RNAseq expression of published ing features from a set of nuclear, cytoplasmic, and membrane immune-cell-specific genesets [1,2], as was Wnt-signaling level [3]. markers. The centroids of each predicted cell instance were Significant associations between immune subpopulations and level used as seed points and, after manual refinement of these seed of infiltration were analyzed. points, watershed segmentation was performed to determine Results boundaries between instances. Both the summed intensity of a Manually annotated positive tumor regions were accurately digitally marker as well as a weighted cell score which accounts for the masked as >83% tumor or lymphocyte. Wnt signaling was strongly spatial distribution of a marker’s expression throughout a cell associated with overall stromal content (Rho=0.47, p<0.0001). Strong instance were calculated and were used for cell type anti-correlation was observed between levels of sTILs and iTILs classification. (Rho=-0.42, p<0.0001), and remained significant when including Results overall stroma area as a covariate. Digital lymphocyte masks some- Cell population and densities were calculated for a number of what correlated with RNAseq-based deconvolution of lymphocyte different cell types, including T cells, B cells, and macrophages classes (Rho=0.30, p=0.0001) in line with reports from others [4], based on a combination of one or more coexpressed biomarkers however this decreased when comparing lymphocyte count within present within segmented cells. Figure 1 shows an example FOV annotated tumor regions only (Rho=0.17, p=0.03), despite high con- with several cell types classified in a single image. Expression of cordance of lymphocyte counts within and outside of annotated re- immunoregulatory proteins including PD-1 and PD-L1 were quan- gions overall (Rho=0.82, p<0.0001). RNAseq-based lymphocyte levels tified and assigned to specific cell types. Finally, nearest neighbor were more associated with sTILs than iTILs (Rho=0.19 vs. -0.28, p< distances between various cell types were determined to 0.01 respectively). characterize the spatial organization of cell populations within Conclusions each tissue image. Adaptive response effectors such as NK and T-cells are found Conclusions more resident in surrounding stromal tissue than infiltrating The ability to characterize the many different cell types within tumor tissue. Increased Wnt/B-catenin signaling in stromal re- the tumor microenvironment is made possible by the highly mul- gions, reported by others as immunosuppressive, may sequester tiplexed nature of MIBI data, the subcellular spatial resolution of immune effectors and aid in immune escape. the image data, and downstream analysis tools, including com- puter vision approaches, which enable cell segmentation, classifi- References cation, and spatial analysis. 1. Bindea G, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 2013; 39:782-795. References 2. Danaher P, et al. Gene expression markers of tumor infiltrating 1. Keren L, Bosse M, Marquez D, Angoshtari R, Jain S, Varma S, Yang S, leukocytes. J Immunother Cancer. 2017; 5:18. Kurian A, Van Valen D, West R, Bendall S, Angelo M. A Structured 3. Slattery ML,et al. Expression of Wnt-signaling pathway genes and Tumor-Immune Microenvironment in Triple Negative Breast Cancer their associations with miRNAs in colorectal cancer. Oncotarget. 2018; Revealed by Multiplexed Ion Beam Imaging. Cell. 2018; 174:1373- 9:6075. 1387. 4. Pai SG, et al. Wnt/beta-catenin pathway: modulating anticancer immune 2. He K, Gkioxari G, Dollár P, Girshick R. Mask R-CNN. IEEE International Con- response. J Hematol Oncol. 2017; 10:101. ference on Computer Vision (ICCV). 2017; 2961-2969. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 34 of 272 combinations for a seven-color panel. Using this method, the number of slides was reduced to three per target (18) plus con- firmation seven-color slides resulting in a panel containing CD3, CD8, CD68, Cytokeratin, FOXP3 and PD-L1 (Figure 2). Conclusions Multiplex IF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interaction/co-localization of multiple targets within a single tissue specimen. MIF panels can take eight or more weeks to optimize, however, researchers can save time and resources using validated antibodies and this antibody order guide. Fig. 1 (abstract P62). See text for description Fig. 1 (abstract P61). See text for description Table 1 (abstract P62). See text for description P62 Bringing the tumor microenvironment into focus: Simplified development of seven-color multiplex immunohistochemistry- immunofluorescence (mIF) panels Melissa Whiteman, PhD, Eric McIntush, PhD, Mike Spencer Bethyl Laboratories, Montgomery, TX, United States Correspondence: Mike Spencer (mspencer@bethyl.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P62 Background For the advancement of immunotherapeutics, the need to under- stand the tumor microenvironment has never been more press- ing. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers. This is critical in instances where sample is limited, such as a tumor bi- opsy or other clinical specimen. The applications of mIF are nu- merous, and span clinical, translational, and basic research applications. A seven-color mIF can take eight weeks, or more, to develop. Herein, we describe a simplified, faster approach. Methods FFPE human tissue was stained with PathPlex™ Panel 4 IHC vali- dated primary antibodies (Bethyl Laboratories [A810-004]), mouse or rabbit HRP-conjugated secondary antibodies (Bethyl Laborator- ies [A90-116P, A120-501P]) and detected using Opal™ Polaris 7- color IHC kit fluorophores (Perkin Elmer [NEL861001KT]). Primary antibody order was optimized utilizing tissue microarray serial sections, and three slides per target by staining after the first, third, or sixth heat-induced epitope retrieval (HIER). All three slides were imaged using the same exposure time and analyzed Fig. 2 (abstract P62). See text for description for target/nucleus counts, signal intensity, and background. Fi- nally, the order was tested in the seven-color mIF and compared to single stain for confirmation. Whole slide scans were gener- ated using the Vectra Polaris® and analyzed using InForm® image analysis package. P63 Results Clinical assay development and validation of multiplex Development time of a seven-color mIF was reduced using IHC immunofluorescent (mIHC) marker panel for evaluation of tumor validated antibodies and the optimized dilution. Antibody order infiltration myeloid cells in FFPE tissue sections 1 1 2 was guided by results of three slides stained after first, third or Lan Yi, PhD , Jonathan Juco, MD , Ashhad Mahmood, MD , Omar 1 1 1 sixth HEIR. The ratio of target staining/DAPI nuclear counts, aver- Laterza , Charo Garrido , Lan Yi, PhD 1 2 age intensity and overall background predicts the optimal order Merck & Co., Inc, Hillsborough, NJ, United States; Diagnostic Pathology of staining. Some targets reveal larger average area staining, Services, LLC, Skillman, NJ, United States higher intensity and lower background when stained last, for ex- Correspondence: Omar Laterza (omar_laterza@merck.com); Charo ample FOXP3 (Table 1, Figure 1), while the inverse may be true, Garrido (charo.garrido@merck.com) or no effect for other targets. There are 720 possible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P63 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 35 of 272 Background staining pattern of more than 20 parameters. The high-parameter anti- Myeloid-derived suppressor cells (MDSCs) are a group of leuco- body panel used was optimized with human FFPE tonsil tissues. Human cytes with myeloid origin and immune-suppressive function. lung cancer FFPE tissues were then analyzed with this same panel. Cell Ample recent evidence supports key contributions of MDSC to clustering using the MAV software identified tens of cell types within tumor progression through immune-mediated mechanisms. the tumor tissues. Immune cell sub-types were mapped onto the ori- MDSCs include two major subsets based on their phenotypic and ginal image data to assess infiltration and spatial associations. morphological features: polymorphonuclear (PMN)-MDSC and Conclusions monocytic (M)-MDSC. However, these cells remain less studied Unlike other cyclic IF approaches involving multiple antibody staining than T lymphocytes as their phenotypical, morphological and and stripping steps, the CODEX platform involves a single initial functional heterogeneity generate confusion in investigation and staining step and subsequent gentle and relatively fast manipulation analysis of their role. of the tissue thereafter. This provides a superior workflow and pre- Methods vents tissue degradation. CODEX data from various normal and can- With the progresses on multiplex IHC assay technology, we are now cer human FFPE tissue types is shown here with corresponding able to develop multiplex immunofluorescent marker panels to single-cell analysis of key tissue features. Overall, the CODEX platform evaluate the expression and localization of the main subpopulations is an accessible and versatile technology for high parameter, spatial of myeloid cells in the tumor microenvironment. profiling of tissue specimens. Results We have developed and validated CD14, CD66b, CD163 and P65 MHCII (HLA-DR) IHC multiplex marker panel to evaluate the main Mutation-targeted T cell responses in blood from patients with populations of myeloid cells, along with their activation status. solid tumors prior to treatment and which evolve with clinical After completing the validation for individual markers in a single benefit from anti-PD-1 therapies chromogenic IHC platform, we optimized the incorporation of 1 2 1 1 Benjamin Yuen, PhD , Fangfang Yin, PhD , Duo An, PhD , Boi Quach , each marker into the multiplex platform. In parallel, a multiplex 1 2 1 Linlin Guo, PhD , Joanne Tan, PhD , Songming Peng, PhD , Zheng Pan, image analysis algorithm (APP) was generated and validated to 1 1 1 1 PhD , Olivier Dalmas, PhD , Robert Bao, PhD , Kyle Jacoby, PhD , Barbara quantify each subpopulation of myeloid cells in the tumor area. 1 1 2 Sennino, PhD , Stefanie Mandl, PhD , Matt Walters, PhD , Juan Jaen, Last, fit for purpose analytical validation, including sensitivity, spe- 2 1 1 PhD , Alex Franzusoff, PhD , Benjamin Yuen, PhD cificity and precision was successfully carried out. 1 2 PACT Pharma, South San Francisco, CA, United States; Arcus Conclusions Biosciences, Hayward, CA, United States This validated assay is currently being used to support multiple on- Correspondence: Songming Peng (speng@pactpharma.com) going and future clinical trials. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P65 P64 Background Highly multiplexed single-cell spatial analysis of FFPE tumor T cells targeting tumor-exclusive neoepitopes (neoE) have been pos- tissues using CODEX® tulated to represent the primary mediators of clinical benefit for pa- Jessica Yuan, PhD, Gajalakshmi Dakshinamoorthy, Joseph Kim, Sejal tients with solid tumors treated with immunotherapies. Identifying Mistry, Nadya Nikulina, Roya Bashier, Cassandra Hempel, Maria Elena and tracking these T cells in patients can help to understand the Gallina, Julia Kennedy-Darling, Jessica Yuan, PhD mechanism for immune checkpoint inhibitor therapies, as well as Akoya Biosciences, Menlo Park, CA, United States provide new therapeutic candidates for personalized adoptive cell Correspondence: Julia Kennedy-Darling (j.kennedy@akoyabio.com) therapies. However, this has been hampered by the low frequency of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P64 neoE-specific T cells in peripheral blood. To this end, we demonstrate the use of the imPACT Isolation Technology®, an ultra-sensitive high- Background throughput technology, to capture neoE-specific CD8 T (neoE-T) cells Characterizing the complexities of the tumor microenvironment is elem- from peripheral blood. In addition, this technology can be utilized to ental to understanding disease mechanisms. The spatial relationships be- quantify and monitor neoE-T cells longitudinally during therapy. We tween infiltrating immune cells and the remodeling of the cellular matrix show here preliminary data applying the imPACT technology to clin- is widely recognized as a key component to defining tumor heterogen- ical trial samples for the characterization of mutation-targeted T cell eity. Current methodologies for analyzing the spatial dimension in tissues, responses from patients associated with clinical benefit. like traditional immunofluorescence (IF) and immunohistochemistry (IHC), Methods are limited to a few parameters at a time, restricting the scope of identifi- Peripheral blood mononuclear cells (PBMC) from patients with non- able cells. Conversely, single-cell technologies like mass cytometry and small cell lung cancer (NSCLC) and treated with combinations con- NGS-based tools provide multiplexing capabilities, but at the expense of taining an anti-PD-1 antibody were analyzed. Briefly, tumor-exclusive the associated spatial information. Here, we present the analysis of hu- neoE-HLA target candidates were predicted and barcoded snare li- man lung cancer FFPE tissues with CODEX using a panel of more than 20 braries comprising personalized neoE-HLA reagents were produced markers targeting the tumor microenvironment. for capture of neoE-specific CD8+ T cells from PBMCs. Longitudinal Methods analysis of neoE-T cells responses throughout the duration of treat- The CODEX technology, developed by Akoya, is comprised of a fluidics ment was performed to obtain valuable information on neoTCR se- instrument that interfaces with existing microscope hardware, as well as quences and neoE-T cell quantification & phenotype. a suite of reagents and associated control and analysis software. The Results CODEX technology involves labeling antibodies with oligonucleotide- A baseline neoE-specific CD8 T cell profile was identified in all sub- based Barcodes followed by a single staining step. Around 40 parameters jects prior to treatment. Among NSCLC subjects exhibiting objective can be measured within a single tissue through fully-automated, iterative responses to therapy, some neoE-T cell clones identified at baseline cycles of adding and removing corresponding dye-conjugated Reporters. persist in the blood and/or diversify in clonality over the course of Here, we apply this technology using a panel of antibodies targeting im- treatment. In some circumstances, new neoE-T cell clones have mune, cancer and other architectural features to measure cell subsets in emerged on treatment with anti-PD-1. Furthermore, phenotype ana- cancer FFPE tissues. Image data is processed using the CODEX analysis lysis suggested the neoE-T cells captured from blood have been acti- pipeline, including clustering, annotation and mapping of cell types to vated, indicating previous encounter with their respective neoE-HLA the original image data with the Multiplexed Analysis Viewer (MAV). targets. Results Conclusions The CODEX technology was used to ascertain complex cellular niches The imPACT technology was used to assess the phenotype & quan- and spatial associations between multiple cell types based on the tity of neoE-specific T cells in blood of trial participants over time. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 36 of 272 This approach revealed the evolution of mutation-targeted T cell re- Conclusions sponses in participants with clinical benefit and may prove to be a Future plans include analyzing the remainder of patients enrolled in powerful tool to provide mechanistic understanding of immune re- the trial and the utilization of CD8+ and MDSC-specific CyTOF panels sponses associated with clinical benefit. These data support further to further classify the aforementioned subpopulations to further elu- testing of the neoE-T cell capture technology, with the potential to cidate this relationship. This methodology offers insight into the pro- uncover the identity of neoE-specific T cells pre-existing in the blood gression of vaccine-induced patient immune responses and exhibits of patients and the evolution of immune attack to cancer. promise as a platform that may be extrapolated to other Ethics Approval immunotherapies. The study was approved by the institutional review boards or ethics committees of the participating sites in Arcus Biosciences’ clinical References studies. 1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW. The 2016 World Health Organization Classification of Tumors of the Cen- P66 tral Nervous System: A summary. Acta Neuropathol. 2016; 131:803– A novel mass cytometry-based immunomonitoring platform for characterizing the peptide vaccine-induced immune response of 2. Chheda Z, Kohanbash G, Okada K, Jahan N, Sidney J, Pecoraro M, Yang X, HLA-A*0201+ patients with K27M+ diffuse midline gliomas Carrera D, Downey K, Shrivastav S, Liu S, Lin Y, Lagisetti C, Chuntova P, 1 1 1 Jared Taitt, BA , Payal Watchmaker, PhD , Takahide Nejo, MD, PhD , Neil Watchmaker P, Mueller S, Pollack I, Rajalingam R, Carcaboso A, Mann M, 2 1 1 Almeida , Kaori Okada , Sabine Mueller, MD PhD , Hideho Okada, MD, Sette A, Garcia C, Hou Y, Okada H. Novel and shared neoantigen derived 1 1 PhD , Jared Taitt, BA from histone 3 variant H3.3K27M mutation for glioma T cell therapy. J University Of California, San Francisco, San Francisco, CA, United States; Exp Med. 2017; 215:141-157. George Washington University, Washington DC, United States Ethics Approval Correspondence: Sabine Mueller (sabine.mueller@ucsf.edu); Hideho The study was approved by UCSF IRB #: 16-20574 Okada (hideho.okada@ucsf.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P66 P67 Background Use of a regional integrated health record data network to identify Diffuse midline glioma, including diffuse intrinsic pontine glioma patients who received checkpoint therapy following cancer (DIPG) constitutes up to 20% of pediatric brain cancer and has a me- diagnosis as a foundation for exploring immunotoxic events dian survival of 9-10 months. Given the proximity of DIPG to paren- Theresa Walunas, PhD, Carlos Galvez, Saya Jacob, Jeffrey Sosman, MD, chymal regions that play vital homeostatic functions, surgical Abel Kho resections are often restricted in size and scope, leaving irradiation Northwestern University, Chicago, IL, United States and chemotherapy as the primary management options. The on- Correspondence: Theresa Walunas (t-walunas@northwestern.edu) going development of immunotherapy has shown significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P67 promise in many fields, including that of gliomas. Genetic studies revealed that greater than 70% of DIPG cases harbor an amino Background acid substitution from lysine (K) to methionine (M) at position 27 Immune related adverse events (irAE) occur in >80% of patients of histone 3 variant 3 (H3.3). We previously identified a novel receiving immune checkpoint inhibitors (ICI). Currently, most HLA-A*02:01-restricted neoantigen epitope encompassing the data about the incidence of irAE comes from clinical trials with H3.3K27M mutation. Accordingly, we have implemented a pilot restrictive eligibility requirements. With the wide use of ICI ther- vaccine through the Pacific Pediatric Neuro-Oncology Consortium apy as standard of care for many cancers, it is important to as- (PNOC). sess incidence of irAE in a general patient population. The Methods Chicago Area Patient Reported Outcomes Research Network Twenty-nine newly diagnosed DIPG patients who are HLA-A2+ and (CAPriCORN) is a clinical data research network containing med- H3.3K27M+ underwent radiation therapy, and then received the ical records for >9.5M patients who receive care in 11 institu- H3.3K27M peptide vaccine and tetanus toxoid (TT) peptide emulsified in tions spanning diverse patient populations and healthcare Montanide in combination with poly-ICLC every 3 weeks for a total of 24 settings [1]. Using CAPriCORN, we wanted to determine whether weeks. Our objective is to characterize vaccine-induced H3.3K27M-spe- we could identify a large, diverse cohort of patients who re- cific CD8+ T-cell and myeloid-derived immunosuppressive subpopula- ceived ICIs as a foundation for exploring the incidence of irAE tions in peripheral blood mononuclear cells utilizing a novel H3.3K27M- in a real-world data source. specific dextramer-based mass cytometry (CyTOF) method [1,2]. Methods Results We identified all patients within CAPriCORN who were 19-88 Through this approach, the temporal expansion of vaccine-reactive years old, had a diagnosis for an ICI-approved cancer, and re- CD8+ T-cells was observed in all patients who completed a minimum ceived an ICI from 1/1/2011 through 12/31/2018. Clinical experts of 24 weeks on the study (n = 4). Simultaneously, this expansion was identified the International Classification of Disease 9 and 10 not observed in 4 of 5 patients who withdrew from the regimen due codes used to document cancer diagnosis and the RxNorm [2] to progression. These T-cells were clustered on a tSNE plot using ca- codes for each ICI documented as a medication ordered in the nonical CD8+ T-cell activation markers and further classified by their medical record (Table 1). The query was developed against the expression profiles, revealing distinct effector memory, central mem- PCORnet Common Data Model version 4.1 [3], validated on the ory and transitional effector subpopulations. Chronological monitor- Northwestern University site node in CAPriCORN and distributed ing of these groups indicates the time course-dependent to all CAPriCORN sites. Six of 9 sites returned counts. Data was development and persistence of vaccine-reactive exhausted and ef- centrally aggregated and stratified by age, race, sex and therapy. fector memory CD8+ T-cells in 3 of the 4 initial patients analyzed. All data are aggregated counts. Furthermore, an analogous clustering and phenotyping approach Results was used for myeloid cells, allowing for the identification of myeloid- As shown in Table 2, we identified 6,541 patients within CAPri- derived suppressor cell (MDSC) subpopulations. A comparative ana- CORN who received ICI therapy for cancer. 45% are female, 75% lysis revealed a positive correlation between two monocytic myeloid- identify as white, 13% African American, 2% Asian and 1% Native derived suppressor cell (M-MDSC) subpopulations and progression- American, and 86% are 51-83 years of age. The most well repre- free survival. sented cancers were Non-small Cell Lung Carcinoma (50%) and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 37 of 272 Metastatic Melanoma (18%). Overall, 67% received anti-PD1 ther- P68 apies, followed by combination ICI therapies, anti-PDL1 and anti- Innate inflammatory pathways are associated with TIL growth and CTLA4, though usage varied within cancer types. response to adoptive immunotherapy 1 1 1 Conclusions Suhendan Ekmekcioglu, PhD , Dai Ogata, MD, PhD , Caitlin Creasy, MS , 1 1 1 Our results demonstrate that a large cohort of cancer patients who Marie Forget, PhD , Sun-Hee Kim, PhD , Jason Roszik, PhD , Mike 3 1 1 have received ICI therapy can be identified in an integrated medical Spencer , Patrick Hwu, MD , Elizabeth Grimm, PhD , Chantale 1 1 record data environment that spans 11 institutions in a major urban Bernatchez, PhD , Suhendan Ekmekcioglu, PhD center. This population is racially diverse, represents both sexes, a The Univeristy of Texax MD Anderson Cancer Center, Houston, TX, wide range of ages and includes all cancer types approved for ICI United States; Bethyl Laboratories, Inc, Montgomery, TX, United States therapy as of 2018. This real-world cohort will be an effective founda- Correspondence: Suhendan Ekmekcioglu tion on which to explore the incidence of irAE, particularly rare irAE (sekmekcioglu@mdanderson.org); Chantale Bernatchez that require large sample sizes to investigate. (cbarnatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P68 Acknowledgements The investigators would like to acknowledge the CAPriCORN network for Background query support. This work was supported by Patient Centered Outcomes Immune infiltration of T cells (TIL) into the melanoma microenviron- Research Institute (PCORI) CDRN-1306-04737. ment has been associated with improved survival for some patients, and also has been exploited to grow TIL in vitro for adoptive therapy. References However, prognostic significance of immune infiltrating cells in melan- 1. Kho AN, Hynes DM, Goel S, et al. CAPriCORN: Chicago Area Patient- oma and other tumors remains a relatively new concept, and markers Centered Outcomes Research Network. J. Am Med Inform Assoc. 2014; related to suppressive versus active functional TIL remain unclear. We 21:607-611. previously reported that in Stage III melanoma patients’ tumors, posi- 2. https://pcornet.org/download/pcornet-common-data-model-v4-1- tive expression of CD74 together with low or absent Macrophage Mi- specification-5-may-2018/?wpdmdl=1919&refresh= gration Inhibitory Factor (MIF) associates with favorable prognosis [1]. 5d42596fab3e11564629359 Methods 3. https://www.nlm.nih.gov/research/umls/rxnorm/ From an ongoing clinical trial using TIL intended for adoptive immuno- Ethics Approval therapy, we have studied the melanoma patient tumors specimens The study was approved under the CAPriCORN IRB, CHAIRb: Research Protocol (FFPE) from 20 patients whose autologous TIL lines grew to sufficient #14120201 “CAPriCORN Clinical Data Research Network Master Protocol”. number for possible use clinically. We also examined another 20 sets of melanoma tumor from which the TIL did not grow or not grow well. We analyzed the differences in the two groups of tumors (40 total FFPE) for Table 1 (abstract P67). See text for description CD74 regulated pathway features and inflammatory marker expression. Results CD74 regulated markers included CD44, MIF, and downstream in- flammatory targets including inducible Nitric Oxide Synthase (iNOS) and Nitrotyrosine (NT). Our findings confirm our previous report in that tumor CD74 expression significantly associates with favorable OS and PFS (both, p=0.0038) and provides new data that in this set of patients the CD74 also correlates with best irRC of TIL treated pa- tients. New findings include that the NT expression in tumor cells as- sociated with poor TIL growth (p=0.014), as well as lack of clinical response to TIL treatment (p=0.02). We have also found that tumor cell-derived MIF and iNOS expression correlate with unfavorable prognosis for both OS and PFS (p=0.016 and 0.018, respectively). Conclusions We have identified the protein expression of CD74, MIF and of iNOS as providing survival information, and proposed that CD74+/MIF-/iNOS- to- gether be considered to form a "signature" of good prognosis in general melanoma outcomes as well as TIL growth and favorable responses for these patients. Use of this signature for selecting patients for entry into TIL and possibly other immunotherapy trials, as well as research on the differential pathways of IFN-γ signaling in melanoma appear as important areas for future mechanistic research to improve patient outcome. Table 2 (abstract P67). See text for description Reference 1. Ekmekcioglu S, Davies MA, Tanese K, Roszik J, Shin-Sim M, Bassett RL Jr, Milton DR, Woodman SE, Prieto VG, Gershenwald JE, Morton DL, Hoon DS, Grimm EA.Inflammatory Marker Testing Identifies CD74 Expression in Melanoma Tumor Cells, and Its Expression Associates with Favorable Sur- vival for Stage III Melanoma. Clin Cancer Res. 2016 Jun 15;22(12):3016-24. P69 Highly multiplexed single cell spatial analysis of the tumor microenvironment in lymphoma 1 1 2 Monirath Hav, MD, PhD , Anthony Colombo , Erik Gerdtsson , Mohan 2 2 2 2 Singh , Denaly Chen , Imran Siddiqi, MD PhD , James Hicks , Peter Kuhn, 2 1 1 PhD , Akil Merchant, MD , Akil Merchant, MD 1 2 Cedars Sinai Medical Center, Los Angeles, CA, United States; University of Southern California, Los Angeles, CA, United States Correspondence: Akil Merchant (akil.merchant@cshs.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P69 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 38 of 272 Background Diffuse large B cell lymphoma (DLBCL) being the most subtype of non-Hodgkin lymphoma. Despite evidence of expression of PDL-1 on lymphoma cells, less than 10% of DLBCL patients re- spond to PD1 therapy [1]. We hypothesize that a better characterization of spatial architecture of the tumour microenvir- onment (TME) in lymphoma will help explain differences in re- sponses to PD1/PDL-1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cy- tometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [2]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for rele- vant immunophenotypes, the spatial architecture of those pheno- types and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype (Figure 1A). Immune cell represented 33% of the cells represented by CD4 (36%), CD8 (30%), macro- phages (26%) and TREG (8%) (Figure 1B). Immune cell infiltra- tion in individual tumor samples ranged from 7% to 75% with marked heterogeneity. (Figure 1C-D. Analysis of immune Fig. 1 (abstract P69). See text for description marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure 1E) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macro- phages, and tumor cells (Figure 2A). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure 2B). Meta-clusters 2, 6, 8 and 11 were the 4 most dominant patterns of CD8 spatial interaction in the TME. Each CD8 spatial interaction pattern is dis- tinctive with case to case heterogeneity (Figures 2C-D). Risk as- sessment analyses of spatial clusters 1, 2 and 4 (“hazardous”)had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 (“protective”) (Figure 2E). In the “protective” spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREG pheno- types, with opposite in “hazardous” areas (Figures 3A-B). TIM-3 expression was high both on T cells and tumor cells in the “haz- ardous” neighborhoods. Conclusions Our novel approach to spatial analysis of the immune architecture re- veals clinically relevant insights into the TME. References 1. Ansell, S. M. et al. Nivolumab for Relapsed/Refractory Diffuse Large B-Cell Lymphoma in Patients Ineligible for or Having Failed Autologous Trans- plantation: A Single-Arm, Phase II Study. J. Clin. Oncol. 37, 481–489 (2019). 2. Giesen, C. et al. highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. methods | 11, 417 (2014). Ethics Approval Fig. 2 (abstract P69). See text for description The study was approved by USC IRB, approval number HS10-260 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 39 of 272 of PD-L1+ immunoreactivity in epithelial/tumor cells, increased im- munoreactivity for PD-L1 in CD68+ cells, and a closer relationship be- tween intra-epithelial and stromal CD8+ cells with PD-L1+ cells. In addition, areas in CRC and CD heavily infiltrated by immune cells or with tertiary lymphoid structures contained clusters of PD-L1+ cells that were negative for both CD68 and pan-Cytokeratin. Most of the CD3+ cells in non-lesional CD were PD-1 negative, except around ter- tiary lymphoid structures. In contrast, a greater percentage of CD3+ cells were also PD-1+ in CRC, and more so in lesional CD tissue. Conclusions The Ultivue UltiMapper multiplex fluorescence immunohisto- chemistry platform was effective in characterizing the PD-L1/PD- 1 axis and T cell exhaustion environment in FFPE tissue, in part due to the ability to clearly identify more complex immune cell phenotypes than traditional multiplex IHC. The application of the UltiMapper assays demonstrated many similarities between marker and cell type distribution between lesional colonic CD and CRC. P71 Refining tumor mutation burden values using variant expression Shannon Bailey, PhD, Muhammad Ekram, PhD, Jim Lund, Jeffrey Gulcher WuXi NextCODE Genomics, Arlington, MA, United States Correspondence: Jeffrey Gulcher (jgulcher@wuxinetcode.com) Fig. 3 (abstract P69). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P71 Background P70 Tumor mutation burden (TMB) is used as a surrogate marker for the Tissue-based characterization of T cell exhaustion in inflammatory neoantigen load of a tumor, and many studies have shown that TMB bowel disease and colorectal cancer using multiplex IHC predicts the success of immune-oncology (IO) treatments for cancers, 1 1 1 Marina Bleck, PhD, Diane Mierz , Ania Mikucki , Marie Marcher , Sidharth such as anti-PD-1 or anti-CTLA4 therapy. While IO treatment of pa- 1 1 2 Kerkar, MD , Gerald Nabozny, PhD , Sean Downing, PhD , Alexander tients with high TMB has led to success and excitement in the field, 1 1 Klimowicz, PhD , Alexander Klimowicz, PhD not all high TMB patients respond to IO treatment. TMB can be de- 1 2 Boehringer Ingelheim, Ridgefield, CT, United States; Ultivue, termined using next-generation sequencing, and both panel and Cambridge, MA, United States whole-exome DNA sequencing have been used to measure the mu- Correspondence: Alexander Klimowicz tation load of a tumor. (alexander.klimowicz@boehringer-ingelheim.com) Because all genes are not expressed in every cell, inclusion of the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P70 mutations found in unexpressed genes may confound the utility of TMB to predict neoantigen load. In this study, we explore improving Background TMB by taking into account both DNA mutation and RNA expression T cell exhaustion and the PD-L1/PD-1 checkpoint axis has been ex- with the hypothesis that using both criteria will lead to a TMB bio- tensively characterized in peripheral blood mononuclear cells and in marker that correlates better with neoantigen load. human tumor tissues. This has provided a better understanding of Methods the role this pathway plays in tumor immunology and of its clinical We examined DNA and RNA sequencing data for different cancer types in utility in predicting responsiveness to checkpoint inhibitor therapies. The Cancer Genome Atlas (TCGA) to determine refined TMB values. The T cell exhaustion is not only associated with tumor progression, but data were assessed to identify mutations with and without expression. has recently been associated with better prognosis and milder course Results of disease for a number of autoimmune and autoinflammatory disor- We found that a significant faction mutations included in a standard ders. We set out to characterize and contrast the T-cell exhaustion TMB calculations reside in genes that are not expressed, and this frac- environment between colonic Crohn’s disease (CD) and colorectal tion varies significantly among samples. A corrected TMB that incorpo- cancer (CRC). We applied the Ultivue UltiMapper multiplex fluores- rates gene expression is likely to be a better predictor of neoantigen cence IHC platform to capture complex immune cell phenotypes and load. In addition, our group has previously identified TCGA samples provide a more in depth characterization than traditional IHC. with allele specific expression in which up to 25% of the tumor muta- Methods tions are not expressed despite significant expression of the gene. The Commercially sourced FFPE surgical resections from n=5 colonic CD pa- fraction of unexpressed mutations is much higher in some cancer tients (matched lesional and non-lesional tissue) were compared to n= types. Adding this correction to the TMB calculation, including only var- 5 CRC tumor resections (3 hot and 2 cold tumors) using the Ultivue Ulti- iants that are expressed in the tumor in the TMB calculation, changes Mapper multiplex fluorescence immunohistochemistry platform. Two the average TMB values found among different cancer types, and cor- UltiMapper kits were used to evaluate the T cell environment in these rects the TMB in individual samples. This refined TMB value provides a tissues: UltiMapper I/O PD-L1 panel included the markers CD8, CD68, biomarker that reclassifies samples scored as high TMB to low TMB and PD-L1, and pan-Cytokeratin/Sox10; UltiMapper I/O PD-1 panel included is likely to better predict response to IO treatment. the markers CD3, CD45RO, PD-1, and pan-Cytokeratin/Sox10. All assays Conclusions were stained on Leica BOND RX autostainers. Whole-slide images were As our results suggest, adding RNA sequencing can be used to im- acquired on a ZEISS Axio Scan.Z1 slide scanner. Image analysis was per- prove the TMB biomarker to better separate treatment groups. The formed using Indica Labs HALO software. initial analysis was performed with TCGA exome data and is now be- Results ing extended to refine TMB values generated from gene panels in- Contrasted to non-lesional CD tissues, several similarities were ob- cluding TSO 500. We are also evaluating differences in tumor- served between CD lesional tissue and CRC, including the presence infiltrating lymphocytes in based on the refined TMB value. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 40 of 272 P72 attractive targets for immunotherapy. Identifying the full comple- Comprehensive and accurate prediction of presented neoantigens ment of peptides derived from a protein presented on a major class-I using ImmunoID NeXT and advanced machine learning algorithms HLA restriction provides a vital step toward increasing the speed and Dattatreya Mellacheruvu, PhD, Rachel Pyke, Charles Abbott, PhD, Nick viability of many immunotherapeutic strategies. Advances in next- Phillips, Rena McClory, John West, MBA, Richard Chen, Sean Boyle, PhD, generation sequencing (NGS) and single-cell technologies have en- Dattatreya Mellacheruvu, PhD abled the accurate capture of somatic mutations accumulated by a Personalis Inc., Menlo Park, CA, United States tumour, yet a significant hurdle remains how this information can be Correspondence: Sean Boyle (sean.boyle@personalis.com) utilized for immunotherapeutic benefit. Identifying which somatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P72 mutations produce neoantigens is crucial in providing the link be- tween genetic change and immunological impact. Background Methods Comprehensive detection of potential neoantigens and accurate pre- Directly identifying potential neoantigens using mass spectrometry diction of their MHC presentation are critical prerequisites for selecting offers a significant improvement over traditional approaches based neoepitopes that can be used for creating personalized cancer vac- on prediction. However, the relatively high sample requirement of cines. However, prediction models developed using in-vitro MHC- this approach inherently limits the depth of analysis that can be per- peptide binding assays cannot model upstream presentation machin- formed, with a significant risk that low abundance neoantigens are ery, such as proteasome cleavage and peptide loading. Advances in not detected. immuno-affinity purification followed by mass spectrometry (IP-MS) By integrating multiomics data from over 1000 experiments in 200 have enabled direct detection of MHC-bound peptides and can there- immortalised cell lines, we have generated a database of over two mil- fore be used for modelling native MHC-peptide presentation. Further, lion unique HLA-peptide sequences that offers near total coverage of genetically engineered cell lines that express a single HLA allele enable the protein-coding genome. Our comprehensive HLA class-I peptide unambiguous HLA-peptide assignment. Here, we present an overview atlas has been used as a reference tool to aid direct identification of of our MHC presentation prediction framework based on a large collec- neoantigens by targeted mass spectrometry, to probe indirectly for the tion of such mono-allelic cell lines and discuss its utility in conjunction presence of neoantigens, and to explore how many common driver with ImmunoID NeXT, our commercially available exome scale DNA mutations associated with cancer interact with the immunopeptidome. and RNA sequencing and analytics platform specifically designed to en- Results able the development of immuno-therapies. We have identified hundreds of neoantigens directly by mass spec- Methods trometry and found that mutated proteins follow the same pattern Mono-allelic cell lines were generated from K-562 null-HLA parental of antigen processing and presentation as their unmutated equiva- cells by transfecting each of the selected alleles. Cells were grown, lents. As a result, our HLA peptide atlas offers significant value in pre- screened for surface expression, lysed and immuno-affinity purified dicting the likelihood of a somatic mutation creating a neoantigen. using a column coated with HLA class I (W6/32) antibody. Peptides Comparing predicted neoantigens with those directly identified by were gently eluted and analyzed using LC-MS/MS. Peptide-to-spectrum mass spectrometry, we show effective prioritization of mutations by assignment was performed and filtered at 1% false discovery rate. accurately predicting the presence and relative abundance of neoan- Results tigens. Applying this process toward the five most commonly mu- The training data for our MHC presentation prediction framework were tated genes in cancer reveals a marked bias toward mutations that generated using a large collection of genetically engineered mono- either act negatively or are in ‘quiet’ areas of the immune landscape. allelic cell lines, encompassing approximately 60 HLA Class I alleles that As all mutated peptides contain novel amino acid sequence, and are are frequently present across various populations. The resulting hence able to elicit an immune response, this ability to convert ‘po- immuno-peptidomics data were comprehensive and of high quality - tential’ into ‘actual’ is crucial in establishing a mechanism for identify- the peptide yields were high (median of approx. 1600 unique peptides ing false positive results observed in cell-based assays. per allele) and the dominant motifs were in agreement with published Conclusions motifs. Our prediction framework is based on multiple modelling algo- An integrative multiomics approach to neoantigen identification rithms, including a multi-layer neural network, and uses proprietary and has delivered a powerful reference for developing novel standard features such as peptide sequence, peptide length, binding immunotherapies pocket sequence and abundance (measured by transcripts per million). We created allele-specific and pan-allele models and evaluated them P74 on an independent hold-out dataset. Both our allele-specific and pan- Integrating CD8 and CD4 effector neo-epitope content with allele models had superior performance compared to other public regulatory T cell epitope exclusion is a superior prognostic tools, with a higher precision across a range of recall (sensitivity) values. biomarker for bladder cancer patient compared to their tumor Conclusions mutation burden Our integrated pipeline for neoepitope discovery, which includes the 1 2 1 Guilhem Richard, PhD , Randy Sweis, MD , Matthew Ardito, BA , comprehensive profiling of putative neoantigens using ImmunoID 2 1 3 Tzintzuni Garcia , Leonard Moise, PhD , Michael Princiotta, MS, PhD , NeXT and accurate and sensitive prediction of MHC presentation of 3 1 3 Dominique Bridon , William Martin, BA MD , Gad Berdugo, MSc, MBA , such neoantigens across all HLA Class I alleles (using our pan-allelic 4 4 1 Arjun Balar , Gary Steinberg , Anne de Groot, MD models) enables the effective generation of neoepitopes that are crit- 1 2 EpiVax, Inc., Providence, RI, United States; University of Chicago, ical for developing personalized cancer vaccines. Chicago, IL, United States; EpiVax Oncology, New York, NY, United States; NYU Langone Health, New York, NY, United States P73 Correspondence: Gad Berdugo (gberdugo@epivaxonco.com) Large scale multiomics reveals a marked bias in driver mutations Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P74 toward areas not reliably presented to the immune system Alex Powlesland, PhD, Michael Cundell, PhD, Floriana Capuano, PhD, Background Brandon Higgs, David Lowne, BS, Ricardo Carreira, PhD We hypothesized that neo-epitope-based prediction using an ad- Immunocore Ltd, Abingdon, United Kingdom vanced in silico T cell epitope screening system (Ancer™) may better Correspondence: Alex Powlesland (alex.powlesland@gmail.com) identify patients with improved prognosis than tumor mutation bur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P73 den. Analysis of genomic data derived from the muscle-invasive bladder cancer (BLCA) cohort of The Cancer Genome Atlas (TCGA) Background database for CD4, CD8, and Treg neo-epitopes was performed to de- The repertoire of HLA-peptides presented to the immune system termine whether Ancer™ would improve prognostic stratification which derive from cancer-associated, viral, and mutated proteins are compared to tumor mutational burden (TMB). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 41 of 272 Methods BLCA patient mutanomes (n=412) were retrieved from the TCGA and evaluated with Ancer™, an innovative and automated neo-epitope screening platform that combines proprietary machine learning- based HLA I and HLA II neo-epitope identification tools with removal of inhibitory regulatory T cell epitopes for neo-epitope ranking and personalized cancer vaccine design. BLCA patients were separated based on median TMB or neo-epitope burdens. We investigated the effect of integrating both CD8 and CD4 neo-epitope burdens as most mutanome pipelines exclusively focus on the identification of Class I neo-epitopes. Overall survival was analyzed using the Kaplan-Meier method and differences analyzed by log-rank testing. Results Compared to low TMB, high TMB was significantly associated with improved survival (p = 0.0001, difference of 38.5 months in median survival, Figure 1). Improved differentiation of median survival times was obtained when separating patients based on their Class I neo- epitope content, as estimated by Ancer™ (p < 0.0001, difference of 59.8 months in median survival). Adding Class II neo-epitope burden further increased separation of OS times, showcased by a 69.6-month increase in median survival for BLCA patients with both high CD8 and high CD4 neo-epitope contents compared to other patients (p = 0.0001). Since we discovered that Class II neo-epitopes can induce in- hibitory responses, we further evaluated whether the screening of these detrimental sequences could improve our analysis. Upon iden- tifying Class II neo-epitopes likely to induce T effector (Teff) re- Fig. 2 (abstract P74). See text for description sponses, we found that the median survival of patients with high CD8 and high CD4 Teff contents was extended by nearly 4 months to 73.4 months compared, to the remainder of the cohort (p < 0.0001, Figure 2). P75 Conclusions targetSCAPE and ultraSCAPE: Simultaneous identification and deep Our analysis suggests that optimal host-immune recognition of profiling of human antigen-specific T cells and other immune cell CD8+, CD4+, and Treg epitopes plays a key role in cancer sur- subsets by mass cytometry 1 1 1 vival. While defining CD8 neo-epitope burden enhanced associa- David Roumanes, PhD , Faris Kairi , Alessandra Nardin, DVM , Evan 2 1 tions with OS, the inclusion of CD4 Teff neo-epitope burden Newell, PHD , Michael Fehlings 1 2 substantially helped identify long-term survivors. These results immunoSCAPE, Cambridge, MA, United States; Fred Hutchinson suggest that defining the number of true neo-epitopes using Cancer Research Center, Singapore, Singapore Ancer™ may represent a novel prognostic or predictive Correspondence: Alessandra Nardin biomarker. (alessandra.nardin@immunoscape.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P75 Background During clinical trial immune monitoring, especially in the field of im- munotherapy, it is critical to collect in-depth phenotypic information from multiple immune cell populations in order to assess the biological activity of the immunotherapy, to identify biomarkers of response or progression, and/or to identify new drug targets. However, patient sam- ples, for example peripheral blood mononuclear cells (PBMC) or tissues, are often only available in small amounts and current methods face limitations in either depth of analysis and/or cell throughput. Methods In order to identify therapy-relevant antigens and to facilitate a concur- rent in-depth characterization of cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling cap- abilities of cytometry by time of flight (CyTOF) and a unique method- ology allowing the identification and characterization of rare antigen- specific T-cell subsets (targetSCAPE). By implementing a new technol- ogy (ultraSCAPE) that combines flow and mass cytometry together with a combinatorial live cell barcoding strategy, we further increased the high-dimensional phenotyping capacities to over 100 different marker molecules through simultaneous in-depth profiling of up to three add- itional immune cell subsets from the same sample. Results We isolated 4 different immune cell populations from a single sample and combined 3 different phenotypic panels consisting of 35 makers each together with a combinatorial tetramer multiplex and phenotyp- ing panel for deep profiling of myeloid cells, NK cells, B cells and T cells. We demonstrate the potential of this novel immuno-phenotyping method, by tracking virus-specific T cells while simultaneously charac- Fig. 1 (abstract P74). See text for description terizing 4 immune cell subsets with over 100 distinct phenotypic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 42 of 272 markers from a single sample, which is currently impossible employing P77 modern flow cytometers or classical mass cytometry methods. Comprehensive profiling of tumor-immune interaction in anti-PD-1 Conclusions treated melanoma patients reveals subject-specific tumor escape With its ability to provide an unprecedented picture of the immune mechanisms 1 1 1 1 status within a single sample, including T cell specificity information Charles Abbott, PhD , Eric Levy, PhD , Rachel Pyke , Rena McClory , 2 1 1 and in depth profiling of relevant immune cell subsets, ultraSCAPE in Sekwon Jang, MD , Richard Chen, PhD , Sean Boyle, PhD 1 2 combination with targetSCAPE can provide detailed insights on the Personalis, Menlo Park, CA, United States; Inova, Fairfax, VA, United effects of immunotherapy on the immune cell population. Informa- States tion learned from in-depth immune phenotyping of several immune Correspondence: Sean Boyle (sean.boyle@personalis.com) cell subsets such as T, NK and myeloid cell subsets can be leveraged Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P77 for the development of novel diagnostics, for biomarker discovery and for monitoring therapeutic strategies in immunotherapy. Background Checkpoint inhibitor therapy has demonstrated meaningful antitu- mor activity for many patients, though the majority fail to achieve P76 complete response. Thus, it is of particular interest to identify bio- Development of immunopeptidomic platform for human leucocyte markers and mechanisms that promote positive response to im- antigens class I using microflow liquid chromatography and munotherapy. In the present study, we apply our comprehensive quadrupole time-of-flight mass spectrometry tumor immunogenomics platform (ImmunoID NeXT), integrating data 1 1 Takashi Shimada, PhD , Noriko Iwamoto, PhD , Yoshinobu Koguchi, MD, from the tumor, tumor microenvironment and immune system to 2 2 2 2 PhD , John Cha , Brian Piening, PhD , Eric Tran, PhD , Hong-Ming Hu, create a comprehensive biological signature of patient response to 2 2 2 PhD , Bernard Fox, PhD , William Redmond, PhD therapy. 1 2 Shimadzu Scientific Instruments, Bothell, WA, United States; Providence Methods Cancer Center, Portland, OR, United States We characterized the immunogenomics of 52 unresectable, stage III/ Correspondence: Takashi Shimada (tashimada@shimadzu.com) IV melanoma patients who underwent anti-PD-1 therapy to assess Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P76 factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 12 months. Background For each patient, a single paired FFPE tumor and normal blood sam- The highly complex population of peptides associated with human ple was collected and profiled using Personalis’ ImmunoID NeXT plat- leucocyte antigens (HLA) is the human immunopeptidome. Compre- form; an augmented exome/transcriptome platform and analysis hensive characterization of the immunopeptidome is key in predict- pipeline, which produces comprehensive tumor mutation informa- ing immunotherapeutic responses by evaluating targets of T cell tion, gene expression quantification, neoantigen characterization, interaction and in developing the next generation of cancer im- HLA typing and LOH, TCR repertoire profiling and tumor microenvir- munotherapies. Mass spectrometry (MS) is a technology that holds onment profiling. Tumor molecular information was then analyzed significant promise for untargeted and complete identification of the together with clinical outcome. immunopeptidome. MS acquisition is mainly used an electrospray Results ionization (ESI) combined with nanoflow liquid chromatography (LC). Comprehensive profiling demonstrated that elevated pretreatment However, the analysis time and retention reproducibility could be is- neoantigen burden was predictive of response to PD-1 blockade, and sues. Therefore, we tried to develop an MS platform that can ensure significantly associated with progression-free survival. Additionally, the coverage while increasing throughput using a microflow LC. we observed increased response to anti-PD-1 therapy in patients Methods with elevated pretreatment TCR clonality. Patients with high neoanti- HLA class I complexes were purified from A431 cell lysate by W6/32 im- gen burden and TCR clonality that failed to achieve complete re- munoaffinity. Purified HLA peptides were eluted with 5% formic acid. sponse revealed potential resistance mechanisms to anti-PD-1 The peptides were fractionated by 10 kDa ultrafiltration. HLA peptides therapy. Specifically, we identified two patients with high expression were separated with L-column2 ODS (0.3x150 mm) using a trap-elute of IDO1 or CTLA4, which may facilitate PD-1-independent immune protocol of microflow LC (Nexera-Mikros) and quadrupole time-of-flight escape. Additionally, we found two patients with antigen presenta- MS (LCMS-9030). Flow rate was set at 5 μl/min with a gradient of aceto- tion machinery (APM) mutations. The first patient had independent nitrile in 0.1% formic acid for 18 min. MS/MS spectra were acquired HLA-A and HLA-B mutations, likely leading to loss of surface expres- using a data-dependent manner of top-10 precursor intensities. The sion of the proteins. In the second APM mutation patient we ob- precursor scan was first set from 400 to 600 Da. The charge states of served a high frequency (80% AF) frameshift variant in B2M, which precursors were set between 1 to 4, and MS/MS scan was from 200 to potentially prevents proper HLA class I folding and antigen presenta- 1200 Da. The data were analyzed by Mascot proteome server and tion. These APM mutations suggest reduced neoantigen presentation PEAKS sequencing software on SwissProt database. The mass toler- in these patients, which are probable mechanisms for tumor escape. ances of precursors and fragments were set at 0.05 Da and 0.3 Da. Min- By integrating neoantigen burden, HLA-LOH and APM mutational imal peptide length was set to 8 amino acids. data into a corrected neoantigen burden, we were able to increase Results the predictive strength of this biomarker. An initial round of optimizations was performed to establish optimal Conclusions parameters for immunopeptidome identifications. Using tryptic pep- In summary, our comprehensive cancer immunogenomic analyses tides from A431 lysate, we optimized the 50-100 msec repeat of MS/ demonstrate that genomic and immune profiling of pretreatment pa- MS scanning, top-10 of data-dependent acquisitions per scan, 50 Da tient samples can identify biomarkers and resistance mechanisms to scan range, 35V±10V spread of collision electrode voltage, and 3.0 kV immune checkpoint blockade, suggesting the potential efficacy of of electrospray voltage. These parameters were then applied for these as an integrated biomarker to optimize anti-PD-1 therapy pa- identification of HLA-associated peptides from A431 cells. From this, tient selection. we identified 4,217 MS/MS and 801 sequences from 34,042 spectra. Conclusions From these data, we demonstrate that similar sensitivity can be suffi- P78 ciently achieved with microflow platform as has been demonstrated Optimization of tumor nutation burden measurement in FFPE DNA preciously for nanoflow LC-MS. This has significant advantages in Janice Au-Young, PhD, Iris Casuga, PhD, Vinay Mittal, Dinesh Cyanam, terms of throughput, instrument maintenance, and widespread ap- MS, Elaine Wong-Ho, Fiona Hyland, Seth Sadis, Warren Tom, PhD plicability. Our future directions are to determine whether cancer Thermo Fisher Scientific, South San Francisco, CA, United States neoepitopes identified by these approaches may be recognized and Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) therapeutically targeted by patient T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P78 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 43 of 272 Background demonstrated [1]. Solid tumors systemically reprogram the lung Tumor mutation burden (TMB) measures the number of somatic mutations unique immune environment, dominated by intravascular neutro- and is a positive predictive factor for response to immune-checkpoint in- phil functions [2], to colonize this site. The concept of ‘oligopro- hibitors in multiple cancer types. While whole exome sequencing (WES) is gression’ has recently received mounting attention, due to its thegoldstandardfor TMBmeasurement,itisnot practicalfor routineuse. relevance and because it represents an interesting in vivo model TMB values measured using targeted sequencing have been shown to to study TIME, although the specific mechanisms of oligometa- have good correlation with WES. However, during FFPE preservation, DNA static process are relatively underinvestigated [3, 4]. may undergo cytosine deamination, resulting in false C>T substitutions Methods and elevated TMB values. We have assessed the effect of DNA damage RNA sequencing was performed on a retrospective collection of tis- andrepaironTMB values usingthe Oncomine TumorMutationLoad sue samples from primary renal cell carcinoma, melanoma, and Assay (OTMLA), a targeted next generation sequencing assay. NSCLC and paired lung oligometastases of untreated patients (Figure Methods 1). Enrichment of tumor-related pathways and transcripts that reflect We measured TMB from 37 FFPE colon, lung, endometrial and gastric tu- the enrichment of immune cell subsets was assessed by single sam- mors usingthe OTMLApanel on IonGeneStudiowith20ngofinput DNA ple gene set enrichment analysis. Differentially expressed genes be- from tumor only samples. The informatics workflow utilizes a custom vari- tween primary tumors versus the corresponding lung metastases ant calling and germline variant filtering algorithm to accurately estimate were used for pathway analysis. Neutrophils extracellular traps (NETs) somatic variants in tumor tissue. In parallel, TMB was measured by Whole were revealed by immunofluorescence, assessing extracellular DNA Exome Sequencing (WES) targeting 50Mb using 100ng of tumor and and citrullinated H4 histone co-localization and/or myeloperoxidase matched normal DNA on a HiSeq X instrument. We examined factors [5]. Autophagy was assessed the CYTO-ID® kit [5]. that affect OTMLA measurements: Deamination signature, degree of de- Results amination and allele ratio identify DNA samples with high levels of dam- While tumor-related pathway enrichment differed mostly according age due to FFPE preservation. A Uracil-DNA glycosylase (UDG) repair step to the primary tumor histology, perturbations of immune-regulatory was introduced to eliminate damaged targets and improve usable TMB pathways was observed during oligoprogression in the lung. Decon- values of DNA from FFPE tumor tissue. At the variant level, samples with volution of immune cell subpopulations identified increased imma- high deamination scores were analyzed dynamically as a function of al- ture dendritic cells and reduced T cell abundance in oligometastatic lele frequency to study TMB values for correlation with WES. lesions. Strikingly, a large proportion of differentially modulated path- Results ways were “immune” rather than “cancer-cell”-related. Core analysis OTMLA TMB values showed good correlation with WES-derived TMB; confirmed that the main transcriptomic network that is affected dur- however ~10% of tumor DNA samples had high TMB and deamination ing disease progression is immune-based, centered on a cross-link values outside the expected range. These samples were included as a between innate and adaptive immunity. Specifically, it was associ- subset of samples tested with and without the UDG repair step. UDG ated with decreased HLA, iCOS, IL-9, and IL-17 pathway activity and treatment decreased TMB and deamination scores, resulting in higher downregulation of interferon signaling. During progression, we ob- correlation with WES TMB values. Some samples with very high de- served coherent modulation of transcripts associated with NET gen- amination scores were unable to be rescued; however, TMB values in eration, related to upregulation of key autophagic genes, to samples with low deamination and minimal damage were not affected. competition of the HMGB1 molecule with CXCL12 and CXCR4 and Conclusions RAGE receptor (AGER) activation. Accordingly, NET expression was We show that deaminated cytosine bases can be enzymatically re- strikingly more abundant in lung metastases than in primary tumors. moved by treatment with UDG. In a subset of FFPE samples tested, Conclusions UDG treatment was demonstrated to reduce the OTMLA estimated Our results identify evident molecular mechanisms associated with SNP proportion consistent with deamination. This results in consist- suppression of the immune milieu during disease oligoprogression in ent and effective reduction of C>T artifacts without affecting true the lung across different tumors. They include innate-adaptive im- variants and can provide TMB values in a biologically relevant range. mune dysfunction HLA-mediated, and interferon dysregulation asso- ciated with neutrophil-mediated immune suppression. Since these tumors are targeted by immune checkpoint blockade (ICB) our data P79 highlights the relevance of characterizing the TIME composition in Molecular comparison of tumor microenvironment in primary paired primary and oligometastatic lesions during ICB treatment to lung, melanoma, and kidney tumors versus paired lung metastases optimize treatment approaches. reveals shared perturbations of the immune milieu during oligoprogression Acknowledgements 1 1 Davide Bedognetti, MD, PhD , Jessica Roelands, Master , Angelo Paola Nistico' and Gennaro Ciliberto are co-last authors. 2 2 3 3 Manfredi , Norma Maugeri , Francesca De Nicola , Ludovica Ciuffreda , 3 3 3 Matteo Pallocca , Maurizio Fanciulli , Francesca Di Modugno, PhD , References 3 3 3 Paolo Visca , Barbara Antoniani , Gabriele Alessandrini , Darawan Rinchai, 1. Angelova M, Mlecnik B, Vasaturo A, Bindea G, Fredriksen T, Lafontaine L, 1 1 3 PhD , Wouter Hendrickx, PhD , Paola Nistico', MD , Gennaro Ciliberto, et al. Evolution of Metastases in Space and Time under Immune MD Selection. Cell. 2018 Oct;175(3):751-765.e16. 1 2 Sidra Medicine, Doha, Qatar; IRCCS Ospedale San Raffaele, Milano, Italy; 2. Granton E, Kim JH, Podstawka J, Yipp BG. The Lung Microvasculature Is a Istituto Nazionale Tumori Regina Elena, Rome, Italy Functional Immune Niche. Trends Immunol. 2018 Nov;39(11):890-899. Correspondence: Gennaro Ciliberto (gennaro.ciliberto@ifo.gov.it) 3. Weichselbaum RR. The 46th David A. Karnofsky Memorial Award Lecture: Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P79 Oligometastasis-From Conception to Treatment. J Clin Oncol. 2018 Sep27:JCO1800847. Background 4. Stephens SJ, Moravan MJ, Salama JK. Managing Patients With The importance of tumor-host interactions during cancerogenesis Oligometastatic Non-Small-Cell Lung Cancer. J Oncol Pract. 2018 and metastatic progression has been now widely appreciated. Jan;14(1):23-31. More recently, the impact of the tumor immune microenviron- 5. Maugeri N, Campana L, Gavina M, Covino C, De Metrio M, Panciroli ment (TIME) to mold tumor evolution was convincingly C, Maiuri L, Maseri A, D'Angelo A, Bianchi ME, Rovere-Querini P, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 44 of 272 Manfredi AA.Activated platelets present high mobility group box 1 to Results neutrophils, inducing autophagy and promoting the extrusion of Overall survival (N=141, median 6.05 months) was not associated neutrophil extracellular traps. J Thromb Haemost. 2014 with PD-L1 positive (>=1% tumor or tumor+immune cells)/nega- Dec;12(12):2074-88. doi: 10.1111/jth.P689. tive, diffuse/non-diffuse, gastric/gastroesophageal, prior therapy Ethics Approval (median=2), tumor IFNgamma signature or CD8+ tumor infiltrate. The study was approved by IFO Institution’s Ethics Board, approval number Differential gene expression analysis identified GRB7, a down- 561/03. stream mediator of HER2 signaling and part of the HER2/ERBB2 amplicon in breast cancer [4], as one of two genes associated with survival >1 year (FDR=0.027). HER2-positivity (medical record) was associated with a 3.5-fold higher median expression of GRB7. Prolonged survival was associated with both HER2-positivity (n= 43/132; HR=0.58, p=0.01) and the top quartile of GRB7 expression (n=25/94; HR=0.48, p=0.007). The median survival for HER2- positive patients was 10.1 months versus 5.95 months for HER2- negative. HER2 status was not associated with PD-L1 status or CD8+ infiltrate. Nearly all HER2-positive (n=40/43) and 2 HER2- negative patients received trastuzumab (median 62 days post- trastuzumab). Prior or best response was not related to 1 year survival and 2 of 3 HER2-positive patients that did not receive trastuzumab had >1 year survival. TMB was also evaluated and significantly associated with HER2-positivity (N=61, p=0.041). In the subset of 61 patients with TMB data, patients with high TMB plus HER2-positivity had the longest median survival of 15.4 months compared to all other patients at 6.7 months (N=14 vs 47; HR=0.47; p=0.04). Conclusions HER2 was associated with improved survival with checkpoint blockade in advanced GEA patients, regardless of response to Fig. 1 (abstract P79). See text for description prior trastuzumab. This study was limited by the lack of pre- treatment biopsies, but consistent with a recent report on Asian GEA patients [5]. The combination of HER2-positivity and relatively higher TMB in a limited dataset led to the greatest observed me- dian survival time, suggesting an interaction between HER2 and P80 TMB that warrants exploration in future GEA studies involving HER2 is associated with prolonged survival in advanced checkpoint inhibition. gastroesophageal adenocarcinoma patients treated with checkpoint blockade Acknowledgements 1 1 2 Carrie Brachmann, PhD , Emon Elboudwarej , Manish Shah, MD , David The authors gratefully acknowledge the patients and their families who 3 4 5 Cunningham , Jean-Philippe Metges , Eric Van Cutsem, MD, PhD , Zev participated in this study. 6 1 1 1 Wainberg, MD , Jingzhu Zhou , Dung Thai , Pankaj Bhargava , Daniel Trial Registration Catenacci, MD Clinicaltrials.gov NCT02862535 1 2 Gilead Sciences, Foster City, CA, United States; Weill Cornell Medicine, NY Presbyterian, New York, NY, United States; Sutton and London References Hospital, London, United Kingdom; Brest University Hospital, Brest, 1. Shah M, Metges J-M, et al. A phase 2, open-label, randomized study to France; University Hospitals Leuven & KU Leuven, Leuven, Belgium; evaluate the efficacy and safety of andecaliximab combined with nivolu- 6 7 UCLA School of Medicine, Santa Monica, CA, United States; University mab versus nivolumab alone in subjects with unresectable or recurrent of Chicago Medical Center, Chicago, IL, United States gastric or gastroesophageal junction adenocarcinoma. ASCO Gastrointes- Correspondence: Carrie Brachmann (carrie.brachmann@gilead.com) tinal Cancers Symposium. 2019. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P80 2. Metges J-M, Elboudwarej E, et al. Exploratory evaluation of baseline tumor biomarkers and their association with response and survival in pa- Background tients with previously treated advanced gastric cancer treated with ande- The benefit of checkpoint blockade in advanced gastric cancer caliximab combined with nivolumab versus nivolumab. ASCO is limited and patient selection biomarkers are needed. In a ran- Gastrointestinal Cancers Symposium. 2019. domizedphase 2study in >=2ndlineadvancedgastroesopha- 3. Brachmann C, Zhang Y, et al. Evaluation of intratumoral T cells in geal adenocarcinoma (GEA) cancer in Europe, US and Australia, biopsies from advanced gastric patients treated with andecaliximab and there was no clinical benefit for the addition of andecaliximab nivolumab. ASCO Gastrointestinal Cancers Symposium. 2019. to nivolumab in the total population or evaluated subgroups 4. Ferrari A, Vincent-Saloman A, et al. A whole-genome sequence and tran- (including PD-L1) [1,2]. Pharmacodynamic analyses demon- scriptome perspective on HER2-positive breast cancers. Nature Commu- strated little to no impact of andecaliximab [3]. This exploratory nications. 2016. biomarker analysis included all patients as a nivolumab-treated 5. Satoh T, Kang Y-K, et al. Exploratory subgroup analysis of patients with prior population. trastuzumab use in the ATTRACTION-2 trial: a randomized phase III clinical Methods trial investigating the efficacy and safety of nivolumab in patients with ad- Evaluation of archival tumor tissue was described [2,3]. Tumor muta- vanced gastric/gastroesophageal junction cancer. Gastric Cancer. 2019. tion burden (TMB) was evaluated by whole exome sequencing with Ethics Approval matched normal. Survival analyses (cox proportional hazards) were This study was approved by the institutional review board or independent adjusted for age and sex. ethics committee appropriate for each site. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 45 of 272 P81 Table 1 (abstract P81). Associations between TMB and Baseline Association of tumor mutational burden with clinical, genomic, Characteristics and treatment characteristics in advanced non-small cell lung cancer 1 1 2 Connor Willis, PharmD , Hillevi Bauer, PharmD , Trang Au, PharmD ,Sudhir 1 1 3 Unni, PhD, MBA , Wallace Akerley, MD , Ashley Sekhon, MD , Firas Badin, 4 5 6 7 MD , John Villano, MD, PhD , Matthew Schabath, PhD , Bing Xia, MD , 8 9 9 Beth Gustafson, PharmD , Komal Gupte-Singh, PhD , Beata Korytowsky , 9 9 John-Michael Thomas, PharmD , Gabriel Krigsfield, PhD , Solomon 9 1 10 Lubinga, PhD , Diana Brixner, PhD , David Stenehjem, PharmD 1 2 University of Utah, Salt Lake City, UT, United States; Long Island University, Brooklyn, NY, United States; MetroHealth Medical Center, Cleveland, OH, United States; Baptist Health, Lexington, KY, United 5 6 States; Markey Cancer Center, Lexington, KY, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; University of Southern California, Los Angeles, CA, United States; Saint Luke's Cancer Institute, Kansas City, MO, United States; Bristol-Myers Squibb, Princeton, NJ, United States; University of Minnesota, Duluth, MN, United States Correspondence: David Stenehjem (stene032@d.umn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P81 Background Tumor mutational burden (TMB) is emerging as a potential predictor of response to immunotherapy in various tumor types. However, the association of TMB data with clinical, demographic, genomic, and treatment characteristics warrants further investigation. Methods Nine U.S. Comprehensive Cancer Centers participated in this observa- tional, cohort study; five centers are members of the Oncology Re- search Information Exchange Network (ORIEN). Adult patients with stage IV non-small cell lung cancer (NSCLC) with tissue-based TMB data from any testing platform were included and their treatment in- formation was abstracted using a standardized case report form. TMB reporting ranged from September 2014 through March 2019. TMB-High and TMB-Low were defined as >10 mutations/megabase (mut/Mb) and <10 mut/Mb, respectively. Clinical, demographic, gen- omic, and treatment characteristics were compared by TMB level. Results There were 426 patients enrolled in the study across seven of the Table 2 (abstract P81). Associations between TMB and Select nine sites. TMB results from comprehensive genomic profiling (CGP) Oncogene Mutations were available for 354 patients. CGP vendors included Foundation Medicine (79.9%), Caris Life Sciences (17.0%), Tempus (2.8%), and NantHealth (0.3%). The median time from diagnosis to CGP testing was 45 days. A comparison of clinical and demographic characteris- tics by TMB is presented in Table 1. TMB-High status was associated with male gender (p<0.01), and positive smoking history (p<0.01). No correlation was found between TMB and PD-L1 (Table 2). TMB-High was positively associated with multiple oncogenes including STK11, LRP1B, TP53, and KDM5C (Table 2). In addition, there were significant negative associations between TMB-High and individual occurrences of altered ALK (p=0.03), EGFR (p<0.01), and ROS1 (p=0.03). The pro- portion of patients receiving first-line immunotherapy increased yearly from 8.5% in 2015, 19% in 2016, 40% in 2017, and 46% in Conclusions These interim results demonstrate the feasibility of conducting multi- site observational electronic health record-based studies with CGP and TMB across a national cohort of comprehensive cancer centers. P82 Immunotherapy utilization has been increasing in the first-line set- High-throughput pairing of single T-cell α and β chains along with ting. Associations between TMB status and driver mutations are indi- phenotypic expression profiling 1 2 3 cative of cancer etiology and informative for treatment decision- Brittany Brown, BS , Miranda Byrne-Steele, PhD , Wenjing Pan, PhD , 2 2 2 4 making. Updated results will be presented with an expanded cohort Song Li , Mary Eisenhower, BS , Daniel Weber , Mollye Depinet, MS , 2 2 2 of patients and future publications with the final cohort (n~1000) will Xiahong Hou, PhD, MD , Alex Moore , Jian Han, MD PhD 1 2 explore treatment, survival and response data. iRepertoire, Inc., Huntsville, AL, United States; iRepertoire, Huntsville, AL, United States; HudsonAlpha Institute for Biotechnology, Huntsville, AL, Acknowledgements United States; iRepertoire.com, Huntsville, AL, United States This study was sponsored by Bristol-Myers Squibb. Recruitment efforts were Correspondence: Jian Han (jhan@irepertoire.com) supported by Mikaela Larson (Huntsman Cancer Institute) and M2Gen® Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P82 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 46 of 272 Background panel was compared to whole-exome sequencing (WES) derived The T-cell receptor (TCR) is responsible for recognizing antigens as TMB. LOH-MHC status reported from PGDx elio tissue complete was peptides bound to a major histocompatibility complex. TCRs typically compared to MHC status determined by WES for accuracy. Addition- contain both an alpha (α) and beta (β) chain that contribute to anti- ally, TMB and MHC status were analyzed together in FFPE samples gen specificity; however, we have seen multiple cases of a single cell from ICI treated patients and correlated to clinical outcomes. containing dual α or dual β chains as well. When analyzing bulk rep- Results ertoires, information about endogenous pairing of α and/or β chains 115 pan-cancer samples were analyzed for microsatellite status and is lost after bulk lysis of T-cell populations. Pairing α and β chains demonstrated an overall agreement of 100.0% with a PCR-based from a single cell while also analyzing the phenotypic expression al- method. TMB was determined in 118 pan-cancer samples and dis- lows us to track TCR specificity and T cell function. This information played a high level of concordance with WES-derived TMB (Pearson can provide direct calculations of clonal frequency in various cell sub- correlation, p=0.903) across a range of TMB scores (0.2-89.7 muts/ sets, allow tracking of specific lymphocytes with treatment, and reveal Mbp). FFPE tissue samples from 98 cancer patients previously treated paired information for both chains of the receptor for downstream Car- with ICIs were then tested for LOH-MHC and demonstrated 88% ac- T development. curacy of detection when compared to WES. Furthermore, analysis of Methods TMB and MHC status in tandem found that patients with both high Here, we developed a method for high-throughput pairing of TCR α TMB and normal MHC status were found to have a significantly and β chains along with expression profiling. We examined, on aver- higher PFS, suggesting greater efficacy of ICI therapy. age, around 15,000 CD4+ cells loaded onto the BD Rhapsody Express Conclusions system. The receptor information is amplified from the same cDNA These data demonstrate the feasibility of measuring MSI, TMB, and using iRepertoire’s proprietary method that incorporates a multiplex evaluating LOH-MHC with high accuracy in a single >500 gene NGS mix of primers associated with both the TCR α and β loci; phenotyp- assay. Additionally, the results presented herein suggest that measur- ing of the cell is obtained using the BD Rhapsody RNA-seq kit. Along- ing TMB and MHC status concurrently can provide added utility in side the high throughput data, we also performed FACS-based single predicting patient response to ICI therapy. cell sequencing on the same individual’s samples through our iPair method (presented previously) and examined the overlapping recep- P84 tor sequences between both methods. Panel-derived tumor mutational burden (TMB) correlates with Results immune checkpoint inhibitors (ICIs) response in gastrointestinal With this mid throughput method, we are able to accurately assess cancers the frequency of single cells containing dual alpha or dual beta TCRs, 1 2 1 San-chi Chen, MD , Kien-Thiam Tan, PHD , Ming-Huang Chen , Yi-Ping which can help to evaluate the high throughput data. 1 2 2 1 Hung, MD , Yi-Lin Hsieh , Yi-Hua Jan , Yee chao Conclusions Taipei Veterans General Hospital, Taipei, Taiwan, Province of China; The described high throughput application should be applicable to ACT Genomics Co., Ltd., Taipei, Taiwan, Province of China any oligo-dT based single cell strategy. Correspondence: Yee chao (ychao@vghtpe.gov.tw) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P84 This study was approved by New England IRB, IRB number 120160202 Background P83 Tumor mutational burden (TMB) has been emerging as a relatively Pan-cancer assessment of composite genomic biomarkers in new biomarker that is independent of PDL1 for the prediction of re- immuno-oncology to predict responses and resistances to immune sponse to the immune checkpoint inhibitor (ICI) treatment. A recent checkpoint inhibitor therapy study has shown that whole exome sequencing (WES)-derived TMB Gustavo Cerqueira, Laurel Keefer, Kelly Gerding, PhD, Kenneth correlates well panel-derived TMB that is estimated using targeted Valkenburg, Christina Oliveras, James White, Leila Ettehadieh, Christopher sequencing. Here, we evaluate the correlation between panel- Gault, James Hernandez, Eric Kong, Isabell Loftin, Samuel Angiuoli, derived TMB with response to ICI treatment in gastrointestinal Abigail McElhinny, John Simmons, PhD cancers. Personal Genome Diagnostics, Baltimore, MD, United States Methods Correspondence: Eric Kong (ekong@pgdx.com) FFPE tumor and normal tissues from 18 patients with gastrointestinal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P83 cancers who had previously received ICI therapy at Taipei Veterans Gen- eral Hospital were retrospectively underwent targeted next-generation Background sequencing (ACTOncoTM) for the identification of somatic variants across Therapeutic response to immune checkpoint inhibitors (ICIs) requires 440 genes and the calculation of TMB. NetMHC and IEDB were used to a prior, suppressed immune response that is released via the inter- predict neopeptide bound to patient-specific HLA class one genotype. action of the checkpoint receptors with their cognate ligands. Micro- RECIST criteria were used to categorize tumor response. satellite instability (MSI) and tumor mutation burden (TMB) have Results emerged as composite genomic metrics that may better predict pa- Patients were grouped into responder (PR or CR, n=10) and non- tient response to ICI treatment, compared to conventional PD-L1 ex- responder (PD or SD, n=7). Among all patients, responders had sig- pression. However, testing for MSI and TMB separately is labor nificantly higher TMB than non-responders (mean 7.37 muts/Mb vs. intensive, increases turnaround time, and consumes valuable tumor 1.24 muts/Mb, p=0.0007). The number of predicted neopeptides tissue samples. Furthermore, recent studies have demonstrated that were significantly higher in responders than in non-responders antigen presentation mechanisms may also play a role in predicting (mean 10.8 vs. 3.6, p=0.0226). Notably, a non-responder harboring outcomes, wherein loss of heterozygosity in MHC class I genes (LOH- EGFR gain-of-function mutation did not respond to the ICI treatment MHC) suggests low likelihood of ICI benefit. despite high TMB. Furthermore, patients harboring MUC16 mutation Methods demonstrated higher TMB than patients without MUC16 mutation Here, we propose that these varied immune-oncology metrics can be (mean 17.38 muts/Mb vs. 3.9 muts/Mb, p=0.0005) (Figure 1). measured together in a single assay, utilizing next-generation se- Conclusions quencing (NGS) technologies. To test this hypothesis, we analyzed Although the cohort size is small, our study showed that panel-based >200 pan-cancer FFPE tumor tissue samples using the PGDx elio™ tis- TMB is predictive of response to ICI. As in lung cancers, EGFR muta- sue complete assay (currently in development; >500 genes panel) to tion is associated with decreased efficacy of ICI in the GI cancers. measure MSI, determine TMB (across 1.3 Mb), and assess MHC status Ethics Approval in a single assay. Detection of MSI was assessed for accuracy against The study was approved by TPEVGH intuition’s Ethics Board, approval a validated PCR approach and TMB determination from our targeted number 2015-07-002BC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 47 of 272 of TMB using publicly available whole-exome cancer sequencing data resulted in high correlation (R2 = 0.902, 0-40 mutations/Mb) and was parallel to the performance of our existing Oncomine Tumor Muta- tion Load Assay (R2 = 0.901, 0-40 mutations/Mb). Empirical analysis and performance of the assay on a common set of cell lines based on a universal reference standard also resulted in a positive correlation. Conclusions A larger tumor only NGS assay was developed to support compre- hensive genomic profiling and routine clinical research in oncology. The assay design and informatics workflow support characterization of mutational signatures and provide normalized TMB estimates. Min- imal input material requirement and rapid sample to report time will have a high impact on clinical research. More detailed information on the assay and an update on performance will be presented. P86 Predictive Immune Modeling enables biomarker discovery in NSCLC patients treated with second line immunotherapy 1 1 2 Natalie LaFranzo, PhD , Steve Daniel, PhD , Walt Carney, PhD , Milan 3 1 Bhagat , Natalie LaFranzo, PhD 1 2 Cofactor Genomics, St. Louis, MO, United States; Walt Carney Biomarkers Consulting, LLC, Boston, MA, United States; TriStar Technology Group, LLC, Washington, DC, United States Fig. 1 (abstract P84). See text for description Correspondence: Natalie LaFranzo (natalie_lafranzo@cofactorgenomics.com) P85 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P86 Development of a pan-cancer NGS assay for detection of tumor mutational burden and targeted biomarkers from FFPE samples Background Dinesh Cyanam, MS, Vinay Mittal, Nickolay Khazanov, Paul Williams, While cancer checkpoint inhibitors have garnered much attention Janice Au-Young, Gary Bee, Sameh El-Difrawy, Aren Ewing, Jennifer due to their ability to generate durable responses and improved sur- Kilzer, Anelia kraltcheva, PhD, Scott Myrand, MS, Yu-Ting Tseng, Cristina vival, the actual number of patients who are eligible for, receive Van Loy, Elaine Wong-Ho, Chenchen Yang, Dinesh Cyanam, MS, treatment with, and respond to these therapies remains modest [1]. Santhoshi Bandla, Warren Tom, PhD, Seth Sadis This is driven by a dependency on legacy diagnostics, built on Thermo Fisher Scientific, Ann Arbor, MI, United States single-analyte biomarkers such as PD-L1, which have failed to cap- Correspondence: Seth Sadis (Seth.Sadis@thermofisher.com) ture the complexity of disease [2]. Even in the case of non-small cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P85 lung cancer (NSCLC), an indication where the benefit of IO therapies is considered significant, there is much to be learned about the biol- Background ogy of the patients who respond, or do not respond to these Next-generation sequencing (NGS) is being applied to support routine therapies. clinical research in oncology with a primary focus on evaluating known Methods oncogenic variants. However, the advent of cancer immunotherapies Multidimensional RNA models have emerged to move beyond requires that clinical research solutions must also address biomarkers these legacy methods to reveal the full scope of disease com- such as Tumor Mutational Burden (TMB) and Microsatellite Instability plexity, resulting in increased predictive accuracy. Leveraging a (MSI) for immune checkpoint inhibitors. Therefore, we developed a re- database of gene expression models built using Predictive Im- search use NGS solution for FFPE tissues that expanded upon our mune Modeling, immune context of the tumor microenvironment current Oncomine Tumor Mutation Load Assay by measuring bio- is quantified. In this study, a cohort of NSCLC patients who re- markers for both targeted and immune checkpoint therapies. ceived second-line immunotherapies (checkpoint inhibitors) were Methods evaluated retrospectively. Pre-treatment solid tumor FFPE tissue Gene content was prioritized based on the relevance and variant samples were processed using the ImmunoPrism immune profil- prevalence of biomarkers in solid tumors. Additional genomic regions ing assay to generate comprehensive, individual immune profiles. were added to supplement the coding sequence footprint to support Pathological, demographic, and survival data (including overall TMB. The assay used Ion AmpliSeq™ technology with automated survival and progression-free survival, indicative of therapy re- templating on the Ion Chef™ system and sequencing on the Ion Gen- sponse), was used to group patients for predictive biomarker eStudio™ S5 sequencing platform. An automated tumor-only work- discovery. flow for variant calling, TMB and MSI estimation and sample quality Results reporting was provided within Ion Reporter Software. Decision sup- Individual immune profiles of the patients are compared, both within port tools were used for variant interpretation and evaluation of po- and between relevant cohorts, and statistically-significant biological tential variant relevance. signals are reported. Machine-learning derived multidimensional Results biomarkers were also generated, which are defined by the optimal Over 500 genes with known DNA and RNA alterations were included. combination of all analytes measured in the assay, enabling improve- The panel has broad capability for variant calling, fusion detection, ments in predictive accuracy. This study represents the first data MSI status, in addition to TMB. Specifically, for TMB, DNA repair path- generated using the ImmunoPrism assay with patients receiving ways were comprehensively represented as alterations in these checkpoint inhibitor therapies. genes may lead to high mutation burden. A coding sequence foot- Conclusions print to support TMB was generated. In development studies, the Predictive Immune Modeling enables us to build multidimensional assay displayed high uniformity and consistent read depth to sup- models of disease. When combined with well-curated patient co- port robust variant calling. The automated workflow required min- horts, such as the NSCLC patients described here, predictive bio- imal input of FFPE tumor only DNA and RNA material. Sample to markers may developed which capture more facets of the complex report turnaround time was less than five days. In-silico assessment immune contexture than previously possible. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 48 of 272 References P88 1. Haslam A, Prasad V. Estimation of the Percentage of US Patients Evaluation of a tumor-only pan-cancer targeted semi-conductor With Cancer Who Are Eligible for and Respond to Checkpoint based next-generation sequencing (NGS) test for microsatellite Inhibitor Immunotherapy Drugs. JAMA Netw Open. 2019 May instability in FFPE samples 1 2 2 3;2(5):e192535. Sameh El-Difrawy, Ph D , Anelia kraltcheva, PhD , Vinay Mittal , Elaine 2 2 2 2 2. Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune- Wong-Ho , Dinesh Cyanam, MS , Seth Sadis , Jennifer Kilzer , Cristina 2 2 2 2 checkpoint blockade: response evaluation and biomarker development. Van Loy , Janice Au-Young, PhD , Aren Ewing , Sameh El-Difrawy Nat Rev Clin Oncol. 2017 Nov;14(11):655-668. ThermoFisher Scientific, South San Francisco, CA, United States; Ethics Approval Thermo Fisher Scientific, Carlsbad, CA, United States The human tissue samples utilized for this study were provided by Correspondence: Sameh El-Difrawy (Sameh.El- TriStar Technology Group and have written, informed donor consent Difrawy@thermofisher.com) permitting academic and commercial research for publication, as Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P88 well as approval from a competent ethical committee. Background Comprehensive genomic profiling using next-generation sequencing (NGS) has become an essential tool to support routine clinical research P87 in oncology. Advent of cancer immunotherapies also requires assess- All-in-One, quantitative immune repertoire profiling of PBMC and ment of immune checkpoint inhibitor biomarkers such as microsatellite FFPE for renal cancer treatment evaluation instability (MSI) and tumor mutational burden (TMB). 1 1 2 Mollye Depinet, MS , Wenjing Pan, PhD , Sang-gin Wu, MD, PhD , MSI arises from defects in the mismatch repair (MMR) system and is as- 1 1 1 Xiaohong Hou, MD PhD , Brittany Brown, BS , Mary Eisenhower, BS , sociated with hypermutability of short DNA sequence repeats, micro- 1 1 3 Daniel Weber , Miranda Byrne-Steele, PhD , Michael Lotze , Jian Han, satellite locations, throughout the genome. Such defects are commonly MD PhD observed in colorectal, gastric and endometrial cancers and have been 1 2 iRepertoire, Huntsville, AL, United States; National Taiwan University, shown to be predictive of response to immunotherapy treatment. Trad- Taipei City, Taiwan, Province of China; UPMC Hillman Cancer Center, itionally MSI testing has been done using single biomarker tests such Pittsburgh, PA, United States as PCR/fragment analysis or immunohistochemistry (IHC) that require Correspondence: Jian Han (jhan@irepertoire.com) high sample input and are time consuming. Therefore, we developed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P87 an RUO NGS solution appropriate for FFPE tissues that addresses bio- markers for targeted and immune checkpoint therapies. Background Methods Next generation sequencing of the immune repertoire is a com- The performance of our RUO NGS based MSI approach was tested in prehensive immune profiling methodology that allows detailed, the context of a large Ion AmpliSeq™ panel composed of more than sequence-specific insight into the adaptive immune response. 13,000 amplicons covering 500+ genes. The content includes a di- While immune repertoire analysis of bulk RNA typically focuses verse set of microsatellite markers targeting MSI locations comprised on a single receptor chain, understanding of the variable rear- of mono- and di-nucleotide repeats that range from 7 to 34 bp. Se- rangements of the immune repertoire as a whole provides a quencing was carried out on the Ion 550™ chip and the Ion GeneStu- broader view of the immune landscape with potential prognostic dio™ S5 system. In-sample standards were designed and value. This is accomplished through the study of all seven TCR incorporated as internal references utilized by the analysis pipeline and BCR chains together (i.e., TCR-alpha, TCR-beta, TCR-delta, and a novel algorithm was developed that leverages the unique sig- TCR-gamma, and BCR-IgK and -IgL). One of the key challenges nal processing properties inherent in semi-conductor sequencing. during immune receptor amplification is the formation of dimers, The test provides results for individual microsatellites and generates which can compete with the immune amplicons of interest dur- an MSI score and status for the sample of interest. ing library preparation. Results Methods The performance of the MSI solution was tested using a set of over We therefore developed a novel PCR technique, dimer avoided multi- 400 FFPE and cell-line samples from different tissue types and plex PCR (dam-PCR), that effectively avoids dimer formation during showed excellent concordance with orthogonal tests. We report on PCR and incorporates unique molecular identifiers for direct RNA the sensitivity and specificity of our tumor only approach and quantification and error removal. With one sample, dam-PCR allows propose ideas to utilize generated MSI score in combination with for the amplification of all seven TCR and BCR loci in a single, quanti- other bio markers. tative multiplex reaction. Here, we apply this method to the amplifi- Conclusions cation of both PBMC and FFPE RNA from renal cancer patients An NGS assay was developed to support comprehensive genomic undergoing treatment. profiling and routine clinical research in oncology. The assay design Results and unique informatics workflow support precise characterization of We found that both TCR-alpha and -beta diversity prior to treatment mutational signatures and provides normalized MSI and TMB esti- along with the expression ratio between B cells and T cells are good mates. The performance of the assay was verified over a large cohort predictors of treatment efficacy. of colorectal, gastric and endometrial cancer samples with MSI status Conclusions independently assigned by orthogonal tests. [For Research use Only. Our study suggests that examining multi-chain immune reper- Not for use in diagnostic procedures] toire composition can be valuable for predicting treatment re- sponse and evaluating treatment protocols. Additionally, this method shows promise for future applications in both clinical P89 settings and basic research, as it allows for a cost effective, all- Obesity related changes in AXL-driven inflammatory signaling inclusive, and quantitative immune-profiling analysis of immune impact survival in melanoma repertoires from a range of sample types, including FFPE, where Alicia Gingrich, MD, Kylie Abeson, BS, Alexander Merleev, PhD, Robert sample RNA may be both limited in quantity and degraded in Canter, MD, MAS, FACS, Emanual Maverakis, Amanda Kirane, MD, Alicia quality. Gingrich, MD Ethics Approval University of California, Davis, Sacramento, CA, United States This study was approved by the University of University of Pittsbur- Correspondence: Alicia Gingrich (agingrich@ucdavis.edu) gh's Ethics Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P89 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 49 of 272 Background The TYRO3, AXL and MERTK (TAM) receptor tyrosine kinase (RTK) family have been associated with a number of human cancers, including melanoma.[1-3] Effects attributed to oncogen- esis and metastasis (epithelial-to-mesenchymal transition) of the TAM receptors have been described.[2] Recent evidence correl- ating obesity with a paradoxical improved response to immuno- therapy in melanoma suggests both tumor microenvironment and clinical phenotype play a role in response.[4] Therefore, we sought to build a predictive model of response to therapy from biomarkers, using TAM receptors and conventional markers of checkpoint inhibition such as PD-1. This model was tested in the normal weight, overweight and obese populations. Methods TCGA-SKCM melanoma tumor mRNA expression and clinical data for metastatic melanoma patients were downloaded from the GDC legacy archive (https://portal.gdc.cancer.gov/legacy-archive) (n = 471).[5] Bio- markers were defined as “high” or “low” expression in each patient. Dif- ferences in Kaplan-Meier survival curves based on level of expression were tested using G-rho family tests. Strength of relationships between biomarkers were measured using Pearson’s correlation. All statistical analysis were performed using R package “survival”. Results Normal weight, overweight and obese patients had markedly dif- ferent biomarker profiles associated with survival (Figure 1). In Fig. 1 (abstract P89). KM curves by clinical phenotype the normal weight population, high CD8 (p=0.0093), PD1 (p= and biomarker 0.0093) and CD84 (p=0.022) were associated with improved sur- vival. In the overweight population, high CD8 (p=0.0098), PD1 (p=0.0004) and CD84 (p=0.0081) were associated with improved P90 survival, while high Gas6 (p=0.029) and MERTK (p=0.043) were as- Unique tumor immune microenvironments of potentially PD-L1/ sociated with decreased survival. And in the obese population, TGF-β trap responsive tumors high AXL expression was associated with improved survival (p= Sean Glenn, PhD, Sarabjot Pabla, MSc, PhD, BS, Erik Van Roey, Jonathan 0.004), while CD8 (p=0.91) and PD1 (p=0.89) demonstrated no as- Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, sociation. In correlation analysis, AXL expression was most closely Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, associated with macrophage markers CD163 (r=0.52), CD84(r= Yirong Wang, MS, Carl Morrison, MD, DVM 0.56) and MS4A4A(r=0.53) in the obese but not the normal OmniSeq, Inc., Buffalo, NY, United States weight population. Correspondence: Sean Glenn (sean.glenn@omniseq.com) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P90 Taken together, these data suggest that immunologic response in metastatic melanoma patients is driven by separate immune Background profiles for obese and non-obese populations. AXL appears to Tumors often do not respond to PD-1/PD-L1 axis inhibitors due to im- mediateresponsein theobesepopulation by a macrophage- mune escape mechanisms present in the tumor microenvironment. Bi- driven mechanism as opposed to T cell mediation. Collectively, functional antibody-based immunotherapies that simultaneously target the significant differences in the transcriptomic profiles between immune checkpoints and immunosuppressive cells are being devel- obese and non-obese patients suggest potential clinical implica- oped to slow tumor growth. Anti-PD-L1/ TGF-β trap fusion proteins are tions regarding targets for treatment and application to patients one approach being tested to counter the traditional immune check- based on clinical phenotype. point inhibition via PD-1/PD-L1 axes and simultaneously inhibit the pro-tumor/anti-inflammatory effects of TGF-β.Inthis study, we not only References describe the tumor immune microenvironment of tumors expressing 1. Dransfield I, Farnworth S. Axl and Mer receptor tyrosine kinases: distinct PD-L1 and TGF-β, but also describe potential patient selection strat- and nonoverlapping roles in inflammation and cancer? Adv Exp Med egies based on gene expression measurements of these tumor im- Biol. 2016;930:113-32. mune microenvironments from clinical samples. 2. Verma A, Warner SL, Vankayalapati H, et al. Targeting Axl and Mer kinases Methods in cancer. Mol Cancer Ther. 2011;10:1763-73. RNA-seq was performed for 395 immune transcripts on 1323 FFPE tu- 3. Wu X, Liu X, Koul S, et al. AXL kinase as a novel target for cancer therapy. mors of diverse histologies. To find true TGF-β high expressing tu- Oncotarget. 2014;5:9546-9563. mors, TGF-β gene expression was normalized by a tumor 4. McQuade JL, Daniel CR, Hess KR, et al. Association of body-mass index inflammatory score (average expression rank of 161 inflammation and outcomes in patients with metastatic melanoma treated wtih tar- genes derived from co-expression signature of 1323 tumors spanning geted therapy, immunotherapy, or chemotherapy: a retrospective, multi- 35 tumor histologies). Proportion of PD-L1 IHC positive, tumor muta- cohort analysis. Lancet Oncol. 2018;19:310-322. tional burden (TMB) high and cell proliferation categories was esti- 5. Guan J, Gupta R, Filipp FV. Cancer systems biology of TCGA SKCM: mated for TGF-β high expressing tumors. Inclusion and exclusion efficient detection of genomic drivers in melanoma. Sci Rep. criteria were developed based on PD-L1 and normalized TGF-β 2015;5:7857. expression. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 50 of 272 Results Results Gene expression revealed varying degrees of TGF-β high tumors RNA-seq libraries from control RNAs at both 10 and 100 ng input in all tumor types studied. Sarcoma, pancreatic cancer and breast have less than 10% rRNA reads, replicate correlations greater cancer had the highest proportion of TGF-β high tumors. Within than 99%, and gene-level and transcript-level detection rates that these TGF-β high tumors, 41% were PD-L1 IHC+ (TPS≥1%), and are highly concordant with a conventional library preparation kit. 28% were TMB-high. 11% (n=147/1323) tumors were both TGF-β Additionally, the data confirms comparable dynamic range and high and PD-L1 high making these tumor microenvironments linearity of response of the ERCC spike-in controls. Libraries pre- ideal for a potential PD-L1/TGF-β trap treatment. Interestingly, pared from FACS-sorted T cells show differential expression pro- 47% (n=69/147) of these tumors presented with strong/moderate files consistent with the expected patterns. inflammation, with 53% (n=78/147) being non-inflamed tumors. Conclusions Conversely, there were 11.7% (n=155/1323) tumors that were The Advanta RNA-Seq NGS Library Prep workflow simplifies the high- TGF-β low and PD-L1 low presenting suboptimal tumor micro- throughput generation of RNA-seq libraries, significantly minimizing environment for a potential treatment. Notably, only 26% (n=40/ hands-on time and costly reagent consumption, which will facilitate 155) of these tumors presented with strong/moderate inflamma- the incorporation of RNA sequencing into the immune-oncology tion with clear majority (74%; n=115/155) being strongly or mod- research toolkit. erately inflamed tumors. For Research Use Only. Not for use in diagnostic procedures. Conclusions This large clinically tested tumor cohort suggests an immune phenotype of potentially PD-L1/TGF-β trap responsive tumors ex- ists across multiple histologies. PD-L1/TGF-β high tumors have distinct immune profiles compared to PD-L1/TGF-β low tumors. A P92 clinical immune gene expression assay described in this study Survival benefits of comprehensive genomic profiling and could not only improve patient selection for anti-PD-L1/TGF-β treatment in metastatic non-small cell lung cancer 1 2 trap treatment, but for other bi-specific fusion protein based Alison Sexton Ward, PhD , Jennifer Johnson, MD ,Komal Gupte- 3 3 immunotherapies. Singh, PhD , Mohammad Ashraf Chaudhary, PhD ,Devender 3 1 1 Ethics Approval Dhanda, PhD , Oliver Diaz, PhD , Katherine Batt, MD, MSc , John De-identified specimens and data were analyzed by OmniSeq under Fox 1 2 IRB approved protocol BDR 080316 (Roswell Park Comprehensive Precision Health Economics, Oakland, CA, United States; Jefferson Cancer Center, Buffalo, NY). University Hospital, Philadelphia, PA, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Priority Health, Grand Rapids, MI, United States Correspondence: Komal Gupte-Singh (Komal.Singh@bms.com) P91 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P92 Expression profiling of T cells using nanoscale automation with a full-length RNA sequencing library preparation kit on a Background microfluidic circuit platform 1 1 1 Metastatic non-small cell lung cancer (mNSCLC) patients who receive Thomas Goralski, PhD , Sangpen Chamnongpol , Michael Phelan , 2 1 1 comprehensive genomic profiling (CGP) at diagnosis may be more Jennifer Snyder-Cappione , Julie Alipaz , Joel Brockman ,Brian 1 1 1 1 likely to receive optimal first line (1L) therapies than patients who re- Fowler , Jennifer A. Geis ,Christopher Kubu ,Raphael Kung , 1 1 1 ceive panel testing (PT) with the enhanced ability to identify bio- Benjamin Lacar , Naveen Ramalingam, PhD ,Mandi Wong ,Charles 1 1 markers with associated therapies. The incremental survival benefits Park ,David King 1 2 of receiving optimal treatments following CGP testing at diagnosis Fluidigm Corp, South San Francisco, CA, United States; Boston have yet to be estimated. University School of Medicine, Boston, MA, United States Methods Correspondence: Christopher Kubu (chris.kubu@fluidigm.com) A Markov simulation model of biomarker testing and treatment Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P91 assignment for mNSCLC was built to estimate the survival out- comes associated with CGP versus PT. Biomarkers identified with Background PT were EGFR, ALK, ROS1, BRAF, and PD-L1 (≥50%). All biomarker RNA sequencing (RNA-seq) provides hypothesis-free profiling of tests were tested simultaneously using single gene testing or transcript levels and isoforms. This profiling captures a compre- assay. CGP, which employed Next-Generation Sequencing, identi- hensive view of the peripheral immune system or the tumor fied all the above biomarker changes and estimated tumor muta- microenvironment. The resulting profiles can be used to tional burden (TMB). The model assumed that PD-L1 testing was characterize differential gene expression patterns that can further conducted together with CGP. Biomarker identification, except for the understanding of the immune system. To further enable TMB and PD-L1, was assumed to be mutually exclusive and to these types of studies we have developed a highly cost-effective, occur at published prevalence rates. Incremental false-negative nanoliter-volume microfluidics-based workflow and chemistry rates of each genetic test in PT relative to CGP were applied. compatible with Illumina® sequencing instruments to simultan- Treatment pathways followed NCCN guidelines and current pub- eously generate RNA-seq libraries from up to 48 samples. This lished clinical trial results. Key inputs and assumptions were method fully automates solid-phase capture of polyadenylated tested in sensitivity analyses. RNA, reverse transcription, and index PCR within a compact nano- Results scale integrated fluidic circuit (IFC) on our Juno™ system. The Patient overall survival for each biomarker test within each test- workflow includes reagents necessary to generate full-length, ing strategy are shown in Table 1. Patients receiving CGP had random-primed RNA-seq libraries from as little as 10 ng of total 8.5% (1.4 months) longer survival on average than those who re- RNA, while preserving strandedness information. ceived PT. Patients receiving CGP testing at presentation spent Methods more time on 1L therapies (40% vs. 33%), thereby less time on Multiple replicates of 10 ng and 100 ng of total RNA from con- 2L therapies (23% vs. 26%) compared to patients receiving PT at trol samples spiked with ERCC RNA Spike-In Mixes were used to presentation. prepare RNA-seq library using the Advanta™ RNA-Seq NGS Library Conclusions Prep Kit. The performance was compared to a conventional li- CGP testing among mNSCLC patients at the time of diagnosis re- brary preparation kit. We also used our platform to profile total sulted in survival gains in comparison to PT due to higher proportion RNA purified from FACS-sorted CD3+, CD8+, CD28–,and CD25+/ of patients receiving optimal 1L treatment. hi T suppressor cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 51 of 272 Table 1 (abstract P92). Overall survival (months) by testing strategy & analyzed using Adaptive immunoSEQ in 125 samples. Tumor im- biomarker mune phenotype classification was done using unsupervised consen- sus clustering based on the expression of ICR genes. Results We have built one of the most extensive high-quality datasets for immunogenomic alterations available so far in colon cancer. Our pre- liminary data supports a positive impact of ICR gene expression in colon cancer cohort: patients with a Th-1 polarized microenviron- ment display better survival. Integrative analysis encompassing som- atic mutation, copy number variations, and transcriptome is ongoing and will be presented at the conference (Figure 1). Conclusions This newly generated immune centric NGS dataset, generated in Qatar, will contribute dramatically to elucidating the genetic determi- nants of immune responsiveness in cancer. Acknowledgements This work was supported by Qatar National Research Fund (QNRF) with grant JSREP07-012-3-005 References 1. Wang, E., Worschech, A. & Marincola, F. M. The immunologic constant of rejection. Trends Immunol. 29, 256–262 (2008). 2. Spivey, T. L. et al. Gene expression profiling in acute allograft rejection: challenging the immunologic constant of rejection hypothesis. J. Transl. Med. 9, 174 (2011). 3. Bertucci, F. et al. The immunologic constant of rejection classification P93 refines the prognostic value of conventional prognostic signatures in The advanced immune-centric NGS cohort for colon cancer breast cancer. Br. J. Cancer (2018). doi:10.1038/s41416-018-0309-1 1 1 2 Wouter Hendrickx, PhD , Jessica Roelands, Master , Peter Kuppen , 4. Galon, J., Angell, H. K., Bedognetti, D. & Marincola, F. M. The Continuum 3 1 1 Francesco Marincola, MD , Najeeb Syed , Davide Bedognetti, MD, PhD of Cancer Immunosurveillance: Prognostic, Predictive, and Mechanistic 1 2 Sidra Medicine, Doha, Qatar; Leiden University Medical Center, Leiden, Signatures. Immunity 39, 11–26 (2013). Zuid-Holland, Netherlands; Refuge Biotechnologies, Half Moon Bay, CA, 5. Roelands, J. et al. Genomic landscape of tumor-host interactions with dif- United States ferential prognostic and predictive connotations. bioRxiv 546069 (2019). Correspondence: Davide Bedognetti (dbedognetti@sidra.org) doi:10.1101/546069 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P93 6. Pagès, F. et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. The Background Lancet 391, 2128–2139 (2018). The immune system has a substantial effect on the progression of 7. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with colon cancer. Typically, an immune response defined by a polarized low memory requirements. Nat. Methods 12, 357–360 (2015). Th1 phenotype, characterized by expression of chemokine-receptor 8. Risso, D., Schwartz, K., Sherlock, G. & Dudoit, S. GC-Content Normalization ligands, activation of interferon-stimulated genes, production of cyto- for RNA-Seq Data. BMC Bioinformatics 12, 480 (2011). toxic molecules by effector immune cells, and upregulation of im- Ethics Approval mune regulatory genes, has been associated with immune-mediated Sidra Medicine IRB approval : #1602002725 tumor rejection. We have previously introduced a gene signature, called Immunology Constant of Rejection (ICR), that reflects these im- mune components.[1–4] This signature was able to differentiate quite well the patients with an active immune environment and improved survival vs those who did not[5]. Virtually, all correlative analyses integrating exome and transcrip- tomic data in colon cancer based on publicly available date use the TCGA cohort. Although it is broadly accepted that T-cell infiltration influences prognosis in colon cancer[6], the association between transcriptomic immune signature and patient survival could not be observed in the TCGA colon cancer cohort. This is likely due to the per protocol exclusion of samples with low tumor purity (i.e., higher stromal/immune infiltration), as at that time TCGA consortium fo- cused on defining cancer genetic makeup. To gain more insight into the underlying mechanism of cancer tissue rejection by the immune system in colon cancer, we build an extensive data repository from high quality snap frozen colon cancer samples unbiased for tumor purity. Methods RNA and DNA were isolated from a cohort of 366 colon cancer pa- tients collected over the last decade at the University of Leiden Med- ical Center (LUMC), Netherlands. Tissue sections flanking the corresponding samples were hematoxylin- and eosin-stained. RNA- seq (HiSeq4000) data was obtained using HISAT2 alignment[7] and quantile normalized after GC-correction of the raw counts.[8] Whole Fig. 1 (abstract P93). The advanced immune-centric NGS cohort for Exome Sequencing (WES) (>100X) was performed for normal and colon cancer cancer tissue (366 RNA-seq and 608 WES). T-cell repertoire was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 52 of 272 P94 A potential mechanism of anti-cancer immune response activated by immune-related adverse events (irAEs) in urological cancer patients 1 1 1 Taigo Kato, MD, PhD , Motohide Uemura , Koji Hatano , Atsunari 1 1 1 2 Kawashima , Takeshi Ujike , Kazutoshi Fujita , Kazuma Kioytani , Norio Nonomura 1 2 Osaka University, Osaka, Japan; Japanese Foundation for Cancer Research, Tokyo, Japan Correspondence: Taigo Kato (kato@uro.med.osaka-u.ac.jp) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P94 Background With the spread of usage of Immune checkpoint inhibitors (ICIs), a certain number of patients face discontinuation of ICIs due to severe immune- related adverse events (irAEs). Recently, some reports have shown en- couraging efficacy among patients who discontinued ICIs, leading to the hypothesis that irAEs-experienced patients have strong and long-lasting anti-cancer immune responses. So far, the molecular mechanisms of the immune response, particularly for T cells that play pivotal roles in attack- ing cancer cells, still remain unclear. Thus, characterization of T cell reper- Fig. 2 (abstract P94). Expanded T cells in metastatic site are toire and immune signatures in peripheral blood mononuclear cells detected in systemic (PBMCs) and tumors before and after ICIs treatment should contribute to better understanding of irAEs-related anti-cancer immune responses. Methods P95 In this study, we collected PBMCs from 4 urological cancer patients, Single-cell RNA-sequencing from clinically relevant core needle before ICIs treatment and at the onset of severe irAEs. For 1 kidney biopsies for evaluation of tumor-immune cell interactions in the cancer patient who had long durable response after discontinuation tumor microenvironment of ICIs, we also collected metastatic tissue sample and applied a next Namit Kumar, PhD, Mohan Bolisetty, Peter Szabo, PhD, Xuan Li, Becky generation sequencing approach to characterize T cell receptor (TCR) Penhallow, Ryan Golhar, Alice Walsh, Saumya Pant repertoires using RNAs isolated from tumors and PBMCs. We also Bristol-Myers Squibb, Princeton, NJ, United States measured mRNA expression levels of immune-related genes in the Correspondence: Namit Kumar (Namit.Kumar@bms.com) PBMCs of pre- and post-ICIs treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P95 Results We found that elevated transcriptional levels of CD3, CD4, CD8, GZMA, Background PRF1, andFOXP3 alongwithhighGZMA/CD3and PRF1/CD3 ratiointhe Elucidating biomarkers associated with immunotherapy response peripheral blood at the onset of irAEs. TCR repertoire analysis revealed and resistance will allow for better informed patient selection and drastic expansion of certain T cell clones in metastatic tissue after irAEs treatment decisions as well as enhanced drug development strategy. (Figure 1). Interestingly, some of these abundant TCR clonotypes were Current biomarker strategies are based on cellular markers (eg, im- also increased in peripheral blood at the onset of irAEs (Figure 2). munohistochemistry) or bulk molecular averages (eg, whole-exome Conclusions sequencing). However, there is limited ability to integrate cellular Our findings revealed that a certain number of expanded- and irAEs- and molecular data. Single-cell RNAseq (scRNAseq) is a promising related T cell clones in cancer tissue may also circulate systemically technology allowing for an unbiased analysis of the tumor microen- and then attack tumor cells in distant regions, leading to durable re- viroment (TME) at cellular resolution. Despite the immense potential, sponse in the patients with irAEs. implementation of this technology in clinical trials has been limited due to lack of methodologies applicable to clinically relevant speci- mens such as core-needle biopsies (CNB). Here, we describe the de- velopment of clinically applicable scRNAseq technology and analysis. Methods Treatment-naïve commercially sourced tumor resections were used to gen- erate ex-vivo CNB for scRNAseq analysis with 10X genomics. Post-clustering, unsupervised cell-type identification was performed (SingleR), and down- stream analyses were carried out (Seurat v2, custom R). Cells from multiple patients/tumor types (endometrial, TNBC, NSCLC, ccRCC, gastrointestinal; n= 8), and healthy donors (peripheral blood mononuclear cells; n=3) were com- bined, batch-corrected and aligned using canonical correlation analysis (CCA); and differential gene expression was performed (MAST algorithm). Results CNB scRNAseq was optimized across 5 tumor types, and the resulting data from ~43,000 cells allowed for the unbiased identification of TME cellular components (stromal, epithelial, immune-cell subtypes). The cellular resolution of this dataset allowed us to identify cell pop- ulations with distinct gene signatures. For example, we identified 2 macrophage subclusters—a lung tumor-specific cluster and a tumor- independent cluster. Lung-specific macrophages showed upregula- tion of genes including SPP1, G0S2, RGCC, PHLDA1, and TREM. Differ- ential gene expression analysis evaluated similarities and differences between TME vs healthy PB cells and allowed for surrogate pharma- codynamics marker assessment. In our analysis, 1197 genes were dif- Fig. 1 (abstract P94). Clonal T cell expansion in ferentially expressed; the most enriched genes in tumor-derived pancreatic metastasis monocytes included HSPA1A, IL8, APOE, and SPP1 whereas PB Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 53 of 272 monocytes were enriched for genes including LGALS2, S100A12, P97 S100A9, AHNAK, and CSTA. TCRB repertoire convergence and clonal expansion define the Conclusions NSCLC tumor microenvironment of responders to anti-PD-1 We have demonstrated the feasibility of scRNAseq from single CNB monotherapy 1 2 2 through the development of protocols to enable identification of bio- Timothy Looney, PhD , Katharina Leonards , Ilaria Alborelli , Luca 1 2 markers related to pharmacodynamics, therapeutic response, or disease Quagliatta , Philip Jermann 1 2 progression. Further, we have optimized the bioinformatics workflow to Thermo Fisher Scientific, Austin, TX, United States; University of Basel, derive meaningful biological insights from these scRNAseq datasets, Basel, Switzerland such as mechanisms involved in immune response or resistance that Correspondence: Philip Jermann (philipmartin.jermann@usb.ch) are tumor extrinsic or intrinsic. Our pilot study sets the groundwork to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P97 explore including scRNAseq in future prospective clinical studies. Background Acknowledgements There is an outstanding need to identify predictive biomarkers for re- Bristol-Myers Squibb. sponse to anti-PD-1 monotherapy for NSCLC. Here we investigated TCRB clonal expansion and TCR convergence within the pretreatment tumor microenvironment as predictors of response in a cohort of 37 P96 FFPE-preserved biopsies. For context, we compared the predictive T-cell receptor alpha and beta repertoire profiling using an value of these features with TMB values from the same tumors. augmented transcriptome Methods Eric Levy, PhD, Pamela Milani, Sean Boyle, PhD, Gabor Bartha, Charles Total RNA from FFPE-preserved pretreatment NSCLC biopsies (11 re- Abbott, PhD, Robert Power, Rena McClory, Robin Li, John West, MBA, sponders, 14 non-responders) was extracted for TCRB repertoire se- Richard Chen quencing via the Oncomine TCRB-SR assay (15-265ng RNA input; Personalis, Inc., Menlo Park, CA, United States average 164ng) and the Ion Torrent Gene Studio S5. TMB values Correspondence: Richard Chen (richard.chen@personalis.com) were obtained from FFPE-preserved gDNA from the same biopsies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P96 using the Oncomine Tumor Mutation Burden Assay. TCR conver- gence and clonal expansion were evaluated independently or in a Background combined model as predictors of response. The promise of immunotherapy has revealed the need for compre- Results hensive profiling of the tumor and its immune microenvironment, in- TCRB sequencing revealed increased TCR convergence (p = .02, Wil- cluding analysis of the T-cell receptor (TCR) repertoire. To address coxon) and clonal expansion (p = .06, Wilcoxon) in those who bene- this challenge, we developed ImmunoID NeXT to provide a more fited from anti-PD-1 therapy. A logistic regression classifier comprehensive view of the tumor and tumor microenvironment combining both features was able to discriminate responders from (TME) from limited FFPE tumor biopsies. This includes profiling both non-responders with a sensitivity of .91 and specificity of .71 at the the TCR alpha and beta chains. We show that ImmunoID NeXT accur- optimal cutoff, per the Youden’s J method. The TCR-based classifier ately and reproducibly profiles abundant clones and provides infor- was able to identify responders who otherwise had low to intermedi- mation on the diversity of T-cells in tumor samples. ate (<10muts per Mb) TMB. Methods Conclusions We first analyze the reproducibility of ImmunoID NeXT using repli- TCRB clonal expansion and convergence warrant further evaluation cates of PBMCs. Then, we compare the concordance of clones from as potential predictive biomarkers of response. Importantly, TCRB se- ImmunoID NeXT to the top clones from a standalone TCR sequen- quencing may allow for identification of responders who are other- cing approach. We also analyze the reproducibility of clones in wise missed by TMB-based stratification. patient-derived FFPE samples, and compare to IHC quantification of CD3+ cells to highlight the intra-sample heterogeneity of T-cell abun- dance and diversity. We then analyze the clonal diversity of pre- P98 treatment tumor samples in a cohort of melanoma patients who Automated rarefaction analysis for precision human and mouse B underwent PD-1 blockade. Finally, we use ImmunoID NeXT to profile and T cell receptor repertoire profiling from peripheral blood and the clonal diversity across over 100 solid tumor samples. FFPE-preserved specimens Results Timothy Looney, PhD, Geoffrey Lowman, PhD, Michelle Toro, Jayde Abundances of clones shared between replicates of PBMC samples Chang, Denise Topacio-Hall, BS, MA, Loni Pickle, PhD, Fiona Hyland, have a very high concordance (R2>0.99 with both TRA and TRB). Timothy Looney, PhD Compared to the standalone TCR approach, we identify over 96% of Thermo Fisher Scientific, Austin, TX, United States the top 1000 TRA clones, and over 99% of the top 1000 TRB clones, Correspondence: Timothy Looney (timothy.looney@thermofisher.com) both with highly concordant abundances (R2>0.95 and R2>0.94 in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P98 TRA and TRB, respectively). Subsequent curls of a tumor FFPE sample also have a high concord- Background ance of clonal abundances (R2>0.89 and R2>0.91 in TRA and TRB, re- Identifying the optimal input amount and sequencing depth for B spectively). TCR sequencing also provides a view of the clonal diversity and T cell receptor repertoire profiling is challenging owing to vari- of T-cells in a sample, which is not available with quantification via IHC. ation in material quality and lymphocyte diversity in blood and FFPE Finally, in a melanoma cohort, clonality based on either TRA or TRB is preserved specimens. Rarefaction analysis has emerged as a potential significantly different in responders to checkpoint inhibition. approach for assessing whether immune repertoire libraries have Conclusions been sequenced to saturation. Here we present a novel automated The ImmunoID NeXT platform can provide insight into the diversity method for saturation analysis of IGH and TCRB chain libraries de- of the immune repertoire, highlighting the platform’s ability to pro- rived from sequencing of peripheral blood leukocytes (PBL) and vide comprehensive analysis of both the tumor and tumor micro- FFPE-preserved RNA and DNA. environment. We demonstrate that ImmunoID NeXT is reproducible, Methods sensitive, and accurate at profiling high-abundance TRA and TRB Human TCRB and IGH repertoire libraries were generated using the clones, as well as feasible with FFPE samples. We also highlight how Oncomine TCRB-SR and BCR IGH-SR assays from: (1) 25ng PBL total immune repertoire results from ImmunoID NeXT can be used to gain RNA (2) 500ng PBL gDNA (3) 150ng RNA from FFPE preserved NSCLC understanding about the immunological composition of the TME. Fi- and (4) 200ng gDNA from FFPE preserved brain tissue. Mouse TCRB nally, we show how ImmunoID NeXT can profile the diversity of the and IGH libraries were generated using the Ion Ampliseq TCRB-SR TCR repertoire in tumor samples. and BCR IGH-SR assays and 25ng RNA or 500 ngDNA derived from Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 54 of 272 spleen or lymph node. Libraries were sequenced on the Ion Torrent P100 Gene Studio S5 then analyzed with Ion Reporter to identify clono- Impact of obesity on immunity in gastroesophageal types, quantify clonal expansion and diversity, and for IGH chain li- adenocarcinoma [GEAC] 1 2 1 braries, identify B cell clonal lineages and assess isotype usage. We Sarbajit Mukherjee, MD, MS , Sami Ibrahimi , Yali Zhang , Jianmin 1 1 then repeated clonotyping and analysis of secondary repertoire fea- Wang , Pawel Kalinski, MD, PhD tures using data that had been downsampled to fixed read depths. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Results University of Oklahoma, Oklahoma City, United States We observed an asymptotic relationship between the sequencing depth Correspondence: Sarbajit Mukherjee and the number of B and T cell clones detected, clone Shannon diversity, (sarbajit.mukherjee@roswellpark.org) and B cell clonal lineage richness and diversity, indicating that libraries Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P100 had been sequenced to saturation. By contrast, T and B cell normalized Shannon entropy appeared robust to sequencing depth. Background Conclusions Obesity is associated with an elevated risk of GEAC [1], but the Automated downsampling analysis may serve as a convenient tool molecular mechanism remains unknown. Paradoxically, however, for optimizing sequencing depth and input amount for B and T cell obesity is associated with a superior response to anti-PD-1 treat- repertoire sequencing studies. We expect this approach to become a ment [2,3]. This may be explained by our recent observations routine component of immune repertoire analysis. that obesity enhances PD-1 mediated T-cell dysfunction in a mechanism involving leptin signaling. Prompted by this data, we aimed to identify obesity/leptin-regulated molecular biomarkers in P99 GEAC. TMBler: a bioinformatic tool for measuring and optimizing Tumor Methods Mutational Burden quantification from targeted sequencing panels Based on the body-mass index (BMI), we categorized patients Laura Fancello, Luca Mazzarella, MD PhD, Alessandro Guida, Arnaud into normal (BMI 18-24.9), overweight (BMI 25-29.9) and obese Ceol, Piergiuseppe Pelicci, Luca Mazzarella, MD PhD (BMI ≥30). We then retrospectively analyzed the clinical report of IEO Istituto Europeo di Oncologia IRCCS, Milano, Italy PD-L1 staining by IHC_22C3/Keytruda from metastatic GEAC pa- Correspondence: Luca Mazzarella (luca.mazzarella@ieo.it) tients treated at our institution between 2014-2019. Chi-squared Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P99 test was used to determine the association between categorical variables. Next, we performed RNA-seq analysis of 13 gastric can- Background cer FFPE specimens (8 obese and 5 normal weight) to identify Tumor mutational burden (TMB) is increasingly proposed as a predict- differential gene expression between these two groups. Gene ex- ive biomarker for immunotherapy response in cancer patients. pression was quantified by log-fold changes. Differentially TMB assessed by Whole Exome Sequencing (WES) is considered the gold expressed genes were identified by using DESeq2. Then we standard but remains confined to research settings. Targeted enrichment looked at the association between the expression of leptin and panels of various genomic sizes are emerging as a more sustainable method- immune-related genes from those specimens, using generalized ology for assessing TMB in the clinical setting. However, panel-based TMB linear model implemented in DESeq2. TCGA gastric cancer data- quantification has not been adequately standardized to date, leading to base (TCGA -STAD) was used to validate these associations (using major heterogeneities in TMB measurement and a lack of uniformly accepted Pearson test) independently. A p-value of <0.05. cutoff values, thus limiting the possibility to transfer results across settings. In Results particular, the choice of variants to include in TMB calculation (synonymous, Our analysis of the clinical report of 77 patients with metastatic cancer driver genes or low-allelic frequency mutations, or other features) may GEAC revealed that patients with a BMI =>25 were more likely to strongly affect results and in particular TMB predictive value [1] express PD-L1 than normal-weight individuals (p = 0.03)(Table 1). Methods Our RNA-seq analysis identified the following genes to be up- We developed "TMBler", an R package to calculate TMB from targeted regulated in the obese group: NOS2, FOXP3, IDO1, EOMES, sequencing panels. TMBler allows to select multiple filters on mutation CD160, and CXCR5 (p<0.05). Expression of these genes was posi- counts for TMB quantification. It also includes a set of functions to tively correlated with leptin in our database; however, these asso- simulate custom panels on WES and calculate predictive value based ciations did not reach statistical significance; possibly due to our on available data on immunotherapy response matched with sequen- small sample size. The same analysis within the TCGA-STAD data- cing data [2,3]. Finally, it allows to measure panel-based TMB concord- base identified a strong positive correlation between the expres- ance with WES-based TMB and its predictive value using Receiver sion of all six genes and leptin (p <0.05)(Figure 1). GSEA Operating Characteristic (ROC) curves. identified several up-regulated immune-related pathways (Adap- Results tive Immune System, Antigen Processing Cross Presentation etc.) By simulating custom and commercially available panels, we show that the in the obese group. application of specific filter combinations can significantly influence TMB cal- Conclusions culation and its predictive value, and we identify instances where risk of erro- Our preliminary data suggest that obesity, and specifically lep- neous assignment of patients to responder/nonresponder groups is highest tin, is associated with several immune markers in GEAC. Our Conclusions mechanistic studies will explore how obesity/ leptin regulates TMBler is a useful tool for quantifying TMB from targeted panels. It the immune system and promotes cancer. These studies may can analyze performance of existing panels, optimize analytical pipe- allow us to identify new leptin regulated pathways as thera- line and design novel custom panels through simulations. peutic targets. References References 1. Fancello L, Gandini S, Pelicci PG, Mazzarella L. Tumor mutational burden 1. Garai J, Uddo RB, Mohler MC, Pelligrino N, Scribner R, Sothern MS et al. quantification from targeted gene panels: major advancements and At the crossroad between obesity and gastric cancer. Methods Mol challenges. J Immunother Cancer. 2019 Jul 15;7(1):183 Biol.2015; 1238:689-707. 2. Hellmann MD, Nathanson T, Rizvi , et al. Genomic Features of Response 2. Ibrahimi S, Mukherjee S, Roman D, King C, Machiorlatti M, Aljumaily R. to Combination Immunotherapy in Patients with Advanced Non-Small- Effect of Body Mass Index and Albumin level on Outcomes of Patients Cell Lung Cancer. Cancer Cell. 2018 Receiving Anti PD-1/PD-L1 Therapy. J Clin Oncol 36, 2018 (suppl 5S; abstr May 14;33(5):843-852 213). 3. Samstein RM, Lee CH, Shoushtari AN. Tumor mutational load predicts 3. Wang Z, Aguilar EG, Luna JI, Dunai C, Khuat LT, Le CT et al. Paradoxical survival after immunotherapy across multiple cancer types. Nat Genet. effects of obesity on T cell function during tumor progression and PD-1 2019 Feb;51(2):202-206 checkpoint blockade. Nat Med. 2019 Jan; 25(1):141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 55 of 272 Ethics Approval P101 The study was approved by the Institutional Review Board at Roswell Park Immune-based classification of pleural malignant mesothelioma by Comprehensive Cancer Center, approval number STUDY00000894 / BDR using integrative transcriptome analysis 1 2 1 2 109419. Ernest Nadal, MD, PhD , Ania Alay , David Cordero , Elisabeth Aliagas , 1 1 3 3 José Ruffinelli , Ramón Palmero , Ricard Ramos , Ivan Macía , Anna 3 3 1 Ureña , Fran Rivas , Xavier Solé 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; Bellvitge Biomedical Research Institute, L'Hospitalet, Spain; Bellvitge University Hospital, Table 1 (abstract P100). See text for description L'Hospitalet, Spain Correspondence: Xavier Solé (x.sole@iconcologia.net) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P101 Background Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia. Immune checkpoint inhibitors in MPM demonstrated modest efficacy, partly due to lack of predictive biomarkers of clinical benefit from immunotherapy. The aims of this work were: to identify immune fractions associated with clinical outcome; to stratify MPM patients based on their immune contexture and to characterize the immune-based groups at the genomic and transcriptomic levels. Methods Seven gene-expression datasets of MPM were used to assess theimmunemicroenvironmentof516 samples. The abundance of 20 immune fractions in each sample was inferred using Gene Set Variation Analysis. Identification of clinically-relevant frac- tions was performed with Cox Proportional-Hazards Models ad- justed for age, stage, sex, and tumor histology. Results T-Helper 2 (Th2, HR=2.14, p=1.5x10-4) and cytotoxic T cells (CTC; HR=0.57, p=9.1x10-3) were found to be consistently asso- ciated with overall survival in multiple datasets. Three immune clusters (IG) were subsequently defined based on Th2 and CTC immune infiltration levels: IG1 (54.5% of samples) had high Th2/ low CTC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) had low Th2/high CTC levels. Immune clusters were associated with overall survival independently of tumor histology, with an improving survival from IG1 to IG3 (HR IG2=0.52, 95% CI 0.39–0.69; HR IG3=0.32, 95% CI 0.19–0.53; p= 8.4x10-8; Figure 1). IG3 was significantly enriched in epithelioid tumors (90% IG3 vs. 62% IG1, p=0.001) and patients were youn- ger compared to the other groups(60 yearsIG3 vs. 66years IG1, p=0.021). These groups showed differential molecular pro- files, being IG1 enriched for CDKN2A and IFN-related genes de- letions. No statistically significant differences in the tumor mutational burden was observed, howerver IG3 tumours had fewer mutations than IG1 and IG2 groups. At the transcriptional level, IG1 samples showed upregulation of cell proliferation and DNA repair-related gene-sets, while IG3 samples presented up- regulation of immune checkpoint inhibitors (Figure 2) and inflammation-related pathways. Finally, integration of gene ex- pression with functional signatures of in vitro drug response showed that IG3 patients are more likely to respond to immune checkpoint inhibitors, while IG1 patients might be more sensi- tive to PARP inhibitors. Conclusions Analysis of publicly available gene-expression data of MPM reveals three major immune-based groups, based on Th2 and CTC composition. These clusters are associated with distinct genomic profiles and clinical outcome. Further validation of this classification is warranted in an independent cohort of Fig. 1 (abstract P100). See text for description MPM. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 56 of 272 P102 CT antigens are frequently expressed non-inflamed tumors Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS, Sarabjot Pabla, MSc, PhD, BS , Erik Van Roey, Sean Glenn, PhD, Jonathan Andreas, MS, Blake Burgher, BS, RN, Jeffrey Conroy, BS, Mary Nesline, MS, Antonios Papanicolau-Sengos, MD, Vincent Giamo, BS, MS, Felicia Lenzo, Yirong Wang, MS, Carl Morrison, MD, DVM OmniSeq, Inc., Buffalo, NY, United States Correspondence: Sarabjot Pabla (sarabjot.pabla@omniseq.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P102 Background Cancer testis (CT) antigens are tumor antigens that have a highly tis- sue restricted expression in germ cells but are often expressed in di- verse malignancies. With their highly immunogenic expression limited to tumor cells, CT antigens have become a prime target for cancer vaccinations and T-cell based therapy with chimeric T-cell re- ceptors. In this study, we investigated the association of two CT anti- gens (NY-ESO-1 and LAGE-1a) with the immune microenvironment of real-world clinical tumors spanning multiple histologies. Furthermore, we describe the association of CT antigens with traditional bio- markers of immunotherapy such as PD-L1 immunohistochemistry (IHC) and tumor mutational burden (TMB), with inflammatory status and cell proliferation status with confirmatory studies performed on a large TCGA pan-cancer cohort of 11,001 tumors. Methods Unsupervised clustering was performed on gene-expression data of 395 immune transcripts of 1323 FFPE tumors to reveal three inflam- matory patient clusters and three distinct gene groups; CT-antigen, inflammatory and neoplastic clusters. Test for proportions was per- formed using Pearson’s chi-squared test to describe association of NY-ESO-1 and LAGE-1a with PD-L1 IHC, TMB, inflammatory cluster and cell-proliferation. A retrospective cohort (n=242) of checkpoint Fig. 1 (abstract P101). Overall survival analysis according to the inhibition (CPI) treated tumors was utilized to perform overall survival immune groups (Kaplan-Meier curves) and response to CPI therapy for CT antigen+ tumors. Survival analysis was confirmed against the Pan-Cancer TCGA cohort (n=11,001). Results Unsupervised clustering showed clear co-expression sub-clustering of CTA genes differentiated from “immune” and from “neoplastic expres- sion”. PD-L1 IHC status was not associated with NY-ESO-1 (p=0.71) or LAGE-1a (p=0.52) status. Interestingly, LAGE-1a positive cases were over-represented in TMB high cases (p=0.016), whereas, NY-ESO-1 sta- tus was not associated with TMB. NY-ESO-1 positive cases were highly over-represented in non-inflamed cluster (p=0.006), whereas, LAGE-1a status was not associated with inflammation status. Both NY-ESO-1 (p= 0.031) and LAGE-1a (p=0.008) were significantly associated with cell- proliferation status. NY-ESO-1 positive tumors have significantly (p= 0.014) higher response rate in retrospective cohort but this was not ob- served for LAGE-1a status. NY-ESO-1 and LAGE-1a status showed trend toward better (p=0.09 and p=0.06 respectively) survival in the retro- spective and TCGA pan-cancer cohort. Conclusions This study presents an in-depth analysis of the immune landscape of CT antigen positive tumors across multiple histologies. CT antigen bearing tumors not only have unique immune profiles but also have significant associations with biologically relevant emerging bio- markers such as inflammatory signature, TMB and cell proliferation. CT antigens are a viable target for non-inflamed tumors for check- point inhibition therapy. Ethics Approval De-identified specimens and data were analyzed by OmniSeq under IRB approved protocol BDR 080316 (Roswell Park Comprehensive Fig. 2 (abstract P101). Expression of immune checkpoint markers Cancer Center, Buffalo, NY). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 57 of 272 P103 Methods Detection of human leukocyte antigen class I loss of Stage I-III subjects (n=24) receiving curative-intent chemo (doxo- heterozygosity in solid tumor types by next-generation DNA rubicin, cyclophosphamide, paclitaxel) were monitored longitudin- sequencing ally (mixed effects linear model) with serial peripheral blood Jason Perera, PhD , Brandon Mapes, PhD, Denise Lau, PhD, Ameen mononuclear cell flow cytometry and quantitative immunose- Salahudeen, Aly Khan, PhD quencing of the T-cell receptor β locus (TCRseq) using the immu- Labs, Chicago, IL, United States noSEQ® assay (Adaptive Biotechnologies, Seattle, WA). To evaluate Correspondence: Jason Perera (jason.perera@tempus.com) for long-term chemo effects, these analyses were repeated in a Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P103 cohort of recurrent breast cancer patients who received chemo >12 months preceding analysis (n=9). Wilcoxon rank sum and Background tests of slope were employed to screen for associations with Human leukocyte antigen (HLA) class I proteins are expressed on the chemo response, defined as complete pathologic response (pCR) surface of all nucleated cells and are vital for immune surveillance. at surgical resection. When tumor-specific mutations (neoantigens) are presented on HLA Results molecules to CD8+ T cells, this recognition can drive immune re- By TCRseq, chemo resulted in an acute decline in T-cell fraction (0-8 sponses against the tumor and lead to tumor destruction. One mech- weeks, p12 months following chemo. anism of immune escape for tumors is loss of heterozygosity in HLA Conclusions genes (HLA-LOH), which reduces the total number of neoantigens avail- Curative-intent chemo is associated with T-cell death followed able for presentation to T cells. Due to the highly polymorphic nature by reconstitution, with the resulting T-cell repertoire being more of HLA, the copy number status of HLA genes is extremely challenging clonal and less abundant in naïve T cells. These findings persist to assess by standard bioinformatics approaches. To investigate the at the time of metastatic recurrence, and therefore may contrib- prevalence of HLA-LOH, we developed a specialized pipeline to detect ute to immunotherapy non-response in metastatic disease. Con- HLA-LOH by DNA next-generation sequencing (NGS). versely, we identified T-cell reconstitution as a potential Methods biologic modifier of chemo response. T-cell reconstitution can A cohort of colorectal and non-small cell lung cancer samples be therapeutically targeted with inhibitors of androgen receptor underwent DNA sequencing on the Tempus xT panel using signaling, which in experimental models enables thymic matur- paired, formalin-fixed, paraffin-embedded tumor and normal ation of naïve T-cell clones and an increase in peripheral T-cell (blood or saliva) samples. To detect HLA-LOH from NGS data, we count. This hypothesis is beingevaluatedinanongoingphase used NGS-based HLA typing to resolve the patient’smostprob- II clinical trial of bicalutamide (androgen receptor antagonist) able HLA haplotype. Based on this haplotype, we adaptively rea- plus ipilimumab and nivolumab in metastatic breast cancer ligned reads, extracted a number of features describing the (NCT03650894). relative allele coverage in the tumor and normal samples, and Ethics Approval used these features to make a confident determination of allelic The study was reviewed and approved by Providence Heath and Ser- loss in the patient’s tumor sample. vices Internal review board, approval number 15-162. Results Evidence of HLA-LOH was detected in 16% of non-small cell lung tumor samples and 17% of colorectal tumor samples. We did not ob- P105 serve a significant association between LOH status and tumor muta- PD-L1 isoform as a potential biomarker to predict response for tional burden or neoantigen load. In the colorectal cancer cohort, anti-PD-(L)1 treatment HLA-LOH was observed in tumor samples classified as microsatellite Kunbin Qu (kunbin.qu@beigene.com) instability-high (MSI-H); however, the association between HLA-LOH BeiGene, USA, San Mateo, CA, United States status and MSI status was not statistically significant. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P105 Conclusions We developed a novel method of determining HLA-LOH by DNA Background NGS and demonstrated that HLA-LOH is a readily detectable feature anti-PD-1/anti-PD-L1 (anti-PD-(L)1) therapies have shown clinical ac- in human tumors. These results highlight the complexity of antigen tivity across different cancers. However, predicting patient response presentation, the potential importance of HLA-LOH as a biomarker of remains challenging. Here we explore PD-L1 splicing isoforms as a immunotherapy response and resistance, and lays the groundwork potential predictive biomarker for anti-PD-(L)1 therapy response. Four for future investigations. PD-L1 splicing isoforms exist, including one dominant wildtype tran- script and an alternative isoform which skips the second exon (del- taExon2_PD-L1). P104 Methods Impact of chemotherapy (chemo) on peripheral T-cell diversity and TCGA normalized mRNA transcript counts were downloaded implications for subsequent immunotherapy response in breast from Genomic Data Commons. anti-PD-1 treated melanoma cancer 1 2 3 RNA-Seq data was from Hugo et. al. [1]. Bioinformatics analyses Joanna Pucilowska, PhD , Paul Fields, PhD , Valerie Conrad, BS , David 3 3 3 were performed in statistical package R. Human wildtype and Page, MD , Alison Conlin, MD , Joanna Pucilowska, PhD , Catherine 2 3 3 3 deltaExon2_PD-L1 isoforms were stably transfected into the Sanders, PhD , Raina Tamakawa, MS , Brie Chun, MD , Isaac Kim, MD , mouse cell-line BW5147. A chimeric PD-1 receptor, P3Z, which Mark Schmidt 1 2 fuses the extracellular and transmembrane domains of human Providence Cancer Center, Portland, OR, United States; Adaptive PD-1 to the cytoplasmic domain of human CD3ζ, was stably Biotechnologies, Seattle, WA, United States; EACRI Providence Cancer transfectedintoHuT78 cellsasa reporter assay for PD-1 signal- Center, Portland, OR, United States ing and IL-2 production [2]. Correspondence: David Page (david.page2@providence.org) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P104 By examining protein crystal structures from Protein Data Bank, we found exon2 occupies the physical interface between PD-1 and PD- Background L1. It is also the interface between anti-PD-L1 therapeutics and PD- Immune checkpoint blockade is only modestly effective in metastatic L1. Therefore, anti-PD-L1 molecules may not effectively target PD-L1 breast cancer. One potential contributing factor is chronic lymphode- in patients harboring the deltaExon2_PD-L1 isoform and may lack pletion associated with preceding curative-intent chemo. Here, we clinical activity. The prevalence of the deltaExon2_PD-L1 isoform evaluate the short and long-term effects of chemo on peripheral T- across TCGA tumors is shown in Figure 1. There are 8 cancers where cell counts and clonal diversity in a cohort of breast cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 58 of 272 the isoform is present above 5%, including liver and endometrial cancers. The deltaExon2_PD-L1 isoform was successfully transfected into BW5147 as demonstrated by mRNA expression. In co-cultures of HuT78/P3Z with BW5147/PD-L1 (both the wildtype and deltaEex- on2_PD-L1), IL-2 was secreted from the wildtype but not from the deltaExon2_PD-L1. Incubation with anti-PD-1 reduced IL-2 in a dose- dependent manner with the wildtype only (Figure 2), indicating del- taExon2_PD-L1 does not support PD-1 signaling. Patients expressing only deltaExon2_PD-L1 or a higher ratio of deltaExon2_PD-L1/wildtype may not have optimal PD-(L)1 axis signaling and be less responsive to anti-PD-1 intervention. To test this hypothesis, a ratio metric between deltaExon2_PD-L1 and wildtype was applied to an anti-PD-1 treated melanoma cohort GSE78220. This biomarker ratio stratified responders from non- responders with a p-value of 0.027 (non-responders with no del- taExon2_PD-L1 isoform were excluded, Figure 3), whereas PD-L1 expression did not. Conclusions Patients with deltaExon2_PD-L1 isoform lack the interface be- tween PD-L1 and PD-1, the same interface necessary for anti-PD- L1 therapeutic binding. This may lead to non-optimal signaling through the PD-(L)1 axis. Suboptimal signaling and inability to bind anti-PD-L1 potentially could reduce response to both anti- PD-1 and anti-PD-L1 treatments. This hypothesis needs to be fur- ther validated in additional anti-PD-L1 and anti-PD-1 treated Fig. 2 (abstract P105). See text for description cohorts. Acknowledgements The authors would like to thank Vanitha Ramakrishnan and Jessica Li for scientific discussions. References 1. Hugo W. et. al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. Cell 2016; 165:35- 2. Zhang T. et. al. The binding of an anti-PD-1 antibody to FcγRΙ has a pro- found impact on its biological functions. Cancer Immunol Immunother. 2018; 67:1079-1090. Fig. 3 (abstract P105). See text for description P106 Tumor mutational burden profile (TMB) of oncogenic driver mutations in non small cell lung cancer Paul Walker, MD, Nitika Sharma East Carolina University, Greenville, NC, United States Correspondence: Nitika Sharma (sharman@ecu.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P106 Background Tumor mutational burden has emerged as a potential biomarker pre- dictive of response to Immune checkpoint blockade (ICB) in lung cancer. The utility of this biomarker in oncogenic driver mutations, that account for nearly 20-50% of NSCLC, is still unknown. KRAS mu- tation in lung cancer is a prognostic biomarker whereas EGFR and Fig. 1 (abstract P105). See text for description BRAF pathogenic mutations are predictive of response to tyrosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 59 of 272 kinase inhibitors (TKI). ICB with bevacizumab has demonstrated clin- TMB was higher in current and former smokers (median TMB 10.7 v ical benefit in EGFR mutated lung cancers per IMpower150 clinical 6.4 mutations/megabase(Mb); p < 0.0001), patients without an identi- trial [1]. TMB analysis between actionable/pathogenic EGFR muta- fiable oncogenic driver mutation (median TMB 14.5 v 8.5 mutations/ tions (i.e. exon 19 del, exon 21 L858R, T790M) and EGFR uncommon/ Mb; p = 0.004), and patients with locoregional disease at the time of variants mutations may provide therapeutic implications [2]. To ex- diagnosis (median TMB 10.8 v 8.0 mutations/Mb, p = 0.02). TMB cor- plore the immunological basis for these findings, we evaluated the related closely across all matched tumor pairs (Pearson’s r = 0.85, Fig- immune biomarker profile of NSCLC patients using Caris next- ure 1). Significant increases or decreases in TMB were uncommon in generation sequencing (NGS) platform. paired samples, and we observed no significant change in median Methods TMB with increasing time between specimen collection or with inter- We studied tissue samples on 446 patients with NSCLC from 2016- vening chemotherapy, immunotherapy, radiation therapy, or tar- 18. TMB was measured by counting all non-synonymous somatic mu- geted therapy. tations per megabase of the genome coding area using targeted Conclusions NGS (592 genes). High TMB was defined as ≥ 10 mut/Mb. The ana- In NSCLC, TMB correlated closely across tumor pairs, and increasing lysis was conducted using SAS 9.4. Variables were tested using a Wil- time between sample collections and intervening treatments were coxon signed-rank test. not correlated with significant changes in TMB. Results Ethics Approval KRAS mutations were found in 85 pts (19%), BRAF in 9 pts (2%), EGFR This study was conducted under Dana-Farber/Harvard Cancer Center mutation in 36 pts (8%), EGFR pathogenic mutation in 22 pts (5%), Protocol 02-180. EGFR variants in 14pts (3%). The median TMB of KRAS mutant vs KRAS wt (wild type) was 10 vs 7 mut/Mb (range 0-31, p<0.01). Conclusions This study highlights the unique immune profile of certain oncogenic driver mutations in NSCLC. Our results show that KRAS and BRAF mu- tant subsets have a significantly higher TMB than KRAS and BRAF wild type. In addition, EGFR variants have a higher TMB as compared to actionable pathogenic EGFR mutations. These findings could have therapeutic implications in guiding patient selection for ICB and merit a prospective investigation. References 1. Socinski M, Jotte R,Cappuzzo F. Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC. N Engl J Med. 2018; 378:2288-2301. 2. Offin M, Rizvi H,Tenet M.Tumor Mutation Burden and Efficacy of EGFR- Tyrosine Kinase Inhibitors in Patients with EGFR-Mutant Lung Cancers. Clin Cancer Res. 2018;1102. Ethics Approval The study was approved by ECU Institutional Review Board, approval number UMCIRB 15-001311. P107 Changes in tumor mutational burden in serially biopsied non-small cell lung cancer 1 1 1 James Smithy, MD, MHS , James Smithy, MD, MHS , David Hwang, MD , 2 2 3 1 Yvonne Li , Liam Spurr , Andrew Cherniack, PhD , Lynette Sholl , Mark Awad, MD PhD 1 2 Brigham and Women's Hospital, Boston, MA, United States; Dana- Farber Cancer Institute, Boston, MA, United States; Broad Institute of MIT and Harvard, Boston, MA, United States Fig. 1 (abstract P107). See text for description Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P107 P108 Background Working towards precision medicine of the tumor High tumor mutational burden (TMB) has been associated with re- microenvironment 2 2 2 2 sponse to checkpoint blockade in non-small cell lung cancer (NSCLC) Kyung Kim, PhD , Jeeyun Kim , Seung-Tae Kim , Jung-Yong Hong , 1 1 and other malignancies. However, the degree to which TMB changes Laura Benjamin , Kristen Strand-Tibbitts, PhD 1 2 over time, across anatomical sites, and with intervening treatment re- Oncologie Inc, Waltham, MA, United States; Samsung Medical Center, mains unknown. To evaluate TMB changes across time points, we Seoul, Republic of Korea compared TMB in tissue specimens from patients with serially- Correspondence: Kristen Strand-Tibbitts biopsied NSCLC. (kristen@oncologie.international) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P108 Clinicopathologic characteristics and changes in TMB were analyzed from patients with NSCLC and more than one tissue specimen that Background had undergone targeted next generation sequencing (NGS, OncoPa- Immune therapies for cancer have generated an enhanced focus on nel) at the Dana-Farber Cancer Institute. Those representing distinct controlling cancer through modulation of biologies associated with primary tumors by histologic or genomic analysis were excluded. the tumor microenvironment, rather than the traditional approach of Results targeting cancer cell biology. As more and more targeted therapies 193 NSCLC patients with more than one interpretable NGS result are designed to modulate the tumor microenvironment, we need a were identified; 30 were excluded due to separate primary tumors. better understanding of microenvironmental heterogeneity in human Of the 163 remaining patients included in the analysis, the median cancer. Similar to what has been done to describe patient subsets time between samples was 14 months (range: 0 to 114 months). based on their cancer biology using DNA and RNA signatures, we are Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 60 of 272 working to describe patient subsets based on their microenviron- nivolumab treatment. The CD8 signature may be used alone and/or in ment biology. The ultimate goal is to find effective means of identify- combination with other relevant and potentially independent bio- ing patients for novel therapeutic treatments that target biological markers such as PD-L1 expression or TMB to identify patients likely to pathways that regulate the non-neoplastic cells and drive cancer benefit from anti–PD-1 therapies. progression. Methods Acknowledgements RNA from publicly available sources including microarray and RNA- Bristol-Myers Squibb. Professional medical writing and editorial assistance Seq were analyzed with respect to gene signatures that describe four were provided by Katerina Pipili, PhD, and Jay Rathi, MA, of Spark Medica Inc, different microenvironmental phenotypes. funded by Bristol-Myers Squibb. Results Trial Registration These four microenvironmental subtypes are prognostic, but also NCT02387996 show evidence of being predictive to existing modalities of cancer drugs when analyzed in retrospective analysis. We examined the im- References pact of cancer stage on the distribution of these subtypes and find 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene little variation. signature with CD8 expression by immunohistochemistry (IHC) in Conclusions multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. Future clinical trials to prospectively test these four unique signatures 2. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- as predictive biomarkers for therapy need to be designed. lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. 3. Galsky M, Saci A, Szabo P, et al. Impact of tumor mutation burden on P109 nivolumab efficacy in second-line urothelial carcinoma patients: explora- Predictive performance of a CD8-derived signature by gene tory analysis of the phase II CheckMate 275 study. Ann Oncol. 2017; expression profiling in patients with urothelial carcinoma from 28(Suppl 5): Abstract 848PD. CheckMate 275 Ethics Approval 1 2 1 Peter Szabo, PhD , Padmanee Sharma, MD, PhD , George Lee, PhD , The protocol was approved by site institutional review boards or 1 1 1 1 Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , Robin independent ethics committees and conducted according to Good 1 1 1 1 Edwards, PhD , Saumya Pant , Abdel Saci , Neeraj Adya , Matthew Clinical Practice guidelines, per the International Conference on Galsky, MD Harmonisation. Patients provided written informed consent based on 1 2 Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD Declaration of Helsinki principles. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Institute, New York, NY, United States Correspondence: Peter Szabo (Peter.Szabo@bms.com) P110 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P109 Tumor CD8+ T-cell infiltration assessed by gene expression profiling alone or by immunohistochemistry plus epithelial- Background mesenchymal transition gene expression in urothelial carcinoma in Gene expression profiling (GEP) has been used to identify biomarkers CheckMate 275 1 1 2 of response to immunotherapy. Using a GEP-based inflammation Peter Szabo, PhD , Abdel Saci , Padmanee Sharma, MD, PhD , George 1 1 1 1 1 assay, we derived and analytically validated a CD8 signature to assess Lee, PhD , Scott Ely , Vipul Baxi, MS , Keyur Desai, PhD , Lisu Wang , 1 1 1 T-cell infiltration in the tumor microenvironment (TME) [1]. Here, we Scott Chasalow , Michael Montalto , Robin Edwards, PhD , Saumya 1 1 1 3 retrospectively explore the association of the CD8 signature, alone Pant , Neeraj Adya , Bruce Fischer, MD , Matthew Galsky, MD 1 2 and in relation to established biomarkers PD-L1 and tumor muta- Bristol-Myers Squibb, Lawrence Township, NJ, United States; MD tional burden (TMB), with clinical response to nivolumab treatment. Anderson Cancer Center, Houston, TX, United States; Tisch Cancer Methods Institute, New York, NY, United States In the phase 2 CheckMate 275 trial, 270 patients with platinum- Correspondence: Peter Szabo (Peter.Szabo@bms.com) resistant metastatic urothelial carcinoma (UC) and evaluable tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P110 PD-L1 expression received nivolumab treatment. Responses were de- termined by blinded, independent review committee assessments Background [2]. Minimal follow-up time for the current analysis was ~3 years. T- Close proximity of CD8+ T cells to cancer cells has been associated with cell infiltration in the TME was assessed using the CD8 signature and improved outcome with immunotherapy. Using a gene expression pro- by immunohistochemistry (IHC) using an automated commercial pro- filing (GEP)-based inflammation assay, we previously derived gene sig- prietary assay (Dako mouse clone C8/144B; Agilent Technologies Co) natures that defined CD8+ T-cell infiltration (CD8 signature) and [1]. PD-L1 expression on tumor cells was independently assessed by localization to tumor parenchymal and stromal compartments (CD8- IHC using the PD-L1 IHC 28-8 pharmDx assay (Dako). TMB was mea- topology signatures) in multiple tumor types [1,2]. In patients with sured by whole exome sequencing [3]. Cox proportional-hazards re- urothelial carcinoma (UC), high stromal/epithelial-mesenchymal transi- gression assessed the dependence of progression-free survival (PFS) tion (EMT) gene expression has been associated with T-cell exclusion or overall survival (OS), and logistic regression assessed the depend- and poor response to immunotherapy [3]. Here, we assess three CD8- ence of objective response (OR) on biomarker values. The linear ef- derived signatures and compare them with a CD8 immunohistochemis- fects of biomarkers and their multiplicative interaction were included try (IHC)-derived score combined with EMT gene expression (CD8.IH- when multiple biomarkers were evaluated. Likelihood-ratio tests (2- C_EMT) to evaluate associations between these biomarkers and with sided) were used to assess biomarker interaction effects. Associations response to nivolumab in patients with UC in CheckMate 275 [4]. with PFS and OS were investigated using Kaplan–Meier analyses with Methods biomarker scores categorized by tertile. 270 patients with platinum-resistant metastatic UC received nivolumab, Results with response assessed by blinded central review [4]. CD8+ T-cell infil- GEP was evaluable in 205 (76%) and GEP+TMB in 113 (42%) of 270 tration in the TME (assessed using the CD8 signature [1], CD8-topology treated patients. Baseline characteristics, OR, PFS, and OS were similar signatures (parenchymal, stromal) [2], and by IHC using a proprietary between all treated patients and the GEP-evaluable cohort. CD8 signa- commercial assay [Dako mouse clone C8/144B antibody; Agilent Tech- ture scores showed a positive association with OR (P=0.005), PFS (P= nologies Co]) and PD-L1 expression on tumor cells (Dako PD-L1 IHC 28- 0.005), and OS (P 8 pharmDx) were assessed on baseline tumor samples. Predictive per- Conclusions formance of the CD8 signature and CD8-topology signatures individu- These results suggest that the GEP-based CD8 signature may have util- ally, the combined CD8-derived signatures (triple CD8), and ity as a potential biomarker for predicting clinical response to CD8.IHC_EMT, was evaluated using Cox proportional-hazards regression Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 61 of 272 for overall and progression-free survival (OS, PFS) and with logistic re- (NGS). Although the 0.4 cutoff identifies MSI-high status, there is in- gression for objective response (OR). Odds ratios were scaled to reflect sufficient data for this score's repercussion for MSI-stable patients [1]. the difference between the 75th and 25th biomarker percentiles. Two- MSI-high status rarely occurs in lung cancer patients representing sided likelihood-ratio tests were used to assess biomarker and inter- less than 1% of the cases. Therefore, we aim to identify how MANTIS action effects. Associations with PFS and OS were also investigated score correlates with immune profile and clinical outcomes in MSI- using Kaplan–Meier analyses with biomarker scores categorized by stable lung cancer. tertile. Methods Results MANTIS score was calculated for two TCGA (The Cancer Genome GEP was evaluable in 205/270 (76%) patients. Baseline characteristics Atlas) cohorts: squamous cell carcinoma (SqCC, n= 501) and adeno- and clinical outcomes were similar in the overall population and the carcinoma (ADC, n=517). After excluding MSI-high patients (n=3 and GEP-evaluable cohort. Response and survival predictions from the 1, respectively) we stratified each cohort into quartiles. The highest triple CD8 and CD8.IHC_EMT overlapped, and both biomarkers pre- quartile was named MANTIS-high (M-H) and the lowest quartile dicted benefit from nivolumab independent of PD-L1 expression. MANTIS-low (M-L). Immune profile (immune cell infiltration and PD- Odds ratios for OR were 2.59 (95% CI, 1.59–4.21) for triple CD8, 2.12 L1 expression), tumor mutational burden (TMB), neoantigen burden (1.47–3.07) for CD8.IHC_EMT, 2.51 (1.42–4.43) for the CD8 signature, and survival outcomes were compared between M-H and M-L. Tumor and 1.74 (1.22–2.49) for the parenchymal CD8-topology signature. immune landscape was identified using signatures from immune Conclusions metagenes predicting infiltration for 31 immune cells. Combined CD8 and CD8-topology gene signatures (triple CD8) Results showed similar performance to CD8.IHC_EMT for predicting response M-H was associated with higher activated CD4, gamma delta and and survival in nivolumab-treated patients with UC. These data sug- Th17 T cell infiltration when compared with M-L in lung SqCC (all gest potential utility of testing biomarker combinations and support p <0.05). No statistically significant difference in tumor T cell infil- further evaluation of gene signatures associated with parenchymal vs tration was found in ADC (Figure 1,2). M-H patients had a higher stromal CD8+ T-cell localization for predicting response to immuno- TMB when compared with M-L patients in ADC (p<0.05) and the therapy in patients with cancer. same tendency was observed for SqCC (p=0.10) (Figure 3). Add- itionally, M-H correlated with lower PD-L1 (CD274) expression in Acknowledgements both SqCC and ADC (each p<0.05) when compared with M-L. No Bristol-Myers Squibb. Professional medical writing and editorial assistance significant differences in neoantigen burden were demonstrated. were provided by Bernard Kerr, PGDipSci, and Jay Rathi, MA, of Spark Medica M-H patients showed a trend towards lower median overall sur- Inc, funded by Bristol-Myers Squibb. vival in SqCC and ADC (75 vs 63 months p=0.21; 53 vs 50 Trial Registration months, p=0.14, Figure 3C,3D). NCT02387996 Conclusions This is the first report that illustrates the implications of a microsatel- References lite instability score on immune landscape, PD-L1 expression, TMB 1. Szabo PM, Qi Z, Zerba K, et al. Association of an inflammatory gene and clinical outcome from a pool of more than a thousand MSS non- signature with CD8 expression by immunohistochemistry (IHC) in small cell lung cancer patients. multiple tumor types. J Clin Oncol. 2019; 37(Suppl): Abstract 2593. 2. Szabo PM, Lee G, Ely S, et al. CD8+ T cells in tumor parenchyma and Reference stroma by image analysis and gene expression profiling: potential 1. Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam biomarkers for immuno-oncology therapy. J Clin Oncol. 2019; 37(Suppl): AM, Waldner MJ, Bindea G, Mlecnik B, Galon J, Trajanoski Z. Abstract 2594. Characterization of the immunophenotypes and antigenomes of 3. Wang L, Saci A, Szabo PM, et al. EMT- and stroma-related gene expres- colorectal cancers reveals distinct tumor escape mechanisms and novel sion and resistance to PD-1 blockade in urothelial cancer. Nat Commun. targets for immunotherapy. Genome Biol. 2015; 16:64. 2018; 9:3503. 4. Sharma P, Retz M, Siefker-Radtke A, et al. Nivolumab in metastatic urothe- lial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. Lancet Oncol. 2017; 18:312-322. Ethics Approval The protocol was approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation. Patients provided written informed consent based on Declaration of Helsinki principles. P111 Clinical and immunologic implications of a microsatellite instability score in lung cancer 1 1 1 Pedro Viveiros, MD , Misuk Lee , Bhoomika Sukhadia, MD , Kyunghoon 1 2 2 1 Rhee , Victor Wang , Jeffrey Chuang , Young Kwang Chae, MD 1 2 Northwestern University, Chicago, IL, United States; Jackson Laboratory For Genomic Medicine, Farmington, CT, United States Correspondence: Misuk Lee (misuklee55@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P111 Background Microsatellite instability status is currently used to predict susceptibil- Fig. 1 (abstract P111). Immune landscape in squamous ity to immunotherapy. MANTIS score was originally developed to cell carcinoma identify microsatellite instability through next-generation sequencing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 62 of 272 Conclusions This rich and large dataset illustrates the power and scalability of the 10x Genomics Chromium Single Cell Immune Profiling Solution with Feature Barcoding technology and presents an exciting opportunity for researchers to explore and draw further conclusions about the mechanisms of TCR–pMHC interaction. Furthermore, this experiment serves as the next step on the path toward the even larger-scale ex- periments that will be necessary to fully comprehend the rules of antigen recognition in the adaptive immune system in response to cancer and infectious diseases and will be key in the development of successful immunotherapies. Acknowledgements This study was performed in collaboration with our 10x Genomics partners Fig. 2 (abstract P111). Immune landscape in adenocarcinoma Immudex and Biolegend. P112 P113 A new way of immunity exploration by linking highly Dynamic analysis and visualization of the immune infiltration in multiplexed antigen recognition to immune repertoire and human cancer by integrating TCGA data phenotype Mingchao Xie, PhD, Bolan Linghu, PhD, Zhongwu Lai, PhD, Jonathan 1 1 1 Dagmar Walter, PhD , Stephane Boutet , Michael Stubbington, PhD , Dry, Ben Sidders 1 2 1 1 Katherine Pfeiffer , Josephine Lee , Luz Montesclaros , Julia Lau , Daniel AstraZeneca, Waltham, MA, United States 1 1 3 Riordan , Alvaro Martinez Barrio , Liselotte Brix, PhD , Kivin Jacobsen, Correspondence: Jonathan Dry (Jonathan.Dry@astrazeneca.com); Ben 3 4 4 1 PhD , Bertrand Yeung , Xinfang Zhao , Tarjei Mikkelsen Sidders (benjamin.sidders@astrazeneca.com) 1 2 10x Genomics, Pleasanton, CA, United States; 10x Genomics.com, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P113 Pleasanton, CA, United States; Immudex, Copenhagen, Denmark; Biolegend, San Diego,CA, United States Background Correspondence: Tarjei Mikkelsen (tarjei@10xgenomics.com) Understanding of the complex interplay between tumors and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P112 their immunologic microenvironment is critical for immune- oncology (IO) studies, which can facilitate the discovery of Background novel prognostic biomarkers, identification of new drug targets, Recent progress in cancer immunotherapy emphasizes the and determination of drug resistance mechanisms. However, importance of understanding immune-regulatory pathways in due to a lack of proper analysis tools and datasets, systematic- cancer. It has been shown that immune cells play a crucial role ally exploring the tumor–immune interaction is still a big in the tumor microenvironment and can be used for targeted challenge. therapeutics. Therefore, it is important to understand and Methods characterize T cells and their antigen binding specificity and di- Here, we deconvoluted the immune cell compositions and per- versity in order to develop effective targeted immunotherapies. formed IO-related pathway/signature enrichment analysis for 9,721 Recent technological advancements have enabled the integra- primary tumor samples from 33 TCGA cancer types using transcrip- tion of simultaneous cell-surface protein, transcriptome, immune tomic data, and developed a web-based application, IO Browser. repertoire and antigen specificity measurements at single cell Results resolution, providing comprehensive, scalable, high-throughput The browser allows the user to visualize the immune infiltrations of a characterization of immune cells. sample or cohort, and to define disease segments or “immuno-types” Methods based on the presence of single/multiple immune cell types or IO- Using the 10x Genomics Single Cell Immune Profiling Solution related pathway/signatures. Users can then perform survival compari- with Feature Barcoding technology in conjunction with Biolegend sons, explore gene expression of key cancer and IO genes as well as oligo-conjugated antibodies and Immudex DNA barcoded generate oncoprints in the different segments. The browser also pro- peptide-MHC Dextramer® (pMHC), we performed multi-omic vides statistical analysis to identify the gene or mutations enriched in characterization of CD8+ T cell recognition of various virus and the immuno-typed disease segment, and correlate gene expression common cancer antigens in normal patients. Next generation se- or mutations with specific immune cell types in tumor microenviron- quencing libraries were made following the 10x Genomics work- ment (TME). flow, where gene expression and immune repertoire libraries are Conclusions generated alongside libraries from DNA barcodes conjugated to In summary IO Browser enables comprehensive analysis and antibodies or pMHC, allowing quantification of cell surface pro- visualization of the dynamic interactions between tumor and im- teins and identification of T cell receptor (TCR) specificities. Ana- mune landscape, and can aid our understanding of the interplay be- lysis was performed using the latest version of Cell Ranger (v3.0). tween tumor genomics and immune biology to facilitate line of sight The TCR-dist algorithm was used to identify clusters of related and disease segmentation. TCR sequences and enriched CDR3 motifs. Results P114 We performed multi-omic characterization of ~100,000 CD8+ T cells Tissutal immune profile and pathological complete response in from four MHC-matched donors. The multi-omic combination of triple negative breast cancer gene expression, paired alpha/beta T cell receptor (TCR) repertoire, 1 1 1 Andrea Botticelli, MD , Bruna Cerbelli , Simone Scagnoli , Maria Ida cell surface proteins and pMHC binding specificity allowed the identi- 1 1 2 2 Amabile , Alessandro De Luca , Lucio Fortunato , Leopoldo Costarelli , fication of CD8+ T cell subpopulations with specificity for pMHCs 1 1 1 Marianna Nuti, PhD , Giulia D'Amati , Paolo Marchetti within our panel. Within our data, we observed TCRs with cognate 1 2 Sapienza University of Rome, Rome, Italy; San Giovanni Addolorata antigens that had been reported previously, while also identifying Hospital, Rome, Italy entirely new TCR–pMHC interactions. In addition, we observed spe- Correspondence: Bruna Cerbelli (bruna.cerbelli@uniroma1.it) cific expanded non-naïve T cell clones along with more diverse bind- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P114 ing in the naïve compartment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 63 of 272 Background spectrometry to deeply characterize global tumor proteomes to In the neoadjuvant setting, pathological complete response (pCR) is identify proteins and pathways that are associated with pre- more frequently achieved by triple negative breast cancer subtype treatment response to anti-PD-1 immunotherapy. and patients who attain this status show improved survival; However, Methods standard neoadjuvant therapy results in pCR rates slightly over 30% Unbiased, data-independent acquisition (DIA) mass spectrometry of cases. The mechanism underlying the resistance to chemotherapy was used to analyze formalin fixed paraffin imbedded (FFPE) is still unclear and could be related both to the molecular heterogen- tumor tissue samples from subjects with Stage III-IV melanoma eity of cancer cells and to the activation of the treatment-related im- which were resected prior to initiation of first-line anti-PD-1 ICI mune response. For this reason, the search for immune biomarkers therapy. The selected samples represent two distinct clinical sub- able to predict the response to chemotherapy represents a new groups; those who received clinical benefit, with a partial re- promising frontier. Recent reports underscore the role of TILs, PDL-1 sponse or better (PR, SD and CR, n = 13), and those with no and CD73. The aim of our work is to define a novel tissutal immune clinical benefit (PD, n = 9) and no observable response to ther- profile (TIP) able to predict pCR [1,2]. apy. Samples were prepared for mass spectrometry using stand- Methods ard procedures. All samples were analyzed using 2-hour gradients We enrolled 61 pts who received NAC (EC for 4 cycles followed by on a LC-MS/MS setup operated in DIA mode. Data was extracted Paclitaxel q7 for 12 cycles or q21 for 4 cycles) between Jan 2011 and using Spectronaut (Biognosys) with a sample specific spectral li- June 2017 at Policlinico Umberto I and San Giovanni Addolorata Hos- brary which was combined with a large human tissue resource li- pital of Rome. We performed, in basal paraffin-embedded biopsies, brary. Statistical analysis was conducted to identify proteins that stromal TILS evaluation and immunohistochemistry for PD-L1 (Ven- are either up- or down-regulated with respect to benefit group. tana SP142 clone) evaluated both on tumor cells (TC) and tumor- Pathway analysis was also conducted to highlight dysregulated infiltrating immune cells (IC) and CD-73 assessed on TC. We defined biological functions and pathways. “positive tissutal immune profile” (TIP+) the pts with “high TILS” Results (>50%), “PD-L1 positive” ( >1% both on TC and IC ) and “low CD73” 7,590 proteins were quantified across all samples, with 6,627 (<40%), and the others as “negative tissutal immune profile” (TIP- quantified on average per sample. Univariate statistical testing ).Statistical analysis was performed with T di Student test and χ2 test. between groups identified 254 proteins that are dysregulated Results (120 up-regulated and 134 down-regulated) in subjects who re- We enrolled 61 females (median age: 50 y; range 28-75) affected by ceived clinical benefit. Through partial least squares discriminant TNBC. The clinical stage before NAC was as follow: 3 pts cT3 (5%), 3 analysis (PLS-DA) a set of 25 proteins was identified that describe pts cT4 (5%) and 28 pts were cN+ (38%). Twenty-three patients the variance between the two sample groups. When annotated (38%) showed pCR. No significant associations were found between to their sub-cellular location, all up-regulated species are identi- pR and cT, cN, age, and KI-67. Seven patients (11%) were TIP+ and fied as mitochondrial proteins, indicating an enhanced metabolic achieved pCR in 100% of cases; 54 patients were TIP- and pCR were environment, and the down-regulated species are cytosolic, lyso- showed in 16/54 of cases (30%) (p< 0,001). somal or membrane associated. This observation was also Conclusions reflected in pathway analysis which identified up-regulation of ar- TIP+ seems to be associated with higher pCR rate in TNBC patients ginine and citrulline metabolism and down-regulation of adhesion .These preliminary results suggest the possibility of using novel pro- related processes driven by MHC-II and integrins. files combining multiple immune- biomarkers. Conclusions Global profiling of the tumor proteome provides a unique References characterization of melanoma tumor biology. A pathway level ana- 1. Matsumoto H, Koo SL, Dent R, Tan PH, Iqbal J. Role of inflammatory lysis shows increased metabolic processes combined with decreases infiltrates in triple negative breast cancer. J Clin Pathol. 2015;68:506–510. in adhesion related proteins may underly the differences in benefit doi: 10.1136/jclinpath-2015-202944 139. related to ICI therapy. 2. Jiang T, Xu X, Qiao M et al. Comprehensive evaluation of NT5E/CD73 Ethics Approval expression and its prognostic significance in distinct types of cancers. The study was approved by the Istituto Nazionale Tumori IRCCS Fon- BMC Cancer. 2018 Mar 7;18(1):267. doi: 10.1186/s12885-018-4073-7. dazione “G. Pascale” of Napoli Institutution‘s Ethics Board, approval PubMed PMID: 29514610; PubMed Central PMCID: PMC5842577. number 33/17. Ethics Approval Consent CE 4181 Sapienza University of Rome Written informed consent was obtained from the patient for pub- lication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this P115 journal. Deep proteomic characterization of FFPE tumor samples from late- stage melanoma subjects treated with anti-PD-1 immunotherapy 1 1 Nicholas Dupuis, PhD , Jakob Vowinckel, PhD , Domenico Mallardo, P116 2 2 2 2 MD , Mariaelena Capone, MD , Madonna Gabriele , Antonio Sorrentino , Centrifuge-free red blood cell lysis and immunostaining of whole 2 1 2 Vito Vanella , Daniel Heinzmann , Paolo Antonio Ascierto, MD blood for flow cytometry using Laminar Wash™ system 1 2 Biognosys AG, Schlieren, Switzerland; Istituto Nazionale Tumori IRCCS, Ira Kim, Melvin Lye, Chyan Ying Ke, Nadiezda Fernandez Oropeza, Naples, Italy Sigeeta Rajaram, Kong Leong Cheng, Ih Chin Kon, Royce Pek, Namyong Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) Kim, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P115 Curiox Biosystems, Boston, MA, United States Correspondnce: Namyong Kim (namyong@curiox.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P116 Immune checkpoint inhibitors (ICI) have improved the treatment options for patients with advanced stage melanoma, with im- Background proved clinical responses and overall survival compared to stand- Blood cells are prime indicators of immuno-surveillance, and the ease ard systemic therapies. However, a large percentage of melanoma of blood sampling makes blood analysis a key interest for clinical patients do not respond to ICIs, highlighting the need for a and research applications. While current flow cytometry methods are greater understanding of the tumor environment and host im- high-throughput and provide fine resolution in the segregation of mune response. Here, we apply unbiased discovery proteomics, white blood cell (WBC) populations, WBC enrichment involving red based on label-free data independent acquisition (DIA) mass blood cell (RBC) lysis are laborious and typically performed manually, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 64 of 272 contributing to experimental variability especially as blood cells are Clonality assessments ; dualplex and multiplex immunohistochemis- sensitive to physical and chemical stress. try coupled to digital pathology analyses to assess Immune Cells Infil- Methods tration and PD-L1 mediated inhibition (Immunoscore® IC), Immune We describe RBC lysis and leukocyte immunostaining on a Suppression through Regulatory T cells and Myeloid-derives suppres- centrifuge-less platform Laminar Wash™, using a novel wall-less plate sor cells quantification, T-Cell Exhaustion status ; standardized and laminar flow washer. The Laminar Wash™ 24-well plate consists methods for assessment of endothelial activation markers ; flow cy- of an array of hydrophilic spots surrounded by hydrophobic surface, tometry for circulating immune cell subtypes quantification; ICI which functions as a virtual wall that separates each spot. Each well plasma exposure levels. A multimodal integrative Immunogram pres- is capable of staining and lysing 100uL of whole blood. During lysis, entation is proposed for each patients. WBCs settle to the surface of the spot, allowing the spent lysis buffer Conclusions to be removed by a gentle and continuous laminar-flow washing This preliminary study shows that multimodal immune profiling is process on the Laminar Wash™ system, eliminating centrifugation feasible and could be a new tool to understand the biology and and resuspension that may stress cells and disrupt antibody binding. pharmacology of lung cancer resistance to anti-PD1/L1 ICIs and po- Results tentially guide patient management décisions. We observed improved retention of CD45+ lymphocytes while lysing on Laminar Wash™ plates compared to conventional centrifuge Acknowledgements tubes. In studies comparing mouse whole blood lysis and antibody This work is supported by the French National Cancer Agency, Agence staining by conventional tube centrifuge and Laminar Wash, Laminar Nationale du Cancer, through the PIONeeR project financing. Wash achieved dramatically higher staining index and improved Trial Registration resolution of cell cluster by flow cytometry. ClinicalTrials.gov Identifier: NCT03493581 Conclusions Ethics Approval In summary, Laminar Wash system provides gentle, fast and convenient The study was approved by the French Ethic Comitee CPP Ouest II Angers, blood lysis, while improving data quality with superior antibody staining. approval number 2018/08. P117 P118 Immunogram to decipher PD1/L1 ICI resistance: a proof of concept HYDRA platform development to investigate Siglec-engaging in advanced Non-small cell lung cancer patients of the PIONeeR tumor immunosuppressive glyco-codes Project Li Peng, PhD, Adam Petrone, Adam Shoemaker, Jillian Prendergast, PhD, 1 2 3 Florence Monville, PhD , Frederic Vely , Joseph Ciccolini , Florence Zakir Siddiquee, Jenny Che, Lihui Xu, BS, Karl Normington, PhD, MBA, 2 2 1 4 Sabatier , Stephane Garcia , Vanina Leca , Marion Fabre , Christelle James Broderick, Li Peng, PhD 4 1 4 1 Piperoglou , Pernelle Outters , Laurent Arnaud , Laurent Vanhille, PhD , Palleon Pharmaceuticals, Waltham, MA, United States 1 1 1 Caroline Lauge, BA , Anna Martirosyan, Dr , Aurelie Collignon , Marie Correspondence: Li Peng (lpeng@palleonpharma.com) 5 6 7 2 Roumieux , Julien Mazieres , Maurice Perol , Françoise Dignat-George , Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P118 2 2 1 Eric Vivier , Fabrice Barlesi, MD, PhD , Jacques Fieschi, PhD 1 2 3 HalioDx, Marseille, France; AMU, APHM, Marseille, France; AMU, APHM, Background 4 5 IPC, Marseille, France; APHM, Marseille, France; AMU, Marseille, France; The glyco-immune checkpoint (Siglec/sialoglycan axis) has emerged 6 7 Toulouse Universitary Hospital, Toulouse, France; Centre Leon Berard, as a new mechanism of cancer immune escape and offers new thera- Lyon, France peutic interventions to overcome resistance to current immunother- Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) apies. Siglecs (sialic acid-recognizing Ig-superfamily lectins) are type I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P117 transmembrane sialoglycan binding proteins expressed on various immune cells (innate and adaptive). Humans express at least four- Background teen unique Siglecs which have distinct preferred sialoglycan ligands. In the management of advanced Non-Small Cell Lung Carcinoma Tumors upregulate certain sialoglycan patterns to facilitate immune (NSCLC), PD1/L1 immune checkpoint inhibitors (ICIs) have increased cell evasion by engaging these inhibitory Siglec receptors. This tumor overall survival (OS) over standard second-line chemotherapy. While inhibitory “glyco-code” consists of a heterogenous mixture of numer- this long-term increase in OS is driven by about 20% of patients, others ous sialoglycans, binding to Siglecs through low affinity and high display disease progression during the first weeks. PIONeeR workpack- avidity interactions. age 2 aims to understand and eventually predict response and/or re- Methods sistance to those ICIs in stage IV or recurrent NSCLC patients. For that Deciphering the hypersialylation glyco-code of tumors is key to iden- purpose, an Immunogram was designed that integrates a comprehen- tifying cancer patients for glyco-immune checkpoint blockade ther- sive set of biomarkers measured in the tumor microenvironment. apies. However, the heterogeneity and complexity of sialoglycans Methods make characterization of the tumor surface sialoglycome difficult The immune contexture from the PIONeeR trial’s patients is being with current technologies. To overcome this challenge, we developed characterized in a prospective manner and will be confronted to clin- a proprietary sialoglycan-probing reagent, HYDRA, to functionally de- ical data at the end of the study. This multi-modal approach, encom- tect inhibitory tumor sialoglycans engaging Siglecs. HYDRA mimics passing a range of immune scoring assays, is applied to blood and this natural avidity driven Siglec-sialoglycan interaction, consisting of tumor biopsy from each patient, both sampled before and through- multimeric fusions of a Siglec N-terminal extracellular domain con- out anti-PD1/L1 ICI treatment. This work aims at describing pre- taining the carbohydrate recognition domain (CRD), a trimerization treatment samples profiling. motif, and a Fc dimerization domain. Results Results We assessed the feasibility of such a profiling and provide descriptive We have generated several HYDRA constructs with robust expression multi-modal immune profiles for the 10 first PIONeeR-included pa- using a mammalian HEK293 system. Size-exclusion chromatography tients. These profiles combine raw results from more than ten tests, profiles of HYDRA demonstrate high purity and confirmed multimeric corresponding to the following technologies and biomarkers: Gen- assembly. HYDRAs have greater than fifteen-fold increase in binding omic Next Generation Sequencing for Tumor Mutational Burden affinity compared to Siglec-Fc dimers as measured using bio-layer (TMB), DNA mismatch-repair deficiency (MSI/MSS status) and T Cell interferometry Octet. HYDRA also demonstrates sialoglycan-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 65 of 272 binding, as its binding was eliminated when cells were treated with P120 sialidase (which removes terminal sialic-acids of sialoglycan) or using Evaluation of CD8 score by automated quantitative image analysis cells lacking sialoglycans from knocking out UDP-GlcNAc 2- in metastatic melanoma treated with PD1 blockade: preliminary Epimerase. Glycan array binding of HYDRA confirmed similar sialogly- results can preferences of its Siglec counterpart as described in the litera- Anjali Rohatgi, MD PhD, Douglas Hartman, Arivarasan Karunamurthy, ture, suggesting engineering did not alter glyco-recognition Julie Burkette, Yana Najjar, MD, John Kirkwood, MD, Hassane Zarour, MD, properties. These high-affinity and sialoglycan-specific HYDRAs en- Liron Pantanowitz, Diwakar Davar, MD abled us to develop a robust immunohistochemistry (IHC) assay to UPMC, Pittsburgh, PA, United States analyze cancer patient samples. A cohort of tissues (>2,500 patients) Correspondence: Diwakar Davar (davard@upmc.edu) from various indications were analyzed to enable indication Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P120 prioritization for glyco-immune checkpoint therapies. HYDRA IHC on healthy and cancerous human tissues demonstrate unique binding Background patterns with concordance between duplicate primary tumor cores PD1 blockade produces responses in 30-40% of metastatic melan- and primary tumor versus metastatic cores from the same patient in oma (MEL) with durable relapse-free benefit [1,2]. Pre-existing tumor- non-small cell lung, kidney and colon cancer samples. infiltrating CD8+T cell infiltrates (TIL), neoantigen burden and IFN-γ Conclusions gene expression signature (GES) correlate with clinical anti-tumor re- In summary, the HYDRA technology distills the structural hetero- sponse [3-5] to PD1 blockade. However, neoantigen burden and IFN- geneity of tumor surface sialoglycans to a straightforward func- γ GES are cost-prohibitive and time-consuming assays that are not tional readout of immunosuppressive glyco-codes engaging available for clinical use; while CD8 T cell analysis by immunohisto- inhibitory Siglecs, which may allow patient stratification based on chemistry (IHC) is cost-effective and operator-independent. The aim deciphering a tumor-specific surface glycan pattern. of this study is to develop and validate an image analysis algorithm to automatically quantify CD8+ T cells (CD8 score) in patients with metastatic MEL treated with PD1 blockade. P119 Methods Analytical validation of run-to-run and site-to-site performance of Included patients had advanced metastatic MEL treated with PD1 a human immune profiling assay and automated data analysis blockade. Radiographic response assessed using RECIST v1.1. For the solution for CyTOF mass cytometry technology purposes of this analysis, patients were defined as responders (R; Clare Rogers (clare.rogers@fluidigm.com) complete, partial response, stable disease) or non-responders (NR; pro- Fluidigm, South San Francisco, CA, United States gressive disease). Pre-treatment tumor biopsies from 58 patients were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P119 utilized. Brightfield image analysis results were cross-validated with fluorescence-based quantification (AQUA™). A nuclear image algorithm Background designed to run on whole slide images was optimized to manual count. Immune profiling is an essential method for quantifying changes The algorithm was locked down and used on a cohort of whole tissue in immune population numbers and states over time in health sections from MEL patients. All images were reviewed by independent and disease. A cornerstone in translational and clinical research, pathologist blinded to clinical outcomes. Response and outcomes were it is frequently used to investigate chronic inflammation, infec- statistically correlated with image analysis results. tious disease, autoimmune diseases, and cancer. The diversity of Results immune populations demands a high parameter approach to There were 40 R patients and 18 NR patients. Median CD8 score was more fully and efficiently quantify these changes. Mass cytometry, 101 cells/mm3 in R and 48.7 cells/mm3 in NR (p=0.098). Median PFS which utilizes CyTOF® technology, is a single-cell analysis platform were greater in R compared to NR (18 months vs. 2 months, p100 that hasusedasmanyas50 metal-taggedantibodies[1] to re- cells/mm3 (64%). solve discrete cell populations, all in a single tube of sample. It is Conclusions an ideal solution for routine enumeration of immune cell We report the successful technical development and clinical valid- populations. ation of an image algorithm to automate CD8 score for metastatic Methods MEL treated with PD1 blockade. Preliminary results demonstrate We have developed a sample-to-answer solution for human im- CD8 score was directly associated with response and improved mune profiling using mass cytometry: the Maxpar® Direct™ Im- PFS. CD8 score is an assay that could be carried out using exist- mune Profiling System. It includes an optimized 30-marker ing technology in pathology departments. Further analysis will immune profiling panel provided in a dried single-tube format, focus on validating these results in a larger cohort to permit clin- validated SOPs for human whole blood and PBMC staining, an in- ical use. strument data acquisition template, instructions for data acquisi- tion on a Helios™ system, and automated Maxpar Pathsetter™ References software for data analysis. 