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32nd Annual Meeting and Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2017): Part Two

32nd Annual Meeting and Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC... Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 DOI 10.1186/s40425-017-0288-4 MEETING ABSTRACTS Open Access 32nd Annual Meeting and Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2017): Part Two National Harbor, MD, USA. 8-12 November 2017 Published: 7 November 2017 About this supplement These abstracts have been published as part of Journal for ImmunoTherapy of Cancer Volume 5 Supplement 2, 2017. The full contents of the supplement are available online at https://jitc.biomedcentral.com/articles/supplements/volume-5-supplement-2. Please note that this is part 2 of 2 Poster presentations 0-1; adequate organ function; no active autoimmune disease. Patients will be randomly assigned 1:1 to receive chemotherapy + pembrolizumab Clinical Trials (In Progress) (arm 1) or chemotherapy + placebo (arm 2). Stratification factors are geo- graphic region (Asia vs Non-Asia), primary tumor location (stomach vs P218 GEJ), and tumor stage (II/III vs IVa). All patients will receive neoadjuvant KEYNOTE-585: randomized, phase 3 study of chemotherapy + (preoperative) chemotherapy + pembrolizumab every 3 weeks (Q3W) for pembrolizumab vs chemotherapy + placebo as neoadjuvant/ 3 cycles or chemotherapy + placebo Q3W for 3 cycles followed by surgery adjuvant treatment for patients with gastric or gastroesophageal and then adjuvant chemotherapy + pembrolizumab Q3W for 3 cycles or junction (G/GEJ) cancer 1 2 3 4 chemotherapy + placebo Q3W for 3 cycles followed by monotherapy with Yung-Jue Bang , Eric Van Cutsem , Charles Fuchs , Atsushi Ohtsu , Josep 5 6 7 6 pembrolizumab or placebo Q3W for 11 cycles; treatment will continue for Tabernero , David Ilson , Woo Jin Hyung , Vivian Strong , Thorsten 8 9 6 10 10 up to 17 cycles overall. Chemotherapy consists of cisplatin 80 mg/m intra- Goetze , Takaki Yoshikawa , Laura Tang , Linda Sun , Aisha Hasan , 11 4 venously + either capecitabine 1000 mg/m twice daily orally or 5- Minori Koshiji , Kohei Shitara 1 2 fluorouracil 800 mg/m intravenously (investigator’s choice). Pembroli- Seoul National University Hospital, Seoul, Republic of Korea; University zumab 200 mg was administered intravenously. Adjuvant monotherapy Hospitals Gasthuisberg Leuven and KU Leuven, Leuven, Belgium; Yale consists of pembrolizumab (arm 1) or placebo (arm 2). Primary end points Cancer Center, New Haven, CT, USA; National Cancer Hospital East, are overall survival (OS), event-free survival, and rate of pathologic Chiba, Japan; Vall d'Hebron University Hospital and Institute of complete response (defined as no invasive disease and histologically Oncology (VHIO), Barcelona, Spain; Memorial Sloan Kettering Cancer negative nodes) per central review. Adverse events (AEs) are graded per Center, New York, NY, USA; Yonsei Cancer Hospital, Yonsei University National Cancer Institute Common Terminology Criteria for Adverse Events Health System, Seoul, Republic of Korea; Institute of Clinical Cancer v4.0 and will be monitored for 30 days after treatment end (90 days for Research, UCT University Cancer Center, Frankfurt, Germany; Kanagawa serious AEs). Patients are followed up for survival every 12 weeks until Cancer Center, Kanagawa, Japan; Merck & Co., Inc., Kenilworth, NJ, USA; death, withdrawal from study, or study termination. Planned enrolment is Merck & Co., Inc., New York, NY, USA approximately 800 patients. Correspondence: Yung-Jue Bang (bangyj@snu.ac.kr) Trial Registration Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P218 ClinicalTrials.gov, NCT03221426 Background In KEYNOTE-012 (NCT01848834) and KEYNOTE-059 (NCT02335411), pem- brolizumab demonstrated manageable safety and promising antitumor P219 activity alone or in combination with chemotherapy in patients with ad- Phase 1/1b, first-in-human study of the PI3K-gamma inhibitor IPI- vanced G/GEJ cancer. Compared with chemotherapy alone, chemother- 549 as monotherapy and combined with nivolumab in patients apy combined with pembrolizumab in the neoadjuvant/adjuvant setting with advanced solid tumors 1 2 3 4 can provide additional benefit to patients with locally advanced, resect- Antoni Ribas , David Hong , Anthony Tolcher , Ryan Sullivan , Geoffrey 5 1 6 6 able G/GEJ cancer. KEYNOTE-585 is a phase 3, randomized, double-blind Shapiro , Bartosz Chmielowski , Les Brail , Lucy Lee , Suresh 6 6 7 study of chemotherapy combined with pembrolizumab versus chemo- Mahabhashyam , Claudio Dansky Ullmann , Michael Postow , Jedd therapy combined with placebo as neoadjuvant/adjuvant treatment for Wolchok 1 2 locally advanced resectable G/GEJ cancer. University of California, Los Angeles, Los Angeles, CA, USA; MD Methods Anderson Cancer Center, Houston, TX, USA; South Texas Accelerated Key eligibility criteria in KEYNOTE-585 (NCT03221426) include age ≥18 Research Therapeutics (START), San Antonio, TX, USA; Massachusetts years; previously untreated G/GEJ adenocarcinoma (Siewert type 2 or 3 tu- General Hospital, Boston, MA, USA; Dana-Farber Cancer Institute, mors; Siewert type 1 tumor eligibility limited to those for whom planned Boston, MA, USA; Infinity Pharmaceuticals, Inc., Cambridge, MA, USA; treatment is perioperative chemotherapy and resection), with no evidence Memorial Sloan Kettering Cancer Center, New York, NY, USA of metastatic disease; planning to undergo surgery after preoperative Correspondence: Les Brail (Les.Brail@infi.com) chemotherapy; Eastern Cooperative Oncology Group performance status Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P219 © The Author(s). 2017 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 2017, 5(Suppl 2):87 Page 118 of 244 Background Background IPI-549 is a potentially first-in-class, oral, potent, selective PI3K-gamma Adoptive cell therapy (ACT) may be effective in treating immunogenic inhibitor being developed as an immuno-oncology therapeutic in mul- tumors with high mutational load such as melanoma and virally- tiple cancer indications. Preclinical solid tumor research has shown that associated tumors like cervical cancer with several patients in studies PI3K-gamma blockade in tumor-associated macrophages by IPI-549 re- performed by other institutions achieving durable complete response sults in transcriptional reprogramming of the pro-tumor macrophage for years. HPV infection increases mutational load, thus providing add- phenotype (M2) to its anti-tumor counterpart (M1). Within this setting, itional neoantigen targets ideal for the polyclonal nature of ACT. As out- IPI-549 has demonstrated activity as monotherapy and still greater ac- comes for patients with recurrent, metastatic or persistent cervical tivity when combined with checkpoint inhibitor therapies. Importantly, cancer remain extremely poor, there is an enormous need for novel im- the latter approach has been found to overcome checkpoint inhibitor munotherapeutic approaches with curative potential such as ACT. resistance in specific resistant models. Methods Methods Clinical trial C-145-04 (NCT03108495) is a prospective, phase 2 multi- This study is being conducted to evaluate the safety, tolerability, center, open-label study evaluating the efficacy of a single autolo- pharmacokinetics, and pharmacodynamics of IPI-549 to ultimately de- gous tumor infiltrating lymphocyte infusion (LN-145) followed by IL-2 termine its recommended Phase 2 dose and activity, both as mono- after a non-myeloablative lymphodepletion (NMA-LD) regimen in pa- therapy and in combination with nivolumab, in patients with advanced tients with recurrent, metastatic, or persistent cervical cancer who solid tumors. As shown in Fig. 1, the design includes four parts: 1) dose have failed at least one prior systemic therapy. The clinical trial escalation (DE) of IPI-549 monotherapy; 2) DE of IPI-549 with fixed-dose protocol requires resection of a tumor lesion which is then shipped nivolumab; 3) monotherapy expansion; and 4) combination expansion to a central GMP manufacturing facility for TIL extraction, expansion, in specific tumor types, including non-small cell lung cancer, melan- and preparation of the final infusion product (LN-145). One week oma, and squamous cell carcinoma of the head and neck, with de novo prior to LN-145 shipment and infusion, patients undergo NMA-LD or acquired resistance to checkpoint inhibitors (Fig. 1). consisting of cyclophosphamide (60 mg/kg) daily x 2 days followed Results by fludarabine (25 mg/m ) daily x 5 days. LN-145 is infused 24 hours This trial is currently in progress. No results are available. after the last dose of fludarabine followed by up to 6 doses of IL-2 Progress update: Monotherapy DE enrollment is complete for once- (600,000 IU/kg) every 8-12 hours. Simon’s two-stage optimal design daily (QD) IPI-549 doses up to 60 mg, while monotherapy expansion with one-sided alpha level=0.025 and 80% power will be used to enrollment has been initiated at 60 mg QD. Combination DE enroll- compare an objective response rate (ORR) of 5% vs. 20% in the first ment is complete for QD doses of 20 mg and 30 mg IPI-549 com- stage (n=15 subjects). If two or more ORR are observed, trial will expand bined with fixed-dose nivolumab (240 mg once every 2 weeks). to Stage 2 (n=47). The primary endpoint is the ORR per RECIST v1.1. Sec- Conclusions ondary endpoints include complete response, duration of response, dis- The collective preclinical data highlight the key role of PI3K-gamma ease control rate, progression free- and overall survival; and the safety in the immuno-suppressive tumor microenvironment and provide a summarization of treatment-emergent adverse events (AEs) including strong rationale for the ongoing clinical study of IPI-549. serious AEs, AEs leading to discontinuation, and clinical laboratory tests. Trial Registration Patients must have been treated with at least 1 systemic chemotherapy ClinicalTrials.gov: NCT02637531 or immunotherapy treatment for recurrent, metastatic, or persistent cer- vical cancer and, in addition to the tumor targeted for excision and TIL manufacture, must have an additional measurable lesion for assessment of response. Other major eligibility criteria include amongst others: adequate bone marrow, liver, pulmonary, cardiac and renal function; ECOG performance status of 0 or 1. Systemic steroids greater than 10 mg/day prednisone equivalents are prohibited as are a history of serious immunotherapy-related adverse events. Trial Registration ClinicalTrials.gov identifier: NCT03108495 P221 A phase 2 study to evaluate the safety and efficacy using autologous tumor infiltrating lymphocytes (LN-145) in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck 1 2 3 3 Rom Leidner , James Ohr , Robert Brown , Sam Suzuki , 3 3 2 Igor Gorbatchevsky , Maris Fardis , Robert Ferris Earle A. Chiles Research Institute – Providence Cancer Center, Portland, 2 3 OR, USA; Hillman Cancer Center at UPMC, Pittsburgh, PA, USA; Iovance Fig. 1 (abstract P219). See text for description Biotherapeutics, San Carlos, CA, USA Correspondence: Rom Leidner; Robert Brown (robert.brown@iovance.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P221 P220 A phase 2, multicenter study to evaluate the efficacy and safety Background using autologous tumor infiltrating lymphocytes (LN-145) in patients Adoptive cell therapy (ACT) may be effective in treating immuno- with recurrent, metastatic, or persistent cervical carcinoma 1 2 3 4 genic tumors with high mutational load such as melanoma and Amir Jazaeri , Robert Edwards , Emese Zsiros , Robert Brown , 4 4 4 5 virally-associated tumors like cervical cancer with several patients in Igor Gorbatchevsky , Sam Suzuki , Maria Fardis , Robert Wenham studies performed by other institutions achieving durable complete University of Texas MD Anderson Cancer Center, Houston, TX, USA; response for years. Despite the heterogeneity of squamous cell car- Magee-Women's Hospital of University of Pittsburgh Medical Center, cinomas of the head and neck (HNSCC), most tumors are either Pittsburgh, PA, USA; Roswell Park Cancer Institute, Buffalo, NY, USA; 4 5 virally-associated (e.g., HPV in oropharyngeal) or carry high muta- Iovance Biotherapeutics, San Carlos, CA, USA; Moffitt Cancer Center, tional load (e.g., tobacco-related) providing an increased diversity of Tampa, FL, USA potential targets ideal for the polyclonal nature of ACT. Furthermore, Correspondence: Robert Brown (robert.brown@iovance.com) outcomes for patients with recurrent and/or metastatic HNSCC Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P220 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 119 of 244 remain poor. Therefore, a clear rationale exists for the potential appli- melanoma with an activating BRAF mutation. The primary endpoint of cation of ACT in patients with HNSCC. the study is tolerability as measured by dose-limiting toxicities seen in Methods the first 5 weeks of treatment. Key secondary endpoints include Clinical trial C-145-03 (NCT03083873) is a prospective phase 2 multicen- progression-free survival, objective response rate, change in tumor bur- ter, open-label study evaluating the efficacy of a single autologous tumor den, time to response, and duration of response among responders. infiltrating lymphocyte infusion (LN-145) followed by IL-2 after a non- Tumor-level responses in injected and uninjected tumors, and myeloablative lymphodepletion (NMA-LD) regimen in patients with characterization of immune markers in pre-study and on-study biopsies recurrent and/or metastatic HNSCC. Study-related therapy begins with will be exploratory endpoints. Key eligibility criteria include unresect- resection of a tumor lesion that is then shipped to a central GMP manu- able stage IIIB-IV BRAF mutant melanoma, presence of measurable and facturing facility where TIL are extracted, expanded, packaged, and injectable disease, no active cerebral metastases or autoimmune dis- shipped for administration (LN-145). One week prior to LN-145 infusion, eases, and any number of prior lines of therapy but no prior receipt of 6 8 patients undergo NMA-LD consisting of cyclophosphamide (60 mg/kg) T-VEC. T-VEC (10 PFU/mL first dose, 10 PFU/mL subsequent doses) will daily x 2 days followed by fludarabine (25 mg/m ) daily x 5 days. LN-145 be administered by intralesional injection into cutaneous, subcutane- is infused 24 hours after the last dose of fludarabine followed by up to 6 ous, or nodal lesions on week 1 day 1, week 4 day 1, and every 2 weeks doses of IL-2 (600,000 IU/kg) every 8-12 hours. Simon’s two-stage optimal thereafter until disappearance of injectable lesions, complete response, design with one-sided alpha level=0.025 and 80% power will be used to progressive disease, intolerance of study treatment, or 24 months after compare an objective response rate (ORR) of 5% vs. 20% in the first starting therapy, whichever occurs first. Dabrafenib and trametinib will stage (n=15 subjects). If two or more ORR are observed, trial will expand be given until progression or intolerance. Twenty subjects are to be en- to Stage 2 (n=47). The primary efficacy endpoints are the objective re- rolled at a single U.S. institution. sponse rate per RECIST v1.1 and the safety summarization of treatment- Trial Registration emergent adverse events (AEs) including serious AEs, AEs leading to NCT03088176 discontinuation, and clinical laboratory tests. Secondary efficacy end- points include CR, DOR, PFS, and OS. Patients must have been treated P223 with at least one systemic chemotherapy or immunotherapy treatment KEYNOTE-199: Phase 2 nonrandomized study of pembrolizumab in for recurrent and/or metastatic HNSCC and, in addition to the tumor patients with PD-L1+ and PD-L1– metastatic castration-resistant targeted for excision and TIL manufacture, must have an additional prostate cancer measurable lesion for assessment of response. Additional eligibility cri- 1 2 3 4 Johann de Bono , Josep Piulats , Marine Gross-Goupil , Jeffrey Goh , teria include amongst others: adequate bone marrow,liver,pulmonary, 5 6 7 Kristiina Ojamaa , Christopher Hoimes , Ulka Vaishampayan , cardiac, and renal function; ECOG performance status of 0 or 1. Systemic 8 9 10 11 Raanan Berger , Ahmet Sezer , Ronald De Wit , Charles Drake , steroids greater than 10 mg/day prednisone equivalents are prohibited 12 12 13 Haiyan Wu , Christian Poehlein , Emmanuel S. Antonarakis as are a history of serious immunotherapy-related adverse events. Royal Marsden Hospital and the Institute of Cancer Research, London, Trial Registration United Kingdom; Instituto Catalan de Oncologia, Hospital Duran i ClinicalTrials.gov identifier:NCT03083873 Reynals, Hospitalet de Llobregat, Barcelona, Spain; Institut Bergonie, Bordeaux, France; Royal Brisbane & Women’s Hospital, Brisbane, 5 6 P222 Australia; East Tallinn Central Hospital, Tallinn, Estonia; University Clinical trial in progress: A phase 1b trial of talimogene Hospitals Seidman Cancer Center, Cleveland, OH, USA; Karmanos laherparepvec (T-VEC) in combination with dabrafenib and Cancer Institute, Detroit, MI, USA; Chaim Sheba Medical Center, Ramat- trametinib in advanced melanoma with an activating BRAF Gan, Israel; Baskent Üniversitesi Adana Uyg. ve Arast, Adana, Turkey; 10 11 mutation Erasmus MC Cancer Institute, Rotterdam, Netherlands; Columbia 1 2 1 2 12 Kenneth Byrd , Nibedita Chakraborty , Mehmet Kocak , Alisa Harber , University, New York, NY, USA; Merck & Co., Inc., Kenilworth, NJ, USA; 2 13 Ari Vanderwalde Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA University of Tennessee Health Science Center, Memphis, TN, USA, Correspondence: Johann de Bono (Johann.DeBono@icr.ac.uk) Memphis, TN, USA; West Cancer Center, Germantown, TN, USA, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P223 Germantown, TN, USA Correspondence: Kenneth Byrd (kbyrd@westclinic.com) Background Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P222 Metastatic castration-resistant prostate cancer (mCRPC) treatment has in- cluded suppression of androgen receptor signaling, palliative radiation Background therapy, and chemotherapy. As expression of the programmed death 1 Patients with BRAF-mutant advanced melanoma have been shown to (PD-1) receptor and its ligand PD-L1 is present in a subset of mCRPC respond at high rates to combination BRAF plus MEK inhibition. lesions, targeting this pathway may be an attractive treatment option. Response rates to single-agent immune therapies tend to be modest, KEYNOTE-199 (NCT02787005) is a nonrandomized, multinational, multi- but are often durable, while response rates to BRAF plus MEK inhibition cohort open-label phase 2 study to evaluate the anti–PD-1 antibody tend to be high but transient. While early attempts at combining tar- pembrolizumab in patients with mCRPC. geted and immune therapy resulted in dose-limiting hepatic toxicity, Methods more recent attempts with newer agents have shown significant prom- Patients must be ≥18 years old with histologically or cytologically con- ise. T-VEC is an oncolytic viral immunotherapy which was designed to firmed prostate adenocarcinoma without small-cell histology, measur- selectively replicate in tumors resulting in lytic cell death, antigen able disease (RECIST v1.1) or detectable bone metastases by whole-body release, and production of GM-CSF to enhance systemic immune re- bone scintigraphy and no RECIST v1.1 measurable tumors, supplied sponse. In a prior randomized, phase 3 study of T-VEC versus GM-CSF, tumor sample for PD-L1 expression (new or archived), disease progres- durable response rate was statistically improved with T-VEC, with a sion within 6 months before screening, and ECOG performance status 0- strong trend towards improved overall survival. Dabrafenib and trame- 2. As of June 2017, 2 additional cohorts were added called cohorts 4 tinib, inhibitors of BRAF and MEK, respectively, have been shown in and 5. For these cohorts patients had to fail or show signs of failure on combination to result in response rates of up to 75% and improvement current prechemotherapy enzalutamide; patients could fail abiraterone in progression-free and overall survival compared to single-agent tar- treatment before enzalutamide. For cohort 4, patients will be enrolled geted therapy and chemotherapy. Combining T-VEC with dabrafenib with RECIST v1.1-measurable disease (n=80) and for cohort 5 patients and trametinib may further enhance antitumor immune responses in will be enrolled with bone metastases only or bone-predominant dis- addition to preserving the targeted effect. ease (n=40). For both cohorts patients will receive pembrolizumab 200 Methods mg every 3 weeks (Q3W) plus current enzalutamide regimen. For the This is a Phase Ib, prospective, single arm study of T-VEC given in com- original cohorts (cohorts 1-3), patients must have been treated with ≥1 bination with standard doses of dabrafenib and trametinib in advanced targeted endocrine therapy (abiraterone or enzalutamide) and ≤2 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 120 of 244 chemotherapy regimens; one must have contained docetaxel. Pa- Conclusions tients also must be undergoing androgen deprivation with serum This interim analysis demonstrated that administration of Rocapuldencel- testosterone <50 ng/dL. Patients will be enrolled based on PD-L1 T resulted in an increase in antigen-specific memory T cells and that the status and RECIST v1.1 measurability and will receive pembrolizu- magnitude of the induced memory T cell response after seven doses cor- mab 200 mg Q3W: PD-L1–positive, RECIST v1.1 measurable disease related with OS. Additionally, the amount of IL-12 secreted by each pa- (cohort 1), PD-L1–negative, RECIST v1.1 measurable disease (cohort tient’s immunotherapeutic correlated with both the magnitude of the 2; cohorts 1 and 2 combined, n=200), and bone metastases and induced memory T cell response and OS. Therefore, the level of IL-12 RECIST v1.1 nonmeasurable disease (cohort 3, n=50). All patients secretion may serve as a predictive biomarker for T cell response to will continue until documented confirmed disease progression, un- Rocapuldencel-T and favorable clinical outcome, warranting further acceptable adverse events (AEs), or illness that prevents further investigation. treatment. Imaging response will be assessed every 9 weeks for Trial Registration approximately 1 year and every 12 weeks thereafter, per central ClinicalTrials.gov identifier-NCT01582672 imaging vendor review (RECIST v1.1) and the Prostate Cancer Clinical Trials Working Group 3 guidelines. AEs will be monitored P225 throughout the study. Primary end points are overall response rate Functional reversal of Foxp3+ T regulatory activity in patients for cohorts 1 and 2 combined and for cohorts 1, 2, and 4. Key enrolled in the Phase 3 ADAPT Trial evaluating Rocapuldencel-T secondary end points include safety and tolerability, duration of re- for the treatment of patients with metastatic renal cell carcinoma sponse, disease control rate, radiographic progression-free survival, (mRCC) and overall survival. Mark DeBenedette, Joe Horvatinovich, Elizabeth Grogan, Larissa Trial Registration Benavente, Irina Tcherepanova, Charles Nicolette ClinicalTrials.gov, NCT0278700 Argos Therapeutics Inc, Durham, NC, USA Correspondence: Mark DeBenedette P224 (mdebenedette@argostherapeutics.com); Joe Horvatinovich Immunological correlates observed in an Interim analysis of the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P225 Phase 3 ADAPT Trial evaluating Rocapuldencel-T (AGS-003), for the treatment of patients with metastatic renal cell carcinoma (mRCC) Background Mark DeBenedette, Alicia Gamble, Ana Plachco, Marcus Norris, Charles Rocapuldencel-T is an investigational patient-specific immunothera- Nicolette peutic comprised of autologous dendritic cells programmed with Argos Therapeutics Inc, Durham, NC, USA RNA from the patient’s tumor to express tumor-specific antigens and Correspondence: Mark DeBenedette induce a memory-T cell response. The Phase 3 ADAPT trial is designed (mdebenedette@argostherapeutics.com) to evaluate overall survival (OS) in subjects with newly diagnosed Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P224 mRCC receiving Rocapuldencel-T in combination with standard-of-care (SOC) versus SOC alone. Current SOC first-line treatment with sunitinib Background is shown to decrease regulatory T cells (Tregs), thus potentially modu- Rocapuldencel-T is an investigational patient-specific immunothera- lating anti-tumor activity. Therefore, FoxP3+ Treg cell counts in patients peutic comprised of autologous dendritic cells programmed with receiving Rocapuldencel-T were measured ex vivo in whole blood sam- RNA from the patient’s tumor to express tumor-specific antigens ples and the relationship to OS was assessed for each arm of the study. and thereby induce a memory T cell response. Rocapuldencel-T is Methods engineered to secrete IL-12, a critical cytokine for memory-T cell For this interim analysis, whole blood samples collected at intervals formation, to induce an antigen-specific memory T cell response. during the course of the ADAPT trial were stained immediately We previously reported results from a Phase 2 study showing a cor- ex vivo by multi-color flow cytometry to determine the number of relation between the magnitude of the induced memory-T cell re- CD4+/CD25+/CD127-/Foxp3+ Treg cells. The correlation between the sponse and overall survival (OS) in advanced RCC patients. We number of Tregs present and overall survival was determined for the therefore measured the antigen-specific memory T cell response combination arm subjects (N=177) and the SOC arm subjects (N=80) and the level of secreted IL-12 in patients receiving Rocapuldencel- for whom data was available. We further studied the impact of T to assess the relationship of these parameters to each other and Rocapuldencel-T on Treg phenotype and function after in vitro stimu- to overall survival (OS). lation of PBMCs from treated subjects. Methods Results The ADAPT trial is designed to evaluate OS in subjects with newly As reported elsewhere, data from this interim analysis showed a de- diagnosed mRCC receiving Rocapuldencel-T in combination with cline in the number of Tregs after the first cycle of sunitinib. Surpris- standard-of-care (SOC) versus SOC alone. As part of this interim ingly, the number of Tregs at baseline or at any time point measured, analysis, immune monitoring was performed on patients (N=146) positively correlated with OS in the combination arm, but negatively enrolled in the United States and randomized to the combination correlated with OS in the SOC arm. Furthermore, in vitro stimulation of arm. The change in the number of CD28+/CD45RA- antigen-specific autologous PBMCs with Rocapuldencel-T resulted in conversion of memory T cells present after in vitro stimulation of PBMCs with Tregs to a proinflammatory cell phenotype that proliferates in culture, Rocapuldencel-T was measured with multi-color flow cytometry. thus providing a possible explanation for the positive correlation be- The amount of IL-12 secreted by each patient’s immunotherapeutic tween the number of Tregs and OS in the combination arm. was measured by a cytokine bead capture method (N=179). The Conclusions increase in the number of antigen-specific memory T cells after In this interim analysis, baseline Tregs positively correlated with sur- administration of Rocapuldencel-T and the concentration of IL-12 vival only in the Rocapudencel-T arm and not in the SOC arm. These secreted by each patient’s immunotherapeutic where correlated data suggest that Rocapudencel-T exerts a biologic effect catalyzed with OS. by the presence of Tregs, potentially by conversion to T effector cells, Results resulting in favorable clinical outcome. This is consistent with the Data from this interim analysis revealed a statistically significant in- in vitro observation that Rocapudencel-T can convert Tregs to T effector crease in the number of CD28+/CD45RA- memory T cells after as few cells. Therefore, the level of Tregs in whole blood samples may serve as as three doses of Rocapuldencel-T administered three weeks apart. The a useful baseline biomarker predictive of favorable long-term clinical increase above baseline became a statistically significant correlate with outcome in patients treated with Rocapudencel-T while also predicting OS after seven doses. Furthermore, the amount of IL-12 each patient’s poor outcome in patients receiving conventional SOC. immunotherapeutic produced showed a statistically significant correl- Trial Registration ation with both the magnitude of the memory T cell response and OS. ClinicalTrials.gov identifier-NCT01582672. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 121 of 244 P226 of patients require additional therapy [1, 2]. Strategies to improve FRACTION-RCC: a randomized, open-label, adaptive, phase 2 study the benefit of these agents are currently focused on combination of nivolumab in combination with other immuno-oncology agents with other systemic agents and adjunctive local treatment mea- in patients with advanced RCC sures. We are evaluating the combination of pembrolizumab with a 1 2 3 4 Corina Candiani Taitt ,RobertJ.Motzer , Toni K. Choueiri , Bernard J. Escudier , vaccine incorporating 6 peptides that induces CD4+ helper T cell 5 6 7 8 Timothy Kuzel ,Michael A.Carducci ,SureshNair , Scott S. Tykodi , (T ) responses (6MHP) and has proven activity in patients with ad- 1 1 1 Sarah Tannenbaum-Dvir , Megan Wind-Rotolo ,KatyL. Simonsen , vanced melanoma [3]. The primary end point is determination of Paula M. Fracasso the safety and tolerability of the combination of 6MHP vaccine plus 1 2 Bristol-Myers Squibb, Princeton, NJ, USA; Memorial Sloan Kettering Pembrolizumab and estimation of the CD4+ T cell response rate to Cancer Center, New York, NY, USA; Dana-Farber Cancer Institute, 6MHP in the blood and sentinel immunized node. Secondary end- Boston, MA, USA; Gustave Roussy Cancer Centre, Villejuif Cedex, France; points include evaluation of the induction of epitope-spreading 5 6 Rush University Medical Center, Chicago, IL, USA; Sidney Kimmel and the T cell infiltration of the tumor microenvironment. Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, Methods 7 8 USA; Lehigh Valley Health Network, Allentown, PA, USA; University of This is an open-label, phase I/II study to evaluate the safety, immuno- Washington and Fred Hutchinson Cancer Research Center, Seattle, WA, genicity and clinical activity of the 6MHP vaccine and pembrolizumab USA (MEL64, NCT02515227). Candidates may be resistant or naïve to sys- Correspondence: Corina Candiani Taitt (ea@chrysalismedical.com) temic immunotherapy agents. Patients with prior PD-1 antibody expos- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P226 ure are included if the patient failed to experience a clinical response after 12 weeks or progressed on treatment. All subjects will receive Background 6MHP on days 1, 8, 15, 43, 64, and 85. Pembrolizumab will be adminis- Nivolumab, a fully human IgG4 monoclonal antibody (mAb) that tar- tered intravenously (IV) every 3 weeks for up to 2 years. Biopsy speci- gets the PD-1 receptor, is approved for patients with advanced renal mens will be collected from the tumor (days 1 and 22) and a sentinel cell carcinoma (RCC) after prior antiangiogenic therapy based on su- immunized node (SIN; day 22). Biopsy and peripheral blood specimens perior overall survival vs everolimus (CheckMate 025; Motzer RJ, et al. will be used in the immunologic analyses. Overall target sample size is N Engl J Med. 2015). Nivolumab has also shown promising antitumor based upon having sufficient information to assess whether combined activity in combination with ipilimumab (a fully human IgG1 mAb treatment with 6MHP vaccine plus pembrolizumab increases the im- that targets cytotoxic T-lymphocyte antigen 4) in patients with meta- munogenicity of 6MHP alone in the entire study population. Maximum static RCC, supporting the rationale that nivolumab in combination target sample size is 40 patients. Trial is currently enrolling patients. with other immuno-oncology (IO) agents or targeted therapies may Results improve outcomes in patients with advanced RCC. Given the rapid Trial in Progress. development of novel IO agents, traditional study designs may not Conclusions efficiently evaluate all possible IO-IO and IO-targeted therapy combi- Trial in Progress. nations. Fast Real-time Assessment of Combination Therapies in Trial Registration Immuno-ONcology (FRACTION) is an innovative clinical trial program NCT02515227 with a rolling, adaptive platform design that allows for the addition of new regimens as well as the withdrawal of ineffective regimens. Here References we describe the study concept, key design components, and first IO 1. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus Ipilimumab treatment combinations of FRACTION-RCC, a phase 2, randomized, in Advanced Melanoma. N Engl J Med. 2015;372(26):2521-32. open-label, adaptive study in advanced RCC (NCT02996110). 2. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined Nivolumab and Methods Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. FRACTION-RCC is envisioned to accelerate the development of the 2015;373(1):23-34. next generation of IO combinations for patients with metastatic RCC. 3. Slingluff CL Jr, Petroni GR, Olson W, et al. Helper T cell responses and Patients with advanced RCC with a clear-cell component will be en- clinical activity of a melanoma vaccine with multiple peptides from rolled based on prior IO treatment and randomized to receive nivolu- MAGE and melanocytic differentiation antigens. J Clin Oncol. mab plus BMS-986016 (a fully human IgG4 mAb that targets 2008;26(30):4973-80. lymphocyte activation gene 3) or nivolumab plus ipilimumab. Enroll- ment is continuous and may offer patients consecutive treatment op- P228 tions based on treatment exposure and response. Primary endpoints Pembrolizumab and decitabine for relapsed and refractory acute include objective response rate, duration of response, and progression- myeloid leukemia (PD-AML) free survival rate at 24 weeks. The secondary endpoint is safety. Bio- Catherine Lai, Oetjen Karolyn, Katherine Lindblad, Christin Destefano, marker analyses will also be performed. New treatment combinations Meghali Goswami, Hong Yuen Wong, Therese Intrater, Debbie Draper, will be added over time to explore their potential benefits and provide Laura Dillon, Christopher Hourigan a continuous flow of treatment options for patients whose cancer pro- National Heart, Lung and Blood Institute, National Institutes of Health, gresses on existing treatments. Bethesda, MD, USA Trial Registration Correspondence: Christopher Hourigan (hourigan@nih.gov) ClinicalTrials.gov, NCT02996110 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P228 P227 Background A trial to evaluate the safety, immunogenicity and clinical activity While a variety of different treatment regimens have been studied of a helper peptide vaccine plus PD-1 blockade for patients with relapsed/refractory acute myeloid leukemia (AML) Craig Slingluff, Jr, Elizabeth Gaughan, William Grosh, Gina Petroni, clinical outcomes unfortunately remain dismal. There appears to be Kimberly Bullock no single superior therapeutic approach and the current standard of University of Virginia, Charlottesville, VA, USA care is referral to an appropriate clinical trial. The unique com- Correspondence: Elizabeth Gaughan (egaughan@virginia.edu) bination of pembrolizumab and decitabine used in this trial was Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P227 selected for investigation based on several factors: 1) Largely non- overlapping adverse reaction profiles for these agents. 2) Both agents Background already FDA approved potentially allowing for rapid translation/adop- PD-1 blocking antibodies are standard first line therapy for man- tion. 3) Both agents have previously evaluated dosing schedules that agement of advanced melanoma. Monotherapy response rates to are compatible with one another. 4) Theoretical possibility of synergy these agents are reported between 30-40%; therefore, the majority given respective mechanisms of action. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 122 of 244 Methods such as IL-12. Furthermore, CD40 activation cause macrophages to PD-AML (17-H-0026, NCT02996474) is an investigator sponsored, develop a more tumoricidal phenotype and tumor cells to increase single-institution, single-arm open-label ten subject pilot study to MHC I expression. evaluate the feasibility of a novel combination of pembrolizumab Direct intratumoral (IT) immune modulation utilizes the tumor as a and decitabine in adults with relapsed/refractory AML. Secondary “vaccine site” to generate a tumor specific immune response. We objectives will explore efficacy and determine time to first re- hypothesize that (IT) injection of a CD40 agonist such as APX005M, sponse, best response and duration of best response. Laboratory will “immunize” patients against melanoma neoantigens through “li- objectives include investigation of changes in AML clonal compos- censing” of tumor infiltrating APCs for tumor specific T cell priming itionand diseaseburdenduringtherapy, and measurementof and activation. In preclinical mouse models, we have shown that IT changes in immune parameters associated with clinical efficacy administration of the recombinant adenovirus encoding the dendritic and/or toxicity. Up to eight 21 day cycles of pembrolizumab are cell-activating CD40L induces CD8 T cell-mediated systemic activity given with decitabine given on days 8-12 and 15-19 on alternative against B16 melanoma. Importantly, IT rAdCD40L also augmented cycles (ie: cycles 1, 3, 5 and 7) (Fig. 1). the activity of anti-PD-1. Results Methods This clinical trial opened in February 2017 and is currently in pro- This phase I/II trial (NCT02706353) evaluates the safety, efficacy, and gress. Currently seven patients have reached response and toxicity immunological impact of IT administration of APX005M (CD40 agon- assessment time-points. Updated results will be presented at the istic mAb) in combination with systemic pembrolizumab in pts with meeting. MM. An accelerated 3+3 design was used for the phase 1 dose escal- Conclusions ation portion of this study. Pts will receive IT APX005M at escalating Acute myeloid leukemia is a heterogeneous group of diseases with doses every 3 weeks for a total of 4 doses. Image guidance will allow distinct molecular and phenotypic characteristics. Even within a single for injection of visceral, nodal, and soft tissue metastases. The single- patient AML may be polyclonal at any examined time-point, and this arm phase 2 expansion will evaluate the overall response rate (ORR) clonal composition can change over time with the clone predominant of this regimen 12 weeks after initiation of treatment. Some key inclu- at presentation not necessarily the one responsible for relapse and sion criteria: confirmed cutaneous or mucosal melanoma; measurable, death. We hypothesize that effective pembrolizumab therapy for refrac- unresectable stage-III or stage-IV disease, and at least 2 injectable tory/relapsed AML may be associated with changes in the leukemic lesions. Key exclusion criteria include: prior immunotherapy, uveal mel- clonal composition due to differences in immunogenicity between anoma, active autoimmune disease, or active immunodeficiency. A clones. The oligoclonal nature of AML biology, together with a blood sample size of 26 patients will have 75% power to detect an improve- and bone marrow distribution highly amenable to repeated sampling ment from a null ORR of 33% to 55%, using a one group chi-square test of the sites of disease burden, provides a near unique opportunity to and assuming a one-sided α–level of 5%. Immune analysis will be per- investigate fundamental mechanisms underpinning treatment efficacy formed on pre and on-treatment tumor/liquid biopsies including but of this new combination of immunotherapeutic drugs. not limited to quantification of dendritic cells and T cells both in Trial Registration injected and non-injected tumors. [https://clinicaltrials.gov/ct2/show/NCT02996474] Trial Registration FDA-IND: 131826 NCT02706353 P231 KEYNOTE-590: randomized, phase 3 study of chemotherapy + pembrolizumab vs chemotherapy + placebo as first-line therapy for patients with advanced esophageal or esophagogastric junction (E/EGJ) cancer 1 2 3 4 5 Ken Kato ,Manish Shah , Peter Enzinger , Jaafar Bennouna , Lin Shen , 6 7 7 7 8 Antoine Adenis ,YingZhu ,Pooja Bhagia , Minori Koshiji , Toshihiko Doi 1 2 National Cancer Center Hospital, Tokyo, Japan; Weill Cornell Medical Fig. 1 (abstract P228) See text for description College, New York Presbyterian Hospital, New York, NY, USA; Dana Farber Cancer Institute, Boston, MA, USA; CHU de Nantes, Nantes, 5 6 France; Beijing Cancer Hospital, Beijing, China; Institut du Cancer de P229 Montpellier, Montpellier, France; Merck & Co., Inc., Kenilworth, NJ, USA; Withdrawn National Cancer Center East, Chiba, Japan Correspondence: Ken Kato (kenkato@ncc.go.jp) P230 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P231 Phase I/II safety and efficacy study of image guided intratumoral CD40 agonistic monoclonal antibody APX005M in combination Background with systemic pembrolizumab in metastatic melanoma patients No chemotherapeutic regimens or targeted agents are approved Daniel Johnson H Johnson, Srisuda Lecagoonporn, Chantale Bernatchez, specifically for esophageal cancer; available options have limited Cara Haymaker, Salah Bentebibel, Marc Uemura, Cassian Yee, Rodabe benefit and substantial toxicity. In the phase 1b KEYNOTE-028 study Amaria, Sapna Patel, Hussein Tawbi, Isabella Glitza, Michael A. Davies, (NCT02054806), pembrolizumab monotherapy demonstrated manage- Michael K. Wong, Wen-Jen Hwu, Patrick Hwu, Willem Overwijk, Adi Diab able safety and durable antitumor activity in heavily pretreated patients UT-MD Anderson Cancer Center, Houston, TX, USA with PD-L1–positive advanced esophageal carcinoma. In KEYNOTE-059 Correspondence: Daniel Johnson H Johnson (NCT02335411), combining chemotherapy (cisplatin and 5-fluorouracil) (dhjohnson@mdanderson.org); Adi Diab and pembrolizumab as first-line treatment for patients with advanced Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P230 gastric or gastroesophageal junction cancer resulted in encouraging ef- ficacy and manageable safety. This suggests chemotherapy plus pem- Background brolizumab as a potential therapeutic strategy for esophageal cancer. Checkpoint blockade has become a major modality in the treatment KEYNOTE-590 is a randomized, double-blind, multicenter phase 3 study of metastatic melanoma (MM). However, long-term survival and dur- of cisplatin and 5-fluorouracil plus pembrolizumab versus cisplatin and able remission rates remain low and new treatment options are 5-fluorouracil plus placebo in patients with advanced E/EGJ carcinoma. needed. CD40 activation on antigen presenting cells (APCs) initiates Methods their maturation and ability to prime and activate CD8+ T cells Eligible patients are ≥18 years of age; have locally advanced unresect- through upregulation of co-stimulatory molecules (CD80, CD86, able or metastatic adenocarcinoma or squamous cell carcinoma of CD70, 4-1BBL, OX40L, and GITR-L) as well as expression of cytokines Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 123 of 244 esophagus or metastatic Siewert type 1 adenocarcinoma of EGJ, meas- also performed at multiple pre-defined timepoints. In addition to asses- urable disease per RECIST 1.1, ECOG performance status 0-1, adequate sing the feasibility, safety, and preliminary efficacy (as measured by organ function; have had no prior therapy for advanced disease, no pathologic complete remission rate) of this novel combination, analyses autoimmune disease, no active infection; and can provide a newly ob- of tumor tissue and blood will be performed, including Tissue Multiplex tained or archival tissue sample. Patients will be randomly assigned 1:1 Immunohistochemistry and flow cytometry for both APC and T cell to cisplatin 80 mg/m IV every 3 weeks (Q3W) for 6 cycles plus 5- activation, as well as T cell receptor sequencing for T cell repertoire fluorouracil 800 mg/m continuous IV on days 1-5 Q3W IV plus pem- diversity. brolizumab 200 mg IV Q3W or cisplatin 80 mg/m IV Q3W for 6 cycles plus 5-fluorouracil 800 mg/m continuous IV on days 1-5 Q3W plus pla- P233 cebo Q3W IV. Treatment will continue up to 2 years. Response will be A phase I study to evaluate the safety of multi-antigen stimulated assessed using CT (preferred) or MRI every 9 weeks by central imaging tumor specific cellular therapy (MASCT-I) in patients with per RECIST v1.1. Adverse events will be graded per NCI CTCAE v4.0 and advanced solid tumors up to at least 30 days after the end of treatment. Primary end points 1 2 2 1 Ruihua Xu , Xiaoshuang Li , Yanjun Kong , Jianchuan Xia , Desheng are PFS per RECIST v1.1 and OS in all patients and in patients with PD- 1 1 1 Weng , Xiaoshi Zhang , Xing Zhang L1 positive or negative tumor expression (combined positive score Sun Yat-sen university affiliated oncology hospital, Guangzhou, China; ≥10% or <10% using immunohistochemistry). Secondary end points in- HRYZ Biotech Company, Shenzhen, China clude ORR per RECIST v 1.1, duration of response, safety, and health- Correspondence: Ruihua Xu (kyj8@sina.com) related quality of life. PFS and OS will be compared between groups Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P233 using a stratified log-rank test; hazard ratios will be estimated using a Cox regression model. The Kaplan-Meier method will be used to esti- Background mate event rates within groups. Enrollment is planned for approxi- Tumor-specific immune responses are known to be initiated by mately 700 patients. tumor associated and/or specific antigen-sensitized dendritic cells Trial Registration (DCs), that can effectively process and present tumor antigens to ClinicalTrials.gov, NCT03189719 CD4+ and CD8+ T cells. MASCT-I is a sequential immune cell therapy for solid tumor, which included multi-antigen loaded DC vaccines P232 followed by the adoptive transfer of anti-tumor specific T cells. DC A pilot study to evaluate the clinical and immunological effects of vaccines are produced from patients’ autologous PBMC-derived DC incorporating a CD40-agonistic antibody into the multimodality loaded with multiple tumor associated antigen peptides and are treatment of resectable esophageal and GE junction cancers injected into patients to induce active anti-tumor immunity. Anti- Andrew H. Ko, Lawrence Fong tumor specific T cells are stimulated from the same patient by co- University of California San Francisco, San Francisco, CA, USA cultured with DC vaccines and Anti-PD1 antibody in vitro, are then Correspondence: Andrew H. Ko (andrew.ko@ucsf.edu infused into the patient to target tumor cells. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P232 Methods This is a single center, three stages phase I study. Stage1 and 2 will Background enroll (3+3 design) patients with advanced (unresectable) or recur- Targeting CD40, a member of the TNF receptor superfamily found on rent bladder cancer or soft tissue sarcoma who have failed all stand- antigen presenting cells (APCs), represents a promising cancer immu- ard therapies, Patients will be treated with MASCT-I. If the dose notherapeutic strategy. Activation of this costimulatory molecule re- limitation toxicity (DLT) in the first cycle of MASCT-I is <33.3%, the sults in improved antigen processing and presentation and cytokine stage 2 will begin. Patients who have advanced recurrent or meta- release from activated APCs, enhancing T cell responses. Additionally, static bladder cancer with Gemcitabine and Cisplatin (GP) chemo- CD40 is expressed on many tumor cells that, when activated, results therapy achieving clinical benefit (group 1) will be treated with in tumor cell apoptosis and inhibition of tumor growth. APX005M MASCT-I as maintenance therapy. Also, patients who have advanced (Apexigen, San Carlos, CA) is a humanized IgG1 anti-CD40 agonistic recurrent or metastatic sarcoma with achieving clinical benefit after antibody that binds to CD40 with high affinity. In a FIH phase I clin- MAID or CAV/IE (predominant Doxorubicin regimens) (group 2) will ical trial in patients with advanced solid tumors, APX005M was rela- be treated with MASCT-I combined with Ifosfamide as maintenance tively well tolerated, with cytokine release syndrome (CRS) as the therapy too. During the stage 2, if, in the first cycle of MASCT-I treat- DLT at doses above the RP2D. Importantly, correlative studies show ment, the DLT is < 33.3%, stage 2 will extend to stage 3. Approxi- that APX005M produces dose-dependent activation of APCs, T cell mately additional 24 patients will be enrolled in group 1 and 2. The activation, and increases in circulating cytokine levels. primary objective is safety and tolerability. Secondary objectives in- Methods clude DCR, PFS, TTP, OS. As of 17 May 2017, Three patients were en- This pilot trial represents the first to evaluate a CD40-agonistic anti- rolled and completed Stage 1 without any DLT and treatment body in esophageal/GE junction cancer, a disease in which IO related SAEs. Five AEs are related with treatment, they are pain on agents (particularly PD-1 mAbs) have demonstrated promising ac- the injection site, fatigue, pruritus and arthralgia, which were all tivity. The study is also the first to explore combining IO with che- grade 1. Recruitment is ongoing for dose expansion (stage 2). Clinical moradiation in the neoadjuvant setting, as this multimodality trial information: NCT030343 approach represents the standard of care for patients with resect- able esophageal/GE junction cancer and is optimally conducive for serial tumor tissue acquisition. A total of 16 patients with resectable (uT1-3N0-1) squamous cell or adenocarcinoma of the esophagus or P234 GE junction will be enrolled. Chemoradiation consists of radiation AST-VAC2: An allogeneic dendritic cell cancer immunotherapy (5040cGy in 28 daily fractions) and low-dose carboplatin plus pacli- entering clinical trials in patients with lung cancer in the advanced taxel weekly x 5, as per standard of care. APX005M 0.3 mg/kg (1 and adjuvant setting 1 2 2 3 dose level below single-agent MTD) is given every 3 weeks for a Christian Ottensmeier , Hayley Farmer-Hall , Gary Acton , Heike Lentfer , 4 4 4 4 total of 4 doses, with the first dose administered two weeks prior Kevin Nishimoto , Uzma Shoukat-Mumtaz , Erik Whiteley , Rob Allen , to the initiation of concurrent chemoradiation. APX005M administration Jane Lebkowski 1 2 is offset by 2-3 days from chemotherapy to avoid the steroid pre- University of Southampton, Southampton, United Kingdom; Cancer medication administered with paclitaxel. Tumor tissue is acquired via Research UK, London, United Kingdom; Cancer Research UK, Potters endoscopic biopsy at baseline and following the single-dose “run-in” of Bar, United Kingdom; Asterias Biotherapeutics, Fremont, CA, USA APX005M; and then again at esophagectomy, which occurs 1-2 months Correspondence: Jane Lebkowski (rallen@asteriasbio.com) following completion of chemoradiation. Serial blood collections are Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P234 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 124 of 244 Background Methods Primary lung cancer is the most common malignancy after non- This EAP program included subjects with histologically or cytologic- melanocytic skin cancer with deaths exceeding those from any other ally documented NSCLC who have relapsed after systemic treatment type of malignancy worldwide. In advanced (TNM stage IIIA, IIIB and IV) with a minimum of 1 prior systemic treatment for stage IIIB/stage IV disease, only palliative therapy is available which carries a high risk of disease. Subjects were treated with 3 mg/kg of nivolumab IV every 2 toxicity and limited potential to extend life. Intradermal delivery of weeks for a maximum of 24 months. Each 14-day dosing period con- dendritic cells (DCs) carrying an immunogenic cargo offers a novel ap- stituted a single cycle. Patients included in the analysis had received proach to address malignancy mediated through upregulated telomer- ≥ 1 dose of nivolumab and were monitored for adverse events (AEs). ase expression and decreased cell death. AST-VAC2 is a mature, Results allogeneic DC vaccine derived by differentiating H1 human embryonic As of June 30, 2017, eight patients participated. Median age was 63.0 stem cells (hESCs) into mature DCs, transfected to express the tumor years. All participants were male. Except for 1 (12.5%) current smoker associated antigen human telomerase reverse transcriptase (hTERT) and 1 (12.5%) never smoker, other 6 (75%) patients were former and lysosomal associated membrane protein 1 (LAMP-1) [1,2]. LAMP-1 smokers. The average of smoking period was 27 pack-years. 2 had fusion proteins enable target hTERT peptides to be directed to HLA squamous and 6 had non-squamous histology. At the time of enroll- (Human Leukocyte Antigen) II and HLA I receptors on the surface of en- ment, 4 had bone, 3 had brain and 3 had brain metastases. Best re- dogenous DCs, invoking dual, antigen-specific CD4+ and CD8+ re- sponse rates was 12.5% and disease control rate was 50%. Median sponses [3]. Preclinical investigations have shown that AST-VAC2 progression free survival was 99.5 days (95% CI 50.1-174.7). During the phagocytose, process, and present antigen upon maturation. Further- nivolumab chemotherapy, 2 patients had pneumonia and 1 had stroke. more they produce immunostimulatory cytokines, migrate in response Other 5 patients had no critical complication during the treatment. to cytokines, and activate antigen specific T cell responses. Conclusions Methods Our study showed that EAP participants had some lower response The first-in-human trial will evaluate safety, tolerability, immunogen- rate and more prolonged PFS compared to previously reported in icity and therapeutic potential in adult NSCLC patients in advanced Checkmate-017 and in Checkmate-057. And nivolumab EAP showed (metastatic or locally advanced disease) and adjuvant (currently good safety profiles. In conclusion, treatment with Nivolumab is safe radiologically disease free) settings. Patients who are positive for the and effective for patients who have previously received heavily HLA-A2 allele (expressed by AST-VAC2) will receive 6 weekly doses of chemotherapy. 1x10 AST-VAC2 cells and will be monitored for one year as the primary follow-up point and subsequently for up to five years for P236 long-term follow-up. Safety assessments as well as immunological Innate immunotherapy of neuroblastoma and PD-1 checkpoint monitoring for the generation and maintenance of hTERT specific T blockade cells will be key endpoints. Results will be used to expand the clinical 1 1 1 1 Holger Lode , Maxi Zumpe , Madlen Juettner , Sascha Troschke-Meurer , indications for assessment and support future combination immuno- 2 2 2 1 Evelyne Janzek , Romana Schaefer , Hans Loibner , Nikolai Siebert therapy approaches. 1 2 University Medicine Greifswald, Greifswald, Germany; Apeiron Biologics, Vienna, Austria References Correspondence: Holger Lode (lode@uni-greifswald.de) 1. Tseng SY, Nishimoto KP, Silk KM, Dawes GN, Waldmann H, Fairchild PJ, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P236 Lebkowski JS, Reddy A. Generation of immunogenic dendritic cells from human embryonic stem cells without serum or feeder cells. Regen. Med. Background 2009; 4(4):513-526. Passive immunotherapy of cancer is established for a variety of ma- 2. Nishimoto KP, Tseng S-Y, Lebkowski JS, Reddy A. Modification of human lignant diseases. Anti-GD antibody (Ab) ch14.18/CHO (dinutuximab embryonic stem cell-derived dendritic cells with mRNA for efficient anti- beta) showed activity for the treatment high-risk neuroblastoma (NB) gen presentation and enhanced potency. Regen. Med. 2011;6(3):303–318. patients and received recently marketing approval in the EU. Here 3. Su Z, Dannull J, Yang B, Dahm P, Coleman D, Yancey D,Sichi S, Niedzwiecki we demonstrate that one important mechanism of action in patients D, Boczkowski D, Gilboa E, Vieweg J. Telomerase mRNA-Transfected Den- is antigen specific Ab-dependent cellular cytotoxicity (ADCC) and we dritic Cells Stimulate Antigen-Specific CD8+and CD4+T Cell Responses in report that ADCC impacts on PD-1/PD-L1 checkpoint regulation that Patients with Metastatic Prostate Cancer. J Immunol. 2005.174: 3798–3807. can be targeted by co-treatment with an inhibitor. Methods 2 6 2 P235 53 patients received 100 mg/m ch14.18/CHO (d8-17), 6x10 IU/m Nivolumab in patients with advanced or metastatic non-small cell sc IL-2 (d1-5; 8-12 and 160 mg/m oral 13-cis-RA (d19-32) in a closed lung cancer (Stage IIIb/IV) who have received at least one prior single center program (53 pts). Polymorphisms in Fcγ-receptor genes systemic chemotherapeutic regimens 2A (H131R), -3A (V158F) and -3B (NA1/NA2) were determined by real- Sung Yong Lee, Sang Mi Chung, Ju Whan Choi, Young Seok Lee, time PCR. Expression of PD-L1 and PD-1 was analyzed by RT-PCR and Jong Hyun Choi, Jee Youn Oh, Kyung Hoon Min, Gyu Young Hur, flow cytometry. Effect of PD-1/PD-L1 blockade and ch14.18/CHO-me- Jae Jeong Shim, Kyung Ho Kang diated anti-NB immune response was evaluated using anti-PD-1 Ab Korea University Medical Center, Seoul, Republic of Korea both in vitro (Nivolumab) and in the syngeneic GD NB NXS2 mouse Correspondence: Sung Yong Lee (syl0801@korea.ac.kr) model (anti-mouse PD-1). Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P235 Results We identified 33/53 patients with low affinity FCGR alleles (FCGR2A- Background H131R/R and/or FCGRA3A-V158 F/F). These patients showed lower Nivolumab, a human programmed death 1 (PD-1) immune check- PFS rates compared to 20/53 patients with high affinity polymor- point inhibitor antibody, has been shown to increase overall survival phisms (p < 0.01). ADCC levels on day 15 of cycle 1 in pts with high in non-small cell lung cancer (NSCLC) patients. This immune checkpoint affinity polymorphisms showed an ADCC increase of 20±6% com- blockade has been approved in Korea, United States, the European pared to 11±2% in the control. The correlation with functional Union, and other countries for the treatment of advanced NSCLC that immune parameter ADCC and clinical outcome confirm its role for has progressed after platinum-based chemotherapy. Nivolumab has clinical efficacy. demonstrated longer overall survival than docetaxel among the pre- Interestingly, tumor specific ADCC in the presence of LA-N-1 neuro- viously treated NSCLC patients. We have assessed the safety of ni- blastoma cells, leukocytes and sub-therapeutic ch14.18/CHO concen- volumab in 8 patients with previously treated, locally advanced or trations (10 ng/ml) results in a strong increase of the PD-L1 metastatic NSCLC who were enrolled in the NSCLC Expanded Access expression and incubation with IL-2 further enhanced this effect. Program (EAP) in Korea University Guro Hospital. Blockade with Nivolumab reversed the PD-L1-dependent inhibition Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 125 of 244 of ADCC. Finally, mice treated with ch14.18/CHO in combination with Results PD-1 blockade showed strongest reduction of tumor growth, longest To date, one patient has been started on therapy and is in week 3 survival rate as well as the highest level of NB cell lysis mediated by monitoring period, is tolerating treatment well, with no significant serum and leukocytes of treated mice compared to controls. toxicities thus far. Data regarding the peripheral blood iNKT cells re- Conclusions sponse will be presented. Patient studies clearly reveal ADCC as mechanism of ch14.18/CHO Trial Registration against neuroblastoma, and this upregulates the inhibitory check- ClinicalTrials.gov identifier NCT03175679. point PD-1/PD-L1. Combination of ch14.18/CHO with PD-1/PD-L1 blockade results in synergistic treatment effects. Similar upregulation P238 of PD-L1 expression by suboptimal ADCC was also seen with the hu- Phase I study of adoptive transfer of specific hepatocellular man GD2+ osteosarcoma cell line MG63. This suggests a broader ap- carcinoma antigens CD8 T cells for treating patients with plicability and to consider combinations of passive immunotherapy relapsed/advanced HCC of cancer with PD-1/PD-L1 checkpoint inhibition. 1 1 1 2 2 1 Jun Lu , Xuli Bao , Yanpin Ma , Hui Chen , Wenfeng Sun , Jia Guo , Xiongwei Cui Beijing YouAn Hospital, Capital Medical University, Beijing, China; P237 Beijing Gene Key Life Technology Co.Ltd., Beijing, China Phase I study of adoptive transfer of iNKT cells for treating Correspondence: Jun Lu (lujun98@ccmu.edu.cn) patients with relapsed/advanced hepatocellular carcinoma Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P238 1 1 1 2 2 1 Jun Lu , Xuli Bao , Jia Guo , Wenfeng Sun , Hui Chen , Yanpin Ma , Xiongwei Cui Background Beijing YouAn Hospital, Capital Medical University, Beijing, China; Hepatocellular carcinoma (HCC) is one of the most prevalent cancers Beijing Gene Key Life Technology Co.Ltd., Beijing, China for neoplastic deaths and shows a high recurrence rate. Adoptive T Correspondence: Jun Lu (lujun98@ccmu.edu.cn) cell therapy, involving the ex vivo selection and expansion of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P237 antigen-specific T cell clones by MHC/peptide tetramer sorting, pro- vides a method of augmenting antigen-specific immunity against Background HCC. To generate of a high number of cytotoxic T lymphocytes Invariant Natural Killer T (iNKT) cells represent a distinctive subset of (CTLs), kind of effective T cells that specific recognizing and killing T lymphocytes characterized by an invariant receptor Vα24/Jα18 in antigen targeted cells through cloning amplification after receiving human, which play critical roles in regulating anti-tumor immunity antigen information from antigen presented cells, is a promising by bridging innate and adaptive immune responses. We have previ- strategy for adoptive therapy. ously demonstrated that the accumulation of circulating iNKT cells Methods provide a better prognosis for hepatocellular carcinoma (HCC) pa- The phase I trial enrolls patients who have HCC tumor relapsed or tients, the adoptive transfer of ex vivo expanded human iNKT cells in metastasized through the body after standard treatment or the pa- HCC tumor-bearing NOD/SCID mice led to the decrease in tumor tients cannot receive standard treatment under current conditions. size, revealing the iNKT immunotherapy may bring clinical benefits The purpose of this study is to evaluate the safety and tolerance as for the HCC patients. well as the potential clinical efficacy of an adoptive transfer of CD8 Methods T cells, sorted with human leukocyte antigen (HLA)-peptide multi- The phase I trial enrolls patients who have relapsed/advanced mers and specific for Glypican (GPC)-3 /New York Esophageal HCC tumor relapsed or metastasized through the body after Squamous-1 (NY-ESO-1) /alpha-fetoprotein (AFP) antigens and cul- standard treatment or the patients cannot receive standard tured in vitro. Key eligibility criteria include age ≥18 years, with HCC treatment under current conditions. The purpose of this study is (BCLC, stage C) proved by histopathology or proved by CT or MRI im- to find the biggest dose of iNKT cells that is safe and tolerance, aging system, relapsed after previous therapy and no effective ther- to see how long they last in the body, to learn the immune- apies known at this time and life expectancy of ≥ 12 weeks. Three response, the side effects and if the iNKT cells will help people different dosing schedules will be evaluated. Three patients will be with relapsed/advanced HCC. Key eligibility criteria include age evaluated on each dosing schedule. The following dose levels will be 7 2 7 2 ≥18 years, with HCC (BCLC, stage C) proved by histopathology evaluated: Loading Dose 1: 3x10 /m ; Loading Dose 2: 6x10 /m ; 7 2 or proved by CT or MRI imaging system, relapsed after previous Loading Dose 3: 9x10 /m . The doses are calculated according to the therapy and no effective therapies known at this time and life actual number of GPC3/NY-ESO-1/AFP CTLs. Human Interleukin-2 will expectancy of ≥ 12 weeks. Three different dosing schedules will be given at a dose of 25,000 IU/kg/day for 5-14 days. Tegafur will be be evaluated. Three patients will be evaluated on each dosing given at a dose of 40~60 mg bis in die (BID) for 2 weeks. Immune re- schedule. The following dose levels will be evaluated: Loading sponses were measured by Elispot and ELISA, flow cytometry assays 7 2 7 2 Dose 1: 3x10 /m ; Loading Dose 2: 6x10 /m ;Loading Dose 3: were performed to evaluate the effects on immune cell subsets. 7 2 9x10 /m . The doses are calculated according to the actual Tumor biopsies were evaluated for CTLs by immunohistochemistry. number of iNKT cells. Human Interleukin-2 will be given at a Incidence of treatment-emergent adverse events were defined as dose of 25,000 IU/kg/day for 5-14 days. Tegafur will be given at signs/symptoms, laboratory toxicities, and clinical events that are a dose of 40~60 mg bis in die (BID) 2 weeks. immune re- possibly, likely, or definitely related to study treatment adverse sponses were measured by Elispot and ELISA; flow cytometry events assessed according to NCI-CTCAE v4.0. HCC progression was assays were performed to evaluate the effects on immune cell evaluated by imaging according to the irRC standard. subsets. Tumor biopsies were evaluated for iNKT cells by immu- Results nohistochemistry. Incidence of treatment-emergent adverse To date, 5 patients have been enrolled and 2 of them are in week 2 events were defined as signs/symptoms, laboratory toxicities, monitoring period, with no significant toxicities thus far. Data regard- and clinical events that are possibly, likely, or definitely related ing the peripheral blood antigen-specific CTL cells response will be to study treatment adverse events assessed according to NCI- presented. CTCAE v4.0 criteria 2. HCC progression was evaluated by im- Trial Registration aging according to the irRC standard. ClinicalTrials.gov identifier NCT03175705 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 126 of 244 P239 reported outcomes. Recruitment will continue until ~780 patients are Phase 3 study of Pembrolizumab plus Chemoradiation (CRT) vs enrolled. CRT alone for locally advanced head and neck squamous cell Trial Registration carcinoma (LA-HNSCC): KEYNOTE-412 ClinicalTrials.gov, NCT03040999. 1 2 3 4 Jean-Pascal Machiels , Chia-Jui Yen , Lisa Licitra , Danny Rischin , 5 6 1 7 John Waldron , Barbara Burtness , Vincent Gregoire , Sanjiv Agarwala , 8 9 10 11 Yun Gan Tao , Jeffrey Yorio , Sercan Aksoy , Sadakatsu Ikeda , P240 12 13 13 13 Ruey-Long Hong , Joy Yang Ge , Holly Brown , Behzad Bidadi , Phase 1b trial of cabozantinib in combination with atezolizumab in Lillian Siu patients with locally advanced or metastatic urothelial carcinoma 1 2 Cliniques Universitaires Saint-Luc, Brussels, Belgium; National Cheng or renal cell carcinoma 1 2 3 Kung University Hospital, Tainan City, Taiwan; Fondazione IRCCS Istituto Manuel Caitano Maia , Neeraj Agarwal , Bradley McGregor , Ulka 4 3 5 6 Nazionale dei Tumori, Milan, Italy; Peter MacCallum Cancer Centre, East Vaishampayan , Toni K. Choueiri , Marjorie Green , Colin Hessel , 6 6 7 1 Melbourne, Australia; Princess Margaret Cancer Centre, Toronto, ON, Christian Scheffold , Gisela Schwab , Thomas Powles , Sumanta Pal 6 7 1 2 Canada; Yale University School of Medicine, New Haven, CT, USA; St. City of Hope, Duarte, CA, USA; Huntsman Cancer Hospital, Salt Lake Luke's Cancer Center–Anderson, Easton, PA, USA; Institut Gustave City, UT, USA; Dana-Farber Cancer Institute, Boston, MA, USA; 4 5 Roussy, Villejuif, France; Texas Oncology–Austin Central, Austin, TX, USA; Karmanos Cancer Center, Detroit, MI, USA; Genentech, South San 10 11 6 7 Hacettepe Universitesi Tip Fakultesi, Ankara, Turkey; Medical Hospital, Francisco, CA, USA; Exelixis Inc., South San Francisco, CA, USA; Barts Tokyo Medical and Dental University, Tokyo, Japan; National Taiwan Cancer Institute, London, United Kingdom University Hospital, Taipei City, Taiwan; Merck & Co., Inc., Kenilworth, Correspondence: Manuel Caitano Maia (hannah.welz@fishawack.com) NJ, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P240 Correspondence: Jean-Pascal Machiels (Jean- pascal.machiels@uclouvain.be) Background Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P239 Cabozantinib is an oral receptor tyrosine kinase inhibitor targeting MET, VEGFR, and TAM family receptors (TYRO3, AXL, and MER). It is Background approved for use in patients with advanced renal cell carcinoma CRT with cisplatin is the standard of care for patients with LA-HNSCC (RCC) after prior therapy with antiangiogenic/VEGFR-targeted ther- not treated by surgery. Preclinical data in murine cancer models apy, and has demonstrated clinical activity in urothelial carcinoma show improved tumor growth control and survival when RT is com- (UC). In clinical studies, cabozantinib exposure resulted in an increase bined with a programmed death 1 (PD-1) inhibitor. Pembrolizumab in circulating CD8+ T cells and reduction of immune-suppressive has been found to be effective for treating recurrent/metastatic monocytes and Tregs. In preclinical tumor models, treatment with HNSCC, and initial results from a phase 1b study suggest that pem- cabozantinib resulted in an increase of MHC class 1 expression on brolizumab plus CRT is tolerable in patients with LA-HNSCC. tumor cells and a reduction of myeloid-derived suppressor cells. KEYNOTE-412 (NCT03040999) is a phase 3, randomized, placebo- These observations support that cabozantinib may facilitate an controlled, double-blind trial to determine the efficacy and safety of immune-permissive tumor environment and may enhance the re- pembrolizumab given concomitantly with CRT and as maintenance sponse to immune checkpoint inhibitors. Atezolizumab, an anti-PD- therapy versus placebo plus CRT in LA-HNSCC. L1 monoclonal antibody, is approved for use in locally advanced or Methods metastatic UC for patients who are either cisplatin-ineligible or have Eligibility includes patient age ≥18 years; newly diagnosed, disease progression during or following platinum-containing chemo- treatment-naive, oropharyngeal p16 positive (any T4 or N3), oropha- therapy. It is also approved for use in patients with metastatic non– ryngeal p16 negative (any T3-T4 or N2a-N3), or larynx/hypopharynx/ small cell lung cancer who have disease progression during or oral cavity (any T3-T4 or N2a-N3) SCC; evaluable tumor burden following platinum-containing chemotherapy. Here, we present the (RECIST v1.1); ECOG performance status 0-1; results available from study design of an ongoing phase 1b study combining cabozantinib local testing of human papillomavirus status for oropharyngeal can- with atezolizumab in patients with locally advanced or metastatic UC cer; eligible for definitive CRT and not considered for primary surgery or RCC. per investigator decision; tissue from a core or excisional biopsy for Methods programmed death ligand 1 (PD-L1) biomarker analysis. Patients will This multicenter, phase 1b, open-label study aims to assess safety, be randomly assigned (1:1) to receive pembrolizumab 200 mg every tolerability, preliminary efficacy, and pharmacokinetics of cabozanti- 3 weeks plus CRT, including radiotherapy (RT; accelerated [70 Gy, six nib in combination with atezolizumab (NCT03170960). The study 2 Gy fractions/wk] or standard [70 Gy, five 2 Gy fractions/week] frac- will enroll patients with advanced UC (including bladder, renal tionation) plus cisplatin 100 mg/m Q3W for 3 cycles only, or placebo pelvis, ureter, urethra) or RCC. It consists of two stages: a dose- Q3W plus CRT. Treatment will be stratified by RT regimen (acceler- escalation stage and an expansion-cohort stage. In the dose-escalation ated vs standard), tumor site/p16 status (oropharynx p16 positive vs stage (3+3 design), a recommended cabozantinib dose for the combin- p16 negative or larynx/hypopharynx/oral cavity), and disease stage ation will be established. In the expansion stage, four tumor-specific co- (III vs IV). A priming dose of pembrolizumab or placebo will be given horts will be enrolled, and the primary objective is to determine the 1 week before CRT, followed by 2 doses during CRT, and an add- objective response rate in each cohort. The four expansion cohorts are itional 14 doses after CRT, for a total of 17 pembrolizumab or pla- (1) patients with UC who have progressed on or after platinum- cebo infusions. Treatment will be discontinued upon centrally containing chemotherapy; (2) chemotherapy-naïve patients with UC confirmed disease progression, unacceptable toxicity, or patient/ who are ineligible for cisplatin; (3) chemotherapy-naïve patients with physician decision to withdraw. Disease status will be assessed by UC who are eligible for cisplatin; and (4) previously untreated patients computed topography or magnetic resonance imaging 12 weeks with RCC with clear cell histology. Exploratory objectives include correl- after CRT, every 3 months for 3 years, then every 6 months for years ation of tumor and plasma biomarkers, and changes in immune cell 4 and 5. Safety will be monitored throughout the study and for 30 profiles with clinical outcome. The study has been initiated and enroll- days after treatment. The primary end point is event-free survival. ment target is up to 120 patients across the 4 cohorts in the Secondary end points include overall survival, safety, and patient- expansion-cohort stage. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 127 of 244 Trial Registration Trial Registration ClinicalTrials.gov: NCT03170960. ClinicalTrials.gov identifier NCT03013218. References P241 1. Weiskopf K. Cancer immunotherapy targeting the CD47/SIRPα axis.Eur J A first-in-human study of ALX148: CD47 blockade to enhance Cancer. 2017;76:100-109. innate and adaptive immunity for advanced solid tumor malignancy and non hodgkin lymphoma 1 2 3 Nehal Lakhani , Patricia LoRusso , Anuradha Krishnamurthy , 1 4 5 4 Timothy O'Rourke , Philip Fanning , Yonggang Zhao , Hong Wan , P242 4 4 3 Jaume Pons , Sophia Randolph , Wells Messersmith A phase 1 multicenter, dose escalation study of CBT-501, a novel 1 2 START Midwest, Grand Rapids, MI, USA; Yale Cancer Center, New anti-PD-1 inhibitor in subjects with select advanced or relapsed/ Haven, CT, USA; University of Colorado Cancer Center, Aurora, CO, USA; recurrent solid tumors 4 5 Alexo Therapeutics Inc., South San Francisco, CA, USA; Skyview Purvi Patel, Mamatha Reddy, Melissa Lopez, Neil Sankar, Sarath Kanekal, Research, Norristown, PA, USA Mike Li, Sanjeev Redkar, Gavin Choy Correspondence: Sophia Randolph (srandolph@alexotherapeutics.com) CBT Pharmaceuticals, Inc., Pleasanton, CA, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P241 Correspondence: Sanjeev Redkar (sanjeev.redkar@cbtpharma.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P242 Background CD47, a marker of self, is upregulated by tumors to evade the im- Background mune system. Blocking the interaction between CD47 and SIRPα, its Programmed death-1 (PD-1, CD279) is an inhibitory co-receptor receptor on myeloid cells, disrupts a key immune checkpoint and expressed on antigen-activated and exhausted T and B cells. PD-1/ may enhance innate and adaptive immunity against cancer. ALX148 PD-L1 axis inhibition by targeted-antibodies, increases the T cell pro- is a high affinity, engineered fusion protein containing the N- liferation and cytotoxicity. This represents a promising mechanism to terminal D1 domain of SIRPα, which binds and blocks CD47, and is stimulate the anti-tumor activity of the immune system. CBT-501, genetically linked to an inactive human Fc domain to minimize tox- genolimzumab (GB226) is a novel humanized IgG4 monoclonal anti- icity. ALX148 enhanced activity of multiple anti-cancer targeted anti- body targeting the PD-1 membrane receptor on T lymphocytes and bodies and checkpoint inhibitors with minimal effect on normal other cells of the immune system. CBT-501 demonstrated highly spe- blood cells in nonclinical models. This phase 1 study evaluates the cific binding to PD-1 of human (Kd=505 pM) and cynomolgus safety, tolerability, pharmacokinetic (PK) and pharmacodynamic pro- (Kd=7.2 nM). CBT-501 efficiently inhibited the binding of PD-L1/L2 to files of ALX148 in patients with advanced malignancy. PD-1 for both human and monkey and enhanced human T cell acti- Methods vation in the Mixed Lymphocyte Reaction (MLR) assay. CBT-501 has The primary study objective is to characterize the safety profile of demonstrated anti-tumor activity in the in vivo animal model and no ALX148 first as a single agent and then in combination with estab- abnormal drug-related toxicity has been observed in the GLP toxicol- lished anti-cancer antibodies. Cohorts (3-6 pts) with advanced malig- ogy studies. Data from all pre-clinical pharmacodynamics and toxicol- nancy receive escalating doses of ALX148, intravenously, once ogy studies of CBT-501 indicate pharmacological activity at effective weekly or once every other week. Tumor response, PK, and target oc- doses with a wide margin of safety. Based on these findings, a Phase cupancy (TO) are characterized as secondary objectives. Preliminary 1 study has been initiated with CBT-501 in Australia. single agent data are reported from the data cutoff, July 21, 2017 Methods and will be updated at the time of presentation. CBT-501-01 is a Phase 1, multicenter, dose escalation study of CBT- Results 501 in subjects in select advanced or relapsed/recurrent solid tumors. Ten patients received ALX148 (4 males/6 females; 0.3 mg per kilogram The primary study objective is to identify the overall safety and toler- (mpk), 3; 1.0 mpk, 4; 3.0 mpk, 3) as of data cutoff. Median age was 63 ability, including any dose limiting toxicities (DLT), and determine the (37-76) yrs and ECOG PS 0/1: 1/9. Four patients experienced treatment recommended Phase 2 dose (RP2D) in subjects with advanced solid related adverse events (AEs) which were predominantly low grade and tumors. Secondary objectives include assessing efficacy by overall re- included 1 each at 0.3 mpk (G1 Headache, Rash, Fatigue); 1.0 mpk (G3 sponse rate (ORR), best overall response rate (BOR) per RECIST v1.1 Anemia, G1 Dysgeusia); and at 3.0 mpk (G2 Decreased Appetite, Hyper- and irRECIST, time to response, duration of response (DOR), disease sensitivity). As of the data cut-off no pts have experienced a dose- control rate (DCR) by RECIST v1.1 and irRECIST, progression free sur- limiting toxicity. One patient achieved SD (0.3 mpk; leiomyosarcoma) vival (PFS), and determining the pharmacokinetic (PK) parameters. for 16 weeks. ALX148 initial PK showed increased exposure with in- Exploratory objectives involve the assessment of PD-1 and PD-L1 ex- creasing dose and noticeable accumulation with repeated dosing, likely pression, receptor occupancy and the host immune response (im- driven by target saturation. Dose dependent TO on CD47 by ALX148 mune modulation) in blood peripheral-blood mononuclear cells was observed on RBCs and T cells. The magnitude and duration of TO (PBMCs) or formalin-fixed paraffin-embedded (FFPE) samples. This is increased with repeat dosing. a 2-part study with a dose-escalation segment and dose and disease Conclusions expansion cohorts of CBT-501. In Part 1, dose escalation (3+3 design) ALX148 is well tolerated in patients with advanced solid tumors with will occur among 3 cohorts to determine the RP2D. The tumor favorable PK/TO characteristics and no significant hematologic tox- type(s) with the most robust clinical signal relative to response rate icity at doses evaluated. Accrual is ongoing. When the maximum tol- and safety/tolerability will be selected for further evaluation in the erated dose/optimal biological dose of single agent ALX148 is expansion cohort (Part 2). Approximately 32 subjects will be enrolled established, patients with advanced malignancy will be evaluated in the dose and disease expansion and treated at the RP2D, as deter- with ALX148 in combination with anticancer antibodies. mined in Part 1. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 128 of 244 Trial Registration P245 Clinical Trial Registry Number: NCT03053466. CAPRA: A Phase 1b study of intratumoral Coxsackievirus A21 (CVA21) and systemic pembrolizumab in advanced melanoma patients 1 1 2 1 P243 Ann Silk , Howard Kaufman , Nashat Gabrail , Janice Mehnert , Jennifer 1 1 1 1 Withdrawn Bryan , Jacqueline Norrell , Azra Haider , Daniel Medina , Praveen 3 4 4 1 Bommareddy , Darren Shafren , Mark Grose , Andrew Zloza P244 Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; 2 3 A phase 1, first-in-human, open-label, dose escalation study of Gabrail Cancer Center, Canton, OH, USA; Rutgers University, MGD013, a bispecific DART® protein binding PD-1 and LAG-3 in Piccataway, NJ, USA; Viralytics Limited, Sydney, Australia patients with unresectable or metastatic neoplasms Correspondence: Ann Silk (ann.w.silk@rutgers.edu) 1 2 3 4 Sadhna Shankar , Manish Patel , George Blumenschein , Erika Hamilton , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P245 5 1 1 1 Jason Luke , Ross La-Motte Mohs , Kalpana Shah , Lisa Adali-Piston , 1 1 1 1 1 Syd Johnson , Ezio Bonivini , Paul Moore , Jon Wigginton , Jim Vasselli Background 1 2 MacroGenics Inc., Rockville, MD, USA; Florida Cancer Specialists, Sarah Coxsackievirus A21 (CVA21) is a novel bio-selected oncolytic, immu- Canon Research Institute, Sarasota, FL, USA; MD Anderson Cancer notherapeutic agent. Intratumoral (i.t.) CVA21 injection can induce Center, Houston, TX, USA; Sarah Cannon Research Institute, Nashville, selective tumor-cell infection, immune-cell infiltration, IFN-g response TN, USA; University of Chicago, Chicago, IL, USA gene up-regulation, increased PD-L1 expression, tumor cell lysis and Correspondence: Sadhna Shankar (shankars@macrogenics.com) systemic anti-tumor immune responses. A clinical trial evaluating Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P244 combination CVA21 and pembrolizumab in patients with melanoma was initiated and preliminary data on a pre-established futility end- Background point are presented here. Lymphocyte-activation gene 3 (LAG-3) is a membrane protein in Methods the immunoglobulin superfamily that binds to major histocom- This is a single-arm, multi-institutional open-label phase Ib clinical patibility complex class II (MHC-II). LAG-3 engagement negatively trial of i.t. CVA21 and i.v. pembrolizumab for treated or untreated regulates T cell proliferation and differentiation. Blockade of PD-1 unresectable Stage IIIB-IVM1c melanoma. Subjects with injectable and LAG-3 in animal tumor models enhanced antitumor immun- disease receive up to 3 x 10 TCID CVA21 i.t. on Days 1, 3, 5, 8, ity via distinct, non-redundant signaling pathways that fostered and then every 3 weeks for up to 19 injections. Subjects also the accumulation of functionally competent CD8 T cells in mice receive pembrolizumab (2mg/kg) i.v. every 3 weeks starting on Day [1]. Dual targeting of PD-1 and LAG-3 may help reverse effector 8. The primary endpoint is safety/tolerability by incidence of dose- cell exhaustion and increase the response rates and/or effective- limiting toxicity. Secondary endpoints include best ORR by ness of immunotherapy beyond that observed with single agents immune-related response criteria, progression-free survival, overall alone. MGD013 is an Fc-bearing bispecific tetravalent (bivalent for survival, quality of life. each antigen) DART® protein engineered as a hinge-stabilized im- Results munoglobulin G4 molecule and designed to concomitantly bind To date, 22 subjects have started on protocol therapy. Overall, the PD-1 and LAG-3, thereby contributing to sustain or restore the adverse events have been low-grade constitutional symptoms related function of exhausted T cells. MGD013 may enhance T cell to CVA21 and expected pembrolizumab-related side effects. One sub- activation in a synergistic fashion beyond that observed with the ject had Grade 3 increased hepatic enzymes that was considered re- anti–PD-1 and anti–LAG-3 monoclonal antibodies alone or in lated to pembrolizumab. No DLT’s have been reported. Currently, 19 combination. A bispecific format for target engagement may con- patients are evaluable for investigator response assessment. Among fer biologic advantages that may translate to clinical advantages the evaluable subjects (n=19), the ORR was 63% (12/19). The DCR (CR over antibody combinations. +PR+SD) is currently 84% (16/19). In subjects with stage IVM1c disease, Methods the ORR is 78% (7/9). The study has met its primary statistical futility This is an open-label, dose escalation / cohort expansion phase 1 endpoint of achieving ≥2 confirmed objective responses (CR or PR) in study (NCT03219268) designed to characterize the safety, toler- the first 12 patients enrolled. One of the 12 responders displayed early ability, pharmacokinetics, pharmacodynamics, and preliminary an- pseudo-progression and later developed a partial response. titumor activity of MGD013. Patients with unresectable, locally Conclusions advanced or metastatic solid tumors of any histology are enrolled Based on these initial results, the sample size has now been ex- in the dose escalation phase. Sequential escalating flat doses panded to enroll up to 50 patients including subjects refractory to ranging from 1 mg to 1600 mg every 2 weeks are evaluated in anti-PD1 therapy. Combination therapy of CVA21 and pembrolizu- successive cohorts of 1 to 6 patients each. A single patient dose mab may represent a new approach for the treatment of patients escalation design is utilized in the lower dose cohorts. The escal- with injectable advanced melanoma. ation approach transitions to a conventional 3+3 design after the Trial Registration first three cohorts. Occurrence of a drug-related Grade 2 adverse NCT02565992. event in a single patient cohort will lead to enrollment of 3 add- Consent itional patients at that dose level. Occurrence of a DLT in a single Written informed consent was obtained from all of the patients for patient cohort will trigger transition to a conventional 3+3 design. participation in the study and use of the data for publication. Response is first determined at 8 weeks. Patient management is guided by response assessment according to irRECIST. MGD013 dosing may continue up to 2 years based on response. Cohort P246 expansion phase will start after maximum tolerated dose is deter- Phase 1/2 study of in situ vaccination with tremelimumab + mined and will be restricted to 5 tumor types, including solid intravenous (IV) durvalumab + poly-ICLC in patients with select tumors and hematological malignancies. relapsed, advanced cancers with measurable, biopsy-accessible Trial Registration tumors 1 2 3 3 Clinicaltrials.gov- NCT03219268. Craig Slingluff, Jr. , Sunita Hack , Paul Schwarzenberger , Toni Ricciardi , 3 3 3 4 Mary Macri , Aileen Ryan , Ralph Venhaus , Nina Bhardwaj 1 2 References University of Virginia, Charlottesville, VA, USA; Ludwig Institute for 1. Grosso JF, Kelleher CC, Harris TJ, Maris CH, Hipkiss EL, De Marzo A, Cancer Research, New York, NY, USA; Ludwig Cancer Research, New et al.LAG-3 regulates CD8+ T cell accumulation and effector function in York, NY, USA; Icahn School of Medicine at Mt Sinai, New York, NY, USA murine self- and tumor-tolerance systems. J Clin Invest. Correspondence: Craig Slingluff, Jr. (cls8h@virginia.edu) 2007;117(11):3383-92. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P246 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 129 of 244 Background progression-free survival, overall survival) and translational research Immunotherapy has demonstrated promising antitumor activity in assessments. Patients are screened under a separate protocol various advanced cancers. Combined tumor targeting from multiple (NCT03132922). Those who are HLA-A*02 positive (with the excep- drugs with unique mechanisms may provide further improved out- tion of A*02:05) and have inoperable or metastatic (advanced) comes. Tremelimumab (TRE) is a CTLA-4 antibody and durvalumab NSCLC, urothelial cancer, melanoma, or squamous cell head and (DUR) blocks PD-L1. Poly-ICLC is a toll-like receptor 3 agonist. Intratu- neck, ovarian, gastric or esophageal tumors with MAGE-A4 expres- moral (intra-T) injection of poly-ICLC directly alters the tumor micro- sion and meet all other entry criteria are eligible for treatment. Sub- environment (TME), and by creating an in situ vaccination, may jects must have prior treatments as described in the table (Table 1). trigger a clinically effective systemic anti-tumor response when also Patients must have received standard of care therapies and have combined with DUR and TRE. measurable disease. Methods Following leukapheresis, the T cells are isolated, transduced with a c1032 This is an ongoing Phase 1/2, open-label, multicenter study lentiviral vector containing the MAGE-A4 TCR, and expanded (NCT02643303). The study evaluates the use of intra-T administra- with CD3/CD28 beads. Subjects are given lymphodepleting chemo- tion of TRE and IV DUR + poly-ICLC (intra-T and intramuscular [IM]) therapy (fludarabine 30 mg/m /day and cyclophosphamide 600 mg/ to determine the safety, preliminary efficacy and immune activity m /day, on days -7, -6 and -5) prior to infusion of transduced cells. of this regimen in patients with advanced, measurable, biopsy- Groups 1, 2 and 3 will consist of 3-6 subjects, and the transduced cell 9 9 accessible tumors: head and neck squamous cell carcinoma, breast doses will be as follows, respectively: 0.1 × 10 (±20%), 1 × 10 9 9 cancer, sarcoma, merkel cell carcinoma, cutaneous T cell lymphoma, (±20%), and 5 × 10 (range: >1.2 – 6×10 ). The DLT observation melanoma, genitourinary cancer, and other solid tumors. Phase 1 period will be during the first 30 days following the infusion of determines the recommended combination dosing (RCD) for the MAGE-A4 SPEAR T cells for each patient in all groups. Following the regimen with dose de-escalation based on dose limiting toxicities dose escalation, up to 10 patients will be enrolled at the target dose. (DLTs) and standard 3 + 3 rules. Starting doses are: DUR, 1500 mg Disease assessments will be conducted at week 6, 12, 18 and 24, and IV; TRE, 75 mg IV; TRE, 10 mg intra-T; poly-ICLC, 1 mg intra-T/IM. then every 3 months until confirmation of disease progression or at Phase 1 starts with Cohort 1A (DUR + poly-ICLC). Upon demonstra- 2 years post-infusion. On study tumor biopsies and blood samples tion of tolerability, enrollment proceeds with Cohort 1B (DUR + IV will be evaluated to compare the pre- and post-T cell infusion im- TRE + poly-ICLC) and Cohort 1C (DUR + intra-T TRE + poly-ICLC). mune profile for association with treatment outcome. The RCD is the highest dose at which < 2/6 patients have DLTs. In Trial Registration Phase 2, up to 66 evaluable patients are treated using the RCD regi- NCT03132922. men, with enrollment of 6 patients per tumor type initially, and en- rollment of 6 additional patients per 3 tumor types contingent upon at least 1 response among the initial 6 patients. Study end- Table 1 (abstract P247). Eligibility Criteria points are RCD and safety, objective response rate, progression-free survival, and overall survival. Exploratory endpoints are biological activity, including effects on the TME and immunological responses. Enrollment opened on 28 Dec 2016. Results Trial in Progress. Conclusions Trial in Progress. Trial Registration Clinicaltrials.gov: NCT02643303. P247 Study Design: Phase 1 dose escalation, multi-tumor study to assess safety, tolerability and antitumor activity of genetically engineered + + MAGE-A4 SPEAR T cells in HLA-A2 subjects with MAGE-A4 tumors 1 2 3 4 David S Hong , Melissa Johnson , Anthony J Olszanski , Marcus Butler , 5 5 5 5 Connie Erickson-Miller , Malini Iyengar , Trupti Trivedi , Karen Chagin , Rafael Amado The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2 3 Sarah Cannon, Nashville, TN, USA; Anthony.Olszanski@FCCC.edu, Philadelphia, PA, USA; Princess Margaret Cancer Centre, Toronto, ON, Canada; Adaptimmune, Philadelphia, PA, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P247 Background MAGE-A4 is a cancer/testis antigen that has been identified in 13- 48% of non-small cell lung cancer (NSCLC), urothelial, melanoma, P248 head and neck, ovarian, gastric and esophageal tumors. This study Study Design: An open-label randomized pilot study of NY-ESO-1 (NCT03132922) will evaluate the safety and tolerability of genetically SPEAR T cells alone or in combination with pembrolizumab in engineered autologous specific peptide enhanced affinity receptor HLA-A2 subjects with relapsed and refractory multiple myeloma c1032 (SPEAR) T cells (MAGE-A4 T cells) directed towards a MAGE-A4 (NCT03168438) 1 2 3 4 peptide expressed on tumors in the context of HLA-A *02. Antitumor Aaron P Rapoport , James E Hoffman ,Myo Htut , Taiga Nishihori , Karen 5 5 5 5 5 activity will also be assessed. Chagin ,Thomas Faitg , Elliot Norry , Trupti Trivedi ,Rafael Amado Methods University of Maryland School of Medicine, Baltimore, MD, USA; 2 3 This first-in-human T cell dose escalation study utilizes a modified 3 Sylvester Cancer Center at the University of Miami, Miami, FL, USA; City +3 design to evaluate safety, including dose limiting toxicities (DLT). of Hope, Duarte, CA, USA; Moffit Cancer Center, Tampa, FL, USA; Secondary objectives include anti-tumor activity (overall response Adaptimmune, Philadelphia, PA, USA (per RECIST v1.1), duration of response, time to response, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P248 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 130 of 244 Background Methods NY-ESO-1 and LAGE-1a are cancer/testis antigens that are expressed This phase 1, dose escalation study will characterize the safety, frequently in multiple myeloma (MM) and are often associated with tolerability, PK/PD, immunogenicity, and preliminary anti-tumor ac- poor prognosis. This two-arm randomized study will evaluate the safety tivity of MGA012 administered IV every two or four weeks in pa- and efficacy of genetically engineered autologous specific peptide en- tients with advanced solid tumors. MGA012 has been evaluated in c259 hanced affinity receptor (SPEAR) T cells (NY-ESO-1 T cells) directed sequential dose escalation cohorts (1-10 mg/kg) of 3 to 6 patients towards a NY-ESO-1/LAGE-1a peptide expressed on tumor cells in the each, using a 3+3 design. Four tumor-specific expansion cohorts context of HLA-A*02, alone and in combination with pembrolizumab. will be treated at the maximum tolerated dose of MGA012. Select- Methods ive cohort expansion was allowed during escalation to gather fur- This open label randomized pilot study will evaluate safety, efficacy ther safety and PK/PD data. (using the International Myeloma Working Group Uniform Response Results Criteria), and translational research endpoints. Patients must meet these At the data cutoff, 33 patients (12M/21F, median age 63 years) with criteria: ≥ 18 yrs old; HLA-A*02:01, A*02:05 or A*02:06 positive; have his- diverse tumor types were treated at doses from 1-10 mg/kg, includ- tologically confirmed diagnosis of MM with either primary refractory or ing 21 patients on treatment at the time of data cutoff. MGA012 has relapsed/refractory disease expressing NY-ESO-1 and/or LAGE-1a; prior demonstrated acceptable tolerability with no dose-limiting toxicities therapies including IMiD and a proteasome inhibitor as separate lines (DLTs). Treatment-related adverse events (AEs) occurred in 20/33 or a combined line of therapy; and adequate organ function. Subjects (61%) patients, most commonly fatigue (n=9) and nausea (n=5). who have relapsed after autologous hematopoietic cell transplantation Treatment-related Grade ≥3 AEs occurred in 3/33 (9%) patients and (HCT) or are unable to receive autologous HCT are eligible. Target en- include increased lipase (n=2) and vaginal ulceration/inflammation rollment for this study is 20 subjects, with 10 in each arm; patients will (n=1). A single treatment-related serious adverse event (SAE) has be randomly assigned to a treatment arm. Eligible subjects who do not been reported (aphasia occurring in conjunction with the emergence receive the T cell infusion may be replaced. of new brain metastases). Immune-related AEs (irAEs) were limited to Following apheresis, the T cells are isolated and expanded with CD3/CD28 rash (n=3), infusion-related reaction (n=1), and vaginal ulceration/in- c259 beads, transduced with a lentiviral vector containing the NY-ESO-1 flammation (n=1). MGA012 has PK features consistent with other TCR, and 1– 8×10 transduced T cells are infused intravenously on day 1 IgG4 monoclonal antibodies, as well as full and sustained receptor after lymphodepletion with fludarabine 30 mg/m /day and cyclophospha- occupancy at all dosing levels tested, consistent with its known bind- mide 600 mg/m /day on days -7 to -5 and granulocyte-colony stimulating ing characteristics. Twenty-two patients were response evaluable at factor support starting on day -4. In Arm 1, SPEAR T cell infusion is the the data cutoff. Three patients have experienced unconfirmed partial only investigational product administered. Subjects in Arm 2 will receive responses (including ovarian, MSI-high colorectal and uterine papil- SPEAR T cell infusion followed by an initial 200 mg dose of pembrolizu- lary serous carcinoma), and 4 additional patients experienced stable mab on day 22 (week 3). If toxicities preclude week 3 treatment, the first disease as a best response. Others had radiographic progressive dis- dose may be given at week 6. Subsequent doses of pembrolizumab will ease or clinical progression. be given every 3 weeks up to week 108 post T cell infusion. Conclusions In both arms, safety will be assessed at each clinic visit. Disease re- MGA012 has demonstrated an acceptable safety profile, predictable sponse is assessed at weeks 1 and 3, every 3 weeks until week 24, every PK/PD, and early evidence of anti-tumor activity. Subsequent to dose 6 weeks until week 72, and then every 12 weeks until confirmed pro- escalation, patients will be enrolled on tumor-specific monotherapy gression of disease. On study biopsies and blood samples will be evalu- expansion cohorts. Future trials also are planned for combination ated to compare the pre- and post-T cell infusion immune profile for testing of MGA012 with T cell directed, CD3-based DART® molecules. association with treatment outcome. Trial Registration Trial Registration NCT03059823. NCT03168438. P250 P249 LTX-315, an oncolytic peptide converts “cold” tumors to “hot” in a A phase 1 study of the safety, tolerability, and pharmacokinetics majority of patients with advanced cancer: results from an (PK) of MGA012 (anti-PD-1 antibody) in patients with advanced ongoing phase I study 1 2 3 solid tumors Aurelien Marabelle , Jean-Francois Baurain , Ahmad Awada ,Rebecca 1 2 3 4 4 5 5 6 Nehal Lakhani ,Janice M. Mehnert ,Drew Rasco , Michael Gordon , Joanna Kristeleit , Dag-Erik Jøssang , Nina-louise Jebsen , Delphine Loirat , 5 5 5 5 5 7 7 7 7 Lohr , Pepi Pencheva , Sharad Sharma ,Hua Li , Ross LaMotte-Mohs ,Paul Andrew Saunders , Wenche Olsen , Berit Nicolaisen , Baldur Sveinbjornsson , 5 5 5 5 6 7 7 8 9 Moore ,Jichao Sun , Bradley Sumrow , Jon Wigginton , John Powderly Vibeke Sundvold Gjerstad ,Øystein Rekdal ,PalBrunsvig , Jerome Galon , 1 10 5 11 12 START Midwest - South Texas Accelerated Research Therapeutics, LLC, Fabienne Hermitte , Bjorn-Tore Gjertsen , Anna Armstrong , James Spicer 2 1 2 Grand Rapids, MI, USA; Rutgers Cancer Institute of New Jersey, New Gustave Roussy, Villejuif, France; Saint-Luc University Hospital, Brussel, 3 3 4 Brunswick, NJ, USA; START - South Texas Accelerated Research Belgium; Institut Jules Bordet, Brussel, Belgium; Univerity College 4 5 Therapeutics, LLC, San Antonio, TX, USA; Honor Health Research London Hospital, London, United Kingdom; Haukeland Univeristy 5 6 7 Institute, Scottsdale, AZ, USA; MacroGenics, Inc., Rockville, MD, USA; Hospital, Bergen, Norway; Institut Curie, Paris, France; Lytix Biopharma, 6 8 9 Carolina BioOncology Institute, Huntersville, NC, USA Oslo, Norway; Oslo University Hospital, Oslo, Norway; Laboratory of Correspondence: Bradley Sumrow (sumrowb@macrogenics.com) Integrative Cancer Immunology, INSERM, Paris, France; INSERM, Paris, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P249 France; Christie Hospital, Manchester UK, Manchester, United Kingdom; King’s College London, London, United Kingdom Background Correspondence: Øystein Rekdal MGA012 is a humanized, IgG4κ monoclonal antibody (mAb) that rec- (baldur.sveinbjornsson@lytixbiopharma.com) ognizes human programmed cell death protein 1 (PD-1). MGA012 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P250 binds to PD-1 expressing T cells, inhibits PD-1 and PD-L1/PD-L2 inter- actions, and disrupts the negative signaling axis to restore T cell Background function. The biological activity of MGA012 is comparable to replicas LTX-315 disintegrates cytoplasmic organelles (e.g mitochondria) and of approved anti-PD-1 mAbs when assessed in vitro, including block- induces immunogenic cancer cell death in preclinical in vivo models. ade of PD-1 and PD-L1/PD-L2 interactions, inhibition of PD-1 signal- Intratumoral LTX-315 increases tumor-infiltrating lymphocytes (TILs) ing, and enhancement of T cell effector function. and induces complete tumor regression in several rodent models. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 131 of 244 Systemic (abscopal) anti-tumor immune responses can be enhanced upregulation of PD-L1 on tumor infiltrating monocytes and macro- upon combination with immune checkpoint inhibitors (ICI). Here we phages, thereby promoting a negative feedback loop, which hampers report preliminary results of the phase 1 trial which evaluates intratu- CD40 induced T-cell responses. This resistance mechanism was success- moral LTX-315 in monotherapy or in combination with ICI. fully circumvented by co-administration of PD-1/PD-L1 blocking anti- Methods bodies. To this end, Apexigen Inc., is developing APX005M a Patients with advanced solid tumors were immunologically primed humanized monoclonal IgG1 CD40 agonistic antibody that stimulates with weekly intratumoral LTX-315 into a single accessible lesion both innate and adaptive immune response. APX005M recognizes a over 6 weeks. Additional injections could be administered thereafter unique epitope that overlaps with the CD40 ligand binding sites and every 2 weeks. ICI combinations included ipilimumab (melanoma co- uses FcRγIIb to cluster CD40, thus mimicking CD40L engagement. As a hort) and pembrolizumab (TNBC cohort). Biopsies of injected lesions result of antigen presenting cell (APC) activation, APX005M enhances T- were taken at baseline and after LTX-315 treatment. Immunoprofiling cell response to tumor antigens. APX005M combined with antibodies was performed using immunohistochemistry and Nanostring analysis. against PD-1 or PD-L1 synergistically enhances T-cell responses. In a Twenty nine patients have been treated with LTX-315 monotherapy. phase 1 trial, APX005M was administered IV every 21 days to adult sub- LTX-315 monotherapy was administered at doses of 2-7mg to a median jects up to 1mg/kg with an acceptable safety profile and has demon- of 1.8 tumor lesions (range 1-6) for a median of 9 weeks (range 1-33). strated a dose-dependent activation of APCs and T cells and increases Results in circulating levels of cytokines. In 29 patients, all LTX-315-related adverse events were CTC grade 1 Methods or 2, most commonly local erythema, flushing, pruritis and Study APX005M-002 is a Phase 1b–2 study of APX005M administered hypotension, usually resolving within minutes of injection. Related in combination with nivolumab every 21 days to adult subjects with grade 3 (3 patients) or 4 (1) allergic/anaphylaxis adverse events oc- platinum pre-treated immunotherapy naïve non-small cell lung can- curred and resolved without sequelae. Of 44 injected lesions in 20 cer (NSCLC) or metastatic melanoma after failure of anti-PD-1/PD-L1 evaluable LTX-315 monotherapy patients, 2 lesions regressed com- therapy (MM). The Phase 1b portion will establish the maximum tol- pletely, > 50% regression was seen in 5 lesions, and 20 remained erated dose and the recommended Phase 2 dose of APX005M with stable. Significant increases in CD8+ TILs occurred in 67% (14 of 21) nivolumab. The Phase 2 portion will evaluate safety and efficacy of patients with evaluable biopsies. The HalioDx Immune Gene Signa- the combination in each of the two distinct tumor types. Inclusion ture analysis of LTX-315-treated tumors showed upregulation of criteria include: age ≥ 18 years, histologically documented NSCLC or genes involved in immune-mediated tumor regression (effector T MM, measurable disease by RECIST 1.1, ECOG performance status 0- cells, TH1 orientation, chemokines and cytokines). Regression of dis- 1, adequate organ function. Exclusion criteria include: concomitant tant non-injected tumors (irRC) criteria has been observed in 11 of anti-cancer therapy, history of bone marrow transplantation, active 30 tumors in 9 patients. Stable disease (SD) of at least 7 weeks dur- coagulopathy, previous immune mediated disorders, active infections ation in non-injected tumors (median duration 11 weeks) by or uncontrolled intercurrent illness. Recruitment is ongoing, with a immune-related RECIST criteria (irRC in evaluable patients) occurred target enrollment of approximately 100 subjects across 7 centers in in 50% of LTX-315 monotherapy patients (4 melanoma, 3 sarcoma, the United States. and 1breast cancer patients). Trial Registration Conclusions Clinical trial information: NCT03123783. This phase 1 study demonstrates that intratumoral LTX-315 is gener- ally safe and tolerable. Intratumoral LTX-315 triggers an increase in References TILs as assessed by IHC, and generates a transition from a cold to a 1. Zippelius A, Schreiner J, Herzig P, Müller P. Cancer Immunol Res. hot tumor transcriptome as assessed by Immunosign® gene signa- 2015;3:236-44. ture. Moreover, local and systemic clinical benefit (achievement of SD in non-injected sites per irRC criteria) was observed. Trial Registration P252 Clinical trial information: NCT01986426. Phase 1 study to evaluate the safety and tolerability of the CD40 agonistic monoclonal antibody APX005M in subjects with solid tumors 1 2 3 4 P251 Marwan Fakih , David Bajor , Ronac Mamtani , Thomas Tremblay , 4 3 A study to evaluate the safety and efficacy of the CD40 agonistic Ovid Trifan , Robert Vonderheide 1 2 antibody APX005M in combination with nivolumab in subjects City of Hope, Duarte, CA, USA; University Hospitals Cleveland Medical with non-small cell lung cancer and subjects with metastatic Center, Cleveland, OH, USA; Abramson Cancer Center, University of melanoma Pennsylvania, Philadelphia, PA, USA; Apexigen Inc., San Carlos, CA, USA 1 2 3 4 Linda Garland , Ravi Salgia , Melissa Johnson , Amy Weise , Correspondence: Ovid Trifan (otrifan@apexigen.com) 5 6 6 7 Gerald Linette , Thomas Tremblay , Ovid Trifan , Martin Edelman Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P252 1 2 University of Arizona Cancer Center, Tucson, AZ, USA; City of Hope, Duarte, CA, USA; Sarah Cannon Research Institute, Tennessee Oncology, Background Nashville, TN, USA; Karmanos Cancer Center, Detroit, MI, USA; APX005M is a humanized monoclonal IgG1 CD40 agonistic antibody Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, developed by Apexigen, which mimics the natural ligand CD154. 6 7 USA; Apexigen Inc., San Carlos, CA, USA; Fox Chase Cancer Center, APX005M binds with high affinity to human CD40 leading to antigen Philadelphia, PA, USA presenting cell (APC) activation and subsequent T cell activation. Correspondence: Martin Edelman (otrifan@apexigen.com) APX005M enhances T cell proliferation, IFN-γ production and T-cell Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P251 response to tumor antigens. In comparison with other anti-human CD40 agonistic antibodies, such as CP-870,893/RG7876, SGN-40, and Background ADC-1013/JNJ-64457107 analogs, APX005M is the most potent CD40 Blocking immune checkpoint PD-1, PD-L1 and CTLA-4 function en- agonist and outperforms all others in many measures of immune ac- hance antitumor immunity, leading to durable clinical responses for a tivation. In a first in human clinical trial APX005M was administered subset of patients with melanoma, lung cancer and other tumor types. IV every 21 days and has demonstrated a dose-dependent activation However, the majority of patients with melanoma or lung cancer con- of APCs and T cells and increases in circulating levels of IL-12, IFN-γ, tinue to have short or no response to checkpoint blockade therapies TNFα and IL-6. APX005M was escalated up to 1mg/kg with a good and thus require novel approaches to stimulate the antitumor immune safety profile. response such as immune stimulatory antibodies. Recently, Zippelius Methods and co-authors [1] showed in preclinical models that CD40 engage- Study APX005M-001 was originally designed as a multicenter Phase ment with an agonistic mAb leads to a T cell and IFN-γ dependent 1 dose escalation study of APX005M administered every 3 weeks to Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 132 of 244 subjects with solid tumors and was amended to introduce two new adenocarcinoma; and head and neck squamous cell, renal clear dosing schedules for APX005M (every 2 weeks and every 1 week). cell, hepatocellular, and esophageal carcinomas. Currently, primary objectives of the study are to evaluate the safety Conclusions of APX005M administered intravenously (IV) every 2 weeks and every Preclinical data from syngeneic tumor models and bioinformatic ana- week and to determine the maximum tolerated dose (MTD) and rec- lyses of TCGA database prioritized 7 tumors for LYC-55716 monother- ommended Phase 2 dose (RP2D) of APX005M for the every 2 weeks apy. Our findings support the inclusion of these tumor types in the and every 1 week schedules. Secondary objectives include determin- Phase 2a clinical trial. ing the pharmacokinetics (PK) of APX005M and preliminary assess- ment of clinical response. Inclusion criteria include: age ≥ 18 years, P254 histologically or cytologically documented diagnosis of urothelial car- A phase 1b/2 study of CD40 agonistic monoclonal antibody cinoma, melanoma, squamous cell carcinoma of the head and neck, (APX005M) together with gemcitabine and nab-paclitaxel with or non-small cell lung cancer, or any solid tumor with high microsatel- without nivolumab in untreated metastatic pancreatic lite instability status (MSI-high), measurable disease by RECIST 1.1, adenocarcinoma patients ECOG performance status 0-1, adequate organ function. Exclusion 1 2 3 3 Mark O’Hara , Rosemarie Mick , Jaclyn Lyman , Jingying Xu , criteria include: concomitant anti-cancer therapy, history of bone 3 3 3 Maryam Hosseini , Theresa LaVallee , Pier Federico Gherardini , marrow transplantation, active coagulopathy, previous immune me- 1 4 5 6 Barbara Vance , Ovid Trifan , Ute Dugan , Aiman Shalabi , diated disorders, active infections or uncontrolled intercurrent illness. 3 1 Ramy Ibrahim , Robert Vonderheide Recruitment is ongoing, with a target enrollment of approximately 1 2 University of Pennsylvania, Philadelphia, PA, USA; University of 20 subjects across 3 centers in the United States. Pennsylvania Perelman, Philadelphia, PA, USA; Parker Institute for Trial Registration Cancer Immunotherapy, San Francisco, CA, USA; Apexigen, San Carlos, Clinical trial information: NCT02482168. 5 6 CA, USA; Bristol-Myers Squibb, New York City, NY, USA; Cancer Research Institute, New York, NY, USA P253 Correspondence: Ramy Ibrahim; Robert Vonderheide Prioritizing tumor types for treatment with a novel (jxu@parkerici.org) immunotherapy: LYC-55716 a small-molecule RORγ agonist Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P254 1 1 1 1 1 Xiao Hu , Xikui Liu , Hongxiu Li , Madhumita Bogdan , Yilin Gao , 2 3 1 Brian Fox , H. Jeffrey Wilkins , Laura Carter Background 1 2 Lycera Corp., Ann Arbor, MI, USA; Celgene Corp., Seattle, WA, USA; Pancreatic cancer is one of the most lethal malignancies of the Lycera Corp., Plymouth Meeting, PA, USA gastrointestinal tract. While check point inhibitors such as αCTLA4 Correspondence: H. Jeffrey Wilkins (autumn@ahkcommunications.com) and αPD-1 have been effective in melanoma and lung cancer, clinical Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P253 benefit of immunotherapy in the management in pancreatic cancer subjects has not been yet established. A recent study using a genet- Background ically engineered mouse model of pancreatic ductal adenocarcinoma RORγ is the master transcription factor for Type 17 effector T cell differ- (PDA) demonstrated that despite robust expression of PD-1 and PD- entiation and function. RORγt expression is induced by cytokines such L1 in the tumor microenvironment, treatment with αPD-1 with or as IL-6, TGF-β, IL-1b, and IL-23 and is activated by endogenous ligands without αCTLA-4 failed to improve the survival of mice or slow the derived from the cholesterol biosynthetic pathway. Synthetic RORγ ag- growth of PDA tumors. However, administration of αCD40, gemcita- onists augment the activity of this transcriptional regulator by modulat- bine (Gem) and nab-paclitaxel (NP), induces T cell immunity in mice ing a gene expression program in immune cells, resulting in enhanced with PDA, controls tumor growth and significantly improves survival effector functions and decreased immunosuppression. LYC-55716 and in a CD8 T cell dependent manner. In particular, αCD40/Gem/NP other RORγ agonists have shown promise as monotherapy and com- plus αPD-1 nearly double the median overall survival in genetically bination therapy in syngeneic tumor models. During Phase 1 clinical engineered KPC mice with pre-established spontaneous pancreatic testing of this compound, preclinical and bioinformatics assessments tumors. Moreover, the capability of treated mice to reject second were undertaken to prioritize tumor types that may respond to RORγ and third subcutaneous tumor challenges in a CD8+ T cell-dependent agonist therapy, for possible inclusion in a Phase 2a trial. fashion thereby rendering long-term survival suggests the establish- Methods ment of antitumor immune memory with curative potential. An RORγ agonist signature was derived from transcriptional profiling Methods of primary murine and human T cells treated with or without RORγ This is a multi-center, open label study evaluating the combination agonists. Using a panel of murine syngeneic models and The Cancer of APX005M with Nivolumab and standard chemotherapy (Gem and Genome Atlas (TCGA) dataset, a series of bioinformatic analyses were NP). The Phase 1b will define the recommended Phase 2 dose of conducted to provide information across tumor types on (a) RORγ ex- APX005M when combined with the standard dose of Gem and NP, pression, (b) RORγ biology, including sterol mobilizing genes and cor- with or without Nivolumab. The second part of the study is a 3-arm relations with prognosis, and (c) general immune parameters. Public randomized Phase 2 (35 subjects per treatment arm) aimed to evalu- data sets were also assessed for correlations of RORγ signature genes ate the activity (overall survival) of APX005M combined with Gem and prognosis. For each assessment, tumor types were prioritized, and NP, with or without Nivolumab with Gem and NP. Main inclusion then compared across categories to determine a final ranking. criteria include: age ≥ 18 years, documented diagnosis of pancreatic Results adenocarcinoma with metastatic disease, measurable disease by Target expression: 15 tumors types were identified, with >20% of RECIST 1.1, ECOG performance status 0-1, normal organ function. samples expressing RORγt. However, based on lack of correlation be- Main exclusion criteria include: concomitant anti-cancer therapy, previous tween baseline RORγt expression and efficacy of RORγ agonists in exposure to CD40, PD-1, PD-L1, CTLA-4 mAbs or any other immunomod- preclinical models, these criteria were extended to include additional ulatory agents, previous immune mediated disorders, active infections or tumors that express mRNA for factors known to induce RORγ expres- uncontrolled intercurrent illness. The study will be exploring different sion. Target biology: As a surrogate for endogenous ligand levels, doses of APX005M in combination with approved doses of Nivolumab TCGA analysis revealed differentially expressed sterol synthesis and and chemotherapy. The main safety endpoints include the frequency of efflux genes across tumor types. RORγt expression correlated signifi- DLT and incidence of AEs. The main efficacy endpoints include overall cantly with patient survival in 5 tumor types. Immune parameters: survival, and 1-year OS rate in each treatment arm. Consideration of tumors with immune infiltrates, high mutational Results burden, and reports of prior immunotherapy success highlighted This trial is open for enrollment. 8 tumor types. After combining these findings, 7 tumor types Conclusions met all selection criteria: non–small-cell lung; ovarian; stomach Clinical trial information: NCT02482168. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 133 of 244 Combination Therapy (IO/IO, IO/Standard of Care, Background Glioblastoma (GBM) is a malignant brain tumor with an overall IO/Other) survival of < 3.3% at 5 years. Novel immunotherapies are being explored but face limitations due to low infiltration of activated T P255 cells. ZVex is an integration-deficient lentiviral vector-based plat- Depleting blood arginine with AEB1102 (Pegzilarginase) exerts form that targets dendritic cells in vivo to generate tumor-specific additive anti-tumor and synergistic survival benefits when T cells. Intratumoral injections of G100, which contains glucopyr- combined with immunomodulators of the PD-1 pathway ansosyl lipid A (GLA, a synthetic TLR4 agonist), modulate the Giulia Agnello, Mark Badeaux, Susan Alters, David Lowe, Scott Rowlinson tumor microenvironment through induction of proinflammatory Aeglea BioTherapeutics, Inc., Austin, TX, USA responses. We have previously shown in the B16 model that Correspondence: Giulia Agnello (gagnello@aegleabio.com); Scott G100 induces T cell homing chemokines CXCL9 and CXCL10, in- Rowlinson creases number of T cells in the tumor, enhances intratumoral Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P255 antigen presentation, and results in antigen spreading of the T cell response. Combined systemic and in situ immunization with Background ZVex/OVA and G100, respectively, completely eradicated estab- Tumor dependence on specific amino acids for survival and prolifera- lished B16/OVA melanomas in mice. Here, we report that, in an tion is well recognized and has been exploited effectively in the orthotopic GBM model, the combined regimen with ZVex/OVA clinic through the use of asparaginases for the treatment of acute and G100 induced near complete regression of established GL261 lymphoblastic leukemia. Sensitivity of tumors to L-Arginine (L-Arg) syngeneic gliomas in C57BL/6 mice. deprivation results from an impaired ability to synthesize L-Arg, most Methods commonly due to decreased functional expression of argininosucci- Female C57BL/6 mice were stereotactically cannulated in the left stri- nate synthase. Native human arginase 1 is not a viable drug candi- atum. At study initiation, 2 x 10 GL261 glioma cells expressing ovalbu- date due to low activity and low stability in serum. We have min (GL261/OVA) were inoculated through the cannula. Tumor growth developed a novel cobalt substituted, PEGylated human arginase 1 was monitored via imaging of luciferase activity, twice weekly. On Day (AEB1102, Pegzilarginase) with enhanced pharmacological properties. 7, mice with tumors (6.8 x 10 avg. radiance) were immunized by a sin- We and others have successfully utilized arginase 1 to impart an gle subcutaneous injection of ZVex/OVA. G100 (2 μg GLA) was adminis- anti-tumor effect through L-Arg starvation in multiple tumor types tered through the cannula, also on Day 7, and then once weekly for in vitro and in vivo (e.g. AEB1102 single agent efficacy in melanoma, the duration of the study, for a total of three injections. Animals were SCLC, sarcoma, large cell NSCLC, Merkel cell carcinoma). Given that sacrificed when displaying signs of cachexia and > 20% weight loss, arginase 1 has been reported to be immune suppressive, immune usually around 25 days after tumor inoculation. neutral (PMID: 23717444), or immune promoting (PMID: 27043409) in Results different experimental settings and by different groups, we have GL261/OVA-bearing mice treated with G100 or ZVex/OVA alone ex- investigated the impact of systemic depletion of L-Arg on the anti- hibited delayed tumor growth or modest tumor regression, respect- tumor efficacy of immune checkpoint inhibitors. ively (mean reduction of 17% and 72%, respectively, ranging from Methods none to 83%), while mice treated with ZVex/OVA and G100 combin- Murine syngeneic models (e.g. CT26, MC38) were dosed with AEB1102 ation exhibited significantly greater reduction in tumor size, aver- alone and in combination with immunomodulatory anti-PD-L1 mono- aging 98% (74%-99.8%). clonal antibody (mAb). Conclusions Results This study demonstrates that ZVex/OVA and G100 combination ther- Combination therapy of AEB1102 with anti-PD-L1 resulted in an addi- apy was very successful in controlling growth of GL261 gliomas, pre- tive anti-tumor effect with improved survival benefit (increased life sumably in part by chemotactically directing peripheral T cells to the span (ILS) 55-129%) compared to AEB1102 (ILS 29-33%) and anti-PD-L1 brain, and suggest the potential for treatment of patients with GBM. (ILS 7-33%) monotherapies. In addition, in the CT26 model, complete The precise mechanism of synergy in this GBM model is currently tumor regression (non-palpable tumors) was observed in 37% of the under investigation. Both ZVex-based therapies and G100 are being mice; importantly, complete responses were observed only in the com- evaluated in multiple Phase 1 and 2 clinical trials, and their combin- bination therapy group. When the complete responders were re- ation is being investigated in a Phase 1 trial in soft tissue sarcoma challenged with fresh CT26 cells, tumors failed to establish, suggesting patients. the development of an immune memory response as a result of the previously administered combination therapy of AEB1102 and anti-PD- L1. Administration of AEB1102 as a monotherapy or in combination with anti-PD-L1 in the CT26 model was associated with an increase in tumor-infiltrated CD45+ cells, indicating that AEB1102 promotes T-cells P257 accumulation in the tumor microenvironment. Phase II basket study of olaparib and durvalumab: Biomarker Conclusions analysis in germline BRCA-mutated (gBRCAm) HER2-negative Collectively, these results demonstrate that in addition to tumor metastatic breast cancer (MBC) and relapsed small-cell lung cancer growth inhibition, L-Arg depletion in the tumor microenvironment (SCLC) patients enhances the effectiveness of immunotherapy. AEB1102 is currently 1 1 2 1 Helen Angell , Vidalba Rocher Ros , Nathan Standifer , Zhongwu Lai , in Phase 1 (monotherapy) clinical trials. These data open the possibil- 1 3 4 5 Christopher Gresty ,Jean-Pierre Delord ,MajaDeJonge , Sophie Postel-Vinay , ity of clinical combination of AEB1102 with immunomodulators of 6 7 8 9 Antoine Italiano ,Matthew GKrebs , Bella Kaufman ,YeonHee Park , the PD-1 pathway to further improve outcomes in cancer patients. 10 11 1 Susan Domchek , Pia Herbolsheimer , Darren Hodgson 1 2 AstraZeneca, Cambridge, United Kingdom; MedImmune, Mountain View, CA, USA; Institut Claudius Regaud-Oncopole, Toulouse, France; 4 5 P256 Erasmus MC Cancer Institute, Rotterdam, Netherlands; Gustave Roussy G100 and ZVex®-based combination immunotherapy induces near Cancer Campus, Villejuif, France; Institut Bergonié, Bordeaux, France; complete regression of established glioma tumors in mice The University of Manchester and The Christie NHS Foundation Trust, Tina C. Albershardt, Jordan E. Krull, Reice D. James, Peter Berglund, Jan Manchester, United Kingdom; Sheba Medical Centre, Ramat Gan, Israel; ter Meulen Samsung Medical Centre, Sungkyunkwan University School of Medicine, Immune Design, Seattle, WA, USA Seoul, Republic of Korea; Hospital of the University of Pennsylvania, Correspondence: Tina C. Albershardt Philadelphia, PA, USA; AstraZeneca, Gaithersburg, MD, USA (tina.albershardt@immunedesign.com) Correspondence: Helen Angell (Helen.Angell@astrazeneca.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P256 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P257 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 134 of 244 Background P258 Poly(ADP-ribose) polymerase inhibitors (PARPi) trap PARP at sites of Resource use and cost implications associated with treatment-free single-strand DNA breaks, and generate genomic instability and cell interval experienced on immunotherapies 1 2 3 death in homologous recombination deficient tumour cells. Both in- Michael Atkins , Ahmad Tarhini , Apoorva Ambavane , David 4 5 3 5 trinsic and PARPi-induced DNA repair defects may attract tumour- McDermott , Komal Gupte-Singh , Josh Weng , Corey Ritchings , 7 6 5 infiltrating lymphocytes (TILs), upregulate programmed cell death Meredith Regan , Agnes Benedict , Sumati Rao ligand-1 (PD-L1) and release tumour neo-antigens upon cell death. Georgetown-Lombardi Comprehensive Cancer Center, Washington, DC, USA; The MEDIOLA study (NCT02734004) assessed the effect of the PARPi, University of Pittsburgh School of Medicine and Cancer Institute, Pittsburgh, 3 4 olaparib, alone and in combination with durvalumab (anti-PD-L1), on PA, USA; Evidera, Inc., Bethesda, MD, USA; Beth Israel Deaconess Medical 5 6 immune cell pharmacodynamics, and the correlation of candidate Center, Boston, MA, USA; Bristol-Myers Squibb, Princeton, NJ, USA; Evidera, predictive markers on disease control rate (DCR). Inc., Budapest, Hungary; Dana-Farber Cancer Institute, Boston, MA, USA Methods Correspondence: Michael Atkins (mba41@georgetown.edu) Patients with HER2-negative, gBRCAmMBC (n=25), or relapsed SCLC Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P258 (n=38) received olaparib (300 mg PO BID) for 4 weeks, followed by a com- bination of olaparib and durvalumab (1.5 g IV every 4 weeks) until pro- Background gression. At 4 weeks after initial olaparib, paired biopsies were conducted Immunotherapies, including ipilimumab, nivolumab, and nivolumab for immuno-oncology profiling. Primary endpoints were DCR at 12 weeks, +ipilimumab, are recommended for the treatment of advanced mel- safety and tolerability. Biomarker endpoints included archival (Fig. 1) and anoma. Patients treated with immunotherapies often experience clin- paired biopsy analysis of tumour cell (TC) and immune cell (IC) PD-L1 ex- ical benefit beyond treatment discontinuation, resulting in a pression (SP263), densities of CD3 and CD8 T-cells (cells/mm ), tumour significant treatment-free interval (TFI) until subsequent treatment mutation status and peripheral blood immunophenotyping. is needed. However, resource use during TFI is not well under- Results stood. The objective of this study was to estimate the resource Olaparib caused small reductions in circulating T, B and Natural Killer use and costs associated with the TFI experienced after first-line (NK) cells on day 29 and thereafter in 86–93% of MBC and 81–91% of immunotherapies. SCLC patients, followed by evidence of post-durvalumab 2-fold or Methods greater increases in Ki67+CD8+ T-cells in 46% of MBC and 50% of SCLC Patient-level data (N=882) from the phase 3 CheckMate 067 and patients on day 57. In MBC (n=25) 5/5 (100%) PD-L1 TC≥1% patients phase 2 CheckMate 069 trials were analyzed to estimate the health- had controlled disease at 12 weeks, compared with 9/14 (64.3%) PD-L1 care resource use associated with the management of advanced mel- TC<1% patients (no data for 6/25 patients). In SCLC (n=38) PD-L1 anoma. TFI is defined as time from first-line discontinuation to TC≥1% patients had a DCR of 1/6 (16.7%) compared with a PD-L1 second-line initiation. Concomitant medications, laboratory tests, pro- TC<1% DCR of 9/27 (33.3%) at 12 weeks (no data for 5/38 patients). At cedures, consultations, hospitalizations, and surgeries were analyzed. the time of analysis, 1/2 MBC with paired biopsy data had a partial re- The annual rate of resource use was estimated for ipilimumab, sponse (PR) and demonstrated dramatic increases in CD3/CD8 densities nivolumab, and nivolumab+ipilimumab, and stratified by disease and PD-L1 TC positivity 4 weeks post-olaparib monotherapy. 1/3 SCLC phase (progression-free or progressed defined by RECIST v.1.1). Mean paired biopsies had PRs and showed increases in CD3/CD8 densities, annual total costs and 95% confidence intervals (CIs) were estimated but no PD-L1 TC expression, compared with no change in 2/3 patients by applying unit costs from publicly available sources in the United with stable disease. Updated results will be presented. States. Conclusions Results Olaparib results in modest reductions in circulating T, B and NK cells: Annual costs per patient during the TFI on various immunotherapies however, this effect does not disrupt durvalumab-based increases in are presented below (Table 1) Although confidence intervals are circulating, proliferating cytotoxic T-cells. Preliminary data suggest wide, ipilimumab is associated with higher total cost/year compared that olaparib treatment may increase levels of TILs, but more data to nivolumab+ipilimumab and nivolumab in the progression-free are required. Data collection and analysis is ongoing. phase (difference in costs $5,624-$6,481), and compared to nivolu- mab+ipilimumab and nivolumab in the progressed phase (difference in costs $354-$3,834). Progression increases the costs of melanoma management across all treatments by $12,486-$16,823. Key resources utilized across all treatments were hospitalizations (34-62% of total costs) in the progression-free phase, due to management of adverse events, and hospitalizations and surgeries (>94% of total costs) in the progressed phase, due to disease worsening. Conclusions Immunotherapies for advanced melanoma, particularly nivolumab +ipilimumab, may lead to a long TFI before starting second-line ther- apy. The estimated annual cost of managing advanced melanoma is low in the TFI prior to disease progression, especially with nivolumab and nivolumab+ipilimumab. These results require comparison with real-world evidence to measure the impact of bias due to the con- trolled clinical trial setting. Table 1 (abstract P258). See text for description *Includes costs associated with concomitant medications, laboratory tests, Fig. 1 (abstract P257). Biomarker analysis summary procedures, and consultations Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 135 of 244 P259 P260 Pushing the accelerator and releasing the brake: testing the Glembatumumab vedotin (GV), an anti-gpNMB antibody-drug soluble LAG-3 protein (IMP321), an antigen presenting cell conjugate (ADC), in combination with varlilumab (V), an anti-CD27 activator, together with pembrolizumab in unresectable or antibody, in advanced melanoma 1 2 3 4 metastatic melanoma Omid Hamid , Anna C. Pavlick , C. Lance Cowey , Lowell Hart , 1 2 3 4 5 6 7 8 Victoria Atkinson , Andrew Haydon , Melissa Eastgate , Amitesh Roy , Douglas B. Johnson , Jose Lutzky , Aaron Alizadeh , David Spigel , 5 6 6 9 10 11 12 Adnan Khattak , Christian Mueller , Tina Dunkelmann , Chrystelle Neal Rothschild , April Salama , Robert Weber , Jason J. Luke , 7 7 13 13 13 13 14 Brignone , Frederic Triebel Ying Wang , Michael Yellin ,Yi He , Rebecca G. Bagley , Patrick Ott 1 2 1 2 Princess Alexandra Hospital, 4102 Woolloongabba, Australia; Alfred The Angeles Clinic and Research Institute, Los Angeles, CA, USA; New 3 3 Hopital, 3004 Melbourne, Australia; Royal Brisbane and Women's York University School of Medicine, New York, NY, USA; Baylor 4 4 Hospital, 429 Herston, Australia; Flinders Medical Centre, Bedfork University Medical Center, Dallas, TX, USA; Florida Cancer Specialists, 5 6 5 Park, Australia; Fiona Stanley Hospital, Murdoch, Australia; Prima Fort Myers, FL, USA; Vanderbilt University Medical Center and Vanderbilt 7 6 BioMed GmbH, Berlin, Germany; Immutep S.A.S., Châtenay-Malabry, Ingram Cancer Center, Nashville, TN, USA; Mount Sinai Comprehensive France Cancer Center, Miami Beach, FL, USA; Northside Hospital, Atlanta, GA, 8 9 Correspondence: Victoria Atkinson USA; Tennessee Oncology, PLLC, Nashville, TN, USA; Florida Cancer (Victoria.Atkinson@health.qld.gov.au) Specialists, West Palm Beach, FL, USA; Duke University, Durham, NC, 11 12 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P259 USA; St. Mary's Medical Center, San Francisco, CA, USA; University of Chicago, Chicago, IL, USA; Celldex Therapeutics, Inc., Hampton, NJ, Background USA; Dana-Farber Cancer Institute, Boston, MA, USA IMP321 is a recombinant soluble LAG-3Ig fusion protein binding Correspondence: Omid Hamid (rbagley@celldex.com) to MHC class II molecules and mediating antigen presenting cell Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P260 (APC) activation followed by CD8 T-cell activation. The activa- tion of the dendritic cell network and the subsequent T cell re- Background cruitmentatthe tumorsitewithIMP321 mayleadtostronger gpNMB is an internalizable transmembrane glycoprotein expressed anti-tumor CD8 T cell responses than observed with pembrolizu- in multiple tumor types. The ADC GV delivers the potent cytotoxin mab alone. This combination of an APC activator with an im- MMAE to gpNMB+ cells and showed promising activity as monother- mune checkpoint inhibitor (ICI) is aiming to increase efficacy apy in advanced melanoma refractory to checkpoint inhibitors (CPI). without additional toxicity. We report initial results of the first 2 Preclinical results showed synergistic antitumor activity between ADC cohorts of a dose escalation phase I trial (TACTI-mel, with CPI [1, 2], via dendritic cell (DC) activation; ADC-MMAE aug- NCT02676869) with pembrolizumab and IMP321 at different mented the immune response induced by V, an agonist monoclonal dose levels. antibody against the T cell costimulatory molecule CD27. This Phase Methods 2 study was conducted to evaluate the activity and safety of the GV/ In this study, melanoma patients treated with pembrolizumab V combination in advanced melanoma. being without a complete response or fast progression after 3 Methods cycles received pembrolizumab per standard dosing plus either Patients with advanced melanoma, progressive after ≤1chemotherapy, 1 mg (n=6; cohort 1) or 6 mg (n=6, cohort 2) IMP321 injections ≥1 checkpoint inhibitor (CPI), and if BRAF mutation ≥1 BRAF or MEK + s.c. (every 2 weeks for 6 months) from cycle 5 onwards. Pa- BRAF inhibitor, were treated with GV (1.9 mg/kg q3w until progression/ tients without progressive disease (PD) at the end of the 6 intolerance) in combination with V (3.0 mg/kg) on day 1 of weeks 1, 3, months combination therapy continue on pembrolizumab 9, 15, 21, and 27. Retrospective analysis on pre-study tumors and skin monotherapy. biopsies include gpNMB expression and infiltrating lymphocytes by im- Results munohistochemistry plus gene expression profiling. Primary objective In total, 12 patients (11 male, 1 female) with a median age of 62 is to evaluate objective response rate (ORR) (RECIST 1.1). years (range 48-85) were enrolled between Apr 2016 and Feb Results 2017. Four patients completed the 6 months combination treat- Thirty-four patients enrolled: median age of 61 years; 59% male; 18% V600 ment. One patient discontinued due to serious adverse event BRAF mutated; 59% ≥3 lines prior therapy; 76% prior anti-CTLA-4; (SAE), unrelated to both study drugs. Two patients withdrew con- 100% prior anti-PD-1/PD-L1 inhibitor; 97% Stage IV; 71% M1c. Of 30 sent and five discontinued prematurely due to PD. No dose limit- response evaluable patients, emerging tumor response data shows 2 ing toxicities for the combination have been reported. No SAE confirmed partial responses (PR) (ORR = 7%, CI: 0.8, 22.1), and 1 single were found related to IMP321. All patients were evaluable accord- time point PR before patient withdrew consent. 48% patients had ing to irRC and 6 showed a reduction of ~50 % after IMP321 ini- tumor shrinkage. Median PFS = 2.6 months and median OS = 4.4 tiation. This includes one patient with a confirmed complete months; 3 patients remain on treatment and 18 patients in survival fol- response while having PD on pembrolizumab monotherapy be- low up. Most common treatment related toxicities include alopecia, fore study start. rash, fatigue, neuropathy, nausea, and vomiting. Tumor cells in 84% of Conclusions patients with samples (n=25) tested to date are 100% gpNMB+. One (1) and 6 mg IMP321 given s.c. every 2 weeks in combination Conclusions with pembrolizumab are safe and testing of the highest dose (30 The GV/V combination was well tolerated without evidence of additive mg) is underway. The 6 late tumor responses seen after the combin- toxicity. There was no apparent enhanced clinical benefit of GV/V over ation was started may point out to the benefit of adding a systemic GV alone in this patient population, perhaps because immune check- APC activator to an ICI. point molecules remained unblocked and/or of a dearth of antigen Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 136 of 244 presenting cells in tumors. Correlative biomarker analyses are ongoing P262 and will be presented. For further insights into the synergy of ADC and Phosphatidylserine targeting antibody in combination with tumor immunotherapy, a cohort evaluating GV with anti-PD-1 in CPI-refractory radiation and immune checkpoint blockade promotes anti-tumor melanoma is enrolling and a cohort investigating GV with the DC activity in mouse B16 melanoma 1 1 1 growth factor FLT3L (CDX-301) is planned. Sadna Budhu ,RachelGiese ,Olivier De Henau ,Roberta 1 1 1 Zappasodi , Luis Felipe Campesato , Aditi Gupta , Christopher References Barker , 2 1 1 1. Muller P, et al.Cancer Immunol Res. 2014; 2:741-755. Bruce Freimark ,Jedd D. Wolchok , Taha Merghoub 1 2 2. Muller P, et al. Sci Transl Med. 2015; 7:315rfa188. Memorial Sloan Kettering Cancer Center, New York, NY, USA; Peregrine Pharmaceuticals, Inc., Tustin, CA, USA Correspondence: Jedd D. Wolchok (budhus@mskcc.org); Taha P261 Merghoub Molecular signatures of combination immunotherapy of prostate Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P262 cancer using a Listeria-based PSA vaccine and radiation 1 1 1 1 Emily Bongiorno , Trevor Baybutt , Carla Portocarrero , Adam Snook , Background 1 2 1 Adam Dicker , Sandra Hayes , Ulrich Rodeck Phosphatidylserine (PS) is a phospholipid that is exposed on sur- 1 2 Thomas Jefferson University, Philadelphia, PA, USA; Advaxis face of apoptotic cells, viable tumor cells, tumor endothelium and Immunotherapeutics, Inc., Princeton, NJ, USA activated immune cells. It has been shown to promote immuno- Correspondence: Emily Bongiorno (emily.bongiorno@jefferson.edu) suppressive signals in the tumor microenvironment. In a mouse Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P261 B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than Background either agent alone. This combination was shown to enhance CD4 Radiotherapy (RT) has the potential to amplify immune responses trig- + and CD8+ T cell infiltration and activation in the tumors of gered by tumor vaccines, including ADXS-PSA, a live-attenuated Listeria treated animals. Radiation therapy (RT) is an effective focal treat- monocytogenes (Lm)-based vector expressing human PSA. Earlier obser- ment of primary solid tumors, but is less effective in treating vations suggest that the two treatment modalities cooperatively induce metastatic solidtumorsasa monotherapy. RT inducesimmuno- regression of syngeneic mouse prostate cancer cells expressing human genic tumor cell death and enhances tumor-specific T cell infiltra- PSA (TPSA23), though immune correlates of efficacy and tumor recur- tion in treated tumors. The abscopal effect, a phenomenon in rence are poorly understood. which tumor regression occurs outside the site of RT, has been Methods observed in both preclinical and clinical trials when RT is com- We compared efficacy of different sequencing regimens of combin- bined with immunotherapy. ation RT/vaccine treatments on TPSA23 tumor growth in syngeneic Methods mice. Using the optimal sequencing protocol, tumors were collected Mice were injected intradermally on the hind limb with 10 B16F10 post-implantation to assess immune infiltrate and function during initial melanoma cells. 