1. Ribas A, Hamid O, Daud A, Hodi FS, Wolchok JD, Kefford R, Joshua AM, Results Patnaik A, Hwu WJ, Weber JS, Gangadhar TC, Hersey P, Dronca R, Joseph Here we present assay analytical validation data on repeatability, re- RW, Zarour H, Chmielowski B, Lawrence DP, Algazi A, Rizvi NA, Hoffner B, producibility, software precision, software accuracy, and site-to-site Mateus C, Gergich K, Lindia JA, Giannotti M, Li XN, Ebbinghaus S, Kang reproducibility. The repeatability of eight identical donor samples ac- SP, Robert C. Association of Pembrolizumab With Tumor Response and quired on a single Helios instrument resulted in CVs 5% in fre- Survival Among Patients With Advanced Melanoma. JAMA. 2016; quency). Reproducibility of three identical samples acquired on three 315:1600-9. different Helios instruments resulted in CVs 2. Larkin J, Lao CD, Urba WJ, McDermott DF, Horak C, Jiang J, Wolchok JD. Conclusions Efficacy and Safety of Nivolumab in Patients With BRAF V600 Mutant and We conclude that this assay provides a robust solution for broad im- BRAF Wild-Type Advanced Melanoma: A Pooled Analysis of 4 Clinical Tri- mune profiling using mass cytometry, reducing sources of variability als. JAMA Oncol. 2015;4:433-40. and subjectivity in sample preparation and data analysis. 3. Tumeh P, Harview C, Yearley J, Shintaku I, Taylor E, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West A, Carmona M, Kivork C, Seja E, Reference Cherry G, Gutierrez A, Grogan T, Mateus C, Tomasic G, Glaspy J, Emerson 1. Simoni Y, Becht E, Fehlings M et al. Bystander CD8+ T cells are abundant R, Robins H, Pierce R, Elashoff D, Robert C, Ribas A. PD-1 blockade in- and phenotypically distinct in human tumour infiltrates. Nature. 2019; duces responses by inhibiting adaptive immune resistance. Nature. 557:575–579. 2014;515:568-71 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 66 of 272 4. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu to identify patient-specific patterns which might improve the predic- XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber tion of the response to therapy. AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Conclusions Tomassini JE, Loboda A, Kaufman D. Pan-tumor genomic biomarkers for We believe that the Cancer Immunogram has the potential to facili- PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362:197 tate drug development by providing a 360° vision of the tumour im- 5. Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee mune contexture and may also help clinicians to personalize W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, advanced cancer patient care. Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN, Chan TA. Mutational References landscape determines sensitivity to PD-1 blockade in non-small cell lung 1. Galon J, Bruni D. Approaches to treat immune hot, altered and cold cancer. Science. 2015;348:124-8. tumours with combination immunotherapies. Nat Rev Drug Discov. Ethics Approval 2019;18:197-218. The study was approved by University of Pittsburgh‘s Institutional Review 2. Pagès F et al. International validation of the consensus Immunoscore for Board, approval number PRO18080253. the classification of colon cancer: a prognostic and accuracy study. Consent Lancet. 2018; 391:2128-2139. Written informed consent was obtained from the patient for publication of 3. Mlecnik B et al. Integrative Analyses of Colorectal Cancer Show this abstract and any accompanying images. A copy of the written Immunoscore Is a Stronger Predictor of Patient Survival Than consent is available for review by the Editor of this journal. Microsatellite Instability. Immunity. 2016; 44:698-711. 4. Blank CU et al. The "cancer immunogram". Science. 2016;; 352:658-60. 5. Sbarrato T et al, Combining multimodal biomarkers as an immunogram P121 to guide immunotherapy use: A proof of concept. Proceedings: AACR Cancer Immunogram: combining multi-parameter approach and Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. machine learning to capture the complexity of tumor immune contexture 1 1 1 Thomas Sbarrato, PhD , Laurent Vanhille, PhD , Mounia Filahi , Anna P122 1 1 1 Martirosyan, PhD , Véronique Frayssinet , Caroline Davin, BA , Caroline Microfluidic-based cell separation method improves workflow for 1 1 1 1 Laugé , Assil Benchaaben , Alboukadel Kassambara , Felipe Guimaraes , evaluation of rare lymphocytes from cancer patient samples 1 2 1 1 1 1 1 1 Régis Perbost , Jérôme Galon , Hélène Girardi , Jacques Fieschi, PhD Jodi Stone, BS , Megan Nichols , Amy Austin , Kala Bradshaw , Jessica E. 1 2 1 1 2 HalioDx, Marseille, France, Centre de Recherche des Cordeliers, Paris, Norris, BS, MT , Jennifer Montague, PhD , Peng Meng Kou , Nitin 2 2 2 2 France Kulkarni , Nirav Sheth , Anya Manning, MBA , Sarah M. Mickool , Kyle 2 2 1 Correspondence: Jacques Fieschi (jacques.fieschi@haliodx.com) Smith , Ravi Kapur , John Powderly, MD, CPI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P121 Carolina BioOncology Institute, Huntersville, NC, United States; MicroMedicine, Waltham, MA, United States Background Correspondence: John Powderly (jpowderly@carolinabiooncology.org) To tailor clinical care and personalized treatment of cancer patients, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P122 the scientific community together with the practitioners have fo- cused into refining our understanding of cancer biology and resist- Background ance to treatments. In that perspective, the concept of Immunoscore Isolation of rare lymphocyte populations from peripheral blood prod- proposed by Galon et al [1, 2, 3] has highlighted the crucial role of ucts of cancer patients can be challenging due to technician variabil- immune response to the tumor. In parallel, immunotherapies by im- ity and substantial cell loss through standard cell separation mune checkpoint inhibitors (ICI) anti-PD-1/PD-L1 were approved in methods such as Mononuclear Cell Preparation Tubes™ (CPTs). An several cancer indications, such as Non-Small Cell Lung Cancer or automated microfluidic approach was evaluated to determine melanoma, even if only a minority of these patients respond posi- lymphocyte recovery, processing time, and ease of use. Furthermore, tively to the treatment. In addition, ICI are far less effective for other increased yields of rare cells from cancer patients’ peripheral blood high-incidence indications like colorectal cancer (CRC), thus suggest- could potentially substitute for leukapheresis when leukapheresis is ing that multiple factors may be critical for capturing the exact na- not a viable option. ture of the tumour microenvironment (TME). In this context, the Methods comprehensive identification and assessment of these factors could White blood cells (WBCs) or peripheral blood mononuclear cells be key to stratify patients and allow the selection of the optimal (PBMCs) were isolated from human peripheral blood using either treatment. MicroMedicine’s Microfluidic System (MS) or CPTs. Cell viability and Methods lymphocyte recovery were compared using a hematology analyzer In order to support clinical researchers and biopharmaceutical com- and flow cytometry. Further, a rare lymphocyte population was posi- panies in the evaluation of the efficacy of candidate drugs, HalioDx tively immunomagnetically selected from healthy volunteers and has developed the Cancer Immunogram, a solution based on Blank cancer patients. Immunophenotyping was performed pre- and post- CU et al. [4]. Our multi-parameter approach encompassing a unique cell selection, followed by in vitro expansion of the rare lymphocytes. range of immune scoring assays is based on the analysis and the un- Results derstanding of the immune contexture of tumors and offers a per- Using cells collected from healthy volunteers, the automated MS sonalized and dynamic “fingerprint” of tumor-immune system prototype consistently recovered 83.5 ± 10.1% lymphocytes in a total interaction. To address this, the Cancer Immunogram combines dif- of 31 ± 5.8 minutes, including hands-on time, compared to the ferent technologies and biomarkers to assess 1) the tumor character- standard CPT process, which recovered 43.2 ± 7.6% lymphocytes in istics (Tumor foreignness, MSI, PD-L1 expression, common mutation 72.2 ± 4.1 minutes, from 32 – 34 mL blood samples (n = 5). The via- drivers), 2) the immune infiltration (Immunoscore®, CD8/PD-L1 prox- bility was comparable at 97.9 ± 1.6% (MS) and 96.7 ± 3.0% (CPT). In imity, TCR clonality, immune expression signature), 3) the immune further studies with both healthy donors and cancer patients, a rare checkpoint status (T Cell Exhaustion BrightPlex panel) and 4) the im- lymphocyte population was successfully selected from WBCs isolated mune suppression status (Treg, MDSC and M1/M2 macrophage with the MS, enabling immunophenotyping of the rare cell popula- BrightPlex panels). tion and subsequent in vitro expansion. Expansion of this lymphocyte Results population from a colon adenocarcinoma patient was found to be Here, we consolidate our Proof of Concept for the Cancer Immuno- suppressed post-immunotherapy compared to pre-treatment. Cells gram in the context of CRC [5] by leveraging this meta-analysis on a isolated from patients with other malignancies were successfully ex- 20-patients cohort. Using machine learning algorithms to extract the panded. Finally, peripheral blood collected from cancer patients most relevant features, we show that the Cancer Immunogram allows yielded a greater number of rare lymphocytes using the MS for the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 67 of 272 cell expansion study in comparison to the CPT isolation method, NK cells, in mediating antitumor response after immune check- which correlates to having a higher lymphocyte recovery. point inhibition. Conclusions NanoString nCounter is Intended for Research Use Only. Not for Use The MS consistently recovered approximately twice the number in Diagnostic Procedures. of lymphocytes in half the time compared to the traditional CPT Ethics Approval method. The automated cell separation process improves the The study was approved by Yale University Human Investigation consistency of cell isolation while freeing up technician time. Rare Committee, approval number 9505008219. lymphocyte populations could be reliably recovered from periph- eral blood with significantly higher yield compared to the CPT P124 method. While leukapheresis enriches for MNCs and MS isolates A novel cell-mediated immunotherapy for treatment of lung and all WBCs, these results suggest that peripheral blood collection- breast cancer based MS has the potential to complement leukapheresis, espe- Indu Venugopal, PhD, Kathlynn Brown, Michael McGuire, Claire Gormley cially for small- to medium-scale studies. SRI International, Harrisonburg, VA, United States Correspondence: Kathlynn Brown (kathlynn.brown@sri.com) P123 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P124 Identification of mRNA signatures that predict response to immunotherapy in melanoma patients Background 1 2 2 Ioannis Vathiotis, MD , Amy Sullivan , Sarah Warren, PhD , Nicole Cancer Immunotherapies designed to generate a cell-mediated 1 1 1 Gianino , Sandra Martinez-Morilla, PhD , Pok Fai Wong, MD, MPhil , immune response against tumors are emerging as frontline 1 3 1 Harriet Kluger, MD , Konstantinos Syrigos , David Rimm, MD, PhD treatment options for cancer; however, concerns regarding effi- 1 2 Yale University, New Haven, CT, United States; NanoString cacy, safety and cost efficacy have limited the use of these Technologies, Seattle, WA, United States; University of Athens, Athens, treatments. Greece Methods Correspondence: David Rimm (david.rimm@yale.edu) To address these weaknesses, we developed a novel immunother- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P123 apy capable of delivering previously encountered antigenic pep- tides specifically to cancer cells and facilitating their presentation Background through the MHC class I pathway. It utilizes a synthetic nanoparti- Currently, there is no diagnostic test that can accurately predict cle delivery system comprised of three components: a neutral response in melanoma patients treated with immunotherapy. stealth liposome, encapsulated synthetic immunogenic HLA class I NanoString® nCounter® PanCancer IO 360™ panel (Research Use restricted peptides derived from measles virus (MV), and a tumor- Only) measures mRNA from 770 genes related to the tumor and targeting peptide on the external surface of the liposome. The host immune response. Here, we used this panel to assess the targeting peptide results in accumulation of liposomes specifically predictive value of individual genes and weighted gene signa- inside cancer cells, and facilitates presentation of MV-derived im- tures in a cohort of immunotherapy (ITx) treated melanoma munogenic peptides in HLA class I molecules (Figure 1). We refer patients. to this system as TALL (Targeted Antigen Loaded Liposomes). Methods Therefore, TALL can generate a secondary immune response spe- We used pretreatment, formalin-fixed paraffin-embedded (FFPE) cifically against the targeted tumor cells in a patient who has whole tissue sections from 59 melanoma patients that received been previously vaccinated against or infected by MV. In short, single agent or combination immunotherapy (pembrolizumab, we are attempting to trick the immune system into responding nivolumab, or nivolumab plus ipilimumab). Two slides from each as though the cancer cell is infected with MV without the use of patient were macrodissected and RNA was extracted. The mRNA viral particles. transcripts were hybridized and tagged by unique probes for the Results 770-plex PanCancer IO 360 panel and then measured on the We synthesized liposomes encapsulating H250, an immunogenic nCounter platform. RNA counts were correlated with best overall HLA class I restricted peptide identified from measles response (BOR), clinical benefit (CB), progression free survival hemagglutinin protein. These liposomes were targeted to breast (PFS) and overall survival (OS). and lung cancer cells via our targeting peptide, which was identi- Results fied using phage-display methodology. Treatment of lung cancer Indoleamine 2,3-dioxygenase 1 (IDO1) was the best single gene cells with TALL results in functional presentation of H250 in both predictor of BOR (Area under the curve (AUC) = 0.73) and CB MHC and HLA class I molecules. Our in-vitro and in-vivo studies (AUC =0.70).Among othergenes,IDO1 mRNAwas also foundto indicate that presentation of H250 is dependent on the cancer be significantly associated with longer PFS (P < 0.01, False dis- targeting peptide; liposomes that lack the targeting peptide did covery rate (FDR) = 0.18) and OS (P < 0.01, FDR = 0.052). The not accumulate in the cancer cells and presentation of H250 was previously described 18-gene tumor inflammation score (Ayers abrogated. Treatment with TALL substantially reduced growth of TIS) validated for the prediction of BOR (AUC = 0.68), PFS (P < LLC1 and 4T1 tumors in vaccinated C57BL/6 and Balb/c mice 0.05, FDR = 0.18) and OS (P < 0.001, FDR = 0.025). TIS also pre- respectively. dicted CB (AUC = 0.67). Its predictive value remained the same ir- Conclusions respective to immunotherapy agent administered. Nevertheless, it The outcome of our therapy is a robust cytotoxic T lymphocyte re- decreased for patients harboring the BRAF and NRAS mutations sponse directed specifically against the tumor. It's advantages in- (AUC = 0.76 versus 0.51 and 0.44 for patients with BRAF and clude: 1) Bypassing the need to identify tumor-associated antigens or NRAS mutations respectively). The best signatures for this cohort educate the immune system through a primary immune response; 2) were for Cytotoxicity, Immunoproteasome and CD56dim Cells It is anticipated to be effective against tumors with a low mutational which were predictive for BOR (AUC = 0.72, 0.71 and 0.70 re- load, making it efficacious on early-stage and metastatic cancer; 3) It spectively), CB (AUC = 0.69, 0.68 and 0.70 respectively), and OS does not use a live virus or biologically-derived material, allowing for (all FDRs < 0.05). Further work is underway to compare these complete synthetic manufacturing. It also does not require isolation Yale melanoma results with other cohorts. or ex-vivo manipulation of patient’s cells, reducing production time Conclusions and costs. Pretreatment mRNA counts of single genes or weighted signature scores are related to immunotherapy outcomes in melanoma pa- Acknowledgements tients. This work validated the Ayers TIS signature and Research reported in this work was supported by DOD under grant number highlighted the role of the immune microenvironment, especially W81XWH-16-1-0262 and SRI internal research funds Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 68 of 272 receptors. In contrast, ciforadenant inhibited induction of the AdenoSig and AMPSig in all experimental settings at the tran- script and protein level. Conclusions A2AR agonists and AMP induce specific GEPs dominated by im- munosuppressive mediators of MDSC and monocyte/macro- phage biology. These GEPs may be used as biomarkers for patient selection. CD73 antagonists alone may be limited by the induction of compensatory immunosuppressive pathways medi- ated by AMP accumulation. Combination ciforadenant and CPI- 006 treatment may synergize to activate anti-tumor immunity by 1) blocking adenosine production and signaling, 2) directly activating immune cells, and 3) blocking a compensatory induc- tion of AMPSig. This combination strategy is being evaluated in an ongoing Ph1/1b clinical trial in patients with advanced solid tumors. Trial Registration NCT02655822 and NCT03454451 Fig. 1 (abstract P124). TALL mechanism of action P125 Adenosine and AMP gene expression profiles predict response to adenosine pathway therapies and indicate a need for dual blockade of CD73 and A2AR with CD73 inhibitors Stephen Willingham, PhD, Drew Hotson, PhD, Jessica Hsieh, Brian Munneke, Long Kwei, PhD, Joseph Buggy, Richard Miller, MD Corvus Pharmaceuticals, Burlingame, CA, United States Correspondence: Stephen Willingham (swillingham@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P125 Background Extracellular adenosine in the tumor microenvironment gener- ates an immunosuppressive niche that promotes tumor growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. Various agents targeting the adenosine path- way are now in clinical trials as cancer therapies. Ciforadenant is a selective A2AR antagonist and CPI-006 is an anti-CD73 anti- body (Fc-mutant IgG1) that blocksthe enzymaticconversion of AMP to adenosine and directly stimulates immunity. Both agents are now being studied in clinical trials (NCT02655822 and NCT03454451). In this report, we evaluate the role of ad- Fig. 1 (abstract P125). See text for description enosine and AMP-related gene expression profiles (GEPs) that may predict the response of patients receiving adenosine path- way therapies. Ex vivo studies reveal a requirement for dual P126 blockade of CD73 and A2AR for optimal neutralization of AMP Discovery of biomarkers associated with benefit from PD-1 mediated immunosuppression. checkpoint blockade in non-small-cell lung cancer (NSCLC) using Methods high-plex digital spatial profiling Normal human PBMCs were stimulated ex vivo with NECA (stable ad- 1 1 2 Jon Zugazagoitia, MD, PhD , Swati Gupta, PhD , Kit Fuhrman, MS PhD , enosine analog) or AMP. RNA from tumor biopsies and PBMC was an- 1 1 1 Scott Gettinger, MD , Roy Herbst, MD, PhD , Kurt Schalper, MD, PhD , alyzed using NanoString. Renal cell cancer (RCC) tumor biopsies David Rimm, MD, PhD collected from patients treated with ciforadenant (100 mg BID) either Yale University School of Medicine, New Haven, United States; as a single agent (n=18) or in combination with atezolizumab (n=14). Nanostring, Inc., Seattle, WA, United States Results Correspondence: David Rimm (david.rimm@yale.edu) Ex vivo A2AR agonism resulted in dose-dependent increases in CXCR2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P126 ligands (CXCL1,2,3,5,8) and key mediators of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory media- Background tors such as IL-1beta and CCL2,3,7,8, 20 were also observed, as were in- Only a minority of patients with advanced NSCLC truly benefit from creases in SERPINB2, S100A8, PTGS2, THBS1. Preliminary biomarker single-agent PD-1 checkpoint blockade, and more robust predictive analysis suggests ciforadenant anti-tumor activity in RCC was associ- biomarkers are needed to optimally deliver these therapies. The ated with increased expression of select analytes (AdenoSig) in pre- GeoMx Digital Spatial Profiler (DSP) (NanoString, Inc.) allows high- treatment biopsies (Figure 1). plex protein expression analysis in a quantitative and spatially- Ex vivo AMP or AMPalphaS (a non-hydrolyzable AMP analog) resolved manner from single formalin-fixed paraffin embedded tissue stimulation induced a similar GEP (AMPSig), but included specific sections. Here we use this technology as a discovery tool to find pro- decreases in OAS3, BIRC5, CDK1, MX1, IFI27, and IFIT1. CD73 anti- tein markers associated with benefit from single-agent PD-1 check- body and small molecule antagonists amplified the AMPSig by point blockade in NSCLC. preserving AMP, which itself directly stimulates adenosine Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 69 of 272 Methods biopsies on the Leica Biosystems BOND RX autostainer. The tissues We used the GeoMx DSP in a cohort of 63 immunotherapy-treated were then imaged in five distinct fluorescent channels (DAPI, FITC, NSCLC cases represented in a tissue microarray, 52 of whom had TRITC, Cy5, Cy7) in 3 rounds of image acquisition on the ZEISS Axio pre-treatment samples and received single-agent PD-1 checkpoint Scan.Z1. HALO analysis software was used to identify cell phenotypes blockade. A panel of 40 photocleavable oligonucleotide-labeled pri- and spatial interactions across the whole slide images. UMAP and mary antibodies (NanoString Human IO panel) was used for protein PSDM were also used to characterize cellular phenotypes and similar- detection. Proteins were measured in 4 independent molecularly- ities amongst samples in the cohorts. Downstream H&E staining was defined tissue compartments by fluorescence co-localization (tumor performed on the same slides with a fourth imaging round to pro- [panCK+], leucocytes [CD45+], macrophages [CD68+], and non- duce a fused 12-plex fluorescent and brightfield image. immune stromal cells [CK-/CD45-CD68-/DNA+]). The photocleaved Results oligos were hybridized and digitally counted with the nCounter plat- The multiplex panel was able identify all 12 markers in FFPE samples form. Two cut-points (median and top tertile) were explored for each and immunophenotype single cells through co-expression of several maker. All statistical testing was performed using a two-sided signifi- biomarkers. Of the 4,096 possible phenotypes, the relevant pheno- cance level of α=0.05 without correction for multiple hypothesis types mapped included, but were not limited to: T cells, T-regs, Cyto- testing. toxic T-cells, Exhausted T-cells, B cells, NK cells, M1 and M2 Results macrophages, tumor cells, and expression along the PD-L1 and PD-1 160 protein variables were generated per case (normalized counts immune checkpoint axis. Distance mapping and infiltration indexes within molecularly defined compartments). In univariate analyses were measured in tumor regions, stroma compartments, and along using pre-specified cut-points, 10 markers were associated with clin- invasive margins. ical benefit (CB) or non-CB, 6 markers with PFS, and 13 markers with Conclusions OS. Of these, CD56 (top tertile) and CD4 (median) measured in the In this work, we introduce a tumor and immune cell phenotyping CD45 compartment were the only markers that significantly pre- multiplex immunofluorescence panel for the comprehensive dicted either CB (OR 6.7, p = 0.014 and OR 8.5, p = 0.014, respect- characterization of the tumor microenvironment and its applicability ively) longer PFS (HR 0.38, p = 0.011 and HR 0.33, p = 0.002, across a range of carcinoma and melanoma FFPE tissue samples for respectively) and longer OS (HR 0.44, p = 0.044 and HR 0.31, p = support of deep pathology assessment in drug discovery research. 0.002, respectively). After adjusting for 3 baseline clinical prognostic The ability to colocalize markers in the same compartment for the factors (performance status, liver metastasis, dNLR) in a multivariate identification of thousands of phenotypes when combined with Cox proportional hazard model, both CD56 and CD4 remained pre- brightfield pathological assessment within a single sample has the dictive for PFS (HR 0.39, p = 0.020 and HR 0.37, p = 0.017, respect- potential to accelerate immunotherapy research. ively), while only CD4 was predictive for OS (HR 0.28, p = 0.006). Conclusions P128 This pilot scale, discovery study shows the potential of the DSP tech- Pooled analysis of Programmed Death Factor Ligand 1 (PD-L1) nology in the identification of spatially-informed biomarkers of re- expression as a predictive biomarker using individual data on sponse to PD-1 checkpoint blockade in NSCLC. This works highlights 7,918 randomized study patients a previously undescribed role for CD56+ immune cells and CD4+ T- Andrea Arfe, Geoffrey Fell, Brian Alexander, MD MPH, Mark Awad, MD cells as potential predictors of immunotherapy outcomes in NSCLC. PhD, Scott Rodig, MD, PhD, Lorenzo Trippa, Jonathan Schoenfeld, MD, Ethics Approval MPH All tissue samples were collected and used with specific consent or Dana-Farber Cancer Institute, Boston, MA, United States waiver of consent under the approval from the Yale Human Investi- Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) gation Committee protocol #9505008219. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P128 P127 Background Development of a 12-marker immunofluorescence multiplex panel PD-L1 expression is one of the most studied biomarkers to predict for the in-depth investigation of the tumor immune landscape the efficacy of immune checkpoint inhibitors (ICIs), but its clinical sig- analyzing 4,096 phenotypes nificance is controversial. Several factors have limited the study of Courtney Hauck, Aditi Sharma, Monique Johnson, Wenya Yang, Bonnie PD-L1 expression. Most trials use of hazard ratios (HRs) to measure Phillips, PhD, Mark Burton, HTL ASCP, Douglas Wood, PhD, Stephanie treatment effects on survival outcomes, a questionable practice for Hennek, PhD, Mael Manesse, PhD, J Kent Moore, PhD, Katir Patel, PhD, immunotherapy studies. Additionally, trials use different cut-off Jamie Buell, Sean Downing, PhD values to dichotomize PD-L1 scores, complicating meta-analyses. Ultivue, Cambridge, MA, United States Therefore, we performed a pooled analysis to: i) estimate the distri- Correspondence: Sean Downing (sean.downing@ultivue.com) bution of PD-L1 expression scores in clinical populations, and ii) as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P127 sess the relationship between PD-L1 levels and ICIs’ effects on overall survival (OS). Instead of HRs, we used a more robust metric, i.e. differ- Background ences in restricted mean survival times (ΔRMSTs). Current IHC methods limit the depth of information from a single tis- Methods sue sample to a single target in the case of chromogenic staining, or Following PRISMA guidelines, we analyzed individual-level data re- to sample morphology and general cell identification in the case of constructed from the publications of 14 randomized clinical trials of H&E. Multiplex immunofluorescence (mIF) methods provide insights ICIs. We used an imputation-based approach to estimate i) the distri- into a wide number of markers of interest and their spatial context in bution of PD-L1 scores, ii) the survival distribution in different PD-L1 a single sample but limit the level of marker co-localization detection classes, and iii) pooled ΔRMST estimates. We show the advantage possible because of multiple antigen retrieval or photobleaching provided by meta-analytic estimates such as ours for the design of steps. Here, we demonstrate the utility of a new 12-plex mIF panel future studies in a simulation study. We simulated 10,000 NSCLC tri- using InSituPlex technology that can identify thousands of pheno- als (1:1 randomization; sample size: 500 patients) that compared ICIs types and spatial behavior through the co-localization of markers with standard chemotherapy. Simulated trials followed either i) a de- that was once limited to the domain of flow cytometry. sign that does not use prior information on the distribution of PD-L1 Methods levels and their association with ICIs’ effects, or ii) a design tailored The 12-Plex marker panel was developed including: CD3, CD4, CD8, to our meta-analytic estimates. CD20, Granzyme B, CD56, CD68, CD163, FoxP3, PD-1, PD-L1, and pan- Results Cytokeratin/Sox10 used the InSituPlex and DNA-Exchange technol- We reconstructed data on 7,918 individual patients, 3,496 with ogy to perform mIF staining of FFPE samples from tonsil and tumor NSCLC, 4,529 with other tumors. The estimated distribution of PD-L1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 70 of 272 expression is U-shaped, with most patients presenting a low or high Methods expression: only about 7% had an expression in the 5%-50% range. Serial peripheral blood mononuclear cells were obtained from pa- ΔRMST estimates suggest that i) ICIs provide an OS benefit to all pa- tients with RCC undergoing immunotherapy. Samples were obtained tients, and ii) the magnitude of OS benefits increases along with PD- at baseline (cycle 1) and initiation of each subsequent cycle (up to L1 score, although changes in ΔRMSTs were greater in NSCLC (Figure cycle 6). Flow cytometry identified longitudinal changes in T cell sub- 1). In the simulations, the power to detect a positive treatment effect sets. Additionally, recently activated CD8 T cells, identified by surface increased from 80% to 93% using a design tailored to meta-analytic expression of CD38 and HLA-DR, were sorted at baseline, post-cycle information. 1, and post-cycle 2, and analyzed by RNA seq. Clinical responses Conclusions were determined at the first restaging scans using RECIST v1.1 cri- By highlighting that higher PD-L1 scores predict increasing OS bene- teria, to define those with clinical benefit (complete response, partial fits, our findings extend those of recent meta-analyses that evaluated response, or stable disease) or no clinical benefit (progressive PD-L1 expression scores as predictors of ICIs’ efficacy. They also illus- disease). trate how meta-analytic estimates like ours can improve the power Results of future trials to detect ICIs’ benefits. Our findings also suggest that Of 27 patients analyzed, 10 received nivolumab, 7 nivolumab + the practice of dichotomizing the range of PD-L1 expression scores is NKTR-214, and 10 nivolumab + ipilimumab. Median age was 58 years inadequate for patient stratification. (range 33-78) with a male (70%), Caucasian (89%), and solely clear cell histology (83%) predominance. A burst in circulating activated CD8 T cells as defined by a ≥1.8 fold increase in CD38+HLA-DR+ CD8 T cells from baseline to post-cycle 1 (Figure 1A) was observed in 8/12 patients who had clinical benefit and 6/15 patients with no clinical benefit (Figure 1B). Transcriptional analysis revealed that in patients with the aforementioned immunological response, T-cells had upreg- ulated TCR signaling, CD28 signaling, enhanced glycolysis and iron uptake, and reduced TGF-beta signaling compared to patients with- out an immunologic response. Conclusions Peripheral blood immune monitoring of RCC patients while on im- munotherapy may provide an early predictor of response. One im- portant limitation identified is the treatment specific cycle length defined sample collection timing and therefore may miss transient early immunologic changes. This study advances knowledge regard- ing the newly generated effector CD8 T cells that contain important information about the immunobiology underlying response to immunotherapy. Acknowledgements This work was supported by funding from the NCI grant 1-R00-CA197891 and Nektar Therapeutics. We would like to acknowledge The Yerkes NHP Genomics Core which is supported in part by NIH P51 OD011132, the Emory Flow Cytometry Core (EFCC) supported by the National Center for Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378, and NIH/NCI under award number, 2P30CA138292-04. Fig. 1 (abstract P128). See text for description References 1. 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Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 71 of 272 Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, approval of the NYU Institutional Review Board at the NYU Perlmut- Araki K, Ahmed R. Rescue of exhausted CD8 T cells by PD-1-targeted ter Cancer Center with informed consent [11]. therapies is CD28-dependent. Science. 2017;355(6332):1423-1427. Serum samples were run on HuProt Human Proteome Microarrays 5. Huang AC, Postow MA, Orlowski RJ, Mick R, Bengsch B, Manne S, Xu W, containing >19,000 human proteins by CDI Laboratories. Raw serum Harmon S, Giles JR, Wenz B, Adamow M, Kuk D, Panageas KS, Carrera C, IgG signal intensities were processed across staining cohorts via Wong P, Quagliarello F, Wubbenhorst B, D'Andrea K, Pauken KE, Herati interquartile range normalization. RS, Staupe RP, Schenkel JM, McGettigan S, Kothari S, George SM, Pre-existing antibody responses were defined as patient-specific IgG Vonderheide RH, Amaravadi RK, Karakousis GC, Schuchter LM, Xu X, signals >3.5 median absolute deviations above cohort median IgG Nathanson KL, Wolchok JD, Gangadhar TC, Wherry EJ. T-cell invigoration background (modified Z-score). Group statistics were computed to tumour burden ratio associated with anti-PD-1 response. Nature. (GraphPad Prism), and gene ontology enrichment analysis was per- 2017;545(7652):60-65. formed (Enrichr) [12]. Ethics Approval Results Samples are collected under an approved IRB protocol (The Urological Several pre-existing antigen-specific IgG autoantibody targets were Satellite Specimen Bank at Emory University, IRB00055316), and all observed to have associations with good outcomes (SD/PR) or ob- patients provided informed consent. jective clinical responses (PR/CR) versus patients with progressive dis- ease (POD). While final determination of the most predictive subsets is ongoing, many targets represent genes in an axis surrounding im- mune signaling pathways, hereditary neurodegenerative disease, and the ubiquitin proteasome pathway (ie, UBQLN1, UBQLN2). An exemplary example was observed in the autoantibody responses shared by >10% of all patients regardless of clinical outcome. Gene ontology enrichment analysis of these shared melanoma-patient autoantibodies versus KEGG 2019 [12] demonstrates this set of pro- teins is strongly enriched for neurotrophin signaling-associated pro- teins after multi-sample correction (P=0.004) (Table 1). Several other associations were observed cohort-wide for ontologies with tissue- specific enrichment in the brain, neurons, and neuronal processes. Conclusions In this pilot study, we found strong associations across the cohort for autoantibodies against nerve-growth-inducing neurotrophins and genes like UBQLN1 and UBQLN2 which have strong associations with amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson’s, and Alzheimer’s – neurodegenerative diseases that are known to have incidences which correlate with melanoma [14–16]; this hints at a potential immunologic connection between the conditions, per- haps related to an antitumor / autoimmune axis involving the targets reported here. Acknowledgements We thank the patients and their families who consented to participate in this study. Funding support for the study was provided by the NYU Cancer Center and NIH/NCI Cancer Center Support Grant P30CA016087, the Marc Jacobs campaign to support melanoma research, Goldberg Charitable Trust, Fig. 1 (abstract P129). See text for description Wings for Things Foundation and Clayman Family Foundation to I. Osman; the American Medical Association foundation, the Melanoma Research Foundation and the American Skin Association grants to M. Gowen. Trial Registration P130 Patient samples included in this study were not part of a randomized Melanoma patients harbor pre-existing IgG autoantibodies controlled clinical trial. targeting neuronal proteins that associate with differential clinical outcomes following checkpoint blockade 1 2 2 2 References Tyler Hulett, PhD , Keith Giles , Michael Gowen, MD , Danny Simpson , 2 2 2 1. Nagele EP, Han M, Acharya NK, DeMarshall C, Kosciuk MC, Nagele RG. Jeremy Tchack , Una Moran , Zarmeena Dawood , Anna Pavlick, MD, 2 1 2 2 Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human MBA , Shaohui Hu , Hua Zhong , Michelle Krogsgaard , Tomas Kirchhoff, 2 2 Sera, and Their Number Is Influenced By Age, Gender, and Disease. PhD , Iman Osman 1 2 Tsokos GC, editor. PLoS ONE. 2013;8:e60726. CDI Laboratories, Portland, OR, United States; New York University 2. Larman HB, Zhao Z, Laserson U, Li MZ, Ciccia A, Gakidis MAM, et al. School of Medicine, New York, NY, United States Autoantigen discovery with a synthetic human peptidome. Nature Correspondence: Tyler Hulett (tyler.hulett@cdi-lab.com) Biotechnology. 2011;29:535–41. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P130 3. Meyer S, Woodward M, Hertel C, Vlaicu P, Haque Y, Kärner J, et al. AIRE- Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Background Autoantibodies. Cell. 2016;166:582–95. Autoantibody landscapes are very specific to the individual, can remain 4. Graff JN, Puri S, Bifulco CB, Fox BA, Beer TM. Sustained Complete stable for many years, and contain unique features reported in associ- Response to CTLA-4 Blockade in a Patient with Metastatic, Castration- ation with cancer, autoimmunity, infection, neurologic conditions, Resistant Prostate Cancer. Cancer Immunology Research. 2014;2:399–403. CD8+ T cell behavior, and checkpoint blockade adverse events [1–11]. 5. Gnjatic S, Ritter E, Büchler MW, Giese NA, Brors B, Frei C, et al. Seromic The goal of this work was to determine whether pre-existing antigen- profiling of ovarian and pancreatic cancer. Proceedings of the National specific features in melanoma patient autoantibody landscapes would Academy of Sciences. 2010;107:5088–5093. associate with clinical outcomes following checkpoint blockade. 6. Wongkulab P, Wipasa J, Chaiwarith R, Supparatpinyo K. Autoantibody to Methods Interferon-gamma Associated with Adult-Onset Immunodeficiency in Pre-treatment serum samples were collected from 117 melanoma pa- Non-HIV Individuals in Northern Thailand. Rottenberg ME, editor. PLoS tients prior to checkpoint blockade with anti-CTLA4 (N=60), anti-PD1 ONE. 2013;8:e76371. (N=38), or both in combination (N=16). All data was collected with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 72 of 272 7. Anderson KS, Sibani S, Wallstrom G, Qiu J, Mendoza EA, Raphael J, et al. P131 Protein Microarray Signature of Autoantibody Biomarkers for the Early Single cellular interrogation of tumor microenvironment enables Detection of Breast Cancer. Journal of Proteome Research. 2011;10:85–96. diagnosis and prognostication of malignancies 1 1 2 8. Miersch S, Bian X, Wallstrom G, Sibani S, Logvinenko T, Wasserfall CH, Wei Jian Tan , Jian Hang Lam , Mona Meng Wang , Paola Ricciardi- 1 2 3 et al. Serological autoantibody profiling of type 1 diabetes by protein Castagnoli , Anita Sook Yee Chan , Tony Kiat Hon Lim , Joe Poh Sheng 3 1 arrays. Journal of Proteomics. 2013;94:486–96. Yeong , Tong Seng Lim, PhD 1 2 9. Srivastava RM, Lee SC, Andrade Filho PA, Lord CA, Jie H-B, Davidson HC, Menarini Biomarkers Singapore, Singapore, Singapore; Singapopre Eye et al. Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate Research Institute, Singapore, Singapore; Singapore General Hospital, to Trigger Tumor Antigen-Specific T-cell Immunity in Head and Neck Singapore, Singapore Cancer Patients. Clinical Cancer Research. 2013;19:1858–72. Correspondence: Tong Seng Lim (tongseng.lim@mbiomarkers.com) 10. Hulett TW. Coordinated responses to individual tumor antigens by IgG Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P131 antibody and CD8+ T cells following cancer vaccination. 2018;14. 11. Gowen MF, Giles KM, Simpson D, Tchack J, Zhou H, Moran U, et al. Background Baseline antibody profiles predict toxicity in melanoma patients treated Tumor microenvironment contains a diverse array of cell types with immune checkpoint inhibitors. Journal of Translational Medicine with heterogeneous genomic and molecular profiles. Averaging [Internet]. 2018 [cited 2018 Nov 4];16. Available from: https://translational- the characteristics of all cells in a cancerous tissue no doubt ob- medicine.biomedcentral.com/articles/10.1186/s12967-018-1452-4 scures important variations in biomarkers among minority, but 12. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, critical, pathogenic cell populations. High resolution, single-cell Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list analyses are thus needed in the clinic to precisely delineate the enrichment analysis tool. BMC Bioinformatics. 2013;128(14). inherent heterogeneity of tumor microenvironment underlying 13. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, oncogenesis in each patient. Biopsies derived from tumor micro- Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, McDermott MG, environment are routinely used for medical diagnosis or prognos- Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene tications. The number of cells typically available from a biopsy is set enrichment analysis web server 2016 update. Nucleic Acids Research. limited and heterogeneous. The ability to distinguish, select, and 2016; gkw377 . sort rare malignant or pathogenic immune cells from either tissue 14. Olsen JH, Friis S, Frederiksen K. Malignant Melanoma and Other Types of or liquid biopsies poses a unique challenge for single-cell based Cancer Preceding Parkinson Disease: Epidemiology. 2006;17:582–7. diagnosis and prognostication. Heterogeneous cell populations 15. Freedman DM, Curtis RE, Daugherty SE, Goedert JJ, Kuncl RW, Tucker MA. with non-target immunoreactive cells in pausicellular biopsies The association between cancer and amyotrophic lateral sclerosis. Cancer complicate conventional bulk-cell analysis, and could lead to dis- Causes & Control. 2013;24:55–60. ease misdiagnosis or prognostication. 16. Roe CM, Fitzpatrick AL, Xiong C, Sieh W, Kuller L, Miller JP, et al. Cancer Methods linked to Alzheimer disease but not vascular dementia. Neurology. We adopted a state-of-the-art multi-modal strategy including the 2010;74:106–12. real-time imaging-based DEPArray with downstream molecular and Ethics Approval genomic assays [1], and quantitative multiplex immunofluorescent All data collected for this study was collected with approval of the NYU technique [2] in order to predict clinical outcome or direct therapy, Institutional Review Board at the NYU Perlmutter Cancer Center with based on single-cell based diagnostic or prognostic biomarkers de- informed consent. rived from tumor microenvironment. Consent Results No sensitive or patient identifiable information is included in the data We provided proof-of concepts that DEPArray technology enabled presented. automated isolation and recovery of rare malignant or pathogenic immune cells from liquid or tissue biopsies in several malignan- cies including vitreoretinal lymphoma (VRL), hepatocellular carcin- oma (HCC) and colorectal carcinoma (CRC). Rare target B lymphoma cells were distinguished and sorted from pausicellular ocular vitreous biopsies with high resolution and purity required Table 1 (abstract P130). Enrichment of anti-neuronal growth for sensitive single-cell based MYD88 mutational profiling to aid autoantibodies VRL diagnosis [1]. Single cellular imaging revealed the presence of large (>10μm), irregular shaped of a novel population of HCC- infiltrating macrophages in association with improved prognosis after surgery. A unique signature regulatory T-cells (Tregs) popu- lation was identified in both blood circulation and cancerous tis- sues of CRC. These signature Tregs expressed phenotypically distinct surface markers in association with better disease-free and overall survival of CRC patients. Conclusions Using real-time imaging-based, digital sorting DEPArray, we could distinguish, select and sort different types of malignant or target immune cells including B-cells, T-cells and macrophages from het- erogeneous tumor microenvironment or liquid biopsies with low cellularity. Comprehensive genomic and molecular characteriza- tions at single cell resolution revealed crucial biomarkers associ- ated with clinicopathological features that impact clinical outcome of patients. The single cell interrogation using DEPArray technology provides a novel precision medicine tool for diagnos- tics and prognostications of malignancies in future. Acknowledgements This study was supported by research funding from the research collaboration between A. Menarini Biomarkers Singapore Pte Ltd, Singapore Eye Research Institute and Singapore General Hospital. Some data presented here are part of patent filed on 21 August 2018 (#10201807097T). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 73 of 272 References P133 1. Tan, W.J., et al., Single-cell-MYD88 sequencing of isolated B cells High tumor expression of DKK1 is associated with improved from vitreous biopsies aids vitreoretinal lymphoma diagnosis. Blood, clinical benefit and longer progression free survival across 2019. multiple solid tumors when treated with a targeted anti-DKK1 2. Lim, J.C.T., et al., An automated staining protocol for seven-colour im- antibody (DKN-01) munofluorescence of human tissue sections for diagnostic and prognos- Michael Kagey, PhD, Girish Naik, MD, Michael Haas, PhD, Heidi tic use. Pathology, 2018. 50(3): p. 333-341. Heath, Franziska Schurpf-Huber, Walter Newman, PhD, Cynthia Sirard, Ethics Approval MD This study was approved by the SingHealth Institutional Review Board in Leap Therapeutics, Cambridge, MA, United States accordance with the Singapore Guidelines for Good Clinical Practice and Correspondence: Cynthia Sirard (csirard@leaptx.com) the Declaration of Helsinki, approval number #2009/907/B, 2012/104/F Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P133 and #2017/2494 Background Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, con- P132 tributes to an immune suppressive tumor microenvironment Harmony: Integrative tool to analyse and visualise multiplex- and promotes tumor growth, angiogenesis and metastasis. immunofluorescence single-cell data DKN-01, a DKK1 neutralizing antibody, has demonstrated clin- 1 2 3 4 Duoduo Wu , Joe Yeong, MBBS, PhD , Grace Tan , Marion Chevrier , ical activity across multiple solid tumors as both a monother- 5 5 4 Josh Loh , Tony Lim , Jinmiao Chen apy and in combination with checkpoint inhibitors and 1 2 National University of Singapore, Singapore, Singapore; Department of chemotherapies. Nonclinical studies indicate that DKN-01 effi- Anatomical Pathology, Sing, Singapore, Singapore; Nanyang cacy depends on a functioning immune system, notably nat- Technological University, Singapore, Singapore; Agency of Science, ural killer (NK) cells. High tumor expression of DKK1 correlates Technology and Resear, Singapore, Singapore; Singapore General with a worse clinical prognosis in many solid tumors. As such, Hospital, Singapore, Singapore, Singapore we evaluated tumor levels of DKK1 and association with clin- Correspondence: Tony Lim (Lim.Kiat.Hon@singhealth.com.sg); Jinmiao ical outcomes for DKN-01 based therapies. Chen (Chen_Jinmiao@immunol.a-star.edu.sg) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P132 DKK1 mRNA expression in patient tumor biopsies was evalu- ated with a RNAscope in situ hybridization assay. Expression Background levels were semi-quantified with QuPath or manually scored. In the advent of immuno-technology, newer single-cell flow cytome- Data was pooled from three separate clinical trials, DKN-01 as try techniques have greatly increased the capacity for the maximum monotherapy or in combination with paclitaxel or pembrolizu- number of immunological parameters measured. Notably, multiplex- mab in esophagogastric cancer (EGC) (NCT02013154), DKN-01 immunofluorescence (mIF) can perform measurements for 7 markers, as monotherapy or in combination with paclitaxel in epithelial flow cytometry can handle 20, and imaging mass cytometry can endometrial cancer (EEC) or epithelial ovarian cancer (EOC) process up to 37 biomarkers simultaneously. Hence, dimensionality (NCT03395080) and DKN-01 in combination with gemcitabine/ reduction techniques such as t-SNE and UMAP are becoming increas- cisplatin in biliary tract cancer (BTC) (NCT02375880). Survival ingly important for tumour single-cell data analysis. Using human he- analysis was performed by the Kaplan-Meier method and multi- patocellular carcinoma (HCC) tissue samples, we aim to compare and variable Cox proportional-hazards and logistic regression evaluate the use of a new technique, UMAP, as an alternative to t- models were used to study the association of DKK1 H-score SNE in mIF derived single-cell data. cutoffs (tertiles and quartiles) with survival and clinical benefit Methods (CR, PR or SD per RECIST v1.1) outcomes. We adopted an unsupervised clustering approach using FlowSOM to Results identify 8 major cell types present in human HCC tissues by staining Atotal of 120 patients (59EGC,28EEC,20EOC and13BTC) them with 7 markers, including immune-checkpoint molecules and had DKK1 tumor expression with response and survival out- one nuclear counterstain. Following that, UMAP and t-SNE were ran comes. Patients who had an H-score ≥ upper-quartile (≥50) of independently on the dataset to qualitatively compare the distribu- DKK1 expression versus < upper-quartile had a higher-odds of tion of clustered cell types in both dimensionality reduction tools. having clinical benefit/response with an adjusted OR of 4.46 Results (95% CI: 1.78, 11.71) and a longer PFS with an adjusted HR of The key advantage of UMAP is its superior runtime – it takes approxi- 0.49 (95% CI: 0.29, 0.81). Patients who had an H-score ≥ upper- mately one-fifth the time required to run t-SNE. Both techniques pro- tertile (≥35) versus < upper-tertile had a higher-odds of having vide similar arrangements of cell clusters, with the key difference clinical benefit/response with an adjusted OR of 2.82 (95% CI: being UMAP’s extensive characteristic branching. Also, increasing 1.21, 6.67) and a longer PFS with an adjusted HR of 0.53 (95% perplexity values in t-SNE results in a t-SNE visualisation with certain CI: 0.33, 0.85). degrees of branching like that of UMAP’s, albeit limited. When pa- Conclusions rameters such as standard deviation, minimum and maximum inten- Elevated DKK1 tumor expression was associated with a higher sity from the mIF image cytometry data were included, a t-SNE plot clinical benefit/response rate and longer PFS for DKN-01 based with virtually the same morphology as the resulting UMAP plot can treatments across multiple solid cancers. Tumor expression of be visualised. Most interestingly, UMAP’s branching highlighted bio- DKK1 may represent an important patient selection criterion logical lineages, especially in identifying potential hybrid tumour cells for further development of DKN-01 based therapies in solid (HTC). Survival analysis shows patients with higher proportion of HTC cancers. The contribution of high levels of tumor DKK1 expres- have a worse prognosis (p-value = 0.019). sion to an immune suppressive tumor microenvironment and Conclusions the role of NK cells is currently under investigation. We conclude that both techniques are similar in their visualisation capabilities, but UMAP has a clear advantage over t-SNE in runtime, Ethics Approval making it highly plausible to employ UMAP as an alternative to t-SNE Studies were approved by the Institutional Review Boards of each in single-cell data analysis. participating institution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 74 of 272 P134 Background Prognostic value of tumor microenvironment based on PD-L1 Loss of Y chromosome (LOY) is a well know established expression and CD8+ TILs density in locally advanced NSCLC phenomenon associated with cancers and ageing. Recently, LOY treated with concurrent chemoradiotherapy in peripheral blood cells was suggested as a possible biomarker 2 1 1 1 Lukas Käsmann , Kathrin Gennen , Julian Taugner , Chukwuka Eze , for different cancers in males. On the basis of previous findings, 1 1 1 Monika Karin , Olarn Roengvoraphoj , Jens Neumann , Amanda the present case-control study was conducted to evaluate the 3 1 1 1 Tufman , Michael Orth , Simone Reu , Claus Belka association of LOY in peripheral blood cells in prostate (PC) and 1 2 University Hospital, Munich, Germany; University of Munich, Munich, colorectal cancers (CRC) in males [1-4]. Germany; Thoracic Oncology Centre Munich, Munich, Germany Methods Correspondence: Lukas Käsmann (lkaesmann@gmail.com) 30 CRC patients (mean age = 44.03±10.8), 36 PC patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P134 (mean age = 60.8 ± 15.8 yrs) and 36 healthy control male cases (mean age = 54.6± 15.1 years) were recruited. DNA was Background extracted by using a standard phenol-chloroform method. The prognostic role of the tumor immunity microenvironment Multiplex quantitative fluorescent (QF) PCR was used to co- (TIME) in multimodal treatment for locally advanced non-small amplify the homologous sequences present on the Y chromo- cell lung cancer (LA-NSCLC) is unclear. Increasing evidence sug- some and other chromosome followed by their analysis on the gests treatment benefit depending on tumor cell PD-L1 expres- genetic analyzer (ABI 3500) and finally the Y/X ratio was calcu- sion. The purpose of this retrospective single-center study was to lated on the basis of the peak height obtained from the investigate the prognostic value of PD-L1 expression on tumor electropherogram. cells in combination with CD8+ tumor stroma-infiltrating lympho- Results cytes (TILs) density in inoperable LA-NSCLC treated with concur- The mean Y/X ratio was significantly lower in the whole group rent chemoradiotherapy (CRT). of cancer patients (0.709±0.02; p <0.0001) when compared to Methods the controls (0.92±0.044). Also, the Y/X ratio when calculated We collected retrospectively clinical characteristics and initial separately was found to be lower in CRC (0.701±0.078; p < tumor biopsy samples of 31 inoperable LA-NSCLC patients treated 0.0001) and PC (0.717±0.044; p <0.0001) cases, when compared with concurrent CRT. PD-L1 expression on tumor cells (0% versus to controls (0.92±0.044). Multivariate logistic regression was ≥1%), CD8+ TILs density (0-40% vs. 41-100%) and TIME according performed by matching cancer and control subjects with age to classification by Zhang et al. were evaluated for potential and the results suggest that LOY is not influenced by their prognostic value in terms of local control, progression-free (PFS) age. and overall survival (OS) as well as correlations with clinic- Conclusions pathological features investigated. The results support the significant association of LOY in peripheral Results blood cells carcinogenesis in males. LOY can also serve as a non- Median OS was 14 months (range: 3-167 months). The OS rates invasive cancer biomarker to improve the early diagnosis and man- at 1- and 2 years were 68% and 20%. Local control rates for the agement of cancer patients in males. entire cohort at 1 and 2 years were 74% and 61%, respectively. Median PFS and PFS at 1 and 2 years were 13±1.4 months, 58% Acknowledgements and 19%. PD-L1 expression <1% on tumor cells was associated We are highly grateful to Sanjay Gandhi Post Graduate Institute of with improved OS, PFS and local control in patients treated with Medical Sciences, Lucknow, Uttar Pradesh, India for providing the concurrent CRT. Univariate analysis showed a trend for improved infrastructure and lab facilities for research work. The authors also thank OS and local control in patients with low CD8+ TILs density. all the consultant and residents of SGPGIMS, who helped in carrying out Evaluation of TIME appears to be an independent prognostic fac- the study. Dr Ambreen Asim is the first author, who collected data, tor for local control, PFS and OS. The longest and shortest OS carried out all the practical work and drafted this abstract. Prof. Sarita were achieved in patients with type I (PD-L1neg/CD8low) and Agarwal is the corresponding and second author, who helped in type IV (PD-L1pos/CD8low) tumors (median OS: 57±37 vs. 10±5 finalizing, correcting and critical review of the work. Prof.Rakesh Kapoor months, p=0.05), respectively. and Prof. Neeraj Rastogi are the oncologist consultant who has provided Conclusions prostate and colorectal cancer patients blood samples after taking Assessment of the tumor immunity microenvironment (TIME) by informed consent for this study. PD-L1 expression on tumor cells and CD8+ TILs density is a pre- dictive biomarker in patients treated with concurrent CRT for in- References operable LA-NSCLC. 1. Noveski P, Madjunkova S, Stefanovska E. Loss of Y Chromosome in Peripheral Blood of Colorectal and Prostate Cancer Patients. Plos One. Acknowledgements 2016;11(1). The study was funded by the German Center for Lung Research (DZL). 2. Chang Y M, Perumal R, Keat P Y, Rita Y.Y. Yong, Daniel L.C. Kuehn, Ethics Approval Leigh Burgoyne. A distinct Y-STR haplotype for Amelogenin nega- The study was approved by the University Ethics Board, approval number tive males characterized by a large Yp11.2 (DYS458-MSY1-AMEL-Y) 493-16. deletion. Forensic Sci Int. 2007; 166(2-3):115-20. Consent 3. Donaghue C, Mann K, Docherty Z and Ogilvie C M. Detection of Written informed consent was obtained from the patient for publication of mosaicism for primary trisomies in prenatal samples by QF-PCR and this abstract and any accompanying images. A copy of the written consent karyotype analysis.Prenat Diagn .2005; 25: 65–72. is available for review by the Editor of this journal. 4. Plaseski T, Noveski P, Trivodalieva S, Georgi D. Quantitative Fluorescent- PCR Detection of Sex Chromosome Aneuploidies and AZF Deletions /Du- plications. Genetic Teasting, 2008 : 12: 4. P135 Ethics Approval Studying Loss of Y chromosome in colorectal and prostate cancers This study was approved by Sanjay Gandhi Post Graduate Institute of in males for non-invasive cancer biomarkers Medical Sciences Ethics Board; approval number IEC CODE – 2018- 53- Ambreen Asim, PhD, Sarita Agarwal, Rakesh Kapoor, Neeraj Rastogi IMP-103 dated 18th June 2018.” Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Consent India Written informed consent was obtained from the patient for publication of Correspondence: Sarita Agarwal (saritasgpgi@gmail.com) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P135 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 75 of 272 P136 Background Quantitative/spatial analysis of Tregs reveal a prominent PD-L1 protein expression by immunohistochemistry (IHC) meas- biomarker role in human non-small cell lung cancer (NSCLC) urement is the only FDA-approved protein diagnostic biomarker 1 1 1 Richa Gupta , Nicolas Rodriguez-Arriagada , Shruti Desai, PhD , for PD1/PD-L1 immunotherapies [1]. However, the tumor-immune 2 1 Konstantinos Syrigos , Roy Herbst, MD, PhD , Vamsidhar Velcheti, MD interaction is complex: PD-L1 expression alone is not predictive 3 1 1 FACP , David Rimm, MD, PhD , Sarah Goldberg, MD, MPH , Kurt of patient response [2]. This has led to the investigation of other Schalper, MD, PhD PD-1 ligands such as PD-L2 [2]. PD-L2 even in the absence of 1 2 Yale University, Burlington, CT, United States; Athens University, PD-L1 has been associated with clinical response to PD-1 block- Athens, Greece; NYU-Langone Medical Center, Pepper Pike, OH, United ade in multiple tumor types [2]. PD-L2 status has also been inves- States tigated where immunotherapy based on PD-L1 has been less Correspondence: Kurt Schalper (kurt.schalper@yale.edu) successful, such as prostate cancer, where PD-L1 expression is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P136 typically low [3]. Here, significantly higher levels of PD-L2 were associated with multiple survival and response measures [3]. Due Background to the diagnostic and therapeutic potential indicated by the pres- Regulatory T cells (Tregs) mediate potent tolerogenic signals, are involved ence of this key immune checkpoint ligand in patients irrespect- in adaptive anti-tumor immune responses and T-cell reinvigoration using ive of PD-L1 expression [2-4], dependable detection tools for immune checkpoint blockers. Despite their prominent immune suppres- investigating the presence and role of PD-L2 are crucial. To ad- sive role, the tissue distribution and contribution of Tregs to clinical out- dress this need, Abcam have developed and extensively charac- comes in human lung cancer is not well understood. terized and validated a recombinant rabbit monoclonal antibody Methods specific to PD-L2 (CAL28). For checkpoint inhibitors, Abcam The levels and tissue distribution of Tregs and major tumor infiltrating already has research use only versions of three anti-PD-L1 Rab- lymphocyte (TIL) subsets were measured using simultaneous detection MAb® antibodies employed in the clinical setting (73-10, 28-8 and of FOXP3, CD4, CD8, pancytokeratin and DAPI by multiplexed quantita- SP142), co-developed with pharmaceutical and diagnostic tive immunofluorescence in 619 formalin-fixed paraffin embedded companies. (FFPE) NSCLCs from 4 independent cohorts represented in tissue micro- Methods arrays (cohort #1 [Yale, n=210], cohort #2 [Greece, n=192]; cohort #3] A recombinant rabbit monoclonal antibody was generated using [80 immunotherapy-treated NSCLCs]; cohort #4 [Yale, n=137, adenocar- a direct B cell cloning process and characterized for IHC. The cinomas with mutation testing). Markers were measured in different tis- clone was tested using PD-L2-transfected and non-transfected sue compartments and cell phenotypes were used for individual cell HEK293 cells fixed in formaldehyde and processed into paraffin counts and machine-learning-based spatial analysis. We studied the as- wax (FFPE) and further validated alongside In Situ Hybridization sociation between T-cell populations, tissue distribution, clinicopatho- (ISH) for PD-L2 mRNA in FFPE commercial cell lines. Once speci- logic/molecular characteristics and outcomes. ficity was determined, it was tested in positive and negative tis- Results sues and TMAs of Head & Neck Squamous Cell Carcinoma Tregs (DAPI+/CD4+/FOXP3+ cells) were predominantly located in the (HNSCC), Prostate Carcinoma (PC) and Renal Cell Carcinoma stromal compartment and represented 3-10% of the total T-cell popula- (RCC). tion. The level of Tregs was positively associated with higher CD8+ T- Results cell infiltration across the cohorts. There was no consistent association CAL28 demonstrated positive IHC staining on PD-L2- between Treg levels and patient age, gender, smoking status, clinical overexpressed HEK293 cells processed in FFPE with a lack of stage or tumor histology. However, Tregs were significantly higher in staining in the parental line. Additionally, CAL28 demonstrated KRAS mutated lung adenocarcinomas than in EGFR mutant or KRAS/ IHC staining in FFPE cell lines where PD-L2 expression was con- EGFR wild-type cases. As a single marker, the level of Tregs was not sig- firmed with ISH for PD-L2 mRNA. Expression in tumor tissue in nificantly associated with survival. However, the Treg to CD8 signal ratio TMAs from HNSCC, PC and RCC was evaluated with no non- was associated with shorter 5-year overall survival across the cohorts. specific background staining. Reduced survival was also seen in cases with a higher 5-nearest neigh- Conclusions bor (5NN) mean distance between CD4+/Tregs and CD8+/CD4+ cells. We have demonstrated sensitivity, specificity and reproducibility Notably, the survival effect of the Treg-associated metrics was numeric- of a recombinant rabbit monoclonal antibody to PD-L2 in IHC ally higher in patients treated with immune checkpoint blockers. (CAL28). The global, commercial availability of this recombinant Conclusions clone to researchers, pathologists, clinicians and the biopharma- Tregs are prominently less abundant than other TIL subsets in NSCLC ceutical industry will enable further progress to be made in un- microenvironments and they are increased in T-cell inflamed tumors. derstanding the clinical relevance and predictive value that PD-L2 Their positive association with CD8+ cytotoxic TILs suggests their up- promises for cancer immunotherapy. regulation upon adaptive anti-tumor immune pressure and could ex- plain the inconsistent reported relationship between Tregs and References prognosis. Elevated Treg to CD8 signal ratio and reduced spatial clus- 1. Tsao MT, Kerr K, Yatabe Y, et al. PL 03.03 Blueprint 2: PD-L1 Immunohisto- tering between CD4-Tregs and CD8-CD4 are indicative of poor out- chemistry Comparability Study in Real-Life, Clinical Samples. J Thor Oncol. come preferentially in NSCLC patients treated with checkpoint 2017;12(Suppl 2):S1606 blockade suggesting a biomarker role. 2. Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Ethics Approval Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T. All tissues were used after approval from the Yale Human Investiga- Clin Cancer Res. 2017;23:3158-3167 tion committee protocol #9505008219 which approved patient con- 3. Zhao SG, Lehrer J, Chang SL, Das R, Erho N, Liu Y, Sjöström M, Den RB, sent forms or waivers of consent. Freedland SJ, Klein EA, Karnes RJ, Schaeffer EM, Xu M, Speers C, Nguyen PL, Ross AE, Chan JM, Cooperberg MR, Carroll PR, Davicioni E, Fong L, Spratt DE, Feng FY.The Immune Landscape of Prostate Cancer and P137 Nomination of PD-L2 as a Potential Therapeutic Target. J Natl Cancer Inst. Development and high specification validation of two recombinant 2019;111:301-310 rabbit monoclonal antibodies to accurately detect human PD-L2 4. Takamori S, Takada K, Toyokawa G, Azuma K, Shimokawa M, Jogo T, expression in FFPE tissue sections by immunohistochemistry Yamada Y, Hirai F, Tagawa T, Kawahara A, Akiba J, Okamoto I, Nakanishi Simon Renshaw, Will Howat, PhD, Subham Basu, PhD Y, Oda Y, Hoshino T, Maehara Y. PD-L2 Expression as a Potential Predict- Abcam, Cambridge, United Kingdom ive Biomarker for the Response to Anti-PD-1 Drugs in Patients with Non- Correspondence: Subham Basu (Subham.Basu@abcam.com) small Cell Lung Cancer. Anticancer Res. 2018;38:5897-5901 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P137 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 76 of 272 P138 3. Oaknin A, Duska LR, Sullivan RJ, et al. Preliminary safety, efficacy, and Review of evidence for predictive value of microsatellite pharmacokinetic/pharmacodynamic characterization from GARNET, a instability/mismatch repair status in response to non-anti-PD-(L)1 phase I/II clinical trial of the anti–PD-1 monoclonal antibody, TSR-042, in therapies in patients with advanced or recurrent endometrial patients with recurrent or advanced MSI-H and MSS endometrial cancer. cancer Gynecol Oncol. 2019; 154:17. 1 2 2 2 Cara Mathews, MD , Ellie Im , Liliana Alfaya , Karin Travers , Craig 4. Antill YC, Kok PS, Robledo K, et al. Activity of durvalumab in advanced Gibson endometrial cancer (AEC) according to mismatch repair (MMR) status: 1 2 Women and Infants Hospital, Providence, RI, United States; TESARO: A The phase II PHAEDRA trial (ANZGOG1601). J Clin Oncol 37, 2019 (suppl; GSK Company, Waltham, MA, United States abstr 5501). Correspondence: Cara Mathews(cmathews@wihri.org) 5. Djordjevic B, Bruegl A, Fellman B, et al. The prognostic effect of MLH1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P138 loss in endometrial endometrioid adenocarcinoma. Lab Invest. 2014; 94:280A–281A. Background 6. Cohen JG, Goodman MT, Karlan BY, Walsh C. Genomic characterization of Multiple immunotherapies have been evaluated in patients with ad- grade 3 endometrial carcinoma. Gynecol Oncol. 2014; 133:134–135. vanced or recurrent endometrial cancer (EC) using molecular bio- 7. Cosgrove CM, Cohn DE, Hampel H, et al. Epigenetic silencing of MLH1 in markers, including microsatellite instability-high (MSI-H) and stable endometrial cancers is associated with larger tumor volume, increased (MSS) status. Clinical outcomes appear to be different in patients with rate of lymph node positivity and reduced recurrence-free survival. Gyne- MSI-H/mismatch repair (MMR)-deficient status versus MSS/MMR-profi- col Oncol. 2017; 146(3):588–595. doi:10.1016/j.ygyno.2017.07.003. cient status when receiving anti-programmed cell death (ligand) 1 (PD- 8. Kim SR, Pina A, Albert A, et al. Does MMR status in endometrial cancer [L]1) therapies [1,2]. It is unclear if these differences are due to the ther- influence response to adjuvant therapy? Gynecol Oncol. 2018; 151:76–81. apies themselves or to differences inherent to the patient populations. 9. Aghajanian C, Filiaci V, Dizon DS, et al. A phase II study of frontline We sought to evaluate the association between MSI-H/deficient MMR paclitaxel/carboplatin/bevacizumab, paclitaxel/carboplatin/temsirolimus, (dMMR) status and response among patients with advanced or recur- or ixabepilone/carboplatin/bevacizumab in advanced/recurrent rent EC. endometrial cancer. Gynecol Oncol. 2018; 150:274–281. Methods We conducted a systematic review of the Embase, MEDLINE, and P139 Cochrane Central Register of Controlled Trials databases from 2000 Expression of GITR and GITR-L by head and neck squamous cell to present to identify publications (manuscripts and conference pro- cancer ceedings) on studies using chemotherapy, surgery, radiotherapy, hor- 1 2 Rachna Moudgil, MS , Christopher Paustian, PhD , Carmen Ballesteros- monal therapy, or biological therapy (or any combination thereof) in 1 1 1 Merino, PhD , Shawn Jensen, PhD , Hong-Ming Hu, PhD , Walter Urba, adult patients (≥18 years) with stage III or IV advanced or recurrent 1 1 1 MD, PhD , Carlo Bifulco, MD , Marcus Couey, MD, DDS , Traci Hilton, EC, and where MMR or MSI status was identified (by any means). To 2 1 1 1 PhD , Bernard Fox, PhD , Rom Leidner, MD , R. Bryan Bell, DDS, MD better understand the prognostic value of MSI-H/MSS status, we ex- 1 2 Earle A. Chiles Research Institute, Portland, OR, United States; UbiVac, cluded anti-PD-(L)1 therapies from the analysis, as recent evidence Portland, OR, United States suggests that there is a positive predictive value for these agents in Correspondence: Bernard Fox(foxb@foxlab.org) patients with MSI-H/dMMR status [2-4]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P139 Results Our systematic review of MSI/MMR status and recurrence-free survival Background (RFS), progression-free survival (PFS), and overall survival (OS) identified Head and neck squamous cell cancer (HNSCC) ranks as the 6th most a total of 5 studies. One study reported dMMR status was associated common cancer afflicting humans and remains a significant unmet med- with a reduction in RFS (hazard ratio, 2.02) [5], while another study ical need. While interfering with the PD-1/PD-L1 axis improves outcomes, found no significant effect [6]. A third study reported a trend towards a the majority of patients progress and die of their disease. To address this higher rate of recurrence among patients with advanced-stage EC with lack of efficacy our group has explored the immune makeup of HNSCC, dMMR than among patients with MMR proficiency (P value not re- hypothesizing that a better characterization of responders and non- ported) [7]. Two studies reported no significant association between responders will result in improved predictive biomarkers and insights into PFS and dMMR status [8,9]. Three studies found no statistically signifi- strategies to improve outcomes for the majority of patients. cant association between OS and dMMR status [5,6,8]. Methods Conclusions Over the past 7 years we have collected and processed more than This review could not identify a consistent association between 350 HNSCC specimens. When sufficient tumor material was available, dMMR or MSI-H status and recurrence, RFS, PFS, or OS among pa- tumor-infiltrating lymphocytes (TIL) and primary tumor cultures were tients with advanced or recurrent EC receiving therapy other than initiated and characterized for autologous tumor reactivity. Once anti-PD-(L)1. For RFS, where differences were present, they trended established, tumor cell lines were characterized for phenotypic towards worse outcomes for patients with MSI-H/dMMR status. Con- markers by flow cytometry. Flow cytometric analysis and RNASeq has sequently, we have identified no evidence of a prognostic or predict- been performed on some established cell lines and on FFPE tumor ive value of MSI-H or dMMR biomarker status for efficacy outcomes specimens. in patients with advanced or recurrent EC receiving non–anti-PD-(L)1 Results therapy. Further investigation into the prognostic or predictive value Consistent with previous reports increased expression of CD8 T cells of MSI-H/dMMR status is warranted. was associated with improved outcome. In preliminary studies in- creased expression of GITR was also associated with improved out- Acknowledgements come. Initial speculation was that GITR expression was coming from Clinical Trial Registration: N/A immune infiltrates. Subsequently a report suggested that GITR could be expressed by HNSCC. Using flow cytometry we detected low level References GITR expression on 3 HNSCC cell lines and low to high level expres- 1. Segal NH, Wainberg ZA, Overman MJ, et al. Safety and clinical activity of sion of GITR-L on a 8 HNSCC cell lines. Studies are continuing to ex- durvalumab monotherapy in patients with microsatellite instability–high pand on these preliminary observations. (MSI-H) tumors [abstract]. J Clin Oncol. 2019; 37(Suppl 4):670. Conclusions 2. Konstantinopoulos PA, Liu JF, Luo W, et al. Phase 2, two-group, two- Anti-GITR and GITR-L both have the potential to provide positive sig- stage study of avelumab in patients (pts) with microsatellite stable (MSS), nals to immune cells. In addition to APC’s, GITR-L expression by some microsatellite instable (MSI), and polymerase epsilon (POLE) mutated re- HNSCC cells may contribute to the make-up of the immune cells infil- current/persistent endometrial cancer (EC) [abstract]. J Clin Oncol. 2019; trating these cancers. 37(Suppl 15):5502. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 77 of 272 Acknowledgements P141 Funding Support: The Harder Family, Robert and Elsie Franz, Wes and One-year progression-free survival in lung cancer patients treated Nancy Lematta, Lynn and Jack Loacker, the Providence Portland Medical with immune checkpoint inhibitors is significantly associated with Foundation and the Oral and Maxillofacial Surgery Foundation, The Mur- a novel immunomodulatory signature but not PD-L1 staining 1 1 1 2 dock Trust. Harsha Ranganath, MD , Amit Jain , Justin Smith , Julie Ryder , Amina 1 2 2 2 3 Ethics Approval Chaudry , Emily Miller , Felicia Hare , Poojitha Valasareddy , Rob Seitz , 3 3 3 2 The study was approved by the institutional review board of the Providence David Hout , Brock Schweitzer , Tyler Nielsen , Janice Mullins , Gregory Portland Medical Center (12-075A). Vidal University of Tennessee Health Sciences, Indianapolis, IN, United States; 2 3 P140 West Clinic Cancer Center, Memphis, TN, United States; Insight Myeloid cell contexture and IL-8 expression as a candidate Genetics, Nashville, TN, United States immunotherapy target in non-small cell lung cancer (NSCLC) Correspondence: Gregory Vidal (gvidal@westclinic.com) 1 1 Venkata Vamsi Nagineni, MD , Kurt Schalper, MD, PhD , Shruti Desai, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P141 1 2 2 1 PhD , Ignacio Melero, MD , Miguel Sanmamed, MD, PhD , Richa Gupta , 1 1 Roy Herbst, MD, PhD , Venkata Vamsi Nagineni, MD Background 1 2 Yale University, Waukegan, IL, United States; University of Navarra, Immune checkpoint inhibitors (PD-(L)1 inhibitors) have shown Pamplona, Spain promising therapeutic outcomes and have been approved for Correspondence: Kurt Schalper (kurt.schalper@yale.edu) multiple indications. However, widespread use of PD-(L)1 inhibi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P140 tors has been limited by a low response rate and immune- related adverse events. Therefore, an improved method for Background predicting response to the immune checkpoint blockade would Interleukin-8 (IL-8) is a chemokine expressed in multiple cancer types, better identify patients misclassified by conventional testing. We including NSCLC. It exerts various functions in shaping cancer have evaluated a proprietary algorithm which utilizes gene vascularization, cell dedifferentiation and inflammation/immunity. IL-8 expression in solid tumors to assess the presence of an immu- was described as a chemotactic factor for neutrophils and it has been nomodulatory (IM) signature intended to predict immunother- proposed to mediate recruitment of tolerogenic myeloid cells favoring apy response. The purpose of this study was to evaluate the a pro-tumorigenic microenvironment. Although clinical trials targeting performance of the IM signature against progression-free IL-8 are ongoing, its expression and role in NSCLC is unclear. survival (PFS) of patients treated with immune checkpoint Methods inhibitors. We developed a multiplexed quantitative immunofluorescence (QIF) Methods panel for simultaneous and localized measurement of IL-8, myeloperox- In this retrospective study, archival tumor tissue from metastatic idase (MPO), CD15, cytokeratin (CK) and DAPI. We analyzed the expres- lung cancer patients treated with one of three PD-(L)1 inhibitors sion of these markers and their association with PD-L1, CD4 and CD8- (pembrolizumab, nivolumab, and atezolizumab) either as a single positive cells in 3 retrospective NSCLC immunotherapy-naive cohorts agent or in conjunction with standard chemotherapy, from whom represented in tissue microarrays (cohort #1, n=262; #2, n=145; and #3, response data was available, was tested for the IM signature. n=132); 1 cohort of NSCLC patients treated with immune checkpoint Patients were stratified into two groups based on IM signature blockers (#4, n=59) and 1 collection of lung adenocarcinomas (LAC) an- classification as positive or negative, which was compared to im- alyzed for activating mutations in EGFR and KRAS (#5, n=121). We stud- munohistochemistry PD-(L)1 testing with a primary endpoint of ied the level of the targets, their distribution and association with one-year progression-free survival. Additionally, the IM signature immune features, clinicopathological variables and survival. classification was compared with objective response by Spear- Results man’s correlation as a continuous variable. IL-8 protein signal was detected in ~85% of cases with cytoplasmic Results staining pattern and was higher in tumor than in stromal cells. Elevated A total of 71 metastatic lung cancer patients were included in the tumor IL-8 was consistently associated with higher MPO+ neutrophils study with a median follow-up of 29 months. The one-year PFS haz- and CD15+ tumor-associated myeloid cells across the cohorts, but not ard ratio for the IM positive group was 0.31 (95% CI 0.14 to 0.68; with CD4+ and CD8+ T-cells. Increased IL-8 expression was not associ- p=.004 - Figure 1). A total of 62 out of the 71 metastatic lung cancer ated with major clinicopathologic variables. Elevated MPO+ and CD15+ patients had previous PD-L1 staining. Head-to-head analysis of PD-L1 cells was significantly higher in KRAS mutated than in EGFR mutated and IM signature on these patients found the one-year PFS hazard LACs. High MPO and CD15 signal was associated with shorter 5-year ratio for the IM group to be 0.30 (95% CI 0.13 to 0.71; p=0.006) and overall survival in all NSCLC cohorts. The negative prognostic effect of the one-year PFS hazard ratio for PD-L1 positive staining to be 0.76 MPO and CD15 was comparable in both immunotherapy-naïve and (95% CI 0.31 to 1.82; p=0.533). The mean IM correlation value with immunotherapy-treated NSCLC collections. objective response for PD = -0.06; SD = -0.04; PR = 0.14; CR = 0.33; p Conclusions Conclusions IL-8 protein is frequently expressed in NSCLCs associated with increased The IM signature was significantly associated with prolonged one- tumor-associated myeloid cells but independent from intratumor T-cell year progression-free survival among patients treated with PD-L1 in- responses. KRAS mutated LACs have prominent MPO+/CD15+ expres- hibitors while PD-L1 staining failed to be significantly associated. Pa- sion, supporting an immune suppressive role of myeloid cells in these tients classified positive by the IM signature demonstrated a three- malignancies. CD15 and MPO are prognostic markers in NSCLC and IL-8 fold improved hazard ratio compared to those who were negative. blockade could mediate favorable immunomodulatory effects. Funding was provided by Insight Genetics working in cooperation Ethics Approval with West Cancer Center and Research Institute. All tissues were used after approval from Yale Human Investigation Ethics Approval committee protocol #9505008219 which approved the patient con- This study was approved by the West Cancer Clinic Institutional Re- sent forms or waivers of consent view Board. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 78 of 272 above. The classifier delivered stratifications for response vs nonre- sponse with 84% accuracy, 79% sensitivity, 92% specificity, 75% posi- tive predictive value (PPV) and 95% negative predictive value (NPV). The associations of EpiSwitch™ response calls with overall survival (OS) and progressive free survival (PFS) in the independent cohort were significant (OS and PFS: log-rank p Conclusions The established EpiSwitchTM classifier contains strong binary markers of epigenetic deregulation with features normally attributed to gen- etic markers; the binary status of these classifying markers is statisti- cally significant for survival. Altogether, these findings highlight the potential of the EpiSwitchTM approach for identifying responders and non-responders to immuno-oncology therapies. Acknowledgements The authors would like to thank patients enrolled in the EMR000070-001 JAVELIN Solid Tumor trial for agreeing for their samples to be used for research purposes. This work is funded by Merck KGaA, Darmstadt, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. References 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- cer. Front Genet. 2016; 7:194. Fig. 1 (abstract P141). See text for description 2. Carini C, Hunter E; Scottish Early Rheumatoid Arthritis Inception Cohort investigators, et al. Chromosome conformation signatures define P142 predictive markers of inadequate response to methotrexate in early Development and validation of baseline predictive biomarkers for rheumatoid arthritis. J Transl Med. 2018; 16:18. response to avelumab in second-line (2L) non-small cell lung 3. Jakub JW, Grotz TE, Jordan P, et al. A pilot study of chromosomal cancer (NSCLC) using EpiSwitchTM epigenetic profiling aberrations and epigenetic changes in peripheral blood samples to 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , identify patients with melanoma. Melanoma Res. 2015; 25:406-11. 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan 4. Bastonini E, Jeznach M, Field M, et al. Chromatin barcodes as biomarkers 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem for melanoma. Pigment Cell Melanoma Res. 2014; 27:788-800. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir 5. Yan H, Hunter E, Akoulitchev A, et al. Epigenetic chromatin conformation 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins changes in peripheral blood can detect thyroid cancer. Surgery. 2019; 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, 165:44-49. Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; Ethics Approval Mayo Clinic, Rochester, MN, United States The protocol was approved by the institutional review board or independent Correspondence: Parantu Shah (parantu.shah@emdserono.com); ethics committee at each center. Matthew Salter (matthew.salter@oxfordbiodynamics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P142 P143 Development and validation of baseline predictive biomarkers for Background response to immuno-checkpoint treatments in the context of Development of baseline predictive classifiers for response to treatment multi-line and multi-therapy cohorts using EpiSwitchTM epigenetic can provide advantages for programs of targeted immunotherapies, profiling development of successful combination therapies, and identification of 1 2 3 1 Parantu Shah, PhD , Ewan Hunter , Shobha Potluri , Sen Zhang , responder populations to active therapies. Chromosome conformations 2 2 2 2 Mehrnoush Dezfouli , Jennifer Back , Louis James , Navin Jandor , Ryan represent strong systemic cellular network deregulations associated 2 2 2 2 Powell , Matthew Salter , Aroul Ramadass , Jayne Green , Willem with differences in clinical phenotypes and outcomes [1]. 2 4 4 Westra , Haidong Dong, MD, PhD , Roxana Dronca, MD , Svetomir Methods 4 2 1 3 Markovic, MD, PhD , Alexandre Akoulitchev , Ti Cai , Paul Robbins Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a 1 2 EMD Serono, Inc, Billerica, MA, United States; Oxford Biodynamics, business of Merck KGaA, Darmstadt, Germany/Pfizer alliance," has ap- Oxford, United Kingdom; Pfizer, Inc, San Francisco, CA, United States; plied its proprietary technology EpiSwitchTM to monitor systemic Mayo Clinic, Rochester, MN, United States epigenetic biomarkers for chromosome conformation signatures in Correspondence: Parantu Shah (parantu.shah@emdserono.com) baseline blood samples of patients with multiline anti–PD-L1 (avelu- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P143 mab) treatment of NSCLC. This application was based on the pub- lished methodology for validated predictive biomarkers for response Background to treatment [2], systemic blood-based monitoring of oncological Development of baseline predictive classifiers for response to treatment conditions [3-5], and proprietary programs in collaboration with the can provide advantages for programs of targeted immunotherapies, Mayo Clinic for predictive and response biomarkers in melanoma pa- development of successful combination therapies, and identification of tients treated with anti–PD-1 therapy (pembrolizumab). responder populations to active therapies. Changes in chromosome Results conformations represent strong systemic cellular network deregulations A 14-marker classifier was generated with 12 avelumab-treated pa- associated with differences in clinical phenotypes and outcomes [1]. tients in each response group; in this cohort, responders were de- However, questions remain about the applicability of classifiers across fined as patients with complete or partial response, and non- treatment lines, indications, and drug combinations. responders were defined as patients with progressive disease. Valid- Methods ation of the developed predictive markers was performed on an in- Oxford Biodynamics, in collaboration with the EMD Serono, Inc., a dependent cohort of 75 patients treated with avelumab as either business of Merck KGaA, Darmstadt, Germany/Pfizer alliance, has ap- first-line (1L) or 2L therapy. In the validation cohort, patients with plied its proprietary technology EpiSwitchTM to monitor systemic stable disease were also considered as responders in addition to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 79 of 272 epigenetic biomarkers for chromosome conformation signatures at Methods baseline in patients with multiline anti–PD-L1 (avelumab) treatment Tyramide signal amplification detection was used to inform on the of non-small cell lung cancer (NSCLC). Additionally, epigenetic bio- tumor/stroma/immune contexture (CD8, PanCK, FAP, MHC-I, CD31) and markers to predict outcome and response in patients with melanoma to characterize T-cell functions (PD1, CD3, PanCK, GZMB, and PD-L1). treated with anti–PD-1 (pembrolizumab) and its combination with Whole slide digital pathology scoring algorithms were developed to another agent were identified in collaboration with the Mayo Clinic. identify all phenotypes represented by the markers and their specificity Results and sensitivity was verified against the results by expert observers. Three NSCLC classifiers predicting response to avelumab in first-line Results (1L), second-line (2L), and combined 1L + 2L cohorts were built and ap- Assay performance including accuracy, precision, and sequential markers plied to test sets. Average accuracy, positive predictive value (PPV), and detection was validated on > 200 unique cases of Gastric, Pancreatic, negative predictive value (NPV) for 10-fold cross-validation on data Breast, Lung, Urothelial and Colorectal carcinomas. For an early proof-of- splits were reported. An NSCLC patient set treated with 2L pembrolizu- concept, we analyzed paired pre- vs. post treatment tumor biopsies from mab served as an independent test set. The 2L NSCLC classifier three pancreatic cancer patients treated with a combination of atezolizu- achieved high (defined hereafter as > 0.7) predictive power (PPV, NPV, mab and chemotherapy. Digital pathology algorithms identified biologic- and accuracy) in the 2L test set but not in the 1L test set. A reduced ally and clinically relevant features: MHC-I is highly expressed in tumor version of this classifier achieved a PPV of 0.71 in the 2L pembrolizu- cells of the primary lesions and very rare tumor cells express MHC-I in the mab population. The 1L classifier was not applicable in patients who re- liver met samples. A patient with partial response (PR) showed a signifi- ceived 2L treatment for NSCLC. The 1L + 2L composite classifier had cantly increased tumor MHC-I upon treatment, while two patients with high predictive power in both 1L and 2L cohorts and a high PPV for stable disease (SD) did not show significant changes. The PR patient also identifying responders in the 2L pembrolizumab population. A fourth showed an increased density of CD3+, CD8+, and GZMB+ T cells within classifier starting with preselected NSCLC markers had good predictive the tumor post-treatment, indicating an increased tumoral T cell infiltra- power for classifying responders in patients with melanoma treated tion and activation by the treatment. with pembrolizumab. Finally, a 2L NSCLC classifier trained to classify re- Conclusions sponse groups from pembrolizumab-treated patients also identified The automated 5-plex IHC assays and digital pathology algorithms NSCLC responders with a high PPV from patients treated with pembro- developed in this study provide a robust tool for quantitative and lizumab in combination with an epigenetic drug. spatially resolved whole-slide characterization of the tumor-immune Conclusions contexture. Applying these tools in large-scale clinical investigations Collectively, these results suggest that a set of EpiSwitchTM bio- may provide better understanding of the response/resistance mecha- markers correlates with outcome on anti–PD-1/PD-L1 immunother- nisms to cancer immunotherapies. apies. Classifier signatures could be generated to work across treatment lines, indications, and combinations, and could be helpful Cellular Therapies for baseline patient stratification. P145 Acknowledgements Expanding Iovance’s tumor infiltrating lymphocytes (TIL) from core The authors would like to thank patients enrolled in the EMR000070-001 biopsies for adoptive T cell therapy using a 22-day manufacturing JAVELIN solid tumor trial for agreeing to consent usage of samples for process research purposes. This work was funded by Merck KGaA, Darmstadt, Michelle Abelson, PhD, Kenneth D'Arigo, Florangel Hilton, Maria Fardis, Germany, as part of an alliance between Merck KGaA, Darmstadt, Germany PhD, MBA, Cecile Chartier and Pfizer Inc., New York, NY, USA. Iovance Biotherapeutics, Inc., Tampa Bay, FL, United States Correspondence: Cecile Chartier (cecile.chartier@iovance.com) References Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P145 1. Tordini F, Aldinucci M, Milanesi L, et al. The genome conformation as an integrator of multi-omic data: the example of damage spreading in can- Background cer. Front Genet. 2016; 7:194. Iovance’s TIL products Lifileucel and LN-145 have demonstrated re- Ethics Approval markable clinical activity in melanoma and cervical cancer utilizing The protocol was approved by the institutional review board or independent Iovance’s proprietary 22-day manufacturing process and surgically ethics committee at each enrolling center. resected tumor lesions ~ 1.5-cm diameter [1, 2]. Using a core needle biopsy procedure to obtain tumor samples could allow for greater convenience of collecting the tumor from patients [3]. We asked whether a streamlined manufacturing process could be implemented P144 to produce therapeutically relevant TIL from multiple histologies Quantification of tumor-stroma-immune contexture by multiplex starting with a core biopsy. fluorescent immunohistochemistry and whole-slide digital image Methods analysis 1 1 2 Core biopsies obtained from 4 melanoma and 3 pancreatic, 2 breast, Adriana Racolta, PhD , Mehrnoush Khojasteh, PhD , Jennifer Giltnane , 1 1 1 2 ovarian, and 1 lung tumors were processed in vitro, using a 22-day Antony Hubbard, BS , Hongjun Zhang , Miriam Matei , Jessica 1 1 1 expansion method termed ‘Core process’. Core biopsy-derived TIL Baumann , Wenjun Zhang, MD, PhD , Tsu-Shuen Tsao, PhD , Hartmut 2 2 1 1 1 were assessed for expansion, phenotype (lineage, youth/differenti- Koeppen , Lisa Ryner , Xingwei Wang , Jim Martin , Auranuch Lorsakul , 1 1 1 1 ation, activation, and exhaustion markers), function (IFN-gamma and Ilya Ravkin , Smadar Shiffman , Lidija Pestic-Dragovich , Lei Tang, PhD , CD107a mobilization), and TCR repertoire. Yulei Wang, BA PhD 1 2 Results Roche Tissue Diagnostics, Tucson, AZ, United States; Genentech, South Iovance’s Core process successfully generated TIL products from all San Francisco, CA, United States tested samples. One to 2 cores yielded more than 10e9 T cells for 10 Correspondence: Yulei Wang (wang.yulei@gene.com) of the 12 preparations. Phenotypic analyses revealed no significant Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P144 differences in terms of T cell lineages and memory subsets, or ex- pression of activation, differentiation, and exhaustion markers when Background compared to Iovance’s current products. Core-derived TIL products Understanding response to immunotherapies in relation to tumor- responded to PMA and to anti-CD3 stimulations by inducing levels of immune contexture requires a paradigm shift from a single-marker CD107a mobilization and IFN-gamma secretion like those produced test towards multiplexed immunohistochemistry (IHC). Here we re- by TIL derived from excisional biopsies. Preliminary TCR sequencing port the development, early proof of concept of two fully automated data suggest that high-diversity products can be also be obtained 5-plex fluorescent multiplex IHC assays and accompanying digital from small samples, similar to what is obtained from TIL expansion. pathology algorithms. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 80 of 272 Conclusions observed compared with single transduced CAR T-cells (product A or This work demonstrates that the Iovance 22-day Core manufacturing B). Expression of the IL7R_CCR in both AUTO6NG and product A con- method is highly robust and that it is feasible to expand TIL to thera- ferred exogenous-cytokine-independent viability and homeostatic peutically relevant numbers from as little as 1 to 2 core biopsies from proliferation of modified T-cells, without causing autonomous T-cell multiple histologies with this method. Resulting products were shown growth. Furthermore, AUOTO6NG T-cells and product B but not to be phenotypically comparable to, and as potent as, products gener- product A proved resistant to both TGFb- and PD1/PDL1-mediated ated with Iovance’s process from excisional biopsy. Iovance anticipates immunosuppression in vitro due to the presence of dnTGFbRII and implementing this process in the clinic in the near future. dSHP2 in those genetically engineered CAR T-cells. Finally, intraven- ous delivery of AUTO6NG exhibited potent anti-tumour activity and References extended survival in NSG mice with established tumour burden. 1. Jazaeri AA, Zsiros E, Amaria RN, Artz AS, Edwards RP, Robert Michael Conclusions Wenham RM, et al. Safety and efficacy of adoptive cell transfer using These results demonstrate the feasibility, safety, and efficacy of autologous tumor infiltrating lymphocytes (LN-145) for treatment of AUTO6NG T-cells. The addition of IL7R_CCR, dnTGFbRII and dSHP2 recurrent, metastatic, or persistent cervical carcinoma. Clin Oncol. modules to the AUTO6NG product augment its functions by extend- 2019;37:15:2538 (suppl). ing T-cell persistence and rendering modified T-cells resistant to 2. Sarnaik A, Khushalani NI, Chesney JA, Kluger HM, Curti BD, et al. Safety TGFb- and PD1/PDL1-driven immune inhibition. and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients P147 who progressed on multiple prior therapies including anti-PD-1. J Clin Effect of chemotherapy on cellular kinetics of NKG2D-based CAR T- Oncol. 2019;37:15:2518 (suppl). cells in metastatic colorectal cancer patients 3. Ullenhag GJ, Sadeghi AM, Carlsson B, Ahlström H, Mosavi F, Wagenius G, Erik Marcelo Alcantar Orozco, Eytan Breman, MSc, Marie-Sophie Dheur, Tötterman TH, et al. Adoptive T-cell therapy for malignant melanoma pa- PhD, Fabian Borghese, PhD, Emilie Cerf, PhD, Nathalie Braun, Caroline tients with TILs obtained by ultrasound-guided needle biopsy. Cancer Lonez, PhD, Anne Flament, Frederic Lehmann, MD Immunol Immunother. 2012;61:725–732. Celyad, Mont-Saint-Guibert, Belgium Correspondence: Frederic Lehmann (flehmann@celyad.com) P146 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P147 AUTO6NG: Next generation GD2-targeting CAR T-cell therapy with improved persistence and insensitivity to TGFb and checkpoint Background inhibition for relapsed/refractory neuroblastoma Autologous and allogeneic Chimeric Antigen Receptor (CAR) T-cells are 1 1 1 Daniela Achkova, PhD , Adrian Zarzoso , Yusuf Demir , Fernando under thorough investigation to translate their success in B-cell malig- 1 1 1 1 Gallardo , Maria Stavrou , Marco Della Peruta , Saket Srivastava , Mathew nancies to other types of cancer. Previous studies associated the anti- 1 1 1 1 Robson , Shimobi Onuoha , Simon Thomas , Shaun Cordoba , Martin tumour effect of CAR T-cells to their long-term persistence. Most stud- 1,2 Pule ies use cyclophosphamide and fludarabine (CyFlu) preconditioning 1 2 Autolus Ltd, London, United Kingdom; University College London chemotherapy to facilitate CAR T-cell persistence. However, the effect Cancer Institute, London, United Kingdom of CyFlu preconditioning was rarely compared to other chemotherapies Correspondence: Martin Pule (m.pule@autolus.com) or to CAR T-cells alone. The THINK, SHRINK and ALLOSHRINK trials Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P146 evaluate the safety and clinical activity of NKG2D receptor-based CAR T-cells in metastatic colorectal cancer (mCRC) patients. THINK and Background SHRINK utilize autologous CAR T-cells, whereas ALLOSHRINK utilizes Neuroblastoma is the most common extracranial solid cancer in chil- allogeneic CAR T-cells. In THINK, CAR T-cells are injected without pre- dren with poor long-term survival in those with high-risk disease. A conditioning chemotherapy or after CyFlu. In SHRINK and ALLOSHRINK, currently ongoing phase I clinical study of GD2-targeted CART for re- FOLFOX chemotherapy is given before CAR T-cell injections. Herein we fractory/relapsed neuroblastoma (NCT02761915) shows activity present cellular kinetics results from these three trials. against disseminated disease without inducing on target/off tumor Methods toxicity. However, CART persistence was limited and clinical activity Whole blood samples were drawn at various timepoints from pa- transient and incomplete. tients receiving at least one injection of CAR T-cells. Peripheral blood Building on the GD2 CAR used in this study, we have developed a next mononuclear cells (PBMCs) were isolated by ficoll gradient centrifu- generation T-cell product candidate termed AUTO6NG. The AUTO6NG gation at a central laboratory designated by the Sponsor. Genomic product consists of 3 distinct populations of GD2-targeted CAR T-cells, DNA was isolated using a commercially available kit. Engraftment of produced by dual transduction of T-cells with two separate retroviral CAR T-cells was measured by digital droplet polymerase chain reac- vectors. The first vector directs the expression of a GD2-targeting CAR, tion (ddPCR) using transgene-specific primers and reported as trans- co-expressed with a constitutively signalling IL7 cytokine receptor gene copies per microgram of genomic DNA. Long-term persistence (IL7R_CCR) (product A), while the second vector is a tri-cistronic retro- of CAR T-cells was measured by calculating the area under the curve viral vector encoding the same GD2 CAR, co-expressed with dominant (AUC) using the linear trapezoidal rule. negative TGFbRII (dnTGFbRII) and truncated SHP2 (dSHP2) (product B). Results dSHP2 confers resistance to inhibitory signals such as those from PD1. 35 mCRC patients have been treated in THINK (14), SHRINK (9) and Methods ALLOSHRINK (12). Preliminary results are available for 29 subjects. Cell Human T-cells were either dual transduced with both vectors yield- kinetics for subjects having received one injection of autologous CAR T- ing a mix of product A/B/A+B (AUTO6NG) or single transduced with cells show a seven-fold increase in mean peak levels of T-cell engraft- each vector individually giving raise to product A or B. Both single ment with CyFlu compared to FOLFOX. Mean AUC is four times higher and dual transduced CAR T-cells were extensively evaluated in vitro with CyFlu compared to FOLFOX. Peak levels of engraftment and per- for redirected lysis, cytokine secretion, T-cell proliferation and survival sistence observed with FOLFOX and without previous chemotherapy and resistance to immunosuppressive pathways (including TGFb and are similar. Additionally, allogeneic CAR T-cells exhibit a five-fold in- PD1/PDL1 inhibition) in co-culture assays with GD2-positive and crease in mean AUC and a ten-fold increase in mean peak levels com- negative tumour cell lines. Additionally, anti-tumour activity of pared to autologous cells with the same prior chemotherapy regimen. AUTO6NG was evaluated in vivo by intravenous administration in an Additional analyses will be presented during the congress. established neuroblastoma xenograft model in NSG mice. Conclusions Results Analyses of the initial 29 patients receiving either autologous or allo- AUTO6NG T-cells (product A/B/A+B) were highly potent in cytotox- geneic NKG2D-based CAR T-cells demonstrate that CyFlu enhances icity assays against GD2 positive tumour cell lines with no differences peak levels and persistence of adoptively transferred cells. FOLFOX Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 81 of 272 does not appear to influence engraftment or persistence of CAR T-cells. P149 Allogeneic CAR T-cells show higher peaks and time-averaged persist- Silencing PD-1 using self-delivering RNAi PH-762- to improve ence compared to autologous cells. Analysis of the results is ongoing. Iovance TIL effector function using Gen 2 manufacturing method 1 1 Ethics Approval Inbar Azoulay-Alfaguter, PhD , Michelle Abelson, PhD , Krit Ritthipichai, 1 1 1 2 The studies referred to in this abstract were approved by all relevant DVM, PhD , Kenneth D’Arigo , Florangel Hilton , Marcus Machin, BS , 2 2 1 1 ethical committees and authorities. Dingxue Yan , James Cardia , Maria Fardis, PhD, MBA , Cecile Chartier 1 2 Iovance Biotherapeutics, Inc., Tampa, FL, United States; Phio Pharmaceuticals, Tampa, FL, United States P148 Correspondence: Cecile Chartier (cecile.chartier@iovance.com) High affinity NK cells expressing a PD-L1 chimeric antigen receptor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P149 demonstrate anti-tumor activity in head and neck cancer through multiple distinct mechanisms Background 1 1 1 Yevtte Robbins , Jay Friedman, PhD , Sarah Greene , Kellsye Fabian, Adoptive T-cell transfer with tumor infiltrating lymphocytes (TIL) 1 1 2 2 PhD , Michelle Padget , John Lee, MD , Patrick Soon-Shiong, MD , is an investigational immunotherapy for advanced solid cancers. 3 2 2 1 Kayvan Niazi , Lennie Sender , Laurent Boissel , Jeffrey Schlom, PhD , Ongoing Phase II clinical trials of Iovance’s lifileucel and LN-145 1 1 James Hodge, PhD, MBA , Clint Allen, MD TIL products have demonstrated efficacy with ORRs of 38% and 1 2 NIH, Bethesda, MD, United States; NantKwest, Culver City, CA, United 44% in patients with melanoma and cervical cancer, respectively 3 4 States; Nantworks, Culver City, CA, United States; NIH/NIDCD, Bethesda, [1,2]. Anti-PD-1 therapy has been widely used as a first-line ther- MD, United States apy in several types of cancer. TIL infusion products from the pa- Correspondence: Clint Allen (clint.allen@nih.gov) tients previously treated with anti-PD-1 therapy still sustain PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P148 expression, especially the subset of tumor antigen-specific TIL [3]. Building on the therapeutic efficacy of PD-1 blockade, we rea- Background soned that intrinsic silencing of PD-1 in our TIL products, may A significant portion of head and neck cancers (HNCs) harbor gen- provide similar benefits to systemic administration of anti-PD-1 omic alterations that render them insensitive to T cell detection. For therapy, while decreasing the side effects associated with sys- these patients, natural killer (NK) cellular therapy may be an effective temic anti-PD-1 [3]. Self-delivering small interfering RNA (sd- complementary treatment approach. We studied the anti-tumor ac- rxRNA) is a chemically modified siRNA molecule, which has ability tivity of a novel, off the shelf, NK cellular therapy consisting of high to penetrate cell types with high knockdown efficiency of specific affinity NK cells engineered to express a chimeric antigen receptor target genes [4]. Furthermore, a knockdown approach yields a (CAR) targeting PD-L1 (PD-L1 t-haNKs). transient effect, which may prove a more favorable approach Methods when compared with permanent genetic modification. Here, we Irradiated (15 Gy) PD-L1 t-haNK cells were assessed for direct cytotox- tested the silencing efficiency of a PD-1-targeted sd-rxRNA, icity of five human and two murine HNC cell lines by real-time imped- termed PH-762, in TIL and its effect on TIL phenotype and ance analysis. PD-L1 knockout by CRISPR/Cas9 gene editing was function. performed in select cells to assess PD-L1-specific killing. Co-culture as- Methods says with PD-L1 t-haNKs and murine or human peripheral and tumor TIL from melanoma, breast cancer, lung cancer, H&N cancer, infiltrating leukocytes were performed to determine selective elimin- and sarcoma were expanded ex vivo with Iovance’s proprietary ation of cells. Wild-type C57BL/6 (B6) or NSG mice were engrafted with 22-day process in the presence of PH-762. Resulting TIL parental or PD-L1 knockout murine or human tumors and assessed for products were assessed for PD-1 knockdown, cell expansion tumor growth inhibition (TGI) following PD-L1 t-haNK treatment. and viability, phenotype (T-cell lineage, differentiation, activa- Results tion, and exhaustion), and effector functions (IFN-gamma PD-L1 CAR expression on PD-L1 t-haNKs was verified. PD-L1 t-haNKs induction). killed all human and murine HNC cell lines at low effector:target ra- Results tios. Killing of cells was significantly enhanced with increased PD-L1 Average silencing of the PD-1 levels was 85%. Sixteen of the 19 expression following IFN-γ pre-treatment. Baseline killing was par- tumors tested demonstrated >80% silencing at the surface of tially reversed and IFN-γ -enhanced killing was completely abrogated PH-762-treated TIL relative to control sd-rxRNA-treated TIL. The in PD-L1 knockout cells. Ex vivo co-culture of PD-L1 t-haNKs with per- remaining 3 samples had ~70% silencing efficiency. Expression ipheral and tumor infiltrating leukocytes from tumor bearing mice or of T-cell activation markers including 4-1BB and OX40 was sig- with peripheral leukocytes from HNC patients revealed selective elim- nificantly increased in TIL expanded with PH-762. Importantly, ination of PD-L1 high macrophages and myeloid derived suppressor other inhibitory and exhaustion molecules remained unaffected, cells (MDSC) but not lymphocyte subsets. Treatment of B6 mice bear- suggesting that compensatory mechanisms were not triggered ing murine oral cancers with PD-L1 t-haNKs in vivo resulted in ≥50% by PD-1 silencing. Functionally, PD-1 knockdown TIL displayed reduction in PD-L1 high macrophages and MDSC but no reduction in elevated IFN-gamma secretion when co-cultured with autolo- lymphocytes. Treatment of NSG mice bearing parental human HNC gous tumor cells, indicating improved effector function upon or B6 mice bearing parental murine oral cancer resulted in significant specific T-cell re-stimulation. TGI after PD-L1 t-haNK treatment. TGI was completely abrogated in Conclusions mice bearing PD-L1 knockout tumors. sd-rxRNA-mediated silencing of PD-1 with PH-762 in TIL was Conclusions highly efficient and generated TIL products with elevated PD-L1 t-haNKs mediated potent PD-L1-specific cytotoxicity against effector function, providing a strong rationale for clinical HNC cells and selectively eliminate immunosuppressive macrophages testing. and MDSC expressing high levels of PD-L1 from the periphery and tumor microenvironment. PD-L1 t-haNK monotherapy resulted in PD- Acknowledgements L1-specific TGI in xenograft and syngeneic models. These data pro- PH-762 was kindly provided by Phio Pharmaceuticals. vide the pre-clinical rationale for the clinical study of PD-L1 t-haNKs in solid tumors. Evidence that PD-L1 t-haNKs selectively eliminate im- References munosuppressive macrophages and MDSC support the clinical study 1. Sarnaik A. et al. Safety and efficacy of cryopreserved autologous tumor of PD-L1 t-haNKs as a monotherapy or in combination with treat- infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic ments designed to activate T cell immunity. melanoma patients who progressed on multiple prior therapies Ethics Approval including anti-PD-1. J Clin Oncol. 2019;37:2518-2518. The study was approved by the NIH Animal care and Use Committee, 2. Jazaeri A A, et al. Safety and efficacy of adoptive cell transfer using approval number 1464-18. autologous tumor infiltrating lymphocytes (LN-145) for treatment of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 82 of 272 recurrent, metastatic, or persistent cervical carcinoma. J ClinOncol. 2019;37:2538-2538. 3. Gros A, et al. PD-1 identifies the patient-specific CD8(+) tumor- reactive repertoire infiltrating human tumors. J Clin Invest. 2014;124:2246-2259. 4. Ligtenberg M A, et al. Self-Delivering RNAi Targeting PD-1 Improves Tumor-Specific T Cell Functionality for Adoptive Cell Therapy of Malig- nant Melanoma. Mol Ther. 2018;26:1482-1493. P150 1st-in-human CAR T clinical trial for metastatic breast cancers Cynthia Bamdad, PhD , Andrew Stewart, PhD, Pengyu Huang, PhD, Benoit Smagghe, PhD, Scott Moe, PhD, Tyler Swanson, Thomas Jeon, Danica Page, Ketan Mathavan, PhD, Trevor Grant, PhD, Rachel Herrup Minerva Biotechnologies, Waltham, MA, United States Correspondence: Cynthia Bamdad (cbamdad@minervabio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P150 Background Minerva will open a 1st-in-human CAR T clinical trial for meta- static breast cancers at the Fred Hutchinson Center September, 2019. huMNC2-CAR44 targets a novel form of MUC1; no thera- peutic that targets this form has ever been tested in humans. All previous, failed attempts to therapeutically target MUC1 Fig. 1 (abstract P150). huMNC2-CAR44 T cells kill MUC1* have targeted the tandem repeat domains, which are cleaved positive tumors and shed from the surface of cancer cells. Cleavage and shed- ding of the tandem repeat domain increases as tumor stage in- creases. huMNC2-CAR44 targets the truncated extra cellular P151 domain of MUC1* (muk 1 star), also known as MUC1-C, which is Solid tumor cytotoxicity by natural killer cells expressing a HER2- the transmembrane cleavage product that remains after MUC1 directed chimeric antigen receptor enhanced by MyD88/CD40 (MC) is cleaved and the tandem repeat domain is shed from the can- Xiaomei Wang, PhD, Daniel Jasinski, PhD, Jan Medina, David Spencer, cer cells. The MNC2 antibody, which is the targeting head of PhD, Aaron Foster, PhD, Joseph Bayle, PhD the CAR, cannot bind to full-length MUC1. It binds to an ectopic Bellicum Pharmaceuticals, Houston, TX, United States epitope that is only unmasked by cleavage and release of the Correspondence: Aaron Foster (afoster@bellicum.com); Joseph Bayle MUC1 tandem repeat domain. MUC1* growth factor receptor is (jhbayle@bellicum.com) activated when onco-embryonicgrowth factorNME7ABdimer- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P151 izes its truncated extracellular domain. NME7AB and the huMNC2 antibody both competefor thesamebinding site, Background which is masked in full-length MUC1. The potent, innate anti-tumor cytotoxicity of natural killer (NK) cells Methods combined with their low risk of inducing graft-versus-host disease Monoclonal antibody MNC2 was selected because it recognizes have made NK cells an emerging platform for allogeneic, off-the- a conformational epitope within MUC1* that is created by cleav- shelf CAR-based cell therapies. However, adoptive transfers of NK age by MMP9, which is overexpressed in breast cancers and is cells have shown limited expansion and persistence which may im- an indicatorofpoorprognosis. The luminal edge of some nor- pact their ability to induce durable anti-tumor responses. Here, we mal tissues express a cleaved MUC1*-like form; however, on demonstrate that constitutive expression of a novel chimeric costi- normal tissues, MUC1 is cleaved by a different cleavage enzyme, mulatory protein, comprised of the signaling domains from MyD88 which alters the conformation of the truncated extra cellular and CD40 (MC) and secreted IL-15 dramatically improves the prolifer- domain and it is not recognized by the MNC2 antibody. ation and anti-tumor efficacy of HER2 CAR-redirected NK cells. Results Methods huMNC2-scFv recognizes 95% of breast cancers, across all subtypes, Human CD56-positive cells were enriched from PBMCs derived from wherein the average percent staining for each tissue specimen is healthy donors and activated with irradiated K562 cells in the presence ~80%. Despite this robust staining of cancerous tissues, huMNC2- of IL-15. NK cells were subsequently transduced with retroviral vector en- scFv showed almost no binding to normal tissues and no staining of coding inducible Caspase-9 (iC9), a HER2-specific CAR (HER2.ζ), MyD88/ critical organs. In vitro, huMNC2-CAR44 T cells killed cancer cells, but CD40 (MC) [1] and IL-15. Gene-modified NK cells were evaluated for ex- not non-cancer cells even if they expressed MUC1 or a cleaved pansion, cytotoxicity, cell phenotype and cytokine production, in vitro MUC1. In NSG mice (n>300), huMNC2-CAR44 T cells eliminated and in an HER2 positive OE-19 NSG mouse xenograft model. MUC1* positive tumors from implanted naturally occurring breast Results cancer cells. A single CAR T cell injection eliminated tumors for 100 NK cells were efficiently transduced (>50%) and demonstrated robust days; control animals had to be sacrificed at Day 20. Further, ex vivo expansion (150 fold, 14 days post-activation) in culture rela- huMNC2-CAR44 T cell mediated killing increased as MUC1* density tive to transduced NK cells. In coculture assays with HER2-expressing increased (Figure 1). OE19 and SKOV3 tumor cells, MC-enhanced CAR-NK cells showed po- Conclusions tent cytotoxicity with elevated expression of pro-inflammatory cyto- If successful, huMNC2-CAR44 could treat a wide variety of solid kines and chemokines including MIP1α, IFN-γ, and GM-CSF. In tumors. huMNC2-scFv binds to 95% of breast, 83% ovarian, 78% addition, NK cells expressing the iC9 safety switch could be rapidly pancreatic and 71% of lung cancers. ablated by treatment with 1 nM rimiducid to initiate apoptosis. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 83 of 272 animals engrafted with OE-19 tumor cells, iC9-CAR.ζ-MC-IL15 modi- Conclusions fied NK cells demonstrated significantly improved control of tumor Through the direct cytosolic delivery of antigen, we engineered expansion compared with control NK cells. unfractionated PBMCs to function as potent APCs. This strategy has Conclusions demonstrated significant potential to generate CD8+ T cell responses MyD88/CD40 and IL-15 enhance the proliferation and anti-tumor po- in both mouse and human systems and has been scaled for clinical tency of CAR-modified NK cells. Further, inclusion of the iC9 safety implementation. switch can be used to mitigate potential toxicities. These technolo- Ethics Approval gies have the potential to provide a potent, off-the-shelf allogeneic Human samples were supplied by an approved vendor and animal cell therapy to treat solid tumors. studies were conducted in accordance with SQZ Biotech's Animal Care Program and IACUC which operate according to principles set Reference forth in PHS Policy and the Guide for the Care and Use of Laboratory 1. Collinson-Pautz MR, Chang WC, Lu A, Khalil M, Crisostomo JW, Lin PY, Animals - 8th edition. Mahendravada A, Shinners NP, Brandt ME, Zhang M, Duong M, Bayle JH, Slawin KM, Spencer DM, Foster AE. Constitutively active MyD88CD40 costimulation enhances expansion and efficacy of chimeric antigen re- P153 ceptor T cells targeting hematological malignancies. Leukemia. 2019; Memory CD8+ T cells are more resistant to cancer stem cell (CSC) 33:2195-2207. suppression than effector CD8+ T cells and are more effective at Ethics Approval targeting CSC in a murine melanoma model This study was approved by Bellicum's IACUC and performed in its AAALAC 1 2 2 2 Brooke Bredbeck, MD , Shibin Qu , Alicia Kevelin , Ashley Pepple , Amy approved vivarium. 1 2 1 Felsted , Anutosh Ganguly , Clifford Cho, MD, FACS University of Michigan Medical School, Ann Arbor, MI, United States; P152 Ann Arbor VA Medical Center, Ann Arbor, MI, United States Antigen delivery to PBMCs by microfluidic squeezing primes anti- Correspondence: Clifford Cho (cliffcho@med.umich.edu) tumor immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P153 Matthew Booty, PhD, Kelan Hlavaty, Emrah Ozay, PhD, Carolyne Smith, PhD, Katherine Seidl, PhD, Howard Bernstein, MD, PhD, Armon Sharei, Background Scott Loughhead The ability to suppress immune reactivity is a defining hallmark SQZ Biotechnologies, Watertown, MA, United States of cancer [1-3]. Both the administration and disinhibition of Correspondence: Matthew Booty (matt.booty@sqzbiotech.com) CD8+ T cells, through adoptive immunotherapy and checkpoint Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P152 inhibition respectively, have yielded unprecedented responses in patients with advanced melanoma [4-8]. However, a majority Background of patients remain stubbornly unresponsive to T cell-based The presentation of sufficient antigen on major histocompatibility therapy [9,10]. A better knowledge of cancer-induced T cell sup- complex class I (MHC-I) is a potential barrier to generating potent pression is needed improve efficacy. Memory CD8+ T cells cancer immunizations. We use microfluidics-based squeezing to de- (Tmem) are more effective than effector CD8+ T cells (Teff) at liver antigen directly to the cytosol of target antigen presenting cells controlling melanoma growth after adoptive cell transfer (ACT) (APCs) – resulting in the enhanced presentation of antigen on MHC-I. in a murine melanoma model [11,12]. Melanoma cancer stem In addition to facilitating potent CD8+ T cell priming by professional cells (CSC) are primarily responsible for tumor growth and APCs, this approach can make unfractionated peripheral blood metastasis [13,14]. We hypothesized that Tmem are both more mononuclear cells (PBMCs) effective, unorthodox APCs capable of resistant to CSC suppression and more effective at targeting priming CD8+ T cell responses in mouse and human systems. CSC after ACT. Methods Methods Protein and peptide antigens were delivered to the cytosol of murine The B16F10 melanoma cell line was stably transfected to ex- splenocytes or human PBMCs by microfluidic squeezing. The re- press low levels of lymphocytic choriomeningitisvirus(LCMV) sponse to in vivo immunization was assessed by flow cytometry in a peptide antigen GP33 (B16GP33). Ly5.1+/C57BL/6 mice were in- series of experiments in mice. Tumor experiments were conducted fected with LCMV to isolate Teff and Tmem on post-infection with the TC-1 cell line, which expresses the viral antigens E6 and E7 days 8 or > 30, respectively. Ly5.2+/C57BL/6 mice were inocu- from human papilloma virus type 16 (HPV16). lated with subcutaneous B16GP33 tumors followed by either no Human PBMCs were loaded with synthetic long peptides (SLPs) con- treatment or ACT with Teff or Tmem on days 1 or 7. On day taining MHC-I restricted epitopes from cytomegalovirus (CMV) or 18-20, tumors were harvested for flow cytometric analysis HPV16. These PBMCs were co-cultured with epitope-reactive human (FACS) to characterize tumor-infiltrating lymphocytes (TIL) and responder CD8+ T cells, and interferon gamma production was quan- composition of melanoma CSC versus non-CSC (NCSC) based on tified to assess antigen-specific responses in vitro. expression of the CSC-specific marker aldehyde dehydrogenase Results (ALDH). In mice, we demonstrate that microfluidic squeezing enables delivery to Results all cell subsets within the spleen and that delivered protein antigen is Tumor inhibition was observed after ACT, with greatest treatment rapidly processed and presented on MHC-I. In vivo immunization using effect found after Tmem ACT (Figure 1). FACS analysis of CD8+ splenocytes squeezed with a HPV16-derived E7 SLP primes E7-specific re- TIL showed a predominant exhausted and non-activated pheno- sponses. Prophylactic immunization of mice implanted with TC-1 resulted type after Teff ACT; in contrast, CD8+ TIL exhibited a highly acti- in complete protection and these responses were durable, as mice were vated phenotype as well as superior endogenous CD8+ T cell protected upon TC-1 re-challenge. Therapeutic immunization following recruitment (Figure 2) after Tmem ACT. FACS analysis of tumor TC-1 implantation reduced tumor growth and extended survival com- cells after ACT demonstrated that ALDHhigh CSC fractions were pared to unimmunized mice (25 days vs 50 days). Following therapeutic markedly expanded after Teff ACT, but diminished after Tmem immunization, 85% of tumor infiltrating CD8+ T cells were found to be ACT (Figure 3). E7-specific compared to 3% in unimmunized mice. Conclusions In human cells, we demonstrate that squeezing of primary PBMCs en- Tmem-based ACT resulted in optimal tumor growth suppression, a ables delivery to all cell subsets. Delivery of CMV and HPV16 SLPs leads more activated TIL phenotype with superior CD8+ T cell recruitment, to presentation on MHC-I, as demonstrated by in vitro responses of and substantially stronger clearance of CSC compared to Teff ACT both CD8+ T cell clones and patient-derived memory populations. De- and controls. These observations suggest that use of Tmem may en- livery of CMV antigens at the manufacturing scale (~1 x 10^9 cells) also able cellular therapies to more effectively evade the suppressive ef- results in presentation and activation of CD8+ T cells. fects of melanoma while selectively targeting CSC. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 84 of 272 References 1. Marincola F, Wang E, Herlyn M, et al. Tumors as elusive targets of T-cell- based active immunotherapy. Trends Immunol. 2003; 24:335-342. 2. Dunn GP, Old LJ, et al. The three E’s of cancer immunoediting. Ann Rev Immunol. 2004; 22:329-360. 3. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Ann Rev Immunol. 2007; 25:267-295. 4. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell trans- fer immunotherapy. Clin Cancer Res. 2011; 17:4550-4557. 5. Dudley MS, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy folloing non-myeloablative but lymphodepleting chemotehrapy for the treatemnt of patients with refractory metastatic melanoma. J Clin Oncol. 2005; 23:2346-2357. 6. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711-723. 7. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimu- mab or monotherapy in untreated melanoma. N Engl J Med. 2015; 373:23-34. 8. Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013; 369:122-33. 9. Brahmer JR, Tykodi SS, Chow LQM, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012; 366:2455-2465. 10. Royal RE, Levy C, Turner K, et al. Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma. J Immunother. 2010; 33:828-33. Fig. 2 (abstract P153). CD8+ TIL exhibit a more activated 11. Contreras A, Sen S, Tatar AJ, et al. Enhanced local and systemic anti- phenotype after memory ACT melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice. Cancer Immunol Immunother. 2016; 65:601-611. 12. Contreras A, Beems MV, Tatar AJ, et al. Co-transfer of tumor-specific effector and memory CD8+ T cells enhances the efficacy of adoptive melanoma im- munotherapy in a mouse model. J Immunother Cancer. 2018; 6:41. 13. Maccalli C, DeMaria R. Cancer stem cells: perspectives for therapeutic targeting. Cancer Immunol Immunother. 2015; 64:91-97. 14. Pan Q, Li Q, Liu S, Ning N, Zhang X, Yu Y, Chang AE, Wicha MS. Concise review: targeting cancer stem cells using immunological approaches. Stem Cells. 2015; 33:2085-2092. Ethics Approval This study was approved by University of Michigan’s ethics board (IACUC), approval #1608-004. Fig. 3 (abstract P153). Memory CD8+ T cells target melanoma CSC P154 Development of an antigen-presenting bead kit for activation and expansion of human antigen-specific T cells Yelena Bronevetsky, PhD (yelena.bronevetsky@gmail.com) Berkeley Lights Inc, Alameda, CA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P154 Background Immunogenicity validation of peptide neoantigens represents a crit- ical bottleneck in the tumor antigen discovery process. Current bio- informatics platforms for antigen prediction are unsatisfactory, forcing researchers to screen many peptides per protein target in order to identify the few bona fide antigens. Standard immunogen- icity assays also cannot discriminate antigen-Human Leukocyte Anti- Fig. 1 (abstract P153). Memory CD8+ T cells vs effector CD8+ T gen (HLA) binding from T cell receptor (TCR) recognition, leading to cells in melanoma unnecessary screening of non-HLA binders in expensive and lengthy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 85 of 272 T cell reactivity assays that require large numbers of expensive pri- products were stimulated ex vivo with a T cell-specific superantigen. mary cells. Further, the use of donor-derived antigen-presenting cells Dogs received ACT of ex vivo-activated T cells followed by five sub- to assay T cell immunogenicity and expand rare, antigen-specific cutaneous IL-2 injections. Dogs were monitored for development of cells from the endogenous repertoire has inherent variability and a metastases via thoracic radiographs every three months. minimal degree of quality control. Berkeley Lights has developed an Results artificial antigen-presenting bead kit that expands antigen-specific T All 14 patients received autologous vaccinations. Due to early metas- cells from peripheral blood. tasis, 11 dogs received ACT. One dog did not receive adjuvant IL-2; Methods ten dogs completed the entire protocol. Toxicity was minimal after Peptide binding to HLA Class I and stability of the peptide-HLA com- pre-medicants (NSAID, antihistamine, and antiemetic) were instituted plex is assayed by loading peptides onto beads and staining with prior to ACT. With premedication, all toxicities were VCOG grade I/II. antibody. Following validation of peptide-HLA binding, primary CD8+ Median disease-free interval for all dogs was 213 days. Median sur- T cells from peripheral blood are stimulated by antigen-presenting vival time (MST) for all dogs was 415 days. Five dogs have survived beads twice over the course of two weeks. Frequencies of antigen- for over 621 days and are disease-free (Figure 1.) In addition, the re- specific T cells in the resulting cells is assayed by tetramer staining. sults included at least one dog with complete regression of distant Antigen-specific T cells can be loaded onto the Berkeley Lights (BLI) macroscopic metastasis. Lightning platform, a novel microfluidic platform that enables thou- Conclusions sands of single cell experiments in parallel. On the BLI Optoselect This immunotherapy protocol is safe and tolerable. Compared to chip, IFNg secretion and CD137 upregulation of antigen-specific T MST for historical amputation alone with or without adjuvant chemo- cells is assayed in response to antigenic stimulation. Following ana- therapy (MST of 307(1) and 134(2) days, respectively), a significant lysis, single cells can be exported for further analysis. survival benefit is noted in this group of patients. Further prospective Results studies are warranted to gain additional immunologic insight to the Berkeley Lights has developed an artificial antigen-presenting bead protocol, further improve disease response and survival, and evaluate that expands antigen-specific T cells from peripheral blood 10 times the translational impact this treatment could have in advancing hu- more effectively than autologous dendritic cells. This system allows man medicine. users to load peptides of choice onto magnetic beads and use them to assay peptide-HLA binding and stability, and to efficiently stimu- References late and expand antigen-specific T cells. Finally, in conjunction with 1. Phillips B, Powers BE, Dernell WS, Straw RC, Khanna C, Hogge GS, Vail the Berkeley Lights Lightning platform and the T cell Phenotype and DM. Use of single-agent carboplatin as adjuvant or neoadjuvant therapy Functional Analytics workflow, multiple functional parameters can be in conjunction with amputation for appendicular osteosarcoma in dogs. assayed from as few as 1000s of T cells, linking peptide-HLA binding J Am Anim Hosp Assoc. 2009; 45:33-8. and recognition to antigen-specific effector function. 2. Spodnick GJ, Berg J, Rand WM, Schelling SH, Couto G, Harvey HJ, Henderson RA, MacEwen G, Mauldin N, McCaw DL, Moore AS, Morrison W, Norris AM, O’Bradovich, J, O’Keefe DA, Page R, Ruslander D, Klausner J, Straw R, Thompson JP, Withrow SJ. Prognosis for dogs with appendicular P155 osteosarcoma treated by amputation alone: 162 cases (1978-1988). J Am Prospective translational study evaluating vaccine-enhanced Vet Med Assoc. 1992; 200(7):995-9. adoptive T cell therapy for treatment of osteosarcoma in Ethics Approval companion dogs The study was approved by University of Missouri’s IACUC, approval number 1 1 Tammie Wahaus, BSBA , Noe Reyes, DVM , Jeffrey Bryan, DVM, MS, PhD, 2 2 DACVIM-Oncology , Jeffrey Bryan, DVM, MS, PhD, DACVIM-Oncology , 3 2 Gary Wood, PhD , Brian Flesner, DVM, MS, DACVIM-Oncology , Lindsay 2 2 Donnelly, DVM, MS, DACVIM-Oncology , Debbie Tate, RVT, VTS 1 2 ELIAS Animal Health, Olathe, KS, United States; University of Missouri, Columbia, MO, United States; TVAX Biomedical, Olathe, KS, United States Correspondence: Tammie Wahaus (twahaus@eliasah.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P155 Background Canine osteosarcoma (OSA) is an aggressively metastatic primary bone malignancy with a 90% mortality rate. Many naturally occurring canine cancers are genetically and biologically similar to their human counterparts. All cancers express neoantigens and therefore are po- tentially susceptible to vaccine-enhanced adoptive T cell therapy. Syngeneic rodent studies demonstrated that metastases could be permanently eliminated with vaccine-enhanced adoptive T cell ther- apy. Canine OSA studies provide an excellent translational bridge be- tween experimental metastatic rodent cancer studies and metastatic human cancer clinical trials. We hypothesized that dogs with OSA could be safely treated at diagnosis with surgery, autologous cancer cell/P. acnes vaccination, adoptive T cell transfer (ACT) of ex vivo-acti- vated T cells, and low dose human interleukin-2 (IL-2) resulting in im- proved survival compared to carboplatin. We further hypothesized that significant efficacy would be achieved by treating dogs with in- tact immune systems and minimal residual disease [1,2]. Methods 14 client-owned cancer bearing dogs were enrolled in a one-arm prospective trial. Dogs were staged with bloodwork, limb and thor- acic radiographs, histopathology, and bone scans prior to amputation to remove the primary bone tumor. Autologous cancer cell/P. acnes Fig. 1 (abstract P155). Survival Analysis of Dogs vaccinations were administered intradermally weekly for three weeks. Completing Protocol Dogs underwent leukapheresis. Mononuclear white blood cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 86 of 272 P156 2. Hodge G, Barnawi J, Jurisevic C, Moffat D, Holmes M, Reynolds PN, Lung cancer sub-types exhibit differential susceptibility to natural Jersmann H, Hodge S. Lung cancer is associated with decreased killer cell cytotoxicity expression of perforin, granzyme B and interferon(IFN)-γ by infiltrating Jason Cahoon, BS, Shilan Dong, MS, Rafet Amoor, Donna Sonntag, MS, lung tissue T cells, natural killer (NK) T-like and NK cells. Clin Exp Immu- Alexander Spurrell, Rachit Ohri, PhD nol. 2014; 178:79–85. Enable Life Sciences, Worcester, MA, United States Correspondence: Rachit Ohri (rachit@enablelifesciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P156 Background Natural Killer (NK) cells hold great promise in immunotherapy, particularly for lung cancer [1]. However, there is a paucity of literature which organizes the susceptibility of various lung can- cer subtypes to NK cells. We evaluated the cytotoxicity (necrosis and apoptosis) of the NK cell line KHYG-1 (Effector) against cell- lines of 3 different subtypes of lung cancer i.e. H1975, H1703, A549 (Target). We also determined the levels of biomarkers rele- vant to NK cell activation and function [2] i.e. the cell-surface biomarker CD107a and 5 soluble biomarkers [Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha]. Methods Three lung cancer cell lines (H1975, H1703, A549) (ATCC, Vir- ginia) were plated at 100% confluency in 96-well plates (1.25 cells/cm^2, 3.2*10^5 cells/mL), while K562 cells (ATCC, Virginia), used as a positive control, were suspended in the plate wells at 3.2*10^5 cells/mL. KHYG-1 cells (JCRB, Japan) were added at a density of 6.4*10^6 cells/mL, at a 20:1 Effector:Target (E:T) ra- tio. Cells incubated for 5 hours at 5% CO2, 37 °C. Subsequently: [i] Cytotoxicity (necrosis and apoptosis) in target cells were quantified using flow cytometry with PerCP-Cy™5.5 Annexin V and Propidium Iodide (PI). [ii] CD107a expression on KHYG-1 cells was evaluated using Fig. 1 (abstract P156). Necrosis flow cytometry with PE-labeled anti-human CD107a Ab. [iii] Expression levels of 5 soluble biomarkers (Perforin, Granzyme-A, Granzyme-B, IFN-gamma and TNF-alpha) were determined in the cell supernatants using Luminex. Results The cytotoxicity data suggests that for necrosis, all target cell- lines were significantly different (all p values < 0.0001) from each other in terms of susceptibility to the effector cells (A549 > H1703 > H1975 > K562) (Figure 1). K562 cells are significantly higher in late apoptosis than all three lung cancer cell lines (Figure 2). Amongst the 3 lung cancer cell-lines, the H1703 cell line is significantly higher in late apoptosis than H1975 and A549 cells (p-value < 0.05) (Figure 3). Although differences were seen in the necrotic and late apoptotic profiles of target cells, the CD107a expression on the KHYG-1 effector cells was similar across all co-cultures (Figure 4). The soluble biomarker data (Luminex) is being collected. Conclusions In conclusion, cell-lines corresponding to different lung cancer subtypes, i.e. A549 (Carcinoma), H1703 (Squamous Cell) and H1975 (Adenocarcinoma) exhibit significant differences in both their necrosis and late apoptosis susceptibility when co-cultured with NK cells. Such insight could be used to better guide NK cell based immunotherapy development. References 1. Aktaş O, Öztürk A, Erman B, Erus S, Tanju S, Dilege S. Role of Natural Killer Fig. 2 (abstract P156). Late Apoptosis Cells in Lung Cancer. J Clin Cancer Res Clin Oncol. 2018; 144:997-1003. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 87 of 272 Background Adoptive cell transfer (ACT) of tumor-targeted T cells has demonstrated encouraging clinical efficacy in some hematological cancers. However, in solid tumors, targeting a single antigen (e.g., CAR-T and TCR therap- ies) can lead to antigen escape and development of resistance. Further- more, although support provided by lymphodepletion or cytokine administration can enhance responses to ACT, these systemic treat- ments are often associated with significant toxicities. Torque’sSlipstream™ T cell manufacturing platform is a high-efficiency process for generating Deep-Primed™ T cells: polyclonal non- genetically engineered T cells that (1) are targeted against multiple tumor-specific antigens and (2) carry immunomodulating cytokine pay- loads to provide prolonged and locally directed immune support with- out systemic toxicities. The Slipstream™ process is designed to resolve the manufacturing challenge of generating high yields of early memory phenotype tumor-reactive T cells, which are associated with clinical benefit. Here, we show that the Slipstream™ process drives robust ex- pansion while preserving favorable memory characteristics of natural tumor-reactive T cells, and we demonstrate that Deep-Priming™ Tcells with Deep IL-15 or Deep IL-12 improves function. Methods Multi-targeted T cells (MTC) were comparatively generated from do- nors via either a first-generation process or the new Slipstream™ process that leverages ex vivo expansion conditions optimized for MTC production. T cell reactivity against tumor-associated antigens, memory, polyfunctionality, cytotoxicity, and response to Deep IL-15 Fig. 3 (abstract P156). Late Apoptosis minus K562 and Deep IL-12 were measured. The modularity of Slipstream™ was tested by training MTC against antigen cassettes including cancer or viral antigens and measuring reactivity against antigen subsets. Results Compared to a first-generation process, MTC generated with Slip- stream™ exhibited >20-fold improvement in antigen-specific reactiv- ity and a substantial improvement in the yield of memory-phenotype antigen-specific T cells including a 10-fold increase in Tcf1-positive cells. Furthermore, the Slipstream™ process yielded MTC with in- creased polyfunctionality and specificity as measured by cytokine production and TCR sequencing, respectively. Notably, T cells ex- panded using the Slipstream™ process showed potent cytotoxicity against human cancer cells as well as responsiveness to Deep IL-15 and Deep IL-12. The Slipstream™ process can also be adapted for simultaneous training of MTC against different antigens including virus-associated tumor antigens. Conclusions The Slipstream™ process is optimized to produce Deep-Primed™ MTC with substantive increases in characteristics associated with clinical efficacy: antigen reactivity, memory phenotype, and polyfunctionality. Modularity of the Slipstream™ process has been demonstrated by simultaneously training T cell clones reactive to cancer and virus- associated antigens, and Deep-Primed™ MTC with cell-associated Deep IL-15 or Deep IL-12 drives enhanced T cell function in vitro. P158 Suboptimal er stress induced autophagy regulates anti-tumor T cell response Fig. 4 (abstract P156). CD107a Expression Shilpak Chatterjee, Danh Tran, Kim Dosung, Satish Nadig, Carl Atkinson, Hongjun Wang, J. Alan Dieh, Shikhar Mehrotra, PhD, Paramita Chakraborty, PhD Medical University of South Carolina, Charleston, SC, United States P157 Correspondence: Shikhar Mehrotra (mehrotr@musc.edu) Optimized process for manufacturing Deep-Primed™ Tcells Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P158 creates product with improved functional characteristics and reactivity against multiple tumor-associated antigens Background Shawn Carey, PhD, Christine McInnis, PhD, Alicia Worthylake, Angela Endoplasmic reticulum (ER) stress induced by external or internal Forte, Elisabeth Brown, Darren Smith, Kate Sackton, PhD, Rosemary stimuli activates a number of well-orchestrated cellular signaling pro- Soucy, Tap Maniar, MD, Karsten Sauer, PhD, Thomas Andresen, PhD, cesses aimed to promote either cell apoptosis or to restore cellular Andy Rakestraw, PhD function and resolve the stress. In tumor microenvironment, induc- Torque Therapeutics, Cambridge, MA, United States tion of ER stress is known to dampen the antitumor activity of T cells Correspondence: Thomas Andresen (tandresen@torquetx.com) by reducing their mitochondrial function. However, if magnitude of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P157 ER stress governs the T cell fate and function is unknown. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 88 of 272 Methods Results We performed our study on B16 murine melanoma model and used Of the 12 peptides with high predicted score, we confirmed 7 (in- standard immunological techniques like flow cytometry, immunoblot cluding NY-ESO-1 antigen SLLMWITQC) strongly activate human pri- analysis, microscopy, real time PCR mary NY-ESO C259-expressing T cells. These off-target peptides Results include peptides with up to 7 amino acid changes (of 9 possible), Using melanoma antigen gp100 reactive T cells, we found that low which could not be predicted using the recognition motif as deter- level of ER stress enhances T cell stemness and promotes mitochondrial mined by alanine scans. biogenesis, whereas high level of ER stress triggers T cell death. More- Conclusions over, upon adoptive transfer, T cells treated with low dose ER stress in- Thus, this replacement scan assay determines the “TCR fingerprint” ducer are able to form long-lived memory in vivo, express reduced and, when coupled with the algorithm applied to the database of level of co-inhibitory molecule, and demonstrate superior anti-tumor human 9-mer peptides binding to HLA-A*02:01, enables identifica- immunity by increasing overall survival of B16 murine melanoma bear- tion of potential off-target antigens and the tissues where they are ing mouse. Mechanistically, we discovered that, upon ER insult at sub- expressed. This platform enables both screening of multiple TCRs to optimal level, a protective autophagy pathway is induced to promote identify the best candidate for clinical development and identifica- cell survival and maintain stemness through the protein kinase R-like tion of TCR-specific cross-reactive peptide recognition and consti- endoplasmic reticulum kinase (PERK)/ activating transcription factor-4 tutes an improved methodology for the identification of potential (ATF4)-dependent manner. Conversely, knockdown of PERK abrogates off-target peptides presented on MHC class I molecules. We used this autophagy activation, hampers mitochondrial biogenesis in response to platform and demonstrate screening of multiple TCRs targeting suboptimal ER stress, which in-turn compromises the antitumor func- tumor antigens. tion of melanoma antigen specific T cells. Furthermore, we demon- strated that blocking autophagy in T cells hampers T cell anti-tumor P160 activity. Lastly, T cells which initiates autophagic process due to sub- Engineered natural killer cells redirected against adenosinergic optimal ER stress show better potential to control tumor compared to immunometabolic suppression for the immunotherapy of lung those, that do not enter into the process carcinoma Conclusions Andrea Chambers, MS, Kyle Lupo, BS, Jiao Wang, PhD, Sandro Matosevic, Overall, these preclinical data highlights that, low level of ER stress PhD response is important for healthy cellular function and therapeutic- Purdue University, Lafayette, IN, United States ally, ER stress pathways can be manipulated in T cells in order to Correspondence: Sandro Matosevic (sandro@purdue.edu) regulate their antitumor potential. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P160 Ethics Approval The study was approved by Medical University of South Carolina‘s Background Ethics Board, approval number 2018-00628. NK cells are powerful effectors in cancer immunotherapy and have po- tential to treat various cancers; however significant challenges remain P159 in the treatment of solid tumors. Energy availability is compromised TCR fingerprinting and off-target peptide identification surrounding solid tumors and NK cell metabolic reprogramming can Armen Karapetyan, Chawaree Chaipan, Katharina Winkelbach, Sandra occur to inhibit NK effector functions [1]. Accumulation of adenosine in Wimberger, Jun Seop Jeong, Bishnu Joshi, Robert Stein, MD PhD, Dennis the tumor microenvironment (TME) from the activity of ectoenzymes Underwood, PhD, Eleni Chantzoura, PhD, Alvaro Yague, Jan Bergmann, CD39 and CD73 on cancer cells is one mechanism that leads to im- John Castle, PhD, Marc Van Dijk, PhD, Volker Seibert paired NK cell function. Our previously published data has established Agenus Inc, Lexington, MA, United States that the effects of TME adenosine on NK cells cause specific Correspondence: John Castle (john.castle@agenusbio.com); Marc Van reorganization of the cells’ metabolism and effector signatures to sup- Dijk (marc.vandijk@agentustherapeutics.com) press NK cell function, and the cytokine combination of IL-12/15 was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P159 hyperresponsive to adenosine [2]. One way to combat immunosuppres- sion induced by cancer-produced adenosine is to engineer NK cells to Background overcome this inhibition. To that end, we engineered NK cells to dir- Adoptive T cell therapy using patient T cells redirected to recognize ectly target CD73 by imparting NK-specific signaling to enhance anti- tumor-specific antigens by expressing genetically engineered high- tumor activity against CD73+ lung carcinoma. affinity T-cell receptors (TCRs) has therapeutic potential for melanoma Methods and other solid tumors. Clinical trials implementing genetically modified Peripheral blood-derived NK cells were isolated from healthy human TCRs in melanoma patients have raised concerns regarding off-target tox- donors and expanded using feeder cells. NK cells were electroporated icities resulting in lethal destruction of healthy tissue, highlighting the ur- using mRNA or transduced with lentivirus expressing the CD73- gency of assessing which off-target peptides can be recognized by a TCR. targeting construct which bears signaling domains derived from Methods FcγRIIIa. Engineered NK cells expressing the construct were tested for As a model system we used the clinically efficacious NY-ESO-1-spe- their killing ability against lung carcinoma A549 cells. The engineered cific TCR C259, which recognizes the peptide epitope SLLMWITQC NK cells were then adoptively transferred into a CD73+ lung cancer presented by HLA-A*02:01. We investigated which amino acids at xenograft into NSG mice. Circulating CD73-CAR NK cells were quanti- each position enable a TCR interaction by sequentially replacing fied for their expression of activating markers NKG2D, DNAM, and every amino acid position outside of anchor positions 2 and 9 with NKp30 and visualized using immunohistochemistry to determine infil- all 19 possible alternative amino acids, resulting in 134 peptides (133 tration into tumors, and mice were assessed for tumor growth. altered peptides plus epitope peptide). Each peptide was individually Results evaluated using three different in vitro assays: binding of the NY-ESO We showed NK cells can be efficiently redirected against CD73 to C259 TCR to the peptide, peptide-dependent activation of TCR- block the generation of immunosuppressive adenosine and rescue expressing cells, and killing of peptide-presenting target cells. To impaired NK cell anti-tumor immunity. Specifically, primary human represent the TCR recognition kernel, we defined Position Weight NK cells were successfully engineered to express the synthetic CD73- Matrices (PWMs) for each assay by assigning normalized measure- FCyRIIIa construct. Retargeted NK cells showed enhanced anti-tumor ments to each of the 20 amino acids in each position. To predict functions in vitro against CD73-expressing A549 cells. Engineered pri- potential off-target peptides, we applied a novel algorithm project- mary NK cells also showed promise in stunting CD73+ lung cancer ing the PWM-defined kernel into the human proteome, scoring NY- tumor growth for up to 3 weeks in vivo. Current and future studies ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 include evaluation of off target effects and local injection to further binding 9-mer peptides. evaluate infiltration of the NK cells in vivo. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 89 of 272 Conclusions Background The microenvironment of solid tumors is highly immunosuppressive Adoptive cancer antigen-specific T cell therapy currently comprised and adenosine has been shown to impair NK cell anti-tumor immun- of chimeric antigen receptor (CAR-) and T cell receptor (TCR) engi- ity. A novel anti-CD73 targeting construct using NK cell signaling neered T cells. Clinical results from CAR-T cells have demonstrated components has been developed and shown to prevent tumor promising results in treating leukemia, while TCR-engineered T cells growth of CD73+ lung carcinoma. which have the advantage of recognizing intracellular tumor anti- gens is still in very early development. References Methods 1. Chambers A *, Lupo K*, Matosevic S. Tumor-microenvironment-induced Here, we report the development of two CD4+ or CD8+ TCRαβ-KO immunometabolic reprogramming of natural killer cells. Front Immunol. reporter T cell lines for the screening and characterization of trans- 2018; 9:2517. genic TCRs. A TCRαβ-KO reporter T cell line was first developed by 2. Chambers A, Wang J, Lupo K, and Matosevic S. Adenosinergic knocking out the endogenous TCR α and β chains in the reporter T signaling alters natural killer cell functional responses. Front Immunol, cell line using CRISPR/Cas9 and the successful knockout is confirmed 2018; 9:2533. by phenotypic assays and TCR v chain locus sequencing. Ethics Approval Results The study was approved by Purdue University Institution's Review Board, We demonstrated that re-introduction of HA peptide-specific HA1.7 approval number 1804020540. TCR α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Furthermore, P161 the select expression of CD4 or CD8 variants in the TCRαβ-KO re- High-efficiency CAR-T cell manufacturing by improved scalable porter T cell line could enable the development of TCRs for both electroporation MHCI- and MHCII-restricted tumor antigen targets. Jian Chen, PhD, George Sun Conclusions Celetrix LLC, Manassas, VA, United States The CD4+ and CD8+ TCRαβ-deficient reporter T cells can serve as Correspondence: Jian Chen (jchen@celetrix.com) valuable tools for screening and characterization of neoantigen- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P161 specific TCRs Background CAR-T cells are currently manufactured for clinical use by infection of P163 human T cells with viral vectors containing the CAR gene. The Effect of common gamma-chain cytokines on myeloid-derived current viral CAR-T manufacturing process is lengthy and costly and suppressor cell and M2 macrophage suppressive function: electroporation has emerged as a promising alternative. However, Implications for cellular immunotherapy 1 2 2 2 clinical use of electroporation technology in CAR-T has been difficult Anna Cole, BA , Charlotte Rivas , Josue Pineda , Corrine Baumgartner , 2 2 and several clinical trials have met significant problems due to the Stephanie Fetzko , Robin Parihar 1 2 low transfection efficiency and/or high cell mortality. Rice University, Houston, TX, United States; Baylor College of Medicine, Methods Houston, TX, United States Our novel understanding of the electroporation mechanism revealed that Correspondence: Robin Parihar (rxpariha@texaschildrens.org) the current widely-used electroporation methods have significant mis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P163 takes in the physical design as well as electroporation buffer design. The first problem is the electroporation sample container design. It is well Background known that electrochemical reaction generates gas bubbles that are Immunotherapy using antigen-redirected lymphocytes such as harmful to the cells and there was no good solution to the problem. Here chimeric antigen receptor (CAR)-T or -NK cells in patients with solid we used a novel pressurization approach to largely eliminate the effect. tumors has shown poor efficacy. Cell therapies are hindered by im- Results munosuppressive cells such as inhibitory macrophages (M2s) and Combined with other improvements including electroporation myeloid-derived suppressor cells (MDSCs) that contribute to a highly buffer design and post-electroporation cell culture strategy, we suppressive tumor microenvironment (TME) [1]. Researchers have have been able to achieve over 80% plasmid transfection effi- armed redirected lymphocytes with the ability to secrete cytokines in ciency in unstimulated T cells and over 90% plasmid transfection hopes of promoting their proliferation and function in suppressive efficiency in stimulated T cells. The viability in survived cells is TMEs [2,3]. However, the effect of these cytokines on other immune over 95% measured by live/dead staining and the true survival cells within the TME, such as MDSCs and M2s, is unknown. rate measured by survived cell number is over 66%. The new Methods electroporation method can achieve over 90% in gene editing To determine how the human common gamma-chain cytokines, and the method is also widely applicable in electroporation of interleukin(IL)-2, IL-7, IL-15, and IL-21 affect human MDSCs and M2s, NK cells, DC cells and monocytes. we exposed ex vivo enriched M2s and MDSCs to each cytokine sep- Conclusions arately and assessed changes in MDSC and M2 phenotype and ability The new method is also scalable as billions of cells can be processed to dampen T-cell activation and proliferation. To further define in the large volume electroporation setting. Our method can poten- cytokine-induced changes in MDSC/M2 function in a more clinically tially eliminate the need for expensive cell expansion and virus pro- relevant system, we tested the ability of cytokine-exposed MDSCs/ duction altogether, therefore cutting the huge economic burden of M2s to impair CAR-T cell proliferation and anti-tumor activity in a CAR-T therapy. TME co-culture. As a clinical correlate, we assessed common gamma- chain cytokine receptor expression on MDSCs and M2s within neuro- blastoma and sarcoma patient tumors and tested the effects of cyto- P162 kine exposure on their suppressive capacity. Development of CD4+ and CD8+ TCRαβ-deficient bioluminescent Results reporter T cells for screening and characterization of neoantigen- Subsets of ex vivo enriched M2s and MDSCs expressed common specific TCRs gamma-chain cytokine receptors. MDSCs expressed receptors for IL-2 Zhi-jie Cheng, PhD, Jamison Grailer, PhD, Michael Slater, Pete Stecha, Jim (22%, avg. MFI=67, n=3), IL-7 (43%, avg. MFI=375, n=3), IL-15 (23%, Hartnett, Frank Fan, PhD, Mei Cong, PhD avg. MFI=310, n=3), and IL-21 (65%, avg. MFI=124, n=4); whereas Promega Corporation, Madison, WI, United States M2s expressed receptors for IL-2 (17%, avg. MFI=59), IL-7 (98%, avg. Correspondence: Zhi-jie Cheng (jey.cheng@promega.com) MFI=543), IL-15 (36%, avg. MFI=619), and IL-21 (91%, avg. MFI=296). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P162 Exposure of human MDSCs or M2s to IL-2, IL-7, IL-15, or IL-21 did not Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 90 of 272 alter their cell-surface phenotype. Exposure of these suppressive median survival of 24 days. Treatment with PM21-NK cells improved myeloid cells to IL-2, IL-7, and IL-15 did not change their ability to survival over untreated (p=0.0003) and PD-L1 alone (p=0.0002) suppress T-cell proliferation. In contrast, exposure of M2s and MDSCs groups having median survival of 40 days. Combination of PM21-NK to IL-21 increased their ability to suppress T-cell proliferation and ac- cells with anti-PD-L1 further improved of survival over the PM21-NK tivation (98% suppression by IL-21 exposed MDSCs vs. 72% suppres- cells alone group (48 days, p=0.042) with 25% of mice still remaining sion by control MDSCs; 98% suppression by IL-21 exposed M2 vs. in good health at day 58. 79% suppression by control M2 at a 2:1 T cell:MDSC/M2 ratio). Conclusions Conclusions These data support the use of anti-PD-L1 in NK cell therapy, regard- These results suggest that IL-21 increases the suppressive capacity of less of initial tumor PD-L1 status. PM21-NK cells can be used for human MDSCs and M2s. Ongoing experiments will define the mech- tumor treatment and to prime tumors to express PD-L1. The PD-L1 anisms by which IL-21 alters MDSC and M2 suppression and further induced upon NK cell treatment can serve as “universal targetable define the effect of IL-21 exposed MDSCs and M2s on tumor growth ligand” if used with humanized anti-PD-L1 antibodies to cause tumor and CAR-T cell therapeutic efficacy in vivo. killing by ADCC. References P165 1. Martinez M, Moon EK. CAR T cells for solid tumors: New strategies for Robust, reproducible and highly scalable manufacturing of P- finding, infiltrating, and surviving in the tumor microenvironment. Front. BCMA-ALLO1, an allogeneic CAR-T stem cell memory product for Immunol. 2019; 10:128. multiple myeloma, from numerous healthy donors 2. Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T Stacey Cranert, PhD, Maximilian Richter, PhD, Min Tong, MS, Leslie Weiss, cells enhance antitumor efficacy and overcome the tumor MS, Yening Tan, MS, Eric Ostertag, MD, PhD, Julia Coronella, PhD, Devon microenvironment. Sci Rep. 2017; 7:10541. Shedlock, PhD 3. Liu D, Song L, Wei J, Courtney AN, Gao X, Marinova E, Guo L, Heczey A, Poseida Therapeutics, San Diego, CA, United States Asgharzadeh S, Kim E, et al. IL-15 protects NKT cells from inhibition by Correspondence: Devon Shedlock (dshedlock@poseida.com) tumor-associated macrophages and enhances antimetastatic activity. J Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P165 Clin Invest. 2012; 122:2221-2233. Ethics Approval Background Tumor tissue use was approved by Baylor College of Medicine IRB study Autologous Chimeric Antigen Receptor (CAR) T cell therapy for re- protocol #26691, and samples were de-identified prior to laboratory lapsed/refractory Multiple Myeloma (MM), such as Poseida’s anti-B evaluation. cell maturation antigen (BCMA) product candidate, P-BCMA-101, have shown significant efficacy in the clinic. P-BCMA-101 is com- P164 prised of a high percentage of stem cell memory T cells (TSCM), Effect of NK cell treatment on PD-L1 expression and anti-PD-L1 resulting in a product that is much safer and potentially more dur- response able than other anti-BCMA autologous product candidates. However, Alicja Copik, PhD, Jeremiah Oyer, Sarah Gitto, Deborah Altomare, PhD individualized products have expensive and time-consuming manu- University of Central Florida, Orlando, FL, United States facturing and significant variability in input patient T cells characteris- Correspondence: Deborah Altomare (deborah.altomare@ucf.edu) tics. We are developing P-BCMA-ALLO1, an off-the-shelf anti-BCMA Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P164 allogeneic (allo) CAR-T product candidate manufactured from serial healthy donor material that circumvents many of the downsides of Background an individualized CAR-T product. PD-1 axis blockade therapies have shown success but responses are Methods limited to ~15% of cancer patients. These responses correlate with P-BCMA-ALLO1 is produced using two key platform technologies: presence of lymphocyte infiltrated, PD-L1 positive tumors. Strategies the nonviral piggyBac® (PB) DNA Modification System and the that increase PD-L1 expression may improve outcomes of PD-1 axis high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System. blockade. PD-L1 on tumor cells is induced by IFNγ, secreted by NK The PB transposase mRNA and DNA encoding the PB-based cells. We developed a method for producing therapeutic quantities transgene are electroporated along with the components of the (>1,000 fold expansion within two weeks) of hyper-activated NK cells CC system needed to knockout (KO) the T Cell Receptor (TCR) with high anti-tumor cytotoxicity and enhanced IFNγ secretion. The and beta-2 microglobulin, thereby eliminating expression of Major utilizes particles from Plasma Membrane of K562 cells expressing Histocompatibility Complex (MHC) class I. The T cells are then ex- membrane bound IL21 (PM21-particles). Herein, the ability of PM21- panded using our proprietary "booster molecule.” The resulting particle expanded NK cells to induce PD-L1 expression on various tu- product demonstrates expression of the transgene in nearly all mors was tested in vitro and in vivo in ovarian cancer model. Fur- cells, and after a purification step, have eliminated all TCR expres- thermore, the effect of anti-PD-L1 on NK cell anti-tumor activity was sion and most MHC class I expression. tested in vitro and in vivo. Results Methods We have produced P-BCMA-ALLO1 at both research and near- NK cells were expanded with PM21-particles as described. For in vivo clinical scale from >35 donors with >97% manufacturing success. experiments, NSG mice were implanted with 1x10^6 SKOV-3 cells Efficiencies of TCR-KO ranged from ~50-90%, with final product i.p.. Mice were treated with 10^7 PM21-NK cells (n=6) or with vehicle always demonstrating >99% TCR-KO. T cell expansion varied from control (n=6) on days 8 and 13. Mice were sacrificed on day 20 to ~0.5-20 fold. At clinical production scale, this translates to up to collect tumors. Tumors were perfused and retrieved tumor cells were 250 doses of CAR-T per manufacturing run at a dose of 150x10e6 analyzed for PD-L1 expression while infiltrating immune cells were cells/patient. P-BCMA-ALLO1 demonstrated a high-percentage of phenotyped. TSCM cells (CD45RA+CD62L+CD45RO-). Furthermore, P-BCMA- Results ALLO1 generated from multiple donors demonstrated potent effi- PM21-NK treatment induced PD-L1 on >30% of tumor cells across cacy in the RPMI-8226 xenograft model in NSG mice, thus estab- multiple cell lines. PM21-NK cells are negative for PD-1 and addition lishing the feasibility of using serial individual donors in our of anti-PD-L1 had no effect on their cytotoxicity or cytokine produc- manufacturing process. tion. In in vivo experiment, PM21-NK cell treated mice had increased Conclusions PD-L1+ tumors vs. the untreated group (29.7% vs 14.5%, p<0.0001). In summary, these data demonstrate a robust, reproducible and Despite T-cell depletion, T-cells made up ~22% of hCD45+ events in highly scalable manufacturing process. Moreover, this production perfused tumors, 83% of which were Tregs. PM21-NK cells are PD-1-, process can be expanded for use with additional targets for treat- but are inhibited by Tregs. Untreated and anti-PD-L1 alone mice had ment of other heme or solid tumors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 91 of 272 P166 P167 ET140202 T-cell therapy for the treatment of liver cancer is built Improved efficacy leveraging CAR-T therapeutics that produce upon a novel antibody-T cell receptor (AbTCR) ARTEMIS™ T-cell tightly controlled, tumor proximal, immunomodulatory outputs platform Michon Pinnix, Krista McNally, Jay Danao, BS, Melissa Fardy, Rachel Jun Cui, PhD, Pengbo Zhang, Hongruo Yun, Yiyang Xu, Lucas Horan, Hovde, MS, Charlotte Davis, Nicole Grant, Dianna Lester-Zeiner, David PhD, Shaohua Xu, Sean Xu, Hong Liu Mai, Ben Wang, PhD, Gus Zeiner, PhD Eureka Therapeutics, Inc., Emeryville, CA, United States Chimera Bioengineering, Emeryville, CA, United States Correspondence: Hong Liu (hong.liu@eurekainc.com) Correspondence: Gus ZeinerGus Zeiner (gus@chimera.bio) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P166 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P167 Background Background The use of engineered T cells for the treatment of solid cancers remains Current chimeric antigen receptor (CAR) T cell therapies are clinically challenging. Recently, we developed a novel antibody-T cell receptor efficacious against several B cell malignancies, but are less effective (AbTCR) ARTEMIS™ T-cell platform, which combines antibody-based target at eliminating solid tumors. A key contributor to this observed lack recognition with gamma/delta TCR-based cellular activation [1]. In contrast of efficacy is the tumor microenvironment (TME) that is erected by to chimeric antigen receptors (CARs), the AbTCR forms a natural multi- solid tumors to impose immunosuppressive physical and chemical meric receptor with the endogenous CD3 complex, which feeds into a barriers to T cell function and survival. To break TME-driven immuno- network of signaling pathways that regulate T-cell activation. In addition, suppression, attempts have been made to arm CAR-T cells with the the inclusion of gamma/delta TCR chains within the AbTCR avoids the for- ability to produce immunomodulatory payloads that target features mation of mispaired receptors with unknown cross-reactivity, which is a of the TME. By their nature, these immunomodulators (e.g. Interleu- potential risk associated with current alpha/beta TCR-based therapies. kin 12) are frequently toxic when systemically delivered. Due to a Using the core design of the AbTCR ARTEMIS™ T-cell platform, we de- limited repertoire of existing programmable gene regulators, consti- veloped ET140202 for the treatment of hepatocellular carcinoma (HCC). tutive expression and systemic distribution of immunomodulators by ET140202 features an AbTCR targeting alpha-fetoprotein (AFP)peptide/ armed CAR-T cells is common. MHC complexes (specifically AFP158-166/HLA-A2) expressed on HCC Chimera Bioengineering has characterized a novel, post-transcriptional cancer cells. To optimize T-cell activation and expansion, the AbTCR is gene regulatory node that strictly governs effector outputs in human T co-expressed with a CD28-based co-stimulatory molecule engineered cells. This gene regulatory node, termed Gold, has been used to create to target Glypican 3 (GPC3) expressed on HCC cancer cells. enhanced CAR-T therapeutics that produce tightly controlled immuno- Methods modulatory outputs only upon tumor engagement. To test the specificity and potency of ET140202 T cells in vitro, Methods ET140202 T cells were co-incubated with either target-positive or Methods include: design and characterization of a proprietary bicis- target-negative cells. Lactate Dehydrogenase release was used to tronic GoldCAR lentiviral vector to simultaneously deliver both CAR quantify target cell lysis. CFSE assay was used to measure cell prolif- and IL12 transgenes; production of lentivirus and infection of primary eration. Expression of differentiation and exhaustion markers were human T cells; in vitro characterization of GoldCAR-T cell function determined by flow cytometry. The in vivo anti-tumor activity of utilizing cell based assays, immunoassays and flow cytometry; im- ET140202 T cells was tested in an AFP+/HLA-A2+ Hep G2 liver cancer plantation of subcutaneous xenograft tumors in NSG mice with xenograft model. We also engineered the same anti-AFP158-166/ tumor growth monitored by caliper and bioluminescent imaging HLA-A2 binding moiety onto a CD28-based CAR (AFP-CAR) and com- (BLI) to assess in vivo efficacy; and ex vivo analysis of blood, tumor pared AFP-CAR-T cells to ET140202 T cells in various assays. and lymphatic organs to characterize safety profile. Results Results ET140202 T cells specifically lysed AFP-positive tumor cells. Com- CD19 targeted GoldCAR-T cells delivering IL12 demonstrate im- pared to AFP-CAR-T cells, ET140202 T cells displayed enhanced proved efficacy over standard CD19 CAR-T cells in a subcutaneous in vitro cell killing and proliferation even after repetitive antigen Daudi B cell xenograft mouse model. These GoldCAR-T cells exhibit stimulations. ET140202 T cells also display a less exhausted surface comparable efficacy to CAR-T cells with constitutive IL12 expression, phenotype (e.g. lower PD-1 expression) and a higher percentage of but levels of pro-inflammatory cytokines in the peripheral blood are central memory T cells (CCR7+ CD45RA-) after antigen stimulation. In lower in the mice treated with GoldCAR-T cells. vivo, both intravenous and intratumoral single administration of Conclusions ET140202 T cells led to significant tumor growth inhibition. GoldCAR-T cells with tumor proximal IL12 delivery demonstrate en- Conclusions hanced efficacy over standard CAR-T cells and an improved safety ET140202 is built upon our novel AbTCR ARTEMIS™ T-cell platform, profile compared to CAR-T cells with constitutive IL12 expression. which was designed to harness the natural biology of T cells to fight The implications of this study point to an amplified CAR-T cell re- cancer. Both in vitro cellular assays and in vivo mouse studies sup- sponse in an immunosuppressive tumor microenvironment. port the safety and efficacy of ET140202 T cells. Whether these pre- Trial Registration clinical findings for AbTCR-based ET140202 T-cell therapy translate Not applicable into the clinical setting is currently being tested (clinicaltrial.gov, Ethics Approval NCT0399803). The animal study was conducted under the Institutional Animal Care and Use Committee (IACUC) of LumiGenics, LLC, 750 Alfred Nobel Reference Drive, Suite 103, Hercules CA 94547 1. Xu Y, Yang Z, Horan LH, Zhang P, Liu L, Zimdahl B, Green S, Lu J, Morales JF, Barrett DM et al. A novel antibody-TCR (AbTCR) platform combines P168 Fab-based antigen recognition with gamma/delta-TCR signaling to facili- Role and function of T-cell immunoglobulin– and mucin domain– tate T-cell cytotoxicity with low cytokine release. Cell Discovery 2018, containing (TIM)–3 receptor on natural killer cells in solid tumors 4(1):62. Tram Dao, Sandro Matosevic, PhD Ethics Approval Purdue University College of Pharmacy, West Lafayette, IN, United States All animal experiments were conducted according to protocols approved Correspondence: Sandro Matosevic (smatosev@purdue.edu) by their Institutional Animal Care and Use Committee (IACUC) and in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P168 accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Background Washington, DC, 1996) and the Policy on Humane Care and Use of Natural killer (NK) cells are part of the innate immune system, but are Laboratory Animals (Department of Health and Human Services, capable of participating in both innate and adaptive immune Bethesda, MD). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 92 of 272 responses due to their wide range of cytolytic activities, from de- P169 granulation, secretion of cytokines to antibody-dependent cell- Enriching non-genetically modified antigen specific marrow mediated cytotoxicity. These are possible due to the cells’ ability to infiltrating lymphocytes (MILs) to target HPV+ oropharyngeal recognize self and non-self-entities via the net signal generated from squamous cell carcinoma 1 1 2 their activating and inhibitory receptors upon engagement. One such Danielle Dillard , Vanessa Chan , Lakshmi Rudraraju, MS , Elizabeth 2 1 1 1 receptor is TIM-3, which is expressed on various lymphocytes. In T- DeOliveira , Amy Thomas , Ervin Griffin , Megan Heimann , Luca Biavati, 1 1 1 cells, TIM-3 is an exhaustion marker [1], but on NK cells, results are MD , Elizabeth Zawidzka , Marguerrita El Asmar , Drew Pardoll, MD, 1 1 3 1 conflicting in regards to its function as the receptor exhibits both ac- PhD , Kellie Smith, PhD , Carole Fakhry, MD , Ivan Borrello, MD tivating and inhibitory effects depending on disease type and activa- Johns Hopkins School of Medicine, Baltimore, MD, United States; 2 3 tion status [2-6]. WindMIL therapeutics, Baltimore, MD, United States; Otolaryngology, Methods Head and Neck Surgery, Baltimore, MD, United States NK cells were isolated from peripheral blood of healthy donors. Correspondence: Danielle Dillard (danielle.dillard@jhmi.edu) After expansion, they were co-cultured for 4 hours with glioblast- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P169 oma (U87) at effector:target (E:T) ratios of 2.5:1 and 10:1, and various receptors were screened by flow cytometry, including PD- Background 1, NKG2A, LAG-3, CD158b, CEAMCAM-1 and TIM-3. Then, expres- Human papillomavirus positive (HPV+) oropharyngeal squamous cell sion of TIM-3 was measured when in the presence of patient- carcinoma (HPV-OPSCC) accounts for ~80% of OPSCCs in the United derived primary glioblastoma cells (GBM43) and prostate cancer States. Although HPV-positivity confers improved survival relative to (PC3) for 4 hours. To determine the effect of TIM-3 expression, HPV(-) OPSCC, outcomes for metastatic HPV-OPSCC remain dismal. killing assay are being carried out by blocking TIM-3 on NK cells. High tumor infiltrating lymphocytes (TILs) are associated with better Further investigation is being performed by blocking one of TIM- outcomes in HPV-OPSCC and have the promise of a therapeutic role 3’s primary ligands, Galectin-9, on cancer cells to determine its based upon their intrinsic enhanced tumor specificity. However, not impact on NK cell cytotoxicity. Statistical analyses are completed all tumors possess TILs. To date, therapeutic expansions involve sur- in SAS JMP Pro 14. gical excision of the tumor, expansion of TILs with high-dose IL-2 for Results up to 4 weeks and clinical product success rate between 40-60%. We found that TIM-3 is significantly downregulated on primary hu- Marrow infiltrating lymphocytes (MILs) represent a novel and distinct man NK cells, in both frequency and surface density, when exposed T cell population obtained from the bone marrow (BM) of patients to solid tumor cells such as U87, GBM43 and PC3 at multiple E:T ra- that possess significant tumor-specificity over peripheral blood lym- tios. Unlike other inhibitory NK receptors, this downregulation was phocytes (PBLs). Although the early work was done in myeloma, the unique to TIM-3. However, it is not known why the downregulation unique nature of the BM microenvironment makes it a reservoir of occurs with solid tumors, and whether this change in expression af- antigen-experienced memory T cells in numerous solid tumors. As fects NK killing capacity. Here, we report the role of TIM-3 on NK cell such, we sought to determine whether HPV-specific MILs could be cytotoxicity against solid tumor cell lines and the role of Galectin-9 in identified and expanded ex vivo. mediating NK cell activity. Methods Conclusions We obtained bone marrow and peripheral blood from patients with We found that Tim-3 was significantly downregulated on NK cells in localized, HPV-OPSCC. A modified version of the MANAFEST assay response to solid tumor cells. Understanding the complex roles of was used to evaluate proliferation of peripheral and bone marrow- Tim-3 expression on NK cells allows us to better understand the nu- derived CD8+ T cells in response to HPV early 6 (E6) and early 7 (E7) anced immunomodulatory role of Tim-3 on NK cell anti-tumor re- peptides (HPVFEST) in a HPV-OPSCC patient. sponses, and provide a basis for the development of Results immunotherapies targeting impaired NK cell function in solid tumors T cell receptor sequencing and bioinformatic analysis of each peptide- stimulated culture revealed a markedly increased frequency of HPV- References specific T cells in MILs compared to peripheral blood. HPV-specific MILs 1. Sánchez-Fueyo A, Tian J, Picarella D, Domenig C, Zheng XX, Sabatos CA, showed a higher average TCR clone frequency relative to PBLs (p value Manlongat N, Bender O, Kamradt T, Kuchroo VK, Gutiérrez-Ramos JC, = 0.0037). Additionally, of the 5 highest expanded TCR clones for both Coyle AJ, Strom TB. Tim-3 inhibits T helper type 1-mediated auto- and compartments HPV-specific MILs possessed an increased average rep- alloimmune responses and promotes immunological tolerance. Nat resentative clone frequency (p value = 0.0001). To investigate general Immunol. 2003; 4:1093-101. responsiveness to shared tumor antigens, we incubated autologous BM 2. Ndhlovu LC, Lopez-Vergès S, Barbour JD, Jones RB, Jha AR, Long BR, with lysate from an HPV+ OPSCC and then added ex-vivo activated Schoeffler EC, Fujita T, Nixon DF, Lanier LL. Tim-3 marks human natural MILs and PBLs. Tumor specificity was defined as IFN훾 production in killer cell maturation and suppresses cell-mediated cytotoxicity. Blood. CD3 cells. MILs possessed an average of 50.73% IFN훾 production as 2012; 119:3734-43. compared to 0.11% in PBLs to HPV-OPSCC lysate. 3. Jost S, Moreno-Nieves UY, Garcia-Beltran WF, Rands K, Reardon J, Toth I, Conclusions Piechocka-Trocha A, Altfeld M, Addo MM. Dysregulated Tim-3 expression Collectively, these data indicate that HPV-specific T cells exist in the on natural killer cells is associated with increased Galectin-9 levels in HIV- BM of patients with localized disease and possess a greater clonoyt- 1 infection. Retrovirology. 2013; 10:74. pic frequency and functional tumor recognition compared to PBLs. 4. Gleason MK1, Lenvik TR, McCullar V, Felices M, O'Brien MS, Cooley SA, These data provide the rationale for developing this novel adoptive T Verneris MR, Cichocki F, Holman CJ, Panoskaltsis-Mortari A, Niki T, Hira- cell approach using MILs in this patient population. shima M, Blazar BR, Miller JS. Tim-3 is an inducible human natural killer Ethics Approval cell receptor that enhances interferon gamma production in response to This study was approved by Johns Hopkins University Institution Re- galectin-9. Blood. 2012; 119:3064-72. view Board, approval number IRB00128334 and NA_00028682. 5. Ju Y, Hou N, Meng J, Wang X, Zhang X, Zhao D, Liu Y, Zhu F, Zhang L, Sun W, Liang X, Gao L, Ma C. T cell immunoglobulin- and mucin- P170 domain-containing molecule-3 (Tim-3) mediates natural killer cell sup- Sequential anti-CD19, anti-CD22, and anti-CD20 autologous pression in chronic hepatitis B. J Heptatol. 2010; 52:322-9. chimeric antigen receptor T cell (CAR-T) therapies treating a child 6. da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo with relapsed refractory Burkitt lymphoma VK, Osman I, Bhardwaj N. Reversal of NK-cell exhaustion in advanced mel- Juan Du, MD, Yonghong Zhang anoma by Tim-3 blockade. Cancer Immunol Res. 2014; 2:410-22. Beijing Boren Hospital, Beijing, China Ethics Approval Correspondence: Yonghong Zhang (yhzhang58@126.com) This study was approved by Purdue Intuition’s Ethics Board, approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P170 number 1804020540. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 93 of 272 Background Results Currently, the prognosis of children with relapsed refractory(r/r) Burkitt lym- Using this method, we have retrieved multiple TCRs that confer specific phoma(BL) remains dismal . New therapies are exlored to achieve a higher recognition of a public neoepitope derived from a PIK3CA hotspot mu- remission rate such as immunotherapy for these patients .We have success- tation (H1047L). These TCRs are restricted by HLA-A*03:01, an allele fully treated a case by adopting sequential autologous chimeric antigen re- present in 20.5% of the North American population. Immune- ceptor T cell(CAR-T) therapies, targeting antigen CD19,CD22,and CD20. precipitation/tandem mass spectrometry analysis determined that the Methods endogenously processed and presented public neoepitope is a 9 amino An 8-year-old boy was studied,who presented with a mass on the acid sequence containing a His to Leu substitution at position 2. To right side of the neck and was diagnosed with BL by pathology. The understand the mechanistic basis for the immunogenicity of this public child was treated with standard chemotherapy but suffered from re- neoepitope, we generated x-ray crystallography structures of mutant lapse. Subsequently, anti-CD19, anti-CD22, and anti-CD20 autologous and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These stud- CAR-T cell treatments were sequentially administered. We observed ies revealed significant topologic overlap in the bound peptides. By the clinical manifestations and response to the three cycles of CAR-T contrast, the thermal and kinetic stability of the mutant peptide/HLA- treatments, values of peripheral CAR-T cells were also monitored and A*03:01 complex was significantly enhanced relative to the WT com- side effects were assessed. plex, as measured by differential scanning fluorimetry and fluorescence Results anisotropy assays. Peripheral blood T cells genetically engineered with The patient displayed no response to anti-CD19-CART treatment PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells .After CD-22 directed CART the patient got partial remission (PR),but in a HLA/mutation-specific manner, leaving HLA-mismatched or WT tar- relapse occurred quickly. Finally, after the use of anti-CD20 CAR-T cell get cells unperturbed. therapy, the child achieved complete remission (CR) and has cur- Conclusions rently achieved a 6-month event-free survival (EFS). During the CD19 These findings reveal for the first time the existence of an endogen- and CD20 CAR-T cell treatments, only mild cytokine release syn- ously processed and presented public neoantigen derived from a drome (CRS) were observed in the patient (grade 1) while he devel- PIK3CA hotspot mutation. These results open the possibility of target- oped a grade 3 CRS during CD22 CAR-T therapy, the symptoms ing this common driver oncogene using adoptively transferred and included fever and hypoxemia. genetically redirected T cells. Conclusions Ethics Approval Autologous CAR-T cell therapies targeting multplei tumor antigens The study was approved by Smita Chandran's Institution‘s Ethics could be novel and safe treatments for children with r/r BL. Board, approval number IRB 17-250. Consent Written informed consent was obtained from the patient for publica- P172 tion of this abstract and any accompanying images. A copy of the Shape and material properties increase artificial antigen written consent is available for review by the Editor of this journal. presenting cell effectiveness Savannah Est Witte, BS, Kaitlyn Calebrisi, Jordan Green, PhD P171 Johns Hopkins University, Baltimore, MD, United States T cell receptor gene therapy for a public neoantigen derived from Correspondence: Jordan Green (green@jhu.edu) mutated PIK3CA, a dominant driver oncogene in breast and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P172 endometrial cancers 2 1 3 3 Jiaqi Ma , Martin Klatt, PhD , Friederike Dundar, PhD , Paul Zumbo , Background 1 3 1 Matthew Femia , Doron Betel, PhD , David Scheinberg , Brian Baker, Particulate delivery of artificial antigen presenting cells (aAPCs) is a promis- 2 1 1 PhD , Christopher Klebanoff, MD , Smita Chandran, PhD ing cell-free strategy to initiate selective T cell stimulation for immunother- 1 2 MSKCC, New York, NY, United States; University of Notre Dame, Notre apy in vivo. While 2D aAPC strategies aim to optimize T cell proliferation Dame, IN, United States; Weill Cornell Medicine, New York, NY, United States and selection in vitro for subsequent cell therapy, it would be advanta- Correspondence: Christopher Klebanoff (klebanoc@mskcc.org); Smita geous to deliver “off-the-shelf” biodegradable aAPCs directly as an in vivo Chandran (chandrs1@mskcc.org) therapeutic. 3D aAPC effectiveness has been limited due to inefficiencies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P171 in stimulating T cells as well as rapid clearance of delivered particles. To improve bioavailability as well as increase particulate aAPC effectiveness, Background we have developed a soft, biodegradable, microparticle aAPC (Figure 1). “Public” neoantigens represent immunogenic epitopes encompassing To create a new platform technology for immunoengineering, material hotspot mutations in driver oncogenes that are also restricted by and shape are investigated as parameters for improving T cell stimulation. common HLA alleles. In contrast with patient-specific “private” Methods neoantigens, public neoantigens are conceptually attractive because One micron size particles were synthesized using a poly(ethylene gly- they are tumor-specific, clonally conserved, and shared across pa- col) diacrylate (PEGDA) or using a poly(lactic-co-glycolic acid) emulsion tients. Whether PIK3CA, the most common driver oncogene in breast method [1,2]. Anti-CD3 and anti-CD28 conjugated particles were incu- and endometrial cancer, can yield public neoepitopes that may be bated with primary mouse T cells and proliferation was quantified at 3 exploited for cancer immunotherapy is unknown. and 7 days. For macrophage uptake studies, particles were incubated Methods with macrophages at 37 °C to analyze particle uptake and 4 °C to evalu- We have developed a high-throughput, single-cell functional assay for ate binding of particles to cells. To synthesize soft, ellipsoidal aAPCs, a the discovery and retrieval of TCR alpha/beta gene sequences that con- novel thin-film stretching technique was developed where emulsified fer specific recognition of endogenously processed and presented pub- PEGDA droplets were frozen then cast into films and stretched [3]. lic neoantigens and not the corresponding wild type (WT) sequence. In Results this approach, donor-derived T cells are sensitized with autologous Protein conjugation efficiency and T cell proliferation were 10-fold antigen presenting cells (APCs) electroporated with RNA encoding higher for PEGDA particles than PLGA particles (Figure 2a-b). Uptake PIK3CA hotspot mutations. Expanded T cells are subsequently divided studies indicate a ~20-fold decrease in binding and uptake by the into paired daughter wells for short-term co-culture with APCs electro- PEGDA particles (Figure 2c). Ellipsoidal aAPCs stimulate T cells 3 porated with minigenes containing either mutant or WT PIK3CA se- times more effectively than spherical particles (Figure 3b,d). Uptake quences. Acutely re-stimulated T cells from paired wells are subject to studies indicate a ~10-fold decrease in nonspecific uptake of ellips- single-cell alpha/beta TCR VDJ and RNA sequencing. TCR alpha/beta oidal particles (Figure 3c). gene sequences associated with selective upregulation of TCR signaling Conclusions transcripts to mutant but not WT PIK3CA stimulation are subsequently Particle material and shape are significant factors in designing partic- cloned into retroviral vectors to confirm reactivity. ulates as biomimetic aAPCs for in vitro T cell stimulation. Ongoing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 94 of 272 work on further decoupling these parameters and optimizing in vivo efficacy has the potential to unleash a promising biomimetic plat- form technology for immunoengineering of T cells. References 1. Anselmo AC, Mitragotri S. Impact of Particle Elasticity on Particle-Based Drug Delivery Systems. Adv. Drug Deliv. Rev. 2017; 108:51–67. 2. Meyer RA, Sunshine JC. Biodegradable Nanoellipsoidal Artificial Antigen Presenting Cells for T Cell Activation. Small. 2016; 11:1519–1525. 3. Meyer RA, Meyer RS, Green JJ. An Automated Multidimensional Thin Film Stretching Device for the Generation of Anisotropic Polymeric Micro- and Nanoparticles. J. Biomed. Mater. Res. A. 2015; 103:2747–2757. Fig. 3 (abstract P172). See text for description P173 Case reports: Correlates of response following adoptive transfer of ADP-A2M4, affinity-enhanced T-cells targeting MAGE-A4, in synovial sarcoma 1 1 1 Svetlana Fayngerts, PhD , Zohar Wolchinsky , Shravani Shitole , Joana 1 1 1 Senra , Rebecca Dryer-Minnerly, PhD , Ruoxi Wang , Jean-Marc Navenot, 1 1 1 1 PhD , Olga Ochkur , Gareth Betts, PhD , Natalie Bath, MSc , Erin Van 1 1 1 1 Winkle , Tom Holdich , Malini Iyengar, PhD , Rafael Amado, MD , Marcus 2 3 1 1 Butler, MD , David Hong, MD , Alex Tipping, PhD , Samik Basu, MD , Indu Ramachandran, PhD 1 2 Adaptimmune, Philadelphia, PA, United States; Princess Margaret Cancer Centre, Toronto, Ontario, Canada; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Indu Ramachandran (indu.ramachandran@adaptimmune.com) Fig. 1 (abstract P172). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P173 Background ADP-A2M4 is a genetically engineered autologous affinity-enhanced receptor immunotherapy (SPEAR T-cells) directed towards a MAGE- A4 peptide expressed in the context of HLA-A*02 on tumor cells. Clinical responses with ADP-A2M4 have been reported in patients with advanced MAGE-A4+ synovial sarcoma (SS) tumors. Here, we describe intra-tumoral and peripheral correlates associated with clin- ical response and resistance in two patients with SS. Methods Transduced T-cell persistence was determined by qPCR in PBMCs. Serum cytokines were measured via a multiplexed electrochemiluminescence-based immunoassay (MSD). Immunohisto- chemistry for antigen and immune markers was performed on FFPE tumor biopsies collected from patients prior to and following ADP- A2M4 transfer. A digital PCR-based assay was performed on FFPE tumor biopsies to detect the presence of SPEAR T-cells in the tumor. T-cell cytotoxicity assays were performed in vitro using the IncuCyte® platform. Clinical responses were assessed by RECIST v1.1. Results In the first patient, the best overall response (BOR) following ADP- A2M4 treatment was a partial response. Multiple correlates previously shown to be associated with response were observed. The patient’s pre- and post-infusion tumor biopsies expressed high levels of MAGE-A4 protein. Post-infusion, high levels of persisting transduced cells were observed in peripheral blood. Additionally, the patient had a grade 2 CRS event associated with a high level of serum IFN-g and Fig. 2 (abstract P172). See text for description IL-15 induction. In the post-infusion tumor sample, a notable increase Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 95 of 272 in CD3+ T-cell infiltration, including SPEAR T-cells, was observed and simultaneously any potential TAA. RevTMs were able to effi- along with PD-L1 induction. ciently redirect RevCAR-T-cells specifically against different tumor tar- In the second patient, the BOR was stable disease; then the disease pro- gets. Moreover, we show that combinatorial targeting can be gressed. MAGE-A4 protein expression was lower prior to ADP-A2M4 infu- achieved using our RevCAR system. Here, dual-RevCAR-T-cells were sion, compared to the 1st patient. Minimal peripheral induction of IFN-g efficiently activated only after engagement by two RevTMs targeting and IL-15 was observed post-infusion along with a lower level of trans- the activating or costimulatory RevCAR and different TAAs. duced T-cell persistence, compared with the 1st patient. No CRS was re- Conclusions ported in this patient. Both patients’ manufactured products contained Taken together, we developed a switchable RevCAR platform showing transduced CD8+ T-cells capable of killing antigen-expressing targets high effectiveness, increased specificity, improved safety, easy control- in vitro. In the responding patient, effective target killing was observed in lability, and small size facilitating combinatorial tumor targeting. transduced CD8+ T-cells isolated from the tumor site post-infusion. Eight Ethics Approval additional SS patients have been treated, and we continue to analyze The study was approved by local authorities and the Ethics Board. biomarkers in these patients. Conclusions P175 Based on these two cases, we have identified some factors that may PD-L1: A side-effect of T cell engagement or a main player in MDS contribute to the anti-tumor activity of ADP-A2M4. High antigen ex- tumor immune evasion? pression levels, IL-15 and IFN-g cytokine induction, good engraft- 1 1 1 2 Valentina Ferrari, BA , Alison Tarke , Hannah Fields , Tiffany Tanaka , ment, tumor site trafficking, and cytolytic function of SPEAR T-cells 2 1 1 Rafael Bejar , Thomas Lane, MD , Antonella Vitiello, PhD , Maurizio may be associated with favorable responses in SS patients treated Zanetti, MD with ADP-A2M4. 1 2 PersImmune, San Diego, CA, United States; University Of California San Trial Registration Diego, San Diego, CA, United States NCT03132922 Correspondence: Antonella Vitiello (avitiello@persimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P175 P174 Combinatorial tumor targeting using a novel switchable RevCAR Background system Immune checkpoint inhibitors (ICIs) are being tested in myelodys- 1 1 1 Anja Feldmann, PhD , Anja Hoffmann , Ralf Bergmann , Liliana Loureiro, plastic syndromes (MDS) based on pre-clinical data suggesting that 1 2 1 1 PhD , Enrico Kittel-Boselli , Nicola Mitwasi , Stefanie Koristka , Justyna the relevant targets are expressed on tumor and immune cells. Here 3 1 1 1 Jureczek , Nicole Berndt , Claudia Arndt , Michael Bachmann we study both tumor cells and T cells from patients with higher-risk 1 2 Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; University MDS to assess the role of PD-L1. Hospital Carl Gustav Carus, Dresden, Germany; German Cancer Research Methods Center (DKFZ), Heidelberg, Germany Patients’ CD3+ control cells, CD34+ stem cells, and their autologous Correspondence: Anja Feldmann (a.feldmann@hzdr.de) MDS cell lines (MCLs) were analyzed by DNA and RNA sequencing to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P174 identify somatic variants present in the tumor cells and absent from the control cells. From all somatic variants identified, we generated Background and tested neopeptides in vitro for their ability to induce tumor- Although T-cells genetically modified to express chimeric antigen recep- specific T cell responses. A T cell killing assay was performed to as- tors (CARs) are successfully used to treat hematological malignancies, pa- sess which neopeptide-specific T cells were capable of mediating tients still suffer from several drawbacks of conventional CAR (cCAR) tumor cell lysis (Figure 1A). In parallel, tumor PD-L1 expression levels therapy. CAR-T-cells can cause severe to life-threatening adverse reac- were measured by flow cytometry before and after 24-hour incuba- tions like on-target, off-tumor toxicities which cannot be controlled in pa- tion with tumor-specific autologous T cells. As a control for back- tients. Moreover, cCAR therapy often fails to successfully affect solid ground tumor cell lysis and PD-L1 expression, tumor cells were also tumors and bears the risk to encourage tumor escape variants upon tar- incubated with CEF-specific T cells (CEF: CMV, EBV, and flu peptides). geting of only one single tumor-associated antigen (TAA). In order to Results overcome these problems, we have established a novel on/off-switchable Patients’ tumor cells did not express PD-L1 at baseline. Remarkably, RevCAR system facilitating combinatorial targeting strategies. after co-culture, PD-L1 expression on the tumor cells ranged from Methods 20% to 70% (Figure 1B). Tumor cells, when incubated with CEF- For combinatorial targeting one T-cell has to be modified with two separ- specific T cells, did not upregulate PD-L1, suggesting that PD-L1 ex- ate CARs recognizing different TAAs. The first CAR mediates the activation pression may be linked to target recognition by neoantigen-specific and the second CAR the costimulatory signal. In case of ‘AND’ gate tar- T cells (Figure 2). Interestingly, tumor cell lysis was independent of geting, dual-CAR-T-cells have to recognize both TAAs on the surface of PD-L1 expression on tumor cells (Figure 1C). Additionally, IFNg the target cells to get activated. However, such combinatorial targeting neutralization did not affect PD-L1 expression nor ability to lyse strategies are struggling with several challenges including the adjustment tumor cells (Figure 3). These data show that when tumor cells are in- of signal strength and affinity of both split CARs as well as the CAR size cubated with autologous tumor-specific T cells, the tumor cells up- limiting the number of transduced specificities. In order to overcome regulate PD-L1 expression yet do not escape lysis by T cells. these obstacles, our idea was to construct small RevCARs comprising only Since lysis of tumor cells may occur prior to their upregulating PD-L1, we a small peptide epitope as extracellular domain. By removing the extra- pre-incubated tumor cells with soluble IFNg prior to co-culture with T cells. cellular single-chain variable fragment (scFv) of cCARs, RevCARs avoid Tumor cells were 96% PD-L1+, and were lysed by tumor-specific T cells at tonic signaling induced by scFv dimerization. As RevCARs do not have an the same level of target cells that had not been treated with IFNg (Figure 4). extracellular antigen binding moiety, they cannot bind to any antigen Conclusions per se. Thus, actually they are switched off. Only in the presence of a bis- Collectively, these data lend support to the notion that, in this sys- pecific target module (RevTM), RevCAR-T-cells can be redirected to tumor tem, PD-L1 is not a main player in MDS tumor immune evasion, sug- cells and switched on. Finally, short-living RevTMs allow a repeatedly on/ gesting that tumor immune evasion might function in a PD-L1- off-switch and controllability of RevCAR-T-cells and furthermore a flexible independent way. They also suggest that cognate recognition of redirection of RevCAR-T-cells to any target. tumor cells by neoantigen-specific T cells can cause the upregulation Results PD-L1 on target cells via a yet to be identified mechanism. For proof of concept two small peptide epitopes were selected to Ethics Approval construct the respective RevCARs. Additionally, a series of different The study was approved by the University of California, San Diego's RevTMs was generated recognizing one of the two peptide epitopes Institutional Review Board, HRPP #161345. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 96 of 272 P176 Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) for metastatic uveal melanoma: feasibility of treatment using a product generated from the primary tumor Marie-Andree Forget, PhD, Cara Haymaker, PhD, Orenthial Fulbright, BS, Shawne Thorsen, BS, Esteban Flores, BS, Arely Wahl, MSc, Rene Tavera, BS, Benjamin Tintera, BS, Timothy Woody, Michelle Williams, Yun Shin Chun, Patrick Hwu, MD, Dan Gombos, Sapna Patel, MD, Rodabe Amaria, MD, Chantale Bernatchez MD Anderson Cancer Center, Houston, TX, United States Correspondence: Chantale Bernatchez (CBernatchez@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P176 Background MD Anderson Cancer Center’s tumor-infiltrating lymphocyte (TIL) program has expanded TIL from tumor fragments of cutaneous metastatic melanoma using high dose IL-2 from over 900 patients with a growth success averaging 62% [1]. Surprisingly, this growth success plunges to 45% for metastatic uveal melanoma tumor fragments [2] and less than 20% from primary uveal tu- mors. The reason for this drop is unclear as uveal melanomas have an infiltration of CD8+TIL comparable to cutaneous melan- oma [2], which in theory makes it an attractive candidate for im- Fig. 1 (abstract P175). Ferrari, V SITC 2019 abstract slide 1 munotherapy. However, limited success observed with checkpoint therapy prompted us to explore ex vivo manipulation of the TIL. A previous report demonstrated the feasibility of TIL adoptive cell therapy for metastatic uveal patients using a TIL product ex- panded from metastases [3]. Since the primary and metastatic sites of uveal melanoma dis- play preserved gene mutations, indicating a potential shared antigen landscape, one could propose generating a TIL product from the primary tumor when the patient undergoes enucle- ation and to utilize this product for treatment at time of recurrence. Methods Given the challenge of propagating TIL from a primary uveal tumor in high dose of IL-2 only, we hypothesized that our new TIL3.0 method to propagate TIL from tumor fragments (1st phase of expansion), based on the 3-signals required for optimal activa- tion of a T-cell (TCR engagement, costimulation and cytokine ex- Fig. 2 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 posure) would enable TIL growth from a higher percentage of primary uveal tumors given the success obtained with metastatic sites [1]. This product can be banked and accessed later for treat- ment at recurrence. Results The TIL3.0 expansion platform was shown to be optimal for T-cell propagation allowing for successful expansion of TIL from primary uveal melanoma tumors in >90% of the cases (n=20). This expan- sion was rapid (less than 3 weeks) and consistently composed of CD8+CD3+TIL. This later observation is attributed to the use of the agonistic anti-CD137/4-1BB, Urelumab, as of costimulation sig- nal in our TIL3.0 method. The TIL3.0 method applied to primary tumors could be scaled and adapted for GMP. This process, followed by a rapid expan- sion protocol, was applied to treat the first metastatic uveal pa- tient with a TIL product generated from the primary tumor. The patient was infused with a total of 14.4 billion TIL with a viability of 99%. Conclusions This study demonstrates the feasibility of generating a TIL product from a primary uveal tumor to be used for treatment at recurrence. Trial Registration Fig. 3 (abstract P175). Ferrari, V SITC 2019 abstract slide 2 NCT00338377 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 97 of 272 References Deletion of p50 in patient-derived marrow CD34+ cells and subse- 1. Tavera RJ, Forget MA, et al. Utilizing T-cell Activation Signals 1, 2, and 3 quent production of IMC for adoptive transfer may contribute to the for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over therapy of these and additional cancers, alone or with additional the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma. J immunotherapies. Immunother. 2018; 41:399-405. Ethics Approval 2. Qin Y, de Macedo MP, Reuben A, et al. Parallel profiling of immune The study was approved by the Johns Hopkins University Animal infiltrate subsets in uveal melanoma versus cutaneous melanoma unveils Care and Use Committee, protocol MO19M10. similarities and differences: A pilot study. OncoImmunology. 2017; 6:e1321187. 3. Chandran SS, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two- P178 stage, single-arm, phase 2 study. Lancet Oncol. 2017; 18:792-802. Dual inhibition of PI3Kdelta and PI3Kgamma to enhance Ethics Approval mitochondrial mass and ex vivo expansion of central and stem cell Institutional review board (IRB)-approved protocol# 2004-0069 memory T cells from CLL patients Christopher Funk, BS, , Shuhua Wang, MD, Alexandra Waller, BS, Lauren Fleischer, BS, Aditi Sharma, Harold Spencer, PhD, Vikas Gupta, MD, PhD, P177 Sruthi Ravindranathan, PhD, Mala Shanmugam, PhD, Christopher NF-kB p50-deficient immature myeloid cell (p50-IMC) adoptive Flowers, MD, MS, Edmund Waller, MD, PhD, FACP transfer slows the growth of murine prostate and pancreatic Emory University School of Medicine, Atlanta, GA, United States ductal carcinoma Correspondence: Edmund Waller (ewaller@emory.edu) Rahul Suresh, PhD, David Barakat, PhD, Theresa Barberi, PhD, Lei Zheng, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P178 PhD MD, Elizabeth Jaffee, MD, Kenneth Pienta, MD, Alan Friedman, MD Johns Hopkins University School of Medicine, Baltimore, MD, United Background States For chimeric antigen receptor T-cell (CAR T) therapy to treat Correspondence: Alan Friedman (afriedm2@jhmi.edu) chronic lymphocytic leukemia (CLL), recent work associates remis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P177 sions with infusion of sufficient non-exhausted memory CAR T, capable of oxidative phosphorylation [1]. Our work aims to Background modulate metabolic pathways during ex vivo expansion of T-cells NF-kB p50 binds DNA but, unlike p65, lacks a trans-activation do- for translation of these findings to clinical adoptive therapies. main and recruits co-repressors. Macrophages and dendritic cells Class I catalytic PI3K enzymes, such as PI3Kdelta and PI3Kgamma, lacking NF-kB p50 are skewed towards a pro-inflammatory pheno- regulate T-cell differentiation, regulatory T cell formation, and TCR type, with increased cytokine expression and enhanced T cell acti- signaling [2]. In this study, we hypothesized that pharmacological vation; additionally, murine melanoma, fibrosarcoma, colon inhibition of these pathways during ex vivo culture would in- carcinoma, and glioblastoma grow slower in p50-/- mice. Given crease populations of early memory CAR T with enhanced meta- these data, we evaluated efficacy of p50-deficient immature mye- bolic and survival potential. loid cells (p50-IMC) adoptively transferred into tumor-bearing hosts. Methods Immature cells were utilized to maximize tumor localization, and Healthy- and CLL- donor peripheral blood mononuclear cells pretreatment with 5-fluorouracil (5FU) was examined due to its po- were isolated and cryopreserved. Thawed cells were sorted for tential to impair marrow production of myeloid cells, to target CD3 expression prior to culture (or transduction) and expanded tumor myeloid cells, and to potentially release tumor neoantigens. in G-Rex plates using anti-CD3/CD28 beads, 30 U/mL Methods interleukin-2, and investigational drugs: idelalisib, duvelisib and WT-IMC or p50-IMC were generated by culturing lineage-negative ibrutinib for 9 days. marrow cells from WT or p50-/- mice in media containing TPO, SCF, Results and FL for six days followed by M-CSF for one day on ultra-low at- Class I catalytic enzymes in T-cells, PI3Kdelta and PI3Kgamma, tachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) exhibit domain homology (Figure-1A). Accordingly, maximum cell or K-Ras(G12D) pancreatic ductal carcinoma (PDC)-luciferase cells re- yields for both idelalisib (Figure 1B) and duvelisib (Figure 1C) ceived 5FU followed five days later by three doses of 1E7 IMC every occurred upon inhibition of both isoforms. Comparing doses of three to four days. Some groups also received four doses of anti-PD- duvelisib, idelalisib, and ibrutinib that yielded optimal T-cell 1 antibody twice weekly alone or with p50-IMC. expansion, we showed potent PI3Kdelta/gamma dual antagonism Results maximizes live T-cell yields (Figure 1D) out-performing PCa grew slower in p50-/- mice, and absence of host p50 led to interleukin-2-inducible kinase inhibition. PI3K antagonists in- prolonged survival of mice inoculated orthotopically with PDC. creased frequencies of cells expressing co-stimulatory molecules 5FU followed by p50-IMC slowed PCa and PDC tumor growth ~3- (Figure 2A-B). A dose-dependent increase in cells expressing FAS/ fold in contrast to 5FU followed by WT-IMC, 5FU alone, or p50- FAS-L (Figure 2C) and an increased expression of pro-survival IMC alone. Slowed tumor growth was evident for 93% of PCa tu- BCL-2 (Figure 2D) after anti-CD3/28 stimulation suggests the mors but only 53% of PDC tumors. In PCa, p50-IMC predomin- increase in live cells with a TSCM phenotype is likely due to en- antly generated tumor and draining lymph node F4/80+ hanced cell survival. macrophages, but also CD11b+F4/80-CD11c+ conventional den- Next,weconfirmed thepositiveeffectofdualPI3Kdelta/gamma dritic cells. A subset of tumor and nodal macrophages co- inhibition in expansion of T cells from CLL donors (Figure 3A-B). expressed Ly6C and MHCII and had reduced MR compared to PI3K antagonists increased frequencies of CD8 cells (Figure 3C) host macrophages, collectively indicating a pro-inflammatory and co-stimulatory molecule expressing cells (Figure 3D-E). Inter- phenotype. p50-IMC also produced a 5-fold increase in activated estingly, addition of idelalisib to T cell cultures resulted in a PCa tumor CD8 T cells, and antibody-mediated CD8 T cell deple- dose-dependent decrease in immune checkpoint molecules LAG- tion obviated slower tumor growth induced by 5FU followed by 3, Tim-3, and PD-1 (Figure 3F-J). Given the importance of T cells p50-IMC. Anti-PD-1 markedly slowed PCa growth but had little ef- with the stem cell memory (TSCM) phenotype in adoptive T-cell ficacy against PDC, whereas anti-PD-1 combined with p50-IMC therapy, T-cell differentiation was studied. PI3K antagonists in- slowed PDC tumor growth to prolong survival more effectively crease the frequency of early, T-memory, and effector memory than either alone in an initial experiment. cells (Figure 4A). Notably, the frequency of TSCM doubled (Figure Conclusions 4B). Lastly, PI3K antagonists significantly increased the mitochon- 5FU followed by p50-IMC slows the growth of murine prostate and drial mass (Figure 4C) within total CD3 (Figure 4D) and CD8 (Fig- pancreatic ductal carcinoma and depends upon CD8 T cell activation. ure 4E) subsets. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 98 of 272 Conclusions Dual inhibition of PI3Kdelta and PI3Kgamma modulates aspects of T-cell biology relevant to CAR T remissions. PI3Kdelta/gamma antagonism enhances CD27 expression and mitochondrial mass, decreases immune checkpoint expression, and enriches the TSCM phenotype. Acknowledgements The authors thank the patients and healthy volunteers for their blood donations. CR Funk is supported by a Howard Hughes Medical Research Fellowship. The authors thank Emory’s Clinical Lymphoma Research Team for requesting patient consent and aiding in sample collection. References 1. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 2018; 24:563-571. [2]2. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, and Abraham RT. Leading Edge The PI3K Pathway in Human Disease. Cell. 2017; 170:605-635. Ethics Approval The study was approved by Emory University's Institutional Review Committee, approval number 00057236. Fig. 3 (abstract P178). See text for description Fig. 1 (abstract P178). See text for description Fig. 4 (abstract P178). See text for description P179 Checkpoint Cbl-b siRNA-based APN401 adoptive cell therapy: superior efficacy & immune memory induction in murine hepatocellular carcinoma following APN401 monotherapy and synergism with anti-PD1 1 1 1 Anderson Gaweco, MD, PhD , Kathrin Thell , Maria Urban , Julia 1 1 2 3 Harrauer , Isabella Haslinger , Josef Penninger , Vincent Chung , 4 5 Anthony El-Khoueiry, MD , Carlos Becerra, MD 1 2 Apeiron Biologics, Vienna, Austria; University of British Columbia, 3 4 Vancouver, BC, Canada; City of Hope, Duarte, CA, United States; USC Norris Comprehensive Cancer Center, Los Angeles, CA, United States; Baylor University Medical Center, Dallas, TX, United States Correspondence: Anderson Gaweco (anderson.gaweco@apeiron- biologics.com) Fig. 2 (abstract P178). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P179 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 99 of 272 Background combination of immunomodulatory cytokines: IL12 and IL21. Upon The intracellular master checkpoint Cbl-b, an E3 ubiquitin ligase, administration, SENTI-101 innately homes to peritoneal tumors, se- negatively regulates the innate and adaptive anti-tumor immune re- cretes IL12 and IL21 in a localized and sustained fashion, and induces sponses. Selective cell-based targeting of Cbl-b not only induces a robust anti-tumor immune response. anti-tumor activity in vivo but also overrides immune regulation by Methods the PD-L1/PD-1 pathway in vitro [1]. Human APN401 is an autologous Two syngeneic pre-clinical models of disseminated peritoneal carcin- adoptive cellular therapy of ex vivo Cbl-b-silenced human PBMCs omatosis with distinct immune phenotypes were established by currently in a clinical Phase 1b multiple dose study demonstrating implanting cells in the peritoneal cavities of mice (CT26-fLUC = early clinical safety and tolerability in patients with advanced solid immune-inflamed; B16-F10-fLUC = immune-excluded) [3]. A library of tumors. Herein the preclinical Proof of Concept efficacy of murine over 50 murine MSC lines engineered to express immune effectors APN401 immunotherapy is established in the syngeneic mouse hepa- (cytokines, chemokines, growth factors), either individually or in com- tocellular carcinoma Hepa1-6 tumor rechallenge study. bination, was administered intraperitoneally and evaluated for anti- Methods tumor activity via bioluminescence and tumor weight measurements. Hepa1-6-C57/BL6-tumor bearing mice were treated for 19 days with Immune phenotype was characterized by flow-cytometry and multi- murine APN401, ex vivo silenced immune cells with Cbl-b specific plexed immunohistochemistry. siRNA, as monotherapy or in combination with anti-PD1 (clone Results RMP1-14) versus control siRNA on D7 post-inoculation. Mice were MSCs expressing the combination of IL12 and IL21 (SENTI-101) were se- later rechallenged on D29 with Hepa1-6 s.c. on the contralateral flank lected based on significant tumor-burden reduction and immune pro- in the absence of any further APN401 treatment. file changes in both syngeneic models. Notably, the combination Results outperformed each individual cytokine in extending survival (p=0.02). Significant tumor growth inhibition (TGI) was observed following 19 Intraperitoneal administration of SENTI-101 into tumor-bearing mice days of treatment with APN401 alone or in combination with anti- led to preferential co-localization with tumors (>10-fold higher vs. PD1 starting on D7 after s.c. inoculation (p<0.0001). Following rechal- normal tissues, p=0.001). Local concentrations of IL12 and IL21 were lenge, significant TGI of 86% (p<0.001) was observed in prior ~100-fold greater in the peritoneal space vs. serum (p=0.002). SENTI- APN401-treated mice versus control siRNA-treated mice. Mice that re- 101 treatment reduced tumor-burden more than 200-fold (p50% of ceived APN401 in combination with anti-PD1 prior to rechallenge the mice were tumor-free after 90 days, while control groups and demonstrate profound synergistic anti-tumor efficacy (p<0.001) ver- groups treated with anti-PD1 antibody had a median survival of 21 sus anti-PD1 alone with control siRNA. APN401 was well tolerated to 30 days. Surviving mice were able to reject newly implanted and APN401-treated mice were unremarkable with optimal body tumor cells, demonstrating anti-tumor immune memory. conditions. Anti-tumor effects of SENTI-101 are mediated by a multi-modal im- Conclusions mune response. The frequency of antigen-presenting cells in periton- APN401 monotherapy demonstrates striking anti-tumor efficacy in eal tumor-draining lymph nodes was more than doubled vs. controls the murine hepatocellular carcinoma Hepa1-6 model. The preclinical (p=0.01). This correlated with increased T-cell and B-cell tumor infil- synergistic effects of APN401 with anti-PD1 support its therapeutic trates forming tertiary-lymphoid structures, which are associated with utility as a combination therapy with immune checkpoint anti-PD1 improved prognosis in cancer [4]. T-cell activation markers (CD38, treatment. The significant TGI observed following tumor rechallenge IFNg, GranzymeB) were significantly increased locally. indicate that prior selective cell-based Cbl-b-silencing with APN401 Conclusions alone or in combination with anti-PD1 induced systemic and durable SENTI-101 induces localized immune-modulation, regulates multiple anti-tumor immune memory responses. These findings highlight the steps of the cancer immunity cycle, and results in durable anti-tumor potential promise of a selective adoptive cell-based Cbl-b silencing responses. These data warrant further development of SENTI-101 for by APN401 as a novel immunotherapy for cancer. the loco-regional treatment of advanced solid tumors. Reference References 1. Fujiwara M, Anstadt EJ, Clark RB. Cbl-b Deficiency Mediates Resistance to 1. Desai JP, Moustarah F. Cancer, Peritoneal Metastasis. StatPearls Programmed Death-Ligand 1/Programmed Death-1 Regulation. Front Publishing. 2019 [Updated 2019 Jun 30] Immunol. 2017 Jan 26;8:42. 2. Lengyel E. Ovarian Cancer Development and Metastasis. Am J Pathol. 2010; 177(3): 1053–1064 3. Mosely SI, Prime JE, Sainson RC, et al. Rational Selection of Syngeneic P180 Preclinical Tumor Models for Immunotherapeutic Drug Discovery. Cancer SENTI-101, an allogeneic cell product, induces potent and durable Immunol Res. 2017; 5(1):29-41 anti-tumor immunity in pre-clinical models of peritoneal 4. Sautès-Fridman C, Petitprez F, Calderaro J, Fridman WH. Tertiary carcinomatosis lymphoid structures in the era of cancer immunotherapy. Nat Rev Alba Gonzalez Junca, PhD, Gary Lee, PhD, Archana Nagaraja, PhD, Alyssa Cancer. 2019;19(6):307-325 Mullenix, Russell Gordley, PhD, Daniel Frimannson, PhD, Anissa Benabbas, Chen-Ting Lee, PhD, Tiffany Truong, Allison Quach, Mengxi Tian, Rishi Savur, Rowena Martinez, Alyssa Perry-McNamara, Don-Hong P181 Wang, PhD, Ori Maller, PhD, Dharini Iyer, PhD, Ashita Magal, Christina Multi-phenotype CRISPR-Cas9 screens identify p38 kinase as a Huynh, Carmina Blanco, Jack Lin, PhD, Brian Garrison, PhD, Philip Lee, target for adoptive immunotherapies PhD, Timothy Lu, MD, PhD, Sravani Mangalampalli Devikala Gurusamy, PhD, Suman Vodnala, Amanda Henning, Rigel Senti Biosciences Inc., South San Francisco, CA, United States Kishton, Tori Yamamoto, Arash Eidizadeh, Li Jia, Christine Kariya, Mary Correspondence: Gary Lee (gary.lee@sentibio.com) Black, Robert Eil, Douglas Palmer, Zhiya Yu, Jenny Pan, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P180 Madhusudhanan Sukumar, Shashank J. Patel, Nicholas Restifo, MD National Institutes of Health, Bethesda, MD, United States Background Correspondence: Nicholas Restifo (restifo@nih.gov) More effective therapies for disseminated peritoneal carcinomatosis, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P181 including high-grade serous ovarian cancer, remain a major medical need [1]. Although several treatments offer initial responses to local- Background ized disease, patients with disseminated peritoneal tumors face poor Adoptive T cell transfer immunotherapy (ACT) using tumor-infiltrating overall survival [2]. lymphocytes (TIL) and gene-modified T cells can induce complete and SENTI-101 is a novel therapeutic agent comprising allogeneic mesen- durable regression of metastatic human malignancies that are other- chymal stromal cells (MSCs) genetically modified to express a potent wise refractory to treatment. While successful T cell-based treatments Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 100 of 272 of patients with widely metastatic melanoma, synovial sarcoma, cholan- concentration of 1 x 106 cells/ml with high concentration of hIL-2 giocarcinoma and cancers of the breast, colon, and cervix have been and 5% UltraGRO® for 14 days in our original closed system. Then, reported in recent years, most patients with common epithelial cancers we confirmed the expression of surface markers, CD107a fail to respond to treatment. While several factors can contribute to the mobilization and cell-mediated cytotoxicity against various tumor efficacy of ACT, a major inherent limitation is the induction of termin- cells and normal cells with or without monoclonal antibody drugs ally differentiated phenotype coupled with the loss of proliferative cap- in vitro and antitumor effects against peritoneal dissemination model acity in TIL during current ex vivo expansion protocols. Individual gene using SKOV3 in vivo. knockout approaches for enhancing T cell-based cancer immunother- Results apies are low-throughput and can improve one desired function (T cell [Results and Discussion] Importantly, we’ve found that our GAIA-102 memory) at the expense of another equally important function (expan- exhibited CD3-/CD56bright/CD57- immature phenotype that could sion). Thus, there is significant interest in identifying T-cell intrinsic kill various tumor cells efficiently from various origins, including Raji negative regulatory circuits that limit their ability to expand robustly cells that was highly resistant to NK cell killing. More importantly, ex vivo, while dampening their terminal effector differentiation along massive accumulation, retention, infiltration and sphere destruction with the reduction of oxidative stress and genomic damage. by GAIA-102 were affected neither by myeloid-derived suppressor Methods cells nor regulatory T-lymphocytes. GAIA-102 was also effective To identify the T cell intrinsic negative regulatory circuits, we developed a in vivo to murine model of peritoneal dissemination of human ovar- multi-phenotype genetic screen to systematically target 29 major kinases ian cancer. screen to concurrently measure the impacts of individual gene knockouts Conclusions on T cell expansion, differentiation, oxidative stress and genomic stress. Thus, these findings indicate that GAIA-102 has a potential to be an Using CRISPR-Cas9-based gene perturbation combined with high- ‘upward compatible’ modality over CAR-T strategy, and would be a throughput flow cytometry, we developed and validated a multi-phenotype new and promising candidate for adoptive immunotherapy against screen, which identified Mapk14/p38 kinase as a target that improved all solid tumors. We now just started GMP/GCTP production of this new four phenotypes in CD8+ T cells. We used murine and human ex vivo T cell and powerful NK cells and first-in-human clinical trials in use of expansion models to validate the results from our genetic screen. GAIA-102 will be initiated on 2020. Results Ethics Approval Results from our genetic screen identified p38 kinase as a unique [Ethics Approval] Written informed consent was obtained from all multi-phenotypic regulator of cellular differentiation, oxidative, and healthy volunteers, in accordance with the Declaration of Helsinki. genomic stress while achieving improved cellular expansion. Further- Upon the approval of the institutional ethical committee (ap- more, pharmacological inhibition of p38 kinase in murine and human proval no. 29-315) of Kyushu University, peripheral blood samples ex vivo T cell expansion models validated the results from our gen- were collected from healthy volunteers. The animal experiments etic screen. Cells cultured in the presence of a p38 inhibitor had in- were reviewed and approved by the Institutional Animal Care creased capacity for cytokine production, specifically interferon-γ and and Use Committee of Kyushu University (approval nos. A30-234- demonstrated improved in vivo persistence. Additionally, cells cul- 0 and A30-359-0). tured in the presence of the p38 inhibitor demonstrated enhanced in vivo cell-expansion, tumor infiltration, and anti-tumor efficacy in P183 an immunocompetent tumor mouse model. Development of novel chimeric antigen receptor T cells for Conclusions immunotherapy of hepatocellular carcinoma This study establishes p38 inhibition in T cells as a potentially import- Yukai He, PhD, Leidy Caraballo Galva, Xiaotao Jiang ant strategy for improving ACT immunotherapy for cancer patients. Augusta University, Augusta, GA, United States Ethics Approval Correspondence: Yukai He (yhe@augusta.edu) All human samples were isolated in accordance with approved clin- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P183 ical protocols and in accordance with NIH institutional review board approval and informed consent from patients and healthy donors. Background Immunotherapy has a great potential for hepatocellular carcin- P182 oma (HCC). Several human glypican 3 (hGPC3)-specific chimeric GAIA-102: a new class NK cell-like phenotype manufactured in antigen receptor T cells (CARTs) are being tested for HCC. But, accordance with GMP/GCTP that can eliminate solid tumors most, if not all, are constructed from one monoclonal antibody Yui Harada, PhD, Yoshikazu Yonemitsu, MD, PhD (mAb). It is unknown whether targeting different epitopes of Kyushu University, Fukuoka, Japan hGPC3 will create more effective CARTs. Here, we aim to develop Correspondence: Yui Harada (rkfraile@med.kyushu-u.ac.jp) novel CARTs that target different regions of hGPC3. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P182 Methods BalB/C mice were immunized with hGPC3 protein. Hybridomas Background and mAbs were generated and characterized. Then, CARTs were [Background] Cancer immunotherapy has been established as a new built from the novel mAbs and their antitumor effect was therapeutic category since the recent success of immune checkpoint studied. inhibitors and a type of adoptive immunotherapy, namely chimeric Results antigen receptor-modified T cells (CAR-T). Although CAR-T demon- Twenty-two hGPC3-specific mAbs were identified by ELISA. Out of strated impressive clinical results, serious adverse effects (cytokine them, 14 bound HepG2 cells. Five mAbs were further character- storm and on-target off-tumor toxicity) and undefined efficacy on ized by immunohistochemical staining. Three of them (6G11, 8F8, solid tumors are important issues to be solved. We’ve developed a and 12D7) were found to specifically stain HCC tumor but not cutting-edge, simple, and feeder-free method to generate highly acti- adjacent normal tissues. The 3 mAb’s affinity were in the nano- vated and expanded human NK cells from peripheral blood molar range. 6G11 and 8F8 bound to hGPC3 epitope aa25-39 (US9404083, PCT/JP2018/018236, PCT/JP2019/012744), and have and aa463-496, respectively. No specific epitope was identified been conducting further investigation why our new type of NK cells, for 12D7 though it bound to the N-fragment (25-358aa). CARTs named as GAIA-102, are so effective to kill malignant cells. built from the 3 mAbs underwent expansion in response to Methods HepG2 cell stimulation. However, their effector function was sig- [Materials and Methods] Cryopreserved PBMCs purchased from nificantly different. 8F8 CARTs possessed the strongest effector HemaCare Corporation were mixed and processed by using LOVO function. 6G11 CARTs generated the greatest expansion, but with and CliniMACS® Prodigy (automated/closed systems). CD3+ and slightly weaker function. In contrast, 12D7 CART had the weakest CD34+ cells were depleted, and the cells were cultured at a effector function. Soluble hGPC3 did not activate CARTs, nor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 101 of 272 blocked CART activation by tumor cells. Adoptive transfer of 8F8 P185 and 6G11, but not 12D7, CARTs generated potent antitumor ef- T cell antigen presenting cell (tAPC) is a strategy to induce CAR T fects with complete regression of HCC xenografts, which corre- expansion in vivo in the absence of a tumor for on-target toxicity lated to their expansion in vivo. studies Conclusions Yen Ho, BS, Jon Jones, BA, Patrick Carlson, BS, Cyr de Imus, The three novel CARTs that target different hGPC3 regions pos- Rebekah Turk, Dina Alcorn, Kyle Kolaja, Ruth Salmon, PhD, Thomas sess significantly different effector function and antitumor effects. Long Adoptive transfer of CARTs targeting the hGPC3 N- or C-epitope Celgene Corporation, Seattle, WA, United States results in complete eradication of HCC xenografts. Correspondence: Thomas Long (thomas.long@junotherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P185 P184 Background Activating marrow infiltrating lymphocytes in hypoxia enhances Preclinical safety evaluation of chimeric antigen receptor (CAR) their efficacy in adoptive T-cell therapy T cells presents a number of unique challenges. One of those 1 1 1 1 Megan Heimann , Ervin Griffin , Luca Biavati, MD , Amy Thomas , challenges is the development ofaCynomolgus macaquetoxi- 1 1 2 Danielle Dillard , Elizabeth Zawidzka , Brianna Richardson , Robert cology model as a tool to understand potential on-target CAR T 1 2 3 1 Leone , Gregory Szeto , Kimberly Noonan, PhD , Ivan Borrello, MD cell toxicity. This is important for CAR T cell programs where Johns Hopkins University School of Medicine, Baltimore, MD, United the lead binder is cyno cross-reactive and the target has known States; University of Maryland Baltimore County, Baltimore, MD, United normal tissue expression conserved across species. Unlike many States; WindMIL Therapeutics, Baltimore, MD, United States mouse models, non-human primates lack target-expressing Correspondence: Ivan Borrello (iborrell@jhmi.edu) tumors that can drive CAR T cell activation and expansion; it Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P184 would be advantageous to recapitulate that expansion in the context of a toxicity model. Background Methods Marrow infiltrating lymphocytes (MILs) are a promising candidate for T cell antigen presenting cells (tAPCs) have been reported to adoptive cell therapy (ACT) due to their broader anti-tumor specifi- drive measurable CAR T cell expansion in Rhesus macaques [1]. city and persistence. These characteristics are due to intrinsic proper- The advantages of this strategy include the ease of manufactur- ties of the bone marrow (BM); known to be a reservoir for long-lived ing tAPCs in parallel to CAR T cells and co-engraftment of memory T cells. It has also been established that naïve and memory tAPCs with CAR T cells in hematological niches to ensure anti- T cells are metabolically quiescent, favoring oxidative phosphoryl- gen availability. The optimal tAPC dosing strategy to drive a ation (OXPHOS) over glycolysis, while effector T cells favor glycolysis strong and persistent CAR T cell activity has not been deter- to fuel their rapid proliferation. mined. Here, we generated human anti-CD19 CAR T cells Methods expressing firefly luciferase and human T cells expressing a We examined how activation and expansion of MILs in hypoxia truncated human CD19 (CD19t) lacking the intracellular domain could be used to better understand the inherent properties of as tAPCs. We then dosed mice either with CAR and tAPC con- the BM, to exploit these properties, and enhance the efficacy of currently, or with CAR first then followed by tAPCs three days MILs in ACT, especially when compared to that of peripheral later. blood lymphocytes (PBLs). By activating MILs in hypoxia, we can Results select for and/or alter the cells best suited to mount an effective (Figure 1) shows bioluminescent imaging (BLI) measurements anti-tumor response. that indicate concurrent and delayed tAPC dosing have similar Results CAR T cell expansion kinetics; however, the delayed tAPC Activation under hypoxic conditions alters MILs in several unique dosing exhibits a greater magnitude of CAR T cell expansion. In ways. MILs show greater overall expansion, enhanced tumor- both cases, the CAR T cell expansion occurs in a tAPC dose- specificity, and a unique metabolic profile—upregulating both dependent manner. Imaging shows that concurrent dosing OXPHOS and glycolytic machinery. This metabolic profile suggests leads to CAR T cell proliferation primary in the lungs, whereas that hypoxia-activated MILs possess properties of both effector delayed tAPC dosing leads to more systemic CAR T cell and memory cells. PBLs grown under the same conditions fail to expansion. In addition, flow cytometry data show a significant expand significantly and show no metabolic differences or specifi- depletion of tAPCs in the peripheral blood between day 7 and city. Following activation in hypoxia we have found that MILs day 14. have upregulated metabolism-related genes such as CPT1A and Conclusions GLUT1 and 3, as well as anti-apoptotic factors such as BCL2 and Follow-up studies delivering additional tAPC doses three, six, BCL2L1 at an RNA level. The post-expansion MILs product, using and nine days after CAR T cells show that repeated tAPC ad- intracellular staining and FACs analysis, shows an increase in ministration can significantly increase the CAR T cell exposure mitochondrial proteins, including TOMM20, CPT1a, and SDHa, in- over time compared to a single tAPC dose. Overall, these data creased mTOR signaling, and increased glycolytic machinery—HK2 demonstrate that tAPCs can be used to induce CAR T cell ex- and GLUT1. Additionally, while cell-cycle analysis with Ki67 and PI pansion in vivo in the absence of a tumor and will enable us shows that MILs are more resting at baseline and arrested in G0, to design a tAPC strategy for use in a Cynomolgus macaque upon activation in hypoxia they become more proliferative—mov- model to evaluate the safety of CAR T cell candidates. ing into S and G2/M phase—than normoxic MILs or PBLs and maintain this phenotype over time. This finding is supported by our RNAseq data showing a lack of transcriptional activity at baseline but a significant increase by day 3 of activation. Gene Reference expression analysis has also shown that there is a substantial in- 1. Berger C, Sommermeyer D, Hudecek M, Berger M, Balakrishnan A, crease in expression of IL2 and IL15Ra with a significant decrease Paszkiewicz PJ, Kosasih PL, Rader C, Riddell SR. Safety of targeting in the expression of IL10 transcripts. ROR1 in primates with chimeric antigen receptor-modified T cells. Conclusions Cancer Immunol Res. 2015;3(2):206-16. These findings suggest that hypoxia contributes to the unique Ethics Approval properties of MILs through modification of their metabolic profile All animal studies were conducted in accordance with protocols approved and is being uniquely employed to generate more effective MILs, by the Institutional Animal Care and Use Committee. and not PBLs, for adoptive cell therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 102 of 272 P187 Single-cell RNA sequencing and functional assessment of healthy donor- and cancer patient-derived T and CAR-T cells Zinkal Padalia, MS, Konstantinos Karagiannis, PhD, Brigid Mcewan, MA, Vahan Simonyan, PhD, Jonathan Terrett, PhD, Demetrios Kalaitzidis, PhD CRISPR Therapeutics, Cambridge, MA, United States Correspondence: Demetrios Kalaitzidis (d.kalaitzidis@crisprtx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P187 Background Autologous chimeric antigen receptor T (CAR-T) cell therapies have shown remarkable success in treating relapsed/refractory B-cell ma- lignancies. However, even in indications with high complete re- sponse rates, not all patients respond or have durable responses after CAR-T treatment. Furthermore, autologous CAR-T treatments have not yielded the same impressive outcomes in solid malignan- cies to date. A major limitation of autologous CAR-T therapy may be the dysfunctional state of a patient’s T cell populations used for manufacturing of a drug product. Allogeneic therapeutics can bypass this limitation by enabling the use of healthy donor starting material. Moreover, healthy donor material that exhibits specific T cell attri- butes can be selected for drug product manufacturing. Methods Fig. 1 (abstract P185). See text for description To identify attributes that can be associated with improved perform- ance of CAR-T cells we have characterized T cells from healthy do- nors as well as cancer patients, in particular from chronic P186 lymphocytic leukemia (CLL) patients as these have been described Evaluation of antigen-specific T-cell immunity at the single cell previously to be dysfunctional. level using large panels of DNA barcoded MHC multimers Results 1 2 2 Kivin Jacobsen, PhD , Dagmar Walter , Michael Stubbington , Katherine We show impaired function of cancer patient-derived CAR-T cells 2 1 1 2 Pfeiffer , Charlotte Halgreen , Liselotte Brix, PhD , Stephane Boutet , when compared to healthy donor-derived cells utilizing both in vitro Kivin Jacobsen, PhD and in vivo assays. We have performed single-cell RNA sequencing 1 2 Immudex, Copenhagen, Denmark; 10x genomics, Pleasanton, CA, (scRNA seq) on both starting material T cells and CAR-T cells from United States multiple healthy and CLL donors used in functional assays to uncover Correspondence: Liselotte Brix (lb@immudex.com) both gene expression and population differences associated with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P186 CAR-T cell performance. scRNA seq analysis revealed marked hetero- geneity among starting populations as well as CAR-T lots from the Background cancer patient-derived T cells. Identification of disease-specific T-cell epitopes is key to developing Conclusions novel cancer vaccines and immunotherapies. Profiling disease-specific Our analysis has allowed us to associate distinct cellular subpopula- T cells, emerging during an induced cellular immune response is im- tion and gene expression profiles with preclinical functional outputs. portant for understanding anti-tumor immunity and guide personalized therapy. The MHC dCODE™Dextramer® technology enables simultan- P188 eous screening of high numbers of T-cell specificities in the same sam- Enhanced anti-tumor activity of human placental CD34+ derived ple using MHC multimer-specific DNA barcodes and next generation natural killer cells in combination with ACY-241 for multiple sequencing as readout. Combining this technology with 10x Genomics myeloma immunotherapy Chromium single cell assay further enables the simultaneous analysis of Lin Kang, PhD, Xiaokui Zhang, Shuyang He, Vanessa Voskinarian-Berse, antigen specific T-cells, sequencing of the cognate T-cell receptors and Bhavani Stout, Valentina Rousseva, William van der Touw, James Edinger, single cell gene expression profiling. Robert Hariri Methods Celularity Inc., Warren, NJ, United States Panels of up to 50 dCODE™Dextramer® reagents were used for screening Correspondence: Xiaokui Zhang (xiaokui.zhang@celularity.com) antigen-specific T-cells in human blood samples. Single cell sequencing was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P188 performed on the isolated T-cell subpopulations, and their gene expression profile analyzed in combination with cell phenotype and TCR sequences. Background Results Celularity, Inc. is developing human placental CD34+ derived, off-the- The experiment generated a large dataset and we show one example of shelf, and allogeneic natural killer (PNK) cells for various hematologic how such a dataset can be analyzed to generate useful information. By com- malignancies and solid tumors. ACY-241 is an orally bioavailable and bining the gene expression profile, cellular phenotypes and Dextramer specifi- selective histone deacetylase (HDAC) 6 inhibitor in MM clinical devel- city we identified expanded populations of antigen-specific T cells in the opment. ACY-241 has been shown to sensitize MM cells to endogen- memory T cell compartment and characterized their individual TCR clono- ous NK cell killing [1]. Here, we investigated the potential types based on TCR sequence. pMHC-specific T-cells were also detected in augmentation of PNK mediated anti-MM activity by ACY-241 the naïve T cell compartment, showing a more diverse TCR sequence profile. treatment. Conclusions Methods This experiment demonstrates a novel method of exploring antigen- Placental CD34+ cells were cultivated in the presence of cytokines in- specific T-cell responses. Linking TCR sequences with pMHC specifi- cluding thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 for 35 city, cellular phenotypes and gene expression at this scale and reso- days to generate PNK cells. MM cell lines were treated with different lution provide a more comprehensive analysis of the antigen-specific doses (0, 0.1, 0.3, 1, 3, 10 and 30μM) of ACY-241 over 24h, 48h, or T cell response than previously available in a single workflow. Novel 72h. Cytotoxicity of PNK against different doses of ACY-241 pre- biomarkers and improved strategies of T cell based immunothera- treated MM cell lines was assessed by a PKH26/TO-PRO-3 FACS based peutic development will result from T cell analysis at this scale and assay. Ligands to NK activating receptors of ACY-241 treated MM resolution. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 103 of 272 cells were evaluated by flow cytometry. The RPMI8226 subcutaneous- surface, as well as release of damage-associated molecular patterns ly(SubQ) xenografted NOD scid gamma (NSG) mouse model was including HMGB1 and ATP. Moreover, we show that TTFields treated used for in vivo efficacy study. cells promote phagocytosis by DCs, DCs maturation in vitro, and pro- Results mote immune cells recruitment in vivo. We also show that the com- ACY-241 treatment of MM cell lines for >48h resulted in significant bined treatment of TTFields plus anti-PD-1 led to a significant inhibition of cancer cell growth and decreased cell viability at doses decrease in tumor volume and significant increases in CD45+ tumor >10μM. In a dose dependent manner, ACY-241 further enhanced infiltrating cells in both tumor models. In the lung tumors, these infil- cytotoxicity of PNK against MM cells. In a 4h cytotoxicity assay at ef- trating cells, specifically macrophages and DCs, demonstrated upreg- fector to target (E:T) ratio of 10:1, relative to vehicle control, PNK (n= ulation of surface PD-L1 expression following short treatment 3 donors) showed increased cytotoxicity to ACY-241 pretreated MM duration. Correspondingly, cytotoxic T-cells isolated from these cell lines: RPMI8226 (16.3% to 34.9%), MM.1S (15.1% to 26.0%), OPM2 tumors have shown higher levels of IFN-γ production relative to (12.7% to 37.2%), and U266 (0% to 9.1%). Increased expression of li- untreated mice. In the colon cancer tumors, significant increases in gands to activating NK receptors, MIC A/B, CD56, CD54, and CD155 T-cell infiltration was observed following long treatment duration was detected from ACY-241 treated MM cells, suggesting that en- with TTFields plus anti-PD-1. gagement of NKG2D, CD11a or DNAM-1 of NK cells leads to en- Conclusions hancement of the anti-MM effect. Our results demonstrate the potential of TTFields therapy to induce In vivo anti-MM activity of PNK in combination with ACY-241 was ICD. We also demonstrate robust efficacy of concurrent application assessed in a RPMI8226 SubQ xenograft NSG model. Single intraven- of TTFields and anti PD-1 therapy in mouse models of cancer. These ous dosing of 1.0E7 PNK in combination with ACY-241 significantly data suggest that combining TTFields with anti-PD-1 might achieve reduced the tumor growth compared to vehicle control (P<0.001). tumor control by further enhancing antitumor immunity. Conclusions Our data demonstrated that enhanced in vitro anti-MM activity of Acknowledgements PNK in combination with ACY-241. In vivo efficacy of PNK in combin- The authors would like to thank Dr. Kenneth Swanson from Beth Israel ation with ACY-241 was further demonstrated in a RPMI8226 SubQ Deaconess Medical Center, and Dr. Ilan Volovitz from Tel Aviv Sourasky xenograft NSG model. Taken together, our results demonstrate the Medical Center for their helpful and constructive comments. synergistic effects of combining an HDAC inhibitor with an NK cell Ethics Approval therapy for anti-MM enhancement. Further development of a com- This study was approved by Novocure’s Ethics Board and by the Israel binatorial PNK and ACY-241 therapy for MM treatment is warranted. National Ethics Board; approval numbers 160816, 21015, IL-17-3-131 and IL- 19-1-38. Reference 1. Ray A, Das DS, Song Y, Hideshima T, Tai YT, Chauhan D, Anderson KC. P190 Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 anti- Single-day CAR manufacturing platform using mRNA and Flow body enhances anti-tumor immunity and cytotoxicity in multiple mye- Electroporation Technology loma. Leukemia. 2018 Mar;32(3):843-846. 1 1 1 1 Michael Kuo , Robert Keefe, PhD , Linhong Li, PhD , Angelia Viley , Mary 1 2 3 Loveras , Brian Mulhern , Melanie Hartsough , Claudio Dansky Ullmann, 4 1 P189 MD , Dhana Chinnasamy 1 2 Tumor Treating Fields (TTFields) induce immunogenic cell death MaxCyte Inc, Gaithersburg, MD, United States; Scilucent, Washington resulting in enhanced antitumor efficacy when combined with DC, United States; Hartsough Nonclinical Consulting, Gaithersburg, MD, anti-PD-1 therapy United States; MaxCyte, Cambridge, MA, United States 1 1 1 1 Noa Kaynan, PhD , Tali Voloshin , Shiri Davidi , Yaara Porat , Anna Correspondence: Dhana Chinnasamy (dhanac@maxcyte.com) 1 1 1 Shteingauz , Mijal Munster , Rosa Schnaiderman , Catherine Tempel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P190 1 1 1 1 1 Brami , Yaniv Alon , Einav Zeevi , Karnit Gotlib , Roni Blat , Orni Tal 1 1 1 1 Yitzhaki , Shay Cahal , Aviran Itzhaki , Eilon Kirson , Uri Weinberg, MD Background 1 2 1 1 PhD , Adrian Kinzel , Yoram Palti , Moshe Giladi MaxCyte has developed a rapid and potent cell therapy that uti- 1 2 Novocure Ltd., Haifa, Israel; Novocure GmbH, Munich, Germany lizes mRNA in transient Flow Electroporation (FEP) to produce Correspondence: Moshe Giladi (mgiladi@novocure.com) gene-modified cell products, termed CARMA™. This proprietary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P189 CARMA platform modifies peripheral blood mononuclear cells (PBMC) from apheresis to generate a cryopreserved drug product Background in a single-day manufacturing process using the cGMP-compliant, Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic closed MaxCyte GT® Transfection System, dramatically reducing treatment modality delivered via noninvasive application of low-intensity, the labor, facilities investment, and cost of raw materials typically intermediate-frequency, alternating electric fields. In this study we evalu- required for such products. The CARMA one-day manufacturing ated whether TTFields-induced cell death can be immunogenic and process using cGMP grade mRNA has the potential to therefore suitable for combination with anti-PD-1 therapy. revolutionize cell therapy strategies by significantly reducing the Methods wait time for patients receiving treatment. Cancer cells were treated with TTFields using the inovitro(TM) sys- Methods tem. Immunogenic cell death (ICD) was characterized by the expos- We report here the implementation of the CARMA platform to ure of calreticulin on the cell surface, secretion of ATP, and release of manufacture MCY-M11, a PBMC cell therapy product expressing HMGB1. For detection of ER stress, phosphorylation of eIF2α was an anti-mesothelin chimeric antigen receptor (Meso-CAR) de- assessed. TTFields effect on autophagy was evaluated using electron signed to target mesothelin-expressing solid malignancies. MCY- microscopy, and evaluation of LC3. Bone marrow derived dendritic M11 expresses the Meso-CAR in all cells in the PBMC preparation, cells (DCs) were co-incubated with TTFields treated cells and phago- which are processed and cryopreserved without the need for cytosis by DCs and DCs maturation were evaluated. The combination prior activation or selective expansion. MCY-M11 for clinical appli- of TTFields and anti-PD-1 was evaluated in short duration treatment cation is manufactured under the appropriate cGMP quality sys- protocol in orthotopic lung cancer model and long duration treat- tems and controls by MaxCyte at HCATS, a Contract Development ment protocol in subcutaneous colon cancer model. Analysis of infil- Manufacturing Organization (CDMO). Manufacturing release speci- trating cells was performed using flow cytometry. fications are preliminarily assigned, with multiple For Information Results Only (FIO) data points being accumulated during clinical produc- We demonstrate that cancer cells that die during TTFields application tion, while sufficient clinical data is being generated to establish exhibit ER stress leading to calreticulin translocation to the cell meaningful release criteria. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 104 of 272 Results lymph nodes. Given the plasticity of Th17 and Treg cells, we A total of 20 CARMA product development and engineering runs assessed FoxP3 expression within the cell product 10 days post were performed during the technology transfer campaign, with transfer and found that the frequency of FoxP3+ transferred cells analytical test methods and supply chain established. The viable was significantly heightened through IL-6 blockade. cell yield from pre-FEP to post-FEP samples averaged around Conclusions 92%. Meso-CAR expression in T and NK cell subsets in MCY-M11 IL-6 induced by Th17 cell therapy promotes an inflammatory ranged from 42-83% (average 73%) and 28-75% (average 59%), over regulatory phenotype in vivo permitting durable memory respectively. The anti-tumor bioactivity and target specificity of against tumors. The expansion of tumor-specific regulatory cells MCY-M11 was successfully established in vitro by demonstrating from the transferred product is enhanced in the absence of IL-6 antigen-specific cytotoxicity and inflammatory cytokine release in signaling. This work implies that the universal strategy of IL-6 co-culture assays with various mesothelin-expressing human inhibition for cytokine release syndrome may come at the ex- tumor cell lines. Increased survival and efficacy were also demon- pense of long-term efficacy for cell therapy approaches. strated in vivo using a human mesothelin expressing ovarian syn- Ethics Approval geneic mouse tumor model. All animal studies were approved by MUSC's IACUC committee, ap- Conclusions proval number 0488. The CARMA one-day manufacturing process using cGMP grade mRNA has the potential to revolutionize cell therapy strategies by significantly reducing the wait time for patients receiving treat- P192 ment. MCY-M11 is currently being tested in a first-in-human clin- Anti-HLA-G antigen receptor T-cells exhibit potent anti-tumor ical trial for advanced epithelial ovarian cancer and peritoneal effects against human solid tumors mesothelioma (ClinicalTrials.gov Identifier: NCT03608618). Alan Epstein, MD, PhD, Aida Kouhi, Aida Kouhi Trial Registration University of Southern California, Los Angeles, CA, United States NCT03608618 Correspondence: Alan Epstein (aepstein@usc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P192 P191 Background IL-6 fuels durable memory for Th17-mediated responses to tumors 1 1 1 HLA-G is highly expressed on human placenta during pregnancy and has Hannah Knochelmann, BS , Connor Dwyer, PhD , Aubrey Smith, BS , 1 1 1 been found to suppress the NK response to cells that lose their HLA and/ Megan Wyatt, MS , Guillermo Rangel RIvera , Jacob Bowers , Michelle 1 2 3 or beta2-microglobulin expression. [1-2]. In addition, except for pregnancy, Nelson, PhD , Gregory Lesinski, PhD, MPH , Zihai Li, MD, PhD , Mark 1 1 HLA-G is rarely expressed in normal adult tissues. Moreover, roughly 50% Rubinstein, PhD , Chrystal Paulos, PhD of human solid tumors lose their HLA expression to avoid detection by Medical University of South Carolina, Charleston, SC, United States; 2 3 the human immune system. [3] HLA-G is therefore an outstanding target Emory University, Atlanta, GA, United States; Ohio State University, for CAR T-cells since, like the placenta, it is up-regulated in HLA-negative Columbus, OH, United States tumors to suppress NK destruction. [4] We have successfully generated Correspondence: Hannah Knochelmann (knochelm@musc.edu) anti-HLA-G CAR T-cells to treat solid tumors that express HLA-G. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P191 Methods An anti-HLA-G CAR construct was generated by fusing anti HLA-G Background scFv to a second generation CAR containing the CD8α leader se- Accessibility of T cell transfer therapies for most patients is hin- quence, 4-1BB co-stimulation sequence, and CD3ζ signaling domain. dered by cost and time required for product development. Our The CAR vector was then fused with a lentivirus vector in-frame with lab has shown that shortening ex vivo expansion of Th17 cells the CAR backbone, and was used to transduce primary human CD3 licenses a proinflammatory cell product which induces cytokine positive T-cells. After transduction, expanded CAR-T cells were char- stormwithhighlevelsofsystemicIL-6intumor-bearing hosts. acterized for their ability to bind HLA-G antigen and HLA-G positive Despite potential toxicity, briefly expanded Th17 cells eradicate SKOV-3 cells (human ovarian cancer model) using flow cytometry. large established tumors in low doses and generate durable Results memory against tumor rechallenge, suggesting a therapeutic Expanded CAR-T cells were able to bind successfully both the benefit to the inflammatory state. Prior reports show that IL-6 HLA-G antigen and SKOV-3 cells in vitro. Expanded CAR-T cells promotes functional CD4+ T cell memory formation. Given that were then co-cultured with SKOV3-Luc cells and studied for their IL-6 is blocked clinically to manage cytokine release syndrome, epitope-driven cytotoxicity. Anti HLA-G CAR T-cells displayed we addressed the physiologic impact of IL-6 on efficacy and dur- dose-dependent cytotoxicity when co-cultured with tumor cells. ability of Th17 cell therapy. We have recently developed an in vivo model of ovarian cancer Methods that can be used for testing the efficacy of our CAR-T cells. In Th17 cells were expanded ex vivo using the TRP-1 transgenic this model, NSG mice are injected with 2 million SKOV3-Luc cells mouse model in which CD4+ T cells express a TCR that recog- intraperitoneally (ip). Seven-10 days after injection, tumors are nizes tyrosinase-related protein 1 on melanoma. Naïve CD4+ T visible when observed by bioluminescence imaging, at which cells were polarized to the Th17 phenotype and infused into time the treatment group will receive an ip injection of anti HLA- mice with B16F10 melanoma after a nonmyeloablative total G CAR-T cells. Since ovarian cancer rapidly metastasizes to the body irradiation (5 Gy) preparative regimen. Serum cytokine peritoneum, the aforementioned model should provide relevant levels were obtained by multiplex array and IL-6 signaling was clinical data that can be translated to patients, and like inhibited with antibodies targeting the IL-6R and neutralizing IL- hematopoietic cancers, will present antigen quickly after injection 6cytokine. of CAR T-cell to keep them stimulated and functional. Results Conclusions Acute IL-6 blockade post Th17 cell transfer did not impact the We are currently testing the efficacy of anti-HLA-G CAR-T cells primary response against melanoma nor the engraftment of Th17 in vivo using this ip model, and plan to show that HLA-G as a cells. However, blocking IL-6 abrogated long-term responses in- pan tumor target will provide selective and specific cell based creasing the frequency of tumor relapse upon secondary chal- therapy which may in the near future be clinically relevant for lenge and reduced survival. Mechanistically, IL-6 blockade chemotherapy resistant ovarian cancer and other tumors. reduced phosphorylation of STAT3 in transferred T cells associat- ing with diminished Bcl-2 expression. The CD4+ compartment Acknowledgements was reshaped by IL-6 blockade via promoting a greater frequency This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. of FoxP3+ Treg cells in the peripheral blood, tumor and draining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 105 of 272 References Immetacyte is assessing this approach in our current Phase I/II clinical 1. Apps R, Gardner L, Moffett A. A critical look at HLA-G. Trends Immunol. trial of TIL in ovarian cancer patients (EudraCT–2019-000106-30). 2008; 29:313–321. 2. Jurisicova A, Casper RF, MacLusky NJ, Mills GB, Librach CL. HLA-G expres- Acknowledgements sion during preimplantation human embryo development. Proc. Natl. This research was supported by IUK projects 133299 and 104468 Acad. Sci. U. S. A. 1996; 93:161–5. Ethics Approval 3. de Kruijf EM et al. HLA-E and HLA-G expression in classical HLA class I- This study was approved by the South Central Research Ethics Committee : negative tumors is of prognostic value for clinical outcome of early 19/SC/0355 breast cancer patients. J. Immunol. 2010; 185:7452–9. 4. Lin, A. & Yan, W.-H. Human Leukocyte Antigen-G (HLA-G) Expression in P194 Cancers: Roles in Immune Evasion, Metastasis and Target for Therapy. Use of stimulatory cells in conjunction with IL-12 and IL-18 Mol. Med. 2015; 21:782–791. augments NK cell expansion and transduction, drives a Ethics Approval memory phenotype, and improves in vitro and in vivo CAR This study was approved by the IRB of the University of Southern California NK activity protocol #HS-16-00029 on 2-29-16. Anmol Vohra, MS, Katherine Jamboretz, MS, Sasha Lazetic, Denise Gonzalez, Daofeng Liu, PhD, Ivan Chan, PhD, James Trager, PhD P193 Nkarta Inc, South San Francisco, CA, United States CoStAR (Costimulatory Antigen Receptor) enhancement of tumour Correspondence: James Trager (jtrager@nkartatx.com) infiltrating lymphocyte therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P194 Gray Kueberuwa, PhD, John Bridgeman, PhD, Martina Sykorova, Milena Kalaitsidou, Michelle Le Brocq, Robert Hawkins Background Immetacyte, Manchester, Manchester County, United Kingdom NK cells have been expanded on K562 stimulatory cells express- Correspondence: Robert Hawkins (r.hawkins@immetacyte.com) ing membrane-bound (mb) IL-15 and 41BBL for clinical use, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P193 can be genetically modified to express activating chimeric recep- tors [1,2,3]. Engineered NK cells targeting CD19 or ligands of Background NKG2D show in vitro and in vivo cytotoxicity against relevant The efficacy of TIL therapy is limited in some patients due to the tumor targets that can overcome endogenous resistance to NK failure of the cells to respond to tumour sufficiently or persist cells. NK cells activated in the presence of IL-12, IL-15 and IL-18 long enough to have a necessary anti-tumour effect. We have ad- develop cytokine induced memory-like phenotype and function; dressed this issue by developing Co-stimulatory receptors (Co- these cells have shown clinical promise [4]. Here we describe NK StARs) that provide enhanced signaling to tumour-specific T-cells cell function and phenotype achieved by combining the robust upon encountering tumour associated antigens. driven by K562-mbIL15-41BBL with the induction of a cytokine- Since tumour reactivity is determined by natural TCRs that have induced memory phenotype achieved after exposure to IL-12 and undergone thymic selection, this approach does not bear with it the IL-18. risks of other therapies targeting tumour antigens expressed on the Methods cell surface Healthy donor PBMC NK were expanded on K562-mbIL15-41BBL Methods stimulatory cells with IL-2 alone or with IL-2 plus IL-12 and IL-18 In order to identify optimal signalling domain for Co-StAR mole- (12-18). We compared NK cell expansion, cytokine secretion, cyto- cules, several iterations of our prototype receptor were synthe- toxicity against tumor lines at various time points, and persist- sised. The ability of each to enhance T-cell activation, ence in culture over 4 weeks. The expanded NK were transduced proliferation, secretion of cytokines and increase resistance to with CD19 and NKG2D CAR constructs, and the resulting cells apoptosis were assessed. evaluated for CAR expression, cytotoxicity and in vivo efficacy To explore if this approach has the potential of wide applicabil- against relevant cell lines. ity, we went on to assess targeting of two additional ovarian Results cancer tumour associated antigens. To achieve this, the antigen Addition of 12-18 to the K562-mbIL15-41BBL stimulatory cells im- binding moiety was exchanged and signaling domain kept proves NK expansion 2-3 fold [If we have a p value, it’s better to constant. give it:<br>‘…significantly improves NK expansion 2-3 fold (p<x) Results relative to that…’][Will put together later for the poster]relative We show that colorectal cancer specific Co-StAR significantly en- to that achieved using the stimulatory cell line alone, while NK hances the number of T-cells expressing IL2, TNFα,41BB,CD107a cell cytotoxicity is unchanged. IFNγ and TNFα production and and bcl-xL by factors ranging from 2-4 fold in model systems. transduction efficiency are also improved in this setting. Over 3 This shows an increase in activation, effector function and resist- weeks of culture following brief exposure to 12-18, NK cell ance to apoptosis. We also identified an optimal signaling do- phenotype changes, with an increased percentage of CD62L+ main that caused the greatest magnitude of enhancement for and NKG2C+ cells, and increased NKG2C expression per NK cell. the above factors. While the NKG2D-CAR driven cytotoxic activity is unchanged by Observations of Co-StAR enhancement were mirrored in model 12-18 at 14 days post-exposure, cytotoxic activity increases in systems for ovarian cancer, targeting two separate ovarian cancer these cells by day 21. In addition, this improved cytotoxicity at tumour associated antigens. day 21 is reflected by improved CD19-CAR driven in vivo activity In addition, stimulation assays showed that Co-StAR with optimal sig- against the CD19+ tumor target [Raji or Nalm6?]Nalm6 [Oops, naling domain increased T-cell proliferation over 3 weeks in compari- Nalm6, thx.] with an increased presence of circulating NK cells son to prototype Co-StAR, Co-StAR that binds an irrelevant target, or over 4 weeks in the mice. indeed, mock transduced T-cells. Conclusions Conclusions The data demonstrates that the addition of IL-12 and IL-18 to Our optimal Co-StAR provides a means to effectively deliver “signal K562-mbIL15-41BBL stimulatory cells during NK expansion main- 2” to T-cells. Enhancing activation, effector functions and resistance tains in vitro cytotoxicity and improves expansion, transduction, to apoptosis upon contact with target tumour cells. persistence, and in vivo efficacy. Further, IL-12 and IL-18 drive de- Since T-cell activation primarily requires “signal 1”, application to of velopment of a memory phenotype and more sustained NK cell Co-StAR to enhance TIL therapy, which works through natural, thymi- function over the course of several weeks. This activation system cally selected TCRs, provides a means to increase the activity of may allow for development of more robust and potent engi- tumour-reactive TIL without risking severe off-tumour side effects. neered NK cells for clinical use. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 106 of 272 References Conclusions 1. Lapteva N, Durett AG, Sun J, Rollins LA, Huye LL, Fang J, Dandekar V, These promising results infer that adoptive transfer of transgenic T Mei Z, Jackson K, Vera J, Ando J, Ngo MC, Coustan-Smith E, Cam- cells can offer an effective strategy to not only prevent further tumor pana D, Szmania S, Garg T, Moreno-Bost A, Vanrhee F, Gee AP, Roo- growth as rapamycin therapy does, but also to treat and even elimin- ney CM. Large-scale ex vivo expansion and characterization of ate arising tumors. This strategy might offer a cure for patients with natural killer cells for clinical applications. Cytotherapy. LAM, a disease that hits women in the prime of their lives. 2012;14(9):1131-1143 2. Chihaya I, Iwamoto S, Campana D. Genetic modification of primary Acknowledgements natural killer cells overcomes inhibitory signals and induces specific Studies supported by a DoD Tuberous Sclerosis Complex Research Program killing of leukemic cells. Blood. 2005; 106:376-383. Clinical Translational Research Award to CLP. 3. Yang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Ethics Approval Chimeric Receptor with NKG2D Specificity Enhances Natural Killer All animal experiments were approved by the Animal Care and Use Cell Activation and Killing of Tumor Cells. Cancer Res. Committee of Northwestern University and followed the institutional 2013;73(6):1777-1786 guidelines; protocol number IS00008259. 4. Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, Neal CC, Yu L, Oh ST, Lee YS, Mulder A, Claas F, Cooper MA, Fehniger TA. Cytokine-induced P196 memory-like natural killer cells exhibit enhanced responses against mye- The first step toward the universal cell therapy: Simultaneous loid leukemia. Sci Trans Med. 2016;8(357): 357ra123 removal of HLAs (Human leukocyte antigens) using CRISPR- Ethics Approval mediated quadruple genome editing in allogeneic T cells Animal studies were conducted and approved by the Explora IACUC Jeewon Lee, Ph D, Munkyung Kim, Joong Hyuk Sheen, Jihye Ryu, Yu committee. Young Kim, Okjae Lim MOGAM Institute for Biomedical Research, Yongin-si, Gyeonggi-do, Republic of Korea P195 Correspondence: Okjae Lim (blubelle@mogam.re.kr) Loss of function of the TSC1-TSC2 complex renders tumors eligible Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P196 for GD3 CART therapy 1 1 1 Ancy Thomas , Saurav Sumughan , Zhussipbek Mukhatayev , Emilia 1 1 2 2 Background Dellacecca , Nicola Lancki , Levi Barse , Jesus Zamora-Pineda , Suhail 2 2 1 2 Chimeric antigen receptor (CAR) T cell therapy is the revolution- Akhtar , Maria Picken , Denise Scholtens , Daniel Dilling , Richard 3 1 ary treatment of choice for hematologic malignancies. Currently Junghans, PhD, MD , Caroline Le Poole 1 2 approved CAR T therapies require patients’ own immune cells, Northwestern University, Chicago, IL, United States; Loyola University, and this autologous T cell manufacturing process involves certain Maywood, IL, United States; Boston University, Boston, MA, United limitations primarily derived from the nature of individualized States therapy. Thus, engineering allogeneic donor cells to evade host Correspondence: Caroline Le Poole immune rejection is required for a broader clinical application of (caroline.lepoole@northwestern.edu) the therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P195 Methods In this study, we attempted to inhibit expression of both HLA I Background and II through the CRISPR/Cas9 gene editing system to reduce Benign tumors can arise from bi-allelic mutations in a single gene. In tu- allo-reactive immune rejection response. First, we screened 60 berous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), gRNAs targeting B2M and 60 gRNAs each targeting alpha chains tumors do not acquire additional mutations, and patients are not eli- of HLA-II molecules (DP, DQ and DR, respectively) to find gRNA gible for therapeutics that rely on neoantigen formation. However, the sequences efficiently ablate expression of HLA molecules on T affected gene is responsible for several predictable phenotypic cell surface. Next, we investigated whether the absence of HLA-I/ changes. As mTOR hyperactivity resulting from mutations in TSC1 or II expression in donor T cells could alleviate immune response TSC2 is associated with overexpression of some melanoma-associated from allogeneic responders using in vitro mixed lymphocyte reac- antigens, de novo expression of ganglioside D3 expression may render tion (MLR) assays. the resulting, benign tumors eligible for immunotherapy. Results Methods We have identified gRNA sequences highly efficient in targeting We probed the expression of GD3 in human TSC lesions of the lungs, B2M and alpha chains of HLA-II molecules without carrying off- kidneys, skin and brain by immunostaining and monitored anti-GD3 target effects. Selected gRNA sequences for HLA-II ablation cov- titers in serum by ELISA. Infiltration by NK cells and NKT was mea- ered the vast majority of each HLA-II alpha chain allele. HLA-I/II sured to look for natural responses to the cell surface antigen. We double negative T cells generated by simultaneous quadruple next isolated tumors cells from TSC2 heterozygote mice and con- genome editing with the selected gRNAs maintained their pheno- firmed loss of heterozygosity by genotyping before challenging types and cytotoxicity upon TCR stimulations compared to the groups of 10 SCID/beige mice in 2 repeat experiments, and subject- control cells treated with non-target gRNA. Furthermore, the MLR ing them to adoptive transfer by GD3-CART cells and measured assays showed that IFN- and TNF-α production in allo-responder tumor sizes over time. Similarly we treated groups of 8 ageing TSC T cells was significantly decreased in the absence of donor HLA-I mice >16 months of age by adoptive GD3 CART-cell transfer, and alone and was further diminished in response to HLA-I/II double measured surface tumor growth on internal organs. negative donor T cells compared with the control cells, implicat- Results ing prolonged survival of the adoptively transferred immune cells. We found consistent overexpression of GD3 in tissues from TSC pa- Conclusions tients compared to healthy controls. GD3 overexpression was not ac- In conclusion, we have identified novel gRNA sequences ablat- companied by an influx of NK(T) cells, and anti-GD3 titers were ing expression of HLA molecules on donor T cell surfaces to reduced rather than elevated in patients, supporting the concept dramatically reduce donor-derived allo-responses, establishing that slow growing tumors in TSC patients are not immunogenic. an essential cornerstone towardsthe universalTcell therapy. However, CART cells responsive to GD3, supplemented by IL-2, medi- Ethics Approval ated prolonged and significant anti-tumor responses in both immu- Human PBMCs were obtained from healthy volunteers by leukapher- nodeficient and immune competent hosts. The majority of TSC2 esis from the Samsung Medical Center (SMC) under IRB approval heterozygote mice treated by CAR T cells displayed no tumors at (SMC IRB no.2018-01-089). end point, versus all mice treated with untransduced T cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 107 of 272 P197 we developed a novel modular universal CAR platform termed Uni- The development of an autologous neoantigen specific T cell CAR. UniCAR T-cells are exclusively activated in the presence of a tar- product from peripheral blood, NEO-PTC-01, through the ex-vivo get module (TM), which establishes the cross-link between antigen- induction protocol, NEO-STIM™ specific cancer cells and UniCAR T-cells in an individualized time- 1 1 1 Divya Lenkala, MS , Marit Van Buuren, PhD , Brian McCarthy , Jessica and target-dependent manner. The carbohydrate antigen sialyl-Tn 1 1 1 1 Kohler, PhD , Michael Nelson , Flavian Brown , Yvonne Ware, MS , (STn) is a particularly interesting target due to its expression in sev- 1 1 Yuting Huang, MS , Janani Sridar , Yusuf Nasrullah, MS, United eral types of cancer and absence in normal healthy tissues. Given the 1 1 2 Kingdom , Dewi Harjanto , Joost Van Den Berg, PharmD , Matthew small size of such TMs, they are rapidly eliminated and thus, possible 3 1 Goldstein, MD, PhD , Richard Gaynor, MD side effects and activation of UniCAR T-cells can be easily controlled 1 2 Neon Therapeutics, Cambridge, MA, United States; Netherlands Cancer by TM dosing. In late phases of treatment, TMs with extended half- Institute, Amsterdam, Netherlands; Tango Therapeutics, Cambridge, MA, life may play an important role by improving the eradication of re- United States sidual tumor cells. Correspondence: Marit Van Buuren Methods (mvanbuuren@neontherapeutics.com) In this work, a novel longer-lasting TM against STn was developed, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P197 characterized and compared to the previously developed short-lived anti-STn TM [1]. Short-lived TMs are composed of a tumor-specific Background binding moiety fused to the La peptide epitope (E5B9) which is rec- Neoantigens are tumor-specific antigens that have been shown to be ognized by UniCAR T-cells. In extended half-life TMs, these two com- important in the anti-tumor immune response. These antigens are ponents are fused via an Fc domain derived from the human IgG4 not subject to central immune tolerance and are therefore potentially molecule. Functional and pharmacokinetic properties were assessed more immunogenic than tumor-associated antigens. The goal of our using in vitro and in vivo assays. studies is to generate neoantigen specific T cell responses and per- Results form detailed characterization of the induced T cell responses to- The developed anti-STn IgG4-based TM efficiently activates and redi- wards these neoantigen targets to assess the applicability of the rects UniCAR T-cells to STn-expressing tumors in a highly efficient approach for adoptive cell therapy. target-specific and target-dependent manner, promoting the secre- Methods tion of pro-inflammatory cytokines, tumor cell lysis of breast and Patient-specific neoantigens were predicted using our RECON® bio- bladder cancer cells in vitro and of breast cancer cells in experimen- informatics platform, and the predicted high-quality neoantigens tal mice. A comparable or increased killing efficiency was obtained at were utilized in our proprietary ex-vivo stimulation protocol, NEO- a lower concentration range in comparison to the results obtained STIM to assess immunogenicity. NEO-STIM is used to prime, activate for the anti-STn scFv-based TM. Additionally, PET studies demon- and expand memory and de novo T cell responses from both the strate the specific enrichment of the anti-STn IgG4-based TM at the CD4+ as well as the CD8+ compartment. In-depth analysis was per- tumor site presenting a prolonged serum half-life compared to the formed to characterize the specificity, functionality (cytokine produc- scFv short-lived TM. tion and cytolytic capacity) and diversity of the induced T cell Conclusions responses through high throughput flow cytometric analysis. Taken together, these data demonstrate the effective and potential Results application of this CAR T-cell-derived modular system to target STn Here we present the successful induction of memory and de novo in different types of cancer using different TM formats. The use and CD8+ and CD4+ T cell responses in peripheral blood mononuclear combination of such molecules with different formats and half-lives cells isolated by leukapheresis from five melanoma patients using provides highly promising and customized tools for retargeting of NEO-STIM. We then extensively characterized these T cell responses UniCAR T-cells in a flexible, individualized and safe manner at differ- and show that these responses are functional, specific and have cyto- ent stages of treatment. lytic capacity. Conclusions Reference NEO-STIM is a novel platform to understand in detail the immuno- 1. Loureiro L R, Feldmann A, Bergmann R, Koristka S, Berndt N, Arndt C, genic potential of high-quality neoantigen-targets. Moreover, this Pietzsch J, Novo C, Videira P, Bachmann M. Development of a novel platform can be utilized to generate T cell products from peripheral target module redirecting UniCAR T cells to Sialyl Tn-expressing tumor blood for adoptive cell therapy for patients with a variety of solid cells. Blood Cancer J. 2018; 8(9): 81. tumors. Ethics Approval Ethics Approval All animal activities and procedures were performed in accordance with the The samples for the study were collected under ClinicalTrials.gov: protocols approved by the Institutional Review Board at Semmelweis NCT02897765 and N16NEON protocol University - Budapest, approval number PE/EA/50-2/2019. P198 P199 Short-lived and extended half-life target modules for redirecting Generation of functionally and phenotypically mature, allogeneic UniCAR T-cells against sialyl-Tn expressing cancer cells natural killer cells from human induced pluripotent stem cells 1 1 1 Liliana Loureiro, PhD , Anja Feldmann, PhD , Ralf Bergmann , Stefanie under chemically-defined, feeder- and serum- free culture 1 1 2 2 Koristka , Nicole Berndt , Nikolett Hegedüs , Domokos Máthé , Paula conditions 3 1 1 Videira , Michael Bachmann , Claudia Arndt Kyle Lupo, BS, Andrea Chambers, MS, Sandro Matosevic, PhD Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; Purdue University, Lafayette, IN, United States 2 3 Semmelweis University, Budapest, Hungary; Faculdade de Ciências e Correspondence: Sandro Matosevic (sandro@purdue.edu) Tecnologia/UNL, Caparica, Portugal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P199 Correspondence: Liliana Loureiro (l.loureiro@hzdr.de) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P198 Background While targeted immunotherapy with engineered natural killer (NK) Background cells has emerged as a promising approach for the treatment of solid The development of chimeric antigen receptors (CARs) has rapidly tumors, challenges in sourcing, processing, and genetically modifying emerged as a promising approach in cancer immunotherapy. None- blood-derived NK cells limit the potential for developing life-saving theless, drawbacks associated with CAR T-cell therapies include on- treatments for cancer patients. As an alternative, the use of induced target/off-tumor effects and cytokine release syndrome. Aiming an pluripotent stem cells (iPSCs) offers a promising approach to over- increased clinical safety while preserving the efficacy of such therapy, coming existing challenges faced when engineering NK cell-based Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 108 of 272 immunotherapies. However, approaches for generating NK cells from antigen-specificity, higher CD8:CD4 ratio and ability to persist long- iPSCs described so far have several shortcomings: they utilize sera or term [3]. Based on these differences, we hypothesized that MILsTM feeder layers to adapt iPSCs or culture hematopoietic progenitors, would provide a more robust platform for CAR-T therapy compared take months to complete, and rely on individualized, and thus highly to PBLs. We have previously shown that CAR-modified MILsTM (CAR- variable, iPSC reprogramming protocols, limiting their utility. MILsTM) demonstrate superior killing of tumor target cells in vitro Methods compared to CAR-T cells generated from patient-matched PBLs (CAR- We have generated NK cells from iPS cells using a novel feeder-free PBLs) [4]. In this study, we compared, at the single cell level, func- differentiation protocol starting from either centrally-validated and tionality of patient-matched CAR-MILsTM and CAR-PBLs following banked iPSC lines or iPSCs reprogrammed from donor fibroblasts. antigen-specific in vitro stimulation. Our protocol utilizes a two-step, entirely feeder-free procedure in- Methods volving hematopoietic progenitor generation followed by NK differ- CAR-MILsTM and CAR-PBLs engineered to express a BCMA-specific, 4- entiation. These differentiated NK cells have been characterized for 1BB/CD3z-signaling CAR were produced using cryopreserved lympho- inhibitory and activating marker expression, IFN-γ production, de- cytes from the bone marrow and blood of six patients with multiple mye- granulation, and cytotoxicity against a number of solid tumor targets, loma. CD4 and CD8 T cells isolated from the CAR-MILsTM and CAR-PBLs including primary patient-derived glioblastoma cells. Moreover, these products were stimulated with K562 cells transduced with either BCMA cells were expanded in culture and manipulated to generate a cyto- (K562-BCMA) or nerve growth factor receptor (K562-NGFR) at a ratio of toxic infusible cell therapy product. 1:2 for 20 hrs. After 20 hrs of co-culture, T cells were enriched and loaded Results into IsoCode chips containing ~12,000 microchambers pre-patterned iPS cells were differentiated into hematopoietic progenitor cells, with a 32-plex antibody array. Protein secretion from 1000-2000 single T yielding CD34+/CD45+ and CD34+/CD43+ cell populations at yields cells per product was detected by a fluorescence ELISA-based assay and consistent with results described in literature using feeder-based pro- single cell polyfunctional profiles analyzed using IsoPeak (IsoPlexis). tocols [1]. Following four weeks of NK cell differentiation, cells Results showed high expression of several NK cell maturation markers as CD4 and CD8 T cells from both CAR-MILsTM and CAR-PBLs demon- well as inhibitory and activating receptors (CD56+/CD3-, NKG2D, strated an antigen-specific increase in polyfunctionality (secretion of 2+ NKp30, NKp44, NKp46, DNAM-1, CD16, CD94/NKG2A, and CD158b). cytokines per cell) and polyfunctional strength index (PSI) in response iPSC-NK cells derived using our protocol were also similar to blood- to BCMA stimulation compared to NGFR control. When compared to derived NK cells in morphology, expansion rate, and functionality (in CAR-PBLs, CAR-MILsTM demonstrated increased polyfunctionality and terms of cytotoxicity and degranulation potential). By using centrally- increased PSI in both CD4 and CD8 T cells. The enhanced PSI in CAR- validated iPSC lines, we further demonstrate our ability of avoiding MILsTM was predominated by effector, stimulatory and chemoattrac- donor-specific reprogramming protocols. tive proteins associated with antitumor activity including Granzyme B, Conclusions IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and en- We developed a new protocol for the generation of NK cells from hanced secretion of these same proteins was reported to be associated iPSCs that is entirely feeder and serum-free and can be extended to with improved clinical responses in patients with Non-Hodgkin lymph- the use of validated iPSC lines avoiding donor and reprogramming oma treated with CD19-specific CAR-T therapy [5]. variability. iPSC-derived NK cells using our protocol exhibit character- Conclusions istics of mature blood-derived NK cells and powerful cytotoxicity Based on these data and the inherent antitumor properties of against solid tumor targets. Additionally, these cells offer the advan- MILsTM, we speculate that CAR-MILsTM would be more potent tage of increased expansion rates, improved ease of transfection and effective than currently approved CAR-T products derived while in the iPSC state, and are free of contaminating T-cells associ- from PBLs. ated with GvHD risk, overcoming many limitations of existing NK cell based immunotherapies. References 1. Borrello I and Noonan KA, Marrow-Infiltrating Lymphocytes – Role in Biol- Reference ogy and Cancer Therapy. Front Immunol. 2016 March 30; 7(112) 1. Bock A, Knorr D, and Kaufman D. Development, Expansion, and In Vivo 2. Noonan K.A., Huff C.A., Davis J., Lemas M. V., Fiorino S., Bitzan J., Ferguson Monitoring of Human NK Cells from Human Embryonic Stem Cells A., Emerling A., … Borrello I. Adoptive transfer of activated marrow- (hESCs) and Induced Pluripotent Stem Cells (iPSCs). Journal of Visualized infiltrating lymphocytes induces measurable antitumor immunity in the Experiments : JoVE 74 (2013): 50337. bone marrow in multiple myeloma. Sci. Transl. Med. 2015; 7: 288ra78. 3. Noonan KA, Rudraraju L, Hoyos V, Lutz E and Borrello I. Persistence of Non Gene-Modified Adoptively Transferred Marrow Infiltrating Lympho- P200 cytes (MILs) More Than Five Years Post Transfer. Blood 2016 128:4552. CART-engineered Marrow-infiltrating Lymphocytes (MILsTM) are 4. Lutz ER, Hoyos V, Rudraraju L, DeOliveira E, Jana S, Weiss I, Borrello IM, more polyfunctional than their matched peripheral blood Noonan K. Marrow-infiltrating Lymphocytes (MILs) provide a robust plat- counterparts form for CAR-T cell therapy. Blood 2018 132:3337. 1 1 1 Eric Lutz, PhD , Lakshmi Rudraraju, MS , Elizabeth DeOliveira , Srikanta 5. Rossi J, Paczkowski P, Shen Y, … Bot A. Preinfusion polyfunctional anti- 1 2 2 3 Jana , Jing Zhou, MD, PhD , Sean Mackay, MBA , Ivan Borrello, MD , CD19 chimeric antigen receptor T cells are associated with clinical out- Kimberly Noonan, PhD comes in NHL. Blood 2018 132(8):804-814. 1 2 WindMIL Therapeutics, Baltimore, MD, United States; Isoplexis, Ethics Approval Branford, CT, United States; Johns Hopkins University, Baltimore, MD, The study was approved by the Johns Hopkins University IRB. United States Correspondence: Eric Lutz (lutz@windmiltherapeutics.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P200 P201 Comparison of phenotype and anti-tumor profile of CD19-CAR-T Background cells generated from either umbilical cord blood- or peripheral WindMIL Therapeutics is developing Marrow-infiltrating Lymphocytes blood-derived T lymphocytes 1 1 1 (MILsTM), a novel form of adoptive T cell therapy composed of bone Cristina Maccalli, PhD , Dhanya Kizhakayil, PhD , Shilpa Ravindran, BSc , 1 1 1 marrow-derived, patient-autologous, polyclonal CD4 and CD8 T cells Saad Rasool , Rebecca Mathew , Valentina Mattei , Monica Casucci, 2 1 1 1 [1]. Genetically unmodified MILsTM have demonstrated antitumor ac- PhD , Sara Deola, MD, PhD , Chiara Cugno, MD , Damien Chaussabel , 1 1 tivity in patients with multiple myeloma [2] and are being developed Sara Tomei, PhD , Christof von Kalle , 1 2 for several other tumor types. Distinguishing features of T cells from Sidra Medicine, Doha, Qatar; San Raffaele Scientific Institute, Milan, Italy bone marrow compared to T cells from peripheral blood lympho- Correspondence: Cristina Maccalli (cmaccalli@sidra.org) cytes (PBLs) include their memory phenotype, inherent tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P201 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 109 of 272 Background Background T lymphocytes expressing antigen-specific chimeric receptors (CARs) Natural killer (NK) cells are innate immune cells with a critical role in im- have been revealed as a powerful therapeutic approach for aggres- mune surveillance against cell transformation and tumor development. NK sive and refectory childhood and adult B cell malignancies. Umbilical cells express an array of unique activating and inhibitory receptors whose cord blood cells (UCB), with their unique capacity of broad leukocyte aggregate signaling determine activation of NK cell effector function. antigen (HLA)-matching, can represent an appealing starting material Adoptive transfer of NK cells has demonstrated the potential to induce an- for the generation of “off-the shelf” CAR-T cells to render this type of titumor responses in the clinic. Celularity has developed a platform for therapy accessible to a large number of cancer patients. generating cytotoxic NK cells from placental CD34+ cells (PNK cells) for Methods adoptive cancer immunotherapy. Although PNK cells demonstrate cytotox- CAR-T cells have been generated from either UCB (N=5, ALLCELLS, icity against diverse cancer cell types, their activating mechanisms are little USA) and peripheral blood lymphocytes (PBLs; N=2) from healthy do- characterized. In this study, we explore the contribution of specific signal- nors. In vitro enriched T cells have been transduced with CD19- ing pathways and upstream NK cell receptors involved in PNK cell cytotox- CD28z-CD3z and CD19-4-1BBz-CD3z encoding lentiviral vectors (LVs). icity against glioblastoma multiforme (GBM) cell targets. Deep phenotype characterization of these CAR-T cells has been per- Methods formed utilizing an in-house designed IF multiparametric (28 PNK cells were transcriptionally profiled using scRNAseq and qRT-PCR markers) panel. Functional assays have been performed to assess to identify candidate pathways regulating cytolytic function. Expression cytokine (IFN-gamma, IL2, IL-5 and IL-17), perforin and granzyme B of major receptors and intracellular signaling molecules were analyzed release (EliSpot or multicolor FluoroSpot) and cytotoxic activity (Del- using flow cytometry and western blot. PNK cell phenotype was com- fia assay) by CAR-T cells following the co-culture with CD19+ or pared to circulating NK cells. PNK cytotoxicity was evaluated against CD19- target cells. In addition, transcriptomic modular repertoire [1- GBM cell lines (LN-18 and U251) in xCELLigence platform and a de- 3] has been applied by parallel quantitative PCR using the high granulation assay using CD107a staining. The role of key signaling path- throughput BioMark HD platform to determine gene expression pro- ways driving PNK effector functions was analyzed in cytolysis assays file of CAR-T cells described above. using small molecule inhibitors of Src kinases, SYK, PLC-γ,PI3Kand Results MAP kinases, including JNK, p38 and ERK. Efficient LV transduction was achieved for UCB-T cells, although requir- Results ing higher MOI as compared to PBL (25 vs. 5; 66-80 vs. 70-88 % of PNK cells highly express genes mediating NK cell effector functions, includ- transduction, respectively). The frequency of CD4+ transduced T cells ing NCR1, NCR2, NCR3, KLRK1 and CD226. Flow cytometry demonstrated (45-59% of positive cells) was superior in UCB as compared to PBL (27- increased expression of NKp44 (99.7±0.2% vs. 69.3±5.2%), NKG2D (68.7± 36% of positive cells) while transduced CD8+ T cells were 18-20 and 7.9% vs. 44.6±5.8%) and GITR 99.7±0.2% vs. 15.0±3.2%) on PNK cells when 40-67%, respectively. CB-CAR-T cells were enriched of compared to circulating NK cells. PNK cells demonstrated strong cytolytic CD45RA+CCR7+CD27+CD62L+ T cells. These cells co-expressed ICOS activity against multiple GBM cell lines. While inhibitors of Src, SYK, PLC-γ, and 4-1BB but these molecules were not detectable on PBL-CAR-T cells. p38 and ERK did not modulate PNK cytotoxicity, inhibitors targeting JNK Markers associated with late differentiation/exhaustion of T cells, such and PI3K pathways significantly suppressed PNK cell cytotoxicity, specific- as LAG-3 and TIM-3, were found only on PBL-CAR-T cells. PD-1 was ally, 64.9±4.7% inhibition by SP600125 (JNK) and 25.2±3.3% inhibition by expressed at higher levels in CB- vs. PBL-derived CAR-T cells (15-40% Ly294002 (PI3K) on LN-18 cells; 100% inhibition by SP600125 and 45.5.5± and 40-60% in CD45RA+ and CD45RO+ T cells, respectively). 8.9% by Ly294002 on U251. JNK and PI3K inhibitors also reduced degranu- Antigen-specific reactivity was shown by CAR-T cells either isolated lation (70.6±3.2% by SP600125 and 58.4±3.6% by Ly294002). Furthermore, from UCB or PBL against acute lymphoblastic leukemia or EBV-B cells PI3K pathway controlled PNK cytokine production upon coculture with overexpressing CD19. U251 cell line, whereas JNK inhibition had minimal effect. Interestingly, differential gene expression profiles were assessed Conclusions through the comparison of UCB- vs. PBL-derived CAR-T cells and Our results demonstrate the importance of PI3K and JNK pathways in their co-culture with antigen-specific target cells. Genes differentially mediating PNK cytotoxicity to GBM cell line targets. These data com- expressed in CD19-CD28z-CD3z vs. CD19-4-1BBz-CD3z CAR-T cells bined with our receptor profiling on PNK cells establish the rationale were also found. for further investigating receptor-ligand interactions that directly Conclusions modulate PI3K and JNK activity. Taken together, these results proved that anti-tumor early differenti- ated/central memory CAR-T cells can be efficiently isolated from UCB P203 with distinctive phenotype as compared to PBL-CAR-T cells. Discovery and characterization of the first fully human Phosphopeptide Tumor Target-specific T cell receptor References 1 1 2 Xavier Michelet, PhD ,Eleni Chantzoura,PhD , Ekaterina Breous-Nystrom, PhD , 1 Chaussabel, D. and N. Baldwin. Democratizing systems immunology with 2 1 1 1 Alessandra Franchino ,RachelSmith , Daniel Pollacksmith ,Jan Bergmann , modular transcriptional repertoire analyses. Nat Rev Immunol, 2014, 1 2 2 2 Alvaro Sebastian Yague , Paisley Myers, PhD , Erin Jeffrey , Benjamin Wolf , 14:271-80; 2 1 1 Dennis Underwood, PhD ,Marc Van Dijk,PhD ,ArthurHurwitz, PhD 2 Altman MC., Rinchai D., Baldwin N., Whalen E., Garand M., Ahamed 1 2 Agentus Therapeutics, Lexington, MA, United States; Agenus, Basel, Kabeer B., et al. A Novel Repertoire of Blood Transcriptome Modules Switzerland Based on Co-expression Patterns Across Sixteen Disease and Physio- Correspondence: Arthur Hurwitz logical States. 2019, bioRxiv; doi: https://doi.org/10.1101/525709. (andy.hurwitz@agentustherapeutics.com) 3 Altman MC., Baldwin N., Whalen E., Al-Shaikhly T., Presnell S., Khaenam P., Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P203 et al. A Transcriptome Fingerprinting Assay for Clinical Immune Monitor- ing. 2019, bioRxiv; doi: https://doi.org/10.1101/587295. Background AgenTus Therapeutics is developing innovative adoptive cell therap- P202 ies to target a novel class of neoantigens called Phosphopeptide Mechanisms underlying human placental CD34+-derived natural Tumor Targets (PTTs). These post-translational modification-based killer cell cytotoxicity against glioblastoma neo-antigens arise in tumor cells through dysregulated kinase and Tanel Mahlakoiv, PhD, Bhavani Stout, Valentina Rousseva, Irene Raitman, phosphatase activities. PTTs represent one of the most promising cell Lin Kang, PhD, Robert Hariri, Xiaokui Zhang, William van der Touw therapy targets, as they are shared within and between cancer indi- Celularity, Warren, NJ, United States cations. Using a mass-spectrometry-based approach that analyzes Correspondence: William van der Touw MHC I-bound peptides, we have analyzed PTTs from several indica- (william.vandertouw@celularity.com) tions. This approach allows us to survey the TCR ligandome of tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P202 cells and healthy tissues. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 110 of 272 Phospho-ligandome analysis identified the phosphopeptide after injection, the alloreactive CD4 T-cells underwent marked expan- EPRpSPSHSM presented by HLA*B07:02+ cancer cells in a patient sion and produced higher levels of interferon-gamma compared to with Acute Myeloid Leukemia (AML). This phosphopeptide results syngeneic CD4 T-cells. This was accompanied by markedly increased from the phosphorylation of the Mixed Lineage Leukemia-1 (MLL1) infiltration of host macrophages within the tumors as early as four protein, a histone lysine methyl transferase that functions as a tran- hours after injection. These tumor-infiltrating macrophages secreted scriptional regulator and has been associated with tumorigenesis. higher levels of interleukin (IL)-1β, IL-12 and IL-23, which are critical for Methods inducing effector T-cell responses. Indeed, 24 hours after injection of + + Using proprietary platforms consisting of primary T cell expansion alloreactive CD4 T-cells, the infiltration of host effector CD8 T-cells from the central compartment and a mammalian display platform into tumors significantly increased, as evidenced by their production of containing TCR α and β chain libraries from the expanded T cells, we high levels of perforin and granzyme B. Furthermore, the melanoma B6 isolated the first fully-human PTT-specific TCR: agenT-04002. mice that survived alloreactive CD4 T-cell therapy developed host Results memory T-cells specific to the B16 melanoma and acquired complete Functional characterization demonstrated that target recognition by resistance to the tumor rechallenge. agenT-04002 is dependent on the phosphoseryl-moiety. Furthermore, Conclusions agenT-04002 shows potent cytotoxic activity against numerous human Results showed that immune reactions triggered by ex vivo-gener- hematologic tumor cell lines in vitro and AML tumor control in vivo in a ated alloreactive CD4 T-cells disrupt immunosuppressive tumor mi- mouse xenograft model. Activated T cells harboring the recombinant croenvironments and establish long-term host antitumor memory T- TCR display a pro-inflammatory phenotype in vitro and in vivo following cell responses. Our findings may help develop new strategies for sig- tumor challenge. Most importantly, when co-cultured with AML cancer nificantly enhancing the efficacy of cancer immunotherapy. cells from patients, agenT-04002 T cells specifically recognize and kill tumor cells while sparing healthy myeloid cells. Acknowledgements Conclusions This work was supported by JSPS KAKENHI Grant Numbers JP15K09659, and AgenTus is developing the next generation of TCRs by targeting a JP19K07754. unique class of neo-antigens with multi-cancer potential. Our data demonstrate feasibility, specificity, and potency of PTT-specific TCRs. References Targeting PTTs across diverse indications will enable us to have 1. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, Bader P, broader applicability of cellular therapies. MR. Verneris MR, Stefanski HE, Myers GD, Qayed M, Moerloose BD, Hiramatsu H, Schlis K, Davis KL, Martin PL, Nemecek ER, Yanik GA, Peters C, Baruchel A, Boissel N, Mechinaud F, Balduzzi A, Krueger J, June CH, Levine P204 BL, Wood P, Taran T, Leung M, Mueller KT, Zhang Y, Sen K, Lebwohl D, Ex vivo-activated allogeneic CD4 T-cells disrupt Pulsipher MA, Grupp SA. Tisagenlecleucel in Children and Young Adults immunosuppressive tumor microenvironment, and induce host with B-Cell Lymphoblastic Leukemia. NEJM. 2018; 378: 439-448. tumor-specific cytotoxic T-cells in mice 2. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, Brudno JN, Stetler- 1 1 1 Kazuhiro Mochizuki, MD, PhD , Shogo Kobayashi , Nobuhisa Takahashi , Stevenson M, Feldman SA, Hansen BG, Fellowes VS, Hakim FT, Gress RE, 1 1 2 3 1 Hideki Sano , Yoshihiro Ohara , Shin Mineishi, MD , Yi Zhang , Atsushi Kikuta and Kochenderfer JN. T cells expressing an anti–B-cell maturation antigen 1 2 Fukushima Medical University, Fukushima City, Japan; Penn State chimeric antigen receptor cause remissions of multiple myeloma. Blood. Cancer Institute, Hershey, PA, United States; Temple University, 2016;128:1688-1700. Philadelphia, PA, United States 3. Majzner RG, Mackall CL. Tumor Antigen Escape from CAR T-cell Therapy. Correspondence: Kazuhiro Mochizuki (mochi-k@fmu.ac.jp) Cancer Discov. 2018; 8: 1219–1226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P204 4. Bezu L, Kepp O, Cerrato G, Pol J, Fucikovag J, Spisekg R, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: Peptide-based vaccines in anticancer Background therapy. Oncoimmunol. 2018; 7: e1511506 (15 pages). Cancer immunotherapies that target tumor-specific or tumor- 5. Negrin RS. Graft-versus-host disease versus graft-versus-leukemia. associated antigens are promising treatments for patients with incur- Hematology Am Soc Hematol Educ Program. 2015; 2015: 225-230 able cancers [1,2]. However, relapses due to the loss of target anti- Ethics Approval gens challenge the success of these therapies [1,3]. Multitargeted Experimental protocols were approved by the Fukushima Medical immunotherapies, such as cancer vaccinations specific to multiple University’s committee on Use and Care of Animals; approval number cancer-associated peptides, are possible approaches. However, clin- 28054, 29039, and 2019048. ical studies have shown that they have limited efficacy with respect to the induction of objective responses [4]. The graft-versus-leukemia effect observed after allogeneic hematopoietic stem cell transplant- P205 ation (allo-HSCT) is another example of strong multitargeted antitu- Automated, closed bioreactors for T cell processing and dendritic mor immunity mediated by donor T-cells that recognize and react to cell-T cell co-culture multiple allo-antigens [5]. In the present study, we demonstrated a Lekhana Bhandary, BS, PhD, Andrew Kozbial, BS, PhD, Shashi Murthy, BS, novel approach for attaining alloreactive CD4 T-cell-induced multi- PhD targeted cancer immunity that does not utilize allo-HSCT. Northeastern University, Boston, MA, United States Methods Correspondence: Shashi Murthy (s.murthy@northeastern.edu) + + Cluster of differentiation (CD)4 and CD8 T-cells isolated form the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P205 spleen of BALB/c mice were separately activated in cultures by den- dritic cells (DCs) generated from the bone marrow of C57BL/6 (B6) Background mice. The resultant host-reactive donor T-cells were injected into B6 Functionally closed and affordable automated cell culture systems mice bearing pre-established B16 melanoma. Host T-cells activated are critical to the success of cell-based immunotherapy. Despite by syngeneic DCs were used as the control. major advances in these therapies, there are few systems available Results that are practical for use at both the pre-clinical and clinical stages. Whereas the intratumoral injection of host-reactive donor CD4 T-cells To address this need, we have designed a system called BATON elicited potent antitumor immunity against established B16 melanoma which is designed for optimal culture of both adherent and suspen- in an alloantigen-dependent manner, intratumoral injection of host- sion cell types (Fig. 1). Cells are cultured via continual perfusion and reactive donor CD8 T-cells or host-type syngeneic T-cells failed to in- the fluid flow loop also enables automated cell loading and harvest. duce antitumor responses. The number of injected donor-type host- This poster will describe two application areas, namely T cell expan- reactive CD4 T-cells diminished after tumor regression and did not in- sion relevant to autologous CAR-T and TCR therapies and dendritic duce graft-versus-host disease-like complications. Interestingly, early cell (DC)-T cell co-culture for neo-antigen-based T cell therapies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 111 of 272 Methods The T cell expansion capability of the BATON system was evaluated by seeding BATON cartridges each having a surface area of 40 cm2 and volume of 25 mL with 23 million PBMCs along with CD3/28 Dynabeads. Cells were continually perfused with Irvine Scientific Prime XV xeno- free T cell medium with 33 U/mL IL-2 for 9 days. For comparison, a simi- lar culture was performed in a G-Rex 6 well plate. For DC-T cell co- culture experiments enriched monocytes (MOs) were seeded into the BATON system at a seeding density of approximately 600k MOs/cm2 into two cartridges. Monocytes were differentiated into immature DCs by continually perfusing the seeded MOs for 6 days with CellGenix DC Medium supplemented with 350 U/mL IL-4 and GM-CSF (CellGenix). On Day 6, the DCs from one cartridge were harvested for flow cytometry. The other cartridge was drained without removal of the DCs and seeded with approximately 23 million PBMCs. This cartridge was then perfused with Irvine Scientific Prime XV xeno-free T cell medium with 33 U/mL IL-2. Cells were harvested following 7 days of co-culture. In addition to flow cytometry characterization, the cytotoxicity of the T cells was evaluated via co-culture with Jurkat cells. Results BATON achieved high levels of T cell expansion, comparable to G-Rex (Fig. 2-3) and harvested cells showed strong cytotoxic ability (Fig. 4). For DC-T cell co-culture experiments, the BATON system generated DCs from monocytes at high yield (27% of seeded monocytes converted into DCs) (Fig. 5A). Expansion of T cells from the seeded PBMCs was ro- Fig. 2 (abstract P205). See text for description bust, with 26-fold expansion achieved in 7 days (Fig. 5B). Harvested T cells showed strong cytotoxic ability relative to control (Fig. 6). Conclusions The BATON system is an effective platform for reagent- and DC- mediated T cell expansion. Acknowledgements Funding from the NSF via grant 1645205 is gratefully acknowledged. Fig. 1 (abstract P205). See text for description Fig. 3 (abstract P205). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 112 of 272 Fig. 4 (abstract P205). See text for description Fig. 6 (abstract P205). See text for description P206 Transmembrane and linker domain amino acid composition alters chimeric antigen receptor (CAR) membrane residence and may conceal detection of novel functional CAR formats 1 2 1 Dina Schneider, PhD , Virginia Hoglund, MS , Ying Xiong, PhD , Darong 1 1 2 Wu, MS , Boro Dropulic, PhD, MBA , Rimas Orentas, PhD Lentigen Technology, Miltenyi Biotec, Gaithersburg, MD, United States; Seattle Children’s Research Institute, Seattle, WA, United States Correspondence: Rimas Orentas (rimas.orentas@seattlechildrens.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P206 Background The relationship between the structure of the extracellular linker (L) and transmembrane (TM) domains, and CAR-T function has not been fully described. In previous studies we used L and TM domains de- rived from CD8. To better define amino acid sequences governing cell surface expression and anti-tumor activity, we altered the se- quence and length of these domains and tested the impact on CAR T biology. Methods TM domains from glycoproteins expressed on the T cell surface were aligned to CD8 and those with a high degree of similarity were used to create new CARs. In some constructs the extracellular sequence proximal to the membrane of those proteins (L) was also included. CAR function was tested using LV-transduced human T cells. Protein expression was analyzed by flow cytometry and western blot, in vitro function by cytokine release and cell-mediated cytolysis, and in vivo function in xenograft models. CAR protein expression was also ana- lyzed by immunofluorescent microscopy. Results Sequences from T cell-expressed CD antigens, the CD3 complex, acti- vation markers, and members of the tumor necrosis factor receptor superfamily (TNFRSF) were analyzed. Based on sequence conserva- tion we created new CARs expressing combinations of CD4 and CD8 Fig. 5 (abstract P205). See text for description TM domains, as well as TNFRSF9, TNFRSF16, and TNFRSF19 (CD137, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 113 of 272 NGFR, TROY/TAJ). All constructs were detected by western blot. Conclusions Strong T cell surface expression was seen for CD8L/CD4TM, CD8L/ These results demonstrate that 3D-ACT model using ex vivo ex- CD4TM, CD8L/TNFRSF19TM, and TNFRSF16L/TNFRSF16TM. Intermedi- panded TILs and 3D tumoroid models is an effective tool for the ate surface expression was seen for TNFRSF9L/TNFRSF9TM. Con- therapeutic assessment of autologous TILs and indicate that it can structs with TNFRSF19L/ TNFRSF19TM had very poor surface also be used to assess efficacy of other cellular therapy applications. expression. However, these “undetectable” CARs by flow cytometry Furthermore, implementation of this platform in the clinical studies had the highest level of cytotoxicity and cytokine release vs Raji may also allow determining the most effective combinatorial cellular lymphoma. Immunofluorescence studies with transduced T cells on therapy strategies for individual patients. their own, or in the presence of Raji target cells, demonstrated that TNFRSF19 sequence may mediate an intracellular residence profile. Association of CARs with the CD3 complex was also noted. P208 Conclusions Impact of combined blockade of PD1 and activation of CD137 on The production of CARs for clinical use generally requires detection tumor infiltration and tumor cell killing efficacy of TILs in an of the CAR protein on the cell surface. We found that high-activity ex vivo autologous 3D tumoroid model of NSCLC patient samples CAR-T constructs can be created using the linker and TM domains of Jenny Kreahling, PhD, Melba Page, PhD, Mibel Pabon, PhD, Vijayendra TNFRSF19, even though these constructs are expressed on the cell Agrawal, PhD, Soner Altiok, MD, PhD surface at low to undetectable levels. The mechanism by which these Nilogen Oncosystems, Tampa, FL, United States CARs are functionally active, while in a primarily intracellular state, is Correspondence: Soner Altiok (soner@nilogen.com) under investigation. Intracellular residence of CARs may be a novel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P208 mechanism to prevent undesired activation or T cell exhaustion and represents a novel locus of CAR-T activity control. Background Adoptive cell therapy (ACT) with TILs has been of growing interest as anti- cancer treatment in solid tumors. This therapy consists of the outgrowth P207 and expansion of tumor resident T cells from tumor material and their trans- A patient-driven ex vivo 3D tumor organoid model to assess fer back into the same patient to achieve tumor cell killing. However, exist- efficacy of tumor infiltrating T-cell adoptive cell therapy ence of intrinsic immune escape mechanisms may diminish the efficacy of Jenny Kreahling, PhD, Mibel Pabon, PhD, Melba Page, PhD, Vijayendra therapeutic applications of TILs. Here we describe an ex vivo patient derived Agrawal, PhD, Soner Altiok, MD, PhD 3D tumoroid platform utilizing powerful high content confocal imaging mo- Nilogen Oncosystems, Tampa, FL, United States dalities to monitor the impact of PD1 inhibition and CD137 activation on au- Correspondence: Soner Altiok (soner@nilogen.com) tologous TIL infiltration and ACT mediated tumor cell killing. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P207 Methods Human tumor samples were obtained with patient consent and rele- Background vant IRB approval. Fresh patient tumor samples were processed into Adoptive cell transfer (ACT) of ex vivo expanded tumor- tumoroids measuring 100-150 μm in size. For these studies, autolo- infiltrating lymphocytes (TILs) has shown promising therapeutic gous TILs were propagated from each tumor sample. TILs were fluo- efficacy in subsets of patients with several solid tumors including rescently labeled and incubated together with 3D tumoroids in the NSCLC. However, to improve the anti-tumor efficacy of TIL ACT in presence or absence of the PD1 inhibitor nivolumab and/or an agon- solid tumors it is critical to develop rational combination strat- ist anti-CD137 mAb urelumab. TIL infiltration into tumoroids and kill- egies and to identify biomarker(s) predictive of patients who ing of metabolically labeled tumor cells were quantified by advanced would respond favorably to TIL therapy. Here we describe a high confocal microscopy and a custom image analysis algorithm that was content imaging approach using a fresh tumoroid model with in- correlated with flow cytometry and cytokine profiling. tact tumor stroma for quantitative assessment of autologous TIL Results infiltration and target tumor cell killing. We show that nivolumab and urelumab treatments had significant Methods impacts on TIL infiltration in subsets of NSCLC tumoroids. Flow cy- All human tumor samples were obtained with patient consent and tometry analysis demonstrated treatment-mediated activation of TILs relevant IRB approval. For the ex vivo assays 3D tumoroids measuring accompanied by marked changes in the release of pro-and anti- 100-150 micron in size were prepared and cryopreserved during the inflammatory cytokine profiles. Furthermore, we documented the ef- process of ex vivo propagation of autologous TILs. Allogeneic periph- fect of TIL transfer and drug treatment on resident T-cells, Tregs and eral blood mononuclear cells (PBMCs) were used as control. Ex vivo myeloid cell populations within the tumoroids. No correlation was propagated TILs were fluorescently labeled and their growth and found between TIL activity and composition of propagated TILs or functional characteristics in the presence or absence of CD3/CD28 PD-L1 expression on tumor cells. tetramer were assessed via flow cytometry. High content confocal Conclusions analysis was used to quantify TILs infiltration into the tumoroids and This data suggests that combined blockade of PD1 and activation of target tumor cell killing using Nilogen’s 3D-ACT platform. Multiplex CD137 may enhance the therapeutic efficacy of TIL ACT in NSCLC. cytokine assays and flow cytometry analysis were performed to as- Overall, this study also shows that our 3D-ACT tumoroid model al- sess TIL activation upon exposure to tumoroids. lows comprehensive analysis of compensatory mechanisms and se- Results lection of rational combinatorial treatment using adaptive cellular We successfully prepared matched autologous TILs and unpropa- therapy with autologous TILs and likely with other types of cellular gated 3D tumoroids from NSCLC patient tumors. The characteristics therapies. of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved tumor organoids using a cus- tom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of P209 marker-specific cell number, cell viability and apoptosis in addition to Hijacking CAR-CD19 T cells to potently control Her2-positive solid structural and functional analysis of cells in intact 3D tumoroids. High tumors in vitro and in vivo through the use of unique and content confocal imaging analysis demonstrated that CD3/CD28 pre- selective bridging proteins activated TILs with increased activation phenotypes and enhanced Paul Rennert, PhD, Christine Ambrose, PhD, Fay Dufort, PhD, Lihe Dufort, pro-inflammatory cytokine release had marked infiltration into the Alyssa Birt, Thomas Sanford, Lan Wu, Roy Lobb, PhD 3D tumor organoids compared to untreated TILs and PBMCs. The Aleta Biotherapeutics, Natick, MA, United States data was correlated with quantitative tumor cell killing assessment Correspondence: Paul Rennert (paul.rennert@aletabio.com) for tumoroids. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P209 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 114 of 272 Background using antibodies directed against the scFv extracellular region. The Cell therapy success is limited by two critical issues. One is loss of anti- analytic panel included 29 cell surface, activation, exhaustion, and gen expression. This accounts for the ~50% relapse rate seen in CAR- cell-cell adhesion markers to identify/characterize lymphocyte sub- treated B cell malignancies. Solid tumors have highly variable antigen sets; and 9 intracellular markers to characterize functional status and expression and CARs targeting a single antigen fail as antigen-negative activation of signaling pathways. viSNE was used to visualize high- tumor cells escape, driving tumor resistance. dimensional data on a 2D map and quantify CyTOF data. A related issue is that most cell therapeutics fail to persist in the pa- Results tient. This is a particularly true of solid tumor treatment with CAR T Axi-cel products contained a median of 63% (range, 20-86%) trans- cells. The persistence failure may result from unproductive CAR-T inter- duced CAR (CAR+) T cells, and included relatively undifferentiated T cell action with the targeted tumor cell. subsets: a median of 0.4% and 52% of CD4 CAR+ cells were T stem cell We have developed CAR-CD19 T cells (CAR19s) that secrete bridging memory (SCM) and central memory (CM) cells, respectively, and 7% proteins to address these two critical issues. We leverage the ability and 34% of CD8 CAR+ cells were SCM and CM cells, respectively. CAR+ of CAR19s to persist independently of the target tumor cell while T cells in products had significantly higher expression of proliferation, simultaneously endowing these CARs with potent targeting technol- activation, and exhaustion markers (Ki67, CD25, HLA-DR, ICOS, OX-40, ogy. Here we illustrate this technology using the CD19-based bridg- Tim3, LAG3) and higher expression of cell-cell adhesion molecules ing protein that binds both EGFR and Her2. These data demonstrate (CD49d, CD29) compared with CAR-negative (CAR–)Tcells. On day 7, a that CAR19 T cells can be redirected to kill solid tumors in vivo. median of 11% of circulating T cells (range 0.6-58.4%) were CD4 CAR+ Methods and 3% (range 0.6-44.1%) were CD8 CAR+. Both CAR– and CAR+ T cells We cloned a highly stable CD19 extracellular domain (ECD) in frame with showed evidence of activation, but circulating CAR+ T cells expressed an anti-Her2 scFv to create, express and purify CD19-ECD-anti-Her2 bridg- higher levels of Ki67, 4-1BB, Tim3, PD-1, PD-L1, CXCR3, CD29, pZAP70, ing proteins. The sequence was also cloned downstream of a CAR19 do- pSTAT3 and pSTAT5, compared to CAR– Tcells. main and P2A cleavage site in a lentiviral vector. Transduced primary T Conclusions cells expressed the CAR19 and secreted the bridging protein. These CyTOF enables detailed characterization of CAR T cell products com- bridging protein formats were evaluated with in vitro cytotoxicity assays prising heterogeneous T cell subsets. Axi-cel comprises both trans- and Her2+ tumors in vivo. Finally, we added an anti-EGFR scFv sequence, duced and untransduced T cells at various stages of differentiation, creating an extremely potent multi-antigen targeting module. including SCM cells. CAR+ T cells showed higher expression of a broad Results range of proliferation and activation/exhaustion markers, compared to Incorporating a stabilized CD19 ECD in bridging proteins improved pro- CAR– cells, both in axi-cel products and in peripheral blood 7 days after tein expression, including from CAR19 T cells. CAR19 T cells secreting CAR T cell infusion. These data shed light on phenotypic and functional stabilized CD19-anti-Her2 bridging proteins were highly potent in vitro diversity of CAR T cells, pre- and post-infusion, influenced by the manu- and in vivo targeting CD19+ or Her2+ cells. An anti-EGFR scFv was facturing process, conditioning-related homeostatic cytokines and added to the CD19-ECD-anti-Her2 bridging protein. This novel multi- antigen-driven activation. Future studies may explore associations be- antigen targeting bridging protein supports highly potent cytotoxicity tween product composition and clinical outcomes. against single and dual antigen-expressing tumor cells while retaining Trial Registration intrinsic anti-CD19 activity, providing these unique CARs with a tumor- NCT02926833 independent and self-renewing antigen depot in CD19-positive normal B cells. For specific indications a third binding domain is added. P211 Conclusions Combining Deep™ IL-12 Primed and Deep™ IL-15 Primed T cells We have created a robust system for targeting multiple tumor anti- leverages complementary mechanisms to enhance anti-tumor gens simultaneously with a single CAR T cell. Further the use of activity CAR19s supports CAR-T cell persistence independently of tumor anti- Katharine Sackton, PhD, Elena Geretti, PhD, Pengpeng Cao, PhD, Shawn gen expression. This unique technology addresses critical issues in Carey, PhD, Xiaoyan Liang, Jonathan Nardozzi, PhD, Zishu Gui, Alicia cell therapy using a potent technology whose modular nature allows Worthylake, Glenn Leary, Becker Hewes, MD, Tap Maniar, MD, Jonathan for rapid program and pipeline development. Fitzgerald, PhD, Andy Rakestraw, PhD, Karsten Sauer, PhD, Douglas Jones, PhD, Thomas Andresen, PhD P210 Torque Therapeutics, Cambridge, MA, United States Deep phenotypic and functional analysis of transduced anti-CD19 Correspondence: Thomas Andresen (tandresen@torquetx.com) CAR T cells and untransduced T cells in patients treated with axi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P211 cel by single cell mass cytometry 1 1 1 Yohei Arihara, MD, PhD , Caron Jacobson, MD , Philippe Armand, MD , Background 2 2 2 John Rossi, MS , Nathalie Scholler, MD, PhD , Stuart Sievers, PhD , Interleukin-15 (IL-15) and Interleukin-12 (IL-12) play complementary 2 2 2 Edmund Chang, PhD , Mauro Avanzi, MD, PhD , Adrian Bot, MD, PhD , roles as immunomodulators. IL-15 induces T cell memory and supports Jerome Ritz, MD survival, activation and proliferation of CD8+ T and NK cells. IL-12 pro- Dana-Farber Cancer Institute and Harvard Medical School, Boston, motes T cell cytotoxicity and innate immune responses in the tumor MA,United States; Kite, a Gilead Company, Santa Monica, CA, Santa microenvironment. Both cytokines have been explored as cancer im- Monica, CA, United States munotherapies, but clinical success has been limited due to severe side Correspondence: Jerome Ritz (jerome_ritz@dfci.harvard.edu) effects. To limit systemic toxicities, Torque has developed its Deep- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P210 Primed™ T cell therapy technology. Multi-targeted T cells (MTC) specific for multiple tumor antigens are generated from patient apheresis. Cyto- Background kines are tethered to MTCs to support MTC persistence and activity fol- Axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T lowing adoptive transfer into patients, while limiting systemic cytokine cell therapy, has shown high efficacy in relapsed/refractory (R/R) dif- exposure. This study evaluates the combination of cytotoxic T lympho- fuse large B cell lymphoma (DLBCL). Axi-cel contains heterogeneous cytes (CTL) Deep-Primed with Deep™ IL-15 and Deep™ IL-12 to leverage populations of transduced and untransduced T cells. We used single their complementary biology for superior efficacy. cell mass cytometry (CyTOF) to analyze the impact of this heterogen- Methods eity on proliferation and expansion of these cells after infusion. CTLs reactive against MART-1 antigen were generated from healthy Methods donors (MART-1 CTLs). Next, expansion and cytotoxicity of MART-1 CyTOF examined CAR T cell products from 12 patients with R/R CTLs loaded with Deep IL-12, Deep IL-15 or both against MART-1 ex- DLBCL and peripheral blood mononuclear cells obtained 7 days after pressing SKMEL-5 melanoma cells were assessed. In addition, murine axi-cel infusion. We identified anti-CD19 CAR T cells (CAR+ cells) PMEL CD8+ T cells reactive against the B16-F10 melanoma antigen Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 115 of 272 gp100 were loaded with Deep IL-12, Deep IL-15 or both and evalu- Conclusions ated for in vitro expansion, activation and cytotoxicity against B16- Conclusions: These results indicate the viability of TIL ACT for refrac- F10 melanoma cells, as well as for anti-tumor activity in B16-F10 tory ovarian cancer by allowing for the large expansion of anti-tumor tumor-bearing mice. TIL in a short time and consistent manner. A Phase II clinical trial Results based on this work is currently open at UTMDACC to evaluate the Loading with Deep IL-15 promoted MART-1 CTL proliferation and feasibility of TIL ACT in recurrent or refractory OvCa (NCT03610490). preserved antigen reactivity over time. Deep IL-12 loaded MART-1 Ethics Approval CTLs displayed enhanced IFN-γ secretion and cytotoxicity, particularly Ethics approval: Ethical approval and tissue from surgical resections used at low effector:target ratios. Combination of MART-1 CTLs loaded to expand TIL were both obtained under a protocol (PA16-0912 and with Deep IL-12 and Deep IL-15 further enhanced T cell expansion, LAB02-188) approved by the Institutional Review Board of UTMDACC. IFN-γ secretion and cytotoxicity. Similarly, combination of murine PMEL T cells loaded with Deep IL-12 and Deep IL-15 resulted in per- P213 sistent T cell activation, improved memory, and enhanced cytotox- Conversion of peripheral blood mononuclear cells into tumor- icity over individually loaded T cells. Coadministration of Deep IL-12 specific cytolytic cell populations using tumor cells engineered and Deep IL-15 loaded PMEL T cells to B16-F10 melanoma-bearing with multiple immunomodulatory factors mice was well-tolerated, with minimal and reversible body weight 1 1,2 2 Joshua Keegan , James Lederer , Frank Borriello, MD, PhD , Nathan loss, and elicited superior anti-tumor activity. Schomer, MS Conclusions 1 2 BWH Harvard Medical School, Boston, MA, United States; Alloplex Modular tethering of Deep™ IL-12 and Deep™ IL-15 to T cells uniquely Biotherapeutics, Inc., Winchester, MA, United States leverages their complementary functions as immunomodulators to Correspondence: James Lederer (jlederer@alloplexbio.com); Frank maximize anti-tumor activity without notable toxicity in preclinical Borriello (fborriello@alloplexbio.com) models. A Phase I clinical trial of Deep IL-15 Primed MTCs (TRQ15-01) in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P213 solid cancers and lymphoma is enrolling (NCT03815682). Torque is initi- ating clinical evaluation of Deep IL-12 Primed MTCs (TRQ12-01), includ- Background ing a combination arm with TRQ15-01. Numerous studies and two recent clinical approvals have demon- strated the efficacy and safety of cellular immunotherapies to treat P212 diverse cancer subtypes. To date, the only approved cytotoxic cel- Potential clinical application of tumor-infiltrating lymphocyte lular therapies in the U.S. are T-cell based. However, ongoing clin- therapy for ovarian epithelial cancer prior or post-resistance to ical trials using gamma delta T cells and natural killer cells have chemotherapy suggested not only clinical efficacy, but also synergistic beneficial Donastas Sakellariou-Thompson, BS, Marie-Andree Forget, PhD, Emily effects when combined with checkpoint inhibitor antibody therap- Hinchcliff, MD, Joseph Celestino, Patrick Hwu, MD, Amir Jazaeri, MD, Cara ies. Our group developed novel immune stimulatory allogeneic Haymaker, PhD, Chantale Bernatchez tumor cells engineered with multiple immunomodulatory factors as MD Anderson Cancer Center, Houston, TX, United States a novel approach to generate heterologous tumor cell lytic popula- Correspondence: Cara Haymaker (chaymaker@mdanderson.org); tions of human peripheral blood mononuclear cells (PBMCs) for Chantale Bernatchez (cbernatchez@mdanderson.org) cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P212 Methods SK-MEL-2 cells obtained from the NIH were engineered to express a set Background of immunomodulatory proteins with the aim of expanding oncolytic cell Background: Epithelial ovarian cancer (OvCa) is the deadliest populations. Cells were engineered using lentiviral vectors prepared by gynecological cancer, and is estimated to account for almost 14,000 VectorBuilder, and sorted by flow cytometer for high expression of de- deaths in 2018. Traditional management of advanced stage OvCa in- sired immunomodulators. These engineered cells were then mixed with cludes tumor reductive surgery and adjuvant platinum-taxane chemo- freshly thawed human PBMCs and co-incubated for up to 14 days. Cells therapy, which results in high rates of initial complete response. were collected at time points during the incubation period for pheno- However, nearly 90% of patients recur and the 5-year survival rate for typic analysis using mass cytometry (CyTOF). Functional characterization late-stage disease is only 28. Immunotherapy has become a powerful of stimulated PBMCs was conducted using a cytotoxicity assay against treatment option for several solid tumor types. The presence of tumor- targets from several cancer subtypes. infiltrating lymphocytes (TIL) is correlated with better prognosis in ovar- Results ian cancer, pointing at the possibility to benefit from harnessing their CyTOF analysis of stimulated PBMCs revealed expansion of natural anti-tumor activity. The effectiveness of adoptive cell therapy (ACT) killer cells, gamma-delta T cells, and CD8 T cells by 8 days after with TIL has already been shown in metastatic melanoma with object- stimulation (Figure 1). All these populations expressed high levels of ive response rates of 40-50%. This preclinical study explores the feasibil- NKG2D and Granzyme B, suggesting widespread recognition of ity of transposing TIL ACT to OvCa using an improved culture method. tumor antigens and cytotoxic capability. In contrast, PBMCs that were Methods activated and expanded by CD3/CD28 activation beads showed less Methods: High-grade serous ovarian cancer (n=84), pre- or post- heterogenous expansion. PBMCs expanded with immunomodulatory chemotherapy and primary or metastatic, samples were accrued. TIL cell lines demonstrated potent cytotoxic activity towards both the were cultured using either high-dose IL-2 only, high-dose IL-2 with an parental cell line, as well as other melanoma and non-melanoma agonistic antibodies targeting 4-1BB (a41BB), or a combination of IL-2, cancer cell lines (Figure 2). As a control for specific cytotoxicity, both a41BB, and an agonistic anti-CD3 mAb. The cells were phenotyped autologous and allogeneic PBMCs were tested as targets and dis- using flow cytometry in the fresh tissue and after expansion. Tumor re- played no detectable cytotoxicity in our assay. activity was assessed against HLA-matched ovarian cancer cell lines via Conclusions IFN-γ ELISPOT. We developed a novel approach to expand immune cell populations Results from normal human PBMCs demonstrating anti-tumor activity Results: Ovarian cancer is highly infiltrated with CD8+ TIL that are pref- against multiple cancer cell types. This strategy is being developed erentially and robustly expanded with IL-2 and the two agonistic anti- to activate and expand PBMCs from cancer patients that will be used bodies. With a 95% success rate, the TIL are grown to ≥100x10^6 cells for autologous cellular immunotherapy. Taken together, the results in 2-3 weeks without over differentiation. In addition, the CD8+ TIL from this study demonstrate our ability to generate multiple popula- grown with this method showed HLA-restricted tumor recognition. TIL tions of cytotoxic effector cells from PBMCs, which should provide a growth and tumor recognition was independent of surgery site or straight-forward approach to generate clinically-relevant cells for chemotherapy exposure. adoptive cellular immunotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 116 of 272 replace endogenous TCRs with the native neoTCR sequence in each edited T cell for expression at native TCR levels. Precision genome en- gineering of a colon cancer tumor cell line (SW620) was used to express the patient-specific neoantigen (COX6C-R20Q) at native levels. neoTCR- T cells were co-cultured with SW620 expressing the COX6C-R20Q muta- tion or the COX6C wild-type peptide. Functional readouts were T cell proliferation, cytokine secretion and tumor cell killing. Results Seven neoTCR clonotypes against the mutated COX6C peptide (COX6C-R20Q) presented in the context of HLA-A2 were cloned from imPACT-captured neoE-specific CD8 T cells. Primary human T cells were engineered with the 7 different TCR specificities against the COX6C-R20Q. Each of the seven candidate neoTCR-engineered T cells displayed specific cytotoxicity against tumor cells expressing en- dogenous levels of the COX6C-R20Q neoantigen. At 96 hours, using Effector to Tumor cell ratio (E:T) 1:1, 85-90% tumor elimination was observed (p < 0.000001 for each comparison). Significant tumor cell killing was detected with an E:T ratio as low as 1:5. neoTCR-T cells also proliferated and secreted interferon-gamma in response to co- culture with the relevant tumor target. Importantly, neoTCR-T cell ac- tivity was absent when co-cultured with tumor cells expressing wild type COXC6 protein. Conclusions Fig. 1 (abstract P213). See text for description These results demonstrate that the imPACT Isolation Technology used to capture antigen-experienced, neoE-specific T cells from the blood of patients with cancer authenticates that these neoE-HLA tar- gets are relevant for engineering neoTCR-T cells therapies. Lever- aging this approach, PACT is developing autologous personalized adoptive T cell therapy (NeoTCR-P1 product). A Phase 1 clinical trial to test NeoTCR-P1 T cells in subjects with solid tumors is currently ongoing (NCT03970382). References 1. Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, Urbinati F, Foy S, Hunter T, Tai A, Bethune M, Peng A, Dalmas O, Franzusoff A and Mandl S. Abstract 4783: Highly efficient, non-viral preci- sion genome engineering for the generation of personalized neoepitope- specific adoptive T cell therapies. Cancer Res 2019;79(13 Suppl). 2. Sennino B, Conroy A, Purandare B, Litterman A, Jacoby K, Moot R, Lu W, Nguyen D, Urbinati F, Foy S, Hunter T, Dalmas O, Bethune M, Park T, Peng S, Franzusoff A and Mandl S. Abstract 1433: NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with ‘youn- ger’ phenotypes that rapidly proliferate and kill target cells upon recogni- tion of cognate antigen. Cancer Res 2019;79(13 Suppl). Fig. 2 (abstract P213). See text for description 3. Peng S, Quach B, An D, Sandoval S, Bao R, Pan Z, Bethune M, Dalmas O, Yi M, Meadows C, Heeringa K, Guo L, Yuen B, Sorfleet J, Jacoby K, Moot R, Lu W, Nguyen D, Sennino B, Conroy A, Purandare B, Litterman A, P214 Mandl S and Franzusoff A. Abstract 1435: An ultra-sensitive and high- T cells precision engineered to express neoepitope-specific TCRs throughput technology (imPACT) for the identification and isolation of cloned from a patient with colorectal cancer specifically target and intrinsic and emergent neoepitope-specific T cells from the peripheral kill relevant neoantigen-expressing tumor cells blood and TILs of cancer patients. Cancer Res 2019;79(13 Suppl). Barbara Sennino, PhD, Adam Litterman, John Gagnon, Andrew Conroy, Ethics Approval Bhamini Purandare, Zheng Pan, Dalmas Olivier, Kyle Jacoby, Songming Human samples in this study were procured from a commercial vendor who Peng, Alex Franzusoff, Stefanie Mandl, PhD collected them according to their established ethics policies. PACT Pharma, South San Francisco, CA, United States Correspondence: Alex Franzusoff (afranzusoff@pactpharma.com); Stefanie Mandl (smandl@pactpharma.com) P215 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P214 Depletion of CD45RA-positive cells potentiates the reactivation of EBV-specific T-cells from EBV positive lymphoma patients Background Sandhya Sharma, BSc, Naren Mehta, Kathan Parikh, RA, Cliona Rooney, Neoepitopes (neoE) derived from private tumor-exclusive mutations PhD represent compelling targets for personalized TCR-T cell therapy to Baylor College of Medicine, Houston, TX, United States eradicate tumor cells throughout the body. The imPACT Isolation Tech- Correspondence: Sandhya Sharma (me.sandhya@gmail.com) nology® is an ultra-sensitive and high-throughput process for the cap- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P215 ture of mutation-targeted CD8 T cells from patient blood. NeoTCRs of native sequence, cloned from the captured T cells, were evaluated for Background tumor-targeted functionality by non-viral precision genome engineer- Epstein-Barr virus (EBV)-positive lymphomas express viral type-2 latency ing of fresh human CD8 and CD4 T cells for neoTCR expression [1,2]. proteins (T2-Ags). Autologous EBV-specific T-cells (EBVSTs) directed to Methods T2-Ags have produced complete responses in ~50% lymphoma pa- NeoTCRs were isolated from the blood of a patient with colorectal can- tients[1]. However, in our ongoing clinical trial, we failed to generate cer using the imPACT Isolation Technology® [3]. Subsequently, healthy EBVSTs from 24% of the patients procured; manufacturing failures donor CD8 and CD4 T cells were precision genome engineered to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 117 of 272 associated with lack of T-cell expansion and T2-Ag-specificity. Further, Background in lines successfully expanded, many EBVST lines demonstrated low T2- Administration of Toll-like receptor agonists with adoptively trans- Ag specificity and some contained a high frequency of NK-cells. Our ferred T cells augment tumor immunity. However, systemic or local goal is to improve both the manufacturing success rate and clinical effi- administration of TLR agonists could heighten inflammation and lead cacy of EBVSTs. In EBV-exposed individuals, EBVSTs reside in CD45RA- to toxic side effects when coupled with CAR or TIL-based therapies. CD45RO+ memory compartment, while CD45RA-positive population in- Thus, we hypothesized that TLR agonists could be used ex vivo dur- cludes unwanted naïve T-cells, suppressive regulatory-T cells, and NK- ing T cell expansion and ultimately generate a cell product with en- cells[2,3]. We hypothesized that removal of CD45RA-positive cells from hanced anti-tumor properties for adoptive cell transfer therapy while PBMCs prior to EBV-antigen specific stimulation would improve EBVST reducing potential toxicity to patients. generation and specificity by eliminating competing naïve T-cells, while Methods reducing potentially inhibitory cells capable of inhibiting the outgrowth To test our hypothesis, we employed the Pmel-1 transgenic mouse of antigen-specific T-cells. We, therefore investigated the effects of se- model, in which CD8+ T cells harbor a TCR specific for the gp100 epi- lective depletion of CD45RA-positive cells from PBMCs prior to EBV T2- tope expressed on melanoma and healthy melanocytes. Pmel spleno- Ags stimulation. cytes were stimulated with hgp100 peptide in the presence or absence Methods of TLR9 agonist CpG (ODN-1668) and expanded in IL-2 for one week. T EBVSTs were generated from whole PBMCs and CD45RA depleted (RAD) cell phenotype was analyzed via flow cytometry and anti-tumor activity PBMCs and we measured proliferation by counting, T2-Ag specificity assessed in mice bearing B16F10 melanoma tumors. using IFN-gamma ELIspot assays and cell-phenotype by flow cytometric Results analysis. To compare their in-vivo efficacy, EBVSTs were adoptively trans- T cells expanded with CpG possessed a unique phenotype (IL-2Ralpha- ferred into immunodeficient mice bearing autologous EBV-transformed high, ICOS-high, CD39-low) and mediated more potent responses lymphoblastoid tumor cells and tumor clearance was evaluated. against melanoma in vivo than traditionally expanded T cells. Interest- Results ingly, this phenotype and anti-tumor function was dependent on the RAD-EBVSTs produced greater expansion of EBVSTs from PBMCs and presence of B cells at the start of culture, as their removal resulted in a decreased the frequency of NK cells, which dominated some of our pa- loss of this unique phenotype and anti-tumor efficacy in vivo. Con- tient lines. T2-Ag specificity increased by 3-5 fold as measured by versely, removal of CD4+ T cells, NK cells, dendritic cells, or macro- gamma-IFN release in response to T2-Ags stimulation in both healthy phages from culture did not ablate the phenotype or anti-tumor donors and patients. RAD-EBVSTs maintained antigen specificity over activity of CpG-generated T cells. The CpG-elicited T cell effects were multiple rounds of weekly T2-Ags stimulation. Phenotypic analysis dem- also dependent on the peptide-mediated interaction between the T onstrated decreased expression of exhaustion markers in RAD-EBVSTs. cell and APC in culture as activating with plate-bound or bead-bound Most importantly, this approach restored responsiveness to antigen antibody strategies resulted in a T cell population that was similar to stimulation in most unresponsive patients enabling the successful re- the ineffective vehicle treated cells both phenotypically and therapeut- activation and expansion of EBVSTs from lymphoma patients that failed ically. We further found that CpG-treated B cells expressed heightened previously. This advantage extends to VSTs with specificity for HPV, VZV levels of CD40, suggesting that induction of a CD40-CD40L axis be- and adenoviral antigens, suggesting that we have identified a general tween B and T cells may account for the generation of potent IL- approach for improving the activity of VSTs for immunotherapy. EBVSTs 2Ralpha-high, ICOS-high, CD39-low T cells. generated from RAD-PBMCs demonstrated more rapid tumor clearance Conclusions and superior control of metastatic tumors in our xenograft model. The TLR9 agonist CpG can be used in ex vivo culture to potentiate Conclusions an anti-tumor T cell product for ACT. The CpG-associated T cell This approach to the generation of VSTs is being translated to the phenotype (IL-2Ralpha-high, ICOS-high, CD39-low) and anti-tumor clinic for use in multiple clinical trials. In future, we aim to elucidate ability was dependent on the presence of B cells and a direct inter- the mechanisms underlying the inhibitory effects of the CD45RA action between the anti-tumor T cells and APC via peptide activation. positive population in the reactivation and expansion of EBVSTs. Post CpG stimulation B cells express heightened CD40, which may promote the B cell/T cell interaction and thus the generation of a po- References tent T cell product for ACT. 1. Bollard CM, Gottschalk S, Torrano V, et al. Sustained Complete Responses in Ethics Approval Patients With Lymphoma Receiving Autologous Cytotoxic T Lymphocytes This study was approved by the Institutional Animal Care & Use Com- Targeting Epstein-Barr Virus Latent Membrane Proteins. Journal of Clinical mittee of the Medical University of South Carolina, protocol number Oncology. 2016;32(8):798-808. doi:10.1200/JCO.2013.51.5304. 0488. 2. Krzywinska E, Cornillon A, Allende-Vega N, et al. CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity. Bobé P, ed. P217 PLoS ONE. 2016;11(4):e0150434. doi:10.1371/journal.pone.0150434. An NK cell line (PD-L1 t-haNK) engineered to target PD-L1 3. Krzywinska E, Allende-Vega N, Cornillon A, et al. Identification of Anti- efficiently kills tumor cells and myeloid derived suppressor cells tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With 1 1 1 Kellsye Fabian, PhD , Michelle Padget , Renee Donahue, PhD , Kristen Hematological Cancers. EBioMedicine. 2015;2(10):1364-1376. doi:10.1016/ 1 2 3 Solocinski, PhD , Clint Allen, MD , John Lee, MD , Patrick Soon-Shiong, j.ebiom.2015.08.021. 3 1 1 MD , Jeffrey Schlom, PhD , James Hodge, PhD, MBA Ethics Approval 1 2 NCI/NIH, Bethesda, MD, United States; NIDCD/NIH, Bethesda, MD, The study was approved by Baylor College of Medicine's Ethics Board, United States; NantKwest, Culver City, CA approval number H7634 and H7666 for human subjects. Animal protocol Correspondence: James Hodge (jh241d@nih.gov) AN5551. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P217 P216 Background CD8+ T cells break tolerance to tumors in a B cell-dependent The ability of natural killer (NK) cells to lyse tumor targets without manner via TLR9 signaling prior sensitization and without human leukocyte antigen (HLA)-re- Aubrey Smith, BS, Hannah Knochelmann, BS, Connor Dwyer, PhD, striction make them promising candidates for “off the shelf” cell- Megan Wyatt, MS, Guillermo Rangel Rivera, Jessica Thaxton, PhD, MSCR, based immunotherapy. Here we investigate the anti-tumor efficacy Mark Rubinstein, PhD, Bei Liu, MD MPH, Eric Bartee, PhD, Chrystal Paulos, of a novel NK cell platform, the PD-L1-targeted high-affinity NK (PD- PhD L1 t-haNK), which lacks killer inhibitory receptors (KIRs), carries a high Medical University of South Carolina, Mount Pleasant, SC, United States payload of granzyme and perforin granules, and has been designed Correspondence: Aubrey Smith (butchera@musc.edu) with a chimeric antigen receptor (CAR) to target PD-L1 expressing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P216 cells on cells. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 118 of 272 Methods Conclusions Frozen, irradiated (15 Gy) PD-L1 t-haNK cells were thawed and charac- The assay provides a sensitive, simple read-out to support discovery terized via flow cytometry and RNAseq. PD-L1 t-haNK lytic activity was research and/or QC lot release applications. assessed in vitro using MDA-MB-231, BT549, T47D, MCF7, SUM149, H460, H441, HCC4006, SW480, SW620, DU145, HTB1, CaSki, and CH22 P220 cell lines as targets in indium-based and flow cytometry-based killing Targeting neoantigens with immunotherapy: Are we limited to assays. The effect of pre-treating tumor targets with IFNγ on PD-L1 t- pre-existing autologous neoantigen-specific T-cells? haNK targeting was also examined. PD-L1 t-haNK cells were co-cultured Aline Bracher, Slavoljub Milosevic, Daniel Sommermeyer with human peripheral blood mononuclear cells (PBMCs) and purified Medigene Immunotherapies GmbH, Planegg-Martinsried, Germany human myeloid derived suppressor cells (MDSCs) to investigate the ef- Correspondence: Daniel Sommermeyer fects of PD-L1 t-haNK on immune subsets. The therapeutic activity of (d.sommermeyer@medigene.com) PD-L1 t-haNK in vivo was studied by adoptive transfer of PD-L1 t-haNK Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P220 cells to NOD scid gamma (NSG) mice engrafted with parental MDA-MB- 231 (PD-L1+) and MDA-MB-231/PD-L1 CRISPR knockout (PD-L1-null). Background Results Several facts shape our considerations regarding the use of neoanti- Here, we show that irradiated PD-L1 t-haNK cells express PD-L1- gens as highly specific targets for immunotherapy of cancer. First, specific CAR, and high levels of perforin and granzyme B. PD-L1 t- adoptive T-cell therapy using TILs seems to be most successful if the haNKs lysed all 14 human tumor cell lines tested in vitro, and in- TILs include T-cells specific for antigens resulting from individual muta- creased cell lysis corresponded with increasing levels of PD-L1 ex- tions. Second, the success of checkpoint inhibitors is often correlated pression on tumor cells. Increasing PD-L1 expression on tumor cells with the mutational load of tumors. However, a mutation must fulfill through IFNγ treatment improved PD-L1 t-haNK-mediated lysis by up several criteria to be effective as a neoantigen that can be recognized to 100%. In vivo, adoptive transfer of PD-L1 t-haNK cells inhibited the by T-cells. Obviously, the mutation must lead to a novel amino acid se- growth of engrafted parental MDA-MB-231 but not PD-L1 null MDA- quence (e.g. single amino acid substitution, fusion- or frameshift- MB-231 tumors. Finally, when co-cultured with human PBMCs and sequence), and be located in a gene expressed in tumor cells. Further- purified human MDSCs expressing PD-L1, PD-L1 t-haNK cells prefer- more, a peptide spanning the new sequence needs to be efficiently entially lysed the MDSC population but not other PBMC subsets. processed and presented by HLA molecules. Finally, a T-cell response Conclusions must be triggered that can specifically recognize the mutated epitope. This study provides a rationale for the potential use of adoptively trans- Targeting neoantigens as patient-individualized epitopes requires ro- ferred irradiated PD-L1 t-haNK cells as a unique immunotherapeutic plat- bust processes for rational and rapid selection and validation of form against a range of human tumor types. In addition to lysing the neoantigens as T-cell targets. Currently, the most challenging step is tumor cells, MDSC killing may be a novel mechanism of action of PD-L1 t- predicting specific T-cell responses. Huge efforts have been made to haNK cells that may potentiate their impact especially in cancers where analyze the reactivity of patients’ T-cells against mutations. However, MDSC’s limit immune approaches. The safety and clinical benefit of PD-L1 this approach is limited to the T-cell repertoire present in patients at t-haNK cells in cancer patients are being assessed in ongoing clinical trials. the time of tumor resection or blood draw and might miss potential Ethics Approval potent T-cell responses that were lacking or no longer present in the The study was approved by the NCI IRB, NIH protocol 99-CC-0168. patient. In our opinion, only screening the T-cell repertoire of several healthy donors can answer the question if a specific mutation can P218 trigger T-cell responses. We present proof-of-concept data how we A novel, bioluminescent assay for the selective detection of target use our high-throughput T-cell receptor (TCR) platform technologies cell killing in mixed cultures and automated processes for fast and efficient screenings of T-cells Richard Somberg, PhD, Brock Binkowski, Aileen Paguio, Peter Stecha, isolated from several partially HLA-matched healthy donors. Chris Eggers, Braeden Butler, Michael Beck, Julia Gilden, PhD, Jey Cheng, Methods Mei Cong, PhD, Frank Fan, PhD Promising neoantigens were predicted and T-cells responses after Promega, Madison, WI, United States stimulation with antigen-presenting cells either transfected with Correspondence: Brock Binkowski (brock.binkowski@promega.com) minigene constructs or loaded with peptides were compared. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P218 Results Peptide stimulation triggered specific T-cells for most tested muta- Background tions, indicating that T-cell repertoires of healthy donors can Efforts to develop and commercialize cellular immunotherapies recognize neoantigens when they are forced to be presented. Also, would benefit from assays that selectively monitor target cell death with the clinically relevant approach using endogenously processed that are sensitive and easy-to-use. To address this, we have devel- antigen encoded by minigenes, specific T-cell responses against oped an approach to selectively quantify target cell death using a neoantigens presented on different HLAs were efficiently triggered, gain-of-signal assay format and bioluminescence read-out. although only against some of the tested epitopes. Methods Conclusions The method relies on the release of a HiBiT-tagged protein from tar- This screening strategy has the aim to develop future TCR-based get cells following cell lysis. HiBiT, an 11 a.a. peptide tag, binds to therapies and can be used for the identification of promising muta- cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconsti- tions for vaccination or as a source for TCRs for adoptive T-cell ther- tute NanoBiT Luciferase. Target cells are engineered to express a apy. Furthermore, generated data can subsequently improve HiBiT-tagged protein using either ectopic expression or CRISPR/Cas9 algorithms predicting the immunogenicity of neoantigens. to tag endogenous lactate dehydrogenase (LDH), and cell lysis is quantified by adding a detection reagent containing LgBiT and furi- P221 mazine substrate (no medium removal). Superior anti-tumor activity of metabolically enhanced bispecific Results antibody armed CAR-less Bionic T cells The signal is proportional to the amount of target cell death, and 1 2 1 Archana Thakur, PhD , John Scholler , Edwin Bliemeister , Carl June, measurements can be made using endpoint or kinetic formats. Cell 2 1 MD , Lawrence Lum, MD, DSc lines have low rates of spontaneous release and fusion proteins that 1 2 University of Virginia, Charlottesville, VA, United States; University of are stable in the extracellular medium, enabling assays up to 24 Pennsylvania, Philadelphia, PA, United States hours or more. The bright signal from NanoBiT Luciferase allows the Correspondence: Archana Thakur (at2fx@virginia.edu) use of low numbers of target cells per well (e.g. 2,500), and the assay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P221 can detect very low levels of target cell death (e.g. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 119 of 272 Background inevitably leads to excessive T cell differentiation ex vivo, and de- The major challenges of T cell-based therapies in solid tumors are creased T cell functionality and persistence in vivo. Importantly, T cell the metabolic insufficiency of T cells to exhibit effector functions and differentiation is tightly controlled by epigenetic mechanisms that persist in an altered metabolic landscape of the tumor microenviron- could be targeted therapeutically. The BET protein BRD4 has been re- ment (TME). To overcome these challenges, we developed the next ported to regulate expression of the transcription factor BATF that generation of chimeric antigen receptors-less (CAR-less) Bionic T cells drives CD8+ T cells towards a more effector-like phenotype, while for the treatment of solid cancers. We hypothesize that metabolically BRD4 inhibition by the drug JQ1 prevented this and thereby en- enhanced Bionic T cells armed with bispecific antibody (BiAb) will hanced in vivo T cell persistence and function in adoptive immuno- show enhanced anti-tumor responses and controlled off-tumor on- therapy models [1]. Ph-29089 is a chemically modified self-delivery target toxicity in solid cancer. RNA inhibitor containing an asymmetric duplex structure (≤ 15 base Methods pair duplex) and a single-strand phosphorothioate tail targeting the We engineered metabolically enhanced “Bionic T cells” (BTC) that are BET protein BRD4. PH-29089 is efficiently delivered to immune cells, devoid of CAR but contain a transmembrane and an intracellular do- including T cells, without the need for specialized formulations or main of a co-stimulatory molecule and TCR signaling domain of mechanical transfection as is observed with current RNAi’s. CD3z. T cells were transduced with CAR-less constructs without co- Methods stimulatory domain-FLAG-ζ (Control) or with co-stimulatory domain- Purified human CD8+ T cells were expanded using the rapid expan- FLAG-4-1BB-ζ, FLAG-CD28-ζ, FLAG-ICOS-ζ and FLAG-OX40-ζ, FLAG-27- sion protocol (REP) developed by the National Cancer Institute. Flow ζ and were tested for their hypoxic tolerance, anti-tumor activity, cytometry was used to study Ph-29089 for its ability to knock down cytokine production and exhaustion phenotype in the presence or BRD4 at the protein level in expanding T cells, and to determine T tumor targets. cell differentiation status during and immediately after ex vivo ex- Results pansion. Release of IFNγ by T cells cocultured with tumor cells was Our data show that hypoxia differentially affected BTC survival de- assessed by ELISA. pending on the co-stimulatory endodomain. Under normoxia Bionics Results with CD28ζ (28z) endodomain show 5% apoptosis versus 61% apop- Ph-29089 elicited a concentration dependent silencing of BRD4 pro- tosis under hypoxic (5% oxygen) condition. On the other hand, Bion- tein with an IC50 of 1-2 μM. The BRD4 silencing persisted at least 5 ics with 4-1BBζ showed only 13% apoptosis under hypoxic condition days post-treatment, whereas media and non-targeting control (NTC) suggesting enhanced hypoxic tolerance. HER2 and EGFR BiAb armed did not significantly affect BRD4 protein levels. Compared to un- BTC were tested against various low-high HER2 and EGFR expressing treated, NTC-treated and JQ1-treated CD8+ T cells, Ph-29089-treated cancer cell lines. Specific cytotoxicity of anti-HER2 BiAb (HER2Bi) and CD8+ T cells contained higher percentages of central memory and anti-EGFR BiAb (EGFRBi) armed BTC against MDA-MB-231, SK-BR-3, stem cell-like memory T cells, as determined by expression of BT-20, MiaPaCa-2 cell lines measured by real time cell analysis using CD45RA, CCR7, CD62L and CD95. Moreover, Ph-29089-treated CD8+ xCELLigence ranged between 75-100% at 2:1 E/T ratio at 72 hours. T cells displayed superior functionality, as indicated by enhanced Sequential killing by HER2Bi armed BTC followed by (f/b) EGFRBi IFNγ production when exposed to the allogeneic melanoma cell lines armed BTC showed efficient killing against target cells (86.2%) or A375 and ROAL. EGFRBi-BTC f/b HER2Bi-BTC (88.2%) compared to the killing by Conclusions HER2Bi-BTC (49.7%) or EGFRBi-BTC (43.5%) alone at low E/T ratio in These findings support the hypothesis that BRD4 silencing by Ph- the presence of 100 IU/ml IL-2 at 96 hours. Cytokine levels of IFN-γ, 29089 is a viable approach for expanding T-cells with superior anti- IL-15, IL-2R, and GM-CSF were significantly higher in culture superna- tumor potential for adoptive cell therapies. tants of tumor cells (SK-BR-3) and HER2Bi-BTC or EGFRBi-BTC co- cultures compared to the control condition with unarmed BTC. Reference Phenotypic data show increased expression of 4-1BB, ICOS and OX40 1. Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, on CD4+ and CD8+ T cells after short antigenic exposure and con- et al. BET bromodomain inhibition enhances T cell persistence and tinuous antigenic exposure. function in adoptive immunotherapy models. J Clin Invest. 2016 Conclusions 126(9):3479–94. Data show that armed BTC: 1) exhibit superior survival in hypoxic Ethics Approval condition; 2) can effectively kill multiple tumor targets in serial killing This study was carried out in accordance with the recommendations of assay; 3) cytokines and chemokines show immune modulating and Karolinska Institutet review board with written informed consent from all tumor killing profile. subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Stockholm Acknowledgements Regional Ethics Committee, with approval number (2011/143-32/1). This study was made possible by start-up funds for LGL from the University of Virginia. AT is a co-founder of NOVA Immune Platform. CJ is a co-founder P223 of T Immunity. LGL is a co-founder of Transtarget, Inc. and sits on the Withdrawn Scientific Advisory Board for Rapa Therapeutics. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P223 P222 Modulating BRD4 in T cells using self-delivery RNAi to improve P224 adoptive cell therapy of cancer 1 1 1 Case studies of sarcoma and MRCLS following treatment with NY- Jeroen Melief, PhD , Laura Van Leeuwe Kirsch, BSc , Esmeralda Hemme , 2 2 2 ESO-1 TCR T Cells (GSK3377794): correlates of predictable John Barrett, PhD , Simon Fricker, PhD , Gerrit Dispersyn, PhD , Rolf response characteristics Kiessling, MD, PhD 1 2 3 1 2 Brian Van Tine, MD, PhD , Sandra D'Angelo, MD , Alexandra Gyurdieva , Karolinska Institute, Stockholm, Sweden; Phio Pharmaceuticals, 3 3 3 3 Laura Johnson , David Turner , Jenna Tress , M. Phillip DeYoung , Yuehui Marlborough, MA, United States 3 3 4 Wu , Aisha Hasan, MBBS MD , Dejka Araujo, MD Correspondence: Rolf Kiessling (rolf.kiessling@ki.se) Washington University in St. Louis, St. Louis, MO, United States; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P222 Memorial Sloan Kettering Cancer Center, New York, NY, United States; 3 4 GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Background Center, Houston, TX, United States Ex vivo expansion of T cells for adoptive cell therapy (ACT) of cancer Correspondence: Brian Van Tine (bvantine@wustl.edu) is commonly done with cytokines and stimuli for efficient activation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P224 and expansion of large numbers of tumor-specific T cells. This Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 120 of 272 Background Background Genetically engineered NY-ESO-1 specific T cells (NY-ESO-1 T Cells; Chimeric Antigen Receptor (CAR) T cell therapy is a revolutionary GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a cancer treatment that genetically alters T cells to redirect and har- self-inactivating lentiviral vector to express an affinity-enhanced NY- ness their cancer killing potential. Currently FDA approved CAR T cell ESO-1-specific T-cell receptor. Phase 1 and 2 trials are evaluating products are autologous-based, requiring individualized blood apher- GSK3377794 in solid tumors and hematologic malignancies. This study esis and manufacture. Deriving patient-specific CAR T cell products is will review biomarker data for eight patients from two ongoing phase expensive, laborious, and time consuming, with logistical and regula- 1/2 pilot studies of GSK3377794 in synovial sarcoma (SS; NCT01343043; tory challenges. The success rate of autologous CAR T cell therapy is N=7) and myxoid/round-cell liposarcoma (MRCLS; NCT02992743; N=1) also limited by the urgency of acute and aggressive cancers, uncer- with prolonged response and stable disease (SD). tainty over T cell number, and intrinsic differences in T cell function- Methods ality. Generating CAR T cells from induced pluripotent stem cells Patients who were progression free ≥4 months following first infu- (iPSC) holds encouraging prospect for generating ‘off-the-shelf’ CAR sion were selected. All received the same lymphodepletion (30 mg/ T cell products and overcoming these challenges. iPSCs can prolifer- m2 x3D fludarabine, 600 mg/m2 x3D cyclophosphamide) followed ate almost infinitely while keeping their pluripotency and lineage by GSK3377794 infusion. Six patients with SS were eligible for second differentiation potential. However, the complexity of T cell develop- infusion and received higher-dose lymphodepletion (30 mg/m2 x4D ment and disturbance of T cell differentiation by CAR expression cre- fludarabine, 1800 mg/m2 x2D cyclophosphamide) before second in- ates a challenge for successful iPSC-derived CAR T cell generation. fusion. Pretreatment biopsies were analyzed for CD3 infiltration by Previously reported iPSC-derived CAR T cells showed innate like phe- RNAScope. Transduced cell persistence was measured by quantitative notypes with weak antigen-specific cytotoxicity and compromised PCR of transgene vectors peripheral blood mononuclear cell (PBMC) cytokine production [1]. DNA. Cytokine expression was measured by Meso Scale Discovery Methods immunoassay. PBMC phenotypes were characterized by flow In our current study, we generated iPSC lines from healthy donor T cells cytometry. by an integration-free method using iPSC reprograming episomal vec- Results tors. The iPSC cells were transduced with clinical grade lentivirus to ex- Five of seven patients with SS had SD for 17.8–105 weeks; two had press CD19-specific CARs (CD19CAR), sorted and colonized to generate partial response/complete response (PR/CR) per RECIST1.1. The pa- a homogeneous CAR+ iPSC cell bank. By using a 3D co-culture system, tient with MRCLS had PR per RECIST1.1 lasting 8.8 months. Six of we successfully generated iPSC-derived CD19CAR T cells. seven patients with SS received second infusion; 2/7 had SD, 3/7 had Results PR, 1/7 had CR. Immunohistochemistry revealed ≥50% of cells with The produced iPSC-derived CD19CAR T cells have a surface marker 2+/3+ NY-ESO-1 expression; this was maintained before second infu- phenotype (CD3+CD5+CD7+TCRalphabeta+CD8alphabeta+ and sion. Baseline tumor samples consistently showed 10 fold over first CD3+CD5+CD7+TCRalphabeta+CD4+) and gene expression signa- infusion; of these, there was one PR and one CR. Cytokine increases tures typical of natural T cells. These iPSC-derived CD19CAR T cells reflecting immune cell activation (eg, IFN gamma, IL-6, and IL-2R expanded robustly within two weeks (~100 fold), and showed potent alpha were observed 4–7 days after both infusions. Transduced T antigen-specific cytotoxicity against CD19+ parental tumor cells such cells within manufactured product showed increased expression of as NALM6 and Raji comparing to their CD19 knockout control cells. It activation markers (eg, CD28, ICOS, and CD40L) versus T cells from is intriguing that the in vitro cytotoxicity potency of iPSC-derived apheresis. In 2 patients, transduced CD8 cells primarily had T effector CD19CAR T cells was superior to conventional PBMC-derived CAR T memory RA+ (CD45RA+CCR7-) and T effector memory (CD45RA- cells generated from the same donor. These iPSC-derived CD19CAR T CCR7-) phenotype. In one, 34.3% transduced CD8 cells had T stem cells also demonstrated efficient degranulation activity and a Th1 cell memory (CD45RA+CCR7+) phenotype. cytokine profile (e.g. IFNgamma and TNFalpha when challenged with Conclusions CD19+ target cells. Furthermore, these cells demonstrated potent SS and MRCLS tumors initially show low immune cell infiltration. anti-tumor activity in vivo in a NSG mouse model using NALM6 as Upon GSK3377794 infusion, increased expression of activated im- target tumor. mune cell cytokines was observed in serum from selected patients. Conclusions Further analysis can provide insights into clinical response character- Our study demonstrates the feasibility of generating naturalistic istics and identification of predictive biomarkers. and functional CAR T cells from iPSCs, which may provide utility in the development of ‘off-the-shelf’ CAR T cell manufacturing Acknowledgements strategies. Medical writing assistance was provided by provided by Fiona Woodward and Chloe Stevenson of Fishawack Indicia Ltd, UK. These studies Acknowledgements (NCT01343043 and NCT02992743) were funded by GlaxoSmithKline (GSK). This research is supported by Mustang Bio. Inc. Trial Registration NCT01343043 and NCT02992743 Reference Ethics Approval 1. Themeli, M., et al., Generation of tumor-targeted human T lymphocytes This study was approved by the appropriate institutional review boards and from induced pluripotent stem cells for cancer therapy. Nat Biotechnol, independent ethics committees. 2013. 31(10): p. 928-33. Ethics Approval The study was approved by the COH Institutional Review Board (IRB) and P225 Office of Human Subjects Protection. Generating iPSC-derived CAR T cells with an endogenous T cell phenotype and conventional CAR T functionality 1 1 Zhiqiang (Daniel) Wang, PhD , Hellen McWilliams-Koeppen, MS , P226 1 1 1 1 Christian Huynh, BS , Hernan Reza , Vibhuti Vyas , Xiuli Wang, PhD , Iovance Gen2 TIL manufacturing process produces drug products 1 1 1 Wen-Chung Chang, MS , Julie Ostberg, PhD , Renate Starr, MS , Jamie that exhibit favorable quality attributes for adoptive cell transfer 1 1 1 1 1 Wagner , Brenda Aguilar, BS , Xiwei Wu , Jinhui Wang , Wei Chen , Chris across 5 solid tumor indications 2 2 1 1 Seet , Gay Crooks , Christine Brown, PhD , Stephen Forman, MD Seth Wardell, Maritza Lienlaf-Moreno, Lavakumar Karyampudi, Anand 1 2 City of Hope, Duarte, CA, United States; UCLA, Los Angeles, CA, United Veerapathran, PhD, Ian Frank, Michelle Blaskovich, BS, Kenneth Onimus, States Arvind Natarajan, Maria Fardis, PhD, MBA, Joe Wypych Correspondence: Zhiqiang (Daniel) Wang (zhwang@coh.org); Christine Iovance Biotherapeutics, Inc., Tampa, FL, United States Brown (cbrown@coh.org); Stephen Forman (sforman@coh.org) Correspondence: Joe Wypych (joe.wypych@iovance.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P225 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P226 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 121 of 272 Background P227 The Iovance Gen2 manufacturing process is a robust T-cell expansion Co-expression of the metabolic enzyme GOT2 with a GPC3- platform that produces a cryopreserved drug product after a 22-day targeted CAR-T overcomes challenges of the solid tumor manufacturing period. Gen2 represents a flexible closed cell produc- microenvironment, substantially improving therapeutic efficacy in tion process that is scalable to meet commercial demand. Drug prod- solid tumor xenografts ucts generated by this process display favorable quality attributes for Kathleen Whiteman, MS, Tapasya Pai, Eugene Choi, Taylor Hickman, Tyler adoptive transfer and the method is reproducible across 5 solid Johnson, Luke Barron, PhD, Taylor Friedman, Madaline Gilbert, Binzhang tumor indications at clinical scale. Shen, Seth Ettenberg, Kathleen McGinness, Greg Motz Methods Unum Therapeutics, Cambridge, MA, United States Methods to assess proliferation, phenotype, and function were ap- Correspondence: Greg Motz (greg.motz@unumrx.com) plied to in-process and final drug products generated with Gen2 at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P227 clinical scale to determine fit within the internally defined target product profile. TIL expansion was assessed through automated enu- Background meration of total and viable nucleated cells. Culture health was The metabolic demands of cancer cells in the solid tumor microenvir- assessed through cellular viability determined by DAPI exclusion. onment (TME) create an unfavorable T cell environment through deple- Immunophenotyping was performed to determine identity and pur- tion of critical nutrients and amino acids and accumulation of waste ity as well as relative levels of activation and differentiation of the products. This drives T cell dysfunction and inhibits the effectiveness of cell product. Cellular function was evaluated as the ability of the cell immunotherapies. To overcome these and other TME challenges, we product to secrete IFN-ɣ in response to CD3, CD28, and 4-1BB recep- developed the BOXR (bolt-on chimeric receptor) platform in which tor engagement. engineered T cells co-express both a chimeric-targeting receptor and a Results “bolt-on” transgene [1]. In a screen of 100+ genes for enhanced T cell Reported herein is the collective experience at Iovance for expansion function when co-expressed with an anti-glypican-3 (GPC3) CAR, we of TIL from five tumor types. The Iovance Gen 2 manufacturing identified the first candidate of our BOXR platform, BOXR1030, which process achieved doses comparable to lifileucel (LN-144, melanoma) co-expresses the transgene glutamic-oxaloacetic transaminase 2 and previously published methods across 5 primary tumor indica- (GOT2), a critical enzyme involved in mitochondrial metabolism. tions (Melanoma: mean 2.83 x 10e10 viable cells, n=82; Cervical: Methods mean 2.31 x 10e10 viable cells, n=53; Head & Neck: mean 5.82 x We compared functional and phenotypic readouts of second- 10e10 viable cells, n=12; Lung: mean 2.09 x 10e10 viable cells, n=3; generation GPC3 CAR-T cells with BOXR1030. Broad transcrip- Sarcoma: mean 1.12 x 10e10 viable cells, n=5). Quality attributes of tome profiling, metabolic characterization, and comprehensive drug products generated with Gen 2 were comparable across all 5 phenotypic assessments were performed; T cell proliferation and primary tumor indications evaluated in terms of T-cell purity, expres- cytokine production under TME-stress conditions (limiting nutri- sion of costimulatory molecules, and memory subsets. Gen 2 drug ents and hypoxia) were evaluated. In vivo, we assessed T cell products across all 5 additional indications continued to exhibit ro- anti-tumor activity, expansion and phenotype using GPC3- bust capacity to produce INF-ɣ upon reactivation, comparable to expressing solid tumor xenograft models in mice. lifileucel. Results Conclusions The addition of GOT2 had pleiotropic effects on BOXR1030 T The Iovance Gen2 manufacturing process allows for the rapid cells, improving multiple T cell functions relative to parent generation of clinical scale doses for patients in urgent need of GPC3 CAR-T cells. BOXR1030 CD4+ T cells had greater polyfunc- therapy. The cryopreserved drug product introduced critical logis- tionality relative to parent CAR-T. BOXR1030 showed improved tical efficiencies and flexibility in distribution that overcame trad- proliferation in vitro, including against TME-challenges. itional barriers to the commercialization of TIL therapy. Gen 2 BOXR1030 CD8+ T cells had a greater proportion of less differ- drug products exhibit favorable quality attributes for adoptive entiated CD27+ cells following production, and CD8+ T cells transfer including high levels of co-stimulatory molecules, and a evaluated ex vivo from xenograft tumors had substantially di- robust capability to secrete cytokine upon reactivation. These minished level of inhibitory receptors (PD-1, Tim-3) suggesting characteristics are reproducible across a broad range of solid resistance to exhaustion in the TME (Figure 1). Further, tumor indications at a high manufacturing success rate opening BOXR1030 was highly efficacious against GPC3-expressing solid the door for many more patients to benefit from this highly tumor models that resisted parental CAR-T therapy (Figure 2), beneficial therapy. [1,2,3] and activity was associated with improved T cell expansion and persistence in peripheral blood. References Conclusions 1. Donia M, Junker N, Ellebaek E, Andersen MH, Straten PT, Svane IM. Co-expression of a metabolic gene to enhance T cell function is Characterization and comparison of ‘Standard’ and ‘Young’ tumor a novel approach to cell therapy for solid tumors. BOXR1030 infiltrating lymphocytes for adoptive cell therapy at a Danish had substantially improved T cell phenotype and function in di- Translational Research Institution. Scand. J. Immunol. 2011;75:157–67. verse ways relative to the parent GPC3 CAR, and GOT2 con- 2. Besser MJ, Shapira-Frommer R, Treves AJ. et al. Clinical responses in a ferred superior activity against numerous TME challenges both phase II study using adoptive transfer of short-term cultured tumor infil- in vitro and in vivo. These results demonstrate that engineering tration lymphocytes in metastatic melanoma patients. Clin Cancer Res. of T cell immunometabolism is an effective and potent strategy 2010;16:2646–55. to overcome the challenges of the solid tumor microenviron- 3. Jin J, Sabatino M, Somerville R, Wilson JR, Dudley ME, Stroncek DF, et al. ment. IND-enabling studies with BOXR1030 are underway with Simplified method of the growth of human tumor infiltrating the expectation that BOXR1030 will be evaluated clinically in lymphocytes in gas-permeable flasks to numbers needed for patient the treatment of GPC3+ malignancies. treatment. J Immunother 2012;35:283–92. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 122 of 272 Reference P228 1. Barron L, Whiteman K, Gilbert M, Pai T, Snyder M, Fray M, Nelson A, Tumor infiltrating lymphocyte recognition of shared neoantigens Johnson T, Lakeman K, Shin J, Boomer R, Ettenberg S, McGinness K, from mutated DNA repair/remodeling proteins in a patient with Motz G. Select metabolic and costimulatory “bolt-on” transgenes metastatic pancreatic adenocarcinoma 1 1 enhance chimeric receptor-bearing T cell activity against solid tu- Ghanshyam Singh Yadav, PhD , Chetana Bhaskarla, PhD , Smriti 1 1 2 2 mors. J Immunother Cancer. 2018;6(Suppl 1):114:110-111, abstract Chaurasia, PhD , Joshua Tobin , Xinming Zhuo , Yinghong Pan , 2 1 P216. Annerose Berndt , Udai Kammula, MD, FACS 1 2 Ethics Approval University of Pittsburgh, Pittsburgh, PA, United States; UPMC Genome This study was approved by Unum Therapeutics’ Institutional Animal Care Center, Pittsburgh, PA, United States and Use Committee (IACUC); approval number 2016-04-004. Correspondence: Udai Kammula (kammulaus@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P228 Background Defective DNA repair, a hallmark of cancer, results in genomic instability and accumulation of genetic abnormalities in many malignancies. Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TIL) rep- resents a personalized cancer immunotherapy capable of targeting shared and private neoantigens resulting from tumor somatic mutations. We sought to interrogate TIL neoantigen reactivity in a tumor harboring a som- atic mutation in the DNA repair gene, ATM (Ataxia-Telangiectasia Mutated). Methods TIL cultures were generated from a surgically resected pancreatic cancer metastasis harboring a somatic ATM mutation. Anti-tumor re- activity of TIL culture was assessed by coculturing TIL with autolo- gous tumor cells and measurement of IFN-gamma release and upregulation of 4-1BB by flow cytometry. Tumor specific mutations were identified by whole genome sequencing (WGS). DNA fragments encoding the altered gene sequences were synthesized and expressed in autologous dendritic cells by RNA electroporation to en- able neoantigen reactivity screening. T cell receptor (TCR) sequencing was performed after single-cell sorting of tumor reactive TIL followed by primer specific PCR for TCR alpha and beta chains. Results Analysis of the pancreatic cancer TIL revealed high level reactivity against autologous tumor. Tumor WGS identified 141 somatic muta- tions (107 SNVs; 19 frameshifts; 15 other). Screening for neoantigen re- activity identified CD8+ T-cell responses against a missense mutation in Fig. 1 (abstract P227). See text for description ATM (23% of TIL) and a frameshift mutation in ARID1A (32% of TIL) but not against respective wild type gene products. TCR sequencing identi- fied a single unique TCR specific for ATM and ARID1A, respectively. Genes encoding the ATM specific TCR were retrovirally transduced into healthy donor T-cells and found to confer strong ATM mutation reactiv- ity without recognition of wild type ATM. Conclusions Over 50% of the TIL expanded from a patient with metastatic pancre- atic cancer were found to recognize neoantigens from either mu- tated ATM or ARID1A, which play a crucial role in DNA repair and chromatin remodeling. ACT using T-cells genetically engineered with these TCRs represents an attractive immunotherapy for patients har- boring these shared tumor mutations. Acknowledgements The study was supported by UPMC Immune Transplant and Therapy Center (ITTC). Ethics Approval This study was reviewed and approved by University of Pittsburgh Institutional Review Board. IRB#18010273. P229 The next generation “off-the-shelf” universal CAR for adoptive immunotherapy 1 1 1 1 Weichih Yang, PhD , Yun Ji , Xiaobing Luo , Huijuan Cui , Michael 1 1 1 1 1 1 Patrick , Yutian Wei , Shigui Zhu , Jiaqi Huang , Xin Yao , Yihong Yao , 2 2 Aibing Liang , Ping Li 1 2 Cellular BioMedicine Group, Gaithersburg, MD, United States; Tongji Hospital of Tongji University, Shanghai, China Correspondence: Yun Ji (yun.ji@cellbiomedgroup.com) Fig. 2 (abstract P227). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P229 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 123 of 272 Background antigen CD4+ and CD8+ responses and cancer-associated antigen- Adoptive immunotherapy using autologous T cells redirected with specific CD8+ T cell responses in vivo. We observe that prophylactic chimeric antigen receptors (CARs) has emerged as a powerful means and therapeutic AAC administration to mice strongly impedes tumor of treating cancer, such as B-cell malignancy. However, this approach growth and extends survival. Following therapeutic immunization, the is limited by the availability of autologous T cells especially for infant anti-tumor responses correlate with an over 10x increase in antigen- patients or patients undergoing multi rounds of chemotherapy. specific CD8+ tumor-infiltrating lymphocytes compared to untreated Methods mice. Finally, in an in vitro human system, we demonstrate that AACs Here we show that employment of the CRISPR-Cas9 system allows can be highly loaded with antigen and adjuvant using CellSqueeze®, highly efficient multiplex gene editing of T Cell Receptor Alpha Con- and that these AACs can be engulfed by human monocyte-derived stant and Beta-2-Microglobulin in primary human T cells, which is dendritic cells. intended to avoid graft-versus-host-disease and minimize the immuno- Conclusions genicity of transferred cells. Furthermore, redirecting the gene- In summary, these results indicate that antigen and adjuvant delivery engineered cells with a B cell maturation antigen (BCMA) CAR led to to APCs in vivo can effectively prime a potent anti-tumor response in their efficient destruction of BCMA+ tumor targets. To further improve mice and support the further study of SQZ AACs as an immunother- the efficacy of these universal BCMA CAR T cells, we use a strategy to apy for cancer treatment. generate the next generation universal CAR T cells by starting from naive precursors and producing the CAR T cells in conditions favoring T P231 memory stem (Tscm) cell expansion. Invariant natural killer T cells as an allogeneic cell therapy Results platform These Tscm enriched gene-engineered BCMA CAR T cells demonstrated Burcu Yigit, PhD, Xavier Michelet, PhD, Simon Yue, Darrian Moskowitz, superior activity compared to the conventional universal CAR T cells Mark Exley, Burcu Yigit, PhD based on their expansion, phenotype, IFN-gamma release, and cytotox- AgenTus Therapeutics, Lexington, MA, United States icity. An early phase clinical trial using this BCMA CAR in an autologous Correspondence: Burcu Yigit (burcu.yigit@agentustherapeutics.com) setting has demonstrated promising clinical readout (NCT03815383). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P231 Conclusions Therefore, we believe this next generation Tscm-enriched universal Background CAR T cells employing the same BCMA vector will provide another al- AgenTus Therapeutics is developing innovative allogeneic and ‘off- ternative choice for multiple myeloma patients in an “off-the-shelf” the shelf’ cell therapies by utilizing invariant natural killer T cells manner similar to other biological drugs. (iNKT) to target solid and liquid tumors. iNKT cells are innate-like lym- phocytes that bridge innate and adaptive immune responses. They P230 can be activated via their invariant T cell receptor recognizing lipid Activating antigen carriers for cancer therapy: preclinical immune antigens (e.g. alpha-Galactosylceramide) presented on CD1d mole- responses drive tumor regression cules, through NKG2D - NKG2D ligand interactions, and by cytokines. Defne Yarar, PhD, Amritha Ramakrishnan, Katarina Blagovic, PhD, Upon activation, large amounts of IFN-gamma production leads to Katherine Seidl, Howard Bernstein, MD, PhD, Armon Sharei recruitment and activation of T cells and NK cells. iNKT cells also SQZ Biotechnologies, Watertown, MA, United States exert potent direct cytolytic activity. While they are found in very low Correspondence: Defne Yarar (defne.yarar@sqzbiotech.com) numbers in human blood (~ 0.01% of T lymphocytes), some of their Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P230 unique properties make them valuable for cell therapy platforms. Due to their invariant antigen receptor, their ability to cause GvHD is Background minimal, and in fact, they have been demonstrated to suppress Productive activation of the immune system by antigen presenting GvHD in BMT settings. This facilitates the use of iNKT cells in an allo- cells (APCs) loaded ex vivo has proven to be challenging. To over- geneic cell therapy setting. In addition, iNKT cells are very efficient in come this, we have developed an approach that harnesses the nat- infiltrating solid tumors to exert their cytotoxic function and activate ural process of red blood cell (RBC) clearance from the body to other anti-tumor immune cells. activate the immune response in vivo. Using the CellSqueeze® micro- Methods fluidics platform, we have generated activating antigen carriers Due to low frequency of circulating iNKT cells, we have developed (AACs), engineered from RBCs, that are highly loaded with antigen and optimized a method to isolate and generate large numbers of and adjuvant and potently activate APCs in vivo. Here, we show that these cells in vitro for use in ‘off-the-shelf’ and allogeneic setting. AAC-mediated antigen and adjuvant targeting to APCs drives antigen Results presentation in vivo and primes potent anti-tumor T cell responses. We can achieve over 40,000-fold expansion of iNKT cells through Methods stimulation of the invariant TCR in less than 30 days. Importantly, To generate AACs, we loaded proteins or synthetic long peptide anti- after such massive expansion, iNKT cells retain their inherent cyto- gens together with adjuvants into murine or human RBCs with CellS- toxic capacity and cytokine production in response to tumor cells. queeze®. Following intravenous AAC injection into mice, we measured Conclusions AAC clearance kinetics from the blood and characterized the site and AgenTus is applying its proprietary Antigen Receptor platforms to cell type of AAC uptake. In addition, we quantified endogenous im- identify novel CARs and TCRs directed against tumor-specific anti- mune responses to AAC administration by flow cytometry. To deter- gens. We believe that through modification with tumor-targeted mine the ability of AACs to control subcutaneously implanted tumors, CARs and TCRs, iNKT cells will serve as potent allogeneic cell therapy we measured tumor growth rates in mice treated either prophylactic- vehicles. This should enable an ‘off-the-shelf’ approach for improving ally or therapeutically with AACs. Finally, to assess if AACs could be patient access to cell therapy. engulfed by antigen-presenting cells in a human system, we quantified in vitro uptake of human AACs by monocyte-derived dendritic cells P232 using flow cytometry and fluorescence microscopy. Characterization of ADCC resistance in multiple cancer types Results David Zahavi, MS, BS, Yongwei ZHANG, Sandra Jablonski, PhD, Louis We demonstrate that CellSqueeze® loads antigen and adjuvant into Weiner, MD AACs effectively. When administered into a mouse, these carriers are Georgetown University, Washington, DC, United States cleared from circulation within one hour and are engulfed by profes- Correspondence: Louis Weiner (weinerl@georgetown.edu) sional phagocytes in both the spleen and liver. Moreover, we find that Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P232 murine SQZ AACs processed with CellSqueeze® stimulate model Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 124 of 272 Background conjugated with H-2Kb OVA, m4-1BBL and mIL-12. Naïve OT1 Antibody-dependent cell-mediated cytotoxicity (ADCC) is an import- cells were transferred into tumor bearing mice followed by ant mechanism of action in targeted monoclonal antibody (mAb) mRBC-321 treatment. Dramatic dose-dependent expansion of cancer immunotherapy. The majority of patients who receive tar- OT1, comprised of functional effector and central memory pheno- geted mAbs develop resistance to therapy and there remains a great types, was observed in peripheral blood (9468-fold Tem; 146-fold need to understand resistance mechanisms. In vitro modeling of Tcm expansion) and secondary lymphoid organs (3323-fold Tem; ADCC provides an experimental system for uncovering tumor cell 725-fold Tcm expansion in spleen) on day 7. mRBC-321 treatment based immune resistance mechanisms. using an EG7.OVA tumor model resulted in tumor regressions in Methods 12/16 mice, 6 of which were cured, and increased survival (p Utilizing our in vitro model system of continuous selection pressure Conclusions with NK92-CD16V effector cells and the mAbs Cetuximab and Trastu- Based on these results, human RTX-321 expressing h4-1BBL, hIL-12, zumab we have generated three ADCC resistant cell lines from par- and HLA-A*02 with an HPV E7 peptide was generated to activate ental A431, SK-OV-3, and FaDu cells. and expand HPV E7-specific T cells. RTX-321 activated TCR signaling Results in an engineered HPV E7-specific TCR Jurkat line and stimulated 4- We show that the induction of ADCC resistance in all three cells lines 1BB and IL-12R signaling in respective reporter cell lines. These re- involves a loss of target cell adhesion properties required for the es- sults support the clinical development of RTX-321, which is currently tablishment of an immune synapse, NK cell activation, and target cell in IND-enabling studies for the treatment of HPV16+ advanced solid cytotoxicity. Remarkably, ADCC-resistant cells possess reduced cell tumors. surface expression of multiple proteins that contribute to intercellular interactions and immune synapse formation. We have termed the P234 loss of a selection of cell surface proteins which contributes to ADCC DAP10 and DAP12 signaling based CAR circumvents ligand- resistance Testudinidosis. This phenomenon is characterized by dys- dependent tonic signaling and mediates potent anti-tumor regulation of protein trafficking and subcellular localization of the cell response in vivo surface molecules. Additionally, ADCC resistant cell lines exhibit aber- Alan Epstein, MD, PhD, Long Zheng, Long Zheng rant IFN/STAT1 signaling. University of Southern California, Los Angeles, CA, United States Conclusions Correspondence: Alan Epstein (aepstein@usc.edu) Using multiple cell lines to model ADCC resistance has led to the dis- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P234 covery of a shared mechanism of resistance across cancer types that may reveal potential therapeutic targets for combination Background immunotherapy. The Lym-1 antibody which targets a unique, discontinuous epitope on the HLA-Dr protein expressed in human B-cell lymphomas and leukemias de- P233 veloped in the late 1970’s by co-author ALE has been found to be clinically RTX-321, an allogeneic artificial antigen presenting red cell safe and effective as an I-131 radioimmunoconjugate [1-3]. Based upon therapeutic, expressing MHC I-Peptide, 41BBL and IL12, promotes these early clinical data, we have generated a humanized version (huLym- antigen-specific T cell expansion and anti-tumor activity in HPV16+ 1-B) to construct chimeric antigen receptors (CARs) using the single chain tumors variable fragment (ScFv) of huLym-1-B to treat Lym-1 positive tumors. Xuqing Zhang, PhD, Tiffany Chen Methods Rubius Therapeutics, Cambridge, MA, United States The antibody ScFv was ligated to a lentivirus vector in frame with Correspondence: Tiffany Chen (tiffany.chen@rubiustx.com) CAR backbone and CAR lentiviruses were produced and used to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P233 transduce primary CD3+ human T-cells. After transduction, we char- acterized the expansion, the effector function, and the immune- Background phenotypes of CAR-T cells. The in vivo efficacy of the CAR-T cells was Autologous CAR-T therapy has demonstrated efficacy in a small measured in a metastatic Raji lymphoma xenograft 8 days after the subset of hematological cancers. The wider adoption of antigen- iv injection of 1 million tumor cells in NSG mice. specific therapies has been limited by significant toxicity and a Results lack of effectiveness in solid tumors. Manufacturing is costly, huLym-1-BCAR was successfully transduced at an efficiency between time-consuming and difficult to scale. To address these limita- 50-80%. Surprisingly, the CAR transduced T-cells showed limited expan- tions, Rubius Therapeutics has genetically engineered red cells to sion, reaching approximately 8-fold expansion after 11 days of culture create an allogeneic artificial antigen-presenting cell (aAPC), compared to mock T-cells which had a robust 310 to 380-fold expan- called RTX-321, for the treatment of HPV16+ advanced solid tu- sion. In expanded CAR-T cells, over 28% of cells showed phosphory- mors. RTX-321 presents an HPV E7 peptide on major histocom- lated CD3z and increased PD-1 and LAG3 expression indicative of T-cell patibility complex I (MHC I [HPV]), a costimulatory signal (4-1BBL) exhaustion which was not detected in mock T-cells. Since Lym-1 recog- and a membrane-bound cytokine (IL-12) on the cell’ssurface to nizes a conformational epitope on HLA-Dr which may be weakly mimic the human immunobiology of T cell APC interactions. RTX- expressed by activated T-cells, we speculated that the huLym-1-BCAR 321 is designed to activate and expand tumor-specific T cells may stimulate the transduced T-cells to cause tonic signaling and ex- present within the patient and eliminates the need for manufac- pansion failure. This was confirmed by flow cytometry that detected turing patient-derived T cells. huLym-1-B but not huLym-1-Bmut, a huLym-1-B mutant that lost its Methods binding ability, binding on T cells. Meanwhile, neither huLym-1-Bmut As a proof of principle, red cells engineered to express mouse based CAR or huLym-1-BCAR with inactive CD3z domains showed de- MHC I H-2Kb loaded with OVA 257-264 peptide (H-2Kb OVA), creased expansion or increased CD3z phosphorylation. murine 4-1BBL (m4-1BBL) and murine IL-12 (mIL-12) were used to Conclusions stimulate OT1 cells. Although the proliferation issue did not compromise CAR-T cells’ ef- Results fector function, this would impose a potential challenge for large-scale Compared to cells expressing H-2Kb OVA or m4-1BBL alone, manufacture. We discovered that replacing the BB3z in huLym-1-BCAR these cells induced up to a 14-fold expansion of OVA antigen- with signaling domains from DAP10 and DAP12 addressed this prolifer- specific OT1 cells in vivo. These expanded cells displayed a mem- ation issue but enabled potent anti-tumor efficacy in vivo. In addition, ory phenotype and enhanced antigen-specific tumor killing of the expanded huLym-1-B DAPCAR-T cells enabled a lower dose of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate, injected CAR T-cells to induce durable control of metastatic lymphoma mRBC-321, was created using murine red cells chemically in mice with large tumor burdens. Further testing of DAP signaling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 125 of 272 sequences in other CAR T-cells may show that this change also im- suggesting myeloid cell proliferation. Finally, stable or partial re- proves the clinical efficacy of CAR T-cell therapy directed against other sponse to therapy tracked with either a dense T cell- or myeloid-cell antigens targeted in both hematopoietic and solid tumors. infiltrate while patients who progressed displayed low infiltrate levels. Acknowledgements Conclusions This work is supported by Cell Biotherapy, Inc., Los Angeles, CA. In conclusion, immunotherapy associated skin rash contains a com- plex immune infiltrate, with increased cellular proliferation among References sites of dense infiltration. The highest levels of T cell or myeloid cell 1. Epstein AL, et al. Two new monoclonal antibodies, Lym-1 and Lym-2, re- infiltrate were seen in stable or responding patients, suggesting a active with human B-lymphocytes and derived tumors, with immunodi- link between the immune profile of skin rash and therapy response. agnostic and immunotherapeutic potential, Cancer Res. 1987; 47:830-840. Ethics Approval 2. DeNardo GL, et al. Low-dose, fractionated radioimmunotherapy for B-cell The study was approved by The Northwestern University Ethics Board maligancies using 131I-Lym-1 antibody. Cancer Biother Radio. 