7-10 days after implantation, tumors were treated lo- tumor regression (day 20) and upon resumption of tumor growth (day cally with 15 Gy RT. 1 day after RT, mice were given antibodies to PS 38). Correlates of treatment efficacy were determined by transcriptome (mch1N11) and PD-1 (RMP 1-10) intraperitoneally every 3 days. analysis, phenotypic analyses of infiltrates and TCR sequencing. Tumor surface area and overall survival of mice were used to deter- Results mine efficacy of the combinations. For FACS analysis, tissues were We confirmed that combination RT/ADXS-PSA is superior to single mo- collected between 1-10 days after RT. dality treatments. Concurrent administration of RT/vaccine was the most Results effective treatment schedule and was associated with enhanced T cell ac- In this study, we show that irradiation of B16 melanoma causes tivation and robust IFNg signatures in the tumor microenvironment. This an increase in PS expression on the surface of viable tumor and was reflected in increased intratumoral CD4 and CD8 T cell infiltration in immune infiltrates. We subsequently examined the effects of mice receiving RT/vaccine. TCRb chain sequencing revealed elevated and combiningRT withanantibody thattargetsPS andanti-PD-1. We sustained T cell diversity in tumor tissues of RT/vaccine-treated mice, found that treatment with mch1N11 synergizes with RT to im- when compared with mice receiving single modality treatments. In these prove anti-tumor activity and overall survival in tumor bearing residual tumors resident and/or memory T cell phenotypic markers were mice. In addition, the triple combination of mch1N11, RT and increased. Transcriptome analysis of recurring tumors further revealed in- anti-PD-1 treatment displayed even greater anti-tumor and sur- duction of PD-L1 as a function of treatment. Targeting of the PD1/PD-L1 vival benefit. Analysis of the immune response in the tumors of axis via a PD1 blocking antibody administered in addition to radiation treated animals revealed an increase in M1-like macrophages in and ADXS-PSA (triple combination) further amplified tumor growth inhib- the tumors after treatment with RT and mch1N11. In addition, ition in mice receiving dual RT/vaccine therapy. analysis of the systemic immune responses revealed an increase Conclusions in antigen-specific CD8 T cell infiltration in the tumors as well as Combining RT with the ADXS-PSA vaccine leads to effective tumor increased activation, effector function and differentiation in the growth inhibition and induces robust, persistent antitumor immunity triple combination therapy. within the tumor environment. Transcriptome analysis during treat- Conclusions ment revealed increased PD1 expression as a potential resistance This finding highlights the potential of combining these agents to mechanism and a PD1 blocking antibody provided further thera- improve outcome in patients with advanced-stage melanoma and peutic benefit. These results support the rationale for combining Lis- may inform the design of future clinical trials with PS targeting in teria-based vaccines with radiation in the clinic. multiple cancers. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 137 of 244 P263 Background Preclinical development of a Vaccine-Based Immunotherapy Sunitinib is a receptor tyrosine kinase (RTK) inhibitor approved as a Regimen (VBIR) that breaks immunological tolerance and induces monotherapy for the treatment of advanced renal cell carcinoma high titer and long lived T-cell responses to a tumor-associated (RCC), gastrointestinal stromal tumors and advanced pancreatic neu- self-antigen roendocrine tumors. It inhibits angiogenesis and targets RTKs 1 2 1 2 3 Helen Cho , Risini Weeratna , Joe Binder , Bassel Akache , Rajeev Nepal , (PDGFR, VEGFR, KIT, FLT3, and RET) involved in tumor cell growth. 4 1 1 1 Paul Cockle , Marianne Martinic , Michael Dermyer , Stanley Dai , Sunitinib also has been shown, in preclinical studies and patients, to 1 4 James Merson , Karin Jooss have immunomodulatory properties that reduce myeloid derived 1 2 Pfizer Inc., San Diego, CA, USA; National Research Council of Canada, suppressor cells (MDSCs) and restore a Th1 cytokine profile. The ap- 3 4 Ottawa, ON, Canada; Pfizer Canada Inc., Kirkland, QC, Canada; Gritstone proved dosing regimens for RCC have a safety profile that is overall Oncology Inc., Emeryville, CA, USA manageable with the most commonly reported sunitinib-related Correspondence: Helen Cho (Helen.k.cho@pfizer.com) grade 3 adverse events including hypertension, fatigue, diarrhea and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P263 hand-foot syndrome. Although the tolerability of sunitinib as mono- therapy was acceptable in advanced stage, metastatic cancer pa- Background tients, the drug’s side effect profile could potentially pose challenges A successful therapeutic cancer vaccine will activate the immune for use in patients with earlier stage cancer and/or when combined system to rapidly induce potent, balanced and durable CD8 and CD4 with other oncology therapies. Thus, lowering the dose of sunitinib T-cell responses to selected tumor antigens that can reduce tumor might improve the safety of combinations, including combinations growth, lead to tumor regression or prevent/delay the onset of new with cancer vaccines, thus enabling treatment of cancers of all lesions. stages. Methods Methods The magnitude and quality of T-cell responses against self (rhesus The anti-tumor efficacy of lowered doses of sunitinib as a monother- Prostate Specific Membrane Antigen; rhPSMA) and non-self (human apy, and in combination with a cancer vaccine, was investigated in PSMA; hPSMA) antigens delivered by various vaccine platforms in non- immune-competent mouse tumor models. To understand the mechan- human primates (NHPs) given with and without a check point inhibitor, ism of such efficacy, the impact of the treatments on MDSC profiles in anti-CTLA4 monoclonal antibody (tremelimumab; anti-CTLA4), were the periphery and tumors, T cell IFNγ production, and antigen-specific evaluated. immune responses was evaluated. Additionally, adoptive immune cell Results transfer experiments were conducted to monitor survival of MDSCs Assessment of T-cell responses against self (rhPSMA) and non-self and T cells in mice treated with sunitinib. (hPSMA) antigens delivered by various vaccine platforms in NHPs, Results demonstrated that the AdV vector was the most potent delivery plat- At doses below the maximum biologic efficacy dose in mice (< 40 mg/ form for breaking of immune tolerance to a self-antigen. kg q.d.), sunitinib selectively and significantly reduced the survival and Many humans have immunity to adenovirus that could blunt the ability frequency of MDSCs in the periphery and tumors, which resulted in in- of an adenovirus vaccine to effectively prime an immune response. This creased IFNg production by T cells. Importantly, when low dose suniti- limitation can be overcome by utilizing an AdV of a different serotype nib was given concurrently with a rat HER2 (rHER2)-based heterologous for which humans do not have pre-existing immunity. However, even prime-boost vaccine, it significantly reduced the growth of subcutane- with a low/no seroprevalent AdV, an AdV boost vaccination following ous tumors and prolonged survival of tumor-bearing rHER2 transgenic an AdV prime vaccination can only be effective when the neutralizing mice compared to mice receiving only monotherapies. Addition of an immunity to the vector wanes in between the vaccinations, making the anti-CTLA4 monoclonal antibody (anti-CTLA4) significantly enhanced regimen less suitable for patients with high tumor burden or fast pro- the vaccine-induced immune response and prolonged survival. gressive disease. Therefore, plasmid DNA boost vaccinations delivered Conclusions intramuscularly by electroporation were evaluated for durable expan- The combination of the vaccine, sunitinib, and anti-CTLA4 further sion of AdV primed T-cell responses. Administration of anti-CTLA4 deliv- increased efficacy and survival. These results indicate that low dose ered subcutaneously to achieve high local concentrations in the sunitinib, alone or together with either a cancer vaccine or a cancer vaccine draining lymph nodes, given concurrently with the AdV prime vaccine plus anti-CTLA4, maintains its immune modulatory properties and DNA boost vaccinations was shown to induce and expand durable and may be a valuable component of a safe and efficacious vaccine- + + + and polyfunctional (IFNγ ,TNFα and/or IL-2 ) T-cell responses to a based immunotherapy regimen. tumor-associated self-antigen in NHPs. Conclusions P265 In summary, we have developed VBIR that consists of a heterologous Optimizing targeted therapy and immune checkpoint blockade prime-boost vaccine approach given in combination with local deliv- therapy in Kras mutant lung cancer ery of tremelimumab to maximize vaccine potency while minimizing 1 2 1 1 1 Hyejin Choi , Jiehui Deng , Tarik Silk , Ann Powers , Jonathan Boiarsky , the negative impact of neutralizing antibodies to the AdV vector. 3 2 1 Taha Merghoub , Kwok-Kin Wong , Jedd Wolchok These results have encouraged clinical development of this unique 1 2 MSKCC, New York, NY, USA; New York University Langone Medical immunotherapeutic regimen. Center, New York, NY, USA; Medicine, New York, NY, USA Correspondence: Taha Merghoub (choih3@mskcc.org); Kwok-Kin P264 Wong; Jedd Wolchok Immune modulation by low dose sunitinib combined with a cancer Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P265 vaccine based immunotherapeutic regimen provides therapeutic benefit to tumor bearing mice Background 1 1 1 1 1 Helen Cho , Cindy Li , Antonio Boccia , Steve Burgess , Terri Harder , KRAS is the most commonly identified driver oncogene in lung cancer. 2 1 1 1 1 Paul Cockle , Jim Eyles , Marianne Martinic , Bryan Clay , Kam Chan , However, to date there is no effective therapy available for KRAS mu- 1 1 1 3 Stanley Dai , Michael Dermyer , Joe Binder , Steve Kurzyniec , tant lung cancers. To identify the most effective therapy, we studied 1 4 1 1 Terrina Bayone , Joan Guo , Robert Hollingsworth , James Merson , the impact of Kras signaling targeted therapy (MEK inhibition) on the Karin Jooss immune microenvironment, in order to formulate a combinatorial 1 2 Pfizer Inc., San Diego, CA, USA; Gritstone Oncology Inc., Emeryville, CA, therapy using targeted therapy and immunotherapy, with a goal of 3 4 USA; Shimadzu Scientific Instruments, San Diego, CA, USA; Moderna optimally enhancing tumor apoptosis and promoting long-term im- Therapeutics, Cambridge, MA, USA mune response simultaneously. MEK signaling is a downstream of Kras Correspondence: Helen Cho (Helen.k.cho@pfizer.com) signaling pathway and critical for tumor growth and T cell activation. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P264 Conventionally, MEK inhibitor is treated daily/continuously. However, to Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 138 of 244 enhance tumor apoptosis and promote T cell activation on MEK Results inhibition, we hypothesized intermittent administration of MEK ALT-803 significantly promoted exPBNK in-vitro proliferation by in- inhibitor will confer T cells temporal release from MEK signaling creasing the phosphorylation of Akt, Stat3/5 and p38 MAPK. ALT-803 inhibition allowing T cells activated, while tumor growth is sup- increased NK activating receptors expression: NKG2D, NKp30, NKp44, pressed and immunotherapy can be combined based on T cell acti- and NKp46. vation/inhibitory markers change on this treatment schedule. ALT-803 significantly enhanced exPBNK mediated ADCC with dinu- Methods tuximab in a E:T dependent manner (p<0.001) against OS, NB and We have treated T cells from Kras lung cancer bearing mice or tumor GBM cells (Fig. 1). ALT-803 significantly enhanced IFN-γ (p<0.001) bearing mice with Selumetinib or Trametinib in a pulsatile/intermit- and perforin (p<0.001) release from exPBNK when it was combined tent or continuous way, then analyzed T cell phenotype changes, with dinutuximab against OS, NB and GBM cells compared to tumor progression, and survival. exPBNK, ALT-803+exPBNK, or dinutuximab+exPBNK. Results Conclusions Ex vivo T cell study showed that pulsatile treatment of MEK inhibi- ALT-803 significantly enhanced exPBNK ADCC and IFN-γ and perforin tor, Selumetinib and Trametinib, showed highly increased CTLA4 release with dinutuximab against GD2 OS, NB and GBM cells. In vivo expression and mild increase of PD1 in CD8+ T cells and CD4 studies using NOD/SCID human solid tumor xenografts are under +Foxp3- T cells, compared to continuously treated group which is a investigation. standard regimen. This result was confirmed in intermittently treated KrasG12D/+; p53-/- transplantable mouse model and GEMM References as well. Moreover, CD8+ T cells from pulsatile group showed in- 1. Yu AL, Gilman AL, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, crease of Ki67 and 4-1BB expression, suggesting that CD8+ T cells and isotretinoin for neuroblastoma. The New England journal of medi- are more activated in pulsatile group. In vivo tumor study using cine 2010; 363:1324-34. Kras mutant lung cancer animal model showed intermittent treat- 2. Xu W, Jones M, et al. Efficacy and mechanism-of-action of a novel super- ment suppressed tumor growth better than continuous treatment. agonist interleukin-15: interleukin-15 receptor alphaSu/Fc fusion complex Better response, more activated phenotype of CD8+ T cells includ- in syngeneic murine models of multiple myeloma. Cancer Res 2013; ing increased CTLA4 expression in intermittent group lead us to 73:3075-86. test combination of intermittent MEK inhibitor treatment with anti- 3. Chu Y, Hochberg J, et al. Targeting CD20+ Aggressive B-cell Non-Hodgkin CTLA4 antibody to maximize anti-tumor T cell activity and it is Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells currently under investigation. In Vitro and in NSG Mice. Cancer Immunol Res 2015; 3:333-44. Conclusions 4. Denman CJ, Senyukov VV, et al. Membrane-bound IL-21 promotes sus- In summary, we found that pulsatile/intermittent treatment with MEK tained ex vivo proliferation of human natural killer cells. PLoS One 2012; inhibitor showed better response and more activated phenotype of T 7:e3026. cells including increased CTLA4 expression compared to continuous treatment. This study suggests that optimized intermittent schedule of MEK inhibitor treatment is essential to maximize T cell mediated anti-tumor activity in combination with anti-CTLA4 therapy and this will benefit Kras mutant lung cancer patients. P266 Significant enhancement of expanded natural killer cells against GD2 pediatric solid tumors (ST) in combination with ALT-803 (IL-15 superagonist) and dinutuximab 1 1 1 2 Yaya Chu , Nang Kham Su , Jeremy Rosenblum , Hing C. Wong , 3 1 Dean A. Lee , Mitchell S Cairo 1 2 New York Medical College, Valhalla, NY, USA; Altor Bioscience, Miramar, FL, USA; Nationwide Children’s Hospital, Columbus, OH, USA Correspondence: Yaya Chu (yaya_chu@nymc.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P266 Background Children with recurrent and/or metastatic osteosarcoma (OS), neuro- blastoma (NB) and glioblastoma (GBM) have a dismal even-free survival Fig. 1 (abstract P266). See text for description (EFS) (<25%). Dinutuximab is an anti-GD2 monoclonal antibody that has significantly increased EFS in children with GD2 neuroblastoma [1]. ALT-803 is a superagonist of an IL-15 variant bound to an IL- 15RαSu-Fc fusion with enhanced biological activity [2]. Our group has P267 successfully expanded peripheral blood Natural Killer cells (exPBNK) Withdrawn with irradiated feeder cells [3]. In this research, we aim to determine if the combination of ALT-803 P268 and dinutuximab significantly enhances exPBNK cell in vitro cytotox- Targeting the tumor microenvironment with first-in-class icity against GD2 OS, NB and GBM. Semaphorin4D MAb for combination immunotherapy 1 1 1 1 Methods Elizabeth Evans , Holm Bussler , Crystal Mallow , Christine Reilly , 1 1 1 1 PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL Sebold Torno , Maria Scrivens , Cathie Foster , Alan Howell , Leslie 1 1 1 2 3 cells [4]. ExPBNK cells were isolated using Miltenyi NK cell isolation Balch , John E. Leonard , Terrence L. Fisher , David Jenkins , Clint Allen , 3 3 4 5 1 kits as we previously described. ALT-803 was generously provided Paul Clavijo , Siwen Hu-Lieskovan , Antoni Ribas , Ernest Smith , by Altor BioScience Corporation. NK proliferation, NK receptors ex- Maurice Zauderer 1 2 3 pression and cytotoxicity were assessed as we previously described Vaccinex, Rochester, NY, USA; Tesaro, Waltham, MA, USA; NIH/NIDCD, 4 5 [3]. Dinutuximab (generously provided by United Therapeutics) was Bethesda, MD, USA; UCLA, Los Angeles, CA, USA; Vaccinex, Los used for antibody-dependent cellular cytotoxicity (ADCC) assays. IFN- Angeles, CA, USA γ and perforin levels were evaluated by ELISA assays. GD2 OS, NB Correspondence: Elizabeth Evans (eevans@vaccinex.com) and GBM cell lines were used as target cells. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P268 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 139 of 244 Background Background Mechanistic findings in preclinical studies demonstrate that antibody The clinical promise of cancer immunotherapy relies on the immune blockade of Semaphorin 4D (SEMA4D, CD100) reduces expansion of system recognizing and eliminating tumor cells identified as non-self. MDSC and shifts the balance of immune cells within the TME to fa- However, solid malignancies evade host immune surveillance by mul- cilitate tumor rejection. Efficacy is further enhanced when combined tiple mechanisms, including epigenetic deregulation and promoting a with various immunotherapies. tumor microenvironment (TME) that suppresses infiltration and func- Methods tion of immune effector cells. The TME hampers T and NK cell matur- Anti-SEMA4D antibodies were evaluated in combination with other ation, recruitment, and function, ultimately causing their functional immunotherapies in preclinical models. Anti-tumor activity and exhaustion via numerous immunosuppressive pathways, including up- immune response was characterized by immunohistochemistry, flow regulation of immune checkpoints such as PD-L1. Epigenetic silencing cytometry, functional assays, and cytokine, chemokine and gene ex- of genes involved in antigen processing and tumor immune recogni- pression analysis. These data support clinical combination trials of tion has been associated with worse prognosis in a wide spectrum of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with im- malignancies. Hence, there is an unmet clinical need to develop ef- mune checkpoint inhibition. fective therapeutic strategies that can reprogram the TME to re- Results store tumor immune recognition and reverse immune evasion. We SEMA4D restricts migration of monocytes and promotes expansion recently demonstrated that entinostat, a class I histone deacetylase of suppressive myeloid cells in vitro. Strong expression of SEMA4D at (HDAC) inhibitor reverses carcinoma immune escape to T cell- the invasive margins of growing tumors in vivo restricts the infiltra- mediated lysis. tion and modulates polarization of leukocytes in the TME. Antibody Methods blockade of SEMA4D in preclinical models facilitated recruitment of We hypothesize that the immune-mediated tumor elimination pro- activated DCs and T lymphocytes with concurrent reduction in M2 moted by the IL-15/IL-15Ra superagonist ALT803 in combination with macrophage and Treg within TME [1]. MDSCs were significantly re- PD-L1 checkpoint blockade or a therapeutic adenoviral vaccine tar- duced in tumor and blood following treatment and new data charac- geting CEA (Ad-CEA) will be augmented by the epigenetic repro- terizing MDSC function will be described. This significant shift in the graming of the TME induced by entinostat. immune contexture is associated with durable tumor rejection and Results immunologic memory in murine colon, breast, HNSCC, and melan- In preclinical studies, ALT803 has been shown to exhibit potent anti- oma models. New translational data characterizing expression of tumor activity in multiple murine models of cancer through the ex- SEMA4D and its receptors in human tumors will be shown. Import- pansion of NK and CD8+ T cells with high effector function. Here, we antly, anti-SEMA4D treatment can further enhance activity of im- demonstrate that entinostat modulates the cell-surface phenotype of munotherapies and chemotherapy. For example, combinations with murine colon and breast carcinoma cells to become more amenable immune checkpoint inhibitor anti-LAG3 or anti-CTLA-4 cause to immune-mediated elimination. In the MC38-CEA murine model of complete tumor regression in 90% or 100% of mice, as compared to colon carcinoma, proper scheduling of entinostat administration sig- ~20% with monotherapy (p<0.01). New preclinical data include syn- nificantly augmented the antitumor activity promoted by ALT803 ergistic activity of combinations of anti-SEMA4D with anti-LAG3 and plus Ad-CEA, resulting in increased survival. Further, in the 4T1 mur- additional studies combining with epigenetic modulators, including ine model of triple-negative breast cancer, the combination of enti- treatment of established tumors. nostat with ALT803 and a monoclonal antibody (mAb) targeting PD- Conclusions L1 significantly reduced primary tumor weight relative to ALT803 SEMA4D blockade represents a novel mechanism to promote func- plus anti-PD-L1 therapy. Treatment with the triple therapy in the tional immune infiltration into the tumor and enhance immunother- neoadjuvant setting resulted in significant reduction of the number apy. Treatment with VX15/2503 was well tolerated in a Phase I trial of 4T1 tumor-forming cells in the lung, with over 85% of animals in patients with advanced refractory solid tumors [2]. Plans for sev- cured. Current studies aim to elucidate the immune mechanisms as- eral clinical trials will be presented, including a Phase 1b/2 of com- sociated with the antitumor effects observed with these therapeutic bination therapy with avelumab in immunotherapy naïve NSCLC, and interventions. combination with anti-PD-1 or Ipilimumab in various indications. A Conclusions neoadjuvant trial of VX15/2503 with anti-PD-1 in patients with meta- Overall, these studies examine the rationale for combining entinostat static colorectal and pancreatic cancers will be described, as well as with multivalent immunotherapy combinations, including cytokines, introduction of a Phase 1/2 trial of VX15/2503 in pediatric and osteo- mAbs targeting PD-L1, and therapeutic cancer vaccines. sarcoma patients. Trial Registration P270 NCT01313065. Simultaneous PD-1 blockade is detrimental to the anti-tumor effects mediated by the agonist OX40 antibody References 1 2 3 1 Seema Gupta , Rajeev Shrimali , Shamim Ahmad , Vivek Verma , 1. Evans, EE et al. Cancer Immunol Res. 2015 Jun;3(6):689-701. 1 1 1 4 Peng Zeng , Sudha Ananth , Pankaj Gaur , Rachel Gittelman , 2. Patnaik A et al. Clin Cancer Res. 2016 Feb 15;22(4):827-36. 4 4 4 5 Erik Yusko , Catherine Sanders , Harlan Robins , Scott Hammond , 1 3 1 John Janik , Mikayel Mkrtichyan , Samir Khleif 1 2 P269 Georgia Cancer Center, Augusta University, Augusta, GA, USA; Georgia Epigenetic reprogramming of the tumor microenvironment Cancer Center, Augusta University, Dallas, TX, USA; Georgia Cancer increases tumor sensitivity to multivalent immunotherapy Center, Augusta University, South San Francisco, CA, USA; Adaptive combinations with an IL-15 superagonist plus vaccine or immune Biotechnologies, Seattle, WA, USA; MedImmune LLC, Gaithersburg, MD, checkpoint blockade USA 1 1 1 2 Kristin C. Hicks , Karin M. Knudson , Anthony S. Malamas , Frank R. Jones , Correspondence: Samir Khleif (segupta@augusta.edu) 3 4 5 1 Peter Ordentlich , Shahrooz Rabizadeh ,HingC.Wong , James W. Hodge , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P270 1 1 Jeffrey Schlom , Sofia R. Gameiro National Cancer Institute, National Institutes of Health, Bethesda, MD, Background 2 3 USA; Etubics Corporation, Seattle, WA, USA; Syndax Pharmaceuticals, The checkpoint inhibitor antibodies (Abs) have improved the anti- 4 5 Inc., Waltham, MA, USA; NantCell, LLC, Culver City, CA, USA; Altor tumor response although in a limited number of patients [1]. The BioScience Corporation, Miramar, FL, USA combination of these Abs has enhanced the efficacy but with in- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P269 creased adverse events [1]. Since immune-stimulatory agonist Abs Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 140 of 244 like anti-OX40 significantly increase the immune response [2], their Background combination with the anti-PD-1 Abs is being tested in clinical trials CSF1 regulates tumor-associated macrophages and myeloid-derived [3] with at least one clinical trial demonstrating lack of benefit [4] suppressor cells, which are critical tumor microenvironment modula- and the rest are not yet reported. Moreover, the potential immune tors of immune response. Combining a programmed death 1 (PD-1) outcome of such a combination are currently lacking. Therefore, pur- inhibitor with a CSF1R inhibitor in preclinical models shows en- pose of this study was to determine the therapeutic and immune hanced antitumor activity. ARRY-382 is a highly selective oral inhibi- outcome of combining anti-PD-1 with anti-OX40 in an immune- tor of CSF1R. Studies of ARRY-382 monotherapy identified the primed environment. maximum tolerated dose (MTD) as 400 mg once daily (QD), with bio- Methods logic activity at doses ≥200 mg QD. This 3-part, phase 1b/2 study Effects of adding PD-1 blockade to anti-OX40/vaccine treatment on evaluates ARRY-382 in combination with pembrolizumab, a human- tumor growth and survival were evaluated in a TC-1 tumor mouse ized monoclonal antibody targeting PD-1. model using different treatment schedules and immune responses Methods were assessed. In vitro mechanistic studies were carried out in pMel- Phase 1b (part A) determined the MTD and recommended phase 2 1 CD8 T-cells. dose (RP2D) of ARRY-382 plus pembrolizumab in patients with se- Results lected advanced solid tumors. Patients were enrolled in 3 successive Anti-OX40 treatment resulted in significant enhancement of antigen- cohorts of ARRY-382 at doses of 200, 400, or 300 mg QD plus pem- specific CD8 T-cells tumor-infiltration leading to strong anti-tumor brolizumab 2 mg/kg every 3 weeks (Q3W). The primary endpoint was response and prolonged survival of mice. Interestingly, we found that the incidence of dose-limiting toxicities (DLTs). Secondary endpoints simultaneous addition of anti-PD-1 to anti-OX40 completely abro- included safety, pharmacokinetics of ARRY-382, and objective re- gated these effects. Despite an increase in IFNγ-producing E7-specifc sponse rate. CD8 T-cells in the spleens of mice treated with the combination, Results these cells underwent significant apoptosis in both the periphery Twenty patients enrolled in part A; 19 were treated: 200 mg QD, n=6; and the tumor. Consistent with increased apoptosis, immunoSequen- 400 mg QD, n=7; 300 mg QD, n=6. Median age was 59 years; tumor cing analysis of the tumor and spleen T-cell population showed re- types were pancreatic (n=6), colorectal (n=5), ovarian (n=3), gastric duction in T-cell fraction and clonality following the addition of anti- and melanoma (n=2 each), and triple-negative breast (n=1). The PD-1. We further showed that delay in anti-PD-1 addition resulted in mean number of prior therapies was 2.9 (range, 1–5). DLTs of grade no significant change in T-cell apoptosis, however, it did not add to 3 increased aspartate aminotransferase (AST)/alanine aminotransfer- the anti-tumor response of the anti-OX40 treatment. ase/bilirubin and grade 3 increased creatine phosphokinase (n=1 Conclusions each, 400-mg dose) and grade 3 pancreatitis (n=1, 300-mg dose) These results indicate that anti-PD-1 added at the initiation of ther- were observed. The most common (>2 patients across doses) grade apy exhibits a detrimental effect on the positive outcome of anti- 3/4 adverse events (AEs) by dose cohort (200, 300, and 400 mg) were OX40. This may provide an important insight into why some of the increased AST (1, 1, and 2 patients), increased lipase (0, 3, 0), and immune combination clinical trials are not producing the intended rash (1, 1, 1), respectively. One patient in the 200-mg cohort discon- outcome, demonstrating the need to rigorously test the combination tinued treatment because of an AE of pneumonitis in cycle 5. ARRY- partners and sequencing before employing them in the clinic. 382 pharmacokinetics was unaffected by pembrolizumab. Preliminary efficacy data will be presented. References Conclusions 1. Page DB, et al. Immune modulation in cancer with antibodies. Annu Rev ARRY-382 plus pembrolizumab has a manageable safety profile. The Med. 2014;65:185-202. RP2D of ARRY-382 plus pembrolizumab 2 mg/kg Q3W is 300 mg QD. 2. Linch SN et al: OX40 Agonists and Combination Immunotherapy: Putting This combination is currently being evaluated in the study in patients the Pedal to the Metal. Front Oncol. 2015;5:34. with melanoma and non–small-cell lung cancer. 3. “ClinicalTrials.gov.”April 15, 2017 from https://clinicaltrials.gov/ct2/ Trial Registration results?term=ox40&amp;Search=Search">https://clinicaltrials.gov/ct2/ ClinicalTrials.gov: NCT02880371. results?term=ox40&amp;Search=Search. 4. Infante, et al. A phase Ib dose escalation study of the OX40 agonist MOXR0916 and the PD-L1 inhibitor atezolizumab in patients with ad- P272 vanced solid tumors. Presentation in ASCO Annual Meeting, 2016. The anti-tumor effect of radiation therapy is enhanced with the addition of TTI-621 (SIRPαFc), an immune checkpoint inhibitor blocking the CD47 “do not eat” signal P271 Lei Cui, Hui Chen, Alison O'Connor, Debbie Jin, Jeffrey Winston, Phase 1b/2 dose-escalation study of ARRY-382, an oral inhibitor of Robert Uger, Lisa Johnson colony-stimulating factor-1 receptor (CSF1R), in combination with Therapeutics Inc., Mississauga, ON, Canada pembrolizumab for treatment of patients with advanced solid Correspondence: Lisa Johnson (lisa@trilliumtherapeutics.com) tumors Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P272 1 2 3 4 Wael A. Harb , Melissa L. Johnson , Jonathan W. Goldman , Amy Weise , 5 6 7 8 Justin A. Call , Arkadiusz Z. Dudek , Rene Gonzalez , C. Lance Cowey , Background 9 9 9 10 Sybil Zildjian , Kati Maharry , Ashwin Gollerkeri , Justin F. Gainor CD47 is an immune checkpoint that binds to signal regulatory protein 1 2 Horizon Oncology Research, Inc., Lafayette, IN, USA; Sarah Cannon alpha (SIRPα) and delivers a “do not eat” signal to suppress macro- Research Institute, Nashville, TN, USA; UCLA Medical Center, Santa phage phagocytosis. Tumor cells frequently overexpress CD47 to evade 4 5 Monica, CA, USA; Karmanos Cancer Institute, Detroit, MI, USA; Utah macrophage-mediated destruction. TTI-621 (SIRPαFc) is an immune Cancer Specialists, Salt Lake City, UT, USA; Regions Cancer Care Center, checkpoint inhibitor consisting of the CD47 binding domain of human St. Paul, MN, USA; University of Colorado Cancer Center, Aurora, CO, SIRPα linked to the Fc region of human IgG1 designed to both: 1) block 8 9 USA; Baylor Health Care System, Dallas, TX, USA; Array BioPharma Inc., the CD47 “do not eat” signal, and 2) engage macrophage Fcγ receptors Boulder, CO, USA; Massachusetts General Hospital, Boston, MA, USA with IgG1 Fc to enhance phagocytosis and antitumor activity. Correspondence: Wael A. Harb (drharb@me.com) Radiation therapy (RT), a primary mode of cancer treatment, induces Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P271 immunogenic cell death, and is associated with release of tumor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 141 of 244 antigens, induction of pro-inflammatory cytokines, and enhanced mi- first line therapy. Until Jul 30, 2017, 29 pts had evaluable outcomes. gration and infiltration of immune cells, including macrophages, to The ORR (CR+PR) is 48.2% (14/29), DCR (CR+PR+SD) was 69.0% (20/ tumor sites. Thus, the combination of RT and TTI-621 may improve 29), and the median PFS: 184 days (32-332). The common toxicities the therapeutic effect over either treatment modality alone. Herein, were: diarrhea 40%; skin rash 37%; mucositis 29%; hand-foot-skin re- we report the efficacy of the combination of RT and the CD47- action 23%; weight loss 17%; and elevated AST/ALT 9%. Grade >= 3 blocking agent TTI-621 in xenograft tumor models. toxicities happened in 8.6% patients. There was no treatment related Methods pneumonitis in the cohort. The in vivo efficacy of RT, TTI-621, and RT+TTI-621 was evaluated in B Conclusions cell lymphoma (SU-DHL-6) and solid tumor xenografts, including the The addition of afatinib with pembrolizumab showed good efficacy radio-insensitive A549 lung adenocarcinoma. TTI-621 (10 mg/kg) or ve- (ORR: 48.2%, DCR: 69.0%, PFS: 6.0 months) and tolerable toxicities. hicle were administered intratumorally 30 min prior to RT, 3 times per Further confirmatory prospective trial is indicated. week for 4 weeks. Tumors were locally irradiated using an image- guided small animal irradiator (225 kVp, 13 mA) at a dose of 4-6 Gy for References 3 fractions. Tumor volumes were monitored using standard caliper 1. Lanaya, Hanane, et al. EGFR has a tumor-promoting role in liver macro- measurement. Systemic toxicity of the treatments was evaluated by phages during hepatocellular carcinoma formation. Nature cell biology. body weight change. Tumor-associated macrophages were quantita- 2014; 16.10:972. tively assessed using flow cytometry and immunohistochemistry. 2. Concha-Benavente, Fernando, and Robert L. Ferris. Oncogenic growth Results factor signaling mediating tumor escape from cellular immunity. Current RT+TTI-621 had a more profound effect on tumor control in both Opinion in Immunology. 2017;45:52-59. lymphoma and solid tumor models than RT alone. In SU-DHL-6, 88% 3. Li, Chia-Wei, et al. Glycosylation and stabilization of programmed death (7/8) of mice treated with both TTI-621 and radiation were tumor- ligand-1 suppresses T-cell activity. Nature Communication. 2016;7:12632. free at the end of the study whereas none were tumor-free when treated with RT or TTI-621 alone, although there was tumor growth P274 delay with each individual therapy compared to vehicle. RT+TTI-621 Combination of NKTR-214 and radiotherapy (RT) to reverse anergy led to an increased infiltration of macrophages at the tumor sites in and expand tumor-specific CD8 T Cells SU-DHL-6. Significant tumor control was also observed in A549 tumor 1 2 2 2 Joshua Walker , Melissa Kasiewicz , Michael McNamara , Ian Hilgart-Martiszus , bearing mice treated with RT+TTI-621 compared to either treatment 3 3 2 Ute Hock , Deborah Charych , William Redmond alone. No toxicity was observed for any of the treatments. 1 2 Oregon Health & Science University, Portland, OR, USA; Providence Conclusions Portland Medical Center, Portland, OR, USA; Nektar Therapeutics, San The current study demonstrates the combination of TTI-621 and radi- Francisco, CA, USA ation therapy is superior to either treatment alone and provides sup- Correspondence: William Redmond (melissa.kasiewicz@providence.org) portive evidence for dual modality therapy in a variety of tumors. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P274 P273 Background Pembrolizumab and afatinib for recurrent or metastatic head and We investigated therapeutic and mechanistic synergy between neck squamous cell carcinoma single-dose RT and systemic administration of NKTR-214.The effects Hsiang-Fong Kao, Huai-Cheng Huang, Bin-Chi Liao, Ruey-Long Hong of NKTR-214 with and without RT on anergic, tumor-specific CD8 T National Taiwan University Hospital, Taipei, Taiwan cells were characterized. NKTR-214 is a CD122-biased cytokine agon- Correspondence: Hsiang-Fong Kao (hfkao.tw@gmail.com); Ruey-Long ist conjugated with releasable chains of polyethylene glycol. NKTR- Hong 214 provides sustained signaling through the IL2 receptor pathway Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P273 (IL2Rβγ) to preferentially activate and expand effector CD8 T and NK cells over regulatory T cells. Preclinical models demonstrated NKTR- Background 214 preferentially expands effector CD8 T and NK cells in the tumor Head and neck squamous cell carcinoma (HNSCC) is an important resulting in marked tumor growth suppression as a single-agent and malignancy in Taiwan. Anti-PD-1, including nivolumab or pembrolizu- in combination with checkpoint inhibitors. RT can induce antigen- mab, have shown the efficacies against head and neck squamous cell release and epitope spreading, while NKTR-214 activates and ex- carcinoma. Afatinib, an irreversible EGFR tyrosine kinase inhibitor pands antigen-specific effector populations. We hypothesized the (TKI), had showed its activity against HNSCC. The role of afatinib for combination of systemic NKTR-214 and RT would generate better cancer immunotherapy have not been explored. In animal model, therapeutic responses than either treatment alone. A phase I/II trial is afatinib can suppress the carcinogenesis by inhibiting the function of in progress to evaluate NKTR-214 safety and efficacy in an outpatient macrophage [1]. EGFR targeted therapy can increase the MHC ex- setting as monotherapy and in combination with nivolumab. pression, enhance dendritic cell function, and increase the infiltrating Methods T cell in the tumor [2]. In syngeneic mice cancer models, EGFR TKI NKTR-214 was dosed 0.8 mg/kg alone or together with high-dose RT can suppress the glycosylation of PD-L1 and sensitize the mice to (20 Gy x 1) in multiple murine models, including an established CD8 anti-PD-1 therapy [3]. We hypothesized that adding afatinib with T cell anergy model using Nur77-GFP reporter CD8 T cells. Activation pembrolizumab may improve the treatment efficacy for patients with markers on CD4, CD8 and NK cells in blood, lymph and tumor were recurrent or metastatic HNSCC. evaluated by flow cytometry and gene expression (mRNA) profiling Methods of the irradiated and non-irradiated tumors. In addition, immunohis- Patients with locally advanced or metastatic HNSCC were retrospect- tochemistry was utilized to visualize immune infiltration. ively reviewed in a university hospital in Taiwan. For patients taking Results pembrolizumab, the combination with afatinib will be discussed be- Consistent with prior observations, NKTR-214 induced activation tween the physician and the patient. For patients taking pembrolizu- marker expression by CD4, CD8 T and NK cells in the blood, lymph mab and afatinib (P+A), the medical records were reviewed. nodes and tumor. The combination of RT and NKTR-214 resulted in Results unique effects including increased absolute lymphocyte counts, in- From Nov 2016 to Jul 2017, there were 35 patients taking P+A. The creased expression of CD8 activation markers (CD25, PD1) in the median age was 59 years, and 32/35 pts were men. Oral cavity can- blood and tumor, increased intratumoral NK cells, and a significant cer is the most common type of HNSCC in the cohort (29/35 pts). survival benefit over monotherapy. Evaluation of tumor infiltrating The treatment were: pembrolizumab 200mg: 30pts; 2mg/kg: 5pts; lymphocytes (TIL) indicates combination therapy reverses tumor- afatinib 40mg QD: 34pts; 30mg QD: 1 pt. The maximal cycles of pem- associated T cell anergy and results in a higher frequency of recently brolizumab per patient is four cycles. Sixteen patients took P+A as activated CD8 TIL. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 142 of 244 Conclusions Background The combination of NKTR-214 and radiotherapy is synergistic, provides Recent advances in cancer immunotherapy have led to the advent of significantly better anti-tumor responses and reverses T cell anergy. treatments that boost an anti-tumor response, such as checkpoint blockade and co-stimulatory molecules. However, these approaches are effective for only a subset of patients and target non-specific molecules, thus the large doses required result in off-target toxicities. P275 Here, we have developed a nanoparticle that synergizes checkpoint Harnessing the innate and adaptive immune system to eradicate blockade and T cell costimulation on a single injectable therapeutic. treated and distant untreted solid tumors Nanoparticles are conjugated with antibodies against 4-1BB, Saul Kivimae, Werner Rubas, Rhoneil Pena, Marlene Hennessy, expressed by T cells, and anti-PD-L1, expressed by tumor cells and Yolanda Kirksey, Wildaliz Nieves, Myong Lee, Clive Law, Kavitha Bhasi, target these pathways while also physically linking the effector and Phi Quach, Janet Cetz, John L. Langowski, Christie Fanton, target cell to increase efficacy. These dual targeting particles, termed Jode Zandro Aquino, Zhongxu Ren, Haiying Cai, BoLiang Deng, “immunoswitch particles”, localize treatment to the tumor site and Wen Zhang, Neel K. Anand, Jennifer Riggs-Sauthier, Steve Doberstein, are effective at 10-100X lower doses than systemically injected drug Jonathan Zalevsky and thus have the potential to reduce cost and off-target toxicities. Nektar Therapeutics, San Francisco, CA, USA Methods Correspondence: Saul Kivimae (skivimae@nektar.com); Jonathan Immunoswitch particles were synthesized by conjugating 80nm iron- Zalevsky dextran nanoparticles with agonistic anti-4-1BB and antagonistic anti- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P275 hi PD-L1 antibodies. In vitro efficacy was assessed by co-culturing PD-L1 hi tumor cells with 4-1BB/PD-1 CD8+ T cells and measuring cytotoxicity, Background cytokine secretion, and effector-target cell conjugation. In vivo efficacy NKTR-262 is a novel therapeutic, which delivers sustained intratu- was measured by comparing intratumoral immunoswitch and soluble moral engagement of the TLR 7/8 pathway, promoting an immune antibody treatment in multiple murine tumor models. stimulatory environment and tumor antigen release. When NKTR-262 Results is administered in combination with NKTR-214, a CD122-biased cyto- Immunoswitch particles were demonstrated to increase CD8+ T cell kine agonist currently in clinical trials as a monotherapy and in com- cytokine secretion when co-incubated with T cells and cognate B16 bination with nivolumab, the combined effect of innate immune tumor cells compared to an equivalent amount of soluble antibody stimulation and enhanced antigen presentation with sustained T cell in vitro. Immunoswitch particles also increased effector-target cell activation leads to systemic tumor immunity. conjugation both in vitro (Fig. 1) and in vivo. In vivo, immunoswitch Methods treatment, but not an equivalent amount of soluble antibody, de- Balb/c mice were implanted s.c. with bilateral CT26 colon carcinoma layed/reversed tumor growth in both a B16-SIY and MC38-OVA tumors. Once established, one tumor was treated with a single peri- tumor treatment model in the absence of adoptively transferred cells tumoral dose of NKTR-262, while NKTR-214 was administered i.v. on (Fig. 2). This was caused by a change in biodistribution, altered CD8+ q9dx3 schedule. Regression of treated tumors and the abscopal ef- T cell repertoire, and an increase in cytokine secretion by tumor- fect in contralateral tumors was assessed by tumor size measure- specific CD8+ T cells (Fig. 3A). Despite local intra-tumoral treatment, ments. Immune cell activation in both tumors was assessed by flow new data shows that MC38-OVA cured mice have systemic immunity cytometry. Cytokine induction was measured in plasma, blood cells against the OVA antigen. These mice have a systemically circulating and treated tumors by MSD and qRT-PCR. repertoire of tumor-specific cells, demonstrated by increased in vivo Results killing of intravenously injected OVA-expressing cells (Fig. 3B) and Combination treatment with NKTR-262 and NKTR-214 eliminated protection against a re-inoculation of MC38-OVA. both tumors in up to ≥95% of mice. Single agent NKTR-262 or NKTR- Conclusions 214 treatment led to complete responses in ≤30% of mice. Combin- Here, we have demonstrated the efficacy of a new dual-targeting ation treatment of NKTR-262 plus NKTR-214 induced a two-step im- immunoswitch particle for cancer immunotherapy. Immunoswitch mune response in treated and untreated tumors. At early timepoints, particles synergize checkpoint blockade with co-stimulation by con- accumulation of activated neutrophils correlated with tumor cell fining the antibodies to a rigid nanoparticle surface. This new ap- death and dendritic cell activation. The innate response was followed proach can be extended to different disease models and new by increased CD8+ T cells and a reduction of immunorepressive cells. checkpoint or co-stimulatory target molecules. Single agent treatment showed only a subset of the cellular changes observed in the combination. Conclusions We present a designed combination therapy that mimics a natural immune response by activating a broad immune cell network. Com- bining NKTR-262 and NKTR-214 engages the entire immune activa- tion cascade required for systemic tumor clearance from local tumor antigen production to a sustained systemic T cell response. Unlike treatments that stimulate downstream components of select immune pathways without eliciting systemic tumor immunity, a comprehen- sive anti-tumor immune activation by coordinated engagement of in- nate and adaptive immune cells may increase the success of immune therapy for patients P276 Immunoswitch particles target activation of anti-tumor CD8+ T cells to inhibit tumor growth Alyssa Kosmides, John-William Sidhom, Andrew Fraser, Catherine Bessell, Jonathan Schneck Johns Hopkins School of Medicine, Baltimore, MD, USA Correspondence: Alyssa Kosmides (akosmides@gmail.com) Fig. 1 (abstract P276) . See text for description Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P276 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 143 of 244 Results Flow cytometry and NanoString based analysis of tumours collected + + - at endpoint showed higher intra-tumoural PD-1 and CD69 CD62L , CD8 T cells, increased expression of IFN response, antigen presenta- tion and MHCII, genes, respectively, in tumours from STING agonist treated mice compared to those from vehicle treated mice. In addition to significantly decreased ascites accumulation and de- creased tumour burden, survival of mice treated with a combination of Carboplatin + STING agonist + anti-PD-1 therapy was significantly longer compared to Carboplatin + STING agonist, Carboplatin only, STING only and vehicle treated mice. Fig. 2 (abstract P276). See text for description Conclusions Findings from this study are foundational to future clinical trials aimed at combinatorial immunomodulatory therapies to improve chemotherapy response and overall survival of HGSC patients. References 1. Koti M, SiuA, Clément I, Bidarimath M, Turashvili G, EdwardsA, et al. A distinct pre-existing inflammatory tumour microenvironment is associated with chemotherapy resistance in high-grade serous epithelial ovarian cancer. Br J Cancer [Internet]. 