1998; 13:239-254. 3. Hu E, et al. A phase 1a clinical trial of LYM-1 monoclonal antibody ser- otherapy in patients with refractory B cell malignancies. Hematol Oncol. P236 1989; 7:155-166. Nivolumab related side effects based on patient-reported Ethics Approval outcomes: A multicenter study from real-life setting 1 1 This study was approved by the IRB of the University of Southern California, Canan Karadas , Zehra Gok Metin, Assoc Prof, PhD, RN , Nur Izgu, PhD, 1 2 2 protocol number HS-16-00029 approved 2-29-16, and by IACUC protocol RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, Checkpoint Blockade Therapy Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Correspondence: Canan Karadas (karadas.canan@gmail.com) P235 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P236 A dense, proliferative myeloid and T cell-rich, immune infiltrate characterizes immunotherapy-induced skin rash Background Cormac Cosgrove, PhD, Cory Kosche, Suyeon Hong, Caroline Le Poole, Immune checkpoint inhibitors provide an effective treatment option Jennifer Choi for patients with melanoma and other cancer types. Nivolumab, as Northwestern University, Chicago, IL, United States an immune checkpoint inhibitor, acts via blockade of the PD-1 recep- Correspondence: Cormac Cosgrove tor, and limits immune responses against tumors. As reported for (cormac.cosgrove@northwestern.edu) nivolumab, the anti-PD-1 antibodies can induce immune-related ad- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P235 verse events [1, 2]. Nivolumab related adverse effects compose of general, pulmonary, gastrointestinal, neurologic, skin, and infusion re- Background actions. These adverse effects may be life threatening, so following Checkpoint inhibitor immunotherapy is associated with a unique tox- of patients receiving nivolumab is essential for early diagnosis and icity profile, collectively known as immune related adverse events management. Therefore, this study aimed to examine nivolumab re- (irAEs). One of the earliest and most common irAEs is skin rash. Inter- lated adverse effects based on patient- reported outcomes (PROs) estingly, development of rash has been associated with improved from a real-life setting. survival, and is likely an early indicator of a successfully activated im- Methods mune response. More severe toxicities also occur, can affect nearly In total, 40 patients receiving first cycle of nivolumab were included every organ and are clinical justification for dose reduction or termin- from three hospitals located in Turkey. Patient Information Form and ation of immunotherapy. However, the mechanism underpinning Patient Monitoring Checklist-Nivolumab were used for data collection. rash development and its link to disease outcome or toxicities is not Patient Monitoring Checklist-Nivolumab included totally 46 questions: known. Thus, we examined the makeup of immunotherapy- (21-general adverse effects, 3-pulmonary, 8-gastrointestinal, 5- associated skin rash infiltrates with the goal of uncovering mechanis- neurological, 2-skin, and 7-infusion reactions) and two options in each tic insights behind rash development. item that evaluating presence of related adverse effect, as “yes” or “no”. Methods PROs were analyzed by frequency and percentages. Immunohistochemistry was used to describe the immune infiltrate in Results skin biopsies from healthy subjects and from a lesional site of patients The mean age of patients was 55.47±16.48 years, and the majority of who developed rash secondary to immunotherapy. Rash samples were them (82.5%) had diagnosed with cancer longer than one year. The obtained from 7 patients receiving α-PD1, α-CTLA4/α-PD1 combination cancer diagnosis composed of melanoma (52.5%), renal cell carcin- or α-PDL1/α-NKG2A combination. Acetone-fixed sections from frozen oma (17.5%), nasopharyngeal carcinoma (7.5%) and other cancer biopsies were stained for CD3, CD4, CD8, CD68, CD11c, CD1a, CD207, types (23.5%). Considering differences in terms of gender, only two or Ki67. Cell abundance in the dermis was compared among groups. items including 8th item related general adverse effects, quiring loss Results of hair, and 37th item evaluating changes in mental status, percep- : Rash samples showed significant enrichment of T cells (CD3 p= tion, judgement, and memory had a significant difference. Female 0.01), CD8 T cells (p=0.03) and dendritic cells (CD11c p=0.03) in the patients reported higher adverse effects than male regarding afore- dermis vs controls. More moderate enrichment of macrophages mentioned items (p (CD68) was observed in rash versus control samples while the abun- Conclusions dance of dermal Langerhans cells (CD1a or CD207) was comparable PROs related nivolumab in this study included fatigue, rash and itch- among groups. We observed more proliferating cells in the dermis of ing, and diarrhea as consisted with previous reports. A comprehen- rash vs control samples (Ki67 p=0.024), associated with areas of sive approach is needed to reduce and manage nivolumab related dense immune infiltration. Ki67 expression was highly correlated with adverse effects. both CD68 (r=0.89; p=0.012) and CD11c (r=0.786; p=0.048), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 126 of 272 References aware of the rare, debilitating, and possibly previously undescribed 1. Naidoo J, Page DB, Li BT, Connell LC, Schindler K, Lacouture ME, Wolchok paraneoplastic and autoimmune toxicities that may occur. JD. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint anti- bodies. Ann Oncol. 2015; 26:2375–2391. References 2. Spain L, Diem S, Larkin J. Management of toxicities of immune 1. Forster MD, Devlin MJ. Immune Checkpoint Inhibition in Head and Neck checkpoint inhibitors. Cancer Treat Rev. 2016; 44:51–60. Cancer. Front Oncol. 2018; 8:310. Ethics Approval 2. Pollack MH, Betof A, Dearden H. Safety of resuming anti-PD-1 in pa- The study was approved by clinical trials ethics committee of the University tients with immune-related adverse events (irAEs) during combined of Health Sciences Ankara Oncology Training and Research Hospital anti-CTLA-4 and anti-PD1 in metastatic melanoma. Ann Oncol. 2018; (decision number: 2018–06/52) and performed in accordance with the 29:250-255. Helsinki Declaration. 3. Bataller L, Wade DF, Graus F. Antibodies to Zic4 in paraneoplastic neurologic disorders and small-cell lung cancer. Neurology. 2004; 62. 4. Schwab KS, Kristiansen G, Schild HH. Successful Treatment of Refractory P237 Squamous Cell Cancer of the Head and Neck with Nivolumab and A case of anti-Zic4 antibody-mediated cerebellar toxicity induced Ipilimumab. Case Rep Oncol 2018;11:17–20. by dual checkpoint inhibition in HNSCC Consent Sunil Iyer, MD, Nidah Khakoo, MD, Gabriella Aitcheson, MD, Marina Consent was obtained from the patient for publication of this abstract. Kushnirsky, MD, Cesar Perez, MD University of Miami, Miami Beach, FL, United States Correspondence: Sunil Iyer (sunil.iyer@jhsmiami.org) P238 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P237 Longitudinal immune and genomic monitoring reveals signatures of response and Immune-related adverse events in cancer patients Background receiving checkpoint inhibitor therapy Combined checkpoint inhibition therapy targeting the PD-L1 and Shaheen Khan, PhD, David Gerber CTLA4 pathways has been a successful approach in the treatment of UT Southwestern Medical Center, Dallas, TX, United States metastatic melanoma, leading to its investigation in the treatment of Correspondence: David Gerber (david.gerber@utsouthwestern.edu) head and neck squamous cell carcinoma (HNSCC) with PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P238 sion [1]. Despite the potential for excellent responses, an increased rate of autoimmune neurological toxicity and paraneoplastic conditions has Background been observed [2]. We present the case of a patient with metastatic Despite the remarkable success of immune checkpoint inhibitor (ICI) HPV-positive HNSCC treated with ipilimumab/nivolumab who experi- therapy, a significant number of patients develop severe and unpredict- enced severe cerebellar ataxia, with a positive screen for the anti-Zic4 able immune-related adverse events (irAEs) affecting a wide variety of antibody, which has been associated with cerebellar degeneration in organs. Concerns over irAE have led to the exclusion of patients with small cell lung cancer (SCLC) and has not been reported in HNSCC [3]. autoimmune disease from ICI clinical trials. Role of host genetic and im- Results mune factors in mediating irAEs remain unclear and it is not clear if the A 40-year-old man diagnosed with HPV-positive HNSCC with meta- manifestations of irAEs is associated with response to therapy. static recurrence after radiation treatment of the initial tumor was Methods started on a clinical trial of a DNA-PK inhibitor. His disease pro- We used multi-faceted approach to identify blood-based biomarkers gressed, and given his PD-L1 tumor proportion score of 70% he was predictive of irAEs and response to ICI therapy. We characterized initiated on ipilimumab/nivolumab. After his second cycle, he pre- changes in host immune system in 200 patients receiving ICI therapy sented with sudden blurred vision and mild ataxia, which rapidly pro- at baseline and post-immunotherapy (100 with irAEs and 100 without gressed to severe ataxia and dysarthria. Autoimmune toxicity was irAEs). We assessed genetic predisposition to autoimmunity in these suspected; initial brain imaging and serum testing were unremark- patients using the Illumina GSA SNP array and via targeted resequen- able. While awaiting the results of complex autoimmune and para- cing of over 150 immunoregulatory loci including the HLA region. We neoplastic CSF testing, he was treated with multiple modalities in an evaluated serum levels of cytokines/chemokines, Antinuclear auto- escalating fashion with minimal improvement, including pulse-dose antibodies (ANA) and 124 autoantibodies and performed RNA se- corticosteroids, IVIG, and plasmapheresis. The paraneoplastic panel quencing and flow cytometry on peripheral blood mononuclear cells returned negative for common autoimmune culprits in cerebellar en- (PBMCs) at baseline and post immunotherapy in patients with and cephalopathy including anti-Hu and anti-Yo; however, anti-Zic4 was without irAEs. detected at borderline levels. Repeat MRI showed an enhancing le- Results sion in the cerebellum. Finally, rituximab was initiated, and the pa- Our preliminary data analysis identified signatures of autoantibodies tient is slowly improving. Notably, restaging scans show a mixed and cytokines correlating with response and toxicity. We also identi- response with resolution of previously extensive metastatic disease fied HLA haplotypes linked with autoimmunity in selected patients in the thorax, however with worsening osseous lesions. that developed immune-related adverse events. In this meeting, we Conclusions will present immune and genetic correlates of irAEs and response to We present a case of anti-Zic4-mediated cerebellar toxicity in the set- therapy in our patient cohort. ting of dual PD-L1/CTLA4 inhibition in the treatment of metastatic Conclusions HNSCC. Anti-Zic4 has been historically associated with cerebellar- In-depth analysis of immune and genetic datasets is currently under- predominant paraneoplastic neurological disorders in SCLC [3], and way. We hope that our studies can help identify blood-based bio- to our knowledge, has not been described in HNSCC. Although the marker signatures predictive of irAEs and/or response and reveal patient experienced an impressive partial response, he suffered novel insights into the mechanisms underlying irAE. Our current gen- grade 4 cerebellar neurotoxicity. Cases have demonstrated excellent etic data suggests further expanding the genetic studies in larger pa- clinical responses utilizing dual PD-L1/CTLA4 inhibition in HNSCC [4], tient population to identify the role of underlying genetic however high-grade adverse events have also been reported in this predisposition to autoimmunity in mediating irAEs. We hope our regimen’s more established use in metastatic melanoma [2]. Despite findings may ultimately help identify customize therapy, expand use the exciting advances in cancer immunotherapy, clinicians must be of immunotherapy and prevent toxicities. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 127 of 272 P239 Table 1 (abstract P239). ICI-arthritis patient characteristics (total n=372) Immune checkpoint inhibitor-associated arthritis: a systematic literature review of case series and case reports 1 2 3 Michael Tiongson, BA , Nilasha Ghosh, MD , Carolyn Stewart, BA , 2 2 2 Karmela Chan, MD , Bridget Gatto, MLIS , Anne Bass, MD 1 2 Albany Medical College, Nanuet, NY, United States; Hospital for Special Surgery, New York, NY, United States; Weill Cornell Medicine, New York, NY, United States Correspondence: Nilasha Ghosh (GhoshN@HSS.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P239 Background As immune checkpoint inhibitors (ICI) continue to revolutionize can- cer treatment, immune-related adverse events (irAE) are becoming more prevalent. Inflammatory arthritis occurs in approximately 4% of ICI-treated patients [1] but remains poorly characterized. We per- formed a systematic literature review to identify all reports of ICI- associated inflammatory arthritis in order to describe it phenotypic- ally and serologically. Methods PubMed, Embase and Cochrane databases were searched for pub- lications reporting musculoskeletal irAEs secondary to ICI treat- ment through the search date, May 31, 2019. Publications were included if they provided individual patient-level data regarding the pattern of joint involvement. Two reviewers screened all ab- stracts and full texts to extract demographics, clinical features, se- rologies, treatment data and outcomes. Descriptive statistics were used to summarize results. Results 4339 articles were screened, of which 67 were included (42 case reports, 15 case series, 10 retrospective chart reviews) encom- passing 372 patients (Table 1). Mean age was 63 +/- 11 years; 61% patients were male. The majority of patients had metastatic melanoma (57%) and were treated with anti-PD1 or anti-PDL1 P240 therapy (78%). Median time to onset of arthritis was 4 months Quantitative cell-based bioassays to advance immunotherapy (range: 1 day-53 months). 49% had polyarticular arthritis, 17% oli- programs targeting immune checkpoint receptors goarthritis, 3% monoarthritis, 10% arthralgia and 21% polymyalgia Vanessa Ott, PhD, Jamison Grailer, PhD, Jun Wang, Julia Gilden, PhD, rheumatica (PMR). 9% tested positive for rheumatoid factor (RF) Pete Stecha, Denise Garvin, Michael Beck, Jim Hartnett, Frank Fan, PhD, or cyclic citrullinated peptide (CCP) antibodies. 74% required cor- Mei Cong, PhD, Zhi-jie Jey Cheng ticosteroids and 45% required additional therapies, including 5% Promega, Madison, WI, United States requiring a TNF inhibitor. 63% of patients achieved control of Correspondence: Zhi-jie Jey Cheng (jey.cheng@promega.com) their musculoskeletal symptoms with treatment, and 32% were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P240 ultimately able to discontinue anti-rheumatic treatments. ICI were continued in 49%, transiently withheld in 11%, and permanently Background discontinued due to musculoskeletal irAEs in 13%. At last follow- The human immune system is comprised of a complex network of im- up, 27% had progression of their cancer. mune checkpoint receptors that are promising new immunotherapy tar- Conclusions gets for the treatment of a variety of cancers and autoimmune-mediated Half of reported ICI-associated arthritis cases have a polyarthritis disorders. Immunotherapies designed to block co-inhibitory receptors (e.g. (often in an RA distribution) but only 9% are seropositive. PMR is also PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of can- commonly seen. The vast majority of ICI-arthritis cases are in melan- cer. However, not all patients and tumor types respond to this approach. oma patients treated with anti-PD1/PDL1 therapy. Most patients re- This has resulted in broadening of immunotherapy research programs to spond to steroids alone but about half require additional anti- target additional co-inhibitory (e.g. LAG-3, TIM-3) and co-stimulatory (e.g. rheumatic agents. Further studies are needed to determine long- 4-1BB, GITR, OX40, ICOS) receptors individually and in combination. term musculoskeletal outcomes in these patients and the impact of Methods arthritis treatment on cancer survival. A major challenge in the development of biologics is access to quantita- tive and reproducible functional bioassays. Existing methods rely on pri- Reference mary cells and measurement of complex functional endpoints. These 1. Kostine M, Rouxel L, Barnetche T, Veillon R, Martin F, Dutriaux C, assays are cumbersome, highly variable and fail to yield data quality re- Dousset L, Pham-Ledard A, Prey S, Beylot-Barry M, Daste A. Rheum- quired for drug development in a quality-controlled environment. To ad- atic disorders associated with immune checkpoint inhibitors in pa- dress this need, we have developed a suite of cell-based functional tients with cancer—clinical aspects and relationship with tumour bioassays to interrogate modulation of immune checkpoint receptors indi- response: a single-centre prospective cohort study. Ann Rheum Dis. vidually (e.g.PD-1,CTLA-4,LAG-3, TIM-3, GITR, 4-1BB) and in combination 2018; 77:393-8. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 128 of 272 (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) that express luciferase reporters driven by response elements under the and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) precise control of mechanistically relevant intracellular signals. could serve as subpopulations relevant for patient stratification (Figure Results 2). We further validated these results using CyTOF wherein these same The bioassays reflect mechanisms of action for the drug candidates de- subpopulations were differentially abundant between responders and signed for each immune checkpoint receptor and demonstrate high spe- non-responders at the pre-therapy time-point. cificity, sensitivity and reproducibility. For example, using the PD-1 Conclusions blockade bioassay, TCR-mediated luciferase activity is recovered with Collectively, our results revealed that (1) PD-1 blockade-based anti-PD-1 and PD-L1 blocking Abs but not with unrelated control anti- treatment-induced gene expression profiling changes (increase cell bodies. Similarly, TCR and CD28-mediated luciferase activity is recovered proliferation, metabolism and activation) of CD8 T cells are detect- in the CTLA-4 blockade bioassay with an anti-CTLA-4 blocking antibody able as early as EOC1 in the PB; (2) specific subpopulations of plastic (ipilimumab), but not with unrelated control antibodies. In a PD-1+CTLA- CD8 T cells identified have the potential to serve as an actionable 4 combination bioassay, anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimu- biomarker to select AML patients most likely to benefit from such im- mab) blocking antibodies individually induced luciferase activity (3.0- and mune checkpoint therapies. These findings need to be confirmed in 5.6-fold, respectively), the combination of both antibodies resulted in a larger studies with αPD-1 based therapies in AML. synergistic 18-fold increase in luciferase activity. Similar antibody-induced Acknowledgements luciferase activity is observed in a panel of bioassays specific for agonist NIH (R01CA174385), CPRIT (RP180466), MRA Established Investigator Award antibodies, including GITR, 4-1BB, OX40 and CD40. This response can be to NV (509800), Welch Foundation (E1774), NSF (1705464), CDMRP enhanced following Fc receptor cross-linking, reflecting in vivo activity. (CA160591), and Owens foundation. We would like to acknowledge the Conclusions MDACC Flow Cytometry and Cellular Imaging Core facility for the FACS Cell-based reporter bioassays overcome the limitations of primary sorting (NCI P30CA16672), UH Seq-N-Edit core for RNA-sequencing service, cell-based assays for functional characterization of antibody and and Intel for the loan of computing cluster. other biologics drugs targeting individual or combination immune Trial Registration checkpoint receptors. Here we show a portfolio of mechanism of NCT02397720 action-based bioassays for co-inhibitory and co-stimulatory immune References checkpoint receptors that can be used for antibody screening, 1. Daver, N. et al. Efficacy, Safety, and Biomarkers of Response to Azacitidine characterization, potency and stability studies. and Nivolumab in Relapsed/Refractory Acute Myeloid Leukemia: A Nonrandomized, Open-Label, Phase II Study. Cancer Discov. 2019; 9: 370–383. P241 2. Corces, M. R. et al. Lineage-specific and single-cell chromatin accessibility Single-cell deconvolution identifies T-cell correlates of response to charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016; PD-1 blockade treatment in AML 48: 1193–1203. 1 1 2 2 Xingyue An, BS , Jay R Adolacion , Mansour Alfayez , Jairo Matthews , 3. Linsley, P. S., Speake, C., Whalen, E. & Chaussabel, D. Copy number loss of 2 2 1 2 Wilmer Flores , Steven Kornblau, MD , Arash Saeedi , Sreyashi Basu , the interferon gene cluster in melanomas is linked to reduced T cell 2 1 Naval Daver, MD , Navin Varadarajan, PhD infiltrate and poor patient prognosis. PLoS One. 2014; 9. 1 2 University of Houston, Houston, TX, United States; Univ. of Texas MD 4. Szczepanski, M. J. et al. Increased frequency and suppression by Anderson Cancer Center, Houston, TX, United States regulatory T cells in patients with acute myelogenous leukemia. Clin. Correspondence: Navin Varadarajan (nvaradarajan@uh.edu) Cancer Res. 2009; 15: 3325–3332.[5. Knaus, H. A. et al. Signatures of CD8+ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P241 T cell dysfunction in AML patients and their reversibility with response to chemotherapy. JCI Insight 2018; 3. Background Ethics Approval The combination of the αPD-1 (nivolumab) and hypomethylating agent All patients signed an informed consent form approved by the Institutional azacytidine demonstrated encouraging response in R/R acute myeloid Review Board (IRB) from The University of Texas MD Anderson Cancer leukemia (AML) patients, but the percentage of patients who achieved Center. The study was conducted in accordance with the Declaration of IWG 2016 responses was limited[1]. Early predictive biomarkers to facili- Helsinki. The study was approved by the IRB from University of Houston. tate future trials patient selection are desirable. A better understanding of T cells in AML pre-therapy and on-therapy should yield valuable in- Table 1 (abstract P241). See text for description sights on the treatment-induced anti-tumor response. Methods We performed RNA-sequencing on T cells from a cohort of AML pa- tients who were treated with azacytidine and nivolumab (Table 1). By leveraging subset definitions based on single-cell RNA-sequencing results from T cells of cancer patients, we implemented deconvolu- tion of our bulk T-cell RNA-sequencing data to obtain the relative abundance of different T-cell subsets (in-silico dissection). Results For validation purpose, we compared the gene expression of periph- eral blood (PB) T cells from AML patients and healthy donors (HD)[2,3]. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients[4,5] (Figure 1). Compared with HD T cell, circulating AML CD4 T cells consisted of a higher frequency of Treg[4]. PB CD8 T cells from AML patients were with a significantly lower frequency of naïve, and higher frequencies of effector and exhausted phenotypes[5]. Independent of the clinical responses, comparison of the pre-treatment CD8 T cells from bone marrow (BM) and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PB CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression changes in the AML circulating CD8 T cells, characterized by increased cell me- tabolism and cell proliferation. Deconvolution identified that pre- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 129 of 272 contribute to immune suppression in the tumor microenvironment, inhibiting angiogenic pathways may normalize the tumor vasculature and relieve immunosuppression to augment antitumor immunity, es- pecially with concurrent immune checkpoint inhibition. Therefore, we characterized lucitanib’s selectivity and investigated the antitu- mor efficacy and mechanisms of action of lucitanib combined with anti-PD-1 in nonclinical models. Methods The kinase inhibition profile of lucitanib was evaluated using func- tional biochemical assays. Kinase phosphorylation in cancer cells or mouse tissues was assessed by western blot. In vivo efficacy studies were conducted in syngeneic mouse models with monotherapy or combinations of lucitanib (10 mg/kg daily), DC101 (mouse VEGFR2 monoclonal antibody; 40 mg/kg every 2 days), or anti-PD-1 (5−10 mg/kg biweekly). Treated tumors were analyzed for gene and protein expression and immune composition. Results In vitro, lucitanib demonstrated selective and potent inhibition of the tyrosine kinases VEGFR1-3, PDGFRalpha/beta, FGFR1-3, CSF1R, DDR1, and RET. Lucitanib caused dose-dependent inhibition of VEGFR2 phosphorylation in vivo; one 10 mg/kg dose sustained inhibition for 12 hours. Compared with DC101, lucitanib significantly enhanced tumor growth inhibition and survival in MC38 colon tumor-bearing mice. Lucitanib combined with anti-PD-1 significantly increased anti- tumor activity relative to single agents and to DC101 plus anti-PD-1. Fig. 1 (abstract P214). See text for description Lucitanib-treated MC38 tumors exhibited gene expression changes beyond those observed with DC101 treatment. Across multiple syn- geneic mouse models, tumor growth was significantly inhibited 81%−98% by lucitanib combined with anti-PD-1 and 57%−88% by lucitanib alone. The combination significantly extended survival by 90% to >186% and 15% to >35% compared with vehicle or the best mono- therapy, respectively. Lucitanib combined with anti-PD-1 increased gene expression associated with T-cells, cytotoxic cells, and T-cell sig- naling in BR5FVB1-Akt ovarian tumors, relative to each monotherapy. However, lucitanib alone appeared sufficient to modulate innate and adaptive immunity-related gene expression in MC38 tumors. Additional tumor profiling and mechanism of action studies are ongoing. Conclusions Lucitanib, a potent and selective angiogenesis inhibitor, is differenti- ated from DC101 and displays enhanced antitumor activity in com- bination with PD-1 inhibition in multiple syngeneic models. Gene expression changes associated with tumor immune infiltration and increased antitumor immunity were observed in the combination- treated tumors and may contribute to the increased antitumor activ- ity. Results from these studies support the clinical development of the combination of lucitanib and immune checkpoint blockade as a potential treatment for patients with solid tumors. Ethics Approval The studies were conducted in accordance with the Shanghai Medi- cilon Inc.Guidelines for Use and Care of Animals or an approved IACUC protocol at Crown Biosciences. P243 Fig. 2 (abstract P214). See text for description Modulating the immunogenicity of low-mutation soft tissues sarcomas with epigenetic targeted therapy Himavanth Gatla, PhD, Maggie Phillips, Brian Ladle Johns Hopkins Medicine, Owings Mills, MD, United States P242 Correspondence: Brian Ladle (bladle@jhmi.edu) Combination of the angiogenesis inhibitor lucitanib with immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P243 checkpoint blockade augments anti-tumor activity in syngeneic models Background Rachel Dusek, PhD, Liliane Robillard, PhD, Thomas Harding, PhD, Andrew Sarcomas account for 13% of all cancers in young adults under the Simmons, PhD, Minh Nguyen age of 20. For recurrent and metastatic sarcomas, the very poor sur- Clovis Oncology, Inc., San Francisco, CA, United States vival rate despite surgery and chemotherapy warrants better sys- Correspondence: Rachel Dusek (rdusek@clovisoncology.com) temic therapies. As opposed to cancers that show good responses to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P242 immune checkpoint blockade which have high mutation burden, pediatric sarcomas present with low mutation burden, and a Background complete ineffectiveness of immune checkpoint blockade as a mono- Lucitanib is an anti-angiogenic, small molecule multi-tyrosine kinase therapy, suggesting poor immune system activation, and immune inhibitor that has demonstrated potent tumor growth inhibition in infiltration. multiple cancer xenograft models. Because angiogenic factors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 130 of 272 Methods types of DNA damage repair deficiencies and immune evasion have We have used a mutated Kras-driven murine sarcoma syngeneic been lacking. Hence, we have modeled the biology of ovarian cancer tumor model (KP Sarc) and GM-CSF-secreting whole cell tumor cell using patient-relevant mutational landscapes in an immune- vaccine approach (GVAX) to investigate if epigenetic targeted ther- proficient, syngeneic mouse model in order to help us identify the apy induced tumor associated antigens are capable of generating contribution of common driver mutations to the immune repertoire sustained anti-tumor immunity in the tumor microenvironment, and thus to responses of HGSOC tu- Results mors to immunotherapy. We show that sequential combination of epigenetic modifying drugs Methods - DNA methyl transferase inhibitor (decitabine) and HDAC inhibitor We hypothesize that the immune composition and gene expression (entinostat) significantly increases the expression of cancer testis an- signatures of the resulting tumors will vary based on the combin- tigens (CTAs), compared to either drugs alone. In addition, these ation of genetic alterations and the DNA repair proficiency of the drugs modify chemokine expression including increased expression transformed cells. To this end, we have engineered novel syngeneic of CXCL10. Significantly improved immune responses can be gener- mouse models from murine-fallopian-tube epithelium using CRISPR/ ated to decitabine and entinostat pre-treated KP sarc tumor cells as Cas9 technology. These tumors capture the most common combina- assessed by slowed tumor growth, increased T cell infiltrates, and in- tions of co-occurring mutations observed in HR-deficient and -profi- creased cytokine production. Furthermore, immune checkpoint cient patient samples. blockade therapy potentiates the tumor regression mediated by the Results combination of decitabine and entinostat more effectively than the To validate the DNA repair proficiency of the transformed cells, we regression mediated by either drugs alone. This suggests that com- measured Rad51 nuclear focus formation after ionizing radiation (IR) bination therapy induced antigenicity generates better anti-tumor and PARP inhibitor and DNA-damaging agent sensitivity. The HR- immune responses. Rechallenging the mice which rejected epigeneti- deficient cell lines had significantly fewer Rad51 nuclear foci and cally modified KP Sarc tumor formation with similarly treated KP Sarc were more sensitive to PARP inhibition in comparison to HR- cells did not result in tumor formation, whereas untreated KP Sarc proficient cells. Initial immune /stromal analysis using flow cytometry, cells grew uninhibited, suggesting that epigenetic therapy induced scRNA seq transcriptomic and immunofluorescence analysis re- tumor associated antigens are capable of generating a sustained vealed substantial differences in the myeloid and T-cell regulatory memory immune response. By depleting CD4 and CD8 T lympho- compartments between HR-proficient and -deficient primary and cytes, we show that epigenetic targeted therapy induced anti-tumor metastatic tumors and within the ascitic fluid. Preliminary results responses are mediated by both CD4 and CD8 T lymphocytes. Diffi- also suggest that inhibition of the DNA damage response (DDR), culty with in vivo treatment includes the myelosuppressive side ef- checkpoint kinase 1 (Chk1) in combination with immune check- fects of decitabine and entinostat which can inhibit T cell responses point inhibitors, potentiates antitumor effects and augments cyto- immediately after treatment. Proper sequencing of the drugs when toxic T-cell infiltration. given in vivo will be crucial to generate successful adaptive T cell re- Conclusions sponses to newly expressed antigens. Understanding the genetic basis of these complex cellular interac- Conclusions tions will be critical to better tailor combinations of existing targeted Epigenetic targeted therapy induced tumor associated antigens are treatments and immunotherapies in ovarian cancer to fight this dev- capable of generating sustained anti-tumor immune responses. astating disease. P244 P245 The genomic architecture of serous carcinomas shapes the tumor Durvalumab after concurrent chemoradiotherapy in inoperable microenvironment and modulates responses to targeted and stage III non-small cell lung cancer (NSCLC) – a German radiation immunotherapies oncology survey 1 2 3 4 Sonia Iyer, PhD , Shuang Zhang , Anniina Farkkila , Sean Smith , David Lukas Kaesmann, MD, Chukwuka Eze, Julian Taugner, Olarn 5 5 1 1 1 Pepin , Raghav Mohan , Tian Xia , Ferenc Reinhardt , Tony Chavarria , Roengvoraphoj, Claus Belka, Farkhad Manapov 1 6 3 7 Esmee Hoefsmit , Shailja Pathania , Yunlan Zhou , Kevin Elias , Benjamin University Hospital, Munich, Germany 8 1 Neel , Robert Weinberg, PhD Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) Whitehead Institute for Biomedical Research, Cambridge, MA, United Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P245 2 3 States; NYU Langone Health, New York, NY, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Institute of Background Technology, Cambridge, MA, United States; Massachusetts General Consolidation PD-L1 inhibition with durvalumab after platin-based Hospital, Boston, MA, United States; University of Massachusetts, Boston, concurrent chemoradiotherapy (CRT) has become the standard of MA, United States; Brigham and Women's Hospital, Boston, MA, United care in inoperable stage III non-small cell lung cancer (NSCLC) States; NYU-Langone Medical Center, New York, NY, United States based on the excellent PACIFIC trial results. Treatment recommen- Correspondence: Sonia Iyer (iyers@wi.mit.edu); Robert Weinberg dations need time for implementation in nationwide settings and (weinberg@wi.mit.edu) require the close interaction of different medical specialities. In Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P244 this nationwide survey, we questioned the distribution and clin- ical settings of durvalumab treatment after concurrent CRT, ob- Background served side effects of this treatment and summarize follow-up High-grade serous ovarian cancer (HGSOC) is the most frequent and management. most aggressive histologic subtype of ovarian cancer. The corner- Methods stone of the existing treatment of HGSOC is DNA-damaging chemo- We surveyed radiation oncology institutions in Germany via an an- therapy; however, practically all patients eventually develop the onymous online questionnaire sent by e-mail to all members of the progressive disease and the 5-year survival is only 40%. Immunother- German Radiation Oncology Society. apy would seem to be an attractive alternative treatment to chemo- Results therapy, yet existing immunotherapies perform poorly in ovarian We received a total of 255 responses (response rate: 18%). Of cancer, with only ~10% of patients responding to checkpoint block- which 203 (80%) were completed and returned and thus eligible ade. Why this is the case remains poorly understood and there is a for further evaluation. The respondents work in 87 different cities pressing need to understand the underlying biology of immune eva- and 44% in a private medical practice, 29% in university and sion in ovarian cancer. One critical area of interest is the role of hom- 22% in a general hospital. Responses of the same department ology dependent DNA repair (HR) in immune evasion. Unfortunately, were analysed for congruence. Durvalumab was implemented in the preclinical tools required to explore the relationship between the clinical routine by 143 (70%) respondents. Reasons for failed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 131 of 272 implementation in clinical practice were patient ineligibility, deci- months. All patients were alive at the time of evaluation. Four (25%) sion of medical oncologists or absence of updated German evi- patients have developed oligoprogression. Metastastic sites were dence review (S3-guidelines) regarding this treatment approach. bone, brain, adrenal gland and distant lymph nodes. Two patients re- Durvalumab was generally administered according to the re- ceived second-line chemotherapy after distant failure. Another two spondents by private oncological practices in 32%, general or received stereotactic body radiotherapy for all metastatic sites and university hospital in 57% and in the radiation oncology de- continued on durvalumab. Common toxicity during durvalumab was partment, which delivered the CRT in 11% of cases. Importantly, dermatitis (I-II° CTCAE) which occurred earliest after 2 cycles in 10 according to 36% of all respondents initial PD-L1 status was (65%) patients and pneumonitis II° CTCAE in 2 (13%) and III° CTCAE present in ≤30% of all patients. 82% of respondents have treated in 2 (13%) patient between 2-7 months after completion of CRT. In 1-15 patients with durvalumab and 14% of respondents >15 pa- total, 3 (19%) patients discontinued durvalumab treatment after a tients. Furthermore, no respondent had applied durvalumab in median of 4 months due to distant progression or unacceptable less than 14 days after the completion of CRT. 65 (46%)and 49 toxicity. (34%) respondents started durvalumab 14-28 days and later than Conclusions 28 days after CRT, respectively. The majority of respondents Durvalumab was well tolerated with reversible acute toxicity. 25% of (>80%) re-staged the patients with CT (thorax/upper abdomen) patients develop oligoprogression after a mean time of 5.5 months prior to durvalumab. Severe side effects requiring hospital admis- after the end of CRT. sion in more than 10% of all patients were reported by only 12% Ethics Approval of all respondents. The study was approved by the University Ethics Board, approval Conclusions number 17-230. Durvalumab was implemented in the multimodal treatment of inop- Consent erable stage III NSCLC and administered by the absolute majority of Written informed consent was obtained from the patient for publica- respondents. Low testing rates of PD-L1 at initial diagnosis were ob- tion of this abstract and any accompanying images. A copy of the served and should be considered a major barrier to universal adop- written consent is available for review by the Editor of this journal. tion and integration in the clinical work-flow. Durvalumab appears to be well tolerated. However, treatment-related side effects need to be P247 considered during and after multimodal therapy. TLR3-targeting combinatorial chemokine modulation sensitizes “Cold” tumors for the therapeutic effectiveness of immune Acknowledgements checkpoint inhibition We would like to thank the Board of the German Society for Radiation 1 2 Kathleen Kokolus, MS, PhD , Natasa Obermajer, PhD , Per Basse, MD, Oncology (DEGRO) for their approval and their office team for providing the 1 1 PhD , Pawel Kalinski, MD, PhD mailing list. Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States; Ethics Approval UPMC Hillman Cancer Center, Pittsburgh, PA, United States The Board of the German Society for Radiation Oncology (DEGRO) approved Correspondence: Pawel Kalinski (Pawel.Kalinski@roswellpark.org) the survey. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P247 P246 Background Prospective evaluation of outcome and toxicity of durvalumab Immune checkpoint inhibition (ICI) has emerged as life-prolonging treatment after chemoradiotherapy in inoperable stage III non- and occasionally curative treatment for many cancer patients, but small cell lung cancer (NSCLC) patients their activity remains disappointing in many common tumors. ICI Lukas Kaesmann, MD, Julian Taugner, Chukwuka Eze, Olarn therapies are effective against “hot” tumors infiltrated with cytotoxic Roengvoraphoj, Claus Belka, Farkhad Manapov T lymphocytes (CTLs) but inefficient against “cold” tumors lacking University hospital, Munich, Germany CTLs. The importance of CTLs, availability of CTL targets and local ex- Correspondence: Lukas Kaesmann (lkaesmann@gmail.com) pression of PD-L1 and PD-L2 (induced by CTL-produced IFNγ) in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P246 overall effectiveness of ICI remain controversial. We have observed that chemokine-modulatory (CKM) regimens combining TLR3 ligands Background with type-1 IFNs are up to 100-fold more effectiveness in inducing Consolidation PD-L1 inhibition with durvalumab after concurrent CTL attractants (CXCL9, CXCL10, CCL5) compared to either factor chemoradiotherapy (CRT) has become the standard of care in inoper- alone. Moreover, CKM suppresses local Treg attractants, and targets able stage III non-small cell lung cancer (NSCLC) based on the excel- tumor microenvironment (TME) rather than healthy tissues [1-3]. lent PACIFIC trial results. The aim of this prospective single center Thus, we tested whether local or systemic CKM treatments enhance study was to evaluate the outcome and toxicity of durvalumab treat- CTL infiltration in “cold” tumors and determined the feasibility of ment after CRT in a tertiary cancer center. short-term CKM to sensitize poorly immunogenic, αPD-1 resistant, tu- Methods mors to PD-1 blockade. We prospectively collected clinical characteristics, toxicity and out- Methods come of all patients with inoperable stage III NSCLC treated with dur- C57BL/6 mice inoculated with MC38 (colorectal) or ID8 (ovarian) can- valumab after CRT/RT since 9th November 2018. Toxicity was cer cells were treated starting on day three (low-stage disease) or collected using the Common Terminology Criteria for Adverse Events eight (late-stage disease). A two dose course of CKM (IFNα and rinta- version 5 before and during treatment. Re-staging after CRT and be- tolimod [2]) followed by three doses of αPD-1 in two distinct regi- fore the start of durvalumab consisted of a CT scan (thorax/upper ab- mens: 1) Sequentially (following CKM) or 2) Concurrent with CKM. domen). 18F-FDG-PET-CT was performed 3 months and CT 6 months Mice were monitored for intratumoral CTL, tumor growth and after start of consolidation treatment. survival. Results Results Data of 16 patients treated with durvalumab after CRT/RT were eval- We observed strong effectiveness of CKM in promoting intratumoral uated. Three patients (19%) were female and 13 (68%) male, median increases in CTL and PD-L1 expression in the TME. CKM aids in the age at treatment start was 64 years. 10 (53%) patients had T4 or T3 sensitization of the largely αPD1-resistant tumors to PD1 blockade. tumors, four (25%) patients had N3 and 9 (56%) N2 disease. 15 Pa- Both sequential and concurrent CKM allowed antitumor effectiveness tients had CRT with a medium radiation dose of 63.20 Gy and were of αPD-1, resulting in overall prolongation of survival and 30-100% treated with two concurrent cycles of platin-based chemotherapy. cures, depending on treatment initiation. Sensitizing tumors to αPD- One patient was treated with moderate hypofractionated radiother- 1 did not require intratumoral CKM administration and was observed apy without chemotherapy. Median follow-up was 7 (range:2-16) with systemic application at distant sites, consistent with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 132 of 272 preferential activation of tumor tissues by CKM observed in tumor 95% CrI: 0.42-2.73) and avelumab+axitinib (HR=1.49, 95% CrI: 0.76- explant model [3]. Although strong antitumor effects were seen in 2.96) over P+A but were not statistically significant. the absence of any vaccination component, a stronger effect could In the intermediate + poor IMDC risk group, P+A showed a significant OS be observed by vaccination with tumor-loaded dendritic cells. benefit over sunitinib (HR=0.52, 95% CrI: 0.37-0.74) and was favored over Conclusions the other two interventions evaluated, but not statistically significant We demonstrate that local or systemic CKM sensitizes mice with [cabozantinib (HR=0.65, 95% CrI: 0.38-1.11) and N+I (HR=0.79, 95% CrI: poorly immunogenic tumors for subsequent effectiveness of PD-1 0.53-1.18)]. P+A showed a significant PFS benefit over sunitinib (HR=0.67, blockade. Thus, promoting intratumoral CTL accumulation may be 95% CrI: 0.53-0.85) and was favored over N+I (HR=0.87, 95% CrI: 0.65- sufficient for therapeutic effectiveness of ICI against “cold” tumors 1.17), but not statistically significant. PFS benefit favored cabozantinib with low mutational load. Our data provides rationale for clinical test- (HR=1.40, 95% CrI: 0.85-2.31) over P+A, but was not statistically significant. ing of sequential regimens where short-term CKM is followed by rou- Conclusions tine ICI, limiting the inconvenience for patients and facilitating the The results of this analysis suggest that pembrolizumab+axitinib may inclusion of CKM into routine immunotherapy plans. have PFS and OS advantages over most alternative first-line treat- ment options for mRCC, irrespective of IMDC risk groups. Acknowledgements Funded by 1P01CA132714, Rustum Family Foundation and Institutional P249 Support. Time-dependent blood transcriptomic perturbations differentially associated with mono and combination checkpoint inhibitor References therapy 1. Muthuswamy R, Berk E, Junecko BF et al. Cancer Res. 2012; 72:3735-3743. 1 2 1 Darawan Rinchai, PhD , Emily Hinchcliff, MD , Wouter Hendrickx, PhD , 2. Theodoraki MN, Yernei S, Sarkar et al. Cancer Res. 2018; 78:4292-4302. 1 1 Jessica Roelands, Master , Damien Chaussabel , Davide Bedognetti, MD, 3. Obermajer H, Urban J, Wieckowski E et al. Nat Protoc. 2018; 13:335-357. 1 2 PhD , Amir Jazaeri, MD Ethics Approval 1 2 Sidra Medicine, Doha, Qatar; The University of Texas MD Anderson This study was approved by Roswell Park Comprehensive Cancer Center's Cancer Center, Houston, TX, United States IACUC (protocol 1398M). Correspondence: Amir Jazaeri (aajazaeri@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P249 P248 Pembrolizumab plus axitinib (P+A) versus other first-line (1L) Background systemic therapies for advanced/metastatic clear-cell renal cell The rationale for combination immunotherapy is based on presumed carcinoma (ccmRCC) by IMDC Risk Status – a network meta- additive or synergistic properties of each individual drug [1-2]. analysis (NMA) Understanding the molecular mechanisms modulated by a given 1 1 1 Ian McGovern, MPH , Rohan Shirali, MS , Andrew Simon, ScM , Yichen drug is critical to implement more efficient therapeutic approaches. 2 2 1 Zhong, PhD , Rodolfo Perini, MD , Maria Lorenzi, MSc , Oluwakayode We conducted a study to determine 1) whether response to anti- Adejoro, MD, MPH anti-CTLA4 monotherapy (Tremelimumab) or combination of anti- 1 2 Precision Xtract, Boston, MA, United States; Merck & Co. Inc., PDL1(Durvalumab) and anti-CTLA4 (Tremelimumab) therapy could be Kenilworth, NJ, United States measured via blood transcriptome profiling; and 2) whether differ- Correspondence: Oluwakayode Adejoro ences between both treatment groups could be observed. In blood (oluwakayode.adejoro@merck.com) transcriptomic correlative studies performed so far, samples have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P248 been collected at limited time points (i.e., before and after treat- ments), preventing the description of the kinetic and dynamic Background changes associated with specific treatment modalities [3]. The International Metastatic Renal Cell Carcinoma Database Consortium Methods (IMDC) risk group classification is an important prognostic factor for ef- An adaptively randomized phase II trial of sequential versus combination ficacy outcomes of first-line systemic treatment of advanced/metastatic administration of Tremelimumab and Durvalumab (MEDI4736) in patients mRCC. IMDC risk is predictive of outcomes including overall survival with recurrent platinum resistant ovarian, peritoneal or fallopian tube (OS), progression-free survival (PFS) and overall response rate (ORR). cancers at the M.D. Anderson Cancer Center. Peripheral blood samples Pembrolizumab in combination with axitinib showed superior and clin- were collected serially before treatment and at 6 time points post- ically meaningful improvements in OS, PFS and ORR versus sunitinib in treatment from patients receiving Tremelimumab, alone or in combin- subjects with untreated ccmRCC in the KEYNOTE-426 trial. This NMA ation with Durvalumab, administered every 28 days. A total of 91 samples synthesized evidence from randomized clinical trials (RCTs) to indirectly were analyzed. Time points include C1D01 (baseline), C1H12 (12 hours compare the relative treatment effects of P+A vs other therapies in sub- after treatment), C1D08 (cycle one day 8), C1D15 (cycle one day 15), jects with favorable and intermediate + poor IMDC risk groups. C2D01 (cycle 2 day one) and C3D01 (cycle 3 day one). Blood transcrip- Methods tome profiles were generated by RNA-seq (Illumina HiSeq4000) at Sidra Fixed-effect Bayesian NMA was conducted to determine the relative ef- medicine. A set of 382 transcriptional modules was used for the analysis ficacy of treatments. Hazard ratios (HRs) for PFS and OS were estimated of this dataset using a pre-defined framework [4-5]. A module is consid- with 95% credible intervals (CrIs). Analyses were conducted among ered to be “responsive” to the treatment when significant changes in subjects with favorable risk, and intermediate + poor risk disease. abundance are observed for a proportion of its constitutive transcripts Results that is greaterthatwhatcouldbeexpectedbychance[4-5]. Among subjects with favorable IMDC risk, the estimated HRs for OS Results favored P+A over the other 3 interventions evaluated [nivolumab+i- We identified changes in blood transcript abundance in both treatment pilimumab (N+I) (HR=0.53, 95% CrI: 0.18-1.60), sunitinib (HR=0.64, groups, the modular perturbation peaking at cycle 1 day 15 post- 95% CrI: 0.24-1.70) and pazopanib (HR=0.73, 95% CrI: 0.26-2.03)] but treatment (Figure 1). Perturbations of Cell cycle, Protein synthesis and none were statistically significant. For PFS, P+A had statistically sig- Gene transcription modules were observed in both groups. But import- nificant benefit over 2 out of 9 interventions evaluated [interferon- ant qualitative differences were observed as well. Most notably, abun- alpha (IFN) (HR=0.30, 95% CrI 0.15-0.60) and bevacizumab (B)+ tem- dance of gene sets associated with Interferon, Tumor necrotic factor, sirolimus (HR=0.41, 95% CrI 0.18-0.98)]. The results numerically fa- cytotoxic lymphocyte and erythroid signatures was specifically in- vored P+A over 5 out of 9 interventions evaluated [ranging from creased in patients receiving combination immunotherapy B+IFN (HR=0.50, 95% CrI 0.23-1.10) through, atezolizumab, pazopa- Conclusions nib, N+I, to sunitinib (HR=0.81, 95% CrI: 0.53-1.23)], but were not sta- We characterized differential, time-dependent, systemic perturbations tistically significant. PFS benefits favored B+atezolizumab (HR=1.07, associated with mono vs combination immune checkpoint blockade. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 133 of 272 The peak of this immune modulation is observed at day 15 after P250 treatment. The mechanistic and clinical relevance of these findings Combination treatment of the oral CHK1 inhibitor, SRA737 and remains to be explored in a larger group of patients low dose gemcitabine, enhances the effect of PD-L1 blockade by Trial Registration modulating the immune microenvironment in small cell lung NCT03026062 cancer Triparna Sen, PhD (triparnasen@gmail.com) References Memorial Sloan Kettering Cancer Center, New York, NY, United States 1. Naumann RW, Coleman RL. Management strategies for recurrent Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P250 platinum-resistant ovarian cancer. Drugs 2011; 71: 1397-412. 2. Davis A, Tinker AV, Friedlander M. "Platinum resistant" ovarian cancer: what is Background it, who to treat and how to measure benefit? Gynecologic oncology 2014; Small cell lung cancer (SCLC), the most aggressive form of lung cancer, 133: 624-31. shows poor response rates to immunotherapy targeting the pro- 3. Friedlander P, Wassmann K, Christenfeld AM, Fisher D, Kyi C, Kirkwood JM, grammed cell death protein 1 pathway (PD-(L)1). Our group previously Bhardwaj N, Oh WK. Whole-blood RNA transcript-based models can predict discovered that SCLC exhibits high expression of checkpoint kinase 1 clinical response in two large independent clinical studies of patients with (CHK1) and that the CHK1 inhibitor SRA737 activates the innate im- advanced melanoma treated with the checkpoint inhibitor, tremelimumab. mune STING pathway, demonstrating robust anti-tumor activity and J Immunother Cancer 2017 Aug 15;5(1):67. doi: 10.1186/s40425-017-0272-z. synergy in combination with anti-PD-L1 in an SCLC model. 4. Chaussabel D, Baldwin N. Democratizing Systems Immunology with Modular Methods Transcriptional Repertoires Analyses. Nat Rev Immunol. 2014 Apr;14(4):271–80. As SRA737 is being tested in SCLC patients in combination with low 5. Altman MC, Rinchai D, et al. A Novel Repertoire of Blood Transcriptome dose gemcitabine (LDG), we evaluated the efficacy and immune cor- Modules Based on Co-expression Patterns Across Sixteen Disease and relates (including macrophages associated with resistance to immune Physiological States. bioRxiv 525709; doi: https://doi.org/10.1101/525709 checkpoint blockade) of the SRA737+LDG regimen in combination Ethics Approval with anti-PD-L1 in an SCLC model. The study was approved by the MD Anderson Cancer Center Ethics Board, Results approval number IRB 5 IRB00006023 and Sidra Medicine's IRB, approval Trp53, Rb1 and p130 (RPP) triple knockout SCLC cells were implanted 1804022877 into the flank of B6129F1 immunocompetent mice. After the mice developed tumors, they were treated with single agents or various drug combinations. Anti-PD-L1 and LDG demonstrated minimal ef- fect on tumor growth as single agents and only a modest effect as a combination. Moderate to strong anti- tumor activity was however observed with SRA737 monotherapy which directly correlated with dosing intensity. The most profound and synergistic anti-tumor activity was observed when anti-PD-L1 was com- bined with the SRA737+LDG regimen, with all animals showing durable regressions. Analysis of tumor infiltrating immune cells at the end of this treatment regimen showed a dramatic induction of cytotoxic T- cells and a reduction of exhausted and regulatory T cells. Similarly, pro- inflammatory M1 type macrophages and dendritic cells were increased while immunosuppressive M2 type macrophages and MDSC cells were dramatically decreased. As monotherapy, the more dose intensive SRA737 schedule resulted in similar effects on lymphocytes when com- bined with anti-PD-L1. These effects are consistent with our previous data showing that SRA737 treatment leads to an induction of STING and type I interferon signaling in tumors, which is associated with the establishment of an anti-tumor immune microenvironment. Conclusions Our findings suggest that the combination of anti-PD-L1 with the SRA737+LDG regimen may represent the optimal implementation of these agents, leading to a dramatic anti-tumor activity accom- panied by the establishment of a strong anti-tumor immune microenvironment. Given that anti-PD-(L)1 drugs are approved but show limited efficacy in SCLC, our preclinical data provide a strong rationale for combining these agents with the SRA737+LDG regimen to enhance clinical response rates. P251 CTX-8371, a novel bispecific targeting both PD-1 and PD-L1, is more potent than combination anti-PD-1 and PD-L1 therapy and provides enhanced protection from tumors in vivo Diana Albu, PhD, Pearl Bakhru, Wilson Guzman, BS, Michael Ophir, Rachel McCrory, BS, William Carson, Jason Kong, Beata Bobrowicz, Pia Muyot, Amanda Oliphant, Dalton Markrush, Rachel Rennard, Cheuk Lun Leung, Sara Haserlat, Michael Schmidt, Jose Gonzalo, Bing Gong, Robert Tighe, BS, Diana Albu, PhD, Benjamin Wolf Compass Therapeutics, Cambridge, MA, United States Correspondence: Benjamin Wolf (benjamin.wolf@compasstherapeutics.com) Fig. 1 (abstract P249). Modular repertoire analysis Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P251 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 134 of 272 Background Results Monoclonal antibody immunotherapies targeting immune check- From 10/2017 to 2/2019, 83 patients received I+N for mRCC and point receptors have shown great promise for a subset of cancer were included. Demographics are shown in Table 1. By International patients. However, novel combination therapies are still needed Metastatic RCC Database Consortium (IMDC) risk criteria [2], 20.5% to increase the benefit of cancer immunotherapy and bring it to were favorable, 61.4% intermediate, and 18.1% poor risk. 65% were broader patient populations. Here we describe the preclinical stage IV at diagnosis, 63.9% were untreated, and 16.9% patients had evaluation of CTX-8371, which combines PD-1 and PD-L1 target- prior nivolumab exposure. 77.1% of patients had clear cell pathology. ing in one bispecific, tetravalent molecule. 12/83 (14.4%) have sarcomatoid differentiation: 2 have an ongoing Methods response and 7 have died. At the data cutoff date, 44/83 (53%) pa- Our proprietary Stitchmabs®™ bispecific screening platform was used tients have progressed or died. Median PFS was 5.3 months (95% CI to conduct an unbiased screen of bispecifics comprised of various 3-8.5) (Figure 1). OS rates at 6, 12, and 18 months were 76.2%, 63.8%, checkpoint blocking antibodies. This screen yielded the surprising and 51.5%, respectively (Figure 2). Rates of best radiographic re- discovery that a bispecific containing both PD-1 and PD-L1 binding sponse were CR 4.8%, PR 22.9%, SD 18.1%, PD 32.5%, and unknown arms was more potent than the combination of parent monoclonal 21.7%. 44/83 (53%) patients experienced no adverse event (AE). 18/ antibodies. We then generated common light chain bispecifics con- 83 (21.7%) patients experienced a grade 3/4 AE (most commonly taining compatible anti-PD-1 and PD-L1 antibodies and used multiple diarrhea, n=7), 20/83 (24%) patients experiencing a grade 1-2 AE at in vitro assays to identify our lead, CTX-8371. Additional in vitro and worst (most commonly hypothyroidism, n=14), and one grade 5 AE oc- in vivo experiments confirmed CTX-8371’s reactivity across species, curred. 23/83 (27.7%) patients have died, with 10/83 (12.0%) patients in vivo anti-tumor effects, and the underlining mechanisms driving dying within 90 days of receiving the first dose of I+N. 4/83 patients its distinctive activity. have achieved a complete response. Two of these patients discontin- Results ued treatment at 11 and 12 months with a sustained response at 1 and We found that CTX-8371 binds to human and cynomolgus monkey 5 months, respectively. Three other patients remain off therapy for AEs PD-1 and PD-L1 targets with sub-nanomolar affinities and is cross- and have not progressed after 11, 5, and 3 months. reactive to mouse PD-1 and PD-L1. Compared to Keytruda®, CTX- Conclusions 8371 increased T cell activation and tumor cell killing in vitro, signifi- In our real-world cohort of mRCC patients, I+N has similar clinical effi- cantly delayed tumor growth, and prolonged survival in human cell cacy as previously described; however, the cohort is more frail, with transfer tumor models. Additionally, CTX-8371 demonstrated efficacy 16.9% of patients treated in the nivolumab refractory setting. Five in transplantable mouse syngeneic models. Investigation into the patients remain off therapy. This forms the basis for larger prospect- mechanisms responsible for the enhanced efficacy of CTX-8371 unex- ive treatment discontinuation trials (ie. Alliance A031704 phase 3 pectedly found that the bispecific causes a massive loss of PD-1 from trial) with prospective treatment discontinuation for complete re- the T cell surface, which was not observed in response to monoclo- sponse patients at 1-year. nal antibodies alone or combined. This robust PD-1 downregulation, potentially mediated through bridging together the T cell and tumor References cell, may explain the ability of CTX-8371 to reverse PD-1 suppression 1. Motzer RJ, Tannir NM, McDermott DF, et al. Nivolumab plus Ipilimumab more potently than standard blocking antibodies. versus Sunitinib in Advanced Renal-Cell Carcinoma. N Engl J Med. Conclusions 2018;378(14):1277-1290. Taken together, the results demonstrate that the bispecific, tetrava- 2. Heng DY, Xie W, Regan MM, et al. Prognostic factors for overall survival lent antibody CTX-8371 has increased potency in vitro and in vivo as in patients with metastatic renal cell carcinoma treated with vascular compared to clinical checkpoint blockade agents. Some of its effects endothelial growth factor-targeted agents: results from a large, multicen- are likely attributable to its unique mechanism of action, driving ro- ter study. J Clin Oncol. 2009;27(34):5794-5799. bust downregulation of cell-surface PD-1. Thus, CTX-8371 has the po- Ethics Approval tential to increase the number of patients that benefit from PD-1/PD- This study was approved by the Duke University IRB (#Pro00101984) L1 checkpoint blockade. P252 A retrospective study to evaluate real-world clinical outcomes in patients with metastatic renal cell carcinoma (mRCC) treated with Ipilimumab and Nivolumab Landon Brown, MD, Emily Kinsey, MD, Chester Kao, MD, Patrick Healy, Andrew Armstrong, MD, Megan McNamara, MD, Sundhar Ramalingam, MD, Michael Harrision, MD, Daniel George, MD, Tian Zhang, MD Duke University, Durham, NC, United States Correspondence: Landon Brown (landon.brown@duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P252 Background The combination of nivolumab at 3mg/kg plus ipilimumab at 1mg/ kg (I+N) followed by maintenance nivolumab has greatly improved outcomes in patients with intermediate or poor-risk untreated mRCC [1]. Real-world series of patients treated with this combination are scarce. In this retrospective analysis, we present a real-world experi- ence with this combination immunotherapy. Methods A search was performed to identify all mRCC patients treated in the Duke Cancer Institute network with I+N. An extensive chart review was conducted. Patient characteristics are summarized with descrip- tive statistics; Kaplan Meier analysis was performed for progression Fig. 1 (abstract P252). See text for description free survival (PFS) and overall survival (OS). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 135 of 272 Methods We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. Results We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young [3]. αPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibit- ing this property. Efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC dif- fered by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM-3 expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. Conclusions Treatment differences in aged versus young could depend on im- mune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. PD-L2 expression differences could be a mechanism for treatment differences. We are now identi- fying mechanisms for increased PD-L2 and contributions to αPD- L2 efficacy in aged, and testing TCSC effects on treatments (Fig- ure 1-3). Our work can improve cancer immunotherapy in aged Fig. 2 (abstract P252). See text for description hosts and further provide important insights even in young hosts. Table 1 (abstract P252). See text for description Acknowledgements South Texas MSTP training grant (NIH T32GM113896), TL1TR002647, R01 CA231325. References 1. Schildberg FA,Klein SR,Freeman GJ,SharpeAH. Coinhibitory Pathways in the B7-CD28 Ligand-Receptor Family. Immunity. 2016;44(5):955-72. 2. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 ther- apy. Nature. 2016;537(7620):417-21. 3. Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol. 2018;105:146-54. Ethics Approval All animal work was done under UTHSA Institutional Animal Care and Use Committee approved studies in compliance with the Guide for the Care and Use of Laboratory Animal Resources (published by National Research Council of the National Academies), Animal Welfare Act (AWA) (published by USDA), Public Health Service Policy on Humane Care and Use of Laboratory Animals (published by NIH) and US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Approval number: 20180021AR. P253 Distinct clinical and immunological responses to αPD-1, ⍺PD-L1 and ⍺PD-L2 immunotherapy in B16 melanoma in aged versus young hosts includes T-cell stem cell effects and PD-L2 expression differences Myrna Garcia, BS, Alvaro Padron, Yilun Deng, MD, PhD, Harshita Gupta, PhD, Aravind Kancharla, Tyler Curiel, MD University of Texas Health Science Center at San Antonio, San Antonio, TX, United States Correspondence: Tyler Curiel (curielt@uthscsa.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P253 Background Aging is the biggest risk factor for cancer, yet little is known about can- cer immunotherapy effects. αPD-1 can block PD-L1 and PD-L2 while ⍺PD-L1 blocks PD-1 and CD80 [1]. A recent key finding in young hosts Fig. 1 (abstract P253). ⍺PD-L2 treats B16 melanoma in aged mice including humans is that melanoma response to αPD-1/αPD-L1 corre- but not young mice lates with CD8+TCF-1+ T cell stem cell (TCSC) generation [2]. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 136 of 272 functional Fc as monotherapies or in a combination with anti-PD-1 was carried out in order to better understand the effects of anti-TIGIT anti- body with functional Fc at molecular level at different time points. Results Here we demonstrate using mouse tumor models that anti-mTIGIT antibodies require interactions with Fc gamma receptors on myeloid cells in the tumor microenvironment for effective anti-tumor re- sponse. Our observations reveal that the anti-mTIGIT therapeutic ef- fect is not achieved by depletion of intratumoral regulatory T cells, but instead is mediated by “reverse activating signals” through Fc gamma receptors on myeloid cells, inducing expression of various mediators such as cytokines, including TNF-alpha and IL-23, and che- Fig. 2 (abstract P253). PD-L2 expression is significantly higher in mokines, such as CXCL10 and CXCL11, thus generating the condi- aged mice when compared to young tions for potentially promoting immune infiltrates into the tumor microenvironment. In addition, up-regulation of co-stimulatory mole- cules, such as CD80, CD86, and CD40, has been observed, consistent with the heightened anti-tumor activity of Fc gamma receptor binding com- petent anti-mTIGIT antibodies. Furthermore, we discovered induction of a robust and persistent granzyme B and perforin response from the in vivo treatment of anti-mTIGIT antibody with a functional Fc, distinct from a predominantly interferon-gamma-driven anti-PD-1 blockade. Conclusions Our observations for the first time provide mechanistic insights into the requirement for Fc engagement of anti-mTIGIT monoclonal anti- bodies for effective anti-tumor activity in vivo which has implications for the various human antibodies of various isotypes are currently under intense clinical investigations. P255 Preclinical characterization and efficacy of MG1124, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy 1 1 1 Jae-Chul Lee, Master degree , Minkyu Hur , Hye-Young Park , Mi-Young 1 1 1 2 3 Oh , Hye-mi Nam , Hye In Yum , HyungSuk Choi , Jaehwan Kim , 3 1 Byoung Chul Cho, MDphD , Yangmi Lim MOGAM Institute for Biomedical Research, Yong-in, Korea, Republic of; 2 3 GC Pharma, Yongin-si, Korea, Republic of; Yonsei Cancer Center, Seoul, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P255 Fig. 3 (abstract P253). αPD-1, αPD-L2 and αPD-L1 elicit distinct Background TCSC that also differ by age CEACAM1 is one of the several immune checkpoint receptors expressed on T cells and NK cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction in- P254 duces downregulation of ZAP70 phosphorylation in response to T cell re- Requirement of Fc gamma receptor-mediated myeloid-cell ceptor (TCR) stimulation. CEACAM1 is also highly expressed on non-small activation for effective cancer immunotherapy with an anti-TIGIT cell lung cancer (NSCLC) and its expression is correlated with cancer pro- antibody gression and poor prognosis. We developed a fully human monoclonal Jin-hwan Han, PhD, Mingmei Cai, Jeffery Grein, Samanthi Perera, PhD, antibody MG1124, targeting human CEACAM1. Hongmei Wang, Mike Bigler, Roenna Ueda, Thomas Rosahl, Elaine Methods Pinheiro, PhD, Drake LaFace, Wolfgang Seghezzi, Sybil Williams, PhD T cell activation of MG1124 was determined by an NFAT-luciferase Merck Research Laboratories, South San Francisco, CA, United States reporter assay with CEACAM1 overexpressing Jurkat stable cells. Correspondence: Sybil Williams (sybil_williams@merck.com) Evaluation of the homophilic interaction of CEACAM1 or interaction Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P254 of CEACAM1 with CEACAM6 was performed by protein ELISA. In vitro efficacy of MG1124 was examined using an NK-mediated tumor cell Background killing assay. The anti-tumor efficacy of MG1124 alone or in combin- The molecule “T cell immunoreceptor with immunoglobulin and ITIM ation was studied in vivo in a humanized mouse model engrafted domain”, or TIGIT, has recently received much attention as a promis- with NSCLC patient-derived tumor xenografts. ing target in the treatment of various malignancies. In spite of the Results quick progression of anti-TIGIT antibodies into clinical testing both as Anti-CEACAM1 antibody MG1124 bound to CEACAM1 but not to other monotherapy and in combination with programmed death-1 (PD-1)– CEA family members. MG1124 blocked CEACAM1-CEACAM1 homophi- directed immune checkpoint blockade, the molecular mechanism be- lic interaction and CEACAM1-CEACAM6 heteropilic interation by bind- hind the observed therapeutic benefits remains poorly understood. ing to the N domain of CEACAM1. Especially CEACAM1-CEACAM1 Methods homophilic interaction induced downregulation of ZAP70 phosphoryl- Anti-Mouse TIGIT (mTIGIT) blocking antibodies of two distinct isotypes ation in response to TCR stimulation in a CEACAM1 overexpressing Jur- (mouse IgG1 with D265A mutation and mouse IgG2a) and TIGIT- kat stable cell line, which was rescued by MG1124 resulting in deficient mice were generated and used to demonstrate the require- augmentation of NFAT activity and IL-2 expression. NK cell-mediated ment of IgG-Fc gamma receptor interaction for effective anti-tumor re- tumor lysis was increased by MG1124 in a CEACAM1 expression- sponse in vivo studies. Gene expression profiling of whole tumors after dependent manner. In an NSCLC PDX-huNSG mouse model, MG1124 in vivo treatment of anti-mTIGIT antibody with functional or non- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 137 of 272 suppressed tumor progression as a monotherapy and combination Conclusions with pembrolizumab in a CEACAM1 high expressing model. In single Our data validate the therapeutic potential of providing IL-7 signals to mouse trial analysis, MG1124 suppressed tumor progression more than overcome PD-1 resistance. The bifunctional anti-PD1/IL-7 favors the T- 30% as monotherapy (53%, 10/19) as well as in combination (73%, 16/ cell effector over T-regulatory immune balance by stimulating effector 22) with pembrolizumab (5 mpk, 2qW). Moreover, PDXs of adenocarcin- and exhausted T-cells while disarming Tregs suppressive functions. oma origin with more than 50% of CEACAM1 expression were more ef- ficiently prohibited for progression with MG1124, suggesting the P257 potential therapeutic use of MG1124 in patients with NSCLC. Obesity is associated with diminished anti-PD-1-based Conclusions immunotherapy response rates in renal cancer MG1124, an anti-CEACAM1 antibody, blocked CEACAM1-mediated 1 1 1 Rachael Orlandella, BS , Shannon Boi , Justin Gibson, BS , William negative regulation and restored T/NK cell activities. MG1124 showed 1 2 2 1 Turbitt , Gal Wald , Lewis Thomas , Katlyn Norris , Lakshminarayanan effective anti-tumor activity in in vivo mouse models and its combin- 1 1 1 Nandagopal, MD , Peng Li, PhD , Eddy Yang, MD, PhD , Tatiana ation with PD-1 blockade further enhanced treatment efficacy. 1 1 Marquez-Lago, PhD , Lyse Norian, PhD MG1124 is a potential therapeutic candidate for immune checkpoint 1 2 University of Alabama, Birmingham, AL, United States; University of blockade in cancer therapy. Iowa, Iowa City, IA, United States Correspondence: Lyse Norian (lnorian@uab.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P257 P256 A novel bifunctional anti-PD-1 / IL-7 fusion protein potentiates Background effector function of exhausted T cell and disarms Treg suppressive Obesity is a regarded as a major risk factor for developing renal cell car- activity cinoma (RCC). Despite the success of anti-PD-1 checkpoint blockade in 1 1 1 Aurore Morello, PhD , Justine Durand , Caroline Mary , Virginie RCC, response rates remain low (20-30%). Recent studies have observed 1 1 1 2 Thepenier , Margaux Seite , Géraldine Teppaz , Nicolas Poirier that obesity is associated with heightened frequencies of PD-1+ CD8 T 1 2 OSE immunotherapeutics, Nantes, France; Poirier Household, Nantes, France cells [1] and favorable outcomes and responses to immunotherapy in Correspondence: Nicolas Poirier (nicolas.poirier@ose-immuno.com) melanoma [1, 2]. However, the effects of obesity on anti-tumor immun- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P256 ity and immunotherapeutic efficacy in renal cancer remain unknown. Methods Background PD-1 expression on tumor-infiltrating CD8 T cells from treatment-naive Despite the clinical success of anti-PD(L)1 therapies, most patients re- RCC subjects with (BMI >30 kg/m2) or without (BMI < 30 kg/m2) obes- main unresponsive or fail to develop a durable response. We explored ity (n = 18) was determined via flow cytometry. In a separate retro- a second generation of PD-1 antibody by fusing IL-7 cytokine to the Fc spective study, outcome data were queried for RCC patients with (BMI portion. IL-7 is an optimal target for immunotherapy to preferentially >30 kg/m2) or without (BMI < 30 kg/m2) obesity that were treated with stimulate effector T-cell (Teff) functions over regulatory T-cells (Treg), anti-PD-1 as standard of care and had at least 6 months of follow-up due to the differential expression of IL-7R. Moreover, It has been pub- (n=58). Overall survival (OS) was analyzed using Kaplan-Meier methods lished that PD-1 blockades increase IL-7R expression and improve IL-7 and Cox proportional hazards regression after controlling for patients’ signaling in exhausted T-cells rationalizing our combinatorial approach. age, sex, and number of prior treatments. For murine studies, BALB/c Methods mice were randomized to and maintained on either standard chow or Proliferation (H3 thymidine), IFN-γ, IL-7R signaling (pSTAT5) and NFAT (PD- high-fat diet for 20 weeks to generate age-matched lean or diet- 1 bioassay, Promega) assays were tested to determine anti PD-1/IL-7 effi- induced obese (DIO) mice. Mice were then given an orthotopic renal cacy on naïve and/or exhausted-like T-cells. For the suppressive assay, CD4 tumor challenge with syngeneic Renca cells and treated with an anti- Treg and autologous CD8 Teff were co-cultured (1:1) and proliferation was PD-1-based combination immunotherapy or saline. assessed on Day 5. Tumor infiltrating T cells (TILs) were isolated from orthotopic tumor-bearing mice (Hepa1.6, LLC-1,AK7) and subjected to IL-7 Results ex vivo, IL-7R signaling pSTAT5 was determined by flow cytometry. Obesity was associated with reduced frequencies of intratumoral PD- Results 1highCD8+ T cells in treatment-naive murine and human renal tumors. Our anti PD-1/IL-7 bispecific antibody efficiently blocks the PD-1/PD- Although the majority (73%) of lean mice responded to immunotherapy, L1 and PD-L2 interactions and the PD-1-mediated inhibitory signal DIO mice exhibited a reduced response rate (44%). Lean and DIO re- (pSHP1). Importantly, we observed that the IL-7 portion synergizes sponders exhibited favorable ratios of activated CD8+ T cells to myeloid- with the anti-PD-1 to enhance TCR mediated signaling (NFAT). Al- derived suppressor cells (MDSC), reduced PD-1 expression on CD8+ T though IL-7R expression on T cells decrease over repeated antigen cells, and elevated concentrations of CCL5 in renal tumors. Neutralization stimulation, we demonstrated that IL-7 still efficiently activate par- of CCL5 in lean immunotherapy-treated mice yielded a reduced response tially and fully-exhausted human T-cells (pSTAT5) and maintain their rate (43%), unfavorable ratios of activated CD8+ T cells to MDSCs, and di- proliferation capacity. We next characterized sensitivity of TILs to IL-7 minished IFNg secretion from intratumoral CD8+ T cells. The translational in multiple orthotopic mouse models. In PD-1 sensitive tumor (Meso- relevance of our murine findings was reflected in metastatic RCC patients, thelioma), only 10% of TILs express IL-7R whereas in PD-1 resistant as patients with obesity had a trending reduction in OS following stand- model (Hepatocarcinoma and Lung carcinoma), 40-60% of TILs (CD4 ard of care nivolumab (p= 0.06) and a 10.2 month reduction in OS. and CD8) express IL-7R and respond to IL-7 stimulation ex vivo as Conclusions measured by pSTAT5 signaling. These data suggest that the anti PD- Our data suggest that obesity is associated with reduced responses to 1/IL-7 bispecific can reactivate TILs that are resistant to PD-1 therapy. anti-PD-1 based immunotherapies in the context of renal cancer. Contin- Knowing that Tregs have a key suppressive function, we also ex- ued study of this critical issue is needed to better inform patient care. plored the possibility that the anti-PD-1/IL-7 fusion protein affect Treg functions. In a human Treg/Teff coculture assay, we observed Acknowledgements that the anti PD-1/IL-7 molecule abrogate the Treg capacity to inhibit Financial support was provided by NIH grant R01CA181088 to LAN; proliferation and IFN-gamma secretion of CD8+ Teff. Moreover, IL-7 CPCTP T32 fellowship #T32CA047888 to RMO; CPCTP R25 fellowship and the anti-PD-1/IL-7 does not stimulate Treg proliferation, in con- #R25CA047888 to SKB; CMDB T32 fellowship #T32GM008111 to JTG; and trast to IL-2 and IL-15 cytokines. Oncology T32 fellowship #T32CA183926 to WJT. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 138 of 272 References 1. McQuade, J.L., et al., Association of body-mass index and outcomes in patients with metastatic melanoma treated with targeted therapy, immunotherapy, or chemotherapy: a retrospective, multicohort ana- lysis. Lancet Oncol, 2018. 19(3): p. 310-322. 2. Wang, Z., et al., Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): p. 141-151. Ethics Approval Human subject studies were approved by the UAB IRB (protocol # X151013003); murine studies were approved by UAB IACUC (protocol #20233). P258 Combination of NK Cells and anti-PD-L1 Ab with ADCC Fig. 1 (abstract P258). See text for description enhances the anti-tumor effects in PD-L1 high cancer cells 1 2 1 Ji-Eun Park, BS , Bhumsuk Keam, MD, PhD , Ha-ram Park , Soyeon Kim, 3 2 2 2 PhD , Chan-Young Ock, MD, PhD , Miso Kim , Tae Min Kim, MD, PhD , P259 2 2 Dong-Wan Kim, MD PhD , Dae Seog Heo Hydrogel-enabled intratumoral co-delivery of anti-PD-1 antibody Seoul National University Cancer Research Institute, Seoul, Korea, and adenosine deaminase in a mouse model of renal cell Republic of; Seoul National University Hospital, Seoul, Korea, Republic carcinoma of; Biomedical Research Institute, SNUH, Seoul, Korea, Republic of Ketki Velankar, MS, Ngoc Pham, BS, Wilson Meng, PhD, Ellen Gawalt, Correspondence: Bhumsuk Keam (bhumsuk@snu.ac.kr) Nathan Schueller Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P258 Duquesne University, Pittsburgh, PA, United States Correspondence: Wilson Meng (meng@duq.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P259 Although Programmed cell death-1 (PD-1)/ Programmed death-ligand 1 (PD-L1) inhibitors showed remarkable antitumor activity, a large por- Background tion of cancer patients do not response to PD-1/PD-L1 inhibitors even It is hypothesized that tumor resistance to anti-PD-1 monoclo- in the PD-L1 high tumor. Most of PD-L1 inhibitors were modified in FcR nal antibodies is due in part to the accumulation of adenosine binding site to prevent antibody-dependent cellular cytotoxicity (ADCC) (ADO) generated in the tumor microenvironment (TME). ADO against PD-L1 expressing non-tumor cells. IMC-001, developed by impairs the activation and proliferation of effector T cells while ImmuneOncia, is a fully human PD-L1 recombinant monoclonal anti- expanding regulatory T cell (Treg) population, which is in- body that did not modify FcR binding and preserved ADCC. Therefore, versely related to the overall survival of cancer patients, includ- IMC-001 would be synergistic with NK compared to other PD-L1 mono- ing those with renal cell carcinoma (RCC). We propose to clonal antibodies (mAbs). We evaluate anti-tumor efficacy of IMC-001 develop an injectable system by which ADO are degraded in and NK cells against several PD-L1 high cancer cell lines through ADCC. the TME in order to enhance the efficacy of anti-PD1 treat- Methods ment. To this end, we have developed a hydrogel to co-deliver PD-L1 expression was measured by flow cytometry. Standard 51Cr-release anti-PD-1 antibody with adenosine deaminase (ADA), which ca- and CD107a degranulation assays were performed to evaluate the in vitro tabolizes ADO. The hydrogel contains a bioaffinity module ADCC efficacy of 3 groups: control, anti-PD-L1 Ab without ADCC (atezolizu- (named “Z15_EAK”) to retain the anti-PD-1 antibody in tumors mab), anti-PD-L1 Abs with ADCC (IMC-001, Anti-hPD-L1-hIgG1 [atezolizu- while limit the diffusion of ADA in TME for extended durations. mab with wild type FcR binding site, hPD-L1mab]). Various cancer cell We have previously shown that Z15_EAK hydrogel can retain lines were used as target cells, including head and neck squamous carcin- IgG at subcutaneous injection site for at least two weeks [1]. oma (HNSCC), lung cancer, stomach cancer, ovarian cancer, bladder cancer The expectation is that persistent co-localization of anti-PD-1 and lymphoma cell lines. 51Cr-release assay was performed using NK-92- and ADA in the TME will expand Th1 T cells and reduce Treg CD16 as an effector cell with effector to target ratio (E:T) of 30:1. CD107a in draining lymph nodes (DLN) and systemic lymphoid tissues. degranulation assay was performed using peripheral blood mononuclear This postulation was tested in an immunocompetent mouse cells (PBMC) from healthy donors with E:T ratio of 1:1. PBMC was activated model of RCC. by IL-15 and grouped by CD16 V158F genotyping individually. Methods Results A mouse RCC cell line (RENCA) was cultivated for in vitro assays and The expression of PD-L1 is high in several cell lines including SNU- in vivo inoculation into BALB/c mice. Beginning three days after 1076 (8.8±1.3), FaDu (15±0.1), HN31 (21.8±1.1) and H1975 (11.3±1.7). tumor inoculation, the hydrogel loaded with an anti-PD-1 IgG anti- NK cell cytotoxicity in PD-L1 high cell lines was more potent in IMC- body and ADA was injected subcutaneously in the peri-tumoral re- 001 or anti-hPD-L1-hIgG1 compared to control treatment or atezoli- gion for three doses three days apart. DLN, spleen, and tumors were zumab. The PD-L1 high or PD-L1 low tumor cell specific lysis was de- collected for flow cytometric analysis and ELISA measurements. tected by 51Cr-release assay in control group (isotype and Results atezolizumab) vs. and anti-PD-L1Ab with ADCC groups (IMC-001 vs. After three doses, DLN in mice received the hydrogel loaded with Anti-hPD-L1-hIgG1) (Figure 1). Besides, in CD107a degranulation anti-PD-1 antibody and ADA were five times larger than those in assay, activated PBMC cytotoxicity was increased when target cells mice received saline control (Figure 1). In addition, the lymph nodes are opsonized by anti-PD-L1 Abs with ADCC. NK cells that character- in treated mice contained fewer CD4+CD25+FoxP3+ Treg cells com- ized with CD16 high affinity genotype (V/F) are more enhancing pared to controls (Figure 2). After ex vivo re-stimulation with RENCA ADCC than (F/F) low affinity genotype. Two genotyped (V/F vs. FF) for expansion, lymphocytes in treated mice exhibited higher NK cell lysis induced by IMC-001 in FaDu cells was 39.3% vs. 12.8%. interferon-gamma levels than controls, indicating elevated Th1 Conclusions phenotype in the DLN. Anti-PD-L1 Abs with ADCC, such as IMC-001, enhanced the cytotoxic Conclusions activity of NK cells on various PD-L1 high cell lines. This study provides The preliminary data indicate that the local delivery of anti-PD- rationale that NK92-CD16 or NK cell immunotherapy for PD-L1 high 1 and ADA with the hydrogel shifted the local T cell popula- tumor through combination with ADCC preserved anti-PD-L1 Ab. tion toward an effector phenotype (Th1) while limiting the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 139 of 272 Treg expansion. An extended co-localization of anti-PD-1 and P260 ADA in the TME not only modulates immune events in the Characterization of AB154, a humanized, non-depleting α-TIGIT local lymphoid tissues but can also enhance the anti-tumor re- antibody undergoing clinical evaluation in subjects with advanced sponse systemically. Furthermore, the localized delivery reduces solid tumors off-target toxicities of anti-PD-1 antibody. Alejandra Lopez, BSc, Joanne Tan, PhD, Amy Anderson, PhD, Akshata Udyavar, PhD, Nell Narasappa, MSC, Susan Lee, PhD, Daniel DiRenzo, Reference PhD, Kristen Zhang, BS, Hema Singh, Sharon Zhao, Kimberline Gerrick, 1. Pham, N.B., et al. Toward reducing biomaterial antigenic potential: a Adam Park, Lisa Seitz, MA, Nigel Walker, PhD, Matthew Walters, PhD miniaturized Fc-binding domain for local deposition of antibodies. Biomaterials Arcus Biosciences, Inc., Hayward, CA, United States Science. 2019: 7(3): 760-772. Correspondence: Joanne Tan (jtan@arcusbio.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P260 The animal study was approved by Duquesne University's Ethics Board, approval number 180604. Background TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and regulatory T cells (Treg). CD226 is an activating receptor found on NK cells, monocytes and a subset of T cells. TIGIT and CD226 are paired receptors that compete for shared ligands CD155 and CD112, which are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression, whereas binding of the same ligand to CD226 promotes immune activation. AB154, designed to lack FcɣR binding, blocks human TIGIT with minimal risk of depleting intra-tumoral antigen-experienced CD8+ T cells. Methods Translational studies quantifying TIGIT, CD226 and CD155 expression in vari- ous tumor types and normal tissues were performed using flow cytometry, immunohistochemistry (IHC) and by mining publicly available RNASeq data- sets. TIGIT occupancy (RO) and Ki-67 levels from Ph1 dose escalation cohorts were quantified by flow cytometry. Downstream transcriptional effects of TIGIT/CD155 interaction in CD8+ T cells and Treg were assessed using Nano- string®. AB154, AB154 modified to restore wild-type (wt) IgG1 effector func- tion or to display enhance FcɣR binding via Fc mutations, were used in functional assays and antibody-dependent cell cytotoxicity (ADCC) studies. Results AB154, regardless of IgG1 variant, effectively abrogated the TIGIT- mediated inhibitory effects on activated T cells. In contrast, only non- depleting AB154 lacked ADCC activity in mixed cultures containing NK cells and activated T cells. Data assembled from TCGA, confirmed by flow cytometry as well as IHC, identified multiple tumor types bearing high TIGIT and CD155 expression. In particular, antigen-experienced T cells isolated from late stage head and neck squamous cell carcinoma tumors express higher levels of TIGIT and PD-1 than of CD226. Levels of Fig. 1 (abstract P259). See text for description TIGIT expression in this subset was equivalent, if not higher, than intra- tumoral Treg. Preliminary results from our Phase-1 dose escalation study demonstrated near complete target engagement by AB154 in T cells, NK cells, and NKT cells, coupled with concomitant increases in Ki- 67 expression within the aforementioned subsets. Conclusions Blockade of multiple immune checkpoint proteins can confer effective and durable responses in the treatment of cancer. The data presented here provide: 1) rationale for clinical development of a non-depleting a- TIGIT blocking antibody (AB154), 2) evidence of AB154-related immune activation in subjects with advanced solid tumors, 3) evidence supporting AB154 as a rational combination partner with a-PD-1 (AB122). Trial Registration NCT03628677 P261 Recruitment of CD103+ DCs via tumor stroma-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy John-Michael Williford, PhD, Jun Ishihara, PhD, Ako Ishihara, Aslan Mansurov, BChen, Tiffany Marchell, Melody Swartz, PhD, Jeffrey Hubbell University of Chicago, Chicago, IL, United States Correspondence: Jeffrey Hubbell (jhubbell@uchicago.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P261 Background Checkpoint inhibitor antibody (CPI) therapy has demonstrated signifi- cant clinical benefit in a number of tumor types. Unfortunately, certain Fig. 2 (abstract P259). See text for description tumor characteristics, such as the lack of immune cell infiltration, often Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 140 of 272 correlate with poor responses to CPI therapy. Studies have identified C- P262 C Motif Chemokine Ligand 4 (CCL4) as a key molecule necessary for Schweinfurthins Cause Rapid Induction of Ecto-calreticulin Expression the recruitment of cross-presenting, CD103+ dendritic cells (DCs) to the Raymond Hohl, MD, Jeffrey Neighbors, PhD, Ruoheng Zhang, MBBS tumor; tumors lacking CCL4 expression exhibit a “cold tumor” pheno- Penn State College of Medicine, Hershey, PA, United States type and respond poorly to immunotherapy [1]. Based on these results, Correspondence: Raymond Hohl (rhohl@pennstatehealth.psu.edu) we hypothesized that tumor-targeted CCL4 could enhance immune cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P262 infiltration into the tumor and synergize with CPI therapy. Methods Background We generated a fusion protein comprised of CCL4 and a collagen bind- Our previous study demonstrated that schweinfurthin analogs im- ing domain (CBD) derived from von Willebrand factor, a tumor-stroma prove anti-PD-1 immunotherapy in a murine melanoma model by in- targeting strategy developed in our lab [2]. Anti-tumor efficacy studies ducing sustainable in vivo anti-tumor immunity.[1] We speculate that were performed in mouse syngeneic models, including B16F10 melan- the induction of immunogenic cell death (ICD) contributes to these oma, EMT6 breast cancer, and PyMT breast cancer. Flow cytometry was effects. The release of immunogenic danger-associated molecular employed to evaluate the tumor immune infiltrate. patterns (DAMPs) from tumor cells during ICD directly activate anti- Results cancer immunity.[2] Cell surface exposure of calreticulin (ecto-CRT) is Utilizing exposure of collagen in leaky tumor vasculature due to its disor- the major determinative DAMP because it stimulates cancer cell dered structure, we observed that intravenous (i.v.) infusion of CBD-CCL4 phagocytosis by dendritic cells and further activates anti-cancer im- fusion proteins, but not native CCL4, can enhance infiltration of CD103+ munity.[3] Phosphorylation of eukaryotic translation inhibition factor DCs, CD8+ T cells, and natural killer cells and slow B16F10 tumor growth eIF2α, an indicator of ER stress, is highly correlated to CRT exposure when combined with CPI therapy consisting of anti-cytotoxic T- on the cell surface in some forms of ICD.[4] Previous studies of the lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand schweinfurthin family of compounds have shown that they increase 1 antibody (PD-L1) (Figure 1, A-B) Further analysis showed strong correla- the phosphorylation of eIF2α and have complex effects on lipid tions between the presence of CD103+ DCs and CD8+ T cells and tumor homeostasis.[5] We hypothesize that schweinfurthin analogs enhance regression. Similarly, in the EMT6 breast cancer model, tumor-targeted CRT exposure during induction of ICD via eIF2α phosphorylation to CCL4 in combination with CPI, but not native form CCL4, enhanced recruit- improve anti-cancer immunity. ment of CD103+ DCs, CD8+ T cells, and led to a reduction in tumor Methods growth. To confirm the importance of CD103+ DCs in mediating anti- B16.F10 murine metastatic melanoma cells were cultured with increas- tumor responses, we utilized Batf3 knockout mice bearing B16F10 tumors; ing concentrations of the schweinfurthin analog, TTI-3114 or vehicle for in this instance, anti-tumor efficacy of CPI + CBD-CCL4 was completely lost. 24 hours, followed by measurement of ecto-CRT expression by flow cy- Efficacy studies in PyMT breast cancer models highlighted the therapeutic tometry. Kinetics of ecto-CRT exposure was also assessed. To determine benefit of CBD-CCL4 delivery (Figure 1C); CPI therapy alone led to the importance of lipids in this process, studies were carried out with complete tumor remission in only 10% of mice, whereas combination normal or charcoal-stripped lipid-free media. In addition, the role of therapy of CPI + CBD-CCL4 cured 50% of the treated mice (Figure 1D). eIF2α phosphorylation in TTI-3114-induced CRT exposure was evalu- Conclusions ated using western blots for total and phosphorylated eIF2α with thap- These results highlight the utility of recruiting CD103+ DCs to the sigargin acting as a positive control for the induction of ER stress. tumor to improve the efficacy of CPI therapy. This engineered chemo- Results kine delivery strategy demonstrates significant translational potential by targeting the tumor stroma following systemic administration. TTI-3114 induces rapid surface calreticulin exposure on B16.F10 murine melanoma cells in a concentration-dependent manner, References starting at 30nM. 1. Spranger S, Gajewski T. Impact of oncogenic pathways on evasion of Lipid depletion sensitizes melanoma cells to TTI-3114-induced antitumor immune responses. Nat Rev Cancer. 2018; 18:139-147. calreticulin exposure. 2. Ishihara J et al. Targeted antibody and cytokine cancer immunotherapies TTI-3114 causes eIF2α phosphorylation before CRT exposure in through collagen affinity. Sci Transl Med. 2019; 11:eaau3259. melanoma cells, indicating potential ER stress response. Conclusions Based on the above results, we hypothesize that schweinfurthins in- duce CRT exposure on the cell surface likely by promoting a form of ER-stress, and this effect is augmented by lipid depletion. Future ex- periments will investigate the function of various lipids in schweinfurthin-induced CRT exposure and subsequent immunogenic cell death. We will also explore the exact mechanisms which are re- sponsible for the CRT cell surface exposure phenomena. References 1. Kokolus, K. M. et al. Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity. Oncoimmunology 8, 1–13 (2019). 2. Zhou, J. et al. Immunogenic cell death in cancer therapy: Present and emerging inducers. J. Cell. Mol. Med. 4854–4865 (2019). doi:10.1111/ jcmm.14356 3. Obeid, M. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61 (2007). 4. Bezu, L. et al. eIF2α phosphorylation: A hallmark of immunogenic cell death. Oncoimmunology 7, 1–3 (2018). 5. Kuder, C. H. et al. Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification. Mol. Fig. 1 (abstract P261). See text for description Pharmacol. 82, 9–16 (2012). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 141 of 272 P263 either alone or in combination with other immune checkpoint therap- Targeting EZH2 enhances antigen presentation, antitumor ies. We hypothesized that an antibody with a unique binding mode immunity and circumvents anti-PD-1 resistance in head and neck could activate T cells in an Fc effector-less format. Hence, we developed cancer 7A5, a CD137 agonist monoclonal antibody which potentially has a 1 2 1 Liye Zhou, PhD , Ravindra Uppaluri, MD, PhD , Tenny Mudianto, BS , ligand-like structural binding mode and demonstrated that it effectively 1 1 Xiaojing Ma, PhD , Rachel Riley, BS engages the CD137 receptor in preclinical studies. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Methods Women's Hospital/DFCI, Boston, MA, United States 7A5 was identified from a human Fab phage display library screen Correspondence: Ravindra Uppaluri and engineered to an IgG1 Fc effector null antibody. Solid phase (ravindra_uppaluri@dfci.harvard.edu) binding assays with recombinant CD137 protein and cell-based as- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P263 says in CD137 expressing cells were used to evaluate binding and functional activity in vitro. To assess agonist activity, 7A5 was tested Background in NF-kB luciferase reporter, PBMC co-stimulation and Treg suppres- Anti-programmed death-1 (PD-1) receptor-based therapeutics im- sion assays. To determine antitumor activity in vivo, human tumor prove survival in recurrent head and neck squamous cell carcinoma xenograft mouse models (NSG mice harboring human NCI-H292 or (HNSCC) patients but many do not benefit due to a low response HCC827 NSCLC tumors) reconstituted with human PBMCs or T cells, rate. Multiple mechanisms of immunoevasion have been identified in were used. Treatments included 7A5 monotherapy and the combin- HNSCCs including in the antigen presentation machinery. Herein, we ation with anti-PD-L1 antibody in these models. identified enhancer of zeste homolog 2 (EZH2) as a therapeutic tar- Results get in HNSCCs that enhanced tumor cell antigen presentation and In this study, we characterized 7A5, a fully human IgG1 Fc effector subsequently sensitized resistant tumors to anti-PD-1 therapy. null monoclonal antibody. We showed 7A5 binds CD137 and the Methods binding epitope overlaps with the CD137 ligand binding site. 7A5 en- EZH2 regulation of antigen presentation was defined using EZH2 in- gages the CD137 receptor and activates signaling independent of hibitors (GSK126 and EPZ6438) in human and mouse HNSCC cell cross-linking or Fc effector function. It binds to activated primary T lines. Mechanistic dissection of EZH2 in regulation of antigen presen- cells and leads to T cell stimulation in cell-based assays. Monother- tation was investigated using flow cytometry, qRT-PCR, ELISA and apy with 7A5 inhibits tumor growth in humanized mouse models chromatin-immunoprecipitation assays. EZH2 deficient cell lines were and this activity is enhanced when combined with a PD-L1 antagon- generated using CRISPR-CAS9. GSK126 and anti-PD-1 blocking anti- ist antibody. Furthermore, changes to the intra-tumoral immune body were used in testing combinatorial therapy in vivo. gene expression signature in response to 7A5 is highly suggestive for Results a mechanism of enhanced T cell infiltration and activation. EZH2 expression was negatively correlated with antigen processing Conclusions machinery (APM) pathway components in HNSCC TCGA datasets. In summary, CD137 antibody 7A5 represents a differentiated agonist EZH2 inhibition resulted in significant upregulation of MHC class I ex- with preclinical biological properties that support its further develop- pression in both human and mouse HNSCC lines and increased anti- ment as an anti-cancer immunotherapy. gen presentation in mouse models. This increased antigen presentation on the tumor cell by EZH2 inhibitors or CRISPR medi- P265 ated EZH2 deficiency, increased antigen specific CD8+ T cell prolifer- Immune checkpoint inhibitors induce response in a dose- ation, IFNγ production and tumor cell cytotoxicity. Mechanistically, dependent manner while their immune related adverse events are EZH2 inhibition reduced the histone H2K27me3 modification on the dose-independent, a meta-analysis β-2-microglobulin promoter to regulate antigen presentation. Finally, 1 2 1 Osama Rahma, MD , Joshua Reuss, MD , Anita Giobbie-Hurder, MS , in an anti-PD-1 resistant model of HNSCC, combination EZH2 inhib- 3 4 5 Ghazaleh Razavi, MD , Pooja Mehra, MD , Seema Gupta , Rawad Elias, ition with anti-PD-1 suppressed tumor growth at least partially due 6 5 MD , Samir Khleif, MD to the upregulation of antigen presentation capacity of tumor cells. 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; John Hopkins, Conclusions Baltimore, MD, United States; Georgia Cancer Center, Augusta, GA, Our results demonstrated that targeting EZH2 enhanced antigen presen- United States; University of Virginia, Charlottesville, VA, United States; tation and circumvented anti-PD-1 resistance. Thus, combining EZH2 tar- 5 6 Georgetown University, Washington, DC, United States; Hartford geting with anti-PD-1 may increase therapeutic susceptibility in HNSCC. Healthcare, Hartford, CT, United States Correspondence: Samir Khleif (snk48@georgetown.edu) P264 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P265 Characterization of a human CD137 (4-1BB) receptor binding monoclonal antibody with differential agonist properties that Background promotes antitumor immunity Despite the expansion of Immune Checkpoint Inhibitor (ICI) indi- Helen Kotanides, Rose Marie Sattler, Maria Lebron, Carmine Carpenito, cations, the relationship between ICI dose-escalation and toxicity PhD, Juqun Shen, Jingxing Li, David Surguladze, Jaafar Haidar, Colleen or response has not been established. To understand this correl- Burns, Leyi Shen, Ivan Inigo, BS, Anthony Pennello, Amelie Forest, MSc, ation, we performed a meta-analysis of all available clinical trials Xinlei Chen, Darin Chin, Andreas Sonyi, Michael Topper, Lauren Boucher, investigating ICIs. Prachi Sharma, Yiwei Zhang, Douglas Burtrum, Ruslan Novosiadly, Dale Methods Ludwig, Gregory Plowman, Michael Kalos We searched PubMed and abstracts presented at (inter)national Eli Lilly and Company, Indianapolis, IN, United States meetings for trials (T) using FDA-approved ICIs including ipilimumab, Correspondence: Helen Kotanides (helen.kotanides@lilly.com) atezolizumab, nivolumab, and pembrolizumab. The reported rates of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P264 treatment-related grade 3-5 adverse events (G3-5AEs), immune- related adverse events (irAEs), and response were collected. For each Background ICI, comparisons of incidence rates between doses or diseases were CD137 (4-1BB) is a member of the TNFR receptor superfamily that plays based on marginal, exact generalized linear models. a key role in mediating immune response through costimulatory sig- Results nals that promote T cell proliferation, survival and memory. CD137 A total of 74T (7469 patients (pts)) published between 1/2010 – 1/2017 agonism has the potential to reinvigorate potent antitumor immunity were included (15T-ipilimumab (1058 pts), 30T-nivolumab (2281 pts), Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 142 of 272 29T-pembrolizumab (4130 pts)) (Figure 1). For ipilimumab, the overall incidence of G3-5AEs was 34%. A significant 27% reduced risk of G3- 5AEs was seen with 3 mg/kg compared to 10 mg/kg (p=0.002) (Figure 2). However, there was no relationship observed between dose of ipili- mumab and incidence of irAEs or response to therapy (Figure 3). With nivolumab, the overall incidence of G3-5AEs was 20.1%. Incidence of G3-5AEs was significantly lower in NSCLC, with risk reductions of 24- 38% when compared to RCC or melanoma (p≤0.05). No dose-toxicity relationship was seen for G3-5AEs or irAEs (Figure 4). In both melanoma (6T) and NSCLC (7T), a dose-response association was observed, with significantly decreased odds of response of 17% and 64% for 1mg/kg compared to 3mg/kg in melanoma and NSCLC, respectively (Figure 5,6) with no further increase in response for doses above 3 mg/kg. This as- sociation was not observed in RCC (Figure 7). For pembrolizumab, the overall incidence of G3-5 AEs was 13.3%. Risk of G3-5AEs was 17% lower in melanoma than in NSCLC (p=0.03). No dose-toxicity relation- ship was seen for G3-5AEs or irAEs (Figure 8). In melanoma (7T), 2mg/ kg every 3 weeks (q3w) had 22% decreased odds of response com- pared to 10mg/kg (q2w) (p=0.01) (Figure 9). For NSCLC (5T), no dose- response relationship was noted (Figure 10). Conclusions We found no correlation between dose of ipilimumab and odds of G3-5iAEs or response. For pembrolizumab and nivolumab, no dose-toxicity correlation was seen but a dose-response correlation was observed suggesting that, for the PD-1 inhibitors, efficacy ap- Fig. 2 (abstract P265). Bootstrap analysis for G3-4 AEs pears to be dose-dependent while toxicity does not. Accordingly, of ipilimumab future clinical trial design of ICIs should use a dose escalation method with a primary objective of identifying an effective dose rather than a maximum tolerated dose. Acknowledgements Merck and BMS for providing input Fig. 1 (abstract P265). Consort Diagram of Literature Search Fig. 3 (abstract P265). Bootstrap analysis for ORR for ipilimumab Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 143 of 272 Fig. 6 (abstract P265). Bootstrap analysis for ORR for nivolumab Fig. 4 (abstract P265). Bootstrap analysis for G3-4 AEs in NSCLC for nivolumab Fig. 5 (abstract P265). Bootstrap analysis for ORR for nivo Fig. 7 (abstract P265). Bootstrap analysis for ORR for nivolumab in melanoma in RCC Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 144 of 272 Fig. 10 (abstract P265). Bootstrap for ORR for pembrolizumab in NSCLC Fig. 8 (abstract P265). Bootstrap for incidence of G3-4 AE P266 in pembrolizumab Evaluation of immunomodulatory receptor/ligand expression on matched human biospecimens Shawn Fahl (shawn.fahl@dls.com) Discovery Life Sciences, Huntsville, AL, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P266 Background The integration of immunomodulatory receptor signaling is crucial for the activation status of responding T cells, and modulation of these re- ceptors, and their ligands, may be of therapeutic benefit. Indeed, recent breakthroughs in checkpoint inhibitor therapies, and in particular those that target the PDL1/PD1 interaction, have demonstrated success in nu- merous oncological indications. Understanding the expression of these receptors and their cognate ligands within the complex cellular archi- tecture of solid tumors will be fundamentally important to the design on the next-generation of immunotherapies. Methods Bulk RNASeq analysis of primary human tumor tissue revealed the expression of numerous co-stimulatory (LIGHT/HVEM, 41BB/41BBL, OX40/OX40L, GITR/GITRL) and co-inhibitory (Lag3, VISTA, PVR/PVRL2/ TIGIT, Tim3/Galectin-9) receptors and ligands within the tumor micro- environment. Using multiparametric flow cytometry, we have profiled the expression of these immunomodulatory receptors and their re- spective ligands on the major cellular components of the tumor microenvironment and correlated it with expression on cellular sub- sets within matched peripheral blood. P267 Phenotyping of TIGIT pathway members may be used for cancer selection in the clinical application of anti-TIGIT antibody EOS884448 Noemie Wald, PhD, Julia Cuende, PhD, Marjorie Mercier, Florence Nyawouame, MSc, Margreet Brouwer, MSc, Erica Houthuys, PhD, Gregory Driessens, PhD, Veronique Bodo, PhD, Catherine Hoofd iTeos Therapeutics, Gosselies, Belgium Correspondence: Gregory Driessens Fig. 9 (abstract P265). Bootstrap for ORR for pembrolizumab (gregory.driessens@iteostherapeutics.com) in melanoma Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P267 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 145 of 272 Background The complement system consists of a network of more than 50 dif- TIGIT is a T cell co-inhibitory receptor that drives tumor cell mediated ferent plasma and membrane associated proteins. It is a part of the immunosuppression. Predominantly expressed on CD4+ Tregs, CD8+ T innate immune system and plays a key role in host defense against and NK cells in healthy individuals, TIGIT is further upregulated in these pathogens as well as in tissue homeostasis. The anaphylatoxin C5a is cells in cancer patients. In patients, it is frequently co-expressed with formed upon cleavage of C5 during the process of complement acti- exhaustion markers such as PD-1. DNAM-1/CD226, a co-stimulatory re- vation. C5a is the most potent chemoattractant and induces recruit- ceptor, is expressed on NK and T cells and competes with TIGIT for ment and activation of different immune cells to inflamed tissue, PVR/CD155 binding, but with a lower affinity. Since cancer cells express among which are neutrophils, eosinophils, monocytes, basophils, and high level of CD155 and because TIGIT expression is increased on TILs, mast cells. C5a binds to the seven transmembrane-spanning recep- the TIGIT pathway represents a major mechanism for immunosuppres- tors C5aR1 (CD88) and C5aR2 (C5L2). Blocking C5aR1 thus appears as sion within the tumor. We developed EOS884448, an antagonist anti- a potent mean to control the myeloid suppressive cells in the TME. TIGIT antibody, to prevent TIGIT-mediated immunosuppression in can- In this context we developed IPH5401, a fully human blocking anti- cer patients. C5aR1 monoclonal antibody that prevents binding to C5a. Methods Results To support selection of indications for clinical application of We first explore further the expression profile of C5aR1 in the TME EOS884448, we used flow cytometry and immunohistochemistry both at mRNA and protein levels in several solid cancer indications (IHC) to characterize peripheral and tumoral expression of TIGIT, showing various levels of infiltration by C5aR1 positive immune cells. CD155 and CD226 in healthy or cancer donors. Then, we demonstrated that IPH5401 can block activation and migra- Results tion of Human neutrophils and macrophages in vitro. TIGIT is expressed on multiple immune subsets in healthy donors. Simi- Conclusions lar analysis on matched PBMCs and TILs from 15 cancer donors Altogether, these results support our ongoing multi-center, open highlighted the overexpression of TIGIT on cells from those samples. label, dose-escalation and dose expansion Phase I/II clinical trial Interestingly, ex vivo polyfunctional analysis of cytokine production (STELLAR-001) evaluating the safety and efficacy of IPH5401 in com- demonstrated immunosuppression of TIGIT+ TILs versus their TIGIT- bination with durvalumab, an anti-PD-L1 immune checkpoint inhibi- counterparts. Among PBMCs and TILs assessed by flow cytometry, tor, as a treatment for patients with advanced solid tumors. tumor-infiltrating Tregs exhibit the highest TIGIT expression (frequency of positive cells and receptor density). This finding was confirmed by P269 IHC on tumor samples, supporting the potential value of an ADCC- IL-15 together with TIGIT blockade reverses PVR-mediated NK cell competent antibody targeting preferentially tumor-infiltrating Tregs. dysfunction in melanoma Finally, intrinsic expression of TIGIT on tumor cells was detected on 1 1 1 Joe-Marc Chauvin, PhD , Mignane Ka , Ornella Pagliano , Carmine several haematological malignancies, opening the potential for 1 1 1 2 Menna , Quanquan Ding , Cindy Sander , Jiajie Hou , Soldano Ferrone, EOS8844488 to directly kill tumor cells in addition to its activities to re- 1 1 1 MD, PhD , Diwakar Davar, MD , John Kirkwood, MD , Robert Johnston, invigorate immunity. The expression of the TIGIT ligands CD155 and 3 3 2 1 PhD , Alan Korman, PhD , Mark Smyth , Hassane Zarour, MD CD226 co-receptor were also analysed by IHC in tissues (n=284-307) 1 2 University of Pittsburgh, Pittsburgh, PA, United States; QIMR Berghofer from 9 cancer indications. CD155 is mostly expressed by tumor cells, Medical Research Institute, Queensland, Australia; Bristol-Myers Squibb, ranging from a median of 2% of CD155high tumor cells for cervix to Redwood City, CA, United States 50% for pancreatic cancer. CD155 expression is highest in pancreatic, Correspondence: Joe-Marc Chauvin (chauvinj@upmc.edu) prostate, kidney, gastric and colon cancers. CD226 is detected on im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P269 mune cells infiltrating tumors. The median percentage of tissue area positive for CD226 ranged from 0.07% in head and neck to 0.98% in Background gastric cancer, with gastric, lung and renal cancer showing the highest Natural Killer cells (NKs) play a critical role in tumor immunosurveil- CD226 expression. lance. Multiple activating and inhibitory receptors regulate NK cell- Conclusions mediated tumor cytotoxicity. The inhibitory receptor TIGIT and its Together, these findings strongly support the relevance of targeting counter-receptor CD226 exert opposite effects on NK cell function, TIGIT with an ADCC-competent antibody and provide a method to with TIGIT blockade reinvigorating NK cell-mediated tumor reactivity. select cancer types that may benefit from treatment with Whether and how the manipulation of the TIGIT/CD226/PVR axis may EOS884448. reactivate NK-cell mediated antitumor activity in melanoma patients Ethics Approval (MPs) has not yet been thoroughly evaluated. The study was approved by UCL‘s Ethics Board, approval number Bio- Methods bank2019/09MAI/005 Flow cytometry was used to evaluate the phenotype and function of NKs in the periphery (cNKs) and tumor sites (TiNKs) in MPs. CD226 P268 mRNA was evaluated by RT-PCR. CD226 and TIGIT internalization was IPH5401 anti-human C5aR antibody targets suppressive myeloid evaluated on isolated cNKs by Imagestream analysis. Melanoma lung cells in the TME metastasis tumor models in WT and TIGIT-/- mice were used to Joanna Fares, Léa Simon, Caroline Soulas, Marion Loivet, Elodie Bonnet, evaluate in vivo effects of TIGIT and CD226 blockades on NK- Luciana Batista, Romain Remark, PhD, Cécile Bonnafous, Robert Zerbib, mediated tumor control with or without IL-15 treatment. MSc, Mathieu Bléry Results Innate Pharma, Marseille, France In sharp contrast with CD8+ T cells, TiNKs downregulated both TIGIT Correspondence: Robert Zerbib (robert.zerbib@innate-pharma.fr) and CD226 expression as compared to cNKs. TiNKs exhibited de- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P268 creased expression of activation markers, lytic potential and melan- oma killing capacity as compared to cNKs in MPs. Membrane-bound Background PVR, but not soluble PVR, triggered CD226 internalization and deg- A hallmark mechanism of synergy in immunotherapy is the elimin- radation, leading to NK dysfunction. IL-15 stimulation increased ation of immunosuppressive cells, such as myeloid cells and neutro- CD226 and TIGIT expression levels and NK cell function, and TIGIT phils, to allow for the reactivation of effector cells. These blockade further increased TiNK proliferation and function against immunosuppressive cells are indeed associated with poor prognosis MHC class-I deficient tumors as compared to IL-15 or TIGIT blockade in many cancer types as well as resistance to checkpoint blockade. alone. TIGIT blockade promotes tumor antigen-specific CD8+ T cells From a therapy perspective, we aimed to specifically target the re- proliferation independently of NK cells. TIGIT blockade impeded me- cruitment of these major mediators of pro-tumoral inflammation into tastasis in mice in a CD226-dependent manner and only in presence the tumor microenvironment (TME). of IL-15. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 146 of 272 Conclusions P271 Membrane-bound PVR plays a critical role in the tumor microenviron- Anti-SIRPalpha antibodies exert anti-tumor activity in both CD47- ment by modulating TIGIT/CD226 expression and the function of dependent and CD47-independent manners TiNKs. IL-15 together with TIGIT blockade, counteracts PVR-mediated Hongtao Lu, PhD, Xiaofeng Niu, PhD, Qinglin Du, PhD, Jingfeng Yu, TiNK dysfunction in melanoma, and prevent metastasis occurrence in Roumei Xing, Yanfen Hu, Jinfeng Zhao, Fengli Wang, Zhihao Wu, PhD, mice in a CD226 dependent manner. Our findings support the devel- Yangsheng Qiu, Hongtao Lu, PhD opment of novel combinatorial immunotherapy with IL-15 and TIGIT Elpiscience Biopharma, Shanghai, China blockade to promote NK cell-mediated destruction of MHC class I- Correspondence: Hongtao Lu (hongtaolu@elpiscience.com) deficient melanoma, which are refractory to CD8+ T cell-mediated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P271 immunity and PD-1 blockade. Background Signal-regulatory protein alpha (SIRPα), is an inhibitory receptor P270 expressed on myeloid cells and dendritic cells. Ligation of CD47 Expression and clinical significance of the CD47/SIRPα pathway as to SIRPα delivers a “don’teat me” signal to suppress phagocyt- a candidate immunotherapy target in non-small cell lung cancer osis. Tumor cells frequently overexpress CD47 to evade (NSCLC) macrophage-mediated destruction. Currently, agents targeting 1 1 1 Shruti Desai, PhD , Franz Villarroel-Espindola , Patricia Gaule, PhD , Adam CD47 have proceeded to clinical trials and demonstrated promis- 1 2 2 Ducler , Marisa Peluso, MS , Benjamin Lee, MD PhD , Kurt Schalper, MD, ing anti-tumor results. However, these agents have been associ- 1 2 PhD , Pamela Holland, PhD ated with hemolytic anemia and thrombocytopenia. In addition, School of Medicine, Yale University, New Haven, CT, United States; universal expression of CD47 causes antigen sink, which leads to Surface Oncology, Cambridge, United States reduced efficacy. We therefore consider targeting SIRPα to Correspondence: Kurt Schalper (kurt.schalper@yale.edu) achieve an improved efficacy with a better safety profile. We Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P270 have developed 2 classes of anti-SIRPα antibodies: CD47-SIRPα interaction “blocker” and “non-blocker”. Both groups of antibody Background functionally stimulate phagocytosis of multiple cancer cell types Immunostimulatory therapies have revolutionized the treatment of by macrophages. NSCLC. Multiple studies show that activation of the CD47/SIRPα path- Methods way can mediate cancer immune evasion by blocking phagocytic ac- Using SIRPα extracellular domain (ECD), SIRPα overexpression tivity of macrophages. Although early stage clinical trials blocking stable cell line and plasmid encoding SIRPα as immunogens, this pathway are ongoing, the expression, tissue distribution and clin- anti-SIRPα antibodies were generated by traditional hybridoma ical significance of the CD47 axis in NSCLC remains unknown. technology. Pan-allele/SIRP family homologue binding proper- Methods ties, and species cross-reactivity profile were evaluated by ELISA Using control tissue samples/cell line transfectants, we validated anti- and FACS. In vitro function activity was determined by phago- bodies to reliably detect CD47 and SIRPα protein in FFPE tissue and cytosis assay. In vivo safety profile was assessed in hCD47/ standardized a multiplexed quantitative immunofluorescence panel for hSIRPα double knock-in mice. Lead clone was humanized via simultaneous measurement of DAPI, pan cytokeratin, CD8/CD47/SIRPα. CDR grafting and back mutation screening. Stress tests were We used this panel to interrogate two retrospective NSCLC Yale cohorts carried out to evaluate the developability of candidate represented in tissue microarrays (#1: n=297 and #2: n=175). Cohort #3, antibody. n=139 adenocarcinomas with mutation testing were also analyzed. We Results studied the levels of the targets, tissue distribution, association with A panel of anti-SIRPα antibodies that stimulate phagocytosis of clinicopathologic/molecular variables and survival. multiple cancer cells were developed. One lead clone (B4) was se- Results lected from the “Blocker” group based on ranking of in vitro bind- Predominant tumor CD47 expression (cytoplasmic/membranous) was ing/function properties. Subsequently, ES004-B4 was identified as recognized in 82 -88% of cases in the NSCLC cohorts. SIRPα protein the candidate antibody after humanization, affinity maturation, was detected in 94- 98% of cases located in the stroma. Elevated ex- and property characterization. It recognized pan-allele human pression of CD47/SIRPα was significantly associated with high CD8+ SIRPα with high affinity (KD hSIRPα V1/V2, 0.86nM/1.43nM). ES004- tumor infiltrating lymphocytes in the cohorts. The targets showed no B4 was shown to stimulate phagocytosis of multiple cancer cell consistent associations between major clinicopathologic variables. types. Another lead antibody (N4) was selected from the CD47/ Lung adenocarcinomas with KRAS mutation showed significantly SIRPα interaction “non-blocker” group. ES004-N4 also induced lower levels of CD47 than EGFR mutated or EGFR/KRAS WT tumors. strong phagocytosis of multiple cancer cell types by macrophages, Although individual CD47/SIRPα levels were not prominently associ- albeit it did not disrupt CD47/SIRPα interaction, suggesting an ated with outcome, their co-localization in stroma was positively as- unique mode of action. ES004-B4 and ES004-N4 didn’t trigger sociated with longer 5-year overall survival. hemagglutination and had no negative impact on T cell prolifera- Conclusions tion in vitro. No severe hemolytic anemia and thrombocytopenia CD47 and SIRPα are frequently expressed in NSCLCs with higher were observed in hCD47/hSIRPα double knock-in mice treated levels in CD8+/T-cell inflamed tumors, suggesting adaptive upregula- with 10mg/kg of each antibody, suggesting a low safety risk tion of this pathway associated with anti-tumor immune pressure. in vivo. CD47 levels are associated with major oncogenic signaling events in Conclusions lung adenocarcinomas. Localized measurement of stromal CD47/ In summary, we have developed 2 anti-SIRPα antibodies with “Best- SIRPα co-expression in intact tumor specimens could provide valu- in-Class” potential: 1) CD47/SIRPα interaction “Blocker” ES004-B4, 2) able information about the pathway activation. CD47/SIRPα interaction “Non-Blocker” ES004-N4. Both antibodies Ethics Approval greatly enhance macrophage-mediated tumor cell destruction, pos- All tissues were used after approval from the Yale Human Investiga- sibly through different mechanisms of action. We are currently ad- tion committee protocol #9505008219 which approved the patient vancing the development of ES004-B4 and ES004-N4 into clinical consent forms or waiver of consent. candidates. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 147 of 272 P272 P273 SRF231, a fully human high-affinity anti-CD47 antibody, exerts Blockade of glyco-immune checkpoint using EAGLE to potentiate potent preclinical antitumor activity through engagement of the anticancer immunity Fc receptor (FcR), CD32a Lizhi Cao, Jenny Che, Abhishek Das, PhD, Sujata Nerle, Wayne Gatlin, Marisa Peluso, MS, Kshama Doshi, PhD, Caroline Armet, BS, Li Zhang, Robert Leblanc, Zakir Siddiquee, Hui Xu, Karl Normington, PhD, MBA, Rachel O'Connor, BS, Matthew Rausch, PhD, Jonathan Hill, PhD, Weiguo Yao, James Broderick, Li Peng, PhD Benjamin Lee, MD PhD, Pamela Holland, PhD, Vito Palombella, PhD, Palleon Pharmaceuticals, Waltham, MA, United States Alison Paterson, PhD Correspondence: Li Peng (lpeng@palleonpharma.com) Surface Oncology, Inc., Cambridge, MA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P273 Correspondence: Marisa Peluso (mpeluso@surfaceoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P272 Background The glyco-immune checkpoint (Siglec/sialogylcan) axis has recently Background emerged as a new mechanism of cancer immune evasion. We have CD47 is a transmembrane protein that acts as a “Don’t Eat Me” signal previously described a bifunctional antibody-sialidase fusion platform to evade immune recognition. It is overexpressed in multiple cancer named EAGLE (Enzyme-Antibody Glyco-Ligand Editing) to inhibit this subtypes and is associated with poor prognosis. Several anti-CD47 axis by selectively removing the terminal sialic acids of sialoglycans molecules designed to antagonize the CD47 axis are being tested in on tumor cells. Using a bacterial sialidase for proof-of-concept stud- the clinic. Preclinical characteristics and antitumor mechanisms of ies, we have shown that EAGLE leads to enzyme-dependent robust the investigational agent SRF231, a fully human antibody targeting monotherapy efficacy with complete regressions and immune mem- CD47, are described. ory in syngeneic mouse tumor models. Methods Methods SRF231 monovalent affinity and binding properties were evaluated Since the bacterial sialidase poses immunogenicity concerns, we by Surface Plasmon Resonance (SPR) technology and in vitro agglu- engineered and optimized a human sialidase for EAGLE overcoming tination assays. Directly labeled SRF231 was used to profile tumor vs the low expression yield and poor stability of human sialidases. We normal cell binding. SRF231-mediated antitumor activity was confirmed the antitumor activity of human sialidase-containing assessed in tumor using macrophage coculture systems designed to EAGLE in vitro and in vivo using coculture of cancer cells and primary evaluate the impact of SRF231 on tumor cell phagocytosis, cell death, human immune cells and immunocompetent tumor models. Further- and cell depletion. Receptor occupancy (RO)/activity relationships more, we explored and identified predictive and correlative pharma- and dependency on FcR were also assessed. A xenograft tumor codynamic (PD) biomarkers to EAGLE treatment in preclinical tumor model was used to characterize the pharmacokinetic (PK)/pharmaco- models. dynamic (PD)/tumor exposure/efficacy relationship of SRF231 follow- Results ing single dose administration. The expression yield of human sialidase was improved by ~400 fold Results compared to the wild type through protein engineering, which en- SRF231 is a fully human, high-affinity anti-CD47 antibody with a slow ables the production of human sialidase-based EAGLE. We con- off-rate and binding mode that does not lead to agglutination of structed EAGLE-408, consisting of the engineered human sialidase patient-derived red blood cells or tumor cells. Increased binding to and anti-HER2 antibody trastuzumab, and confirmed its robust desia- several tumor vs normal cells is observed with SRF231. Analyses lylation efficiency using various HER2-expressing tumor cells in vitro. probing the relationship between FcR and SRF231 activity revealed EAGLE-408 enhanced macrophage-mediated phagocytosis of tumor that SRF231 leads to antitumor activity through both phagocytosis cells in vitro. EAGLE-408 also demonstrated enzyme-dependent and cell death mechanisms in a manner largely dependent on the monotherapy efficacy with complete regressions and immune mem- activating receptor FcγRIIa (CD32a), predominantly expressed by ory in syngeneic EMT6-HER2 mouse tumor models. Furthermore, in myeloid cells. Additionally, SRF231-mediated antitumor activity is the PD study, we observed EAGLE-408 treatment enhanced CD8+ T retained in longer-term assay conditions and in washout conditions. cell infiltration into tumors, increased CD8+ T cells in the draining Moreover, submaximal SRF231 RO is sufficient for maximal phagocyt- lymph nodes, and augmented NK cells and myeloid cells in osis induction in vitro. In a B-cell lymphoma xenograft model, a sin- circulation. gle dose of SRF231/mouse yields tumor stasis out to 21 days with Conclusions submaximal tumor exposure. Antitumor activity is associated with an In summary, EAGLE with engineered human sialidase offers a increase of host macrophage infiltration and cytokine induction sug- new immunomodulatory approach to overcome resistance to gestive of an innate immune response. current immunotherapies by effectively inhibiting Siglec/sialogly- Conclusions can axis in the tumor microenvironment. SRF231 is a high affinity, CD47-targeting antibody that delivers an ac- tivating signal to myeloid cells via CD32a and displays favorable pre- P274 clinical characteristics regarding its RO/tumor exposure/efficacy Antibody targeting of tumor associated macrophages in relationship. SRF231 is currently being evaluated in a Phase 1 clinical pancreatic cancer and melanoma remodel the tumor trial [NCT03512340] in advanced solid tumors and lymphomas. Un- microenvironment and revives immune targeting of tumor cells derstanding these PK/PD/tumor exposure/efficacy relationships, as Dhifaf Sarhan, PhD (dhifaf.sarhan@ki.se) well as the role of target vs FcR affinity, offers guidance on the devel- Karolinska Institute, Stockholm, Sweden opment of CD47 antagonists for patients with cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P274 Ethics Approval Mice were used in compliance with protocols approved by the IACUC Background of Mispro Biotech Services, Cambridge, MA (#2017-03-21SUR-1), Immunotherapy for cancer has revolutionized clinical practice and Charles River Accelerator and Development Lab, Cambridge, MA enabled cures for previously lethal cancers. However, the clinical re- (#CR-008), Charles River Laboratories, Worcester, MA (#I023), or MI sponses are variable and highly influenced by immune regulatory Bioresearch, Ann Arbor, MI (VUF #26). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 148 of 272 compartments in the tumor microenvironment (TME). This is espe- tumor types. Immunotherapeutic agents have a mechanism of action cially true for pancreatic cancer (PC) where clinical trials aiming to re- distinct from chemotherapy and are being used across a broad array cover T cell anti-tumor activity have been disappointing. Thus, in PC of tumor types. A standardized, universal scoring system for patho- and other cancers there is a clinical need for alternative treatments logic response that encompasses features characteristic for immuno- We have previously shown that antibodies targeting the scavenger therapy and spans tumor types is needed. receptor MARCO expressed on tumor-associated macrophages Methods (TAMs), reduces tumor growth and impair metastasis in murine can- Hematoxylin and eosin-stained slides from neoadjuvant surgical re- cer models. Here we investigated targeting of the scavenger receptor sections and on-treatment biopsies were assessed for features of MARCO on human TAMs in pancreatic ductal adenocarcinoma immune-related pathologic response (irPR). 258 specimens from pa- (PDAC) and hypothesized that targeting this receptor will remodel tients with 11 tumor types as part of ongoing clinical trials for anti- the suppressive environment and relive the anti-tumor responses to PD-1 were evaluated. An additional 98 specimens from patients re- increase the efficacy of immunotherapy. ceiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types. Methods Results To test our hypothesis, analysis of MARCO gene expression data Common irPR features (immune activation, cell death, tissue repair, from the Human Protein Atlas (HPA) project was performed in- regression bed) were present in all tumor types reviewed, including vestigating pancreatic tumors (n=176), as these consist of up to melanoma, non-small cell lung, head and neck squamous cell, Merkel 80% stroma, compared with healthy pancreatic tissues (n=171). In cell, and renal cell carcinoma, amongst others. Features were consist- vitro, cytokine differentiated macrophages alternatively cultured ent across primary tumors, lymph nodes, and distant metastases. with PC cell lines under hypoxia and normoxia conditions were Specimens from patients treated with anti-PD-(L)1 in combination co-cultured with cytotoxic cells to mimic their interaction in the with another agent also exhibited irPR features. TME. Later, macrophages were treated with anti-MARCO Abs and Conclusions their phenotype and function were examined prior and following irPR features are consistent across tumor types and treatment set- interaction with immune effector cells. Subsequently, anti-MARCO tings. Standardized, pan-tumor immune-mediated pathologic re- ab anti-tumor efficacy was tested in vitro and in vivo in PC and sponse criteria (irPRC) are defined and associated specimen-handling melanoma models. considerations are described. Future, prospective studies are merited Results to validate irPRC in larger datasets and to associate pathologic fea- We found a 30-fold increase in MARCO expression in pancreatic tu- tures with long-term patient outcomes. mors compared to healthy tissues. Also, a significant (p=0.03) correl- Ethics Approval ation between high expression and decreased survival was noted in The study was approved by the Johns Hopkins University Institu- pancreatic cancer patients. Furthermore, pancreatic cancer cell lines tional Review Board. induced MARCO expression on macrophages and dedifferentiated them towards myeloid-derived suppressor cells (MDSC). This ef- fect was amplified by hypoxic condition. Notably, MARCO+ MDSC P276 in contrast to control monocytes and macrophages suppressed T- Efficacy of PD-1/PDL-1 Immune Checkpoint Inhibitors in and Natural Killer (NK) cell anti-tumor activities, which was re- Elderly Patients with Non-Small-Cell Lung Cancer; A Subgroup versed by treatment with anti-human MARCO Abs. In addition, Meta-analysis of Randomized Controlled Trials targeting TAMs with anti-MARCO Abs, abolished their anti- 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD inflammatory phenotype in vitro and in vivo and normalized their 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of metabolism towards pro-inflammatory. Moreover, in B16 melan- Medicine at Mount Sinai, New York, NY, United States; King Edward oma tumor model the anti-MARCO Ab mediated an anti-tumor Medical University, Lahore, Pakistan; The University of Texas, effect that was dependent on NK cells and their TRAIL-mediated Houston, TX, United States killing mechanism. Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P276 Thus, our study reveals a novel interaction between TAMs and cyto- toxic cells as a result of TAM targeting with monoclonal Ab demon- Background strating a promising approach as immunotherapy that remodel the PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as immune TME. an efficacious drug class for the treatment of non-small-cell lung can- Ethics Approval cer (NSCLC). The efficacy of PD-1/PD-L1 ICPI therapy in the elderly The study was approved by Institutional Ethics Board, approval num- (patients age ≥65 and ≥75) has not been thoroughly investigated. ber Dnr 2013.977-31.1. The aim of this study was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy in elderly patients with NSCLC. Methods P275 A systematic review of the literature to identify randomized con- Pan-tumor pathologic scoring of response to PD-(L)1 blockade trolled trials (RCTs) which reported overall survival (OS) and progres- Julie Stein, Evan Lipson, MD, Tricia Cottrell, MD, PhD, Patrick Forde, sion free survival (PFS) of elderly patients with NSCLC who were MD, Robert Anders, MD, PhD, Ashley Cimino-Mathews, Elizabeth randomized to receive PD-1/PDL-1 ICPIs or docetaxel/investigator’s Thompson, MD PhD, Mohamad Allaf, MD, Mark Yarchoan, Josephine choice chemotherapy. The hazard ratios (HR) of OS and PFS in elderly Feliciano, MD, Elizabeth Jaffee, MD, Drew Pardoll, MD, PhD, Suzanne patients (along with their 95% confidence intervals; CI) were ex- Topalian, MD, Janis Taube, MD, MSC tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Johns Hopkins University School of Medicine, Baltimore, MD, United 1 versus chemotherapy stratified by patient age (≥65 and ≥75). A States random effects model was employed only when there was significant Correspondence: Janis Taube (jtaube1@jhmi.edu) heterogeneity among studies (>40%, as assessed by I-squared). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P275 Results Screening of 15,092 studies yielded four RCTs (two with patients age Background ≥75) which enrolled a total of 2,429 patients. No significant difference Pathologic response assessment of tumor specimens from patients in PFS with PD-1/PDL-1 treatment versus chemotherapy was found receiving systemic treatment provide an early indication of thera- among patients aged ≥65 (HR 0.8, 95% CI 0.53-1.06, I2 78.30%) or pa- peutic efficacy and predict long-term survival. Grading systems for tients aged ≥75 (HR 1.1, 95% CI 0.43-1.77,I2 0.00%). Patients aged ≥65 pathologic response were first developed for chemotherapy in select had an improved OS with PD-1/PD-L1 ICPI treatment versus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 149 of 272 chemotherapy (HR 0.67, 95% CI 0.51-0.84, I2 62.50%). Patients aged 1 versus chemotherapy stratified by the degree of PD-L1 expression. ≥75 did not show any significant difference in OS when treated with A random effects model was employed only when there was signifi- PD-1/PD-L1 versus chemotherapy (HR 1.02, 95% CI 0.35-1.69,I2 0.00%). cant heterogeneity among studies (>40%, as assessed by I-squared Results Conclusions Screening of 15,092 studies yielded four RCTs (two reporting only Patients aged ≥65 show an improved OS with PD-1/PD-L1 therapy. How- PD-L1 expression ≥50%) which enrolled a total of 2,429 patients. An ever, NSCLC patients ≥75 do not show a significant difference in OS or improved PFS with PD-1/PDL-1 treatment versus chemotherapy was PFS when treated with PD-1/PD-L1 ICPI versus chemotherapy (Figure 1). found among patients with PD-L1 expression ≤1% (HR 0.78, 95% CI This may be a consequence of aging and its impact on individuals’ cap- 0.57-0.98, I2 25.30%) ≥1% (HR 0.62, 95% CI 0.46-0.78, I2 0.00%), ≥5% ability to mount an anti-tumor response. Further studies assessing the ef- (HR 0.46, 95% CI 0.31-0.6, I2 0.00%), and ≥10% (HR 0.44, 95% CI ficacy of PD-1/PD-L1 ICPI use in patients ≥75 are warranted. 0.29-0.58, I2 0.00%). Similarly, an improved OS was observed with PD-1/PDL-1 treatment versus chemotherapy was found among patients with PD-L1 expression ≥1% (HR 0.7, 95% CI 0.52-0.87, I2 0.00%), ≥5% (HR 0.54, 95% CI 0.38-0.69, I2 0.00%), and ≥10% (HR 0.51, 95% CI 0.35-0.68, I2 0.00%), but not with PD-L1 expression of ≤1%, ≤5%, and ≤10% (figure 1). No significant difference in OS and PFS was observed with PD-1/PD-L1 treatment versus chemotherapy among patients with ≥50% PD-L1 expression. Of the two RCTs which reported ≥50% PD-L1 expression, one used Nivolumab (and reported a negative outcome in OS and PFS) whereas the other used Pembrolizu- mab (and reported a favorable OS and PFS). Conclusions NSCLC patients who express PD-L1 have an improved OS and PFS with PD-1/PD-L1 ICPI use versus chemotherapy. However, high PD-L1 expression (≥50%) may not translate into a better response to all PD- 1/PD-L1 ICPIs (Figure 1). Further studies are needed to assess the intra-class efficacy of different PD-1/PD-L1 ICPIs in NSCLC patients with a high PD-L1 expression. Fig. 1 (abstract P276). OS and PFS in Elderly Patients with NSCLC P277 Utility of PD-L1 Expression in Non-Small-Cell Lung Cancer Patients Treated with PD-1/PDL-1 Immune Checkpoint Inhibitors; A Subgroup Meta-analysis of Randomized Controlled Trials 1 2 3 4 Faisal Ali , Maryam Hussain , Arafat Farooqui , Syed Jafri, MD 1 2 Saint Joseph Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States; King Edward Medical University, Lahore, Pakistan; The University of Texas, Houston, TX, United States Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P277 Background Fig. 1 (abstract P277). OS and PFS Stratified by PD-L1 Expression PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an efficacious drug class for the treatment of non-small-cell lung can- cer (NSCLC) evident by findings of multiple randomized controlled P278 trials (RCTs). The utility of PD-L1 expression analysis in treatment PD-1/PDL-1 immune checkpoint inhibitors in smokers with non- planning and prognosis remains questionable. The aim of this study small-cell lung cancer; a subgroup meta-analysis of randomized was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemo- controlled trials therapy in patients with NSCLC stratified by PD-L1 expression 1 2 3 4 Faisal Ali , Bibek Pannu , Maryam Hussain , Arafat Farooqui , Taha Methods 1 1 5 Alrifai , Phyo Myint , Syed Jafri, MD A systematic review of the literature to identify RCTs which reported 1 2 Saint Joseph Hospital, Chicago, IL, United States; John H Stroger Jr overall survival (OS) and progression free survival (PFS) of patients Hospital, Chicago, IL, United States; Icahn School of Medicine at Mount with NSCLC who were randomized to receive PD-1/PDL-1 ICPIs or do- Sinai, New York, NY, United States; King Edward Medical University, cetaxel/investigator’s choice chemotherapy and underwent PD-L1 ex- Lahore, Pakistan; The University of Texas, Houston, TX, United States pression analysis. The hazard ratios (HR) of various degrees of PD-L1 Correspondence: Syed Jafri (syed.h.jafri@uth.tmc.edu) expression (along with their 95% confidence intervals; CI) were ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P278 tracted to compute a pooled (HR) to report the efficacy of PD-1/PDL- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 150 of 272 Background P279 PD-1/PDL-1 immune-checkpoint inhibitors (ICPIs) have emerged as an effi- PDL-1 is highly expressed in ovarian germ cell tumor and cacious drug class for the treatment of non-small-cell lung cancer (NSCLC) associated with cancer stem cells population expressing CD44 evident by findings of multiple randomized controlled trials (RCTs). How- Salmah Alamri, Miral Mashhour, Kholoud Alwosaibai, PhD ever, the efficacy of PD-1/PDL-1 ICPIs in patients with NSCLC who are King Fahad Specialist Hospital, Dammam, Saudi Arabia current or former smokers remains to be debated. The aim of this study Correspondence: Kholoud Alwosaibai (kh20978@gmail.com) was to assess the efficacy of PD-1/PDL-1 ICPIs compared to chemotherapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P279 in patients with NSCLC who were active or former smokers Methods Background A systematic review of the literature to identify RCTs which reported Immunotherapy using checkpoint inhibitors have proposed beneficial overall survival (OS) and progression free survival (PFS) among effects for some types of cancer such as lung cancer and melanoma. former or active smokers with NSCLC who were randomized to re- However, using PD-1/PDL-1 inhibitors to treat ovarian cancer was lim- ceive PD-1/PDL-1 ICPIs or docetaxel or investigator’s choice chemo- ited and further studies are needed to identify the patients that will therapy. The hazard ratios (HR) of these subgroups (along with their benefit from this treatment. In this study we have explored predictive 95% confidence intervals; CI) were extracted to compute a pooled biomarkers in ovarian cancer that might associate with checkpoint (HR) to report the efficacy of PD-1/PDL-1 versus chemotherapy in treatment outcome. For the first time, we have investigated the role of former or active smokers with NSCLC. A random effects model was PDL-1 expression in the tumor microenvironment cells that includes employed only when there was significant heterogeneity among immune cells and cancer stem cells in different types of ovarian cancer. studies (>40%, as assessed by I-squared) Methods Results 66 surgical samples of different types of ovarian cancer have been Screening of 15,092 studies yielded six RCTs which enrolled a total of collected from pathology department. IHC staining has been per- 3,584 patients. PFS did not differ significantly with PD-1/PDL-1 treat- formed using PD-L1 IHC 22C3 pharmDx to detect PDL-1, CD8 and ment or chemotherapy among non-smokers (HR 1.33, 95% 0.80-1.80; I2 CD4 to detect tumor infiltrating lymphocyte (TIL), and CD44, CD117 20.6%) and former or active smokers (HR 0.83, 95% CI 0.63-1.04; I2 and OCT3/4 to detect stem cell markers. 67.5%). Survival analysis revealed that former or active smokers tended Results to have an improved OS when treated with PD-1/PDL-1 inhibitors ver- We found that 47% of ovarian cancer patients express PDL-1. The expres- sus chemotherapy (HR 0.72, 95% CI 0.54-0.90; I2 70.5%), whereas OS sion of PDL-1 have been detected in different types of ovarian cancer in- did not differ significantly among non-smokers when treated with PD- cluding, serous carcinoma, germ cell tumor, endometrioid. The majority 1/PDL-1 inhibitors versus chemotherapy (HR 0.76, 95% CI 0.48-1.04; I2 (73%) of germ cell tumor tissues express PDL-1 whereas serous cancer 0.0%) and endomitrioid express PDL-1 in 46% and 50% of the cancer tissue, re- Conclusions spectively. However, PDL-1 protein was undetectable in some histological NSCLC patients who are former or active smokers have an improved type of ovarian cancer such as granulosa tumor and mucinous tumor. OS when treated with PD-1/PDL-1 ICPIs, though the PFS does not dif- Also we determined the expression levels of TIL in the ovarian cancer fer significantly (Figure 1). This may be a consequence of a higher tissue that either PDL-1 positive or negative. We found that 81% of mutation burden among smokers. Further research into the inter- ovarian cancer samples have TIL that express CD8 and 92% of these action between carcinogens, toxins and proinflammatory substances ovarian cancer samples are associated with PDL-1 expression.TIL that of smoking and ICPIs are warranted. express CD4 have been found in 66% of all ovarian cancer samples and 77% of these samples are associated with PDL-1 expression. Further, we have studied the association between PDL-1 expression and cancer stem cell markers such as CD44, CD117, OCT 3/4. We found that all PDL-1 positive samples are expressing CD44 and also, we found strong association between CD117 expression and PDL-1 expression. Conclusions Immunotherapy treatment using PDL-1 inhibitor could be considered for ovarian cancer patients that expressing PDL-1 particularly, germ cell ovarian cancer. In addition, PDL-1 expression is strongly associated with CD44. Inhibiting PDL-1 using immunotherapy might downregulate stem cell populations and decrease the chemotherapy resistance and recurrence that derived by stem cell residual in the cancer tissue. Ethics Approval The study was approved by the IRB at King Fahd Specialist Hospital- Dammam. approval number ONC0340 P280 Impact of tumor mutational burden on overall survival in patients with non-small cell lung cancer treated with immunotherapy 1 2 1 Mark Awad, MD PhD , Navin Mahadevan , Andrew Polio , Natalie 1 1 1 1 Vokes , Elizabeth Aguilar , Biagio Ricciuti, MD , Giuseppe Lamberti, MD , 1 1 1 Gonzalo Recondo, MD , Giulia Leonardi , Anika Adeni , Pasi Janne, MD 1 1 1 1 PhD , Eliezer Van Allen, MD , Adem Albayrak, MS , Renato Umeton , Lynette Sholl 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Women’s Hospital, Boston, MA, United States Correspondence: Mark Awad (Mark_Awad@DFCI.harvard.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P280 Background In non-small cell lung cancer (NSCLC), tumor mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint in- hibitors, but the optimal TMB cutpoint associated with a survival benefit Fig. 1 (abstract P278). OS and PFS in Smokers and Non-Smokers remains unclear. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 151 of 272 Methods We collected clinicopathologic data from patients with NSCLC se- quenced by the OncoPanel NGS platform at the Dana-Farber Cancer Institute. The relationship between TMB and clinical outcomes after treatment with immune checkpoint inhibitors was investigated in the subset of patients treated with immunotherapy. Results Among 2690 patients identified, median TMB was significantly higher Fig. 3 (abstract P280). See text for description among current smokers compared to former (P<0.0001) and never smokers (P<0.0001) and among squamous tumors compared to smoking-related nonsquamous tumors (P=0.01) (Figure 1). Patients without oncogenic drivers and those harboring BRAF or KRAS mutation had the highest median TMB (10.6, 11.1 and 9.8 mutations/megabase [mut/Mb], respectively), while tumors with ROS1, MET, RET and ALK al- terations had the lowest median TMB (6.7, 6.1, 5.3, 5.3 mut/Mb, respect- ively) (Figure 2). Among patients treated with immunotherapy (N=489), a recursive partitioning algorithm identified an optimal TMB cut-off for PFS and OS of 18.5 mut/Mb, which represents the 88th percentile for TMB in this cohort. Baseline clinicopathologic characteristics were well- balanced between patients with a TMB of ≥18.5 and <18.5 mut/Mb (Table 1). Patients with a TMB of ≥18.5 mut/Mb had a significantly Table 1 (abstract P280). See text for description higher response rate (43.3% vs. 17.5%, P<0.0001), a longer median progression-free survival (8.2 vs. 2.7 months, HR:0.52 [95%CI:0.38-0.72], P<0.0001), and a longer median overall survival (20.7 vs. 10.2 months, HR:0.55 [95%CI:0.38-0.79], P=0.001) compared to those with a TMB < 18.5 mut/Mb (Figure 3). After adjusting for performance status, smoking history, and PD-L1 expression, a TMB of ≥18.5 mut/Mb was associated with a significantly longer PFS (HR:0.56 [95%CI: 0.41-0.78], P =0.001) and OS (HR: 0.57 [95%CI: 0.40-0.82], P =0.003) in multivariate analysis. Conclusions Tumor-only NGS identifies clinical and genomic correlates of high TMB in NSCLC. Patients with a TMB ≥88th percentile are most likely to experi- ence a survival benefit when treated with immune checkpoint inhibitors. Fig. 1 (abstract P280). See text for description Fig. 2 (abstract P280). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 152 of 272 P281 L1+ tumors, do not benefit. The magnitude of immune activation Durable response after immunotherapy discontinuation: a promoted by PD-1/PD-L1 axis blockade can be further enhanced multicenter real-life experience through concomitant T-cell co-stimulation such as that achieved 1 2 3 Maria Bassanelli, MD, PhD , Diana Giannarelli, MD , Marco Russano , through CD137 agonism; however, clinical applications of such an 2 4 5 Fabiana Letizia Cecere , Maria Rita Migliorino , Silvana Giacinti , Viola approach may be limited by toxicity associated with the systemic ef- 4 6 5 4 Barucca , Emilio Bria , Enzo Maria Ruggeri , Fabio Calabrò , Alain fects of CD137 agonists. Here we characterize PD-L1 and CD137 7 3 1 Gelibter , Daniele Santini , Anna Ceribelli tumor expression supporting the development of a PD-L1xCD137 1 2 San Camillo de Lellis Hospital, Rieti, Italy; Regina Elena National Cancer bispecific molecule that provides PD-1 axis blockade coupled with 3 4 Institute, Rome, Italy; University Campus Bio-Medico, Rome, Italy; San tumor-targeted CD137 co-stimulation. Camillo Forlanini Hospital, Rome, Italy; Belcolle Hospital, Viterbo, Italy; Methods 6 7 Fondazione Pol. Universitario A.Gemelli, Rome, Italy; Policlinico In situ hybridization (ISH) and multicolor flow cytometry was per- Umberto I, Rome, Italy formed to characterize PD-L1 and CD137 expression in tumor biop- Correspondence: Maria Bassanelli (maria.bassanelli@yahoo.it) sies. A PD-L1xCD137 bispecific molecule (PD-L1xCD137) was Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P281 constructed based on PD-L1 blocking mAbs and CD137-engaging mAbs and was evaluated for binding to respective antigens. Its func- Background tional activity was evaluated in CD3 or SEB-driven T-cell activation Immune checkpoint inhibitors (ICIs) have significantly improved over- systems, MLR assays and tumor microenvironment models. Anti- all survival (OS) in several cancer types. Unlike chemotherapy, the tumor activity in vivo was evaluated in combination with tumor tar- optimum treatment duration with ICIs is not clearly established [1,2] geted anti-CD3 based bispecific DART® molecules. Methods Results We conducted an observational, retrospective analysis of 46 consecu- ISH revealed expression of PD-L1 in significant proportion of surgi- tive patients (pts) with advanced solid tumors who discontinued cally resected carcinomas; noteworthy, many such tumors contained immune-based therapies for any reason except progressive disease. CD137+ immune infiltrate adjacent to PD-L1+ cells. Moreover, The aim of this study was to assess the outcome and the antitumor ex vivo co-incubation of tumor and immune cells in the presence of activity of ICIs after treatment discontinuation. Treatment-free sur- CD3-based bispecifics or Fc-enhanced antibodies further induces PD- vival (TFS) was defined as the time from interruption of immunother- L1 and CD137 expression. PD-L1xCD137 binds and blocks PD-L1, re- apy for any reason except progressive disease to start of subsequent versing PD-1-mediated T-cell inhibition equipotently to the effect of anticancer therapy or best supportive care or death. Median OS, pro- approved PD-L1 benchmark mAbs; it also binds CD137, but absent gression free survival (PFS), TFS and the 95% confidence interval (CI) clustering supported by PD-L1+ cells, fails to induce CD137 signaling. were estimated with the Kaplan -Meier method In the presence of PD-L1-expressing cells, however, PD-L1xCD137 Results drives CD137 activation and immune cell co-stimulation. Robust T- 46 pts (median age 68 years [range 41-86]; male: 65.2%) with advanced cell activation and cytokine secretion was induced by PD-L1xCD137, cancer (n.39 non-small-cell lung cancer, n.15 renal cell carcinoma and with significantly greater activity than that observed with the com- n.2 melanoma) were treated with ICIs, as clinically indicated, at eight bination of PD-L1 blocking and CD137 agonistic mAbs. Notably, Italian institutions: 44 pts received programmed death 1 (PD-1) inhibi- when combined with tumor targeted immunotherapies, PD- tors (n.31 nivolumab, n.13 pembrolizumab) and 2 pts programmed L1xCD137 supports enhanced activation of effector cells and anti- death ligand 1 (PD-L1) (n.1 durvalumab, n.1 atezolizumab). A median of tumor activity. 8 cycles were administered [range 1 to 52]. 36 pts discontinued ICIs Conclusions due to toxicities (diarrhoea, pneumonitis, hepatotoxicity)and 10 pts for These data show that an investigational PD-L1xCD137 bispecific can reasons non immune-related. The median PFS from the beginning of switch on CD137 co-stimulation in a PD-L1-dependent fashion. While ICIs was 12.4 months (mo) [95% CI: 8.2-16.6] and the median OS was tumor adaptive resistance via PD-L1 induction promotes tumor im- 20.0 mo (95% CI: 11.8-28.2). Median PFS from ICIs completion was 5.0 mune escape, PD-L1xCD137 can exploit the checkpoint ligand up- mo [95% CI: 2.7-7.3] and median OS was 16.1 mo (95% CI: 5.4-26.8). Me- regulation by contributing a co-stimulatory signal in addition to dian TFS was 7.4 (95% CI: 5.8-8.9) mo checkpoint blockade. PD-L1xCD137 provides a potential therapeutic Conclusions approach to overcome limitations of existing PD-1/PD-L1-targeting This study shows the durable cancer-specific immune response in pts strategies either as monotherapy or in combination with comple- with advanced cancer even after stopping the ICIs for any reason ex- mentary immune based therapeutic modalities, such as CD3 based cept progressive disease bispecifics or Fc-enhanced mAbs. References P283 1. Kim PS, Ahmed R. Features of responding T cells in cancer and chronic Generation and characterization of a PD-1 resistant mouse tumor infection. Curr Opin Immunol 2010; 22: 223–230. model 2. Schadendorf D, Hodi FS, Robert C et al. Pooled analysis of long-term sur- Marie Bernardo, PhD, Tatiana Tolstykh, Yu-An Zhang, Dinesh Bangari, Hui vival data from phase II and phase III trials of ipilimumab in unresectable Cao, PhD, Joon Sang Lee, PhD, Natalia Malkova, Jack Pollard, PhD, or metastatic melanoma. J Clin Oncol 2015; 33: 1889–1894. Fangxian Sun, Dmitri Wiederschain, Timothy Wagenaar Sanofi Oncology Research, Cambridge, MA, United States P282 Correspondence: Timothy Wagenaar (timothy.wagenaar@sanofi.com) Tumor-targeted T-cell activation via an investigational PD-L1 x Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P283 CD137 bispecific molecule Alexey Berezhnoy, PhD, Ling Huang, Daorong Liu, Jennifer DiChiara, Background Jonathan Li, PhD, Douglas Smith, PhD, Jill Rillema, BS, Valentina Immune checkpoint blockade elicits durable anti-cancer responses Ciccarone, PhD, James Tamura, PhD, Ralph Alderson, PhD, Gundo in the clinic, however a large proportion of patients do not bene- Diedrich, Ezio Bonvini, PhD, Paul Moore, PhD fit from treatment. Several mechanisms of innate and acquired MacroGenics, Inc, Rockville, MD, United States resistance to checkpoint blockade have been defined and include Correspondence: Paul Moore (moorep@macrogenics.com) mutations of MHC I and IFNg signaling pathways. However, such Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P282 mutations occur in a low frequency of patients and additional mechanisms have yet to be defined. In an effort to better under- Background stand acquired resistance to checkpoint blockade, we generated Blockade of the PD-1/PD-L1 axis can improve outcome in a variety of a mouse tumor model exhibiting in vivo resistance to anti-PD-1 cancers; yet, many patients, including subsets of patients with PD- antibody treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 153 of 272 Methods CD8+ T cells in the blood of E2 supplemented huNOG mice. Tumor A PD-1 resistant mouse tumor model was generated by serially pas- immunohistochemistry showed low number of TILs and very low ex- saging MC38 tumors in mice treated with anti-PD-1. The resistant pression of programmed death-ligand 1 (PD-L1). No clear anti-tumor tumor line was characterized using a range of molecular techniques. effects were observed with pembrolizumab treatment compared to Results the isotype control group. MC38 tumors acquired resistance to PD-1 blockade following serial Conclusions in vivo passaging. Lack of sensitivity to PD-1 blockade could not be In conclusion, estrogen supplementation was needed to support attributed to dysregulation of PD-L1 or B2M expression, as both were orthotopic ER+ breast cancer growth. However, estrogen decreased expressed at similar levels in parental and resistant cells. Similarly, survival of female huNOG mice. Estrogen had immunomodulatory ef- IFNg signaling and antigen processing and presentation pathways fects and induced adverse effects including anemia. Due to these were functional in both parental and resistant cell lines. Unbiased deleterious effects and decreased number of immune cells, no direct gene expression analysis was used to further characterize potential conclusions can be drawn for the possible anti-tumor effects of pem- resistance mechanisms. RNA-sequencing revealed substantial differ- brolizumab in this orthotopic ER+ breast cancer model. Caution ences in global gene expression with PD-1 resistant tumors display- should be taken when evaluating efficacy of immunotherapies in ing a marked reduction in expression of immune-related genes hormone-dependent preclinical cancer models, and using ER+ breast relative to parental MC38 tumors. Transcriptomic data revealed that cancer models where tumor growth is supported by local microenvir- PD-1 resistant tumors exhibit reduced immune infiltration across onment, such as in bone metastasis models, should be considered. multiple cell types, including T and NK cells, while pathway analysis Ethics Approval identified activation of two major tumor promoting signaling path- This study was approved by the National Animal Experiment Board ways in PD-1 resistant tumors. Pharmacological inhibition of these in Finland; license number ESAVI-2331-04 10 07-2017. pathways in combination with PD-1 blockade inhibited tumor growth and extended the survival of mice bearing resistant tumors. P285 Conclusions Integrated molecular characterization of primary resistance This study describes a novel PD-1 resistant mouse tumor model and mechanisms to immune checkpoint blockade in advanced non- underscores the importance of two well defined signaling pathways small cell lung carcinoma (a-NSCLC) to response to immune checkpoint blockade. 1 2 2 Benjamin Besse, MD PhD , Caroline Fraslon, PhD , Hui Cao, PhD , Joon 2 2 2 Sang Lee, PhD , Stephanie Malyszka, MS , Marielle Chiron, PhD , Anne 2 2 1 P284 Caron, PhD , Souâd Naimi, PhD , Laura Mezquita, MD , David Planchard, 1 1 1 Pitfalls in preclinical development of immunotherapies for ER+ MD, PhD , Jean-Yves Scoazec, MD , Ludovic Lacroix, PharmD , Etienne 1 1 breast cancer: estrogen as an immunomodulator potentially Rouleau, MD , Naima Imam-Sghiouar, PhD , Aurélien Marabelle, MD 1 1 1 2 influencing pembrolizumab efficacy in a breast cancer model in PhD , Eric Angevin, MD , Christophe Massard, MD , Jack Pollard, PhD 1 2 humanized mice Gustave Roussy Cancer Center, Villejuif, France; Sanofi, Vitry sur Seine, 1 1 1 2 Tiina Kähkönen , Mari Suominen , Jenni Mäki-Jouppila , Emrah Yatkin , France 3 3 1 1 Philip Dube , Azusa Tanaka , Jussi Halleen , Jenni Bernoulli, PhD Correspondence: Caroline Fraslon (caroline.fraslon@sanofi.com) 1 2 Pharmatest Services, Turku, Finland; University of Turku, Turku, Finland; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P285 Taconic Biosciences, Rensselaer, NY, United States Correspondence: Jenni Bernoulli (jenni.bernoulli@pharmatest.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P284 Reinvigoration of anti-tumor immunity via immune checkpoint block- ade (ICB) has transformed outcomes in a-NSCLC. However, a majority Background of patients are innately resistant to ICB, and a better understanding Immunotherapies have the potential to improve outcomes in triple- of the resistance mechanisms may guide the development of new negative breast cancer patients but evidence is less consistent in es- treatment strategies and patients therapies. trogen receptor -positive (ER+) patients. To advance preclinical devel- Methods opment and understand the effects of immunotherapies against ER+ Biopsies performed immediately before treatment with single agent breast cancer, we aimed to establish a novel orthotopic ER+ breast ICB in patients with a-NSCLC (MATCH-R trial [NCT02517892]) were an- cancer model in humanized mice and to study efficacy of pembroli- alyzed. The stromal microenvironment and immune context was zumab in the model. characterized via an integrated analysis of whole transcriptome Methods (RNA-seq), whole exome sequencing (WES), and immunohistochemis- Female CIEA NOG® (NOG) mice and NOG mice engrafted with human try (IHC) of CD3, CD8, FOXP3 and PDL1. Specifically, the immune con- CD34+ hematopoietic stem cells (huNOG, Taconic Biosciences) were text and the relative abundance of 10 immune and stromal cell types implanted with 5 μg/day estradiol (E2)-releasing implants, and one were assessed with integrated IHC and Cell Populations-counter week later inoculated with ER+ MCF-7 human breast cancer cells into (MCP-counter) [1] analysis of the RNA-seq. Somatic mutations and the mammary fat pad. One group of huNOG mice did not receive E2 Tumor Mutation Burden (TMB) were evaluated. The transcriptional implants. Orthotopic tumor growth was followed by caliper mea- state of the tumor and its microenvironment was assessed by GSVA surements. At study week 2, the E2 supplemented mice were strati- analysis [2] of the MSigDB collection [3]. Patient’s outcome was asso- fied to receive human IgG4 isotype control or pembrolizumab (5 ciated to molecular data. Primary resistance to ICB was defined as PD mg/kg, i.p., Q5D) until the end of the study. The study was termi- in the first radiological examination, or a median PFS inferior to 3 nated at study week 7 and tumors were processed for histological months. and immunohistochemical analysis of tumor infiltrating lympho- Results cytes (TILs). Changes in blood cell counts were assessed by flow cy- Thirty-one patients with adenocarcinoma were enrolled: Median age tometry and hematology. was 60 (34-80), 13 were female, 28 were smokers, 10 were re- Results sponders, and 21 were non-responders. Median tumor cellularity was ER+ orthotopic breast tumors grew only in the presence of E2 sup- 60% (30%-90%). plement. However, general condition of huNOG mice started to de- Responders had higher TMB and immune infiltration compared to crease after 3 weeks on E2 supplementation and their survival was non-responders. Non-responders could be divided into two classes: decreased compared to huNOG mice without E2 supplement. those with equal infiltration to responders “hot tumors” and those Hematological analysis indicated that estrogen decreased the levels with less infiltration “cold tumors”. Neutrophil infiltration was associ- of white and red blood cells, hemoglobin and hematocrit that were ated with resistance to ICB in both “hot” and “cold” resistant tumors. further decreased by concomitant pembrolizumab treatment. Flow Mutations in the IFN-γ and/or KRAS/STK11/KEAP pathways were asso- cytometry analysis confirmed lower numbers of CD3+, CD4+ and ciated to ICB resistance. Increased activation of hypoxia-response, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 154 of 272 transforming growth factor (TGF-β) and MYC pathways from the partial responders and non-responders (high expression), thereby GSVA analysis were also associated to ICB resistance. TGF-β pathway suggesting that the identified target is relevant to the clinical activation was associated to the “cold tumor” ICB-resistant tumor situation. class. Conclusions Conclusions The mAb is currently under preclinical development as a novel im- ICB sensitivity was associated to TMB, IFN-γ pathway mutation, and munotherapeutic antibody to overcome resistance to anti-PD-1 im- immune infiltration. We have further refined our understanding of mune checkpoint blockade. the primary ICB resistance mechanisms in that there are both “hot” and “cold” non-responsive tumors, which suggests that different References therapeutic approaches be may be required in these two subtypes, 1. Perrot I, Michaud HA, Giraudon-Paoli M, et al. Blocking Antibodies i.e. targeting the TGF-β pathway in the “cold” non-responding tumor Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Im- class. We are continuing to increase our baseline cohort size and will mune Responses in Combination Cancer Therapies. Cell Rep. 2019 include post-treatment biopsies collected with this protocol. May 21;27(8):2411-2425.e9. doi: 10.1016/j.celrep.2019.04.091 2. Hugo W, Zaretsky JM, Sun L et al. Genomic and Transcriptomic References Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma. 1. Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, Selves J, Cell. 2016 Mar 24;165(1):35-44. doi: 10.1016/j.cell.2016.02.065. Epub Laurent-Puig P, Sautès-Fridman C, Fridman WH, de Reyniès A. Estimating 2016 Mar 17. the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol. 2016; 17(1):218-238. P287 2. Hänzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for A novel bispecific checkpoint inhibitor antibody to preferentially microarray and RNA-seq data. BMC Bioinformatics. 2013;14:7-22. block PD-1 and LAG-3 on dysfunctional TILs whilst sparing Treg 3. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, activation Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 1 1 2 Laura Codarri Deak , Patrick Weber, Mr , Stefan Seeber, Dr , Mario Perro, 2011; 27(12):1739-40. 1 1 1 1 Dr , Xavier Miot, Mr , Heidi Poulet, Mrs , Iryna Dekhtiarenko, Dr , Ethics Approval 3 3 4 Matthias Füth , Henry Kao, Dr , Christine McIntyre, Dr , Francesca This study was approved by Gustave Roussy Scientific Board, the French 3 1 1 Michielin, Dr , Christian Klein, Dr rer nat , Pablo Umana, PhD , Lin-Chi National Health Agency and Ethical Committee (ID-RCB : 2014-A01147- 5 2 3 Chen, Dr , Christoph Markert, Dr , Merlind Mücke, Dr 40) 1 2 Roche Innovation Center Zurich, Zurich, Switzerland; Roche Innovation Center Munich, Munich, Germany; Roche Innovation Center Basel, Basel, P286 Switzerland; Roche Innovation Center Welwyn, Welwyn, United In vivo genetic screens in PD-1 resistant mouse models identified Kingdom; Roche Innovation Center New York, New York, NY, United modulators of anti-PD1 response with relevance to States pembrolizumab-treated human metastatic melanoma Correspondence: Laura Codarri Deak (laura.codarri_deak@roche.com) Aurélie Docquier, PhD, Cécile Déjou, Gilles Alberici, Armand Bensussan, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P287 PhD, Jeremy Bastid, Nathalie Bonnefoy, PhD OREGA Biotech, Ecully, France Background Correspondence: Jeremy Bastid (jeremy.bastid@orega-biotech.com) Check point inhibitors targeting PD-1 have shown unprecedented Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P286 clinical efficacy in several cancer indications and therefore have, revolutionized the standard of care. However, despite this ad- Background vancement in cancer immunotherapy, only ~20-30% of the pa- We previously reported the identification of CD39 as a regulator of tients derive durable benefit from such a treatment. One of the anti-PD-1 response in combination with oxaliplatin in resistant suggested reasons for this limited success is the expression/acti- models using our screening approach [1]. vation of compensatory inhibitory pathways such as LAG-3 on Methods tumor-reactive T cells. These pathways compensate for the loss Similar approach was used to identify novel targets that modulate of function of PD-1 upon its blockade. Therefore, it is envisioned anti-PD-1 response. Here we report the discovery of a novel potent that simultaneous antagonism of PD-1 and LAG-3 receptors regulator of anti-PD-1 therapy in MCA205 fibrosarcoma and B16K1 would overcome this adaptive resistance mechanism and allow a melanoma syngeneic mouse models harboring no or low response more profound reinvigoration of dysfunctional tumor-reactive T rates to PD-1 immune checkpoint inhibitor. cells. Conversely, a recent report has highlighted that blockade of Results LAG-3 on regulatory T cells (Tregs) increases their suppressive Remarkably, the Knock-Out (KO) of the identified target had limited, function and, therefore may off-set its benefit on the reinvigor- if any, impact on tumor growth in various mouse models. Whereas ation of dysfunctional tumor-reactive T cells. the mouse models used are broadly resistant to anti-PD1 therapy, Methods treatment in the KO background markedly improved the efficacy of We therefore developed a 1+1 PD1-LAG3 bispecific antibody (BsAb) anti-PD-1 mAb by increasing the response rates and by increasing with a 10-20 fold higher affinity for PD-1 than for LAG-3, allowing an the rate of complete vs partial responses, which translated into im- avidity driven selectivity gain to PD-1 and LAG-3 double positive T proved mouse survivals. We generated various human-mouse cross- cells. reactive blocking antibodies to the target, including a humanized Results mAb. The neutralizing antibodies mimicked the KO phenotype and Hence, PD1-LAG3 BsAb is assumed to have the following advantages markedly improved the response to anti-PD1 therapy in preclinical over monospecific and other bispecific aPD/L1 and aLAG-3 anti- mouse models. The mechanism of action is being investigated. We bodies: 1) improved targeting to dysfunctional T cells rather than found that the expression of target within the tumor induces an im- Tregs due to the selectivity gain and different expression patterns of munosuppressive tumor immune microenvironment by upregulating PD-1 and LAG-3 on these two T cell types, 2) reduced internalization, several immunoregulatory cytokines and chemokines. Interestingly, 3) Fc silent-mediated resistance to drug-shaving by macrophages. we re-analyzed transcriptomic data from 28 metastatic melanoma tu- Conclusions mors prior to anti-PD-1 pembrolizumab therapy [2] to validate our These characteristics translated in a significant increase in 1) in vitro target and related signaling pathways. We found a stepwise in- T cell effector functions even in the presence of Tregs, and 2) in vivo creased expression of our target, its receptor and the identified tar- tumor control/eradication in mouse models compared to combin- gets of the pathway from complete responders (low expression) to ation of monospecific anti-PD1 and anti-LAG3 antibodies. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 155 of 272 P288 P289 CCR5+CTLA4+ Treg cell subset characterizes renal tumors (RCC) Macrophages modulate patient response to immune checkpoint immunosuppressed by PD1 blockade inhibition in a novel lung tumour explant model 1 1 1 1 1 Agathe Dubuisson , Agathe Dubuisson , Charles Bayard , Sebastien Lauren Evans, BSc, MSc , Kate Milward, DPhil , Richard Attanoos, BSc, MB 1 1 1 1 2 1 1 Lofek , Nicolas Voisin , Séverine Mouraud , Delphine Bredel , Sandrine BS, FRCPath , Aled Clayton, PhD , Rachel Errington, PhD , Zsuzsanna 1 1 1 2 1 Susini , Mathieu Rouanne, MD , Hervé Beaumert , Bastien Parier , Tabi, PhD 1 1 1 2 Aurélien Marabelle, MD PhD , Laurence Zitvogel, MD, PhD , Mélodie Cardiff University, Cardiff, United Kingdom; University Hospital Wales, 1 1 Bonvalet , Mélodie Bonvalet Cardiff, United Kingdom 1 2 Gustave Rousy, Villejuif, France; APHP, Le Kremlin Bicetre, France Correspondence: Lauren Evans (Evansl57@cardiff.ac.uk) Correspondence: Mélodie Bonvalet (bonvaletmelodie@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P289 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P288 Background Background The tumour microenvironment (TME) consists of a dynamic interplay A small subset of patients undergoing PD1 blockade therapy develop between the tumour and stroma. Various stroma-resident and a hyper-progressive disease (HPD), corresponding to an acceleration tumour-infiltrating immune cells are associated with pro-tumour ac- of tumor growth rate [1]. However, the mechanisms underlying HPD tivity. Whilst immunotherapies may trigger anti-tumour immune re- are unknown, and the identification of predictive biomarkers remains sponses, anti-inflammatory M2-like tumour-associated macrophages an unmet clinical need. We aimed at better understanding the mech- (TAMs) within the TME present an obstacle for effective treatment anism of hyper-progression following nivolumab treatment by exam- [1]. Pre-clinical research has predominantly used mouse models to ining the tumor immune infiltrate of fresh RCC using an ex vivo represent the in vivo TME. Unfortunately, such models do not faith- model system. fully replicate the human immune system therefore, providing an in- Methods adequate measure of immunotherapy response. Human tumour- Fifteen fresh primary RCC were processed according to methods pre- derived explants maintain the original 3D tumour architecture and viously reported [2] and stimulated for 3 days with nivolumab. At combination of multiple cell types. Therefore, we established an baseline, we monitored the phenotype of the immune infiltrate by ex vivo tumour explant model of non-small cell lung cancer (NSCLC) flow cytometry. After 3 days of culture, we measured 26 soluble fac- incorporating TAMs, to determine their role in immunotherapeutic tors which were released into the supernatant. We then examined response. the relationship between baseline phenotype and functional immune Methods reactivity to nivolumab, considering variations of at least 2 independ- Tumour explants (ca. 1mm³) were generated from fresh tumour tis- ent soluble factors in the range of sue. Autologous CD14 peripheral blood mononuclear cells (PBMCs) Results were added to explants for 48h followed by flow cytometry pheno- Nivolumab induced a strong inhibition of soluble factor release in 5 out typing using a 7-marker macrophage panel (CD14; CD64; PDL-1; of 15 RCC. The decreased soluble factors were IL1RA (3 out of 5 tumors CD163; CD206; CD23; CD200R). The functional contribution of macro- (3/5)), IFNg, CXCL10, IL10, G-CSF, GM-CSF (2/5), IL-4, IL-5, IL-6, IL-8, IL-9, phages to explant-mediated immunosuppression was assessed + + CCL4, CCL5, PDGFbb (1/5). In these hypo-sensitive tumors (HS), nivolu- through measuring IFNγ/TNFα production from CD4 /CD8 T cells by mab induced a significant decrease of IL-4, IL-8, G-CSF (p<0.05). flow cytometry. T cells, in the presence of explants, were stimulated Conclusions with a viral peptide pool and incubated for 6 days ±250μg/mL Atezo- The TME of HS renal tumors that are immunosuppressed by nivolu- lizumab, prior to intracellular cytokine staining. Culture supernatants mab display high levels of the CCR5 ligands and CCR5+ CTLA4+ Treg were collected and the Th1/Th2 cytokine profile determined using a cells, compared to NHS tumors. As reported by Kamada et al. [3], dis- LEGENDplex bead-based immunoassay (BioLegend). Transcriptomic tinct Treg subset might be involved in the deleterious effects of nivo- analysis was performed on patient tumour tissue and peripheral lumab observed in HPD. Prospective clinical studies should blood using the NanoString PanCancer IO360 gene expression panel. determine if nivolumab-associated HPD is caused by a pre-existing Results pool of highly immunosuppressive CCR5+ CTLA4+ Treg cells. Tumour explants significantly promoted M2-like macrophage differ- entiation and suppressed T cell activity ex vivo. The PDL-1 inhibitor, Acknowledgements Atezolizumab significantly improved T cell function in some patients This work was supported by Bristol-Myers Squibb. and reduced explant-mediated immunosuppression, particularly in the presence of macrophages. This suggests that response or resist- References ance to anti-PDL-1 therapies may be partially TAM dependent. The 1. Champiat S, Dercle L, Ammari S, Massard C, Hollebecque A, Postel-Vinay differential effect of Atezolizumab observed in the presence of S, Chaput N, Eggermont A, Marabelle A, Soria JC, Ferté C. Hyperprogres- patient-derived tumour explants indicates the potential of this model sive Disease Is a New Pattern of Progression in Cancer Patients Treated in predicting immunotherapy responses. Ongoing transcriptomic by Anti-PD-1/PD-L1. Clin Cancer Res. 2017;23(8):1920-1928. analysis of lung cancer tissue aims to reveal associations between 2. Jacquelot N, Roberti MP, Enot DP, Rusakiewicz S, Ternès N, Jegou S, the TME and T cell function. Variations in the cytokine secretion pro- Woods DM, Sodré AL, Hansen M, Meirow Y, Sade-Feldman M, Burra A, file of tumour explant co-cultures are also being studied under differ- Kwek SS, Flament C, Messaoudene M, Duong CPM, Chen L, Kwon BS, An- ent experimental conditions. derson AC, Kuchroo VK, Weide B, Aubin F, Borg C, Dalle S, Beatrix O, Conclusions Ayyoub M, Balme B, Tomasic G, Di Giacomo AM, Maio M, Schadendorf D, Using the tumour explant model, we found that alleviation of Melero I, Dréno B, Khammari A, Dummer R, Levesque M, Koguchi Y, Fong explant-mediated immunosuppression by Atezolizumab may be L, Lotem M, Baniyash M, Schmidt H, Svane IM, Kroemer G, Marabelle A, macrophage dependent. This model could be used to predict patient Michiels S, Cavalcanti A, Smyth MJ, Weber JS, Eggermont AM, Zitvogel L. response to anti-PDL-1 immunotherapy, and explore combination Predictors of responses to immune checkpoint blockade in advanced therapies. Ongoing research aims to improve immunotherapy re- melanoma. Nat Commun. 2017 ;8(1):592. sponses through reprogramming TAMs using immune-modifying 3. Kamada T, Togashi Y, Tay C, Ha D, Sasaki A, Nakamura Y, Sato E, Fukuoka drugs. S, Tada Y, Tanaka A, Morikawa H, Kawazoe A, Kinoshita T, Shitara K, Sakaguchi S, Nishikawa H. PD-1+ regulatory T cells amplified by PD-1 Acknowledgements blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A. Study supported by research funds from Cardiff University, School of 2019 May 14;116(20):9999-10008. Medicine. We would like to thank the Wales Cancer Bank and Lung Ethics Approval Multidisciplinary Research Group for their ongoing involvement in patient The study was approved by the Comité de Protection des Personnes Ile de recruitment and sample acquisition, and to the patients who donated their France III Ethics Board, approval number 2016-A00732-49. samples for our research. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 156 of 272 Reference P291 1. Cassetta L, Kitamura T. Targeting Tumor-Associated Macrophages as a Po- Driving T cell dysfunction in vitro for rational immunotherapy tential Strategy to Enhance the Response to Immune Checkpoint Inhibi- design tors. 2018;6. Matthew Hancock, Cailin Joyce, PhD, Thomas Horn, Simarjot Pabla, Ethics Approval Benjamin Duckless, Andrew Basinski, Dhan Chand, PhD, Jeremy Waight, Ethical approval for this project was provided through the Wales Cancer PhD, Mariana Manrique, PhD, Nicholas Wilson, Alex Duncan, PhD, Bank (WCB). The WCB has ethics approval as a Research Tissue Bank from Jennifer Buell, PhD, David Savitsky, PhD, Lukasz Swiech, John Castle, PhD the Wales Research Ethics Committee 3, reference 16/WA/0256 (previous Agenus Inc, Lexington, MA, United States approval references – 06/MRE09/26 and 11/WA/0279). This approval Correspondence: John Castle (john.castle@agenusbio.com) covers the collection of samples (including consent), processing and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P291 storing samples across multiple collection and storage sites. The approval also allows release of anonymised samples to researchers carrying out Background cancer related activity, following successful application approval from the Immune checkpoint blockade (ICB) elicits durable responses in some WCB External Review panel (project 17/016). Sample collection of blood cancer patients, but novel targets and combination approaches are and tissue, from non-small cell lung cancer patients undergoing surgical needed to address resistance and broaden clinical benefit. Here, we resection, was performed at the University Hospital of Wales, Cardiff. present a system for characterizing mechanisms of T cell dysfunction in the tumor microenvironment (TME) and apply the system to un- cover novel approaches to ICB resistance. P290 Methods Interferon gamma production by regulatory T cells is necessary for We developed a long-term human co-culture system comprised of response to cancer immunotherapy primary T cells and cancer cells that enables controlled differentiation Angela Gocher, PhD, Dario Vignali, PhD, Creg Workman, PhD, Sanah of naïve T cells to effector, memory and dysfunctional states. We lon- Handu gitudinally monitored T cell effector functions, protein and RNA ex- University of Pittsburgh, Pittsburgh, PA, United States pression across states and single cells. Finally, we challenged the Correspondence: Dario Vignali (dvignali@pitt.edu) system with PD1 antibody to uncover biomarkers and mechanisms Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P290 of therapeutic resistance. Results Background T cells in our system become activated and then gradually progress Our lab has shown that murine regulatory T cells (Tregs) produce the to a terminally dysfunctional state driven by multiple cancer antigen cytokine interferon gamma (IFNγ) during anti-PD1 therapy and that exposures. T cell cytotoxicity is maintained over several antigen ex- Treg response to IFNγ is necessary for tumor eradication by anti-PD1 posures before sharply decreasing whereas cytokine secretion begins therapy [1]. However, the role of this cellular source of IFNγ in the to decrease with only one prior antigen exposure. The expression of tumor microenvironment (TME) and how Treg derived IFNγ dictates known T cell regulators and novel factors is altered over the time response to other cancer immunotherapies has yet to be studied. In course, with known factors reflecting previous observations in vivo. addition, it has been shown that IL-12-induced production of IFNγ is Anti-PD1 prolongs cytotoxic capacity but T cells eventually fail to re- necessary for response to anti-PD1 therapy [2]. However, it has yet to spond. Single cell mapping in the presence of anti-PD1 reveals an ex- be determined whether Tregs are a key responder to IL-12 during panded population of T cells that co-express PD1, TIGIT and cancer immunotherapy. Thus, elucidating the interplay of IL12, IFNγ activation markers. Consistent with this, the combination of PD1 and and Tregs in the TME is essential for maximizing efficacy and minim- TIGIT blockade enhances cytotoxicity relative to monotherapies. izing the clinical resistance to current cancer immunotherapies. Conclusions Methods These findings demonstrate the utility of our system to deeply inter- Our lab has generated two novel murine models that allow for Treg- rogate therapeutic response and resistance. Moreover, its scalability restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/ and modularity enable genome-scale screening to discover novel tar- LFoxp3Cre-YFP). These murine models were validated and used to gets that could enhance antitumor activity of both natural and adop- assess the contribution of Treg-derived IFNγ on the growth of syn- tively transferred T cells. geneic MC38 (colon adenocarcinoma) and B16 (melanoma) tumors and response to a various cancer immunotherapies (checkpoint blockade, vaccination and tumor-specific antibodies). Flow cytometry P292 was conducted on tumor and control non-draining lymph nodes to Tissue site of tumor growth dictates anti-tumor immunity and phenotype the immune infiltrate in response to immunotherapy. response to checkpoint blockade Results Brendan Horton, PhD, Stefani Spranger, PhD Mice with Tregs either unable to generate IFNγ (IfngL/LFoxp3Cre- Massachusetts Institute of Technology,, Cambridge, MA, United States YFP) or respond to the IFNγ-inducing cytokine IL-12 (Il12rb2L/ Correspondence: Stefani Spranger (spranger@mit.edu) LFoxp3Cre-YFP) were unable to eradicate tumors in response to im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P292 munotherapy. In addition, preliminary results suggest that Il12rb2- deficient Tregs may be more suppressive as indicated by an increase Background in tumor growth compared to control. An emerging area of clinical importance is differential responsiveness Conclusions to checkpoint blockade immunotherapy across different tissues sites These data suggest that IFNγ producing Tregs are necessary to shift of tumor growth, leading to partial responses and ultimately cancer- the balance from an immunosuppressive TME to one that favors the related deaths [1, 2]. However, tissue-specific anti-tumor immune re- reinvigoration of the anti-tumor response generated by current can- sponses are not well understood. We used mouse models to deter- cer immunotherapeutic agents. Future studies will determine mine how anti-tumor immune responses differ across tissue sites and whether the capacity of Tregs to produce IFNγ can predict response how this relates to immunotherapy efficacy. to immunotherapy. Methods Mice were inoculated subcutaneously or intravenously with syngen- References eic KP lung cancer cells, then treated with anti-CTLA-4 and anti-PD- 1. Overacre-Delgoffe A, Chikia M. Interferon-γ Drives Treg Fragility to Pro- L1 antibodies, and analyzed for tumor burden. Response to check- mote Anti-tumor Immunity. Cell. 2017; 169: 1130-1141. point blockade was correlated with tumor-infiltrating T cells (TIL) and 2. Garris C, Arlauckas S. Successful Anti-PD1 Cancer Immunotherapy Re- systemic immune responses using flow cytometry and immuno- quires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL- histochemistry. Mice were also tested for their ability to generate sys- 12. Immunity. 2018; 49: 1148-1161. temic and protective immunity against a second tumor challenge. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 157 of 272 Results ~500 cancer causing genes) as the number of nonsynonymous coding Comparing lung and subcutaneous tumors, we observed striking dif- mutations per megabase and the optimal cut-off for TMBhigh was de- ferences within the TIL compartment. Lung tumors contained more termined using the 98th percentile of newly-diagnosed tumors and TIL with higher expression of PD-1. Despite this, lung tumors did not segmented linear regression analysis. Patients were stratified by histo- respond to anti-CTLA-4 + anti-PD-L1, while subcutaneous tumors did. molecular subtype, IDH mutation, 1p/19q co-deletion, TMB, and ICB TIL expanded in subcutaneous tumors after immunotherapy, while treatment under clinical trials and expanded access. Overall (OS) and lung tumors had no change in the number of TIL. We further tested progression-free (PFS) survival were estimated by the Kaplan-Meier for concomitant immunity and found that subcutaneous KP tumors method and evaluated using multivariable Cox regression. generated an immune response that could protect against a second Results tumor. However, KP lung tumors failed to generate a protective sys- We identified 1,223 HGG patients with genomics, including 64 temic immune response. These results were reproduced using a pan- hypermutated tumors. Overall the cohort consisted of 79% newly- creatic cancer cell line, indicating that tumors growing in the lung diagnosed and 21% recurrent gliomas and subclass distribution generated weaker systemic immune responses than subcutaneous was 75% IDH-wildtype glioblastomas/anaplastic astrocytomas,16% tumors. Protective concomitant immunity required CD8+ T cells, and IDH-mutant glioblastomas/anaplastic astrocytomas, and 6% 1p/19- to a lesser extent CD4+ T cells, but not NK cells. Therefore, we inves- codeleted anaplastic oligodendrogliomas. Hypermutated HGG tigated differences in the generation of systemic, antigen-specific were predominantly seen in the setting of recurrence (18% of re- CD8+ T cell responses between subcutaneous and lung KP tumors. current HGGs vs 2% of newly diagnosed, 5% overall incidence). Using KP.SIY tumors, elispot assays found that lung tumors gener- Comparisons of biomarkers and genomics in hypermutated versus ated fewer antigen-specific T cells in the spleen. Transferred 2C T non-hypermutated HGGs showed distinct characteristics in glioma cells proliferated less in the spleens of lung tumor-bearing hosts than subtypes with implications for differential mechanisms of in hosts with subcutaneous tumors, even though proliferation in TMBhigh acquisition in HGGs with the most important biomarkers draining lymph nodes was similar. Consistently, KP.SIY lung tumors of differential hypermutation risk being MGMT promoter methyla- generated weaker concomitant immunity than subcutaneous KP.SIY tion (22% of methylated vs. 6% of unmethylated cases, p=0.02) tumors. and IDH1/2 mutation (25% of IDH-mutant vs. 12% of IDH- Conclusions wildtype HGGs, p=0.007). The tissue site of tumor growth determines the number and pheno- 129 (11%) of HGGs (mostly IDH-wildtype GBMs/AAs) received PD-1/ type of TIL, the generation of systemic immunity, and the response PD-L1 ICB therapy, including 13% (n=8) of hypermutated HGGs. to checkpoint blockade. Response to immunotherapy correlates not Immunohistopathologic assessment of tumor responses and immune with the number of TIL, or their phenotype, but with the ability of infiltrates was conducted and correlated with the genomic profiling. the host to mount a systemic anti-tumor CD8+ T cell response. Because the majority of ICB-treated cases were IDH-wildtype HGGs, this subset was further evaluated in analyses which were risk- References adjusted by age, sex, histomolecular subgroup, MGMT promoter 1. Khoja, L., et al., Patterns of response to anti-PD-1 treatment: an explora- methylation, and prior therapy (i.e. RT/TMZ/CCNU/bevacizumab). Cor- tory comparison of four radiological response criteria and associations relations between TMB, molecular genotypes, and clinical response with overall survival in metastatic melanoma patients. Br J Cancer, 2016. to ICB in HGGs will be presented. 115(10): p. 1186-1192. Conclusions 2. Lee, J.H.J., et al., Metastasis-specific patterns of response and progression Our study represents the largest set of genomically characterized gli- with anti-PD-1 treatment in metastatic melanoma. Pigment Cell Melan- omas and gliomas with hypermutation with data related to re- oma Res, 2018. 31(3): p. 404-410. sponses to PD-1/PD-L1 ICB therapy. Our data support systematic TMB Ethics Approval characterization and clinical molecular genotyping to assist therapy- The animal work in these experiments was reviewed and approved by the decision making in gliomas and provide foundational data essential MIT Committee on Animal Care. The approved animal protocol number to future clinical trial designs of immunotherapeutics. for this work is 0117-012-20. Ethics Approval This study was approved by the Partners HealthCare (#2015P002352) and Dana-Farber Cancer Institute (10-417) institutional review boards. P293 Tumor mutational burden (TMB) and genomic predictors of clinical outcomes to PD-1/PD-L1 checkpoint blockade in high-grade P294 gliomas Long survival associated with receipt of anti-CTLA-4 in patients 1 1 1 1 Bryan Iorgulescu, MD , Mehdi Touat , Yvonne Li , Liam Spurr , Gareth with metastatic melanoma from acral lentiginous, mucosal and 1 1 1 1 Grant , William Pisano , Mary Jane Lim-Fat , Eudocia Lee , Lakshmi uveal primary tumors 1 2 2 3 1 Nayak , E Chiocca , Raymond Huang , Andrew Cherniack , Patrick Wen , Nicholas Klemen, MD, Melinda Wang, BS, Kelly Olino, MD, James Clune, 1 4 1 1 Ahmed Idbaih , Franck Bielle , David Reardon, MD , Keith Ligon MD, Stephan Ariyan, MD, Charles Cha, MD, Sarah Weiss, MD, Harriet 1 2 Dana-Farber Cancer Institute, Boston, MA, United States; Brigham and Kluger, MD, Mario Sznol, MD Women's Hospital, Boston, MA, United States; Broad Institute, Yale University School of Medicine, New York, NY, United States Cambridge, MA, United States; Sorbonne Université, Paris (UPMC), Paris, Correspondence: Mario Sznol (mario.sznol@yale.edu) France Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P294 Correspondence: David Reardon (david_reardon@dfci.harvard.edu); Keith Ligon (Keith_Ligon@dfci.harvard.edu) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P293 Metastatic melanoma from acral lentiginous, mucosal and uveal pri- mary tumors responds poorly to checkpoint inhibitors (CPI), poten- Background tially due to a low burden of immunogenic neoantigens [1-7]. Long- High tumor mutational burden (TMB) is an emerging biomarker for term outcomes of these patients after treatment with CPI have not predicting response to PD-1/PD-L1 immune checkpoint blockade been established. (ICB) in a spectrum of cancer patients, however, its clinical value and Methods therapeutic implications in high-grade gliomas (HGG) is not yet We performed a retrospective review of a single institutional experi- established. ence using antibodies against CTLA-4, PD-1 and PD-L1 for patients Methods with stage IV melanoma. Primary tumor histology was categorized as We retrospectively reviewed all HGGs at our institutions from 2013- cutaneous, acral, mucosal or uveal. Patients with unknown primary 2018 that underwent genomic characterization. TMB was determined were excluded. We measured overall survival (OS) after the first dose from clinical targeted exome next-generation sequencing (DFCI-Profile, of CPI using the Kaplan-Meier method. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 158 of 272 Results We treated 428 patients with metastatic melanoma from 2007-2019. Pri- mary tumors were cutaneous in 283 (66%) patients, unknown in 55 (13%), acralin22(5%), mucosalin38(9%)and uvealin30(7%)(Table 1).Mucosal patients had a slight female preponderance. The proportion staged M1c was higher in mucosal and uveal patients. Patients with cutaneous primary tumors had median OS after CPI of 45 months, compared with 17 months for acral (P = 0.047), 18 months for mucosal (P = 0.003) and 12 months for uveal (P < 0.001)(Figure 1). Five-year survival for cutaneous, acral, mucosal and uveal patients was 46%, 34%, 21% and 22% respectively. Next we combined the patients with acral, mucosal and uveal melan- oma (n = 90) and performed survival analysis stratified by the first type of CPI treatment. Median OS after anti-PD-1 or anti-PD-L1 was 9 months, compared with 18 months after anti-CTLA-4 (P = 0.010) and 20 months after combination therapy with anti-CTLA-4 plus anti-PD-1 (P = 0.003)(Figure 2). While 21 of 31 (68%) patients treated with anti- CTLA-4 later were treated with anti-PD-1, only 5 of 18 (28%) patients Fig. 1 (abstract P294). Overall survival stratified by histology treated with anti-PD-1 later received anti-CTLA-4 (P = 0.02). There were 21 patients who survived at least three years after CPI, all of whom were treated with anti-CTLA-4 with or without anti-PD-1. Of the 10 patients with actual five-year survival, 3 had complete re- sponses while the other 7 all required local and/or regional therapies to control progressive disease (Table 2). Conclusions Long survival in patients with metastatic melanoma from acral, mu- cosal and uveal primary tumors was uniformly associated with re- ceipt of anti-CTLA-4. Our experience shows that while acral, mucosal and uveal melanomas have worse outcomes than cutaneous melan- oma, with an aggressive multidiscliplinary approach five-year survival is still possible for 25-32% of these patients. References 1. Luke JJ, Callahan MK, Postow MA, Romano E, Ramaiya N, Bluth M, et al. Clinical activity of ipilimumab for metastatic uveal melanoma. Cancer. 2013;119:3687–95. 2. Algazi AP, Tsai KK, Shoushtari AN, Munhoz RR, Eroglu Z, Piulats JM, et al. Clinical outcomes in metastatic uveal melanoma treated with PD-1 and PD-L1 antibodies. Cancer. 2016;122:3344–53. 3. D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy Fig. 2 (abstract P294). Overall survival stratified by treatment and Safety of Nivolumab Alone or in Combination With Ipilimumab in Patients With Mucosal Melanoma: A Pooled Analysis. JCO. 2017;35:226–35. 4. Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for Patients With Advanced Mucosal Melanoma. The Table 2 (abstract P294). Characteristics of five-year survivors Oncologist. 2013;18:726–32. 5. Bello DM, Chou JF, Panageas KS, Brady MS, Coit DG, Carvajal RD, et al. Prognosis of acral melanoma: a series of 281 patients. Ann Surg Oncol. Springer US; 2013;20:3618–25. 6. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. Nature Publishing Group; 2012;44:1006–14. 7. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al. Mutational heterogeneity in cancer and the search for new cancer- associated genes. Nature. 2013;499:214–8. Ethics Approval The study was approved by the Yale-New Haven Hospital Institutional Review Board, approval number 2000021595. Table 1 (abstract P294). Demographics and first treatment P295 The impact of metastatic sites on checkpoint inhibitor outcomes in patients with cutaneous and unknown primary melanoma Penina Krieger, MPhil, Francisco Sanchez-Vega, Nikolaus Schultz, Alexander Shoushtari, MD Memorial Sloan Kettering Cancer Center, Bronx, NY, United States Correspondence: Penina Krieger (peninakrieger@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P295 Background Clinical biomarkers of response to Programmed Death-1 (PD-1) based therapies are sorely needed in melanoma. The American Joint Committee on Cancer (AJCC) 8th Edition Staging system is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 159 of 272 derived from the pre-checkpoint inhibitor era and divides patients into locally advanced or soft tissue disease (M0/M1a), lung metastases (M1b), other visceral metastases (M1c), and brain metastases (M1d). It is unclear whether this prognostic classification remains valid in the mod- ern therapeutic era. The number of metastatic sites influences out- comes with BRAF-MEK therapy [1], but its prognostic importance with PD-1-based therapy is unknown. Methods All patients with melanoma who had prospective tumor molecu- lar profiling at a single center (MSK-IMPACT) and subsequently received frontline PD-1 blockade as single agent (Nivolumab or Pembrolizumab) or combination therapy with Ipilimumab (combo) were included. Demographic and clinical data were collected, in- cluding metastatic sites present at time of PD-1 therapy. Overall survival (OS) and time to treatment failure (TTF) were calculated from the onset of PD-1 therapy using Kaplan-Meier methodology. The impact of specific metastatic sites on TTF and OS was determined using Cox Proportional Hazards Regression Models. Tumor mutational burden (TMB) was calculated as previously described [2]. Results Fig. 1 (abstract P295). See text for description 309 patients received frontline PD-1 monotherapy (n=179) or PD-1 combo (n=130). Overall survival varied by AJCC stage (p<0.0001, Fig- ure 1); M1b and M0/M1a groups had similar OS (median=NR, p = 0.27) followed by M1c (median=39 mo) and M1d (median=28 mo). Patients with 3+ metastatic sites had worse median OS than those with 0-2 metastatic sites (45 vs 39 mo, p<0.0001, Figure 2). Among patients with M1c disease, those with bone or liver me- tastases had worse median OS than those without them (39 vs NR mo, Liver HR=2.4, p=0.036, Bone HR=2.6, p=0.021, Figure 3). Among patients with M1d disease, those with liver metastases had shorter TTF than those without when treated with PD-1 com- bination therapy (HR=2.4, p=0.044). There was no significant dif- ference in median TMB by AJCC M stage at treatment or by site of metastasis. Conclusions AJCC 8th edition M1b disease has a similar prognosis to M0/M1a dis- ease in the era of PD-1-based therapy. The presence of liver or bone metastases at time of PD-1 therapy portends worse OS and TTF even within M1c and M1d disease. Patients with 0-2 metastatic sites live longer than those with 3+ sites. These clinical observations are not explained by differences in TMB. Future trials of PD-1 blockade in ad- vanced melanoma should account for these differences. Fig. 2 (abstract P295). See text for description Acknowledgements Research reported in this abstract was supported by the National Cancer Institute of the National Institutes of Health under Award Number R25CA020449 and by National Cancer Institute Cancer Center Core Grant P30CA008748, Kravis Center for Molecular Oncology. The content is the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1. Long G, Grob J, Nathan P, Ribas A, Robert C, Schadendorf D, Lane S, Mak C, Legenne P, Flaherty K, Davies M. Factors predictive of response, disease progression, and overall survival after dabrafenib and trametinib combination treatment: a pooled analysis of individual patient data from randomised trials. Lancet Oncol. 2016; 17:1743–54. 2. Samstein RM, Lee CH, Shoushtari AN, Hellmann MD, Shen R, Janjigian YY, Barron DA, Zehir A, Jordan EJ, Omuro A, Kaley TJ, Kendall SM, Motzer RJ, Hakimi AA, Voss MH, Russo P, Rosenberg J, Iyer G, Bochner BH, Bajorin DF, Al-Ahmadie HA, Chaft JE, Rudin CM, Riely GJ, Baxi S, Ho AL, Wong RJ, Pfister DG, Wolchok JD, Barker CA, Gutin PH, Brennan CW, Tabar V, Mel- linghoff IK, DeAngelis LM, Ariyan CE, Lee N, Tap WD, Gounder MM, D’An- gelo SP, Saltz L, Stadler ZK, Scher HI, Baselga J, Razavi P, Klebanoff CA, Yaeger R, Segal NH, Ku GY, DeMatteo RP, Ladanyi M, Rizvi NA, Berger MF, Riaz N, Solit DB, Chan TA, Morris LGT. Tumor mutational load predicts sur- vival after immunotherapy across multiple cancer types. Nat Genet. 2019; 51:202–206 Ethics Approval The study was approved by Memorial Sloan Kettering's Ethics Board, Fig. 3 (abstract P295). See text for description approval number 18-244. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 160 of 272 P296 BMI, irAE and gene expression signatures predict resistance and survival to immune-checkpoint inhibition in renal cell carcinoma 1 1 1 2 Brian Labadie, MD , Ping Liu, PhD , Riyue Bao, PhD , Michael Crist, MD , 3 4 5 Ricardo Fernandes, MD , Laura Ferreira Freire , Scott Graupner , Andrew 5 4 3 6 Poklepovic, MD , Ignacio Duran , Saman Maleki Vareki , Arjun Balar, MD , Jason Luke, MD, FACP 1 2 University of Chicago, Chicago, IL, United States; NYU School of Medicine, New York, NY, United States; Western University, London, 4 5 Ontario, Canada; HUMV, Santander, Spain; Virginia Commonwealth University, Richmond, VA, United States; New York University, New York, NY, United States; University of Pittsburgh, Pittsburgh, PA, United States Correspondence: Jason Luke (lukejj@upmc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P296 Background Treatment with immune-checkpoint inhibition (ICI) has changed the treatment paradigm in ccRCC however many do not respond to these treatments and no reliable molecular biomarker exists to pre- dict response to ICI in individual patients. Clinical variables may cor- relate with lack of response to treatment (primary resistance) or clinical benefit. Methods Via an international multi-institution collaboration, clinical charac- Fig. 1 (abstract P296). Kaplan-Meier curves depicting teristics from patients with ccRCC treated with anti-PD-1/L1 ther- survival outcomes apy were collected. Patients with primary resistance (defined as progression on initial computed tomography scan) were com- pared to patients with clinical benefit. Multivariable analysis was performed to identify factors associated with improved time to progression or death. The Cancer Genome Atlas Kidney Renal P297 Clear Cell Carcinoma cohort (TCGA-ccRCC) was examined for the A semi-mechanistic platform model to capture individual animal correlation between gene expression patterns, clinical factors, and responses to checkpoint inhibitors in a syngeneic mouse model 1 2 1 2 2 survival outcomes. Lin Lin, PhD , Alison Betts , Carissa Young , Wendy Qiao , Jatin Narula , 2 2 2 2 Results Peter O’Brien , Derek Bartlett , Andrea Hooper , Jason Williams , John 1 1 1 1 Of 90 patients, 38 (42.2%) had primary resistance and 52 (57.8%) Burke , Joshua Apgar , Lore Gruenbaum, PhD , Fei Hua, PhD 1 2 had clinical benefit. Compared with the cohort of patients with Applied BioMath, LLC, Concord, MA, United States; Pfizer Worldwide initial benefit, primary resistance was more likely to occur in pa- R&D, Cambridge, MA, United States tients with worse ECOG performance status (p=0.03), earlier stage Correspondence: Fei Hua (fei.hua@appliedbiomath.com) at diagnosis (p=0.04), no prior nephrectomy (p=0.04) and no Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P297 immune-related adverse events (irAE) (p=0.02). In the entire co- hort, improved overall survival was significantly correlated with Background lower International Metastatic RCC Database Consortium risk Syngeneic mouse models have been widely employed in preclinical score (p=0.02) and lower neutrophil:lymphocyte ratio (p=0.04). In discovery of checkpoint inhibitors as they enable study of drug im- patients with clinical benefit, improved progression free survival pact on the intact immune system. However, the interpretation of was significantly associated with increased BMI (p=0.007) and irAE such studies remains challenging partly due to the large variability in occurrence (p=0.02) while improved overall survival was signifi- individual animal responses to drug treatment. cantly correlated with overweight BMI (BMI 25-30)(p=0.03) and no Methods brain metastasis (p=0.005). In the TCGA-ccRCC analysis, higher ex- In this work, we describe the generation of a model platform that pression of angiogenesis gene signature was found to be corre- captures essential aspects of the pharmacokinetics, cellular and lated with lower neoplasm histologic grade and better survival (p tumor growth effects of murine surrogates of two checkpoint thera- < 0.05). Angiogenesis and T cell-inflamed gene signatures were peutic antibodies, anti-PD1 and anti-CTLA4, in the CT26 syngeneic inversely correlated in tumors of high T cell-inflamed gene ex- tumor model. The model describes individual animal responses with pression (p=0.008), a pattern not observed in non-T cell-inflamed regard to drug exposure, key intra-tumoral cell kinetics and tumor tumors. (Figure 1) volume changes and provides biologically plausible explanations for Conclusions the observed differences between good and poor responders to Identification of BMI, performance status and prior nephrectomy as treatment with anti-PD1 or anti-CTLA4. predictors of response to PD1/L1 in ccRCC may help inform treat- Results ment selection. The inverse association of angiogenesis gene signa- We used the model to predict the antibody dose-response relation- tures with ccRCC histologic grade highlight opportunities for ships for individual animals and to identify dose thresholds above adjuvant combination VEGFR2 TKI and ICI. which complete tumor elimination can be achieved in good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 161 of 272 responders. In contrast, our models predict that poor responders demonstrated higher expression of angiogenesis-related profiles and would not achieve complete response even with much higher drug increased CD31 expression. Furthermore, Pbrm1 deficient Renca sub- doses. The parameters in our model that impact the response in cutaneous tumors in mice demonstrated longer latency but more re- poor responders are not drug-related. This finding suggests that sistance to programmed death-1 (PD-1) blockade. Analysis of the immune-cell related barriers have to be crossed in order to achieve a IMmotion150 cohort revealed that ccRCC patients with PBRM1 muta- therapeutic response in these animals - possibly via combination tions were associated with decreased immune infiltrates and a re- therapy. duced response rate to atezolizumab monotherapy or combination In addition, we identified the net tumor cell doubling rate, one po- therapy with bevacizumab. tential parameter that contributes to individual variability in response Conclusions to treatment, as the most sensitive biological parameter determining Pbrm1 and PBRM1 loss reduced IFN gamma-STAT1 signaling. Pbrm1 tumor volume changes upon treatment with anti-PD1 or anti-CTLA4. inactivation was associated with a less immunogenic tumor micro- Measuring individual animal tumor cell growth characteristics may environment in animal and human tissue samples. Response to PD- help with the experimental design and qualification of animals for L1 blockade is reduced in patients with PBRM1 mutations in the studies (in addition to absolute tumor volume), and thereby reduce IMmotion 150 study. This study forms a framework for future mech- inter-animal variability and enhance the interpretability of study re- anistic and clinical studies on the interaction between genomic fea- sults, especially in combination with a model such as the one pre- tures in RCC and response to immunotherapy. sented here. Conclusions Acknowledgements This model platform can be adapted to capture and compare check- We acknowledge the TCGA Research network. This work was supported by point drug effects in different syngeneic tumor models. Moreover, it funding from DOD grant W81XWH-17.1.0307, DOD grant CA160728P1, UT can be expanded to add additional drug mechanisms and can serve MD Anderson Cancer Center CCSG grant 5 P30 CA016672 (Biostatistics as a tool to inform the experimental design of mouse studies. shared resource group) and the Adopt-a-Scientist Foundation. Ethics Approval The animal protocols (2018-0376) were approved by Institutional Animal P298 Care and Use Committee (IACUC) of The Health Science Center, Texas A&M PBRM1 loss defines distinct tumor phenotype associated with University. Human subject protocol (2007-0511) was approved by immunotherapy resistance in renal cell carcinoma Institutional Research Board at M.D. Anderson Cancer Center. 1 1 1 1 Xiande Liu, PhD , Wen Kong , Christine Peterson , Daniel McGrail , Anh 1 1 1 1 Hoang , Xuesong Zhang , Truong Lam , Patrick Pilie, MD , Haifeng Zhu, 1 2 2 3 PhD , Kathryn Beckermann , Scott Haake , Sevinj Isgandrova , Margarita P299 3 1 2 Martinez-Moczygemba , Nidhi Sahni , W. Kimryn Rathmell , Eric Jonasch, Neuropilin-1 is a T cell memory checkpoint limiting long-term MD tumor immunity 1 2 1 2 3 MD Anderson Cancer Center, Houston, TX, United States; Vanderbilt Chang Liu, PhD , Ashwin Somasundaram, MD , Sasikanth Manne , 3 1 1 1 University Medical Center, Nashville, TN, United States; Texas A&M Angela Gocher, PhD , Andrea Szymczak-workman , Kate Vignali , Daniel 2 1 1 Health Science Center, Houston, TX, United States Normolle, PhD , Robert Ferris, MD, PhD , Tullia Bruno, PhD , E. John 3 1 1 Correspondence: Eric Jonasch (ejonasch@mdanderson.org) Wherry, PhD , Creg Workman, PhD , Dario Vignali, PhD 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P298 University of Pittsburgh, Pittsburgh, PA, United States; UPMC Hillman Cancer Center, Pittsburgh, PA, United States; University of Pennsylvania, Background Philadelphia, PA, United States Polybromo-1 (PBRM1), encoding a mammalian specific subunit of the Correspondence: Dario Vignali (dvignali@pitt.edu) switch/sucrose non fermenting (SWI/SNF) chromatin remodeling Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P299 complex, is the second most frequently mutated gene in clear cell renal cell carcinoma (ccRCC). Data thus far on the effect of PBRM1 Background loss on immune responsiveness are inconsistent. The impact of Robust CD8+ T cell memory is essential for long-term protective im- PBRM1 mutation on response to immunotherapy in patients with munity but is often impaired in cancer due to T cell exhaustion, which renal cell carcinoma (RCC) has become a topic of intense debate. causes a loss of memory precursors. Immunotherapy via checkpoint RCC-specific mechanistic and large-scale clinical data are needed to blockade does not effectively reverse this defect in the majority of pa- precisely further characterize the influence of PBRM1 loss on re- tients, potentially underlying disease relapse. Resistance mechanisms sponse to immunotherapy. that underlie poor CD8+ memory development remain unknown. Methods Methods An immunocompetent murine RCC model was applied to investigate The development of post-surgical tumor immunity [1] was interrogated in the response to anti-PD-1 therapy. Multiple human RCC datasets the CD8+ T cell-restricted Neuropilin-1 (Nrp1)-deficient mice (E8ICreNrp1L/ (TCGA, IMmotion150 and ICGC), a murine pre-malignant dataset and L), by surgically removing the primary B16F10 tumor followed by re- a Renca tumor dataset were used to perform gene signature enrich- challenge 30- or 60-days post resection. The synergy between CD8-specific ment analysis (GSEA). Immunohistochemistry and Multiplex Opal Im- Nrp1 deficiency and anti-PD1 blockade was investigated with the MC38 munofluorescence were performed to assess immune cell infiltration. tumor model. In a competitive setting, the Nrp1–/– and Nrp1+/+ pMel-T Real-time PCR, western blot, ELISA and chromatin immunoprecipita- cells [2] were co-transferred into the same host (CD45.1) followed by tion (ChIP) were used to study the activity of the interferon gamma gp100-B16 tumor inoculation, where the in vivo long-term persistence of signaling pathway. the donor cells was assessed. The transcriptomic modulation by Nrp1 defi- Results ciency was studied using bulk population RNA sequencing (bpRNAseq) on Pbrm1 knockout in murine RCC Renca cells impaired the binding of the pMel-T cells (Nrp1–/– vs. Nrp1+/+) recovered from various phase of brahma-related gene 1 (BRG1), the adenosine-triphosphate- in vivo activation (effector, memory and recall). Lastly, the physiological rele- dependent enzyme subunit of the SWI/SNF complex, to the promoter vance of NRP1 expression on CD8+ T cells in cancer patients was studied of IFN gamma receptor 2 (Ifngr2) and reduced Ifngr2 expression. in a cohort of peripheral blood leukocyte (PBL) samples from treatment- PBRM1/Pbrm1 deficiency impaired IFN gamma-induced phosphoryl- naïve patients with head and neck squamous cell carcinoma (HNSCC). ation of Janus kinase 2 (JAK2) and STAT1, and the subsequent ex- Results pression of downstream target genes involved in tumor The E8ICreNrp1L/L mice exhibited substantially enhanced protection from microenvironment (TME) modulation, such as Cxcl9, Icam1, Irf1, and tumor re-challenge, despite unchanged primary tumor growth. Enhanced Stat1 itself. In both human and murine RCC tumors, PBRM1/Pbrm1 responsiveness to anti-PD1 immunotherapy was also observed. NRP1 was loss was associated with lower expression of immune-related profiles co-expressed with multiple inhibitory receptors (IRs) on CD8+ T cells and and reduced T cell infiltration. PBRM1/Pbrm1 loss tumors also restrained memory differentiation and development by repressing an Id3- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 162 of 272 dependent transcriptional program. NRP1 was also highly expressed on medications received before recruitment to INT-NA had significant the exhausted CD8+ T cells found in the HNSCC patients and negatively relevance in the outcome. Naïve patients or previously challenged associated with the size of memory T cell pool and disease prognosis. by Immunotherapy had the best clinical benefit compared to Conclusions those receiving first line BRAF/MEK inhibitors (RR 40% vs 10%; p= These data reveal NRP1 as a unique “immune memory checkpoint” 0.001). Again, after disease progression treatment with target ther- with a mode of action that is distinct from other immune check- apy in naïve and immunotherapy previously treated patients, points. NRP1 blockade may promote the establishment of long-term showed a different beneficial pattern compared to patients re- T cell memory that is essential for durable anti-tumor immunity. ceived BRAF/MEK inhibitors before immunotherapy. The clinical parameters correlated with an increase of clinical benefit were References genus (female vs male), age (older [+60] vs younger), LDH score 1. Zhang, P., et al., Induction of postsurgical tumor immunity and T-cell mem- (normal vs high and very high), neutrophil/lymphocyte ratio (ele- ory by a poorly immunogenic tumor. Cancer Res, 2007. 67(13): p. 6468-76. vated vs normal). The introduction of an algorithm including the 2. Overwijk, W.W., et al., Tumor regression and autoimmunity after reversal of a clinical variables above mentioned could define four predictable functionally tolerant state of self-reactive CD8+ T cells. J Exp Med, 2003. 198(4): cohorts of benefit with a 95% of accuracy. p. 569-80. Conclusions Ethics Approval From the real-life analysis, we generated a simple algorithm that All animal experiments were performed in the American Association for the might drive clinical decision. Our finding clearly showed how previ- Accreditation of Laboratory Animal Care-accredited, specific-pathogen-free ous treatment impacted outcome: patients treated with iBRAF/iMEK facilities in Division of Laboratory Animal Resources, University of Pittsburgh after treatment with ICI showed better clinical outcome respect pa- School of Medicine (UPSOM). Animal protocols were approved by the tients treated with iBRAF/iMEK before treatment with ICI. Institutional Animal Care and Use Committees of University of Pittsburgh. Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) Acknowledgements electing to undergo treatment were offered the option to participate in the The study was supported by the Institutional Project "Ricerca Corrente" of University of Pittsburgh Cancer Institute (UPCI) protocol for research. All Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy patients signed an informed consent that was approved by the Institutional Review Board (IRB) of the University of Pittsburgh. References 1. Balar AV, Weber JS. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother 2017; 66(5):551–564.2. P300 2. Larkin J, Minor D, D’Angelo S et al. Overall survival in patients with Real World data analysis related to metastatic melanoma patients advanced melanoma who received nivolumab versus investigator’s treated with immunotherapy from 2012 to 2018 at Istituto choice chemotherapy in CheckMate 037: a randomized, controlled, Nazionale Tumori IRCCS Fondazione “G. Pascale” of Napoli, Italy open-label phase III trial. J Clin Oncol 2018; 36(4): 383–390. Gabriele Madonna, Medical Biotechnology (LS) , Mariaelena Capone, 3. Schachter J, Ribas A, Long GV et al. Pembrolizumab versus ipilimumab 1 1 1 1 MD , Marilena Tuffanelli , Marcello Curvietto, PhD , Miriam Paone, PhD , for advanced melanoma: final overall survival results of a multicentre, 1 1 1 Assunta Esposito, PhD , Antonio Sorrentino , Marco Palla , Luigi randomised, open-label phase 3 study (KEYNOTE-006). Lancet 2017;390: 1 1 1 Scarpato , Domenico Mallardo, MD , Ester Simeone, MD , Antonio 1853–1862.4. 1 2 2 2 Grimaldi, MD , Kristina Viktorsson , Lisa Villabona , Rolf Lewensohn , 4. Larkin J, Chiarion-Sileni V, Gonzalez R et al. Combined nivolumab and ipi- 2 1 Giuseppe Masucci, MD, PhD , Paolo Antonio Ascierto, MD limumab or monotherapy in untreated melanoma. N Engl J Med2015; Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, Naples, Italy; 373: 23–34.5. Karolinska Institutet, Stockholm, Sweden 5. Robert C, Long GV, Brady B et al. Nivolumab in previously untreated Correspondence: Paolo Antonio Ascierto (paolo.ascierto@gmail.com) melanoma without BRAF mutation. N Engl J Med 2015; 372(4):320–330. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P300 6. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbé C, et al: Guidelines for the evaluation of immune therapy activity in solid tumors: Background immune-related response criteria. Clin Cancer Res 2009, 15:7412–20. Immuno checkpoint inhibitors (ICI) have improved the prognosis for Ethics Approval patients with advanced malignancy [1-5]. Their real-life application The study was approved by the internal ethics board of the Istituto may give different outcome compared to the benefit presented by Nazionale Tumori IRCCS Fondazione “G. Pascale” in Napoli Italy, approval clinical trials as the inclusion and exclusion criteria might be selective number of registry 33/17 and give overoptimistic survival rates. Here we present the analysis of cutaneous metastatic melanoma patients treated with check-point inhibitors at Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” P301 of Napoli Italy (INT-NA). Expression of tumor matrix metalloproteases ADAM10 and Methods ADAM17 correlates with low PD-L1 protein-to-mRNA ratio in We investigated retrospectively, from 2012 to 2018, 578 stage IV multiple tumors, predicting poor outcomes melanoma patients received ipilimumab, pembrolizumab or nivo- Aaron Mansfield, MD, Jacob Orme, MD PhD, Roxana Dronca, MD, lumab as monotherapy at the INT-NA. Ipilimumab was adminis- Haidong Dong, MD, PhD tered intravenously at the dosage of 3 mg/kg every 3 weeks for Mayo Clinic, Rochester, MN, United States four doses, pembrolizumab at the dosage of 200 mg every 3 Correspondence: Jacob Orme (orme.jacob@mayo.edu) weeks and nivolumab at the dosage of 3 mg/kg every 2 weeks Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P301 until disease progression or unacceptable toxicity appeared. Dis- ease evaluation was performed at baselineand then every 12 Background weeks until progression or the discontinuation of treatment ac- ADAM10 and ADAM17 portend poor prognosis in many malig- cording to the Response Evaluation Criteria in Solid Tumors nancies [1]–[6]. We previously showed these proteases cleave Pro- (RECIST 1.1) [6]. Survival analysis was performed using the Kaplan grammed death-ligand 1 (PD-L1) from tumors in soluble form Meier method and with the log-rank test. Cox regression was (sPD-L1) [7]. sPD-L1 engages immune cell Programmed death 1 used in the univariate and multivariate analysis. The results were (PD-1) to inhibit tumor immunity. It is unknown how broadly this considered significant if p mechanism occurs in solid malignancies. We hypothesized (1) Results ADAM10 and/or ADAM17 may be elevated in tumors with low Patients treated at INT-NA with nivolumab and pembrolizumab PD-L1 protein despite high PD-L1 (CD274) mRNA and (2) this low showed comparable better clinical benefit toward patients treated tumor PD-L1 protein-to-mRNA ratio may predict lower overall with ipilimumab (RR 44.5% vs 20.7%; p=0.01). The anti-tumoural survival. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 163 of 272 Methods immunity – a resistance mechanism to PD-1 checkpoint blockade We queried the Cancer Genome Atlas (TCGA) for all solid tumors in melanoma,” in CRI-CIMT-EATI-AACR International Cancer Confer- with Level 3 reverse phase protein array (RPPA) PD-L1 protein ence, 2018. levels and RNA-seq sequence per million mapped fragments Ethics Approval (FPKM) PD-L1 (CD274), ADAM10, and ADAM17 mRNA levels. We Lab studies involving human subjects are approved by Mayo Clinic‘s calculated a PD-L1 protein-to-mRNA ratio for each sample. Groups Institutional Review Board (IRB), approval number 15-000934. of high and low PD-L1 protein-to-mRNA ratios were evaluated for (1) ADAM10 and ADAM17 expression and (2) overall survival by Cox proportional hazards modeling, adjusting for age and stage at diagnosis. Results Tumor samples demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly more ADAM10 and/or ADAM17 in 23 of 25 cancer types (Table 1). Cox proportional hazards ratios for Table 1 (abstract P301). See text for description death in each group were calculated and reported as a forest plot including hazard ratios and 95% confidence intervals for overall survival for each cancer subtype adjusted for patient age and tumor stage (Figure 1). Patients with tumors demonstrating low PD-L1 protein-to-mRNA ratio experienced significantly worse outcomes in 8 of 25 tumor types and improved outcomes in 2 tumor types (Table 2). Conclusions In this work we report that reduced human PD-L1 protein-to-mRNA ratios are associated with (1) high ADAM10 and/or ADAM17 expres- sion and (2) poor outcomes in multiple cancers. We previously showed in multiple cell lines that ADAM10 and ADAM17 cleave PD- L1 from the surface of tumor cells [7]. Our results suggest that ADAM10 and/or ADAM17 may cleave PD-L1 to cause a low PD-L1 protein-to-mRNA ratio in these tumors. This process may explain poorer survival of patients with low PD-L1 protein-to-mRNA ratios in some cancers. Our findings may explain why some tumors that do not have detect- able PD-L1 expression on immunohistochemistry respond to PD-(L)1 inhibitor therapy given the solubilization of PD-L1 and its subsequent systemic negative regulatory effects on T cells. Further, ADAM10/ ADAM17 inhibition may prevent PD-L1 shedding and sensitize tu- mors to therapy. While this work is correlative in nature, studies ex- ploring this mechanism of tumor immune system evasion are ongoing. Table 2 (abstract P301). See text for description Acknowledgements The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. References 1. M. Uhlen et al., “A pathology atlas of the human cancer transcriptome.,” Science, vol. 357, no. 6352, p. eaan2507, Aug. 2017. 2. P. C. Buchanan et al., “Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17.,” J. Biol. Chem., vol. 292, no. 15, pp. 6339–6351, Apr. 2017. 3. M. E. Powers, H. K. Kim, Y. Wang, and J. Bubeck Wardenburg, “ADAM10 Mediates Vascular Injury Induced by Staphylococcus aureus α- Hemolysin,” J. Infect. Dis., vol. 206, no. 3, pp. 352–356, 2012. 4. S.-S. Ni, J. Zhang, W.-L. Zhao, X.-C. Dong, and J.-L. Wang, “ADAM17 is overexpressed in non-small cell lung cancer and its expression correlates with poor patient survival,” Tumor Biol., vol. 34, no. 3, pp. 1813–1818, Jun. 5. Y.-Y. Wang, Z.-Y. Ye, L. Li, Z.-S. Zhao, Q.-S. Shao, and H.-Q. Tao, “ADAM 10 is associated with gastric cancer progression and prognosis of patients,” J. Surg. Oncol., vol. 103, no. 2, pp. 116–123, Feb. 2011. 6. B. You, Y. Shan, S. Shi, X. Li, and Y. You, “Effects of ADAM10 upregulation on progression, migration, and prognosis of nasopharyngeal carcinoma,” Cancer Sci., vol. 106, no. 11, pp. 1506–1514, Nov. 2015. 7. J. J. Orme et al., “Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 164 of 272 “responders” and “non-responders” to ICI drugs and is also being used to screen co-stimulatory agonists, peptide biologics and other drug classes. Conclusions Taken together, our data indicate that the recall antigen potency assay we established is highly potential to screen the drug candidates for im- mune checkpoint inhibitor and enhancer drug candidates. P303 PD-1 checkpoint blockade in advanced melanoma patients: Neutrophils, NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates Yago Pico de Coaña, Maria Wolodarski, MD, Irene van der Haar Àvila, Takahiro Nakajima, Stamatina Rentouli, Andreas Lundqvist, PhD, Giuseppe Masucci, MD, PhD, Johan Hansson, Rolf Kiessling, MD, PhD Karolinska Institute, Stockholm, Sweden Correspondence: Yago Pico de Coaña (yago.pico.de.coana@ki.se) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P303 Background Blockade of the PD-1 receptor has revolutionized the treatment of meta- static melanoma, with significant increases in overall survival and a dra- matic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of benefitting patients is limited and there is a need for predictive biomarkers and a deeper mechanistic ana- lysis of the cellular populations involved in a clinical response. Methods Fig. 1 (abstract P301). See text for description With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients undergoing treatment with pem- P302 brolizumab (n=7) or nivolumab (n=30) treatment at Karolinska Uni- A reversible T cell exhaustion-like in vitro assay to screen versity Hospital. Blood samples were collected from patients at the candidate drugs following time points: Before treatment and at the time of the sec- 1 2 2 Wushouer Ouerkaxi , Eden Kleiman , Pirouz Daftarian ond and fourth doses. Peripheral blood mononuclear cells (PBMCs) 1 2 MBL International, Woburn, MA, United States; JSR Lifesciences, were isolated by density gradient centrifugation and stained for flow Sunnyvale, CA, United States cytometric analysis within two hours of sample collection. Correspondence: Pirouz Daftarian (pdaftarian@jsrlifesciences.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P302 Two distinct cellular populations were inversely correlated with survival: Neutrophils, and Monocytic myeloid derived suppressor cells (MDSCs). Background Furthermore, overall survival and progression free survival were also T cells undergo different layers of suppression, with a spectrum of events found to be inversely correlated with the activation status of NK cells. that may overlap such as dysfunction, anergy, unresponsiveness, toler- Finally, PD-L1 expression in different monocytic subsets was signifi- ance, and exhaustion. Many factors have been attributed to this including cantly increased in patients with shorter progression free survival and multiple co-inhibitory receptor surface expression, altered transcription was correspondingly correlated inversely with overall survival. factor expression, epigenetic rewiring and dysregulated metabolism. Anti- Conclusions gen persistence is necessary for driving TEX maintenance in both the Our results suggest that cellular populations other than T cells can chronic viral infection setting and cancer. In addition to persistent antigen be critical in the outcome of PD-1 blockade treatment. Specifically, exposure, tumor-infiltrating lymphocytes (TILs) within the tumor micro- the frequencies of activated NK cells and monocytic MDSCs are in- environment (TME) encounter numerous tumor-mediated immunosup- versely correlated with survival and clinical benefit and their role as pressive metabolic byproducts, suppressive cytokines, hypoxia and predictive biomarkers should be further evaluated. cellular debris which converge to suppress T cell function and uniquely Ethics Approval alter is transcription factor profile. These suppressed or dysfunctional T The protocol was approved by the local Ethics Committee and the In- cells are incapable of mounting an optimal anti-tumor response in part stitutional Review Board at Karolinska Institute (approval number due to lack of fitness in competing for glucose and oxygen. Our team 2015/1862-32) and all patients provided written informed consent in has utilized the recall antigen potency assay as a tool for function-based accordance with the Declaration of Helsinki. screening of immune checkpoint inhibitor (ICI) drug candidates. Methods P304 A recall antigen potency assay has been used as a tool for function- DNA damage response gene alterations are associated with high based screening of immune checkpoint inhibitor (ICI) drug candidates. In tumor mutational burden and clinical benefit from programmed this assay, healthy human PBMCs are stimulated with peptide(s) derived death 1 axis inhibition in non-small cell lung cancer from either viruses or tumor proteins and grown in culture for one week. 1 1 1 Biagio Ricciuti, MD , Gonzalo Recondo, MD , Renato Umeton , Giuseppe Day 4 supernatants are functionally assayed by ELISA for IFN-γ secretion 1 2 2 1 Lamberti, MD , Mizuki Nishino , Lynette Sholl , Michael Cheng , Mark and cells are assayed on day 7 by flow cytometry for CD8+ or CD4+ T cell Awad, MD PhD expansion by using a single or cocktail of pMHC tetramers. 1 2 Dana Farber Cancer Institute, Boston, MA, United States; Brigham and Results Women’s Hospital, Boston, MA, United States We have shown that in roughly 30% of donor PBMCs, ICI drugs such as Correspondence: Biagio Ricciuti (biagio_ricciuti@dfci.harvard.edu) pembrolizumab are able to boost both IFN-γ secretion and antigen-specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P304 recall. One potential explanation for this effect is that the observed increase in T cell co-inhibitory receptor expression and presumed co-inhibitory re- Background ceptor downstream signaling is ameliorated with ICI drugs releasing these DNA damage response (DDR) gene alterations are associated with in- T cells from the repressive effects of these co-inhibitory receptors. Further, creased tumor infiltrating lymphocytes, higher genomic instability, this assay has the potential to screen for donors who are in vitro Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 165 of 272 and higher tumor mutational burden (TMB) in cancer. Whether DDR Table 1 (abstract P304). See text for description alterations are associated with benefit from immune-checkpoint in- hibitors (ICIs) in non-small cell lung cancer (NSCLC) is unknown. Methods Clinicopathologic and genomic data were collected from patients (pts) with advanced NSCLC at the Dana-Farber Cancer Institute (DFCI) treated with PD-(L)1 inhibitors. Targeted next-generation sequencing (NGS) by OncoPa- nel was used to determine DDR gene mutation status and TMB. Patients were categorized based on the presence or absence of deleterious DDR gene alterations in a panel of 53 DDR genes. All loss-of-function alterations in DDR genes (including nonsense, frameshift, or splice site) were classified as pathogenic. Missense mutations were evaluated using the Catalogue of Somatic Mutations in Cancer (COSMIC) [1], and ClinVar databases [2], as well as the PolyPhen-2 (Polymorphism Phenotyping v2) functional predic- tion tool [3]. Missense mutations were classified as pathogenic if annotated as pathogenic by either COSMIC or ClinVar and damaging by Polyphen-2. Because only tumor tissue was sequenced, common single nucleotide poly- morphisms (SNPs) were filtered if present at ≥0.1% in Genome Aggregation Database (gnomAD) version 2.1.1 [4]. Clinical outcomes to immunotherapy were evaluated according to DDR mutation status. Results Among 256 pts with successful NGS who received ICIs, 134 (52.3%) were identified as having deleterious DDR mutations (DDR-positive). DDR-positive and DDR-negative groups were well balanced in terms of baseline clinico- pathological characteristics (Table 1). The median TMB was significantly higher in the DDR-positive group compared to the DDR-negative group (12.17 vs 8.36 mutations/megabase, P< 0.0001), as well as among never smokers (9.40 versus 5.70 mut/Mb, P = 0.035, Figure 1B). Compared to DDR-negative pts (N=122), DDR-positive pts had a significantly higher ob- jective response rate (28.6% vs 16.4%, P=0.025, Figure 2A), longer median progression-free survival (4.2 vs 2.2 months, HR: 0.64 [95%CI: 0.49-0.84], P= 0.001, Figure 2B) and overall survival (17.5 vs 9.9 months, HR: 0.60 [95%CI: 0.43-0.82], P=0.002, Figure 2C) with PD-(L)1 therapy. DDR-positive status was associated with significantly longer PFS (HR: 0.70 [0.51-0.95], P=0.024) and OS (HR: 0.61 [95%CI: 0.43-0.85], P=0.004) in multivariate analysis (Table 2). Conclusions Deleterious DDR alterations are frequent in NSCLC and are associated with higher TMB and improved clinical outcomes in NSCLC pts treated with PD-1 axis inhibition. References 1. Forbes SA, Beare D, Boutselakis H, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic acids res. 2017; 45:D777-D783. 2. Landrum MJ, Lee JM, Benson M, et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic acids res. 2016; 44:D862-D868 3. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat. Methods. 2010; 7:248 4. https://gnomad.broadinstitute.org Fig. 1 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 166 of 272 Table 2 (abstract P304). See text for description Response to Pembrolizumab and tumor microenvironment composition is associated with IL8 expression in a head and neck squamous-cell carcinoma cohort 1 2 2 1 Arun Khattri, PhD , Jason Reeves , SuFey Ong , Riyue Bao, PhD , Arya 2 1 2 Bahrami, PhD , Yi-Hung Carol Tan, PhD , Andrew White, BSc , Michael 2 2 2 Bailey , Heather Brauer, PhD , Sarah Warren, PhD , Joseph Beechem, 2 3 PhD , Tanguy Seiwert, MD 1 2 University of Chicago, Chicago, IL, United States; NanoString Technologies, Seattle, WA, United States; Johns Hopkins University, Baltimore, MD, United States Correspondence: Tanguy Seiwert (tseiwert@jhmi.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P305 Background A subset of head and neck squamous-cell carcinomas are known to re- spond to immune checkpoint inhibitors. To better understand response to therapy in these tumors, a cohort of samples treated with Pembrolizumab was examined to determine if specific cell types are associated with re- sponse to intervention by combined profiling of standard bulk expression assays, ISH staining, and spatially resolved multiplexed protein analysis. Methods RNA was extracted from archival FFPE samples (n = 107) col- lected prior to therapeutic intervention and profiled using the NanoString® nCounter® PanCancer IO 360™ panel (Research Use Only). Gene expression signatures were calculated for immune cell subsets, as well as the Tumor Inflammation Signature (TIS; Ayers 2017 JCI). Individual gene expression and signatures were compared to patient outcome. Subsequently, expression of IL8 was validated by RNAScope in a subset of responders and non- responders (n = 9). To determine whether the IL8 staining pat- tern observed was consistent with specific cell types, a set of six tumor samples (three IL8+ and three IL8-) were further character- ized by multiplexed protein expression analysis on the GeoMx™ digital spatial profiling (DSP) platform to quantitate expression of 40 antibodies. IL8 staining was used to guide DSP selection of re- gions of interest (ROI) within the tumors that were either IL8+ or IL8-. Protein expression was specifically measured from tumor or stromal areas based on Pan-cytokeratin immunofluorescence. Results Initial analysis of the head and neck cohort found that previously reported signatures of response, including TIS, were not associ- ated with patient outcome in this cohort. In contrast, IL8 expres- sion was observed to be highest in patients with progressive disease. Further investigation demonstrated that IL8 expression was most specifically associated with neutrophil markers/expres- sion signatures. DSP profiling confirmed that tumor and stromal segments from IL8+ regions were associated with high expression of CD66B and ARG1 and lower expression HLA-DR consistent with neutrophil/granulocytic MDSCs presence. Furthermore, these re- gions were shown to have lower expression of T-cell markers in- cluding CD3, CD8 and CD4. Conclusions These results demonstrate that, in addition to previously reported biomarkers, IL8 expression and neutrophil presence may be related to response to checkpoint therapy in head and neck cancers. De- creased T-cell marker expression in IL8+ regions may reflect de- creased response in the larger cohort. Ethics Approval The study was approved by the University of Chicago‘s Ethics Board, approval number 8980 and 16-1269 P306 Innate immune cells play a role in therapy resistance to anti-PD1 in Hu-mice melanoma model Raj Somasundaram, PhD, Meenhard Herlyn, DVM PhD P305 The Wistar Institute, Philadelphia, PA, United States Correspondence: Raj Somasundaram (shyam@wistar.org) Fig. 2 (abstract P304). See text for description Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P306 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 167 of 272 Background constituted the majority of cases in both groups. The overall re- Immune checkpoint inhibitor therapy is rapidly emerging as a front- sponse rates of the CPI+M group were significantly higher than those line treatment option for many solid tumors. However, only a third of treated with CPI only (75% vs 53.3%, p = 0.05). melanoma patients respond to immune checkpoint blockade. Cur- Conclusions rently available mouse models have many short comings and are un- The data from this chart review shows an apparent benefit with respect able to address the basis of therapy resistance and immune non- to overall response rates in patients treated with M and CPIs. The cohorts responsiveness that are observed in patients. remain small in this study, but the data is significant. These results align Methods well with data from mouse studies and two small retrospective human Our laboratory has developed a novel humanized mouse melanoma studies in melanoma [2] and lung cancer [3], hence, demonstrating an in- model. Immuno-deficient NSG mice were reconstituted with human creased immune response and higher response rates. Currently, we are CD34+ cells and after 8-12 weeks, mice are fully reconstituted with hu- expanding our cohort number and data-set as we gained access to a big- man innate and adaptive immune cells. Humanized mice were then ger data warehouse. More prospective data are awaited from an ongoing challenged with HLA-matched melanoma cells and the functional abil- phase Ib (UMIN000028405), and phase II (NCT03800602, NCT03048500) ity of human immune cells to restrict tumor growth was monitored. trials that should shed more light on the effects of metformin on Results immuno-oncologic therapies. Restricted tumor growth was observed in humanized mice indicating in vivo sensitization of human immune cells to melanoma. References In therapy studies, tumor-bearing humanized mice treated with anti- 1. Scharping N, Menk A, Whetstone R, Zeng X, Delgoffe G. Efficacy of PD-1 PD-1 showed restricted tumor growth. Anti-PD-1 therapy resulted in blockade is potentiated by metformin-induced reduction of tumor hyp- enhanced infiltration of T-cells that correlated with tumor response. oxia. Cancer Immunol Res. 2017;5(1):9-16. MassCyTOF studies was performed using a panel of immune markers 2. Afzal M, Mercado R, Shirai K. Efficacy of metformin in combination with to understand the mechanism of therapy non-responsiveness in immune checkpoint inhibitors (anti-PD-1/anti-CTLA-4) in metastatic some tumors. Results indicated downmodulation of HLA-class I mole- malignant melanoma. J Immunother Cancer. 2018;6(1):64. cules and increased presence of mast cells cells in the tumor region. 3. Afzal M, Dragnev K, Sarwar T, Shirai K. Clinical outcomes in non-small-cell In tumor-bearing mice, combination of therapy drugs targeting c- lung cancer patients receiving concurrent metformin and immune kit+ mast cells and anti-PD1 caused complete regression of tumor le- checkpoint inhibitors. Lung Cancer Manag. 2019:1-12 (online publication). sions. Tumor free mice were able to reject freshly challenged melan- Ethics Approval oma cells indicating the presence of memory T-cell responses. The study was approved by Louisiana State University Health Science Center Conclusions of Shreveport’s Institutional Review Board separately for each site, IRB Our results suggest that humanized mouse melanoma model can be numbers are STUDY00000891, and STUDY00001017. explored further to understand the therapy resistance mechanisms to immune-based treatments. Further, model will be useful for devel- P308 oping new therapeutic strategies for treating melanoma patients. The impact of obesity on the response rates of checkpoint inhibitor (CPI) cancer immunotherapy 1 2 P307 Philip Haddad, MD, MPH , David Sommerhalder, MD The impact of metformin (M) on the response rates of checkpoint Overton Brooks VA Medical Center/LSUHSC-S, Shreveport, LA, United inhibitors (CPI) States; Louisiana State University Health Science, Bossier City, LA, United 1 2 Philip Haddad, MD, MPH , David Sommerhalder, MD States 1 2 Overton Brooks VA/ LSUHSC, Shreveport, LA, United States; Louisiana Correspondence: Philip Haddad (haddad8838@msn.com) State University Health Science, Bossier City, LA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P308 Correspondence: Philip Haddad (haddad8838@msn.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P307 Background With durable responses, improved clinical benefit, and relatively fewer Background toxicities, CPIs targeting cytotoxic T-lymphocyte-associated protein 4 Immunotherapies in oncology have brought significant change and (CTLA4), programmed death-1 (PD-1), and its ligand (PD-L1) have estab- hope to the field over the last several years. Responses, however, re- lished themselves as essential components of cancer immunotherapy main unpredictable and low in most cases. One of the aspects thought across multiple cancer types. Obesity is a known risk factor for several to be limiting immunotherapies is the tumor microenvironment which cancer types. It is associated with increased progression and cancer- favors tumor growth and immunosuppression. It has been hypothe- related death. This is thought to be the result of inflammaging and PD- sized that dysregulated tumor metabolism creates a hypoxic tumor 1 mediated immune suppression. Recently, two large retrospective microenvironment which acts as a barrier to antitumor immunity. Met- studies found that obesity conferred a survival advantage for cancer formin has been shown to reduce oxygen consumption and subse- patients treated with CPIs which may be independent of sex [1,2]. How- quently reduce the microenvironment hypoxia, leading to improved ever, the mechanistic explanation of this observed obesity paradox, as- response rates of checkpoint inhibitors in murine in vitro and in vivo suming it is real, has been the subject of many scientific conjectures. models [1]. We performed a retrospective review to evaluate response These studies focused on the impact of obesity on CPI overall survival rates in our patients who were treated with CPI+M. which can be affected by many confounding variables. Instead, we ex- Methods plored the effect of obesity on CPI response rates. We reviewed all adult cancer patients who were treated with a CPIs. Pa- Methods tients treated with M and CPIs were compared to those who were We retrospectively reviewed every cancer patient that received CPIs at treated with CPIs only. All tumor types and all CPI drugs were included. Overton Brooks VA Medical Center (OBVAMC) between 2015 and 2019. The primary endpoint was overall response rate, which included stable Patient’s BMI scores at the beginning of CPI therapies were calculated. disease, partial response, and complete response. Additional data was Based on the WHO definition, the patients were grouped according to captured for subgroup analysis. Patients were excluded if they had never their BMIs into overweight and obese (Group A) versus normal and received the treatment, or if they were never assessed for response. underweight (Group B). Our primary outcome of interest was defined as Results the presence or absence of CPI response. Patients who attained stable As of this date, 144 patients had been included in the study data-set. disease, partial response, and complete response were categorized as re- Of those, 24 patients were treated with M and CPIs and 120 patients sponders. Those who progressed on CPI were labeled as non-responders. received CPIs only. Both groups were comparable with respect to Thesignificanceofthe associationbetween the grouped BMI categories sex. However, the M+CPI group was slightly older. Lung cancer and the occurrence of any response was analyzed statistically. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 168 of 272 Results Results Between 2015 and 2019, 65 patients were treated with CPIs and had In the neoadjuvant ICB cohort, increasing age correlated with better documented responses. Both groups were comparable with respect to response to immunotherapy (OR= 0.91, P=0.039) and remained a sig- age, sex, race, and types of CPIs. Lung cancer constituted the majority nificant predictor of response in the multivariable model (OR=0.88, of cases in both groups. Head and neck cancers were more prevalent P=0.03) after adjusting for sex, tumor site, stage, prior therapies, and in Group B while renal and bladder cancer and melanoma were more toxicity; similar results were observed in the OpACIN neoadjuvant prevalent in Group A. Group B had a significantly higher response rate trial study (NCT02977052). Tumor mutational load was positively cor- (80% vs 50%, p=0.01). Furthermore, a higher response rate was ob- related with age (r=0.4) but did not reach statistical significance (P= served between normal BMI and overweight patients (p=0.001) and 0.1). Differential gene expression analysis revealed genes associated normal BMI and obese patients (p=0.06). No difference in response with MHC class II antigen expression (HLA-DQB1, HLA-DRB1) as sig- rates was observed between underweight and obese patients. nificantly overexpressed in younger patients upon treatment with Conclusions ICB. Furthermore, MHC class II regulator interferon-gamma signaling This is the first report to show a detrimental effect of overweight was also observed upregulated in younger patients (P=0.03). PD-1 ex- and obesity on CPI response rates in a retrospective cohort of pression (by IHC) was lower in older patients upon treatment with non-selected consecutive cancer patients in a real-world clinical ICB (r=-0.47, P=0.035). In keeping with human cohort, mice injected setting. with Yumm1.7 melanoma cells showed significant increase in im- mune cell populations expressing MHC class II antigen in the young References mice versus aged mice (P=0.0048). 1. McQuade J, et al. Association of body-mass index and outcomes in pa- Conclusions tients with metastatic melanoma treated with targeted therapy, immuno- Increased age was associated with improved outcomes in melanoma therapy, or chemotherapy: a retrospective, multicohort analysis. Lancet patients receiving neoadjuvant ICB, similar to results in stage IV. The Oncol. 2018;19(3):310-322. mechanisms behind this association are likely multifactorial, and may 2. Xu H, Cao D, He A, Ge W. The prognostic role of obesity is independent relate in part to MHC class II antigen expression mediated immune of sex in cancer patients treated with immune checkpoint inhibitors: A evasion in younger patients, though further studies are needed to pooled analysis of 4090 cancer patients. Int Immunopharmacol. delineate contributing factors. 2019;74:1-10. Trial Registration Ethics Approval NCT02519322 The study was approved by the Louisiana State University Health Science Ethics Approval Center of Shreveport Institutional Review Board, IRB number This trial was approved by the MD Anderson Cancer Center Institu- STUDY00001017. tional Review Board. The trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and with adherence to the Good Clinical Practice guidelines, as defined by the Inter- P309 national Conference on Harmonization. This protocol was conducted Older age predicts better outcome to neoadjuvant immune with compliance with all relevant ethical regulations. checkpoint blockade in metastatic melanoma Consent 1 2 1 Rohit Thakur , Stephen Douglass, PhD , Beth Helmink, MD PhD , Rodabe Written informed consent was obtained from all participants. The MD 1 1 1 Amaria, MD , Hussein Tawbi, MD, PhD , Jennifer McQuade , Eliza Anderson Data Safety Monitoring Board reviewed the data at 12- 3 1 4 Rozeman , Elizabeth Burton , Sangeetha Reddy, MD, MSci , John month increments. 5 3 6 Wherry , Christian Blank, MD PhD , Georgina Long , Jeffrey Gershenwald, 1 1 1 MD , Michael Davies, MD, PhD , Michael Tetzlaff, MD PhD , Ashani 7 1 Weeraratna , Jennifer Wargo, MD, MMSc P310 1 2 MD Anderson Cancer Center, Houston, TX, United States; The Wistar Optimal priming prevents the induction of dysfunctional CD8 T- Institute, Philadelphia, PA, United States; The Netherlands Cancer cells in subprimed conditions, reversing resistance to anti-PD-1 Institute, Amsterdam, Netherlands; UT Southwestern Medical Center, Vivek Verma, PhD, Rahul Nandre, PhD, Jose Lopez, Seema Gupta, PhD, Houston, TX, United States; University of Pennsylvania, Philadelphia, PA, Samir Khleif, MD 6 7 United States; Melanoma Institute Australia, Sydney, Australia; Johns Georgetown University Medical Center, Washington, DC, United States Hopkins School of Medicine, Baltimore, MD, United States Correspondence: Samir Khleif (snk48@georgetown.edu) Correspondence: Jennifer Wargo (JWargo@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P310 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P309 Background Background Suboptimal-priming of lymphocytes by low-affinity antigens is a mechan- There is a growing appreciation for studying the impact of host and ism for prevention of generation of strong immune responses against environmental factors on response to immune checkpoint blockade self-antigens. However, we recently found that the suboptimally-primed (ICB). Recently it was reported that older age correlated with better CD8 cells have a pre-disposition to develop a dysfunctional phenotype outcome in stage IV melanoma patients treated with ICB. We exam- that is marked by expression of CD38 on PD1+CD8+T-cells. Interestingly, ined the association of age with response to ICB and anti-tumor im- the number of these dysfunctional cells were significantly increased upon munity in melanoma patients treated neoadjuvantly (NCT02519322) PD-1 blockade in these suboptimally-primed CD8 cells that served as a and interrogated underlying mechanisms in a murine model. reason for resistance to anti-PD-1 therapy. On the other hand, anti-PD-1 Methods blockade of optimally-primed CD8 cells did not generate these dysfunc- Tumor samples were obtained from neoadjuvant ICB trial patients tional cells and led to cell activation and generation of effector functions pre-treatment, on-treatment and on-surgery. Transcriptome profil- [1]. Hence, here we investigated the effect of optimal-priming on revers- ing was performed using Illumina NextSeq platform and whole ing the resistance to anti-PD-1 therapy. exome sequencing using Illumina HiSeq 2500 platform. Univari- Methods able and multivariable analyses were performed using logistic re- Mice were inoculated with TC-1 cells (a mouse lung epithelial cell- gression modeling. Immune profiling was performed by line, expressing human papillomavirus-specific E7-peptide) in the Immunohistochemistry (IHC). The effects of age on anti-tumor im- presence or absence of concomitant priming with gp100 peptide, a munity were examined by implanting 2x105 Yumm1.7 cells sub- non-cognate tumor vaccine. Seven days later mice were treated with dermally in young (8wks) and aged (>12months) male mice. anti-PD-1 either alone or followed by combination of tumor-specific Tumors were harvested after 30 days of growth and immune sur- E7-peptide vaccine+anti-PD-1. Tumor growth rates, mice survival, face markers were analyzed by flow cytometry. and immune responses in the TME were estimated. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 169 of 272 Results results in anti-tumor efficacy. TPST-1495 is a first-in-class, orally avail- We found that in suboptimally-primed TC-1 tumor-bearing mice, able, small molecule, selective dual antagonist of the human PGE2 anti-PD-1 treatment did not show any anti-tumor effects. Therefore, receptors EP2 and EP4, currently under development by Tempest. to check if the optimal priming of CD8 T-cells could reverse this re- Methods sistance, we vaccinated mice with gp100 at the time of tumor im- The effects of TPST-1495 as monotherapy or in combination with plantation with TC-1 cells. We found that compared to suboptimally- anti-PD1 were evaluated in the syngeneic mouse colon models CT26 primed mice, anti-PD-1 treatment of primed-mice resulted in signifi- and Apcmin/+. The mechanism of anti-tumor immunity of TPST-1495 cant retardation of tumor growth and a prolonged mice survival. was evaluated using in vitro primary dendritic cell (DC) differentiation Interestingly, tumor-specific vaccination (E7-peptide) of these and activation assays. Characterization of in vitro differentiated im- primed-mice at the time of anti-PD-1 treatment further enhanced the mune cells or tumor infiltrating lymphocytes were performed using therapeutic efficacy of PD-1 blockade. Moreover, in gp100-primed- flow cytometry. ELISA was used for measurement of cytokine mice we found a significant reduction in the number of PD-1+CD38hi production. dysfunctional cells compared to suboptimally-primed mice. The num- Results ber of these dysfunctional cells was further reduced upon anti-PD-1 Treatment with TPST-1495 reversed PGE2 immune suppression treatment of primed-mice. In addition, the priming state also affected in vitro and in vivo compared to antagonism of EP4 alone or all 4 EP the functionality of CD8 T-cells. Although gp100 alone prevented the receptors. TPST-1495 prevented PGE2 inhibition in vitro of DC differ- induction of these dysfunctional cells, the functionality of CD8 T-cells entiation and activation from human donor monocytes; single EP2 or was only increased when anti-PD-1 was given subsequent to priming EP4 antagonists were sub-optimal in this assay. Significantly, combin- with gp100 peptide. ation with EP1 and/or EP3 antagonists reversed the effect of dual Conclusions EP2 and EP4 blockade on PGE2 immune suppression, suggesting that Here we demonstrate that optimal priming of CD8 cells reverses re- COX-2 inhibition is not optimal for blocking the effects of PGE2. TPST- sistance to anti-PD-1 therapy. The suboptimal-priming of the CD8+ T- 1495 induced potent anti-tumor immune responses and significant cells induces higher numbers of dysfunctional PD-1+CD38hi CD8+ T- tumor regression as a monotherapy in two different murine tumor cells and their frequency further increases upon anti-PD-1 therapy, treatment models of colon cancer, CT26 and Apcmin/+. CT26 tumors leading to therapeutic failure. Since in most tumors, T-cells are analyzed from mice treated with TPST-1495 alone revealed a significant suboptimally-primed [2,3], our mouse data demonstrate the import- increase of infiltrating effector T cells. TPST-1495 combination with anti- ance of appropriately primed T-cells in responding to anti-PD-1 PD1 synergistically inhibited CT26 tumor progression. treatment. Conclusions TPST-1495 is a differentiated highly potent selective dual antagonist References of EP2 and EP4 that overcomes prostaglandin-mediated immune 1 Verma, V. et al. PD-1 blockade in subprimed CD8 cells induces dysfunc- suppression and promotes anti-tumor efficacy. tional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nat Immunol, doi:10.1038/s41590-019-0441-y (2019). P312 2 Vonderheide, R. H. The Immune Revolution: A Case for Priming, Not Ipilimumab treatment immunophenotypic changes are associated Checkpoint. Cancer Cell 33, 563-569, doi:10.1016/j.ccell.2018.03.008 (2018). with progression of disease with sequential nivolumab therapy in 3 Vonderheide, R. H., Domchek, S. M. & Clark, A. S. Immunotherapy for metastatic melanoma Breast Cancer: What Are We Missing? Clin Cancer Res 23, 2640-2646, 1 1 2 David Woods, PhD , Andressa Sodre Laino, PhD , Aidan Winters , Jason doi:10.1158/1078-0432.CCR-16-2569 (2017). 1 1 1 Alexandre , Jeffrey Weber, MD, PhD , Pratip Chattopadhyay 1 2 NYU Langone Health, New York, NY, United States; UCSF, San P311 Francisco, CA, United States Dual antagonism of prostaglandin receptors EP2 and EP4 by TPST- Correspondence: Pratip Chattopadhyay 1495 suppresses tumor growth and stimulates anti-tumor (Pratip.Chattopadhyay@nyulangone.org) immunity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P312 1 2 2 Chan Whiting, PhD , Kim Fischer, PhD , Bryan Laffitte, PhD , Lisa 2 2 2 Rahbaek, PhD , Nick Stock, PhD , Davorka Messmer, PhD , Austin Chen, Background 2 2 2 1 PhD , Traci Olafson , Natalie Nguyen , Amanda Enstrom, PhD , Derek Nivolumab (nivo) and ipilimumab (ipi) combination immunotherapy has 1 1 3 Metzger , Brian Francica , Dingzhi Wang, PhD , Raymond Dubois, PhD, a ~60% response rate in metastatic melanoma patients. However, the im- 3 1 2 1 MD , Ginna Laport, MD , Peppi Prasit, PhD , Thomas Dubensky, PhD pact of these therapies on immune cell phenotypes and the relationship 1 2 Tempest Therapeutics, San Francisco, CA, United States; Inception of those changes to patient outcomes remains under-investigated. Sciences, San Diego, CA, United States; Medical University of South Methods Carolina, Charleston, SC, United States High dimension flow cytometry on baseline and at week 13 (e.g. Correspondence: Brian Francica (bfrancica@tempesttx.com) after initial nivo or ipi therapy) was performed for peripheral blood Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P311 samples from 33 metastatic melanoma patients receiving sequential nivo-ipi or the reverse sequence. We used a novel computational ap- Background proach to analyze the data through semi-comprehensive Boolean Progression of diverse malignancies is promoted by elevated levels gating in which immune cell lineages (e.g. CD3+CD4+) were evalu- of Prostaglandin E2 (PGE2). High PGE2 levels results from dysregula- ated for all possible combinations for up to 15 markers. tion of Cyclooxygenase-2 (COX-2), the enzyme that produces this Results lipid. PGE2 stimulates tumor cell proliferation, survival, evasion and 3,844 measured immunophenotypes were significantly altered post-nivo, metastasis along with host angiogenesis. PGE2 suppresses anti-tumor and 7,133 immunophenotypes were altered post-ipi. The frequency of immunity through inhibiting the function of critical immune effectors 584 immunophenotypes were significantly changed in both treatments, such as NK and T cells, and M1 macrophages, while promoting the with 59 of those changing in opposing directions. In the nivo-ipi cohort, activity of suppressive immune cells including myeloid derived sup- 260 baseline and 662 post-nivo immunophenotypes were significantly as- pressor cells, M2 macrophages, and regulatory T cells. PGE2 signals sociated with response and survival (outcomes). In the ipi-nivo cohort, 432 through a family of four homologous E-prostanoid (EP) G-coupled re- baseline and 668 post-ipi immunophenotypes were associated with out- ceptors, known as EP1, EP2, EP3 and EP4; which,are activated via dis- comes. Two highly similar immunophenotypes associated with outcomes tinct signal transduction pathways. Published literature and overlapped between the cohorts, CD14+CD11C+CD33+CD15-CD19-PDL1- experimental results presented here demonstrate that selective an- PDL2+CD163+GAL9-CD80-CD86-41BBL+CD40+OX40L+ cells. While lower tagonism of both EP2 and EP4 receptor signaling, but not EP1 and levels of these cells were associated with response and improved survival EP3, effectively overcomes PGE2-mediated immune suppression and in nivo-ipi treated patients, lower levels were associated with better Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 170 of 272 outcomes in the ipi-nivo treated patients. Of the 3,844 immunopheno- Specifically, these data show that changes in secretome production types altered post-nivolumab, 100 were also associated with ipi-nivo re- potential of T-cells after treatment with the PD1 blocking antibody sponse. Of these 100, 97% were altered in a manner positively associated nivolumab are associated with metastatic melanoma patient out- with response (e.g. upregulated by nivolumab and higher in responders). comes. We identified a signature of secreted molecules that were as- For example, nivo upregulated CD4+CD45RO-CCR7+ frequencies, which sociated with patient outcomes, providing rationale for targeting were associated with response and longer survival in ipi-nivo treated pa- these molecules to increase the efficacy of nivolumab. Work is under- tients. Of the 7,133 immunophenotypes altered post-ipi, 110 were also as- way to validate the observed associations in an independent set of sociated with nivo-ipi response. Of these, 95% were altered in a manner patient samples. negatively associated with response. This includes ipi associated upregula- Ethics Approval tion of a population of CD4+CD38+CD39+CD127-GARP- cells that are The study was approved by NYU Langone Health's IRB, approval negatively associated with coutcomes in nivo-ipi treated patients as well number S16-00035. as downregulation of a CD4+CD127+CD45RO+CD95+CCR7+ population of cells positively associated with outcomes. P314 Conclusions Alterations of DNA damage response signaling in the development These results demonstrate that nivo and ipi altered the peripheral im- of antibody-dependent cellular cytotoxicity (ADCC) resistance mune landscape in distinct ways. While immunophenotypic changes Louis Weiner, MD, Yongwei Zhang, Dalal Aldeghaither, David Zahavi, MS, BS post-nivo favored response in ipi-nivo treated patients, changes associ- Lombardi Cancer Center, Washington, DC, United States ated with ipi treatment favored progression with ipi-nivo. These data Correspondence: Louis Weiner (weinerl@georgetown.edu) suggest that the immunophenotypic impact of ipi alters the immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P314 landscape in a manner that may impair a subsequent response to nivo. These data also highlight several novel immune cell populations that Background are associated with both treatment effects and patient outcomes. Accumulating evidence has shown that DNA damage response Ethics Approval (DDR) is closely associated with immune response. Innate im- The study was approved by NYU Langone Health's IRB, approval mune responses, such as Natural killer (NK) cell-mediated killing, number S16-00035. are dependent on the DDR essential kinases Ataxia telangiectasia mutated (ATM) or ATM- and RAD3-related (ATR) [1]. However, P313 DNA damage inducing agents activate the NF-kappaB-regulated Single-cell secretome assessment of metastatic melanoma patient interferon immune response pathway [2]. Moreover, DDR inhib- peripheral T-cells reveals a pharmacokinetic signature of patient ition resulting from DNA repair deficiency or cell cycle check- response to nivolumab therapy point inhibition can potentiate efficacy of antibody-based David Woods, PhD, Andressa Sodre de Castro Laino, Daniel Freeman, immunotherapy, such as immune checkpoint blockade and ADCC Jeffrey Weber, MD, PhD, Pratip Chattopadhyay [3-5]. However, the mechanistic role of DNA damage response NYU Langone Health, New York, NY, United States signalinginthe developmentofimmunotherapy resistance re- Correspondence: David Woods (David.Woods@nyumc.org) mains unclear. A NK cell-mediated cetuximab-dependent killing Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P313 in vitroADCCmodel wasusedtostudy this involvement. Our previous studies have suggested a loss of cell surface adhesion Background molecules in ADCC resistant cells [6]. Here we study the alter- Therapies targeting T-cell co-inhibitory molecules (e.g. PD1) have dem- ations of DNA damage response signaling in the process of onstrated unprecedented efficacy in the treatment of metastatic melan- ADCC resistance development. oma. However, not all patients respond to checkpoint inhibition, so Methods there is an unmet need to identify mechanisms of resistance/response. A431, a human epidermoid carcinoma cell line, develops resist- Methods ance to cetuximab and NK92-CD16v cell-mediated killing after Using IsoLight, a platform for assessing the secretion of 32 analytes 35 challenges of continuous exposure. Surviving cells following at single-cell resolution, we evaluated previously frozen peripheral each ADCC challenge were collected and studied for protein ex- blood T-cells from six responding and six progressing patients (ac- pression by western blot, immunofluorescence and flow cytome- cording to RECIST 1.1 criteria) treated with nivolumab. Baseline and try. Neutral comet assay was used to measure the level of DNA week 13, post-treatment CD4+ and CD8+ T-cell samples were double strand breaks. Fluorescence-based cytotoxicity methods assessed for each patient. T-cells were stimulated with CD3 and were used to determine the activity of ADCC. Apoptosis was CD28 activating antibodies overnight and subsequently placed on measured by Annexin-V-PI staining. Small interference RNA capture chips for 20 hours. Approximately 400 single-cell events were (siRNA) were used to knockdown gene expression. assessed for each sample. Results Results Levels of gammaH2AX, a marker of DNA damage signaling, CD4+ T-cells from progressing patients, compared to those from were significantly increased with exposure to ADCC, but no in- responding patients, had significantly (p<0.05) higher mean pro- creased DNA breaks were detected in ADCC resistant cells. p53, duction of IL-17F post-nivolumab and an increased proportion of phosphorylated-p53 and signal transducer and activator of tran- cells secreting IL-13, RANTES, IL-6, soluble CD137, TNF, MIP1a and scription 1 (STAT1) were also enhanced during the process of MIP1b relative to baseline. Using an elastic net machine learning ADCC resistance development. Interestingly, phosphorylated- algorithm with cross validation, we assessed the ability of a STAT1 reached a peak prior to the emergence of ADCC resist- manually curated list of analytes to predict patient outcomes. ance and then decreased until cells became entirely resistant. Delta values (post-treatment minus baseline values) were used for There was less apoptosis induction, no caspase activation, less 15 parameters. A receiver operating characteristic with an area induction of gammaH2AX and no activation of p53 in response under the curve of 0.898 was achieved. The most important fea- to ADCC in resistant cells. Inhibition of p53 or STAT1 by siRNA, tures in these models were the percentage of CD4+ T-cells ex- and ATM/ATR inhibitors enhanced ADCC activity in A431 cells pressing IL-6, MIP1a and soluble CD137 along with the but not in ADCC resistant cells.TP53knockdown activated percentage of CD8+ T-cells expressing IL-13. STAT1 in A431 cells, but reduced activation of STAT1 in ADCC Conclusions resistant cells. These results demonstrate the ability of single-cell, high-dimension Conclusions technologies coupled with machine learning to reveal complex asso- DNA damage response signaling was altered during the develop- ciations between immune cell function and clinical outcomes. ment of ADCC resistance, which is involved in ADCC activity Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 171 of 272 regulation and might become be a signature of cell sensitivity in PD1/PD-L1 therapies. PD-L2 expression is observed in multiple tumor response to immunotherapy. Further DDR-related multiplex mech- types, and animal studies suggest PD-L2 may be involved in T-cell anisms are being investigated. suppression. However, the role of PD-L2 expression in the TME and the role as a predictive biomarker in immunotherapy is not as well References understood as PD-L1. Here, we investigated the expression of PD-L2 1. Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathway in multiple cellular components of the TME and its potential impact regulates innate immune system ligands of the NKG2D receptor. Nature. on the efficacy of PD-L1 blockade in cancer patients. 2005;436(7054):1186-90. Methods 2. Brzostek-Racine S, Gordon C, Van Scoy S, Reich NC. The DNA damage re- Single-cell RNA sequencing (scRNAseq) was performed using 10X sponse induces IFN. J Immunol. 2011;187(10):5336-5345. genomics for two commercial NSCLC samples and ~4,500 cells were 3. SenT,Rodriguez BL,ChenL,Corte CMD, Morikawa N, Fujimoto J, clustered by their expression pattern using shared nearest neighbor. Cristea S,Nguyen T, Diao L, Li L, Fan Y, Yang Y, Wang J, Glisson BS, Gene expression data for lung adenocarcinoma (LUAD) and squa- Wistuba II, Sage J, Heymach JV, Gibbons DL, Byers LA. Targeting mous carcinoma (LUSC) in TCGA were analyzed. Baseline tumor tran- DNA Damage Response Promotes Antitumor Immunity through scriptomes were profiled for 97 1L+ NSCLC patients treated with PD- STING-Mediated T-cell Activation in Small Cell Lung Cancer. Cancer L1 inhibitor durvalumab (NCT01693562). PD-L1 and PD-L2 immuno- Discov. 2019,9(5):646-661. staining was performed on baseline samples in CP1108. Gene expres- 4. Fenerty KE, Padget M, Wolfson B, Gameiro SR, Su Z, Lee JH, sion signatures for macrophage, fibroblast, dendritic cells, cancer Rabizadeh S, Soon-Shiong P, Hodge JW. Immunotherapy utilizing associated fibroblast (CAF) and interferon gamma were curated in- the combination of natural killer- and antibody dependent cellu- house or adopted from previous studies. lar cytotoxicity (ADCC)-mediating agents withpoly (ADP-ribose) Results polymerase (PARP) inhibition. J Immunother Cancer. 2018,6(1):133- In scRNAseq data, PD-L2 mRNA was found in over 10% of macro- 136. phage and fibroblast cells, and 5. Germano G, Lamba S, Rospo G, Barault L, Magrì A, Maione F, Russo M, Conclusions CrisafulliG, Bartolini A, Lerda G, Siravegna G, Mussolin B, Frapolli R, PD-L2 mRNA expression is mainly in immunosuppressive cellular Montone M, MoranoF, de Braud F, Amirouchene-Angelozzi N, Marsoni S, components of the TME in NSCLC, including macrophage and cancer D'Incalci M, Orlandi A,Giraudo E, Sartore-Bianchi A, Siena S, Pietrantonio associated fibroblast cells and low PD-L2 expression in PD-L1 high F, Di Nicolantonio F,Bardelli A. Inactivation of DNA repair triggers neoan- NSCLC patients may associate with improved OS. tigen generation and impairstumour growth. Nature. 2017,552(7683):116- Trial Registration 120. NCT01693562 6. Aldeghaither DS, Zahavi DJ, Murray JC, Fertig EJ, Graham GT, Zhang Ethics Approval YW,O'Connell A, Ma J, Jablonski SA, Weiner LM. A Mechanism of This study was conducted according to the Declaration of Helsinki Resistance to Antibody-Targeted Immune Attack. Cancer Immunol Res. and approved by the independent ethics committee/institutional re- 2019;7(2):230-243. view board at each participating center, with informed consent ob- tained from all patients. P316 MG1131, a novel TIGIT-targeted monoclonal antibody, induces T cell activation and anti-tumor immune response and suppresses Treg cell activity 1 1 2 1 Hyemi Nam, MS , Hye-Young Park , Eun Jung Song , Eunhee Lee , Hye 1 1 1 1 1 In Yum , Munkyung Kim , Jeewon Lee, Ph D , So Jung Lim , Okjae Lim , Yangmi Lim MOGAM Institute for Biomedical Research, Yongin-si, Korea, Republic of; GC Pharma, Yongin-si, Gyeonggi-do, Korea, Republic of Correspondence: Yangmi Lim (ymlim@mogam.re.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P316 Background Fig. 1 (abstract P314). Enhancement of DDR signaling T cell immunoreceptor with Ig and ITIM domain (TIGIT) is a co- pathway molecules inhibitory receptor expressed on CD8+ T cells, CD4+ T cells, NK cells, and regulatory T cells (Treg). TIGIT binds two ligands PVR (CD155) and PVRL2 (CD112) and these ligands are expressed by P315 T cells, APCs, and tumor cells. As malignancies progress, PVR The expression of programmed death ligand 2 (PD-L2) in over-expressed in tumor cells interacts with TIGIT expressed on immunosuppressive tumor microenvironment of non-small cell tumor infiltrating lymphocytes (TIL) and suppresses TIL activity lung cancer (NSCLC) and its potential association with by sending an inhibitory signal to immune cells, which is an im- immunotherapy 1 2 2 2 mune escape mechanism in cancer. In cancer, TIGIT blockade Qu Zhang, PhD , Stefan Bentink , Vinay Pawar , Farzad Sekhavati, PhD , 1 1 1 results in improved effector CD8+ T cell and NK cell function as Keith Steele, DVM, PhD , Jason Hipp , Song Wu, PhD 1 2 well as decreased Treg-cell-mediated suppression. Therefore, we AstraZeneca, Gaithersburg, MD, United States; Definiens AG, Munich, developed MG1131, a novel anti-TIGIT antibody, to modulate Germany the tumor microenvironment towards a more effective anti- Correspondence: Qu Zhang (zhangq@medimmune.com) cancer response. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P315 Methods TIGIT-targeting antibody candidates were screened out of a Background phage display library. The TIGIT antigen binding affinity and PVR In the tumor microenvironment (TME), programmed death-1 receptor blocking of anti-TIGIT antibodies were evaluated through both (PD-1), combined with its ligands PD-L1 and PD-L2, play an important protein-based and cell-based assays. Functional consequences of suppressive role in the immune response to cancer. PD-L1 expression MG1131 were determined using a cell-based reporter assay for T has been shown to be a key predictive biomarker of response to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 172 of 272 cell activation, a Treg cell functional assay and an NK-mediated group were hypertension (32%), anemia (29%), colitis/enterocolitis tumor killing assay. Treg cells and NK cells were isolated from (26%), and nausea/vomiting (25%). Median survival was signifi- healthy donor PBMC. cantly longer in the IO group relative to the CT group (18.6 Results months and 15.1 months, respectively; adjusted HR 0.73, 95% CI We screened out a few of the clones which stood out showing 0.66-0.81, Figure 1). high affinity binding to TIGIT and significant blocking of the Conclusions TIGIT-PVR interaction. Among the tested antibodies, MG1131 was Among the veteran population in real-world settings, patient identified to have the strongest affinity for human TIGIT and sig- characteristics were similar for 2L IO or CT therapy, with the ex- nificant blocking activity, resulting in competition with PVR in a ception of age and geographic region. Rates of common AEs dose dependent manner. Furthermore, MG1131 was cross-reactive were as expected. Our findings indicate improved survival among with cynomolgus monkey TIGIT, but not with mouse TIGIT. Our patients receiving IO versus CT in the 2L setting. More in vitro efficacy data demonstrated that MG1131 significantly en- population-based studies are needed to confirm these findings in hances T cell activation and NK-mediated tumor killing activities other healthcare settings. in a PVR-dependent manner and MG1131 induces IFN-γ secretion and proliferation of CD8+ T cells by inhibiting Treg suppressive Acknowledgements function. The team would like to thank Daniel Lane, PharmD, PhD, MBA for his Conclusions support of this study. We developed an anti-TIGIT antibody, MG1131, with pronounced in- Ethics Approval hibitory activity on the TIGIT-PVR signaling axis. In this study, This study was approved by the Durham VA Institutional Review Board (IRB MG1131 significantly enhanced T cell activation and NK-mediated #02009). tumor killing activity, and efficiently suppressed Treg cell function. Therefore, MG1131 is a potential candidate for cancer immunotherapy. P317 Utilization of second-line immuno-oncology agents and associated health outcomes among united states veterans with advanced non-small cell lung cancer 1 2 3 Mina Allo, PharmD, MPH , Lin Gu, MS , Vishal Vashistha, MD , Ashlyn 2 2 2 Press, MPH , Michael Kelley, MD , Christina Williams, PHD, MPH 1 2 Bristol-Myers Squibb, Princeton, NJ, United States; Durham Veterans Affair, Duke Cancer Ins, Durham, NC, United States; Dept of Medicine, Duke University, Durham, NC, United States Correspondence: Mina Allo (mina.allo@bms.com); Christina Williams (christina.williams4@va.gov) Fig. 1 (abstract P317). KM curve of overall survival of patients in 2L Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P317 Background Studies describing treatment safety, effectiveness and patterns of use are needed to evaluate the real-world impact of immuno-oncology P318 (IO) in advanced non-small cell lung cancer (NSCLC) relative to Large-scale evaluation of concordance of genomic scores in whole chemotherapy (CT). This retrospective cohort analysis assessed exome sequencing and Foundation Medicine comprehensive utilization of IO and CT agents and associated outcomes in second- genomic platform across cancer types 1 1 1 1 line (2L) treatment among stage IV NSCLC patients receiving care in Deepti Aurora-Garg , Andrew Albright, PhD , Ping Qiu, PhD , Yongjin Li , 1 2 1 1 the Veterans Affairs (VA). Xiaoqiao Liu , David Fabrizio, PhD , Lixin Lang , Jared Lunceford, PhD , Methods Razvan Cristescu, PhD 1 2 The VA Corporate Data Warehouse (CDW) oncology database was Merck & Co., Inc., Kenilworth, NJ, United States; Foundation Medicine, used to determine survival and 14 common adverse events (AE) Cambridge, MA, USA, Cambridge, MA, United States of adult patients with stage IV NSCLC diagnosed from 2012 to Correspondence: Deepti Aurora-Garg (deepti.aurora-garg@merck.com) 2017 who received systemic non-targeted (ALK, EGFR) therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P318 within 120 days of diagnosis and were followed until death or end of the study period in June 2019. Descriptive statistics were Background used to summarize treatment and AE occurrence. Kaplan-Meier Whole exome sequencing (WES) is a comprehensive method to methodology and multivariate Cox regression were used to evalu- evaluate the clinical relevance of DNA molecular characteristics, in- ate survival. cluding pan-exomic genomic scores (eg, tumor mutational burden Results [TMB] and homologous recombination deficiency–loss of heterozy- We identified 1655 patients who received 2L therapy, with 42% gosity [HRD-LOH]) and individual alterations (eg, BRCA1/2). Although (n=695) receiving IO monotherapy (nivolumab, pembrolizumab, comprehensive targeted genomic panels are available to measure atezolizumab, and durvalumab), 56.5% (n=935) receiving CT only, TMB and HRD-LOH, including FoundationOne® CDx (F1CDx), imple- and 1.5% (n=25) receiving IO+CT (not included in the current mentation of WES as a diagnostic approach in clinical practice can analysis due to limited sample). Greater than 99% of 2L IO users be challenging. To assess the feasibility of translating findings using used CT only in 1L setting, and >96% of 2L CT only group used WES as an exploratory tool into a practical diagnostic device such as CT in 1L (~ 3% used IO monotherapy, 0.6% IO+CT). Median age F1CDx, we evaluated the concordance of genomic scores (TMB and was 67 vs. 65 years in the IO and CT groups, respectively (p= HRD-LOH) and single-gene alterations between WES and F1CDx in a 0.006). No statistically significant differences between the IO and large pan-tumor data set. CT groups were observed by sex (~97% male), race (~77% White), Methods ethnicity (~98% non-Hispanic), smoking history (~95% current/ This analysis used solid tumor samples from patients with advanced former smoker), or histology (~58% adenocarcinoma). The most disease who received pembrolizumab monotherapy in the second common AEs in the IO group were dyspnea (50%), colitis/entero- line or later during single-arm clinical trials. WES and F1CDx (Dx1 colitis (40%), and anemia (28%); most common AEs in the CT baitset) were used to analyze samples from 436 patients across 22 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 173 of 272 tumor types. Spearman rank-order correlation and linear regression June 2019. Patient Information Form, Brief Fatigue Inventory (BFI), were used to determine concordance and cutoff equivalence for Functional Living Index-Cancer (FLIC), and Dermatology Life Quality TMB and HRD-LOH, each calculated by both WES [1] and F1CDx Index (DLQI) were used for data collection at 1st, 2nd, 3rd, and 4th (Foundation Medicine proprietary pipeline QSR_F1Dx_v1.0.3). cycles of nivolumab. Descriptive statistics, Mann Whitney U and Results Friedman tests were utilized for data analysis. Using WES and F1CDx, high concordance was observed in the pan- Results tumor assessment of TMB (Spearman correlation, 0.7; n=413) and The majority of the patients was male (60%), and the mean age of HRD-LOH (Spearman correlation, 0.5; n=364). When individual indica- patients was 54.10±18.88 years. The mean time of diagnosis for pa- tions were considered, the concordance was further improved for in- tients with melanoma was 40.50±47.84 months (range 2-168). There dications with higher distribution medians. TMB concordance was were no significant differences between patients’ BFI, FLIC, and DLQI higher when restricted to non–small cell lung cancer (Spearman cor- total scores in terms of age and gender (p>.05). The mean scores of relation, 0.8; n=38). HRD-LOH concordance was higher when re- BFI scores were 4.15±2.90 at the 1stcycle, and 3.75±2.98 at the 4th stricted to ovarian cancer (Spearman correlation, 0.7; n=54) and cycle; FLIC scores were 88.15±9.69 and 95.26±12.07; and DLQI scores breast cancer (Spearman correlation, 0.6; n=80). Regression analysis were 2.60±6.30 at 1.90±3.11, respectively. Considering the changes of TMB using both platforms identified F1CDx (Foundation Medicine within time in terms of all scale scores, no significant differences proprietary pipeline QSR_F1Dx_v1.0.3) TMB cutoffs of 10 and 13 mu- were found in BFI (p=.29), and DLQI (p=.49). With regard to FLIC tations/megabase to correspond to WES TMB of ~150 and ~175 mu- scores a significant difference was found from the 1st to the 4th tations/exome, respectively. Assessment of BRCA1/2 deleterious cycle of nivolumab (p=.05). mutations also demonstrated agreement between WES and F1CDx, Conclusions with 305 of 309 (98.7%) samples showing agreement; 282 samples The present study may be the first effort to evaluate changes in BFI, showed wild-type status by both methods and 23 samples showed FLIC and DLQI scores during nivolumab treatment in patients with mel- mutant status by both methods. anoma from the 1st to the 4th cycle. The study findings revealed that Conclusions improvements in BFI, and DLQI scores following nivolumab, even not The high level of concordance between WES and F1CDx suggests statistically significant. Lastly supporting literature [3,4], significant in- that molecular biomarker discoveries, including clinically relevant cut- crease has been found in FLIC scores with nivolumab treatment. offs and molecular epidemiology findings evaluated on the transla- tional WES platform, may be translated successfully in the diagnostic References setting. To our knowledge, this is the first evaluation of concordance 1. Dine J, Gordon R, Shames Y, Kasler MK, Barton-Burke M. Immune checkpoint of genomic scores performed in the context of clinical trial data inhibitors: an innovation in immunotherapy for the treatment and manage- across many indications and in a large data set. ment of patients with cancer. Asia Pac J Oncol Nurs. 2017; 4(2):127-135. 2. Shepherd FA, Douillard JY, Blumenschein GR. Immunotherapy for non- Reference small cell lung cancer: Novel approaches to improve patient outcome. J 1. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for Thorac Oncol. 2011; 6(10): 1763-1773. PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362. pii: 3. Ramirez RA, Lu J, Thomas KE. Quality of life for non-small cell lung cancer eaar3593. patients in the age of immunotherapy. Transl Lung Cancer Res. 2018; Ethics Approval 7(Suppl 2):149-152. The studies in which patient samples were collected were approved by an 4. Schadendorf D, Larkin J, Wolchok J, Hodi FS. Chiarion-Sileni V, Gonzalez independent ethics committee before being initiated at each site. R, Wagstaff J. Health-related quality of life results from the phase III Check Mate 067 study. Eur J Cancer. 2017; 82: 80-91. Ethics Approval P319 The study was approved by clinical trials ethics committee of the University Changes in fatigue severity, health related, and dermatology of Health Sciences Ankara Oncology Training and Research Hospital related quality of life in melanoma patients receiving nivolumab: (decision number: 2018–04/52) and performed in accordance with the Preliminary results of a prospective study from real-life experience Helsinki Declaration. 1 1 Canan Karadas , Nur Izgu, PhD, RN , Zehra Gok Metin, Assoc Prof, PhD, 1 2 2 RN , Canan Porucu, MSc, RN , Nuri Karadurmus, Prof Dr, MD , Sadettin 3 4 Kilickap, Prof Dr, MD , Umut Demirci, MD P320 1 2 Hacettepe University, Ankara, Turkey; Gulhane Training and Research Novel in vivo preclinical humanized models for the evaluation of Hospital, Ankara, Turkey; Hacettepe University Cancer Institute, Ankara, human specific immune checkpoint inhibitors 4 1 2 1 1 Turkey; Dr. A.Y. Ank Onco Tra and Res Hospital, Ankara, Turkey Anya Avrutskaya , Fabienne Sonego , Jacob Hauser , Emily O’Koren , 1 2 2 1 1 Correspondence: Canan Karadas (karadas.canan@gmail.com) Robin Ball , Gaëlle Martin , Julie Chaix , Thi Bui , Ian Belle , Elizabeth 1 1 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P319 Reap , Patrick Fadden, PhD , Chassidy Hall , Kader Thiam , Paula Miliani de Marval 1 2 Background Charles River Discovery, Wilmington, MA, United States; Genoway, lyon, Previous studies have reported that nivolumab, as an immune check- France point inhibitor, may enhance survival, reduce therapy related toxic- Correspondence: Paula Miliani de Marval ities and improve quality of life (QoL) among melanoma patients (paula.milianidemarval@crl.com) compared with traditional cytotoxic chemotherapy [1,2]. However, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P320 nivolumab is frequently associated with fatigue, increased risk of skin toxicity and autoimmune-related adverse events. Thus, patients may Background experience important changes in daily living activities, health related In the last few years, there has been an increasing demand for suitable pre- QoL, and skin integrity during nivolumab treatment. In view of this clinical mouse models for evaluating the efficacy of checkpoint inhibition- issue, studies are needed evaluating these important changes related based cancer immunotherapies. During tumor progression, immune cells to nivolumab, concurrently. Therefore, this study aimed to investigate can become unresponsive and evade immune surveillance upon chronic fatigue severity, health related QoL, and dermatology related QoL in activation and expression of the programmed cell death protein-1 (PD-1) melanoma patients receiving nivolumab. the ligand PD-L1 on tumor cells or expression of the T lymphocyte associ- Methods ated antigen 4 (CTLA4) in T-cells cells resulting in tumor immune-tolerance. A total of 20 patients, scheduled to receive first dose of nivolumab, We have previously demonstrated that murine anti-PD-1, anti-PD-L1 and in three leading hospitals located in Ankara, was included in this de- CTLA-4 blockade can effectively enhance immune normalization and re- scriptive, prospective and multicenter study. All the patients received activate the antitumor response against multiple syngeneic tumor models. at least four cycle of nivolumab infusion between October 2018 and While these models proved instrumental for evaluating murine immune- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 174 of 272 checkpoint inhibitors (ICI), there is a clear need for additional mouse models Conclusions to evaluate the efficacy of ICI specific for human targets. While heterogeneous patient characteristics may have influenced Methods the results of this study, the trends observed suggest favorable To address this need, we describe the development of humanized PD- outcomes in patients with aNSCLC treated with IO monotherapy 1 and CTLA-4 knock-in (KI) mouse models. The main advantage of in the 1L setting. Further research should explore whether this is these models is that human PD-1 or CTLA-4 proteins are expressed in related to a predominance of patients with high PD-L1 expression the context of a fully functional immune system. To validate these among those who received IO monotherapies. TTD for IO-based models we evaluated the response to pembrolizumab or ipilimumab in therapies in this real-world setting appears to be shorter than a colorectal carcinoma and a glioblastoma preclinical tumor models. PFS reported in previous trials, indicating an unmet need may re- Results main and needs to be explored. However, these results could be We observed significant tumor growth inhibition and growth delay in the influenced by effects of informative censoring or other underlying MC38 tumor model with either monotherapy, but not when treated with clinical factors. Additionally, future studies should investigate dif- the murine counterparts: anti-PD-1 (clone RPM1-14) or CTLA-4 (clone 9H10). ferences in the tolerability profiles of 1L regimens, as well as To extend our validation studies to other tumor models, we implanted how treatment sequences contribute to outcomes. GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembro- Acknowledgements lizumab compared to both the control group and the group treated with This study was funded by Merck KGaA, Darmstadt, Germany, as part of an alliance murine anti-PD-1 antibody. Furthermore, we found that pembrolizumab between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, NY, USA. and ipilimumab therapy results in enhanced effector functions of CD4+and CD8+ T cells associated with increased expression of Granzyme B. Reference Conclusions 1. Blumenthal GM, Gong Y, Kehl K, et al. Analysis of time-to-treatment dis- In summary, the results shown here underscore the value of resour- continuation of targeted therapy, immunotherapy, and chemotherapy in cing to humanized knock-in (KI) mouse models as tools to evaluate clinical trials of patients with non-small-cell lung cancer. Ann Oncol. human specific immune-checkpoint based therapeutics alone and in 2019;30(5):830-8. combination with other agents. Ethics Approval The study was reviewed and granted exception and waiver of consent by the US Oncology, Inc. Institutional Review Board. P321 Real-world clinical outcomes among patients with advanced non- small cell lung cancer who initiated first-line regimens 1 2 3 3 Eric Nadler, MD , Bhakti Arondekar, PhD , Kathleen Aguilar , Jie Zhou , Table 1 (abstract P321). See text for description 2 4 4 Jane Chang , Xinke Zhang , Vivek Pawar 1 3 Texas Oncology, Medical Oncology, Dallas, TX, United States; Pfizer Inc., New York, NY, United States; McKesson Life Sciences, The Woodlands, TX, United States; EMD Serono, Inc., Billerica, MA, United States Correspondence: Eric Nadler (eric.nadler@USONCOLOGY.COM) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P321 Background While clinical trials have demonstrated the clinical benefit of immuno-oncology (IO) regimens for the treatment of advanced non- small cell lung cancer (aNSCLC), either in combination or as mono- therapies, limited research has evaluated clinical outcomes with these therapies in a real-world setting. This retrospective observa- tional study evaluated time to treatment discontinuation (TTD) and overall survival (OS) in patients with aNSCLC receiving care in US community oncology clinics. Previous research suggests that TTD is a pragmatic real-world efficacy endpoint, as TTD and progression-free survival (PFS) are associated across different types of therapy in NSCLC clinical trials [1], therefore TTD was explored in this study. Methods Patients with aNSCLC who initiated first-line (1L) treatment with sys- temic chemotherapies, targeted therapies, or IO regimens in the US Oncology Network between 3/1/15 and 8/1/18 were included in the study population. Electronic health record data for these patients was captured through 2/1/19. Descriptive analyses were performed to assess baseline characteristics and treatment patterns, and the Kaplan-Meier method was used to evaluate TTD and OS from the start of 1L treatment. Results In total, 7,746 patients were included in this analysis (Table 1): 5,859 (75.6%) initiated 1L systemic chemotherapies, 656 (8.5%) targeted therapies, 907 (11.7%) IO monotherapies, and 324 (4.2%) IO combin- ation regimens (with chemotherapies or targeted therapies). Of these, 51.8%, 50.3%, 21.7%, and 17.6%, respectively, proceeded to a subsequent treatment following 1L discontinuation. Median TTD ranged from 2.0 months (95% CI 1.9-2.1) in patients who received systemic chemotherapies to 3.5 months (95%CI: 2.8-4.2) in patients who received IO monotherapies (Figure 1). Similarly, median OS was longest in patients who received IO monotherapies (19.9 months Fig. 1 (abstract P321). See text for description [95%CI: 16.6-24.1]; Figure 2). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 175 of 272 Conclusions We describe the development of a very high affinity antagonistic TIGIT antibody, COM902, that is currently in preclinical development. Co-expression of TIGIT with PVRIG in TILs and their non-redundant in- hibitory effects on T cell activation suggest a potential therapeutic advantage in clinical combinations targeting both pathways. Towards this end we are planning a trial that will eventually incorporate com- binations of COM902 with the anti-PVRIG antibody, COM701. P323 IPH5301, a CD73 blocking antibody targeting the adenosine immunosuppressive pathway for cancer immunotherapy Ivan Perrot, Caroline Denis, PhD, Marc Giraudon-Paoli, Severine Augier, Rachel Courtois, Diana Jecko, Violette Breso, Thomas Arnoux, Nicolas Gourdin, PhD, Romain Remark, PhD, Cecile Bonnafous, Ariane Morel, PhD, Eric Vivier, Yannis Morel, PhD, Pascale Andre, Carine Paturel, PhD Innate Pharma, Marseille, France Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P323 Fig. 2 (abstract P321). See text for description Background CD73 is an extracellular ectonucleotidase highly expressed by tumoral P322 or stromal cells in the tumor microenvironment. By inducing tumor cell COM902, a novel therapeutic antibody targeting TIGIT augments T death, conventional anti-cancer therapies induce extracellular release cell function and the activity of PVRIG pathway blockade in vitro of adenosine triphosphate (ATP), which is degraded by CD39 into ad- and in vivo enosine monophosphate (AMP) and then by CD73 into adenosine, an Maya Kotturi, BS, PhD, Eran Ophir, PhD, Sarah Whelan, PhD, Spencer inhibitor of immune response. Blockade of CD73-mediated degradation Liang, Kathryn Logronio, BS PhD, Kyle Hansen, BS, Zoya Alteber, PhD, of AMP may therefore stimulate anti-tumor immunity across a wide Mark White, BS, PhD range of tumors through preventing the production of adenosine. Compugen, South San Francisco, CA,United States IPH5301 is a humanized effector-silent IgG1 monoclonal antibody that Correspondence: Eran Ophir (erano@cgen.com) selectively binds to and inhibits the activity of both membrane-bound Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P322 and soluble human CD73. IPH5301 is designed to enhance anti-tumor immune responses by inhibiting the enzymatic activity of CD73 in the Background tumor microenvironment, thus releasing tumor-infiltrating lymphocytes TIGIT is a coinhibitory receptor that is highly expressed on tumor in- from adenosine-mediated suppression. Here, we described the expres- filtrating lymphocytes (TILs), including effector and regulatory (Treg) sion of CD73 in several human solid tumors, characterized IPH5301 CD4+ T cells, effector CD8+ T cells, and NK cells. Engagement of antibody properties and its efficacy in vitro. TIGIT with its cognate ligand PVR directly suppresses lymphocyte ac- Methods tivation. TIGIT and PVR are broadly expressed in different types of CD73 expression was assessed by immunochemistry on cohorts of solid tumors, suggesting that TIGIT-PVR signaling may be a dominant solid tumors i.e breast, ovarian, lung, melanoma, pancreatic and head immune escape mechanism for cancer. Utilizing COM902, a thera- and neck cancer. In vitro efficacy of IPH5301 was evaluated (1) in hu- peutic antibody targeting TIGIT, we demonstrate that co-blockade of man T cell proliferation assays; and (2) in enzymatic assays with lym- TIGIT and a new checkpoint inhibitor, PVRIG, augments T cell re- phocytes and serum from healthy donors and human CD73-knock-in sponses in vitro and in vivo. (huCD73KI) mice. To get more insight into the mechanism of action Methods of IPH5301, CD73-IPH5301 complex was analyzed using electron mi- Multi-color flow cytometry analysis of dissociated tumors was used croscopy and the crystal structure of IPH5301 Fab in complex with to quantify TIGIT and PVRIG expression on TILs. Membranous PVR CD73 ectodomain was determined. and PVRL2 expression was characterized by immunohistochemistry. Results The ability of COM902 to promote T cell responses in vitro, alone Whereas inter-patient variability was observed in all tested indications, and in combination with an anti-PVRIG antibody, COM701, was eval- CD73 expression was always detected mainly on tumor cells and did not uated in a primary TIL assay. To examine the in vivo effects of TIGIT correlate with the expression of CD39 or PD-L1. In vitro IPH5301 effi- blockade with COM902 a chimeric antibody with the constant region ciently restored T cell proliferation and blocked adenosine-mediated sup- of mouse IgG1 was generated. The anti-tumor activity of the chimeric pression of Tcellproliferationinamixedlymphocytereactioninadose- COM902 antibody in combination with an anti-mouse PVRIG anti- dependent manner. IPH5301 did not induce CD73 down-modulation and body was assessed in the mouse CT26 colon carcinoma model. did not directly activate B cells. Furthermore, IPH5301 efficiently blocked Results CD73 enzymatic activity in human serum and whole blood as well as in COM902 is a fully human antibody that binds TIGIT with high affinity serum and splenocytes from huCD73KI mice. Finally, we showed that and specificity and disrupts the binding of TIGIT to PVR. This anti- IPH5301 contrains CD73 in an intermediate inactive form. body binds to TIGIT on human CD8+ T cells with higher affinity than Conclusions tested benchmark antibodies. In dissociated tumor samples, TIGIT ex- These results indicate that IPH5301 blocks CD73 with a differentiated pression was highest on TILs in endometrial, head and neck, kidney mechanism of action compared to benchmarked anti-CD73 clinical and lung tumors, and directly correlated with PVRIG expression. Ex- candidates and support the clinical development of IPH5301 for can- cept for breast tumors, PVR was moderately to highly expressed in cer immunotherapy, potentially in combination with chemotherapy all tumor types examined, while PVRL2 expression was highest in or immune checkpoint inhibitors. prostate, ovarian, liver and endometrial tumors. Combination of COM902 and COM701 resulted in enhanced CD3+ TIL activity Acknowledgements in vitro. Furthermore, the combination of chimeric COM902 and anti- The research leading to CD73 results were obtained within the TumAdoR PVRIG resulted in significant CT26 tumor growth inhibition and en- collaborative consortium that received funding from the European hanced overall survival, which was comparable to the combination Community's Seventh Framework Program (FP7/2007-2013) under grant of chimeric COM902 and anti-PD-L1. agreement n°602200. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 176 of 272 Clinical Trial Completed outcomes in other cancer types and in randomized settings will pro- vide additional insight into their prognostic or predictive character. P324 Pan-tumor analysis of the association of cancer and immune Acknowledgements biology-related gene expression signatures with response to Joanne E Tomassini for writing support and Sheila Erespe for editorial pembrolizumab monotherapy support, both employees of Merck & Co., Inc. 1 1 Razvan Cristescu, PhD , Michael Nebozhyn, PhD , Chunsheng Zhang, Trial Registration 1 1 1 1 PhD , Andrew Albright, PhD , Julie Kobie, PhD , Lingkang Huang , Qing NCT01295827, NCT01866319, NCT02335411; NCT02335424; NCT01848834; 1 1 1 1 1 Zhao, MD PhD , Anran Wang , Hua Ma , Andrea Webber , Petar Jelinic , NCT02255097; NCT02447003; NCT02674061; NCT02853344 1 1 1 1 Mohini Rajasagi , Sandra Souza , Raluca Predoiu , Z. Alexander Cao , 1 1 1 1 Junshui Ma , Michael Morrissey , Clemens Krepler, MD , Stephen Keefe , References 1 1 1 Jonathan Cheng, MD , Vassiliki Karantza , Sukrut Shah , Rodolfo Perini, 1. Ayers M, Lunceford J, Nebozhyn M, et al. IFN-gamma-related mRNA pro- 1 2 3 4 MD , Antoni Ribas, MD, PhD , Petros Grivas, MD, PhD , David Cescon , file predicts clinical response to PD-1 blockade. J Clin Invest 1 1 1 Terrill McClanahan, PhD , Alexandra Snyder, MD , Mark Ayers, PhD , 2017;127:2930-2940. 1 1 Jared Lunceford, PhD , Andrey Loboda, PhD 2. Cristescu R, Mogg R, Ayers M, et al. Pan-tumor genomic biomarkers for 1 2 Merck Inc., Boston, MA, United States; University of California, Los PD-1 checkpoint blockade-based immunotherapy. Science Angeles, Los Angeles, CA, United States; University of Washington, 2018;362:eaar3593. Seattle, WA, United States; Princess Margaret Cancer Centre, Toronto, 3. Ayers M, Nebozhyn M, Cristescu R, et al. Molecular Profiling of Cohorts of Canada Tumor Samples to Guide Clinical Development of Pembrolizumab as Correspondence: Razvan Cristescu (razvan_cristescu@merck.com) Monotherapy. Clin Cancer Res 2019;25:1564-1573. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P324 Ethics Approval The clinical trials included in this analysis were approved by the appropriate Background ethics committees at each participating study center. RNA sequencing (RNASeq) data on baseline tumor biopsies from pa- tients in pembrolizumab monotherapy studies were used to explore Table 1 (abstract P324). See text for description potential relationships between key biological gene expression signa- tures and objective response rate (ORR) in the trials. Methods A canonical set of 10 consensus signatures representative of key tumor biology and tumor microenvironment (TME) elements beyond the 18- gene T-cell inflamed gene expression profile (GEP [1]) was defined using the independent Merck-Moffitt and TCGA databases [2,3], exter- nal to any pembrolizumab trial and prior to relating RNASeq data to clinical outcomes from studies evaluated. These signatures (Angiogen- esis, Hypoxia, Glycolysis, Proliferation, MYC, RAS, gMDSC, mMDSC, Stroma/EMT/TGFβ, WNT) were evaluated in the trial dataset blinded to clinical outcome, to test the association with ORR (RECIST 1.1; where re- sponse=PR or CR). Studies with available RNASeq data (N=1188) in- cluded: KN001/KN006-Melanoma (N=476; pembrolizumab-treated and ipilimumab-naïve), KN052-urothelial (N=186), KN012/KN055-HNSCC (N= 147; HPV-negative by whole exome sequencing), KN086-TNBC (N=132), KN059-Gastric (N=92), and KN427-RCC (N=78), KN100-Ovarian (N=77). Pan-cancer logistic regression analysis of ORR for consensus signatures included terms adjusting for cancer type, ECOG performance status, and the T-cell inflamed GEP, an approach equivalent to evaluating asso- ciation between ORR and the residuals of consensus signatures after detrending them for their relationship with the T-cell inflamed GEP and cancer type. Testing of the 10 pre-specified consensus signatures for P325 negative association (except Proliferation with a hypothesized positive- Tumor Infiltrating Lymphocytes (TILs) in triple-negative breast association) with ORR was adjusted for multiplicity. cancer: High Immunoscore is associated with pathological CR in Results patients receiving neoadjuvant chemotherapy. 1 2 3 4 Covariance patterns of the 11 signatures (including GEP) in Merck- Bernardo Rapoport, MD , Simon Nayler , Jerome Galon, Dr , T Mlecnik , 1 1 4 5 Moffitt and TCGA showed highly concordant co-expression patterns Teresa Smit , Jacqui Barnard-Tidy , Aurelie Fugon , Marine Martel , 6 2 in the RNASeq data from pembrolizumab trials. As anticipated, the T- Ronald Anderson, Professor , Carol Benn cell inflamed GEP demonstrated the strongest association with ORR The Medical Oncology Centre of Rosebank, Johannesburg, South Africa; 2 3 to pembrolizumab. Beyond the positive association seen with T-cell Prof., Johannesburg, South Africa; LABORATORY OF INTEGRATIVE inflamed GEP, three other RNA signatures, Angiogenesis, mMDSC CANCER IMMUNOL, France, France; Luminy Biotech enterprises 163 Ave and Stroma/EMT/TGFβ, exhibited negative associations at the 0.05 de Lu, Marseille, France; HalioDx, Marseille, France; level after adjusting for multiple testing (Table 1). Immunology,University of Pretoria, Pretoria, South Africa Conclusions Correspondence: Teresa Smit (pharmacist@rapoport.co.za) Pan-cancer testing of exploratory gene expression signatures using Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P325 the RNASeq platform in 1188 patients from single-arm pembrolizu- mab trials suggests that features beyond interferon gamma-related Background T-cell inflammation may be relevant to response to anti-PD1 mono- The presence of high levels of tumor infiltrating lymphocytes (TILs) has therapy and may define other axes of tumor biology as rational can- been associated with better prognosis in early triple-negative breast didates for pembrolizumab combinations. These features cancer (TNBC). Immunoscore is a prognostic tool, which categorizes the (Angiogenesis, mMDSC and Stroma/EMT/TGFβ) have been previously densities of spatially positioned CD3 and CD8 cells in both invasive hypothesized to represent immune-suppressive axes with potential margins (IM) and the center of the tumor (CT) yielding a five-tiered clas- negative impact on immunotherapy efficacy. Future evaluation of sification (0–4). High immunoscores have been reported to be associ- the association of these signatures with response and survival ated with improved outcomes in patients with colorectal cancer. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 177 of 272 Methods low initial immunity, characterized as 0.05. Furthermore, patients with We performed the Immunoscore in a cohort of 103 breast cancer low initial immunity who received the DNA-based vaccine showed (BC) patients previously receiving neo-adjuvant chemotherapy. There improved immunogenicity towards the unvaccinated extracellular were triple-negative (TNBC)=53, Luminal=32, Her2+=18 who received domain of HER2 at 1 month and 6 months, p treatment with anthracycline and/or taxane- and/or trastuzumab- Conclusions based neo-adjuvant chemotherapy. Pre-treatment tumor samples A comparison of DNA-based and peptide-based vaccines targeting were immune-stained for CD3 and CD8 T-cell markers. Quantitative HER2 intracellular domain epitopes revealed a more robust and long- analysis of the immune cells was carried out using a computer- lasting immunogenic response with the DNA-based vaccine while assisted image analysis in different tumor locations. maintaining an excellent safety profile. Additionally, immune re- Results sponses to extracellular domain regions of HER2 demonstrate the The pathological complete response (pCR) rate of the entire cohort intra-epitope spreading potential in the DNA-based vaccine. was 44%. On univariate analysis factors associated with higher pCR included primary tumor size (T1=43.48% vs. T2=52.31% vs. T3+T4 Acknowledgements 6.67%, Chi2=10.3201, p40=56.86% vs.15-39=40.54% vs. A special thanks to the Boyd Scholarship for financial assistance during my T-cell density subsets (CD3, CD8) and Immunoscore were significantly research. higher in TNBC compared to non-TNBC patients. Receiver-operating Trial Registration characteristic (ROC) curve analysis was used to determine the opti- Clinicaltrials.gov: NCT00436254 and NCT0034310 mal cut-off points for CD3 and CD8. A high density of CD3 (> than 800mm2) and CD8 (> than 400mm2) positive T-cells in the CT was References associated with higher pCR (CD3 CT:60% vs.25%, p=0.00035 and CD8 1. Datta J., et. al. Progressive loss of anti-HER2 CD4+ T-helper type 1 re- CT: 64% vs.27%, p=0.00016). Analysis of CD3 (> than 1400mm2) (CD3 sponse in breast tumorigenesis and the potential for immune restoration. IM:63% vs.19%, p=0.0001) and CD8 densities in the IM (> than Oncoimmunology. 2015;4(10):e1022301. 500mm2) was also significantly associated with pCR (CD8 IM:63% vs. 2. Mittendorf EA, et. al. Clinical trial results of the HER-2/neu (E75) vaccine 15%, p=0.00003). High immunoscore (24/38 pts (63%)) vs. intermedi- to prevent breast cancer recurrence in high-risk patients: from US Military ate (17/48 pts (35%)) vs. low (4/17 pts (24%)) was significantly associ- Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer. ated with pCR (p=0.00674). In a logistic regression model, Ki-67 (p 2012;118(10):2594-602. Conclusions 3. Disis, M.L., et al., Generation of T cell immunity of the HER-2/neu protein The results of this study show a significant prognostic and potentially after active immunization with HER-2/neu peptide based vaccine. J. Clin predictive role for the Immunoscore and Ki-67 in BC patients, particu- Onc, 2002;20(11): 26424-2632 larly in the TNBC subset. 4. Disis, M.L., et al., Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based Acknowledgements vaccine. Clin Cancer Res, 1999;5(6):1289-97. Dr Ronwyn van Eeden 5. Disis ML, et. al. A phase I trial of the safety and immunogenicity of a Ethics Approval DNA- based vaccine encoding the HER-2/neu (HER2) intracellular domain Ethics Approval was obtained from Pharmaethics SA and University of PTA, in subjects with HER2+ breast cancer. ASCOAnnual Meeting. 2014; approval no 517/2017 6. Disis ML, et. al. Phase II study of a HER-2/neu (HER2) intracellular domain Consent (ICD) vaccine given concurrently with trastuzumab in patients with newly Written informed consent was obtained from the patient for publication of diagnosed advanced stage breast cancer. SABCC. 2009; this abstract and any accompanying images. A copy of the written consent 7. Aurisicchio L., Ciliberto G. Genetic cancer vaccines: Current status and is available for review by the Editor of this journal. perspectives. Expert Opin. Biol. Ther. 2012;12:1043–1058. P326 P327 A retrospective analysis of DNA plasmid and peptide-based Evaluation of PD-L1 and cutoff selection to define a predictive vaccine therapy in treatment of HER-2/neu+ breast cancer biomarker for pembrolizumab monotherapy in esophageal cancer 1 1 1 Aaron Stewart, BS , William Gwin, MD , Mary Disis, MD, FACP , Jennifer using KEYNOTE-180 1 2 1 1 Childs , James Dai , Doreen Higgins, RN, BSN, OCN , Angela Kask Mary Savage, Serafino Pantano, Qi Liu, Jared Lunceford, PhD, Peter Kang, 1 2 University of Washington, Seattle, WA, United States; Fred Hutchinson Pooja Bhagia, MBBS, MD, Kenneth Emancipator, MD Cancer Research Center, Seattle, WA, United States Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United States Correspondence: William Gwin (wrgwin@medicine.uw.edu) Correspondence: Kenneth Emancipator Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P326 (kenneth.emancipator@merck.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P327 Background Patients with HER-2/neu+ overexpressing breast cancer often lose Background immunity toward the HER2 antigen [1]. Vaccines are capable of indu- Interim analysis of the KEYNOTE-180 study (NCT02559687) was used cing a cytotoxic T lymphocyte immune response toward overexpress- to establish a relationship between PD-L1 expression levels and ob- ing antigens, leading to targeted tumor destruction [2-4]. Clinical jective response rate (ORR) in patients with esophageal cancer whose trials have explored peptide-based and DNA-based vaccines as pos- disease progressed after ≥2 lines of therapy and to select a PD-L1 sible vehicles for vaccine delivery, though a direct comparison of cutoff for further validation in the randomized setting (ie, KEYNOTE- safety and immunogenicity has not been previously studied[5-6]. We 181). hypothesize that the DNA-based vaccine will produce superior im- Methods munogenicity due to stable plasmid persistence within the tissue KEYNOTE-180 was a single-arm, open-label, phase 2 study of pem- leading to prolonged HER2 immune response[7]. brolizumab in patients with previously treated, advanced esophageal Methods cancer. Pembrolizumab 200 mg was given intravenously every 3 We retrospectively analyzed adverse events and ELISpot data from weeks. The primary objective was ORR. Patients were required to 104 patients treated with vaccines targeting the intracellular domain provide a tumor sample for retrospective analysis of biomarkers, of HER2/neu using either DNA or peptide fragments. which may predict response to pembrolizumab. PD-L1 expression Results was measured using the PD-L1 IHC 22C3 PharmDx assay and evalu- Adverse event profiles of the 104 patients analyzed were similar with ated using a combined positive score (CPS). CPS is the ratio of PD- no reported grade 3, 4, or 5 events. There was no significant effect L1–expressing cells (tumor cells, lymphocytes, macrophages) to vi- on left ventricular ejection fraction (p=0.88 and p=0.59). Patients with able tumor cells. Testing for a relationship between CPS and ORR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 178 of 272 (per RECIST v1.1 by central review) was conducted using logistic re- Background gression. Cutoff selection proceeded by joint evaluation of the ORR BL-8040 (BL), a CXCR4 antagonist, increases T cell entry into the enrichment profile, the sensitivity and specificity profile (receiver op- bloodstream and thereafter into tumor in humans. We hypothesized erating characteristic analysis), the prevalence of patients with late- that BL in combination with pembrolizumab (P) may thereby pro- line esophageal cancer selected by the cutoff, and trends in overall mote efficacy in metastatic pancreatic cancer (mPC). survival (OS) by Kaplan-Meier (KM) curves. Methods Results Methods: This phase IIb open label study enrolled patients with There were 8 responders among 105 patients with available PD-L1 progression after at least one prior chemotherapy for mPC. Two results at the time of interim analysis (March 1, 2017). CPS was statis- weeks of single agent BL (1.25 mg/kg) was followed by 3-week tically significantly associated (P=0.03) with probability of response. A cycles of P (200 mg IV d1) plus BL (d1,4,8,11). Biopsies for tumor cutoff of CPS 1 did not show enrichment of ORR, whereas higher cut- biology were performed before treatment, after BL monotherapy offs did (Table). CPS 10 showed >3-fold enrichment in ORR above (optional), and after BL/P combination versus below the cutoff. Although higher cutoffs indicated further Results enrichment for ORR, attendant drops in sensitivity and prevalence As of July 2019, 20 pts enrolled; 15 were evaluable for the pri- occurred. Separation between KM OS curves for CPS ≥10 versus CPS mary endpoint of radiologic response. Baseline characteristics: Conclusions median age 66, 10M/10F, median 2 prior lines of therapy (range CPS was useful for identifying patients who responded to pembroli- 1-3). Best overall response includes 1 PR, 2 SD, 12 PD yielding zumab monotherapy. Through the evaluation of several clinical utility 21.4% disease control (1PR+2SD). Median TTP was 2 months over- dimensions, CPS 10 was chosen for further validation in the random- all and 7 months for the PR/SD pts. Median OS was 7 months ized setting based on its ability to enrich for ORR and simultaneously overall and 12 months in PR/SD pts. The combination was well to preserve sensitivity. The preservation of sensitivity, along with tolerated with most AEs being injection site discomfort. Five pa- prevalence, was considered particularly important because of the tients experienced grade 3/4 toxicities. Grade 3 toxicities included safety profile of pembrolizumab and the paucity of treatment op- HTN (n = 1), Alk Phos (n = 1), N/V (n = 2), ascites (n = 1), dys- tions in this patient population. pnea (n = 1), and abd pain (n = 2). One pt had grade 4 dyspnea. Trial Registration Paired biopsies have been analyzed for six patients (1 PR 2 SD 3 ClinicalTrials.gov, NCT02559687 PD). Patients with PR/SD had, at baseline, trends towards greater Ethics Approval T cell, especially cytotoxic CD8+ T cell counts within the tumor The study and the protocol were approved by the Institutional Re- niche than patients with PD (T cells: 188-627 cells/mm2 vs 7-41 view Board or ethics committee at each site. cells/mm2, Cytotoxic CD8+ T cells: 18-137 cells/mm2 vs 0-2 cells/ Consent mm2). The PR patient demonstrated an increase in cytotoxic All patients provided written informed consent to participate in the CD8+ T cell number in the tumor niche and reduction in the clinical trial. stroma following treatment. Additional molecular profiling data from multiplex IF will be available at the meeting. Conclusions Table 1 (abstract P327). See text for description This combination of immunotherapy with pembrolizumab plus BL-8040, without cytotoxic chemotherapy, shows clinical activity in patients with pancreatic cancer even in this heavily pretreated population. The combination was well tolerated. We noted a trend towards greater CD8+ T cell infiltrate at baseline within the tumor cell niche among patients who demonstrated clinical bene- fit. OS for all comers was longer than expected in this heavily pretreated patient group suggesting that P + BL might have a salutary effect on survival time if used earlier in the disease course. Trial Registration NCT02907099 Ethics Approval This study was approved by the M.D. Anderson Institutional Review Board, approval number 2016-0410. P329 PolyPEPI1018 off-the shelf vaccine as add-on to maintenance P328 therapy achieved durable treatment responses in patients with A phase IIB study of Pembrolizumab plus BL-8040 in metastatic microsatellite-stable metastatic colorectal cancer patients (MSS pancreatic cancer: Clinical outcomes and biological correlates 1 1 1 mCRC) David Fogelman, MD , Michael Overman, MD , Robert Wolff , Milind 1 1 1 2 1 2 1 Joleen Hubbard, MD , Chiara Cremolini, MD , Rondell Graham, MD , Javle, MD , Shubham Pant, MBBS , Gauri Varadhachary , Rachna Shroff , 1 1 2 1 1 Roberto Moretto, MD , Jessica Mitchell, CNP , Jaclynn Wessling , Eniko Ignacio Wistuba, MD , Carmelia Barreto, PhD , Renganayaki 1 3 3 3 3 3 3 Toke , Zsolt Csiszovszki, PhD , Orsolya Lőrincz , Levente Molnár , Eszter Pandurengan, MS , Sandesh Subramanya, PhD , Debora Ledesma, PhD , 4 4 4 3 3 3 3 3 Somogyi , Mónika Megyesi , Kata Pántya , József Tóth , Péter Páles , Abi Vainstein-Haras, MD , Ella Sorani, PhD , Tzipora Lustig , Osnat 4 4 5 6 3 2 1 István Miklós , Alfredo Falcone, MD , Joleen Hubbard, MD Kashtan , Yosi Gozlan , Steven Townson, PhD , Jeanne Fahey, PhD , 1 1 2 Mayo Clinic, Rochester, MN, United States; Azienda Ospedaliera James Yao, MD 1 2 3 Universitaria Pisana, Pisa, Italy; Treos Bio, Budapest, Hungary M.D. Anderson Cancer Center, Houston, TX, United States; University of Correspondence: Joleen Hubbard (joleenhubbard@gmail.com) Arizona, Tuscon, AZ, United States; MD Anderson Cancer Center, 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P329 Houston, TX, United States; Bioline Rx, Modi'in, Israel; Merck & Co., Inc., Shoreline, WA, United States; Merck & Co., Inc, Boston, MA, United Background States PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine against CRC, Correspondence: David Fogelman (dfogelman@mdanderson.org) containing 12 immunogenic epitopes derived from 7 conserved Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P328 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 179 of 272 cancer antigens frequently expressed in mCRC based on the analysis tologous cell products. Whilst feasible in such relatively small of 2,931 biopsies. Here we report the results of the phase I study of patient populations, delivering an autologous product to large PolyPEPI1018 vaccine as an add-on to maintenance therapy in MSS cohorts of patients is likely beyond the current logistical cap- mCRC patients. abilities. In the phase 1 alloSHRINK study, we tested the first-in- Methods class non-gene edited allogeneic CAR T-cell therapy, CYAD-101, 11 patients with MSS mCRC in the first-line setting were vaccinated administered concurrently with chemotherapy, for the treatment with PolyPEPI1018 just after the transition to maintenance therapy of metastatic colorectal cancer (mCRC). The NKG2D-based CAR with a fluoropyrimidine and a targeted agent (bevacizumab). (Part A: of CYAD-101 targets eight ligands present at high frequencies n= 5, single dose, 12 weeks follow-up; Part B: n= 6, 3 doses, Q12W). in mCRC, not only on tumor cells but also cells from the tumor Primary endpoints were safety and immunogenicity. Multiple analysis microenvironment, and co-express a T-cell receptor (TCR) inhi- of vaccine-induced immune responses in blood and tumor were per- biting molecule (TIM) that interferes with TCR signaling in an at- formed. Both immune response and clinical benefit were predicted tempt to avoid the main issue of allogeneic T-cell therapy, the using the autologous HLA-genotype determined from patient’s saliva graftversushostdisease (GvHD). sample. Methods Results The alloSHRINK study (NCT03692429) evaluates the safety and The vaccine was well tolerated; most common side effects were tran- clinical activity of multiple infusions of CYAD-101, administered sient skin reactions and flu-like syndrome. No vaccine-related SAE oc- concurrently with standard of care FOLFOX chemotherapy, in pa- curred. 90% of patients had vaccine-specific CD8+ T-cell responses of tients with non-resectable mCRC who received prior chemother- memory-effector type against at least 2 of the 7 vaccine antigens, 5 apy lines (i.e. rechallenge population). Three dose-levels (DL; on average. Vaccine specific CD4+ T-cell responses were detected in 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated all patients. Ex vivo CD8+ T cell responses of effector type were de- through a 3+3 design. tected in 71% of patients, as well as increased fractions of CRC- Results reactive, polyfunctional, circulating CD8+ and CD4+ T cells in pa- In total 12 patients have been enrolled in the dose escalation tient’s PBMC after vaccination. Among the 11 patients 3 patients had segment, now completed (3 at DL1, 3 at DL2 and 6 at DL3). At objective tumor response according to RECIST v1.1, one of them re- the time of submission, only data from the first two DLs were ceived a single dose and 2 of them received 3 doses. For the Part B available. At DL1 and DL2, there was no report of dose-limiting of the study, the Objective Response Rate (ORR) was 33% (2/6) and toxicity (DLT) and no patient experienced Grade ≥ 3 related ad- the Disease Control Rate (DCR) was 67% (4/6). Notably, one patient verse events (uncleaned database). No clinical evidence of GvHD experienced complete tumor shrinkage on 2 of 3 target lesions and has been recorded. Best overall response ≥ 3monthsinclude 1 partial response on 1 lesion after 25 weeks of treatment, qualifying partial response and 3 stable disease over the first 6 patients for curative surgery. Median duration of disease control was 9 (DL1 and 2). At DL1 and 2, preliminary data show a dose- months (95%CI 6.3-11.5) (mPFS not reached during the study). The dependent effect on the cell kinetics and control of the host- 10 month PFS was 50% (3/6). Predicted vaccine antigen-specific versus-graft response against CYAD-101 cells as evidenced by the CD8+ T cell responses were confirmed in vitro with a PPV of 79% (p= similar levels of CYAD-101 engraftment after 2nd and 3rd 0.01). Predicted multiantigenic immune responses tend to correlate infusions. with both PFS and tumor volume reduction. Conclusions Conclusions As of August 2019, no GvHD has been observed following infu- Treatment with PolyPEPI1018 vaccine and maintenance therapy was sions of non-gene edited allogeneic CAR T-cells to mCRC pa- safe, well-tolerated, and demonstrated evidence of immunological tients at the first two DLs, with preliminary signals of clinical and clinical activity in MSS mCRC tumors. In addition predicted multi- activity. The study will have reached protocol-specified end- antigenic immune responses indicated treatment benefit, which sup- points for analysis at the time of presentation and safety, clin- ports further development of a companion diagnostic together with ical and cell engraftment will be presented. The results from the vaccine. this study, in comparison with a study evaluating the autolo- Trial Registration gous analog of CYAD-101 in mCRC will provide critical informa- NCT03391232 tion to support the development of CAR-T therapy in solid Ethics Approval tumors. This study was approved by Mayo Clinic Institutional Review Board Trial Registration and by Central Ethics Committee, Italy (Protocol number: OBERTO- NCT03692429 101). Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics P330 Boards and authorities. Results from the completed dose-escalation of the alloSHRINK phase I study evaluating the allogeneic NKG2D-based CAR T- cell therapy CYAD-101 in metastatic colorectal cancer patients P331 1 1 2 Hans Prenen, MD , Marika Rasschaert, MD , Alain Hendlisz, MD , Leila Results from the completed dose-escalation phase I SHRINK study 2 3 3 Shaza, MD , Erik Alcantar-Orozco, MD, PhD , Emilie Cerf, PhD , Florence evaluating the autologous NKG2D-based CAR T-cell therapy CYAD- 3 3 3 4 Renard , Caroline Lonez, PhD , Anne Flament , Jeroen Dekervel, MD , 01 in metastatic colorectal cancer patients 4 1 1 1 Eric Van Cutsem, MD, PhD Leila Shaza, MD , Alain Hendlisz, MD , Ahmad Awada, MD, PhD , Jean- 1 2 2 3 University Hospital Antwerp (UZ Antwerp), Edegem, Belgium; Luc Canon, MD , Javier Carrasco , Eric Van Cutsem, MD, PhD , Jeroen 2 3 3 4 4 Institut Jules Bordet, BRUSSELS, Belgium; Celyad, Mont-Saint- Dekervel, MD , Erik Alcantar-Orozco, MD, PhD , Florence Renard , Emilie 4 4 4 4 Guibert, Belgium; University Hospital Leuven (UZ Leuven), Leuven, Cerf, PhD , Caroline Lonez, PhD , Anne Flament , Marc Van den Eynde, 5 5 Belgium MD , Jean-Pascal Machiels, MD, PhD 1 2 Correspondence: Caroline Lonez (clonez@celyad.com) Institut Jules Bordet, Brussels, Belgium; Grand Hôpital de Charleroi Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P330 (GHdC), Charleroi, Belgium; University Hospital Leuven (UZ Leuven), 4 5 Leuven, Belgium; Celyad, Mont-Saint-Guibert, Belgium; Cliniques Background Universitaires Saint Luc, Brussels, Brussels, Belgium Current success of chimeric antigen receptor T-cell (CAR-T) ther- Correspondence: Caroline Lonez (clonez@celyad.com) apy in hematological malignancies has been achieved using au- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P331 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 180 of 272 Background Methods Chimeric antigen receptor T-cell (CAR-Ts) therapies have yet to demon- The antigen-specific T cell response in the patients who received a strate positive results in the context of solid tumors largely due to the vaccine targeting the prostate cancer antigen, TARP (TCRγ alternate lack of suitable target antigens. NKG2D-based CARs target 8 stress ligands reading frame protein)[1] for biochemically recurrent (D0) prostate notably expressed to a very high frequency across the metastatic colorec- cancer on NCT00972309 was assessed. Patients with HLA-A0201 re- tal cancer (mCRC) patient population. The autologous NKG2D-based ceived vaccination at weeks 3, 6, 9, 12 and 15 following 1:1 CAR-T therapy CYAD-01 achieved stable disease in several patients with randomization between a vaccine consisting of TARP peptides, mon- mCRC when given as a monotherapy in a multiple injection setting with- tanide ISA 51 VG and GM-CSF versus autologous dendritic cell (DC) out any other supportive therapy (THINK study). In the SHRINK phase 1 pulsed with TARP peptides. Both vaccines used two types of pep- study, CYAD-01 was given concurrently with FOLFOX chemotherapy. tides; wild type (WT) TARP 27-35 (TARP2735) and epitope-enhanced Methods TARP 29-37 peptides (TARP2937-9V). The peripheral blood mono- The SHRINK phase 1 study (NCT03310008) evaluated the safety and clin- nuclear cells were collected at baseline and study weeks following ical activity of multiple infusions of CYAD-01, administered concurrently vaccination to be stored in liquid nitrogen until analysis. Cells were with FOLFOX chemotherapy in mCRC patients. Three dose-levels (DL; thawed and stimulated in vitro with TARP peptides with cytokine 1x10E8, 3x10E8 and 1x10E9 T-cells per infusion) were evaluated through support for multicolor flow cytometry. a 3+3 design in two different mCRC patient populations: (i) resectable Results liver dominant mCRC with FOLFOX chemotherapy as 1st line treatment CD8+ T cell subsets were analyzed for association with the dis- (i.e. neoadjuvant population), and (ii) non-resectable mCRC with prior ease response in 5 patients each who were responders and non- chemotherapy lines for mCRC including FOLFOX and/or FOLFIRI (i.e. re- responders whereas response was defined as slowing of PSA challenge population). slope log value as previously published by Wood et al [3]. The Results proportion of PD1-expressing CD8+ cells showed statistically im- The three DL have been completed with 9 patients in total (3 at each portant differences from baseline with an increase in responders DL), without any report of dose-limiting toxicity (DLT). Only 1 patient and a decrease in non-responders at week 12 after stimulation experienced Grade 3 related adverse event (AE) and no patient expe- with TARP 2735 (p= 0.016), TARP 2937 (p=0.032) and TARP 2937- rienced Grade 4 related AE (uncleaned database as of August 2019). 9V (p= 0.016) peptides. Other CD8+ subsets with granzyme A, Best overall response ≥ 3 months includes 1 partial response and 6 IFN-γ, IL-2, TNFα and perforin positive cells were investigated. stable disease out of 9 patients. Preliminary data show a dose- CD8+TNFα+ cells at week 12 (p=0.032) and CD8+perforin+ cells dependent effect on the cell kinetics. The study will have reached at week 18 (p=0.032) stimulated with TARP 2937 also showed protocol-specified endpoints for analysis at the time of presentation. statistically large differences with an increase in responders and a Conclusions decrease in non-responders. Early data show preliminary signs of clinical activity with the concurrent Conclusions administration of CYAD-01 and FOLFOX chemotherapy in the present Antigen-specific CD8+ T cells from responders as defined by reduced SHRINK study. Safety, clinical and translational research data (cell engraft- PSA slope log showed statistically greater activation as assessed by ment) will be presented. The results from this study, in comparison with expression of activation marker PD-1 than those from non- the results from a similar Phase I study evaluating the allogeneic analog responders after the vaccination in a first-in-human trial targeting of CYAD-01 in mCRC patients (i.e. CYAD-101), will provide critical informa- TARP. The antigen-specific T cell response to the vaccine peptides is tion to support the development of CAR T-cell therapy in solid tumors. an immune correlate of vaccine-induced protection and may be used Trial Registration to guide the ongoing study NCT02362451 that does not have HLA NCT03310008 restrictions. Ethics Approval The study was approved by all relevant Belgian Institution‘s Ethics Acknowledgements Boards and authorities. This work was supported by the Center for Cancer Research, National Cancer Institute, National Institue of Health. Trial Registration P332 NCT00972309 Cancer vaccine against prostate cancer antigen TARP induces antigen-specific CD8+ T cells with upregulation of activation marker References PD1 in patients with decreased PSA velocity in D0 prostate cancer 1 1 2 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein Hoyoung Maeng, MD , Brittni Moore , Lauren Wood, MD , Seth 1 1 1 expressed in prostate and breast cancer cells derived from an alternate Steinberg , Katherine McKinnon, MS , Masaki Terabe, PhD , Purevdorj 1 1 1 reading frame of the T cell receptor gamma chain locus. Proc Natl Acad Olkhanud , Ira Pastan, MD , Jay Berzofsky, MD, PhD 1 2 Sci U S A. 2000;97(17):9437–9442. National Cancer Institute, Bethesda, MD, United States; PDS 2. Oh S, Terabe M, Pendleton CD et al. Human CTLs to wild-type and en- Biotechnology, Berkeley Heights, NJ, United States hanced epitopes of a novel prostate and breast tumor-associated pro- Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) tein, TARP, lyse human breast cancer cells. Cancer Res 2004;64(7):2610- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P332 3. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated Background with slowing in PSA velocity and decreasing tumor growth rates in With the success of immune checkpoint inhibitors, anti-tumor im- patients with StageD0prostatecancer. Oncoimmunology. munity is at the focus of cancer therapy. The pursuit of the mechan- 2016;5(8):e1197459. ism to boost anti-tumor T-cell responses is critical to improve the Ethics Approval suboptimal response rate to checkpoint inhibitors. Cancer vaccines The study was approved by the National Cancer Institute Ethics Board can be used to induce such tumor-specific T-cell responses as one of assigned a local number 09C0139, approval number P08397.” the solutions to low responses to checkpoint inhibitors. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 181 of 272 P333 Ethics Approval Timed anti-tumor vaccination during chemotherapy induces This study was approved by the Central Committee of Human Investigations strong T-cell immunity and prolonged survival of late stage and by the ethical board of the Leiden University Medical Center (LUMC): cervical cancer patients EudraCT 2013-1804-12. 1 1 Marij Schoenmaekers-Welters, PhD , Marij Welters, PhD , Cornelis Melief, 2 3 1 MD, PhD , Ignace Vergrote , Judith Kroep, MD, PhD , Gemma Kenter, 4 5 6 7 P334 MD,PhD , Nelleke Ottevanger , Wiebren Tjalma , Hannelore Denys , 1 8 8 Concurrent cetuximab (CTX) and nivolumab (NIVO) in patients Mariette van Poelgeest, MD, PhD , Hans Nijman , Anna Reyners , Thierry 9 9 10 1 1 with recurrent and/or metastatic (R/M) head and neck Velu , Frederic Goffin , Roy Lalisang , Nikki Loof , Sanne Boekestijn , 2 2 2 squamous cell carcinoma (HNSCC): Safety results of a phase I/II Willem Jan Krebber , Leon Hooftman , Sonja Visscher , Brent 11 2 5 study Blumenstein, PhD , Richard Stead , Winald Gerritsen , Sjoerd van der 1 1 2 3 Christine Chung, MD , Marcelo Bonomi, MD , Conor Steuer, MD , Burg, PhD 1 2 1 1 1 1 Michael Schell , Jiannong Li , Matthew Johnson , Caitlin McMullen ,J. Leiden University Medical Center, Leiden, ZA, Netherlands; ISA 3 1 1 1 1 Trad Wadsworth , Krupal Patel , Julie Kish, MD , Jameel Muzaffar, MD , Pharmaceuticals, Leiden, Netherlands; University Hospital Leuven, 4 1 4 3 Kedar Kirtane , James Rocco , Nabil Saba, MD Leuven, Belgium; Center for Gynecological Oncology, Amsterdam, 5 1 2 Moffitt Cancer Center, Tampa, FL, United States; Ohio State University Netherlands; Nijmegen University Medical Center, Nijmegen, 6 7 3 Medical Center, Columbus, OH, United States; Emory University, Atlanta, Netherlands; University Hospital Antwerp, Antwerp, Belgium; University 8 4 GA, United States; Ohio State University, Columbus, OH, United States Hospital Gent, Gent, Belgium; University Medical Center Groningen, Correspondence: Christine Chung (christine.chung@moffitt.org) Groningen, Netherlands; Chirec Cancer Institute, Maastricht, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P334 Netherlands; University Medical Center Maastricht, Maastricht, Netherlands; Trial Architecture Consulting, Washington, DC, United Background States Use of anti-Programmed Death-1 (anti-PD-1) inhibitors is a Correspondence: Marij Schoenmaekers-Welters (M.J.P.Schoenmaekers- standard of care for patients (pts) with R/M HNSCC, but only Welters@lumc.nl) limited numbers of pts achieve long term clinical benefits. Im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P333 proving its efficacy and maintaining low toxicity profile are critical in combination strategies. We report the safety results Background of a phase I/II trial of CTX and NIVO in pts with R/M HNSCC. High-risk human papilloma virus type 16 (HPV16) is the major cause Methods of inducing cervical cancer. The oncoproteins E6 and E7 are respon- Pts were treated with CTX 500 mg/m2 IV on Day (D) -14 as a lead-in sible for the cancer development and therefore targeted by the followed by CTX 500 mg/m2 IV and NIVO 240 mg/m2 IV on D1 and therapeutic synthetic long peptide (SLP) vaccine ISA101. Monother- D15 every 28-D cycle (C). Pts with CTX infusion reaction or who did apy induced HPV16 E6/E7-specific T cells and was clinically effective not receive C1D1 for any reason were considered to be non- in half of the HPV16-SLP vaccinated patients with HPV16+ high- evaluable and were replaced. The toxicities with possible, probable, grade premalignant lesions of the vulva. However, in HPV16+ cervical and definite attribution were included in treatment-related adverse cancer patients additional measures need to be taken as the vaccine- events (TRAEs) and immune-related adverse events (IRAEs) analyses. induced T cells encounter an immunosuppressive milieu in the tumor NIVO dose reduction was not allowed. microenvironment. Therefore, in the current study the ISA101 vaccin- Results ation is combined with standard-of-care chemotherapy. For the phase I cohort, 3 pts were enrolled. No dose limiting toxic- Methods ities were observed during 4 weeks of observation period after C1D1, Late stage cervical cancer patients (n=77) were treated 3 times with and no dose reduction was required. An additional 44 pts were en- ISA101 with a 3-week interval in a single arm dose escalation study rolled, and 2 pts were non-evaluable. A total of 45 pts were analyzed. testing 4 different doses of ISA101 and with the addition or not of The median age was 64 (range 24-77), with 37 males and 8 females. pegylated IFN alpha (PegIntron). The start of ISA101 vaccination was The ECOG performance status at baseline was 0 (9 pts, 20%), 1 (33 at day 15 after the second cycle of standard-of-care chemotherapy, pts, 73.3%), and 2 (3 pts, 6.7%). The primary sites were oral cavity 10 which consisted of carboplatin (AUC6)/paclitaxel (175mg/m2). Blood (22%), oropharynx 24 (53%), hypopharynx 3 (7%), larynx 6 (13%), and samples taken during the study were subjected to a set of comple- unknown primary 2 (4%). The p16 status was positive 22 (49%), mentary immune assays to determine the vaccine-induced T-cell negative 11 (24%), and unknown 12 (27%). The p16 status of the responses. subsite, oropharynx, was positive 20 (83%) and negative 4 (17%). The Results smoking status was current 6 (13%); former 27 (60%) and never 12 In 43% of the 72 evaluated patients an objective clinical response (27%) with median pack years of 20 (range 0-185). Prior chemother- was observed. Carboplatin/paclitaxel depleted myeloid suppressive apy was given in 44 (98%). Prior radiotherapy was given in 36 (80%). cells (p The most common grade 3 TRAEs occurring >2% were fatigue 5 Conclusions (11%) and rash-acneiform 2 (4.4%). The only grade 4 TRAE was CTX Our study demonstrates that chemotherapy combined with immuno- infusion reaction in 1 (2.2%). The most common grade 3 and 4 IRAEs therapy, in this case HPV16-SLP vaccination, can be exploited to ef- occurring >2% were fatigue 2 (4.4%). No grade 5 TRAEs or IRAEs oc- fectively treat HPV16+ cervical cancer patients and warrants curred. TRAEs led to CTX dose reduction in 4 (9%) of pts: infusion re- confirmation in a randomized controlled trial. action, diarrhea, hypomagnesemia, fatigue (one each). Conclusions Acknowledgements The combination of CTX and NIVO is well tolerated and remains to This work was financially supported by the Dutch Cancer Society grant 2009-4400 be an option for future studies. (to C.J.M. Melief and S.H. van der Burg). ISA Pharmaceuticals sponsored the trial. Ethics Approval Trial Registration The study was approved by Advarra CIRBI, approval number 00000971 ClinicalTrials.gov NCT02128126. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 182 of 272 P335 Surprisingly, FOXP3+, and CD68+ staining cells were positively as- The tumor immune microenvironment and its association with pCR sociated with pCR. Additional analyses currently being conducted in NRG Oncology/NSABP B-52: Quantification of PD-1, PD-L1, CD8, to assess during treatment biopsies and the spatial relationships FOXP3, and CD68 by multiplex fluorescent-immunohistochemistry between different immune markers may provide further mechan- 1 1 1 1 1 Marion Joy, PhD , Ying Wang , Rim Kim , Nan Song , Ashok Srinivasan , istic insights. 1 1 2 1 Huichen Feng , Corey Lipchik , Reena Cecchini , Samuel Jacobs , Joseph 2 3 4 5 Costantino , Sandra Swain , Eleftherios Mamounas , Priya Rastogi , Acknowledgements 6 7 2 8 Soonmyung Paik , C. Kent Osborne , Norman Wolmark , Peter Lucas , U10CA180868; U24CA196067; UG1CA189867; Genentech, BCRF 9 1 Mothaffar Rimawi , Katherine Pogue-Geile Trial Registration 1 2 NRG Oncology/NSABP, Pittsburgh, PA, United States; NRG Oncology/ NCT02003209 NSABP and the University of Pittsburgh, Pittsburgh, PA, United States; Ethics Approval NRG Oncology/NSABP and Georgetown Lombardi Comprehensive Chesapeake IRB: Samples are exempt based on the Determination for NSABP Cancer Center, Georgetown University Medical Center, Washington, Foundation, Inc. Protocol TB-2 “NSABP TB-2: Comprehensive Survey of DC, United States; NRG Oncology/NSABP and Orlando Health, UF Prognostic and Predictive Markers for Breast and Colon Cancer” Health Cancer Center, Orlando, FL, United States; NRG Oncology/ (Pro00005069). All patients provided written informed consent to the NSABP NSABP and the University of Pittsburgh Cancer Institute, Pittsburgh, B-52 Clinical Study, which was reviewed and approved by the NCI CIRB. No PA, United States; NRG Oncology/NSABP and Yonsei University personal identifiable information is included. College of Medicine, Pittsburgh, PA, United States; NRG Oncology/ NSABP and The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States; NRG Table 1 (abstract P335). See text for description Oncology/NSABP and the University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; NRG Oncology/NSABP The Baylor College of Medicine/Dan L Duncan Comprehensive Cancer Center, Houston, TX, United States Correspondence: Katherine Pogue-Geile (katherine.pogue- geile@nsabp.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P335 Background The NRG Oncology/NSABP B-52 neoadjuvant clinical trial was conducted to test whether addition of estrogen deprivation (ED) would improve pCR rate in HER2+/ER+ breast cancer patients treated with docetaxel, carboplatin, trastuzumab, and pertuzu- mab (TCHP). A numerical increase in pCR rate was observed with ED (46.1% v 40.9%), but the difference was not statistically significant. B-52 provided the opportunity to explore potential predictive markers of pCR to possibly guide new treatment strategies. We examined the tumor immune microenvironment (TME) of B-52 baseline biopsy tumors to determine its associ- ation with pCR. Methods Pretreatment (N=238) biopsies were assessed for CD8, FOXP3, P336 CD68, PD-L1, and PD-1, with multiplex fluorescent immunohisto- Impact of cytokine release syndrome on cardiac function following chemistry (mf-IHC) utilizing the Vectra® Quantitative Pathology CD19 CAR-T cell therapy in children and young adults with acute Imaging System and inForm® Advanced Image Analysis software. lymphoblastic leukemia 1 1 2 Tumor and stromal regions were defined with a panCK antibody Amita Kulshrestha , Haneen Shalabi, Do , Vandana Sachdev , Douglas 2 2 3 4 (included in the same multiplex) and annotated by a patholo- Rosing , Stanislav Sidenko , Crystal Mackall, MD , Brandon Wiley , Daniel 5 6 gist. Digital quantitation of all markers was assessed in the Lee , Nirali Shah 1 2 tumor and stromal regions (defined by panCK). In our pre-specified, National Institutes of Health, Bethesda, MD, United States; National CTEP-approved analysis, we tested the association of PD-L1 in tumor+- Heart, Lung and Blood Institute, Bethesda, MD, United States; Stanford, stroma with a cut-point optimized by ROC curves (Table1). In explora- Stanford, CA, United States; Mayo Clinic, Rochester, MN, United States; 5 6 tory analyses, we also tested a clinically meaningful PD-L1 cut-point of University of Virginia, Charlottesville, VA, United States; National Cancer >1%; other markers were tested for associations with pCR using chi- Institute, Kensington, MD, United States square tests and a median cut-off (Table 1). Correspondence: Nirali Shah (nirali.shah@nih.gov) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P336 Based on our pre-specified analysis, total PD-L1 (assessed in both stroma+tumor) was positively associated with pCR across Background trial arms (45% v 30%, p=0.038). PD-L1 was not significantly as- Cytokine release syndrome (CRS) is the main toxicity of CAR-T cell sociated with pCR with a clinically utilized cut-off of >1% within therapy, which may require hemodynamic support. The impact of the stromal-immune cell compartment (CD8+FOXP3+CD68). Sur- CRS on cardiac function has not been well described. prisingly, in both stromal and tumor cell compartments, CD68 Methods was positively associated with pCR when the two arms are eval- We report on cardiac toxicity seen in children and young adults with uated together. When the treatment arms are examined separ- ALL treated on our phase I trial of CD19 CAR-T cell therapy (clinical- ately, CD68 in the stromal compartment correlates positively trials.gov NCT01593696). All patients had a baseline echocardiogram. with pCR. FOXP3 was also positively correlated with pCR in the Cumulative anthracycline exposure was calculated from prior expos- stromal cell compartment across arms and in the TCHP+ED arm. ure. Additional studies included increased frequency of echocardio- PD-1 and CD8 were not significantly associated with pCR. grams upon ICU transfer, and serial troponin and proBNP. Conclusions Results B-52 showed a positive association of PD-L1 expression with pCR From July 2012 to March 2016, 52 patients, with a median age of 13.4 based on our pre-specified analysis but the extremely low cut-off years (range, 4.2-30.3) were treated on-study; 23 underwent at least (0.05%) makes the clinical utility of this observation doubtful. one prior allogeneic stem cell transplantation. CRS was seen in 37/52 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 183 of 272 (71%), which was grade 3-4 CRS in 8 subjects (21.6%). The median prior anthracycline exposure was 205 mg/m2 (range, 70-620 mg/m2) in doxorubicin equivalents. The median baseline LV ejection fraction (LVEF) was 62% (range 52%-71%). The median LV global longitudinal strain (GLS), at baseline was abnormal: -17 (range, -14 to -24, n=35). ICU transfers occurred in 20 patients, 11 of whom required vasoactive hemodynamic support, with 5 necessitating more than 1 pressor. Seven patients received tocilizumab and 4 patients received steroids. Six (16%) patients developed cardiac dysfunction, amongst whom 4 had grades 3-4 CRS. (Figure 1) Severe cardiac dysfunction, (LVEF < 30%) was seen in 3, with one patient developing cardiac arrest with subse- quent full recovery following placement of an intra-aortic balloon pump, steroids, and tocilizumab. In 2 of these patients, anthracycline exposures exceeded > 360 mg/m2. All but 2 patients had full resolution of cardiac dysfunction by day 28 post CAR. Troponin elevations were seen in 4 of 6 patients with low LVEF. In a limited cohort of patients with pre/post pro-BNP, pro-BNP was higher during CRS, with the high- est levels correlating with more severe cardiac dysfunction. (Figure 2) Conclusions Patients with higher-grade CRS are more likely to experience significant cardiac side effects from CAR T-cell therapy. In most cases, resolution to near baseline was seen coinciding with resolution of CRS, with most having near complete resolution by day 28 post infusion. Implementation of more frequent echocardiogram monitoring and incorporation of BNP and troponin into daily laboratory panel may help to identify those at highest risk of severe cardiac Fig. 2 (abstract P336). Changes in BNP pre and post-CAR infusion dysfunction at an earlier time point, allowing for earlier interven- tion in CRS to potentially limit acute cardiac toxicity. P337 Acknowledgements Survival prolongation by dendritic cell vaccination in combination This research was supported by the Intramural Research Programs of the Center with OK-432, gemcitabine and/or S-1 in patients with advanced of Cancer Research, National Cancer Institute, NIH and the Clinical Center. pancreatic cancer Trial Registration Masahiro Ogasawara, MD, PhD, Shuichi Ota, MD PhD The clinical trial is registered at clinicaltrials.gov NCT01593696 Sapporo Hokuyu Hospital, Sapporo, Japan Ethics Approval Correspondence: Masahiro Ogasawara (ogasawara@hokuyu-aoth.org) This study was approved by the National Cancer Institute Institutional Review Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P337 Board. Background Pancreatic cancer is the most fatal human cancer, with a 5-year overall survival rate of less than 5%. In the current study, we have evaluated the clinical and the immunological responses in patients with advanced pancreatic cancer who received dendritic cell (DC) vaccination in combination with a toll like receptor (TLR) 4 agonist, OK-432 and chemotherapeutic agents, gemcitabine (GEM) and/or S-1. Methods Twenty three patients (13 males, 10 females; aged 37-83 years, me- dian 64 year old) with advanced pancreatic cancer refractory to standard treatment were treated with DC vaccination in combination with OK-432, GEM and/or S-1 from 2012 to 2013 at Sapporo Hokuyu Hospital. Autologous DCs were generated by culturing adherent mononuclear cells with interleukin-4 and granulocyte-macrophage colony stimulating factor. DCs were then loaded with synthetic pep- tides derived from cancer antigens such as Wilms’ tumor 1 (WT1) and MUC1 following maturation by prostaglandin E2 and OK-432. Peptide-loaded mature DCs and OK-432 were administered intrader- mally every 2 weeks, 7 times. The induction of vaccine-induced T cell responses was monitored by using HLA-tetramer and ELISPOT assays. Results The treatment was well tolerated and none of the patients experi- enced more than grade 3 adverse events during the treatment period. Of 23 patients, 1 had partial response (PR), 8 had stable dis- ease (SD) and 14 had progressive disease after one course of vaccin- ation. The median overall survival (OS) was 9.6 months. Survival of patients achieving PR or SD (responders) was longer than those who did not respond to the treatment (non-responders) (median OS; 18.0 Fig. 1 (abstract P336). Change in Ejection Fraction Post CRS Onset vs 5.8 months). An HLA-tetramer assay showed an increase in the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 184 of 272 positivity of WT1-specific CD8+ T cells in both responders and non- Results responders after vaccination. However, the increment in the positivity 792 patients were enrolled (intention-to-treat [ITT] population), includ- was remarkable in responders in comparison with non-responders; ing 529 with PD-L1+ tumors (primary analysis population) and 263 with 46.3 and 10.7 fold in responders and non-responders, respectively. PD-L1− tumors. (n=263). At data cut-off (March 4, 2019) in the PD-L1+ Similarly, an ELISPOT assay showed marked increase in spot-positive population, median duration of follow-up for OS was 35.4 months in cells in responders. The median OS in patients showing the positivity the avelumab arm (n=264) and 34.7 months in the docetaxel arm (n= in both assays was longer than those who were positive in either 265); study treatment was ongoing in 25 (9.5%) vs 0 patients, and 17 assay or who were negative in both assays; a median OS was 18.4 (6.4%) vs 74 (27.9%) had received a posttreatment checkpoint inhibitor months, 9.7 months and 4.7 months, respectively, suggesting a cor- (CPI), respectively. 2-year OS rates with avelumab vs docetaxel in differ- relation between an immune response and a clinical outcome. ent PD-L1+ subgroups are shown (Table 1). Of patients with PD-L1+ tu- Conclusions mors alive at 2 years, 67% had received a posttreatment CPI in the DC vaccination combined with a conventional chemotherapy in pa- docetaxel arm compared with 13% in the avelumab arm. In patients tients with advanced pancreatic cancer was demonstrated to be safe with PD-L1+ tumors who had an objective response with avelumab (50 and can elicit immune responses against tumor antigens, which was [18.9%]) or docetaxel (28 [10.6%]), median duration of response (DOR; correlated with clinical effects. investigator assessed) was 19.1 months (95% CI: 10.8-34.8) vs 5.7 Ethics Approval months (95% CI: 4.1-8.3), and proportions with a response lasting ≥6 This study was approved by the Ethics and Internal Review Board at months were 86.0% (95% CI: 72.9%-93.1%) vs 48.1% (95% CI: 28.7%- Sapporo Hokuyu Hospital, approval number 131111.08 65.2%), respectively. Safety profiles of avelumab and docetaxel were similar to those in previous analyses. Conclusions P338 Updated data from JAVELIN Lung 200 showed that although avelu- 2-year follow-up from JAVELIN Lung 200, an open-label, mab did not significantly prolong OS vs docetaxel in the primary randomized, phase 3 study of avelumab vs docetaxel in patients confirmatory analysis, 2-year OS rates were doubled with avelumab with platinum-treated advanced non-small cell lung cancer vs docetaxel in higher PD-L1+ subgroups, and median DOR was >12 (NSCLC) months longer with avelumab vs docetaxel. 1 2 3 Fabrice Barlesi, MD, PhD , Mustafa Özgüroğlu , Johan Vansteenkiste , 4 5 6 7 David Spigel , James Yang , Hidenobu Ishii , Marina Garassino , Filippo Acknowledgements 8 9 10 11 de Marinis , Aleksandra Szczesna , Andreas Polychronis , Ruchan Uslu , This study was funded by Merck KGaA, Darmstadt, Germany, as part of an 12 13 14 Maciej Krzakowski , Jong-Seok Lee , Luana Calabro, MD , Osvaldo alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, 15 16 17 17 Aren Frontera , Barbara Ellers-Lenz , Marcis Bajars , Mary Ruisi , NY, USA. Keunchil Park Trial Registration Aix-Marseille University, Assistance Publique - Hôpitaux de Marseille, NCT02395172 Livon, France; Cerrahpaşa Medical Faculty, Istanbul University, Leuven, Ethics Approval 3 4 Belgium; University Hospital KU Leuven, Leuven, Belgium; Sarah The study protocol was approved by institutional review boards and ethics Cannon Research Institute, Nashville, TN, United States; National Taiwan committees at each institution. The study was done in accordance with the University Hospital, Taipei, Taiwan, Province of China; Kurume University trial protocol, Good Clinical Practice guidelines, and the Declaration of School of Medicine, Kurume, Japan; Fondazione IRCCS Istituto Helsinki. All patients provided written informed consent. Nazionale dei Tumori, Milan, Italy; Istituto Europeo di Oncologia, Milan, 9 10 Italy; Regional Lung Disease Hospital, Otwock, Poland; Mount Vernon Cancer Centre, Northwood, Middlesex, United Kingdom; Ege University 12 Table 1 (abstract P338). See text for description Hospital, Izmir, Turkey; Centrum Onkologii-Instytut Im. M. Skłodowskiej- Curie w Warszawie, Warszawa, Poland; Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea, Republic of; University Hospital of Siena, Siena, Italy; 15 16 Instituto Nacional del Cáncer, Santiago, Chile; Merck KGaA, Darmstadt, Germany; EMD Serono Inc, Billerica, MA, United States; Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence: Fabrice Barlesi (Fabrice.BARLESI@ap-hm.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P338 Background P339 Avelumab, a human IgG1 anti–PD-L1 monoclonal antibody, is ap- Survival is improved by antigen-specific cytotoxic T lymphocytes proved as monotherapy for metastatic Merkel cell carcinoma and (CTL) responses after treatment with the vaccine Tedopi in HLA-A2 platinum-treated urothelial carcinoma in various countries, and in positive advanced non-small cell lung cancer (NSCLC) patients 1 2 combination with axitinib to treat advanced renal cell carcinoma in Benjamin Besse, MD PhD , Enriqueta Felip, MD PhD , Giuseppe 3 4 5 6 the United States. In the JAVELIN Lung 200 study, avelumab did not Giaccone , Rafal Dziadziuszko , Elisabeth Quoix, MD , Werner Hilgers , 7 8 9 10 significantly prolong overall survival (OS) vs docetaxel in patients Federico Cappuzzo , Christophe Borg , Jordi Remon , Nicolas Poirier , 10 10 11 with platinum-treated PD-L1+ NSCLC (primary objective); however, Dominique Costantini , Bérangère Vasseur , Santiago Viteri 1 2 prespecified exploratory analyses showed longer OS with avelumab Gustave Roussy, Villejuif, France; Vall d'Hebron University Hospital, vs docetaxel in patients with higher PD-L1+ tumors. We report up- Barcelona, Spain; Georgetown University, Washington, DC, WA, United 4 5 dated data from JAVELIN Lung 200. States; Medical University of Gdańsk, Gdańsk, Poland; Nouvel Hôpital Methods Civil, Strasbourg, France; Institut Sainte Catherine, Avignon, France; 7 8 Patients with stage IIIB/IV or recurrent NSCLC and disease progres- AUSL Romagna, Ravenna, Italy; Centre Hospitalier Universitaire, 9 10 sion following platinum-doublet chemotherapy were randomized 1:1 Besançon, France; CIOCC- Barcelona, Barcelona, Spain; OSE to avelumab 10 mg/kg Q2W or docetaxel 75 mg/m2 Q3W. The pri- Immunotherapeutics, Nantes, France; University Hospital Dexeus, mary endpoint was OS; the primary analysis population was patients Barcelona, Spain with PD-L1+ tumors (≥1% tumor cell expression; PD-L1 IHC 73-10 Correspondence: Benjamin Besse (benjamin.besse@gustaveroussy.fr) pharmDx assay). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P339 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 185 of 272 Background Tedopi (OSE2101) is a multiple epitope vaccine restricted to HLA-A2 positive patients (45%), targeting five tumor-associated antigens (TAA) frequently expressed in solid tumors: carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER-2/neu), melanoma-associated antigen type 2 and 3 (MAGE2 and MAGE3), and p53. Tedopi is composed by 2 wild type and 7 chemically modi- fied peptides to increase HLA-A2 or T cell receptor (TCR) affinity. A pan-DR epitope (PADRE) of helper T-lymphocyte (HTL) has been added to increase the Cytotoxic T Lymphocyte (CTL) responses. In previously treated advanced NSCLC patients, Tedopi showed a strong CTL immune response, which correlated with overall survival (OS) [1]. The aim of the current translational study was to explore the predict- ive effect of the epitope type and number of epitopes on OS. Methods Fig. 1 (abstract P339). Overall survival in patients with 3-6 vs 0-2 Out of 64 previously treated HLA2+ advanced NSCLC patients en- CTL responses rolled in a phase II trial testing the efficacy of Tedopi (1mL subcuta- neously Q3W for 6 cycles, then Q8W for the reminder year 1 and Q12W up to year 2), 33 patients were assessed for epitope-specific cytotoxic response and HTL responses using an interferon gamma P340 enzyme-linked immunosorbent assay. Leukapheresis was performed Region-focused deep survival learning on PD-L1 stained tissue at baseline, at week 9, 18 and 30 for immunogenicity assays. Predict- samples for data-driven stratification of durvalumab-treated ive analyses of OS were performed using Cox regression. NSCLC patients Results 1 1 1 Nicolas Brieu, PhD , Ansh Kapil , Armin Meier, PhD , Keith Steele, DVM, Patients were stage IV (64%), or locally advanced stage IIIb (36%). 2 2 1 PhD , Marlon Rebelatto, DVM, PhD, DACVP , Guenter Schmidt, PhD Eleven patients were assessed for all 10 epitopes, and 33 for 6 se- 1 2 Definiens AG, Munich, Germany; AstraZeneca, Gaithersburg, MD, lected epitopes (2 CEA, 1 HER-2, MAGE2, MAGE3, PADRE). Median United States survival was 30 months. Correspondence: Nicolas Brieu (nbrieu@definiens.com) There was at least one CTL response to one vaccine epitope in >90% Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P340 of patients. Eight epitopes were highly immunogenic (from 55% to 91%), while HER-2 wild type and one p53 analogue shown a lower Background response (respectively 36% and 9%). The selection of metastatic non-small cell lung cancer (NSCLC) pa- In patients evaluated for 6 selected epitopes, the best cut-off of num- tients that are likely to respond to an anti-PD-L1 checkpoint mono- ber of CTL responses to discriminate OS were 1-6 versus 0, 2-6 vs 0-1 therapy can be guided by the visual assessment by pathologists of or 3-6 vs 0-2. All of three were statistically significant. As an example, the Tumor Cell (TC) score on PD-L1 stained tissue samples [1]. Deep patients with CTL responses to 3-6 epitopes (n=23) had a median OS learning approaches have recently enabled the computer-based rep- of 38 months compared to 15 months in patients (n=10) with CTL re- lication of this visual TC score [2,3] and of its ability to predict overall sponses to 0-2 epitopes (HR=0.39; p=0.04) (Figure 1). CTL response survival (OS) [4]. Because these methods try to reproduce as close as to HER-2 analogue, MAGE3, PADRE and one p53 analogue were pre- possible the visual scoring methodology, they are built on extensive dictive of better OS. prior hypotheses (e.g. definition of cell positivity, score and cut-off) Conclusions and do not enable the data-driven discovery of novel stratification In NSCLC patients, survival was significantly prolonged in patients immu- rules. We present here a novel region-focused end-to-end deep- nized to epitope specific Tedopi vaccine. HER-2, MAGE3, PADRE and p53 learning approach that enables the data-driven generation of survival were identified as vaccine predictive epitopes for prolonged survival. risk heatmaps and the stratification of patients into two risk groups. Methods Acknowledgements On a subset (N=151) of core needle biopsies and tissue resections from We thank François Montestruc and Constant Josse (eXYSTAT, Malakoff, the NCT01693562 clinical trial (NSCLC), epithelium regions are automat- France) for the statistical analysis ically segmented within the manually delineated tumor area [3]. A patch-based convolutional neural network (CNN) is trained on selected Reference patches in a two-fold pre-validation procedure to maximize a log partial 1. Barve M, Bender J, Senzer N, Cunningham C, Greco FA, McCune D, Steis likelihood derived from the Cox proportional hazards model [5,6]. To R, Khong H, Richards D, Stephenson J, Ganesa P, Nemunaitis J, Ishioka G, avoid a disproportionately large number of patches from tissue resec- Pappen B, Nemunaitis M, Morse M, Mills B, Maples PB, Sherman J and tions, a random subset of up to 10K patches is selected for each patient Nemunaitis JJ. Induction of Immune Responses and Clinical Efficacy in a within the segmented regions. The overall survival risk is predicted and Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, aggregated by mean on the detected epithelium regions only. Patients in Metastatic Non-Small-Cell Lung Cancer. J Clin Oncol. 2008;26(27):4418– are finally stratified based on the cohort median of the resulting aggre- gated risk scores. For baseline comparison, the same steps are repeated Ethics Approval considering the complete delineated tumor area instead of the sole The study protocol and its related documents (including the patient segmented epithelium regions. information and informed consent form) received approval from the Institutional Review Board (IRB), and the Competent Authority prior to Results study initiation. The proposed epithelium-focused and data-driven survival CNN Consent yields similar patient stratification (HR=0.525, p=0.003) as obtained Each patient gave his/her written informed consent prior to study with 25% cut-off on visual (HR=0.574, p=0.01) or automated (HR = enrolment. 0.539, p=0.004) TC score (Figure 1), while releasing prior hypotheses Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 186 of 272 on PD-L1 region positivity, score methodology, and cut-off value. As P341 expected on durvalumab-treated patients, high and low risks are as- Activation of toll-like receptors via PS-targeting monoclonal sociated with low and high PD-L1 staining respectively. No relevant antibodies risk groups are identified if the analysis is performed on the full Rolf Brekken, PhD (rolf.brekken@utsouthwestern.edu) tumor area instead. UT Southwestern Medical Center, Dallas, TX, United States Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P341 Our results suggest, for the first time on core needle biopsies and tis- sue resections, (i) the ability of end-to-end deep survival learning to Background directly learn relevant patient stratification as well as (ii) the neces- Multifocal immune suppression in the tumor microenvironment is a sity, in case of small patient cohorts, to restrict the analysis to auto- major underlying cause for the limited efficacy of immune check- matically detected meaningful regions. point blockade. Persistent immune suppression prevents the devel- opment of a robust T cell response to tumor specific antigens that is References required for effective downstream immune checkpoint blockade. An 1. Rebelatto et al., Development of a programmed cell death ligand-1 im- underappreciated but significant contributor to immune suppression munohistochemical assay validated for analysis of non-small cell lung in tumors is the exposure of the membrane phospholipid phosphati- cancer and head and neck squamous cell carcinoma, Diagnostic Path- dylserine (PS) on the surface of tumor cells and tumor-derived micro- ology 2016 vesicles. PS is recognized by receptors on immune cells where it 2. A. Kapil et al., Deep Semi Supervised Generative Learning for Automated triggers the secretion of immune suppressive cytokines, prevents the Tumor Proportion Scoring on NSCLC Tissue Needle Biopsies, Scientific differentiation of myeloid-derived suppressor cells (MDSCs) and in- reports, 2018 hibits dendritic cell (DC) maturation; events that prevent a productive 3. A. Kapil et al., DASGAN - Joint Domain Adaptation and Segmentation for anti-tumor T cell response. Bavituximab, a chimeric monoclonal the Analysis of Epithelial Regions in Histopathology PD-L1 Images, arXiv antibody (mAb) that targets PS and inhibits PS-mediated im- preprint arXiv:1906.11118, 2019 munosuppressive signaling, drives immune activation by reducing 4. N. Brieu et al., Deep learning-based PD-L1 tumor cell (TC) scoring im- the levels of MDSCs, by polarizing tumor-associated macrophages proves survival prediction compared to pathologists on durvalumab- towards an immune stimulatory phenotype and by promoting treated NSCLC patients, SITC 2018. the maturation of dendritic cells (DCs). Bavituximab and other PS- 5. P. Mobadersany et al., Predicting cancer outcomes from histology and targeting mAbs (2aG4 and 1N11) bind to PS via beta-2 glycopro- genomics using convolutional networks, PNAS 2018. tein 1 (β2GP1). β2GP1, an abundant serum glycoprotein, was re- 6. A. Meier et al., End-to-end learning to predict survival in patients with cently identified as a novel component of innate immunity via gastric cancer using convolutional neural networks, Annals of Oncology activation of Toll-like receptors (TLRs). (ESMO), 2018. Methods Ethics Approval Human β2GP1. Monoclonal-antibodies: Bavituximab and 1N11. qPCR, For the Phase 1/2 durvalumab trial (NCT01693562), the study protocol was WB, IP, ICC/IHC, CD, TEM, MST and ELISA reviewed and approved by the Institutional Review Board of the Results participating centers and informed consent was obtained from all We investigated whether the innate immune changes induced by patients. PS-targeting mAbs is mediated in part by β2GP1-induced activation of TLRs in myeloid cells. β2GP1 has a closed conformation that pre- vents its interaction with PS and cell surface receptors. However, interaction of β2GP1 with LPS can induce an open conformation of the protein that allows interaction of β2GP1 with PS and cell surface receptors, including the TLRs. We found through circular dichroism and transmission electron microscopy that the PS-targeting anti- bodies also induce conformational changes in β2GP1, shifting it to an open conformation. In addition, we demonstrate that PS-targeting mAb-mediated dimerization of β2GP1 stimulated pro-inflammatory polarization of bone marrow-derived macrophages (BMDMs) and in- duced TLR2 signaling. Finally, we investigated the expression of a newly characterized TLR-induced transcription factor, Spi-C in bone marrow progenitor cells after stimulation with TLR specific agonists or PS-targeting mAbs +/- β2GP1. Conclusions These studies demonstrate that the PS-targeting mAbs stimulate Spi- C expression in a β2GP1-dependent manner. Overall these data sup- port that one mechanism of innate immune activation induced by PS-targeting mAbs is through TLR2 stimulation on myeloid cells. Fu- ture studies are focused on validating these results in vivo using Tlr- deficient and β2gp1-deficient mice. Trial Registration NCT01999673, NCT03139916, NCT03519997 Ethics Approval All animal experiments were done according to the Animal Research Center (ARC) at UT Southwestern Medical Center. All clinical trials Fig. 1 (abstract P340). See text for description were conducted in accordance with all Federal and State Laws. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 187 of 272 P342 P343 Long term outcomes of a phase I study with UV1, a second Initial results from a Phase II study (TACTI-002) in non-small cell generation telomerase based vaccine, in patients with advanced lung cancer, or head and neck cancer patients receiving non-small cell lung cancer eftilagimod alpha (LAG-3 fusion protein) and pembrolizumab 1 2 2 1 2 3 Wenche Rasch, PhD , Paal Brunsvig, MD PhD , Martha Nyakas, MD , Clau Julio Peguero, MD , Enriqueta Felip, MD PhD , Bernard Doger , Margarita 2 2 2 4 5 6 7 Reisse, MD , Jon Amund Kyte , Hedvig Vidarsdotter Juul , Steinar Majem , Enric Carcereny , Tim Clay , Pawan Bajaj , Matthew Krebs, MD 1 3 2 8 9 Aamdal , Gustav Gaudernack, PhD , Else Marit Inderberg PhD , Frederic Triebel, MD, PhD 1 2 1 2 Ultimovacs ASA, Oslo, Norway; Oslo University Hospital, Oslo, Norway; Oncology Consultants, Houston, TX, United States; Vall d’ Hebron 3 3 Ultimovacs ASA, Prof. Emeritus, Oslo University Hospital, Oslo, Norway Institute of Oncology, Barcelona, Spain; Fundación Jimenez Díaz, Correspondence: Wenche Rasch (wenche.rasch@ultimovacs.com) Madrid, Spain; Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P342 Institut Català d'Oncologia Badalona, Barcelona, Spain; St John of God Subiaco Hospital, Perth, Australia; Tasman Health Care, Queensland, Background Australia; The University of Manchester and The Christie NHS A first generation hTERT vaccine (GV1001) showed evidence of clin- Foundation Trust, Manchester, United Kingdom; Immutep, Orsay, France ical efficacy in patients with advanced non-small cell lung cancer Correspondence: Frederic Triebel (ftriebel@immutep.com) (NSCLC). We have now tested a second generation hTERT vaccine, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P343 UV1. This vaccine is designed to give high population coverage and is composed of three synthetic long peptides containing multiple Background epitopes identified by epitope spreading data from long-term survi- Eftilagimod alpha (efti; previously IMP321) is a recombinant LAG-3 Ig vors who participated in previous hTERT vaccination trials. fusion protein that binds to MHC class II molecules to mediate anti- Methods gen presenting cell (APC) and CD8 T-cell activation. The stimulation Eighteen non-HLA-typed patients with stage III/IV NSCLC with no evi- of the dendritic cell network and subsequent T cell recruitment at dence of progression after prior treatments, were enrolled in a phase the tumor site with efti may lead to stronger anti-tumor CD8 T cell I dose-escalation study of UV1 vaccination with GM-CSF as adjuvant, responses than observed with pembrolizumab alone. Combining an evaluating safety, immune response, and long term clinical outcome. APC activator with an immune checkpoint inhibitor (ICI) aims to in- The present study also aimed to provide a rationale for combining crease efficacy without additional toxicity. We hereby report initial re- UV1 vaccine with PD-1/PD-L1 blockade. sults of stage 1 of this phase II trial (NCT03625323). Results Methods Treatment with GM-CSF and UV1 was well tolerated with no serious The study is based on a Simon's optimal two-stage design, with ob- adverse events observed. All patients experienced one or more ad- jective response rate (ORR) as primary endpoint. Secondary end- verse events, the majority grade 1, such as injection site reactions points include progression free survival and overall survival. Blood and fatigue. Seventeen patients were evaluable for tumor response; for PK/PD assessments and anti-drug antibody evaluation is col- 15 patients had stable disease as best response, while 2 patients had lected. During the first stage of the study, patients (pts) are recruited progressive disease. The median progression free survival (PFS) was into each of three indications: A: 1st line, PD-X naïve NSCLC; B: 2nd 12.3 months and the median overall survival (OS) was 28.2 months. line, PD-X refractory NSCLC; C: 2nd line PD-X naïve HNSCC. Additional The OS at 3 years was 44%. None of the 7 long-term surviving pa- patients (N2) will be recruited for each part if the pre-specified tients (median survival 4.96 years, range 4.04-5.51) have received threshold for ORR is met. In total 109 patients are planned to be en- checkpoint blockade therapy after UV1 vaccination. UV1-vaccination rolled. Eftilagimod alpha is administered as 30 mg subcutaneous in- induced specific T helper 1 (Th1) immune responses in the majority jection every 2 weeks for the first 8 cycles and every 3 weeks for the (67%) of patients. Both immune responses and OS were dose related. 9 following cycles. Pembrolizumab is administered at a standard Conclusions dose of 200 mg intravenous infusion every 3 weeks for maximum 2 The highest dose of UV1 (700 μg) resulted in the highest proportion years. The study was approved by all relevant ethics committees and of immune responses. These responses occurred more rapidly and institutional review boards. were stronger compared to lower doses and the patients in this Results group had a 3-year OS of 83%. This, together with the safety and Between 05 March and 24 July 2019, 27 pts were enrolled and clinical outcome data, favours 700 μg as the preferred UV1 dose in treated in the study. The mean age was 67 (range 53-84) and 74% this patient population. These results provide a rationale for further were male. The ECOG PS was 0 in 59% of the pts and 1 in 41% of clinical studies in NSCLC with UV1 vaccination in combination with the pts. The treatment has been well tolerated with the most com- immune checkpoint blockade. mon AEs being cough (9%), dyspnea (9%), diarrhea (6%) and asthe- nia (5%). Eleven treatment related SAEs were reported in ten pts. Acknowledgements Thirteen (13) pts of part A are evaluable (data cut-off 24th July 2019) We thank all the patients for their participation in the study for efficacy. The vast majority had only one post-baseline tumor as- Trial Registration sessment. Four pts of 13 (31%) had a partial response and six (46%) The clinical trial UV1/hTERT-L was performed with NoMA approval and is pts had stable disease according to iRECIST at data cut-off. registered with Clinicaltrials.gov on February 11, 2013 (NCT01789099). Conclusions Patients provided written informed consent to participate. Enrollment started Thirty (30) mg efti s.c. every 2 weeks in combination with standard on April 8, 2013. dose of pembrolizumab is safe and shows encouraging antitumor Ethics Approval activity. The study was approved by the institutional protocol board, the regional Trial Registration Ethical Committee (REC 2012/1114, EudraCT 2012- 001852-20) and the EudraCT: 2018-001994-25 Norwegian Medicines Agency (NOMA) and the study was registered at NCT: 03625323 clinicaltrials.gov (NCT01789099). Ethics Approval Consent The study was approved by Advarra IRB (US), approval number N/A, Individual patient consent was not applicable as no information in this approval date: 13/08/2018; London - Harrow Research Ethics Com- abstract/poster can be categorized as identifiable. mittee (UK), approval number 18/LO/1889; Instituto de Investigación Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 188 of 272 Hospital 12 de Octubre (Spain), approval number 18/376; Belberry 3.3mos), or overall survival (p=0.40, overall mOS 11.4 mos); therefore, HREC (Australia), approval number 2018-08-636; St John of God the study closed to enrollment at interim analysis. PD-L1 expression Health Care (Australia), approval number 1450. was not associated with response (p=0.52). IF demonstrated an asso- ciation between intratumoral CD4+/PD1+/Ki67+ cells and response (p=0.02). P344 Conclusions A randomized multi-center phase 2 study of combined PD-L1/ We did not observe a benefit adding targeted radiotherapy concur- CTLA-4 inhibition with or without radiation in non-small cell lung rently with combined PD-L1/CTLA-4 therapy in a PD-(L)1 inhibitor re- cancer patients who progressed on PD-(L)1 directed therapy: fractory NSCLC population. However, across cohorts PD-L1/CTLA-4 ETCTN 10021 was generally tolerable and led to response/disease control in some 1 2 3 Arta Monjazeb, MD, PhD , Anita Giobbie-Hurder, MS , Ana Lako , Mark patients, including responses >6 months. Multiplex IF suggests tumor 3 4 5 6 Awad, MD PhD , Ryan Gentzler, MD , Carrie Lee , Joleen Hubbard , infiltration by Ki-67+/PD-1+ CD4 T cells is associated with response 7 8 9 James Abbruzzese, MD , Salma Jabbour, MD , Nataliya Uboha , Kevin and worthy of further investigation. Additional correlative genomic 10 11 12 13 Stephans , Jennifer Johnson, MD , Haeseong Park , Liza Villaruz, MD , and immune analyses are planned. 