2015;112(7):1215–22. 2. Au KK, Le Page C, Ren R, Meunier L, Clément I, Tryshkin K, et al. STAT1- associated intratumoural T 1 immunity predicts chemotherapy resist- ance in high-grade serous ovarian cancer. J Pathol Clin Res. 2016. 3. Au KK, Peterson N, Truesdell P, Reid-schachter G, Khalaj K, Ren R, et al. Gy- necologic Oncology CXCL10 alters the tumour immune microenviron- ment and disease progression in a syngeneic murine model of high- grade serous ovarian cancer. Gynecol Oncol [Internet]. Elsevier Inc.; 2017;4–13.P278. P278 A Rational Combination of Standard of Care and Immunotherapy Fig. 3 (abstract P276). See text for description Increases Survival Against Glioblastoma Erik Ladomersky, Derek Wainwright Northwestern University, Chicago, IL, USA Correspondence: Derek Wainwright P277 (erik.ladomersky@northwestern.edu) STING agonist treatment increases response to chemotherapy and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P278 immune checkpoint blockade therapy in a syngeneic murine model of high-grade serous ovarian cancer Background 1 1 1 2 Abdi Ghaffari , Nichole Peterson , Kasra Khalaj , Andrew Robinson , Glioblastoma (GBM) is a fatal primary brain tumor that, even after max- 2 1 Julie-Ann Francis , Madhuri Koti imum surgical resection, radiotherapy (RT) and chemotherapy, is associ- 1 2 Queen's University, Kingston, ON, Canada; Kingston Health Sciences ated with a median overall survival (OS) of 14.6 months. Combinatorial Center, Kingston, ON, Canada treatment approaches that simultaneously address tumor growth, as well Correspondence: Madhuri Koti (kotim@queensu.ca) as the immunosuppressive microenvironment, may prove to be more ef- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P277 fective than current clinical strategies. This work aims to determine the therapeutic efficacy of combining the novel, pharmaceutical-grade IDO1 Background enzyme inhibitor, BGB-5777, with PD-1 blockade and/or whole brain radi- Background: High Grade Serous Carcinoma of the ovary is mostly di- ation, in a syngeneic, immunocompetent mouse glioblastoma model. agnosed at late stages and primarily treated with surgery followed Methods by platinum/taxane chemotherapy. Unfortunately, majority of the pa- Mice were intracranially-engrafted with 2x10 GL261 or CT-2A murine tients exhibit resistance to chemotherapy and ultimately succumb to GBM cells, then treated, beginning day 14 post-tumor cell implant- the disease. We previously reported that tumours from patients with ation, with 500μg loading dose IgG control mAb followed by three early recurrence show an immunosuppressed pre-existing tumour 200μg doses, administered every third day (n=7), or 2 Gy WBRT for 5 immune microenvironment with decreased expression of genes in- days, PD-1 mAb (J43) administered the same as IgG control mAb, volved in Type I Interferon (IFN1) and T helper type 1 response [1, 2, and 100mg/kg BGB-5777 BID for 4 weeks (n=9). 3]. We thus hypothesized that response to chemotherapy and overall Results survival of HGSC patients can be improved by stimulating the IFN1 Mice engrafted with the GL261 tumors and treated with the combin- response in the TME post chemotherapy. ation therapy showed a significant increase in overall survival (53 days) Methods compared to IgG treated mice (25 days). This therapy regimen also sig- In this pre-clinical study we tested the efficacy of a novel “Stimulator nificantly increased durable survival (>120 days) (P<0.0001) in mice ic. of Interferon Genes” agonist in immunocompetent mice implanted unmodified GL261 or CT-2A cells. (Fig. 1A) Paradoxically, mice with -/- with ID8-TRP53 mouse ovarian cancer cells. Post treatment tumour GBM cells overexpressing mouse IDO1 cDNA and treated with triple immune cell profiles were measured using a combination of flow cy- therapy showed a similar proportion of durable survivors (40%) vs. mice tometry and CyTOF based profiling. Tumour immune transcriptomic with similar tumors treated with IgG control (P<0.0001). (Fig. 1B) Inter- alterations were measured using the NanoString mouse pan cancer estingly, the survival effects seen in engrafted with unmodified GL261 immune profiling panel. Log-rank test based survival analysis was tumors correlated to a significant decrease in GBM-infiltrating Treg performed to determine significant differences in overall survival levels (P<0.01), however this same effect was not seen in mice with post treatment. GL261 mIDO1 O/E tumors. (Fig. 1C) These results suggest that effective- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 144 of 244 ness of triple therapy is independent of tumor IDO1 expression level P279 -/- and Treg accumulation. Results from the analysis of WT and IDO1 An oral small molecule combination therapy targeting PD-L1, mice with intracranial tumor indicate that in the presence of non- VISTA and Tim-3 immune inhibitory checkpoints exhibits enhanced tumor IDO1, IDO1 inhibition is necessary to confer survival benefit anti-tumor efficacy in pre-clinical models of cancer 1 1 1 2 (P<0.01). (Fig. 2) Adam Lazorchak , Troy Patterson , Yueyun Ding , Pottayil G. Sasikumar , 2 2 Conclusions Naremaddepalli S. Sudarshan , Nagaraj M. Gowda , Raghuveer K. 2 2 2 2 Ultimately, the data suggest that using radiation to induce potential im- Ramachandra , Dodheri S. Samiulla , Mohammed Rafi , Nagesh Gowda , 2 2 2 2 munogenicity and/or inflammation in GBM, while co-inhibiting immuno- Sreenivas Adurthi , Jiju Mani , Rashmi Nair , Murali Ramachandra , David 1 1 suppression, is a rational and potentially clinically-beneficial pursuit. Tuck , Timothy Wyant 1 2 Curis, Inc., Lexington, MA, USA; Aurigene Discovery Technologies Limited, Bengaluru, India Correspondence:Adam Lazorchak (adam.lazorchak@icloud.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P279 Background Antibodies targeting immune inhibitory checkpoint pathways have transformed cancer therapy. However, the majority of pa- tients fail to respond to therapies targeting single immune check- points due, in part, to the compensatory activity of alternative immune suppressive pathways. A therapeutic strategy that targets multiple immune checkpoints may significantly improve the anti- tumor response rate, however this strategy may also increase the risk of severe treatment related immune mediated side effects. Orally bioavailable, small molecule immune checkpoint antago- nists with short in vivo half-lives are ideal candidates for combin- ation cancer immune therapy due to a short drug washout time which may improve safety and flexibility in scheduling which may increase efficacy. Methods The anti-tumor efficacy of orally administered CA-170 (a PD-L1/2 and VISTA/PD-1H antagonist), CA-327 (a PD-L1/2 and Tim-3 antagonist) or the combination of CA-170 plus CA-327 was studied in the mouse syngeneic CT26 colon carcinoma tumor model. Compound dosing Fig. 1 (abstract P278) . See text for description was initiated within 1-3 days of tumor implantation or in mice bear- ing established tumors (90-170 mm ). Populations of immune cells were measured by flow cytometric analysis in tumor or blood. Ex vivo functional assays were performed on tumor derived CD8 T cells to evaluate effector cell function and gene expression profiling was per- formed on tumors. Results Significant anti-tumor efficacy was observed in CT26 tumor bear- ing mice treated with either CA-170 or CA-327 compared to ve- hicle treated animals. Flow cytometric analysis of the tumors taken from these mice revealed that both compounds signifi- cantly increased the total number of CD45 immune cells within the tumor relative to vehicle treated animals. Tumor growth in- hibition positively correlated with increased numbers of activated + + + CD8 T cells and IFN-γ CD8 effector T cells within the tumor. A significant increase in anti-tumor efficacy was observed when CA- 170 and CA-327 were administered together as an oral combin- ation therapy, when compared to animals treated with either compound alone. Conclusions These non-clinical data demonstrate a proof-of-concept showing that a combination therapy consisting of oral small molecules that antagonize the PD-L1/2, VISTA and Tim-3 pathways signifi- cantly enhance anti-tumor efficacy. CA-170 is currently undergo- ing Phase I clinical testing and CA-327 is in pre-clinical development. The results of our study provide a strong rational for the continued development of combination therapies using small molecule immune checkpoint antagonists for the treatment Fig. 2 (abstract P278). See text for description of advanced cancers. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 145 of 244 P280 Background Combination lymphoma immunotherapy using intratumoral The activation of VEGFR-2 by its primary cognate ligand VEGF has virus-like particles containing CpG TLR9 agonist combined with been principally implicated in tumor angiogenesis. However, emer- checkpoint blockade ging data has suggested that the VEGF/VEGFR-2 axis also mediates a 1 1 2 1 Caitlin Lemke-Miltner , Sue Blackwell , Arthur Krieg , George Weiner suppressive effect on the anti-tumor immune response. Inhibition of 1 2 University of Iowa, Iowa City, IA, USA; Checkmate Pharmaceuticals, this pathway has demonstrated the potential to facilitate T cell mi- Cambridge, MA, USA gration into the tumor and to reduce the direct immune inhibitory Correspondence: Caitlin Lemke-Miltner (caitlin-lemke@uiowa.edu) activity promoted by tumor endothelial cells. Clinical studies that Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P280 combine anti-angiogenic antibodies to immune checkpoint inhibitors have shown promising results in a number of solid tumors. However, Background the mechanisms underlying immunomodulatory activity of agents Checkpoint blockade of immune inhibitory pathways is an exciting ap- that target the VEGF/VEGFR-2 axis remain incompletely understood. proach to cancer immunotherapy, however there remains considerable Methods room for improvement through the use of unique immunotherapeutic Ramucirumab is an approved VEGFR-2 targeted antibody currently combinations. We explored the combination of intratumoral injection undergoing clinical trials in combination with PD-1/PD-L1 inhibitory (in situ immunization) with a virus-like particle (VLP) combined with antibodies. The emerging data from these early trials has prompted anti-PD1 antibody therapy. The VLP is designated CMP-001 and is com- us to undertake non-clinical studies with potential to uncover mecha- posed of an immunostimulatory CpG oligodeoxynucleotide TLR9 agon- nisms that mediate the enhancement of anti-tumor immune re- ist encapsulated in the Qb bacteriophage protein. CMP-001 was sponse by the blockade of VEGFR-2. Using murine EMT6 breast and previously evaluated clinically in over 700 subjects in non-cancer trials MC38 colon carcinoma models we investigated the immunomodula- where it stimulated a strong Th1 cytokine response. The current studies tory effects of DC101, a surrogate antibody that blocks mouse were based on the hypothesis that intratumoral CMP-001 can augment VEGFR-2, and its ability to increase the anti-tumor efficacy of check- the development of a tumor-specific T cell response that can be main- point inhibition. Intra-tumor immune-related changes were evaluated tained by anti-PD-1 therapy. by flow cytometry, immunohistochemistry (IHC) and nCounter gene Methods expression analysis (NanoString). The impact of CMP-001 with and without anti-PD1 was assessed in Results both human (in vitro) and mouse (in vitro and in vivo) systems. In Anti-VEGFR2 monotherapy resulted in increased T cell infiltration into vivo therapy studies in mice included the B16F0 melanoma and A20 the tumors. Combination of anti-VEGFR-2 with anti-PD-L1 resulted in B cell lymphoma models. CMP-001, or saline control, was delivered greater anti-tumor efficacy compared to anti-PD-L1 monotherapy in intratumorally starting one week after tumor challenge. Anti-PD-1, or both MC38 and EMT6 tumor models. Mice achieving complete tumor isotype control, was administered systemically starting one week regressions after the combination treatment resisted tumor rechal- after tumor challenge. Tumor growth and survival was followed. In lenge demonstrating the development of immunologic memory. some experiments, tumor inoculation was done bilaterally while a Analysis of changes in the tumor microenvironment by flow cytome- unilateral tumor was injected with CMP-001 to allow for assessment try during combination therapy showed increased myeloid and T cell of response of both the treated and untreated tumor. infiltration. nCounter gene expression analysis confirmed that anti- Results VEGFR-2 treatment enhanced inflammation and immune activation In vitro, CMP-001 stimulated IFNα production, as well as other pro- gene expression signature in monotherapy, and this effect was much inflammatory cytokines, from human and mouse mononuclear cells more pronounced after the combination treatment. Pathway analysis (from the peripheral blood and spleen respectively). This was only highlighted that the combination effect could be attributable to seen when anti-Qβ antibody was present. enhanced innate immune response (e.g. dendritic cell maturation, Intratumoral CMP-001 enhanced survival, and reduced tumor growth antigen presentation) and T cell activation. These results were cor- of treated tumors in the B16F0 model, and of both treated and un- roborated by flow cytometry, showing increased MHCI and MHCII ex- treated tumors in the A20 lymphoma model. Anti-PD-1 enhanced pression on DCs and macrophages, along with increased PD-L1 this effect in the A20 model. Depletion of T cells in mice eliminated expression during anti-VEGFR-2 monotherapy. the anti-tumor effect in both the treated and untreated tumors. Conclusions Conclusions Taken together, these results highlight the potential of anti-VEGFR- We conclude CMP-001 can induce a robust Th1 response in both 2 antibodies to partially ameliorate intra-tumor immune suppres- murine and human systems in a manner that is dependent on sion, providing insights into the mechanisms by which combined opsonization of the VLP with anti-Qb antibody. The combination of VEGFR-2/PD-L1 antibody therapy leads to increased anti-tumor intratumoral CMP-001 and systemic anti-PD-1 leads to development efficacy. of a systemic anti-tumor T cell response in two murine tumor models and is a promising immunotherapy combination worthy of clinical evaluation. A Phase 1 clinical trial of the combination of anti-PD-1 P282 and CMP-001 is underway in advanced melanoma. These results will Immunostimulatory CD40L/4-1BBL Gene Therapy Enhances aPD-1 be presented separately. Antibody Therapy in Experimental Models Jessica Wenthe, Emma Eriksson, Ann-Charlotte Hellström, Angelica Loskog Uppsala University, Uppsala, Sweden Correspondence: Angelica Loskog (angelica.loskog@igp.uu.se) P281 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P282 Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances antitumor efficacy of PD-L1 blockade in Background syngeneic murine tumor models Lung cancer is a deadly disease responding poorly to conventional 1 1 1 1 1 yanxia li , David Schaer , Marguerita O’Mahony , Ivan Inigo ,QiLi , therapy. Checkpoint blockade antibodies inhibiting PD-1/PD-L1 have 1 1 2 Nelusha Amaladas , Erik Rasmussen , Thompson Doman , Jason shown clinical benefit. Since the success of PD-1 blockade therapy 2 1 1 1 1 Manro , Mary Murphy , Macrina Francisco , Gerald Hall , Michael Kalos , prerequisites the presence of tumor-reactive T-cells, we hypothesized 1 1 Ruslan Novosiadly , Bronislaw Pytowski that an activating immunotherapy with high potency to stimulate T- 1 2 Eli Lilly and Company, New York, NY, USA; Eli Lilly and Company, cell responses would be an ideal candidate to combine with anti-PD- Indianapolis, IN, USA 1 antibody treatment. LOAd703 is a replication-restricted adenovirus Correspondence: David Schaer (yanxia.li@lilly.com) serotype 5/35. It is armed with two immunostimulatory transgenes, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P281 4-1BB ligand (4-1BBL) and a trimerized human CD40 ligand (TMZ- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 146 of 244 CD40L). Both molecules are co-stimulatory molecules in the dendritic doxorubicin. Complete blood counts and immunophenotyping were cell (DC)/T-cell synapse. This study aims to evaluate if immunostimu- performed to track changes in immune cell profile as a biomarker of re- + + + latory gene therapy, such as LOAd703, is a good enhancer of check- sponse. TAA-specific IFN-γ CD8 and CD4 T cell responses and point blockade therapy. humoral responses were measured throughout the study. Methods Results The capacity of LOAd703 was tested using the lung cancer cell lines In non-tumor bearing macaques, different chemotherapies impacted A549 and H727. Tumor viability was evaluated in vitro using MTS via- VBIR-induced immune responses with varying degrees of severity. TAA- + + bility assay. Co-culture experiments were analyzed in real time using specific IFN-γ CD8 T cell responses ranged from unaffected with Incucyte technology. Cells were phenotyped with multicolor flow cy- doxorubicin, to moderately reduced with carboplatin-based doublet tometry and cytokine patterns investigated by MesoScale. A20 cells chemotherapies, to almost entirely ablated with paclitaxel/gemcitabine. + + that can be infected with LOAd703 virus were used in a syngeneic TAA-specific IFN-γ CD4 T cell responses, however, were generally un- model in BalbC mice. impaired by chemotherapy: indeed, in many instances chemotherapy Results boosted CD4 T cell responses. TAA-specific humoral responses were Tumor cells were killed by oncolysis post infection with high dose superior in animals treated with carboplatin-based doublet chemother- virus (100MOI) in vitro and in vivo in multiple xenograft mouse apies, while paclitaxel/gemcitabine continued to preclude an effective models (lung, ovary, pancreas, colon, bladder). In tumor cell/PBMC immune response. Doxorubicin treated animals demonstrated a co-culture,addition of anti-PD-1 antibody did not increase tumor cell strongly diminished humoral response, attributed to B cell depletion. apoptosis. If OKT3/IL2 was added apoptosis was confirmed in the cul- The impact of chemotherapy on the VBIR-induced immune response tures peaking at day 5. Combination of OKT3/IL2 with anti-PD-1 anti- was also dependent on antigenic strength, with responses to a weaker body significantly enhanced the effect. LOAd703 was added to antigen more dramatically reduced than were responses to a more im- tumor cell/PBMC co-cultures at a low MOI to delay oncolysis. munogenic antigen. LOAd703 induced tumor cell death, starting already at day 2 and the Conclusions activity was continuously rising throughout the experiment. Combin- Careful clinical trial planning is critical for vaccine-based immuno- ing LOAd703 with anti-PD-1 antibody significantly increased apop- therapy regimen and chemotherapy combinations that will not only tosis induction of tumor cells while a control virus was less effective. permit robust immune response activation and maintenance but also At endpoint, LOAd703 plus anti-PD-1 was twice as effective as OKT3/ provide the adequate conditions for optimal synergistic anti-tumor IL2 plus anti-PD-1. The T cells were increased in the same groups as effect. tumor cell killing was previously noted. IFNg, TNF, IL2 were all en- hanced by LOAd703 and OKT3/IL2. In line with in vitro data, P284 LOAd703 could enhance in vivo tumor control of anti-PD-1 therapy. T cell priming by Toca 511 and 5-FC coupled with T regulatory cell Conclusions depletion by αCTLA-4 synergistically enhances anti-tumor immune In conclusion, LOAd703 is a potent stimulator of T cell responses and memory in a mouse model of glioma potentiates the effect of aPD-1 therapy. Leah Mitchell, Kader Yagiz, Anthony Munday, Fernando Lopez, Daniel Mendoza, Douglas Jolly P283 Tocagen, San Diego, CA, USA Differential impact of chemotherapy on tumor-associated antigen- Correspondence: Leah Mitchell (lmitchell@tocagen.com) specific immunogenicity in cynomolgus macaques Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P284 Maria Josic, Aaron Longworth, Joe Binder, Helen Cho, Erick Gamelin, Siradanahalli Guru, Eugenia Kraynov, Steve Burgess, Bryan Clay, Charlie Background Huang, Jannine Landry, Mark Lesch, Cindy Wei Li, Shangjin Li, Pavinder Toca 511 (vocimagene amiretrorepvec) is a gamma retroviral replicating Kaur, Sophie Muscat-King, Peter Weady, Dan Xu, James Merson, Robert vector that selectively infects cancer cells in vivo and encodes cytosine Hollingsworth, Marianne Martinic deaminase. In combination with the prodrug, 5-fluorocytosine (5-FC), Pfizer, San Diego, CA, USA Toca 511 produces 5-fluorouracil (5-FU) locally in the tumor microenvir- Correspondence: Marianne Martinic (maria.josic@pfizer.com) onment. Prior work has demonstrated a reduction in immunosuppressive Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P283 myeloid cells and an increase in CD4 and CD8 T cells in tumors while T regulatory cells remain unchanged with treatment with Background Toca 511 and 5-FC. Pfizer’s vaccine-based immunotherapy regimen (VBIR) is a cancer Methods treatment which combines a tumor-associated antigen (TAA)-specific This work, in a mouse model of glioma, aimed to determine if the vaccine, delivered via an AdC68 adenovirus and DNA regimen, with addition of a checkpoint inhibitor, αCTLA-4, would provide thera- the immunomodulatory agent tremelimumab (anti-CTLA4 antibody) peutic benefit to Toca 511 and 5-FC. for T cell expansion. Various tumor types that are targets for VBIR, in- Results cluding non-small cell lung, triple negative breast, ovarian, and pan- Initially, we noted that Toca 511 and 5-FC was highly efficacious and creatic cancer, are treated with chemotherapy as the standard of that it provided little room for further improvement and therefore care (SOC). Accordingly, VBIR clinical administration may take place combination with αCTLA-4 did not show additive benefit against the in patients who are currently undergoing chemotherapy. Synergistic primary cancer. However tumor associated Regulatory T cells were effect has been reported between immune therapies and chemother- significantly reduced with αCTLA-4 treatment and long term memory apy, either due to immunogenic cell death or a direct effect on im- was significantly improved with the combination as shown in adop- munosuppressive cells. However, limited information is known about tive transfer studies. Adoptive transfer of immune cells from animals the impact of chemotherapy on a vaccine-induced de novo immune that cleared their primary tumor through Toca 511, 5-FC, and αCTLA- response. 4 showed 100% survival benefit to animals bearing orthotopic gli- Methods omas, significantly greater than the ~50% survival seen with transfer A nonhuman primate model was developed to most precisely from animals that cleared primary tumor through Toca 511 and 5-FC mimic clinical therapy conditions, including pharmacokinetics and alone. Further, αCTLA-4 treatment during clearance of primary tu- dynamics, dosing regimens, and supplemental treatments, allowing mors resulted in a marked reduction of memory T regulatory cells in evaluation of potential chemotherapy impact on the VBIR-induced secondary tumors after adoptive transfer. To determine if αCTLA-4 in immune response. Macaques were dosed with a VBIR targeting a self- combination with Toca 511 and 5-FC could reduce primary tumor version of a human TAA with or without the following SOC treatments: burden we developed a model using a submaximal infection level of paclitaxel/gemcitabine, paclitaxel/carboplatin, docetaxel/carboplatin, or Toca 511. Specifically, restricting Toca 511 infection to only 2% of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 147 of 244 tumor cells limited the tumor growth arrest activity of 5-FC and the P286 loss of efficacy with 2% infection was rescued when 5-FC treatment Efficacy of interleukin 2 and interleukin 15 for in situ vaccination was combined with αCTLA-4. in a mouse melanoma model 1 1 1 Conclusions Alexander Rakhmilevich , Anna Hoefges , Jacob Slowinski , Kayla 1 1 2 3 These data suggest that αCTLA-4, and other compounds that target Rasmussen , Mackenzie Heck , Michael Meagher , Alan Korman , Paul T regulatory T cells, should be evaluated in patients receiving Toca Sondel 1 2 511 and Toca FC to determine if the combination confers additional University of Wisconsin-Madison, Madison, WI, USA; St. Jude Children’s clinical benefit. Research Hospital/Children’s GMP, LLC, Memphis, TN, USA; Bristol-Myers Squibb Company, Redwood City, CA, USA Correspondence: Alexander Rakhmilevich (rakhmil@humonc.wisc.edu) P285 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P286 Antibody and T cell response profiling of pancreatic cancer patients before and after chemotherapy reveals increased Background recognition of antigens suitable for immunotherapy We have previously shown that a direct intratumoral (IT) injection 1 1 1 1 Giorgia Mandili , Moitza Principe , Emanuela Mazza , Sara Bulfamante , of immunocytokine (IC), an anti-GD2 antibody linked to interleu- 1 1 2 1 Laura Follia , Giulio Ferrero , Andrea Evangelista , Daniele Giordano , kin 2 (IL-2), can serve as an in situ vaccine and synergize with 1 1 Paola Cappello , Francesco Novelli anti-CTLA-4 antibody to induce T cell-mediated antitumor effects. 1 2 University of Turin, Torino, Italy; Hospital Città della Salute e della We have also shown a synergy of this approach with activation Scienza di Torino, Torino, Italy of innate immunity with an agonistic anti-CD40 monoclonal anti- Correspondence: Francesco Novelli (franco.novelli@unito.it) body (anti-CD40) and CpG (Rakhmilevich et.al. Journal of Immun- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P285 ology 2017). We used these two immunotherapeutic approaches to test whether IT treatment with a mixture of IL-2 and anti-GD2 Background antibody, hu14.18K322A, will be effective against subcutaneous Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal melanoma. In addition, we hypothesized that IL-15, a cytokine cancer, both for lack of effective screening method and for resistance having several activities similar to those of IL-2 except induction to chemotherapy (CTX) and radiotherapy. At present surgical resec- of T regulatory cells (Tregs), may be as or more effective than IL- tion is the only potentially curative option. Once diagnosed, CTX, ra- 2 for in situ vaccination. diation or combination therapy regimens are used to treat patients, Methods but responses remain poor. However, some chemotherapeutic GD2+ B78 mouse melanoma cells were injected subcutaneously in agents have an immune modulating effect and a combination of C57BL/6 mice. Anti-CD40 and anti-CTLA-4 were given intraperitone- CTX and immunotherapy could increase therapeutic efficacy. Thus, ally, and CpG, IL-2, IL-15 and K322A were given IT. IL-2 and IL-15 more immunogenic antigens can be induced by CTX and targeted were given at the dose of 7.5x104 units per injection daily for 5 days. by passive or active immunotherapy. To discover TAAs that might be Tumor growth and survival were followed. In some experiments, selected for immunotherapy, antibody response in PDA patients’ sera tumors were analyzed by flow cytometry. were analyzed before and after CTX. TAAs selected based on their in- Results creased recognition after CTX were used to evaluate whether PDA Flow cytometric analyses showed that IL-2 treatment increased Tregs patient autologous T cells have an increased TAAs specific response within a tumor whereas IL-15 did not. When the treatments were after CTX. started on day 6 post tumor cell implantation, IL-2 and IL-15 com- Methods bined with K322A induced similar reductions of B78 tumor growth, Antibody response in sera of 29 PDA patients, before and after CTX and complete tumor regression with the addition of anti-CTLA-4. treatments, has been analyzed by Serological Proteome Analysis When the treatments were started on day 22 post tumor cell im- (SERPA). The production of IFN-g and IL-10 by PBMC stimulated plantation, IL-2 and IL-15, combined with K322A, anti-CTLA-4, anti- in vitro with recombinant TAAs was evaluated by ELISA. CD40 and CpG, also induced comparable antitumor effects resulting Results in survival of 40-60% of mice. CTX increases the number of TAAs recognized by PDA patients. Conclusions Specifically, the set of TAAs we identified are proteins whose mRNA In two therapeutic approaches, IL-2 and IL-15, combined with other expression have been found up-regulated in PDA patients. We immunotherapeutic agents, induced similar antitumor effects. observed a positive correlation between patients survival and the increased antibodies production against alpha-enolase (ENO1), P287 glyceraldehyde-3-phosphate dehydrogenase (G3P), tubulin (TUBB5), Immunological effects of checkpoint blockade plus galectin-3 and keratin, type II cytoskeletal 8 (K2C8). Of note, 48% of PDA pa- inhibition with GR-MD-02 in a first-in-human phase I clinical trial tients sera after CTX treatment showed an increase of Complement William Redmond1, Yoshinobu Koguchi1, Christopher Fountain1, Dependent Cytotoxicity (CDC) against human cell lines. Moreover in Peter Traber2, Brendan Curti1 about 50% of the same PDA patient cohort the in vitro specific T 1Providence Portland Medical Center, Portland, OR, USA; 2Galectin cell response to recombinant TAAs that were mostly recognized by Therapeutics, Norcross, GA, USA antibodies after CTX (ENO1, G3P, K2C8 and Far Upstream Binding Correspondence: William Redmond (william.redmond@providence.org) Protein 1 (FUBP1) switched from a pro-tumor regulatory (low IFN-g/ Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P287 IL10 ratio) to anti-tumor effector (high IFN-g/IL10 ratio) phenotype after one or two rounds of CTX. Background Conclusions Immunosuppression of tumor-infiltrating lymphocytes (TIL) is a major Data indicated that in PDA patients CTX induces an increase of IgG obstacle to creating effective therapies for patients with metastatic antibody to ENO1, G3P, TUBB5 and K2C8 whose expression is up- cancer. Galectin-3 (Gal3), a lectin family member, is expressed by nu- regulated in PDA that may have a prognostic role. CTX also in- merous cancers and immune cell subsets. Serum Gal3 expression is creases the ability of IgG kill PDA cells by CDC. Finally , CTX higher in patients with metastatic disease and is associated with re- switches the T cell response to ENO1, G3P, K2C8 or FUBP1 from duced survival in patients with metastatic melanoma. Furthermore, regulatory to effector and thus renders these TAAs promising tar- Gal3 has been implicated in disease progression via the promotion gets for the design of new immunotherapeutic approach to im- of angiogenesis and metastasis. Interestingly, extracellular Gal3 prove the efficacy of CTX. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 148 of 244 induces immune suppression via inhibiting TIL function, promoting using Fabs of KY1044. This antibody, which binds ICOS from human, M2 macrophage polarization and mobilizing myeloid cells from the cynomolgus monkey, rat and mouse with similar affinity, was used in bone marrow to promote a metastatic niche within the tumor. We several in vitro and in vivo assays to refine its mechanism of action hypothesized that Gal3 inhibition in conjunction checkpoint block- and assess the anti-tumour efficacy in pre-clinical models. ade immunotherapy would improve TIL function while inhibiting Results tumor growth and metastasis. Preclinical studies revealed that Gal3 Using in vitro reporter and primary cell assays, we have demon- blockade with GR-MD-02 and agonist anti-OX40, anti-CTLA-4 or anti- strated that KY1044 can affect the immune context via a dual mech- PD-1 mAb therapy enhanced tumor-specific immunity and improved anism of action. On the one hand, KY1044 is extremely potent (low survival in tumor-bearing mice. pM EC50) at depleting ICOShigh Tregs and on the other hand Methods KY1044 was shown to stimulate ICOSInt effector T-cells by increasing We initiated 2 phase I clinical trials at our Institute to evaluate the their IFNγ and TNFα production. Since KY1044 is cross reactive to the safety and immunological effects of GR-MD-02 plus checkpoint rodent orthologue of ICOS, a mouse effector enabled version of blockade immunotherapy. These studies are investigating dose KY1044 (mIgG2a) was generated and tested in syngeneic tumour escalations of GR-MD-02 with the standard therapeutic dose of anti- models. Using this antibody, we have confirmed strong anti-tumour CTLA-4 (ipilimumab/ipi) or anti-PD-1 (pembrolizumab/pembro) in pa- efficacy as monotherapy or in combination with surrogates of tients (pts) with advanced melanoma (ipi; pembro), HNSCC (pembro), “approved” immune checkpoint blockers. Noteworthy, and confirm- or NSCLC (pembro) (NCT02117362, NCT02575404). ing the in vitro data, pharmacodynamic studies demonstrated a Results long-term depletion of Tregs and a significant increase in the effector The GR-MD-02 + ipi study has completed enrollment. Three patients T cell to Treg ratio in response to KY1044. received GR-MD-02 at 1 mg/kg, 3 at 2 mg/kg and 2 at 4 mg/kg. Conclusions There were no DLTs for GR-MD-02; however there was 1 grade 3 AE Altogether, the in vitro and in vivo properties of this novel, fully from ipilimumab (diarrhea) that occurred after dose 4 of ipi. The GR- human anti-ICOS antibody support the continued development of MD-02 + pembro study is ongoing. Six patients were enrolled at the KY1044 as a treatment option to activate an anti-tumour immune 2 mg/kg dose level to gain more data from the immunological moni- response. toring assays. Three patients were treated at 4 mg/kg. Eight of the 9 enrolled patients have melanoma and 1 has HNSCC. There have been P289 no DLTs related to GR-MD-02 or pembro, but there one patient expe- Dual cIAP1/XIAP inhibitor ASTX660 synergizes with radiation rienced transient grade 3 tumor-related pain. Two pts in cohort 1 (2 therapy and PD-1 blockade to enhance anti-tumor immunity mg/kg GR-MD-02) had objective responses (1 PR, 1 CR) and two pts 1,2 1,3 1 1 1 Roy Xiao , Clint Allen , Linda Tran , So-Jin Park , Zhong Chen , in cohort 2 (4 mg/kg) have PR at the first response assessment. All 1 1,3 Carter Van Waes , Nicole Schmitt responses have been observed in patients with melanoma. NIDCD, NIH, Bethesda, MD, USA Conclusions Correspondence: Nicole Schmitt (nicole.schmitt@nih.gov) These data demonstrate that the Gal3 inhibitor GR-MD-02 can be com- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P289 bined safely with checkpoint blockade in patients with metastatic dis- ease and that melanoma regression was observed in multiple patients Background following combination therapy. Comprehensive immunological moni- Head and neck squamous cell carcinomas (HNSCCs) frequently har- toring is being conducted to provide insight into potential mechanisms bor genomic mutations in cell death pathways. Nearly 30% of HNSCC of action. overexpress Fas-Associated Death Domain (FADD), with or without Trial Registration BIRC2/3 genes encoding cellular Inhibitor of Apoptosis Proteins 1/2 NCT02117362, NCT02575404 (cIAP1/2), critical components of the Tumor Necrosis Factor (TNF) Re- ceptor signaling pathways. ASTX660 is a novel dual cIAP1/XIAP an- P288 tagonist in clinical trials for advanced solid tumors and lymphomas. KY1044, a novel anti-ICOS antibody, elicits long term in vivo anti- Methods tumour efficacy as monotherapy or in combination with immune Murine oral cancer 1 (MOC1) cells were used in vitro and in vivo to checkpoint inhibitors investigate the anti-tumor activity of ASTX660 alone and in combin- Richard C.A. Sainson, Anil Thotakura, Nahida Parveen, Gwenoline Bohris, ation with tumor necrosis factor receptor (TNFR) superfamily Robert Rowlands, Miha Kosmac, Jamie Campbell, Ian Kirby, ligands, radiation, cisplatin chemotherapy, and anti-PD-1 check- Volker Germaschewski, Matthew McCourt point blockade. OT-1 T cells were used to investigate the effects of Kymab Ltd, Cambridge, United Kingdom ASTX660 on antigen-specific T cell killing of ovalbumin-expressing Correspondence: Richard C.A. Sainson (richard.sainson@kymab.com); MOC1 (MOC1ova) cells. Matthew McCourt Results Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P288 ASTX660, at nanomolar concentrations, sensitized MOC1 cells to TNFα and stimulated cytotoxic T lymphocyte (CTL) killing of Background MOC1ova. CTL killing was found to be predominantly mediated by The inducible co-stimulator molecule (ICOS/CD278) is a member of perforin/granzyme B during the earliest stages of killing and release the CD28/CTLA-4 family that is up-regulated upon T cell activation. In of death ligands TNFα, TRAIL, and FasL as a sustained mechanism of the tumour microenvironment, ICOS expression levels vary in differ- killing. Using MOC1 cells in vivo, ASTX660 synergized with radiation ent immune cell subtypes, with expression on highly immunosup- therapy (XRT), cisplatin chemotherapy, and PD-1 blockade to signifi- pressive regulatory T cells (Treg: CD4+/FOXP3+) significantly higher cantly delay or eradicate MOC1 tumors. These combination therapies than that on effector CD8+ T cells. The high expression levels on significantly increased CD8+ T cells and dendritic cells, as well as T Tregs highlights the potential of targeting ICOS to deplete these cells cell activity. Depletion of CD8+ T cells and NK cells in vivo revealed and enhance the anti-tumour immune response when used in com- both to be important components of the anti-tumor response en- bination with immune checkpoint blockers. hanced by ASTX660+XRT. Methods Conclusions Using the Kymouse™ platform, we have identified a novel, fully hu- These findings serve to inform future studies of IAP inhibitors and man antibody called KY1044. KY1044 is an anti-ICOS subclass G1 support the potential for future clinical trials investigating ASTX660 kappa monoclonal antibody that selectively binds to dimeric ICOS with XRT and immunotherapies such as PD-1/PD-L1 blockade in (Fc fusion) with an affinity of less than 2nM as measured by SPR HNSCC. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 149 of 244 P290 molecules lose target avidity when engineered to bind multiple targets Agonist redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer with monovalent antigen binding arms. Fusion proteins incorporating immunotherapy the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked Josiah Hornblower, Taylor Schreiber to the hinge-CH2-CH3 domain of antibodies are both functional, des- Shattuck Labs, Inc., Research Triangle Park, NC, USA pite the ECDs being in opposite orientation. We report the generation Correspondence: Taylor Schreiber (tschreiber@shattucklabs.com) of a two-sided fusion protein incorporating the ECD of SIRPα (CD172a) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P290 and the ECD of CD40L, adjoined by a central Fc domain. Methods Background Shattuck synthesizes both murine and human versions of ARC proteins, Current attempts at combination immunotherapy with bispecific and assesses them using a litany of biochemical assays to determine antibodies, linked scFv’s or T cell engagers have not demonstrated MW, subunit composition & binding affinity; molecular assays to that both checkpoint blockade and TNF receptor activation (agon- characterize in vitro/ex vivo binding & functional activity; and anti- ism) can be achieved with a single molecule. This is likely due to the tumor efficacy in syngeneic tumor models. The human SIRPa-Fc-CD40L fact that these molecules lose target avidity when engineered to has completed cell line development and single cell cloning and is in bind multiple targets with monovalent antigen binding arms. Fusion late-stage manufacturing. proteins incorporating the extracellular domain (ECD) of type I mem- Results brane proteins (eg. Enbrel, Orencia) or type II membrane proteins The SIRPα end of the ARC binds immobilized CD47 at 3.59 nM affinity (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of and binds CD47 on the surface of human tumor cells both in vitro and antibodies are both functional, despite the fact that the ECDs are in in vivo, but does not bind human platelets or RBCs. Importantly, no opposite orientation. Here we report the generation of a two-sided hemolytic activity has been observed with the human SIRPa-Fc-CD40L fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, ARC; where significant hemolysis has been reported with comparative adjoined by a central Fc domain. CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at Methods 756 pM affinity and binds CD40 on primary macrophages. The SIRPa- Shattuck synthesizes both murine and human versions of ARC pro- Fc-CD40L ARC stimulates functional activity in NFkB-luciferase reporter teins, and assesses them using a litany of biochemical assays to cells (CD40 driven activation of NFkB) and when added ex vivo to determine molecular weight, subunit composition & binding affinity; human PBMCs primed with the super-antigen SEB; increases secretion molecular assays to characterize in vitro/ex vivo binding, in vitro of IL2 and TNFa. Furthermore, when activated human macrophages functional activity; and anti-tumor efficacy in multiple syngeneic were co-cultured with CD47 positive human tumor cells, SIRPα -Fc- tumor model systems. The human TIM3-Fc-OX40L has advanced into CD40L was shown to enhance phagocytosis of human tumor cells. cell line development and early manufacturing. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established Results murine MC38 and CT26 tumors was superior to either CD47 block- The TIM3 end of the fusion protein binds GAL9 and phosphtidylser- ing antibody, CD40 agonist antibody or combination antibody ine (PS) on the surface of human tumor cells. The OX40L end of the therapy. fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc- Conclusions OX40L activates NFkB signaling in cells engineered to overexpress These data demonstrate feasibility and functional activity of a OX40 and an NFkB-luciferase reporter. Additionally, the TIM3-Fc- novel chimeric fusion protein platform, providing checkpoint OX40L ARC added to primary human PBMCs along with the super- blockade and TNF superfamily costimulation in a single molecule. antigen Staphylococcal enterotoxin B, induced robust secretion of Signal replacement of CD47 by CD40Lmayuniquelypoise macro- the cytokines IL2 and TNFa. In vivo, TIM3-Fc-OX40L stimulates signifi- phages in the tumor microenvironment for activation and cross- cant expansion of antigen-specific CD4 and CD8 T cells in mice adop- presentation of tumor antigens following enhanced tumor cell tively transferred with OT-I/OT-II cells and vaccinated with ova/alum. phagocytosis. Finally, the therapeutic activity of TIM3-Fc-OX40L in established mur- ine MC38 and CT26 tumors was significantly superior to either TIM3 P292 blocking antibody, OX40 agonist antibody or combination antibody Pre-clinical activity of a novel immunotherapy combination of therapy. Importantly, a pharmacodynamic biomarker of tumor rejec- CAVATAK (Coxsackievirus A21), anti-PD1 blockade and an IDO tion was identified by coordinated elevations in serum IFNγ, IL-2, IL- inhibitor in melanoma 4, IL-5, IL-6 and IL-17A. Gough Au, Min Yuan, Yvonne Wong, Darren Shafren Conclusions Viralytics Limited, Sydney, Australia These data demonstrate feasibility and functional activity of a novel Correspondence: Darren Shafren (darren.shafren@viralytics.com) chimeric fusion protein platform, providing checkpoint blockade and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P292 TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the Background same microenvironmental context, at the time in which T cells are TM Coxsackievirus A21 (CAVATAK ) is a bio-selected oncolytic immuno- engaging cognate tumor antigen. therapy virus. A Phase Ib trial of i.v. CAVATAK (NCT01227551) in ad- vanced cancer patients has displayed viral tumor targeting and initial P291 indications of antitumor activity in some lesions. Intratumoral CAVA- Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer TAK injection of melanoma lesions can induce selective tumor-cell in- immunotherapy fection, immune-cell infiltration, IFN-g response gene up-regulation, Suresh de Silva, George Fromm, Arpita Patel, Kellsey Johannes, increased PD-L1 and IDO (indoleamine-pyrrole 2,3-dioxygenase) ex- Josiah Hornblower, Taylor Schreiber pression, tumor cell lysis and systemic anti-tumor immune responses. Shattuck Labs, Inc., Research Triangle Park, NC, USA Blockade of programmed death protein-1 (PD1) and/or IDO inhibition Correspondence: Taylor Schreiber (tschreiber@shattucklabs.com) in many cancer patients has resulted in substantial tumor responses. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P291 We investigated anti-tumor activity in a B16-ICAM-1 melanoma im- mune competent mouse model of a novel combination of CAVATAK, Background an anti-PD1 mAb and an IDO inhibitor. Current attempts at combination immunotherapy with bispecific anti- Methods bodies, linked scFv’s or T cell engagers have not demonstrated that Palpable flank tumor of murine melanoma B16-cells expressing human both checkpoint blockade and TNF receptor activation (agonism) can ICAM-1 were propagated to assess the antitumor activity of CAVATAK, be achieved with a single molecule. This is likely because these an anti-mouse PD1 (mPD1) mAb and an murine IDO inhibitor in an Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 150 of 244 immune competent mouse model. CAVATAK was administered i.v, follow-up time, the data revealed positive signals in the responses of while anti mPD1 mAb was delivered via the i.p route and the IDO in- both on-target and off-target lesions to the combination of T-VEC and hibitor in drinking water. checkpoint inhibitor treatment. In addition, the results show a better re- Results sponse rate than previous publications using T-VEC. Ongoing clinical tri- Notable single agent antitumor activity against the B16-ICAM-1 als will elucidate the true clinical benefit of this combinational therapy. tumors was only observed in mice treated with anti-PD1 blockade relative to saline controls. Significant survival benefits were only ob- P294 served in mice treated with the CVA21-anti-mPD1 doublet or the Cost of adverse events associated with immunotherapy CVA21, anti-PD1 and IDO inhibitor triplet combinations. While not monotherapy versus targeted therapy in elderly metastatic significant, we observed a positive trend in both reduction of overall melanoma patients tumor burden and survival in mice treated with the CVA21, anti- 1 1 2 2 Jackson Tang , Zhiyi Li , Syed Mahmood , Sameer Ghate mPD1 doublet and the CVA21, anti-mPD1 and IDO inhibitor triplet 1 2 Asclepius Analytics, New York, NY, USA; Novartis Pharmaceuticals compared with mice receiving anti-mPD1 blockade and IDO- Corporation, East Hanover, NJ, USA inhibition. All combinations of CVA21, anti-mPD1 and IDO blockade Correspondence: Jackson Tang (jtang@asclepiusanalytics.com) appeared to be generally well tolerated. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P294 Conclusions The notable anti-tumor activity and survival benefit mediated by the Background combination of CAVATAK, PD1 blockade and IDO inhibition observed in The National Comprehensive Cancer Network guidelines recommend the presented melanoma model supports potential clinical evaluation of the use of immunotherapy (IO) or targeted therapy (TT) (if BRAF- such a novel immunotherapeutic combination treatment regimen. mutated) for the first-line treatment of metastatic melanoma (MM). Both IO and TT can lead to treatment-related adverse events (AEs). P293 The objective of the study is to estimate and compare the cost of Talimogene Laherparepvec combined with anti-PD-1 based treatment-related AEs between elderly MM patients receiving IO immunotherapy for unresectable stage III-IV melanoma: a case monotherapy and those receiving TT. series Methods 1 2 2 2 Lillian Sun , Pauline Funchain , Jung-Min Song , Michael McNamara , A retrospective cohort study was conducted using Medicare research Brian Gastman identifiable files from 2006 to 2014. Eligible patients had ≥1MM Cleveland Clinic Lerner College of Medicine of Case Western Reserve diagnosis and ≥1 prescription for an IO (ipilimumab or pembrolizumab) University, Cleveland, OH, USA; Cleveland Clinic Taussig Cancer Institute, or TT (dabrafenib, trametinib, vemurafenib, or dabrafenib+trametinib) Cleveland, OH, USA; Cleveland Clinic, Cleveland, OH, USA that was FDA-approved during the study period. Patients were Correspondence: Brian Gastman (sunl2@ccf.org) assigned to the IO or TT cohort based on the most recent therapy used, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P293 and had a minimum of 3 months follow-up within the study period. 10 categories of AEs were identified based on a review of IO or TT package Background inserts, as well as AEs that were common among MM patients. Cost per Checkpoint inhibitors have become standard of care for treating ad- AE was calculated as the average per-episode cost (inpatient + out- vanced melanoma. By blocking key inhibitory pathways, checkpoint in- patient) incurred in the 30 days after the first occurrence of the AE. hibitors reinvigorate effector T cells to overcome an immunosuppressed Costs were inflated to 2017 USD and compared between the cohorts microenvironment. A key preceding step to effective tumor killing is the using the Wilcoxon rank-sum test, with a significance level of 0.05. release and presentation of tumor antigen. Talimogene Laherparepvec Results (T-VEC) is an oncolytic virus recently approved as an intratumoral therapy The study included 266 IO patients and 159 TT patients. The TT co- for treating unresectable stage IIIB-IV metastatic melanoma. As monother- hort was younger (72.8±14.3 vs. 73.7±12.6 years; p=0.04) and less apy, it confers modest efficacy in controlling disease progression in pa- likely to be male (57.2% vs. 63.2%; p=0.01) compared with the IO co- tients with locoregional lesions. T-VEC is used to directly lyse tumor cells hort. The 30-day AE costs for each cohort are summarized in Table 1. and promote anti-tumor immunity via the release of tumor antigens and The average costs associated with gastrointestinal, respiratory, and virus-encoded GM-CSF. The mechanisms of action for T-VEC and check- pain-related AEs were significantly higher in the IO cohort than the point inhibitors are highly complementary, raising the possibility of syner- TT cohort (difference: $9270, $2654, and $2155, respectively; p<0.01). gistic benefit from combining these two therapies. In contrast, the average costs associated with pyrexia and/or chills, Methods and other AEs (including decreased appetite/anorexia, fatigue, or in- We reviewed 10 consecutive cases of stage IIIC to stage IVM1b mel- fections) were significantly lower in the IO cohort (difference: -$5761, anoma patients that received T-VEC plus either pembrolizumab or -$598 respectively; p<0.05). There was no significant difference in the ipilimumab/nivolumab, treated between January 2016 and July 2017 costs associated with hematologic/lymphatic, central nervous sys- at the Cleveland Clinic with a median follow-up of 7 months (range: tem/psychiatric, metabolic/nutritional, skin/subcutaneous tissue, or 4 to 13 months). Responses of injected (on-target) and uninjected cardiovascular AEs. (off-target) lesions were evaluated according to RECIST 2.0. Conclusions Results The costs associated with treatment-related AEs among elderly MM The overall response rate for on-target lesions was 90%, with 5 patients patients were substantial. Patients treated with IO incurred higher experiencing a complete response of injected lesions. Two patients had costs after gastrointestinal, respiratory, and pain-related AEs, while off-target lesions, both of whom experienced complete response of patients treated with TT incurred higher costs after pyrexia/chills, de- their uninjected distant metastases. Overall survival of this cohort was creased appetite/anorexia, fatigue, or infections. 80%. Of the two patients who died, one died of causes unrelated to melanoma. Checkpoint inhibitor therapy was interrupted for 2 patients due to adverse events, with one patient experiencing Grade 3 nephritis P295 and the other patient experiencing Grade 3 diarrhea. There were 2 pa- Economic burden of adverse events associated with tients who experienced progression of disease. Taking this into account immunotherapy and targeted therapy for metastatic melanoma in with the 2 patients who died, overall progression-free survival for this the US elderly population 1 1 2 2 cohort was 60%. , Zhiyi Li , Syed Mahmood , Sameer Ghate Jackson Tang 1 2 Conclusions Asclepius Analytics, New York, NY, USA; Novartis Pharmaceuticals There may be potential synergistic benefit in combining checkpoint in- Corporation, East Hanover, NJ, USA hibitors with T-VEC injection in patients with unresectable melanoma. Correspondence: Jackson Tang (jtang@asclepiusanalytics.com) Although this study is limited by the lack of randomization and short Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P295 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 151 of 244 Background Exosomes are secreted by cancer cells and can carry dsDNA. Thus, The National Comprehensive Cancer Network guidelines for the first- we also tested whether tumor-derived exosomes (TEX) can deliver line treatment of metastatic melanoma (MM) recommend the use of dsDNA to DCs. immunotherapy (IO) or targeted therapy (TT) (if BRAF-mutated), both Methods of which are associated with treatment-related adverse events (AEs). TEX were purified from supernatants of mouse carcinoma TSA or TSA KI Trex1 The occurrence of AEs is associated with increased healthcare re- knock-in for Trex1 (TSA ) cells that were mock-treated (UT-TEX), source use and costs. The objective of this study is to estimate the or irradiated with 3 doses of 8Gy (RT-TEX). Protein composition and incremental costs of experiencing treatment-related AEs among eld- content of dsDNA was analyzed. TEX were incubated in vitro with erly MM patients receiving IO monotherapy or TT. primary DCs, or used to vaccinate BALB/c mice (n=6) by 3 s.c. injec- Methods tions followed by challenge with TSA cells to evaluate the develop- A retrospective cohort study was conducted using Medicare research ment of protective anti-tumor immunity. Tumor-specific CD8 T cells identifiable files from 2006 to 2014. Eligible patients had ≥1MM were identified using H2-L /AH1 peptide pentamers. diagnosis, ≥1 prescription for an IO (ipilimumab or pembrolizumab) Results or TT (dabrafenib, trametinib, vemurafenib, or dabrafenib+trametinib) Double-stranded DNA content of RT-TEX was significantly higher that was FDA-approved during the study period, and a minimum of than that of UT-TEX. In vitro, RT-TEX but not UT-TEX induced the up- 3 months of follow-up. 10 categories of AEs were identified based on regulation CD40, CD80 and CD86 on DCs, and the production of a review of IO or TT package inserts, as well as AEs that were com- IFNb, which was dependent on STING expression by DCs. When KI Trex1 mon among MM. The incremental cost for each AE category was de- TREX1 is upregulated in TSA cells, dsDNA amount of RT-TEX termined by comparing the 30-day expenditures (inpatient + was markedly reduced, indicating that it is largely derived from cyto- outpatient) between patients with the AE versus patients without the solic dsDNA present in the irradiated parent cells. Most importantly, AE using a generalized linear model with a log-link function and enforced TREX1 expression abrogated the ability to RT-TEX to induce gamma distribution, adjusting for baseline covariates. All costs were IFN-I in recipient DCs. In vivo, vaccination with UT-TEX led to 100% inflated to 2017 USD. tumor outgrowth, while 2/6 mice vaccinated with RT-TEX were pro- Results tected from tumor development. Remaining tumors grew signifi- A total of 425 patients were included. The mean age was 73.4 years cantly slower compared to UT-TEX treated animals. Tumor-specific (SD±13.8), 61% were male, 94% were white, and the mean baseline CD8 T cells were significantly increased in the tumors and spleen of Charlson comorbidity index was 8.5 (SD±2.3). Table 1 summarizes RT-TEX vaccinated mice. the adjusted 30-day incremental cost of each AE. The adjusted 30- Conclusions day incremental cost was highest for respiratory AEs ($24,150; 95% Overall these results identify RT-TEX as a mechanism whereby IFN- confidence interval [CI] $17,630–30,671), followed by central nervous stimulatory dsDNA is transferred from cancer cells to DCs. Import- system/psychiatric disorders ($21,932; 95% CI $16,011–27,854), meta- antly, they also demonstrate that TREX1 in the irradiated cancer cells bolic/nutritional disorders ($19,776; 95% CI $14,239–25,314), skin/ regulate the production of IFN-I by DCs. Findings further support the subcutaneous tissue AEs ($19,183; 95% CI $14,195–24,170), fever use of RT doses that do not induce TREX1 to achieve in situ vaccin- (pyrexia) and/or chills ($18,976; 95% CI $13,663–24,289), pain ation by RT. ($18,406; 95% CI $13,805–23,008), cardiovascular AEs ($16,393; 95% CI $12,131–20,655), hematologic/lymphatic AEs ($15,850; 95% CI P297 $11,888–19,813), gastrointestinal AEs ($13,699; 95% CI $10,138– Predicting the efficacy of combination immunotherapy in animal 17,261), and other AEs (e.g. decreased appetite/anorexia fatigue, in- models using tumor microenvironment immune cell profiling fections (including folliculitis)) ($9,754; 95% CI $7,315–12,192). Ava Vila-Leahey, Genevieve Weir, Alecia MacKay, Valarmathy Kaliaperumal, Conclusions Cynthia Tram, Marianne Stanford Among elderly MM patients in the United States, the incremental Immunovaccine, Halifax, NS, Canada costs of treatment-related AEs are substantial. These findings may Correspondence: Ava Vila-Leahey (avilaleahey@imvaccine.com) help to inform comparisons between treatments, and thereby aid in Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P297 clinical and budgetary decision-making in this population. Background P296 Due to the complex nature of the tumor microenvironment, combi- Exosomes shuttle TREX1-sensitive IFN-stimulatory dsDNA from nations of immune modulating compounds are likely required for irradiated cancer cells to dendritic cells maximal clinical benefit in advanced cancer patients. In our previous 1 1 1 1 Claire Vanpouille-Box , Julie Diamond , Nils Rudqvist , Karsten Pilones , work, we have demonstrated that a therapeutic cancer vaccine com- 1 2 1 Yasmeen Sarfraz , Silvia Formenti , Sandra Demaria bined with metronomic cyclophosphamide (mCPA) can enhance T 1 2 Weill Cornell Medicine, New York, NY, USA; Weill Cornell, New York, cell infiltration of the tumor. This treatment can be added to treat- NY, USA ments such as anti-PD-1 to provide enhanced immune infiltration Correspondence: Sandra Demaria (claire.vanpouille.box@gmail.com) and better tumor control in murine models. A part of this increased Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P296 efficacy was due to the increased levels of these molecules in the tumors of treated animals. In this study, we have conducted a system- Background atic flow cytometry survey of checkpoint molecules expressed on T Radiotherapy (RT) used at immunogenic doses leads to accumulation cells within the tumor microenvironment after immune therapy, and of cytosolic dsDNA in the cancer cells, which activates interferon type utilized this to predict the potential of combining vaccine therapy with I (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is re- other clinical stage antibodies targeting the checkpoint system. quired to recruit BATF3-dependent dendritic cells (DCs) to poorly im- Methods munogenic tumors and trigger anti-tumor immune responses in HPV16 E7-transformed C3 cells were implanted subcutaneously into combination with immunotherapies. Importantly, we have recently C57BL/6 mice, and then treated with mCPA (20mg/kg/day PO), TM demonstrated that TREX1 regulates radiation immunogenicity by de- followed by vaccination with HPV16 E7 peptide in a DepoVax 49-57 grading cytosolic dsDNA (Vanpouille-Box et al., 2017 Nat Commun). platform (DPX-R9F). Additional mice were also treated with anti-PD-1 Tumor-derived dsDNA has also been shown to be critical for cGAS/ (200 mg) or anti-OX40 (100 mg). Tumors were collected for analysis STING-mediated IFN-I by tumor-infiltrated DCs (Woo et al., 2014 Im- 10 days post-vaccine treatment, and digested for staining for flow cy- munity). Here we hypothesized that activation of DCs by tumor- tometry to observe differences in infiltrating immune cells and derived dsDNA is modulated by RT and regulated by TREX1. markers on tumor infiltrating immune cells. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 152 of 244 Results Nonetheless, there are some important challenges with using the We found that expression of the inhibitory checkpoint markers such 4T1 model, in particular with studying the activity of T cell check- as PD-1 and TIM-3 were increased, but activating checkpoint mole- point inhibitors and costimulatory antibodies. Mice bearing 4T1 tu- cules GITR and OX40 were not increased, on T cells after treatment mors develop a fatal pulmonary hypersensitivity upon repeated with DPX/mCPA, and no checkpoint molecules are altered further treatment with rat antibodies to PD-1, PD-L1 or OX40. 4T1 tumors with either anti-PD-1 or OX40 antibody. We performed a tumor chal- are completely resistant to PD-1 pathway blockade and the hyper- lenge study with the C3 model and antibodies targeting either PD-1, sensitivity reaction makes it challenging to perform combination GITR or OX40 to determine whether targeting a molecule upregu- studies with novel test agents. lated by the vaccine would have greater effect than targeting a mol- Methods ecule that is not altered. Antibodies targeting PD-1, GITR or OX40 We have characterized two mammary cancer alternatives to 4T1, had significant synergistic activity with DPX-R9F and mCPA treat- EMT-6 and E0771. The in vivo growth characteristics and baseline im- ment, but anti-PD-1 antibody was most potent in tumor growth in- mune cell profiling have been completed for these models with hibition. (Fig. 1) some notable immune profile differences. Further, we have com- Conclusions pleted an efficacy study of E0771 using an array of immunotherapies. In conclusion, we have found that an extensive tumor immune pro- Results file of treatment regimes in animal models can accurately predict the All three models have a similar proportion of CD4+ T cells and Tregs. efficacy of combination immunotherapy, and activating checkpoint In contrast, 4T1 has a much larger proportion of G-MDSCs while molecules do not necessarily enhance the efficacy of treatment in E0771 is almost entirely lacking in G-MDSCs with EMT-6 in between our tested models. Current studies are exploring additional check- the two. The content of monocytic MDCSs (M-MDSCs) is nearly recip- point molecules in this model that were shown to be elevated in rocal with E0771 having a proportionately high population and 4T1 tumor infiltrating CD8 T cells with vaccine treatment to maximize having a minimal number. These immune profile characteristics can combination immunotherapy potential. be factored into model selection decisions. An efficacy study in the E0771 model showed that it is highly sensi- tive to several checkpoint inhibitors, including anti-PD-1, anti-PD-L1, and anti-CTLA-4. E0771 shows a more modest response to anti-LAG3. Costimulatory agonist antibodies to OX40, CD137 (4-1BB) and gluco- corticoid induced TNFR-related protein (GITR) were highly active. The indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor epacadostat had no impact on the growth of E0771. Conclusions These data enable rational combination strategies and provide alter- natives to breast cancer studies in 4T1. P299 Cell therapy - TRAcking, Circulation, & Safety (CT-TRACS): The Health and Environmental Sciences Institute (HESI)’s new collaborative effort to address the challenges of cell therapies translation 1 1 2 3 Brooke Helfer , Charles O'Hanlon , Patricia Murray , Keiji Yamamoto , Fig. 1 (abstract P297). See text for description 4 5 6 7 Gregory Mullen , Carla Herberts , William Shingleton , Lucilia Mouriès 1 2 Celsense, Inc, Pittsburgh, PA, USA; University of Liverpool, Liverpool L69 3BX, United Kingdom; Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan; King's College London, London, United Emerging Models and Imaging Kingdom; Medicines Evaluation Board, Netherlands, 3531 AH Utrecht, 6 7 P298 Netherlands; GE Healthcare, Amersham, United Kingdom; ILSI Health Utilization of murine breast cancer models in preclinical immuno- and Environmental Sciences Institute, Washington, DC, USA oncology pharmacology Correspondence: Brooke Helfer (brooke@celsense.com) Dylan Daniel, Sumithra Urs, Kevin Guley, David Draper, Alden Wong, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P299 Dan Saims, Scott Wise, Maryland Rosenfeld Franklin MI Bioresearch, Ann Arbor, MI, USA Background Correspondence: Dylan Daniel (ddaniel@molecularimaging.com) Cell therapies show great therapeutic promise in the field of im- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P298 munotherapy. To realize their full clinical potential there is a need for greater understanding of their mode of action, migration after ad- Background ministration, delivery, persistence at sites of action, and whether their The field of immuno-oncology is persistently challenged by the need localization or distribution may cause safety issues. There are several for more syngeneic mouse models in any given tissue type. The most existing and emerging tools available to develop pharmacokinetics prevalently used syngeneic model for breast cancer is the 4T1 mam- data on cell-derived therapies to improve our understanding but mary cancer cell line. The 4T1 cell line has useful traits for immuno- adoption by investigators has been limited. Furthermore, the regula- oncology research including a highly metastatic phenotype that tory landscape is not clearly defined for these emerging therapeutics. leads to extensive lymph node and lung metastasis. The tumors have Methods a highly immunosuppressed microenvironment with regulatory T The Health and Environmental Sciences Institute’s Emerging Issues cells (Tregs) and large numbers of granulocytic myeloid derived sup- Committee recently launched a multi-sector collaborative sub- pressor cells (G-MDSCs). Radiation can induce changes in an im- committee to identify key needs for assessing the safety of cell ther- munosuppressive microenvironment, and focal beam radiotherapy apies and identify opportunities to meet these needs. This program, remains an important therapeutic strategy for the treatment of the Cell Therapy - TRAcking, Circulation, & Safety (CT-TRACS) sub- breast cancer. For this reason, we established a radiation dose re- committee, provides a platform for developers, researchers, regula- sponse on established 4T1 tumors for the purpose of guiding future tors, imaging specialists and other stakeholders to interact, discuss immunotherapy combinations. challenges and identify best practices to ensure that therapies are Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 153 of 244 safe and effective. The sub-committee aims to bring awareness on subcutaneous and secondary disseminated tumor sites. CD19- how existing cell tracking technologies, methods, and best practices tPSMA CAR T cell treatment eradicated Nalm6 tumors from mice up can benefit the clinical translation of these new therapies. to 90 days post-treatment. PET/CT with [ F]DCFPyL visualized infil- Results tration of CAR T cells at primary and metastatic tumor sites, which Since its inception in December 2015, CT-TRACS gathered more than was confirmed by immunohistochemical analyses. 60 members from 25 organizations across the United States, Europe Conclusions and Japan. The sub-committee has convened monthly and the focus These preclinical results establish a new technology for whole body, of the group has been narrowed to Cell Fate, i.e., distribution, sur- non-invasive tracking of CAR T cells and support translation into the vival/engraftment and phenotype, post-administration, in vivo, as clinic. well as evaluating the tumorigenic potential of cell-based therapies. Our initial goals have been to: 1. evaluate current cell-based therap- P301 ies safety assessment practice and tools; 2. develop best practices for A dual in vivo and in silico system to model tertiary lymphoid application of available tools for safety assessment of cell therapies structure formation and anti-tumor immune response in the and/or identify gaps in safety assessment; 3. organize a workshop to murine tumor microenvironment present findings of the sub-teams and develop recommendations for Rana Falahat, Yohsuke Yagawa, Mark Robertson-Tessi, Susan L. Zhou, next steps; and 4. initiate a manuscript describing the needs and Alexander Anderson, James J. Mulé, Adam Mailloux gaps identified, to build confidence in safety assessment approaches H. Lee Moffitt Cancer Center, Tampa, FL, USA for clinical applications. To date, we have held our first scientific ses- Correspondence: Adam Mailloux (adam.mailloux@moffitt.org) sion at an international cell therapy meeting and are compiling the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P301 results of a survey of therapeutic stakeholders’ needs and wishes in the imaging of cellular therapeutics, survey shared herein. Background Conclusions Tertiary Lymphoid Structures (TLS) are highly organized foci of lym- The CT-TRACS project is open to all current HESI members as well as phocytes and antigen-presenting cells that predict increased survival new participants with relevant technical expertise. The program across multiple solid tumors, and are associated with a previously encourages inquiries by those with interest in providing support for identified gene expression signature composed of twelve chemo- these innovative efforts. kines (12-CK-GES) [1]. These chemokines likely result from chronic lymphotoxin-beta receptor activation on resident stromal cells or by P300 paracrine production involving subsequent infiltrates. The exact na- PSMA-associated PET imaging of CAR T cells ture and timing of this induction is unclear due in part to insufficient 1 2 1 2 1 IL Minn , David Huss , Hye-Hyun Ahn , Tamara Chinn , Andrew Park , models of TLS formation. Here, we build upon insights gained from 2 1 1 1 1 Jon Jones , Mary Brummet , Steven Rowe , Polina Sysa-Shah , Yong Du , the 12-CK-GES using an implantable three-dimensional bioscaffold to 2 1 Hyam Levitsky , Martin Pomper study the interaction of developing murine tumor, endogenous infil- 1 2 Johns Hopkins Medical Institutions, Baltimore, MD, USA; Juno trate, and implanted stroma. Concurrently, we use this system to Therapeutics, Seattle, WA, USA parameterize an integrated mathematical model of the microenviron- Correspondence: Hyam Levitsky (david.huss@junotherapeutics.com); ment, which can then re-inform our three-dimensional bioscaffold Martin Pomper model. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P300 Methods Injectable bioscaffolds were prepared using primary lymph node Background reticular fibroblast cells implanted in either Matrigel or chitosan Chimeric antigen receptor (CAR) T cells have demonstrated clinical hydrogels loaded with recombinant Lymphotoxin-α1β2 (LT) or indi- benefit in numerous hematological malignancies and hold promise vidual chemokines from the 12-CK-GES, with or without murine MC- for application in solid tumors. As a “living drug”, CAR T cells traffic 38 colon carcinoma cells. In some experiments, recombinant factors throughout the body with dynamic expansion and contraction kinet- were loaded in lipid-coated silica microparticles to delay release. ics. This dynamic nature limits the utility of standard blood pharma- Preparations were then injected subcutaneously in C57BL/6 mice. Im- cokinetic analyses where the temporal and spatial distribution of plants were resected at later time points and dissociated for flow cy- CAR T cells is only partially captured. Therefore, a non-invasive im- tometry or cryosectioned for histology and immunofluorescent aging technique that allows tracking of CAR T cells may enhance the staining. Resulting data, along with those from in vitro chemotaxis as- understanding of in vivo CAR-T behavior and inform associations with says, were used to parameterize an in silico model of TLS formation safety and efficacy. Previous attempts to track gene modified T cell simulated in two dimensions using fixed stromal cells, three types of therapies with PET imaging have been limited by immunogenicity of immune cells, and discrete chemokine fields. PET reporters, poor tissue penetration of large molecular weight PET Results ligands and/or limited sensitivity. LT, when implanted with stromal cells, can induce organized aggre- Methods gates of endogenous lymphocytes, significantly increase infiltration To improve the ability to track CAR T cells, we engineered CD19- of T cells, B cells, and dendritic cells (all p<0.05), and prevent the directed CAR T cells to express a truncated form of human prostate- growth of MC-38 tumors in resected implants (p<0.001). In silico specific membrane antigen (tPSMA) as a reporter. We then used the model runs predict polarized activation of stromal cells and subse- PSMA-directed small molecule PET ligand [ F]DCFPyL for in vitro quent production of CCL19, CCL21, and CXCL13 is sufficient to and in vivo CAR T visualization. compartmentalize lymphoid aggregates into discrete B and T cell Results zones and promote anti-tumor activity. We demonstrate high level surface co-expression of tPSMA and the Conclusions CD19-directed CAR. The addition of tPSMA to the CAR T cells did This dual-model system has identified an important role for LT activa- not impact in vitro or in vivo anti-tumor functionality. Phantom im- tion of stromal cells in the induction of TLSs and suggests components aging studies demonstrated that PET with [ F]DCFPyL could reli- of the 12-CK-GES are vital for TLS organization. ably detect as few as 4000 CD19-tPSMA CAR T cells in a 50uL volume. We developed a spontaneous metastatic acute lympho- References blastic leukemia model by subcutaneously injecting CD19+ Nalm6- 1. Messina JL, Fenstermacher DA, Eschrich S, et al. 12-Chemokine gene ffLuc-eGFP tumor cells into immunodeficient NSG mice. Metastatic signature identifies lymph node-like structures in melanoma: potential lesions were observed in liver, spleen and bone marrow, providing for patient selection for immunotherapy? Scientific Reports 2012; an opportunity to evaluate CAR T infiltration into primary 2:765-770. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 154 of 244 P302 Background Characterizing immunotherapy-induced lymphocyte infiltration at Melanoma patients develop resistance to both chemo- and TM the single patient level using CANscript ,an ex-vivo human targeted-therapy drugs. Promising pre-clinical and clinical results tumor model with immune checkpoint inhibitors using antibodies directed Munisha Smalley, Basavaraja Shanthappa, Hans Gertje, Mark Lawson, against CTLA-4 and PD-1 have re-energized the field of immune- Baraneedharan Ulaganathan, Allen Thayakumar, Laura Maciejko, based therapies in melanoma. However, only a third of melanoma Padhma Radhakrishnan, Aaron Goldman patients respond to immune checkpoint blockade. Currently avail- Mitra Biotech, Woburn, MA, USA able mouse xenograft and transgenic mouse melanoma models Correspondence: Aaron Goldman (msmalley@mitrabiotech.com) have several short comings and are unable to address the basis of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P302 drug resistance and immune non-responsiveness that are fre- quently observed in melanoma patients. Thus, there is an urgent Background need to establish an in vivo model with a human immune micro- The presence and activity of lymphocytes within the tumor is critical environment that can address issues of therapy resistance. for clinical response to cancer immunotherapy, such as immune check- Methods point blockade. Poor lymphocyte infiltration into the tumor, known as For this, our laboratory has developed a humanized mouse melan- a ‘cold’ phenotype, is associated with modest clinical response. High oma model using patient-derived xenografts (PDX). Immunodeficient baseline infiltration of effector lymphocytes is considered ‘hot’,and pa- NSG mice are reconstituted with human CD34+ cells and after 7-9 tients are predicted to respond more favorably to treatment. Despite weeks, mature human CD45+ cells are observed in circulating blood. these fundamental predictive biomarkers, patient-to-patient response Humanized mice were then challenged with HLA-matched melan- and durability remains highly variable. There is an urgent gap in avail- oma PDX and the functional ability of human immune cells to restrict able methods to study lymphocyte infiltration, trafficking and spatial tumor growth is monitored. heterogeneity induced by different cancer immunotherapies in individ- Results ual patients. Delayed tumor growth was observed in humanized mice indicating Methods in-vivo sensitization of human immune cells to melanoma. This was TM Here, we used CANscript ,an ex-vivo human tumor model that re- confirmed by in-vitro demonstration of human lymphocytes from capitulates and preserves the native, patient-autologous tumor tumor-bearing mice showing enhanced cytokine expression after microenvironment, including peripheral blood mononucleated cells stimulation with melanoma antigen peptides. Further, cytotoxic T- (PBNC). Utilizing tissue from breast cancer patients classified as either cells derived from melanoma peptide stimulation could functionally ‘cold’ (N=5) or ‘hot’ (N=5), we studied lymphocyte infiltration under lyse tumor cells in vitro. In preliminary therapy studies, most tumor- pressure of immune checkpoint blockade for 72h using pembrolizu- bearing humanized mice treated with anti-PD-1 antibody showed mab (a-PD-1) and avelumab (a-PDL-1). Using flow cytometric analysis, restricted tumor growth. Anti-PD-1 antibody therapy resulted in we characterized infiltrating lymphocytes, and coupled these data enhanced infiltration of CD4+ and CD8+ T-cells that correlated with + + + + with multiplex immunohistochemistry (CD3 , CD4 , CD8 , CD56 , tumor response. CD25 ) to map proximity of tumor cells to lymphocytes before and Conclusions after treatment, ex-vivo. Our results suggest that humanized mouse melanoma model can be Results explored further to understand the causes of therapy resistance and We determined that immune checkpoint blockade induced immune non-responsiveness. unique patterns of migration and infiltration of effector T-cells (T ), T-regulatory (T ) cells and natural killer (NK)-cells in ‘hot’ eff reg P304 vs ‘cold’ tumors. Furthermore, we determined that, in some in- Predicting pre-clinical tumour response to anti-PD-1 stances, ‘cold’ tumors can be driven towards a ‘hot’ phenotype immunotherapy with computational modelling characterized by trafficking of active immune lymphocytes follow- 1,2 1,2 3 3 Damijan Valentinuzzi , Urban Simončič , Katja Uršič , Martina Vrankar , ing treatment, which corresponded to differential ratio of T to eff 2 1,2,4 Maruša Turk , Robert Jeraj T compared to baseline. reg 1 2 Jožef Stefan Institute, Ljubljana, Slovenia; Faculty of Mathematics and Conclusions TM 3 Physics – University of Ljubljana, Ljubljana, Slovenia; Institute of Taken together, these data demonstrate the utility of CANscript as Oncology Ljubljana, Ljubljana, Slovenia, Ljubljana, Slovenia; University of a platform to characterize response to immunotherapy in a spatial Wisconsin – Madison (WI) context. Such an advance in our preclinical methods to study Correspondence: Damijan Valentinuzzi (damijan.valentinuzzi@ijs.si) immuno-modulators at the individual patient level can help guide Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P304 treatment decisions for clinicians while simultaneously functioning as a platform to study and discover mechanisms of clinical efficacy for Background emerging drugs. Majority of patients fail to respond to anti-programmed death-1 (anti-PD-1) immunotherapy and the reasons for this remain largely References unknown. The aim of this study was to develop a computational 1. Brijwani N, Jain M, Dhandapani M. Rationally co-targeting divergent model, able to predict antitumour response to anti-PD-1 on the basis pathways in KRAS wild-type colorectal cancers by CANscript technology of key biological properties that might serve as predictive biomarkers reveals tumor dependence on Notch and Erbb2, Sci. Rep. 2017;7:1502-1513. of response. 2. Majumder B, Baraneedharan U, Thiyagarajan S. Predicting clinical response Methods to anticancer drugs using an ex vivo platform that captures tumour Interplay between tumour cells and tumour infiltrating lymphocytes heterogeneity, Nat. Comm. 2015;6:6169-6183. (TILs) is described with deterministic population model. It incorporates intrinsic tumour parameters (growth rate (k)), as well as other biological P303 parameters, such as major histocompatibility complex (MHC) class I and Immune checkpoint inhibitor responses in humanized mouse PD-1 ligand (PD-L1) appearance on tumour cells, PD-1 appearance on melanoma models using patient-derived xenografts TILs, anti-PD-1 pharmacodynamics, dosing and scheduling regimen, etc. Rajasekharan Somasundaram, Marilda Bequiri, Ling Li, Meenhard Herlyn, Most of the parameters were taken from literature. The remaining free Meaghan Kiernan, Kar Muthumani, Hyeree Choi parameters (k, TILs infiltration rate, tumour cells-TILs interaction rate) The Wistar Institute, Philadelphia, PA, USA were fitted to experimental data, where B16-F10 melanoma was treated Correspondence: Rajasekharan Somasundaram (shyam@wistar.org) with anti-PD-1 [1]. Predictive ability of the model was tested on Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P303 independent experiment from literature, namely B16-OVA treated with Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 155 of 244 anti-PD-1 [2]. Simulated tumour growth curves were compared to aimed to characterize the immune infiltrates at different stages of experimental data and sensitivity study of key parameters was carcinogenesis and elucidate the role of different cell subtypes in performed. tumor development and progression and the possible causal rela- Results tionship between the immune phenotypes in pre-neoplastic lesions Simulated tumour growth curves are in good agreement with ex- and tumor progression and patient prognosis. perimental data. Maximum deviation of simulated control tumour Conclusions volume (solid line – blue) to experimental data (blue squares) is Evaluation of the immune contexture and prognostic assessment of −25% (day 13). On the other hand, the model slightly underesti- precancerous lesions of the lung may identify promising new bio- mates the effect of treatment with anti-PD-1. Maximum deviation markers for early detection and targets of novel therapeutic strat- of simulated anti-PD-1-treated tumour volume (solid line – red) to egies for lung cancer. experimental data (red squares) is +40% (day 17). Sensitivity study reveals that dosing and scheduling regimen does not importantly affect treatment outcome (data not shown). The most sensitive par- Immune Modulation, Cytokines, and Antibodies ameter of the model is MHC class I appearance. By modulating MHC class I appearance from 1% to 100%, while keeping other pa- P306 rameters fixed, we are able to simulate responders as well as non- A protein extract from fermented wheat germ promotes NK responders to anti-PD-1. cell-mediated lymphoma eradication in mouse xenografts Conclusions 1 1 1 1 Gustavo Barisone , Yunpeng Ma , Mastewal Abuhay , Robert O'Donnell , Model predictions of antitumour response to anti-PD-1 are within ex- 1 1 2 1 Kathleen Lundeberg , Sonia Gowda , William Murphy , Joseph Tuscano pectations. The predictions might be even more reliable if model pa- 1 2 University of California Davis, Sacramento, CA, USA; University of rameters were, due to their inter-patient variability and presumably California, Sacramento, CA, USA dynamic nature, actually measured for every specific experiment/pa- Correspondence: Joseph Tuscano (gabarisone@ucdavis.edu) tient. The emphasis should be on MHC class I appearance as it might Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P306 be one of the predictive biomarkers of response to anti-PD-1. Background References Proteomic and genomic data has allowed for the development of 1. Kleffel, Sonja, et al. Melanoma cell-intrinsic PD-1 receptor functions promising targeted agents for NHL [1]. Most have acute and chronic promote tumor growth. Cell. 2015; 162.6:1242-1256. toxicities that limit efficacy. The use of complementary and alterna- 2. Sánchez-Paulete, Alfonso R., et al. Cancer immunotherapy with tive medicines has increased during the last decade. However, scien- immunomodulatory anti-CD137 and anti–PD-1 monoclonal antibodies tific evidence of their efficacy is scarce. Fermented wheat germ requires BATF3-dependent dendritic cells. Cancer discovery. 2016; extract (FWGE) has been claimed to have anti-cancer properties in 6.1:71-79. many tumor types. FWGE therapeutic activity has been attributed to its content of benzoquinones [2]. Methods A protein fraction (FWGP) was isolated by FPLC and proteins identi- P305 fied by mass spectrometry. Direct cytotoxic was studied in vitro using Unravelling the immune contexture of pre-invasive lesions of the NHL cell lines. Immunomodulatory properties were evaluated ex vivo lung by multispectral imaging 1 2 1 1 by measure immune cell activation in human PBMCs and isolated NK Angela Vasaturo , Celine Mascaux , Mihaela Angelova ,Benedict Buttard , 3 1 cells. In vivo experiments used nu/nu NHL xenografts with or without Jean-Paul Sculier ,JeromeGalon NK cell depletion; endpoints were tumor volume and toxicity. In vivo Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, immunomodulatory effects were evaluated by treating tumor-free Sorbonne Universités, Paris, France; Aix Marseille University, APHM, BALB/c mice with FWGP and measuring NK cell killing activity and Marseille, France; Institut Jules Bordet, Centre des Tumeurs de l'Université degranulation. Libre de Bruxelles, B-1000 Brussels, Belgium, Bruxelles, Belgium Results Correspondence: Jerome Galon (angelavasaturo@gmail.com) FWGP was cytotoxic in 17 cancer cell lines (IC50 = 20-171 μg/ml in Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P305 NHL, 12-27 μg/ml in colon and 70-144 μg/ml in lung) and induced apoptosis by increasing levels of caspase-3, PARP, BAK, BAD and p53, Background while reducing levels of AKT. FWGP increased % NK cells, production Lung cancer is the leading cause of cancer deaths world- wide and of IFg and GrB, and NK-mediated killing. In vivo efficacy was con- despite advances in therapy, the overall survival rate for lung cancer firmed, with no toxicity, in pre-emptive and established models. In patients remains only 15%. vivo treatment with FWGP+rituximab was as effective as R-CHOP, As most of sporadic cancers, lung cancer emerges from pre-neoplastic with 90% complete remission. NK depletion resulted in no response lesions characterized by morphological and molecular changes. If to FWGP. These results support the hypotheses that FWGP augments morphological changes of pre-invasive bronchial lesions are well char- NK-mediated tumor killing. Proteomic profiling identified 844 pro- acterized, the cause and effect relationship between those changes teins. An active fraction consisted of 169 proteins. and the immune response is still un- known. Though, we identified Conclusions gene expression alterations that suggest a role of the innate and adap- FWGP represents a promising immunomodulatory agent with anti- tive immunity in the transformation towards carcinoma. tumor activity, minimal toxicity and low cost. Our results suggest Methods In order to characterize the evolution of the immune response in FWGP has direct lymphomacidal activity by inducing apoptosis and indirect anti-tumor efficacy by enhancing NK-mediated tumor eradi- pre-invasive bronchial lesions, we have optimized different multi- cation. Further experimental validation will allow translation of al spectral 7 colors immunofluorescence panels, by using the Tyramide “alternative” product into mainstream medicine. Signal Amplification (TSA) technology. Results References We have performed multiplex staining on FFPE human bronchial bi- 1. Abuhay, M., et al. The HB22.7-vcMMAE antibody-drug conjugate has opsies (N=114) at 8 successive morphological stages of lung squa- efficacy against non-Hodgkin lymphoma mouse xenografts with minimal mous carcinogenesis, from normal, to low grades dysplasia, high systemic toxicity. Cancer Immunol Immunother. 2016; 65(10):1169-75. grades, to carcinoma. Images of each biopsy have been acquired 2. Hidvegi, M., et al. MSC, a new benzoquinone-containing natural product multispectrally and digitally analyzed to identify and quantify the with antimetastatic effect. Cancer Biother Radiopharm. 1999. 