3 14 15 Katrina Kao , Elad Sharon, MD, MPH , David Raben, MD , Raymond Trial Registration 3 14 14 Mak , Howard Streicher, MD , Helen Chen, MD , Mansoor Ahmed, ClinicalTrials.gov Identifier: NCT02888743 14 16 3 PhD , Scott Rodig, MD, PhD , F. Stephen Hodi, MD , Jonathan Ethics Approval Schoenfeld, MD, MPH This study was approved by the NCI Central IRB. 1 2 UC Davis Cancer Center, Sacramento, CA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Dana-Farber Cancer Institute, Boston, MA, United States; University of Virginia, Charlottesville, Table 1 (abstract P344). See text for description VA, United States; University of North Carolina, Chapel Hill, United 6 7 States; Mayo Clinic, Rochester, United States; Duke, Durham, United States; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United States; University of Wisconsin, Madison, WI, United States; 10 11 Cleveland Clinic, Cleveland, United States; Jefferson Medical Center, Philadelphia, PA, United States; Washington University in St. Louis, St. Louis, United States; University of Pittsburgh Medical Center, Pittsburgh, PA, United States; National Institutes of Health, Bethesda, MD, United States; University of Colorado, Greenwood Village, CO, United States; Brigham and Women's Hospital, Boston, MA, United States Correspondence: Jonathan Schoenfeld (jdschoenfeld@partners.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P344 Background Preclinical data support combined PD-L1/CTLA-4 inhibition and sug- gest synergy between PD-L1/CTLA-4 inhibition and targeted radi- ation via enhanced systemic anti-tumor immune responses. We aimed to evaluate combined PD-L1/CTLA-4 inhibition in NSCLC pa- tients who progressed on prior PD-(L)1 inhibitors and determine whether high- or low-dose radiation could increase objective re- sponses outside the radiation field. Methods ETCTN 10021 is a multicenter randomized phase 2 study evaluating the addition of repeated low-dose fractionated radiotherapy (0.5 Gy BID x 2 days) or hypofractionated radiation (8 Gy x 3) concurrently P345 with PD-L1/CTLA-4 inhibition (durvalumab 1500mg/tremelimumab PARP inhibition when combined with PD-L1 inhibition has a 75mg q4w for 4 cycles followed by durvalumab monotherapy) in suppressive effect on T cells in patients with relapsed or recurrent NSCLC patients progressive on prior PD-(L)1 inhibitors (intervening small cell lung cancer therapy allowed). Patients were randomized 1:1:1 to durvalumab/tre- Nobuyuki Takahashi, MD, PhD, Vinodh Rajapakse, Min-Jung Lee, Akira melimumab alone or with low-dose or hypofractionated radiother- Yuno, Sunmin Lee, Sehyun Kim, Rasa Vilimas, Samantha Nichols, Jane apy. The primary endpoint was objective response per RECIST v1.1 Trepel, Anish Thomas excluding irradiated lesions with planned interim analysis. Correlative National Cancer Institute, Bethesda, MD, United States analyses were performed on tissue obtained following progression Correspondence: Anish Thomas (anish.thomas@nih.gov) on prior PD-(L)1 inhibitors using PD-L1 immunohistochemistry and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P345 multiplex immunofluorescence (IF) evaluating CD8, CD4, PD1, Ki67, and cytokeratin in tandem. Background Results Poly ADP-ribose polymerase (PARP) inhibition increased PD-L1 ex- We randomized 78 patients (26 per each of 3 arms) who received >= pression, augmented cytotoxic T-cell infiltration and potentiated the 1 cycle of study therapy between August 2017 and March 2019 anti-tumor effect of PD-L1 blockade in small cell lung cancer (SCLC) across 18 sites. Patients received PD-(L)1 inhibitors for a median of 1 in vivo [1]. Yet in clinical studies, PARP inhibitor plus PD-L1 inhibitor cycles (range 1-5) prior to enrollment; 68% had prior radiation. Treat- did not improve responses in relapsed or recurrent SCLC patients ment related adverse events (TRAE) of any grade were observed in compared to historical controls of PD-L1 inhibitor alone [2, 3]. Given 53 subjects (68%), and grade ≥3 events in 18 subjects (23%), includ- the role of PARPs in activating inflammatory gene expression [4], we ing 1 grade 5 respiratory failure. Response rate across all cohorts investigated the effects of PARP inhibition plus PD-L1 blockade on were 10% (n=8, 95% exact CI: 5%-19%), and disease control 19% (n= the adaptive immune system in SCLC patients. 15, 95% exact CI: 11-30%). Median duration of response was 10.3 Methods months (95% CI: 1.4 months – not reached). Response and disease NCT02484404 SCLC cohort is an open label phase 2 study evaluating control weren’t significantly different between arms (Table 1, p=0.99/ the combination of durvalumab (1500 mg iv, Q4W) and olaparib (300 0.52, respectively), nor was time to progression (p=0.88, overall mTTP Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 189 of 272 mg BID) in patients with relapsed or recurrent SCLC [2]. Peripheral Preliminary efficacy from a cohort of NSCLC patients who re- blood lineages (pretreatment [C1D1], 2 weeks [C1D15] and 6 weeks ceived prior immune checkpoint therapy was previously pre- [C3D1] after treatment) were serially assessed by flow cytometry. sented (SITC-2017); baseline tumor samples were collected to Results assess mechanisms of resistance to prior therapy. Here we de- 20 patients were evaluated. Activated Ki67+ HLA-DR+ T cells sig- scribe the baseline tumor microenvironments (TMEs) of 2 co- nificantly decreased post-treatment (median [interquartile range] horts of patients, melanoma and NSCLC, who experienced on C1D1 and C3D1: 3.5% [2.2–5.9] vs. 2.1% [1.8–3.3], p=0.033 progressive disease on prior anti–PD-(L)1 treatment before among CD4+ T cells; 2.6% [1.8–5.1] vs. 1.4% [1.0–2.3], p=0.002 starting bintrafusp alfa administration. among CD8+ T cells). Activated Ki67+ PD-1- T cells also signifi- Methods cantly decreased post-treatment (1.9% [1.2–3.9] vs. 1.3% [1.1–2.4], Fresh tumor biopsies from melanoma (n=29) and NSCLC (n=64) pa- p=0.020 among CD4+ T cells; 3.2% [2.0–5.0] vs. 2.4% [2.0–2.7], p= tients refractory or resistant to prior anti–PD-(L)1 therapy were proc- 0.025 among CD8+ T cells). By contrast, exhausted Ki67- TIM-3+ essed for FFPE and subjected to RNA sequencing. Based on the CD8+ T cells significantly increased post-treatment (0.8% [0.5–1.3] mechanism of action of bintrafusp alfa, genes and gene signatures vs. 1.2% [0.8–2.0], p=0.002). PD-1 expression on regulatory related to immune and TGF-β pathways were evaluated and com- Foxp3+ CD25+ T cells (Treg) and effector regulatory CD45RA- pared between tumor types. Foxp3hi T cells (eTreg) significantly increased post-treatment (me- Results dian [IQR] of mean fluorescence intensity [MFI] ratio on C1D1 Melanoma had elevated CD8+ T-cell gene signatures but low levels and C3D1: 2.2 [1.6–2.8] vs. 3.1 [2.2–3.4], p=0.002 among Treg; 2.4 of predicted tumor neo-antigens. NSCLC had elevated gene signa- [1.7–2.8] vs. 3.4 [2.3–4.2], p=0.002 among eTreg). CTLA-4 expres- tures suggesting increased infiltration of inhibitory immune cells and sion on non-regulatory Foxp3- CD4+ T cells (non-Treg) also in- gene signatures related to the TGF-β pathway. Lastly, certain mesen- creased post-treatment (0.18 [0.16–0.20] vs. 0.21 [0.18–0.23], p= chymal markers were expressed at a higher level in melanoma com- 0.007). pared with NSCLC—in particular, the gene encoding vimentin, which Conclusions is associated with metastasis and poor prognosis, was 4-fold higher The combination of olaparib and durvalumab resulted in significant in melanoma. decrease in peripheral blood activated T cells, whereas exhausted T Conclusions cells and inhibitory markers on Treg, eTreg and non-Treg cells signifi- The results suggest that this cohort of melanoma patients may cantly increased. These findings contrast with the expected changes have resisted prior immune therapy due to a low neo-antigen under PD-L1 inhibitor treatment alone. These paradoxical changes of count and/or a mesenchymal phenotype. In contrast, the mecha- immune subsets likely reflect the anti-inflammatory effect of olaparib, nisms of resistance in NSCLC patients in this study may have which may have attenuated the antitumor immune efficacy of been related to high levels of inhibitory immune and TGF-β path- durvalumab. way genes. Collectively, these results provide novel insights into Trial Registration TMEs of melanoma and NSCLC in patients refractory or resistant NCT02484404 to anti–PD-(L)1 agents. Ethics Approval References This study was performed with IRB (#15C0179) and FDA approval 1. Sen T, Rodriguez BL, Chen L, Corte CMD, Morikawa N, Fujimoto J, et al. and is registered with Clinicaltrials.gov on August 7, 2015 Targeting DNA Damage Response Promotes Antitumor Immunity (NCT02517398). Patients provided written informed consent to through STING-Mediated T-cell Activation in Small Cell Lung Cancer. Can- participate. cer Discov. 2019;9(5):646-61. 2. Thomas A, Vilimas R, Trindade C, Erwin-Cohen R, Roper N, Xi L, et al. Dur- P347 valumab in Combination with Olaparib in Patients with Relapsed SCLC: Immunomodulation in tumor and peripheral blood following Toca Results from a Phase II Study. J Thorac Oncol. 2019. 511 & Toca FC treatment in patients with solid tumors 3. Krebs M, Ross K, Kim S, De Jonge M, Barlesi F, Postel-Vinay S, et al. P1.15- 1 2 3 4 Gerald Falchook, MD , Jordi Rodon , Shree Venkat , Arthur Donahue , 004 An Open-Label, Multitumor Phase II Basket Study of Olaparib and 4 3 5 Peder Horner , Amber Thomassen , William Accomando , Maria Durvalumab (MEDIOLA): Results in Patients with Relapsed SCLC. J Thorac 5 5 5 5 Rodriquez-Aguirre , Cornelia Bentley , Daniel Hogan , Derek Ostertag , Oncol. 2017;12(11):S2044-S5. 5 5 5 5 Sharon Yavrom , Thian Khoeh , Douglas Jolly, PhD , Harry Gruber, MD , 4. Rosado MM, Bennici E, Novelli F, Pioli C. Beyond DNA repair, the 5 3 Jolene Shorr , Jaime Merchan immunological role of PARP-1 and its siblings. Immunology. 1 2 Sarah Cannon Research Institute, Denver, CO, United States; MD 2013;139(4):428-37. Anderson Cancer Center, Barcelona, Spain; University of Miami, Miami, Ethics Approval FL, United States; Diversified Radiology of Colorado, Denver, CO, United The trial was conducted under a National Cancer Institute Center for States; Tocagen Inc., San Diego, CA, United States Cancer Research–sponsored investigational new drug application Correspondence: Gerald Falchook (gerald.falchook@sarahcannon.com) with institutional review board approval; approval number 15-c- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P347 Background P346 Toca 511 (vocimagene amiretrorepvec) is a cancer-selective, gamma- Comparison of TMEs from melanoma and NSCLC patients retroviral replicating vector encoding yeast cytosine deaminase, an refractory or resistant to anti–PD-(L)1 therapies enzyme that converts 5 fluorocytosine (5-FC) into 5-fluorouracil in George Locke, Cherie Taglienti, PhD, Laureen S. Ojalvo, Christoph the tumor microenvironment. Preclinical models indicated that Toca Helwig, MSc, Alex Rolfe, Olaf Christensen, Isabelle Dussault, PhD 511 and 5-FC treatment kills dividing cancer and nearby immunosup- EMD Serono Research & Development, Billerica, MA, United States pressive cells, leading to T-cell priming and antitumor immune activ- Correspondence: Isabelle Dussault (isabelle.dussault@emdserono.com) ity [1]. A Phase 3 trial of Toca 511 & Toca FC (extended-release 5-FC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P346 for treatment of recurrent high grade glioma is ongoing, following Phase 1 observations of prolonged survival and durable complete re- Background sponses in some patients [2]. Bintrafusp alfa (M7824), an innovative first-in-class bifunctional Methods fusion protein composed of the extracellular domain of the This Phase 1b, single-arm, multicenter study (Toca 6) was designed TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb to investigate immunological changes following Toca 511 & Toca FC blocking PD-L1, has shown evidence of clinical activity in treatment in patients with advanced solid tumors. Patients received phase 1 studies of patients with advanced solid tumors. intravenous (IV) Toca 511 for 3 days (Week 1), underwent biopsy of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 190 of 272 metastatic tumor (Week 2), were dosed with oral Toca FC (Weeks 5 Background and 6), underwent follow-up biopsy (~Week 9), and then repeated The anti-tumor responses to immune checkpoint inhibitors (ICI) is oral Toca FC every 4-6 weeks. Longitudinal peripheral blood mono- limited in malignancies such as pancreatic adenocarcinoma (PA), nuclear cell (PBMC) samples were immunophenotyped by flow cy- head and neck squamous cell carcinoma (HNSCC) and castration re- tometry, and tumor biopsies were analyzed by sistant prostate cancer (CRPC) through establishment of inaccessible immunohistochemistry (IHC). hypoxic regions [1,2]. Under hypoxic conditions, evofosfamide (EVO) Results releases the alkylating agent Br-IPM, which decreases hypoxia, re- A total of 21 patients with a median 4 lines of prior chemotherapy duces density of MDSC, restores T cell infiltration and increases were enrolled (17 colorectal cancer, 2 sarcoma, 1 each non-small cell tumor antigen presentation [3]. Across syngeneic models, EVO dem- lung and pancreas cancer). PBMC results suggest T-cell shifts from onstrates strong therapeutic cooperativity with ICI [4]. naïve to effector phenotypes, CD4+ memory T-cell expansion, and/or Methods B-cell increases after Toca FC in 41% of patients with pre- and post- A phase 1, dose-escalation trial using 3+3 design was conducted to Toca FC blood samples. Following treatment with Toca FC, some pa- determine the safety, tolerability and activity of EVO in combination tients showed marked changes in tumor infiltrating immune popula- with ipilimumab (IPI) for the treatment of 4 tumor types: metastatic tions assessed by IHC, including decreases in CD11b+ myeloid cells, or locally advanced PA, HPV negative HNSCC, ICI-refractory melan- Tregs, and exhausted T-cells, and increases in CD8+ T-cells. In oma and CRPC (NCT03098160). The study drugs (EVO, IPI) were given addition, IV Toca 511 led to viral expression in tumor, which was de- at the following doses respectively: level 1 (400mg/m2, 3mg/kg), creased post-Toca FC. Treatment has been generally well tolerated, level 2 (480mg/m2, 3mg/kg), level 3 (560mg/m2, 3mg/kg), level 4 with no related Grade 4 adverse events. At data cut-off, 9 patients (640mg/m2, 3mg/kg). EVO was administered on days 1 and 8 in cy- were alive (median follow-up 10.4 months); median overall survival cles 1 and 2. IPI was administered on day 8 of each 3 week cycle for was 9.6 months (95% CI 6.3, 16.4). A patient receiving concomitant a maximum of 4 doses after which retreatment was allowed in those panitumumab had a partial response. with irCR/ irPR/ irSD or irPD anytime after study initiation. Tumor re- Conclusions sponse was assessed using irRECIST. Change from baseline in periph- Results suggest Toca 511 infects metastatic tumor following IV eral blood and tumor tissue immune and hypoxia parameters were administration, and subsequent Toca FC induces tumor and im- evaluated as potential biomarkers of activity for this combination. munosuppressive cell killing. Preliminary analyses indicate Toca Results 511 & Toca FC treatment may be associated with T-cell mediated Twenty-one patients with a median age of 67 years were enrolled in immune activity in peripheral blood and metastatic tumor, con- the study, of whom a majority had CRPC (n=11) followed by PA (n= sistent with the immunologic mechanism of action observed in 7), melanoma (n=2) and HNSCC (n=1). Three patients were enrolled preclinical models. Preliminary clinical data suggest a signal of ac- at level 1 and six in level 2, 3 and 4 each. The most common any tivity in these heavily pretreated patients warranting further grade adverse events were rash (n=17), anemia (n=16) and investigation. leukopenia (n=12). The most common grade 3 adverse events were Trial Registration transaminitis (n=4), lymphopenia (n=3) and anemia (n=3). One pa- NCT02576665 tient required treatment discontinuation and 3 required EVO dose re- duction for toxicities. Out of 18 patients with measurable disease at References baseline, three (16.7%) had PR (2 with CRPC and 1 with HNSCC) and 1. Mitchell LA, Lopez Espinoza FL, Mendoza D, et al. Toca 511 gene transfer twelve (66.7%) had SD. The best responses were observed at dose and treatment with the prodrug, 5-fluorocytosine, promotes durable anti- level 3 (Figure 1). Reduced hypoxic exposure of myeloid stroma, cor- tumor immunity in a mouse glioma model. Neuro Oncol. 2017;19:930- relating with reduced suppressive polarization was observed. 939. Conclusions 2. Cloughesy TF, Landolfi J, Vogelbaum, et al. Durable complete responses No new or unexpected safety signals were observed with combined in some recurrent high-grade glioma patients treated with Toca 511 + EVO and IPI. The combination showed evidence of activity in heavily Toca FC. Neuro Oncol. 2018;20:1383-1392. pretreated refractory solid tumors. Dose expansion is planned at EVO Ethics Approval 560mg/m2 and IPI 3mg/kg. This study was approved by the institutional review boards of University of Miami Hospitals and Clinics, The University of Texas MD Anderson References Cancer Center, and Sarah Cannon Research Institute at HealthONE. 1. Blank CU, Haanen JB, Ribas A, et al: CANCER IMMUNOLOGY. The "cancer immunogram". Science 352:658-60, 2016 2. Chouaib S, Noman MZ, Kosmatopoulos K, et al: Hypoxic stress: obstacles P348 and opportunities for innovative immunotherapy of cancer. Oncogene A phase 1 dose escalation study to evaluate the safety and 36:439-445, 2017 tolerability of evofosfamide in combination with ipilimumab in 3. Duan JX, Jiao H, Kaizerman J, et al: Potent and highly selective hypoxia- advanced solid malignancies activated achiral phosphoramidate mustards as anticancer drugs. J Med 1 1 1 Aparna Hegde, MD , Priyamvada Jayaprakash, PhD , Elizabeth Sumner , Chem 51:2412-20, 2008 1 1 1 1 Di Nguyen , Hira Zain , Sarina Piha-Paul, MD , Daniel Karp , Jordi 4. Ai M, Budhani P, Sheng J, et al. Tumor hypoxia drives immune 1 1 1 Rodon , Shubham Pant, MBBS , Siqing Fu, MD, PhD , Ecaterina suppression and immunotherapy resistance. J Immunotherapy of Cancer 1 1 1 Dumbrava, MD , Timothy Yap, MD PhD , Vivek Subbiah, MD , Priya 2015;3(Suppl 2):P392. 1 2 2 2 Bhosale, MD , Jack Higgins, PhD , Eric T.Williams , Thomas F. Wilson , Ethics Approval 1 1 1 Funda Meric-Bernstam, MD , Michael Curran, PhD , David Hong, MD The study was approved by University of Texas MD Anderson Cancer The University of Texas MD Anderson Cancer Center, Bee Cave, TX, Center's Ethics Board, approval number IRB00000121. United States; Molecular Templates, Austin, TX, United States Consent Correspondence: Michael Curran (MCurran@mdanderson.org); David Written informed consent was obtained from the patient for publication of Hong (dshong@mdanderson.org) this abstract and any accompanying images. A copy of the written Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P348 consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 191 of 272 to reduce α-SMA expression vs RT, suggesting that bintrafusp alfa can reduce RT-induced fibrosis, presumably via TGF-β blockade. Conclusions Collectively, these preclinical findings support the clinical develop- ment of bintrafusp alfa and RT combination therapy and support the rationale for a clinical trial investigating bintrafusp alfa in combin- ation with chemoradiation (CRT) in stage III non-small cell lung can- cer (NSCLC; NCT03840902). In addition, the enhanced efficacy seen in multiple murine models supports the broad application of this combination for treatment of additional cancer indications. Ethics Approval This study was approved by the Institutional Animal Care and Use Committee at EMD Serono, Inc.; approval number [17-008]. P350 Clinical signal/profile in a phase I study of T cCell receptor (TCR) affinity-enhanced specific T cells (TAEST) in advanced cancer patients 1 1 2 3 Yi Li, PhD , Zhaosheng Han, PhD , Xing Zhang, MD , Jian Zhang , 4 1 2 Chengzhi Zhou , Haiping Gong , Desheng Weng, MD , Jianchuan Xia, 2 5 4 3 PhD MD , Johnson Lau , Shiyue Li , Weiliang Zhu Fig. 1 (abstract P348). See text for description 1 2 Guangdong Xiangxue Life Sciences, Ltd., Guangzhou, China; Sun Yat- sen University Cancer Center, Guangzhou City, Guangdong Provi, Peoples Republic of China; Zhujiang Hospital, Guangzhou, China; 4 5 Guangzhou Institute of Respiratory Healt, Guangzhou, China; Axis Therapeutics Ltd., Hongkong, Hong Kong P349 Correspondence: Shiyue Li (lishiyue@188.com) Effects of bintrafusp alfa (M7824) and radiation combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P350 therapy on antitumor activity, immune response, and radiation- induced fibrosis in multiple cancer models Background Yan Lan, MD, Chunxiao Xu, PhD, Huakui Yu, Guozhong Qin, Bo Marelli, T-cell triggering thresholds can be improved by engineered TCR with Jin Qi, Rachel E. Fontana, Amit Deshpande, George Locke, Alex Rolfe, enhanced binding affinity. TAEST for NY-ESO-1 was designed for po- Molly H. Jenkins, Joern-Peter Halle, Kin-Ming Lo tentially better efficacy and good safety profile. EMD Serono Research & Development, Billerica, MA, United States Methods Correspondence: Yan Lan (yan.lan@emdserono.com) Preclinical: Determined the TCR affinities. Expression of CD3, CD4, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P349 CD8, and TCR were traced by antibodies/tetramer. Specificity/efficacy in vitro/in vivo and TAEST infiltration in tumor/lymph node were Background evaluated. Clinical: Phase I study – 14 advanced cancer patients were We recently reported the enhanced preclinical antitumor activity of bin- treated with TAEST. trafusp alfa (M7824), an innovative first-in-class bifunctional fusion pro- Results tein composed of the extracellular domain of the TGF-βII receptor (a Preclinical: TAEST had higher affinity to its antigen vs wild type T- TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In phase 1 cells and with great expression (80-90% positive engineered TCR-T and 1b expansion studies in patients with advanced solid tumors, bin- cells), with ~5-6X more CD8+ over CD4+ cells. There was great trafusp alfa showed early evidence of clinical activity. Bintrafusp alfa is in vitro and in vivo efficacy with strong evidence of tumor specific a particularly rational combination partner for radiation therapy (RT) be- TAEST infiltration. Clinical: Stage I - TAEST alone was dosed in 3 cause RT induces expression of TGF-β, which can promote epithelial-to- NSCLC patients with demonstrated safety and stable disease (SD) ob- mesenchymal transition (EMT), fibrosis, and metastasis, and the expres- served for 28-165 days (OS: 77-308 days). Stage II - lymphodepletion sion of PD-L1. Furthermore, the induction of abscopal effects requires was added to TAEST in 11 patients (NSCLC 4, thyroid CA 1, liver the combination of RT with immunotherapy in mouse models, and CA 1, breast CA 1, colon CA 1, melanoma CA 1, synovial sarcoma abscopal responses have been reported in patients receiving RT in 1,and fibrotic sarcoma 1,) and treated with 0.8-2.15x1010 TAEST combination with an immune checkpoint inhibitor. cells: the synovial sarcoma patient had PR (> 70% tumor size re- Methods duction) with > 12 months duration; the breast CA patient had a The combination of bintrafusp alfa and RT was compared with bin- > 40% tumor shrinkage with healing of skin metastatic ulcers trafusp alfa monotherapy or RT alone in MC38 colorectal carcinoma, during Rx; Two other patients (liver, thyroid CA) showed SD but GL261-luc2 glioma, and 4T1 breast cancer murine models. Antitumor significant tumor necrosis (>50%) with symptomatic relief of local activity was evaluated via tumor growth, survival, and lung metasta- pain. Three NSCLC patients had SD with 59-188 days (Survival ses. Enzyme-linked immune absorbent spot (ELISpot) and immuno- 129-392 days). The fibrotic patient had SD for 87 days (Survival: histochemistry were used to measure the function and infiltration of 273 days); the melanoma patient had SD with 105 days (Survival: CD8+ T cells and the quantity of α-SMA, a marker of cancer- 176 days); last 2 patients ( NSCLC 1, colon CA 1) had PD at 14- associated fibroblasts. Gene expression signature scores of different 16 days post infusion ( OS: 92-129 days) . pathways were calculated from targeted RNAseq analysis. The treatment was tolerated well with fever (12/14), chills (4/14), neu- Results tropenia (5/14), thrombocytopenia(1/14), diarrhea(2/14), chest pain The combination therapy enhanced antitumor activity in all three mur- (1/14), and skin rash (3/14) observed. Expected cytokine response, ine models, increased tumor-specific and tumor-infiltrating CD8+ T cells TCR-gene detection/persistence (>60 days), were also observed in in the MC38 and 4T1 models, respectively, and potentiated an abscopal the patients above (particularly, >362 day for synovial sarcoma effect in secondary MC38 tumors. In the 4T1 model, combination ther- patient). apy decreased lung metastases vs either monotherapy and decreased the expression of EMT and VEGF pathway gene signatures vs RT. Ex- Conclusions pression of α-SMA significantly decreased with bintrafusp alfa mono- (1) TAEST, with its enhanced TCR binding affinity, is safe and toler- therapy in this model, whereas it significantly increased with RT able in a clinical phase I study; (2) TAEST exhibits encouraging effi- monotherapy. However, the combination with bintrafusp alfa was able cacy (DCR: 85.7%, 12/14) with a near CR for synovial sarcoma patient Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 192 of 272 (duration >12 months), and marked tumor necrosis with two more Conclusions patients (liver CA, thyroid CA); (3) Lymphodepletion pretreatment ap- The discordance between disease control per irRECIST and RECIST peared to be critical for efficacy/cytokine response/persistence of suggests that for approximately one in 12 patients, irRECIST is a bet- TAEST cells. ter indicator of clinical benefit from ICI treatment than RECIST. How- ever, overall no stronger association was observed between OS and irPFS compared with between OS and PFS. Thus, neither RECIST nor Acknowledgements irRECIST showed a clear advantage for predicting OS for clinical deci- The National key R&D Program of China, 2016YFC1303404; The Sciences and sions or regulatory purposes. Technology Program of Guangzhou, No. 201704020220. Trial Registration Acknowledgements ClinicalTrials.gov Identifier: NCT03159585; NCT03029273; NCT03462316 This study was funded by Merck KGaA, Darmstadt, Germany, as part of an Ethics Approval alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, The study of bone sarcoma and soft tissue sarcoma was approved by Sun NY, USA. Yat-sen University Cancer Center, approval number B2017-023-01. Trial Registration The study of NSCLC was approved by The First Affiliated Hospital of All trials were registered at clinicaltrials.gov, trial numbers NCT01772004 and Guangzhou Medical University, approval number 2016 No.63. NCT02155647. The study of multiple solid tumor was approved by Zhujiang Hospital of Ethics Approval Southern Mediacl University, approval number 2017-ZLZX-001. The trials were approved by the institutional review board or independent Consent ethics committee at each participating center. Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. P352 Workflow for Immune Monitoring during Clinical Trials by using unsupervised high dimensional augmented intelligence assisted P351 analysis Association between response assessment using RECIST and Alessandra Metelli, PhD, Carsten Krieg, PhD, Luis Cardenas, BS irRECIST in 1765 patients with advanced solid tumors treated with Medical University of South Carolina, Charleston, SC, United States avelumab monotherapy 1 2 1 Correspondence: Carsten Krieg (KriegC@musc.edu) Juliane Manitz , Peter Eggleton , Marcis Bajars , Oliver Bohnsack, MD, 3 4 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P352 PhD, MBA , James Gulley, MD, PhD, FACP EMD Serono Research and Development Institute, Inc, Billerica, Background Massachusetts, United States; Merck KGaA, Darmstadt, Germany, Following checkpoint inhibitors, which significantly improved cancer Darmstadt, Germany; PAREXEL Informatics, Berlin, Germany, Berlin, treatment, an increasing number of combination therapeutics is be- GERMANY; Center for Cancer Research, National Cancer Institute, ing tested in clinical trials. To find possible clinical or biological corre- National Cancer Institutes of Health, Bethesda, MD, United States lates of response or intervention, high throughput multi-omic Correspondence: Juliane Manitz (juliane.manitz@emdserono.com) approaches are necessary to catch all features of possible immune Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P351 responses during treatment. Methods Background To this aim, we present a portfolio of multi-omic approaches includ- A subset of patients receiving immune checkpoint inhibitor (ICI) ing high-dimensional mass cytometry (CyTOF) and single cell se- treatment may have unconventional response patterns, such as pseu- quencing in combination with unsupervised machine-learning doprogression, which can be classified as best overall response (BOR) bioinformatics to perform in depth characterization of immune re- of progressive disease (PD) by Response Evaluation Criteria in Solid sponses during clinical (immuno)therapy. The analysis is data driven, Tumors (RECIST) v1.1; therefore, immune-related (ir) response criteria, can be adapted to high throughput approaches and can model arbi- irRECIST, have been proposed. This analysis reports the differences in trary trial designs. response assessment by RECIST v1.1 and irRECIST and their associ- Results ation with overall survival (OS) in patients with advanced solid tu- We here show three proof of concept projects using biobanked per- mors treated with avelumab monotherapy (anti–PD-L1). ipheral blood mononuclear cells (PBMCs). In the first study, 51 pa- Methods tients with stage IV melanoma before and after 12 weeks of anti-PD- Data from patients with metastatic or locally advanced solid tumors 1 therapy were analyzed. We observed a clear T cell response on (n=1677, data cutoff, February 15, 2017) enrolled in the phase 1, therapy. The most evident difference in responders before therapy open-label JAVELIN Solid Tumor trial (NCT01772004), and data from was an enhanced frequency of CD14+ CD16+HLA-DRhi classical patients with metastatic Merkel cell carcinoma with disease progres- monocytes. We validated our results using conventional flow and sion after prior chemotherapy (n=88, data cutoff, March 24, 2017) en- found a clear correlation of enhanced monocyte frequencies before rolled in part A of the phase 2 open-label JAVELIN Merkel 200 trial therapy initiation with clinical response such as lower hazard and ex- (NCT02155647) were pooled. Patients with castration-resistant pros- tended progression-free and overall survival. In a second study, we tate cancer from the JAVELIN Solid Tumor study were excluded. All used CyTOF to monitor immune response in 21 non-small cell lung patients received avelumab 10 mg/kg every 2 weeks by intravenous cancer (NSCLC) patients that initially responded and then progressed infusion. BOR, disease control rate, and progression-free survival under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 (PFS) were evaluated. Concordance of disease control rates, Kaplan- plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a Meier, landmark OS, and correlation analyses were performed. response in the CD8+ T cell compartment was observed. Unexpect- Results edly, our high dimensional unbiased analysis was able to detect and A total of 1765 patients were included. All patients had ≥3 months characterize a strong expansion of innate tumor-reactive effector NK of follow-up. The pooled data set included 12 tumor types. The dis- cells starting around day 4 of therapy. In our third unpublished study cordance between the tumor assessment criteria for disease control we were able to identify neutrophils as predictors of outcome in lung rate was 8.3% (n=147), i.e. complete response + partial response + cancer patients. stable disease (SD) per irRECIST and PD + not evaluable per RECIST; Conclusions most patients (n=135) had a BOR of PD by RECIST and irBOR of SD Taken together, our unbiased artificial intelligence-driven immune by irRECIST. The Kaplan-Meier analysis according to Wolchok et al ex- workflow is an extraordinary instrument to monitor immune re- hibited clear separation of the respective (dis)concordant subgroups. sponses during (immuno)therapy and serves as a novel approach for The rank correlations between OS and PFS and OS and irPFS were therapeutic target identification. 0.73 (95% CI, 0.70-0.75) and 0.75 (95% CI, 0.72-0.78), respectively. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 193 of 272 Trial Registration P354 ClinicalTrials.gov Identifier: NCT02523469 Exploring correlates of clinical and immune response to cancer Ethics Approval immunotherapy using FAUST, a novel unbiased cell population This study was approved by the MUSC and Zurich institutional review discovery method, in whole blood flow cytometry board. Steven Fling, PhD, Nirasha Ramchurren, PhD, Leonard D'Amico, Martin Cheever, MD, Evan Greene, Greg Finak, Raphael Gottardo, PhD Fred Hutchinson Cancer Research Center, Seattle, WA, United States P353 Correspondence: Steven Fling (sfling@fredhutch.org) Phase 1 pilot study of RRx-001 + nivolumab in advanced Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P354 metastatic cancer (PRIMETIME) 1 2 1 3 Corey Carter, MD , Bryan Oronsky, MD PhD , Mary Quinn , Jane Trepel , Background 4 5 Nacer Abrouk, PhD , Jeff Skinner, MD The purpose of this study is to describe an immune monitoring ap- 1 2 EpicentRx, Inc., La Jolla, CA, United States; EpicentRx Inc, La Jolla, CA, proach that combines multi-parameter whole blood flow cytometry 3 4 United States; NIH, Bethesda, MD, United States; Clinical Trials with an automated, unbiased, cell population evaluation method to Innovations, Mountain View, CA, United States; Walter Reed National Mil facilitate discovery of informative correlative biomarkers. The Cancer Medical Center, Bethesda, MD, United States Immunotherapy Trials Network (CITN) coordinates multi-center can- Correspondence: Mary Quinn (mquinn@epicentrx.com) cer immunotherapy trials, wherein multiparameter flow cytometry is Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P353 performed in real time on longitudinally collected whole blood samples. Background Methods RRx-001 is a minimally toxic small molecule that downregulates We recently reported results from two CITN multi-center clinical trials. CD47 and repolarizes tumor associated macrophages (TAMs) as well We also reported a non-parametric method for unbiased cell popula- as normalizes aberrant tumor perfusion. On the premise that the tion discovery that annotates cell populations with biologically inter- interaction between a CD-47 downregulator like RRx-001 and an pretable phenotypes through a new procedure called Full anti-PD-1 inhibitor like nivolumab may serve to activate both arms of Annotation Using Shape-constrained Trees (FAUST). We used FAUST the immune system, a phase 1 pilot study was undertaken to deter- to analyze extensive flow cytometry data in these two CITN clinical mine the safety and feasibility of RRx-001 and nivolumab in patients trials and demonstrate that candidate biomarkers can be associated with advanced cancer and no standard options. with clinical outcome. Here we compare flow cytometry data ana- Methods lyzed both by conventional manual gating strategies as well as by This single arm, open-label pilot study (NCT02518958) called PRIME- the FAUST method. TIME was designed to evaluate the safety profile of RRx-001 and Results nivolumab in patients with advanced malignancies and no other We highlight the value of FAUST in identifying predictive biomarkers of standard therapeutic options. A 3+3 trial design was used to estab- clinical responses to immunotherapy within fresh whole blood. By com- lish safety of the combination at each dose level and guide the deci- bining whole blood flow staining with FAUST, our results demonstrate sion to escalate dose. RRx-001 is infused once weekly while the ability to capture important minor cell subpopulations, including nivolumab is given at 3mg/kg once every 2 weeks. The RRx-001 start- within the CD8 T cell compartment, that otherwise are missed by man- ing dose was 2 mg IV weekly with 4 dose level escalations up to 16 ual gating. Manual gating can be biased and limited to characterizing mg IV weekly. From January 2015 to November 2015, twelve patients cell populations considered a-priori to be significant. received treatment for only 4 cycles (total 12 weeks) with the com- Conclusions bination due to unavailability of nivolumab, which was not supplied Our results emphasize the unique value of performing flow cytome- to the Sponsor. Treatment-emergent (all cause, TEAEs) and try in multi-center trials using fresh whole blood which preserves the treatment-related (TRAEs) adverse events that occurred within 16 minor cell populations identified by FAUST which may be lost or weeks of the first dose of RRx-001 and nivolumab were characterized compromised by standard cryopreservation methods. according to CTCAE v4.03. Results Twelve patients received >1 dose of RRx-001 and nivolumab. One P355 discontinuation occurred due to pneumonitis and one to volun- Multicenter, open-label, phase 1 study of DSP-7888 Dosing tary withdrawal after a post-procedural infection. There were no Emulsion (DSP-7888) in patients with advanced malignancies 2 3 4 5 DLTs. The main adverse event related to RRx-001 was infusion re- Morris Groves , Aaron Hansen , Wael Harb , Kelly Curtis, MD , Erina 6 7 7 8 action (33.3%). The main adverse event related to the combin- Koga-Yamakawa , Makoto Origuchi , Zhonggai Li , Jose Iglesias , Walid 9 1 ation was pseudoprogression manifested by larger tumors in Shaib, MD , Alexander Spira, MD, PhD, FACP 1 2 patients that were symptomatically improved (25%). The most Virginia Cancer Specialists, Fairfax, VA, United States; Texas Oncology- common immune-related treatment-emergent AEs were pneu- Austin Midtown, Austin, TX, United States; UHN Princess Margaret monitis (8.3%), and hypothyroidism (8.3%). The objective response Cancer Centre, Toronto, Canada; Horizon Oncology Research, LLC, rate at 12 weeks was 25% and the disease control rate (DCR) Lafayette, IN, United States; Syneos Health, Phoenix, AZ, United States; consisting of > SD was 67% by Response Evaluation Criteria in Sumitomo Dainippon Pharma Co., Ltd, Cambridge, MA, United States; 7 8 Solid Tumors (RECIST) version 1.1 25% of the patients progressed Boston Biomedical, Inc., Cambridge, MA, United States; Former on the combination. employee, Boston Biomedical, Inc., Cambridge, MA, United States; Conclusions Emory University, Atlanta, GA, United States The combination of RRx-001 and nivolumab was safe and well- Correspondence: Alexander Spira (Alexander.Spira@USOncology.com) tolerated with preliminary evidence of anti-cancer activity. Further Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P355 analyses with a larger sample size will be required to confirm the ac- tivity of the combination and to determine the optimum schedule Background for RRx-001 and nivolumab. DSP-7888, a cancer vaccine composed of 2 synthetic peptides de- Ethics Approval rived from Wilms’ tumor 1 (WT1) protein, may induce WT1-specific The study was approved by all the revelant Institution‘s Ethics cytotoxic T-lymphocytes (CTLs) and helper T-lymphocytes–mediated Boards. immune responses against WT1-expressing tumors. This dose- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 194 of 272 escalation study (NCT02498665) evaluated DSP-7888 in patients with infiltrating leukocyte (TIL) analysis. CTL, IgG, and TIL were mea- recurrent or progressive advanced malignancies, despite receiving sured by ELISPOT assay, Luminex assay, and IHC (CD8 positive), standard therapy, or in patients intolerant to standard therapy or for respectively. whom no standard of therapy existed for their malignancy. The pri- Results mary objectives were safety, tolerability, and identification of the rec- Seventeen patients were enrolled in 9mg (n=10) and 27mg (n= ommended phase 2 dose (RP2D). Secondary and exploratory 7) groups; the median age was 65 years, and 53% of the pa- objectives included overall survival and WT1-specific CTLs induction. tients had ECOG PS 1. There was no serious adverse drug reac- Methods tion (ADR) in any patient. All ADRs were of grade 1 or 2, with Patients who failed or were intolerant to prior lines of treatment and the most frequent being dermatological injection site reaction, tested positive for HLA-A*02:01, HLA-A*02:06, or HLA-A*24:02 re- in 7/10 (70%) and 6/7 (86%) patients and pyrexia, in 1/10 (10%) ceived escalating doses of intradermal (ID) or subcutaneous (SC) and 2/7 (29%) for the 9mg and 27mg groups, respectively. The DSP-7888 in a rolling study design: 3.5, 10.5, or 17.5 (ID only) mg best overall response was stable disease, in 2/10 (20%) and 2/7 every week for 4 weeks, then every 1–2 weeks for 6 weeks, and every (28%) patients. One patient with cancer of unknown origin re- 2–4 weeks thereafter until progression or other discontinuation event ceived prolonged administration (over 10 months) of the 9mg was met. Dose-limiting toxicities (DLTs) were evaluated over days 1– dose.Inthe 9mgand 27mggroups, antigenspecific IgG was 29. The dose at which ≤1 of 6 patients had a DLT was eligible to be augmented in 9/10 (90%) and 7/7 patients (100%), antigen spe- the RP2D. WT1-specific CTL inductions were assessed by HLA Tetra- cific CTL was detected in 2/10 (20%) and 3/7 patients (43%), mer with peripheral blood. and TIL counts were increased in 2/3 (67%) and 3/4 patients Results (75%), respectively. Twenty-four patients received ID (3.5 mg, n=4; 10.5 mg, n=3; 17.5 Conclusions mg, n=3) or SC DSP-7888 (3.5 mg, n=9; 10.5 mg, n=5). The most fre- TAS0313 demonstrated safety, tolerability, and immunological re- quent adverse event (AE) was injection site reaction (ISR; n=15; sponses in patients with advanced solid tumors in the 9mg and 62.5% [ID: 100% of patients, SC: 36%]); all were grade 1 or 2. No DLT 27mg groups. A phase II part, evaluating the efficacy of combin- was observed. ID DSP-7888 10.5 mg was determined to be the dose ation therapy with pembrolizumab in patients with urothelial car- level for further study based on the RP2D identified in a phase 1/2 cinoma and monotherapy in glioblastoma patients, is currently study of DSP-7888 in patients with myelodysplastic syndrome underway. (NCT02436252). Four patients (ID 17.5 mg, n=1; SC 3.5 mg, n=1; SC Trial Registration 10.5 mg, n=2) had stable disease, 16 had progressive disease, and 4 JapicCTI-183824 were not evaluable. Twenty-one patients were evaluable for WT1- Ethics Approval specific CTL detection. In evaluable patients, WT1-specific CTL induc- The study was approved by National Cancer Research Center Central tion was observed in 6 of 9 ID patients (66.7%) and 5 of 12 SC pa- Hospital’s Ethics Board, approval number T4499. tients (41.7%). Consent Conclusions Written informed consent was obtained from the patients for publi- DSP-7888 was well tolerated, with no DLTs, in patients with ad- cation of this abstract. A copy of the written consent is available for vanced malignancies, supporting further evaluation of DSP-7888. The review by the Editor of this journal. 10.5 mg ID dose was identified as a dose level and route of adminis- tration for further evaluation. P357 Phase I/II clinical and immune responses for locally advanced or P356 metastatic pancreatic cancer using anti-CD3 x anti-EGFR bispecific First-in-human study of the cancer peptide vaccine, TAS0313, in antibody armed T cells (BATs) 1 1 2 1 patients with advanced solid tumors: phase I dose finding part Lawrence Lum, MD, DSc , Tri Le , Minsig Choi , Archana Thakur, PhD , 1 1 3 1 results Matthew Reilly , Paul Kunk, MD , Abhinav Deol, MD , Karen Ballen, MD , 1 1 1 Noboru Yamamoto, MD, PhD, Jun Sato, MD, PhD, Satoru Iwasa, MD, Tamila Kindwall-Keller, DO , Dana Schalk , Ewa Kubicka , Manley Huang, 1 3 3 3 4 PhD, Kan Yonemori, MD, PhD, Takafumi Koyama, MD, Kenji Tamura, MD, PhD , Philip Philip, MD , Hussein Aoun , Gregory Dyson, PhD , Qin Liu , PhD, Toshio Shimizu, MD, PhD, Syunsuke Kondo, Shigehisa Kitano Anthony Shields, MD PhD 1 2 National Cancer Center Hospital, Tokyo, Japan University of Virginia, Charlottesville, VA, United States; Stony Brook Correspondence: Toshio Shimizu (tosshimi@ncc.go.jp) University, Stony Brook, NY, United States; Karmanos Cancer Institute, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P356 Wayne State U, Detroit, MI, United States; Wistar Institute, Philadelphia, PA, United States Background Correspondence: Lawrence Lum (lawrenceglum@cs.com) TAS0313 is a cancer vaccine cocktail containing three long peptides, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P357 with a total of 12 cytotoxic T lymphocyte (CTL) epitope peptides. These peptides were derived from eight cancer-associated antigens Background that are highly expressed in various cancers. We report the results of Chemotherapy for locally advanced pancreatic cancer (LAPC) and a phase I part examining the tolerability, safety, potential efficacy, metastatic pancreatic cancer (MPC) has poor responses and survival and immunological responses of 9 mg and 27 mg TAS0313 in pa- rates. Retargeting anti-CD3 activated T cells (ATC) by arming them tients with advanced solid tumors. with anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) makes ATC Methods into specific cytotoxic T lymphocytes (EGFR BATs). Targeting pancre- The enrolled patients had ECOG PS 0-1 and at least one of the atic cancer cell lines induces cytokine secretion, proliferation, cyto- following HLA types: HLA-A*02:01, -A*02:06, -A*02:07, -A*11:01, toxicity, and inhibits tumor growth. We present 5 phase I and 13 -A*24:02, -A*31:01, or -A*33:03. Emulsified TAS0313 solution with phase II patients for a total of 18 evaluable patients out of 21 who an immunological adjuvant (Montanide ISA-51) was subcutane- underwent apheresis. ously administered on Days 1, 8, and 15 of Cycles 1 and 2 and Methods on Day 1 of Cycle 3 or later in 21-day cycles until disease pro- In the phase I, LAPC or MPC patients at Karmanos Cancer Institute gression, or unacceptable toxicity.. Tolerability was assessed in at (KCI) on Protocol #2011-025, in a dose escalation, were given 10, 20, least six patients during the first cycle. Tumor response was eval- and 40 x 10^9 BATs/infusion weekly for 3 weeks, followed by a uated using RECIST v1.1. Blood samples were collected pre- and booster infusion 3 months later if patients were stable or better. post-treatment for the analysis of antigen specific CTL and IgG. There were no dose limiting toxicities, and all infusions were given in Optional serial tumor biopsies were performed for tumor the outpatient setting. In the phase II portion, 13 PC patients at KCI Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 195 of 272 (NCT02620865) and University of Virginia (NCT03269526) received Methods twice weekly infusions of 10 x 10^9 BATs/infusion over 4 weeks for a Patients with advanced SS were enrolled to cohorts based on NY-ESO-1 total of 80 x 10^9 EGFR BATs. expression (Cohort 2, low; Cohort 4, high) determined by immunohisto- Results chemistry. Treatment response (RECIST v1.1), safety (CTCAE v4.0), and Eighteen patients were evaluable. Four patients were stable at 6.1, GSK3377794 persistence in transduced PBMCs (transgene vector copies 6.5, 5.3, and 39 months. Two patients developed complete responses measured by qPCR) were assessed. Progression-free survival (PFS) was (CR) when chemotherapy was restarted after their BATs infusions. Pa- defined as the interval between first infusion and first documented dis- tient IT20104 was stable for 1 year on capcitabine, developed “pseu- ease progression or death. Safety was monitored throughout. The study doprogression,” achieved a CR after restarting capcitabine, and was was not designed/powered for cohort comparison. off therapy until 54 months after enrollment when relapse occurred. Results The median overall survival is 14.8 months with a time to progres- As of April 2019, 50 patients were enrolled (N=13 Cohort 2; N=15 Co- sion of 6.6 months. Specific cytotoxicity mediated by peripheral hort 4). Table 2 summarizes response outcomes by Cohort. Median PFS blood mononuclear cells (PBMC) peaked at 31% two weeks after the (95% CI) was 13.1 weeks (7.9, 13.9; Cohort 2) and 22.4 weeks (11.3, 26.6; third infusion, and IFN-γ EliSpots rose from Cohort 4). Median peak (range) persistence of ~64,712 DNA copies/μg Conclusions (13,364–197,546) occurred in Cohort 2 first week post-infusion versus EGFR BATs infusions were safe and induced specific adaptive anti- ~16,468 DNA copies/μg(163–131,175) in Cohort 4. No significant cor- tumor responses. This phase I/II study strongly suggests that multiple relation was observed between peak persistence and best overall re- infusions of EGFR BATs may provide a survival benefit in patients sponse in either cohort (p>0.05). Grade 3/4 adverse events occurring in with pancreatic cancer, and that BATs therapy may increase the ef- ≥40% of patients in both cohorts were leukopenia, neutropenia, fectiveness of subsequent chemotherapy, which will drive the design anemia, thrombocytopenia, lymphopenia, and hypophosphatemia. of future combination trials of BATs and other modalities of therapy. Conclusions Cohorts 2 and 4 showed similar ORRs; more durable responses were Acknowledgements observed in Cohort 4, with prolonged DoR, duration of stable dis- These studies were made possible thanks to philanthropy from Karmanos Cancer ease, and PFS. Peak persistence of GSK3377794 was higher in Cohort Institute and start-up funds for LGL from the University of Virginia. LGL and MH 2, likely due to higher lymphodepletion, but this did not correlate are co-founders of Transtarget, Inc. LGL is a member of the Scientific Advisory with response, unlike data previously reported in other cohorts. Fur- Board for Rapa Therapeutics. AT is a co-founder of NOVA Immune Platform. ther development in SS will be based on previously reported data Trial Registration from Cohort 1. Protocol #2011-025; NCT02620865; NCT03269526 Ethics Approval Acknowledgements These studies were approved by the Karmanos Cancer Institute / Wayne Medical writing assistance was provided by provided by Fiona Woodward State University IRB, approval numbers 2011-25 and 2015-100, and the and Victoria Hunter of Fishawack Indicia Ltd. This study (NCT01343043) was University of Virginia IRB, approval number HSR 19236. funded by GlaxoSmithKline. Trial Registration NCT01343043 P358 Ethics Approval Phase 1 trial of NY-ESO-1-specific adoptive T-cell therapy with This study was approved by the appropriate institutional review boards and GSK3377794 in patients with advanced synovial sarcoma independent ethics committees. 1 2 3 Sandra D'Angelo, MD , George Demetri , Brian Van Tine, MD, PhD , 4 5 6 7 Mihaela Druta , John Glod, MD , Warren Chow , Jenna Tress , M. Phillip 7 7 7 7 7 DeYoung , Aisha Hasan, MBBS MD , Yuehui Wu , David Turner , Ran Ji , 7 8 Alexandra Gyurdieva , Dejka Araujo, MD Table 1 (abstract P358). See text for description Memorial Sloan Kettering Cancer Center, New York, NY, United States; 2 3 Dana-Farber Cancer Institute, New York, NY, United States; Washington University in St. Louis, St. Louis, MO, United States; H. Lee Moffitt Cancer Center, Tampa, FL, United States; National Cancer Institute, Bethesda, MD, United States; City of Hope Comprehensive Cancer Center, Duarte, 7 8 CA, United States; GlaxoSmithKline, Collegeville, PA, United States; MD Anderson Cancer Center, Houston, TX, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P358 Background Genetically-engineered NY-ESO-1 specific T-cells (NY-ESO-1 T-Cells; GSK3377794) are autologous CD4+ and CD8+ T cells transduced with a self-inactivating lentiviral vector to express affinity-enhanced NY-ESO-1- specific T-cell receptors (TCRs). Ongoing phase 1 and 2 trials are evalu- ating GSK3377794 in solid tumors and multiple myeloma. Study NCT01343043 (208466) is a phase 1 clinical trial assessing GSK3377794 in patients with previously treated, advanced metastatic synovial sar- coma (SS), stratified into 4 cohorts (Table 1). Of 12 patients receiving GSK3377794 infusion in Cohort 1, responses were observed in 6 pa- tients (1 complete response [CR]/5 partial responses [PR]), with an over- all response rate (ORR) of 50% (95% confidence interval [CI]: 0.21–0.79). Median progression-free survival (PFS) was 15.2 weeks (95% CI: 7.6– 37.9); median duration of response (DoR) was 30.9 weeks (95% CI: 14– 72). As of October 15, 2018, median overall survival (OS) was 105 weeks (95% CI: 37–NA). This abstract reports data from Cohorts 2 and 4. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 196 of 272 Table 2 (abstract P358). See text for description manageable adverse events. Our study shows feasibility of tracking phenotypic profiles of adoptively transferred tumor-antigen-specific T cells in patients and derive association between adoptive T cell sta- tus and clinical read-outs. Ethics Approval The study was approved by Mie University Ethics Board, approval number H2018-092. P360 The influence of Durvalumab/Tremelimumab Combination Therapy on Sarcomas Immune Microenvironment profile in a phase II clinical trial (NCT02815995) 1 1 1 Edwin Parra, MD, PhD , Carmelia Barreto, PhD , Ruth Salazar, MD , Cara 1 1 1 1 Haymaker, PhD , Heather Lin , Carmen Behrens, MD , Mei Jiang , Luisa 1 1 1 Solis, MD , Krishna Pandurenga, MS , Sandesh Subramanya, PhD , Young 2 1 1 1 Kim, PhD , Chantale Bernatchez , Jack Lee, PhD , Taylor Tate , Teresa 1 1 1 Simmons , Alexander Lazar, MD, PhD , Wei-Lien Wang , Zachary Cooper, 3 3 3 PhD , Jaime Rodriguez-Canales, MD , Jean Soria, MD , Anthony Conley, 1 1 1 MD , Ignacio Wistuba, MD , Neeta Somaiah, MD, MBBS 1 2 MD Anderson Cancer Center, Houston, TX, United States; Translational Molecular Pathology, Houston, TX, United States; AstraZeneca, Gaithesburg, MD, United States Correspondence: Edwin Parra (erparra@mdanderson.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P360 Background To determine the tumor microenvironment (TME) changes after the combination of durvalumab/tremelimumab treatment, longitudinal P359 sarcoma tissue collections were obtained and analyzed for in-depth Tracking and profiling of NY-ESO-1 TCR-transgenic T cells upon immunoprofiling. adoptive transfer in patients with NY-ESO-1-expressing solid Methods tumors: in vivo differentiation associated with response 1 1 1 Sixty-two patients were enrolled and 36 paired samples (Liposarco- Michael Fehlings , Alessandra Nardin, DVM , Faris Kairi , Evan Newell, 2 3 3 3 ma,LS=6; Angiosarcoma,AS=2; Leiomyosarcoma,LMS=2; Osteo/Chon- PHD , Yoshihiro Mihayara , Shinichi Kageyama , Hiroshi Shiku, MD 1 2 drosarcoma,OS/CS=3/1; Undifferentiated Pleomorphic Sarcoma,UPS= immunoSCAPE, Singapore, Singapore; Fred Hutchinson Cancer 3; Alveolar Soft Part Sarcoma,ASPS=8; Synovial Sarcoma,SS=3; Chor- Research Center, Singapore, Singapore; Mie University School of doma,C =2; and other types,OT=6), were evaluable for TME changes Medicine, Tsu, Japan and correlated with clinical benefit (PR or SD). All patients were Correspondence: Hiroshi Shiku (shiku@clin.medic.mie-u.ac.jp) treated with durvalumab/tremelimumab every 4 weeks for four cy- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P359 cles and then continued durvalumab every 4 weeks for up to 1 year. Biopsies were collected prior to-and- during treatment (Wk6). Malig- Background nant cells (MCs) PD-L1+ was studied by immunohistochemistry. NY-ESO-1 is highly expressed in the majority of synovial sarcomas as Tumor-infiltrating-lymphocytes (TILs), and macrophages were interro- well as other solid tumors and may be an effective target for T cell- gated by multiplex immunofluorescence, Figure-1. The combination based therapies. We conducted a clinical study of adoptive transfer of three T-cell phenotypes (CD3+,CD3+CD8+ and CD3+CD8+GZB+) of lymphocytes transduced with NY-ESO-1-specific TCR in refractory greater than the median density as higher TILs (TILs+) was used to cancer patients with preconditioning (TBI-1301). define “inflamed” tumors and ≤ than the median of 1 or 2 of these Methods as lowest TILs (TILs–) to define “non-inflamed” tumors, through base- High-dose of 5 billion autologous transduced and expanded lympho- line to Wk6. To characterize patterns of the TME and changes be- cytes, consisting of >96% T cells, was transferred into 6 patients, three of tween baseline and Wk6 we stratified the tumors in four groups whom with synovial sarcoma. Longitudinal PBMC samples were obtained using an approach similar as Teng's criteria (1). for immunomonitoring. We used high-dimensional mass cytometry and Results combined a 36-antibody panel with a multiplexed combinatorial Overall, all the phenotype median densities increased from baseline to peptide-MHC tetramer staining approach to longitudinally track and Wk6, Table-1. PR or SD was observed in 17/36 with paired samples, phenotypically characterize adoptively transferred HLA-A*02:01-NY-ESO-1 47% (3 LS, 1 LMS, 1 OS, 7 ASPS, 1 SS, 2 C, and 2 OT). Five ASPS showed transgenic TCR T cells 14, 28, and 56 days after treatment. PR and 2 SD out of 8 cases. We categorized as inflamed tumor 1 LPS, 1 Results AS, 1 LMS, 1 OS, and 5 ASPS at baseline. Interestingly 1 AS, 1 OS, 1 CS, Three out of 6 patients with tumors expressing >75% NY-ESO-1 experi- 1ASPS, 1 SS and 2 OT defined as non-inflamed tumors at baseline chan- enced an objective clinical response (PR) and had cytokine-release syn- ged to inflamed tumors at Wk6 (Figure-2) and from those, OS and ASPS drome (CRS) with high-levels of IL-6 and MCP-1 that could be managed showed SD and PR, respectively. Finally, 4/17 inflamed tumors showed with tocilizumab. The infusion products had variable percentages of SD and 3/17 PR. Furthermore, CD3+CD8+CD45Ro+ increase in the in- naive, TEMRA and EM CD8+ cells, with the three clinical responders flamed tumors at Wk6 than non-inflamed tumors (P=0.005). The most having the highest proportion of EM T cells. NY-ESO-1 TCR transgenic T frequent TME pattern detected at baseline and Wk6 was the immuno- cells could be detected in the circulation of 5 out of 6 treated patients, logical ignorance (TILs-PD-L1-, 61% and 47%, respectively). Interestingly, with frequencies peaking at day 14 and day 28; specific T cells were un- adaptive immune resistance pattern changed from baseline to Wk6 detectable in all patients by day 56. In responders, a substantial num- (TILs+PD-L1+, 14% and 22%, respectively), Figure-3. ber of circulating NY-ESO-1-specific CD8+ T cells showed a phenotypic Conclusions profile consistent with antigen-experience, activation and differenti- Combination of durvalumab/tremelimumab influenced the TME in ation into an effector phenotype 28 days post transfusion. the selected sarcomas cohorts. The immunologic score assessment in Conclusions this longitudinal collection demonstrates the capability to distinguish Adoptive transfer of NY-ESO-1 TCR-transgenic T cells has shown signs non-inflamed vs inflamed tumors and relate it with a clinical benefit, of efficacy in patients with high NY-ESO-1 tumor expression, with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 197 of 272 showing the value of use these markers as possible immune prog- nostic markers in sarcomas. Trial Registration This trial is registered with ClinicalTrials.gov (NCT02815995) References 1. Teng MW, Ngiow SF, Ribas A, Smyth MJ. Classifying Cancers Based on T- cell Infiltration and PD-L1. Cancer Res. 2015;75(11):2139-45. Ethics Approval The study was approved by MD Anderson Institution Ethics Board, Clinical Trail number NCT02815995 Fig. 3 (abstract P360). See text for description Table 1 (abstract P360). See text for description P361 Molecular and immunologic profiling of CD8+ T cell responses in patients receiving a multiple antigen-engineered dendritic cell vaccine 1 2 2 Juraj Adamik, PhD , Patricia Santos, PhD , Samuel Du, BS , Lazar 2 1 3 Vujanovic, PhD , Timothy Howes , Sarah Warren, PhD , Andrea 2 2 1 Gambotto, MD , John Kirkwood, MD , Lisa Butterfield, PhD Parker Institute for Cancer Immunotherapy, San Francisco, CA, United States; University of Pittsburgh, Pittsburgh, PA, United States; NanoString Technologies, Seattle, WA, United States Correspondence: Lisa Butterfield (lbutterfield@parkerici.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P361 Background Despite the immunogenicity and safety profile of dendritic cell (DC) vaccines, the importance of vaccine-induced antigen-specific T cell responses is unclear across clinical trials, and therapeutic efficacy re- mains low with limited clinical responses. Our comprehensive characterization of T cell responses, cell-intrinsic and soluble immune checkpoint molecules and immune-related gene expression profiles reveal novel insights into CD8+ T cells specific for melanoma- associated antigens (MAA) from patients who received autologous DC engineered to express three full length melanoma antigens: tyro- sinase, MART-1 and MAGE-A6 [1]. Methods MAA-specific T cell responses were examined by standardized IFNγ Fig. 1 (abstract P360). See text for description ELISPOT assays at baseline, day 43 (post DC vaccines) and d89 (post observation or IFNα). Luminex was used to detect serum checkpoint and costimulatory molecules, and whole blood flow cytometry was used to quantify PBMC subsets. Targeted mRNA and protein expres- sion analyses in circulating lymphocytes and melanoma tumor sam- ples were performed using NanoString nCounter platform (RUO). Results The majority of the 35 patients were successfully vaccinated, and the total vaccine-induced T cell responses were higher among those exhibiting a favorable clinical outcome. Patients who received check- point blockade treatment prior to DC vaccination had higher base- line MAA-specific CD8+ T cell responses, yet they did not respond more strongly to the vaccine. Two patients who received checkpoint blockade post-DC vaccine showed very strong amplification of their MAA-specific T cells. Molecular profiling in circulating lymphocytes and tumor biopsies showed that elevated PD-1 and CTLA-4 protein levels and gene expression signatures representing checkpoint sig- naling, interferon response and T-cell exhaustion were associated with unfavorable clinical outcome. Gene signatures showing positive correlation with PD-1 protein expression included CD28-dependent PI3K-AKT signaling, the IL12/STAT4 pathway and pan-semaphorin re- ceptor interactions. CTLA-4 protein levels correlated with type I inter- feron response and NOTCH signaling genes. Interestingly, B cell receptor pathways negatively correlated with PD-1 expression, while gene signatures downstream of T cell receptor activation and IL-2 signaling were negatively correlated with CTLA-4 expression. Serum levels of PD-1 and PD-L2 were inversely correlated and post-vaccine Fig. 2 (abstract P360). See text for description serum levels PD-L2 correlated with decreased circulating Treg and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 198 of 272 favorable outcome in patients, suggesting that it may serve as a bio- (22.4%). Median follow-up was 21.2 months (range, 14.9-36.6). marker of clinical response. The ORR was 39.7% (95% CI: 30.7%-49.2%), including 19 patients Conclusions (16.4%) with a CR and 27 (23.3%) with a PR. In patients with PD- Collectively, our study shows that specific checkpoint molecular path- L1+ (n=21 [18.1%]) or PD-L1− (n=87 [75.0%]) tumors, ORRs were ways are critical for vaccine outcomes and for the activation of anti- 61.9% (95% CI: 38.4%-81.9%) and 33.3% (95% CI: 23.6%-44.3%), tumor responses in melanoma patients. Comprehensive profiling of respectively. Median DOR was 18.2 months (95% CI: 11.3 months- MAA-specific T cell responses suggests that DC-vaccine immunization not estimable). 35 patients had a response lasting ≥6months followed by immune checkpoint blockade may be an optimal se- (durable response rate, 30.2% [95% CI: 22.0%-39.4%]). 6- and 12- quential therapy to improve antitumor immunity in melanoma. month PFS rates were 41% (95% CI: 32%-50%) and 31% (95% CI: Trial Registration 23%-40%), respectively. Median OS was 20.3 months (95% CI: FDA IND #15044 and NCT01622933. 12.4 months-not evaluable), and the 12-month OS rate was 60% (95% CI: 50%-68%). In PD-L1+ and PD-L1− subgroups, 12-month Reference OS rates were 71% (95% CI: 47%-86%) and 56% (95% CI: 45%- 1. Butterfield LH, Vujanovic L, Santos PM, Maurer DM, Gambotto A, Lohr J, 66%), respectively. Treatment-related adverse events (TRAEs) of Li C, Waldman J, Chandran U, Lin Y, Lin H, Tawbi HA, Tarhini AA, any grade occurred in 94 patients (81.0%), including grade ≥3 Kirkwood JM. Multiple antigen-engineered DC vaccines with or without TRAEs in 21 (18.1%). No treatment-related deaths occurred. IFNa to promote antitumor immunity in melanoma. JITC. 2019; 7:113. Conclusions Ethics Approval Updated results from JAVELIN Merkel 200 confirm that first-line ave- The clinical trial was fully approved by the Univ. Pittsburgh PRC and IRB lumab treatment was associated with durable responses, a clinically (PRO12010416, #09–021). meaningful OS benefit, and an acceptable safety profile in patients with mMCC. P362 Acknowledgements First-line avelumab treatment in patients with metastatic Merkel This study was funded by Merck KGaA, Darmstadt, Germany, as part of an cell carcinoma: primary analysis after ≥15 months of follow-up alliance between Merck KGaA, Darmstadt, Germany and Pfizer Inc., New York, from JAVELIN Merkel 200, a registrational phase 2 trial NY, USA. 1 2 3 Sandra D'Angelo, MD , Celeste Lebbé , Laurent Mortier , Andrew Brohl, Trial Registration 4 5 6 7 8 MD , Nicola Fazio , Jean-Jacques Grob , Natalie Prinzi , Glenn Hanna , Registered at www.clinicaltrials.gov, NCT02155647 9 10 11 12 Jessica Hassel, MD , Felix Kiecker , Barbara Ellers-Lenz , Marcis Bajars , Ethics Approval 12 13 Meliessa Hennessy, MPH , Paul Nghiem, MD, PhD The trial was conducted in accordance with international good clinical Memorial Sloan Kettering Cancer Center, New York, NY, United States; practice standards and approved by the independent ethics committee at 2 3 Saint Louis Hospital, Paris, France; Lille Hospital–Claude Huriez Hospital, each participating center Lille Cedex, France; H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States; European Institute of Oncology (IEO), IRCCS, Milan, Italy; Aix-Marseille University, AP-HM Hospital, Marseille, P364 France; Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; A gp100 targeting TCR-based soluble T cell engaging bispecific 8 9 Dana-Farber Cancer Institute, Boston, MA, United States; Heidelberg induces mobilisation and activation of peripheral T cells in patients University Hospital, Heidelberg, Germany; Charité Universitätsmedizin with metastatic melanoma Berlin, Campus Charité Mitte, Berlin, Germany; Merck KGaA, Darmstadt, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc PhD, Sion Lewis, BSc MSc Germany; EMD Serono Research and Development Institut Inc, PhD, Mariantonella Vardeu, Jacob Hurst, PhD, Cheryl McAlpine, MSN Billerica, MA, United States; University of Washington Medical Center at Immunocore Ltd, Abingdon, United Kingdom South Lake Union, Seattle, WA, United States Correspondence: Sion Lewis (Sion.Lewis@immunocore.com) Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P364 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P362 Background Background ImmTAC® (immune-mobilizing monoclonal TCRs Against Cancer) Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine car- molecules are a new class of bispecific therapeutics, consisting of cinoma with a poor prognosis. In the pivotal phase 2 JAVELIN Merkel a high affinity TCR fused to an anti-CD3 single-chain variable 200 trial (NCT02155647), avelumab, a human anti–PD-L1 monoclonal fragment (scFv) T cell-activating moiety [1]. Tebentafusp (gp100 antibody, yielded durable responses in patients with metastatic MCC antigen specific ImmTAC) can elicit a polyfunctional T cell re- (mMCC) who had received prior chemotherapy (part A), and a high sponse and has demonstrated monotherapy activity in advanced objective response rate (ORR) in an initial subgroup treated in the metastatic melanoma [2-5]. Clinical benefit was associated with a first-line metastatic setting (part B), leading to regulatory approval reduction in peripheral CXCR3+ T cells and concurrent increase in worldwide. Here we report the primary analysis of JAVELIN Merkel serum CXCL-10 [6, 7]. The chemokine receptor CCR5 is under- 200 part B after ≥15 months of follow-up in the full patient stood to drive T cell extravasation and potentialy synsergise with population. CXCR3 to promote tumour infiltration [8, 9]. Therefore the aim of Methods this study was to investigate the effect of tebentafusp on the dy- Eligible patients had no prior systemic therapy for mMCC and were namics of T cell mobilisation and activation in metastatic melan- enrolled irrespective of biomarker status; PD-L1+ status was defined oma patients. as ≥1% expression in tumor cells (PD-L1 IHC 73-10 pharmDx assay). Methods All patients received avelumab 10 mg/kg IV every 2 weeks. The pri- HLA-A2+ patients with metastatic melanoma were enrolled on a mary endpoint was durable response, defined as objective response first-in-human, multicentre, Phase I/II, open-label, dose-finding (complete response [CR] or partial response [PR] per RECIST v1.1, ad- study (NCT01211262). Immunophenotypic analysis was under- judicated by independent endpoint review committee) lasting ≥6 taken to assess baseline levels and pharmacodynamic changes months. Secondary endpoints included best overall response, dur- in peripheral immune subsets. PBMC samples were analysed by ation of response (DOR), progression-free survival (PFS), overall sur- flow cytometry from patients at baseline (n=38) and on- vival (OS), and safety. treatment (n=22) over the first dosing cycle, and from age/sex- Results matched healthy control subjects (n=18). Data is reported on T At data cut-off on May 2, 2019, 116 patients had been treated cell populations, markers of activation (CD25) and function with avelumab. Median treatment duration was 5.5 months (CCR5, Ki67) and represented as mean ± standard deviation of (range, 0.5-35.4), and treatment was ongoing in 26 patients subset frequency or percentage change from baseline, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 199 of 272 relationships with overall survival (OS) assessed by univariate vaccine adjuvants to support T cell responses to peptide vaccines. Cox proportional hazards model. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC and/ Results or low-dose metronomic cyclophosphamide (mCy) would be safe Tebentafusp induced T cell extravasation within 24hrs (p and would support strong and durable CD4+ T cell responses in T cells from patients on-treatment exhibited an increase in activation combination with an incomplete Freund’s adjuvant (IFA). marker expression and an expansion of memory and effector T cell Methods subsets (p<0.05). An adaptive design based upon toxicity and durable immune re- Conclusions sponse (dRsp) was used to assign participants with resected stage Tebentafusp administratation induces the rapid extravasation of che- IIA-IV melanoma to one of four study regimens, including a vaccine mokine receptor expressing T cells and the expansion and activation comprising 6 melanoma peptides restricted by Class II MHC (6MHP), of memory T cells. The association between clinical benefit and base- administered in an emulsion with IFA (Montanide ISA-51), with or line levels of peripheral immune subsets may aid our mechanistic un- without the TLR3 agonist polyICLC and with or without systemic derstanding of its anti-tumour activity in metastatic melanoma mCy. Toxicities were recorded (CTCAE v4). T cell responses were mea- patients. sured in peripheral blood lymphocytes (PBL) and in vaccine-site Trial Registration draining lymph node (sentinel immunized node, SIN) with IFNγ ELI- NCT01211262 spot assay ex vivo. Serum antibody responses to 6MHP were mea- sured by ELISA, and changes in circulating regulatory T cells were References assessed by flow cytometry. 1. Lowe KL, et al. Cancer Treat Rev. 2019. Results 2. Boudousquie C, et al. Immunology 2017;152:425–38. Forty-eight eligible patients were enrolled and treated. Following an 3. Middleton M, et al. Presented at ASCO 2016. Abstract 3016. adaptive design, early safety data and T cell response data favored en- 4. Carvajal R, et al. Presented at SITC 2017. Abstract P208. rollment on arm D. At study conclusion, total enrollment was 3, 7, 6, 5. Middleton M, et al. Presented at ASCO 2019. Abstract 9523. and 32 individuals for arms A-D, respectively. Treatment-related dose- 6. Middleton M, et al. Presented at ASCO 2019. Abstract 9530 limiting toxicities (DLTs) were observed in 1/7 (14%) patients on arm B 7. Mullins et al, Cancer Res 2004; 64, 7697-7701 and 2/32 (6%) on arm D, with no treatment arm exceeding the DLT 8. Hong M et al, Cancer Res 2011; 71, 22:6997-7009 25% threshold for early stopping. Strong and durable T cell responses 9. Harlin, Cancer Res 2009;69(7):3077–85: to 6MHP were detected ex vivo in 0%, 29%, 50%, and 50% of patients Ethics Approval enrolled on arms A-D, respectively (Table 1). IgG antibody responses This study was approved by following institutions’ Ethics Boards: were also induced and were greatest for arms C and D (Figure 1). Circu- lating regulatory T cell frequencies were not altered by use of mCy. Oxfordshire Research Ethics Committee; 10/H0604/47, Approved Conclusions June 4, 2010. Combination vaccine adjuvants with IFA, polyICLC, and mCy were Mary Crowley Cancer Research Center; MCMRC IRB # 12-06, Ap- well-tolerated. The dRsp rate for arm D (IFA + polyICLC + mCy) of proved March 16, 2012. 50% (90% CI: [34,66]) exceeded the 18% dRsp rate (90% CI: [11,26]) Human Investigation Committee, Yale University; HIC Protocol # from prior experience with 6MHP in IFA alone. The regimen with IFA 1302011504, Approved March 22, 2012. + pICLC alone also showed promise for enhancing T cell and anti- IntegReview; Protocol No IMCgp100/01, Approved November 13, body responses. Addition of mCy does not alter circulating T reg fre- 2013. quencies but shows some promise as a systemic vaccine adjuvant. Western Sydney Local Health District; HREC2012/7/4.1 (3552) AU RED HREC/12/WMEAD/237, Approved on October 24, Acknowledgements 2012. We thank the Cancer Research Institute/ Ludwig Institute for Cancer Western Institutional Review Board; Panel 1, Study Num 1147687, Research for providing the polyICLC used in the vaccines. Funding was WIRB Pro Num 20141184, Approved July 15, 2014. provided by NCI R01 CA178846 (CLS); 5K25CA181638 (NW); P30 CA044579 Memorial Sloan Kettering Cancer Center, Institutional Review (Biorepository and Tissue Research Facility, Office of Clinical Research, and Board; Protocol # 14-152, August 28, 2014. Biostatistics Shared Resource). Trial Registration The clinical trial Mel63 is registered with Clinicaltrials.gov (NCT02425306). P365 Ethics Approval A trial to evaluate the immunogenicity and safety of a melanoma The clinical trial Mel63 was performed with IRB (#17860) and FDA approval helper peptide vaccine plus incomplete Freund’s adjuvant, (IND #10825). cyclophosphamide, and polyICLC (Mel63) 1 1 1 Craig Slingluff, MD , Gina Petroni, PhD , Kimberly Chianese-Bullock, PhD , 1 1 1 1 Nolan Wages, PhD , Walter Olson, PhD , Kelly Smith , Lynn Dengel, MD , 1 2 1 Anna Dickinson , Caroline Reed , Elizabeth Gaughan, MD , William 1 1 1 1 Grosh, MD , Varinder Kaur, MD , Nikole Varhegyi , Mark Smolkin , 1 1 Table 1 (abstract P365). T cell responses to 6MHP Nadejda Galeassi , Donna Deacon, BS 1 2 University of Virginia, Charlottesville, VA, United States; Emory University School of Medicine, Atlanta, GA, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P365 Background Cancer vaccines require adjuvants to induce effective and durable protective immunity. However, there is no consensus on optimal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 200 of 272 assay. Circulating antibody (Ab) responses to overlapping NY-ESO-1 peptides were detected by ELISA. Tumor biopsies obtained pre- treatment and day 85 were evaluated for immune infiltrates by multi- spectral immunofluorescence histology. Results Target enrollment was 27; study closed early for slow enrollment. Eight patients enrolled and were treated (Table 1). All had ≥ 1 treat- ment emergent adverse event (TEAE); most common (≥50%): rash, fatigue, injection site reaction, pruritus, and diarrhea. Two patients had Gr3 TEAEs related to IPI but not to vaccine. There were no DLTs. Best responses: SD (n=4); PD (n=4). T cell responses to NY-ESO-1 were detected in 6 of 8 (75%) patients.[1] Both patients without T cell response had PD as best response. Ab responses were detected in 7/8 (88%) patients (Table 1). The patient without Ab response had PD as best response. The breadth of Ab responses to NY-ESO-1 was greater for patients with SD than those with PD (p = 0.02). Evaluation of TME of 5 patients revealed increases in proliferating (Ki67+) CD8 T cells, decreases in RORγt+ CD4+ T cells (Figure 1). Interestingly, there were increases in density of CD8+ and CD4+ cells for those with SD (n=3), but decreases for those with PD (n=2, not shown). Conclusions T cell responses and Ab responses to NY-ESO-1 were induced in most patients and were evident ex vivo, suggesting that IPI may have en- hanced the T cell responses to NY-ESO-1 protein and OLP4. Inte- grated T cell and antibody responses were associated with tumor control. Preliminary data of the TME suggests increased activating and proliferating T cells after vaccination plus IPI, especially in pa- tients with tumor control. Acknowledgements The trial was supported by the Ludwig Institute for Cancer Research, the Fig. 1 (abstract P365). Antibody responses to 6MHP Cancer Research Institute, and by the National Institutes of Health, including support from the University of Virginia Cancer Center Support Grant (NIH/NCI P30 CA44579:, Clinical Trials Office, Biorepository and Tissue Procurement Facility, Flow Cytometry Core, and Biomolecular Core Facility). P366 Earlier presentation of results of this clinical trial [1] has been expanded in A phase 1 study of NY-ESO-1 vaccine + ipilimumab (ipi) in patients the present abstract with additional biologic correlates. with unresectable or metastatic melanoma 1 2 3 Trial Registration Craig Slingluff, MD , Hassane Zarour, MD , Michael Postow, MD , Philip 4 5 1 This trial was registered at ClinicalTrials.gov (NCT01810016). Friedlander, MD PhD , Craig Devoe, MD , Ileana Mauldin, PhD , Kelly 1 6 Smith , Mary Macri, BSc 1 2 Reference University of Virginia, Charlottesville, VA, United States; University of 1. Slingluff CL Jr, Zarour HM, Postow MA, Friedlander P, Devoe CE, Macri M, Pittsburgh Cancer Center, Pittsburgh, PA, United States; Memorial Sloan Ryan A, Venhaus R, Wolchok J. J Clin Oncol. 2018; 36(suppl; abstr e15175). Kettering Cancer Center, New York, NY, United States; Mount Sinai Ethics Approval Medical Center, New York, NY, United States; Northwell Health Cancer The study was approved by each institution's Ethics Board, with approval Institute, Lake Success, NY, United States; Ludwig Institute for Cancer numbers: IRB#12-253 (Memorial Sloan Kettering), HS#13-00471 (Mount Research, New York, NY, United States Sinai), IRB#14-133B (Northwell Health), MOD13030240-02/ Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) PRO13030240(University of Pittsburgh), and HRS#16347(University of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P366 Virginia), and to the FDA with IND 10369. Background Ipilimumab (IPI) is an approved immunotherapy for advanced melan- oma. It can enhance immunity to cancer-testis antigen NY-ESO-1. Vac- Table 1 (abstract P366). Enrollment, immune and clinical responses cines with NY-ESO-1 protein or NY-ESO-1 overlapping long peptides (OLP4) have enhanced immunity when administered with Montanide ISA-51 (Montanide) and/or Poly-ICLC (pICLC) adjuvants. This trial assessed safety, immunogenicity, clinical responses (irRC), and effects of IPI + NY-ESO-1 vaccines on the tumor microenvironment (TME). Methods This Phase 1, open-label study enrolled patients among 3 arms: IPI (3 mg/kg i.v. q3 wks x 4) + NY-ESO-1 protein + pICLC + Montanide (Arm A); IPI + NY-ESO-1 OLP4 + pICLC + Montanide (Arm B); and IPI + NY-ESO-1 OLP4 + pICLC (Arm C). Patients had measurable NY-ESO- 1+ tumors. Treatments were administered days 1, 22, 43, 64. Circulat- ing T cell responses were assessed by ex vivo IFN-gamma ELIspot Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 201 of 272 Conclusions Seviprotimut-L is very well tolerated. Subgroup efficacy analyses identi- fied two populations who may benefit from Seviprotimut-L: those with AJCC stage IIB/IIC melanoma and those under age 60. These data support proceeding to the definitive final part of the MAVIS phase III trial testing seviprotimut-L for stage IIB/C patients, in particular those under age 60. Acknowledgements We acknowledge the support of all investigators and clinical coordinators responsible for enrolling patients to this trial. Trial Registration This trial was registered at ClinicalTrials.gov: NCT01546571. References 1. Bystryn JC, Zeleniuch-Jacquotte A, Oratz R et al. Double-blind trial of a Fig. 1 (abstract P366). See text for description polyvalent, shed-antigen, melanoma vaccine.[see comment]. Clinical Can- cer Research 2001; 7: 1882-1887. P367 2. Dorshkind K, Swain S. Age-associated declines in immune system devel- A multicenter, double-blind, placebo-controlled trial of opment and function: causes, consequences, and reversal. Curr Opin seviprotimut-L polyvalent melanoma vaccine in post-resection Immunol 2009; 21: 404-407. melanoma patients at high risk of recurrence 1 2 3 Ethics Approval Craig Slingluff, MD , Brent Blumenstein, PhD , Karl Lewis, MD , Robert 4 5 The study was approved by the Ethics Board at each participating institution (IRB#), as Andtbacka, MD, CM, FACS, FRCSC , John Hyngstrom, MD , Mohammed 6 7 8 follows: University of Virginia Hospital (16223); Anschutz Cancer Pavilion, UC Milhem, MBBS , Svetomir Markovic, MD, PhD , Omid Hamid, MD , Leonel 9 10 11 Denver(1134601); Huntsman Cancer Institute, / Univ of Utah Health Care (55911); Hernandez-Aya, MD PhD , Tawnya Bowles, MD , Prejesh Philips, MD , 12 13 14 University of Iowa Hospitals and Clinics (1133782); Mayo Clinic Cancer Center / Joel Claveau, MD , Sekwon Jang, MD , Jose Lutzky, MD, FACP , Anna 15 16 Mayo Clinic Rochester (12-002308); The Angeles Clinic and Research Institute Bar, MD , Peter Beitsch, MD 1 2 (1196134); Washington University School of Medicine (201205056); Intermountain University of Virginia, Charlottesville, VA, United States; Tri Arc Medical Center (1024288); University of Louisville (15.0039); CHU de Quebec, Consulting, Washington, DC, United States; University of Colorado, L'Hotel Dieu de Quebec (MP-20-2015-2318); Inova Melanoma and Skin Cancer Aurora, CO, United States; Seven and Eight Biopharmaceuticals, Salt Center (1152774); Mount Sinai Medical Center (12-16-H-03); Oregon Health and Lake City, UT, United States; Huntsman Cancer Institute/ Univ of Utah, Science University (IRB00011848); Cancer Solutions (1197259). Salt Lake City, UT, United States; University of Iowa Hospitals and Clinics, Iowa City, IA, United States; Mayo Clinic Rochester, Rochester, MN, United States; The Angeles Clinic & Research Institute, Los Angeles, Table 1 (abstract P367). Enrollment and adverse events CA, United States; Washington University School of Medicine, Saint Louis, MO, United States; Intermountain Medical Center, Murray, UT, United States; University of Louisville, Louisville, KY, United States; 12 13 CHU de Quebec, L'Hotel Dieu de Quebec, Quebec, Canada; Inova Melanoma and Skin Center, Fairfax, VA, United States; Mount Sinai Medical Center, Miami Beach, FL, United States; Oregon Health and Science University, Portland, OR, United States; Cancer Solutions, Dallas, TX, United States Correspondence: Craig Slingluff (CLS8H@hscmail.mcc.virginia.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P367 Background Seviprotimut-L is a vaccine prepared from antigens shed by 3 human mel- anoma cell lines, administered with alum. Prior formulations showed prom- ising immunogenicity for T cell and antibody responses and improved survival in a small phase II clinical trial[1]. Part B1 of MAVIS (Melanoma Anti- gen Vaccine Immunotherapy Study, a three part, Phase III clinical program), was a multicenter, double-blind, placebo-controlled trial to assess the effi- cacy of seviprotimut-L, with the primary endpoint of relapse-free survival (RFS) in patients at high risk of recurrence after definitive surgical resection. Methods For MAVIS Part B1, patients with AJCC v7 stage IIB-III cutaneous melan- oma, after surgical resection, age 18-75, ECOG PS 0-1, were randomized 2:1 to seviprotimut-L 40 mcg or placebo, administered subcutaneously every 2 weeks x 5, then monthly x 4, then every 3 months to month 24. Patients were stratified by stage (IIB/C, IIIA, IIIB/C). Target enrollment was 325. The study was powered for assessment of RFS, with target hazard ratio (HR) of 0.625, one-sided alpha of 0.10, and power 80%. Results 347 patients were randomized, and arms were well-balanced. Treatment- emergent adverse events (AEs) were similar for seviprotimut-L and placebo patients (Table 1). By intent-to-treat (ITT) analysis, RFS was not significantly enhanced for seviprotimut-L in the full study population, but trended slightly higher (Figure 1A). Analysis of subgroups based on pre-planned stratification suggested enhanced RFS for seviprotimut-L among Stage IIB/IIC patients (HR 0.59, 95% CI[0.33,1.07], Figure 1B). Age has been identified as a cause of de- creased immune competence[2]; thus, outcomes were assessed as a function of age as an effect modifier. Figures 1C and 1D show all randomized patients Fig. 1 (abstract P367). Clinical outcome (Figure 1C) and Stage IIB/IIC subset (Figure 1D) by arm and age split at Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 202 of 272 Clinical Trial In Progress 3. Bernhardt SL, Gjertsen MK, Trachsel S, et al. Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose es- P368 calating phase I/II study. Br J Cancer. 2006;95:1474-1482 ZI-H04 - A novel MHC class II restricted TCR based cellular therapy targeting hTERT to treat solid tumours Jens-Peter Marschner, MD, Mona Welschof, PhD, Miguel Forte, Eva P369 Kristine Klemsdal, Sylvie Pollmann, Namir Hassan Feasibility of a phase I personalized adoptive T-cell therapy in Zelluna, Seeheim-Jugenheim, Germany patients with relapsed/refractory solid tumors 1 3 Correspondence: Jens-Peter Marschner Apostolia Tsimberidou, MD, PhD , Ali Mohamed , Stephen Eck, MD, 4 4 1 1 (jenspeter.marschner@zelluna.com) PhD , Harpreet Singh , Patrick Hwu, MD , Cassian Yee, MD , Borje Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P368 Andersson, MD, PhD 1 2 MD Anderson Cancer Center, Houston, TX, United States; MD Background Anderson, Houston, TX, United States; Immatics Biotechnology, Chimeric Antigen Receptor T-cells (CAR-T) are highly effective in the Tubingen, Germany; Immatics, Houston, TX, United States treatment of some hematological malignancies but solid tumors re- Correspondence: Apostolia Tsimberidou (atsimber@mdanderson.org) main a challenge for cellular therapies. A few T-cell Receptor T- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P369 cells (TCR-T) have been investigated in solid tumors. To our knowledge, there is only one published study and one case re- Background port using MHC Class II restricted TCRs targeting MAGE-A3 and Adoptive cellular therapy (ACT) is limited in solid tumors due to lack of NY-ESO-1, respectively [1,2]. suitable immunotherapy targets with high specificity and frequent re- lapse following immunotherapy to single targets often associated with Methods loss of target expression in the tumor. ACTolog® is a personalized, ZI-H04 represents a novel approach of TCR based therapies. multi-targeted ACT approach in which autologous T-cell products are Autologous T-cells from patients are genetically modified by manufactured against the most relevant tumor target peptides for indi- lentiviral transduction to express the TCR targeting hTERT in vidual patients whose tumors are positive against predefined targets. the context of the MHC Class II allele, HLA-DPB1*04:01. The Methods TCR was isolated from a pancreatic cancer patient who experi- Patients with advanced metastatic cancers and HLA-A*02:01 enced clinical benefit following a peptide-based cancer vaccin- phenotype, undergo a tumor biopsy. Patients whose tumors ex- ation against hTERT [3]. The TCR clone responded to press >1 of 8 cancer targets undergo leukapheresis. Autologous T autologous tumor and preclinical data demonstrate that ZI-H04 cells are primed against the expressed ACTolog targets in the exhibits high sensitivity to hTERT peptide as well as recogni- presence of IL-21 followed by HLA tetramer-guided cell sorting tion of processed antigen. Furthermore, specificity analysis sup- and rapid expansion. Patients who meet criteria for treatment re- ports the safety of the TCR-T. The restricted combined ceive lymphodepletion with Fludarabine 40 mg/m2 i.v. and Cyclo- expression of hTERT plus HLA class II on normal cells limits the phosphamide 500 mg/m2 i.v. (Days, -6 to -3). T-cells are infused potential for on-target off tumor toxicity. A first-in-human on Day 0, followed by low-dose of IL-2 for 14 days (www.clinical- study is designed to treat patients with relapsed/refractory trials.gov NCT02876510). solid tumors lacking an option of further treatments. Patients Results must be tested positive for HLA-DPB1*04:01 and the tumors From 7/2017 to 7/2019, 203 patients signed an informed consent to must express hTERT. Adequate organ function and lab parame- participate in the study, 91 had HLA-A*02:01 phenotype, 52 had a ters are required. CNS involvement, autoimmune diseases, in- tumor biopsy and 34 patients underwent leukapheresis. To date, 9 fections and immunosuppressive medication are main exclusion patients have received treatment (median age, 38 yrs; range, 25-58 criteria. Primary objectives are safety and tolerability. Part 1 of yrs; 2 men and 7 women; breast cancer, 3; sarcoma, 3; ovarian can- the study, starting in 2020, will be a dose finding part, Part 2 cer, 1; nasopharyngeal, 1; anal carcinoma, 1; median time from diag- a dose extension part with 5 cohorts. Prior to adoptive cell in- nosis 4 years, range, 2-18 years; median number of prior therapies 6, fusion patients will receive a low dose conditioning regimen range 3-12). Very high ACTolog cell doses could be administered. Pa- consisting of 2 x 600 mg/m² cyclophosphamide followed by 3 tients received a median of 2 target-specific ACTolog products (range x 25 mg/m² fludarabine. Patients will be observed for safety, 1-3). Treatment was overall well tolerated. The most common ad- efficacy and exploratory biomarkers. verse events were cytopenias and cytokine release syndrome. All pa- Conclusions tients are alive to date. At 6 weeks, restaging imaging studies ZI-H04 is a novel TCR-T with potentially favourable characteristics. demonstrated stable disease in all patients. One patient with squa- The TCR was isolated from an hTERT vaccinated pancreatic cancer mous cell carcinoma of the anus treated with T cells directed to patient that experienced clinical benefit. A lower probability of COL6A3, exon 6, and PRAME had 26% decrease in tumor measure- off-target activity is expected since no engineering was done to ments at week 6 associated with high T-cell frequencies at 2 weeks the TCR. The MHC Class II restriction provides the possibility to but her disease subsequently progressed. Another patient with naso- induce a multi-pronged immune response including antigen pharyngeal cancer treated with COL6A3 tumor stroma-specific T cells spreading as demonstrated in a case report using an MHC Class had resolution of tumor associated pain and has not required further II TCR-T in melanoma [2]. More than 50% of patients are HLA- treatment for 11 months. A recent tumor biopsy demonstrated nec- DPB1*04:01 positive and the expression rate of hTERT is >80% in rotic cells and no tumor cells could be identified. many tumors. Therefore, a substantial population may benefit Conclusions from ZI-H04 treatment. ACTolog IMA101 is well-tolerated and no safety issues have been noted to date. The study is ongoing. References Trial Registration 1. Lu Y-C, Parker LL, Lu T, et al. Treatment of patients with metastatic cancer www.clinicaltrials.gov NCT02876510 using a major histocompatibility complex class II-restricted T-cell receptor Ethics Approval targeting the cancer germline antigen MAGE-A3. J Clin Oncol. The study was approved by MD Anderson's IRB. 2017;35:3322-3329 Consent 2. Hunder NN, Wallen H, Cao J, et al. Treatment of metastatic melanoma Written informed consent was obtained from the patient for publica- with autologous CD4+ T cells against NY-ESO-1. N Engl J Med. tion of this abstract and any accompanying images. A copy of the 2008;358:2698-703 written consent is available for review by the Editor of this journal. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 203 of 272 P370 Trial Registration The positive correlation between baseline absolute eosinophil NCT02791334 count (AEC) in blood and clinical benefit to PD-(L)1 inhibition monotherapy Reference 1 1 1 Anna Szpurka, PhD , Danni Yu, PhD , Michelle Carlsen , Antoine Tanizaki, Junko, Koji Haratani, Hidetoshi Hayashi, Yasutaka Chiba, Yasushi 2 3 4 Hollebecque, MD , Hyun Cheol Chung, MD, PhD , Amita Patnaik , Nakamura, Kimio Yonesaka, Keita Kudo, et al. "Peripheral Blood 5 6 7 Johanna Bendell, MD , Antoine Italiano, MD , Yung-Jue Bang, MD PhD , Biomarkers Associated with Clinical outcome in Non–Small Cell Lung 8 9 10 Chia-Chi Lin, MD, PhD , Marcus Butler, MD , Timothy Yap, MD PhD , Cancer Patients Treated with Nivolumab." Journal of Thoracic Oncology 11 11 María José de Miguel, MD , María José de Miguel, MD , Jean-Pascal 13, no. 1 (2018/01/01/ 2018): 97-105. 12 13 14 Machiels, MD, PhD , Marc Peeters, MD, PhD , Wu-Chou Su, MD , Ethics Approval 15 1 1 Victor Moreno, MD , Yumin Zhao, PhD , Erik Rasmussen, PhD , Xiaojian The study included multiple investigator sites, and some of these had Xu, MD approval dates instead of approval numbers. The following are listed 1 2 Eli Lilly and Company, Indianapolis, IN, United States; University of Paris showing approving committee followed by approval date or approval # Sud, Villejuif, France; Yonsei University College of Medicine, Seoul, (if available): IntegReview, 15Jun2016; IntegReview, 21June2016; MD Korea, Republic of; South Texas Accelerated Research Therape, San Anderson Office of Protocol Approval IRB, 26May2016; Princess Margaret Antonio, TX, United States; Sarah Cannon Research Institute, Nashville, Cancer Centre, University Health Network Research Ethics Board, 16-5824 6 7 TN, United States; Institut Bergonié, Bordeaux, France; Seoul National (initial approval date 01Feb2017); Comite de protection des Personnes University Hospital, Seoul, Korea; National Taiwan University Hospital, (CPP), EudraCT number 2016-000440-33. Taipei, Taiwan, Province of China; Princess Margaret Cancer Center, Toronto, Canada; The University of Texas, Houston, TX, United States; 11 12 START-HM Sanchinarro, Madrid, Spain; jean- pascal.machiels@uclouvain.be, Brussels, Belgium; Antwerp University Hospital,, Antwerp, Belgium; National Cheng Kung University Hospital, Tainan, Taiwan, Province of China; START Madrid-FJD, Madrid, Spain Correspondence: Danni Yu (yu_danni@lilly.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P370 Background Anti-PD-(L)1 immunotherapies have increased the response rate in certain cancer subtypes however, some patients who may have clin- ical benefit are not identifiable with existing predictive biomarkers. Research is ongoing to identify routinely available blood and clinical markers to predict response to PD-(L)1 therapies. In this study, we ex- Fig. 1 (abstract P370). See text for description plored absolute eosinophil count (AEC) as a biomarker in patient’s re- sponse to PD-(L)1 treatment. Methods P371 This is a phase 1a/1b study of an anti-PD-L1 antibody (LY3300054) Cytokine Microdialysis for real-time immune monitoring in administered alone or in combination with other agents in patients Glioblastoma patients undergoing Checkpoint Blockade 1 2 2 with advanced refractory solid tumors. Eligible patients were ≥18 John Lynes, MD , Victoria Sanchez , Anthony Nwankwo , Gifty 2 2 2 2 years old, had ECOG status ≤1 and had at least 1 measurable lesion Dominah , Xiang Wang, MS , Isac Kunnath , Samantha Dill , Gretchen 2 2 2 2 2 per RECIST v1.1. We assessed the association of AEC with confirmed Scott , Christi Hayes , Tianxia Wu , Marta Penas-Prado , Jing Wu , Eric 2 2 2 2 best overall response (BORc). The AEC cutoff 0.155 (10^9/L) maxi- Burton , John Heiss , Christopher Hourigan, MD, PhD , Mark Gilbert , mized the difference in ORR, similar to previous reports (Tanizaki etal, Edjah Nduom, MD 1 2 2018, JTO [13] e85-e86) . The impact of AEC status was demonstrated Georgetown University, Bethesda, MD, United States; National by a 3-dimensional waterfall plot depicting the best change in tumor Institutes of Health, Bethesda, MD, United States size and overall survival (OS). Correspondence: Edjah Nduom (edjah.nduom@nih.gov) We also tested the correlation between AEC and OS/proxy-progres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P371 sion-free survival (PFS; time to next treatment, TTNT) in NSCLC pa- tients (n=455) who received anti-PD-1 therapy with same cutoff from Background an independent Flatiron database. Glioblastoma is the most common primary malignancy of the brain, Results with a dismal prognosis. Immunomodulation via checkpoint inhibition As of 8 December 2017, 30 patients (MSI-H: n=22, M: n=8) were has provided encouraging results in non-CNS malignancies, but predic- treated. There were no deaths due to adverse events. Two patients tion of responders has proven to be challenging in glioblastoma pa- in MSI-H cohort experienced grade 3 treatment-related AEs (TRAEs): tients. OBJECTIVES: To determine the proportion of patients who have diarrhea (n=1, 4.5%), blood creatinine phosphokinase increased (n=1, a measurable increase of interferon gamma levels in brain tumor tissue 4.5%), and hyponatraemia (n=1, 4.5%). No grade 3 events were re- after their first dose of nivolumab; to evaluate the safety of using brain ported in M cohort, and no grade 4/5 TRAEs were reported in either tumor microdialysis to monitor for immune response; to evaluate the cohorts. There were no TRAEs leading to discontinuation of study safety of the combination of anti-programmed death 1 (PD-1) and anti- treatment. Preliminary efficacy data in MSI-H cohort showed ORR of lymphocyte activation gene 3 (LAG-3) checkpoint inhibition in recurrent 36% [CR in 1 pt (5%)(ovarian), PR in 7 pts (32%)(small intestine glioblastoma patients. adenocarcinoma [1 pt], endometrial [3 pts], colon [3 pts])], DCR in Methods 64% [SD in 6 pts (27%)]; mPFS was 7.39 months (95% CI 1.7, NR). In The study design is a single-center, nonrandomized phase 1 clinical the M cohort, DCR was 63% [PR in 1 pt (13%), SD in 4 pts (50%)]. As trial. Up to 20 adult patients with recurrent glioblastoma will be en- of data cut-off, 16 pts (53%) remain on treatment. Preliminary bio- rolled with the goal of 10 patients completing the trial over an antici- marker analysis, including but not limited to, PD-L1 and CD8 expres- pated 18 months. Patients will undergo biopsy; placement of sion and circulating markers will be presented. microdialysis catheters and lumbar drains; treatment with anti-PD-1 Conclusions checkpoint inhibition; comprehensive immune biomarker collection; LY3300054 was well-tolerated and demonstrated antitumor activity tumor resection; and then treatment with anti-PD-1 and anti-LAG-3 in patients with MSI-H solid tumors; combination expansions are checkpoint inhibition until progression (Figure 1). Three patients ongoing. have undergone all study procedures (Figures 2 and 3). There have Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 204 of 272 been no serious adverse events related to the research surgical pro- cedure, nor during the microdialysis portion of the trial. Enrollment is ongoing. EXPECTED OUTCOMES: We expect interferon gamma levels to in- crease in the brain as measured via microdialysis in treated patients. Based on published reports, microdialysis in this patient population is expected to be safe, and anti-LAG-3 and anti-PD-1 combined will likely have a similar side effect profile to other checkpoint inhibitor combinations. The failure of recent trials of immune therapies in glioblastoma un- derscores the need to appropriately measure response in the treated tissue. This trial may provide insight on indicators of which patients will respond to immune therapy. Acknowledgements Funding and support came from: Intramural Research Program of the Na- tional Institute of Neurological Disorders and Stroke Trial Registration Clinicaltrials.gov: NCT03493932 (Registration Date: April 11, 2018) Fig. 3 (abstract P371). See text for description Ethics Approval Institutional Approvals: National Institutes of Health Combined Neurosciences Internal Review Board number - 18-N-0077 P372 Circulating immune cell biomarkers predict response to immune checkpoint inhibitor therapy in metastatic breast cancer Jin Sun Bitar, MD, Colt Egelston, PhD, Susan Yost, Wanqiu Hou, Padam Simran, Paul Frankel, PhD, Mina Sedrak, Jana Portnow, MD, Joanne Mortimer, MD, Christina Yeon, MD, Arti Hurria, MD, Aileen Tang, Norma Martinez, Peter Lee, MD, Yuan Yuan City of Hope, Duarte, CA, United States Correspondence: Yuan Yuan (yuyuan@coh.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P372 Background The role of immune checkpoint PD-1/PD-L1 inhibitor (ICI) in breast cancer (BC) is being investigated in clinical trials. Preclinical evidence Fig. 1 (abstract P371). See text for description strongly supports the synergistic effects of CDK4/6 inhibitor and ICI [1]. A phase II trial is testing the safety and efficacy of the combin- ation of letrozole, palbociclib and pembrolizumab in patients with hormone receptor positive (HR+) BC (NCT02778685). Currently, there is no well-defined circulating biomarker to predict response to ICI. Methods Peripheral blood mononuclear cells (PBMC) were collected at day 1 of cycles 1 (pre-treatment), 2, 4, 6 and 8. The comprehensive characterization of circulating immune cell composition was per- formed using 15-color flow cytometry. Results Preliminary analysis included 9 patients with the following responses by RECIST 1.1: 1 complete response, 4 partial response, 2 stable disease, and 2 progressive disease. Higher baseline frequencies of CD4+ effector memory (p=0.01) and CD8+ CD45RA+ effector memory cells (p=0.01) were observed in patient responders. Additionally, patient responders demonstrated higher fre- quencies of T cells expressing KLRG1, a marker of effector T cell differenti- ation, on both CD4+ (p=0.001) and CD8+ T cells (p=0.004) at baseline. An increase in the frequency of circulating CXCR5+ CD8+ T cells (p=0.01) at cycle 2 was identified in all treated patients and an increase in CCR10+ CD8+ T cells (p=0.03) was detected at cycle 2 in patient responders indi- cating changes in T cell trafficking. Finally, a shift in myeloid cell compos- ition from predominantly classical to non-classical monocytes was observed in patient responders between baseline and cycle 2 (p=0.007). Conclusions High baseline levels of both CD4+ and CD8+ effector T cells indicate the necessity for a pre-existing favorable T cell composition in check- point blockade responders. Temporal changes in T cell trafficking mole- Fig. 2 (abstract P371). See text for description cules and shifts in myeloid cell composition over the course of therapy Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 205 of 272 indicate potential changes in T cell priming and regulation. Further ana- 2. Bernstein V, Ellard SL, Dent SF, et al. A randomized phase II study of lysis is currently ongoing to understand correlates of systemic immune weekly paclitaxel with or without pelareorep in patients with metastatic changes and changes in the tumor microenvironment. breast cancer: final analysis of Canadian Cancer Trials Group IND.213. Breast Cancer Res Treat 2018;167:485-93. Trial Registration 3. Nuciforo, P., Pascual, T., Cortés, J., Llombart-Cussac, A., Fasani, R., Paré, L., NCT02778685 … Holgado, E. (2018). A predictive model of pathologic response based on tumor cellularity and tumor-infiltrating lymphocytes (CelTIL) in HER2- Reference positive breast cancer treated with chemo-free dual HER2 blockade. An- 1. Goel S, DeCristo MJ, Watt AC, et al. CDK4/6 inhibition triggers anti- nals of Oncology. https://doi.org/10.1093/annonc/mdx647 tumour immunity. Nature. 2017;548(7668):471–475. Ethics Approval Ethics Approval This study was approved by the Spanish Health Authority, protocol number The study was approved by City of Hope National Cancer Center‘s Ethics 2018-003345-42. Board, approval number 16058 P373 A window-of-opportunity study of pelareorep in early breast cancer (AWARE-1) 1 2 3 4 Luis Manso , Patricia Villagrasa , Nuria Chic , Juan Cejalvo , Yann 5 5 6 3 Izarzugaza , Blanca Cantos , Salvador Blanch , Manel Juan , Blanca 3 7 8 7 Gonzalez , Rita Laeufle , Gerard Nuovo , Grey Wilkinson, PhD , Matt 7 3 3 2 2 Coffey , Azucena Gonzalez , Patricia Galvan , Laia Paré , Jordi Canes , 9 3 6 Xavier Gonzalez , Aleix Prat, MD PhD , Joaquín Gavilá 1 2 Hospital Universitario 12 de Octubre, Madrid, Spain; SOLTI Breast Cancer Research Group, Barcelona, Spain; Hospital Clinic, Barcelona, 4 5 Spain; Hospital Clínico, Valencia, Spain; Hospital Universitario, Madrid, Spain; Instituto Valenciano de Oncología (IVO), Valencia, Spain; 7 8 Oncolytics Biotech Inc., San Diego, CA, United States; Ohio State University, Columbus, OH, United States; Hospital Universitari, Sant Cugat del Vallés, Spain Fig. 1 (abstract P373). See text for description Correspondence: Aleix Prat (ALPRAT@clinic.cat) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P373 Background P374 Pelareorep is an intravenously delivered (IV) unmodified oncolytic reo- TCR repertoires from peripheral blood correlate with prognostic virus. Clinical studies have demonstrated that IV delivered pelareorep can response in TNBC cancer vaccine immunotherapy replicate in tumor tissue and promote an inflamed tumor phenotype Sadanand Vodala, PhD, Andrew Nguyen, PhD, Noe Rodriguez, Peter characterized the recruitment of CD8+ T cells and upregulation of PD-L1 Sieling, Charles Vaske, Jon Van Lew, Kayvan Niazi, John Lee, MD, Patrick [1]. Consistent with pelareorep’s role in promoting adaptive anti-tumor Soon-Shiong, MD, FRCS, FACS, Shahrooz Rabizadeh immunity, a randomized phase 2 study in metastatic breast cancer dem- ImmunityBio, Inc, Culver City, CA, United States onstrated a statistically significant improvement in overall survival when Correspondence: Kayvan Niazi (kayvan.niazi@immunitybio.com) pelareorep was combined with paclitaxel [2]. We hypothesize that pelar- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P374 eorep mediated anti-tumor immune responses, such as those mediated by T cells, represent a novel strategy for the control or elimination of Background tumor cells in breast cancer. Specifically, in the preoperative setting for TNBC is an aggressive, heterogeneous, and high-grade subtype that rep- early breast cancer, we examined if pelareorep in combination with anti- resents 10-20% of breast carcinomas. Recently, TECENTRIQ and Abraxane PD-L1 therapy, atezolizumab, and other breast cancer therapies offers were approved for the treatment of PD-L1+ unresectable locally ad- clinical benefit in terms of CeLTIL score, a metric for quantifying tumor vanced or mTNBC suggesting a role for immunotherapy in the treatment cellularity (Cel) and tumor-infiltrating lymphocytes (TIL) [3]. of this disease. Growing evidence suggests that chemotherapeutic agents Methods and immunotherapies synergize in patients. Cancer vaccines are also a This exploratory, non-randomized, window of opportunity study, will promising option for TNBC due to the discovery of neo-antigens and evaluate the safety and effect of pelareorep ± atezolizumab on the tumor tumor associated antigens that mobilize anti-tumor T cells. microenvironment in 38 women with early breast cancer. Patients will re- Methods ceive study treatment for ~21 days prior to definitive surgery or neoadju- Here, we characterize T cell receptor repertoires in patients enrolled in a vant chemotherapy. Five cohorts will be examined (Figure 1): Cohort 1: Phase Ib/2 clinical trial (NCT03387085) following treatment by a regimen HR+/HER2-neg (10 patients), pelareorep + letrozole. Cohort 2: HR+/HER2- intended to induce a synchronized, multi-compartment, anti-tumor im- neg (10 patients): pelareorep + letrozole + atezolizumab. Cohort 3: TNBC mune response by mitigating the immune-suppressive effects of the (6 patients): pelareorep + atezolizumab. Cohort 4: HER2+/HR+ (6 pa- tumor microenvironment and activating immunogenic tumor cell death. tients): pelareorep + trastuzumab + atezolizumab. Cohort 5: HER2+/HR- The trial combined metronomic low-dose chemotherapy, SBRT, an allo- (6 patients): pelareorep + trastuzumab + atezolizumab. CelTIL, viral repli- geneic NK cell line expressing high affinity CD16, yeast and adenoviral cation, and other immune-based biomarkers will be used to examine tumor-associated antigen vaccines, an IL15RαFc super-agonist, check- treatment-related changes within the tumor microenvironment. Blood point inhibition, and an anti-angiogenic agent in a manner predicted to and tumor tissue biopsies will be collected at screening, Day 3 (after maximize cytotoxic T-cell mediated immunological recognition of tumor pelareorep but before atezolizumab), and at surgery (Day ~21). cells. Tumor associated antigens included adenoviral vector-based CEA, Trial Registration MUC1, brachyury, and yeast-based brachyury and CEA vaccines. Blood Spanish clinical studies registry: 2018-003345-42 was collected pre- and post-treatment and target lesion analysis was per- formed using irRC and Recist1.1. Total RNA from PBMCs was used to gen- References erate sequencing libraries from each longitudinal sample. Using NGS, 1. Samson A, Scott KJ, Taggart D, et al. Intravenous delivery of oncolytic TCR-α and -β CDR3s were clonotyped and tracked over serial blood reovirus to brain tumor patients immunologically primes for subsequent draws. Additionally the Shannon-Wiener Diversity Index (SWDI) was calcu- checkpoint blockade. Sci Transl Med 2018;10. lated for each time point. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 206 of 272 Results (T+CTX) versus 1 experimental arm (M+CTX) + either MGA012 or Patient samples showing consistent positive responses by irRC/Recist1.1 MGD013, depending on the interim analysis from part 1; with 250 pa- showed emergence and persistence of new TCR clones post-induction. tients each. The primary efficacy endpoint for cohort A (both parts) is This was further reflected in acute surges in SWDI. Furthermore, a high ORR per RECIST 1.1; for cohort B part 2 it is overall survival. SWDI at baseline and post-treatment indicated clinical benefit suggest- ing an inverse correlation between disease severity and peripheral rep- Acknowledgements ertoire diversity. A TNBC super responder showed dramatic increases in The authors thank all the patients, their families, and the entire staff who are mean SWDI index from 74 prior treatment to 1177 at first biopsy post participating in this trial. Professional medical writing support was provided treatment (34% decrease by irRC and 26% by Recist1.1 analysis) and by Meredith Rogers, MS, CMPP, of The Lockwood Group (Stamford, achieved an index as high as 3516 in a subsequent biopsy (83% and Connecticut, USA), in accordance with Good Publication Practice (GPP3) 64% decrease by irRC and Recist 1.1 respectively). guidelines, with funding by MacroGenics, Inc. (Rockville, MD, USA). Trial Registration Conclusions NCT number to come Our findings strongly suggest that peripheral blood TCR repertoires are prognostic indicators/biomarker for TNBC cancer vaccine immunotherapy References and for T cell-based immunotherapy in general. Taken together, these re- 1. Nordstrom JL, Gorlatov S, Zhang W, et al. Anti-tumor activity and toxico- sults strongly indicate activation and expansion of anti-tumor T cell clones kinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with en- following combination therapy. Further functional studies will expand our hanced Fcγ receptor binding properties. Breast Cancer Res. 2011;13:R123. understanding of T cell based cancer vaccine immunotherapy in TNBC. 2. Nordstrom JL, Muth J, Erskine CL, et al. High frequency of HER2-specific immunity observed in patients (pts) with HER2+ cancers treated with margetuximab (M), an Fc-enhanced anti-HER2 monoclonal antibody P375 (mAb). J Clin Oncol. 2019;37(suppl; abstr 1030). Margetuximab combined with anti-PD-1 (MGA012) or anti-PD-1/LAG-3 3. Catenacci DVT, Limet KH, Uronis HE, al. Antitumor activity of (MGD013) +/- chemotherapy in first-line therapy of advanced/metastatic margetuximab (M) plus pembrolizumab (P) in patients (pts) with HER2+ gastroesophageal junction (GEJ) or gastric cancer (GC) 1 2 2 advanced HER2+ (IHC3+) gastric carcinoma (GC). J Clin Oncol. Daniel Catenacci, MD , Minori Rosales , Jon Wigginton, MD ,HyunCheol 3 4 5 6 7 2019;37(suppl 4; abstr 65). Chung, MD, PhD , Harry Yoon ,Lin Shen , Yoon-Koo Kang ,MarkusMoehler 1 2 4. Fuchs CS, Doi T, Jang RW, et al. Safety and efficacy of pembrolizumab University of Chicago, Chicago, IL, United States; MacroGenics, Inc. monotherapy in patients with previously treated advanced gastric and Rockville, MD, United States; University College of Medicine, Seoul, gastroesophageal junction cancer: phase 2 clinical KEYNOTE-059 trial. Korea, Republic of; Mayo Clinic Cancer Center, Rochester, MN, United 5 6 JAMA Oncol. 2018;4:e180013. States; Beijing Cancer Hospital, Beijing, China; Asan Medical, Seoul, 5. Kang YK, Boku N, Satoh T, et al. Nivolumab in patients with advanced Korea, Republic of; University Medical Center Mainz, Mainz, Germany gastric or gastro-oesophageal junction cancer refractory to, or intolerant Correspondence: Daniel Catenacci (dcatenac@bsd.uchicago.edu) of, at least two previous chemotherapy regimens (ONO-4538-12, ATTR Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P375 ACTION-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;390:2461-2471. Background Ethics Approval Trastuzumab (T), a monoclonal antibody (mAb) targeting the human Each investigator’s institutional review/ethics board approved the study. epidermal growth factor receptor 2 (HER2) is the standard of care pal- liative first-line therapy for advanced HER2+ GEJ and GC patients. Mar- getuximab (M) is an Fc-engineered anti-HER2 mAb targeting the same P376 HER2 epitope, but with higher affinity for both 158V (high binding) and NOUS-209: A phase I, first-in-human, multicenter, open-label study 158F (low binding) alleles of the activating Fc receptor CD16A. Even of Nous-209 genetic vaccine for the treatment of microsatellite more, M coordinately enhanced both innate and adaptive immunity, in- unstable solid tumors cluding antigen-specific T-cell responses to HER2 [1,2]. Programmed cell Anna Gorrasi, PhD, Elisa Scarelli, Maria Teresa Catanese, Cinzia Traboni, death receptor 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) are Paola Antonini, Denis Brkic both T-cell checkpoint molecules that suppress T-cell function. MGA012 Nouscom Srl, Rome, Italy (INCMGA00012) is a humanized, hinge-stabilized, IgG4κanti-PD-1 mAb Correspondence: Elisa Scarelli (booking@nouscom.com) blocking binding of PD-L1 or PD-L2 to PD-1. MGD013 is a humanized Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P376 Fc-bearing bispecific tetravalent protein that concomitantly binds to PD-1 and LAG-3, inhibiting their respective ligand-binding. We previ- Background ously reported that a chemotherapy (CTX)-free regimen consisting of MSI-H tumors are caused by a defective DNA mismatch repair M+PD-1 blockade was well tolerated in GEJ/GC patients, and induced a (dMMR) system that leads to the accumulation of mutations within 30% objective response rate (ORR) [3]. This was 2- to 3-fold greater than microsatellite regions. Insertions or deletions (indels) in microsatel- in historical controls with checkpoint inhibitors alone [4,5]. This lites of coding regions can result in the synthesis of tumor-specific registration-directed trial investigates the efficacy, safety, and tolerabil- frameshift peptides (FSPs). FSPs are considered safe and potent ity of M+checkpoint inhibition ± CTX in metastatic/locally advanced, neoantigens because they are not expressed in the normal human treatment-naïve, HER2+ GEJ/GC patients. proteome. We selected shared FSPs among patients with MSI cancers Methods with the aim of developing an off-the-shelf vaccine for the cure of This adaptive open-label phase 2/3 study includes 2 cohorts. In the first MSI tumors. 209 FSPs were assembled into 4 artificial genes and single arm, CTX-free cohort A, M+MGA012 is evaluated in HER2+ (im- cloned into 4 Great Apes Adenoviral (GAd) and 4 Modified Vaccinia munohistochemistry [IHC] 3+) and PD-L1+ (excluding microsatellite in- Ankara (MVA) vectors to generate a viral vectored vaccine called stability high) patients. After 40 patients are evaluated for response/ Nous-209. We showed that treatment of tumor-bearing mice with safety, 60 more patients will be enrolled if the threshold for study con- GAd/MVA-based neoantigen vaccines synergizes with Checkpoint In- tinuation is met. In the randomized cohort B, HER2+ (IHC 3+ or IHC 2+/ hibitors (CPI), resulting in a 3-fold-increase of cured animals over CPI fluorescent in situ hybridization+) patients, are enrolled irrespective of monotherapy. PD-L1 status. Part 1 randomizes patients to 1 of 4 arms (50 patients Methods each): control arm (T+CTX) or 1 experimental arm (M+CTX; A Phase-I, FIH study was designed to evaluate the safety, tolerability, M+CTX+MGA012; M+CTX+MGD013). CTX is investigator’s choice of and immunogenicity of Nous-209 genetic polyvalent vaccine in XELOX or mFOLFOX-6. Part 2 (pick-the-winner) consists of the control Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 207 of 272 combination with the licensed programmed death receptor-1 (PD-1)- Conclusions blocking antibody pembrolizumab and to detect any preliminary evi- The combination of vactosertib plus pembrolizumab was tolerable dence of anti-tumor activity. Nous-209 is administered intramuscu- with no additional safety concern. The activity of this combination in larly, with a heterologous prime/boost regimen composed of 1 prime CRC and GC patients will be further evaluated in the Dose Expansion with the mixture of 4 GAd vectors (GAd20-209-FSP) and 3 boosts part of the study. Clinical trial information: NCT03724851 with 4 MVA vectors (MVA-209-FSP). The target population includes Trial Registration adult patients with unresectable or metastatic dMMR or MSI-H colo- NCT03724851 rectal cancer (CRC), gastric, and gastroesophageal (G-E) junction Ethics Approval tumors. The study was approved by Ethics Board from Asan Medical Center, The study is composed of two sequential cohorts. In the first cohort Samsung Seoul Hospital, Severance Hospital, Seoul National Univer- (dose escalation), the Recommended Phase 2 Dose (RP2D) will be sity Bundang Hospital, and National Cancer Center, with approval established. In the second part (dose expansion), additional patients number 2018-1215, SMC 2018-07-146-006, 4-2018-0728, B-1808/487- will be evaluated to consolidate the safety of the RP2D and establish 003 and NCC2019-0042, respectively. the immunogenicity of the vaccination. NOUS-209 IND has been cleared by the US Food and Drug Adminis- P378 tration (FDA). The trial will be enrolling up to 30 patients at US clin- Phase II study of combination ipilimumab, nivolumab, and ical sites. Preliminary results from the study are expected in early panitumumab in patients with KRAS, NRAS, and BRAF wild-type (WT) microsatellite stable (MSS) metastatic colorectal cancer (mCRC) 1 2 3 P377 Michael Lee, MD , Patrick Loehrer, MD , Iman Imanirad, MD , Stacey 4 5 1 Safety and anti-tumor activity of the transforming growth factor β Cohen, MD , Kristen Ciombor, MD , Cheryl Carlson, MD,PhD , Hanna 1 1 receptor I kinase inhibitor, vactosertib, in combination with Sanoff, MD , Autumn McRee, MD pembrolizumab in patients with metastatic colorectal or gastric Univ of North Carolina at Chapel Hill, Chapel Hill, NC, United States; 2 3 cancer Indiana University, Indianapolis, IN, United States; Moffitt Cancer 1 2 3 4 Keun-Wook Lee, MD , Young Suk Park, MD, PhD , Joong Bae Ahn , Sun Center, Tampa, FL, United States; University of Washington, Seattle, WA, 3 4 4 5 5 Young Rha , Hark Kyun Kim , Park Young Lee , Min-Hee Ryu , Jeeyun United States; Vanderbilt University, Nashville, TN, United States 2 6 6 6 Lee, MD, PhD , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kiim , Tae Correspondence: Michael Lee (michael_s_lee@med.unc.edu) Won Kim, MD, PhD Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P378 Seoul National University Bundang Hospit, Seongnam, Korea, Republic 2 3 of; Samsung Medical Center, Seoul, Korea, Republic of; Severance Background Hospital, Seoul, Korea, Republic of; National Cancer Center, Kyunggi-do, Panitumumab is an IgG2 monoclonal antibody (mAb) targeting the Korea, Republic of; Asan Medical Center, Seoul, Korea, Republic of; epidermal growth factor receptor (EGFR) and is a standard therapy Medpacto, Inc, Seoul, Korea, Republic of for patients with KRAS, NRAS, and BRAF WT mCRC. Preclinical data Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) shows that anti-EGFR mAbs require functional innate and adaptive Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P377 immunity to mediate efficacy. Anti-EGFR therapy causes a tumor- specific adaptive immune response and immunogenic apoptosis Background [1,2], and anti-EGFR antibodies require functional T cells for in vivo Vactosertib is a highly selective and potent inhibitor of transforming efficacy [3]. However, resistance to anti-EGFR therapy inevitably de- growth factor beta (TGF-β) receptor type 1. Recent studies have re- velops and is associated with increased regulatory T cells expressing vealed that inhibition of TGF-β signaling reverses immunosuppres- CTLA-4 [4] and activated immunosuppressive macrophages with up- sive tumor microenvironment and poor responses to cancer regulation of PD-L1 [5]. Thus, resistance to anti-EGFR antibody ther- immunotherapy. To date, antitumor efficacy by immune check point apy is associated with increased expression of both CTLA-4 and PD- inhibitors in colorectal or gastric/gastroesophageal cancer as mono- L1. We hypothesized that treatment with ipilimumab (anti-CTLA-4) therapy is known to be limited. A combination of TGF-β and PD-1 in- and nivolumab (anti-PD-1) synergizes with panitumumab to signifi- hibition may induce immune restoration and improve antitumor cantly improve the response rate in patients with KRAS, NRAS, and responses. We are reporting Dose Finding part of Phase 1b/2a study BRAF WT MSS mCRC. evaluating the combination of vactosertib plus pembrolizumab in Methods metastatic colorectal cancer (CRC) or diffuse gastric cancer (GC). LCCC1632 is a multicenter, single-arm, phase II clinical trial with a Methods pre-specified safety run-in of panitumumab, ipilimumab, and nivolu- Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and have mab in KRAS/NRAS/BRAF WT, MSS mCRC (NCT03442569). Eligible pa- no prior exposure to immunotherapy including anti-CTLA-4, anti-PD-1, tients must have received 1-2 prior lines of therapy and no prior anti-PD-L1, and TGFβR1 kinase inhibitors. The primary objective is to as- anti-EGFR or immune checkpoint inhibitor therapy. A 6-subject safety sess the safety and the recommended dose of vactosertib given 5 days run-in was treated with ipilimumab 1 mg/kg IV q6wk, nivolumab 240 on/2 days off in combination with pembrolizumab 200 mg every 3 mg IV q2wk, and panitumumab 6 mg/kg IV q2wk and observed for weeks. The Dose Finding part starts with vactosertib 200 mg BID plus 12 weeks for dose-limiting toxicities (DLTs), followed by expansion pembrolizumab. Secondary objectives include characterization of vac- into a Simon’s two stage phase II trial, with 26 more subjects enrolled tosertib pharmacokinetics and anti-tumor activity by response rate. in the first phase and 56 total subjects planned. The primary end- Results point is response rate defined by RECIST 1.1. Secondary endpoints in- As of July 8, 2019, among 10 patients enrolled to 200 mg BID cohort, 6 clude response rate by irRECIST, progression-free survival (PFS), were with CRC and 4 diffuse type GC. Median age was 51 (range 31-71), overall survival (OS), and duration of response. Correlative studies in- 50% were male, median number of previous lines of chemotherapy was 4 clude Consensus Molecular Subtype analysis by archival tissue and (range 2-6). All patients were immune checkpoint inhibitor naïve. No dose assessment of peripheral immune cell activation. limiting toxicity was reported. Common adverse events (AE) were anorexia Results (33%), fatigue (33%), abdominal pain (33%), and fever (33%). There were 3 Within the 12-week DLT period, only one grade 3-4 toxicity (grade 3 serious adverse events (SAE) reported; bilirubin elevations (1), pleural effu- increased lipase) and no DLTs were observed. The most common sion (1), and ileus (1). All SAEs were not related to the study drugs. One pa- grade 1-2 treatment-related adverse events within the DLT period in- tient (1/6) with microsatellite stable (MSS) metastatic CRC achieved partial cluded acneiform rash, hypomagnesemia, decreased lymphocyte response. Biomarker data will be presented at the meeting. count, anemia, nausea, vomiting, hypothyroidism, fatigue, cough, oral Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 208 of 272 mucositis, and elevated AST. No dose modifications were required. CD73 levels will derive greater benefit from CD73 inhibition. Immu- Five of the 6 subjects (83%) had disease control (1 unconfirmed par- nohistochemistry analyses were performed on serial sections of tial response, 4 stable disease) at 12 weeks. tumor tissue to correlate protein and gene expression. Conclusions Results The combination of panitumumab, ipilimumab, and nivolumab was The HV study enrolled more than 50 participants, randomized 3:1 (ac- well-tolerated, without unexpected toxicities encountered in the tive: placebo). AB680 exhibited a good safety profile and displayed a safety run-in cohort. The response rate and disease control rate dem- long half-life following a 30-60 minute intravenous (IV) infusion, con- onstrate early signs of clinical activity. Enrollment in the phase II ex- sistent with the intended Q2W dosing schedule in cancer patients. pansion is ongoing. Doses were identified that provided maximal inhibition of peripheral Trial Registration AMP-ase activity. Our bioinformatics analyses identified tumors that ClinicalTrials.gov Identifier: NCT03442569 have high CD73 expression relative to TNAP and identified pan-RAS mutations that correlate with upregulated CD73 and poor prognosis. References Conclusions 1. Garrido G, Rabasa A, Sanchez B, et al. Induction of immunogenic AB680 is the first potent and selective small-molecule CD73 inhibitor apoptosis by blockade of epidermal growth factor receptor activation to be tested in humans. This first-in-human study demonstrates that with a specific antibody. J Immunol 2011;187:4954-66. AB680 is well tolerated and has optimal PK/PD to support its contin- 2. Pozzi C, Cuomo A, Spadoni I, et al. The EGFR-specific antibody cetuximab ued evaluation in cancer patients. combined with chemotherapy triggers immunogenic cell death. Nat Trial Registration Med 2016;22:624-31. ClinicalTrials.gov NCT03677973 3. Garrido G, Lorenzano P, Sanchez B, et al. T cells are crucial for the anti- Ethics Approval metastatic effect of anti-epidermal growth factor receptor antibodies. The study was approved by Bellberry Limited Ethics Board, approval Cancer Immunol Immunother 2007;56:1701-10. number 2018-08-673 4. Jie HB, Schuler PJ, Lee SC, et al. CTLA-4(+) Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cyto- toxicity and Correlate with Poor Prognosis. Cancer Res 2015;75:2200-10. P380 5. Pander J, Heusinkveld M, van der Straaten T, et al. Activation of tumor- Phase 1b/2 study of BXCL701, a small molecule inhibitor of promoting type 2 macrophages by EGFR-targeting antibody cetuximab. dipeptidyl peptidases, with bempegaldesleukin (bempeg, NKTR- Clin Cancer Res 2011;17:5668-73. 214) and avelumab (anti-PD-L1) in unresectable or metastatic Ethics Approval pancreatic cancer 1 1 2 3 The study was approved by the Institutional Review Board of the University of North Louis Weiner, MD , Benjamin Weinberg, MD , Stina Singel , Cedric Burg , 3 2 3 Carolina at Chapel Hill (IRB number 17-1832) and by the IRB of each subsite. Diane Healey , Jonathan Zalevsky, PhD , Chetan Lathia , Willem 2 4 2 Overwijk, PhD , Cristian Massacesi , Joyce Acbay , John MacDougall, 3 3 PhD , Vincent O'Neill P379 Georgetown Lombardi Comprehensive Cancer Center, Washington, DC, Phase 1 safety study in healthy volunteers of AB680, a small- United States; Nektar Therapeutics, San Francisco, CA, United States; 3 4 molecule inhibitor of CD73 and rationale for combination therapy BioXcel Therapeutics, New Haven, CT, United States; Pfizer, New York, in patients with gastrointestinal malignancies NY, United States Devika Ashok, PhD, Irene Luu, Akshata Udyavar, PhD, Lixia Jin, Lijuan Fu, Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) Elaine Ginn, Ken Lawson, Jenna Jeffrey, PhD, Manmohan Leleti, PhD, Jay Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P380 Powers, PhD, Eric Connor, Andy Pennell, Daniel DiRenzo, PhD, Dana Piovesan, MSc, Joanne Tan, PhD, Amanda Garofalo, Wade Berry, BA, Background Matthew Walters, PhD, Steve Young, PhD, Fangfang Yin, PhD, Dominic Treatment of pancreatic cancer continues to have poor outcomes with Lai, Lisa Seitz, MA currently available therapies including checkpoint inhibitors. BXCL701 Arcus Biosciences, Hayward, CA, United States (talabostat, previously PT100) is an orally administered, small molecule Correspondence: Dominic Lai (dlai@arcusbio.com) inhibitor of dipeptidyl peptidases (DPP) specifically DPP4, DPP8 and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P379 DPP9. Inhibition of DPP8 and DPP9 triggers a process in macrophages called pyroptosis leading to innate immune proinflammatory stimula- Background tion of the tumor microenvironment[1,2,3]. BXCL701 also inhibits fibro- Extracellular adenosine, present at high concentrations in the tumor blast activation protein (FAP) releasing the FAP-mediated block of T-cell microenvironment (TME), suppresses immune function. The enzymes migration into the tumor[4]. In syngeneic animal models, significant ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phos- tumor responses were observed when BXCL701 was used with check- phatase (TNAP) catalyze extracellular conversion of adenosine mono- point inhibition[2]. Bempegaldesleukin (bempeg, NKTR-214) is a CD122- phosphate (AMP) into adenosine. Inhibition of CD73 eliminates a preferential interleukin-2 (IL-2) pathway agonist being investigated for major pathway of adenosine production in the TME and can reverse its potential to leverage the clinically validated IL-2 pathway and select- adenosine‐mediated immune suppression. Here we present the first ively stimulate an immune response, without overacting the immune results from a Phase 1 healthy volunteer (HV) study of AB680, a po- system. Bempeg has demonstrated robust anti-cancer activity when tent, reversible and selective small-molecule CD73 inhibitor. This used with checkpoint inhibition in multiple murine tumor models and placebo-controlled HV study assessed the safety, tolerability, pharma- recently in multiple human cancers[5,6]. Avelumab is a checkpoint in- cokinetic (PK) and pharmacodynamic (PD) profile of AB680. hibitor that binds PD-L1 resulting in the release of immune inhibitory Methods effects of this pathway thereby restoring immune responses, including Male or female healthy volunteers aged 18-55 with a body mass anti-cancer immune responses. In a syngeneic mouse model of pancre- index of 18-30 kg/m2 were eligible for enrollment in the atic cancer (Pan02), the triple combination demonstrated potent anti- AB680CSP0001 study (NCT03677973). Escalating doses of AB680 were cancer activity, including long-lasting anti-cancer immunity[7]. These re- evaluated in a single ascending dose (SAD) and repeat dosing study. sults provide therapeutic rationale for testing of this combination in pa- Post dosing, participants were admitted for evaluation and serially tients with pancreatic cancer. assessed for adverse events. Blood samples were collected at various Methods timepoints to elucidate the PK and PD profiles of AB680. AB680 This is an open-label, multicenter study to determine the safety and effi- plasma concentrations were determined using LC-MS/MS and PD ef- cacy of the triple combination therapy of BXCL701, bempeg and avelu- fects were evaluated by monitoring AMP-ase activity in serum. Linear mab. Patients with pancreatic cancer should have received at least 1 line models were used to predict tumor up-regulation of CD73 in mul- of gemcitabine-based therapy and no more than 2 lines of chemother- tiple tumor types with the assumption that tumors expressing higher apy for unresectable or metastatic disease, received no prior anti-PD-1/ Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 209 of 272 PD-L1, IL-2 based or other T-cell directed anti-cancer therapy, and have observed when BXCL701 was used in combination with checkpoint in- ECOG 0-1. Patients must agree to biopsy of metastatic disease. Part 1 hibition[2]. Therefore, it is believed BXCL701 mediated activation of the (Phase 1b), is the 3+3 dose escalation phase designed to evaluate the innate immune system via macrophage pyroptosis inflames the cancer safety of escalating doses of BXCL701 with bempeg and avelumab. Part microenvironment and t-SCNC might become responsive to checkpoint 2 (Phase 2) will begin once the recommended combination dose is de- inhibition combined with BXCL701. termined. A Simon two-stage design will be used in Phase 2, initially en- Methods rolling 13 patients. If 2 or more responses are observed, the cohort will This is an open-label, multicenter study in patients with progressive, expand to 34 patients. The primary efficacy parameter is objective re- metastatic castration resistant prostate cancer (CRPC) as defined by sponse by RECIST 1.1. The study will also assess other parameters meas- PCWG3. Patients should have received at least 1 line of systemic uring clinical benefit and mechanistic effects on the immune system therapy and no more than 2 lines of cytotoxic chemotherapy for and tumor microenvironment. The study is not yet recruiting in the US. CRPC, received no prior anti-PD-1/PD-L1 or other T-cell directed anti- Trial Registration cancer therapy, and have ECOG 0-2. Patients in Phase 2 must also Pending have evidence of SCNC,NEPC by central pathology and agree to bi- opsy of metastatic disease. Phase 1b, is the 3+3 dose escalation References phase designed to evaluate the safety of 0.4 mg and 0.6mg BXCL701 1. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP QD on days 1 to 14 of 21-day cycle plus fixed dose pembrolizumab inhibitor, and immune checkpoint inhibitors discovered using AI and Big 200 mg administered IV on day 1 every 21 days to determine the Data analytics. Cancer Research 2017;77(13 Suppl):Abstract nr 2629. recommended dose for phase 2. A Simon’s two-stage design will be 2. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- used in Phase 2 and initially 15 patients with SCNC/NEPC will be en- caspase-1-dependent monocyte and macrophage pyroptosis. Nature rolled. If more than 2 responses are observed, then the cohort will Chemical Biology. 2017;13(1):46-53 expand to 28 patients. Primary efficacy parameter is the composite 3. Okondo M, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the response defined as achieving 1 or more of the following: • Objective Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 response by RECIST 1.1 • CTC conversion from > 5/7.5 mL to < 5/7.5 4. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- mL per Veridex assay by Week 12 • Greater than 50% PSA decline rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. from baseline by Week 12. The study is open in the US with expan- 2015;75(14):2800-2810. sion to the UK underway. 5. Charych D, Khalili S, Dixit V et al. Modeling the receptor pharmacology, Trial Registration pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically- NCT03910660 controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. EUDRACT:2018-003734-32 PLOS ONE 2017; 12(7): e0179431. 6. Diab A, Tannir N, Bernatchez C, et al. A phase ½ study of a novel IL-2 References cytokine, NKTR-214, and nivolumab in patients with select locally ad- 1. Aggarwal R, Huang J, Alumkal JJ, et al. Clinical and Genomic vanced or metastatic solid tumors. J Clin Oncol. 2017;35(suppl):e14040. Characterization of Treatment-Emergent Small-Cell Neuroendocrine Pros- 7. Rastelli L, Gupta S, Jagga Z, et al. Efficacy and immune modulation by tate Cancer: A Multi-institutional Prospective Study. J Clin Oncol. 2018; BXCL701 and dipeptidyl peptidase inhibitor, NKTR-214 a CD122-biased 36(24): 2492-2505 immune agonist with PD1 blockage in murine pancreatic tumors [ab- 2. Rastelli L, Gupta S, Dahiya A, et al. The synergy between BXCL701, a DPP stract]. J Clin Oncol. 2018;36(suppl):3085 inhibitor, and immune checkpoint inhibitors discovered using AI and Big Ethics Approval Data analytics [abstract]. In: Proceedings of the American Association for This study was approved by Institution Review Boards or Ethics Committees Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. affiliated with participating institutions Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2629. 3. Okondo M, Johnson D, Sridharan R, et al. DPP8/9 inhibition induces pro- caspase-1-dependent monocyte and macrophage pyroptosis. Nature P381 Chemical Biology. 2017;13(1):46-53 Phase 1b/2 study of BXCL701, a small molecule inhibitor of 4. OkondoM, Rao S, Taapazuing C, et al. Inhibition of DPP8/9 Activates the dipeptidyl peptidases (DPP), with pembrolizumab, (anti-PD-1) Nlrp1b Inflammasome. Cell Chemical Biology. 2018;25:1-6 monoclonal antibody, in small cell neuroendocrine prostate cancer 5. Lo A, Wang LC, Scholler J, et al. Tumor-Promoting Desmoplasia Is Dis- (SCNC, NEPC) rupted by Depleting FAP-Expressing Stromal Cells. Cancer Research. 1 2 2 Diane Healey , Rahul Aggarwal, MD , Rahul Aggarwal, MD , Vincent 2015;75(14):2800-2810. 1 1 1 3 O'Neill , Cedric Burg , Diane Healey , Jiaoti Huang , Johann De Bono, 6. cBioPortal version 1.4.3 (dataset accessed on 9th March, 2017) 4 2 MD , Eric Small Ethics Approval 1 2 BioXcel Therapeutics, New Haven, CT, United States; UCSF Helen Diller The study was approved by Institution Review Boards or Ethics Committees Family Comprehensive Cancer Center, San Francisco, CA, United States; affiliated with participating institutions. Duke University School of Medicine, Durham, NC, United States; Institute for Cancer Research Royal Marsden NHS Foundation Trust, London, United Kingdom P382 Correspondence: Diane Healey (dhealey@bioxceltherapeutics.com) KEYNOTE-365 cohort D: phase 1b/2 study of pembrolizumab plus Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P381 abiraterone acetate and prednisone in metastatic castration- resistant prostate cancer 1 2 3 Background Leonard Appleman, MD, PhD , Josep Piulats , Nataliya Mar , José 4 5 6 7 Treatment emergent Small Cell Neuroendocrine Prostate Cancer (t-SCNC) Arranz , Anthony Joshua, MD , Tina Mayer , Neal Shore, MD , Haiyan Wu, 8 8 9 is an aggressive with poor survival outcomes on standard therapies given PhD , Charles Schloss , Evan Yu 1 2 for metastatic castration-resistant disease[1 ]. BXCL701 (talabostat previ- UPMC Hillman Cancer Center, Pittsburgh, PA, United States; Catalan ously PT100) is an orally administered, small molecule inhibitor of dipepti- Cancer Institute, Barcelona, Spain; UC Irvine Medical Center, Orange, CA, dyl peptidases (DPP) specifically DPP4, DPP8 and DPP9 triggering Uunited States; Hospital General Universitario Gregorio Marañon, macrophage cell death via pyroptosis resulting in proinflammatory stimu- Madrid, Spain; St. Vincent’s Hospital Sydney, Sydney, NSW, Australia; lation of the innate immunity pathway[2,3,4]. BXCL701 also inhibits fibro- Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United blast activation protein (FAP) releasing the FAP-mediated block of T-cell States; Carolina Urologic Research Center, Myrtle Beach, SC,United 8 9 migration into the tumor[5]. FAP, DPP8 and DPP9 are expressed and acti- States; Merck & Co. Inc., Kenilworth, NJ, United States; University of vated in neuroendocrine CRPC[6]. Correlation is robust between the ex- Washington, Seattle, WA, United States pression of PD-L1 and the targets of BXCL701, particularly FAP, DPP8 and Correspondence: Leonard Appleman (applemanlj@upmc.edu) DPP9[2]. In syngeneic animal models, significant tumor responses were Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P382 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 210 of 272 Background Background For patients with metastatic castration-resistant prostate cancer HPN424 is a PSMA-targeting T cell engager derived from the TriTAC (mCRPC), additional therapeutic options are needed to improve over- platform (Tri-specific T Cell-Activating Construct). PSMA is a well- all outcomes and to delay the use of chemotherapy. In early-phase validated antigen specific to prostate epithelial cells upregulated trials, pembrolizumab, an anti–PD-1 antibody, showed some activity upon malignant prostate cancer with limited expression in normal as monotherapy in heavily pretreated patients with mCRPC. Cohort D tissues. HPN424 is a recombinant polypeptide of ~50kDa containing of the nonrandomized, multicohort, open-label, phase 1b/2 three humanized antibody-derived binding domains, targeting PSMA KEYNOTE-365 (NCT02861573) study has been designed to evaluate (for tumor binding), albumin (for half-life extension) and CD3 (for T the safety and efficacy of pembrolizumab combined with abiraterone cell engagement). It has been engineered to be a small, globular pro- acetate and prednisone in patients with mCRPC who have not re- tein to enable efficient exposure in solid tumor tissue with prolonged ceived chemotherapy for mCRPC. half-life and excellent stability under physiological conditions. Methods HPN424 binds monomerically to CD3 and PSMA, minimizing non- Adults (≥18 years) with histologically or cytologically confirmed pros- specific T-cell activation. These features are designed to increase the tate cancer, without small cell histology, and who experience pro- therapeutic index compared to earlier generations of T cell engagers gression ≤6 months before screening and have an ECOG PS score of by minimizing off-target toxicities. HPN424 mediates potent target 0 or 1 are eligible. Patients must be chemotherapy naïve for mCRPC tumor cell killing in a PSMA-specific manner in vitro and in xenograft and must not have received second-generation hormonal therapy for models in the presence of T cells, demonstrated at very low antigen mCRPC or must not have experienced failed treatment with enzaluta- densities. Consistent with its mechanism of action (MOA), tumor cell mide or become intolerant to enzalutamide for mCRPC. Patients will killing is accompanied by T cell activation, cytokine induction, and T receive pembrolizumab 200 mg IV every 3 weeks, abiraterone acet- cell expansion. ate 1000 mg once daily, and prednisone 5 mg twice daily. Responses Methods will be radiographically assessed every 9 weeks during year 1 and This is a Phase 1, open-label, multicenter, dose escalation and dose every 12 weeks thereafter. Pembrolizumab treatment will continue expansion study to evaluate the safety, tolerability, clinical activity, for up to 35 cycles (approximately 2 years) or until disease progres- and pharmacokinetics of HPN424 in adult patients with meta- sion, unacceptable toxicity, or patient/physician decision to withdraw. static castrate-resistant prostate cancer (mCRPC) who have pro- Patients who discontinue 1 of the 2 drugs in the combination be- gressed on the prior regimen (per PCWG3 criteria) and received cause of drug-related adverse events can continue with the other at least 2 prior systemic therapies approved for mCRPC. HPN424 combination partner. All patients who discontinue treatment will be is administered once weekly as one-hour IV infusion by single- monitored until trial completion. Primary end points are prostate- patient cohorts until either a Grade ≥2 adverse event (AE) that is specific antigen (PSA) response rate, defined as a PSA decrease of possibly related to HPN424 is observed or an estimated thera- ≥50% from baseline measured on 2 occasions at least 3 weeks apart peutic dose level has been reached. Then a conventional 3+3 de- for confirmation, objective response rate (ORR) per RECIST v1.1 by sign is implemented. Dose escalation will continue until a blinded independent central review (BICR), and safety. Secondary recommended phase 2 dose (RP2D) is determined. In dose ex- end points include time to PSA progression, ORR based on Prostate pansion, up to 18 patients receive HPN424 at the established Cancer Working Group 3 (PCWG3)–modified RECIST v1.1 assessed by RP2D. Additional expansion cohorts may be added. Patients may BICR, duration of response based on RECIST 1.1 and PCWG3-modified continue weekly HPN424 treatment as long as they are receiving RECIST 1.1 assessed by BICR, radiographic progression-free survival clinical benefit. Primary endpoints are number and severity of based on PCWG3-modified RECIST 1.1 assessed by BICR, and overall DLTs following treatment with escalating doses of HPN424 during survival. Recruitment began in December 2018 and will continue escalation, and overall response rate (per PCWG3 criteria) in dose until ~100 patients are enrolled. expansion. Secondary endpoints include AEs, preliminary anti- Ethics Approval tumor activity, pharmacokinetic and pharmacodynamic parame- The study and the protocol were approved by the Institutional Re- ters based on the proposed MOA of HPN424. view Board or ethics committee at each site. Trial Registration Consent NCT03577028 All patients provided written informed consent to participate in the Ethics Approval clinical trial. This study was approved by each participating institution's Institu- tional Review Board. P383 Withdrawn P385 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P383 Pembrolizumab plus enzalutamide versus placebo plus enzalutamide for metastatic castration-resistant prostate cancer: phase 3 KEYNOTE-641 study 1 2 3 4 Julie Nicole Graff , Joseph Burgents , Li Wen Liang , Arnulf Stenzl P384 Knight Cancer Institute, Oregon Health & Science University, Portland, A phase I dose escalation and expansion study of HPN424, a OR, United States; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, 3 4 PSMA-targeting T cell engager, in patients with advanced prostate United States; MSD, China, Beijing, China, Beijing, China; University of cancer refractory to androgen therapy Tuebingen Medical School, Tuebingen, Germany, Tübingen, Germany 1 2 3 Johanna Bendell, MD , Mark Stein, MD , Johann de Bono, MD , Richard Correspondence: Julie Nicole Graff (graffj@ohsu.edu) 4 4 4 Austin, PhD , Sue Hirabayashi , Che-Leung Law, PhD , Bryan Lemon, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P385 4 4 5 PhD , Holger Wesche, PhD , Aaron Weitzman, MD FACP , Lawrence Fong, MD Background Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, Treatment options for patients with metastatic castration-resistant 2 3 United States; Columbia University, New York, NY, United States; Royal prostate cancer (mCRPC) are noncurative, and life expectancy is only Marsden Hospital and The Institute of Cancer Research, Sutton, United about 3 years. Enzalutamide is an androgen receptor inhibitor used Kingdom; Harpoon Therapeutics, South San Francisco, CA, United for the treatment of patients with mCRPC. Pembrolizumab is a pro- States; Weitzman Consulting Group, Los Altos Hills, CA, United States; grammed death 1 (PD-1) inhibitor with antitumor activity as mono- University of California, San Francisco, San Francisco, CA, United States therapy in mCRPC. Results of clinical studies have shown that the Correspondence: Johanna Bendell (jlee@samornbiosciences.com) mechanisms of action of pembrolizumab and enzalutamide may be Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P384 synergistic. In the phase 1b/2 KEYNOTE-365 (NCT02861573) study, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 211 of 272 antitumor activity of pembrolizumab plus enzalutamide was ob- [1]. To further assess the economic impact of NIVO+IPI associated served in mCRPC patients pretreated with abiraterone acetate. Also, with TFS, this study compared healthcare costs among untreated in a single-arm, phase 2 study (NCT02312557) of patients who pro- intermediate/poor-risk aRCC patients with different lengths of TFS. gressed on enzalutamide, some patients had profound anticancer re- Methods sponse when pembrolizumab was added to enzalutamide that lasted This study used individual patient data from the NIVO+IPI arm in Check- years. KEYNOTE-641 (NCT03834493) is a randomized, double-blind, Mate 214 (database lock, August 6, 2018; minimum follow-up, 30 phase 3 trial to evaluate efficacy and safety of pembrolizumab plus months). TFS is defined as the time from last dose of NIVO+IPI to the start enzalutamide versus placebo plus enzalutamide for patients with of subsequent systemic therapy or death, whichever occurs first. All inter- mCRPC. mediate/poor-risk aRCC patients who received NIVO+IPI and provided Methods consent were classified into 3 cohorts based on the length of TFS: cohort Approximately 1200 patients will be randomly assigned 1:1 to receive 1 remained on NIVO monotherapy maintenance; cohort 2 had TFS ≤6 enzalutamide 160 mg/day plus pembrolizumab 200 mg Q3W or months; and cohort 3 had TFS >6 months. Patient characteristics and enzalutamide 160 mg/day plus placebo. Treatment will be stratified overall survival from randomization were described for the 3 cohorts. per prior abiraterone acetate treatment (yes/no), metastases (bone Monthly costs from randomization to last known date alive, including only/liver/other), and prior docetaxel treatment for metastatic study treatment costs, all-cause grade 3/4 adverse event costs, terminal hormone-sensitive prostate cancer (yes/no). Adults (≥18 years) with care costs, and subsequent treatment costs were compared between co- histologically or cytologically confirmed prostate cancer and mCRPC hort 3 versus cohort 1 and cohort 3 versus cohort 2 using Wilcoxon rank- who experienced biochemical or radiographic progression are eli- sum tests. All costs were adjusted to 2019 United States dollars. gible. Patients who received chemotherapy for mCRPC, checkpoint Results inhibition, or any treatment with a second-generation androgen re- Of the 420 eligible patients, 16.4% (N=69) remained on NIVO mono- ceptor inhibitor (eg, enzalutamide, apalutamide, or darolutamide) are therapy maintenance, 60.2% (N=253) had TFS ≤6 months, and 23.3% excluded. Patients intolerant of or experiencing progression with (N=98) had TFS >6 months by the end of patient follow-up. Patient prior abiraterone acetate therapy are included. Patients must have characteristics were mostly similar between cohort 3 versus cohort 1 or ECOG PS 0/1, adequate organ function, and tissue for biomarker ana- 2. By definition, all patients (100%) in cohort 1 were alive at the end of lysis. Responses will be assessed by CT/MRI and radionuclide bone follow-up. The survival probabilities were 94% for cohort 3 and 60% for imaging per PCWG-modified RECIST v1.1 every 9 weeks during the cohort 2 by 18 months and 83% for cohort 3 and 39% for cohort 2 by first year and every 12 weeks thereafter. Treatment will continue with 30 months. Patients with TFS >6 months had significantly lower enzalutamide plus pembrolizumab/placebo until radiographic disease monthly costs ($8,318) compared with those who never discontinued progression, unacceptable toxicity, or consent withdrawal, with a NIVO+IPI ($16,374) and those with TFS ≤6 months ($22,811) (Figure 1). maximum of 2 years of treatment for the pembrolizumab/placebo Conclusions component of the combination. Dual primary end points are overall This retrospective healthcare cost assessment of 3 patterns of patient survival and radiographic progression-free survival by blinded inde- outcomes during and after treatment with NIVO+IPI suggests an eco- pendent central review. The key secondary efficacy end point is time nomic value of achieving prolonged TFS (>6 months). Thus, manage- to subsequent anticancer therapy or death. Additional secondary ment strategies that would lead to prolonged TFS could be end points include objective response rate, duration of response, beneficial both clinically and economically. prostate specific antigen (PSA) response rate, PSA-undetectable rate, time to PSA progression, time to pain progression, and time to radio- Acknowledgements graphic soft tissue progression. Safety and tolerability will also be Writing support was provided by Analysis Group, Inc. and editorial support reported. was provided by Parexel, funded by Bristol-Myers Squibb. Trial Registration Trial Registration ClinicalTrials.gov; NCT03834493 NCT02231749. Ethics Approval The study and the protocol were approved by the Institutional Re- Reference view Board or ethics committee at each site. 1. McDermott DF, Rini BI, Motzer RJ, Tannir NM, Escudier B, Consent Kollmannsberger CK, Hammers HJ, Porta C, George S, Donskov F, Gurney All patients provided written informed consent to participate in the HP. 874P Treatment-free interval (TFI) following discontinuation of first- clinical trial. line nivolumab plus ipilimumab (N+ I) or sunitinib (S) in patients (Pts) with advanced renal cell carcinoma (aRCC): CheckMate 214 analysis. Ann Oncol. 2018; 29(suppl 8):mdy283.083. P386 Ethics Approval Economic benefits associated with treatment-free survival of This trial was approved by the institutional review board or ethics committee immuno-oncology agents among untreated patients with at each site. intermediate/poor-risk advanced or metastatic renal cell carcinoma 1 2 3 Michael Harrison, MD , Meredith Regan, PhD , Michael Atkins, MD , 4 4 4 Sumati Rao, PhD , Shuo Yang, PhD , Jennifer Johansen, PharmD, BCPS , 5 5 5 5 Ella Du, MESc , Chenyang Gu , Ela Fadli , Keith Betts, PhD , David McDermott, MD 1 2 Duke Cancer Institute, Chapel Hill, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Georgetown Lombardi Comprehensive Cancer, Washington, DC, United States; Bristol-Myers Squibb, Princeton, NJ, United States; Analysis Group, Inc, Los Angeles, CA, United States; Beth Israel Deaconess Medical Center, Milton, MA, United States Correspondence: Michael Harrison (Michael.Harrison@Duke.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P386 Background Nivolumab plus ipilimumab (NIVO+IPI) was associated with signifi- cantly longer treatment-free survival (TFS) compared with sunitinib in intermediate/poor-risk patients with previously untreated advanced Fig. 1 (abstract P386). See text for description or metastatic renal cell carcinoma (aRCC) in the CheckMate 214 trial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 212 of 272 P387 Trial Registration A multicenter, open-label, exploratory platform study to evaluate https://clinicaltrials.gov/ct2/show/NCT03835533 biomarkers and immunotherapy combinations for the treatment of patients with metastatic castration-resistant prostate cancer References (PORTER) 1. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin 1 2 3 Leo Nissola, MD , Karen Autio, MS, MD , Nina Bhardwaj, MD, PhD , AV, et al. Signatures of mutational processes in human cancer. Nature 3 4 5 Matthew Galsky, MD , Kristopher Wentzel, MD , Vanessa Lucey , Cheryl 2013;500(7463):415–21. Beer TM, Kwon ED, Drake CG, Fizazi K, Logothetis 1 1 1 Selinsky, PhD , Christopher Perry , Christopher Cabanski, PhD , Ari C, Gravis G, et al. Randomized, Double-Blind, Phase III Trial of Ipilimumab 1 1 6 7 Bitton , Justin Fairchild , Christine Horak, PhD , Jeffrey Skolnik, MD , Versus Placebo in Asymptomatic or Minimally Symptomatic Patients With 8 6 1 Michael Yellin, MD , Ute Dugan, MD, PhD , Ramy Ibrahim, MD , Metastatic Chemotherapy-Naive Castration-Resistant Prostate Cancer. J Lawrence Fong, MD Clin Oncol 2017;35(1):40–7. Parker Institute for Cancer Immunotherapy, San Francisco, CA, United 2. Brahmer JR, Drake CG, Wollner I, Powderly JD, Picus J, Sharfman WH, 2 3 States; Memorial Sloan Kettering Cancer Center, New York, NY; The et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) Mount Sinai Hospital, New York, NY, United States; The Angeles Clinic & in refractory solid tumors: safety, clinical activity, pharmacodynamics, and Research Institute, Los Angeles, CA, United States; Cancer Research immunologic correlates. J Clin Oncol 2010;28(19):3167–75. Institute, New York, NY, United States; Bristol-Myers Squibb, 3. Di Lorenzo G, Buonerba C, Kantoff PW. Immunotherapy for the treatment Lawrenceville, NJ, United States; Inovio Pharmaceuticals, Plymouth of prostate cancer. Nat Rev Clin Oncol 2011;8(9):551–61. Drake CG. Meeting, PA, United States; Celldex Therapeutics, Hampton, NJ, United Prostate cancer as a model for tumour immunotherapy. Nat Rev States; University of California San Francisco, San Francisco, CA, United Immunol States 2010;10(8):580–93. Correspondence: Leo Nissola (lnissola@parkerici.org) 4. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P387 et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. Background 5. Flammiger A, Bayer F, Cirugeda-Kühnert A, Huland H, Tennstedt P, Simon Metastatic castration resistant prostate cancer (mCRPC), the lethal R, et al. Intratumoral T but not B lymphocytes are related to clinical out- form of prostate cancer, has shown limited benefit from immune come in prostate cancer. APMIS 2012;120(11):901–8. checkpoint inhibition as monotherapy, with two randomized phase 3 6. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. trials with ipilimumab failing to show a survival benefit, and a large VISTA is an inhibitory immune checkpoint that is increased after phase 2 trial with pembrolizumab demonstrating an overall response ipilimumab therapy in patients with prostate cancer. Nat Med rate (ORR) of 3-5%. Clearly novel combinations are needed and a 2017;23(5):551–5. deeper understanding of immune resistance. 7. Graff JN, Alumkal JJ, Drake CG, Thomas GV, Redmond WL, Farhad M, Using a multi-arm, multi-stage platform design, the PORTER study et al. Early evidence of anti-PD-1 activity in enzalutamide-resistant pros- will adaptively test multiple immunotherapeutic combinations to ac- tate cancer. Oncotarget 2016;7(33):52810–7. Kantoff PW, Higano CS, tivate the innate and adaptive immune systems. Coupled with deep Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel- immune biomarker profiling, this design will enable rapid insights Timmunotherapy for castration-resistant prostate cancer. N Engl J Med into the immune responses for each combination, providing data for 2010;363(5):411–22. potential larger definitive trials, while generating hypotheses for new 8. Kwon ED, Drake CG, Scher HI, Fizazi K, Bossi A, van den Eertwegh AJM, cohorts. et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after Methods docetaxel chemotherapy (CA184-043): a multicentre, randomised, PORTER is an open-label, non-randomized, exploratory platform double-blind, phase 3 trial. Lancet Oncol 2014;15(7):700–12. study designed to assess the safety and antitumor activity of multiple 9. Lee P, Gujar S. Potentiating prostate cancer immunotherapy with immunotherapy combinations in participants with mCRPC who have oncolytic viruses. Nat Rev Urol 2018;15(4):235–50. received prior secondary androgen inhibition therapy. Each cohort 10. Lopez-Bujanda Z, Drake CG. Myeloid-derived cells in prostate cancer pro- has a two-stage design (initial n = 15, expansion n = 15) with a deci- gression: phenotype and prospective therapies. J Leukoc Biol sion to expand based on the safety, clinical activity, and biomarker 2017;102(2):393–406. results observed in the initial stage. 11. Martin AM, Nirschl TR, Nirschl CJ, Francica BJ, Kochel CM, van Cohort A is open and recruiting, testing the combination of bempe- Bokhoven A, et al. Paucity of PD-L1 expression in prostate cancer: In- galdesleukin (“BEMPEG”, NKTR-214; a CD-122 preferential IL-2 path- nate and adaptive immune resistance. Prostate Cancer Prostatic Dis way agonist) with nivolumab (PD-1 inhibitor), postulating that this 2015;18(4):325–32. will increase PD-L1 expression, intratumoral T and NK cells, and in- Ethics Approval duce an IFN gamma signature. The study was approved by WIRB‘s Ethics Board, IRB Tracking Number: Cohort B will combine CDX-301 (Flt3L), poly-ICLC (PAMP-adjuvant), nivolumab and stereotactic body radiation therapy, in 1-5 metastatic sites, inducing immunogenic cell death, mobilizing and activating dendritic cells increasing tumor antigen presentation, and overcom- P388 ing adaptive immune resistance in mCRPC. Pembrolizumab plus docetaxel and prednisone for enzalutamide- Cohort C will evaluate INO-5151, a DNA vaccine encoding PSA, PSMA, or abiraterone acetate–pretreated patients with metastatic and IL-12 delivered via intramuscular electroporation, in addition to castration-resistant prostate cancer: phase 3 KEYNOTE-921 study 1 2 3 CDX-301 and nivolumab. This is a multi-pronged approach to Neal Shore, MD , Daniel Petrylak, MD , Mostefa Bennamoun , Raffaele 4 5 6 6 7 mobilize and activate dendritic cells, stimulate anti-tumor CD8 T cells, Ratta , Josep Piulats , Ben Li , Charles Schloss , Karim Fizazi and circumvent adaptive immune resistance. Carolina Urologic Research Center, Myrtle Beach, SC, USA, Myrtle Beach, Inclusion criteria include: histologically-confirmed mCRPC that is SC, United States; Smilow Cancer Hospital at Yale University, New measurable or non-measurable by Prostate Cancer Clinical Trials Haven, CT, USA, New Haven, CT, United States; Institut Mutualiste Working Group 3 and progressing despite secondary androgen re- Montsouris, Paris, France, Paris, France; Hopital Foch, Suresnes, France, ceptor signaling inhibitor therapy. Suresnes, France; Catalan Cancer Institute, Barcelona, Spain, Barcelona, The primary endpoint: safety, as assessed by the incidence and se- Spain; Merck & Co., Inc., Kenilworth, NJ, USA, Kenilworth, NJ, United verity of adverse events. Secondary endpoints: Composite ORR (PSA States; Gustave Roussy, Villejuif, France, Villejuif, France reduction >50%, confirmed CR or PR per RECIST 1.1, or change in cir- Correspondence: Neal Shore (nshore@gsuro.com) culating tumor cell (CTC) from >5 cells/7.5 ml to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P388 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 213 of 272 Background (HD) IL-2 regimen has demonstrated superior overall response rate Docetaxel is an established treatment for patients with metastatic (ORR), depth and durability of response versus lower dose alterna- castration-resistant prostate cancer (mCRPC). Pembrolizumab is a tives. Resistance mechanisms exploited by tumors may play a domin- programmed death 1 inhibitor that was found to have antitumor ac- ant role in limiting the effectiveness of T-cell mediated cancer tivity as monotherapy in mCRPC. In the phase 1b/2 KEYNOTE-365 therapies. The PD-1/PD-L1 interaction is a major pathway hijacked by study (NCT02861573), docetaxel plus pembrolizumab and prednisone RCC tumors to suppress immune control. Antibody-mediated PD1 had activity in patients treated with abiraterone acetate or enzaluta- blockade with pembro results in spontaneous and durable regres- mide for mCRPC, warranting further evaluation of this treatment sions for a subset of RCC tumors (SS Tykodi et al., ASCO 2019, ab- combination. KEYNOTE-921 (NCT03834506) is a randomized phase 3 stract #4570). PD1 blockade has entered clinical practice for trial to evaluate the efficacy and safety of pembrolizumab plus doce- advanced RCC as both a front-line and salvage therapy option. A fa- taxel and prednisone in chemotherapy-naïve patients who were pre- vorable safety profile for PD1 blockade has encouraged exploration viously treated with enzalutamide or abiraterone acetate for mCRPC of novel immuno-oncology combinations. and experienced progression while on therapy. Methods Methods Methods: This is an investigator-initiated, phase I trial of IL-2 plus Approximately 1000 patients will be randomly assigned 1:1 to receive pembro in patients with advanced, clear cell RCC. The study will use docetaxel 75 mg/m2 every 3 weeks (Q3W) plus prednisone/prednis- a 3 + 3 trial design to test three IL-2 dose levels in combination with olone 5 mg twice daily (BID) and pembrolizumab 200 mg Q3W or do- pembro given every 3-weeks at 200 mg flat dosing. Cohorts will re- cetaxel 75 mg/m2 Q3W plus prednisone/prednisolone 5 mg BID plus ceive subcutaneous IL-2 given once daily, 5 days per week for 6 placebo Q3W. Treatment will be stratified per previous treatment with weeks (250,000 U/kg week 1, 125,000 U/kg weeks 2-6); or IV bolus a next-generation hormonal agent (abiraterone acetate or enzaluta- dosing at 72,000 U/kg or 600,000 U/kg (HD IL-2) every 8 hours to a mide) and metastases (bone only, liver, other). Adult (≥18 years) maximum of 14 doses on week 1 and 4 of a 12-week treatment patients with chemotherapy-naïve histologically or cytologically con- course. Patients with stable or responding disease and without firmed mCRPC who experienced progression while receiving androgen treatment-limiting toxicity can receive up to 3 courses of therapy. deprivation therapy (or postbilateral orchiectomy) within 6 months be- The HD IL-2 cohort will enroll an additional 9 patients to gain further fore screening were eligible. Patients must have experienced progres- insight into anti-tumor efficacy. The primary objective is to evaluate sion after ≥8weeks (≥14 weeks for those with bone progression) or safety and tolerability for IL-2 plus pembro. The secondary objective become intolerant after ≥4 weeks of abiraterone acetate or enzaluta- is to assess antitumor activity by RECIST 1.1 for ORR, disease control mide treatment (but not both) with androgen-deprivation therapy in rate, and progression free survival. Exploratory endpoints will include the chemotherapy-naïve mCRPC state. Patients must have ECOG PS 0 pretreatment tumor analysis for PD-L1 expression, and quantitation or 1, adequate organ function, and tissue for biomarker analysis. Re- of regulatory T cell frequency in peripheral blood and tumor sponses will be assessed by CT or MRI and radionuclide bone imaging microenvironment. perProstateCancer Working Group–modified RECIST v1.1 by blinded Trial Registration independent central review (BICR) Q9W during the first year and Q12W Trial Registration: ClinicalTrials.gov, NCT03260504 thereafter. Treatment will continue with docetaxel and prednisone for Ethics Approval up to 10 cycles and with pembrolizumab for up to 35 cycles or until Ethics Approval: This study was approved by the Fred Hutchinson radiographic disease progression, unacceptable toxicity, or consent Cancer Research Center Institutional Review Board, approval number withdrawal. Primary end points are radiographic progression-free sur- 9611. vival by BICR and overall survival. The key secondary efficacy end point is time to initiation of subsequent anticancer therapy or death. Add- P390 itional secondary end points include prostate-specific antigen response Pembrolizumab plus olaparib vs enzalutamide or abiraterone in rate (decline of ≥50% from baseline, measured on 2 occasions at least patients with metastatic castration-resistant prostate cancer who 3 weeks apart), time to PSA progression, and objective response rate experienced progression on chemotherapy: phase 3 KEYLYNK-010 and duration of response per PCWG-modified RECIST 1.1 as assessed study by BICR. Safety and tolerability will also be reported. 1 2 3 4 5 Evan Yu , Se Hoon Park , Yi-Hsiu Huang , Mostefa Bennamoun ,Lu Xu , Trial Registration 5 6 Jeri Kim , Emmanuel Antonarakis, MD ClinicalTrials.gov, NCT03834506 1 2 University of Washington, Seattle, WA, United STates; Samsung Medical Ethics Approval Center, Seoul, South Korea, Seoul, Korea, Republic of; Taipei Veterans The study and the protocol were approved by the Institutional Re- General Hospital, Taipei, Taiwan, Taipei, Taiwan, Province of China; view Board or ethics committee at each site. 4 5 Institut Mutualiste Montsouris, Paris, France, Paris, France; Merck & Co., Consent Inc., Kenilworth, NJ, USA, Kenilworth, United States; Johns Hopkins All patients provided written informed consent to participate in the University, Baltimore, MD, United States clinical trial. Correspondence: Evan Yu (evanyu@u.washington.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P390 P389 A phase I trial of Interleukin-2 (IL-2) and Pembrolizumab (Pembro) Background Combination Therapy for patients with advanced renal cell The docetaxel-pretreated, metastatic castrate-resistant prostate can- carcinoma cer (mCRPC) disease state remains an unmet need for new therapeu- Scott Tykodi, MD, PhD, Johanna Whitney, Sumia Dakhil, Eleanor Bergren, tics. The programmed death 1 (PD-1) inhibitor pembrolizumab and Vivian Nguyen, Samantha Kiriluk, Shailender Bhatia, MD, John Thompson, the polyadenosine diphosphate ribose polymerase (PARP) inhibitor ola- MD parib have some independent antitumor monotherapy activity for University of Washington, Seattle, WA, United States mCRPC. In patients with mCRPC who were unselected for homologous Correspondence: Scott Tykodi (stykodi@fredhutch.org) recombination deficiency (HRD), promising activity was seen with the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P389 combination of pembrolizumab and olaparib in the phase 1b/2 KEYNOTE-365 study (NCT02861573), warranting further investigation in Background this population. KEYLYNK-010 (NCT03834519) is a randomized, open- Background: Cellular immune responses play a key role modulating label, phase 3 trial to evaluate the efficacy and safety of pembrolizu- renal cell carcinoma (RCC) progression. IL-2 (aldesleukin) is a potent mab plus olaparib in molecularly unselected enzalutamide-pretreated growth and differentiation factor for T and NK cells with anti-tumor or abiraterone acetate–pretreated patients with mCRPC whose disease activity for advanced RCC across a broad dose range. A high dose progressed on or after taxane chemotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 214 of 272 Methods adjunctive therapy after completion of standard optimal therapy. Key Approximately 780 patients will be randomly assigned 2:1 to receive eligibility criteria are a diagnosis of stage III or IV primary epithelial pembrolizumab 200 mg intravenously Q3W plus olaparib 300 mg or- ovarian cancer, successful establishment of a short-term cancer cell ally twice daily or abiraterone acetate 1000 mg orally once daily plus line, a successful leukapheresis collection of monocytes, and a Kar- prednisone/prednisolone 5 mg orally twice daily (for enzalutamide- nofsky Performance Status of 70 or greater at the time of pretreated patients) or enzalutamide 160 mg/day orally (for abirater- randomization, which takes place shortly after completion of primary one acetate–pretreated patients). Arms will be stratified per prior therapy. Tumor is collected at the time of surgery from which a treatment (abiraterone acetate/enzalutamide) and presence of meas- short-term cell line is derived. Dendritic cells are produced by incu- urable disease (yes/no). Eligible patients (≥18 years) must have histo- bating peripheral blood monocytes in the presence of GM-CSF and logically confirmed mCRPC, experienced progression while receiving IL-4. The antigen source is a lysate of irradiated tumor cells from the androgen deprivation therapy within 6 months before screening, cell culture. Six to seven months after tumor collection, after comple- previously received treatment with abiraterone acetate or enzaluta- tion of concurrent surgery and primary systemic therapy, patients are mide (but not both), and previously received treatment with chemo- stratified by whether they have detectable residual disease, and then therapy (1 prior docetaxel-based regimen). Patients must have an randomized 2:1 to receive the dendritic cell vaccine or autologous ECOG PS of 0 or 1, adequate organ function, and tumor tissue suit- monocytes. Both are admixed with GM-CSF and injected subcutane- able for biomarker analysis. Responses will be assessed by CT/MRI ously at weeks 1, 2, 3, 8, 12, 16, 20, and 24 for up to eight doses. The and radionuclide bone imaging per Prostate Cancer Working Group objective is to achieve a 50% reduction in the risk of death in the (PCWG)–modified RECIST v1.1 by blinded independent central review vaccine arm. (BICR) Q9W during the first year and Q12W thereafter. Treatment will Results continue with up to 2 years of pembrolizumab (35 cycles) and ola- Cell line success rate for submitted tumor samples is 22/22 with 1 in parib or abiraterone acetate/enzalutamide until radiographic disease progress. A satisfactory leukapheresis product has been obtained for progression, unacceptable toxicity, or consent withdrawal. Primary 14/14 patients, but was repeated for 2. 12 of a planned 99 patients end points are overall survival and radiographic progression-free sur- have been randomized. 11 have started treatment; 7 have completed vival. The key secondary efficacy end point is time to initiation of all 8 doses, 1 discontinued early for disease progression, 3 are cur- subsequent anticancer therapy. Other secondary end points are ob- rently in treatment. A total of 77 doses have been administered. No jective response rate and duration of response per PCWG-modified significant toxicity directly attributed to the vaccine has been RECIST v1.1 by BICR, time to prostate-specific antigen progression, reported. time to first symptomatic skeletal event, and safety and tolerability. Conclusions Prognostic or predictive molecular biomarkers (eg, genomic HRD sta- Although logistically complex, this patient-specific vaccine approach tus, microsatellite instability) and patient-reported outcomes will be is feasible, and has been well-tolerated. [NCT02033616] explored. Trial Registration Trial Registration ClinicalTrials.gov NCT02033616 ClinicalTrials.gov, NCT03834519 Ethics Approval Ethics Approval This study was approved by the Western Institutional Review Board The study and the protocol were approved by the Institutional Re- 20171661 view Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the P392 clinical trial. Phase 1 combination study of the CHK1 inhibitor prexasertib (LY2606368) and anti-PD-L1 antibody LY3300054, in patients with high-grade serous ovarian cancer and other advanced solid tumors P391 1 1 1 1 Khanh Do , Claire Manuszak , Sarah Kelland , Allison Powers , Adrienne Randomized phase II trial of autologous dendritic cells loaded with 1 2 1 Anderson , Alona Muzikansky , Andrew Wolanski , Mariano Severgnini, autologous tumor cell antigens from self-renewing cancer cells in 1 1 1 MSc , Geoffrey Shapiro, MD, PhD , Khanh Do, MD patients with newly diagnosed stage III or IV ovarian cancer 1 2 1 2 1 Dana-Farber Cancer Institute, Boston, MA, United States; Massachusetts Lisa Abaid, MD , Richard Friedman, MD, PhD , John Brown, MD , Alberto 1 1 3 4 General Hospital, Boston, MA, United States Mendivil, MD , Tiffany Beck, MD , Bradley Corr , Leslie Randall , James 5 6 6 6 Correspondence: Khanh Do (Khanh_Do@dfci.harvard.edu) Mason , Candace Hsieh , Gabriel Nistor, MD , Robert Dillman, MD, FACP 1 2 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P392 Hoag Hospital, Newport Beach, CA, United States; Disney Family Cancer Center, Burbank, CA, United States; University of Colorado, Background Aurora, CO, United States; University of California Irvine, Orange, CA, Ovarian cancers are characterized by defects in DNA damage repair United States; Scripps Green & Memorial Hospitals, La Jolla, CA, United and high levels of replication stress, creating susceptibility to inhibition States; AiVita Biomedical, Inc., Irvine, CA, United States of checkpoint kinase 1 (CHK1). CHK1 inhibition results in intratumoral Correspondence: Robert Dillman (bob@aivitabiomedical.com) DNA damage that can drive T cell infiltration, as well as PD-L1 expres- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P391 sion. Combined CHK1 inhibition and immune checkpoint blockade therefore has the potential to enhance T cell activation against tumors. Background Methods Despite recent advances, the 5-year survival rate for patients who We conducted an open-label phase 1 study of prexasertib-mediated present with stage III or IV ovarian cancer remains less than 40%. CHK1 inhibition combined with LY3300054-mediated PD-L1 blockade Standard therapy includes surgical debulking and neoadjuvant and/ following a 3+3 design evaluating 3 administration schedules: lead-in or adjuvant combination chemotherapy, with or without bevacizu- of LY3300054 alone (Arm A), lead-in of prexasertib alone (Arm B), mab, with or without intraperitoneal therapy in certain stage III pa- and combined LY3300054 and prexasertib at outset (Arm C). Both tients, and increasingly the use of PARP inhibitors. So far advanced agents were administered on days 1 and 15 of a 28-day cycle. The ovarian cancer has been relatively refractory to anti-checkpoint ther- MTD was defined as the highest dose level at which less than one- apy, presumably because of limited host anti-tumor immune re- third of at least 6 patients experienced a DLT during C0+C1. Flow cy- sponses. Adjunctive treatment with an effective vaccine could tometry of peripheral blood mononuclear cells was performed for increase immune responses and improve survival. analysis of T cell subsets. Plasma cytokine and chemokine analyses Methods were conducted using the Luminex platform. Patients enrolled to the This randomized phase II trial was approved by Western IRB. AV- currently ongoing expansion phase of the study undergo mandatory OVA-1, patient-specific dendritic cell vaccines loaded with autologous tumor biopsies during C0 and on C1D16 after the combination. tumor antigens from self-renewing cancer cells, is administered as an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 215 of 272 Results 41-77) with ECOG 0-1 and a median of 4 prior lines of systemic treat- Fifteen patients have been treated in the dose escalation phase. The ment (ranging from 1 to 8). All 12 patients received platinum-based combination of both agents is tolerable at the RP2D with prexasertib treatment prior to study entry. No DLTs were observed. The most fre- at 105mg/m2 IV on days 1 and 15 in combination with LY3300054 at quent AEs regardless of relationship to study drug were fatigue (7 700mg flat dosing. Two DLTs occurred, including febrile neutropenia patients), nausea (7 patients) and UTI (5 patients). Overall, 7 patients (Arm C) and prolonged grade 4 neutropenia lasting > 5 days (Arm B). had AEs of > grade 3, 4 of which were considered related to study Most common drug-related adverse events included leukopenia, drug by the investigator. Pharmacodynamic assessments included neutropenia, thrombocytopenia, and anemia. Confirmed partial re- immune phenotyping at the periphery where immune activation was sponses have been observed in 2 patients with CCNE1-amplified observed following AGEN2034 treatment. In this subset of recurrent HGSOC ongoing for 9 and 10 months, respectively. One additional ovarian cancer patients, 1 of 12 patients developed a durable partial CCNE-1 amplified HGSOC patient has had prolonged SD for 11 response (42 wks) at the lowest dose level (1 mg/kg), 8 patients months. Preliminary data on T-cell subset analysis and cytokine pro- demonstrated at least stable disease lasting 8.7 -65.7 weeks, with 5 file show immune modulatory effect of prexasertib, confirming of them meeting the DCR criteria of at least 12 weeks of duration. proof-of-mechanism. Four patients demonstrated progressive disease at the first on treat- Conclusions ment tumor evaluation. Full-dose prexasertib in combination with immune checkpoint block- Conclusions ade is tolerable and has preliminary clinical activity in patients with AGEN2034, a PD-1 inhibitor, is well tolerated with no DLTs observed HGSOC with durable responses. An expansion cohort in this popula- at all dose levels evaluated. The clinical activity and safety observed tion is currently being enrolled utilizing schedule B. Comprehensive in the recurrent ovarian cancer subset were consistent with the over- characterization of the immune microenvironment will be performed all Phase 1 study population. Biomarker evaluations (including PD-L1 in paired tumor biopsies, with attention to pharmacodynamic proof- status) are ongoing. of-mechanism endpoints, including T cell infiltration and PD-L1 ex- Ethics Approval pression and their correlation with the induction of DNA damage. 20170314 - IRB tracking for Copernicus Additionally, immune signatures will be correlated with genomic pro- Consent file and response duration. Written informed consent was obtained from the patient for publica- Ethics Approval tion of this abstract and any accompanying images. A copy of the This study was approved by Dana-Farber Cancer Institute's Ethics written consent is available for review by the Editor of this journal. Board. Consent P394 Written informed consent was obtained from the patient for publica- A Phase 1 study of INCMGA00012, a PD-1 inhibitor, in patients tion of this abstract and any accompanying images. A copy of the with advanced solid tumors: Preliminary results for patients with written consent is available for review by the Editor of this journal. advanced cervical cancer (POD1UM-101) 1 2 3 Janice Mehnert, MD , Luis Paz Ares, MD, PhD , Joanna Pikiel, Dr n med , 4 5 6 P393 Udai Banerji, PhD , Anna Kryzhanivska, MD , Nehal Lakhani, MD, PhD , 7 8 Single agent activity of a novel PD-1 inhibitor AGEN2034 in Sebastian Ochsenreiter, Dr med , Tobias Arkenau, MD, PhD , Nawel 9 9 9 recurrent ovarian cancer:Subset analysis of phase I dose escalation Bourayou, MD , Deanna Kornacki, PhD , Chuan Tian, PhD , Thomas 9 10 NCT03104699 study Condamine, PhD , Itziar Gardeazabal González 1 1 2 3 1 David O'Malley , John Hays , Charles Drescher , Jasgit Sachdev , Wilberto Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, United 4 5 6 7 2 3 Nieves-Neira , Breelyn Wilky , Marylin Huang , Kathleen Moore , Waldo States; Hospital Universitario, Madrid, Spain; Szpitale Pomorskie Sp. z 8 8 8 8 4 Ortuzar , Anna Wijatyk , Hagop Youssoufian , Remigiusz Kaleta, MD , o.o., Gdansk, Poland; Royal Marsden NHS Foundation Trust, Sutton, 8 8 8 5 Inbal Sapir , Christopher Dupont, PhD , Irina Shapiro , Debra Richardson, United Kingdom; Regional Clinical Oncology Center, Ivano-Frankivsk, 7 6 7 MD Ukraine; START Midwest, Grand Rapids, MI, United States; Charité 1 8 The James Cancer Center Hospital, Columbus, OH, United States; Comprehensive Cancer Center, Berlin, Germany; Sarah Cannon Institute, 2 3 9 Swedish Cancer Institute, Seattle, WA, United States; HonorHealth London, United Kingdom; Incyte Corporation, Wilmington, DE, United 4 10 Research Institute, Scottsdale, AZ, United States; Northwestern States; Hospital Vall d’Hebron, Barcelona, Spain University, Stroger Hospital, Chicago, IL, United States; University of Correspondence: Janice Mehnert (mehnerja@cinj.rutgers.edu) Colorado Anschultz Medica, Aurora, CO, United States; Sylvester Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P394 Comprehensive Cancer Center, Miami, FL, United States; Stephenson Cancer Center, Oklahoma City, OK, United States; Agenus Inc, Background Lexington, MA, United States Background Correspondence: Christopher Dupont INCMGA00012 is an investigational humanized, hinge-stabilized IgG4 (Christopher.Dupont@Agenusbio.com) monoclonal antibody that binds to PD-1. At all doses tested, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P393 INCMGA00012 has an acceptable tolerability profile with no dose- limiting toxicities or maximum tolerated dose. A dose of 3 mg/kg Background Q2W was initially selected for the tumor specific cohorts. Multiple AGEN2034 is a novel, fully human monoclonal immunoglobulin G4 fixed doses were also examined in this study. The 500 mg Q4W and (IgG4) antibody, designed to block PD-1 from interacting with its li- 375 mg Q3W doses are selected for further development based on gands PD-L1 and PD-L2 with high affinity. The overall objective of favorable pharmacokinetics and safety. the study was to assess safety, MTD, and pharmacokinetic (PK) and Methods pharmacodynamic (PD) characteristics of AGEN2034 monotherapy in Methods patients with advanced, refractory malignancies. The initial expansion phase contained 4 tumor-specific cohorts Methods (endometrial [unselected], cervical, soft tissue sarcoma, and non- Between April 2017 - April 2019, 50 patients with advanced solid tu- small cell lung) treated for up to 2 years. Eligible patients presented mors were enrolled in a phase 1 dose escalation study treated with with a histologically proven, unresectable locally advanced or meta- infusion of AGEN2034 every 2 weeks at the dose range of 1-10 mg/ static tumor, ECOG performance status (PS) ≤1, disease progression kg. Within the study population a subset of patients with heavily pre- during or following ≤5 prior treatments, measurable disease per treated recurrent epithelial ovarian cancer was identified. RECIST v1.1, and no prior treatment with immune checkpoint inhibi- Results tors. The primary endpoint is safety (using CTCAE v4.03 grading). Twelve patients with recurrent epithelial ovarian cancer were en- Confirmed best overall response rate and duration of response were rolled in the Phase I dose escalation. Median age was 58 years (range evaluated by RECIST v1.1 (investigator's assessment). Treatment past Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 216 of 272 progression was allowed for patients experiencing clinical benefit. A recent study of enoblituzumab combined with pembrolizumab Preliminary safety and efficacy results for patients with unresectable showed this combination is feasible and well tolerated with minimal locally advanced or metastatic cervical cancer are presented. additive toxicity [5]. While studies of monotherapy pembrolizumab in Results this population report responses below 17% [6], the overall response Results rate of PD-1/PD-L1 inhibitor-naïve patients (post platinum) receiving As of 24 APR 2019, 35 patients with cervical cancer were treated with 3 enoblituzumab plus pembrolizumab was 33% (6/18) including 1 con- mg/kg INCMGA00012. Median age was 51 (29-81) years, 88.6% were firmed CR and 5 confirmed PRs [5]. This suggests a cooperative white, 48.6% had an ECOG PS of 1. All patients were pretreated with at mechanism and provides a rationale for further development of this least 1 prior platinum-based chemotherapy for recurrent or advanced combination in patients with recurrent/metastatic SCCHN. disease, 91.4% were treated with radiotherapy, and 62.9% underwent MGA012 (also known as INCMGA00012) is an investigational anti-PD-1 surgery. Median drug exposure was 4.4 (0.03-16.0) months. Fourteen monoclonal antibody with a tolerable safety profile and efficacy signal patients (40.0%) experienced Grade (G) 3/4 AEs regardless of causality. consistent with other agents in its class [7] demonstrated in early studies. Seven patients (20.0%) had immune-related AEs (colitis [G2, n=1; G3, Methods n=2], infusion-related reaction [G1, n=1; G3, n=1], diarrhea [G1], hyper- This is a Phase 2/3, randomized, open label study in first-line treatment of thyroidism [G2], and maculopapular rash [G3]). Three patients with col- patients with R/M SCCHN not curable by local therapy (Figure 1). We itis discontinued study treatment. No treatment-related deaths hypothesize that combining enoblituzumab and PD-1 inhibition (with or occurred. Confirmed responses per RECIST v1.1 were observed in 6/31 without chemotherapy) will improve objective response rates and OS (19.4%) response evaluable patients, with 1 patient having a confirmed compared to pembrolizumab/chemotherapy in patients in R/M SCCHN. CR. Median duration of response was not reached as 5/6 patients re- Approximately 200 patients will be randomized in a 1:1:1:1 ratio to main on treatment (10.3, NE months). An additional 12 patients had one of four treatment arms to select the preferred enoblituzumab stable disease for an overall disease control rate of 58.1% (18/31). combination treatment for further evaluation based primarily on Conclusions ORR. In subsequent Phase 3 portion, the selected enoblituzumab/ Conclusions MGA012 regimen (with or without chemotherapy) will be compared INCMGA00012 has been generally well tolerated with evidence of to pembrolizumab and chemotherapy with an endpoint of OS. significant and durable antitumor activity in platinum-refractory cer- Trial Registration vical cancer. These data support further development of To be registered on clinicaltrials.gov INCMGA00012 in cervical cancer. Trial Registration References NCT03059823, 2017-000865-63 1. Siegel R, Naishadham D, and Jemal A, Cancer statistics, 2013. CA Cancer Ethics Approval J. Clin, 2013. 63(1): p. 11-30. The study was approved by institutional review boards or independ- 2. Price KA and Cohen EE, Current treatment options for metastatic head ent ethics committees of participating institutions. and neck cancer. Curr Treat Options Oncol, 2012. 13(1): p. 35-46. 3. Keynote 048. 1200/JCO.2019.37.15_suppl.6000 Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019) 6000-6000. P395 4. Collins M, Ling V, and Carreno BM, The B7 family of immune-regulatory li- Phase 2/3 open-label trial of enoblituzumab in combination with gands. Genome Biol, 2005. 6(6): p. 223. MGA012, with and without chemotherapy, in the treatment of 5. Aggarwal C et al, Open-Label, Dose Escalation Study of Enoblituzumab in patients with recurrent or metastatic head and neck squamous cell Combination with Pembrolizumab in Patients with Select Solid Tumors. carcinoma 1 2 1 33rd Annual Meeting of The Society for Immunotherapy of Cancer Wash- Fernanda Arnaldez , Charu Aggarwal, MD MPH , Scott Currence , Jan 1 1 3 ington, DC, USA November 7–11, 2018 Baughman, MPH , Paul Moore, PhD , George Blumenschein, MD , Jon 1 4 6. Cohen EE, Harrington KJ, Tourneau C, Dinis J, Licitra L, Ahn M, et al., Wigginton, MD , Robert Ferris, MD, PhD 1 2 Head and Neck Cancer, Excluding Thyroid. ESMO, 2017. 28. MacroGenics, Inc., Rockville, MD, United States; Abramson Cancer 3 7. Mehnert J et. At. 33rd Annual Meeting of The Society for Center, Philadelphia, PA, United States; MD Anderson Cancer Center, 4 Immunotherapy of Cancer Washington, DC, USA November 7–11, 2018 Houston, TX, United States; UPMC Hillman Cancer Center, Pittsburgh, Ethics Approval PA, United States Each institution will obtain Ethics Board approval prior to enrollment Correspondence: Fernanda Arnaldez (farnaldez@gmail.