14(4):277-89. density and the tissue distribution of different immune cell types. We Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 156 of 244 P307 Background Expression and function of PD-1 and TIM-3 in non-small cell lung Immunotherapy with the combination of monoclonal antibodies that cancer (NSCLC) block PD-1 and CTLA-4 has shown clinical benefit beyond that ob- Jonathan Travers, Krtisten McEachern, Srimoyee Ghosh, Sridhar served with either mAb alone. A PD-1xCTLA-4 bispecific DART protein Ramaswamy, David Jenkins was designed to induce antitumor immunity through simultaneous TESARO Inc., Waltham, MA, USA targeting of both checkpoint pathways via administration of a single Correspondence: David Jenkins (dianna.bartel@ashfieldhealthcare.com) molecule. The DART protein increases checkpoint blocking activity on Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P307 PD-1/CTLA-4 dually expressing cells, while displaying distinct immuno- logical effects of CTLA-4 blockade in vivo absent evidence of toxicity. Background Methods The use of anti-programmed cell death protein 1 (PD-1) and PD-Ligand A PD-1xCTLA-4 DART protein was engineered as a tetravalent bispecific 1 agents in the treatment of non-small cell lung cancer (NSCLC) has molecule from humanized anti-PD-1 and anti-CTLA-4 mAb sequences been well established but many patients are either intrinsically resistant in a human hinge-stabilized IgG4 backbone. PK, PD and toxicology or become refractory during therapy. One potential resistance mechan- studies were performed in cynomolgus monkeys. ism is the upregulated expression of additional checkpoint receptors Results such as T cell immunoglobulin and mucin domain 3 (TIM-3), a trans- The PD-1xCTLA-4 DART molecule demonstrated binding to immobi- membrane receptor that binds multiple putative ligands, and that has lized PD-1 protein and PD-1-expressing cells lines, inhibition of PD-1 been shown to negatively regulate the function of T cells that co- interaction with PD-L1 or PD-L2, as well as reversal of PD-1-mediated express PD-1 [1]. T-cell signal inhibition in gene-reporter assays comparable to that Methods supported by a replica of nivolumab. Similarly, binding, ligand We examined the immunophenotype and checkpoint receptor ex- blocking and rescue of CTLA-4-mediated T-cell suppression was pression of over 100 NSCLC samples from primary surgical resections. comparable to that supported by a replica of ipilimumab. The DART From a subset of these samples, we evaluated T cell functional status molecule demonstrated activation properties comparable to the by gene expression analysis on sorted PD-1+ and TIM-3+ CD8+ T combination of ipilimumab and nivolumab replicas in a variety of hu- cells as well as ex vivo stimulation assays to evaluate cytokine pro- man primary T-cell assays and showed enhanced B7-ligand binding duction. Furthermore, we used ex vivo and in vivo studies to assess blockade over that mediated by the ipilimumab replica on PD-1/CTLA- the effect of blockade of PD-1 and TIM-3 alone and in combination 4 double-positive cells. In the cynomolgus monkey, the PD-1xCTLA-4 on T cell activation and anti-tumor activity. DART molecule exhibited a PK profile consistent with that of an IgG4 Results and was well tolerated, with no mortality or significant adverse findings We showed that primary NSCLC samples display heterogeneity in up to 75 mg/kg QWx3, the highest dose tested. T-cell expansion in the both their baseline immune infiltrate and also PD-1 and TIM-3 check- peripheral blood and lymphoid organs was observed, which was attrib- point receptor expression. We examined mRNA expression of mul- utable to the CTLA-4 blocking arm, since no such finding was observed tiple immune genes on sorted PD-1+ and TIM-3+ CD8+ T cells, and with similar or higher doses of the anti-PD-1 constituent of the bispeci- found that PD-1/TIM-3 double positive cells express reduced fic molecule. interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFα), but similar Conclusions mRNA levels of interferon gamma (IFNγ) when compared to double negative cells. This phenotype is recapitulated in CD8+ T cells de- PD-1xCTLA-4 DART protein binds and blocks its targets, with rived from patient samples stimulated with PMA and ionomycin, increased activity on dual PD-1/CTLA-4-expressing cells. where we found PD-1 and TIM-3 double positive cells to be signifi- The DART molecule enhances T-cell responses in vitro to the cantly deficient in IL-2, but not IFNγ production. Importantly, in level achieved by a combination of nivolumab and ipilimumab addition to their expression being associated with T-cell dysfunction, replicas. we also found that blockade of PD-1 and TIM-3 was associated with PD-1xCTLA-4 DART protein was well tolerated in cynomolgus increased T cell activation and anti-tumor activity in ex vivo and monkeys, with a safety profile similar to that observed with in vivo models, suggesting a potential functional role for the inhib- PD-1 blockade alone, while demonstrating biological effects of ition of TIM-3, in addition to PD-1, in the enhancement of anti-tumor CTLA-4 antagonism. immunity. Conclusions The favorable safety and tolerability profile of the PD-1xCTLA-4 DART Taken together, these data provide further evidence that TIM-3 may molecule combined with its enhanced activity on PD-1/CTLA-4 double- play a role in intrinsic resistance to single agent anti-PD1 therapy in positive cells suggest a potential for an improved therapeutic window NSCLC and support evaluating the combination of anti-PD-1 and for PD-1/CTLA-4 co-blockade strategies, with the administration of a anti-TIM-3 agents in the clinic. single molecule providing dosing convenience and ease of incorpor- ation into additional therapeutic regimens. References 1. Koyama, et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun. 2016;7:10501. P309 Treatment with heterodimeric IL-15 promotes effector T cell infiltration into tumors 1 1 1 1 P308 Cristina Bergamaschi , Konstantinos Dimas , Dimitrios Stellas , Bethany Nagy , 2 2 1 1 A PD-1 x CTLA-4 bispecific DART® protein with optimal dual Shawn Jensen ,Bernard Fox , Barbara Felber ,GeorgePavlakis 1 2 checkpoint blockade and favorable tolerability in non-human National Cancer Institute at Frederick, Frederick, MD, USA; Earle A primates Chiles Research Institute, Portland Providence Cancer Center, Portland, 1 1 1 1 Alexey Berezhnoy ,Kurt Stahl ,Kalpana Shah ,Tim Gaynutdinov , Gurunadh OR, USA 1 1 1 1 1 Chichili , Daorong Liu , Rebecca Johnson , Ross La Motte-Mohs , Jessica Hill , Correspondence: Cristina Bergamaschi (cristina.bergamaschi@nih.gov) 2 1 1 1 1 Jonathan Li ,Sergey Gorlatov ,Valentina Ciccarone , Ralf Alderson ,Hua Li , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P309 1 1 1 1 1 James Tamura , Jennifer Brown , Jon Wigginton ,EzioBonvini ,PaulMoore , Syd Johnson Background 1 2 Macrogenics, INC, Rockville, MD, USA; Macrogenics, INC, South San The presence of tumor-infiltrating effector T cells is considered the Fancisco, CA, USA most predictive biomarker for clinical benefit in response to immuno- Correspondence: Alexey Berezhnoy (berezhnoya@macrogenics.com) therapies. IL-15 is a cytokine important for the proliferation, activa- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P308 tion and mobilization of lymphocytes, including natural killer and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 157 of 244 CD8 T cells. We have previously shown that bioactive IL-15 in vivo Methods comprises a complex of the IL-15 chain with the IL-15 receptor alpha Western Blotting and multiplex assays of tumors and sera of anti-mouse chain that are together termed heterodimeric IL-15 (hetIL-15). Several CSF1R mAb (clone 2G2)-treated mice showed an association of TAM preclinical models have indicated the ability of IL-15 to enhance the elimination with an early intratumoral and systemic release of a specific response of the immune system against cancer, and based on these set of MMPs, including MMP-2, -3 and -8, in multiple transplant (MC38, results hetIL-15 has advanced to clinical trials. E0771, KPL-4 and PyMT) and de novo (MMTV-PyMT) tumor models. Methods Results We have produced hetIL-15 and tested its anti-tumor activity in In addition, we found that the early increase of MMPs in the face of several murine cancer models. Analysis of lymphocytes in lymphoid CSF1/CSF1R pathway blockade was independent of tumor burden organs and in tumors was performed by flow cytometry and multi- and was accompanied by a significant increase of body weight in color immunohistochemistry. Chemokine and cytokine levels were tumor-free mice following long-term exposure to the antibody. The determined using electrochemiluminescence (MSD) and ELISA assays. body weight gain may therefore indicate the enhanced retention of Results body fluids. Discontinuation of the CSF1R antibody reinstated un- Repeated injections of hetIL-15 in mice were effective in delaying altered body weight and MMP levels. We excluded platelets and neu- tumor growth in the MC38 colon carcinoma, TC-1 cervical carcinoma trophils as sources of MMP release, even if they accumulated in and B16 melanoma models. The combination of hetIL-15 and adop- tumor-bearing mice during CSF1R inhibition. We also examined the tive cell transfer of melanoma specific Pmel-1 cells showed anti- effects of blocking the CSF1R ligand CSF1, which is one of the two tumor efficacy in B16-bearing mice in absence of lymphodepletion. known ligands of the CSF1R [2,4]. CSF1 antibodies had no impact on The E0771 orthotopic breast cancer model showed delay in tumor the binding of IL-34 (the second CSF1R ligand) to CSF1R. Similar to progression and significantly reduced lung metastasis upon hetIL-15 CSF1R blockade, an anti-mouse CSF1 antibody (clone 5A1) provoked treatment. A significantly reduced onset of lung metastasis was also an early systemic surge of a subset of MMPs. observed in 4T1 breast cancer-bearing mice. Flow cytometry and Conclusions multi-color immunohistochemistry assays showed increased traffick- Collectively, our data suggest that CSF1 and CSF1R blocking antibodies ing and persistence of CD8 T cells, including tumor specific T cells, induce the release of a distinct set of matrix-degrading proteases into the tumors and an increased CD8 /Treg ratio, upon hetIL-15 ad- (MMP-2,-3 and -8), but not metastasis-promoting proteases (MMP-9 ministration. Importantly, hetIL-15 treatment led to preferential en- and -12), which may be potentially causative of edema formation. Fur- richment of adoptively transferred tumor-specific CD8 T cells in the ther studies may inform optimized dosing schedules of CSF1/CSF1R tar- B16 tumor in an antigen-dependent manner. Tumor infiltration by geting regimens for cancer patients. CD8 T cells was accompanied by increased plasma levels of CXCL10. Tumor-resident CD8 T cells showed features of activated effector References cells with enhanced proliferation (Ki67 ) and high cytotoxic potential 1. Ries CH, et al. Cancer Cell 2014;25(6):846-59. (Granzyme B ). Upon ex-vivo stimulation, an increased frequency of 2. Cassier PA, et al. Lancet Oncol 2015;16(8):949-56. + + both CD8 and CD4 T cells producing IFNg was observed in the 3. Tap WD, et al. N Engl J Med 2015;373(5):428-37. tumors of mice treated with hetIL-15. 4. Papadopoulos KP, et al. Clin Cancer Res. 2017. Conclusions Our results show that hetIL-15 administration may be a general method to enhance T cell entry in non-inflamed tumors, increasing P311 the success rate of immunotherapy interventions. Preclinical cancer Clinical outcomes of PD-1 inhibition by PD-L1 expression level studies support the use of hetIL-15 in tumor immunotherapy across malignancies in 204 consecutive patients in a real world approaches to promote the development of anti-tumor responses by oncology setting favoring effector over regulatory cells. The effect of hetIL-15 on me- 1 1 2 2 Kenneth Byrd , Tristan Hayes , Mike Martin , Lee Schwartzberg , tastasis establishment in orthotopic models may provide synergies Ari Vanderwalde against metastatic disease. University of Tennessee Health Sciences Center, Memphis, TN, USA; West Cancer Center, Germantown, TN, USA Correspondence: Kenneth Byrd (kbyrd@westclinic.com) P310 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P311 CSF1/CSF1R signaling blockade triggers release of matrix-degrading proteases in mouse models Background 1 2 2 Stefan Bissinger , Martina Schmittnaegel , Ioanna Keklikoglou , The utility of the PD-L1 biomarker in predicting response to anti-PD- 2 1 1 Michele De Palma , Sabine Hoves , Carola Ries 1 agents has been inconsistent across malignancies. In this study, we 1 2 Roche Innovation Center Munich, 82377 Penzberg, Germany; The describe outcomes of patients treated with anti-PD-1 agents by PD- Swiss Institute for Experimental Cancer Research (ISREC), School of Life L1 expression level. Sciences Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Methods Switzerland Molecular profiling of tumors in patients with advanced cancer was Correspondence: Stefan Bissinger (stefan.bissinger@roche.com) performed at West Cancer Center using Caris Molecular Intelligence Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P310 Profile testing, which includes PD-L1 percentage assessed by immu- nohistochemical staining. Patients were included in this retrospect- Background ive analysis if they were treated with a PD-1 inhibitor and had The heterotypic interplay between cancer cells and their microenviron- available PD-L1 results between November 2014 and May 2017. Pa- ment provides an opportunity for therapeutic targeting. The abundant tients were assessed by PD-L1 expression level, defined as negative tumor-associated macrophage (TAM) infiltrate can be substantially (0% expression) or positive (>1% expression), regardless of staining reduced in mouse tumor models and cancer patients by colony- intensity. PD-L1 positive samples were further subclassified into PD- stimulating factor 1 receptor (CSF1R) signaling blockade [1]. Notably, L1 low (1-4%), intermediate (5-49%) and high (≥50%). Best overall TAM depletion provides marked clinical benefits in diffuse-type tenosy- response using RECIST 1.1 criteria was retrospectively assessed novial giant cell tumors [2,3]. However, facial edema is reported as the using 2-physician review of radiologic data. Progression free sur- most common adverse event of TAM elimination in patients [2,4]. We vival (PFS) and overall survival (OS) were assessed using the here sought to gain insight into the molecular mechanisms mediating Kaplan-Meier method. edema formation. To this aim, we characterized antibody exposure, Results CSF1 levels and an array of extracellular matrix-degrading and restruc- 204 patients with quantifiable PD-L1 expression were treated with turing metalloproteinases (MMPs) in tumor-bearing and tumor-free PD-1 inhibitors. Primary tumors included 125 non-small cell lung can- mice treated with an anti-CSF1R antibody. cers, 31 melanomas, 12 renal cell carcinomas, and 36 others. 110 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 158 of 244 (54%) tumors were PD-L1 negative and 94 (46%) were PD-L1 positive in cynomolgus monkeys, including a transient increase in prolifera- (22 [11%] low, 37 [18%] intermediate, and 35 [17%] high). ORR was tion and ICOS (inducible co-stimulator molecule) expression in a 39% for PD-L1 positive versus 17% for PD-L1 negative (p=<0.001). subset of central memory and effector memory T cells. Best response for each PD-L1 level is shown in Table 1. The esti- Conclusions mated median PFS was 6.4 months for PD-L1 positive versus 3.0 The functional attributes of AGEN2034 combined with the favor- months for PD-L1 negative (HR 0.59; p=0.001; 95% CI, 0.43 to 0.81). able pharmacokinetic and pharmacodynamic profile in cynomol- The estimated median OS was 17.3 months for PD-L1 positive versus gus monkeys are ideally suited for clinical development. 6.9 months for PD-L1 negative (Fig. 1; HR 0.64; p=0.021, 95% CI, 0.44 Moreover, AGEN2034 combined effectively with CTLA-4 blockade to 0.94). Increasing PD-L1 expression was associated with a statisti- in a range of preclinical assays to enhance antigen-specific T cell cally significant improvement in PFS and OS. There was a statistically responsiveness and produced a dynamic pharmacodynamic re- significant difference among PFS and OS with increasing PD-L1 levels sponse in non-human primates. AGEN2034 is currently under (PFS p = 0.002; OS p = 0.026). Multivariate analysis did not identify evaluation in a Phase 1/2 study in subjects with advanced tumors tumor type as a predictor of response. and cervical cancer (NCT03104699) and clinical studies to evaluate Conclusions AGEN2034 in combination with AGEN1884 are planned. PD-L1 staining of any level predicted for improvements in ORR, PFS, and OS with PD-1 inhibitors across multiple malignancies. The higher P313 the PD-L1 staining, the greater the likelihood of benefit. These data In vivo effect of albumin binding domains attached to immune provide important real world confirmation for the potential utility of modulators global PD-L1 testing in clinical practice, regardless of malignancy. Haomin Huang, Keneshia Haenssen, Anil Bhate, Supriya Sanglikar, John Baradei, Shan Liu, Senthil Kumar, Zihao Cui, Richard Hampton, Robert Kramer, John Cini P312 Sonnet BioTherapeutics, Cranbury, NJ, USA AGEN2034, a novel anti-PD-1 antibody that combines effectively Correspondence: Haomin Huang (johncini@sonnetbio.com); Keneshia with CTLA-4 pathway blockade to enhance T cell activity Haenssen; Anil Bhate; Supriya Sanglikar; John Baradei; Shan Liu; Senthil 1 1 1 1 Dhan Chand , David Savitsky , Ana Gonzalez , Christopher Clarke , Kumar; Zihao Cui; Richard Hampton 2 1 1 2 Andrea Schuster , Elise E. Drouin , Jeremy D. Waight , Cornelia Mundt , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P313 1 4 4 4 Gerd Ritter ,Taha Merghoub , David Schaer ,Rikke B Homlgaard , 4 5 1 1 Roberta Zappasodi ,Marc van Dijk , Jennifer S. Buell , Jean-Marie Cuillerot , Background 1 1 Robert Stein , Nicholas S Wilson Recombinant therapeutic proteins < 50Kd (eg., receptor ligands, 1 2 Agenus Inc., Lexington, MA, USA; Former employee of Agenus cytokines) exhibit short circulation half-lives (mins/hour vs. days for Switzerland Inc., Basel, Switzerland; Memorial Sloan Kettering Cancer IgGs) which limit their therapeutic utility. A specific way to increase Center, New York, NY, USA; Agenus Switzerland Inc., Basel, Switzerland the pharmacokinetic half-life of these agents is via conjugation to Correspondence: Dhan Chand (dhan.chand@agenusbio.com) circulating albumin. Here we describe the creation of an albumin Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P312 binding single chain fragment antibody (ScFv-ABD) that binds albu- min in circulation, is recycled by binding to the FcRn (similar to IgG’s) Background and then recycled after cellular uptake resulting in increased half-life PD-1 (or CD279) is a co-inhibitory receptor that suppresses T cell of the appended therapeutic protein. A second advantage to linking function upon binding to its ligands, PD-L1 or PD-L2. PD-1 signal- a therapeutic protein to an ScFv-ABD is improved tumor target deliv- ing functions cooperatively with CTLA-4 to limit T cell activation ery as numerous studies have shown that albumin accumulates in during priming by antigen presenting cells, leading to reduced tumors and inflamed tissues. proliferation, cytokine and chemokine production and cell sur- Methods vival. Anti-PD-1 antibody therapies that block the interaction be- Sonnet BioTherapeutics, using a XOMA phage library, has developed tween PD-1 and its ligands have shown durable clinical benefit scFv ABD fusion constructs with several different small therapeutic pro- both as single agents, but particularly in combination with anti- teins (recombinant interleukin proteins and scFvs targeting relevant bodies that antagonize CTLA-4. immune-oncology receptors). These various ABD constructs have high Methods binding affinity to mouse, human & cyno circulating serum albumin AGEN2034 (anti-PD-1; IgG4) was discovered using a proprietary mam- thereby preventing renal clearance and retaining benefits of FcRn malian display technology, Retrocyte Display™. Binding kinetics and mediated recycling of albumin for extended PK. Early studies to investi- affinity to PD-1 were characterized by surface plasmon resonance. gate improved tumor accumulation translates into anti-tumor efficacy Cell-based potency was determined in a Jurkat PD-1 NFAT reporter- in vivo and have shown that ScFv-ABD enhances tumor targeting. based assay where luciferase activity was measured as an endpoint Results of PD-1/PD-L1 antagonism. The pharmacological effects of AGEN2034 Our characterized scFV-ABD constructs have demonstrated that; alone or in combination with CTLA-4 pathway blockade using a novel high affinity anti-CTLA-4 IgG1 antibody, AGEN1884, was 1. biologic activity is retained when the therapeutic protein is assessed in vitro using peripheral blood mononuclear cells (PBMC) attached via the N- or C-terminus suggesting utility for from healthy donors. Prior to human clinical trials, pharmacokinetic delivering more than one therapeutic protein/scFv; and pharmacodynamic profiling of AGEN2034 were performed in cy- 2. half-life in mouse serum in vivo was extended from minutes to nomolgus monkeys both alone and in combination with CTLA-4 hours/days for three different recombinant proteins and scFv blockade. with MWs of 10-80Kd; Results 3. in an established B6F10 melanoma model these ABD constructs AGEN2034 selectively binds to human and cynomolgus PD-1 with have demonstrated markedly superior reductions in tumor high affinity (Kd<1nM) and sub-nanomolar EC . The Fc interactions growth and improved overall survival compared to the same of AGEN2034 are minimized via selection of a human IgG4 Fc re- constructs without the ABD. Superior efficacy was observed gion. In primary human immune cell assays, AGEN2034 showed a with lower doses and with a single dose of the ScFv-ABD dose-dependent increase in T cell cytokine and proliferative re- constructs vs free recombinant protein sponses. Notably, AGEN2034 combined effectively with AGEN1884 in a dose-dependent manner to further enhance T cell responsive- ness. AGEN2034 was well tolerated, and no-observed-adverse-effect Conclusions level (NOAEL) could be established up to 40 mg/kg in non-human We will describe several examples of improved half-life, tumor accu- primates. Furthermore, the combination of AGEN2034 and anti- mulation and efficacy using our albumin linkage approach that is CTLA-4 blockade promoted a dynamic pharmacodynamic response leading to the selection of drug candidates for clinical development. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 159 of 244 P314 patient. Chlorotoxin is a small peptide derived from the venom of Glioblastoma stem-like cell targeting antibodies identified using the deathstalker scorpion that has been shown to be highly selective yeast display biopanning for all GBM cells, does not bind healthy tissue, is non-toxic to Paul Clark, Michael Zorniak, Benjamin Umlauf, Yongku Cho, Eric Shusta, humans, and has been demonstrated to cross the BBB. To this end, John Kuo we have designed a T cell engaging molecule, anti-CD3/chlorotoxin University of Wisconsin - Madison, Madison, WI, USA (ACDClx), composed of the variable heavy (V ) and light (V ) frag- H L Correspondence: John Kuo (clark@neurosurgery.wisc.edu) ments of an anti-CD3 antibody (2C11) tethered to chlorotoxin. Here, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P314 we show that T cells can be selectively activated against GBM cells, only in the presence of ACDClx. Background Methods BACKGROUND: Glioblastoma stem-like cells (GSCs) are hypothesized to The gene sequence encoding for His-tagged ACDClx was cloned into evade current therapies and cause tumor recurrence, contributing to a deconstructed and improved geminiviral vector and introduced poor patient survival. Existing cell surface markers for GSCs are devel- into agrobacteria for needleless infiltration into leaf tissue of Nicoti- oped from embryonic or neural stem cell systems; however, currently ana benthamiana. ACDClx was extracted from tissue 4 days post- available GSC markers are suboptimal in sensitivity and specificity. We infiltration and purified via nickel affinity chromatography. T cell acti- hypothesized that the GSC surface proteome could be mined with a vation was evaluated via calcium flux assay (Fluo-4) using ionomycin yeast display antibody library to reveal novel immunophenotypes. as a positive control and CD69 expression using full length anti-CD3 Methods antibody as a positive control. Activity was measured using freshly MATERIALS AND METHODS: A naïve yeast expression library of isolated mouse splenocytes and mouse GBM cells (GL261-LucNeo). single-chain human antibodies (scFv) was mined using biopanning For flow cytometry, lymphocytes were gated from the splenocyte against patient-derived GSCs. Discovered unique clones were charac- population and further gated based on CD4 and CD8 markers. terized for qualitative binding affinity against 5 patient-derived GSC Results lines, 5 matched non-GSC/GBM lines, and 2 normal cell lines. Pre- Expression and purification from N. benthamiana resulted in a yield sumptive GSC-specific antibodies were purified from yeast, and GSC of 614ug ACDClx per gram of leaf tissue with greater than 99% purity specificity and affinity determined by confocal microscopy and flow (Figure 1A). Expression of CD69 was observed when splenocytes and cytometry. GSC targeting in vitro was evaluated using flow cytome- GBM cells were incubated with ACDClx or anti-CD3 antibody, but not try, and in vivo after intravenous administration of near infrared with media alone (Figure 1B). T cell activation as measured by an in- fluorescent tagged scFv to immunodeficient mice harboring orthoto- crease in calcium flux over baseline was observed when ACDClx was pic GBM xenografts. added to a mixture of splenocytes and GBM cells (Figure 1C). Flux Results was not observed when mock isolated protein from mock-infiltrated RESULTS: Nine rounds of positive selection against patient-derived plants was added to splenocytes and GBM, nor with splenocytes and GSCs enriched for GSC-binding scFv, with selected pools also ACDClx alone. negatively screened against normal human astrocytes, neural stem Conclusions cells, and serum-cultured GBM (i.e. non-GSC). We identified 62 Our results indicate that ACDClx can be expressed to high levels in unique scFv clones from ~600 candidates by differential PCR and plant tissue and has the capacity to selectively activate T cells restriction analysis. Clone 9.7 (heavy chain only, termed VH-9.7) in vitro. These results provide further motivation for our current stud- demonstrated specificity against 5 patient-derived GSC lines, with ies of ACDClx in vitro and in an immunocompetent model of GBM minimal binding to non-GSC and normal controls. Purified VH-9.7, in vivo. produced predominantly in a monomeric form, had a GSC binding affinity (Kd) of 74.3 ± 9.85 nM. Flow cytometry using 5 GSC lines verified VH-9.7 specific GSC labeling: 17-115-fold higher compared P316 to normal astrocytes and 10-65-fold higher compared to non-GSC/ Impact of anti- PD1 on TIL phenotype and function GBM lines. After intravenous injection, VH-9.7 significantly localized 1 2 2 2 Caitlin Creasy , Cara Haymaker , Marie-Andrée Forget , Gopal Singh , to GSC-derived GBM xenografts in mice [92 ± 11 relative fluores- 2 2 2 Coya Tapia , Jeane Marie Painter , Funda Meric-Bernstam , cent units (RFI), n=3, p<0.05], compared to control non-targeting 2 2 Chantale Bernatchez , Aung Naing scFv (11 ± 7.8 RFI). MD Anderson Cancer Center University of Texas Health Graduate Conclusions School of Biomedical Sciences, Houston, TX, USA; University of Texas CONCLUSIONS: Rapid screening via yeast antibody library biopanning MD Anderson Cancer Center, Houston, TX, USA identified human-specific antibodies that demonstrated GSC specifi- Correspondence: Chantale Bernatchez (cacreasy@mdanderson.org); city compared to both non-GSC (i.e. bulk of GBM) and normal neural Aung Naing cells. Identified antibodies could potentially be developed into immu- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P316 notargeted diagnostics and therapeutics in brain cancer. Background Therapeutic antibodies targeting PD-1 have demonstrated efficacy in P315 several solid tumor types with durable responses in a subset of pa- A bispecific fusion protein, ACDClx, can selectively activate T cells tients. However most patients will either not respond to therapy or against glioblastoma cells in vitro progress after an initial response. It is not fully understood how this 1 1 1 1 Rebecca Cook , Sierra Bichler , Andrew Diamos , Braeden Schaefer , treatment may alter the phenotype or function of Tumor-infiltrating 1 1 1 2 Andrew Niemann , Hugh Mason , Tsafrir Mor , Rachael Sirianni , Lymphocytes (TIL). In this study, methods have been optimized to Joseph Blattman derive TIL from a single core biopsy and applied to growing TIL from 1 2 Arizona State University, Tempe, AZ, USA; Barrow Neurological pre-treatment and on-treatment biopsies in patients receiving anti- Institute, Phoenix, AZ, USA PD-1 to investigate drug-induced changes in TIL phenotype and Correspondence: Rebecca Cook (Rebecca.McCall@asu.edu) function. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P315 Methods Tumor samples are obtained from an ongoing Phase II clinical trial of Background anti-PD-1 in cohorts of patients with rare solid tumor types Glioblastoma (GBM) is a highly invasive and fatal form of brain tumor (NCT02721732). Mandatory core biopsies are taken at baseline and on with a median survival of approximately 15 months. Treatment for day 15-21 after the first cycle of anti-PD1 (Pembrolizumab, 200 mg). TIL GBM is hampered by the blood-brain barrier (BBB) and small popula- are propagated ex vivo utilizing IL-2 and an agonistic anti-4-1BB anti- tions of cells that resist conventional therapies. There is an urgent body (Urelumab, BMS), with or without anti-CD3 (clone OKT3). TIL need for innovative new therapies to target all GBM cells within a phenotype and function are evaluated after 2 or 3 weeks of culture. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 160 of 244 Results In CT26 tumors, treatment with CASC-674 but not an IgG1 version TIL cultures were first initiated by mincing one core biopsy into 3-6 of the same variable domains, shifted the immunosuppressive tumor fragments and cultured in media containing IL-2 and the anti- phenotype to an inflammatory anti-tumor phenotype. CASC-674 4-1BB mAb. Observed TIL growth was poor with only 11% of all sam- alone and in combination with anti-PD1 significantly reduced the % ples yielding over 2 million TIL per fragment after 3 weeks (2 /17 of Tregs and exhausted (PD1+ Ki67+) CD8+ cells and increased the samples). The addition of anti-CD3 to the regimen dramatically im- % of cytotoxic CD8+ cells (PD-1- Ki67+) and anti-tumor (CD155+) proved TIL outgrowth, with 78% and 80% of the baseline biopsy and M1 macrophages. on-treatment biopsy samples, respectively, growing over 108 million Conclusions TIL (n=38/49 baseline; 24/30 treatment, range 10 million to 357.7 mil- These results combined with the antitumor effect observed suggest lion). Phenotypic analysis of the expanded TIL showed an effector Fc functionality is critical for the effect of CASC-674. The observed memory differentiation status at both time points. However TIL grown single agent anti-tumor effect in several different tumor models of from samples obtained after anti-PD-1 dosing showed enhanced pro- CASC-674 and its unique human, non-human primate, and murine portion of CD8 TIL and enriched CTLA-4 expression in both CD4 and cross-reactivity supports consideration of CASC-674 as a therapeutic + + CD8 TIL subsets. PD-1 expression on expanded CD8 and CD4 TIL was development candidate. maintained or elevated after therapy. Analysis of 4 patients’ paired + + baseline and on-treatment TIL show that CD4 and CD8 TIL grown P318 post anti-PD-1 treatment secrete significantly more of the effector cyto- Anti-tumor efficacy and enhancement of T cell effector functions kines IFN-γ, IL-13, and TNF-α following anti-CD3 re-stimulation. by EOS084448, an antagonist anti-TIGIT antibody Conclusions 1 1 1 1 Gregory Driessens , Julia Cuende , Sofie Denies , Chaterine Hoofd , Our study highlights phenotypical and functional differences of TIL 1 1 1 2 Florence Lambolez , Shruthi Prassad , Virginie Rabolli , Anthony Cooper , after a single dose of anti-PD-1. Additionally we demonstrate that it is Christophe Quéva possible to grow TIL in numbers that would be sufficient to proceed 1 2 iTeos Therapeutics, Gosselies, Belgium; Adimab, Lebanon, NH, USA with rapid expansion and adoptive cell transfer from one core biopsy. Correspondence: Gregory Driessens Trial Registration (gregory.driessens@iteostherapeutics.com) NCT02721732 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P318 P317 Background In vivo efficacy and mechanism of action of anti-TIGIT monoclonal T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is an ITIM antibody CASC-674 domain- containing co-inhibitory receptor preferentially expressed + + Peter de Vries, Robert Rosler, Janelle Taylor, Scott Peterson by NK, CD8 and CD4 T cells as well as by regulatory T cells (Treg). Cascadian Therapeutics, Inc., Seattle, WA, USA Several ligands are described to bind to TIGIT with PVR (CD155) Correspondence: Peter de Vries (pdevries@cascadianrx.com) showing the highest affinity. CD226 (DNAM-1), a co-stimulatory re- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P317 ceptor also expressed on NK and T cells compete with TIGIT for PVR binding but with a lower affinity. Co-expression of TIGIT and Background CD226 receptors on T and NK effector cells suggests a role in the TIGIT is a coinhibitory immune checkpoint receptor expressed on fine control of their activation regulatory T cells (Tregs), cytotoxic T cells and NK cells. TIGIT ligands Methods include CD155 and CD112, which are expressed on antigen present- Antagonistic anti-TIGIT antibodies were selected by Adimab, LLC. ing cells and a variety of tumors. These ligands also bind the activat- using a synthetic library of human antibodies presented on the ing receptor CD226, often co-expressed with TIGIT, creating a surface of yeast. Anti-TIGIT mAbs were characterized for affinity to re- network that modulates adaptive and innate immune response in a combinant human TIGIT (Biacore), for binding to human primary T manner analogous to the CD28-CTLA4-CD80-CD86 network. We pre- cells, for competition to CD155 binding as well as for functional activ- viously reported on the characterization of a panel of fully human ity on cells engineered to express TIGIT or on human primary T cells. antagonistic monoclonal antibodies that bind with subnanomolar An anti-TIGIT mAb cross-reactive to mouse TIGIT was used to evaluate affinity to mouse, NHP and human TIGIT and block ligand-receptor the anti-tumor efficacy of anti-TIGIT in the CT26 syngeneic model. interactions and signaling in T cells (AACR, 2017). Here we report on Results the in vivo mechanism of action and efficacy of CASC-674 in a num- Anti-TIGIT mAb EOS084448 affinity for human TIGIT was 0.25nM, ber of syngeneic tumor models. translating into potent binding to human primary T cells with an Methods average EC of 0.09nM. EOS084448 inhibited CD155 binding to A lead candidate antibody (CASC-674) was selected and produced as TIGIT at the surface of TIGIT-expressing cells with an EC of mouse IgG2a and tested in syngeneic in vivo mouse tumor models. 0.16nM. EOS084448 antagonistic activity was evaluated in a To explore the mechanism of action in vivo, CT26 tumor bearing TIGIT:CD155 reporter bioassay that resulted in activation of the mice were treated with CASC-674 alone or in combination with anti- IL-2 promoter and by measuring IFNg secretion by minimally PD1/anti-PD-L1 and their tumors and spleens isolated and evaluated stimulated human primary CD8 T cells; EOS084448 potency in by flow cytometry for various lymphoid and myeloid cell subsets. these 2 assays was 8nM and 0.4nM. A surrogate anti-TIGIT mAb Results with a 26,7nM Kd for mouse TIGIT was evaluated in established CASC-674 as single agent has significant anti-tumor activity in the CT26 mouse tumors. Anti-TIGIT monotherapy delayed CT26 tumor anti-PD1/anti-PD-L1 insensitive CT26 colorectal tumor model, result- growth and achieved complete response in few mice. Complete ing in 8/10 complete regressions (CR). Combination treatment with tumor regression occurred in most of the animals receiving the anti-PD-1/PDL-1 did not enhance the anti-tumor effect compared to anti-TiGIT mAb combined with anti-PD-1 mAb. Anti-tumor efficacy CASC-674 alone. Tumor free mice from this study were re-challenged was associated with an increased CD8 T cell : Treg ratio, an in- 8 weeks after the last treatment with CT26 cells in the opposite flank crease TH1/TH2 cytokine ratio and a transcriptional signature indi- of the original inoculum and no CT26 tumor growth was observed in cating an increased cytolytic T cell activity. any of the mice after 30 days, demonstrating that previous treat- Conclusions ments resulted in an immune memory response. In addition to the In vitro and in vivo data demonstrate the potential for EOS084448 to CT-26 model, CASC-674 demonstrated significant anti-tumor effects promote antitumor immunity and efficacy and supports the rationale in EMT6, H22 and MBT-2 syngeneic mouse tumor models. for its clinical evaluation. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 161 of 244 P319 Background Long-term disease-free survival (DFS) of metastatic melanoma Because of its high level of expression on haematological cancers, deplet- (mM) and renal cell cancer (mRCC) patients following high-dose ing antibodies targeting CD38, an ectoenzyme with hydrolase and cy- interleukin-2 (HD IL2) clase activity, have been generated and showed clinical benefits in 1 2 3 4 Joseph Clark , Brendan Curti , Elizabeth Davis , Howard Kaufman , particular against multiple myeloma. Interestingly, CD38 is not restricted 5 6 7 8 9 Asim amin , Ajjai Alva , Theodore Logan ,Ralph Hauke , Gerald Miletello , to haematological cancer cells but also expressed on many different im- 10 3 5 11 + Ulka Vaishampayan , Douglas Johnson , Richard White , Peter Wiernik , mune subsets including NK and effector T cells, exhausted PD-1 T cells, Janice Dutcher suppressive myeloid cells, and regulatory T and B cells. Anti-CD38 anti- 1 2 Loyola University, Maywood, IL, USA; Providence Cancer Center, bodies will therefore not only impact CD38-expressing tumour cells but 3 4 Portland, OR, USA; Vanderbilt University, Nashville, TN, USA; Rutgers also both effector and suppressive immune cells, as illustrated by the in- University, New Brunswick, NJ, USA; Carolinas Health Care System, creased interest for CD38 as a target in Immuno-Oncology. Charlotte, NC, USA; University of Michigan, Ann Arbor, MI, USA; Methods 7 8 University of Indiana, Indianapolis, IN, USA; Midwest Cancer Center, An antibody production and screening campaign has been initiated Omaha, NE, USA; Hematology/Oncology Clinic, LLP, Baton Rouge, LA, resulting in a panel of fully human CD38-binding antibodies. All anti- 10 11 USA; Wayne State University, Detroit, MI, USA; Cancer Research bodies have been screened for their potential to induce ADCC, apop- Foundation of New York, Chappaqua, NY, USA; Cancer Research tosis, ADCP, and CDC. The potential to deplete CD38-expressing cells Foundation of New York, Bronx, NY, USA has been verified in vivo in lymphoma engrafted mice. Additionally, Correspondence: Janice Dutcher (jpd4401@aol.com) all antibodies have been evaluated for their ability to influence ef- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P319 fector T cell and NK cell activation. Results Background We have produced a portfolio of antibodies targeting distinct epi- HD IL2 treatment produces durable complete responses (CRs) and surgi- topes of CD38. These antibodies exert ADCC when directed against cal CRs. Patients achieving partial response (PR) and stable disease (SD) CD38-overexpressing targets, while showing differential capacity to demonstrate improved survival compared with patients who progress. induce ADCP and CDC. This translated into differential inhibition of Methods in vivo tumour growth of human lymphoma tumours in SCID mice. Eleven HD IL2 treatment centers identified patients with survival Most interestingly, some of these antibodies augment TCR-induced > 5 years after HD IL2. DFS was from end of IL2 to June 2017. proliferation and activation of human T cells in vitro, with an activity Treatment courses generally consisted of 2 1-week cycles of HD ranging from strong T cell activators that increase proinflammatory IL2, 600,000-720,000 U/kg IV every 8 hours. We collected data on cytokine release, to medium or weak activators resulting in low or no patients treated with HD IL2 alone, or HD IL2 plus local therapy cytokine release. In addition, in tumour coculture models, anti-CD38 (surgery or radiation (SRS) leading to CR) with survival > 5 years antibodies increased NK cell activation and proliferation. Regarding after HD IL2. CD38 enzymatic function, i.e. hydrolase and cyclase activity, our anti- Results bodies display differential profiles in terms of blockade or augmenta- Ninety-nine patients are reported: 46 mRCC (male 32, female 10, tion of both activities. Effects of anti-CD38 antibodies on immune unknown 4) and 53 mM (male 31, female 22). Median age at HD effector cells, suppressive immune cells as well as tumour cells will IL2 treatment of mRCC patients is 54 years (range, 39-73 years) be further explored in patient-derived ex vivo tumour models. and of mM patients is 53 years (range, 24-76 years). Sites of Conclusions metastatic disease for mM patients were lymph nodes (LN), lungs, We present a portfolio of CD38-targeting antibodies with distinct activity bone, liver, brain, and other organs, and for mRCC patients were profile. The broad expression and multiple functions described for CD38 lung, LN, adrenal, bone, and other organs. The majority of pa- underline the importance of being able to choose from a range of anti- tients received 2-3 courses of IL2 (63 of 99 patients) and 18 re- bodies that can address the different functionalities depending on the ceived 1 course, with the overall number of IL2 courses ranging most prominentroleofCD38ineachdisease setting.The newclass of from 0.5 to 4 courses. Among the 46 mRCC patients, there are anti-CD38 antibodies presented here will be further explored for their po- 38 CRs, 5 surgical CRs and 3 PRs with no further treatment. tential to improve response rates, especially in solid tumours. Among 53 mM patients, there are 42 CRs, 2 near CRs, and 9 Surgical/ SRS CRs without further treatment. DFS in these patients after HD IL2 P321 ranges from 5+ years to 30+ years, median 10+ years. 27 mRCC and 31 Ibrutinib in combination with agonist αOX40 mAb and CTLA-4 mM are alive > 10 years after IL2. Long-term toxicity among these 99 hi blockade induces Eomes CD8 T cells and promotes tumor patients includes hypothyroidism-5 patients, arthralgias/arthritis-6 pa- regression tients, vitiligo-3 patients, and 1 patient each: neuropathy, PVCs, and 1 2 2 Dana Emerson , Michael Mcnamara , Ian Hilgart-Martiszus , Mohammad normal pressure hydrocephalus. Additional patients may be added as 2 2 Farhad , William Redmond records become available. 