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P395 Background Squamous cell carcinoma of the head and neck (SCCHN) accounts for >500,000 new cases and nearly 300,000 deaths annually worldwide as of 2012 [1]. Patients with recurrent/metastatic (R/M) SCCHN have a poor prognosis with median overall survival (OS) of Enoblituzumab is an investigational Fc-modified monoclonal antibody that binds B7-H3, which is over-expressed in a wide range of cancers including SCCHN [4], but not in most normal tissues. It has increased affinity for the acti- vating FcγR IIIA (CD16A) and decreased affinity for the inhibitory FcγRIIB (CD32B). The engineered Fc domain confers enoblituzumab with target-specific antibody-dependent cellular cytotoxicity in vitro and anti-tumor activity in preclinical studies, and in vivo and clinical data suggest that Fc-optimized antibodies such as enoblituzumab can en- gage both innate and adaptive immunity as mediators of anti-tumor activity [6]. Enoblituzumab was well tolerated in a Phase 1 monother- Fig. 1 (abstract P395). Study Schema apy trial with no maximum tolerated dose defined up to 15 mg/kg. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 217 of 272 P396 Key secondary endpoints include occurrence and severity of treatment- A phase 2 efficacy and safety trial of ADU-S100 and emergent adverse events and changes from baseline in safety assess- pembrolizumab in adults with head and neck cancer ments. This trial is currently in the recruitment phase. 1 2 3 Ezra Cohen, MD , Robert Ferris, MD, PhD , Douglas Adkins, MD , Dan 2 4 5 Zandberg, MD , Arkadiusz Dudek, MD, PhD , Matthen Mathew , Lara References 6 7 8 Dunn, MD , Juneko Grilley-Olson, MD , Ammar Sukari, MD , Rebecca 1. Chen PL, Roh W, Reuben A, et al. Analysis of Immune Signatures in 9 10 11 Redman, MD , Julie Bauman, MD, MPH , John Kaczmar, MD , Lisle Longitudinal Tumor Samples Yields Insight into Biomarkers of Response 12 13 14 15 Nabell, MD , Nabil Saba, MD , Eric Nadler, MD , Young Kim, MD , and Mechanisms of Resistance to Immune Checkpoint Blockade. Cancer 16 17 17 Ranee Mehra, MD , Nitya Nair, PhD , Somayeh Honarmand, MS , Discov 2016;6:827-37. 17 18 Richard Cutler Jr. , Barbara Burtness, MD 2. Gajewski TF, Fuertes MB, Woo SR. Innate immune sensing of cancer: University of California at San Diego, La Jolla, CA, United States; clues from an identified role for type I IFNs. Cancer Immunol University of Pittsburgh Medical Center, Pittsburgh, PA, United States; Immunother 2012;61:1343-7. Washington University of St. Louis, St. Louis, United States; 3. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the HealthPartners Regions Cancer Care Center, St. Paul, MN, United States; tumor microenvironment. Nat Immunol 2013;14:1014-22. Columbia University Medical Center, New York, United States; 4. Gajewski TF, Woo SR, Zha Y, et al. Cancer immunotherapy strategies Memorial Sloan-Kettering Cancer Center, New York, NY, United States; based on overcoming barriers within the tumor microenvironment. Curr University of North Carolina at Chapel Hill, Chapel Hill, NC, United Opin Immunol 2013;25:268-76. States; Wayne State University School of Medicine, Detroit, MI, United 5. Woo SR, Corrales L, Gajewski TF. The STING pathway and the T cell- 9 10 States; University of Louisville, Louisville, United States; The University inflamed tumor microenvironment. Trends Immunol 2015;36:250-6. of Arizona Cancer Center, Tucson, United States; MUSC Hollings Ethics Approval Cancer Center, Charleston, SC, United States; University of Alabama at This study was approved or is currently under review by an institutional Birmingham, Birmingham, United States; Emory University, Atlanta, GA, review board at each site. United States; Baylor Charles A. Sammons Cancer Center, Dallas, United States; Vanderbilt University School of Medicine, Nashville, P397 United States; University of Maryland, Greenebaum Comprehensive Interim analysis of the combination of durvalumab and cetuximab Cancer Center, Baltimore, United States; Aduro Biotech, Berkeley, CA, in a phase II trial of patients with recurrent and metastatic head United States; Yale University School of Medicine, New Haven, CT, and neck squamous cell carcinoma United States 2 1 1 1 Shuchi Gulati, MD , Sarah Palackdharry , Layne Weatherford , Sarah Wilson , Correspondence: Richard Cutler Jr. (rcutler@aduro.com) 1 1 1 3 1 Shireen Desai , Aubrey Steele ,KashifRiaz , Vinita Takiar ,Trisha Draper Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P396 1 2 University of Cincinnati, Cincinnati, OH, United States; University of Cincinnati Cancer Institute, Cincinnati, OH, United States; University of Background Cincinnati/Barrett Cancer, Cincinnati, OH, United States Immune checkpoint inhibitors such as the PD-1 blocking antibody Correspondence: Shuchi Gulati (gulatisi@ucmail.uc.edu) pembrolizumab have demonstrated marked improvements in duration Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P397 of response and long-term survival over standards of care (SOC) in head and neck squamous cell carcinoma (HNSCC) and other cancers. Background However, the significant percentage of patients who are nonresponsive Cetuximab ( IgG1 isotype monoclonal antibody) monotherapy is con- to these immunotherapies (primary resistance) or experience disease sidered standard of care therapy for recurrent and metastatic head relapse following an acquired immune resistance mechanism (second- and neck squamous cell carcinoma (HNSCC).[1] Cetuximab results in ary resistance) [1] highlights the need for new therapies. As tumor re- NK cell mediated ADCC and inhibition of the EGFR signaling path- sponsiveness to immunotherapy may depend, in part, on the way.[1] NK cell activation increases secretion of plasma transforming immunophenotype of the tumor microenvironment (TME) [2-5], one ex- growth factor β (TGFβ) and interleukin 10 (IL-10), resulting in in- ploratory approach to establish, re-establish, or enhance active immune creased expression of PD-1 on T cells and PD-L1 expression on tumor surveillance conditions within the TME is to inject innate immune mod- cells. Blocking the PD-1/PD-L1 check-point receptor pathway in- ulators directly into the tumor to promote an adaptive tumor-specific creases the cytotoxic response of NK cells in mice.[2] Therefore, it immune response. ADU-S100 (MIW815) is a novel synthetic cyclic di- was hypothesized that cetuximab and PD-1/PD-L1 blockade would nucleotide that activates the stimulator of interferon genes (STING) be synergistic. Here we report our findings on the combination of pathway within the TME leading to activation of tumor-resident APCs cetuximab with a PD-L1 inhibitor, durvalumab on T cells, NK cells and priming of tumor antigen specific CD8+ T cells. Direct activation of and cytokines from a phase II open-label single site clinical trial in STING via intratumoral injection of ADU-S100 (MIW815) has been HNSCC patients with recurrent or metastatic disease. (NCT03691714.) shown to overcome active tolerance mechanisms through stimulation Methods of resident leukocyte populations. Preclinical models indicate that sur- Interim analysis includes a total of 15 enrolled patients. Using vival and local tumor shrinkage were significantly enhanced when flow cytometry and Luminex, we evaluated the immune cell phe- ADU-S100 (MIW815) was administered with an anti-PD-1 antibody, sug- notypes and cytokine profiles of peripheral blood in patients be- gesting the PD-1 blockade may act synergistically with concomitant fore and after treatment with the combination of cetuximab and STING activation. In phase 1 trials, tumor shrinkage and durable re- durvalumab with respect to overall response rate (ORR). sponses have been observed after treatment with S100 alone or in Results combination with a PD-1 inhibitor. The primary objective of this trial is Fourteen patients who received at least 2 cycles of treatment were to evaluate the clinical efficacy of intratumoral ADU-S100 (MIW815) included in the interim analysis. Median age was 66 years (range 47- when administered in combination with pembrolizumab. 75), majority of patients were male (79%). Eight patients (57%) had Methods received 1 line of prior chemotherapy, while 3 (21%) had received 2 This open-label, multicenter phase 2 clinical trial (NCT03937141) aims to prior chemotherapies. Seven patients (50%) had received prior im- enroll 33 adults with PD-L1 positive, recurrent or metastatic HNSCC for munotherapy. Of the 7 patients who had next generation sequen- which pembrolizumab is indicated as SOC in the first-line setting. Patients cing completed, 1 was PDL1 positive (14%), all had MSI-high tumors with at least one lesion that is accessible for repeat intratumoral injection (100%) and all had TP53 mutations (100%). One patient achieved a and can provide tumor tissue for eligibility determination and biomarker partial response, and 3 were noted to have stable disease; overall re- analyses will receive intravenous infusions of pembrolizumab (200 mg) at sponse rate (ORR) was noted as 27%. No grade 3/ 4 adverse events Day 1 and intratumoral injections of ADU-S100 (MIW815) (800 mcg/le- attributable to study drugs were reported. Results from peripheral sion) at Day 1 and 8 in 21-day dosing cycles up to 35 cycles, or until cri- blood flow cytometry analyses showed an increase in cytokine pro- teria for treatment discontinuation are met. The primary endpoint is the ducing NK cells and CD3+ T cells in all responders. Luminex assay objective response per Response Evaluation Criteria in Solid Tumors v1.1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 218 of 272 revealed that all responders had a drop in their GM-CSF levels, and an increase in their TNF-alpha and CXCL-10 levels. Conclusions The combination of cetuximab and durvalumab results in a pro- tumorigenic profile with a modest ORR. References 1. Ferris, R. L. et al. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation. Cancer Treat. Rev. 63, 48–60 (2018). 2. Hsu, J. et al. Contribution of NK cells to immunotherapy mediated by PD- 1/PD-L1 blockade. J. Clin. Invest. 128, 4654–4668 Ethics Approval The study was approved by University of Cincinnati's Ethics Board, approval no. FWA #: 000003152 P398 miRNA-A and programmed death ligand 1 (PD-L1) expression in oral squamous cell carcinoma 1 2 2 2 Hong Hyung, MD PhD , Yoon Ho Ko , Lee Hee JIn , Sang Hoon Jeon 1 2 Catholi, Seoul, Korea, Republic of; Catholic Universtiy, Uijeongbu-si, Korea, Republic of Correspondence: Yoon Ho Ko (koyoonho@catholic.ac.kr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P398 Background Overexpression of PD-L1 in cancer cells is involved not only in the im- Fig. 1 (abstract P398). A and B. See text for description mune evasion but also in cancer progression. Increasing evidence indi- cates that dysregulation of miRNA(miR)s contributes to the pathogenesis of oral squamous cell carcinoma (OSCC). Here, we identified miR-C that regulate the expression of PD-L1 and elucidated whether miR-C affects chemotherapy responsiveness via regulating PD-L1 expression in OSCC. Methods To further verify the role of miRNAs on PD-L1 in OSCC, we carried out the functional study in human head and neck cancer cell line CAL27 and YD8. After transfection with scrambled miRNA-A, B, C (Scr), miRNAs-A, B, C for 48 h, PD-L1 mRNA and PD-L1 protein levels were assessed by RT-qPCR and western blot analysis. To perform EGFP reporter assay, cells were transfected with miRNA-C and re- porter constructs, containing the putative PD-L1 3’-UTR target sites, along with a control vector, EGFP levels were assessed by western blotting. Cell viability was assessed after treatment with 5-FU for 72 h using by MTT solution. GAPDH mRNA and its protein level were used for normalization and as a loading control. Results We investigated various miRs that were negatively correlated with PD-L1 in The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset and could recognize PD-L1 3'-UTR by analyzing TargetScan. Three miRs (miR-A, miR-B, miR-C) were identi- fied which had not been reported to be as associated with PD-L1 be- fore. EGFP reporter assay of only miR-C out of three miRs showed a decrease in the relative PD-L1 expression. This would indicate that only miR-C can recognize target sites in the 3’-UTR of PD-L1 mRNA in OSCC cells. Overexpression of miR-C induced the decrease of PD-L1 mRNA and protein (Figure 1A,1B). The sensitivity of CAL27 and YD8 cells to 5-FU was increased when miR-C was overexpressed (Figure 2). Also, the level of cleaved PARP, one of the apoptotic markers, was increased according to miR-A overexpression (Figure 3). Conclusions Our data suggest that miR-C can regulate PD-L1 expression by tar- geting PD-L1 mRNA, and our present findings shed new light on the complex regulation of PD-L1 in human tumors, and on miR-C in can- Fig. 2 (abstract P398). See text for description cer immuno-based therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 219 of 272 Trial Registration NCT03246958, NCT03341936, NCT03425331, NCT02971956, NCT03075527, NCT02635061 Ethics Approval The present studies were reviewed and approved by the Dana- Farber/Harvard Cancer Center (DF/HCC) institutional review board (Boston, Massachusetts, USA) and all were performed in accordance with relevant guidelines and regulations. Consent Written informed consent was obtained from all subjects prior to par- ticipation in these studies. Informed consent by patients to DF/HCC protocol 02-180 enabled collection of clinical and demographic data, and genomic characterization. P400 Sitravatinib and Nivolumab for resectable Oral cavity squamous cell carcinoma Window of opportunity study (SNOW) 1 1 2 Marc Oliva Bernal, MD , Douglas Chepeha, MD , Amy Prawira, MD , Fig. 3 (abstract P398). See text for description 1 1 3 Anna Spreafico, MD PhD , Scott Bratman, MD , Tina Shek, MD , John De 1 3 1 1 Almeida, MD , Ivan Yeung, MD , Aaron Hansen , Andrew Hope, MD , 1 1 P399 David Goldstein, MD , Ralph Gilbert, MD , Doug Vines, BSc, MRT(N), 3 1 1 1 Instructive conclusions from performing immune correlatives on IO CNMT , Patrick Gullane , Dale Brown, MD , Ilan Weinreb, MD , Bayardo 1 4 4 trials: a meta-analysis of patient tumor and blood samples Perez-Ordoñez, MD , Trevor Pugh, PhD , Pamela Ohashi, PhD , Ben 4 1 5 Patrick Lizotte, PhD, Megan Cavanaugh, Melissa Jean, Cloud Paweletz, Wang, PhD , Jonathan Irish, MD , Hirak Der-Torossianh, MD , Isan Chen, 5 1 PhD MD , Lillian Siu, MD Dana-Farber Cancer Institute, Boston, MA, United States Princess Margaret Cancer Centre, University of Toronto, Toronto, Correspondence: Cloud Paweletz Canada; The Kinghorn Cancer Centre, St Vincent’s Hospital, Darlinghurst, (CloudP_Paweletz@DFCI.HARVARD.EDU) Australia; Princess Margaret Cancer Centre, University of Toronto; Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P399 Quantitative Imaging for Personalized Cancer Medicine, TECHNA Institute, University Health Network., Toronto, Canada; University of Background Toronto, Toronto, Canada; Mirati Therapeutics, San Diego, CA, United Blood-based immune phenotyping provides a cost-effective, States minimally invasive, longitudinal, and logistically convenient Correspondence: Lillian Siu (lillian.siu@uhn.ca) assay that may provide two critical pieces of information for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P400 cancer patients receiving immunotherapy: 1.) if the therapeutic is working as intended, and 2.) if there will be any benefit to Background the patient. Sitravatinib is a receptor tyrosine kinase inhibitor that blocks TAM Methods and VEGF family of receptors. Based on non-clinical findings, it is pre- Our group has performed multi-parameter flow cytometric immune- dicted to increase M1 macrophage response and decrease immuno- profiling on hundreds of blood and tumor samples from patients suppressive Tregs and MDSCs in the tumor microenvironment. treated with immunotherapy. We have compiled this dataset of im- Sitravatinib combined with nivolumab showed a safe toxicity profile mune correlatives from patients receiving immune checkpoint block- and promising antitumor activity in non-small cell lung cancer pa- ade enrolled on Dana-Farber clinical trials in the following cancers: tients (pts) progressing on anti-PD-1 agents [1]. The CheckMate-358 thyroid cancer (tumor n = 24; blood n = 180) , head & neck squa- study revealed that preoperative nivolumab was safe and active in mous cell carcinoma (tumor n =18; blood n = 118) , mesothelioma oral cavity squamous cell carcinoma (OCSCC) [2]. We hypothesize (tumor n = 41) , non-small cell lung cancer (tumor n = 34) , and that preoperative sitravatinib and nivolumab have synergistic im- gastric-esophageal cancer (tumor n= 55). munogenic and antitumor effects in OCSCC. Results Methods Our meta-analysis of blood and tumor flow-based immune profil- SNOW is an investigator-initiated, single-center, non- ing confirms that tumor tissue remains the benchmark for deter- randomized, window-of-opportunity study evaluating pre- mining therapeutic efficacy to immune checkpoint blockade. operative sitravatinib and nivolumab in pts with resectable, However, our profiling of blood has produced several previously untreated OCSCC. Pts with T2-4a, N0-2 or T1 generalizable findings: 1.) IO-relevant markers are generally (>1cm)-N2 tumors as per AJCC 8th edition, ECOG >/=1, ad- expressed at very low levels by circulating T cells and only subtly equate organ function and no autoimmune disorders are eli- change after treatment, 2.) the abundance of different leukocyte gible. Figure 1 summarizes study design and treatment. lineages is also largely static, 3.) serial blood profiling at time- Primary objective is to evaluate the immune and pharmaco- points later than three weeks after initiation of treatment are dynamic effects of the treatment combination. Secondary ob- minimally informative, 4.) some immune parameters significantly jectives are: (a) safety, including rate of treatmen-related correlated with therapeutic efficacy are simply indicative of adverse events (TRAEs), surgery completion within the immune-related adverse events, which are historically associated planned window and postoperative complications; (b) antitu- with improved therapeutic efficacy and also easy to diagnose mor activity, including clinical and pathologic responses; rate without immune correlatives, and 5.) it is impossible to delineate of pathological extranodal extension (ENE) and positive mar- between reinvigorated or activated tumor-specific circulating T gins; (c) pharmacokinetics/pharmacodynamics of sitravatinib cells and global reinvigoration or activation of, for instance, by- alone and combined with nivolumab. Correlative studies in- stander T cells without more sophisticated and costly TCR clude: immune biomarkers by multiplex immunohistochemis- deconvolution. try, tumor and blood immunophenotyping; tumor genome Conclusions and transcriptome analyses; intratumoral hypoxia changes We conclude that blood-based immune phenotyping can be, in using 18FAZA-PET. Preliminary results as of June 30th, 2019 some contexts, highly informative, but invites over-analysis. are reported. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 220 of 272 Results P401Neoadjuvant Seven out of the 12 planned evaluable pts have been en- and adjuvant pembrolizumab plus standard of care (SOC) in rolled: 1 pt is currently undergoing study treatment and thus patients with resectable, locally advanced head and neck excluded from this analysis. Median follow-up: 19.5 weeks. All squamous cell carcinoma (HNSCC): the phase 3 KEYNOTE-689 pts completed study treatment and had surgery within the study 1 2 3 planned window. None required sitravatinib dose reduction/ Ravindra Uppaluri, MD, PhD , Nancy Lee, MD , William Westra , Ezra 4 5 6 hold or nivolumab delay. No G3/G4 TRAEs occurred pre- Cohen, MD , Robert Haddad , Stephane Temam , Christophe Le 7 8 9 10 surgery. One pt had G3 neck infection and G3 bleeding from Tourneau , Rebecca Chernock , Sufia Safina , Arkadiy Klochikhin , 11 12 13 the tracheostomy site 11 days post-surgery, both resolved Amichay Meirovitz , Irene Brana, MD , Joy Yang Ge , Ramona Swaby, 13 13 8 and deemed possibly related to study drugs. Tumor reduction MD , Cecilia Pinheiro , Douglas Adkins, MD as per investigator’s assessment was observed in all pts. Five Dana-Farber Cancer Institute and Brigham and Women’s Hospital, pts had pathological downstaging, including 1 complete Boston, MA, United States; Memorial Sloan Kettering, New York, NY, pathological response (Table 1); all pts had clear margins and USA, Sylmar, CA, United States; Icahn School of Medicine, New York, no ENE. All pts received standard of care postoperative radio- NY, USA, New York, United States; University of California San Diego, La therapy based on clinical stage. None required postoperative Jolla, CA, USA, La Jolla, CA, United States; Dana-Farber Cancer Institute chemotherapy. All pts are alive with no recurrence to date. and Brigham and Women's Hospital, Boston, MA, United States; 6 7 Conclusions Gustave Roussy, Villejuif, France, Villejuif, France; Institut Curie, Paris, These preliminary results suggest that preoperative sitravatinib France, Paris & Saint-cloud, France; Washington University School of and nivolumab is a safe and active combination in OCSCC. On- Medicine, St. Louis, MO, United States; Republican Dispensary of going biomarker and tumor immunophenotyping analyses will Tatarstan MoH, Kazan, Russia, Kazan, Russian Federation; Yaroslavl be presented Regional Clinical Oncology, Ulitsa Chkalov, Yaroslavl, Russia, Yaroslavl, Russian Federation; Hadassah-Hebrew University Medical Center, Acknowledgements Jerusalem, Israel, Jerusalem, Israel; Hospital Vall d’Hebron, Barcelona, The authors would like to thank patients and their families for their Spain, Barcelona, Spain; Merck & Co., Inc., Kenilworth, NJ, USA, participation and Mirati Therapeutics for drug supply and their support of Kenilworth, NJ, United States this study. Correspondence: Ravindra Uppaluri Trial Registration (ravindra_uppaluri@dfci.harvard.edu) NCT03575598 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P401Neoadjuvant References Background 1. Leal et al. ESMO Meeting 2018, Abstract 1129O. Neoadjuvant and adjuvant pembrolizumabshowedevidenceof 2. Ferris et al. ESMO Meeting 2017, Abstract LBA46. pathological response (PR) and acceptable safety in patients Ethics Approval with high-risk, resectable, locally advanced (LA) HNSCC in phase This study was approved by the University Health Network Research Ethics 2 studies (NCT02296684 and NCT02641093). KEYNOTE-689 Board (Study number: 18-5537) on July 12th 2018. (NCT03765918), a randomized, open-label, phase 3 trial, will as- sess efficacy and safety of neoadjuvant pembrolizumab and ad- juvant pembrolizumab plus SOC in patients with previously untreated, resectable LA HNSCC. Methods Eligible patients are adults with newly diagnosed, resectable HNSCC (stage III oropharyngeal p16-positive disease [T4 (N0-N2), M0]; stage III/IVA oropharyngeal p16 negative; or stage III/IVA larynx or hypo- pharynx or oral cavity, independent of p16 status) [1] and ECOG per- Fig. 1 (abstract P400). SNOW study design and treatment plan formance status 0 or 1. Patients will be randomly assigned 1:1 to arms A and B, with randomization stratified by primary tumor site (oropharynx/oral cavity vs larynx vs hypopharynx), tumor stage (III vs Table 1 (abstract P400). Tumor downstaging following study IVA), and PD-L1 status defined by tumor proportion score 50% treatment (TPS≥50% vs TPS Trial Registration ClinicalTrials.gov, NCT03765918 Reference 1. American Joint Committee on Cancer. AJCC Cancer Staging Manual, Eight Edition. Amin MB, ed. Chicago, IL: American College of Surgeons; Ethics Approval The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Consent All patients provided written informed consent to participate in the clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 221 of 272 P402 BELINDA : A phase 3 study evaluating the safety and efficacy of tisagenlecleucel versus standard of care in adult patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma 1 3 4 5 Michael Bishop, MD , Ian Flinn, MD , Peter Borchmann , Ulrich Jaeger , 6 7 8 9 Jason Westin, MD , Nada Hamad , Duncan Purtill , Richard Greil , 10 11 12 13 Simone Thomas , Takanori Teshima , Hideo Harigae , Carlos Garcia , 14 15 16 17 Pere Barba , Abhinav Deol, MD , Paul Shaughnessy , Jessie Gu , 18 17 17 Giovanna Andreola , Marcela Martinez Prieto , Lida Pacaud , Stephen Schuster, MD 1 2 University of Chicago, Chicago, IL, United States; Univeristy of Chicago, Chicago, IL, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University Hospital of Cologne, Cologne, Germany; 5 6 Medical University of Vienna, Vienna, Austria; University of Texas, MD Anderson Cancer Center, Houston, TX, United States; St Vincent’s Hospital, Sydney, Australia; Fiona Stanley Hospital, Murdoch, Australia; 9 10 Paracelsus Medical University, Salzburg, Austria; University Hospital Fig. 1 (abstract P402). See text for description Regensburg, Regensburg, Germany; Hokkaido University, Hokkaido, Japan; Tohoku University Graduate School of Med, Miyagi, Japan; 13 14 University Hospital "12 de Octubre", Madrid, Spain; Universitat Autònoma de Barcelona, Barcelona, Spain; Karmanos Cancer Institute, P403 Wayne State University, Detroit, MI, United States; Texas Transplant Serum soluble CD25 may predict the early therapeutic response in Institute, San Antonio, TX, United States; Novartis Pharmaceuticals pediatric patients with B-cell non-Hodgkin's lymphoma(B- Corporation, East Hanover, NJ, United States; Novartis Pharma AG, Basel, NHL)during autologous chimeric antigen receptor T cell(CAR-T) Switzerland: Abramson Cancer Center, University of Pennsylvania, therapy Philadelphia, PA, United States Jing Guo, Yonghong Zhang, Professor, Juan Du, MD Correspondence: Michael Bishop Beijing Boren Hospital, Beijing, China (mbishop@medicine.bsd.uchicago.edu) Correspondence: Yonghong Zhang (yhzhang58@126.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P402 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P403 Background Background Around one-third of aggressive B-cell non-Hodgkin lymphoma (NHL) CAR-T therapies have been widely employed in B-NHL.Immune acti- patients will not respond to, or will relapse or progress after frontline vation induced by CAR-T therapies includes significant changes of treatment; >50% of treatment failures occur within one year. Progno- some inflammatory cytokines. In this study we analyzed the changes sis is particularly poor in these patients, regardless of salvage chemo- of serum cytokine levels in 12 pediatric patients with B-NHL during therapy and autologous hematopoietic stem cell transplant (auto- CAR-T therapy, so as to explore the relationship between serum cyto- HSCT). Novel therapies are therefore needed for refractory or early- kine levels and early treatment response. The association between relapsed patients with NHL. cytokine levels and cytokine-release syndrome(CRS) grade was also Methods investigated. BELINDA (NCT03570892) is a randomized, open-label, multicenter, Methods phase 3 study to compare the safety and efficacy of two treat- 12 B-NHL pediatric patients aging from 4 to 14 in stage II to IV ac- ment strategies: tisagenlecleucel with standard of care (SOC) cording to st.jude stage were enrolled .Each patient received 1 to 3 immunochemotherapy followed by auto-HSCT in adult patients rounds sequential CAR-T treatment, consisting of CD19, CD20, CD22 with aggressive B-cell NHL whose disease relapsed or progressed CAR-T treatment. A total of 18 rounds CAR-T were performed, includ- after frontline immunochemotherapy. Eligible patients are aged ing 12 CD19, 3 CD20 and 3 CD22. Early tumor response were evalu- ≥18 years, have histologically confirmed aggressive B-cell NHL re- ated on day 15, 30 and 60 of each round of CAR-T and early side lapsed/refractory to frontline therapy containing rituximab and effect known as CRS were observed and graded. Serum samples anthracycline within one year of last dose, and are eligible for were collected at baseline and on day 3,7,11,15,20,30,60 of each auto-HSCT. Patients are apheresed prior to enrollment and ran- round of CAR-T. Levels of Interleukin-6 (IL-6), soluble CD25 (sCD25) domized 1:1 to receive tisagenlecleucel (Arm A) or SOC (Arm B) and interferon-γ (IFN-γ) interleukin-10 (IL-10), tumor necrosis factor- (Figure 1). Randomization is stratified by remission duration (re- α(TNF-α)were measured by ELISA(enzyme-linked immunosorbent fractory or relapse assay). We analyzed the cytokine changes in different treatment out- Trial Registration comes, explored the predictive value of different cytokines using NCT03570892 ROC curve .The relationship between cytokine level and CRS grade Ethics Approval was also analyzed. Tumor response was assessed per RECIST 1.1. The The study is done in accordance with the principles of Good Clin- ROC curve take” response “and “no response” as the outcome indica- ical Practice, the Declaration of Helsinki, and all local regulations. tors ,stable disease (SD) and progressive disease (PD)were defined as The study protocol and all amendments were reviewed and ap- “no response”, complete response(CR) and partial response(PR )were proved by independent ethics committees or institutional review defined as “response”. Adverse Event (AE) grade categorization is ac- boards for each center. All patients provided written informed cording to CTCAE 4.0. consent. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 222 of 272 Results with B cell malignancies for which there are no effective therap- There were statistically significant differences in sCD25 values among ies that induce durable remissions. different treatment responses (P<0.01), with an average decrease in SD patients and an average increase in PR patients ,it is speculated P405 that sCD25 may play a predictive role in treatment response. The CASSIOPEIA: A phase 2 study evaluating efficacy and safety of ROC curve analysis shows that sCD25 had a predictive effect on the tisagenlecleucel in first-line therapy for high-risk pediatric and response to treatment, and the AUC was 0.719,95%ci =(0.516, 0.922) young adult patients with B-ALL who are MRD positive at the EOC ,excluding 0.5, indicating that the difference is statistically significant. 1 1 Shannon Maude, MD, PhD , Hunger Stephen, MD , Jochen Buechner, In this study, other cytokines were not found to be predictive 2 1 3 4 5 MD , Stephen Grupp, MD , Susana Rives , Andre Baruchel , John Levine , markers of therapeutic response. Besides,Spearman rank correlation 6 7 8 9 Joerg Krueger , Theodore Laetsch , Marianne Ifversen , Aiesha Zia , coefficient showed that there was a positive correlation between 10 10 11 Jaclyn Davis , Eric Bleickardt , Mignon Loh sCD25 and CRS grade (r=0.693,P=0.001). University of Pennsylvania; Children’s Hospital of Philadelphia, Conclusions Philadelphia, PA, United States; Oslo University Hospital, Oslo, Norway; sCD25 may be a useful predictive marker for early response of CAR-T 3 4 Sant Joan de Deu Hospital, Barcelona, Spain; Hôpital Robert and level of sCD25 are correlated with CRS grade. Clinical trial Debré&Université de Paris, Paris, France; Mount Sinai School of information:ChiCTR18000144 Medicine, New York, NY, United States; The Hospital for Sick Children, Toranto, Canada; UT Southwestern Medical Center and Child, Dallas, TX, P404 United States; Copenhagen University Hospital Rigshospi, Copenhagen, 9 10 Developing canine CART-19 to fully leverage comparative Denmark; Novartis Pharma AG, Basel, Switzerland; Novartis oncology and inform human clinical trials Pharmaceuticals Corporation, East Hanover, NJ, United States; Kumudhini Haran, MS, Ailian Xiong, Enrico Radaelli, Patrick Savickas, University of California, San Francisco, CA, United States Avery Posey, Donald Siegel, Nicola Mason, BVet Med PhD Correspondence: Shannon Maude (maude@email.chop.edu) University of Pennsylvania, Springfield, PA, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P405 Correspondence: Nicola Mason (nmason@vet.upenn.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P404 Background Survival is compromised for patients with high-risk (HR) B-cell acute Background lymphoblastic leukemia (B-ALL) who have a poor response to first- CD19 specific chimeric antigen receptor T cell (CART-19) therapy has line chemotherapy. A Children’s Oncology Group (COG) phase-3 resulted in unprecedented durable clinical responses in adult and study for HR B-ALL, AALL0232, showed poor 5-year disease free sur- pediatric patients with B-cell malignancies. However, poor quality of vival (DFS) of 39% in patients with minimal residual disease (MRD) patient T cells, failed persistence, reduced effectiveness within an im- ≥0.1% at end of induction (EOI) and MRD ≥0.01% at the end of con- munosuppressive microenvironment and target antigen loss, repre- solidation (EOC) [1]. The objective of this trial is to determine the effi- sent some of the challenges to improving CART19 efficacy. cacy and safety of tisagenlecleucel in pediatric and young adult Furthermore, correlative biomarkers that predict CART-19 response patients with de novo HR B-ALL who received first-line treatment remain elusive. and remain MRD-positive after the EOC therapy. Pet dogs spontaneously develop B-NHL and B cell leukemias that Methods share oncogenic pathways and similar immunosuppressive features CASSIOPEIA (NCT03876769) is a phase-2, single-arm, global, multicen- to human B cell malignancies. Therefore, they provide an immuno- ter, open-label study being conducted in collaboration with COG. Pa- logically intact, parallel patient population in which to evaluate next tients aged 1-25 years with de novo National Cancer Institute generation CAR T cell strategies and combination approaches that defined HR B-ALL (presenting white blood count >50,000/μL or over address current CART19 challenges. Previously, we have demon- the age of 10 years) who are in first complete remission (CR1) but re- strated the ability to generate functional CD20-targeting canine CAR main MRD-positive (≥0.01% by flow cytometry determined at a cen- T cells. Their use in client owned animals with B-NHL can lead to the tral reference laboratory) at EOC are eligible. Prior to screening, development of canine anti-mouse antibody (CAMA) formation and patients will complete a standard of care first-line 4-drug induction, target antigen escape. To address these issues and provide a parallel MRD assessment at EOI, a Berlin-Frankfurt-Münster phase-1b consoli- reagent that can inform human CAR T cell strategies, we have devel- dation, and MRD assessment at EOC. Eligible patients undergo leuka- oped a fully canine CD19 targeting CAR and confirmed its function pheresis either at the EOI or EOC. Prior to tisagenlecleucel infusion, in vitro against CD19 expressing targets. patients will receive interim maintenance including high-dose Methods methotrexate. Following lymphodepleting chemotherapy, patients We employed a canine scFv phage display library to isolate canine receive a single infusion of tisagenlecleucel based on body weight; CD19-specific scFvs following 3-4 rounds of panning against the sol- 0.2-5.0x10^6 chimeric antigen receptor (CAR)-positive viable T-cells uble extracellular domain of canine CD19. Twelve unique scFvs were per kg in patients ≤50 kg or 0.1-2.5x10^8 CAR-positive viable T-cells isolated and their binding to soluble canine CD19 and cell surface in patients >50 kg. Patients may receive a second infusion based on expressed CD19 was confirmed by ELISA and flow cytometry respect- B-cell recovery and MRD status. Efficacy will be assessed at day 29, ively. One of the highest binding candidates was cloned into a fully then every 3 months for the first year, every 6 months for the second canine CD28ζ CAR in a pMX retroviral plasmid. Retroviruses pseudo- year, then yearly until the end of study. The primary outcome is 5- typed with both RD114 and VSV-G envelope proteins were generated year DFS rate by local investigator assessment, defined as the time using standard protocols and used to transduce primary canine T from tisagenlecleucel infusion to morphologic relapse, occurrence of cells activated using anti-canine CD3/CD28 beads in the presence of secondary malignancy or death from any cause, whichever occurs RetroNectin®. Successful transductions of canine T cells were ob- first. Secondary outcomes include percentage of patients in remis- tained with 45% of T cells expressing CAR on their cell surface by sion without allogeneic transplantation at 1 year, MRD negativity at flow cytometry. Canine CART-19 cells demonstrated antigen-specific month 3, overall survival, cellular kinetics, and safety. The primary proliferation and cytokine production in vitro. We now aim to per- analysis of DFS will be undertaken when 40 DFS events are observed form a pilot study to evaluate the safety and efficacy of this ap- or 6 years after first-patient-first-treatment, whichever occurs later. proach in canine patients with relapsed, refractory B-NHL. This work The estimated enrollment for this study is 160 patients (with 140 in- will serve the dual purpose of enabling pet dogs with spontan- fused). The study is currently enrolling patients in the U.S., Europe eous B cell malignancies to accelerate application of next gener- and Canada. ation CART cell therapies into the human clinics as well as Trial Registration provide much needed immunotherapeutics for canine patients NCT03876769 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 223 of 272 Reference 2. Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, 1. Borowitz MJ, Wood BL, Devidas M et al. Prognostic significance of Sadelain M. CAR T cell-induced cytokine release syndrome is mediated minimal residual disease in high risk B-ALL: a report from Children's On- by macrophages and abated by IL-1 blockade. Nat Med. 2018 cology Group study AALL0232. Blood. 2015;126(8):964-971. Jun;24(6):731-738. Ethics Approval Ethics Approval The study is done in accordance with the principles of Good Clinical UCLA IRB #19-000604 Practice, the Declaration of Helsinki, and all local regulations. The study protocol and all amendments were reviewed and approved by P407 independent ethics committees or institutional review boards for each Phase 3 KEYNOTE-937: adjuvant pembrolizumab versus placebo in center. All patients provided written informed consent. patients with hepatocellular carcinoma and complete radiologic response after surgical resection or local ablation 1 2 3 4 P406 Masatoshi Kudo, MD, PhD , Andrew Zhu , Arndt Vogel , Thomas Yau , 5 6 6 6 Interleukin-1 blockade to prevent severe immune effector cell- Jian Zhou , Erluo Chen , Usha Malhotra , Abby Siegel , Ann-Lii Cheng, associated neurotoxicity syndrome; Trial in progress MD PhD Caspian Oliai, MD, Anna Crosetti, John Timmerman, MD Kindai University School of Medicine, Osaka, Japan, Osaka-Sayama, UCLA, Los Angeles, CA, United States Japan; Massachusetts General Hospital Cancer Center, Harvard Medical Correspondence: John Timmerman (jtimmerman@mednet.ucla.edu) School, Boston, MA, United States; Medizinische Hochschule, Hannover, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P406 Germany, Hannover, Germany; University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong PRC; Zhongshan Hospital, Fudan Background University, Shanghai, China, Shanghai, China; Merck & Co., Inc., CAR T-cell therapy targeting CD19 is a promising new treatment for Kenilworth, NJ, USA, Kenilworth, NJ, United States; National Taiwan relapsed/refractory B-cell lymphomas and leukemias. However, se- University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan vere grade 3 neurotoxicity (immune effector cell-associated neuro- Correspondence: Masatoshi Kudo (m-kudo@med.kindai.ac.jp) toxicity syndrome, or ICANS) is seen in up to one-third of patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P407 Recently, preclinical animal studies of human CD19 CAR T-cell therap- ies have shown that while IL-6 and IL-1 receptor antagonists could Background prevent cytokine release syndrome (without impairing anti-tumor ef- For patients with hepatocellular carcinoma (HCC) who are undergo- ficacy), only IL-1 blockade could prevent neurotoxicity [1,2]. The re- ing potentially curative surgical resection or local ablation, 5-year re- combinant IL-1 receptor antagonist Anakinra was used successfully currence rates are up to 50%-80%; there is no standard of care for to avert lethal neurotoxicity in mice. Anakinra crosses the blood brain adjuvant treatment. The programmed death 1 inhibitor pembrolizu- barrier and has been shown to be safe and efficacious in rheumatologic mab is approved for the treatment of patients with HCC previously conditions driven by high levels of monocyte lineage-associated IL-1 in- treated with sorafenib. There is no direct evidence of benefit with cluding rheumatoid arthritis and neonatal onset multisystem inflamma- pembrolizumab in the HCC adjuvant setting, but a favorable benefit/ tory disease, for which it is FDA approved. We are conducting the first risk profile is anticipated based on data from other indications. human trial of Anakinra to treat ICANS in B-cell lymphoma patients KEYNOTE-937 (NCT03867084) is a randomized, double-blind, phase 3 treated with anti-CD19 CAR T-cells. The trial has been approved by the trial to examine the safety and efficacy of adjuvant pembrolizumab U.S. FDA under an investigator-sponsored IND, and Anakinra is supplied versus placebo in patients with complete radiologic response after by the agent’s manufacturer (Sobi Pharmaceuticals). surgical resection or local ablation of HCC. Methods Methods Patients with diffuse large B-cell lymphoma receiving standard of Eligible patients are aged ≥18 years and have confirmed HCC, complete care CAR T-cells are eligible for enrollment. The primary objectives radiologic response after complete resection or local ablation, Eastern are to: 1) Evaluate the effectiveness of IL-1 blockade in reducing the Cooperative Oncology Group performance status of 0, and class A incidence and duration of severe ICANS in participants receiving anti- Child-Pugh score. Patients with past or ongoing HCV or controlled HBV CD19 CAR T-cells, 2) Assess the safety of Anakinra in CAR T-cell pa- are eligible if they meet certain criteria. Patients (N=~950) will be ran- tients, 3) Measure cytokines (including IL-1, IL-6, IL-15, TNF-alpha, domly assigned 1:1 to receive pembrolizumab 200 mg or placebo every interferon-gamma) and nitric oxide in the serum and CSF of treated 3 weeks and stratified by geographic region, prior local therapy (resec- patients prior to and during CAR T-cell therapy for correlation with tion vs ablation), recurrence risk, and alpha-fetoprotein level at diagno- ICANS events, and 4) Determine the tumor response rate in compari- sis. Treatment will continue for up to 17 cycles (~1 year) or until son to historical controls. Upon development of grade 1 ICANS, or documented disease recurrence, unacceptable toxicity, or investigator/ grade 3 CRS (which is often followed by ICANS), participants will re- patient decision to withdraw. Dual primary end points are recurrence- ceive Anakinra 100 mg subcutaneously every 6 hours for at least 12 free survival (RFS) and overall survival. Secondary end points are safety, doses, or until ICANS returns to grade 1 in participants who develop tolerability, and quality of life. Exploratory end points include distant grade 2 neurotoxicity. Patients will be continuously evaluated for tox- metastases–free survival (DMFS); time to recurrence (TTR); and gen- icity, and assessed for overall tumor response by day 120 with PET/ omic, metabolic, and/or proteomic biomarkers. RFS, DMFS, and TTR will CT scanning. Thirty-six participants will be treated in this multi- be assessed radiographically by the investigator and/or by subsequent center trial, at four centers within the University of California biopsy and confirmed by blinded independent central review. Adverse Hematologic Malignancies Consortium (UC Los Angeles, UC San events (AEs), graded per National Cancer Institute Common Termin- Francisco, UC San Diego, and UC Davis). The trial is powered to ology Criteria for Adverse Events version 4.0, will be recorded up to 30 detect a 50% reduction in the rate of severe ICANS compared to days after last dose (90 days for serious AEs). historical rates. Trial Registration ClinicalTrials.gov, NCT03867084 References Ethics Approval 1. Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, Sanvito The study and the protocol were approved by the Institutional Re- F, Ponzoni M, Doglioni C, Cristofori P, Traversari C, Bordignon C, Ciceri F, view Board or ethics committee at each site. Ostuni R, Bonini C, Casucci M, Bondanza A. Monocyte-derived IL-1 and Consent IL-6 are differentially required for cytokine-release syndrome and neuro- All patients provided written informed consent to participate in the toxicity due to CAR T cells. Nat Med. 2018 Jun;24(6):739-748. clinical trial. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 224 of 272 P408 P409 LEAP-002: phase 3 study of first-line lenvatinib plus A case report of personalized neoantigen peptide vaccine in pembrolizumab for patients with advanced hepatocellular treating patients with biliary tract cancer 1 2 1 3 3 carcinoma Fang Yong , Fan Mo , Jiawei Shou , Huimin Wang , Lin Chen , Shanshan 1 2 3 4 1 1 1 4 Josep Llovet , Masatoshi Kudo, MD, PhD , Ann-Lii Cheng, MD PhD , Zhang , Hongsen Li , Weidong Han , Hongming Pan , Shuqing Chen 4 5 6 7 1 2 Richard Finn , Peter Galle , Shuichi Kaneko, MD PhD , Tim Meyer , Sir Run Run Shaw Hospital, Hangzhou, China; Vancouver Prostate 8 9 10 10 3 Shukui Qin , Corina Dutcus , Erluo Chen , Leonid Dubrovsky , Abby Centre, UBC, Hangzhou, China; Hangzhou Neoantigen Therapeutics Co., 10 11 4 Siegel , Andrew Zhu Hangzhou, China; Zhejiang University, Hangzhou, China 1 2 Icahn School of Medicine at Mount Sinai, New York, NY, USA; Kindai Correspondence: Shuqing Chen (chenshuqing@zju.edu.cn) University School of Medicine, Higashiosaka, Japan, Osaka-Sayama, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P409 Japan; National Taiwan University Hospital Cancer Center, Taipei, Taiwan, Taipei, Taiwan; David Geffen School of Medicine at UCLA, Los Background Angeles, CA, USA, Los Angeles, CA, United States; University of Mainz Despite recent advance in immune checkpoint blockade therapies in Medical Center, Mainz, Germany, Mainz, Germany; Kanazawa University cancer, the overall response rate is still low in malignancy treatment. Hospital, Kanazawa, Japan, Kanazawa, Japan; University College London Arisen from tumor somatic mutations, neoantigens provide tumor Cancer Institute, London, United Kingdom, London, United Kingdom; specific targets for developing personalized cancer vaccines, further 8 9 Jinling Hospital, Nanjing, China, Nanjing, China; Eisai Inc., Woodcliff eliciting strong T cell-mediated immune response. A single-arm, Lake, NJ, USA, Woodcliff Lake, NJ, United States; Merck & Co., Inc., open-labelled, investigator-initiated clinical study was carried out to Kenilworth, NJ, USA, Kenilworth, NJ, United States; Massachusetts examine the safety and efficacy of personalized peptide vaccine General Hospital Cancer Center, Harvard Medical School, Boston, MA, (iNeo-Vac-P01). Total of 22 patients with solid tumors had been en- USA, Boston, MA, United States rolled in the trial from Feb 7th, 2018 to May 31st, 2019. A biliary tract Correspondence: Josep Llovet (Josep.Llovet@mountsinai.org) cancer patient achieved unique neoplastic changes. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P408 Methods The 63-year-old male, initially diagnosed with intrahepatic biliary Background tract cancer in 2013, was treated with surgical excision in Jun. 2013 Lenvatinib, a multikinase inhibitor, is approved for first-line treatment and postoperative chemotherapy in Apr. 2014 respectively. Both of unresectable hepatocellular carcinoma (HCC). Pembrolizumab, a tumor recurrence and metastases were confirmed with CT scan in programmed death 1 inhibitor, is approved for second-line treatment Nov. 2017. And then, he was treated with 6 cycles of PD-1 antibody of advanced HCC in patients previously treated with sorafenib. The in a clinical trial and dropped out due to disease progression. Under phase 1b KEYNOTE-524 trial showed that lenvatinib plus pembrolizu- his consent, his biopsy and blood samples were obtained for whole mab was well tolerated and demonstrated promising antitumor ac- exome sequencing (WES), RNA sequencing (RNA-seq), and neoanti- tivity in patients with unresectable HCC. LEAP-002 (NCT03713593) is gen identification [1-4]. Finally the total of 7 peptides were synthe- a phase 3 study to evaluate the safety and clinical benefit of lenvati- sized and pooled into 2 groups. nib plus pembrolizumab in patients with previously untreated ad- On Mar 22th, 2018, he started to receive iNeo-Vac-P01 subcutane- vanced HCC. ously (s.c.). The injection sites were the two upper arms. He was Methods scheduled to receive vaccinations with GM-CSF as adjuvant on Eligible patients are aged ≥18 years and have confirmed HCC, East- day 1, 4, 8,15 and 22 (i.e.priming phase),aswellas 6 subse- ern Cooperative Oncology Group performance status (ECOG PS) 0 or quent boosters [5-8]. 1, Barcelona Clinic Liver Cancer stage C or stage B disease not amen- Results able to locoregional or curative therapy, class A Child-Pugh score ≤7 After the last booster vaccination, a grade 3~4 allergic reaction days before study day 1, and ≥1 measurable lesion (per RECIST v1.1 (under NCI-CTCAE 4.03) happened, while clinical manifestations were by blinded independent central review [BICR]). Past or ongoing HCV nausea, vomiting and rash. The treatment-relating allergic reaction infection and controlled HBV are allowed. Patients will be randomly maybe result from peptide-specific antibody accumulation, however, assigned 1:1 to receive oral lenvatinib 12 mg (body weight [BW] ≥60 this hypothesis needs experimental validation by enzyme-linked im- kg) or 8 mg (BW 400 ng/mL); and ECOG PS (0 vs 1). Tumor imaging munosorbent assay. The CT scans indicated an evident increase of will be performed every 9 weeks. Dual primary end points are tumor size at 5th month, and a surprisingly decrease of tumor size at progression-free survival (PFS), assessed per modified RECIST v1.1 by 8th month, implying a pseudo-progression. The duration of stable BICR, and overall survival. Secondary end points are objective re- disease was 14.5+ months, and he had been keeping progression- sponse rate (ORR), duration of response (DOR), disease control rate free since then. The results of IFN-γ ELISPOT assay shows that the (DCR), and time to progression (TTP) per RECIST v1.1 by BICR, efficacy neoantigen peptides stimulated highest number of IFN-γ spots and outcomes (PFS, ORR, DOR, DCR, and TTP) per modified RECIST v1.1 induced significant peptide-specific T-cell response. TCR sequencing by BICR, pharmacokinetics, and safety. Exploratory end points are ef- demonstrated the evident increase of peripheral T cells with three ficacy outcomes evaluated per RECIST v1.1 and iRECIST assessed by TCRs (data unshown). the investigator. Adverse events (AEs), graded per National Cancer In- Conclusions stitute Common Terminology Criteria for Adverse Events version 4.0, The preliminary results demonstrated that iNeo-Vac-P01 treatment will be monitored throughout the treatment period and for 90 days was feasible and safe, and can prolong progression-free survival and after the last dose (120 days for serious AEs). overall survival. Trial Registration ClinicalTrials.gov, NCT03713593 Acknowledgements Ethics Approval The authors would like to gratitude all the patients who participated in the The study and the protocol were approved by the Institutional Re- trial and their families, as well as the Sir Run Shaw clinical site. This study was view Board or ethics committee at each site. funded by Hangzhou Neoantigen Therapeutics Co. Consent Trial Registration All patients provided written informed consent to participate in the This trial had been registration on ClinicalTrials.gov, the identifier number clinical trial. was NCT03662815. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 225 of 272 References dose finding part. Secondary objectives include characterization of 1. Chen F, Zou Z, Du J, Su S, Shao J, Meng F, Yang J, Xu Q, Ding N, Yang Y vactosertib pharmacokinetics and anti-tumor activity by response et al. Neoantigen identification strategies enable personalized rate (RECIST v1.1). immunotherapy in refractory solid tumors. J Clin Invest. 2019; 130. Results 2. Hundal J, Carreno BM, Petti AA, Linette GP, Griffith OL, Mardis ER, Griffith As of July 18, 2019, 13 patients were enrolled to the study (7 in 100 M. pVAC-Seq: A genome-guided in silico approach to identifying tumor mg BID cohort and 6 in 200 mg BID cohort). Median age was 66 neoantigens. Genome Med. 2016; 8(1):11. (range 45-76), 62% were male, median number of previous lines of 3. Ng AWR, Tan PJ, Hoo WPY, Liew DS, Teo MYM, Siak PY, Ng SM, Tan EW, chemotherapy was 4 (range 2-8). All patients were PD-L1 less than Abdul Rahim R, Lim RLH et al. In silico-guided sequence modifications of 25% by SP263 antibody assay. At 100 mg BID cohort, no dose limit- K-ras epitopes improve immunological outcome against G12V and G13D ing toxicity was observed. The most frequently reported adverse mutant KRAS antigens. PeerJ. 2018; 6:e5056. events (AE) were skin rash (30.8%), nausea (23.1%), and pruritis 4. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, Zhang W, Luoma A, (23.1%). There were 3 serious adverse events (SAE) reported; pleural Giobbie-Hurder A, Peter L et al. An immunogenic personal neoantigen effusion (1), skin eruption (1), and empyema (1), and no patients with vaccine for patients with melanoma. Nature. 2017; 547(7662):217-221. reported cardiotoxicity. Among 7 tumor response evaluable patients, 5. Gjertsen MK, Buanes T, Rosseland AR, Bakka A, Gladhaug I, Soreide O, best responses to treatment were SD in 3 patients; 5.9%, 10.4%, and Eriksen JA, Moller M, Baksaas I, Lothe RA et al. Intradermal ras peptide 26.4% decreases from baseline. Biomarker data will be presented at vaccination with granulocyte-macrophage colony-stimulating factor as the meeting. adjuvant: Clinical and immunological responses in patients with pancre- Conclusions atic adenocarcinoma. Int J Cancer. 2001; 92(3):441-450. The combination of vactosertib plus durvalumab has been tolerated 6. Keskin DB, Anandappa AJ, Sun J, Tirosh I, Mathewson ND, Li S, Oliveira G, thus far with no safety concerns; the study is ongoing. The anti- Giobbie-Hurder A, Felt K, Gjini E et al. Neoantigen vaccine generates tumor activity of this combination in patients with advanced NSCLC intratumoral T cell responses in phase Ib glioblastoma trial. Nature. 2019; will be further explored. Clinical trial information: NCT03732274 565(7738):234-239. Trial Registration 7. Weden S, Klemp M, Gladhaug IP, Moller M, Eriksen JA, Gaudernack G, NCT 03732274 Buanes T. Long-term follow-up of patients with resected pancreatic can- Ethics Approval cer following vaccination against mutant K-ras. Int J Cancer. 2011; The study was approved by Ethics Board of Severance Hospital (ap- 128(5):1120-1128. proval number 4-2018-0892) and National Cancer Center (approval 8. Kirner A, Mayer-Mokler A, Reinhardt C. IMA901: a multi-peptide cancer number NCC2019-0057) vaccine for treatment of renal cell cancer. Hum Vaccin Immunother. 2014; 10(11):3179-3189. P411 Ethics Approval Treating advanced non-small lung cancer (NSCLC) patients after This study was approved by the institutional review board and independent checkpoint inhibitor treatment failure with a novel combination of ethics committee of Sir Run Run Shaw Hospital, Zhejiang University Viagenpumatucel-L (HS-110) plus nivolumab School of Medicine; approval number 20180109-9. 1 1 Daniel Morgensztern, MD , Saiama Waqar, MD , Lyudmila Bazhenova, Consent 2 3 4 MD , Rachel Sanborn, MD , Lori Mcdermott, RN, MSc , Jeff Hutchins, Written informed consent was obtained from the patient for publication of 4 5 6 PhD , Luis Raez, MD, FACP, FCCP , Corey Langer, MD , Roger Cohen, this abstract and any accompanying images. A copy of the written MD consent is available for review by the Editor of this journal. Washington University School of Medicine, St. Louis, MO, United States; 2 3 Moores Cancer Center, La Jolla, CA, United States; Earle A. Chiles P410 Research Institute, Portland, OR, United States; Heat Biologics, Tampa, Safety and anti-tumor activity of the transforming growth factor β FL, United States; Memorial Cancer Institute, Pembroke Pines, FL, United receptor I kinase inhibitor, vactosertib, in combination with States; Perelman School of Medicine, Philadelphia, PA, United States durvalumab in patients with advanced non-small cell lung cancer Correspondence: Lori Mcdermott (lmcdermott74@yahoo.com) (NSCLC) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P411 1 2 2 Ji-Youn Han, MD, PhD , Kyoung-Ho Pyo , Jea Hwan Kim , Chun-Feng 2 3 3 3 Xin , Jin Kyung Lee , Sunjin Hwang , Seong-Jin Kim , Byoung Chul Cho, Background 2 2 MDphD , Byoung Chul Cho, MDphD Viagenpumatucel-L (HS-110) is an allogeneic cellular vaccine derived 1 2 National Cancer Center, Goyang-si, Korea; Severance Hospital, Seoul, from a human lung adenocarcinoma cell line transfected with the Korea, Republic of; Medpacto, Inc, Seoul, Korea, Republic of gp96-Ig fusion protein that functions as an antigen chaperone for Correspondence: Byoung Chul Cho (cbc1971@yuhs.ac) cross presentation and dendritic cell activation. DURGA is a multi- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P410 cohort study evaluating the combination of HS-110 and anti-PD-1 monoclonal antibodies in patients with advanced NSCLC. We report Background on Cohort B, which enrolled patients with progressive disease (PD) TGF-β signaling is known to be associated with poor response to single- after receiving a minimum of 4 months of treatment with a check- agent immune checkpoint inhibitors by immunosuppressive microenviron- point inhibitor (CPI) at any time prior to study entry. ment through strong epithelial-mesenchymal transition (EMT) induction. Methods Combined inhibition of immune checkpoint and TGF-β signal is anticipated Patients with previously treated NSCLC received weekly HS-110 (1 X as a promising therapeutic strategy because these two key pathways have 107 cells) intradermally for 18 consecutive weeks and nivolumab IV independent and complementary immunosuppressive functions. We are 240 mg every 2 weeks, followed by nivolumab maintenance until reporting the Dose Finding part of a Phase 1b/2a study evaluating the tumor progression or intolerable toxicity. Tissue was tested at base- combination of vactosertib, a highly selective and potent TGF-β inhibitor, line for PD-L1 expression (≥ 1% or < 1%) and tumor infiltrating lym- with durvalumab in patients with advanced non-small cell lung cancer phocytes (TILs). TIL high was defined as >10% CD8+ lymphocytes in (NSCLC) who progressed following platinum-based chemotherapy. the tumor stroma. The primary endpoint was objective response rate Methods (ORR) by RECIST 1.1. Secondary endpoints included ORR and clinical Eligible patients (pts) are ≥19 years old, have ECOG status ≤1, and benefit rate using iRECIST, progression-free survival (PFS), overall sur- have no prior exposure to immune checkpoint inhibitors, or TGFβ R1 vival (OS) and adverse events (AEs). kinase inhibitors. The primary objective is to assess the safety and Results the recommended dose of vactosertib given 5 days on/2 days off in As of March 2019, 56 patients were enrolled and evaluated for efficacy. combination with durvalumab 1500 mg every 4 weeks. Two dose The median number of prior treatment lines was 2 [range 1 to 6]. Seven levels of vactosertib (100 mg BID and 200 mg BID) were tested in the patients (13%) achieved partial response and 26 patients (46%) had Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 226 of 272 stable disease. Median PFS and median OS were 3.2 months and 11.8 Hossein Borghaei et al., Nivolumab versus Docetaxel in Advanced months, respectively. Immune ORR and clinical benefit rate by iRECIST Nonsquamous Non–Small-Cell Lung Cancer. The New England Journal of were 14% and 61%, respectively. Patients experiencing injection site re- Medicine. 2015; 373:1627-1639 actions (ISR) had improved PFS (3.7 vs 1.8 months; HR 0.21, p =0.0021) Achim Rittmeyer et al., Atezolizumab versus docetaxel in patients with and improved OS (12 vs 5 months; HR 0.16, p=0.0005) compared to previously treated non-small-cell lung cancer (OAK): a phase 3, open- those without ISR. 96% of patients experienced at least one adverse label, multicentre randomised controlled trial. The Lancet. 2017; 389 event, and 92% of all AEs were grade 1 or 2. The most common AEs (10066): 255-265 were fatigue (34%), hypocalcemia (18%), cough (16%) and diarrhea and Ethics Approval dyspnea (14% each). There were four grade 4 events: QTc prolongation, The last amendment was approved in June 2019 by WIRB and Copernicus stroke, pericardial tamponade, and hyponatremia, none of which were IRB, approval number 420160463 deemed related to treatment. There were no grade 5 AEs. Conclusions P413 The combination of HS-110 and nivolumab is well tolerated, and A Phase 1b/2 study of galunisertib in combination with nivolumab does not appear to increase the incidence of immune-related AEs as in solid tumors and NSCLC compared to CPI monotherapy. Patients continue to be enrolled into 1 2 3 Ernest Nadal, MD, PhD , Mansoor Saleh, MD , Santiago Ponce Aix, MD , this cohort. Data suggest that re-challenging the immune system 4 5 6 Maria Ochoa de Olza , Sandip Patel, MD , Scott Antonia, MD, PhD , with nivolumab and HS-110 after CPI treatment failure restores re- 7 7 7 Yumin Zhao, PhD , Ivelina Gueorguieva, PhD , Michael Man, PhD , sponsiveness and clinical benefit for some patients. 7 7 8 7 Shawn Estrem, PhD , Emin Avsar , Wen Hong Lin , Karim Benhadji , 7 9 1 Susan Guba , Inmaculada Ales Diaz , Ernest Nadal, MD, PhD Acknowledgements 1 2 Catalan Institute of Oncology, L'Hospitalet, Spain; University of Thank you to the Investigators, their staff, and the patients and family Alabama, Birmingham, AL, United States; Hospital 12 de Octubre, members that made this research possible. Madrid, Spain; Hospital Universitario Vall d'Hebron, Barcelona, Spain; Trial Registration 5 6 University of California, La Jolla, CA, United States; H. Lee Moffitt NCT 02439450 Cancer Center and, Tampa, FL, United States; Eli Lilly and Company, Ethics Approval Indianapolis, IN, United States; Bristol-Myers Squibb, New York, NY, This study was approved by Advarra IRB, Western IRB, Washington University United States; Hospital Universitario Regional de Mala, Malaga, Spain IRB, Cleveland Clinic IRB, UCSD IRB, Providence Portland IRB, NYU Winthrop Correspondence: Ernest Nadal (esnadal@iconcologia.net) IRB, Baptist Health Louisville IRB, Lifespan IRB, and US Oncology IRB. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P413 P412 Background Validation of a single-blinded (patients only) study design for the TGF-β promotes immune suppression. In this study, both TGF-β and prevention of premature patient consent withdrawal in the PD-1 were targeted in patients with advanced refractory solid tumors immuno-oncology trial DUBLIN-3 and recurrent/refractory NSCLC using galunisertib, an oral small mol- Ramon Mohanlal, MD, PhD, MBA, Huang Lan, PhD ecule inhibitor of TGF-β receptor I, in combination with nivolumab, a BeyondSpring Pharmaceuticals, Inc., New York, NY, United States monoclonal antibody that binds PD-1. Correspondence: Ramon Mohanlal Methods (rmohanlal@beyondspringpharma.com) This is a Phase 1b/2 open-label study. Eligible patients were ≥18 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P412 years old, had ECOG status ≤1, and were treatment-naive for anti-PD- 1/PD-L1, or TGFβ R1 kinase inhibitor. Patients had advanced solid tu- Background mors that were refractory to standard systemic therapy (Phase 1b). Patients (pts) generally prefer immunotherapy (IO) over chemother- NSCLC patients (Phase 2) were required to have received prior apy (Chemo) in clinical trials and may prematurely withdraw consent platinum-based treatment. Phase 2 portion of the trial evaluated the if allocated to Chemo. This may impact study outcome (Barlesi Lan- safety of 150 mg BID galunisertib administered on a 14 days on, 14 cet Onc 2018). Due to pts awareness of their treatment allocation in days off dosing schedule in combination with nivolumab given at 3 unblinded IO trials, ‘premature’ consent withdrawal (thus before re- mg/kg Q2W. Efficacy, pharmacokinetics (PK) and pharmacodynamic ceiving first dose of study drug) is consistently and significantly (p data were also evaluated. Methods Results ‘Premature’ pts consent withdrawal rate was calculated for the Plin/ 15 patients were enrolled in Phase 1b and 25 in Phase 2. No dose- Doc (n=174) and Doc (n=181) arms in DUBLIN-3 (NCT02504489) limiting toxicities were observed in the Phase 1 portion of the study. around the time of the first pre-planned Interim Analysis (IA). In the Phase 2 NSCLC cohort, the most frequent treatment-related Results grade 3 AEs included immune-related encephalitis, diarrhea, fatigue, ‘Premature’ consent withdrawal rate in DUBLIN-3 was 1.1 % for Doc ALT/ AST/GGT increase, blood alkaline phosphatase increase, abdom- and 2.3 % for Plin/Doc (p=0.53; NS). Premature consent withdrawal inal distension, cutaneous rash (n=1 each), and cholestasis (n=2) that rate of the Doc arm was significantly (p resolved or were resolving at the time of data cutoff. Two deaths on Conclusions treatment (multi-organ failure and myocardial infarction), both unre- A single-blinded design (for pts only) is effective in preventing pre- lated to study treatment, were observed. 6 (24%) patients had con- mature and imbalanced patient consent withdrawal. This finding firmed partial response (PR) and 4 (16%) had stable disease; 1 may have relevance for the design of future IO trials. A second pre- patient had confirmed PR after initial pseudo-progression. Among planned IA for DUBLIN-3 to evaluate OS is projected for end 2019. the 6 responders, 5 had low or negative PD-L1 expression (≤50%). Trial Registration Median PFS was 5.26 months (95% CI: 1.77, 9.20) and median OS was NCT02504489 11.99 months (95% CI: 8.15, NR). Phase 1b PK data showed rapid ab- sorption (1-3h) and elimination of galunisertib within 48h. Additional References biomarker data including tumor mutational burden and gene- Fabrice Barlesi et al., Avelumab versus docetaxel in patients platinum-treated expression data will be presented. advanced non-small-cell lung cancer (JAVELIN Lung 200): an open-label, Conclusions randomised, phase 3 study. The Lancet. 2018; 19 (11): 1468-1479 Combination treatment of galunisertib at the RP2D of 150 mg BID Roy S Herbst et al., Pembrolizumab versus docetaxel for previously treated, for 14 days on 14 days off schedule with nivolumab 3 mg/kg Q2W PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a was well tolerated. Preliminary efficacy was observed in a subset of randomised controlled trial. The Lancet. 2016; 387 (10027): 1540-1550 patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 227 of 272 Trial Registration Conclusions NCT02423343 Preliminary data suggest the combination of pepinemab plus avelu- Ethics Approval mab is well tolerated and shows initial signals of antitumor activity in The study was performed in accordance with the Declaration of patients with IO failure. We will present updated clinical response Helsinki and was approved by ethics committees in multiple investi- data, as well as additional immunophenotyping of tissue biopsies, in- gator sites. cluding but not limited to activated T cells, regulatory T cells, DCs, monocytes, macrophages, and importantly myeloid-derived suppres- sor cells (MDSCs). P414 Interim results from CLASSICAL-Lung, a phase 1b/2 study of Acknowledgements pepinemab (VX15/2503) in combination with avelumab in All of the CLASSICAL-Lung investigators, site staff, and patients advanced non-small cell lung cancer patients Trial Registration 1 2 2 Michael Shafique, MD , Terrence Fisher, PhD , Elizabeth Evans, PhD , NCT03268057 2 2 2 John Leonard, MD PhD , Desa Rae Pastore , Crystal Mallow, BS , Ernest 2 3 4 5 Smith, PhD , Andreas Schroeder, MD, PhD , Kevin Chin , Joseph Beck , References 6 7 Megan Baumgart, MD , Ramaswany Govindan, MD , Nashat Gabrail, 1. Evans EE et al. Antibody blockade of semaphorin 4D promotes immune 8 9 10 MD , Jonathan Goldman, MD , Rachel Sanborn, MD , Alexander Spira, infiltration into tumor and enhances response to other 11 12 13 MD, PhD, FACP , Nagashree Seetharamu , Yanyan Lou, MD , Aaron immunomodulatory therapies. Cancer Immunol Res. 2015; 3: 689-701 13 2 2 Mansfield, MD , Maurice Zauderer, PhD , Terrence Fisher, PhD 2. Clavijo PE et al. Semaphorin4D inhibition improves response to immune 1 2 Moffitt Cancer Center, Tampa, FL, United States; Vaccinex, Inc, checkpoint blockade via attenuation of MDSC recruitment and function. 3 4 Rochester, NY, United States; Merck KGaA, Darmstadt, Germany; EMD Cancer Immunol Res. 2019; 7(2):282-291. Serono, Rockland, MA, United States; Highlands Oncology Group, Ethics Approval Fayetteville, AZ, United States; University of Rochester, Rochester, NY, This protocol and its amendments were approved by the appropriate IRBs at United States; Washington University School of Medicine, St. Louis, MO, each site. 8 9 United States; Gabrail Cancer Center, Canton, OH, United States; UCLA Medical Center, Paramus, NJ, United States; Earle A. Chiles Research Institute, Portland, OR, United States; Virginia Cancer Specialists, Fairfax, P415 VA, United States; Northwell Health, New York, NY, United States; Tumor Treating Fields (TTFields, 150 kHz) concurrent with Mayo Clinic, Jacksonville, FL, United States standard of care treatment for stage 4 non-small cell lung cancer Correspondence: Terrence Fisher (tfisher@vaccinex.com) (NSCLC) in Phase 3 LUNAR Study Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P414 Ori Farber, Moshe Giladi, Ze'ev Bomzon, Eilon Kirson, Uri Weinberg, MD PhD Background Novocure Ltd., Haifa, Israel Despite progress of immune checkpoint blockade therapies, many Correspondence: Uri Weinberg (weinberg@novocure.com) patients with non-small cell lung cancer (NSCLC) do not receive dur- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P415 able clinical benefit from these agents, and even in those who do re- spond initially, acquired resistance and tumor recurrence can Background develop. Pepinemab is an IgG4 humanized monoclonal antibody tar- Tumor Treating Fields (TTFields) are a non-invasive, anti-mitotic treat- geting semaphorin 4D (SEMA4D, CD100). In vivo preclinical studies ment that disrupts the formation of the mitotic spindle and disloca- demonstrated antibody blockade of SEMA4D promoted immune infil- tion of intracellular constituents. TTFields plus temozolomide tration and reduced function and recruitment of immunosuppressive significantly extended survival in newly diagnosed glioblastoma. Effi- myeloid cells within the tumor [1,2]. Importantly, preclinical combina- cacy of TTFields in NSCLC has been shown in preclinical models, and tions of anti-SEMA4D with various immunotherapies enhanced T cell safety in combination with pemetrexed in a pilot study. In the Phase infiltration and activity, as well as durable tumor regression. 3 LUNAR study [NCT02973789], we investigated if the addition of Methods TTFields to immune checkpoint inhibitors or docetaxel increases The CLASSICAL-Lung clinical trial evaluates the combination of overall survival (OS). pepinemab with anti-PD-L1 antibody avelumab to couple benefi- Methods cial modifications of the immune microenvironment via pepine- Trial Design: mab with immune activation via checkpoint inhibition. This Patients (N=534), with squamous or non-squamous NSCLC, are strati- ongoing phase 1b/2, open label, single arm, first-in-human com- fied by their selected standard therapy (immune checkpoint inhibitors bination study is designed to evaluate the safety, tolerability and or docetaxel), histology and geographical region. Key inclusion criteria efficacy of the combination in patients with advanced (stage IIIB/ are disease progression, ECOG 0-2, no electronic medical devices in the IV) NSCLC, including a dose escalation cohort and expansion co- upper torso, and absence of brain metastasis. TTFields (150 kHz) are ap- horts consisting of 1) 17 immunotherapy-naïve patients and 2) 33 plied to the upper torso for at >18 hours/day until progression in the patients whose tumors progressed during or following immuno- thorax and/or liver. The primary endpoint is superiority in OS between therapy (IO failure). patients treated with TTFields in combination with the standard of care Results treatments versus standard of care treatments alone. Key secondary The combination was well tolerated with no concerning safety sig- endpoints compare the OS in patients treated with TTFields and doce- nals identified to date. No patient experienced a treatment-related taxel versus docetaxel alone, and patients treated with TTFields and im- adverse event leading to permanent treatment discontinuation or mune checkpoint inhibitors vs those treated with immune checkpoint death and the most frequent related AEs were grades 1 or 2 fatigue, inhibitors alone. An exploratory analysis will test non-inferiority of pyrexia, or chills. Interim analysis focused on the IO failure cohort TTFields with docetaxel compared to checkpoint inhibitors alone. Sec- which included 22 evaluable patients. Two patients experienced a ondary endpoints include progression-free survival, radiological re- partial response (PR) with 49% and 37% tumor reduction on study sponse rate, quality of life based on the EORTC QLQ C30 questionnaire. following acquired resistance to prior treatment with pembrolizu- The sample size is powered to detect a HR of 0.75 in TTFields-treated mab. In addition, stable disease of at least 8 weeks was observed in patients versus control group. In January 2019, an independent Data 11 patients and 4 patients have remained on study for ≥20 weeks. Monitoring Committee (DMC) performed a review of the LUNAR trial Analysis of pre- and on-treatment lung biopsies demonstrated no or data collected to that point. The DMC concluded that no unexpected low tumor burden detected in 2 patients with PR, and interestingly safety issues could be found in patients treated with the combination no detectable tumor was observed in the biopsies from 3 of 4 pa- of immune checkpoint inhibitors and TTFields, and recommended to tients with stable disease. continue the LUNAR study as planned. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 228 of 272 Trial Registration P417 NCT02973789 ATLAS™ identifies relevant neoantigens for therapeutic anti-tumor vaccination and may serve as a biomarker for efficacy of immunotherapy of solid tumors P416 Parul Agnihotri, Tulin Dadali, Parul Agnihotri, PhD A phase 1 dose escalation with expansion study to evaluate the Genocea Biosciences Inc, Cambridge, MA, United States safety, tolerability, pharmacokinetics, and efficacy of AMV564 in Correspondence: Tulin Dadali (tulin.dadali@genocea.com) subjects with advanced solid tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P417 1 1 2 Raghad Abdul Kairm, MD , Anthony Tolcher , Victoria Smith , Sterling 2 2 2 Eckard, PhD , Jeanmarie Guenot , Patrick Chun Background NEXT Oncology and Texas Oncology, San Antonio, TX, United States; Mutation-derived neoantigen cancer vaccines are promising as Amphivena Therapeutics, Inc., South San Francisco, CA, United States next generation cancer therapies. However, the success of vaccin- Correspondence: Patrick Chun (pchun@amphivena.com) ation is dependent on the ability to identify the right neoanti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P416 gens for vaccine inclusion, which remains a critical challenge. Computationally-identified neoantigens do not necessarily gener- Background ate immunogenic responses. Recently, we reported interim im- Overcoming the suppressive tumor microenvironment is a major munogenicity results from the ongoing GEN-009 personalized challenge in immune therapy. The critical cellular effectors of immunotherapy Phase 1/2a clinical trial (NCT03633110). For GEN- the suppressive tumor microenvironment are the myeloid- 009, ATLAS, an ex vivo, cell-based assay selects neoantigens for derived suppressor cells (MDSC). MDSC are elevated in both the vaccine inclusion based on a patient’s own pre-existing T cell re- tumor microenvironment and periphery in cancer patients and sponses. The interim results revealed that vaccination elicited T are associated with immune dysfunction, repression of anti- cell responses to over 98% of administered peptides. Here, we tumor immunity and poor response to immunotherapy. MDSC explore the relationship between ATLAS readouts and immuno- secrete a variety of immunosuppressive factors that directly in- genicity outcomes in the same subjects. hibit both the cytolytic activity and proliferative capacity of Methods anti-tumor T cells. AMV564 is a bivalent, bispecific antibody that Antigens were profiled by expressing each mutation, identified by engages both CD3 and CD33. Preferential binding of AMV564 to whole exome sequencing, as individual clones in E. coli which regions of high CD33 density enables the selective targeting of are subsequently processed by each subject’sown antigenpre- MDSC. Data from both ex vivo studies [1] and an ongoing clin- senting cells and presented to autologous CD4+ or CD8+ T cells. ical trial in acute myeloid leukemia (AML) support the ability of Antigen-specific responses were determined based on cytokine AMV564 to selectively deplete monocytic and granulocytic secretion in the supernatant after overnight incubation. GEN-009, MDSC while sparing monocytes and neutrophils. composed of 4 pools of 1-5 unique ATLAS-identified neoantigen- Methods specific peptides combined with Hiltonol® was administered to AMV564-301 is an open label, phase 1, multicenter, dose- each subject. Both ex vivo and ten day in vitro stimulated Fluoro- escalation with expansion trial of AMV564 in patients with ad- Spot assays were performed on unsorted PBMC and CD4- and vanced solid tumors for which no recognized standard curative CD8-sorted T cells at baseline and 50 days post vaccination to therapy options are available. The key objectives of the dose- identify peptides to which T cells from the vaccinated patients escalation stage of the study are to characterize the safety and responded. tolerability of AMV564 and identify a maximum tolerated dose Results (MTD) or a recommended phase 2 dose (RP2D) for further study. In the first cohort of six patients, ATLAS identified neoantigens In the dose expansion stage of the study, the safety and toler- by recalling both stimulatory and inhibitory neoantigen-specific T ability of AMV564 will be further characterized in addition to cell responses. One subject, who had a greater proportion of in- evaluating the preliminary efficacy of AMV564. Other objectives hibitory to stimulatory responses detected, progressed prior to include characterization of AMV564 pharmacokinetics, pharmaco- vaccination while no vaccinated patients have experienced pro- dynamics, and immunogenic potential. gressive disease. Compared to NetMHCPan results, more than half Approximately 90 patients with locally advanced or metastatic of the ATLAS-identified neoantigens were not predicted. More- solid tumors will be enrolled. The Dose Escalation Stage will in- over, the predicted epitopes did not result in better immunogen- clude up to approximately 40 patients, depending on the dose icity outcomes post-vaccination than the non-predicted ATLAS- at which the MTD/RP2D is determined, and approximately 50 identified neoantigens. Comprehensively profiling T cell responses additional patients will be enrolled in the Expansion Stage. over time shows consistency of results in patients with no evi- AMV564 will be administered once daily as a subcutaneous in- dence of disease. jection for 14 days in each 21-day cycle. Patients will be treated Conclusions until disease progression, unacceptable toxicity, or withdrawal Neoantigens selected by immune response data from ATLAS and of consent. included in the GEN-009 vaccine were immunogenic and many Trial Registration were not algorithm-predicted, confirming that ATLAS identifies NCT pending relevant neoantigens. ATLAS will be useful for profiling epitope spread in tumor-bearing subjects post-vaccination. The proportion Reference of inhibitory to stimulatory neoantigen-specific responses may be 1. Cheng P, Eksioglu E, Chen X, Wei M, Guenot J, Fox J, List A, Wei S. a biomarker of immunotherapy success. Combination of GEN-009 Immunodepletion of MDSC by AMV564, a novel tetravalent bispecific with standard-of-care checkpoint blockade therapy is currently CD33/CD3 T cell engager restores immune homeostasis in MDS in vitro. ongoing. Blood. 2017;130:51. Trial Registration Ethics Approval ClinicalTrials.gov NCT03633110 This study will be approved by the Institutional Review Board (IRB) or Ethics Approval Independent Ethics Committee (IEC) at each participating institution The study was approved by Western Institutional Review Board, ap- prior to patient enrollment. proval number 1-1078861-1. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 229 of 272 P418 paired peripheral blood analysis revealed a treatment-related in- Intratumoral IL-12 plus pembrolizumab combination therapy in crease of KLRG1+/CD127- SLECs as well as a treatment-related reduc- treatment refractory solid tumors: a safety and biomarker analysis tion of MDSCs in the periphery predominantly in responding Pablo Fernandez-Penas, MD, PhD , Matteo Carlino, MBBS, PhD, BMedSC, patients. 1 2 3 4 5 F , Victoria Atkinson, MD , Melinda Telli , Rohit Joshi , Sajev Thomas , Conclusions 6 4 7 Katy Tsai, MD , Rachel Roberts-Thomson , Andrew Haydon, MBBS PhD , TAVO + pembrolizumab continues to be well tolerated in patients 8 9 10 11 Andrew Mant , Tom Van Hagen , Katharine Cuff , Bianca Devitt , Igor with advanced solid tumors and peripheral blood analyses demon- 12 13 14 Puzanov, MD, MSCI, FACP , Marcus Butler, MD , Catalin Mihalcioiu , strates both local and most importantly, systemic signals of IL-12 me- 15 16 17 Hatem Soliman, MD , John Hyngstrom, MD , Mecker Moller , Gregory diated anti-tumor immunity in the absence of clinical signs of 18 19 20 Daniels, MD, PhD , Eric Whitman, MD, FACS , Erica Browning, BS , systemic IL-12 exposure. Thus, TAVO acts as an in situ vaccine to po- 20 20 20 20 Reneta Hermiz , Lauren Svenson , Jack Lee , Donna Bannavong , tentiate the anti-tumor activity of pembrolizumab with a favorable 20 20 20 20 Jendy Sell , Kellie Malloy , David Canton, PhD , Christopher Twitty , toxicity profile. 6 6 Adil Daud, MBBS MD , Alain Algazi, MD Trial Registration 1 2 Westmead Hospital, University of Sydney, Westmead, Australia; Princess NCT03132675; NCT03567720 Alexandra Hospital, University of Queensland, Woolloongabba, Australia; Ethics Approval Stanford University Medical School, Stanford, CA, United States; These studies were approved by the appropriate ethics committees. 4 5 Adelaide Oncology and Haematology, Adelaide SA, Australia; UF Consent Health Cancer Center, Orlando Health, Orlando, FL, United States; Written informed consent was obtained from the patient for publica- University of California San Francisco, San Francisco, CA, United States; tion of this abstract and any accompanying images. A copy of the 7 8 The Alfred Hospital, Melbourne, Australia; Box Hill Hospital, Box Hill, written consent is available for review. 9 10 Victoria, Australia; St. John of God Hospital, Subiaco, Australia; Princess Alexandra Hospital, Woolloongabba QLD, Australia; Eastern Health P419 Clinical School, Box Hill, VIC, Australia; Roswell Park Cancer Institute, A phase 1/2 study of GB1275, a novel CD11b modulator, as Buffalo, NY, United States; Princess Margaret Cancer Centre, Toronto, monotherapy and with an anti-PD-1 antibody in specified Canada; McGill University Health Centre, Montreal, QC, Canada; 15 16 advanced solid tumors or with chemotherapy in metastatic Moffitt Cancer Center, Tampa, FL, United States; Huntsman Cancer pancreatic cancer (mPDAC) Institute and Hospital, Salt Lake City, UT, United States; Sylvester 1 2 3 Johanna Bendell , Drew Rasco, MD , Wungki Park, MD , Lei Zhou, MD, Comprehensive Cancer Center, University of Miami, Miami, FL, United 18 4 4 4 4 MS , Anna Galkin, PhD , Debbie Slee, PhD , Laura Carter, PhD , David States; University of California San Diego, La Jolla, CA, United States; 19 20 4 4 4 4 Nickle, PhD , Rebecca Tran, MS , Jack Li, PhD , Beatrice Ferguson, MS , Atlantic Health System, Morristown, NJ, United States; OncoSec 4 5 3 Jakob Dupont, MD , Vineet Gupta, PhD , Eileen O'Reilly Medical Inc., San Diego, CA, United States 1 2 Sarah Cannon Research Institute, Nashville, TN, United States; The Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) START Center for Cancer Care, San Antonio, TX, United States; Memorial Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P418 Sloan Kettering Cancer Center, New York, NY, United States; Gossamer Bio, San Diego, CA, United States; Rush University Medical Center, Background Chicago, IL, United States Intratumoral inflammation, including IL-12 expression and intratu- Correspondence: Johanna Bendell (ttobore@samornbiosciences.com) moral T cell infiltration, is a prerequisite for response to anti-PD-1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P419 therapies. Previously, we demonstrated that enhanced intratumoral IL-12 expression via injection of plasmid IL-12 (tavokinogene telse- Background plasmid; TAVO) followed by electroporation (IT-tavo-EP) can increase Tumor influx of CD11b-expressing Myeloid-Derived Suppressor Cells TIL infiltration, ratios of CD8+ T cell:suppressive immune subsets, and (MDSCs) and M2 Tumor-Associated Macrophages (TAMs) creates an IFN-gamma gene signatures, converting weakly immunogenic tumors immunosuppressive tumor microenvironment that is associated with into highly inflamed, immunologically active lesions that regress with resistance to anti-PD-1 antibody therapy [1, 2, 3]. GB1275 is a novel, anti-PD-1 antibody therapy. Here, we present further support for our first-in-class, CD11b modulator that in vivo led to reduced MDSCs hypothesis that local IT-tavo-EP induces local and systemic immune and TAMs at the tumor site, repolarized M2 immuno-suppressive modulation with minimal systemic toxicity. TAMs towards an M1 phenotype, and subsequently increased tumor Methods infiltration of activated CD8+ T cells [4]. In combination settings with Melanoma (KEYNOTE-695) and mTNBC (KEYNOTE-890) patients were an anti-PD-1 antibody or chemotherapy, these immunomodulatory treated every three weeks with IT-tavo-EP on days 1, 5, and 8 of effects translated into potent anti-tumor effects and prolonged sur- every odd numbered cycle. Clinical toxicity was assessed at 3-week vival in orthotopic PDAC models [4]. We hypothesize that GB1275 ad- intervals and graded by CTCAE v4. In addition, pre- and post- ministration can alleviate myeloid cell-mediated immunosuppressive treatment tumor biopsies and peripheral blood samples were interro- effects and improve cancer treatment outcomes. gated for treatment-related changes in the frequency of CD8+ TIL Methods and other key IL-12-driven peripheral immune cell populations. In This is an open-label, first-in-human study consisting of a Phase 1 particular, we examined circulating short-lived effector T cells (SLECs, Dose Escalation phase with Regimen A using GB1275 monotherapy KLRG1+/CD127-), which are induced by IL-12 exposure, and granulo- and Regimen B using GB1275 with an anti-PD-1 antibody in pts with cytic myeloid derived suppressor cells (gMDSCs or PMN-MDSCs), pancreatic, esophageal, gastric/GEJ, triple negative breast, castration which serve a regulatory function, inhibiting effective anti-tumor im- resistant prostate, or Microsatellite Stable Colorectal Cancer (MSS mune responses. CRC) and Regimen C (GB1275 with Nab-paclitaxel + Gemcitabine Results (Nab-P+Gem)) in mPDAC, followed by a Phase 2 Expansion phase 62 patients were assessed including 46 patients with anti-PD-1 with three cohorts planned: newly diagnosed stage IV mPDAC, MSS antibody-refractory melanoma, and 16 patients with chemotherapy- CRC and PD-L1+ gastric/GEJ cancer. The study starts with Regimen A refractory mTNBC. TAVO in combination with pembrolizumab was with Regimen B starting after the completion of the first few cohorts well tolerated with only 2 of 46 (4.3%) patients from KEYNOTE-695 of Regimen A. Regimen C will start when Regimen A is completed. (cellulitis and presyncope) and 1 of 16 (6.3%) from KEYNOTE-890 Key Inclusion Criteria: Age ≥18 years, histologically confirmed locally (acute renal failure) experiencing grade 3 treatment-related adverse advanced/metastatic tumor specified, ECOG 0-1, prior immunother- events (TRAEs). Paired biopsies were available from both advanced apy is permissible in Dose Escalation phase for Regimen A and B, but melanoma patients and mTNBC patients. Flow cytometry on fresh bi- not for the expansion or Regimen C. Key Exclusion Criteria: untreated opsies from the KEYNOTE-695 revealed significant increases in CD8+ or symptomatic CNS metastasis, received prior myeloid targeting T cells after 1 cycle of treatment. Despite previous data demonstrat- agent or other prohibited medications, history of clinical significant ing non-detectable circulating IL-12 levels after treatment with TAVO, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 230 of 272 cardiovascular disease. Pts with active autoimmune disease requiring consistent with those expected from the poly-ICLC adjuvant alone, systemic therapy will be excluded from Regimen B. Primary objec- and no DLTs. ATLAS results show high interpatient variability as de- tives for the Dose Escalation phase are to determine the MTD/RP2D scribed previously. In an interim analysis of patients, vaccination has and PK profile of GB1275 monotherapy and in combination with an generated both CD8 and CD4 T cell responses measured by ex vivo anti-PD-1 antibody, and safety in combination with Nab-P+Gem. The fluorospot (Table 1). Ten-day in vitro stimulation (IVS) fluorospot as- primary objective for the Basket Expansion phase is to assess says confirm even broader immune responses. Overall, T cell re- efficacy. sponses were measured to 98% of administered peptides. Statistical Considerations: 3+3 design for the Dose Escalation Phase Conclusions and Simon’s 2-stage design for Expansion Phase. AEs graded per GEN-009 is a neoantigen vaccine that targets tumor specific immune CTCAE v5.0, responses per RECIST v1.1. The study is open for recruit- antigens recognized by the individual patient’s lymphocytes and ment and clinical trial registration on clinicaltrials.gov is pending likely expressed by tumor cells. Immunogenicity data show that (NCTxxxxx). ATLAS can, with very high frequency, identify relevant neoantigens and exclude putatively deleterious (immune inhibitory) antigens. References Clinical vaccination together with Standard of Care PD-1 blockade- 1. Fleming, V., Hu, X., Weber, R., Nagibin, V., Groth, C., Altevogt, P., et al. based regimens is in progress. Targeting myeloid-derived suppressor cells to bypass tumor-Induced im- Trial Registration munosuppression. Front Immunol. 2018; 9: 398. ClinicalTrials.gov NCT03633110 2. Kumar, V., Patel, S., Tcyganov, E. and Gabrilovich, D. I. The nature of Ethics Approval myeloid-derived suppressor cells in the tumor microenvironment. Trends The study was approved by Western Institututional Review Board, ap- Immunol. 2016; 37(3): 208-220. proval number 1-1078861-1. 3. Mantovani, A., Sozzani, S., Locati, M., Allavena, P. and Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized Table 1 (abstract P420). See text for description M2 mononuclear phagocytes. Trends Immunol. 2002; 23(11): 549-555. 4. Panni R., Herndon J., Zuo C., et al. Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. Sci Transl Med. 2019; 11: eaau9240. Ethics Approval The study was approved by the local IRB at each participating study site. P420 Broad immunogenicity from GEN-009, a neoantigen vaccine using ATLAS™, an autologous immune assay, to identify immunogenic and inhibitory tumor neoantigens 1 2 3 Roger Cohen, MD , Melissa Johnson, MD , Przemyslaw Twardowski, MD , P421 4 5 6 Mark Stein, MD , Ulka Vaishampayan, MD , Maura Gillison, MD, PhD , Lisa Phase 1 study of the safety, tolerability and preliminary anti-tumor 7 7 7 McNeil, PhD , Louisa Dowal, PhD , James Foti, PhD , Parul Agnihotri, activity of COM701 monotherapy in patients with advanced solid 7 7 7 7 PhD , Daniel DeOliveira, PhD , Manish Jain, MS , Jessica Price , Richard tumors 7 7 7 1 2 3 Hernandez , Arthur DeCillis, MD , Narinderjeet Singh, MS, MBA , Thomas Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD , Ryan 7 7 4 5 5 Davis, MD , Jessica Flechtner, PhD Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 1 2 6 7 7 University of Pennsylvania, Philadelphia, PA, United States; Sarah Adam ElNaggar, MD , John Hunter, PhD , Judy Olweny , Adeboye 3 7 8 9 Cannon Research Institute, Nashville, TN, United States; John Wayne Adewoye, MD , Bartosz Chmielowski, MD, PhD , Dale Shepard, MD PhD , 4 10 11 6 Cancer Institute, Duarte, CA, United States; Columbia University Medical Manish Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD , Drew 5 5 Center, New York, NY, United States; Karmanos Cancer Institute, Detroit, Rasco, MD 6 1 2 MI, United States; MD Anderson Cancer Center, Houston, TX, United The MD Anderson Cancer Center., Houston, TX, United States; The 7 3 States; Genocea, Cambridge, MA, United States University of Chicago, Chicago, IL, United States; Sarah Cannon Correspondence: Thomas Davis (tom.davis@genocea.com) Research Institute/TN Oncology, Nashville, TN, United States; 4 5 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P420 Massachusetts General Hospital, Needham, MA, United States; The START Center for Cancer Care, San Antonio, TX, United States; West Background Cancer Center and Research Institute, Memphis, TN, United States; 7 8 Tumor-specific neoantigens provide personalized targets for im- Compugen USA Inc, South San Francisco, CA, United States; University munotherapy. Vaccines against epitopes predicted by in silico ap- of California Los Angeles, Los Angeles, CA, United States; Cleveland proaches very rarely induce CD4+ and CD8+ ex vivo T cell responses Clinic, Cleveland, OH, United States; START - Midwest Cancer Center, regardless of formulation. ATLAS selects neoantigens for vaccine in- Chicago, IL, United States; Columbia University Medical Center, New clusion using ex vivo screening of all patient-specific mutations to York City, NY, United States identify pre-existing CD4+ or CD8+ T cell responses and to exclude Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) inhibitory peptides that may suppress immunity and potentially ac- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P421 celerate tumor progression. Preliminary data suggest that the inhibi- tory peptide profile may predict tumor response to immunotherapy. Background Methods COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of GEN-009-101 is a phase 1/2a study testing safety, immunogenicity poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It in- and clinical activity in immune responsive tumors (NCT03633110). hibits the binding of PVRIG with its ligand, PVRL2. PVRIG is a member After next-generation tumor sequencing and ATLAS testing of au- of the DNAM/TIGIT signaling axis regulating the activity of T/NK-cells. tologous leukocytes, each personalized vaccine is created using up In preclinical experiments we have demonstrated that PVRIG inhib- to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC. ition alone and in combination with anti-PD-1 and/or TIGIT blockers The immunogenicity pilot includes 8 patients in remission (NED), leads to activation of T cells in the tumor microenvironment generat- who received a course of GEN-009 monotherapy. ing an anti-tumor immune response and tumor growth inhibition [1]. Results Although ICI revolutionized cancer treatment, there is an urgent Eight patients have participated and reached the primary immuno- need to develop treatments for patients who are refractory or relapse genicity readout at day 50 (some data pending). The 24 doses given after treatment with ICI. We hypothesized that COM701 will be safe, across all patients have induced only grade 1/2 adverse events tolerable and demonstrate preliminary anti-tumor activity. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 231 of 272 Methods inhibits the binding of PVRIG with its ligand, PVRL2. Nivolumab is an A phase 1a, dose-escalation of COM701 monotherapy utilizing a hybrid anti-PD-1 antibody approved in patients with several malignancies accelerated and 3+3 study design was conducted to determine safety, [2]. PVRIG is a member of the DNAM/TIGIT signaling axis regulating tolerability, to assess the pharmacokinetics (PK), pharmacodynamics, to the activity of T/NK-cells. PD-1 inhibitors play an important role in determine the recommended phase 2 dose and to evaluate preliminary this axis by modulating DNAM activation [3]. In preclinical experi- anti-tumor activity of COM701. Patients with performance status ECOG 0- ments we have demonstrated that PVRIG inhibition alone and in 1 and advanced solid tumors who failed standard of care treatments combination with anti-PD-1 leads to activation of T cells in the tumor were eligible for inclusion. Prior ICIs were permissible. COM701 0.01, 0.03, microenvironment generating an anti-tumor immune response and 0.1, 0.3, 1, 3 and 10 mg/kg IV every 3 weeks were administered until pro- tumor growth inhibition [1]. Although ICI revolutionized cancer treat- gression, intolerable toxicity or investigator or patient discretion. Adverse ment there is an urgent need to develop treatments for patients events were reported per CTCAE v4.03 and anti-tumor activity was evalu- who are refractory or relapse after treatment with ICI. We hypothe- ated using RECIST v1.1. Dose-limiting toxicities (DLTs) were evaluated sized that COM701 will be safe and tolerable and demonstrate pre- within a 21-day window. Data cutoff date was August 09, 2019. liminary antitumor activity as monotherapy and in combination with Results nivolumab in patients with advanced solid tumors. A total of 13 patients were enrolled and treated during dose escalation Methods of COM701, including 6 patients with metastatic colorectal cancer (CRC), This is a phase 1 study with single patient cohorts and 3+3 study design 5 with microsatellite stable status (MSS) and 1 unknown. Patients were of COM701 in escalating doses as monotherapy IV Q3 weeks and in com- heavily pretreated with a median of 7 prior anticancer therapies (range 2- bination with nivolumab 360mg IV Q3 weeks. Key Inclusion Criteria: Age 15). No DLTs have been reported up to 10 mg/kg COM701 dose level. ≥18 years, histologically confirmed advanced solid tumor, performance The most frequent toxicities were fatigue (8%), abdominal pain (6%). status ECOG 0-1, prior anti-PD-1, anti-PD-L1, anti-CTLA-4, OX-40, CD137 Likely immune-related adverse events: elevated TSH and rash were ob- treatments are permissible. Key Exclusion Criteria: Active autoimmune dis- served in 2 patients. Overall 7/13 patients (54%) maintained best re- ease requiring systemic therapy in the last 2 years, symptomatic intersti- sponse of stable disease (SD) ≥12 weeks (13.6 – 43 weeks), including 5/6 tial or inflammatory lung disease, untreated or symptomatic central (83%) of patients with CRC. Five patients continue on study treatment. nervous system metastases. Primary objectives: to evaluate the safety Peripheral PVRIG receptor occupancy (≥90%) was demonstrated at and tolerability of COM701 monotherapy and in combination with nivo- ≥1mg/kg dose of COM701 and PK profile supports Q3 weekly dosing. lumab measured by the incidence of adverse events and dose-limiting Conclusions toxicities (21-day window), to evaluate the pharmacokinetics of COM701, COM701 monotherapy demonstrates an acceptable safety and toler- and to identify the maximum tolerated dose and/or the recommended ability profile with preliminary anti-tumor activity in a patient popula- dose for expansion as monotherapy and in combination with nivolumab. tion that had received multiple prior anti-cancer therapies. Updated Secondary objectives: to characterize the immunogenicity and prelimin- data will be presented at the conference. ary antitumor activity of COM701 in combination with nivolumab. Statis- Trial Registration tical Considerations: AEs will be reported as per CTCAE v4.03 and tumor Clinical trial identification: NCT03667716. responses will be evaluated per RECIST v1.1. Analyses of objectives are descriptive and hypothesis generating. Reference Results 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody At the time of submission no DLTs have been observed up to dose targeting the novel immune checkpoint PVRIG, for the treatment of level 7 of COM701 monotherapy and dose level 1 of COM701 in cancer. J Clin Oncol. 2017; (suppl; abstr 3074) combination with nivolumab 360mg IV Q3 weeks. Ethics Approval Conclusions The study was approved by each site's ethics board. Assessment of safety and tolerability is ongoing for all patients. Up- Consent dated results will be presented at the congress. Written informed consent was obtained from the patient for publication of Trial Registration this abstract and any accompanying images. A copy of the written Clinical trial identification: NCT03667716. consent is available for review by the Editor of this journal. References 1. Spencer L, Ofer L et al, Discovery of COM701, a therapeutic antibody P422 targeting the novel immune checkpoint PVRIG, for the treatment of Phase 1 study of COM701 monotherapy and in combination with cancer. J Clin Oncol. 2017; (suppl; abstr 3074) nivolumab in patients with advanced solid tumors 2. Nivolumab package insert. http://packageinserts.bms.com/pi/pi_opdivo.pdf. 1 2 3 Ecaterina Dumbrava, MD , Gini Fleming, MD , Erika Hamilton, MD ,Ryan Accessed 07/22/2019. 4 5 5 Sullivan, MD , Amita Patnaik, MD FRCP(C) , Kyriakos Papadopoulos, MD , 3. Wang B, Zhang W et al., Combination cancer immunotherapy targeting 6 7 7 Adam ElNaggar, MD ,JohnHunter, PhD ,JudyOlweny , Adewoye Adewoye, PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory 7 8 9 MD , Bartosz Chmielowski, MD, PhD ,DaleShepard,MDPhD ,Manish phenotype. Sci. Immunol. 2018; Nov 2:3(29). 10 11 6 5 Sharma, MD , Emerson Lim, MD , Daniel Vaena, MD ,DrewRasco,MD Ethics Approval The University of Texas MD Anderson Cancer Center, Houston, TX, The study was approved by the Investigational Review Board/Ethics United States; The University of Chicago, Chicago, IL, United States; Committee of the participating sites. Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United States; Massachusetts General Hospital, Harvard Medical School, Needham, MA, United States; The START Center for Cancer Care, San P423 Antonio, TX, United States; West Cancer Center and Research Institute, SURPASS trial design: A phase 1 dose escalation trial to assess Memphis, TN, United States; Compugen USA Inc, South San Francisco, safety and efficacy of ADP-A2M4CD8 in HLA-A2+ patients with CA, United States; University of California Los Angeles, Los Angeles, CA, MAGE-A4+ tumors 9 10 2 3 4 United States; Cleveland Clinic, Cleveland, OH, United States; The David Hong, MD , Marcus Butler , Melissa Johnson, MD , Tanner 5 1 1 START-Midwest Center for Cancer Care, Chicago, IL, United States; Johanns , Francine Brophy , Rebecca Dryer-Minnerly, PhD , Trupti 11 1 1 1 Columbia University Medical Center, New York City, NY, United States Trivedi, MS , Rafael Amado, MD , Paula Fracasso, MD, PhD 1 2 Correspondence: Ecaterina Dumbrava (EEIleana@mdanderson.org) Adaptimmune, Philadelphia, PA, United States; MD Anderson Cancer Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P422 Center, Houston, TX, United States; Princess Margaret Cancer Centre, Toronto, Canada; Sarah Cannon, Nashville, TN, United States; Background Washington University in St. Louis, St. Louis, MO, United States COM701 is a novel first-in-class immune checkpoint inhibitor (ICI) of Correspondence: Paula Fracasso (paula.fracasso@adaptimmune.com) poliovirus receptor related immunoglobulin domain (PVRIG) [1]. It Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P423 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 232 of 272 Background tumors are presented. The study (NCT03548467) was approved by ADP-A2M4CD8 specific peptide enhanced affinity receptor (SPEAR) T- Central Ethics Committee in Heidelberg, Germany. cells are genetically engineered to target MAGE-A4+ tumors in the Methods context of HLA-A*02. ADP-A2M4CD8 are autologous CD4+ and CD8+ Patients with melanoma, NSCLC, clear RCC, urothelial cancer or SCCHN T-cells that express a high affinity MAGE-A4-specific T-cell receptor who did not reach complete responses after >12 weeks of immune (TCR) and an additional CD8α co-receptor. The ADP-A2M4 TCR is be- checkpoint inhibitor (CPI) therapy as standard of care were eligible. ing explored in a pilot study (NCT03132922), where clinical responses After patient-specific vaccine production, patients receive up to 14 vac- have been observed and the TCR has been well tolerated in doses cinations as intramuscular jet injections over a one-year period. CPI up to 10 × 10^9 transduced cells. Because CD4+ T-cells have a weak treatment continued. CT/MRI scans were performed according to hospi- effector function in response to class I antigens, a CD8α co-receptor tals’ routine. Immune responses were assessed by IFN-γ ELISpot. was genetically engineered alongside the TCR in ADP-A2M4CD8, to Results increase TCR binding avidity and enhance the polyfunctional re- At July 22, 2019 data cut-off, 15 patients (9 RCC, 4 SCCHN, 1 Melan- sponse of engineered CD4+ T-cells against MAGE-A4+ tumors. This oma, 1 NSCLC) had received ≤ 11 VB10.NEO vaccinations. Most com- approach is intended to widen the immune response to the tumor mon AEs were injection site reactions which all subsided within days. and improve depth and durability of clinical responses. 6 patients reported ≥ Grade 3 AEs, the most frequent ones were Methods injection-related hypertensive episodes normalizing within hours. In This phase 1, dose-escalation, open-label trial (SURPASS Trial) will the 4 patients assessed with low TMB (2 RCC, 2 SCCHN) strong T-cell characterize safety, tolerability, and antitumor activity across multiple responses towards 63% of selected neoepitopes were observed after tumor types. Patients who are HLA-A*02+ with MAGE-A4+ advanced 3-6 vaccinations. An amplification of existing neoepitope-specific T- esophageal, esophagogastric junction, gastric, head and neck cancers, cells (average of 250-fold increase) as well as de novo responses non-small cell lung, ovarian and urothelial carcinoma, melanoma, myx- were observed suggesting that VB10.NEO increases both the breadth oid/round cell liposarcoma, or synovial sarcoma and who meet all other and strength of the immune responses. inclusion criteria are eligible. Up to 30 patients will be enrolled. 10 patients were evaluable with >1 scan after VB10.NEO start (after Following apheresis, T-cells are isolated, transduced with CD8α_- being on CPI for 9-32 months). 4 patients (3 low TMB, 1 medium MAGE-A4c1032TCR, and expanded. Prior to ADP-A2M4CD8 infusion, TMB) started VB10.NEO with progressive disease (PD) development, patients will receive lymphodepletion consisting of fludarabine (30 of which 3 showed as stable disease (SD) in target lesions after vac- mg/m2/day x 4 days) and cyclophosphamide (600 mg/m2/day x 3 cination (followed up to 7 months), one developed PD after 5 days). During dose escalation, patients will be treated in one of three months. New lesions were detected in 2 patients. 6 patients (5 low ADP-A2M4CD8 dose groups. The initial dose of ADP-A2M4CD8 will TMB, 1 medium TMB) had SD at VB10.NEO start, 5 remained SD be 0.8 × 10^9 - 1.2 × 10^9 to be escalated to 1.2 × 10^9 - 3 × 10^9 (followed up to 9 months), while one had a best target lesion reduc- and then to 3.0 × 10^9 - 6.0 × 10^9 transduced cells in a modified 3 tion of 40%. Updated data will be presented. + 3 dose escalation scheme. Patients will be monitored for dose- Conclusions limiting toxicities (DLTs). Once the tolerability and safety of the lym- Vaccinations with VB10.NEO in addition to CPI were well tolerated. phodepletion regimen and cell dose has been demonstrated, the VB10.NEO induces strong T cell responses towards personalized dose range will increase to a maximum of 10 × 10^9 transduced neoepitopes; both novel T cell specificities and amplification of pre- cells in the expansion phase. Disease will be assessed per RECIST existing T cell responses were observed. Clinical signs of effect on v1.1 by CT/MRI at weeks 4, 8, 16, and 24, and every 3 months for 2 tumor size will continuously be monitored in the trial and early signs years, then every 6 months up to 15 years or until progression. Blood are promising. and tumor biopsy samples will be obtained pre- and post-infusion to Trial Registration evaluate safety, monitor persistence of transduced T-cells, and iden- NCT03548467 tify tumor-intrinsic correlates of response or resistance to therapy. Ethics Approval Trial Registration The study was approved by Central Ethics Committee in Heidelberg. NCT04044859 Ethics Approval P425 This trial is under review by the local institutional review boards for Phase 1b study of GX-I7, a long-acting interleukin-7, evaluating the each proposed study center. safety, pharmacokinetics and pharmacodynamics profiles in patients with advanced solid cancers 1 1 2 P424 Minkyu Heo , Minkyu Heo , Tae Won Kim, MD, PhD 1 2 Preliminary safety, efficacy and immunogenicity results from a Genexine, Inc, New York, NY, United States; Asan Medical Center, phase 1/2a study (DIRECT-01) of cancer neoantigen DNA vaccine Seoul, Korea VB10.NEO in patients with locally advanced or metastatic solid Correspondence: Tae Won Kim (twkimmd@amc.seoul.kr) tumors Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P425 1 2 3 1 Jürgen Krauss , Angela Krackhardt , Elke Jaeger , Anja Williams , Reza 3 4 4 4 Rafiyan , HEDDA WOLD, MSc , LIsa Gerner , Monika Sekelja , Agnete Background 4 4 4 Fredriksen, PhD , Karoline Schjetne , Mads Axelsen , Agnete Fredriksen, Cancer and treatment-related lymphopenia is associated with 4 4 PhD , Hedda Wold, MSc higher mortality in patients with various oncologic malignancies. 1 2 NCT, University Hospital Heidelberg, Heidelberg, Germany; Klinicum Interleukin-7(IL-7), a homeostatic cytokine of T lymphocytes, plays rechts der Isar, TUM, Munich, Germany; Krankenhaus Nordwest, a critical and non-redundant role in T cell development and Frankfurt, Germany; Vaccibody AS, OSLO, Norway homeostasis of mature T lymphocytes. IL-7 is a potent amplifier Correspondence: Hedda Wold (hwold@vaccibody.com) of naïve and memory T cells, thereby correcting T cell deficiency Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P424 and contributing to immune reconstitution. This may result in sig- nificant clinical benefit when combined with lymphopenia- Background inducing radiation/chemotherapy or immunotherapy where anti- Generation of potent neoantigen-specific T-cell responses has shown tumor effects are mediated by T cells. promising preclinical efficacy as well as clinical responses, especially Methods in patients with high tumor mutational burden (TMB). VB10.NEO is a A phase 1b study was conducted to assess the safety, pharmacokin- DNA vaccine with intrinsic adjuvant designed for delivery of 20 per- etics and pharmacodynamics of single-agent GX-I7(human IL-7 fused sonalized neoepitopes to antigen presenting cells. Preliminary results to the half-life extension hyFcTM) administered intramuscularly q3w from the ongoing phase 1/2a study treating patients with solid to advanced solid cancer patients who have no available effective Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 233 of 272 treatments (n=21). The dose escalation phase followed the 3+3 de- (NCT03416335). Primary objectives are to evaluate DSP-0509 safety/ sign of GX-I7 doses ranged from 60 to 1,200 μg/kg. Adverse events, tolerability, determine the maximum tolerated dose (Part A mono- PK, and subset analysis of peripheral blood monocytes(PBMCs) were therapy), and identify DSP-0509 monotherapy recommended phase evaluated. 2 dose (RP2D) and the RP2D of DSP-0509 in combination with pem- Results brolizumab for future studies. Secondary objectives include pharma- GX-I7 was well tolerated without DLT and cytokine release syn- cokinetics and antitumor activity. drome. Injection site reactions were the most common Methods treatment-emergent adverse events, which were Grade1 or 2 and Eligible patients are aged ≥18 years with advanced solid tumors (Part resolved. GX-I7 was slowly but steadily absorbed with a Tmax A and C) or melanoma or head and neck squamous cell carcinoma range of 12-48 hours with delay in higher doses. Following GX-I7 with acquired immune checkpoint inhibitor resistance (grouped high administration, up to 4-fold increase in absolute lymphocyte or low CD8+ cell density in tumor tissue; Part B). In Part A, approxi- count(ALC) were demonstrated. Importantly, the number of vari- mately 21–30 patients will be enrolled in each of the monotherapy ous subsets(naïve, TEM, TCM and TEMRA) of both CD4+ and and combination arms. DSP-0509 will be given as a constant rate IV CD8+ T cells was in a greater magnitude than that of ALC, infusion over 3 minutes at a fixed dose. Five provisional dose levels coupled with enhanced expression of Ki-67 peaked at day 7. of DSP-0509 may be tested, with approximately 3–6 patients at each Among T cell subsets, increase in naïve CD4+ and CD8+ T cells level (3 escalation levels at target doses of 0.3, 1, and 3 mg; 2 de- was most prominent. IL-7 receptor alpha(CD127) expression was escalation levels at target doses of 0.6 and 1.8 mg). During induction reduced during the first week, and recovered to baseline after treatment, patients will receive 5 doses of DSP-0509 every week for 4 2~3 weeks post GX-I7 administration. CCR5 expression in both weeks on days 1, 8, 15, 22, and 29 followed by every 2-weeks until CD4+ and CD8+ T cells increased transiently in a dose dependent discontinuation. In the combination arm, pembrolizumab will be ad- manner, suggesting GX-I7 promotes migration of T cell to tumor ministered IV at 200 mg every 3 weeks (Q3W). Dose limiting toxicities environment. No apparent increases in the number of other im- will be monitored within the first 6 weeks of dosing. The mono- and mune cells (NK cell, monocytes, B cells) were observed. combination DSP-0509 RP2Ds will be determined using a Bayesian Conclusions logistic regression model. In Part B, approximately 20–40 patients will A 3-week interval repeated IM administration of GX-I7 appears to receive DSP-0509 at the RP2D using the same dosing schedule as be well tolerated in dose range of 60 – 1,200 μg/kg in advanced Part A. In Part C, approximately 3–6 patients will be treated using the solid cancer patients. Following GX-I7 administration, dose- RP2D with the same induction treatment schedule as Part A, but dependent increase of ALC and T cell subsets(not Treg) were ob- followed by Q3W maintenance dosing. This study is currently recruit- served. These findings suggest that GX-I7 can be an excellent ing patients. combination partner for chemo-radiation, cancer vaccines and im- mune checkpoint inhibitors such as anti-PD-1/PD-L1 antibodies, P427 by increasing T lymphocytes and thereby contributing to en- A phase 1 study of IMC-001, novel anti-PD-L1 antibody, in patients hanced anti-tumor effects. with advanced solid tumors Trial Registration 1 1 Bhumsuk Keam, MD, PhD , Tae Min Kim, MD, PhD , Do-Youn Oh, MD, ClinicalTrials.gov Identifier: NCT03478995 1 1 2 PhD , Chan-Young Ock, MD, PhD , Won Ki Kang, MD, PhD , Yeon Hee Ethics Approval 2 2 3 Park, MD, PhD , Jeeyun Lee, MD, PhD , Ji Hye Lee, MD , Yun Jeong The study was approved by the Severance Hospital, Asan Medical 3 2 Song, MD , Young Suk Park, MD, PhD center, and Catholic Medical Center Institutional Review Board, proto- 1 2 Seoul National University Hospital, Seoul, Korea, Republic of; Samsung col number GX-I7-CA-003. Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, Republic of; ImmuneOncia Therapeutics Inc., Gyeonggi, South P426 Korea A first-in-human phase 1, multicenter trial of toll-like receptor Correspondence: Young Suk Park (pys27hmo@skku.edu) (TLR) 7 agonist DSP-0509 as monotherapy and in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P427 with pembrolizumab in adult patients with advanced solid tumors 2 3 4 Jared Weiss, MD , Anthony Olszanski, MD, RPh , Jordan Berlin, MD , Background 5 5 5 5 Makoto Origuchi , Zhonggai Li , Bella Ertik , Hongliang Cai , Daniel IMC-001 is a fully human IgG1 monoclonal antibody that binds to hu- 5 6 7 1 Clancy , Jose Iglesias , Vivek Subbiah, MD , Shadia Jalal, MD man PD-L1 and mediate the antibody-dependent cell-mediated cyto- Indiana University School of Medicine, Indianapolis, IN, United States; toxicity. The main objectives of this study were to evaluate the 2 3 UNC School of Medicine, Chapel Hill, NC, United States; Fox Chase safety, pharmacokinetics, and pharmacodynamics of IMC-001 in pa- Cancer Center, Phildelphia, PA, United States; Vanderbilt University tients with advanced solid tumors. Additional objectives were to ex- Medical Center, Nashville, TN, United States; Boston Biomedical Inc, plore the anti-tumor activity and identify the maximum tolerated Cambridge, MA, United States; Former employee, Boston Biomedical dose (MTD) of IMC-001. Inc, Cambridge, MA, United States; University of Texas, Houston, TX, Methods United States This is a phase 1, open-label study of IMC-001 in patients with meta- Correspondence: Shadia Jalal (sjalal@iu.edu) static or advanced solid tumors. IMC-001 was administered intraven- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P426 ously every two weeks with a standard 3+3 dose-escalation design until disease progression or unacceptable toxicity. Dose limiting tox- Background icity (DLT) window was defined as 21 days from the first dose. Ad- DSP-0509 is a TLR7 agonist designed to have high water solubility verse events (AEs) were assessed using CTCAE v4.03, and tumor allowing for intravenous (IV) administration and has rapid elimin- response was assessed by the Response Evaluation Criteria In Solid ation, partially due to excretion via organic anion transporting pep- Tumors, version v1.1. tide transporters. In preclinical models, DSP-0509 suppressed tumor Results volume and lung metastasis versus vehicle control. Furthermore, Fifteen patients (8 Male, 7 Female; Median age: 58 [range 39-69]) DSP-0509 in combination with anti–programmed cell death protein 1 were included in 5 dose escalation cohorts, dose ranging from 2 (PD-1) antibody suppressed tumor growth versus vehicle, DSP-0509 to 20 mg. Of the 15 subjects, 5 colorectal cancers, 3 biliary tract alone, or anti–PD-1 antibody alone. This 3-part dose escalation (Part cancers and 2 thymic cancers were included. No DLT was ob- A), dose expansion (Part B), and maintenance dose schedule evalu- served and the maximum tolerated dose was not reached. Most ation (Part C) study will investigate DSP-0509 alone or in combin- common AEs were decreased appetite, pyrexia, and cough. No ation with PD-1 inhibitor pembrolizumab (Part A only) Grade 4 or 5 treatment emergent AEs were reported during the Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 234 of 272 study and no TEAE or serious AE led to treatment discontinuation Acknowledgements or death. There were no infusion-related reactions during this This study was funded by OSE Immunotherapeutics (the sponsor of the study. Two grade 2 serious AEs suspected to be related to IMC- study) in collaboration with Boehringer Ingelheim. 001 were seen in one subject at 2mg/kg cohort. Over the dose Trial Registration range 2 to 20 mg/kg IMC-001, AUC ,AUC ,and C gen- This study is registered under ClinicalTrials.gov Identifier: NCT03990233 0-14d 0—∞ max erally appeared to increase in a dose proportional manner for each step of dose escalation. Efficacy evaluation is ongoing and Reference will be presented in the future. 1. Gauttier V, Pengam S, Durand J, Morello A, Conchon S, Vanhove B and Conclusions Poirier N. Selective SIRPa blockade potentiates dendritic cell antigen IMC-001 demonstrated a favorable safety profile up to 20mg/kg cross-presentation and triggers memory T-cell antitumor responses. Can- given IV every 2 weeks in patients with advanced solid tumors. cer Res 2018;78(13 Supplement): abstr 1684. doi:10.1158/1538- Trial Registration 7445.AM2018-1684. Clinical trial identification : NCT03644056 Ethics Approval Ethics Approval The study protocol and its related documents (including the patient This study was approved by Institutional Review Board; approval information and informed consent form) received approval from the Ethics number SMC 2018-01-007-001 and H-1801-042-913. Committees, and the Competent Authority prior to study initiation. Each patient gave his/her written informed consent prior to study enrolment. P428 A phase 1 study evaluating BI 765063, a first in class selective P429 myeloid SIRPa inhibitor, as stand-alone and in combination with BI A phase 1/2a dose escalation and expansion study of HPN536, a 754091, a PD-1 inhibitor, in patients with advanced solid tumours mesothelin-targeting T cell engager, in patients with advanced 1 2 3 Nuria Kotecki, MD , Philippe Cassier , Jean-Pierre Delord, MD , Stéphane cancers expressing mesothelin who have failed standard therapy 4 1 2 3 1 2 2 Champiat , Christiane Jungels , Armelle Vinceneux , Iphigenie Korakis , Erika Hamilton, MD , Richard Austin, PhD , Sue Hirabayashi , Che-Leung 5 6 6 2 2 2 Richard Huhn , Nicolas Poirier , Dominique Costantini , Bérangère Law, PhD , Bryan Lemon, PhD , Holger Wesche, PhD , Debra Richardson, 6 4 3 Vasseur , Aurélien Marabelle MD 1 2 1 Institut Jules Bordet, Brussels, Belgium; Centre Léon Bérard, Lyon, Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN, United 3 4 2 3 France; IUCT, Oncopole, Toulouse, France; Gustave Roussy, Villejuif, States; Harpoon Therapeutics, South San Francisco, CA, United States; Sarah 5 6 France; Boehringer Ingelheim, Ridgefield, CT, United States; OSE Cannon Research Institute/Stephenson Cancer Center at the University of Immunotherapeutics, Nantes, France Oklahoma Health Sciences Center, Oklahoma City, OK, United States Correspondence: Nuria Kotecki (nuria.kotecki@bordet.be) Correspondence: Erika Hamilton (ehamilton@tnonc.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P428 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P429 Background Background Signal Regulatory Protein α [SIRPα] is a polymorphic protein, strongly HPN536 is a mesothelin-targeting T cell engager derived from the TriTAC expressed on myeloid suppressive cells. BI 765063 (OSE172), a hu- platform (Tri-specific T Cell-Activating Construct). Mesothelin (MSLN) is a manized IgG4 monoclonal antibody (mAb), is a selective antagonist tumor antigen overexpressed in malignant mesothelioma, ovarian carcin- of SIRPα/CD47 interaction, it does not bind to SIRPɣ, known to assist oma, pancreatic carcinoma, lung cancer, and triple negative breast cancer T cell co-stimulation and migration. BI 765063 strongly binds V1 al- with limited expression in normal tissues. HPN536 is a recombinant poly- lele, one of the 2 major functional allele of SIRPα expressed in more peptide of ~50kDa containing three humanized antibody-derived binding than 80% of general population and Asian (in 60%). domains, targeting mesothelin (for tumor binding), albumin (for half-life ex- Anti-tumor effect was shown in various in vivo cancer models using tension) and CD3 (for T cell engagement). It has been engineered to be a the validated anti-mouse SIRPα mAbs surrogate, as single agent. The small, globular protein to enable efficient exposure in solid tumor tissue effect was more pronounced in combination with T checkpoint inhib- with prolonged half-life and excellent stability under physiological condi- itors [1]. BI 765063 mechanism of action includes promotion of tions. HPN536 binds monomerically to CD3 and MSLN, minimizing non- tumor-antigen-presentation while preserving T-cell activation and in- specific T-cell activation. These features are designed to widen the thera- crease tumor phagocytosis. peutic index compared to earlier generations of T cell engagers by minimiz- The trial plans to assess the safety profile and preliminary efficacy of ing off target toxicities. HPN536 mediates potent target tumor cell killing in BI 765063, a first in class myeloid check point inhibitor antagonist of a MSLN-specific manner in vitro and in xenograft models in the presence of SIRPα on myeloid cells. T cells. Consistent with its mechanism of action (MOA), tumor cell killing is Methods accompanied by T cell activation, cytokine induction, and T cell expansion. This study comprises a dose escalation (step 1) to determine the Methods Dose-Limiting Toxicities, Maximum Tolerated Dose (MTD), and Rec- This is a Phase 1/2a, open-label, multicenter, dose escalation and ex- ommended Phase 2 Dose (RP2Ds) of BI 765063 monotherapy and pansion study to evaluate the safety, tolerability, clinical activity, and with BI 754091, and dose-confirmation expansion cohorts (step 2). pharmacokinetics of HPN536 in adult patients with advanced cancers In Step 1, ascending dose of BI 763063 once every 3 weeks intraven- expressing mesothelin who have failed standard available therapy. This ously (iv) using a Bayesian approach with overdose control are tested. study will be divided into 2 parts: Dose Escalation (Part 1) and Expan- When MTD determined, BI 763063 will be tested with BI 754091, a PD- sion (Part 2). Eligible patients with ovarian cancer will be enrolled in 1 mAb inhibitor. In step 2, 2 parallel randomized, non-comparative Dose Escalation. Dose expansion will include patients with ovarian can- mono and combination cohorts will further confirm the RP2Ds and as- cer, pancreatic carcinoma and mesothelioma. HPN536 is administered sess the safety and preliminary efficacy (RECIST 1.1 and iRECIST). once weekly as one-hour IV infusion by single-patient cohorts until ei- Patients ≥ 18 years, PS:0-1, with advanced solid tumor who failed or ther a Grade ≥2 adverse event (AE) that is possibly related to HPN536 is are not eligible to standard therapy will be included. V1/V1 and V1/ observed or an estimated therapeutic dose level has been reached. V2 patients (central testing) are evaluated in separate cohorts in step Then a conventional 3+3 design will be implemented. Dose escalation 1. In step 2, a selected population of V1/V1 patients with advanced- will continue until a recommended phase 2 dose (RP2D) is determined. stage cancers (e.g. non-small cell lung cancer, triple negative breast In dose expansion, up to 20 patients per group receive HPN536 at the cancer, or gastro-intestinal cancers) will be included. established RP2D based on a Simon 2-stage design. Patients may con- Pharmacokinetics (PK), SIRPα receptor occupancy (RO) and a compre- tinue weekly HPN536 treatment cycles until disease progression. Pri- hensive translational program (in blood and tumour) will assess PK/ mary endpoints are number and severity of DLTs following treatment PD profile and biomarkers of activity. with escalating doses of HPN536 during escalation, and overall re- A total of 116 (56 instep1 and60 in step2) patients willbe enrolled. sponse rate (by RECIST v1.1 for ovarian and pancreatic, mRECIST v1.1 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 235 of 272 for mesothelioma) in dose expansion. Secondary endpoints include with a PD-1 inhibitor. After defining the MTD and/or RP2D, additional AEs, preliminary anti-tumor activity, pharmacokinetic and pharmacody- subjects may be enrolled at the respective dose schedules to further namic parameters based on the proposed MOA of HPN536. evaluate safety, pharmacodynamic effects, and anti-tumor activity Trial Registration Trial Registration NCT03872206 NCT04009681 Ethics Approval This study was approved by each participating institution's Institu- Table 1 (abstract P430). See text for description tional Review Board. P430 Open-label, multicenter phase 1/2 dose escalation and expansion study of THOR-707 as a single agent and in combination with a PD-1 inhibitor in adult subjects with advanced or metastatic solid tumors 1 2 3 David Luo , Raghad Abdul-Karim, MD , Arun Azad, MD , Joanna Bendell, 4 5 6 7 MD , Hui Gan, MBBS PhD , Filip Janku, MD, PhD , Shiraj Sen, MD, PhD , 8 9 1 1 Tira Tan, MBBS , Judy Wang, MD , Lisa Schechet , Lauren Baker, PhD , 1 10 Joseph Leveque, MD , Tarek Meniawy, MBBS FRACP 1 2 Synthorx Inc, La Jolla, CA, United States; NEXT Oncology, Texas Oncology, San Antonio, TX, United States; Peter MacCallum Cancer Centre, Melbourne, Austrailia; Sarah Cannon Research Institute, Nashville, TN, United States; Austin Hospital, Melbourne, Victoria, Australia; University of TX, MD Anderson Cancer Center, Houston, TX, United States; Sarah Cannon Research Institute at HealthONE, Houston, TX, United States; National Cancer Centre Singapore, Toronto, Canada; 9 10 Florida Cancer Specialists, Sarasota, FL, United States; Linear Clinical Research, Nedlands, WA, Australia Correspondence: Lauren Baker (lbaker@synthorx.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P430 Background Recombinant interleukin-2 (aldesleukin), an approved immunotherapy in metastatic melanoma and renal cell carcinoma, can induce complete durable responses in some patients. The anti-neoplastic properties of IL-2 are mediated by activation of effector memory T cells and newly recruited naïve CD8+ T cells against the tumor. The widespread use of P431 IL-2 has been limited due to its high affinity bias for the IL-2 receptor Semi-mechanistic PK and target-occupancy modeling to support alpha chain (IL-2R⍺) on regulatory CD4+ T cells, leading to immunosup- dose justification for anti-PD-L1 clinical candidate CK-301 (TG- pression and, eosinophilic recruitment and activation on innate lymph- 1501) in oncology patients oid cells in the vascular endothelium causing vascular leak syndrome 1 1 2 3 Lin Lin, PhD , James Hilbert , Leonid Gorelik , Jian-Ping Tang , James (VLS). THOR-707 is a recombinant human IL-2 variant that is site- 4 1 1 1 Oliviero , Joshua Apgar , Lore Gruenbaum, PhD , John Burke specifically pegylated, providing a “not alpha” pharmacologic profile 1 2 Applied BioMath, LLC, Concord, MA, United States; Fortress Biotech, designed to prevent engagement of IL-2R⍺, thereby providing an im- Waltham, MA, United States; TG Therapeutics, New York, NY, United proved safety profile while still promoting newly recruited and effector States; Checkpoint Therapeutics, New York, NY, United States memory T cell anti-tumor activity In preclinical studies, eosinophilia was Correspondence: Lore Gruenbaum (lgruenbaum@gothamtx.com) not observed at a doses 10-fold higher than the dose responsible for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P431 eliciting maximal expansion of peripheral CD8+ T cells. Based on these findings, a first-in-human study of THOR-707 was started in June 2019. Background Methods Mathematical modeling was used in conjunction with in vitro, pre- This open-label, multicenter, dose escalation and expansion study in clinical and clinical data to facilitate dose selection of CK-301 (also adult subjects with advanced or metastatic solid tumors will evaluate known as TG-1501), an anti-PD-L1 monoclonal antibody (mAb), for THOR-707 as a single agent and in combination with a PD-1 inhibitor. ongoing and future clinical trials in oncology patients. Study objectives are to define the maximum tolerated dose (MTD) and/ Methods or recommended phase 2 dose (RP2D) of THOR-707 as single agent and A semi-mechanistic pharmacokinetic/target-occupancy (PKTO) model in combination with a PD-1 inhibitor; and to evaluate the overall safety was developed to predict pharmacokinetics (PK) of CK-301 at steady state and tolerability as well as, pharmacokinetics, pharmacodynamics, and and its tumor target occupancy (TO) under various dosing regimens. The preliminary anti-tumor activity. The study will be conducted in 3 parts. model captures the interactions between CK-301, PD-L1, soluble PD-L1 and PD-1 in 3 compartments: tumor, circulation (central) and other tissues (per- Part 1 will evaluate THOR-707 as a single agent across different dos- ipheral). The model was calibrated with CK-301 PK data from the first 5 pa- ing schedules (e.g., dosing every 2 weeks [Q2W] or 3 weeks [Q3W]). tients in a clinical study, CK-301-101, and PK data from published Phase 1 Part 2 will evaluate THOR-707 (Q3W) in combination with a PD- studies of 3 marketed anti-PD-L1 mAbs: atezolizumab, avelumab and durva- 1 inhibitor. lumab. Additionally, the model incorporated experimentally determined Part 3: Dose expansion will begin after the RP2D for THOR-707 as binding affinities for the 3 marketed anti-PD-L1 mAbs and CK-301. a single agent or in combination with a checkpoint inhibitor has Results been determined and will enroll selected populations (e.g., spe- Using the PKTO model, plasma Ctrough values and tumor TO of CK-301 at cific tumor types, treatment history, and/or biomarker profile). steady-state with 800 and 1200 mg q2w or q3w were projected. The TO of CK-301 were compared with predicted steady-state Ctrough TOs of atezoli- Between 50-100 subjects may be enrolled in the dose escalation phase zumab, avelumab and durvalumab at their marketed doses. The steady- to determine the MTD and/or RP2D as a single agent and in combination state Ctrough values of CK-301 are predicted to give >99% tumor TO for Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 236 of 272 patients with a nominal or a 10-fold greater than nominal PD-L1 tumor bur- P433 den. This is similar to predicted TO for atezolizumab and durvalumab. The Initial results of the phase 1 portion of an ongoing phase 1/2 PKTO model was used to simulate PK and TO of CK-301 in 1000 virtual pa- study of RP1 as a single agent and in combination with nivolumab tients. The simulations predicted that, at 800 and 1200 mg q2w or q3w, in patients with solid tumors 1 2 3 ≥93.0% of patients with a nominal PD-L1 tumor burden or ≥80.1% of pa- M Middleton, MD PhD , Joseph Sacco , Jaime Merchan , Amber 3 4 5 tients with 10-fold higher than nominal PD-L1 tumor burden would have a Thomassen , Brendan Curti, MD , Ari VanderWalde, MD, MPH, MBioeth , 2 1 >99% tumorTOatsteady-stateCtrough. Anna Olsson-Brown, MBChB (Hons), BSc (Hons) , Francesca Aroldi , Nicos 6 5 7 Conclusions Fotiadis , Scott Baum , Howard Kaufman, MD, FACS , Kevin Harrington, At the proposed CK-301 dosing regimens of 800 and 1200 mg q2w or MD 1 2 q3w, a >99% TO is expected throughout the dosing interval. Relative to University of Oxford, Childrey, United Kingdom; University of Liverpool, atezolizumab and durvalumab treatments, similar percentages of pa- Liverpool, United Kingdom; University of Miami, Miami, United States; 4 5 tients would possibly benefit from CK-301 treatment. Providence Medical Center, Portland, OR, United States; West Cancer Center, Germantown, TN, United States; The Institute of Cancer Research, London, United Kingdom; Replimune, Woburn, MA, United States P432 Correspondence: Howard Kaufman (Howard.Kaufman@replimune.com) A phase 1 dose-escalation study of safety, tolerability, and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P433 pharmacokinetics (PK) of ABBV-368 monotherapy and combination in patients (pts) with locally advanced or metastatic solid tumors Background 1 2 3 Christophe Le Tourneau , Wu-Chou Su, MD , Ki Chung, MD , Patricia Background: RP1 is an enhanced-potency oncolytic HSV-1 expressing 4 5 6 LoRusso, DO , Chia-Chi Lin, MD, PhD , Fabrice Barlesi, MD, PhD , Her- a fusogenic glycoprotein (GALV-GP R-) and GM-CSF which is being 7 8 9 Shyong Shiah , Eric Angevin, MD , Alexander Spira, MD, PhD, FACP , tested in a Phase 1/2 clinical trial in ~150 patients with a range of 10 11 Amita Patnaik, MD FRCP(C) , John Powderly, MD, CPI , Dimitrios solid tumors (NCT03767348). 12 13 14 Colevas , Helen Chew, MD , Maulik Patel, PharmD, PhD , Stacie Methods 14 14 14 14 Lambert , Yan Li , Daniel Da Costa , Martha Blaney, PharmD , Methods: The objectivesweretodefinethesafety of RP1 aloneand with 14 15 Michael McDevitt, MD, PhD , Philippe Cassier nivolumab, determine the recommended phase 2 dose (RP2D), and in 1 2 Institut Curie, Paris & Saint-cloud, France; National Cheng Kung University 30 patient phase 2 cohorts, assess efficacy in melanoma, non-melanoma Hospital, Tainan, Taiwan, Province of China; GHS Cancer Institute, skin cancer, urothelial carcinoma and MSI-H tumors. Initial phase 1 re- Spartanburg, SC, United States; Yale Cancer Center, New Haven, CT, United sults will be reported where patients were treated by intra-patient dose States; National Taiwan University Hospital, Taipei, Taiwan, Province of China; escalation of RP1 (up to 10mL of 104-108PFU/mL) by intratumoral injec- 6 7 Aix Marseille Univ Hôpitaux de Marseille, Livon, France; Taipei Medical tion into a single tumor Q2W up to 5 times followed by 12 patients University, Taipei, Taiwan, Province of China; Gustave Roussy, Villejuif, France; dosed 8 times at the RP2D combined with nivolumab (240mg Q2W for 9 10 Virginia Cancer Specialists Research Ins, Fairfax, VA, United States; South 4 months from the second RP1 dose, then 480 mg Q4W for 20 months). Texas Accelerated Research Thera, San Antonio, TX, United States; Carolina Clinically accessible lesions were directly injected, with imaging guidance BioOncology Institute, Huntersville, NC, United States; Stanford University, for deep/visceral lesions. Pre- and on-treatment tumor biopsies were ob- 13 14 Stanford, United States; UC Davis, Sacramento, United States; Abbvie Inc, tained for biomarker analysis. Viral shedding and anti-HSV antibody titers North Chicago, IL, United States; Léon Bérard Cancer Center, Lyon, France were also monitored. Correspondence: Michael McDevitt (michael.mcdevitt@abbvie.com) Results Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P432 Results: 22 heavily pretreated patients with advanced tumors were enrolled into the dose-rising phase with largely low-grade adverse Background events, including febrile and other constitutional symptoms, local in- ABBV-368 is a humanized anti-OX40 monoclonal antibody. OX40 flammation and erythema. No clear differences were seen between is a member of the TNF receptor superfamily, which exerts its ac- superficial and visceral dosing. RP1 was detected at the injection site tion via activating T effector cells and inhibiting the suppressive and in blood for up to 14 days (next injection), suggesting virus repli- capacity of T reg cells. Preclinical data have shown ABBV-368 an- cation. All HSV seronegative patients seroconverted after three injec- titumor activity in animals. tions. Biological activity was demonstrated including tumor necrosis Methods and shrinkage, with extended clinical benefit and delayed (post-treat- This is a multicenter, phase 1, dose-escalation study (NCT03071757) ment termination and initial PD) systemic reduction in multiple tu- in pts (≥18 years; Eastern Cooperative Oncology Group performance mors in two patients (ipilimumab/nivolumab-refractory melanoma status 0–2) with locally advanced or metastatic solid tumors. The and chemotherapy-refractory cholangiocarcinoma) without interven- study consisted of 3 parts: 1) dose escalation (DE1); 2) cohort expan- ing treatment. The RP2D was selected as up to 10mL of 106PFU/mL sion (CE2); and 3) imaging substudy (IA3). Specific inclusion criteria followed Q2W by multiple doses of 107PFU/mL. Twelve evaluable pa- and dosing schedules for each cohort are shown in the table (Table tients (6 direct injection, 6 image-guided) were then enrolled into 1). In DE1, the primary endpoints are safety, tolerability, and PK of the phase 1 expansion combined with nivolumab. This demonstrated ABBV-368 monotherapy to establish the maximum tolerated dose or tolerability and clinical activity, including complete and partial re- reach the maximally administered dose; the secondary endpoint is sponses in patients with chemotherapy-refractory cutaneous squa- preliminary antitumor activity. Preliminary results for DE1 have been mous carcinoma, and ipilimumab/nivolumab-refractory melanoma. reported (Powderly et al. ESMO 2018). For CE2, the primary end- Treatment remains ongoing, and current data will be presented, in- points are safety, tolerability, and PK of ABBV-368 monotherapy and cluding biomarker data (CD8, PD-L1 staining and Nanostring analysis in combination with ABBV-181 (a humanized anti-programmed cell from tumor biopsies). death 1 monoclonal antibody), and to establish the recommended Conclusions phase 2 dose; the secondary endpoint is preliminary antitumor activ- Conclusions: The Phase 1 clinical data supports the safety and effi- ity of ABBV-368 monotherapy and combination therapy. The primary cacy of RP1 alone and when combined with nivolumab, including endpoints of the IA3 part are safety and tolerability. For all cohorts, demonstration of abscopal anti-tumor effects in patients refractory to AEs will be assessed according to the NCI CTCAE v4.03; response will prior checkpoint inhibitors. The Phase 2 portion of this clinical trial is be assessed Q2 months (mo) for 12 mo, and Q3 mo thereafter, as open in the US and the UK. per the immunotherapy Response Evaluation Criteria in Solid Tumors Trial Registration (iRECIST), and RECIST v1.1. As of 12 Jul 2018, enrollment into DE1 NCT03767348 was completed and CE2 has started. Ethics Approval Trial Registration The study was approved by applicable institutional review or ethics NCT03071757 boards. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 237 of 272 P434 2. Piccione et al. Preclinical and initial phase I clinical characterization of A phase 1/1b study to evaluate the humanized anti-CD73 CPI-006: an anti-CD73 monoclonal antibody with unique immunostimula- antibody, CPI-006, as a single agent, in combination with CPI-444, tory activity. Presented at Society for Immunotherapy of Cancer Meeting; and in combination with pembrolizumab in adult patients with November 7-11, 2018; Washington, DC, USA: Abstract P205. advanced cancers Ethics Approval 1 1 1 1 Mehrdad Mobasher , Richard Miller , Brian Munneke , Deborah Strahs , The study was approved by Western IRB, approval number 1-1066703-1. 1 1 1 Gabriel Luciano , Emily Piccione, PhD , Suresh Mahabhashyam , Jaime 2 3 4 Merchan , John Powderly, MD, CPI , Lauren Harshman, MD , Minal 5 6 7 8 Barve , Walter Stadler, MD , Patricia LoRusso, DO , Melissa Johnson , 9 10 Abhishek Tripathi, MD , Sumanta Pal, MD , Ben Markman, MBBS 11 12 13 FRACP , Jason Luke, MD, FACP , Thomas Marron, MD PhD 1 2 Corvus Pharmaceuticals Inc, Burlingame, CA, United States; University of Miami, Miami, United States; Carolina BioOncology Institute, Huntersville, NC, United States; Dana-Farber Cancer Institute, Boston, MA, United States; Mary Crowley Cancer Research Center, Dallas, TX, United States; University of Chicago Comprensive Cancer Center, Chicago, IL, United States; Yale University School of Medicine, New Haven, CT, United States; Sarah Cannon Research Institute, Nashville, TN, United States; University of Oklahoma, Stephenson Cancer Center, Oklahoma City, OK, United States; City of Hope, Duarte, CA, United 11 12 States; Monash Health, Melbourne, Australia; UPMC, Pittsburgh, PA, United States; Icahn School of Medicine at Mount Sinai, New York, NY, United States Correspondence: Mehrdad Mobasher (mmobasher@corvuspharma.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P434 Background CD73 expression is elevated in tumors and contributes to increas- ing levels of immunosuppressive adenosine in the tumor micro- environment. CD73 knockout mice exhibit reduced tumor growth and resistance to experimental metastasis. Inhibition of CD73 ac- tivity with an anti-CD73 antibody blocks adenosine production, Fig. 1 (abstract P434). See text for description shown to inhibit tumor growth in syngeneic models. Dual inhib- ition of CD73 and A2aR improves anti-tumor immune responses in mouse tumor models[1]. CPI-006 is a humanized IgG1 Fc gamma receptor binding-deficient anti-CD73 antibody that has a P435 dual mechanism of action. It blocks CD73 catalytic activity and A window of opportunity trial using intratumoral injection of adenosine production. In addition, it has immunomodulatory ac- glatiramer as an immune modulator in patients with resectable tivity on CD73 positive immune cells including B cells, T cells and head and neck and cutaneous squamous cell cancer antigen presenting cells. CPI-006 relieves adenosine-mediated im- Ghulam Rehman Mohyuddin, MD, Joaquina Baranda, MD, Andres Bur, munosuppression in vitro as a single agent and in combination Lisa Shnayder, Kiran Kakarala, Terry Tsue, Prakash Neupane, Gregory Gan, with ciforadenant[2]. CPI-006 is now being investigated in this Joshua Mammen, Daniel Aires, Sufi Thomas, Stephen Williamson, Nelli Phase 1/1b multicenter, open label trial as single agent (SA), in Lakis, Rashna Madan, Prabhakar Chalise, Scott Weir, Andrew Godwin, combination with ciforadenant, an oral, small molecule, selective Greg Reed, Cory Berkland A2aR antagonist and in combination with pembrolizumab, an Kansas University Medical Center, Kansas City, KS, United States anti-PD1 indicated for the treatment of patients across a number Correspondence: Joaquina Baranda (gioncol@gmail.com) of malignancies (NCT03454451). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P435 Methods Up to 462 subjects will be enrolled at approximately 35 sites in the Background US, Canada and Australia. Eligible patients with: non-small cell lung Immunotherapy using checkpoint inhibition improves outcome of cancer (NSCLC), renal cell carcinoma cancer (RCC), urothelial bladder patients with melanoma, lung, bladder, microsatellite instability-high cancer, cervical cancer, colorectal cancer, ovarian cancer, pancreatic and other tumors[1]. However, systemic administration of immuno- cancer, prostate cancer, head and neck cancer, triple-negative breast therapy may have limited activity in some tumors partly due to fail- cancer, endometrial cancer, select sarcomas and non-Hodgkin lymph- ure of activated T cells to migrate to tumor[1]. Intratumoral injections oma (NHL) who are relapsed, refractory or intolerant to 1 to 5 stand- (ITI) may allow high concentration of immunostimulatory products lo- ard therapies; aged ≥ 18 yo; with adequate organ function and cally while using small amounts of drugs. This may also facilitate mul- measurable disease. Study details is presented in Figure 1. tiple combination therapies and avoid systemic off-target toxicities. The primary objective of the dose escalation is to assess safety/ tolerabil- By capitalizing on existing data and experience, repurposing ap- ity, MTD or MDL of CPI-006 SA, in combination with ciforadenant and proved drugs for cancer represents an opportunity to rapidly ad- with pembrolizumab in ascending dose levels. Secondary objectives are vance promising therapies. to evaluate the PK of CPI-006 as SA or in combinations and analyze po- Glatiramer acetate is an agent commonly used for multiple scler- tential predictive biomarkers. In dose escalation, the primary objective is osis[2]. It acts as an immunomodulator and has essentially no sys- to assess the safety and tolerability of CPI-006 SA and in combinations in temic bioavailability but exhibits a high prevalence of injection site patients with selected advanced cancers. Secondary endpoints include reactions[2]. It upregulates the activity of natural killer cells in efficacy; PK of CPI-006 as SA and in combinations and to evaluate the re- leukemia cell lines [3], suggesting potential for immunostimulatory lationship between biomarkers and clinical activity. effect for ITI. For percutaneously accessible tumors for which the standard of care is surgical resection without any neoadjuvant ther- References apy, there exists a window of opportunity where ITI of glatiramer can 1. Young et al. Co-inhibition of CD73 and A2AR adenosine signaling im- be performed before surgery. proves anti-tumor immune responses. Cancer Cell. 2016; 30(3):391-403. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 238 of 272 Methods blood pressure, and must have provided a tumor sample evaluable for This is a proof-of-concept, investigator-initiated, window of opportunity PD-L1; and must not have immunodeficiency, active central nervous trial in subjects with percutaneously accessible head and neck or cuta- system metastases (except in GBM cohort), or be on steroid therapy neous squamous cell cancer who are to undergo surgery. Subjects will (patients with GBM may be on dexamethasone ≤2 mg/day orally or receive glatiramer 40 mg by ITI 3 times a week prior to surgery. Sub- equivalent and stable for 5 days at the time of enrollment). Patients will jects will receive at least one dose and up to 3 doses of glatiramer. receive lenvatinib 20 mg daily and pembrolizumab 200 mg every 3 About 10 eligible patients will be included in this trial. Safety data will weeks (Q3W) for ≤2 years, or until confirmed disease progression (may be collected. Tumor tissue at the time of diagnosis and at the time of continue lenvatinib if receiving clinical benefit), unacceptable toxicity, surgery will be collected and compared for biomarkers. Primary end- or study withdrawal. Patients with confirmed complete response may point is safety. Secondary endpoint is effect of ITI of glatiramer on bio- discontinue after ≥24 weeks combination therapy and ≥2pembrolizu- marker levels. Pre- and post-treatment tumor samples will be tested mab doses after initial complete response date. Tumor imaging will using an immunology panel that profiles immunology genes and pro- occur at baseline, Q9W (or for GBM patients: Q6W until 18 weeks, then teins including major classes of cytokines, interferons, KIR family, and Q9W) for the first 54 weeks, Q12W until week 102 (~2 years), and TNF-receptor. Tumors samples will also be evaluated for the Ki-67 pro- Q24W thereafter using RECIST v1.1/RANO by investigator assessment in liferative index and for caspase-3 and cleaved caspase-3 immunoex- the initial cohorts and by BICR after cohort expansion. Primary end- pression. Paired T-test or the Wilcoxon signed rank test will be used to points are objective response and safety (adverse events graded using assess the changes in the variables. We hypothesize that this approach NCI CTCAE v4.0, and discontinuation due to adverse events). Secondary will break the immunosuppressive tumor microenvironment as evi- endpoints include disease control, duration of response, progression- denced by an increase in inflammatory cytokines, chemokines, and im- free survival, and overall survival. Initially, approximately 180 patients mune cell infiltration. Decline in Ki-67 and increase in caspase-3 may be will be enrolled (30/cohort; each cohort may be expanded to 100 after a signal of anti-tumor activity. planned interim analysis). Enrollment is ongoing at 44 sites in 10 coun- Trial Registration tries across North America, South America, Europe, Asia, and Australia. NCT03982212 Acknowledgements References Writing support was provided by Shilpa Aggarwal, PhD, of C4 MedSolutions, 1. Whiteside, T.L., et al., Emerging Opportunities and Challenges in Cancer LLC (Yardley, PA, USA), a CHC Group company, funded by Eisai Inc. and Merck Immunotherapy. Clin Cancer Res, 2016. 22(8): p. 1845-55. Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. 2. Wingerchuk, D.M. and J.L. Carter, Multiple sclerosis: current and emerging Legal Entity Responsible for the Study: Eisai Inc. and Merck Sharp & Dohme disease-modifying therapies and treatment strategies. Mayo Clin Proc, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA 2014. 89(2): p. 225-40. 3. Maghazachi, A.A., K.L. Sand, and Z. Al-Jaderi, Glatiramer Acetate, Dimethyl Funding Source Fumarate, and Monomethyl Fumarate Upregulate the Expression of Funding for this research was provided by Eisai Inc. and Merck Sharp & CCR10 on the Surface of Natural Killer Cells and Enhance Their Chemo- Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. taxis and Cytotoxicity. Front Immunol, 2016. 7: p. 437. Trial Registration Ethics Approval NCT03797326 This study was approved by Kansas University Medical Center Institutional Ethics Approval Review Board, approval number: HSC00144030 An independent institutional review board or ethics committee approved the protocol at each study site, and the trial is being conducted in compliance with Good Clinical Practice guidelines and the Declaration of Helsinki. P436 Phase 2 study of lenvatinib plus pembrolizumab in previously treated patients with solid tumors: LEAP-005 P437 1 2 3 4 5 Ravit Geva , Seock-Ah Im , Zarnie Lwin , Susan Weil , Lei Xu , Anne Disease-related biomarkers are associated with extended 5 5 6 Morosky , Kevin Norwood, MD , Hyun Cheol Chung, MD, PhD progression free survival after treatment with NEO-PV-01 in 1 2 Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; Seoul National combination with anti-PD1 in patients with metastatic cancers 3 1 2 University Hospital, Seoul, Korea, Republic of; University of Queensland, Patrick Ott, MD, PhD , Ramaswamy Govindan, MD , Aung Naing, MD, 4 3 4 5 6 Queensland, Australia; Eisai Inc., Woodcliff Lake, NJ, United States; FACP , Terence Friedlander, MD , Kim Margolin, MD , Jessica Lin, MD , 5 6 7 8 Merck & Co., Inc., Kenilworth, NJ, United States; Yonsei University Nina Bhardwaj, MD, PhD , Matthew Hellmann, MD , Mark Awad, MD 1 9 9 9 College of Medicine, Seoul, Korea, Republic of PhD , Amy Wanamaker , Lisa Cleary , Michael Rooney , Julian Scherer, 9 9 9 9 Correspondence: Ravit Geva (ravitg@tlvmc.gov.il) PhD , Meghan Bushway , Melissa Moles , Zakaria Khondker , Richard 9 9 9 9 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P436 Gaynor, MD , Lakshmi Srinivasan, PhD , Andrew Chi , Joel Greshock , Siwen Hu-Lieskovan, MD, PhD 1 2 Background Dana Farber Cancer Institute, Boston, MA, United States; Washington Lenvatinib (multiple receptor tyrosine kinase inhibitor of vascular endo- University, Saint Louis, MO, United States; MD Anderson Cancer Center, thelial growth factor receptors 1–3, fibroblast growth factor receptors 1– Houston, TX, United States; University of California San Francisco, San 4, platelet-derived growth factor receptor α,RET,and KIT) andanti–PD-1 Francisco, CA, United States; City Of Hope, Duarte, CA, United States; 6 7 inhibitor pembrolizumab have shown clinical benefit as monotherapies Massachusetts General Hospital, Boston, MA, United States; Mt. Sinai across multiple cancers. In preclinical studies, lenvatinib plus PD-1 block- Medical Center, New York, NY, United States; Memorial Sloan Kettering ade improved antitumor activity vs either agent alone. LEAP-005 Cancer Center, New York, NY, United States; Neon Therapeutics, (NCT03797326) evaluates the efficacy and safety of lenvatinib plus pem- Cambridge, MA, United States; Huntsman Cancer Institute, Los brolizumab in patients with previously treated selected advanced tumors. Angeles, CA, United States Methods Correspondence: Joel Greshock (jgreshock@neontherapeutics.com) This global, open-label, phase 2 study enrolls patients ≥18 years with Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P437 the following previously treated histologically/cytologically confirmed advanced tumors: triple negative breast, ovarian, gastric, colorectal Background (non-MSI-H/pMMR), glioblastoma multiforme (GBM), or biliary tract (ex- Neoantigens arise from mutations in cancer cell DNA and are import- cluding ampulla of Vater). Patients must have progressed on or since ant targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a last treatment; have measurable disease per RECIST v1.1 (modified to personal neoantigen vaccine of up to 20 peptides designed by the follow ≤5 target lesions/organ [10 total]) or the RANO criteria (GBM RECON® bioinformatics platform using patient neoantigen and HLA only), assessed locally and confirmed by blinded independent central profiles. Here we report biomarker correlates of clinical benefit for review (BICR); ECOG performance score 0–1, adequately controlled NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 239 of 272 with nivolumab in anti-PD1 naïve metastatic melanoma, NSCLC and antigen recall studies (Hanson 2018) showed that soluble vopra- bladder cancer patients (NCT02897765). telimab stimulated a polyfunctional cytokine response only in Methods CD4 T cells that were ICOS hi, further supporting the hypothesis Patients received 12 weeks of nivolumab monotherapy (240 mgs that vopratelimab induces activation and proliferation of CD4 T Q2W), then NEO-PV-01 in a prime-boost format spanning 12 weeks, effector cells only after an initial priming event induces an ICOS nivolumab continued for up to 2 years. The primary objective was hi CD4 T cell phenotype. Furthermore, in melanoma patients safety, secondary objectives were overall response rate (ORR), treated with ipilimumab, a sustained increase in the frequency of progression-free survival (PFS), and overall survival. Comprehensive ICOS-positive CD4 T cells correlated with clinical benefit and sur- comparisons of baseline and serial molecular and immunological vival (Carthon 2010). In contrast, emergence of these ICOS hi cells characteristics between patients with vs. without durable PFS were has not been noted with PD-1/PD-L1 inhibitors (Hanson 2018). performed for all tumor cohorts. We hypothesized that the combination of vopratelimab with ipili- Results mumab will enhance the presence and functionality of ICOS hi A total of 34 melanoma, 27 NSCLC and 21 bladder cancer patients re- CD4 T effector cells, thereby potentially increasing the likelihood ceived nivolumab therapy, of which 27, 18 and 15 initiated vaccine of clinical benefit. respectively. The median follow up time was 13.4, 12.0 and 14.7 Methods months for melanoma, NSCLC and bladder cancer respectively. No This open label, multi-center, phase 2 study will evaluate efficacy, treatment-related serious adverse events were noted. The median safety, PK, and exploratory pharmacodynamics of vopratelimab in PFS for the melanoma cohort was not reached (95% CI: 3.3, NE), and combination with ipilimumab in adult patients with non-small cell the ORR was 47%. The median PFS in both the NSCLC and bladder lung cancer or urothelial cancer who have been previously cohort was 5.6 months (95% CI’s: 2.3, 8.7; 2.0, 8.1 respectively) with treated with PD-1/PD-L1 inhibitors. We expect to enroll approxi- ORR’s of 22% and 24% respectively. RECON tumor neoantigen abun- mately 200 evaluable subjects in total. Primary endpoint is ORR. dance was predictive of durable PFS in melanoma patients. Analyses Secondary endpoints include safety and tolerability, PFS, OS as of pre-treatment peripheral TCR repertoires reveal a more clonal T well as PK/PD. cell population in melanoma patients with extended PFS. Other fac- Trial Registration tors that associated with durable PFS included the abundance of B ClinicalTrials.gov NCT03989362 cells and CD8+ T cells in the tumor microenvironment. Finally, across Ethics Approval cohorts, longitudinal tumor biopsies from patients with extended Study was approved by the applicable Institution Ethics Boards PFS showed higher rates of initial pathologic responses after vaccin- ation vs. biopsies from patients with shorter PFS, suggesting vaccine- P439 related anti-tumor responses in this subset. Phase 1 first in human study of programmed cell death receptor- Conclusions 1(PD-1) inhibitor monoclonal antibody (mAb) JTX-4014 in adult NEO-PV-01 in combination with nivolumab is safe and leads to post- subjects with advanced refractory solid rumor malignancies vaccine immune and pathologic responses, indicating further clinical 1 2 Kyriakos Papadopoulos, MD , Gerald Falchook, MD , Nehal Lakhani, MD, evaluation is warranted. The association of baseline disease charac- 3 4 4 4 4 PhD , Gosia Riley , Jian Xu, PhD , Johan Baeck , Gilad Gordon , Elizabeth teristics with prolonged PFS suggests future patient enrichment 4 5 Trehu, MD , Judy Wang, MD strategies. 1 2 START, San Antonio, TX, United States; Sarah Cannon Research Inst. at Trial Registration Healthone, Denver, CO, United States; START-Midwest, Grand Rapids, NCT02897765 MI, United States; Jounce Therapeutics, Cambridge, MA, United States; Ethics Approval Florida Cancer Specialists - SCRI, Sarasota, FL, United States This trial has been approved by all institutional Review Boards of Correspondence: Kyriakos Papadopoulos every clinical trial site involved with this study. (Kyri.Papadopoulos@startsa.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P439 P438 Phase 2 Multicenter Trial of ICOS agonist vopratelimab and a Background CTLA-4 inhibitor in PD-1/PD-L1 inhibitor experienced adult JTX-4014 is a fully human mAb consisting of 2 identical hinge- subjects with Non-small Cell Lung Cancer or Urothelial Cancer stabilized immunoglobulin gamma 4 (IgG4, S228P) heavy and two (EMERGE) identical kappa (Igκ) light chains, that specifically binds to PD-1. The 1 1 2 Russell Pachynski, MD , Ramaswamy Govindan, MD , Ellen Hooper, MD , mechanism of action of JTX-4014 is to block the interaction of PD-1 2 2 2 Christopher Harvey, PhD , Amanda Hanson , Sean Lacey, MA , Rachel with its ligands, PD-L1 and PD-L2, and augment anti-tumor T-Cell ac- 2 2 2 2 McComb , Courtney Hart , Haley Laken , Johan Baeck , Elizabeth Trehu, tivity. This Phase 1 trial objectives were to evaluate the safety and MD tolerability of the drug along with its maximum tolerated dose (MTD) Washington University School of Medicine, St. Louis, MO, United States; and recommended Phase 2 dose. Jounce Therapeutics, Cambridge, MA, United States Methods Correspondence: Russell Pachynski (rkpachynski@wustl.edu) Key inclusion criteria included age ≥18 yrs, histologically or cytologic- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P438 ally confirmed extracranial solid tumor refractory to at least one prior line of therapy, no concurrent anticancer treatment, no prior anti-PD- Background 1 or anti-PD-L1 therapy, no requirement for selection based on PD- ICOS is a costimulatory molecule upregulated on activated T cells. L1 expression, no history of immune-mediated conditions, and ad- Vopratelimab (JTX-2011) is an IgG1 ICOS agonist monoclonal anti- equate renal, hepatic, and bone marrow function. The trial was a body known to activate and proliferate primed CD4 T effector standard 3+3 design with 5 fixed dose levels ranging from 80 mg cells in vitro, with established preclinical efficacy in multiple Q3wk to 1200 mg Q3wk given by IV infusion. In addition, there was tumor models. In the Phase 1/2 ICONIC trial (NCT02904226), one arm of 800 mg Q6wk. vopratelimab in patients with advanced solid tumors (Yap 2019) Results has shown to be safe and well tolerated as monotherapy and in 18 patients were enrolled in the trial (10 males, 8 females) with an combination with nivolumab. The ICONIC study showed no correl- average age of 66.3 yrs. Tumor types included ovarian (n=4), salivary ation between tumor reductions and ICOS and PD-L1 levels in gland, sarcoma, prostate and mesothelioma (n=2 each). The max- pre-treatment tumor samples by IHC. However, emergence of imum administered dose was 1200mg; MTD was not reached. There peripheral blood ICOS High (hi) CD4 T effector cells following were no deaths, no dose limiting toxicities. One treatment-related treatment with vopratelimab +/- nivolumab was associated with serious adverse event of pneumonitis occurred after the second dose tumor reductions and improved PFS and OS. In addition, ex vivo at 1200 mg Q3wk. Adverse events occurring in > 15% of patients Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 240 of 272 included fatigue, anemia, AST increased, dizziness, and tumor pain. 1 SA arm and 3 combination arms in which TPST-1120 is combined Only fatigue was noted as related in more than one patient (all with nivolumab, docetaxel or cetuximab. The RP2D of TPST-1120 to Grade 1 and 2). Grade 3 related AEs included increase alkaline phos- proceed to DEx will be determined by safety and biomarkers includ- phatase and pneumonitis. At time of data cutoff, median number of ing analysis of FAO/PPARα gene expression in the peripheral blood doses administered was 3 (range 1-11). Preliminary investigator and in tumor biopsies. The DEx arms will follow a 2-stage expansion assessed antitumor activity included: confirmed partial response (PR) design. This trial began accrual in May 2018 at U.S sites and is cur- in 1 patient with salivary gland carcinoma, unconfirmed PR in 1 pa- rently enrolling into the Monotherapy/Dose Escalation cohort. Expan- tient with ovarian cancer (both PD-L1+ by IHC) and best response of sion cohorts are projected to open in early 2019. The total sample stable disease in 6 patients. Systemic exposure of JTX-4014 increased size is up to 338 pts. dose proportionally; mean terminal half-life ranged from 11 to 17 Trial Registration days. JTX-4014 pharmacokinetics was comparable to other approved NCT03829436 anti-PD-1 mAbs. No anti-drug antibodies were observed. Ethics Approval Conclusions This study is being conducted in accordance with Good Clinical Prac- JTX-4014 is well-tolerated and appears to have similar qualities to tice and the Helsinki Declaration and has been approved by the known anti-PD-1 inhibitors in terms of pre-clinical and clinical charac- Western IRB/Copernicus Group, tracking # 20190182. teristics. Antitumor activity was observed in the difficult to treat population enrolled. Phase 2 testing JTX-4014 is planned. P441 Trial Registration ARTISTRY-2: a phase 1/2 study of subcutaneously administrated NCT03790488 ALKS 4230 as monotherapy and in combination with Ethics Approval pembrolizumab in patients with advanced solid tumors The study was approved by the relevant Institutions' Ethics Board 1 2 3 John Powderly, MD, CPI , Bradley Carthon, MD, PhD , Marc Ernstoff, MD , 4 5 6 Anthony Olszanski, MD, RPh , Stephen Liu, MD , Kelly Curtis, MD , 7 7 7 7 P440 Yangchun Du, PhD , Lei Sun, PhD , Emily Putiri, PhD , Yan Wang, PhD , 7 7 8 Phase 1/1b multicenter trial of TPST-1120, a peroxisome Heather Losey, PhD , Bruce Dezube, MD , Ulka Vaishampayan, MD 1 2 proliferator-activated receptor alpha (PPARα) antagonist as a Carolina BioOncology, Huntersville, NC, United States; Emory University, single agent (SA) or in combination in subjects with advanced Atlanta, GA, United States; Roswell Park Comprehensive Cancer Center, cancers Buffalo, NY, United States; Fox Chase Cancer Center, Phildelphia, PA, 1 2 2 5 John Powderly, MD, CPI , Saurin Chokshi, MD , Johanna Bendell, MD , United States; Georgetown University, Washington, DC, United States; 3 3 4 6 7 Leisha Emens, MD, PhD , Jason Luke, MD, FACP , Brian Francica , Chan Syneos Health, Phoenix, AZ, United States; Alkermes, Inc., Waltham, 4 4 4 8 Whiting, PhD , Thomas Dubensky, PhD , Ginna Laport, MD MA, United States; Barbara Ann Karmanos Cancer Institute, Detroit, MI, 1 2 Carolina BioOncology, Huntersville, NC, United States; Sarah Cannon United States Research Institute, Nashville, TN, United States; University of Pittsburgh, Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Pittsburgh, PA, United States; Tempest Therapeutics, South San Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P441 Francisco, CA, United States Correspondence: Thomas Dubensky (tdubensky@tempesttx.com); Background Ginna Laport (glaport@tempesttx.com) ALKS 4230 is an engineered fusion protein of circularly permuted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P440 interleukin-2 (IL-2) and IL-2 receptor α (IL-2Rα) designed to selectively bind the intermediate-affinity IL-2R for selective expansion of natural Background killer (NK) and CD8+ T cells (Figures 1 and 2). Compared with recom- Tumor cells initially rely on glucose consumption via aerobic glyco- binant human IL-2, ALKS 4230 exhibited enhanced pharmacokinetic lytic pathways. However, as tumor cells proliferate and metastasize in and selective pharmacodynamic properties in mice, resulting in im- an increasingly hypoxic tumor microenvironment (TME), tumors in- proved antitumor efficacy [1]. Intravenous dosing of ALKS 4230 is be- creasingly utilize fatty acid oxidation (FAO) as glucose stores are de- ing studied in the ARTISTRY-1 trial of patients with advanced solid pleted. FAO supports both tumor growth and suppressive immune tumors (NCT02799095), which has more than 50 patients enrolled cells in the TME, facilitating tumor progression. PPARα is a ligand- to date [2]. Here, we present a study investigating ALKS 4230 ad- activated nuclear transcription factor which regulates lipid metabol- ministered subcutaneously. Potential advantages of subcutaneous ism, FAO and inflammation. TPST-1120 is a first in class, oral, selective dosing over intravenous include: (i) lower peak serum drug con- PPARα antagonist that blocks transcription of PPARα target genes centrations with a prolonged exposure profile, which may result leading to a metabolic shift from FAO to glycolysis. Antagonism of in a milder safety profile and improved tolerability; (ii) lymphatic FAO in the TME leads to direct killing of tumor cells dependent on absorption, which may facilitate direct immunologic effects; and FAO and facilitates the cytotoxicity of effector cells. Preclinical studies (iii) a more convenient dosing schedule than daily inpatient intra- with various tumor models demonstrate efficacy of TPST-1120 as venous dosing. monotherapy and in combination with anti-PD1 antibodies and Methods chemotherapy. TPST-1120 has an IC50 of 0.04 nM with a >35 fold se- ARTISTRY-2 (NCT03861793) is a phase 1/2 study of ALKS 4230 ad- lectivity over other PPAR isoforms. ministered subcutaneously as monotherapy and in combination Methods with pembrolizumab in patients with advanced solid tumors. The We have initiated a phase 1/1b multicenter, open label trial to evalu- study will be conducted in 2 parts. In the first part (dose escal- ate TPST-1120 as a SA and in combination (combo) with other sys- ation; phase 1), multiple ascending doses of ALKS 4230 will be temic therapies including nivolumab, an anti-PD1 monoclonal administered subcutaneously every 7 days (q7d) or every 21 days antibody; docetaxel, a cytotoxic chemotherapeutic agent and cetuxi- (q21d) during a 6-week lead-in period. Injection site locations will mab, an anti-EGFR monoclonal antibody. The objectives are to 1) include the back of the arm, the thigh, or the abdomen. If the evaluate safety and tolerability of continuous dosing of TPST-1120 2) patient has tolerated ALKS 4230 monotherapy treatment, combin- identify a recommended phase 2 dose (RP2D) 3) evaluate efficacy, ation therapy with pembrolizumab (200 mg) administered as an and 4) evaluate PK/PD parameters. Eligibility criteria: 1) patients with intravenous infusion over 30 minutes q21d will be added to the advanced non-small cell lung, hepatocellular, renal cell, triple- ongoing ALKS 4230 regimen. In the second part (dose expansion; negative breast, urothelial, pancreatic, gastro-esophageal, castration- phase 2), ALKS 4230 will be administered subcutaneously at the resistant prostate, head and neck, or MSS colorectal cancer, or chol- selected recommended phase 2 dose (RP2D) and dosing schedule angiocarcinoma, or sarcoma; and 2) 1-5 prior therapies for metastatic from phase 1 in combination with pembrolizumab in 5 tumor- disease. This phase 1/1b adaptive design is composed of Dose Escal- specific cohorts of patients with non-small-cell lung cancer, small- ation (DEs) and Dose Expansion (DEx) cohorts. DEs consist of 4 arms, cell lung cancer, hepatocellular carcinoma, squamous cell Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 241 of 272 carcinoma of the head and neck, and squamous cell carcinoma Background of any tissue origin. Additional eligibility criteria for the study in- T-cell activation requires effective co-stimulation along with T-cell re- clude Eastern Cooperative Oncology Group performance status of ceptor (TCR) engagement. CD80 provides a well-characterized costi- 0 to 1 and adequate bone marrow, liver, and kidney function. mulatory signal by binding to CD28 on the surface of T cells. Outcomes include RP2D, safety, pharmacokinetics/pharmaco- Following activation, T cells upregulate CTLA4 on the cell surface dynamics, immunogenicity, and antitumor activity. Efficacy end- which binds to CD80 with higher affinity than CD28, disrupts effect- points include overall response rate, disease control rate, duration ive CD80-CD28 signaling, and inhibits T-cell activation. FPT155 is a of response, time to response, and progression-free survival and novel CD80 extracellular domain-Fc fusion protein that directly in- overall survival at 6- and 12-month milestones. duces T-cell activation and cytokine production by binding to CD28 and de-represses endogenous CD80-CD28 activity in the tumor Acknowledgements microenvironment by binding to CTLA4. FPT155 has potent efficacy The study is sponsored by Alkermes, Inc. Medical writing and editorial in syngeneic preclinical tumor models, including some that are not support was provided by Parexel and funded by Alkermes, Inc. responsive to agents targeting PD-1. FPT155 is not a superagonist as Trial Registration it requires separate, concurrent TCR engagement. ClinicalTrials.gov NCT03861793 Methods FPT155 is being investigated in a multi-center, open-label, first- References in-human phase 1 trial. The dose escalation portion of the trial 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of is currently enrolling patients with advanced solid tumors that ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic have progressed after treatment with available therapies. A tumor models alone and in combination with immune checkpoint minimum anticipated biological effect level (MABEL) based ap- inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. proach was used to select the initial dose in humans. Patients 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, receive a fixed dose of FPT155 every three weeks with single- McDermott DF, et al. Intravenous administration of ALKS 4230 as patient accelerated titration cohorts through the first four dose monotherapy and in combination with pembrolizumab in a phase I levels of 0.07, 0.21, 0.7 and 2.1 mg and a standard 3+3 dose- study of patients with advanced solid tumors. J Clin Oncol. escalation design for the subsequent 7, 21, 42, and 70mg dose 2019:37(Suppl). Abstract TPS2649. levels. The primary objective of the phase 1a portion of the trial Ethics Approval is to determine the recommended dose and evaluate the safety This study was approved by Ethics and Institutional Review Boards (IRBs) at and tolerability of FPT155. all study sites; IRB reference numbers 20182543 (Western IRB), 00006731 Results (Roswell Park Comprehensive Cancer Center), STUDY00000056 As of June 17, 2019, 7 patients have been treated on study with (Georgetown University, MedStar Health Research Institute). FPT155 doses ranging from 0.07-7mg; median age was 58 years, 57% had ECOG PS 1 and median number of prior therapies was 4 (range: 2-8). To date, no dose-limiting toxicities or ≥Grade 3 treatment- emergent adverse events (TEAEs) from causes other than disease progression have been reported. There have been no serious adverse events or ≥grade 3 TEAEs attributed to FPT155 and the only TEAE at- tributed to FPT155 in more than one patient has been fatigue (Gr1, Gr2; 1 pt each). 2/7 patients continue on treatment. Conclusions FPT155 as monotherapy has been well tolerated to date. Enrollment Fig. 1 (abstract P441). ALKS 4230 is a fusion of IL-2 and IL-2Rα of accelerated titration cohorts is complete with dose-escalation in 3+3 cohorts ongoing. Trial Registration ACTRN12618001955202 Ethics Approval The study was approved by IRBs at all participating study sites. P443 Expansion cohorts of non-small cell lung cancer (NSCLC) and castration resistant prostate cancer (CRPC) in COSMIC-021, a phase 1b study of cabozantinib plus atezolizumab 1 1 2 Nick Salgia, PhD , Sumanta Pal, MD , Santiago Ponce Aix, MD , Yohan 3 4 5 6 Loriot , Robert Dreicer , Ulka Vaishampayan, MD , Toni Choueiri , Patrick Fig. 2 (abstract P441). Cell activation by IL-2 and ALKS 4230 7 8 8 9 10 Schöffski , Giri Ramsingh , Amy Liu , Farah Lim , Joel Neal, MD, PhD , Neeraj Agarwal, MD 1 2 City of Hope, Duarte, CA, United States; University Hospital 12 de Octubre, Madrid, Spain; Institut de Cancérologie Gustave Roussy, Villejui, P442 France; University of Virginia School of Medicin, Charlottesville, VA, A phase 1 study of FPT155, a first-in-class CD80 extracellular United States; Karmanos Cancer Institute, Detroit, MI, United States; domain-Fc fusion protein, in patients with advanced solid tumors 2 3 4 5 6 7 Dana-Farber Cancer Institute, Boston, MA, United States; Leuven Jermaine Coward , Hui Gan, MBBS PhD , James Kuo , Michael Millward , 6 7 7 7 8 Cancer Institute, Leuven, France; Exelixis, Alameda, CA, United States; Gary Richardson , Wei Deng , Siddhartha Mitra , Maike Schmidt , Hong 7 8 1 9 10 Barts Cancer Institute, London, United Kingdom; Stanford University Xiang, PhD , Lisa Horvath , Amy Prawira, MD 1 2 11 Medical Center, Standford, CA, United States; Huntsman Cancer St. Vincent’s Hospital Sydney, Darlinghurst, Australia; Icon Cancer Institute, Salt Lake City, UT, United States Centre, Brisbane, Australia; Olivia Newton-John Cancer Center, Correspondence: Nick Salgia (jennifer.humbert@fishawack.com) Melbourne, Victoria, Australia; Scientia Clinical Research, Randwick, 5 6 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P443 Australia; Linear Clinical Research, Nedlands, Australia; Cabrini Hospital, Malvern, Australia; Five Prime Therapeutics, Inc, South San Francisco, Background CA, United States; Chris O’Brien Lifehouse, Camperdown, Australia Cabozantinib inhibits tyrosine kinases involved in tumor growth, Correspondence: Amy Prawira (amy.prawira@svha.org.au) angiogenesis, and immune regulation, including MET, VEGFR, RET, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P442 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 242 of 272 ROS1, and TAM family kinases (TYRO3, AXL, MER). Encouraged by with minimal toxicities at the dose levels studied (1×108 HPVSTs/m2) preclinical and clinical studies that suggested that cabozantinib pro- so far. However, most patients remained with disease after infusion. motes an immune-permissive environment, the safety and efficacy of A great challenge for HPVST therapy is to generate more specific and cabozantinib or cabozantinib in combination with atezolizumab are potent HPVSTs as ~30% of our HPVST products failed the potency being evaluated in the COSMIC 021 phase 1b study (NCT03170960) criterion, evaluated by γ-IFN ELISpot assay. The aim of this work was in solid tumors including NSCLC and CRPC. Cabozantinib has demon- to increase the potency and success rate of HPVST manufacturing. strated clinical activity as monotherapy in advanced NSCLC and in Methods previously treated CRPC [1,2]. Here we provide updated trial details The current manufacturing strategy uses peripheral blood mono- for expansion cohorts of NSCLC and CRPC patients. nuclear cells (PBMCs) as starting material for the enrichment and ex- Methods pansion of HPVSTs, in the presence of dendritic cells and cytokines. The dose-escalation stage of this global, open-label trial is com- Either low frequency or anergy of HPVSTs, even in HPV-exposed do- pleted; in the expansion stage, 20 combination cohorts are being en- nors, impede growth, and manufacturing failure is largely attributed rolled at the recommended dose of cabozantinib 40 mg QD PO + to non-specific T cell outgrowth. To overcome this problem, we eval- atezolizumab 1200 mg Q3W IV. uated CD45RA depletion of PBMCs to remove the bulk of non- NSCLC cohorts include patients with: (1) nonsquamous (nsq)NSCLC specific cells (naïve T cells and natural killer (NK) cells). The CD45RA with prior immune checkpoint inhibitor (ICI) therapy (anti–PD-1 or fraction also contains B-cells and T regulatory cells that may inhibit anti–PD-L1); (2) nsqNSCLC without prior systemic anticancer therapy specific outgrowth. We also evaluated the use of an HLA-negative for metastatic disease; (3) EGFR-mutant nsqNSCLC with prior EGFR- universal lymphoblastoid cell line (uLCL), developed in our center, as targeting therapy. An additional exploratory cohort will assess cabo- a co-stimulatory cell line to rapidly expand the cells while maintain- zantinib monotherapy (60 mg) in nsqNSCLC patients with prior ICI ing HPV specificity. therapy. Results CRPC cohorts include patients with: (1) metastatic CRPC adenocarcin- HPVSTs manufactured using CD45RA negative PBMC populations as oma with measurable disease and prior enzalutamide and/or abira- starting material consistently displayed overarchingly higher specifi- terone therapy; (2) high-risk (measurable visceral metastasis or city than HPVSTs manufactured from PBMCs. Interestingly, uLCLs not prostate-specific antigen doubling time of only supported exponential growth of HPVSTs, but increased their The study allows an initial enrollment of 30 patients in each cohort HPV specificity, opening the possibility of producing sufficient with potential for expansion per recommendation by the Study Over- HPVSTs for higher dose levels. We reported successful production sight Committee. Based on preliminary efficacy per RECIST v1.1 and using PBMC from HPV-exposed healthy donors and cancer patients, safety, the original cohorts of nsqNSCLC with prior ICI therapy and and greatly improved HPVST specificity in all. metastatic CRPC adenocarcinoma with measurable disease and prior Conclusions enzalutamide and/or abiraterone therapy are being expanded to 80 These changes will be incorporated in our HPVST manufacturing patients each. protocol with the goal of improving the anti-tumor activity of our The primary endpoint of the expansion stage is the objective re- product. sponse rate for each cohort. Exploratory objectives include correl- ation of tumor and plasma biomarkers and immune cell profiles with References clinical outcome. 1. Centers for Disease Control and Prevention https://www.cdc.gov/cancer/ Trial Registration hpv/index.htm NCT03170960 2. Forastiere AA, Ang KK, Brizel D, et al. Head and neck cancers. J Natl Compr Canc Netw. 2008; 6:646–695. References 3. 11. Greer BE, Koh WJ, Abu-Rustum N, et al. Cervical cancer. J Natl Compr 1. Smith DC, Smith MR, Sweeney C, Elfiky AA, Logothetis C, Corn PG, Canc Netw. 2008; 6:14–36 Vogelzang NJ, Small EJ, Harzstark AL, Gordon MS, Vaishampayan UN. J Ethics Approval Clin Oncol. 2013; 31:412-419. This study was approved by Baylor College of Medicine Institutional Review 2. Drilon A, Rekhtman N, Arcila M, Wang L, Ni A, Albano M, Van Board; approval number H7634, H7666 and HESTIA and HESTIA IND. Voorthuysen M, Somwar R, Smith RS, Montecalvo J, Plodkowski A. Lancet Oncol. 2016; 17:1653-60. P445 Preliminary results of a Phase 1 trial with a personalized P444 neoantigen vaccine (ADXS-NEO) in advanced and refractory cancer The quest for highly potent human papillomavirus-specific T patients 1 2 3 Lymphocytes for Adoptive Immunotherapy of HPV-associated Frank Tsai, MD , Jonathan Goldman, MD , Marc Matrana , Sumitra 4 4 4 4 malignancies Sheeri , John Heyburn , Megan Parsi , Andres Gutierrez, MD PhD , Joel 1 2 2 2 2 Pei Yun Teo, PhD , Sandhya Sharma, BSc , Alex Salyer , Dimitrios Hecht , Joel Hecht 2 2 2 3 1 Wagner , Benjamin Shin , Sachin Thakar , Li-Chun Huang , Shian Jiun Honor Health Virginia Piper Cancer Care, Scottsdale, AZ, United States; 3 2 2 2 Shih , Carlos Ramos , Cliona Rooney, PhD UCLA Jonsson Comprehensive Cancer Center, Paramus, NJ, United 1 3 4 Tessa Therapeutics/ Baylor College of Medicine, Houston, TX, United States; Ochsner Cancer Center, New Orleans, LA, United States; Advaxis 2 3 States; Baylor College of Medicine, Houston, TX, United States; Tessa Inc, Princeton, NJ, United States Therapeutics, Singapore, Singapore Correspondence: Joel Hecht (JRHecht@mednet.ucla.edu) Correspondence: Cliona Rooney (crooney@bcm.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P445 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P444 Background Background ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based im- The human papillomavirus is linked to 42,700 new cases of cancers munotherapy. This vaccine is a bioengineered Lm vector that se- each year [1]. While many HPV-associated cancers can be eradicated cretes an antigen-adjuvant fusion protein consisting of up to 40 by multimodal therapies, recurrent diseases have dismal prognosis unique (personal) neoantigens and a truncated fragment of listerioly- [2,3]. HPV-positive tumors express viral antigens (E6 and E7) that are sin O (tLLO), which has adjuvant properties. Preliminary clinical and recognized by HPV-specific T cells (HPVST). We are evaluating the immunogenicity results from two dose-levels of ADXS-NEO mono- adoptive transfer of ex vivo expanded autologous HPVSTs for the therapy evaluated in the ongoing Phase 1 trial are herein reported. treatment of HPV-positive cancers in a phase I clinical trial (HESTIA). Methods To date, 12 patients have been treated and promising outcomes ADXS-NEO-02 is a phase 1 dose-escalation study of ADXS-NEO mono- have been attained- 1 complete response and 1 partial response, therapy in subjects with advanced and refractory metastatic Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 243 of 272 microsatellite stable-colon cancer (MSS-CRC), metastatic squamous was also examined. Furthermore, the possibility of combination treatment histology head and neck cancer, and metastatic non-small cell lung of TAS-116 and anti-PD-1 mAb were investigated in animal models cancer (NSCLC). Manufacturing of ADXS-NEO starts with whole ex- Results ome sequencing of each pt-matched normal and tumor samples to TAS-116 significantly reduced Treg cells, particularly effector Treg cells in detect genetic alterations in the coding regions of the genome both peripheral blood and the TME, resulting in augmentation of tumor followed by its production under GMP specifications. ADXS-NEO is in- antigen-specific CD8+ T cells. STAT5, one of the HSP90 client proteins fused intravenously every 3 weeks until disease progression or limit- that is important for Treg cell development, maintenance and function ing toxicity. Main endpoints include safety, tolerability, preliminary was degraded by TAS-116, thereby reducing FoxP3 expression in effector efficacy and immune-correlative data. Treg cells. TAS-116 augmented tumor antigen-specific CD8+ T cells in Results animal models with reduction of Treg cells in the TME. Additionally, com- The turnaround time for manufacturing ADXS-NEO has consistently bination treatment with PD-1 blockade exhibited a far stronger antitumor been 7-8 weeks from biopsy to first dose. Two pts treated at 1X109 effect than either treatment alone. Moreover, in a phase I trial, the com- CFU (dose level 1) experienced dose limiting toxicities (i.e., Gr 3 hyp- bination of an anti-PD-1 mAb and TAS-116 exhibited a notable clinical ef- oxia ± Gr 3 hypotension) within 4 hours of completing the infusion ficacy in patients with microsatellite-stable (MSS) colorectal cancer of the second dose. These acute adverse events were manageable accompanied by effector Treg cell reduction in the TME. and reversible with tocilizumab and/or steroids. A de-escalated dose Conclusions of 1X108 CFU, has been found to be safe, tolerable and immuno- We propose a novel concept to control eTreg cells by targeting a genic in a cohort of 3 pts. ADXS-NEO at both doses induced: 1) acti- Treg cell-critical signaling pathway and the potential as a combin- vation and proliferation of CD4+ / CD8+ T cells; 2) neoantigen- ation therapy with PD-1 blockade. specific T cell responses -including hotspot mutations- after 1 week Trial Registration of the initial priming dose in pooled ELISPot analysis and 3) T cell re- UMIN000032801 sponses to neoantigens found in the pts’ tumor, but not included in Ethics Approval the construct (i.e., antigen spreading). Deconvolution ELISPot data This study was approved by the institutional review boards of the Na- from the first MSS-CRC pts. analyzed, showed T cell responses to tional Cancer Center and was conducted in accordance with ethical 90% of the targets in the ADXS-NEO construct. Two out of 4 initial guidelines, including the Declaration of Helsinki.All mouse experiments pts treated had stable disease. were approved by the Animals Committee for Animal Experimentation Conclusions of the National Cancer Center, Japan, and Taiho Pharmaceutical Co. Ltd. A safe and tolerable dose of ADXS-NEO monotherapy has been established (1X108 CFU) which elicited fast and broad antitumor im- P447 munity, including T cell responses to neoantigens and antigen ALKS 4230, an engineered IL-2 fusion protein, in monotherapy spreading. Enrollment in a combination therapy arm with pembroli- dose-escalation and combination therapy with pembrolizumab in zumab is due to start in 4Q2019. patients with solid tumors: ARTISTRY-1 trial Trial Registration 1 2 Ulka Vaishampayan, MD , Jameel Muzaffar, MD , Vamsidhar Velcheti, MD, NCT03265080 3 4 5 FACP , Christopher Hoimes, DO , Lucy Gilbert, MD , David McDermott, Ethics Approval 6 7 8 9 MD , Anna Spreafico, MD PhD , Quincy Chu, MD , Kelly Curtis, MD , This clinical tria has been performed in accordance with the Declar- 10 10 10 Yangchun Du, PhD , Harald Mackenzie, MB , Lei Sun, PhD , Emily ation of Helsinki and has been approved by appropriate ethics com- 10 10 10 Putiri, PhD , Heather Losey, PhD , Bruce Dezube, MD , Marc Ernstoff, mittee at UCLA LA, Ochsner Cancer Center LA and Honor Health MD Virginia G Piper Cancer Care AX. Barbara Ann Karmanos Cancer Institute, Detroit, MI, United States; 2 3 Moffitt Cancer Center, Tampa, FL, United States; Perlmutter Cancer P446 Center, NYU Langone Health, New York, NY, United States; UH A novel regulatory T Cell-Targeted Immunotherapy by targeting Cleveland Medical Center, Cleveland, OH, United States; Cedars Cancer their crucial signal by HSP90 inhibitors Center, Montreal, QC, Canada; Beth Israel Deaconess Medical Center, Ayaka Tsuge, MD, Yosuke Togashi, MD, PhD, Kohei Shitara, MD, Hiroyoshi Milton, MA, United States; Princess Margaret Cancer Centre, Toronto, Nishikawa, MD, PhD ON, Canada; Cross Cancer Institute, University of Alberta/Alberta Health National Cancer Center, Kashiwa, Japan Services, Edmonton, AB, Canada; Syneos Health, Phoenix, AZ, United 10 11 Correspondence: Hiroyoshi Nishikawa (hnishika@east.ncc.go.jp) States; Alkermes, Inc., Waltham, MA, United States; Roswell Park Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P446 Comprehensive Cancer Center, Buffalo, NY, United States Correspondence: Bruce Dezube (bruce.dezube@alkermes.com) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P447 Cancer immunotherapy, particularly immune checkpoint inhibitors opened a new era of cancer therapy. Yet, the clinical efficacy is limited Background due to the complexed immune suppressive mechanisms in the tumor ALKS 4230 is an engineered fusion of IL-2 and IL-2Rα designed to se- microenvironment (TME). Regulatory T (Treg) cells, an immune suppres- lectively expand NK and CD8+ T cells (Figures 1 and 2). In preclinical sive subset of CD4+ T cells, are abundant in tumor tissues and play a key studies, ALKS 4230 exhibited enhanced pharmacokinetic and select- role as an immune suppressive mechanism in the TME via inhibiting ef- ive pharmacodynamic properties with improved antitumor efficacy fective antitumor immunity. While various Treg cell-targeted reagents is relative to IL-2 [1]. under development, none of them have not been translated into the Methods clinic due to the difficulty of specific Treg cell depletion in the TME. The ARTISTRY-1 (NCT02799095) is a phase 1/2 study investigating major obstacle to develop effective Treg cell-targeted reagents was the ALKS 4230 as monotherapy and in combination with pembrolizu- lack of molecules specifically expressed by Treg cells in the TME. We mab in adults with advanced solid tumors [2]. For monotherapy therefore focused on the specific signal(s) used in Treg cells in the TME. dose escalation, ALKS 4230 is administered intravenously over 30 Methods minutes once daily for 5 days every 14 or 21 days. For combin- We focused on HSP90 inhibitor, TAS-116 as a Treg cell regulator, especially ation therapy, the same regimen of ALKS 4230 is administered terminally-differentiated effector Treg cells. Peripheral blood mononuclear with pembrolizumab every 21 days in cohorts based on tumor cells (PBMCs) were treated with TAS-116, and the changes in T cell popula- type, prior anti-PD-1 therapy, and rollover from monotherapy. tions including Treg cells were analyzed. In addition, we explored the Outcomes include the monotherapy recommended phase 2 dose mechanism(s) of Treg cell reduction using PBMCs and FoxP3+ T cell lines. (RP2D), safety, pharmacodynamics, and antitumor activity (RECIST The effect of TAS-116 on tumor antigen (NY-ESO-1)-specific CD8+ T cells 1.1). Results of the completely enrolled cohorts of dose-escalation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 244 of 272 phase and of combination therapy in anti-PD-1-unapproved tu- mors as of June 21, 2019, are presented. Results For dose escalation, 36 patients received ALKS 4230 monotherapy ≤6 μg/kg/d. Maximum tolerated dose has not been reached. Most fre- quent adverse events (AEs), regardless of relationship, were pyrexia (75%) and chills (72%); the majority were grades 1 or 2. Grade ≥3 AEs related to ALKS 4230 occurred in 11 patients (31%) and were mainly transient leukopenia. One death from aspiration pneumonia was considered unrelated to ALKS 4230 by the investigator. ALKS 4230 induced dose-dependent increases in circulating NK and CD8+ T cells with minimal, non-dose-dependent effects on regulatory T Fig. 2 (abstract P447). ALKS 4230 structure and activity cells (Tregs). At 3 and 6 μg/kg/d, 8 of 14 patients with evaluable scans had stable disease. One patient with heavily pretreated pancre- atic adenocarcinoma had prolonged stable disease with 6+ months of monotherapy; CA19-9 decreased from 2571 U/mL (pretherapy) to P448 673 U/mL (nadir). Data from 20 patients enrolled in the combination Phase I study of Veliparib and Nivolumab in adults with refractory therapy cohort of PD-1-unapproved tumors indicate no new toxic- advanced solid tumor and lymphoma ities; 7 of 11 patients with evaluable scans had stable disease or bet- Young Kwang Chae, MD, Pedro Viveiros, MD, Sheetal Kircher, Valerie ter. One patient (ovarian cancer) had confirmed partial response; CA- Nelson, Aparna Kalyan, Devalingam Mahalingam 125 normalized from a peak of 282 to 24.5 U/mL after 2 months of Northwestern University, Chicago, IL, United States therapy. Correspondence: Young Kwang Chae (ychae@nm.org) Conclusions Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P448 ALKS 4230 is a promising agent with acceptable tolerability and pre- liminary clinical benefit. It selectively expanded CD8+ T cells and NK Background cells with minimal Treg expansion. The intravenous monotherapy Tumors with high mutation burden often respond to immunother- RP2D was established as 6 μg/kg/d. Safety and pharmacodynamic apy. Veliparib, a Poly (ADP-ribose) polymerase (PARP) inhibitor carries data enabled selection of the 3 μg/kg dose for initial evaluation in anti-neoplastic activity by accumulating DNA damage, possibly en- combination with pembrolizumab. hancing the effect of checkpoint inhibitors. Here we report safety and preliminary efficacy of veliparib and nivolumab combination Acknowledgements from the dose escalation phase of [NCT03061188] clinical trial. The authors would like to thank all the patients who are participating in this Methods study. The study is sponsored by Alkermes, Inc. Medical writing and editorial We conducted a phase I study of veliparib in combination with nivo- support was provided by Parexel and funded by Alkermes, Inc. lumab in chemo-refractory stage IV/ unresectable solid cancer pa- Trial Registration tients. Nivolumab 240mg IV day 1 and 15 q28 days for 4 cycles; ClinicalTrials.gov NCT02799095 480mg IV q28 days Cycle 5 onwards, combined with veliparib start- ing 300mg PO bid 3+3 dose escalation until maximum tolerated References dose (MTD) was established. Treatment continued until disease pro- 1. Losey HC, Lopes JE, Dean RL, Huff MR, Moroso RA, Alvarez JC. Efficacy of gression or limiting toxicity. Primary objective was establishing MTD ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic for veliparib, defined as the highest dose causing dose-limiting tox- tumor models alone and in combination with immune checkpoint icity (DLT) in < 2 of 6 patients. Secondary objective was to assess inhibitors. Cancer Res. 2017;77(13 Suppl). Abstract 591. safety, tolerability and early efficacy of combination. 2. Vaishampayan UN, Fishman MN, Cho DC, Hoimes CJ, Velcheti V, Results McDermott DF, et al. Intravenous administration of ALKS 4230 as Nine patients with adequate end-organ function and performance monotherapy and in combination with pembrolizumab in a phase I status were enrolled. Tumor types included colon cancer (n = 3) and study of patients with advanced solid tumors. J Clin Oncol. pancreatic cancer (n = 2). Four patients (44%) had BRCA-related som- 2019:37(Suppl). Abstract TPS2649. atic mutations (BRCA1, BRCA2, ATM and BRIP1). Most common ad- Ethics Approval verse events categorized as possibly or probably related to treatment This study was approved by Ethics and Institutional Review Boards (IRBs) at were fatigue (n = 6, 67%), anemia (n = 5, 56%), nausea (n = 4, 44%) all study sites; IRB reference numbers 16-229 (Dana-Farber Cancer and diarrhea (n = 3, 33%). Grade 3 and 4 events were anemia (n = 3, Institute), MOD00003422/PH285316 (Roswell Park Comprehensive Cancer 33%), fatigue and alkaline phosphatase elevations (n = 2, 22% each), Center), 20160175 (Western IRB), i15-01394_MOD23 (New York University AST, ALT elevations, low platelet count, hypokalemia and hyperten- School of Medicine), STUDY20190090 (Cleveland Clinic), 0000097 sion (n = 1, 11% each). One patient experienced DLT, grade 3 fatigue, (ADVARRA). at dose level 2 (400 mg BiD) and the MTD was established as 400mg bid. Disease control rate after 24 week follow up was 11%. Five pa- tients presented disease progression (56%). One patient withdrew consent at Cycle 3 and another developed limiting fatigue at Cycle 3, both had stable disease (SD). A patient succumbed due to complica- tions of disease before first assessment. One patient with refractory metastatic pancreatic carcinoma harboring a BRIP1 L680fs*9 muta- tion had SD after a 35-week follow-up. Median progression-free sur- vival and overall survival were 9 and 25 weeks, respectively. Conclusions The recommend phase 2 dose of Veliparib is 400mg bid when com- bined with Nivolumab. The side effect profile is on par with the ones Fig. 1 (abstract P447). ALKS 4230 structure and activity previously described for veliparib and nivolumab in monotherapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 245 of 272 We are expanding the cohort to now include tumors harboring DNA Effects on tumor infiltrating immune cells and molecular signatures repair defects. in correlative biomarker analyses provide insights to ALX148’s mech- Trial Registration anism as a myeloid checkpoint inhibitor. NCT03061188 Ethics Approval Acknowledgements The study was approved by Northwestern University Ethics Board. We would like to thank all of the participating patients and their families as STU00204250. well as site research staff. Trial Registration ClinicalTrials.gov identifier NCT03013218. P449 Pharmacodynamic biomarker characterization of ALX148, a CD47 References blocker, in combination with established anticancer antibodies in 1. Kauder et al., ALX148 blocks CD47 and enhances innate and adaptive patients with advanced malignancy antitumor immunity with a favorable safety profile. PLOS ONE. 2018 1 2 3 Hong Wan, PhD , Laura Chow, MD , Justin Gainor, MD , Nehal Lakhani, 13(8): e0201832 4 5 6 MD, PhD , Hyun Chung, MD, PhD , Keun-Wook Lee, MD , Jeeyun Lee, 2. Lahkani et al., A phase 1 study of ALX148, a CD47 blocker, alone and in 7 8 9 MD, PhD , Patricia LoRusso, DO , Yung-Jue Bang, MD PhD , Stephen combination with established anticancer antibodies in patients with 10 11 1 12 Hodi , Wells Messersmith, MD , Philip Fanning, PhD , Pierre Squifflet , advanced malignancy and non-Hodgkin lymphoma. Journal of Clinical 1 1 1 1 Feng Jin , Tracy Kuo , Sangeetha Bollini , Jaume Pons, PhD , Sophia Oncology 2018 36:15_suppl, 3068-3068 Randolph, MD, PhD 3. Lahkani et al., A phase 1 study of ALX148: CD47 blockade in combination 1 2 ALX Oncology, Burlingame, CA, United States; University of with anticancer antibodies to bridge innate and adaptive immune Washington, Seattle, WA, United States; MGH Cancer Center, Boston, responses for advanced malignancy. Journal for ImmunoTherapy of MA, United States; START Midwest, Grand Rapids, MI, United States; Cancer 2018 6 (Suppl 1):114. Abstract 335. 5 6 Yosei Cancer Center, Seoul, Korea, Republic of; Seoul University 4. Chow et al., A phase I study of ALX148, a CD47 blocker, in combination Bundang Hospital, Seongnam, Korea, Republic of; Samsung Medical with established anticancer antibodies in patients with advanced Center, Seoul, Korea; Yale Cancer Center, New Haven, CT, United States; malignancy. Journal of Clinical Oncology 2019 37:15_suppl, 2514-2514. 9 10 Seoul National University Hospital, Seoul, Korea; Dana Farber Cancer Ethics Approval Center, Boston, MA, United States; University of Colorado Cancer The study was approved by institutional review boards or independent Center, Aurora, CO, United States; International Drug Development ethics committees of participating institutions (approval numbers on file Institute, Brussels, Belgium at ALX Oncology). Correspondence: Hong Wan (Hong@alxoncology.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P449 P450 Background A phase I/IIa, open-label, dose-escalation and expansion study to CD47 is a myeloid checkpoint upregulated by tumor cells to investigate the safety, tolerability, pharmacokinetics and evade immune destruction. ALX148 (A) is a fusion protein com- pharmacodynamics of TJ107 in Chinese patients with advanced prised of a high affinity CD47 blocker linked to an inactive hu- solid tumors 1 1 1 1 1 1 man immunoglobulin Fc region [1]. We have previously shown in Jin Li , Ye Guo , Wei Peng , Junli Xue , Wei Zhao , Xiaoxiao Ge , Liqiong 1 1 1 1 2 the first-in-human clinical trial, that ALX148 is well tolerated in Xue , Wenbo Tang , Li Zhou , Min Zhang , Bingshi Guo , Liping Wang, 2 2 2 2 combination with trastuzumab (T) or pembrolizumab (P) with no MD , Jiyuan Guo , Feifei Cui, PhD , Haiyun Suo 1 2 maximum tolerated dose (MTD) identified [2, 3]. Antitumor activ- Shanghai East Hospital, Shanghai, China; I-Mab Biopharma, Shanghai, ity of ALX148 in combination with T or P was observed in pa- China tients with advanced gastric/ gastroesophageal junction (G/GEJ), Correspondence: Jin Li (lijin@csco.org.cn) head and neck squamous cell carcinoma (HNSCC) and non-small Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P450 cell lung cancer (NSCLC) [4]. The objective of this exploratory analysis was to characterize tumor infiltrating immune cells and Background molecular signatures from tumor biopsies and peripheral blood TJ107, an immuno-oncology agent also known as Hyleukin, is a samples obtained from this trial. T cell amplifier comprising a homodimer of engineered human Methods interleukin-7 (IL-7) fused with Genexine’s proprietary long-acting Patients with HER2-positive malignancy (including G/GEJ cancers pro- platform hybrid Fc. IL-7 is a critical homeostatic factor for T gressed on T + fluoropyrimidine and platinum-based therapy) re- cells, acting on T cells to increase their number, diversity and ceived A+T. Patients with advanced malignancy including NSCLC functionality. TJ107 could play a pivotal role in reconstitution [checkpoint inhibitor (CPI)-resistant/refractory or PD-L1 tumor propor- and reinvigoration of T cell immunity in cancer patients, provid- tion score (TPS) ing unique opportunities for immuno-oncology combination Results strategies. The aim of this study (NCT04001075) is to determine Eighty-two patients received A+T (n=30) or A+P (n=52) as of the safety, tolerability and PKPD profile of TJ107 in Chinese can- April 18, 2019. In dose expansion cohorts (N=60), anticancer ac- cer patients. tivity was observed in response-evaluable patients [G/GEJ (n= Methods 18) 4PR, 5SD; HNSCC (n=19) 3PR, 6SD and NSCLC (n=18) 3SD]. This ongoing study is to evaluate the safety, tolerability, PK profile, Near complete CD47 TO was maintained throughout the dosing and anti-tumor activity of TJ107 in patients with advanced solid tu- interval. No dose-dependent changes were apparent in mors who failed standard therapy. Patients receive TJ107 every 4 peripheral lymphocyte populations. Preliminary results from weeks by intramuscular (IM) injection. Dose escalation is aided by a paired biopsies (n=15) demonstrated increased tumor-associated 3+3 scheme from 240μg/kg to 1200μg/kg. A dose expansion cohort macrophages and lymphocytes in both intra-tumoral and peri- is being planned after the RP2D is determined. Safety is assessed by tumoral regions after treatment with ALX148 combinations. monitoring AEs and the associated grades per NCI CTCAE v5.0. Gene expression signatures of tumor inflammation and immune Tumor response will be assessed per RECIST v1.1. Samples will be cell subsets are being investigated. Results will be updated at collected for PK, PD, ADA, immunophenotyping and TCR repertoire presentation. analysis. Conclusions Results ALX148 demonstrates excellent tolerability with objective responses Three patients with colorectal cancer were enrolled in the first observed in patients with advanced G/GEJ cancer and HNSCC that cohort (240μg/kg).TJ107 was well tolerated and no DLTs were re- have progressed on prior systemic and HER2-targeted therapies. ported during the first cycle at this dose level. The preliminary Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 246 of 272 PK results shows that TJ107 was rapidly absorbed and reached limiting toxicities (injection-site reaction [ISR], lymph node pain; serum peak concentration around 24 hours post-dose. TJ107 was ISR). Among all treated patients, the most common adverse slowly cleared from the body and remained detectable in serum events (AEs) were ISR (89.5%), fatigue (39.5%), nausea (28.9%), until Day 14 post-dose. A substantial increase in absolute constipation, and decreased appetite (23.7% each). Nine (23.7%) lymphocyte count (ALC) from baseline was observed and peaked patients had MEDI5083-related ≥G3 events, most commonly ISR. around 3 to 4 weeks post first dose. FACS analysis revealed in- Six (15.8%) patients discontinued due to a MEDI5083-related AE. creases in CD3+, CD4+ and CD8+ T cells. The numeric increase in There were 4 (10.5%) deaths due to AEs (sequential cohort, T cells is consistent with increased Ki67 expression on Day 8 post MEDI5083 5mg, n=2 and MEDI5083 7.5mg, n=1; concurrent co- first dose. There were no notable changes in B cells, monocytes, hort, MEDI5083 4mg, n=1), 3 unrelated to treatment and 1 pos- NK cells, neutrophils, nor Tregs, as expected. sibly related to MEDI5083. The maximum tolerated dose for Conclusions MEDI5083 was 5mg. In the response evaluable population (N=36), Preliminary results from this trial suggest that TJ107 activated a PR was observed (head and neck squamous cell carcinoma; se- IL-7 pathway and expanded T cells in cancer patients in a simi- quential cohort, MEDI5083 3mg; time to response 5.7 months) lar way to data previously reported in healthy subjects. TJ107 and 11 (30.6%) patients had SD (sequential cohort, n=7; concur- exhibits a promising safety and tolerability profile in cancer pa- rent cohort, n=4). Six patients had SD ≥24 weeks. The ORR (95% tients under current dose. These findings support further clinical CI) was 2.8% (0.1–14.5%). MEDI5083 showed dose-dependent investigation. pharmacological activity in the mobilization of peripheral blood B Trial Registration cells, and induced measurable increases in activated proliferative Investigate the Safety, Tolerability, Pharmacokinetics and Pharmaco- Ki-67+ CD8+ T cells in peripheral blood. dynamics of TJ107 in Chinese Patients With Advanced Solid Tumors. Conclusions ClinicalTrials.gov Identifier: NCT04001075 Subcutaneous administration of MEDI5083 caused high rates of injec- Ethics Approval tion site reactions. The toxicity profile does not support further devel- The study was approved by the Ethics Committee Shanghai East Hos- opment of the subcutaneous formulation of this drug. pital's, approval number 2018 (058). Trial Registration ClinicalTrials.gov NCT03089645 Ethics Approval P451 This study was approved by the Institutional Review Board/Independ- First-in-human study of CD40 agonist MEDI5083 in advanced solid ent Ethics Committee at each investigational site participating in the tumors with durvalumab administered sequentially or concurrently study 1 2 3 Ben Tran, MBBS FRACP , Mark Voskoboynik , Johanna Bendell, MD , 4 5 Martin Gutierrez, MD , Charlotte Lemech, MBBS BSc(med) MD(res) , 6 6 7 Daphne Day , Sophia Frentzas , Ignacio Garrido-Laguna , Chris P452 8 8 8 8 DelNagro , Fujun Wang , Charles Ferte, MD, PhD , Mayukh Das , SPEARHEAD-1 trial design: A phase 2, single arm, open-label Benedito Carneiro, MD clinical trial of ADP-A2M4 SPEAR T-cells in patients with advanced 1 2 Peter MacCallum Cancer Center, Melbourne, Australia; Nucleus synovial sarcoma or myxoid/round cell liposarcoma 3 1 2 3 4 Network, Melbourne, Australia; Sarah Cannon Research Institute, Dejka Araujo, MD , Jean-Yves Blay , Sandra Strauss , Claudia Valverde , 4 5 5 5 Nashville, TN, United States; Hackensack University Medical Center, Erin Van Winkle , Malini Iyengar, PhD , Rafael Amado, MD 5 1 2 Hackensack, NJ, United States; Scientia Clinical Research, Sydney, MD Anderson Cancer Center, Houston, TX, United States; Leon Berard, 6 7 3 Australia; Monash Medical Centre, Clayton, Australia; Huntsman Cancer Lyon, France; University College London Hospitals, London, United 8 4 Institute, Salt Lake City, UT, United States; AstraZeneca, San Francisco, Kingdom; Vall D'Hebron University Hospital, Barcelona, Spain; 9 5 CA, United States; The Warren Alpert Medical School, Providence, RI, Adaptimmune, Philadelphia, United States United States Correspondence: Erin Van Winkle (erin.vanwinkle@adaptimmune.com) Correspondence: Mayukh Das (mayukh.das@medimmune.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P452 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P451 Background Background ADP-A2M4 specific peptide enhanced affinity receptor (SPEAR) T-cells MEDI5083 is a homodimeric fusion protein of three single-chain are genetically engineered to target MAGE-A4+ tumors in the con- CD40L domains linked to an immunoglobulin G fragment text of HLA-A*02. MAGE-A4 has been described as having high ex- crystallizable domain, and activates the CD40 pathway to promote pression in synovial sarcoma (SS) and myxoid/round cell liposarcoma immune responses. This first-in-human study evaluated the safety (MRCLS). In recent studies [1, 2], immunohistochemistry (IHC) ana- and clinical activity of MEDI5083 given sequentially or concurrently lyses showed that 82% of SS samples and 68% of MRCLS samples with the PD-L1 antibody durvalumab in patients with advanced solid expressed MAGE-A4. A pilot study (NCT03132922) of ADP-A2M4 in- tumors. duced clinical responses in patients with SS. Methods Methods Eligible patients with metastatic or recurrent tumor types progressing This phase 2, open-label trial (SPEARHEAD-1 Trial) will evaluate the on or refractory to prior therapy were enrolled in multiple cohorts of efficacy, safety and tolerability of ADP-A2M4 SPEAR T-cells. Patients MEDI5083 (3mg, 4mg, 5mg, 6mg, and 7.5mg) administered subcuta- who are HLA-A*02+ (excluding A*02:05, and A*02:07 and A*02 null neously (SC) Q2W for 4 doses. In the sequential-treatment cohort, as sole A*02 alleles), who have advanced/metastatic SS or MRCLS MEDI5083 was followed by a 4-week wash-out, then durvalumab who have received prior chemotherapy, and have MAGE-A4 expres- 1500 mg intravenously (IV) Q4W. In the concurrent-treatment cohort, sion assessed by IHC at ≥2+ in ≥ 30% of tumor cells, and who meet MEDI5083 was administered with concurrent durvalumab 1500 mg IV all other inclusion criteria are eligible for treatment. Up to 60 patients Q4W for 2 doses, followed by durvalumab 1500 mg IV Q4W. The pri- will be treated. mary endpoint was safety. Secondary endpoints included pharmaco- Following apheresis, T-cells are isolated, transduced with MAGE- kinetics, immunogenicity, and efficacy based on investigator- A4c1032TCR, and expanded. Prior to infusion, patients will receive assessed RECIST V1.1. lymphodepletion consisting of fludarabine (30 mg/m2/day x 4 days) Results and cyclophosphamide (600 mg/m2/day x 3 days). Patients will re- As of June 30, 2019, 38 patients were treated; 29 received se- ceive 1 – 10 × 10^9 transduced T-cells. Futility analysis will be con- quential treatment (MEDI5083 3mg, n=4; 4mg, n=4; 5mg, n=18; ducted after 15 patients are dosed and have been followed for at 7.5mg, n=3) and 9 patients received concurrent treatment least 4 months from the time of T-cell infusion. An independent Data (MEDI5083 3mg, n=3; 4mg, n=6). Two patients (sequential cohort, Safety Monitoring Board will review ongoing safety and benefit:risk MEDI5083 5mg and 7.5mg) had MEDI5083-related G3/4 dose- during the interventional phase of the study. Disease will be assessed Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 247 of 272 by independent review per RECIST v1.1 by CT/MRI at weeks 4, 8, 12, Results 16, 24, and every 2 months thereafter until confirmed disease pro- The primary objective of sub-study 2 is to evaluate GSK3377794 gression. Once disease progression is established, patients will enter efficacy by overall response rate per RECIST v1.1 (central inde- the long-term follow-up phase of the study, with visits every 6 pendent review). Secondary objectives include: time to and dur- months through Year 5, and annually thereafter for Years 6-15. ation of response; disease control rate; progression-free survival; Trial Registration overall survival; potential immune response to GSK3377794; safety NCT04044768 and tolerability. Exploratory objectives include: correlation of T- cell persistence with safety, clinical responses, and phenotype of References infused T cells. Impact on quality of life and daily functioning will 1. Iura K, et al. Cancer-testis antigen expression in synovial sarcoma: NY- also be assessed. ESO-1, PRAME, MAGEA4, and MAGEA1. Human Pathology 2017a; 61:130- Conclusions 139. Based on the encouraging clinical activity of GSK3377794 observed 2. Iura K, et al. MAGEA4 expression in bone and soft tissue tumors: its utility in earlier trials, this larger clinical trial is being initiated to establish as a target for immunotherapy and diagnostic marker combined with and further discern the efficacy and safety of GSK3377794 in this NY-ESO-1. Virchow Archiv 2017b;471:383–392. biomarker-selected metastatic SS patient population. This innovative Ethics Approval Master Protocol study design permits evaluation of GSK3377794 This trial is under review by the institutional review board of the trial sites treatment in other NY-ESO-1+ tumor types in HLA A*02+ patients within separate sub-studies. P453 Acknowledgements Autologous T cells with NY-ESO-1-specific T-cell receptor Medical writing assistance was provided by Fiona Woodward of Fishawack (GSK3377794) in HLA-A*02+ previously-treated and -untreated Indicia Ltd, UK, funded by GlaxoSmithKline (GSK). This study (NCT03967223) advanced metastatic/unresectable synovial sarcoma: A master was funded by GSK. protocol study design Trial Registration 1 2 3 4 Sandra D'Angelo, MD , Jean-Yves Blay , Warren Chow , George Demetri , NCT03967223 5 6 7 Fiona Thistlethwaite, MD, PhD , Michael Wagner , David Loeb , Steven 8 9 10 Attia , Albiruni Razak , John Haanen, MD PhD , Aisha Hasan, MBBS References 11 11 11 11 11 MD , Julia Billiard , Laura Pearce , Yuehui Wu , Ran Ji , Laura 1. Riedel RF, Jones RL, Italiano A, et al. Systemic anti-cancer therapy in syn- 11 11 11 12 Johnson , Chandra Srinath , Aiman Shalabi , Sandra Strauss , ovial sarcoma: A systematic review. Cancers 2018; 10:E417. 4 13 1 Katherine Thornton , Crystal Mackall, MD , William Tap , Brian Van Tine, 2. Vlenterie M, Litière S, Rizzo E, et al. Outcome of chemotherapy in MD, PhD advanced synovial sarcoma patients: Review of 15 clinical trials from the Memorial Sloan Kettering Cancer Center, New York, NY, United States; European Organisation for Research and Treatment of Cancer Soft Tissue 2 3 Centre Léon Bérard, Lyon, France; City of Hope Comprehensive Cancer and Bone Sarcoma Group; setting a new landmark for studies in this Center, Duarte, CA, United States; Dana-Farber Cancer Institute, Boston, entity. Eur J Cancer 2016; 58:62–72. MA, United States; The Christie NHS Foundation Trust, Manchester, Ethics Approval United Kingdom; Fred Hutchinson Cancer Research Center, Seattle, WA, This Master Protocol will be conducted under approval by the appropriate United States; Montefiore Medical Center, New York, NY, United States; institutional review boards and independent ethics committees. 8 9 Mayo Clinic in Florida, Jacksonville, FL, United States; Princess Margaret Cancer Centre, Toronto, Canada; Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, Netherlands; GlaxoSmithKline, Collegeville, PA, United States; University College London Hospitals, London, United Kingdom; 13 14 Stanford University, Stanford, CA, United States; Washington University in St. Louis, St. Louis, MO, United States Correspondence: Sandra D'Angelo (dangelos@mskcc.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P453 Background Synovial sarcoma (SS) comprises ~5%–10% of soft-tissue sarcomas [1]. Anthracycline-based chemotherapy is a 1st-line treatment in advanced metastatic/unresectable disease, but response rates are low Fig. 1 (abstract P453). Study Design Methods A clinical trial is underway utilizing a Master Protocol design allowing investigation of GSK3377794 in multiple tumor types (NCT03967223). The first 2 sub-studies are single-arm trials evaluating treatment in previously-untreated (sub-study 1) and previously-treated (sub-study P454 2) HLA A*02+ patients with NY-ESO-1+ metastatic SS. Sub-study 1 is Induction of serum CXCL10 by tebentafusp, a gp100-CD3 a pilot study evaluating efficacy as 1st-line treatment (N=10). Sub- bispecific fusion protein, was associated with survival in uveal study 2 plans to enroll 55 patients with metastatic/locally advanced melanoma in a Phase I/II Study unresectable SS who have progressed following anthracycline-based 1 2 2 Marcus Butler, MD , Brandon Higgs , Cheryl McAlpine, MSN , Joseph chemotherapy. Inclusion criteria include: ≥10 years of age; measur- 3 4 5 Sacco , Jessica Hassel, MD , Shaad Abdullah, MD , Koustubh Ranade, able disease; adequate organ function; ECOG performance status 0– 5 6 PhD , Richard Carvajal, MD 1. Exclusion criteria include: CNS metastases; clinically significant sys- 1 2 Princess Margaret Cancer Centre, Toronto, Canada; Immunocore, Ltd, temic illness; prior gene therapy with integrating vector or NY-ESO-1- Oxfordshire, United Kingdom; Clatterbridge Cancer Centre, Liverpool, specific T cells, vaccine or targeting antibody; prior autoimmune dis- United Kingdom; University Hospital Heidelberg, Heidelberg, Germany; ease or allogeneic hematopoietic stem-cell transplant. Patients will 5 6 Immnuocore, Ltd, Rockville, MD, United States; Columbia University undergo eligibility screening; leukapheresis and manufacture of Medical Center, New York, NY, United States GSK3377794; lymphodepletion and infusion of GSK3377794 followed Correspondence: Brandon Higgs (brandon.higgs@immunocore.com) by safety follow-up and disease assessments; and a 15-year follow-up Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P454 under a separate protocol (Figure 1). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 248 of 272 Background P455 Tebentafusp (formerly IMCgp100) is a TCR–anti-CD3 bispecific fusion A randomized phase 2 study of neoadjuvant talimogene protein targeting melanocyte-expressed gp100 antigen; Phase I/II laherparepvec (T-VEC) plus surgery vs surgery for resectable stage clinical studies showed monotherapy activity in metastatic melanoma IIIB-IVM1a melanoma: 2-year primary analysis of recurrence-free including uveal (UM) [1]. Historical 1-year survival (OS) rate for 2L survival (RFS) 1 2 3 metastatic UM is ~35% [2]. In a phase I clinical study of tebentafusp Reinhard Dummer, MD , David Gyorki , John Hyngstrom, MD , Adam 4 5 6 7 in cutaneous and 19 UM patients, serum IFNg-induced chemokines, Berger, FACS, MD , Robert Conry, MD , Lev Demidov , Anjali Sharma , 7 7 7 8 particularly CXCL10, were induced by tebentafusp and high induced Sheryl Treichel , Kevin Gorski , Abraham Anderson, PhD , Mark Faries , levels correlated with OS and tumor reduction. In a subsequent Merrick Ross, MD 1 2 phase I/II trial (NCT02570308) of 2L UM, we sought to confirm this University Hospital of Zurich, Zurich, Switzerland; Olivia Newton-John and increase mechanistic understanding with blood mRNA analysis. Cancer Centre, Melbourne, Australia; University of Utah Huntsman Methods Cancer Inst, Salt Lake City, UT, United States; Rutgers Cancer Institute of NCT02570308 was conducted in HLA-A*0201+ patients with ad- New Jersey, New Brunswick, United States; University of Alabama vanced 2L UM; this exploratory analysis focused on further investiga- School of Medicine, Birmingham, AL, United States; N.N. Blokhin Russian tion of an initial 40 patient cohort [3]. Intra-patient escalation dosing Cancer Research Ce, Moscow, United States; Amgen Inc, Thousand regimen used low initial dosing at Cycle1, Day1 (C1D1, 20 mcg) and Oaks, United States; John Wayne Cancer Institute, Santa Monica, United C1D8 (30 mcg). From C1D15, 19 patients received between 54-73 States; University of Texas MD Anderson Cancer, Houston, TX, United mcg , 22 patients received 68 mcg (expansion phase). Sera from 18 States and 22 patients in escalation and expansion phases, respectively Correspondence: Reinhard Dummer (Reinhard.Dummer@usz.ch) were profiled pre-treatment and post first and third dose with 11 im- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P455 mune markers (Luminex); whole blood from 19 escalation phase pa- tients was analysed for gene expression (NanoString). Low/High Background groups were defined at the median for OS and Mann-Whitney tests Risk of recurrence and death after resection of stage IIIB-IVM1a mel- were used for time contrasts. anoma is high. In the previous 1-year interim analysis, T-VEC plus sur- Results gery demonstrated a pathological complete response rate of 22.8% In an updated analysis of rash (on-target, off-tumor toxicity), 31 of 40 and improved RFS compared to upfront surgery (Dummer et al, patients with Grade 2+ rash had 1-year OS rate ~77%; 1-year OS in ASCO 2019). Here, we present results from the primary 2-year RFS remaining patients was ~35%. Tebentafusp induced a transient re- and biomarker analyses (CT.gov identifier: NCT02211131). sponse in cytokines, reaching maximal changes at 8-24 hours post Methods first and third doses. High levels of CXCL10 induced at first dose cor- Patients with resectable stage IIIB-IVM1a melanoma, ≥ 1 injectable cuta- related with improved OS (HR=0.37 95%CI=[0.15, 0.89]); high induced neous, subcutaneous, or nodal lesions ≥10 mm, and no systemic treat- CXCL9 levels also trended with OS (HR=0.52 95%CI=[0.21,1.3]). ment 3 months prior were randomized 1:1 to 6 doses/12 wks of CXCL9/CXCL10 transcripts increased at the same time points, as did neoadjuvant T-VEC followed by surgery during weeks 13-18 (Arm 1) signatures for neutrophils, type I IFN, and eosinophils (folds>1.5, p<- versus surgery during weeks 1-6 (Arm 2). T-VEC was given at standard 2, p<-1.5, p dosing until surgery, no remaining injectable tumors, or intolerance. Conclusions The primary analysis estimated a between-group difference in 2-yr RFS In this exploratory analysis, high CXCL10 levels induced by tebenta- on the intent-to-treat set. RFS event was defined as the first local, re- fusp associated with improved OS in UM. Tebentafusp reduced CD8+ gional, or distant recurrence or death due to any cause after surgery. and CD4+ gene signatures in the blood and induced systemic cyto- Per protocol, patients who withdrew prior to surgery or had an R1 or kine and gene expression responses, consistent with T cell redirec- R2 resection were counted as an RFS event at randomization. An add- tion and immune activation. Patients who develop tebentafusp- itional analysis calculated RFS from randomization to the date of first induced Grade 2+ rash appear to have better survival than those post-surgery event regardless of surgical margin status. who do not. Results Trial Registration 150 pts were randomized (76 arm 1, 74 arm 2). Median (range) NCT02570308 follow-up time was 31.2 (0.1–49.9) months. 75% in Arm 1 and 93% in Arm 2 had surgery as planned. In the per protocol analysis, 29.5% of References patients in Arm 1 and 16.5% of patients in Arm 2 remained recur- 1. Middleton MR. J Clin Oncol 37, 2019 (suppl; abstr 9523) rence free (HR: 0.75, P=0.07). In the additional analysis, 50.5% of pts 2. Rantala ES, Hernberg M, Kivelä TT. Overall survival after treatment for in Arm 1 and 30.2 % in Arm 2 remained recurrence free (HR: 0.66, P= metastatic uveal melanoma: a systematic review and meta-analysis. Mel- 0.038). 2-year overall survival rates were 88.9% in Arm 1 and 77.4% anoma Res. 2019 Jan 16. doi: 10.1097/CMR.0000000000000575. in Arm 2 (HR: 0.49, P=0.050). In arm 1, T-VEC treatment resulted in a 3. Sato T DOI: 10.1200/JCO.2017.35.15_suppl.9531 Journal of Clinical 3-fold increase (P Oncology 35, no. 15_suppl (May 20 2017) 9531-9531. Conclusions Ethics Approval Neoadjuvant T-VEC improved 2-year RFS and OS in resectable stage This study was in accordance with the Declaration of Helsinki and was IIIB-IVM1a melanoma. T-cell influx and PD-L1 upregulation after T- approved by all IRBs/ethics committees from each clinical sites VEC treatment support a role for the adaptive immune system con- participating in the study. Specific approval numbers can be provided sistent with the mechanisms of action. Additional biomarker results upon request. including clinical correlations will be presented at the congress. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 249 of 272 Trial Registration Ethics Approval CT.gov identifier: NCT02211131 The study and the protocol were approved by the Institutional Review Board or ethics committee at each site. Reference Consent 1. Dummer R, et al. Presented at The American Society of Clinical All patients provided written informed consent to participate in the clinical Oncology; May 31–June 3, 2019; Chicago IL, USA. J Clin Oncol 37, 2019 trial. (suppl; abstr 9520) Ethics Approval P457 The study was approved by participating institutions' Ethics Board. Randomized phase II neoadjuvant study: PD-1 inhibitor TSR-042 vs. combination PD-1 inhibitor TSR-042 and Tim-3 inhibitor TSR- P456 022 in borderline resectable stage III or oligometastatic stage IV KEYNOTE-630: phase 3 study of adjuvant pembrolizumab versus melanoma 1 2 2 placebo in patients with high-risk, locally advanced cutaneous Zahra Kelly, DO , Yana Najjar, MD , Hassane Zarour, MD , John Kirkwood, 2 3 2 4 squamous cell carcinoma MD , Suthee Rapisuwon, MD , Hong Wang , Marc Ernstoff, MD , Joseph 1 2 2 5 2 6 Jessica Geiger , Gregory Daniels, MD, PhD , Ezra Cohen, MD , Joy Yang Drabick, MD, FACP, FIDSA , Diwakar Davar, MD , Mohan Bala 3 3 3 1 Ge , Burak Gumuscu, MD PhD , Ramona Swaby, MD , Anne Lynn University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 4 2 3 Chang University of Pittsburgh, Pittsburgh, PA, United States; Georgetown 1 2 4 Cleveland Clinic, Cleveland, OH, United States; University of California, University, Washington, DC, United States; Roswell Park Comprehensive 3 5 San Diego, La Jolla, CA, United States; Merck & Co., Inc., Kenilworth, NJ, Cancer Center, Buffalo, NY, United States; Penn State Cancer Institute, 4 6 United States; Stanford University Medical Center, Stanford, CA, United Palmyra, PA, United States; Tesaro, Inc, Wltham, MA, United States States Correspondence: Diwakar Davar (davard@upmc.edu) Correspondence: Jessica Geiger (GEIGERJ@ccf.org) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P457 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P456 Background Background Neoadjuvant PD-1 blockade produces pathological responses in Despite undergoing current standard-of-care surgical resection and ~30% of patients (pts) with high-risk resectable melanoma (MEL) adjuvant radiotherapy, ~20% of patients with high-risk, locally ad- with durable relapse-free benefit, and increased circulating acti- vanced cutaneous squamous cell carcinoma develop local recurrence vated CD8+ T cells (1,2). TIM-3 is an inhibitory immune check- within 5 years [1]. Recent data show effective antitumor activity and point and mediates immune escape; blockade of which produces acceptable safety of programmed death 1 inhibitors in patients with anti-tumor immune responses and synergizes with anti-PD-1 (3– locally advanced or metastatic cutaneous squamous cell carcinoma. 5). TSR-042/dostarlimab is an IgG4 humanized monoclonal anti- KEYNOTE-630 (NCT03833167), a randomized, double-blind, placebo- body that binds with high affinity to PD-1, inhibiting binding to controlled phase 3 trial, will evaluate the efficacy and safety of adju- PD-L1 and PD-L2. TSR-042 has been studied in patients with ad- vant pembrolizumab in patients with high-risk locally advanced or vanced non-small cell lung (NSCLC) and endometrial cancers with metastatic cutaneous squamous cell carcinoma. promising results (6). TSR-022 is an IgG4-k isotype humanized Methods monoclonal antibody that binds with high affinity to TIM-3, thus Patients with high-risk locally advanced cutaneous squamous cell enhancing T cell activity. TSR-042/TSR-022 combination has been carcinoma who have undergone surgical resection and radiother- studied in a phase I/II study that demonstrated promising efficacy apy will be randomly assigned 1:1 to intravenous pembrolizumab of combination in PD-1 refractory melanoma and NSCLC (7). We (400 mg every 6 weeks) or placebo for up to 9 cycles (~1 year) hypothesized that neoadjuvant therapy with TSR-042/TSR-022 or until disease recurrence, unacceptable toxicity, or investigator combination may improve pathologic response rates compared to or patient decision to withdraw. Randomization will be stratified TSR-042 monotherapy in high-risk resectable MEL. by extracapsular extension (yes vs no), cortical bone invasion (yes Methods vs no), and prior systemic therapy (yes vs no). Eligible patients Pts with regionally advanced (stage IIIB-D) or oligometastatic are adults with histologically confirmed locally advanced cutane- (stage IVA-B) melanoma who have yet to undergo definitive sur- ous squamous cell carcinoma with ≥1 high-risk features at the gery are eligible. Primary endpoints are rate of major pathologic primary site of malignancy and macroscopic resection with or response (MPR), safety, and incidence of dose-limiting toxicities without microscopic positive margins who completed adjuvant (DLT). Secondary endpoints are radiographic response, relapse- radiotherapy, were disease free ≤28 days from randomization, free survival (RFS) and overall survival (OS). Pathological response and have Eastern Cooperative Oncology Group performance sta- will be assessed depending on residual volume of tumor (RVT) tus 0 or 1. The primary efficacy end points are investigator- using prior cutoffs: 0% (complete response, pCR); 0%<RVT< assessed and biopsy-confirmed recurrence-free survival. Secondary RVT50% (non-response, pNR) (8–10). Sample size is 28 patients end points are overall survival, health-related quality of life, and per arm. Patients will receive neoadjuvant therapy for 6 weeks safety. To assess treatment response, radiographic imaging will prior to planned surgery. Surgery will occur 1-3 weeks after com- be performed at least every 12 weeks in year 1, then every 6 pletion of pre-operative therapy. After surgery, subjects will re- months until the end of year 5. All patients meeting crossover or ceive additional maintenance TSR-042 for approximately 48 weeks retreatment criteria at first disease recurrence may receive pem- (Figure 1). Sample size provides 80% power to detect an im- brolizumab 400 mg every 6 weeks for up to 18 cycles. Adverse provement of 30% upon MPR rate of 30% with anti-PD-1 events will be recorded until 30 days (90 days for serious adverse monotherapy. events) after study end and will be graded per NCI CTCAE v4.0. Enrollment of ~570 patients is planned, and recruitment is on- References going in 18 countries. 1. Amaria, R. N. et al. Neoadjuvant immune checkpoint blockade in high- Trial Registration risk resectable melanoma. Nature Medicine 24, 1649–1654 (2018). ClinicalTrials.gov, NCT03833167 2. Huang, A. C. et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nature Medicine 25, 454–461 Reference (2019). 1. Porceddu SV, Bressel M, Poulsen MG, et al. Postoperative concurrent 3. Fourcade, J. et al. Upregulation of Tim-3 and PD-1 expression is asso- chemoradiotherapy versus postoperative radiotherapy in high-risk cuta- ciated with tumor antigen–specific CD8+ T cell dysfunction in melan- neous squamous cell carcinoma of the head and neck: the randomized oma patients. The Journal of Experimental Medicine 207, 2175–2186 phase III TROG 05.01 trial. J Clin Oncol. 2018;36:1275-1283. (2010). Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 250 of 272 4. Chauvin, J.-M. et al. TIGIT and PD-1 impair tumor antigen–specific CD8+ signals and tumor antigens that can be captured and processed by T cells in melanoma patients. Journal of Clinical Investigation 125, 2046– IT co-administered CD1c (BDCA-1)+ myDC, reinvigorating the cancer 2058 (2015). immunity cycle. 5. Woo, S.-R. et al. Immune Inhibitory Molecules LAG-3 and PD-1 Synergis- Methods tically Regulate T-cell Function to Promote Tumoral Immune Escape. Patients with advanced melanoma who failed standard therapy were Cancer Research 72, 917–927 (2012). eligible for IT injections of ≥1 non-visceral metastasis with T-VEC 6. Moreno, V. et al. Abstract CT053: Preliminary safety, efficacy, and PK/PD (10^6 PFU/mL; max total volume of 4 mL) on day 1 followed by IT in- characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 jection of autologous, non-substantially manipulated CD1c (BDCA-1)+ monoclonal antibody, TSR-042, in patients with recurrent or advanced myDC on day 2. Injection of T-VEC (10^8 PFU/mL; max total volume NSCLC and MSI-H endometrial cancer. Cancer Research 78, CT053–CT053 of 4 mL) was repeated on day 21 and every 14 days thereafter. Pa- (2018). tients were treated with 0.5x10^6, 1x10^6, or 10x10^6 CD1c (BDCA- 7. Davar, D. A phase 1 study of TSR-022, an anti-TIM-3 monoclonal anti- 1)+ myDC in cohort-1, -2, and -3, respectively. Primary objectives body, in combination with TSR-042 (anti-PD-1) in patients with colorectal were safety and feasibility. Repetitive biopsies of treated lesions were cancer and post-PD-1 NSCLC and melanoma. (2018). performed. 8. Cottrell, T. et al. Pathologic Features of Response to Neoadjuvant Anti- Results PD-1 in Resected Non-Small Cell Lung Carcinoma: A Proposal for Quanti- In this ongoing trial, 2 patients were treated in cohort-1, 2 patients in tative Immune-Related Pathologic Response Criteria (irPRC). Annals of on- cohort-2, and 3 patients in cohort-3. Patients received a median of 6 cology : official journal of the European Society for Medical Oncology (range 3-10) injections of T-VEC. All patients are evaluable for re- (2018). doi:10.1093/annonc/mdy218 sponse. The best overall tumor response (according to iRECIST) was a 9. Stein, J. et al. Major pathologic response on biopsy (MPRbx) in CR (pathologic CR) and one PR (confirmation pending; pathologic CR patients with advanced melanoma treated with anti-PD-1: evidence of treated lesions). Both patients were treated in cohort-3 and had for an early, on-therapy biomarker of response. Annals of oncology : previously progressed on anti-PD-1 checkpoint inhibition, and one official journal of the European Society for Medical Oncology 30, patient also on anti-CTLA-4 therapy. Adverse events include G1 fever 589–596 (2019). in 4 patients, G1-2 flu-like symptoms in 5 patients, transient G1-2 10. Tetzlaff, M. et al. Pathological assessment of resection specimens after local pain and redness at the injection-site in 3 patients, and G1 neoadjuvant therapy for metastatic melanoma. Annals of oncology : gastrointestinal symptoms in 4 patients. The patient with CR devel- official journal of the European Society for Medical Oncology 29, 1861– oped an asymptomatic G3 eosinophilia during treatment; the patient 1868 (2018). with PR developed a transient purpuric rash at the site of skin metas- tases after the first treatment. Multiplexed immune-profiling (Ultivue) of baseline and on-treatment tumor biopsies is ongoing. Conclusions IT co-injection of autologous CD1c (BDCA-1)+ myDC with T-VEC is feasible and tolerable and resulted in encouraging early signs of anti- tumor activity in patients with immune checkpoint inhibitor refrac- tory melanoma who received high dose CD1c (BDCA-1)+ myDC. Trial Registration ClinicalTrials.gov: NCT03747744 Ethics Approval This study was approved by the Ethics Board of Universitair Zieken- huis Brussel. Consent Written informed consent was obtained from the patient for publica- tion of this abstract and any accompanying images. A copy of the written consent (NL,FR) is available for review by the Editor of this journal. P459 New generation chimeric antigen receptor T-Cell Therapy Fig. 1 (abstract P457). See text for description (CoupledCAR) induces high rate remissions in solid tumor Chengfei Pu, Lei Xiao Innovative Cellular Therapeutics, Shanghai, China Correspondence: Lei Xiao (xiaolei@ictbio.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P459 P458 Background A phase I clinical trial on intratumoral administration of Conventional CAR T cell therapy showed weak CAR T expansion in autologous CD1c (BDCA-1)+ myeloid dendritic cells plus patients, thus achieved no or little response for treating solid tu- talimogene laherparepvec (T-VEC) in patients with advanced mors. Here, we generated "CoupledCAR" T cells including an anti- melanoma TSHR CAR molecule. Compared with conventional CART cells, "Cou- Julia Katharina Schwarze, MD, MSc, Gil Awada, Louise Cras, Inès Dufait, pledCAR" T cells successfully improved the expansion of CART cells Ramses Forsyth, Ivan Van Riet, Bart Neyns more than 100 times and enhanced CAR T cells’ migration ability, UZ Brussel, Brussels, Belgium allowing the CAR T cells to resist and infiltrate the tumor microenvir- Correspondence: Julia Katharina Schwarze onment and killed tumor cells. (juliakatharina.schwarze@uzbrussel.be) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P458 We designed a “CoupledCAR” lenti-vector containing a scFv targeting hTSHR. Patient‘s CD3 T cells were isolated and transduced with the Background lentivirus. Then,transduction efficiency was evaluated. After infusion, Intratumoral (IT) myeloid dendritic cells (myDC) play a pivotal role in peripheral blood samples were collected to analyze expansion and initiating antitumor immune responses and re-licensing of anti-tumor cytokine release. The evaluation of response level for patients were cytotoxic T-lymphocytes within the tumor microenvironment. IT in- performed at month 1,month 3,and month 6 by PET/CT. jection of the oncolytic virus T-VEC leads to the release of maturation Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 251 of 272 Results and documented D0 prostate cancer whose PSADT is between 3 and To verify the effect of “CoupledCAR” T cells on solid tumors, we have 15 months are eligible. The participant should have normal organ func- completed several clinical trials for different solid tumors, including tions, ECOG 0-1. Patient should not be on any other cancer treatment two patients with thyroid cancer. Immunohistochemistry (IHC) results at enrollment. The primary objective is to assess the PSA slope log showed thyroid stimulating hormone receptors (TSHR) were highly change at week 24 and 48 compared to baseline. Secondary objectives expressed in thyroid cancer cells. In vitro co-culture experiments are 1) to assess the safety of ME-TARP DC vaccines 2) To characterize showed TSHR CAR T cells specifically recognized and killed TSHR- cellular and humoral immune responses associated with vaccination. positive tumor cells. Animal experiments showed TSHR CAR T cells Results inhibited the proliferation of TSHR-positive tumor cells. Therefore, we The lead-in safety cohort (N=6) completed the treatment without designed "CoupledCAR" T cells expressing a binding domain against DLT and we are currently enrolling the randomized arms (N=66). TSHR. Further,we did clinical trials of two group patients that were Conclusions successfully treated using conventional TSHR CAR T cells and "Cou- Multi-Epitope TARP Peptide Autologous Dendritic Cell Vaccination in pledCAR" T cells, respectively. In the group using conventional Men with Stage D0 Prostate is safe and open for further accrual in TSHR CAR T cells, patients showed weak cell expansion and less mi- randomized arms to compare vaccine versus placebo. gration ability. In the group using TSHR "CoupledCAR" T cells, pa- tients showed rapid expansion of CAR T cells and killing of Acknowledgements tumor cells. One month after infusion (M1), the patient was evalu- This study is supported by the Center for Cancer Research, National Cancer ated as PR(Partial Response): the lymph node metastasis disappeared, Institute, National Institute of Health. and thoracic paratracheal tumors decreased significantly. Three Trial Registration months after infusion (M3), the patient was evaluated as a durable NCT02362451 response, and the tumor tissue was substantially smaller than M1. Further, two patients with colorectal cancer were enrolled in this References trial and infused "CoupledCAR" T cells. One patient achieved PR and 1. Wolfgang CD, Essand M, Vincent JJ et al. TARP: a nuclear protein the other one achieved SD. expressed in prostate and breast cancer cells derived from an alternate Conclusions reading frame of the T cell receptor gamma chain locus. Proc Natl Acad “CoupledCAR” T cells can effectively promote expansion, migration Sci U S A. 2000;97(17):9437–9442. and killing ability of CAR T cells in patients with thyroid cancer. “Cou- 2. Wood LV, Fojo A, Roberson BD, et al. TARP vaccination is associated pledCAR” T cell technology is a technological platform, which may with slowing in PSA velocity and decreasing tumor growth rates in be used to treat other cancer types. Next, we are recruiting more pa- patients with StageD0prostatecancer. Oncoimmunology. tients for clinical trials using “CoupledCAR” T cells. 2016;5(8):e1197459. Ethics Approval The study was approved by National Cancer Institute Ethics Board, approval P460 number P121083 and assigned a local number 15C0075. A randomized, placebo-controlled phase II study of multi-epitope TARP peptide autologous dendritic cell vaccination in men with stage D0 prostate cancer P461 1 3 1 Hoyoung Maeng, MD , Lauren Wood, MD , Seth Steinberg , David Phase 1b study of INCMGA00012, a programmed cell death-1 (PD- 2 1 1 Stroncek, MD , Masaki Terabe, PhD , Jay Berzofsky, MD, PhD 1) inhibitor, in combination with chemotherapy in patients with 1 2 National Cancer Institute, Bethesda, MD, United States; National advanced solid tumors (POD1UM-105) 3 1 2 2 Institute of Health, Bethesda, MD, United States; PDS Biotechnology, David Planchard, MD, PhD , Jill Bowman , Nawel Bourayou, MD 1 2 Berkeley Heights, NJ, United States Gustave Roussy Institute, Villejuif, France; Incyte Corporation, Correspondence: Hoyoung Maeng (hoyoung.maeng@nih.gov) Wilmington, DE, United States Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P460 Correspondence: David Planchard (david.planchard@gustaveroussy.fr) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P461 Background TARP is a 58 amino acid protein expressed in normal and malignant Background prostate tissue [1]. TARP is highly expressed in 95% of prostate cancers Background: The combination of PD 1/programmed death-ligand 1 including all Gleason types and in both castration sensitive (CSPC) and (PD-L1) checkpoint inhibitors with chemotherapy has proven clinic- castration resistant prostate cancer (CRPC). In the pilot study of 1st gen- ally meaningful efficacy and manageable safety profile based on sev- eration TARP peptide-pulsed autologous dendritic cell vaccination eral randomized trials in treatment-naive advanced non-small cell (TARP DC vaccine, NCT00972309, N= 41) utilizing TARP WT 27-35 and lung cancer (NSCLC) [1,2,3] and encouraging data are emerging in epitope enhanced EE29-37-9V in HLA-A*0201 positive men with stage unresectable advanced malignant pleural mesothelioma (MPM) [4]. D0 prostate cancer (PSA biochemical recurrence) published by Wood et INCMGA00012 is an investigational humanized immunoglobulin G4 al, the vaccine was found to be immunogenic and safe [2]. TARP vaccin- (IgG4) monoclonal antibody against human PD-1. In the phase 1 ation was also associated with a decreased slope log PSA in more than POD1UM-101 study, INCMGA00012 has demonstrated acceptable tol- 70 % of the patients at 24 and 48 weeks and a decrease in calculated erability with clinical activity observed in multiple tumor types, in- tumor growth rate constant. Standard of care in D0 prostate cancer cluding a confirmed objective response rate (by Response Evaluation ranges from watchful waiting, salvage radiation and anti-androgen Criteria in Solid Tumors [RECIST] version1.1) of 19% in an interim ana- therapy without strong evidence to support one or the other. In the lysis of a cohort of patients with platinum-refractory NSCLC [5]. The current study of multi-epitope (ME)-TARP DC vaccines, 5 overlapping POD1UM-105 trial aims to investigate INCMGA00012 in combination 18-20-mer peptides encompassing the full sequence of TARP are added with standard-of-care chemotherapy regimens in patients with ad- to 1st generation TARP peptides to pulse the autologous DCs. The use vanced NSCLC or MPM. of synthetic long peptides can increase the chance of a durable multi- Methods valent anti-TARP response. Methods: POD1UM-105 is a phase 1b, global, multicenter study, in pa- Methods tients with histologically or cytologically confirmed advanced/meta- This is a single-blinded, randomized, placebo-controlled phase II study static NSCLC or unresectable MPM and regardless of PD-L1 expression, in men with Stage D0 prostate cancer. Men with a PSADT between 3 not previously treated with systemic therapy. Key eligibility criteria in- and 15 months will be randomized 2:1 to receive a ME TARP DC vac- clude no prior systemic treatment (except for patients [with known sen- cine or a monocyte placebo. HLA restriction is not required. Patients sitizing mutations] who have disease progression on or following an will receive a total of 6 doses of vaccine or a placebo, 20e6 cells/dose approved targeted tyrosine kinase inhibitor, or chemotherapy com- intradermally every 3 weeks. Males older than 18 with adenocarcinoma pleted > 6 months before enrollment), no prior checkpoint inhibitor Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 252 of 272 therapy, measurable or nonmeasurable disease by RECIST version 1.1, Combination Immunotherapies and Eastern Cooperative Oncology Group performance status ≤1. P462 Patients will be assigned to 1 of 4 treatment groups (12-24 patients ImmTAC®-chemotherapy combination: A preclinical evaluation each), and will receive INCMGA00012 every 3 weeks for up to 2 years shows potential benefits in combination with standard doses of chemotherapy agents (4 to 6 Filipa Bravo-Lopes, Nora Rippaus, Kristina Petrovic, Francesca Amicarella, cycles) (Treatment Group A: gemcitabine/cisplatin; Group B: peme- Adam Taylor, Laure Humbert, Rupert Kenefeck, Adel Benlahrech, BSc trexed/cisplatin; Group C: pemetrexed/carboplatin; Group D: pacli- PhD taxel/carboplatin) (Figure 1). Immunocore Ltd, Abingdon, United Kingdom The primary study objective is to evaluate safety, tolerability (DLTs), and Correspondence: Rupert Kenefeck (rupert.kenefeck@immunocore.com); determine a recommended phase 2 dose of INCMGA00012 in combin- Adel Benlahrech (adel.benlahrech@immunocore.com) ation with chemotherapy. Secondary objectives include determining Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P462 preliminary clinical activity (measured by objective response rate, dur- ation of response, and disease control rate) and pharmacokinetics. Ex- Background ploratory objectives include assessment of additional efficacy measures ImmTAC molecules are bispecific T cell redirectors comprised of an (progression-free survival and overall survival), and relevant biomarkers. affinity‐enhanced TCR recognizing tumour antigen in the context of HLA molecules and a T cell engaging anti‐CD3 domain. Paclitaxel is a Acknowledgements chemotherapy drug which stabilises microtubules and it is widely This study is sponsored by Incyte Corporation (Wilmington, DE). used in combination with platinum-based chemotherapies against Trial Registration multiple indications. Building on the recent evidence that chemo- NCT03920839 therapy positively combines with immunotherapy [1], this study was designed to evaluate the potential of combining Paclitaxel with References ImmTAC-mediated T cell activation. 1. Gandhi L, Rodriguez-Abreu D, Gadgeel S, et al. Pembrolizumab plus Methods chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. In vitro assays to assess T cell-mediated lysis, T cell expansion, 2018;378:2078-2092. and cytokine release in response to ImmTAC-mediated T cell re- 2. Langer CJ, Gadgeel SM, Borghaei H, et al. Carboplatin and pemetrexed direction were performed with a range of ImmTAC concentrations with or without pembrolizumab for advanced, non-squamous non-small- and/or Paclitaxel. T cell-mediated lysis of antigen positive cells cell lung cancer: a randomised, phase 2 cohort of the open-label was monitored in real time while Granzyme A and B, IL-2, IL-8, KEYNOTE-021 study. Lancet. 2016;17:1497-1508. IL-10, IFN-γ, TNF-α,MIP-1α, IP-10, and MIG were measured in cell 3. Socinski MA et al. Atezolizumab for first-line treatment of metastatic non- culture supernatants. T cell expansion was measured by flow squamous NSCLC. N Engl J Med. 2018;378:2288-2301 cytometry. 4. Nowak AK, Lesterhuis WJ, Hughes BGM, et al. DREAM: a phase II study of Results durvalumab with first line chemotherapy in mesothelioma─first results. J ImmTAC molecules were highly effective at redirecting T cells to Clin Oncol. 2018;36(suppl):8503 proliferate, produce pro-inflammatory cytokines, chemokines and 5. Mehnert JM, Joshua AM, Lakhani N, et al. First-in-human phase 1 study of granzymes, and to lyse antigen positive targets. ImmTAC- INCMGA00012 in patients with advanced solid tumors: interim results of the co- Paclitaxel combination showed substantially enhanced lysis of tar- hort expansion phase. J Immunother Cancer. 2018;6(suppl 1):115. Abstract P669. get cells compared to either agent alone (in both magnitude and Ethics Approval kinetics). With 100 pM ImmTAC and 25 nM Paclitaxel, the median The study was approved by institutional review boards or independent cytolysis area under the curve using the compounds in combin- ethics committees of participating institutions. ation was 6269 compared to 3782 for Paclitaxel alone or 3196 for ImmTAC alone. Additionally, the time it took to lyse 80% of target cells decreased from 84 hours for ImmTAC alone to 51 hours when in combination with Paclitaxel. Despite enhanced kill- ing, some reduction of cytokine release and T cell proliferation were observed when ImmTAC and Paclitaxel were combined. Not- ably, cytokine secretion and T cell proliferation were restored, and improved killing maintained by pre-treating target cells with Paclitaxel. Similarly, pre-treatment of effector T cells with Pacli- taxel did not impact their ability to kill and proliferate in re- sponse to ImmTAC. Conclusions These in vitro studies show enhanced ImmTAC-mediated tumour cell lysis with Paclitaxel when administered either in combination with ImmTAC or sequentially. These data support the premise that Pacli- taxel positively combines with ImmTAC and provide strong rationale for further clinical investigation. Reference 1. Liu SV, Camidge DR, Gettinger SN, Giaccone G, Heist RS, Hodi FS, Ready NE, Zhang W, Wallin J, Funke R, Waterkamp D, Foster P, Iizuka K, Powderly J. Long-term survival follow-up of atezolizumab in combination with platinum-based doublet chemotherapy in pa- tients with advanced non-small-cell lung cancer. Eur J Cancer. 2018;101:114-122. Fig. 1 (abstract P461). See text for description Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 253 of 272 P463 melanoma efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine, correlating Immunotherapy combinations for betel-nuts related HNSCC: one with increases of T-cells and CD8α+ DCs institutional experiences in Taiwan Methods Jo-Pai Chen, MD, Ruey-Long Hong, MD, PhD, Wei-Chen Lu, MD Utilizing the B16F10 syngeneic mouse melanoma model, vaccina- National Taiwan University Hospital, Taipei, Taiwan, Province of China tions are administered by intramuscular electroporation with CpG ad- Correspondence: Ruey-Long Hong (rlhong@ntu.edu.gov.tw) juvant three times at one-week intervals beginning five days post Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P463 lethal tumor implantation. Aza is given intraperitoneally at 1mg/kg, and IFNα therapy is given in a series of one high followed by three Background low doses, as noted. Tumor sizes, growth, and survival were all Betel-nuts chewing might contribute to (1) strong inflammation, in- assessed. Tumor-infiltrating lymphocytes (TILs) were assessed by vasion, and angiogenesis; (2) poor response to traditional therapeis. stimulating the purified lymphocyte fraction of tumors with vaccine In the 1st line setting of R/M HNSCC, EPF offers survival benefit and antigens followed by intracellular cytokine staining flow cytometry. immune checkpoint inhibitor(anti-PD1 monoclonal antibody), like Dendritic Cells were assessed by flow cytometry. nivolumab or pembrolizumab, has already brought survival benefit in Results the 2nd line treatment. We demonstrate that the addition of IFNα and Aza treatments to Methods mice vaccinated with the MIP-3α-Gp100-Trp2 vaccine led to signifi- From 2016 to early 2019, 30 R/M HNSCC patients receiving cantly reduced tumor burden and overall increases in mouse survival, immunotherapy-containing regimens in Yun-lin Branch of National increasing median survival by 39% over vaccine. Importantly, this in- Taiwan University Hospital were reviewed. crease in efficacy was dependent on the presence of all three com- Results ponents, as vaccine plus IFNα or vaccine plus Aza did not differ These patients consisted of 1 HPV and 29 non-HPV; 18 pembrolizu- significantly from vaccine alone. The addition of Aza and IFNα to the mab and 12 nivolumab; 10 with afatinib(6 pembrolizumab & 4 nivo- vaccine increased T-cell tumor infiltration, altered the proportion of lumab); 5 with bevacizumab; 6 with chemotherapy. The objective CD8+T-cells, and increased CD8α+ DC infiltration. response rate was 47%(14/30) and clinical benefit was 80%(24/30). Conclusions 16 patients were still under use(4 afatinib with pembrolizumab; 4 afa- Efficient targeting of antigen to immature dendritic cells with a tinib with nivolumab). 1 patient under afatinib and pembrolizumab chemokine-fusion vaccine offers a potential alternative approach presented hyperprogression but then got pCR after bevacizumab to classic and dendritic cell-based vaccines currently undergoing combined with strong chemotherapy. 1 patient under low dose nivo- clinical investigation. Combining this approach with IFNα and Aza lumab in 20 mg biweekly with oral metronomic cyclophosphamide treatments significantly improved vaccine efficacy, with efficacy had mild tumor response. 1 patient under nivolumab with high dose correlating with changes in TILs and tumor infiltrating DCs. Fur- ifosfamide developed nephritis. 1 patient had rapid skin metastasis ther potential therapy optimization currently undergoing investi- over previous radiation fields after pembrolizumab, bevacizumab, gation offers promise for this line of investigation to become a and chemotherapy. 3 patients under afatinib & pembrolizumab de- novel melanoma therapy. veloped autoimmune cholestasis(2 also with pneumonitis). Afatinib & Ethics Approval nivolumab had similar efficacy but less toxicity. 10 pateints receiving All procedures performed in studies involving animals were in ac- afatinib combined with anti-PD1(8 faliling EPF, 7 with pleural/pericar- cordance with the ethical standards of the IACUC of the Johns Hop- dial/skin metastasis. 5 rapid progression within 3 months after defin- kins University under Protocols #MO16H147 and MO19H319. ite CCRT) had 70% response rate(7/10) and 90% clincial benefit(9/10). Post-progression use of anti-PD1 with other treatments were seen in P465 4 patients(esp. 1 with nivolumab & ipilimumab for sarcomatous Immune-mediated mechanisms involved in the synergistic anti- change). 3 patients got benefits and had longer survival. tumor efficacy of NHS-IL12 combined with the class I HDAC Conclusions inhibitor entinostat Immunotherapy-containing combinations are of clinical significance Kristin Hicks, PhD, Yohei Ozawa, MD, PhD, Karin Knudson, PhD, Jeffrey in refractory betel-nuts related HNSCC in Taiwan. Afatinib has several Schlom, Sofia Gameiro, PharmD, PhD immuno-modulatory effects. In high risk patients(pleural/pericardial/ National Cancer Institute, NIH, Bethesda, MD skin metastasis failing EPF and rapid progression within 3 months Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) after definite CCRT) in Taiwan, afatinib with anti-PD1 may be a good Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P465 option to avoid hyperprogression. Earlier use, well biomarkers, best combinations, and optimal sequencing will be future goals. Background Combining epigenetic agents with immunotherapy has shown P464 clinical promise. Preclinically, we and others have shown that the IFN-α and 5-Aza-2’-deoxycytidine enhance the anti-tumor efficacy class I histone deacetylase (HDAC) inhibitor entinostat can po- of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by tentiate the anti-tumor efficacy of immunotherapies. Mechanistic- affecting T-cell and Dendritic Cell recruitment into tumor ally, this is partially mediated by tumor MHC Class I upregulation, James Gordy, PhD, Richard Markham, BS MD impairing regulatory CD4+ T cells (Tregs) and monocyte derived Johns Hopkins Bloomberg School of Public, Baltimore, MD, United States suppressive cells (MDSCs) and/or enhancing CD8+ T cells and Correspondence: Richard Markham (rmarkha1@jhu.edu) granzyme B levels in the tumor microenvironment (TME). In these Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P464 studies, we observed that entinostat induced murine tumor necrosis. Background Methods The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic Therefore, we investigated if this treatment-induced necrosis cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated anti- could be targeted by NHS-IL12, an IL-12 NHS76 conjugate that gens have shown improved efficacy and immunogenicity, compared binds free DNA in regions of tumor death/necrosis. NHS-IL12 has to vaccines lacking the chemokine. To optimize the therapy, our la- been shown to have significant anti-tumor efficacy in preclinical boratory has added agents designed to further enhance the T cell ac- murine models and has been demonstrated to be safe in pa- tivating function of DCs and overcome immunoregulatory tients. We hypothesize that increasing the pro-inflammatory, im- mechanisms of the tumor microenvironment. Here, we report that mune stimulatory cytokine IL-12 in the TME will synergize with the combination of type-I interferon therapy (IFNα) with 5-Aza-2’- the entinostat-mediated immune effects to induce significant deoxycitidine (Aza) profoundly enhanced the therapeutic anti- tumor control. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 254 of 272 Results paclitaxel was 54.5% (6/11), the median PFS was 6.4 months (95% CI In the EMT6 breast cancer murine model, administration of NHS- 3.7 to 9.2 months), and the median OS was 14.1 months (95% CI 3.2 IL12 at the onset of entinostat-induced necrosis synergized to to 25.0 months). produce significant anti-tumor efficacy, including resolving well- Conclusions established tumors and enhancing survival. These studies are Our preliminary data suggest that carboplatin and paclitaxel often in- now being extended to additional murine models of solid carcin- duce objective responses in patients with prior treatment with anti- omas. Examination of immune subsets in the TME by flow cytom- PD1/PD-L1 antibodies and that the clinical outcomes compare favor- etry revealed that the combination increased infiltration of ably to those seen in checkpoint inhibitor naïve patients. If con- granzyme B+ CD8+ T cells and M1-like CD38 expressing tumor firmed, these data suggest that there is no “opportunity cost” for macrophages. Currently, we are using depletion studies to assess administering immunotherapy in the first line. the relative contribution of these immune subsets to the anti- Ethics Approval tumor efficacy. Further, we are examining the effect of this com- Approval by the UCSF IRB has been requested and will be obtained bination on CD8+ T cell function and macrophage polarization, before this work is presented. phenotype, and function. Conclusions Overall, the combination of NHS-IL12 and entinostat is showing en- P467 couraging results in a preclinical solid tumor model, providing a ra- ALPN-202, a conditional CD28 costimulator and dual checkpoint tionale to examine this combination clinically. inhibitor, enhances the activity of multiple standard of care modalities Katherine Lewis, PhD, Mark Maurer, BS, Sherri Mudri, BS, Kayla Susmilch, Acknowledgements MS, Fariha Ahmed-Qadri, MS, Chelsea Gudgeon, BS, Steven Levin, PhD, The authors thank Curtis Randolph for excellent technical assistance. Martin Wolfson, BS, Stacey Dillon, PhD, Kristine Swiderek, PhD, Stanford Peng, MD, PhD Alpine Immune Sciences, Seattle, WA, United States P466 Correspondence: Katherine Lewis Carboplatin and paclitaxel after anti-PD1 or anti-PDL1 therapy: A (katherine.lewis@alpineimmunesciences.com) retrospective study in patients with squamous cell carcinoma of Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P467 the head and neck Audrey Humphries, BS, Madeleine Welsh, BA, Alain Algazi, MD Background UCSF, San Francisco, CA, United States Checkpoint inhibitors targeting the PD-1 axis have transformed Correspondence: Alain Algazi (Alain.algazi@ucsf.edu) cancer treatment. However, objective response rates remain low, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P466 suggesting that novel therapeutics and/or combination treat- ments are needed. At the same time, non-immuno-oncology Background therapeutic approaches such as chemotherapy remain standard Historically, cytotoxic chemotherapy has been the first line stand- of care for many malignancies. ALPN-202 is a variant CD80 ard of care for patients with RM-SCCHN. More recently, a ran- vIgD™-Fc fusion that mediates PD-L1-dependent CD28 costimula- domized phase 3 clinical trial demonstrated improved overall tion and inhibits the PD-L1 and CTLA-4 checkpoints. This novel survival in RM-SCCHN patients receiving pembrolizumab in the mechanism of action provides potent single agent immunomodu- first line versus those receiving the prior standard carboplatin, latory activity in mouse tumor models, and thus has the potential 5FU, cetuximab. Additional studies have suggested that sequen- to complement other therapeutic modalities, such as checkpoint cing of therapies impacts outcomes. Several small case series inhibitors or chemotherapies. have described clinical outcomes in patients treated with chemo- Methods therapy after immunotherapy, but these have often included pa- Mice were implanted subcutaneously with human (hu) PD-L1- tients treated with a variety of cytotoxic regimens such that transduced MC38 colon carcinoma and B16-F10 melanoma cell lines. comparison to historical controls is difficult. Here we present clin- Once measurable tumors were established, mice were treated with ical outcomes in patients treated with carboplatin and paclitaxel anti-mouse checkpoint (i.e. PD-1 or CTLA-4) blocking monoclonal immediately following disease progression on anti-PD1 or anti- antibodies (mAbs) or oxaliplatin, a platinum-based chemotherapeutic PDL1 therapy. agent, alone or in combination with ALPN-202, to evaluate compati- Methods bility of the novel ALPN-202 protein with existing cancer therapies. A chart review was performed to identify all patients with RM- Anti-tumor responses were evaluated by serial tumor volume measure- SCCHN, including tumors originating in the oral cavity, oropharynx, ments and RNA-Seq analysis of tumors isolated from treated mice. hypopharynx, larynx, and sinuses treated with an anti-PD1 or anti- Results PDL1 antibody immediately followed by carboplatin and paclitaxel Anti-PD-1, anti-CTLA-4, or oxaliplatin alone were only modestly ef- through December 2018. Patients were required to have both pre- fective as monotherapy in huPD-L1+ MC38 tumor-bearing mice, treatment and post-treatment imaging associated with treatment while ALPN-202 has potent anti-tumor activity in this model. with both immunotherapy and chemotherapy or documented death When the checkpoint inhibitors or chemotherapy were adminis- from disease after administration of at least 1 cycle of chemotherapy. tered in combination with ALPN-202, significantly greater reduc- Anti-PD1 / PDL1 treatment history, p16 IHC (for OPC), and baseline tions in tumor growth over time were observed than with any of PD-L1 IHC (where available) were assessed. The best overall response these agents alone (Figure 1 and Figure 2,respectively).Further- was assessed by RECIST v1.1 and PFS and OS were determined using more, ALPN-202 was extremely effective (92% tumor growth in- the Kaplan-Meier method. hibition) in improving the anti-tumor activity of anti-PD-1 mAb in Results mice bearing huPD-L1+ B16-F10 tumors, a tumor that is known A total of 11 patients meeting the inclusion criteria were identified to be poorly immunogenic and treatment-recalcitrant (Figure 3). including 5 patients with p16+ SCC of the oropharynx, and 3 with RNA-Seq analysis of tumors from the MC38 studies was per- SCC of the oral cavity. 5 patient has detectable staining for PD-L1 at formed to explore in-depth the mechanisms, including enhance- baseline, 2 did not, and 3 patients had no PD-L1 IHC available. 6 pa- ment of T cell effector transcript expression, that play a role in tients receive pembrolizumab alone or in combination, 4 received the ability of ALPN-202 to provide anti-tumor immunity and to durvalumab, and 1 patient was treated with nivolumab. The median enhance the activity of checkpoint inhibitors and the chemother- duration of prior PD1 / PDL1 exposure prior to chemotherapy was apeutic oxaliplatin. 2.7 months (range 1.4 to 18.2 months). The BORR to carboplatin and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 255 of 272 Conclusions P468 ALPN-202 demonstrates potent anti-tumor efficacy as monother- Targeting the ICOS pathway in combination with apy and significantly improves the anti-tumor activity of other chemotherapy to enhance T-cell mediated anti-tumor immune only modestly effective treatment modalities, such as checkpoint- responses only blockade mAbs and chemotherapy. ALPN-202 has the poten- Tamer Mahmoud, PhD, Jeffrey Riggs, Madhu Ramaswamy, PhD, Leigh tial to be significantly effective as a monotherapy, and its com- Hostetler, Brian Naiman, Ilyssa Ramos, Sean Turman, Fernanda Pilataxi, patibility with checkpoint inhibitors and chemotherapeutics Christopher Morehouse, MD, Alex Alfaro, Norman Peterson, Ryan suggest versatility in its potential to improve outcomes in the Fleming, Nazzareno Dimasi, Kyle Kuszpit, Ronald Herbst, Gianluca frontline setting alone and/or in combination with standard of Carlesso, PhD care of multiple cancer types. A first-in-human clinical study with AstraZeneca, Gaithersburg, MD, United States ALPN-202 is in preparation. Correspondence: Gianluca Carlesso (carlessog@medimmune.com) Ethics Approval Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P468 The study was approved by the vivarium's International Animal Care and Use Committee (IACUC), IR# 17-01. Background ICOS (Inducible T-cell Costimulator) is a member of the CD28 superfamily that is detected on activated and memory T lym- phocytes. Treatment with Immune checkpoint inhibitors (ICI) in clinical studies has shown that expansion of ICOS-expressing T cells is associated with positive patient outcome. Preclinical studies have validated the rationale for targeting ICOS and the clinical investigation of ICOS agonist antibodies (mAbs) com- bined with ICI is under way. To enhance tumor immunogenicity and increase the response rate to immunotherapy, we examined the efficacy of combination therapy of ICOS mAbs with stand- ard of care chemotherapy using a colorectal tumor syngeneic animal model. Methods For the microarray analysis, freshly isolated normal primary hu- man T cells were stimulated with sub-optimal concentration of Fig. 1 (abstract P467). See text for description anti-CD3 mAb with either ICOS, OX40 mAbs, or GITRL-FP for 4 hours. ¬In-vitro proliferation and cytokine secretion assays were performed using primary human T cells stimulated with anti- CD3 mAb and ICOS mAb in the presence of different inhibitors. For in-vivo studies, CT26 tumor cells were subcutaneously injected into BALB/c mice and ICOS or control mAbs were ad- ministered intraperitoneally (ip) or in combination with an intra- venous injection of 5-Fluorouracil (5-FU) at day 10 post tumor implantation. For the biodistribution study, CT26 tumor-bearing mice were dosed ip with 89Zr-labeled anti-ICOS or control mAb and imaged using positron emission tomography (PET)/SCAN. For the depletion study, CT26 tumor-bearing mice were injected ip with either CD4 or CD8 mAb twice a week starting one day before tumor implantation, whereas sphingosine-1 phosphate re- ceptor (S1PR) modulator (FTY720) was orally administered daily starting at day 9 post-implantation. Tumor growth was assessed three times a week. Results Fig. 2 (abstract P467). See text for description Compared to other T-cell agonists, in-vitro co-stimulation of human T cells with ICOS mAb lead to enhanced metabolic T cell reprogramming, activation of the PI3K/mTOR pathways, and secretion of IFN-gamma and IL-10. Immuno-PET scan imaging analysis revealed ICOS expression across tumor and secondary lymphoid tissues. ICOS mAb and 5-FU combination therapy on CT26 tumor-bearing mice resulted in a significant increase in anti-tumor responses compared to single-arm treatment or con- trol groups. Moreover, the efficacy of either anti-ICOS or 5-FU monotherapy or combination required cytotoxic T cell activity and was dependent on the S1P-mediated egress of immune cells from peripheral lymph nodes. Conclusions Collectively, the results of these studies show that ICOS agonism in combination with chemotherapy may provide an effective therapeutic option for the activation of T cells in solid tumor Fig. 3 (abstract P467). See text for description indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 256 of 272 P469 4. Cappello P, et al. Vaccination with ENO1 DNA prolongs survival of Chemotherapy enhances effector T cell responses to tumor genetically engineered mice with pancreatic cancer. Gastroenterology. associated antigens in human and mouse pancreatic cancer 2013;144:1098–106. 1 1 1 Giorgia Mandili, PhD , Claudia Curcio, PhD ,SaraBulfamante, MS , Ethics Approval 1 1 2 Laura Follia, MS , Daniele Giordano, MD , Rossella Spadi, MD ,Maria The study was approved by the local research ethical committee 2 1 Antonietta Satolli, MD , Paola Cappello, PhD MS , Francesco Novelli, (Azienda Ospedaliera Città della Salute e della Scienza di Torino, PhD Turin) and investigations were performed according to the Helsinki 1 2 University of Turin, Turin, Piedmont, Italy; Azienda Ospedaliera Declaration principles. Universitaria Città della Salute e della Scienza di Torino, Turin, Piedmont, Italy Correspondence: Francesco Novelli (franco.novelli@unito.it) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P469 P470 Impact of treatment-induced necrosis in the anti-tumor Background efficacy of Entinostat combined with the immunocytokine Pancreatic Ductal Adenocarcinoma (PDA) is one of the most NHS-IL12 lethal cancer, both for lack of effective screening method and Yohei Ozawa, MD, PhD, Kristin Hicks, PhD, Karin Knudson, PhD, Jeffrey resistance to chemotherapy (CT). Immunotherapy (IT) trials with Schlom, Sofia Gameiro, PharmD, PhD immune check-point inhibitors did not achieve significant gain National Cancer Institute, NIH, Bethesda, MD, United States of survival yet [1]. However, some CT agents, such as gemcita- Correspondence: Sofia Gameiro (Sofia.Gameiro@nih.gov) bine (GEM), have several immune modulating effects [2] and Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P470 starting from the hypothesis that more immunogenic antigens can be induced by CT treatment, its ability to increase the sus- Background ceptibility of PDA to IT was evaluated. Tumor necrosis resulting from hypoxia, inflammation, or ab- Methods normal angiogenesis is associated with poor outcome in sev- Sera from 28 PDA patients before and after CT (BCT and ACT eral solid malignancies. However, theroleoftreatment- respectively) were profiled by serological proteome analysis induced necrosis is still controversial. The class I HDAC inhibi- (SERPA) [3]; the recognized TAAs were identified by mass spec- tor entinostat (Syndax) has been shown to promote significant trometry and confirmed by ELISA. The proliferation, phenotype tumor control in combination with immunotherapies through and cytokine production of T cells were evaluated on patients’ immune-mediated mechanisms. In addition, we observed that PBMCs from the same cohort after in vitro stimulation with entinostat induces tumor necrosis in murine solid tumors. We four selected TAAs (ENO1, G3P, K2C8 and FUBP1). Mice that hypothesize that entinostat-induced necrosis could be tar- spontaneously develop PDA (KC) were treated with GEM prior geted by NHS-IL12 (M9241, Merck KGaA), an IL-12 NHS76 con- of DNA vaccination against ENO1 [4]. Tumor lesions, immune jugate designed to bind free DNA in regions of tumor death/ infiltration and the titer of TAAs-specific antibody were evalu- necrosis, therefore promoting a synergistic anti-tumor effect. ated. TAAs-specific IFNγ production from splenocytes was NHS-IL12 has demonstrated significant anti-tumor efficacy in analyzed. preclinical solid tumor models and has been safely adminis- Results tered to cancer patients. The number of TAAs recognized by IgG in PDA patients’ sera, Methods as well as their ability to induce a complement dependent cyto- The amount of necrosis entinostat induced over time was exam- toxicity against PDA cells, was increased in ACT sera. Some ined in three distinct murine solid tumor models: MC38 (colon), identified TAAs showed a positive correlation between the in- 4T1 (triple-negative breast) and EMT6 (breast). Necrosis was crease of ACT antibody titer and longer patients’ survival. An in- evaluated by histological analysis of H&E staining and a ter- creased T cell TAAs-specific proliferative response after CT and minal deoxynucleotidyl transferase dUTP nick end labeling the evaluation of IFNγ/IL10 ratio was detected, revealing that (TUNEL) assay. To assess if NHS-IL12 could bind to necrotic ATC T cells shifted TAAs-specific responses from regulatory to tumor regions, tumor sections from mice treated with PBS or effector one. After stimulation with TAAs, in mostly patients the entinostat were incubated in vitro with NHS-IL12. The binding ratio between CD8 and CD4 Treg cells was increased in ATC T and distribution of NHS-IL12 in the tumor was then examined cells. The role of CT to enhance the TAAs-specific adaptive re- by immunofluorescence staining usingananti-humansecondary sponse prompt us to exploit its effect in combination with the antibody. DNA vaccination. Of clinical relevance, KC mice treated with Results GEM prior of ENO1 DNA vaccination displayed smaller tumor le- Entinostat promoted tumor control in all three tumor models, sions together with an increase of tumor-infiltrating CD4 and albeit to different degrees. The onset of entinostat-induced ne- CD8, in comparison to mice vaccinated or GEM-treated only. crosis was observed after two weeks of continuous dosing. In Furthermore, CT increased specific antibodies and IFNγ- EMT6 tumors, areas of tumor necrosis identified via H&E stain- producing T cells in vaccinated mice not only against ENO1, the ing also displayed DNA fragmentation measured by the TUNEL target TAA of vaccination, but also against G3P, suggesting an assay. Of note, in the entinostat-treated tumors there were antigen spreading effect of the combinatory treatment. areas of tumor death/necrosis with increased immune cell infil- Conclusions tration identified by H&E staining. Furthermore, initial immuno- Overall these data indicated that in pancreatic cancer CT effect- fluorescence studies indicate that NHS-IL12 binds to these areas ively ameliorates T cell responses against TAA and it might be in the entinostat-treated tumors in vitro. These preliminary find- reconsidered to render them suitable targets for IT. ings are being confirmed utilizing specimens from mice treated with all the agents. Additionally, in the EMT6 model, entinostat and NHS-IL12 syner- References gized to produce significant anti-tumor efficacy, including 1. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients resolving well-established tumors and enhancing survival. The with advanced cancer. N Engl J Med. 2012;366:2455–65. combination therapy promoted tumor infiltration of CD8 T cells 2. Cappello P, et al. Next generation immunotherapy for pancreatic and M1-like macrophages. Ongoing correlative studies in tumor cancer: DNA vaccination is seeking new combo partners. Cancers. specimens are examining the spatial distribution between 2018;10(2):51 entinostat-induced tumor necrosis, NHS-IL12 binding, and CD8 3. Tomaino B, et al. Autoantibody signature in human ductal pancreatic and M1 infiltration. adenocarcinoma. J Proteome Res. 2007;6:4025–31. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 257 of 272 Conclusions Journal of Clinical Oncology, ASCO, 2017 35, e14544. Overall, our results suggest that entinostat-induced necrosis may 2. Pankaj Gaur et al. CXCR4 antagonist (BL-8040) to enhance antitumor promote a tumor microenvironment conducive to NHS-IL12 bind- effects by increasing tumor infiltration of antigen-specific effector T-cells. ing, resulting in significant anti-tumor efficacy. These results in- Journal of Clinical Oncology, ASCO, 2018, 36, 73. form a rationale to examine this combination in the clinic for 3. Manuel M. Hidalgo et al. Evaluation of pharmacodynamic (PD) patients with solid carcinomas. biomarkers in patients with metastatic pancreatic cancer treated with BL- 8040, a novel CXCR4 antagonist. Acknowledgements Journal of Clinical Oncology, ASCO, 2018 36, 88-88. The authors thank Curtis Randolph for his excellent technical assistance. Ethics Approval The study was approved by the Hebrew University Ethics Board, approval number 18-15644-4. P471 Combination of BL-8040, anti PD-1 and chemotherapy significantly reduced pancreatic tumor growth and changed the balance between CD4+/FOXP3+ cells and CD8+ cells in the tumor Amnon Peled, PhD (peled@hadassah.org.il) Hadassah University Hospital, Jerusalem, Israel Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P471 Background Cancer cells shape the tumor microenvironment (TME) to support their growth by recruiting immune suppressing cells such as T regulatory cells, as well as inhibiting the recruitment and activa- tion of effector CD8+ T cells. In this study, we investigated the effect of combining the CXCR4 antagonist BL-8040 [1, 2], anti PD- 1 immune checkpoint inhibitor and chemotherapy of Irinotecan, Fluorouracil and Leucovorin) IFL (on pancreatic tumor immune cell composition and growth. Methods The effect of BL-8040, anti PD-1 and IFL on tumor growth and im- mune cell constitution was assessed using the syngeneic Panc02 tumor mouse model. Tumors were established by s.c. injection . The Fig. 1 (abstract P471). Tumor growth accumulation of immune cells in the TME was assessed by immuno- histochemical staining for CD8, CD4, Foxp3, and CD69. Results Treatment of tumors with anti PD-1 or BL-8040 alone, had no effect on tumor growth, whereas, IFL treatment had significant effect on tumor growth (67% inhibition). Combination of anti PD-1 + IFL, had no significant better effect on tumor growth compared to IFL (p<0.09), whereas, BL-8040 + IFL, had a signifi- cantly better effect on tumor growth compared to IFL (p<0.04). Moreover, IFL + BL-8040 + anti PD-1, had a highly significantly better effect on tumor growth, compared to IFL alone (p<0.004) (Figure 1). The triple combination treatment (TCT), further re- duced tumor growth, compared to chemotherapy alone, by 58%.Inthe TCT, 3out of 8micedid notdevelop tumoratall, compared to 1 mouse that did not develop tumor in the IFL alone group. The TCT did not significantly change the number of CD8+ T cells accumulating in the tumor but increased their activation status (Figure 2A,B). Only in the TCT, the CD8+ T cells Fig. 2 (abstract P471). A, B, C. See text for description were larger in size inside the tumor parenchyma (p<0.01, Fig. 2C) and expressed CD69. Interestingly, we found that tumors treated with the TCT, had significantly reduced numbers of CD4+ and CD4+, Foxp3+ cells (Figure3). Conclusions TCT reduced significantly the number CD4+ and CD4+FOXP3+ cells and increased the numbers of activated CD8+, CD69+ cells in the TME. We hypothesize, that the ability of BL-8040 to modulate the TME may allow better activation of immune effector cells, contributing to the effect of chemotherapy and immunotherapy on tumor growth .A Phase IIa, multicenter, open label trial in patients with metastatic pancreatic cancer (the COMBAT study cohort II, [3]) is currently ongoing to as- sess the effect of the triple combination with BL-8040, +Pem- brolizumab and ILF on disease progression. References 1. Michal Abraham et al. Effect of BL-8040, high-affinity CXCR4 antagonist, on T-cell infiltration, tumor growth, and synergy with immunomodula- Fig. 3 (abstract P471). See text for description tory agents. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 258 of 272 P472 Background STAT3 ASO and Cisplatin combination sensitizes protected tumor While immune checkpoint blockade has revolutionized cancer care, microenvironments to checkpoint-mediated therapy many patients remain refractory to checkpoint inhibition. Converting Theresa Proia, PhD, Maneesh Singh, PhD, Nanhua Deng, Minwei Ye, checkpoint-refractory tumors into checkpoint-responsive tumors is a Frank McGrath, Douglas Ferguson, Simon Barry major challenge for cancer immunotherapy. Interleukin-12 (IL-12) is a AstraZeneca, Waltham, MA, United States potent cytokine that holds potential to reshape the immune environ- Correspondence: Simon Barry (simon.t.barry@astrazenea.com) ment in solid tumors. Its clinical utility, however, has been limited by Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P472 severe toxicities both from systemic administration and from expres- sion by adoptively transferred gene engineered T cells. We report Background here that adoptive cell therapy (ACT) with T cells carrying surface- Chemotherapy-immunotherapy (chemo-IO) combinations are being tethered DeepTM IL-12 overcomes these challenges, enhances T cell explored in the clinic. Immune responses mediated by PD-1 or PD-L1 therapeutic efficacy, activates immune cells in the tumor and over- inhibition may be enhanced by the immunogenic effects of cytotoxic comes resistance to checkpoint blockade. agents, which can increase tumor antigens. Chemo-IO combination Methods strategy relies on drug dose and schedule optimization, to minimize Activity of Deep IL-12 Primed T cells were evaluated in B16-F10 melan- direct T cell killing with chemotherapy, enhance antigen presenta- oma tumors, a cell line resistant to checkpoint inhibition. Mouse PMEL tion, and promote T cell activation. CD8 T cells, which are reactive against the B16-F10 melanoma antigen Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that se- gp100, were surface-tethered with Deep IL-12 and evaluated for anti- lectively targets STAT3, a master regulator of immune suppression, tumor activity in mice bearing B16-F10 melanoma. We additionally and is currently in Ph 1/2 clinical trials in combination with an anti- evaluated effects in the tumor of Deep IL-12 Primed T cells and PMEL T PD-L1 antibody, durvalumab. In preclinical tumor models, we demon- cells alone or co-administered with IL-12. Further studies evaluated strated that mouse surrogate STAT3 ASO remodels the suppressive combinations of Deep IL-12 Primed T cells with checkpoint inhibition. tumor microenvironment to enhance cytotoxic T cell activity in com- Results bination with anti-PDL1 (Singh et al. SITC 2019). ACT of Deep IL-12 Primed PMEL T cells significantly improved tumor Methods growth inhibition and overall survival of B16-F10 tumor bearing mice We sought to maximize the therapeutic benefits of chemo-IO by compared to PMEL T cells alone or combined with systemic co- dose and schedule optimization of adding cisplatin to STAT3 ASO administration of IL-12. In the tumor, Deep IL-12 repolarized im- and anti-PDL1. In patients, cisplatin dose ranges from 50mg/m2- munosuppressive monocytic myeloid-derived suppressor cells (M- 100mg/m2. We modeled relevant preclinical doses in the range of 5- MDSC) into an immune-activating antigen-presenting cell (APC) 10 mg/kg based on mouse plasma exposure, and proceeded with 5 phenotype. Consistent with an anti-tumor role for the repolarized M- mg/kg to represent a low clinical therapeutic (~60mg/m2) dose. MDSC, administration of an antibody to deplete these cells reduced Using the immunogenic MC38 model, various schedules of chemo-IO the efficacy of Deep IL-12 Primed T cells. Further evaluation revealed were explored; (1) Cisplatin priming on day 3 (to increase antigens), high expression of the checkpoint ligand PD-L1 on the repolarized followed by STAT3 ASO/anti-PDL1 on day 7. (2) STAT3 ASO pre- M-MDSC. To test the hypothesis that this limits efficacy of Deep IL-12 treatment on day 3 (to remodel the suppressive tumor microenviron- Primed T cell ACT, Deep IL-12 Primed T cells were co-administered ment), followed by cisplatin/anti-PDL1 on day 7. (3) All 3 agents with PD-L1 blockade. This further improved anti-tumor efficacy, dosed simultaneously on day 7 post implant. resulting in durable long-term responders. Efficacy was further im- Results proved by repeat dosing of Deep IL-12 Primed PMEL T cells. Cisplatin treatment in MC38-tumor bearing mice resulted in variable Conclusions tumor growth inhibition, with one tumor regression. Flow cytometry Our data demonstrates that ACT with tumor-specific T cells carrying analysis confirmed that cisplatin treatment had no detrimental effect surface-tethered Deep IL-12 repolarizes suppressive M-MDSC in the on T cell number or functionality, and showed a trend of increased tumor, enhances anti-tumor efficacy, and synergizes with checkpoint CD11b+/Ly6C+ dendritic cells, providing confidence to explore effi- inhibition in a checkpoint refractory cancer model. Torque is apply- cacy of the triplet combination. ing this approach to develop a novel adoptive cell therapy for can- Regardless of schedule, we observed 20% complete response rate in cer, Deep IL-12 Primed multi-targeted T cells (TRQ12-01), which is the triplet combinations, compared with 0 complete responses in expected to start clinical evaluation in 2019. any other treatment group; but interestingly, schedule 1 required dose holidays. Flow cytometry analysis of the triplet compared with P474 vehicle revealed enhanced CD4 T cell functionality (1.6x increase Efficacy and toxicity evaluation of anti-human CD47 and SIRPa IFNγ, p<0.001, and 1.2x increase IL-2, p=0.001), and enhanced NK antibodies in genetically humanized B-hSIRPa/hCD47 mice functionality (1.3x increase Granzyme B+, p<0.01; 4.3x increase TNFα, Frank An, Yanan Guo, Jie Xiang, Chaoshe Guo p<0.001). Biocytogen, Boston, MA, United States Conclusions Correspondence: Jie Xiang (info@biocytogen.com); Chaoshe Guo Collectively, we have generated data to support the combination of (info@Biocytogen.com) low-dose chemotherapy and immunotherapy to enhance the anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P474 tumor immune response. As chemo-IO combinations are being ex- plored in the clinic, it will be important to optimize dose and sched- Background ule to minimize toxicity and maximize therapeutic benefit. CD47 is a transmembrane protein expressed ubiquitously on the sur- face of human cells, including. SIRPa, one of the binding partners of P473 CD47, is a member of the signal-regulatory-protein (SIRP) family which Adoptive transfer of Deep IL-12 Primed T cells increases sensitivity is expressed on many myeloid cells including phagocytic cells. Engage- to PD-L1 blockade for superior efficacy in checkpoint refractory ment of SIRPa by CD47 elicits the “do not eat me” signal to prevent tumors phagocytosis of “self” cells by macrophages. Anti-CD47 and anti-SIRPa 1 1 2 Gulzar Ahmad, PhD , Jonathan Nardozzi, PhD , Lars Petersen, PhD , antibodies that interfere with the CD47-SIRPa interaction have demon- 2 2 1 Esben Christensen, MSc , Ditte Jaegher , James Suchy, PhD , Karsten strated promise in clinical trials as new class of therapeutics. Despite 1 1 1 Sauer, PhD , Douglas Jones, PhD , Thomas Andresen, PhD such exciting potential, side effects associated with CD47/SIRPa block- 1 2 Torque Therapeutics, Cambridge, MA, United States; Denmark ade such as anemia may represent a significant toxicity concern. There- Technical University, Kgs. Lyngby, Denmark fore, evaluation of both efficacy and toxicity of human CD47/SIRPa Correspondence: Thomas Andresen (tandresen@torquetx.com) antibody candidates emerges as one of most actively investigated Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P473 areas in immuno-oncology in recent years. However, lack of animal Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 259 of 272 models that enable expedient testing of anti-human CD47 or anti- further increased with loss of both PD1 and LAG3, as a result of en- human SIRPa antibodies in vivo has been a limiting factor for CD47/ hanced proliferation (Ki67/BrdU) – a phenotype demonstrated to be SIRPa antibody development. intrinsically regulated in the co-adoptive transfer system. Although Methods expression of TIM3, TIGIT and 2B4 IRs that normally co-express with To accelerate direct efficacy and toxicity testing of anti-human CD47 PD1 are maintained, CD8+ TIL isolated from Pdcd1L/L E8ICre.GFP and SIRPa antibodies, Biocytogen has generated the double human- and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP show increased functionality ized mice, B-hSIRPa/hCD47, where the human extracellular domains (IFNg, TNFa and GzmB release) by flow cytometry. Moreover, CD8+ of SIRPa and CD47 replace their respective murine counterparts. TIL polyfunctionality is evident with PD1/LAG3 loss that was largely Homozygous B-hSIRPa/hCD47 mice express humanized but not the driven by effector and chemoattractive secretions by analysis with a wild type mouse SIRPa and CD47. Further, we also created two triple 28-plex single-cell cytokine response panel (Isoplexis). humanized mouse strains, B-hPD1/hSIRPa/hCD47 and B-hPD-L1/ Conclusions hSIRPa/hCD47, where humanized PD1 and PD-L1 extracellular do- Overall, these data suggest that PD1 and LAG3 synergize to have a mains replace their mouse counterparts, respectively, in the B- dominant effect on CD8+ TIL and limit antitumor immune effects, as hSIRPa/hCD47 background. intrinsic removal of both IRs results in reduced B16-F10 tumor Results growth which has a substantive impact on the development of sys- We present here that B-hSIRPa/hCD47 mice were successfully used temic anti-tumor immunity. These results are encouraging for the for screening anti-human CD47 and anti-human SIRPa antibodies for continued development of LAG3 targeting agents in the clinic, which efficacy and toxicity in tumor models of the engineered MC38- would hopefully yield improved clinical responses in combination hCD47 cell line that expresses human CD47 in MC38 cells. Anti- with anti-PD1. human CD47 and anti-human SIRPa antibodies were efficacious in controlling MC38-hCD47 tumor growth in B-hSIRPa/hCD47 mice. Var- P476 ied toxicity profiles were observed in terms of body weight loss, The combination of a STING agonist with cytokines results in blood cell counts, and blood liver enzyme levels in anti-human CD47 robust anti-tumor effects in autochthonous tumor models antibody treatments. Anti-human PD-1 and anti-human CD47 anti- 1 1 1 2 Cristina Blaj, PhD , Yingjoy Li , Allen Chen , Anthony Descien, PhD , bodies showed single agent and combination anti-tumor effect in B- 2 2 3 Brian Francica, PhD , Sarah McWhirter , Lora Picton , K. Christopher hPD1/hSIRPa/hCD47 mice. So did anti-human PD-L1 and anti-human 3 1 Garcia , David Raulet, PhD CD47 antibodies in B-hPD-L1/hSIRPa/hCD47 mice. 1 2 University of California Berkeley, Berkeley, CA, United States; Aduro Conclusions Biotech Inc, Berkeley, CA, United States; Stanford University School of Taken together, we have validated three double and triple human- Medicine, Stanford, CA, United States ized CD47/SIRPa mouse models and demonstrate that these human- Correspondence: Cristina Blaj (cristina.blaj@berkeley.edu) ized mice are useful tools in facilitating development of therapeutics Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P476 targeting human CD47/SIRPa. Background P475 Cancer immunotherapies based on immune checkpoint blockade are PD1 and LAG3 converge to limit polyfunctionality and systemic highly effective, but only in a limited number of tumor types. Even in immunity transplanted preclinical models, monotherapy rarely results in cures. 1 2 2 Lawrence Andrews, PhD , Sasikanth Manne , E. John Wherry, PhD , Creg Moreover, preclinical subcutaneous models appear to be more respon- 1 1 Workman, PhD , Dario Vignali, PhD sive to therapies than preclinical carcinogen or genetically engineered 1 2 University of Pittsburgh, Pittsburgh, PA, United States; University of mouse (GEM) autochthonous models of cancer, or human tumors. To Pennsylvania, Philadelphia, PA, United States extend immunotherapy to a broader range of tumor types, our strategy Correspondence: Dario Vignali (dvignali@pitt.edu) is to rationally design combination immunotherapies with the potential Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P475 to boost innate and adaptive immune responses and overcome im- munosuppressive environments characteristic of human tumors. Our Background regimen includes a stimulator of interferon genes (STING) agonist, cyto- Targeting PD1 has yielded clinical success across a variety of tumor kines and checkpoint inhibitors. types, yet a significant proportion of patients remain unresponsive to Methods treatment. Thus, overcoming inhibitory receptor (IR)-mediated toler- Autochthonous tumors induced by a carcinogen or in GEM models, ance is essential to improve immunotherapeutic responses. Co- as well as subcutaneous models derived from the GEM models, were expression of PD1 and LAG3 on CD8+ tumor-infiltrating T cells (TIL) is treated with combination immunotherapies. We used flow cytometry, associated with an exhausted phenotype, exemplified by a severe de- immunofluorescence, Luminex assays and qPCR to characterize the fect in cytokine production, cytolytic activity and inability to proliferate. immune cell infiltration, activation status, receptor expression and se- In a number of murine tumor models, dual PD1/LAG3 blockade syner- cretion of cytokines in response to therapy. gistically limits tumor growth greater than targeting PD1 alone, yet the Results relative and synergistic contributions of PD1 and LAG3 on CD8+ T cells Intratumoral injection of cyclic dinucleotides (CDNs, specifically ADU- in preventing effective anti-tumor immunity is unknown. S100, a STING agonist) resulted in ~20% stable regressions of estab- Methods lished transplanted tumors and immunity to re-challenge in a sub- To understand the cellular and mechanistic basis for PD1/LAG3 syn- cutaneous sarcoma model. The same protocol resulted in significant ergy, conditional knockin mice “surgically dissect” Pdcd1 and/or Lag3 tumor growth delay in autochthonous GEM models and a carcinogen floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. To allow model. Antibody-mediated depletion studies revealed that natural for intrinsic analysis of PD1 and/or LAG3 on antigen-specific CD8+ T killer (NK) cells as well as CD4 and CD8 T-cells played an important cells, these mice have been generated as a pmel-1 transgenic back- role in mediating the anti-tumor effects. The combination of CDNs ground, with each mutant strain uniquely congenically marked for use and an IL-2 superkine resulted in synergistic anti-tumor efficiency in in a co-adoptive transfer system allowing analysis of PD1 and/or LAG3- all models tested. deficient CD8+ T cells, and controls, in the same host. Autochthonous tumors are clinically more relevant, as they resemble Results the tumor resistance signature observed in human cancers. Our sar- Mice with CD8+ T cells deficient in PD1 or PD1 and LAG3 (Pdcd1L/L coma models represent excellent platforms to dissect the differences E8ICre.GFP and Pdcd1L/L Lag3L/L-yfp E8ICre.GFP, respectively) show between the refractory GEM models and the more responsive trans- attenuation of B16-F10 tumor growth with improved survival, com- planted tumors. Flow cytometry and immunofluorescence analysis pared to LAG3-deficient mice (Lag3L/L-yfp E8ICre.GFP) and controls revealed the prevalence of tumor associated macrophages in the (E8ICre.GFP). CD8+ TIL frequency is increased with loss of PD1, and GEM models, consistent with the established role of macrophages in Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 260 of 272 promoting an immunosuppressive environment. Additionally, we dis- played by TGFb1 in this model. Based on these promising data, we covered a number of differentially secreted cytokines that might play have generated novel humanized 13A1 variants with in-vitro charac- a role in the anti-tumor response. The roles of macrophages and cy- teristics similar to the parental antibody. tokines are being tested. Conclusions Conclusions Isoform-specific blockade of active TGFb1 with antibody 13A1 is as The combination of cyclic dinucleotides with an IL-2 superkine efficacious or better than pan-TGFb blockade in enhancing the anti- produced strong antitumor effects in transplanted and autoch- tumor efficacy of PD-L1 checkpoint therapy in mice with established thonous tumor models and may translate into an efficacious ap- EMT6 tumors. Humanized 13A1 antibodies that maintain the in-vitro proach to treat human cancers. Our studies provide indications features of the biologically active parental murine 13A1 antibody are for additional targets that might modulate the tumor microenvir- therefore attractive clinical candidates for combination therapy with onment to enhance antitumor responses and synergize to en- checkpoint antibodies, especially in patients with low response rates hance immunotherapy effects. to current anti-checkpoint mono-therapies. Ethics Approval The study was approved by the University of California Berkeley Insti- References tutional Animal Care And Use Committee, approval number AUP- 1. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, de 2015-1-8058-1. Gramont A. Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther. 2015;147:22-31; Colak S. and Ten Dijke P. Targeting TGFb signaling in cancer. Trends Cancer 2017; 3(1):56-71.; Tauriello, D.V.D., Palomo-Ponce P477 S., et al. TGFb drives immune evasion in genetically reconstituted colon Isoform-specific blockade of active TGFb1 with mAb 13A1 cancer metastasis. Nature, 2018; 554:538-543 enhances the efficacy of PD-L1 checkpoint therapy in a EMT6 2. Poniatowski LA, Wojdasiewicz P, Gasik R, Szukiewicz D. Transforming mouse tumor model growth factor beta family: Insight into the role of growth factors in 1 2 2 Matteo Brioschi , Jacques Van Snick, PhD , Catherine Uyttenhove , regulation of fracture healing biology and potential clinical applications. 3 4 5 Pamela Cheou , George Coukos, MD, PhD , Gerd Ritter , Steven Dunn, Mediators Inflamm. 2015; 2015:137823 PhD 3. Uyttenhove C, Marillier RG, Tacchini-Cottier F, Charmoy M, Caspi RR, UNIL/Ludwig Cancer Research - Lausanne, Epalinges, Switzerland; Damsker JM, Goriely S, Su D, Van Damme J, Struyf S, Opdenakker G, Van 2 3 Ludwig Cancer Research - Brussels, Brussels, Belgium; University of Snick J. Amine-reactive OVA multimers for auto-vaccination against cyto- Louvain, Brussels, Belgium; CHUV/Ludwig Cancer Research - Lausanne, kines and other mediators: perspectives illustrated for GCP-2 in L. major Epalinges, Switzerland; Ludwig Cancer Research, New York, NY, United infection. J Leukoc Biol. 2011 Jun; 89(6):1001-1007 AND Brioschi M., States Cheou P., van Snick, J., Uyttenhove C., Coukos, G., Ritter, G., Dunn, S. Jour- Correspondence: Steven Dunn (steven.dunn@chuv.ch) nal for ImmunoTherapy of Cancer 2018, 6(Suppl 1):115 Abstr. Nr. 482 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P477 4. Gupta A, Budhu S, Giese R, van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Isoform specific TGF-β inhibition in combin- Background ation with radiation therapy as a novel immune therapeutic ap- TGFb is a highly pleiotropic cytokine implicated in tumor escape proach to cancer therapy. Journal for ImmunoTherapy of Cancer and progression. Targeting TGFb has recently emerged as an ex- 2017; 5 (suppl 2):87 Abstract 326 AND Gupta A, Budhu S, Giese R, citing new approach to overcome TGFb-mediated resistance to van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Target- checkpoint cancer immunotherapy [1]. In humans, three isoforms ing specific TGF-β isoforms in combination with radiation therapy of TGFb (TGFb1, -2 and -3) have been shown to individually drive leads to differential antitumor effects in mouse models of cancer. context-dependent physiological and phenotypic responses [2]. Cancer Res. 2018; 78(13 suppl): Abstract 4716 Most current therapeutic TGFb reagents do not adequately distin- 5. Mariathasan S., Turley S.J. et al. TGFb attenuates tumor response to PD-L1 guish among the three TGFb isoforms and could give rise to on- blockade by contributing to exclusion of T cells. Nature, 2018; 554:544- target off-tumor toxicity, undesirable inflammatory adverse events or lack of activity. To address this unmet need for more selective therapeutic reagents, we generated a panel of murine antibodies with mono-isoform specificity for TGFb1 (13A1) or TGFb3 (1901) P478 and successfully humanized these antibodies, carefully maintain- SEMA4D antibody blockade overcomes mechanisms of immune ing their selective specificity and neutralization potency [3]. The suppression and combination immunotherapy including TGFβ murine TGFb isoform-specific antibodies enhanced anti-tumor effi- blockade promotes efficient tumor regression 1 1 1 cacy in-vivo in B16 melanoma and 4T1 breast cancer models [4]. Terrence Fisher, PhD , Crystal Mallow, BS , Holm Bussler, PhD , Sebold 1 1 1 2 We now expanded our in-vivo studies into the immune-exclusion- Torno, BS , Desa Rae Pastore , Alan Howell, MS , Luis Ruffolo, MD , 2 2 1 1 type tumor model EMT6, in which a pan-specific TGFb antibody Nicholas Ullman , Brian Belt, JD , Joe Bucukovski , Christine Reilly, BS , 2 1 2 was shown to overcome TGFb mediated resistance to PD-L1 Benjamin Dale , Ernest Smith, PhD , David Linehan, MD , Maurice 1 1 checkpoint therapy [5]. Zauderer, PhD , Elizabeth Evans, PhD 1 2 Methods Vaccinex, Rochester, NY, United States; University of Rochester, Efficacy of the murine TGFb1 antibody 13A1 and a pan-TGFb anti- Rochester, NY, United States body (1D11) in combination with anti-mPD-L1 were evaluated in Correspondence: Elizabeth Evans (eevans@vaccinex.com) established orthotopic EMT6 murine breast carcinomas. Antibody Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P478 humanization was performed using molecular engineering, combin- ing framework grafting, competitive screening and selective back Background mutations guided by assaying for TGFb neutralization potency in Despite progress of immune checkpoint blockade therapies, resist- TMLEC reporter cells. Further humanized 13A1 variants with distinct ance mechanisms including myeloid suppression and upregulation kinetic properties were generated by error-prone mutagenesis and of TGFβ signaling prevent durable clinical benefit in many cancer pa- selective library screening. tients. Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody pro- Results motes immune infiltration, reduces immunosuppression, and We found that isoform-specific neutralization of active TGFb1 with enhances T cell activity in the tumor microenvironment (TME), result- mAb 13A1 was highly efficacious in overcoming the low efficacy of ing in increased tumor control in preclinical models when combined PD-L1 checkpoint mono-therapy, enhancing control of tumor growth with various immunotherapies [1,2]. Clinical trials of immune check- and increasing survival of mice with established EMT6 tumors. Fur- point inhibitors (ICI) in combination with pepinemab (VX15/2503), a ther, 13A1 appeared at least as potent as the pan-TGFb antibody humanized anti-SEMA4D antibody, are currently underway in several when combined with anti-PD-L1, highlighting the dominant role cancer indications. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 261 of 272 Methods Background Activity of anti-SEMA4D antibody in combination with immune Tumor necrosis factor receptor 2 (TNFR2) is central to immune bal- checkpoint inhibitors and TGFβ blockade was evaluated in preclinical ance control in humans and mice. We created human-directed anti- mouse tumor models. Ongoing clinical trials of immune checkpoint TNFR2 antibodies as a therapeutic approach in cancer, with positive inhibitors (ICI) in combination with pepinemab include: (i) a Phase findings [1]. We have now identified a murine-directed surrogate an- 1b/2a combination trial of pepinemab with avelumab in ICI naïve or tagonistic antibody to TNFR2. This antibody shares traits identified in ICI refractory or relapsed NSCLC (CLASSICAL-Lung) (NCT03268057, N= our human-directed antibodies as critical to limiting regulatory T cell 65); (ii) neoadjuvant integrated biomarker trials in patients with (Treg) expansion and activating T effector (Teff) cells. Here we metastatic melanoma (NCT03769155, n=36), metastatic colorectal, present data on this surrogate anti-TNFR2 antibody in two syngeneic pancreatic (NCT03373188, n=32) and head and neck (NCT03690986, mouse models of colon cancer. n=36) cancers treated with pepinemab in combination with nivolu- Methods mab or ipilimumab. Gene expression and immunohistochemical ana- We studied the therapeutic effects of solo and combined immuno- lysis are employed to evaluate changes in immunophenotype as well therapy using the murine-directed anti-TNFR2 antibody in CT26 and as TGFβ-induced effects on TME and tumor progression. MC38 colon tumor models. We compared the new mouse surrogate Results anti-TNFR2 antibody therapy, a commercially available anti-PD1 ther- Anti-SEMA4D antibody enhanced tumor regression when combined apy, and anti-TNFR2/anti-PD1 combination immunotherapy. Mice with antibodies targeting CTLA-4, PD-1, PD-L1, LAG3, and TGFβ in sev- were dosed bi-weekly (100ug/mouse antibody). Antigen-specific CD8 eral preclinical models. For example, anti-SEMA4D plus anti-TGFβ treat- and Treg infiltrates were also studied. ment resulted in maximal tumor growth delay (TGD) of 239% (p<0.01) Results and 10/15 complete tumor regressions (CR) (p<0.05), compared to 10% In the CT26 model, anti-TNFR2 antagonism alone or co-treatment TGD and 0/13 CR with single agent anti-TGFβ or 29% TGD and 1/10 CR with anti-PD1 and anti-TNFR2 was highly efficacious (55-62% of mice with anti-SEMA4D alone in MC38 colon carcinoma model. SEMA4D cured). Anti-PD1 alone was less efficacious (25% cured). In the MC38 blockade reversed expression of genes related to EMT. Additionally, the model, therapy with anti-TNFR2 alone showed some efficacy (20% combination of anti-SEMA4D, folfirinox, and ICI improved survival in cured) and anti-PD1 alone had the least efficacy (10% cured), but KP2-tumor bearing mice, a KPC-derived pancreatic adenocarcinoma anti-PD1 in combination with anti-TNFR2 yielded the best overall sur- model of immune exclusion, myeloid suppression and active TGFb sig- vival (70% cured). Sequential antibody dosing with anti-PD1 followed naling. In clinical trials, pepinemab was well-tolerated and analysis of by anti-TNFR2 yielded no synergy. In contrast, sequential treatment pre- and on-treatment biopsies revealed increased CD8:FoxP3 ratios with anti-TNFR2 first followed by anti-PD1 or the combination of and reduced presence of myeloid derived suppressor cells within TME. anti-TNFR2 plus anti-TNFRF2 showed synergy and highest efficacy. Conclusions Anti-TNFR2 therapy was distinct from anti-PD1 therapy in showing SEMA4D antibody blockade modulates the TME to enhance anti- pronounced regulatory Treg depletion and enhanced Teff infiltration tumor immunity and combination therapies further enhance anti- in the tumor microenvironment, demonstrating in vivo specificity for tumor activity and overcome important resistance mechanisms. Pre- disease-causing cells only in the tumor. liminary data suggest the combination of pepinemab plus immune Conclusions checkpoint therapy is well tolerated and shows initial signals of anti- Anti-TNFR2 immunotherapy provides benefits in two colon cancer tumor activity in patients. Ongoing analysis of various therapeutic models, both as a single agent and when administered in combin- combinations and immunophenotyping of tissue biopsies will shed ation with anti-PD1. Anti-PD1 before anti-TNFR2 was associated with light on mechanism of action of SEMA4D antibody blockade in sev- poor outcomes for survival, histology and lack of long-term cure, eral combination therapies. suggesting that non-specific unleashing of the immune system with anti-PD1 destroys the tumor microenvironment specificity of anti- Acknowledgements TNFR2. These results highlight the value of anti-TNFR2 antagonism We would like to thank the clinical and research teams at Emory University, in vivo in mouse tumor models as solo therapy or as a combination including Doctors Greg Lesinsky, Christina Wu, Conor Steuer, Nabil Saba, therapy, administered first or concurrently with anti-PD1. This study Michael Lowe, Ragini Kudchadkar, and Brian Olson. We also extend gratitude of new immunotherapy combinations highlights the need to test to the Avelumab team at EMD Serono, as well as clinical investigators and both single agent therapy as well as the sequencing of combination their teams related to the CLASSICAL-Lung trial. therapy as new agents are brought forward to the clinic. Trial Registration NCT03268057 References NCT03769155 1. Torrey H, Butterworth J, Mera T, et al.Targeting TNFR2 with antagonistic NCT03373188 antibodies inhibits proliferation of ovarian cancer cells and tumor- NCT03690986 associated Tregs. Sci Signal. 2017;10(462). pii: eaaf8608. Ethics Approval References Mice were tested and monitored for tumor growth by either Champions 1. Evans EE, et al. Antibody Blockade of Semaphorin 4D Promotes Immune Oncology (Hackensack, NJ) or a third-party pharmaceutical company in Infiltration into Tumor and Enhances Response to Other accordance with their animal welfare guidelines. Immunomodulatory Therapies. Cancer Immunol Res. 2015;3(6):689-701. 2. Clavijo PE, Friedman J, Robbins Y, Moore EC, Smith ES, Zauderer M, Evans P480 EE, Allen CT. Semaphorin4D inhibition improves response to immune Heterologous prime-boost vaccination safely enhances antitumor checkpoint blockade via attenuation of MDSC recruitment and function. immunity to the colorectal antigen GUCY2C Cancer Immunol Res. 2019;7(2):282-291 John Flickinger, BS, Robert Carlson, Jagmohan Singh, Trevor Baybutt, BS, Elinor Leong, Alicja Zalewski, Amanda Pattison, Jeffrey Rappaport, Joshua P479 Barton, Scott Waldman, Adam Snook, PhD Evaluation of a TNFR2 antibody with and without anti-PD-1 Thomas Jefferson University, Philadelphia, PA, United States therapy in two murine colon cancer models Correspondence: Adam Snook (adam.snook@jefferson.edu) Katie Case, Lisa Tran, Hui Zheng, Michael Yang, Denise Faustman, MD, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P480 PhD Massachusetts General Hospital/Harvard Medical School, Charlestown,, Background MA, United States The transmembrane receptor guanylyl cyclase C (GUCY2C) is an Correspondence: Denise Faustman (faustman@helix.mgh.harvard.edu) emerging target for colorectal cancer immunotherapy. Recently, an Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P479 adenovirus-based vaccine against GUCY2C was tested in a phase I Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 262 of 272 clinical trial where it was found to safely induce GUCY2C- Although paradoxically, obesity has been found to improve re- specific immune responses [1]. However, GUCY2C immune re- sponse to immunotherapy in a subgroup of patients with mel- sponses following immunization wane over time and optimal anoma [4,5]. GUCY2C immunity may require multiple GUCY2C vaccinations. Methods Moreover, repeated vaccination utilizing adenovirus-based Wildtype C57BL/6femalemicewererandomizedto ahigh-fat vectors is hindered by the production of adenovirus-specific (60%) or low-fat standard chow (14%) diet for 16 weeks to gen- antibodies following first vaccination. For this reason, we have erate diet-induced obese (DIO) or age-matched lean controls, generated a recombinant strain of Listeria monocytogenes respectively. Animals were then challenged with the syngeneic secreting GUCY2C (Lm-GUCY2C) to boost GUCY2C immune E0771 mammary carcinoma cell line. Tumor outgrowth was responses. These studies assess the immunogenicity, thera- quantified by caliper measurements, bioluminescent imaging peutic efficacy, and safety of a heterologous prime-boost (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, immunization utilizing adenovirus and Listeria monocytogenes and endpoint tumor weights. Once tumors were palpable, ani- vectors. mals were randomized to receive no therapy or immunotherapy Methods consisting of intratumoral CpG co-administered with non- T-cell responses following vaccination were assessed by IFNy ELISpot. replicative adenovirus (Ad) encoding murine TNF-related apop- Tumor protection following vaccination was assessed by challenging tosis inducing ligand (TRAIL; AdT). Whole tumor immunogenetic mice with a luciferase-expressing CT26 colorectal cancer cell line. Lu- gene expression profiles were evaluated using nanoString and minescence following luciferin injection and overall survival were immune populations were assessed via multi-parameter flow cy- quantified. Safety was assessed by histopathologic evaluation of tometry. T cell cytokine production was evaluated via flow cy- known GUCY2C-expressing tissues following vaccinations. tometry following ex vivo CD3/CD28 stimulation. Results Results Construction of Lm-GUCY2C was validated by GUCY2C western DIO mice had significantly increased body weights at tumor blot on J774A.1 macrophage cells infected with Lm-GUCY2C. challenge versus lean controls (45 versus 25 grams, p <0.0001) Optimal GUCY2C immunogenicity was achieved utilizing All methodologies demonstrated that obesity significantly in- adenovirus-GUCY2C to ‘prime’ GUCY2C immune responses with creases primary mammary tumor outgrowth and alters cellular Lm-GUCY2C to ‘boost’ and was found to be superior to homolo- and immunogenetic profiles within the tumor microenviron- gous administration using either adenovirus-GUCY2C or Lm- ment. Notable alterations include significant reductions in the GUCY2C vectors. Similarly, anti-tumor studies found heterol- frequency of CD4+ T cells, CD8+ T cells, and CD19+ B cells; ogous administration of adenovirus-GUCY2C and Lm-GUCY2C to with a simultaneous increase in the frequency of myeloid- be superior to homologous administrations. Importantly, histo- derived suppressor cells (MDSCs). Following immunotherapy ad- pathologic evaluation of mice following heterologous prime- ministration, lean animals controlled tumor growth whereas DIO boost revealed no toxicity. animals experienced progressive tumor growth. Despite these Conclusions differential tumor outcomes, both lean and DIO animals dis- Heterologous prime-boost vaccination utilizing adenovirus and played robust intratumoral effector CD8+ T cell accumulation Listeria vectors expressing the tumor antigen GUCY2C demon- and ex vivo function. In contrast, immunotherapy reduced the strate superior immunogenicity and antitumor efficacy over intratumoral accumulation of monocytic and granulocytic MDSCs homologous immunization with either vector, a strategy that only in lean animals. Both MDSC populations persisted in the can be translated to colorectal cancer patients. tumors of animals with DIO, resulting in less favorable effector CD8+ T cell to MDSC ratios. Acknowledgements Conclusions The authors thank the Center for Cell and Gene Therapy, Baylor College of Our data implicate obesity as a causal factor in impairing im- Medicine for assistance in adenovirus vaccine manufacturing. munotherapeutic efficacy in a pre-clinical model of breast can- cer, potentially via accumulation of MDSCs. Our data suggest Reference that clinical investigation and consideration is needed for fac- 1. Snook AE, BaybuttTR, XiangB,Abraham TS,Flickinger JC,HyslopT, tors such as body composition and body mass index when Zhan T, Kraft WK, Sato T, and Waldman SA. Split tolerance permits treating breast cancer patients with immunotherapy. safe Ad5-GUCY2C-PADRE vaccine-induced T-cell responses in colon cancer patients. J. Immunother Cancer. 2019;7, 104. Acknowledgements Ethics Approval This study was supported in part by NIH-NIGMS training grant T32GM008111 Studies were approved by the Thomas Jefferson University IACUC (Protocol # to JTG. 01956). References P481 1. Incio, J., et al., Obesity promotes resistance to anti-VEGF therapy in breast Obesity impairs immunotherapeutic efficacy in pre-clinical breast cancer by up-regulating IL-6 and potentially FGF-2. Sci Transl Med, 2018. cancer 10(432). Justin Gibson, BS, Rachael Orlandella, BS, William Turbitt, Robert Sorge, 2. Sheng, X., et al., Adipocytes Sequester and Metabolize the PhD, Lyse Norian, PhD Chemotherapeutic Daunorubicin. Mol Cancer Res, 2017. 15(12): p. 1704- University of AlabamaBirmingham, AL, United States 1713. Correspondence: Lyse Norian (lnorian@uab.edu) 3. Lehuede, C., et al., Adipocytes promote breast cancer resistance to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P481 chemotherapy, a process amplified by obesity: role of the major vault protein (MVP). Breast Cancer Res, 2019. 21(1): p. 7. Background 4. McQuade, J.L., et al., Association of body-mass index and outcomes in Obesity has long been known to worsen prognosis and survival patients with metastatic melanoma treated with targeted therapy, im- for breast cancer patients. Recent reports further indicate that munotherapy, or chemotherapy: a retrospective, multicohort analysis. obesity negatively impacts response to targeted anti-VEGF ther- Lancet Oncol, 2018. 19(3): p. 310-322. apy [1] and efficacy of chemotherapeutics [2,3]. However, no 5. Wang, Z., et al., Paradoxical effects of obesity on T cell function during studies have yet investigated the impact of obesity on re- tumor progression and PD-1 checkpoint blockade. Nat Med, 2019. 25(1): sponse to immunotherapy in the context of breast cancer. p. 141-151. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 263 of 272 P482 P483 STACT: A novel therapeutic platform that delivers TLR enhanced GVAX elicits tumor-specific tissue resident memory immunomodulatory payloads to tumor-resident myeloid cells After T cells independent of T cell priming IV dosing and demonstrates potent anti-tumor efficacy in Michael Korrer, Young Kim, MD, PhD, David Taylor preclinical studies Vanderbilt University Medical Center, Nashville, TN, United States Laura Glickman, PhD, Christopher Rae, PhD, Alexandre Iannello, PhD, Correspondence: Young Kim (young.j.kim@vanderbilt.edu) Anastasia Makarova, PhD, Haixing Kehoe, MS, John Faulhaber, Bill Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P483 Hanson, Christopher Thanos, PhD Actym Therapeutics, Inc, Berkeley, CA, United States Background Correspondence: Christopher Thanos (cthanos@actymthera.com) GVAX, a genetically modified whole cell vaccine, is proposed to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P482 work by recruiting and activating antigen-presenting cells which then traffic to the draining lymph node and elicit tumor-specific Background Tcells.Inmouse models GVAX haslittle therapeutic benefit as Many experimental therapies developed to promote proper T- a monotherapy, but when combined with a Toll-like receptor cell infiltration in immune-excluded tumors are too toxic for sys- (TLR) 4 adjuvant, TLR Enhanced GVAX (TEGVAX) significantly re- temic administration, which will be required in a metastatic dis- duces tumor burden. Paradoxically, the increased therapeutic ease setting. These include innate targets such as STING and benefit of TEGVAX corresponds with a decrease in delivery of TLR agonists, co-stimulatory receptor agonists, and type I/II tumor antigen to the draining lymph node. In order to improve cytokine receptor combinations. To address these limitations, the efficacy of the TEGVAX platform, it is critical to understand we have engineered a highly attenuated, microbial-based im- the mechanism by which it induces anti-tumor immune re- munotherapy platform called STACT (S. Typhimurium Attenuated sponses. Since TEGVAX requires T cells to work, yet significantly Cancer Therapy). Upon IV administration, the microbe traffics to reduces antigen delivery to the draining lymph node, we and enriches in the tumor microenvironment. There, it is specif- hypothesize that TEGVAX functions independent of lymph node ically phagocytosed and lysed by tumor-resident myeloid cells, priming. enabling efficient delivery of plasmids encoding immunomodu- Methods latory payloads. Using our proprietary platform, we have gener- B16-mOVA cells injected s.c. into B6 mice. TEGVAX (1e6 B16- ated multiple systemically-administered therapies that target mOVA + 1e5 B78H1-GM Irradiated cells + 20ug MPLA) injected several well-characterized, yet intractable immune pathways. s.c. into opposite flank 5 days after tumor injection. 10ug daily Characterization of STACT microbes encoding constitutively ac- FTY720 i.p. on day 4. tive STING variants (STACT-STING) and IL-2 (STACT-IL2) are pro- Results vided as examples. To determine if TEGVAX alters the priming of CD8 T cells, we Methods performed longitudinal studies of OT-1 CD8 T cell proliferation The STACT platform strain has been engineered using precision in vivo comparing TEGVAX to GVAX. We found that GVAX in- genome modifications for enhanced tolerability, reduced im- duced rapid proliferation of OT-1 cells, whereas TEGVAX failed to munosuppressive inflammation, and tumor specificity. STACT- induce OT-1 cell proliferation as shown by FACS and in vivo im- mediated delivery of immunomodulatory proteins in primary aging. We then determined if TEGVAX required myeloid or NK mouse and human cells was confirmed by in vitro functional as- cells to reduce tumor burden. We found that TEGVAX reduced says. STACT strains were evaluated in vivo for tumor-specific en- tumor burden in NK depleted, but not myeloid cell depleted richment, payload delivery, tolerability, and therapeutic efficacy mice. These results raised the question if priming in the draining following IV administration in several subcutaneous syngeneic lymph node was required for therapeutic efficacy of TEGVAX. To tumor studies. determine this, we performed tumor growth studies with mice Results administered FTY720, which sequesters circulating T cells in STACT was found to be 100,000-fold enriched in tumors, relative lymph nodes. We demonstrated that TEGVAX significantly re- to spleen, after tail vein injections in mice. Flow cytometry duced B16-mOVA tumor growth even in the presence of FTY720 staining revealed that STACT does not infect stromal or tumor treatment, suggesting T cell priming was not required. Immune cells and is specifically targeted by tumor-resident myeloid cells phenotyping of TEGVAX treated mice, showed a significant in- (TAMs, DCs, and monocytes). STACT is rapidly phagocytosed crease in tumor infiltrating tissue-resident memory (Trm) T cells. and then destroyed by these cells, delivering its plasmid DNA Conclusions contents encoding immunomodulatory protein expression cas- Our results demonstrate that combining TLR4 agonist with GVAX settes. We have measured highly efficient heterologous gene (TEGVAX) completely alters the immune response to vaccination. transfer and protein expression within primary mouse and hu- TEGVAX does not prime naïve T cells nor require trafficking of T cells man M2 macrophages treated with STACT, at levels comparable from LN to tumor to function. We observed an increased number of to DNA transfection. Therapeutically relevant levels of IL-2 were Trm CD8 T cells infiltrating the tumor leading us to conclude that measured in the tumor microenvironment of STACT-IL2 treated TEGVAX is functioning by eliciting a tumor-specific Trm T cell re- mice several weeks after dosing. For STACT-STING, significant sponse independent of lymph node priming. tumor growth inhibition, including complete tumor regressions Ethics Approval were observed, and the therapy was well tolerated. Immune The study was approved by Vanderbilt University Animal care and correlates were consistent with on-target expression in the use board tumor microenvironment, and the anti-tumor effect was adap- tive immune mediated. P484 Conclusions CB-708, an orally bioavailable small molecule inhibitor of CD73 STACT is a highly attenuated, microbial-based therapeutic platform with immunostimulatory and anti-tumor activity engineered to deliver immunomodulatory payloads, alone or in com- Clarissa Lee, PhD, Deepthi Bhupathi, Roland Billedeau, Jason Chen, Lijing bination, to phagocytic cells of the solid tumor microenvironment Chen, Rosalyn Dang, Matthew Gross, Tony Huang, Weiqun Li, PhD, Yong after systemic administration. The goal of STACT therapy is to pro- Ma, Andrew MacKinnon, Gisele Marguier, MS, Silinda Neou, MS, mote immune-mediated tumor clearance of T-cell excluded solid tu- Francesco Parlati, PhD, Natalija Sotirovska, MS, Sandra Spurlock, Timothy mors and elicit durable anti-tumor immunity. Stanton, Susanne Steggerda, PhD, Jing Zhang, Winter Zhang, Jim Li Ethics Approval Calithera Biosciences, South San Francisco, CA, United States All animals were used according to protocols approved by an Institu- Correspondence: Jim Li (jli@calithera.com) tional Animal Care and Use Committee and maintained in specific Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P484 pathogen-free conditions in a barrier facility. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 264 of 272 Background Methods High adenosine (ADO) in the tumor microenvironment suppresses CT26 tumor measurement seven days after treatment was used to the immune response against cancer cells by inhibiting immune ef- define tumors as KD033-surrogate responders (decreasing tumor vol- fector functions and promoting the development of immunosuppres- umes), non-responders (no change or increasing tumor volumes) and sive cells. Extracellular ADO can be generated from ATP released by non-targeted IL-15 best responders. RNA was isolated from these tu- cells undergoing stress or death through the combined actions of mors and analyzed using the Nanostring PanCancer IO 360 Gene Ex- the ectonucleotidases CD39 (ATP to AMP) and CD73 (AMP to ADO). pression Panel for immune cell responses. Combination therapy with Inhibition of ADO production via CD73 is a promising therapeutic ap- genes identified through Nanostring analysis was evaluated in a proach for the treatment of cancer. tumor model where KD033-surrogate monotherapy showed minimal Methods efficacy such as 4T1, an aggressive breast carcinoma murine model We developed CB-708, a potent and selective small molecule inhibi- involving spontaneous metastases to other organs. 4T1 cells were tor of CD73. The potency of CB-708 was evaluated against recombin- injected into the mammary gland of Balb/c mice and grown to 100 ant CD73 and CD73-expressing cells using a malachite green assay. mm3 prior to treatments. Tumors and metastasis nodules in the lung Selectivity against related ectonucleotidases was also assessed. Inhib- were evaluated. ition of CD73 in plasma was measured using LC/MS to assess conver- Results sion of 15N5-AMP into 15N5-ADO. Reversal of AMP-mediated Transcriptional analysis showed that CTLA-4 was one of the top immune suppression of human CD8+ T cells was determined by genes that was differentially upregulated after KD033-surrogate treat- measuring T cell activation in the presence of exogenous AMP. T cell ment in comparison to non-targeting IL-15. In the 4T1 tumor model, proliferation was assayed by flow cytometry and cytokine levels were monotherapies of both KD033-surrogate or anti-CTLA-4 did not have measured by ELISA. The EG7 and CT26 syngeneic tumor models were any effect on 4T1 tumor growths; however, the combination therapy used to assess the therapeutic effect of CB-708. with single dose of KD033-surrogate and repeat dose of anti-CTLA-4 Results showed a decrease in the average number of lung metastases and a CB-708 potently and completely inhibited soluble human CD73 (IC50 significant tumor growth inhibition compared to vehicle-treated = 170 pM) and cell-bound human CD73 (IC50 = 210 pM), but did not animals. inhibit human CD39, ENTPD2, or ENTPD3. CB-708 retained high po- Conclusions tency in the presence of whole human plasma (IC50 = 380 pM) and Analysis of murine tumors treated with KD033 surrogate in vivo re- reversed AMP-mediated suppression of human CD8+ T cell prolifera- sulted in combination strategies, including KD033 in combination tion and production of IFNγ and granzyme B in vitro. Oral administra- with CTLA-4, that can be exploited in targeting resistant and refrac- tion of CB-708 was well-tolerated in tumor-bearing mice, resulted in tory cancers. Based on the therapeutic activity and improved safety sustained exposure above mouse plasma IC50, and exhibited single- of the fusion protein, Kadmon plans to initiate clinical studies of agent tumor growth inhibition in syngeneic tumor models including KD033 in 2019. established EG7 tumors. Efficacy in the EG7 model was dependent Ethics Approval on CD8+ T cells and was correlated with pharmacodynamic inhib- Animal studies were conducted for Kadmon by Crown Bioscience Inc. ition of CD73. Enhanced tumor growth inhibition was observed when with approved SOP and IACUC protocol. CB-708 was combined with checkpoint inhibition (anti-PD-L1) or with chemotherapy (oxaliplatin, doxorubicin, docetaxel) in the EG7 model. P486 Conclusions Releasing the break on T cell activation through novel small CB-708 is an orally bioavailable and highly potent small molecule in- molecule inhibition of HPK1 hibitor of CD73. CB-708 reverses the immunosuppressive effects of Minhui Shen, Gayathri Bommakanti, PhD, Deanna Mele, PhD, Neil AMP-derived ADO in vitro and in vivo and has anti-tumor activity. Grimster, PhD CB-708 is expected to enter clinical development in 2019. AstraZeneca, Waltham, MA, United States Correspondence: Deanna Mele (Deanna.Mele@astrazeneca.com) P485 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P486 Synergistic efficacy of anti-PD-L1/IL-15 fusion protein in combination with anti-CTLA-4 antibody in a murine orthotopic 4T1 Background breast carcinoma model Loss of immune surveillance is required for cancer cell growth and 1 2 2 2 Stella Martomo, PhD , Dan Lu, MA , Jeegar Patel , Zhanna Polonskaya , metastasis, tumors co-opt suppressive mechanisms to evade detec- 2 2 Xenia Luna , Kevin McCracken tion by the immune system. The immune system is equipped with 1 2 Kadmon Corporation, New York, NY, United States; Kadmon, New York, multiple feedback mechanisms to limit inflammation in order to pre- NY, United States vent autoimmunity, tumors activate these pathways to escape im- Correspondence: Stella Martomo (stella.martomo@kadmon.com) munity. HPK1 (hematopoietic progenitor kinase 1) is a negative Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P485 regulator of T cell activation, the kinase activity limits T cell signaling and tumoricidal cytokine production. Recent literature has shown in- Background activation of the kinase function of HPK1 prevents tumor progression Administration of immune checkpoint inhibitors anti-PD1/PD-L1 have in murine tumor models [1]. led to durable objective responses in select cancers. However, a sub- Methods stantial number of patients fail to respond or become resistant to these We used lentiviral delivered shRNAs and CRISPR/Cas9 technology to therapies. We have generated a therapeutic fusion protein (KD033) by delete HPK1 in Jurkat cells and primary human T cells, respectively. combining a proprietary high affinity anti-human-PD-L1 (or anti- Jurkat T cells (wt/KO) were activated with anti-CD3 antibody and cell murine-PD-L1, (KD033-surrogate)) antibody with human IL-15. Initial as- lysates were assessed by western blot to examine signaling events sessment of this fusion antibody showed enhanced tolerability relative downstream of the T cell receptor (TCR). Primary human T cells or to a non-targeted IL-15 fusion protein in addition to its potent anti- CRISPR/Cas9 KO T cells were activated in vitro using anti-CD3/anti- tumor activity. In the CT26 murine colorectal tumor model, a single CD28 antibodies in the presence or absence of HPK-1 inhibitors +/- dose of KD033-surrogate consistently resulted in antitumor response prostaglandin E2 (PGE2). Cell viability and numbers were assessed by that included tumor clearance and long-term tumor-free survival. Initial flow cytometry. Cytokines were quantified by ELISA. analysis of KD033-surrogate treatment showed robust adaptive and Results cytotoxic immune gene signatures in tumors leading to tumor inhib- We present novel findings demonstrating that the role of HPK1 is ition and memory responses. We further analyzed tumors from KD033- conserved in human Jurkat cells as well as in primary human T cells. surrogate responders and non-responders to evaluate possible thera- Loss of HPK1 enhanced T cell receptor signaling in Jurkat cells. In peutic combinations to broaden the response of KD033. CRISPR/Cas9 KO HPK1 primary human T cells, TCR activation resulted Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 265 of 272 in enhanced cytokine secretion and proliferation concomitant to a 100% lethality by 60 days, but 3 doses of AgN2a 4P leads to 100% decrease in pSLP76, the target molecule phosphorylated by HPK1. survival at 50 days after both allogeneic and syngeneic BMT. Consistent with the KO phenotype, our HPK1 inhibitors resulted in Conclusions enhanced T cell activation, cytokine secretion and proliferation. In Co-culture of NK cells with an engineered costimulatory vaccine is an addition, the inhibitors were able to rescue T cells from PGE2 medi- effective strategy to induce apoptosis of neuroblastoma tumor cells ated suppression. In vivo studies are currently underway to examine by increasing NK-mediated cytokine production and cytotoxicity, and anti-tumor activity of these compounds in various syngeneic models. enhances anti-tumor effects after BMT. Usage of cell-based vaccines Conclusions after BMT could be an effective strategy to augment NK cell activity In summary our small molecule inhibitors of HPK1 could enhance against neuroblastoma. anti-tumor immunity through increased T cell function overcoming suppressive signals in the tumor microenvironment and thus Acknowledgements broaden the response to check point inhibitors for cancer AgN2a 4P was a gift from Dr. Bryon Johnson at Medical College of immunotherapy. Wisconsin. This work was supported by grants from the St. Baldrick’s – Stand up to Cancer Pediatric Dream Team Translational Research Grant SU2C- Acknowledgements AACR-DT-27-17, NCI/NIH R01 CA215461, American Cancer Society Research Minhui Shen1, Gayathri Bommakanti1, Kevin Xu1, Kun Song1, Rob Ziegler1, Scholar grant RSG-18-104-01-LIB, Hyundai Hope on Wheels and the MACC Jason Kettle2, Adelphe Mfuh1 Jason Sheilds2, Neil Grimster1, Lisa Drew1, Fund (C.M.C). We would like to thank the UWCCC core facilities, who are Stephen Fawell1, Deanna A. Mele1 supported in part through NCI/NIH P30 CA014520. Stand Up to Cancer is a 1AZ Discovery Early Oncology, Boston, MA, 2AZ Discovery Early Oncology, division of the Entertainment Industry Foundation. Research Grants are Cambridge UK, MA administered by the American Association for Cancer Research, the Scientific Partner of SU2C. Reference Ethics Approval 1. Hernandez S, Qing J et al. The Kinase Activity of Hematopoietic The study was approved by University of Wisconsin-Madison Animal Care Progenitor Kinase 1 is Essential for the Regulation of T cell Function and Use Committee, approval number M005915. Cancer. Cell. 2018; 25: 80-94. P488 P487 IPH5201, a blocking antibody targeting the CD39 Combining an engineered costimulatory vaccine with NK cells immunosuppressive pathway, unleashes immune responses in induces an anti-tumor effect against murine neuroblastoma combination with cancer therapies 1 1 1 1 in vitro and after bone marrow transplant in vivo Pascale Andre , Ivan Perrot , Caroline Denis, PhD , Marc Giraudon-Paoli , 1 1 1 1 Nicholas Mohrdieck, BS, Paul Bates, Sean Rinella, Katharine Tippins, Severine Augier , Rachel Courtois , Diana Jecko , Thomas Arnoux , 1 1 2 Christian Capitini, MD Violette Breso , Nicolas Gourdin, PhD , Nadia Luheshi, PhD , Ariane 1 1 1 1 University of Wisconsin-Madison, Madison, WI, United States Morel, PhD , Yannis Morel, PhD , Eric Vivier , Carine Paturel, PhD 1 2 Correspondence: Christian Capitini (ccapitini@pediatrics.wisc.edu) Innate Pharma, Marseille, France; AstraZeneca, Milton, Cambridge, Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P487 United Kingdom Correspondence: Pascale Andre (pascale.andre@innate-pharma.fr) Background Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P488 High risk neuroblastoma remains a challenge to cure with only 50% survival, despite multi-modality treatment. Natural killer (NK) cells Background have been previously shown to have activity versus neuroblastoma CD39 is an extracellular ectonucleotidase highly expressed in the but have not been consistently successful in clinical trials. NK cell ac- tumor microenvironment, by stromal cells and some immune in- tivation via co-culture with a vaccine engineered to express CD54, filtrating cells. CD39 contributes to the production of adenosine, CD80, CD86, and CD137L, called AgN2a 4P, was studied to investi- an inhibitor of immune response, via sequential hydrolysis of ad- gate NK cells’ ability to induce cytotoxicity of murine neuroblastoma enosine triphosphate (ATP) and adenosine diphosphate into ad- tumor cells in vitro and in vivo. enosine monophosphate, which then is degraded into adenosine Methods by CD73 enzyme. In contrast, ATP has immune-stimulatory activ- NKs and irradiated AgN2a 4P were co-cultured in ratios of 1 (NKs):0.5 ity through promoting dendritic cell (DC) maturation. Blockade of (AgN2a 4P) and 1:1, and compared to NK only and AgN2a 4P only CD39-mediated degradation of ATP may therefore stimulate anti- controls, with all groups receiving IL-15/IL-15Ralpha, and then ana- tumor immunity across a wide range of tumors by preventing lyzed by flow cytometry, multiplex cytokine analysis, and cytotoxicity production of immunosuppressive adenosine and by promoting in vitro after 1, 3, 5, 7, and 9 days. To study the efficacy of in vivo accumulation of immunostimulatory ATP in the tumor microenvir- vaccination with AgN2a 4P after bone marrow transplant (BMT), onment. IPH5201 is a humanized monoclonal antibody that se- C57BL/6 or B6AJ recipients were lethally irradiated, followed by trans- lectively binds to and inhibits the activity of both membrane- plantation of T-cell depleted C57BL/6 donor bone marrow on day +0. bound and soluble human CD39. Here, we explored the efficacy BMT recipients were then treated with the AgN2a 4P vaccine for 2 of IPH5201 in vitro and in vivo in immunocompetent human versus 3 weekly doses, and with or without adoptive transfer of CD39 knockin (huCD39KI) mouse model in combination with im- donor NK cells to accelerate immune reconstitution. All recipients mune checkpoint inhibitor. were then challenged with NXS2 neuroblastoma tumor, and followed Methods for tumor growth and survival. In vitro, efficacy of IPH5201 was evaluated (1) on the phenotypic Results changes and stimulatory potential of monocyte-derived DC, (2) on The NK:AgN2a 4P co-culture at 1:0.5 and 1:1 increases Ly49A+ NKs the inflammasome pathway by assessing interleukin-1b secretion from 6% to 21%, and Ly49D+ NKs from 3% to 30% from day +0 to from in vitro-derived M1 macrophages, and (3) on T cell proliferation. day +9. pSTAT1 activation remains consistently high between 80%- HuCD39KI mice were characterized for the expression and function 98%, and pSTAT3 activation remains 20%-50%, across the co-culture of human CD39. To assess CD39 blockade in vivo, a mouse IgG1 ver- period. NK cells release increased levels of IFN-gamma and IL-6 at sion of IPH5201 was produced (moIPH5201), which contained key the co-cultured ratios of 1:0.5 and 1:1, and CXCL1 at the 1:1 ratio, as point mutations in the Fc region to abrogate Fc receptor interactions. compared to the NK with IL-15/IL-15Ralpha controls. The NK:AgN2a Antitumor efficacy of CD39 blockade was assessed in huCD39KI mice 4P ratios of 1:0.5 and 1:1 induce significantly increased apoptosis of grafted with mouse tumor cells not expressing mouse CD39. Neuro2a neuroblastoma cells than NK cells with IL-15/IL-15Ralpha HuCD39KI mice were treated with blocking anti-human CD39 Ab, ei- alone. In vivo, injection of 2 doses of AgN2a 4P vaccine leads to ther alone or in combination with a blocking anti-mouse PD-L1 Ab. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 266 of 272 Results the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and As hypothesized, in vitro IPH5201 enhanced the phenotypic 5 mg/kg induced significant tumor growth inhibition (55% and 57%, maturation and the activation of DCs and macrophages by inhi- respectively) as compared to vehicle control or Fc-silenced huIgG1 biting ATP hydrolysis. IPH5201 also efficiently restored T cell controls. Additionally, 2 out of 9 animals in the 5 mg/kg MCLA-145– proliferation in a dose-dependent manner to the levels ob- treated group had complete tumor regression. MCLA-145 increased served in the absence of ATP addition. Thus, IPH5201 preserved the number of infiltrating CD8+ T cells, as well as the percentage of extracellular ATP, thereby promoting the activation of DCs and central memory CD8+ T cells. macrophages and limiting adenosine accumulation and its im- Conclusions munosuppressive effect on T cells. Furthermore, moIPH5201 MCLA-145 is currently undergoing clinical development mice treatment efficiently inhibits membrane and soluble hu- (NCT03922204). man CD39, from huCD39KI mice ex vivo without mediating CD39-expressing cell depletion in vivo. Finally, blockade of P490 CD39 potentiates the anti-tumor efficacy of anti-PD-L1 Ab Antibody derived from an elite responder to checkpoint inhibitor monotherapy. therapy relieves immunosuppression by tumor associated Conclusions macrophages Together these data indicate that blocking CD39 in conjunction with Randi Simmons, Siddarth Chandrasekaran, Melissa Conerly, Tyrel Smith, PD-1/PD-L1 checkpoint inhibitors provides increased anti-tumor effi- Sam Lam, Jacqueline Pham, Ray Fox, Darbie Whitman, Meghan Zuck, cacy and support the rational for assessing this combination in clin- Sara Carbonetti, Kamal Puri, PhD ical trials. Oncoresponse Inc, Seattle, WA, United States Correspondence: Kamal Puri (kpuri@oncoresponseinc.com) P489 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P490 Identification and characterization of MCLA-145 (CD137 x PD-L1): a bispecific antibody that requires PD-L1 binding to activate CD137 Background 1 1 1 Simon Plyte , Cecile Geuijen , John de Kruif , Pieter Fokko van Loo, Tumor-associated macrophages (TAM)s in the tumor microenviron- 1 1 1 1 PhD , Paul Tacken , vanessa Zondag-vander Zande , Rinse klooster , ment (TME) contribute to tumor immune evasion by suppressing 1 1 1 1 hans van Maaden , Erik Rovers , steef engels , floris franzen , abdul anti-tumor immune responses and by promoting a tumorigenic mi- 1 1 1 2 basmeleh , willem bartelink , Mark Throsby , Patrick Mayes , Horacio lieu. High infiltration of immunosuppressive myeloid cells generally 2 2 2 2 2 Nastri , shaun stewart , jing zhou , steve wang , Chen-yen Huang , predicts unfavorable prognosis. Reduction or repolarization of sup- 2 2 2 2 thomas codamine , ashwini kularni , yao bin lui , arpita mondal , leslie pressive myeloid cells is an attractive strategy to enhance clinical re- 2 2 2 2 2 hall , soeon kim , marina martinez , shaun o'brien , edmund moon , sponses to immune checkpoint inhibitor (CPI) therapy. Cancer steven albelda patients who achieved durable response to CPI therapy (elite re- 1 2 Merus, Utrecht, Netherlands; Incyte Research Institute, Wilmington, DE, sponders) may harbor antibodies that contribute to clinical response United States by promoting an anti-tumor TME. Correspondence: Simon Plyte (simon.plyte@merus.nl) Methods Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P489 B cells derived from elite responders were cloned and screened for IgG antibodies binding to myeloid derived suppressor cells. Background Hits were prioritized based on myeloid binding profiles and their CD137 (4-1BB) is costimulatory receptor on T and NK cells that re- variable-regions sequenced, cloned, and expressed as recombin- quires clustering to elicit its effects on target cells and enhance adap- ant IgG1. Cloned antibodies underwent further characterization to tive immune responses against tumors. The development of CD137 evaluate their ability to reverse the immunosuppressive effects of targeted agents for cancer therapy has been hampered by on-target myeloid cells in assays modelling the TME. Primary human mono- off-tumor toxicity in the case of agonistic monospecific antibodies, or cytes and T cells were used to interrogate antibody-dependent limited antitumor activity in the case of Fcγ-mediated crosslinking of immunomodulatory responses in vitro. A humanized mouse mAbs. model was used to evaluate the anti-tumor activity of the lead Methods antibody, OR2805. To address the issues of toxicity and efficacy, we have identified a Results highly selective and potent CD137xPD-L1 bispecific antibody, MCLA- The target of OR2805 is highly expressed on TAMs and M2-like 145. Collections of common light chain Fabs recognizing CD137 and macrophages. OR2805 does not bind to other hematopoietic PD-L1 were produced based on antibody panels from immunized cells nor a panel of human primary non-immune cells. The anti- MeMo® mice. Unbiased, combinatorial, functional screening was then body stains positively on M2-like TAMs from primary human performed on a large and diverse panel of CD137xPD-L1 bAbs to lung tumor samples. OR2805 treatment reduces expression of identify those for which CD137 mediated activation is dependent on cell-surface markers associated with tumor-promoting M2c-like the presence of PD-L1 on a neighboring cell macrophages. In co-culture assays, OR2805 relieves the suppres- Results sive effect of M2 macrophages and resulted in increased T cell Both the CD137 and PD-L1 Fab arms block the interaction with their activation and proliferation, upregulation of T cell activation respective ligands as demonstrated in competition flow cytometry or markers, and enhanced T cell-mediated tumor cell killing. Ad- ELISA assays, respectively. MCLA-145 drives transactivation of CD137 ministration of OR2805 in humanized NSG-SGM3 mouse tumor in the vicinity of cells expressing PD-L1 and the degree of CD137 ag- models resulted in approximately 50% reduction in A549 tumor onistic activity in T cells correlated with the expression level of PD-L1 growth and a 60% reduction in H1975 tumor growth. In this on neighboring cells. CD137 signaling was induced by MCLA-145 in model, OR2805 treatment significantly increased the proportions multiple primary human immune cell assays and reversed T cell sup- of human CD8+ T cells and human CD11b+ myeloid cells in the pression mediated by M2 macrophages or Tregs, in vitro. In one hu- spleen as well as significantly enhanced expression of activation manized mouse tumor model, human T cells expressing NY-ESO markers (ICOS, OX-40) by human CD8+ T cells. specific TCR were adoptively transferred to mice bearing A549 tu- OR2805 reduces TAM-mediated immunosuppression and enhances mors, which expressed NY-ESO antigen and human PD-L1. MCLA-145 anti-tumor immune responses. OR2805 treatment induces robust treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) anti-tumor activity in lung cancer xenograft models in humanized as compared to T cell only–treated mice. In the tumors of MCLA- mice. This data justifies further development of OR2805 as anti- 145–treated mice, the percentage of NY-ESO specific CD8+ T cells cancer therapy in combination with other CPI treatments. OR2805 were significantly increased compared with controls. In a second has the potential to increase the number of patients who may bene- model, mice engrafted with human CD34+ cells were implanted with fit from current CPI therapy. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 267 of 272 P491 References Preclinical development of a novel TNFRSF25 agonist antibody, 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. PTX-35, for cancer immunotherapy combinations Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 Matthew Seavey, PhD, Jayalakshmi Miriyala, MS, Jason Rose, MS, Vikas 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg Tahiliani, PhD, Patrick Dillon, PhD, Elena Gorovits, PhD, Jeff Hutchins, expansion in mice by TNFRSF25 prevents allergic lung inflammation. J PhD, Rahul Jasuja, PhD Clin Invest.; 120(10):3629-3640 Heat Biologics, Inc., Durham, NC, United States 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed Correspondence: Matthew Seavey (mseavey@heatbio.com) M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P491 That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell Background responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Tumor Necrosis Factor Receptor Super Family 25 (TNFRSF25), 5. Schreiber T.H., et al.; (2014). Comparative combination cancer also known as Death Receptor 3 (DR3), is preferentially immunotherapy with vaccination and TNFRSF stimulation. Society of expressed by activated and antigen-experienced T-cells. Immunotherapy of Cancer (SITC) Conference, Poster: https:// TNFRSF25 is a potent costimulatory molecule, similar to OX40, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ 4-1BB and GITR. PTX-35 was developed as a humanized, affinity heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf matured, IgG2 mAb against TNFRSF25 for use with current can- 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ cer immunotherapy options, including cancer vaccines. All regulatory T cells and the severity of acute graft-versus-host disease. pharmacology and IND-enabling PK and toxicology has been Blood 128 (24), 2846-2858 completed for PTX-35 [1-6]. Methods Previous proof-of-concept studies were completed elsewhere. P492 Pharmacology studies described here were completed with a sur- A new generation anti-TGFβ antibody, SAR439459, relieves rogate antibody, mouse-IgG1-PTX-35 (mPTX-35). Regulatory T-cell immunosuppression and improves anti-tumor efficacy of PD1 expansion studies were completed with Foxp3-RFP+ transgenic blockade mice (FIR). CD8+ T-cell expansion studies were conducted with Rita Greco, Hongjing Qu, Joachim Theilhaber, Gary Shapiro, Richard adoptively transferred OVA-specific, TCR-transgenic, CD8+ T-cells Gregory, PhD, Christopher Winter, Natalia Malkova, Lily Pao, Mikhail Levit, (OT-1). Human PTX-35 was tested in 28-day mouse, and 2-week Alexei Protopopov, Jack Pollard, PhD, Tun Tun Lin, MD, Dmitri and 8-week non-human primate, toxicology studies. Human, Wiederschain, Sharad Sharma, PhD mouse, and monkey, in vitro, tissue-cross reactivity tests were Sanofi, Cambridge, MA, United States also performed to check species cross-reactivity. Human PTX-35 Correspondence: Sharad Sharma (sharad.sharma@sanofi.com) was also tested in a human PBMC stimulation assay, in vitro, to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P492 check for impact on proliferation and cytokine release. Background Results TGFβ is a potent immunosuppressive cytokine that acts on TNFRSF25-engagement in mice expanded antigen specific CD8+ multiple cell types of the innate and adaptive arms of the im- T-cells when given in the context of vaccination (6 and 19-fold mune system within the tumor microenvironment. Emerging increase in CD8+ T-cells in blood over vaccination alone at peak data implicates role of TGFβ in tumor immune evasion, resist- and boost, respectively), and Tregs were expanded in vaccine ance to cancer therapy and poor prognosis. We report preclin- absence in FIR animals (2-fold increase in CD4+ Foxp3+ T-cells ical data on SAR439459, a new pan anti-TGFβ antibody, which in blood).The MABELand NOEL,inFIR mice,using mPTX-35, is capable of neutralizing all active isoforms of human and was determined to be 0.01 mg/kg and 0.001 mg/kg, respect- murine TGFβ. ively. The 2-week PTX-35 treatment of cynomolgus monkeys confirmed findings in mice, that treatment results in the expan- Methods sion of Teff and Treg cell subsets. Intravenous bolus injection Cancer patient databases were analyzed for various gene signa- once every 2-weeks over an 8-week period was well tolerated tures. In vitro experiments were performed to examine the effi- giving a NOAEL of 100 mg/kg. Species cross-reactivity was ob- cacy of SAR439459 in preventing TGFβ-mediated suppression of served for mouse, human and monkey tissues. No adverse primary human T and NK cells. To evaluate anti-tumor efficacy, events were recorded for the 28-day mouse toxicology study. MC38 and EMT6 mouse tumor models were treated with Testing PTX-35 in a human PBMC, anti-CD3 proliferation assay, SAR439459, anti-PD1, or combination. MC38 model was used to in vitro, showed that TNFRSF25-engagement provided the ne- examine immune cell functions ex vivo. cessary costimulation to drive cellular division at sub-optimal Results concentrations of anti-CD3 (2-fold increase), providing the ne- TGFβ was found to be upregulated in cancer patients and its cessary in vitro, human proof-of-concept data, which was con- increased activation correlated with reduced overall survival (OS) sistent across four different human blood donors. in PD-1 refractory cancer patients. SAR439459 blocked TGFβ- Conclusions mediated suppression of human T and NK cells. TGFβ impaired PTX-35 is a potent costimulatory agonist targeting a novel pathway the activity of anti-PD1 mediated T cell response, while that can work in concert with cancer vaccines. Due to the ability of SAR439459 restored this activity. In MC38 and EMT6 tumor PTX-35 to possibly stimulate both pro-inflammatory and anti- models, treatment with SAR439459 resulted in anti-tumor efficacy; inflammatory pathways, depending on treatment context, thera- the combination of SAR439459 with anti-PD1 enhanced this activ- peutic modulation can provide numerous opportunities for cancer ity and resulted in complete tumor regression and elicited a pro- and inflammatory diseases. longed anti-tumor response. Conclusions Acknowledgements This data demonstrates that combination of SAR439459 with anti- Pelican Therapeutics would like to thank the Cancer Prevention and PD1 generates a strong anti-tumor immune response and im- Research Institute of Texas (CPRIT) that helped fund these studies. We would proves anti-tumor efficacy in several tumor models. The preclin- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for ical data presented here formed the basis for the ongoing clinical mouse TL1A-Ig and advice, both located at the University of Miami. investigation of SAR439459 in cancer patients. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 268 of 272 P493 Background High-dimensional analysis delineates modulation of myeloid and Heat (Heat) Biologics has developed a next generation cellular vac- lymphoid compartments with STAT3 ASO and PDL-1 combination cine platform that incorporates a tumor antigen chaperone (gp96-Ig) therapy in a tumor cell line and a host of over-expressed cancer associated Theresa Proia, PhD, Maneesh Singh, PhD, Larissa Carnevalli, PhD, Gayathri neoantigens. Viagenpumatucel-L (HS110), a human lung adenocar- Bommakanti, PhD, Nanhua Deng, Matthew Griffin, Lukasz Magiera, Adina cinoma cell line, stably transfected to express gp96-Ig, is being tested Hughes, Laura Prickett, Patricia McCoon, PhD, Corinne Reimer, Simon in a phase 1/2 clinical trial (NCT#02439450) for NSCLC. Heat has re- Barry, PhD cently developed HS130, an allogeneic cell-based vaccine, designed AstraZeneca, Waltham, MA, United States to secrete tumor-associated antigens along with a costimulatory mol- Correspondence: Theresa Proia (theresa.proia@astrazeneca.com); ecule, OX40L. Preclinical results of mouse HS130 (mHS130) in com- Gayathri Bommakanti (gayathri.bommakanti@astrazeneca.com) bination with mouse HS110 (mHS110) has shown a potent anti- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P493 tumor effector and memory CD8+ T cell response, followed by tumor regression. In our current study, we further characterized the role of Background mHS110 and mHS130 in combination with an agonist TNFRSF25 STAT3 is a ubiquitously expressed transcription factor and master monoclonal antibody, PTX35. PTX35 is a potent stimulator of effector regulator of immune suppression in the tumor microenvironment and memory CD8+ T cell responses, which taken-together with (TME). Danvatirsen, a therapeutic antisense oligonucleotide (ASO) HS110 and HS130 has the potential of treating human cancers [1-7]. that selectively targets STAT3, has shown clinical benefit alone and in Methods combination with durvalumab (anti-PDL1) and is currently in Phase To study expansion, contraction, and maintenance of tumor-specific 1/2 clinical studies. CD8+ T cell responses, mHS110 and/or mHS130, in combination with Methods different doses of mouse-IgG1-PTX35 (mPTX35) was administered to To gain mechanistic insight into the therapeutic response induced C57BL/6 mice that were adoptively transferred with syngeneic OVA- by mouse surrogate STAT3 ASO in the CT26 syngeneic mouse tumor specific T cells (OT-I). Mice were then challenged with murine melan- model, we have used two complementary forms of high-dimensional oma tumors (B16F10-OVA) to characterize the tumor-specific immune profiling; mass cytometry (CyTOF) and flow cytometry. We supported cells in the periphery, spleen, and tumor-microenvironment that the in vivo mouse findings with in vitro studies in human macro- were involved in tumor regression. phages treated with danvatirsen. Results Results Combination of mPTX-35 with mHS-110 and mHS130 increased the Multidimensional immune profiling studies provided key mechan- expansion of tumor-specific CD8+ T-cells, in a mPTX-35 dose- istic observations: (1) Robust reduction of total STAT3 protein in dependent manner. This cellular expansion was significantly higher myeloid lineage cells, including an 80% reduction in macro- in the 1 mg/kg dose of mPTX-35 and far exceeded the additive value phages and 50% reduction in dendritic cells, but not in CD8+ T of mPTX-35, mHS130, and mHS110 treatment alone. Systemic admin- cells. (2) In the combination treatment arm, STAT3 ASO treatment istration of mPTX35, in combination with mHS110 and mHS130, led promoted a two-fold reduction of intratumoral immunosuppres- to a significant increase in the expansion of activated CD8+ T cells in sive macrophages, doubling of iNOS positive activated macro- the blood and stimulated activation of KLRGhi IL7Rlo short-lived ef- phages and enhanced proliferation and IFNγ production from fector cells (SLECs). Importantly, this combination resulted in higher tumor antigen specific T cells. (3) The tumor-associated mono- frequencies of tumor infiltrating lymphocytes (TILs), which enhanced cyte/macrophage compartment is highly complex and dynamic regression of established B16F10-OVA tumors and increased overall and displays a spectrum of activation states ranging from a pre- survival. dominantly anti-inflammatory phenotype (F4/80+ CD206+ IL4r+ Conclusions MerTK+; six fold higher) in progressively growing control tumors These results strongly suggest that mPTX35 synergizes with mHS110 to a predominantly proinflammatory phenotype (F4/80+ iNOS+ and mHS130 to amplify activated tumor-specific CD8+ T cells, pro- CCR2+; three fold higher) in responding tumors from combination gram a strong memory response, and allow for tumor regression. treated groups. The combinations of these three treatments in the clinic may trans- In vitro, human macrophages were highly sensitive to danvatirsen late into an efficacious approach to treating human cancers. treatment, with an IC50 of 60nM for total STAT3. Human ‘M2-like’ macrophages were generated in the presence of IL10 and M-CSF and Acknowledgements treated with danvatirsen, which promoted an increase in IFNγ, IL-12, Pelican Therapeutics would like to thank the Cancer Prevention and and TNFα, as well as increased CD80/CD86 expression, consistent Research Institute of Texas (CPRIT) that helped fund these studies. We would with polarization from a suppressive phenotype to a pro- also like to thank Dr. Natasa Strbo for 4C12 antibody and Dr. Robert Levy for inflammatory phenotype. mouse TL1A-Ig and advice, both located at the University of Miami. Conclusions Our data support the hypothesis that STAT3 reduction in the myeloid References lineage results in activation of macrophages entering the TME and 1. Schreiber TH, Podack ER. Immunobiology of TNFSF15 and TNFRSF25. enhanced effector T cell responses in combination with checkpoint Immunologic Research, December 2013, Volume 57 (issue 1-3), pp. 3-11 inhibition. Our ongoing work is focused on exploring the effects of 2. Schreiber T.H., Levy R.B., and Podack E.R.; (2010). Therapeutic Treg STAT3 reduction in other key immune cells in which we have ob- expansion in mice by TNFRSF25 prevents allergic lung inflammation. J served robust knockdown including Tregs, endothelial cells, and Clin Invest.; 120(10):3629-3640 CAFs. 3. Melero I., Hirschhorn-Cymerman D., Morales-Kastresana A., Sanmamed M.F., and Wolchok J.D.; (2013). Agonist Antibodies to TNFR Molecules That Costimulate T and NK Cells. Clin Cancer Res.; 19(5); 1044–53 P494 4. Slebioda T.J. et al.; (2011). Triggering of TNFRSF25 promotes CD8+ T-cell A novel TNFRSF25 agonist, PTX35, synergizes with Gp96-Ig/OX40L- responses and anti-tumor immunity. Eur J of Immunol 41 (9), 2606-2611 Ig to enhance effector and memory anti-tumor CD8+ T cell 5. Schreiber T.H., et al.; (2014). Comparative combination cancer responses and delay tumor growth immunotherapy with vaccination and TNFRSF stimulation. Society of Vikas Tahiliani, Patrick Dillon, PhD, Jayalakshmi Miriyala, MS, Jason Rose, Immunotherapy of Cancer (SITC) Conference, Poster: https:// MS, Anh Trinh, Rahul Jasuja, PhD, Jeff Hutchins, PhD, Matthew Seavey, d1io3yog0oux5.cloudfront.net/_675812118f795f2435017262bf9d3804/ PhD heatbio/db/527/5588/pdf/2014-AACR_gp96-Ig-and-Costim-Combos.pdf Heat Biologics, Inc., Durham, NC, United States 6. Nishikii H. et al.; (2016). DR3 signaling modulates the function of Foxp3+ Correspondence: Matthew Seavey (mseavey@heatbio.com) regulatory T cells and the severity of acute graft-versus-host disease. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P494 Blood 128 (24), 2846-2858 Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 269 of 272 7. FrommG,deSilva S, GiffinL,Xu X,Rose J,Schreiber TH.Gp96-Ig/ P496 Costimulator (OX40L, ICOSL, or 4-1BBL) Combination Vaccine Im- Inhibition of autophagy enhances multifunctional genetically- proves T-cell Priming and Enhances Immunity, Memory, and Tumor engineered NK cell-based immunotherapy of glioblastoma Elimination. Cancer Immunol Res. 2016;4(9):766-778. doi:10.1158/2326- Jiao Wang, PhD, Sandro Matosevic, PhD 6066.CIR-15-0228 Purdue University, West Lafayette, IN, United States Correspondence: Sandro Matosevic (sandro@purdue.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P496 P495 Secondary immune resistance mechanisms induced by therapeutic Background cancer vaccines which prevent tumor regression and foster Despite aggressive treatments, the median life expectancy for GBM recurrences patients is only around 15 months, highlighting the need for new Sjoerd Van der Burg, PhD, Elham Beyranvand Nejad, Camilla Labrie, therapeutic approaches. NK cells are showing potential for immuno- Suzanne van Duikeren, Ing, Ramon Arens, PhD, Thorbald van Hall, PhD, therapy of GBM. However, NK cells struggle to cross the blood brain Sjoerd van der Burg, PhD barrier (BBB) and infiltrate into GBM [1]. Moreover, the immunosup- Leiden University Medical Center, Leiden, Netherlands pressive tumor microenvironment (TME) impairs NK cell activity, for Correspondence: Sjoerd van der Burg (shvdburg@lumc.nl) instance due to adenosine-mediated downregulation of NKG2D ex- Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P495 pression [2]. Methods Background We developed an innovative NK cell-based immunotherapy for Immunotherapy may induce complete tumor regressions but often GBM that targets multiple “checkpoints” simultaneously, by tumors partially regress followed by tumor recurrence. Here, we fo- combining 1) multifunctional NK cells which consist of a cleav- cused on the underlying mechanisms. able scFv targeting CD73 alongside dual chimeric antigen recep- Methods tors directed against GD2 and NKG2D ligands and 2) inhibition The TC-1 mouse tumor model in which different formulation and ap- of autophagy in GBM cells to sensitize them to NK cell lysis and plication of an HPV16 SLP vaccine results in full cure or tumor recur- promote NK cell infiltration into GBM via the secretion of GBM- rence and therapy resistance after initial full tumor regression. specific chemoattractants. Tumors, spleens and lymph nodes were analyzed by mass- and flow- Results cytometry. Mice were treated with antibodies to PD-1, PD-L1, OX-40, We have designed and synthesized a multifunctional CAR con- 4-1BB, NKG2A and TGFβ. Cell-sorted tumor cells were RNA se- struct that expresses an anti-CD73 scFv which is cleavable by quenced. Immune parameters were assessed in 5-10 mice, survival GBM-associated proteases, and a dual CAR that enables NK cells analyses were performed on at least 10 mice per group. All experi- to avoid antigen escape common to GBM (Figure 1A). We have ments were performed 2-3 times. generated engineered NK-92 or primary human NK cells that effi- Results ciently express the construct, from which the anti-CD73 scFv Optimal vaccination resulted in about 7% circulating tumor- could be functionally released via uPA treatment (Figure 1B and specific CD8+ T cells and complete cure of all mice, whereas sub- C). Engineered NK cells showed a significantly higher in vitro abil- optimal vaccination led on average to 1.7% tumor-specific T cells ity to kill patient-derived GBM43 targets (Figure 1D and E). To and tumor regression followed by recurrence in all mice. Neither target autophagy, BECN1- GBM43 cells were generated, and their booster vaccinations, which increased the numbers of circulating in vivo subcutaneous growth in RAG-1-/- mice validated the crit- tumor-specific type 1 cytokine-producing CD4+ and CD8+ T cells ical role of autophagy in GBM onset and progression (Figure 1F). (p<0.01), nor the co-administration of (combinations of) anti- We further showed that targeting autophagy inhibited the bodies against PD-1, PD-L1, 4-1BB, or OX-40 prevented tumor re- in vitro proliferation of GBM43 itself (Figure 1G), sensitized GBM currence or improved survival after vaccination. Immune escape to NK cell lysis (Fig. 1H), and induced elevated chemokine secre- was intrinsic to the tumor cells as the direct reinjection of ex- tion (CCL5), which in turn increased NK cell migration across the vivo cell-sorted recurrent tumor cells into groups of 10 naïve BBB using an in vitro BBB model (Figure 1I). hosts did not result in any response to vaccination while in all Conclusions cases the reinjected ex-vivo cell-sorted nontreated tumor cells We have generated multifunctional NK cells that can target mul- displayed vaccine-induced tumor regression followed by relapse tiple “checkpoints” at once showing improved cytotoxicity against (p<0.01). Ex-vivo analyses of escaped tumor cells showed no al- GBM through increased resistance to the immunosuppressive terations in the expression of MHC-I or the E7 tumor-antigen or TME via adenosinergic CD73 blockade and the ability to avoid their sensitivity to tumor-specific CTL mediated killing. RNA se- antigen escape by GBM via dual CARs. We have also found that quencing on bulk sorted (CD45-) tumor cells from non-treated blocking the autophagy pathway in GBM displayed potent syn- (n=4) and escaped (n=4) tumors, revealed a specific vaccine- ergy with NK cell-mediated immunotherapy. Based on these re- induced downregulation of the TNF- and P53-signaling pathways sults, to achieve combined therapeutic effects in vivo, we are and upregulation of TGFβ signaling. Indeed, more TGFβ positive currently evaluating this immunotherapy in an orthotopic GBM fibroblasts surrounded the escaped tumors (p<0.05). Recurrent tumors mouse model. Taken together, this approach provides a promis- displayed strongly reduced numbers of infiltrated CD8+ T cells (p< ing platform for the combination treatment of GBM with engi- 0.001), whereas this was not the case for escaped tumor cells- neered NK cells. reinjected tumors. However, both types of escaped tumors displayed lower numbers of tumor-infiltrating Ly6C+MHCI-II+ inflammatory mac- rophages (p<0.01). TGFβ-blockade delayed but did not prevent relapses References to recur (p=0.07). The use of inflammation inducing chemotherapy rein- 1. Kmiecik J, Zimmer J, Chekenya M. Natural killer cells in intracranial stalled the infiltration of tumors with inflammatory myeloid cells after neoplasms: presence and therapeutic efficacy against brain tumours. J vaccination, prevented relapse and reinstalled sensitivity of escaped tu- Neurooncol. 2014 ;116(1):1-9. mors to therapeutic vaccination (p=0.01). 2. Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting Conclusions enhances immunotherapy of CD73+ solid tumors with piggyBac- The sequential clinical phases during non-curative immunotherapy engineered chimeric antigen receptor natural killer cells. J Immunother may involve several distinct secondary escape mechanisms. Cancer. 2018;6(1):136. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 270 of 272 not show tumor growth inhibition in this model, DRP-104 significantly inhibited tumor growth and the combination further enhanced efficacy, illustrated by extended survival for both DRP-104 alone (50 days) and combination (96 days) treatment groups compared to vehicle (33days) and anti-PD-L1 alone (33days). Combination treatment also resulted in long term durable cures in 50% of the mice. Conclusions DRP-104 treatment results in dramatic remodeling of the tumor micro environment, leading to enhanced function of multiple immune cells distinct from activities obtained by anti-PD-1 Ab. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti- tumor efficacy including long-term durable cures even in checkpoint in- hibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in com- bination with PD-1/PD-L-1 checkpoint inhibitors. P498 Blockade of PD-1 and LAG-3 on CD8+ T cells, induced by vaccination, elicits superior anti-tumor efficacy Christopher Zahm, PhD, Douglas McNeel, MD, PhD, Laurne Delmastro UW Carbone Cancer Center, Madison, WI, United States Correspondence: Douglas McNeel (dm3@medicine.wisc.edu) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P498 Fig. 1 (abstract P496). See text for description Background T cell immune checkpoint receptors (ICR) and their ligands have emerged as a major mechanism by which tumors avoid immune de- tection. ICR blockade targeting PD-1/PD-L1 and/or CTLA-4 have revo- P497 lutionized the treatment of many cancer types. However, not all DRP-104, a novel broad acting glutamine antagonist, induces cancers respond to ICR blockade, in large part mediated by the pres- distinctive immune modulation mechanisms and synergistic ence or absence of tumor infiltrating CD8+ T cells. We have focused efficacy in combination with immune checkpoint blockade on tumor vaccines as a means to increase the number of tumor- Yumi Yokoyama, PhD, Michael Nedelcovych, PhD, Robert Wild, PhD specific CD8+ T cells. We have previously demonstrated that activa- Dracen Pharmaceutical, New York,, NY, United States tion of CD8+ T cells by vaccination leads to increased expression of Correspondence: Robert Wild (rwild@dracenpharma.com) specific ICR, and that blockade of these ICR with vaccination leads to Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P497 better anti-tumor response than either alone. Differences in expres- sion of specific ICR, notably PD-1 and LAG-3, appeared dependent on Background presentation of antigen by professional versus non-professional APC, Glutamine is an essential amino acid for rapidly proliferating cancer hence we hypothesized that blockade of both of these ICR with vac- cells, thus depriving the same fuel from immune cells and contributing cination should be superior to either alone. to tumor immune evasion. DRP-104 was designed as a novel prodrug Methods of the broad acting glutamine antagonist 6-Diazo-5-oxo-L-norleucine In these studies we directly assessed the expression of PD-1, LAG-3, (DON). DRP-104 is inert in its prodrug form, affords high levels of CTLA-4, and TIM-3 on CD8+ T cells following activation in the pres- plasma and gastro-intestinal (GI) tissue stability; has high tumor cell ence or absence of professional APC. Next, we transferred these cells permeability and preferential tumor versus plasma/GI tissue distribution into tumor bearing mice, alone or in combination ICR blocking anti- for DON. Here we sought to (1) compare immunological modulation of bodies, to directly evaluate their anti-tumor efficacy. Finally, we im- DRP-104 to anti-PD-1Ab, and (2) evaluate the combination effect of munized tumor-bearing HLA-A2-transgenic mice with different anti- DRP-104 with PD-1/PD-L1 checkpoint inhibitors. tumor DNA vaccines that have previously been shown to elicit CD8+ Methods T cells preferentially expressing either PD-1 or LAG-3, and used each Immunomodulatory effects of DRP-104 as a single agent and com- vaccine alone or in combination with ICR blockade. bination with anti-PD-1Ab was evaluated in the CT26 mouse colon Results carcinoma model by flow cytometry and Luminex assay. In vivo anti- We found that PD-1, LAG-3, CTLA-4 and TIM-3 are all increased on tumor efficacy of combination with anti-PD-1/PD-L1Ab was evaluated CD8+ T cells after activation by professional APC, however LAG-3 in CT26 and H22 hepatocellular carcinoma models. alone was increased on CD8+ T cells activated in the absence of pro- Results fessional APC (Figure 1). When these cells were adoptively trans- DRP-104 treatment showed broad immune cell modulation effects in- ferred into tumor bearing mice, LAG-3 blockade improved the anti- cluding increased T, NK, and macrophages; while anti-PD-1Ab affected tumor efficacy of CD8+ T cells activated without APC, and PD-1 mainly CD8+T cells. Cytokine modulation in tumor and plasma revealed blockade improved the anti-tumor efficacy of CD8+ T cells activated that DRP-104 decreased pro-tumorigenic cytokines such as VEGF and by APC (Figure 2). Immunization with different DNA constructs [1-3] KC(IL-8) while anti-PD-1Ab showed either no change or slight increase in combination with ICR blockade led to improved anti-tumor re- in these cytokines. CT26 bearing mice treated with anti-PD-1Ab alone, sponses, however combining LAG-3 blockade with PD-1 blockade DRP-104, and the combination showed tumor growth inhibition at day showed no benefit over PD-1 blockade alone (Figure 3). 12 of 48%, 90%, and 94%, respectively. Median survival days were 31.5, Conclusions 36, and 56 days, respectively (vehicle; 17.5 days). Notably 9 mice These data support our previous finding that PD-1 blockade improves treated with combination of anti-PD-1 with DRP-104 were tumor free at the efficacy of CD8+ T cells activated by vaccination. In this model, we end of the experiment (day 77) and 100% of these mice rejected a detected no additional benefit to concurrent LAG-3 blockade. The role of CT26 tumor re-challenge. In the H22 model, mice were treated with ei- other ICR in limiting anti-tumor immunity, and strategic blockade of these ther anti-PD-L1 Ab, DRP-104, or combination. While anti-PD-L1Ab did receptors following T-cell activation, is an area of active investigation. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 271 of 272 References 1 Smith, H. A., Rekoske, B. T. & McNeel, D. G. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses. Vaccine 32, 1707-1715, doi:10.1016/j.vac- cine.2014.01.048 (2014). 2 Rekoske, B. T., Smith, H. A., Olson, B. M., Maricque, B. B. & McNeel, D. G. PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization. Cancer Immunol Res 3, 946-955, doi:10.1158/2326-6066.CIR-14-0206 (2015). 3 Colluru,V. T., Zahm,C.D.&McNeel,D.G.Mini-intronicplasmid vac- cination elicits tolerant LAG3+ CD8 T cells and inferior anti-tumor re- sponses. OncoImmunology, 0-0, doi:10.1080/2162402X.2016.1223002 (2016). Fig 3 (abstract P498). Some vaccines only effective when combined with ICR blockade P499 Nano-Pulse Stimulation in combination with the TLR 7/8 agonist, resiquimod, synergizes to eliminate murine melanoma through innate and adaptive immune responses Joel Benjamin, PhD, Amanda McDaniel, BA, Kristin von Rothstein, Sasha Farina, Bruce Freimark, PhD, Richard Nuccitelli, MS, PhD Pulse Biosciences, Inc, Hayward, CA, United States Correspondence: Richard Nuccitelli (rnuccitelli@pulsebiosciences.com) Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P499 Fig. 1 (abstract P498). Priming with professional APCs leads to Background ICR expression Nano-Pulse Stimulation (NPS) is a non-thermal treatment modality that provides high amplitude electrical energy pulses in the nanosec- ond range that is focal and directly acts on cellular structures and membranes to initiate regulated cell death. Previous work has shown that NPS induces release of tumor antigen and stimulates an in situ anti-tumor immune response [1, 2, 3]. The TLR 7/8 agonist resiqui- mod (RES) has been used as an immune adjuvant in previous cancer vaccine treatments in murine models to aid in antigen processing and presentation [4]. We have evaluated the combination of NPS and RES treatment to inhibit tumor growth and induce innate and adaptive immune responses. Methods The B16-F10 melanoma in C57BL/6j mice was used to investigate the potential combined effects of NPS and RES. B16-F10 tumor cells (2x10e5) were injected intradermally on the left flank and treated with NPS 5 days after inoculation. RES (50 μg) was then dosed in multiple combinations and timing to assess optimal tumor cell elimination and immune stimulation from the combin- ation treatment. Tumor efficacy (volume) was measured twice per week. Immune biomarkers included flow cytometry and IHC of T cell and myeloid immune cells from tumors, draining lymph nodes and spleens. Results Low energy NPS and up to 3 doses of RES as monotherapies partially inhibit tumor growth. However, combination of NPS with 24-hour, post treatment of RES resulted in complete regression in a large frac- tion of treated animals. This efficacy is concomitant with increased antigen-specific and memory CD8 populations in the spleen and lymph node, as well as an increase in certain innate immune cell Fig. 2 (abstract P498). APC-induced ICR signaling compromised populations. Tumor regressions showed no sign of regrowth 90 days anti-tumor response after treatment. Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):282 Page 272 of 272 Conclusions Methods Compared to monotherapy treatments, the combination of NPS and Three models of colon tumorigenesis were used to conduct the RES treatments induce stronger tumor growth inhibition which per- present study (IACUC#3408), which included a colitis (AOM/DSS –in- sists as long-term tumor regression. Persistent tumor regression by duced), a spontaneous (APCMin-driven), and a syngeneic (engrafted combination treatment is associated with increases in B16F10 with MC-38 or CT-26 cells) model. Several mutant mice were used in antigen-specific and memory CD8 populations, as well as increases in these models including PARP-1-/-, PARP-1-/+ APCMin/+, APCMin/ innate immune populations with potential for antigen presentation. +PARP-1-/-+/−, APCMin/+PARP-1-/+ as well as WT mice. Mice were These data support a mechanism by which NPS combined with randomized and assigned to the different experimental groups. TLR7/8 agonists activates an enhanced immune response. Some groups of mice were administered olaparib, anti-mouse PD-1 antibodies, or a combination of the two agents. Mice were scarified References according to the requirements of each model and tumor and tissues 1. Nuccitelli R, Berridge JC, Mallon Z, Kreis M, Athos B, Nuccitelli P. were collected for the analysis. MDSCs were generated by incubating Nanoelectroablation of murine tumors triggers a CD8-dependent inhib- bone marrow cells with GM-CSF, G-CSF, and IL-6. Tumor MDSCs were ition of secondary tumor growth. PLoS ONE. 2015; 10(7): e0134364 1-17. generated by enzymatic digestion of MC-38-engrafted tumors 2. Nuccitelli R, McDaniel A, Anand S, Mallon Z, Berridge JC, Uecker D. Nano- followed by positive selection. The suppression assay was performed Pulse Stimulation is a physical modality that can trigger immunogenic by co-cultured with CD3/CD28-stimulated CFSE-labeled T cells and tumor cell death. J Immunotherapy Cancer. 2017; 5:32 DOI 10.1186/ proliferation was assessed by FACS. s40425-017-0234-5. Results 3. Skeate JG, DaSilva DM, Chavez-Juan E, Anand S, Nuccitelli R, Kast W.M. Here, we show that partial PARP-1 inhibition via gene heterozygosity or Nano-Pulse Stimulation induces immunogenic cell death in human a moderate olaparib dose was sufficient to protect against colitis- or papillomavirus-transformed tumors and initiates an adaptive immune re- APCMin-mediated intestinal tumorigenesis, while extensive inhibition sponse. 2018; PLoS ONE. 13(1): e0191311 via gene knockout or a high olaparib dose was ineffective or aggra- 4. Caisova V, Vieru A, Kumzakova Z, Glaserova S, Husnikova H, Vacova N, vated the burden despite anti-inflammatory effects and promotion of a Krejcova G, Padoukova L, Jochmanova I, Wolf KI, Chmelar J, Kopecky J, tumor-suppressive microenvironment. A sub-IC50 metronomic dose of Zenka J. Innate immunity-based cancer immunotherapy: B16-F10 murine olaparib or PARP-1 heterozygosity was also sufficient to block tumori- melanoma model. 2016; BMC Cancer.16(1); 940; DOI:10.1186/s12885-016- genesis in syngeneic colon cancer models by modulating the suppres- 2982-x. sive function, but not differentiation or intratumoral migration, of myeloid-derived suppressor cells (MDSCs). These effects occurred through a reduction of arginase-1, iNOS, and COX-2 expression but in- P500 dependently of PARP-1-trapping on chromatin. Interestingly, the metro- Targeting PARP-1 with metronomic therapy as a new approach to nomic olaparib dose increased the intratumoral numbers, but not modulate MDSC function and enhance anti-PD1 immunotherapy in percentages, of CD8+ T cells. Adoptive transfer of WT bone marrow- colon cancer derived MDSCs abrogated the protective effects of PARP-1 heterozy- 1 1 1 Salome Valentina Ibba , Mohamed Ghonim , Abdelmetalab Tarhuni , gosity against the tumor burden. A metronomic olaparib dose was 1 1 1 Matthew Dean , Hamid Boulares, PhD , Augusto Ochoa, MD , Youssef highly synergistic with anti-PD1-based immunotherapy leading to al- 1 1 1 1 Errami , Ali Elbahraway , Ilyes Benslimane , Dorota Wyczechowska , Luis most complete eradication of tumors on mice. 1 2 1 Del Valle, MD , Amir Al-Khami , Hanh Luu Conclusions Louisiana State University-Health Science Center, New Orleans, LA, Our results support a paradigm-shifting concept that expands the United States; Tanta University, Tanta, Egypt utility of PARPi and encourage testing metronomic dosing of PARPi Correspondence: Hamid Boulares (hboulr@lsuhsc.edu) to enhance efficacy of check-point inhibitor-based immunotherapies Journal for ImmunoTherapy of Cancer 2019, 7(Suppl 1):P500 not only in cancer of the colon but also that of other tissues ultim- ately benefiting a large proportion of cancer patients. Background Ethics Approval PARP inhibitors (PARPi) have important anti-tumor effects in BRCA- IACUC#3408 defective cancers but efficacy requires their use at/near maximum- tolerated-doses to achieve trapping of the enzyme on damaged Publisher’s Note chromatin. However, the benefits of targeting non-DNA repair as- Springer Nature remains neutral with regard to jurisdictional claims pects of PARP with low metronomic doses have not been explored. in published maps and institutional affiliations.

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