1 2 Oregon Health & Science University, Portland, OR, USA; Earle A. Chiles Conclusions Research Institute, Portland, OR, USA We document long-term DFS (>5 years) after CR or PR from HD IL2 Correspondence: William Redmond (emersoda@ohsu.edu) alone. Surgical or SRS conversion of PR to CR can produce durable Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P321 CRs. Long-term DFS was observed in patients with visceral and bone metastases, not only those with LN or lung sites of metastases. Nei- Background ther age, sex, nor number of courses of IL2 predicted long-term DFS. Antagonist monoclonal antibodies (mAb) targeting T cell checkpoints Chronic toxicity due to IL2 is uncommon in long-term survivors. such as CTLA-4 or PD-1 have shown efficacy in treating a subset of patients with metastatic disease. In preclinical models, CTLA-4 block- P320 ade synergized with an agonist anti-OX40 mAb to enhance the Targeting CD38 beyond haematological malignancies: a panel of expansion and effector function of tumor-specific T cells. Previous anti-CD38 antibodies with unique functional properties studies demonstrated that combined aOX40/aCTLA-4 therapy also in- 1 1 1 Nina Eissler , Pascal Merchiers , Simone Filosto , Rahul Chaitanya duced the generation of CD8 T cells expressing high levels of Eome- 1 1 1 1 Khanolkar , Beatriz Goyenechea , Dominic Smethurst , Kevin Moulder , sodermin (Eomes), a transcription factor known to regulate CD8 T 2 1 Sergio A. Quezada , Anne Goubier cell differentiation and memory. Eomes expression is negatively regu- 1 2 Tusk Therapeutics, Stevenage, United Kingdom; UCL Cancer Institute, lated in T cells by the T cell signaling kinase ITK (interleukin-2 indu- London, United Kingdom cible T cell kinase). Importantly, the FDA-approved Bruton’s tyrosine Correspondence: Anne Goubier (nina.eissler@tusktherapeutics.com) kinase inhibitor ibrutinib also blocks ITK, thus providing a potential Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P320 means of modulating Eomes expression. We sought to characterize Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 162 of 244 hi these Eomes CD8 T cells and investigate the mechanisms regulating +L2mAb+IL2), tumor growth was significantly reduced as compared the generation of this novel subset. to mice treated with either RT alone (p<0.001) or with RT+L2 Methods (p<0.001). In our preliminary study, only the combination of RT Wild-type C57BL/6 mice were challenged by 1x10 TRAMP-C1 tumor +L2mAb+IL2 yielded any mice that were tumor free (2 tumor-free cells in the right flank. Wild-type BALB/C mice were challenged by mice out of 5); treatment with RT+L2mAb, or RT+IL2 did not gener- 5x10 4T1 tumor cells orthotopically in the mammary fat pad. Mice ate any tumor-free mice. were treated starting day 7 with 200ug aCTLA-4 (clone 9D9), 200ug Conclusions aOX40 (Clone OX86), or 150ug Ibrutinib. All treatments were injected Tem8-targeted therapy given in conjunction with other anti-cancer i.p. Tumor growth (area) was assessed with microcallipers every 2 to agents has shown promising effects. Prior to this study, anti-Tem8 anti- 3 days. Mice were killed when tumors exceeded 175mm for experi- body given in combination with RT or IL2 had not been investigated. ments tracking tumor growth and survival. Lymph nodes and tumors Here, we show that RT followed by Tem8-specific antibody significantly were harvested on day 14 to assess and characterize T cell responses improved outcome, and that addition of IL2 further improved outcome. by flow cytometry. Inhibition of tumor-angiogenesis via anti-Tem8 antibodies may block Results the function of the Tem8 that is expressed on tumor endothelial cells, hi Our data revealed that these Eomes CD8 T cells expressed signifi- slowing tumor growth. However, since RT+L2mAb treatment resulted lo cantly less PD-1 on their surface compared to Eomes CD8 T cells in improved outcome when given in combination with IL2, antibody- (97% vs. 57%, respectively), while maintaining high levels of IFN-g dependent-cell-mediated cytotoxicity may also contribute to the mech- production within the tumor. Additionally, we confirmed that clinical anism of action of this therapy. concentrations of ibrutinib do not inhibit T cell receptor signaling in CD8 T cells. Next, demonstrated that the combination of ibrutinib/ hi P323 aOX40/aCTLA-4 therapy enhanced the frequency of Eomes CD8 T Novel treatment of cutaneous T cell lymphoma: Targeting TNFR2, cells in 4T1 tumor-bearing mice, a model of triple negative breast an oncogene and marker of potent Tregs, with anti-TNFR2 cancer. Additionally, this triple therapy significantly enhanced IFN-g antibodies and TNF-a expression by CD4 and CD8 T cells in the tumor and 1 1 1 2 Heather Torrey , Audrey Defusco , Danielle Baum , Ziba Rahbar , draining lymph nodes, which was associated with tumor regression 2 2 1 Michael Khodadoust , Youn H. Kim , Denise Faustman and enhanced survival in TRAMP-C1 and Myc-CaP models of prostate Massachusetts General Hospital/Harvard Medical School, Charlestown, adenocarcinoma. MA, USA; Stanford University School of Medicine/Cancer Institute, Conclusions Stanford, CA, USA Taken together, these data demonstrate that combined ibrutinib/ hi Correspondence: Denise Faustman (faustman@helix.mgh.harvard.edu) aOX40/aCTLA-4 therapy induced a robust population of Eomes CD8 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P323 T cells with enhanced effector function capable of mediating tumor regression in multiple pre-clinical tumor models. Background Tumor necrosis factor receptor 2 (TNFR2) is a lymphoid marker of the P322 most potent regulatory T cell (Treg) subtype, which is enriched in the Antitumor effects of radiation combined with intratumoral anti- tumor microenvironment, and a commonly expressed oncogene in Tem8 mAb and IL2 human tumors. Anti-TNFR2 antibodies can inhibit both Tregs and tu- 1 1 1 1 Amy K. Erbe , Kathryn Komro , Arika Feils , Mackenzie Heck , Sabrina mors with specificity for the tumor microenvironment [1], and recent 1 1 1 1 VandenHeuvel , Manasi Mohan , Peter M. Carlson , Jacquelyn A. Hank , data shows that TNFR2 is a candidate oncogene in cutaneous T cell 1 1 2 Alexander Rakhmilevich , Zachary S. Morris , Amit Chaudhary , lymphoma (CTCL) with recurrent point mutations and gain of func- 3 1 Brad St. Croix , Paul M. Sondel tion alteration of TNFR2, resulting in abnormal expression of TNFR2 1 2 University of Wisconsin-Madison, Madison, WI, USA; Meso Scale on CD4+CD26- tumor cells [2]. Diagnostics, Rockville, MD, USA; National Cancer Institute, Frederick, MD, Methods USA We screened novel TNFR2-directed antagonistic antibodies for their Correspondence: Paul M. Sondel (aerbe@wisc.edu) ability to kill leukemic cells in Stage IV CTCL (Sézary syndrome) subjects Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P322 with failure on diverse drug regimens, as well as their ability to induce killing of tumor-associated Tregs and unleash effector T cell (Teffector) Background proliferation. Studies were performed in vitro on sorted CD4+CD26- Tumor Endothelial Marker 8 (Tem8) is overexpressed on endothelial Sézary cells or V-beta specific populations when a tumor was typed. cells that line the tumor vasculature. Chaudhary et al. [Cancer Cell, Results 2012] showed in several tumor models that the anti-Tem8 monoclonal At baseline, CTCL blood samples showed significant burdens of antibody, L2mAb, augmented the activity of anti-cancer agents leading tumor cells within the CD26- subset of CD4 cells (averaging 45-95% to slowed tumor growth. Tem8 is also overexpressed on tumor cells of this subfraction), vs control blood (18% CD26- cells on average). In themselves in several tumor types, including melanoma. We have re- CTCL subjects, Treg numbers (CD4+CD25hiFoxp3) were elevated at cently shown that radiation (RT) given in combination with intra- baseline (11% vs 7% control, p< 0.05), Teffectors were depressed (3% tumoral injection (IT) of a tumor-specific antibody and interleukin-2 vs 8% control, p< 0.05), and Treg/Teffector ratios were abnormally (IL2) can elicit an in situ vaccine effect for mice bearing a B78 syngeneic elevated (8% vs 1% control, p< 0.05). melanoma. Here, we show that RT+L2mAb+IL2 treatment resulted in Regardless of underlying therapy used in vivo, TNFR2 antagonism improved outcome for mice bearing a Tem8+ B78 melanoma. showed dose responsive killing of tumor cells within the CD26- Methods fraction of peripheral CD4 T cells. TNFR2 antagonism also had B78 melanoma cells were transduced to overexpress Tem8 and specificity for tumor cells vs CD26- cells from paired controls. In injected into C57BL/6 mice. Mice bearing a ~200mm tumor received vivo treatment of CTCL subjects with anti-proliferative agents external beam RT (12Gy), followed by IT injection of L2mAb (2mg/kg) such as methotrexate hindered TNFR2 antagonism driven killing, and/or IL2 (150,000U) on days 5-9 post-RT. Mice were monitored for demonstrating the specificity of TNFR2 antagonism for rapidly tumor growth and overall survival. Statistical differences in tumor vol- proliferating cells. ume were determined by Two-Way ANOVA followed by Tukey’stest for Dose response experiments in vitro showed TNFR2 antagonism also multiple comparisons, and Log-rank/Mantel-Cox test was used for over- had the desired dual effect of Treg killing combined with unleashing all survival. Chi-Square test was used to compare response rates. of Teffector proliferation at 48-72 hours. Results Conclusions Treatment with RT+L2mAb resulted in slower tumor growth as com- TNFR2 is a recurrent genomic gain alteration in CTCL that can poten- pared to RT alone (p<0.001). By including IL2 to RT+L2 (i.e. RT tially be targeted to directly stop tumor cell growth by antibody- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 163 of 244 induced cell death, eliminate Tregs of the tumor microenvironment P325 and unleash Teffector proliferation. Characterization of the anti-CTLA-4 antibody AGEN1884, including toxicology and pharmacology assessments in non-human primates 1 1 1 1 References Randi Gombos , Ana Gonzalez , Mariana Manrique , Dhan Chand , 1 1 2 1. Torrey H, Butterworth J, Mera T, et al. Targeting TNFR2 with antagonistic David Savitsky , Benjamin Morin , Ekaterina Breous-Nystrom , 1 1 3 4 antibodies inhibits proliferation of ovarian cancer cells and tumor-associated Christopher Dupont , Rebecca Ward , Cornelia Mundt , Benjamin Duckless , 4 1 3 1 Tregs. Sci Signal. 2017;10(462):pii: eaaf8608. Hao Tang , Mark Findeis ,AndreaSchuster , Jeremy Waight , 1 1 5 6 2. Ungewickell A, Bhaduri A, Rios E, et al. Genomic analysis of mycosis Dennis Underwood , Christopher Clarke ,GerdRitter ,Taha Merghoub , 6 2 1 1 fungoides and Sézary syndrome identifies recurrent alterations in TNFR2. David Schaer , Marc van Dijk ,Jennifer Buell , Jean-Marie Cuillerot , 1 1 1 Nat Genet. 2015;47(9):1056-60. Robert Stein , Elise Drouin ,NicholasWIlson 1 2 Agenus Inc., Lexington, MA, USA; Agenus Switzerland Inc., Basel, Switzerland; Former employee of Agenus Switzerland, Inc., Basel, P324 Switzerland; Former employee of Agenus Inc., Lexington, MA, USA; 5 6 Co-targeting of mesothelin and CD47 with bispecific antibodies for Ludwig Institute for Cancer Research, New York, NY, USA; Memorial efficient elimination of solid tumors Sloan Kettering Cancer Center, New York, NY, USA Krzysztof Masternak, Limin Shang, Vanessa Buatois, Stefano Majocchi, Correspondence: Randi Gombos (randi.gombos@agenusbio.com); Eric Hatterer, Xavier Chauchet, Valéry Moine, Lucile Broyer, Nicholas WIlson Marie H. Kosco-Vilbois, Nicolas Fischer, Walter G. Ferlin Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P325 Novimmune S.A., Geneva, Switzerland Correspondence: Walter G. Ferlin (wferlin@novimmune.com) Background Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P324 Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an important negative regulator of T cell function. Together with CD28, these receptors exem- Background plify a co-inhibitory and co-stimulatory signaling axis that dynamically Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in sculpts the interaction of antigen-specific T cells with antigen present- several human cancers, including mesothelioma, pancreatic-, ovarian- ing cells (APCs). Preclinical studies have demonstrated that anti-CTLA-4 , lung- and gastric cancer. MSLN overexpression is associated with antibodies can enhance tumor-specific immunity through a variety of poor prognosis, with serum levels of soluble MSLN a biomarker of mechanisms including: i) blockade of CD80 or CD86 binding to disease severity in mesothelioma patients. While a promising target CTLA-4; ii) preventing CTLA-4-expressing regulatory T cells from in cancer, monoclonal antibodies (mAbs) targeting MSLN have demon- physically removing CD80 and CD86 from the surface of APCs; and strated limited efficacy in clinical trials. Current MSLN-targeted ap- iii) selective elimination of CTLA-4-expressing intratumoral regula- proaches in development have thus incorporated novel modalities to tory T cells by an Fcg receptor-dependent mechanism. enhance tumor-killing potential (e.g., Antibody Drug Conjugates and Methods CAR-T cells). CD47, an immune check-point, interacts with SIRPα provid- Here we describe the pharmacological and toxicological ing a ‘don’teatme’ signal that allows healthy cells to limit elimination characterization of a novel human IgG1 anti-CTLA-4 antagonist anti- by immune cells, in particular macrophages. CD47 upregulation in solid body, AGEN1884. Binding, blocking, T cell activation as well as cancers is correlated with poor clinical prognosis, almost certainly by Fcgamma receptor-mediated activity of AGEN1884 were evaluated allowing tumor cells to escape immune surveillance by phagocytes. in vitro. The activity and tolerability of AGEN1884 was further Clinical development of mAbs to CD47 is hindered by the ubiquitous assessed in vivo using a non-human primate model. Our in vitro and expression of CD47 leading to rapid drug elimination and significant in vivo assessments extended to a direct comparison of AGEN1884 hematological toxicity including anemia. To address these concerns, we with an IgG2 Fc variant, AGEN2041. have employed a bispecific antibody (biAb) approach that pairs a high Results affinity anti-MSLN targeting arm to an anti-CD47 arm of an optimized AGEN1884 potently enhanced T cell responsiveness in vitro,and com- affinity that drives the efficacious binding only on MSLN-positive cells. bined effectively with other immunomodulatory antibodies targeting This MSLN/CD47 biAb approach, therefore, is designed to target the co-inhibitory and co-stimulatory receptors on T cells. AGEN1884 was CD47-SIRPα pathway in the tumor microenvironment to more effi- well-tolerated in non-human primates and was confirmed to modulate ciently harness the immune system for tumor eradication. cellular and humoral immune responses to co-administered reporter Methods vaccines. In addition to the activity of AGEN1884 as a monotherapy, a Fully human biAbs targeting both MSLN and CD47 were generated. An memory T cell proliferative response was observed in peripheral blood array of biAbs coupling MSLN-targeting arms binding to different epi- of animals when co-administered with an anti-PD-1 antibody. Finally, topes on MSLN with a common CD47-targeting arm have been tested we provide a comparison of the in vitro and in vivo functional proper- in antibody dependent cellular phagocytosis (ADCP) and antibody ties of an IgG2 variant of AGEN1884, revealing important antibody iso- dependent cellular cytotoxicity (ADCC) assays in vitro plus in mouse type differences that may have an impact on the design of optimal xenograft experiments. The following human cell lines were used: NCI- dosing regimens in patients. N87 (gastric), HPAC (pancreatic), OVCAR3 (ovarian), Caov-3 (ovarian), Conclusions NCI-H226 (mesothelioma) and MSLN-transfected HepG2 (hepatic). Taken together, the pharmacologic properties of AGEN1884 support Results its clinical investigation as both a single therapeutic agent and in The MSLN/CD47 biAbs significantly enhanced macrophage-mediated combination therapies. ADCP of NCI-N87, HPAC, OVCAR3 and Caov-3 as compared to the anti- MSLN mAb, Amatuximab. In addition, the biAbs also demonstrated superior ADCC of NCI-N87 cells and NCI-H226. When tested in a xeno- P326 graft tumor model using MSLN-transfected HepG2 cells, therapeutic Isoform specific TGF-β inhibition in combination with radiation treatment with MSLN/CD47 biAbs significantly prevented tumor therapy as a novel immune therapeutic approach to cancer development, while Amatuximab and B6H12, an anti-CD47 mAb, only therapy 1 1 1 2 slightly delayed tumor growth. Aditi Gupta , Sadna Budhu , Rachel Giese , Jacques van Snick , 2 3 1 1 Conclusions Catherine Uyttenhove , Gerd Ritter , Jedd Wolchok , Taha Merghoub 1 2 Using a novel biAb approach that focuses the blockade of the innate Memorial Sloan Kettering, New York, NY, USA; Ludwig Institute for immune checkpoint receptor CD47 to MSLN-expressing tumors sub- Cancer Research Ltd Brussels Branch, Brussels, Belgium; Ludwig Institute stantially enhances their elimination in vitro and in vivo. Thus, MSLN/ for Cancer Research, Ltd, New York, NY, USA CD47 targeting biAbs are a potentially superior strategy in managing Correspondence: Taha Merghoub (guptaa@mskcc.org); Jedd Wolchok MSLN-positive solid tumors. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P326 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 164 of 244 Background to radiation or IT-IC alone, radiation + IT-hu14.18 antibody, or radi- TGF-β is a pleotropic cytokine, which has emerged as a potential ation + intravenous-IC. In those studies, radiation + IT-IC induced an target in cancer treatment due to its dual role in tumorigenesis and in situ vaccine effect, resulting in a tumor-specific memory T cell re- homeostasis. There are three isoforms of TGF-β (TGF-β1, TGF-β2 and sponse. Here we test whether IT administration of non-fused tumor- TGF-β3), which are secreted by immune and non-immune cells as a specific antibody and IL2 may elicit an in situ vaccination response latent complex. Depending on the local context, TGF-β adopts op- following local radiation. posing roles in carcinogensis and in modulating the immune system. Methods These dueling roles of TGF-β are dependent on its secretion and acti- C57BL/6 mice were flank engrafted with syngeneic GD2+ B78 mel- vation. Local radiation therapy (RT) can activate TGF-β via reactive anoma and 5-week tumors (~200 mm ) were treated with single oxygen species. Such TGF-β expression is linked to radioresistance fraction 12 Gy radiation, IT-IL2 (150,000U), and/or IT-hu14.18K322A and dose-limiting toxicities, reducing the effectiveness of RT. In these (50μg). IT injections were given daily on days 6-10 after radiation. studies, we aim to characterize the effect of RT on the temporal and Outcomes included tumor response and rates of complete regres- cell-specific expression patterns of TGF-β isoforms in mouse tumor sion, overall survival, tumor-specific memory (tested in disease-free models. This will inform treatment regimens combining isoform spe- mice by contralateral flank injection with B78 melanoma >90 days cific anti-TGF-β therapy with RT. after radiation), and immunohistochemistry on tumors resected at Methods day 12 after radiation. Fluorescence-activated cell sorting (FACS): C57BL/6 mice were im- Results planted on the hind limb with B16-F10 melanoma cells. On day 10, The combination of local radiation + IT-hu14.18K322A + IT-IL2 resulted tumors were irradiated locally with 15 Gy. Expression of TGF-β iso- in greater tumor regression compared to radiation + IT-hu14.18K322A forms was measured at 1, 3 and 5 days post-RT by FACS. or radiation + IT-IL2 [50% (19/38) aggregate complete tumor regression In-vivo: C57BL/6 mice were implanted with tumors and irradiated as vs 0% (0/11) and 25% (3/12), respectively, p<0.001]. No mice were ren- described. Mice were treated (10/group) with anti-TGF-β1, anti-TGF- dered disease-free with these IT-treatments in the absence of radiation. β3 or a pan-TGF-β antibody beginning 1 day after RT given intraperi- Kaplan-Meier analysis demonstrated improved survival with radiation + toneally (200 ug/mouse) every other day for 8 doses. Tumor growth IT-hu14.18K322A + IT-IL2 compared to radiation + IT-hu14.18K322A and overall survival were monitored. A similar experiment was con- and radiation + IT-IL2 (log-rank p<0.0001; 80% alive at day 65 vs 0% ducted in the 4T1 breast cancer model, in which mice were treated 1 and 50%, respectively). Thus far,100% (6/6) of mice rendered disease- day prior to radiation. free by combined radiation + IT-hu14.18K322A + IT-IL2 have rejected Results subcutaneous B78 re-implantation at >90 days after radiation, com- FACS data indicated that TGF-β1 and TGF-β3 expression increases on pared to 0/10 naïve control mice. Preliminary immunohistochemistry most immune cells in the tumor 1 day after RT, decreases 3 days analyses suggest that IT-IL2 may increase tumor infiltrating CD8+ T cells after RT and reaches a peak 5 days after RT. Preliminary in-vivo stud- in this preclinical model. ies demonstrate that both αTGF-β1 and αTGF-β3 as monotherapies Conclusions have activity against B16 melanoma. In combination with RT, αTGF- We present evidence of a cooperative anti-tumor effect with the com- β3 trends towards inhibiting tumor growth. Similar observations bination of local radiation and IT injection of both tumor-specific anti- were obtained in a 4T1 breast model; however, αTGF-β3 alone and in body and IL2. Given the widespread availability of tumor-specific combination with RT as well as αTGF-β1 + RT showed a significant antibodies, this may offer a viable approach to pursuing in situ tumor delay against tumor growth. No significant differences in survival vaccination in many diverse types of cancer using off-the-shelf reagents. were seen in either tumor model. Conclusions P328 TGF-β1 and TGF-β3 are expressed on numerous lymphoid and myeloid Therapeutic application of radiation-induced anti-tumor antibody cells in B16 tumors and spleens. TGF-β isoform expression peaks 5 days Tsuguhide Takeshima, Raquibul Hannan post-RT. Anti-TGF-β therapy is effective in delaying tumor growth and UT Southwestern Medical Center, Dallas, TX, USA may synergize with RT in certain cancers. This demonstrates rationale Correspondence: Raquibul Hannan for the use of anti-TGF-β therapy to enhance the effectiveness of RT in (Raquibul.Hannan@utsouthwestern.edu) cancer. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P328 P327 Background Intratumor injection of tumor-specific antibody and IL2 triggers in Radiation therapy (RT) has significant immune modulatory properties situ vaccination following local radiation therapy that, when strategically integrated with immunotherapy, may improve 1 1 1 1 1 Emily I. Guy ,SaraBusche , Peter Carlson ,Clinton Heinze ,Ciara N.Schwarz , cancer treatment outcomes. It has been previously reported that RT in- 1 1 2 1 Raghava N. Srirameneni , Jasdeep Kler , Michael M. Meagher ,Amy K.Erbe , duces tumor-specific antibodies [1]; however, this response needs to be 1 1 1 Jacquelyn A. Hank , Alexander L. Rakhmilevich ,Paul M.Sondel , better characterized and its therapeutic potential evaluated. Antibodies Zachary S. Morris binding to the Fc receptor of dendritic cells (DC) have been shown to University of Wisconsin School of Medicine and Public Health, Madison, direct DC to the tumor site [2]. We hypothesize that radiation-induced WI, USA; St. Jude Children's Research Hospital/Children's GMP, LLC, anti-tumor antibodies (RT-Ab) increase dendritic cell (DC) trafficking to Memphis, TN, Memphis, TN, USA the irradiated tumor to increase tumor antigen presentation. Here, we Correspondence: Emily I. Guy (eguy@wisc.edu) explore the therapeutic application of RT-Ab. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P327 Methods Multiple syngeneic murine cell lines were used (EG7, B16, LLC, 4T1), Background along with their respective murine strains. Fluorescence tagged anti- + + In murine models of GD2 melanoma, GD2 neuroblastoma, and mouse antibodies (eBiosciences) and anti-CD11c antibodies (eBios- EGFR head and neck cancer, we have reported a cooperative inter- ciences) were used for flow cytometry. action between radiation and intratumor (IT) injection of tumor- Results specific antibody (anti-GD2 hu14.18K322A or anti-EGFR cetuximab). Multiple syngeneic murine tumor grafts (B16, LLC, 4T1) were grown Consistent with a process mediated by antibody-dependent cell- in the hind legs of respective murine strains and focally irradiated mediated cytotoxicity, this interaction required the Fc portion of the with a single 15Gy RT dose, with post-RT sera (pRTs) obtained at antibody, host Fcʏ receptor, NK cells, and tumor expression of the different time intervals. Flow cytometric analysis using anti-mouse antibody-targeted antigen. In this GD2+ melanoma model, combined antibodies of the pRTs incubated with the respective tumor cell treatment with RT + IT-hu14.18-IL2 immunocytokine (a fusion protein showed a significant increase in RT-Ab that peaked between days 7- of hu14.18 antibody and IL2) markedly enhanced response compared 13. This increase was more pronounced for a single fraction of 15Gy Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 165 of 244 when compared to 4.5Gy on 5 consecutive days while the tumor an equimolar mixture of monovalent component antibodies that com- growth delay was equal between the two groups. Next, flow cytome- pose XmAb20717 lead to inferior activity compared to XmAb20717, try was performed on bone marrow-derived dendritic cells (BMDC) suggesting that binding selectivity of the bispecific antibody contrib- from the respective murine strains co-cultured with pRTs, which utes to function. XmAb20717 also exhibited anti-tumor activity in pre- showed an increase in the binding of RT-Ab to DCs when compared liminary anti-tumor studies in mice. to normal or non-irradiated tumor bearing mice sera. When an Fc re- Conclusions ceptor blocking agent was used, this binding was significantly inhib- Dual blockade of PD1 and CTLA4 with XmAb20717 resulted in T cell ited, which suggested Fc receptor-mediated binding of RT-Ab to the activation that is comparable to a combination of bivalent antibodies BMDC. Lastly, we demonstrated that RT-Ab-bound-BMDC (RT-DC), targeting PD1 and CTLA4. Specific targeting of human lymphocytes when administered as an autologous DC therapy in conjunction with positive for both PD1 and CTLA4 with XmAb20717 may promote simi- focal RT, increased the therapeutic effect of focal tumor RT signifi- lar efficacy compared to a combination of bivalent antibodies while re- cantly compared to multiple control conditions. ducing adverse events. These data suggest that clinical development of Conclusions XmAb20717 is warranted for the treatment of human malignancies. RT induces a tumor-specific antibody response that increases DC traf- ficking to the tumor via Fc receptor-mediated targeting. This likely P330 contributes to RT-mediated tumor antigen presentation and adaptive T cell immunotherapies trigger innate immunity and aseptic anti-tumor immunity. Amplification of the RT-Ab response may have inflammation leading to potent anti-tumor and off-targets effects therapeutic applications. 1 1 1 1 Daniel Hirschhorn , Jacob Ricca , Bilel Gasmi , Levi Mark Mangarin , 1 1 1 1 1 Olivier De Henau , Sadna Budhu , Yanyun Li , Czrina Cortez , Yang Xia , References 1 1 1 1 Hong Zhong , Cailian Liu , Roberta Zappasodi , Travis Hollmann , 1. Nesslinger NJ et. al. Standard treatments induce antigen-specific immune 1 2 2 1 Mario Lacouture , Jean Albrengues , Mikala Egeblad , Wolchok Jedd , responses in prostate cancer. Clin Cancer Res. 2007 Mar 1;13(5):1493-502. Taha Merghoub PubMed PMID: 17332294. 1 2 Memorial Sloan Kettering Cancer Center, New York, NY, USA; Cold 2. Franki SN et. al. Dendritic cells loaded with apoptotic antibody-coated Spring Harbor Laboratory, Cold Spring Harbor, NY, USA tumor cells provide protective immunity against B-cell lymphoma in vivo. Correspondence: Wolchok Jedd (hirschhd@mskcc.org); Taha Merghoub Blood. 2008 Feb 1;111(3):1504-11. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P330 P329 Background Dual blockade of PD1 and CTLA4 with bispecific antibody Mobilizing the immune system to treat advanced cancers is now a clin- XmAb20717 promotes human T cell activation and proliferation ical reality. Successful immune-based therapies that treat tumors are 1 1 1 1 Michael Hedvat , Matthew Bernett , Gregory Moore , Christine Bonzon , often accompanied by immune-related adverse events (irAE) including 1 2 1 1 1 Rumana Rashid ,Seung Chu ,KendraAvery , Alex Nisthal , Umesh Muchhal , toxicities that can occasionally present with severe and lethal symp- John Desjarlais toms. The primary immunotherapies currently in clinic include agents 1 2 Xencor, Inc., Monrovia, CA, USA; schu@xencor.com, Monrovia, CA, USA that activate T cell responses such as checkpoint blockade of inhibitory Correspondence: John Desjarlais (mhedvat@xencor.com) pathways and infusion of ex-vivo tumor-derived or T cell receptor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P329 (TCR)-transgenic or chimeric antigen receptor (CAR)-modified T cells. While the beneficial and toxic effects of T cell-based immunotherapies Background in the clinic are being extensively explored, the precise mechanisms of Treatment of melanoma patients with nivolumab plus ipilimumab tumor elimination and irAE remain the subject of intense investigation. increases progression-free-survival compared to each monotherapy. Methods The increase in efficacy of the combination regimen is accompanied In the present study, we treated established tumors with melanoma- + + by an increase in adverse events. Since PD1 CTLA4 tumor- specific adoptive CD4+ T cell transfer and costimulation via OX40 or infiltrating-lymphocytes are dysfunctional in the tumor microenviron- checkpoint blockade with anti-CTLA-4. + + ment, we developed XmAb20717 to selectively target PD1 CTLA4 Results double-positive T cells in an effort to recapitulate efficacy of the We found that, in spite of co-opting the adaptive immune response combination regimen while reducing toxicity. to treat cancer, acute local inflammation, resembling delayed-type Methods hypersensitivity, plays a fundamental role in tumor elimination and XmAb20717 component antibodies binding to PD1 and CTLA4 with related toxicities in a model of irAE. While OX40 or CTLA-4 antibodies favorable stability and functionality were assembled in a bispecific stimulated T cells are necessary for initiating a therapeutic response, antibody platform with substitutions in the Fc domain to suppress ef- activation of endogenous neutrophils constitutes an important and fector function. XmAb20717 was evaluated in vitro by measuring necessary effector mechanism of tumor destruction and irAE. Upon antibody binding and de-repression of super-antigen stimulated per- closer examination, we found extensive neutrophil extracellular traps ipheral blood lymphocytes (PBMCs) and in vivo by monitoring the (NETs) in ear pinnae of treated mice and in melanoma patients suf- engraftment of human PBMCs in NSG mice (huPBMC-NSG) by flow fering from immunotherapy-induced irAE. cytometry. To evaluate anti-tumor efficacy we monitored the growth Conclusions of established cancer cells in huPBMC-NSG following treatment. Our results illustrate the involvement of innate immunity in promot- Results ing tumor elimination and subsequent side effects with immunother- Optimized candidate single-chain Fvs were confirmed to bind PD1 and apies that engage T cells. functionally block PDL1 and PDL2 binding to PD1. We also generated optimized anti-CTLA4 Fabs. Anti-PD1 and anti-CTLA4 targeting compo- nent antibodies were assembled into XmAb20717, which displayed P331 favorable biophysical and manufacturing properties. XmAb20717 en- Involvement of estrogen and progesterone in the modulation of hanced IL2 secretion in vitro 4.1-fold relative to a negative control anti- indoleamine 2,3 dioxygenase - IDO – expression in cultured body (p < 0.01, n = 15 donors) in response to antigenic challenge of mammary carcinoma cells of female dog previously stimulated T cells, with 1.6-fold superior activity compared Jose Roberto Kfoury Jr, Pedro Bianchi, Flavia Ponce, Rafael Leandro, to an anti-PD1 bivalent antibody (p < 0.01, n = 15 donors). XmAb20717 Tulio Yoshinaga enhanced T cell engraftment (9.6-fold) and IFNγ secretion (2.9-fold) School of Veterinary Medicine and Animal Sciences, University of Sao compared to vehicle controls in huPBMC-NSG mice (p < 0.001, n =10/ Paulo, Sao Paulo, Brazil group), with equivalent activity to a combination regimen of anti-PD1 Correspondence: Jose Roberto Kfoury Jr (jrobertok@usp.br) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P331 and anti-CTLA4 bivalent antibodies. Blockade of PD1 and CTLA4 with Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 166 of 244 Background Methods Indoleamine 2,3 dioxygenase - IDO is an enzyme that prevents the To assess the pharmacodynamic effects of NKTR-255 in mice and establishment of an immune response in the microenvironment in non-human primates NKTR-255 was delivered intravenously and which it is expressed by catabolizing the amino acid tryptophan. The whole blood was collected at the indicated timepoints; flow cytom- deprivation of tryptophan and the generation of its metabolites, etry was used to measure signaling activity (STAT5 phosphoryl- mainly kynurenine, impairs effector T- cells proliferation leading them ation), proliferative status (Ki-67 expression) and absolute frequency to apoptosis [1] and, by its interaction with the aryl-hydrocarbon re- of various lymphocyte subpopulations. Cytotoxicity assays were ceptor (AhR) in CD4+ T-cells, favors the expansion of T-regulatory performed by incubating NK cells purified from spleens of NKTR- cells [2]. It is known that several cancer cells and leucocytes in the 255-treated mice with dye-labelled YAC-1 target cells and assessing tumor microenvironment express IDO and are sensitive to hormones. extent of cell-killing by flow cytometry. Efficacy was studied using Steroidal hormones, such as estrogen and progesterone, are capable the CT-26 lung metastasis model; briefly, 10 CT-26 cells were of altering immune functions in cells and may influence IDO expres- injected into the tail-vein of mice, NKTR-255 treatment was initiated sion; nonetheless, the mechanisms involved are still poorly under- the following day, lungs were harvested on day 13 and metastases stood. Our group previous data have shown that the progesterone were counted. was directly involved in IDO expression modulation via its receptor in Results dendritic and CD4+ T cells from the maternal-fetal interface of Wistar NKTR-255 induces rapid and sustained signaling in lymphocytes fol- rats [3]. Therefore, this study aims to investigate whether this mech- lowing intravenous administration in mice and non-human primates. anism is present in the female dog mammary carcinoma microenvir- This sustained signal results in proliferation of CD8 T-cells and a pref- onment and if it occurs in a similar way with estrogen. erential expansion of the CD8 central memory population. NK cells Methods increase in number and in Granzyme B expression, concomitant with Cells of mammary carcinoma from bitches were treated with exogen- an increase in cytotoxic potential. The robust induction of CD8 and ous progesterone and estrogen and their respective receptor antago- NK cell proliferation is maintained upon repeat dosing. In a mouse nists tamoxifen and mifepristone. IDO expression was analyzed by model of tumor metastasis to the lungs, NKTR-255 treatment results immunohistochemistry, flow cytometry and the mRNA by real-time in an 85% reduction in the number of metastases. This efficacy is PCR. IDO quantification was obtained by western blot technique. dependent on NK cells but not CD8 T-cells, as demonstrated in cell- Results depletion studies. Toxicology assessments demonstrate that NKTR- IDO quantification exhibited the same pattern as mRNA expression. 255 is well tolerated at efficacious dose levels. There was an increase of the enzyme expression and mRNA in the es- Conclusions trogen treated group, in contrast to the decrease observed in the pro- NKTR-255 provides a sustained IL-15 signal, resulting in profound gesterone group. When the cells were subjected to the hormonal and sustained immune activation and anti-tumor activity. NKTR-255 inhibitors, an evident decrease of IDO expression percentage and the is well tolerated and its pharmacokinetic properties and pharmacody- respective mRNA was verified following the supplementation of tam- namic effects translate well from rodents to non-human primates, oxifen and a restoration of IDO expression values and the mRNA after supporting further development. the addition of the progesterone inhibitor, mifepristone. Conclusions These findings strongly suggest that progesterone and estrogen par- P333 ticipate indirectly in the modulation of IDO through their membrane MPL-5821, an ESM™-p38 MAPK inhibitor, enhances tumor immune receptors. response and M1 macrophage polarization in a 3D Ex Vivo system utilizing fresh tumor microspheroids of lung cancer patients 1 1 1 References Melanie Mediavilla-Varela , Melba Marie Page , Jenny Kreahling , 2 2 1 1 1. Munn DH., Mellor AL: IDO in the Tumor Microenvironment Inflammation, Martin Perry , David Moffat , Scott Antonia , Soner Altiok 1 2 Counter-Regulation, and Tolerance. Trends in Immunology. 2016; Nilogen Oncosystems, Tampa, FL, USA; Macrophage Pharma Limited, 37(3):193-207. Windsor, United Kingdom 2. Harden JL, Egilmez NK: Indoleamine 2,3-Dioxygenase and Dendritic Cell Correspondence: Soner Altiok (jenny@nilogen.com) Tolerogenicity. Immunol Invest. 2012; 41:738-764. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P333 3. Bianchi PKF, Leandro RM, Poscai AN, Yoshinaga T, Gonçalez PO, Kfoury Junior JR: Progesterone Decreases in vitro Indoleamine 2, 3-dioxygenase Background Expression in Dendritic and CD4+ Cells from Maternal-Fetal Interface of Lung cancer is one of the most common causes of death worldwide. Rats. Immunol Invest. 2017; 46(5):1–13. Immunotherapy has demonstrated durable responses and tolerability in subsets of lung cancer patients. Macrophages are a significant component of the tumor microenvironment and the predominant P332 phenotype (M2-like) is frequently associated with one supportive of Pre-clinical efficacy and tolerability of NKTR-255, a polymer-conjugated tumor immune evasion. However, the plasticity of macrophages of- IL-15 for immuno-oncology fers the opportunity for therapeutic intervention to repolarize the Peter Kirk, Peiwen Kuo, Murali Addepalli, Takahiro Miyazaki, Jane phenotype to one that is non-immunosuppressive and supportive of Gunther, Laurie VanderVeen, Ping Zhang, Palakshi Obalapur, Mekhala an anti-tumor immune response (M1-like). MPL-5821 is a p38 MAPK Maiti, Amol Murkar, Arunasree Lanka, Werner Rubas, inhibitor employing Esterase Sensitive Motif (ESM™) technology [1] to Jonathan Zalevsky principally target myelomonocytic cells. This study evaluates the immu- Nektar Therapeutics, San Francisco, CA, USA nomodulatory effect of MPL-5821 in a 3D ex vivo assay of lung cancer. Correspondence: Peter Kirk (pkirk@nektar.com) Methods Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P332 Fresh tumor tissues obtained from consented patients with non- small cell lung cancer at the time of surgical resection were utilized Background in a proprietary 3D ex vivo tumor microsphere assay with intact IL-15 is a cytokine that activates T-cells and NK cells and has long tumor immune microenvironment. Tumor microspheres were treated been recognized for its potential as an immunotherapeutic agent for with MPL-5821 for 36 hours. At the end of the treatment, flow cy- the treatment of cancer. Exploiting this potential has been challen- tometry analysis was performed to assess M1/M2 plasticity and TIL ging due to unfavorable pharmacokinetic properties. NKTR-255 is a proliferation (CD3+/Ki-67+). Additionally, multiplex human cytokine polymer-conjugated IL-15 that shows improved plasma exposure assay was used to simultaneously analyze the differential release of while retaining potency and high affinity for IL-15Rα. Here we investi- cytokines in culture media and gene expression analysis was per- gate the pharmacodynamics, pre-clinical efficacy and tolerability of formed using the NanoString PanCancer Immune Profiling panel NKTR-255. which contains probes to quantitate 770 immune function genes. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 167 of 244 Results in tumor growth (CCL25) and increased expression of antitumor im- MPL-5821 at 10nM and 50nM effectively inhibited the p38 signaling mune response genes (CXCL14, CCL19 and CCL21). These studies pathway in the tumor samples and led to profound inhibition of the showed epacadostat at an effective concentration of 1μM induced immunosuppressive cytokine IL-10 and pro-inflammatory cytokines specific changes in the microenvironment and increased immune re- TNFα and IL-6. Evidence of enhanced T-cell proliferation and activa- sponse. Furthermore, the autologous patient derived cell line in vitro tion was observed in several of the tumor samples. The most statis- assay determined that epacadostat overcame CAF induced inhibition tical significant up-regulated genes were associated with antigen of TIL activity. processing/presentation and MHC class II protein binding and Conclusions included CD4, CR1, CD74 and multiple HLA genes. CD40 and ICOSLG This patient-derived 3D ex vivo approach demonstrated the immu- genes were up-regulated in several tumors as were CXCL9 and 10. nomodulatory activity of epacadostat in NSCLC and indicates that Chemokine genes CXCL3, 5, 7 and 8, associated with a pro- inhibition of IDO activity may overcome stroma-induced immuno- angiogenic macrophage phenotype, were the most statistically down suppression in lung cancer. Studies on the effects of epacadostat in regulated genes along with the M2 phenotype associated genes combination with anti-PD1 in the same culture systems are cur- CCL13, 23 and 24. Down-regulation of PD-L1 and IDO gene expres- rently ongoing. sion was also seen in several tumors. Conclusions P335 This lung patient derived ex vivo approach indicates that MPL-5821 Characterization of a novel differentiated anti-CTLA-4 antibody may alleviate myelomonocytic cell induced immunosuppression and (ADU-1604) in vitro and in vivo enhance antigen responsiveness suggesting potential clinical implica- 1 1 1 1 Maaike Hendriks , Joost Kreijtz , Paul Vink , David Lutje Hulsik , Imke tions in the treatment of lung cancer. 1 1 1 1 Lodewijks , Astrid Bertens , Jos van de Crommert , Maurice Habraken , 1 1 2 Wout Janssen , Judith Stammen-Vogelzang , Weiwen Deng , References 2 2 2 Laura Hix Glickman , Meredith Leong , Sarah McWhirter , 1. Needham LA, et al, J Pharmacol Exp Ther. (2011), 339 :132-42. 2 1 1 Thomas Dubensky , Hans van Eenennaam , Andrea van Elsas 1 2 Aduro Biotech Europe, Oss, Netherlands; Aduro Biotech, Inc., Berkeley, P334 CA, USA Inhibition of IDO activity by epacadostat (INCB024360) activates Correspondence: Joost Kreijtz (jkreijtz@aduro.com) tumor infiltrating lymphocytes in a patient-derived 3D ex vivo Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P335 system of lung cancer and alleviates stromal immunosuppression 1 1 1 Melanie Mediavilla-Varela , Melba Marie Page , Jenny Kreahling , Background 2 1 1 Peggy Scherle , Scott Antonia , Soner Altiok Targeting the CTLA-4 immune checkpoint with antibodies, either 1 2 Nilogen Oncosystems, Tampa, FL, USA; Incyte Corporation, alone or in combination with PD-1/PD-L1 inhibitors shows clinical activ- Wilmington, DE, USA ity and durable responses in advanced cancer. The use of anti-CTLA-4 Correspondence: Soner Altiok (jenny@nilogen.com) may augment other immunotherapies. Indeed, in syngeneic mouse Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P334 tumor models anti-CTLA-4 strongly enhanced anti-tumor efficacy of live attenuated double-deleted Listeria monocytogenes (LADD) immuno- Background therapy and of the stimulator of interferon genes (STING) pathway acti- Immune evasion is one of the major hallmarks of cancer and identify- vator ADU-S100, indicating combination potential. Activity of anti- ing mechanisms by which cancer cells evade the immune system have CTLA-4 is enhanced by Fc receptor engagement and tumor-selective become a major strategy against cancer. IDO (indoleamine 2,3-dioxy- depletion of regulatory T cells in mice, but this is unproven as of yet for genase) is a tryptophan catabolizing enzyme expressed constitutively cancer patients. Here we present a novel, humanized anti-CTLA-4 anti- by tumor cells and different components of immune cells present body originating from Aduro’s proprietary B-Select platform. within the tumor microenvironment. It has been shown that high ex- Methods pression of IDO increases the number of Tregs and blocks the prolifera- Among a panel of mouse anti-human CTLA-4 antibodies, 27A was tion of effector T cells. Thus, inhibiting the IDO pathway is a promising humanized and functionally characterized on an IgG1 and IgG4 back- strategy to restore immune system responses to more easily identify bone. Both variants, named hCTLA4.27H6L1.i1 and .i4 were character- and destroy cancer cells. This study evaluates the immunomodulatory ized in vitro for binding to human and cynomolgus CTLA-4, blocking effect of an IDO inhibitor epacadostat (INCB024360) on the immuno- of CD80/CD86 and functional activity. Both antibodies are compared suppressive effect of cancer-associated fibroblasts and activation of for their capacity to induce tumor rejection in patient-derived tumor tumor infiltrating lymphocytes in a 3D ex vivo assay utilizing fresh pa- graft models in humanized mice. tient tumor samples Results Methods The IgG1 and IgG4 variants bound recombinant human CTLA-4 with 3D ex vivo studies were performed with fresh tumor tissue obtained a Kd of 2.7 ± 2.9 nM and 1.3 ± 0.5 nM as measured by Bio-Layer from consented NSCLC patients. Tumor samples were treated with Interferometry, and cell-expressed CTLA-4 with an EC50 of 0.042 ± IDO inhibitor at 1μM for 48 hours. HPLC analysis on kynurenine and 0.000 nM and 0.048 ± 0.002 nM, respectively. Binding to endogenous tryptophan was performed to verify target inhibition in the ex vivo CTLA-4 was demonstrated by flow cytometry using primary activated model. A multiplex human cytokine assay was used to simultan- human and non-human primate lymphocytes. Both IgG1 and IgG4 eously analyze the differential release of cytokines in culture media. variants potently blocked the interaction of CD80 and CD86 with hu- Additionally, NanoString PanCancer Immune Profiling platform con- man CTLA-4 (IC50 ranged from 1-2 nM). Using swap mutants of taining probes to quantitate 770 immune function genes was used mouse and human CTLA-4 and cross-competition assays by Bio-Layer to determine positive and negative associations between expression Interferometry, these antibodies showed a unique binding profile in- of immune function genes and TIL activation by ex vivo treatment. dicating a previously undiscovered epitope on CTLA-4. Functional Furthermore, autologous patient-derived cell lines (CAF and TILs) characterization demonstrated that both antibodies enhanced activa- were utilized in an in vitro assay to determine the role of IDO inhib- tion and IL-2 production by human primary T cells or peripheral ition on CAF-mediated immunosuppression. blood mononuclear cells (PBMCs) co-stimulated by Raji cells or Results Staphylococcal enterotoxin B, in a dose-dependent manner. The IgG1 3D ex vivo studies showed a significant decrease in kynurenine dem- variant (in contrast to IgG4) bound to low and high-affinity Fcγ re- onstrating that epacadostat effectively inhibited the enzymatic activ- ceptors inducing antibody-dependent cell-mediated cytotoxicity ity of IDO in the tumor microenvironment accompanied by increased (ADCC) mediated by natural killer (NK) cells and CD16+ monocytes release of pro-inflammatory cytokines such as IFNγ. Treatment with towards CTLA-4+ cells. Similarly, complement-dependent cytotoxicity epacadostat demonstrated decreased expression of genes involved was differentially induced in vitro. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 168 of 244 Conclusions Trial Registration Overall, both hCTLA4.27 derivatives compared favorably to bench- ClinicalTrials.gov NCT03059823. marks in their biophysical and functional characteristics. The IgG1 variant, designated ADU-1604, is progressing through preclinical P337 development. Preclinical characterization of MGD013, a PD-1 x LAG-3 bispecific DART® molecule 1 1 1 2 P336 Ross La Motte-Mohs , Kalpana Shah , Jennifer G. Brown , Doug Smith , 1 3 1 1 Preclinical characterization of MGA012, a novel clinical-stage PD-1 Sergey Gorlatov , Valentina Ciccarone , James K. Tamura , Hua Li , 1 1 1 2 monoclonal antibody Leilei He , Farha Vasanwala , Christine Shoemaker , Jonathan Li , 1 1 1 1 2 2 2 1 1 Ross La Motte-Mohs , Kalpana Shah ,JenniferG.Brown , Ralph F. Alderson , Shereen Saini , Jill R Rillema , Monica Licea , Jessica Hill , Arin Whiddon , 2 1 1 1 1 2 2 Doug Smith , Sergey Gorlatov , Valentina Ciccarone ,JamesK.Tamura , Massimiliano Pascuccio , Francine Z. Chen , Anushka De Costa , Ann 1 1 1 2 2 2 2 1 1 1 Hua Li ,LeileiHe , Farha Vasanwala , Jill R. Rillema , Monica Licea , Easton , Peter Lung , Kurt Stahl , Jeffrey Nordstrom , Scott Koenig , Ezio 1 1 1 2 1 1 1 Jessica Hill , Christina Wolff , Massimiliano Pascuccio ,FrancineZ.Chen , Bonvini , Syd Johnson , Paul A. Moore 2 2 2 1 1 2 Yan Chen , Anushka De Costa , Ann Easton , Alexey Berezhnoy , MacroGenics, Inc., Rockville, MD, USA; MacroGenics, Inc., South San 2 1 1 1 3 Jonathan Li , Jeffrey Nordstrom ,Scott Koenig , Ezio Bonvini , Francisco, CA, USA; MacroGenics, Inc., Rockville, CA, USA 1 1 Syd Johnson ,Paul A. Moore Correspondence: Ross La Motte-Mohs 1 2 MacroGenics, Inc., Rockville, MD, USA; MacroGenics, Inc., South San (lamottemohsr@macrogenics.com) Francisco, CA, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P337 Correspondence: Ross La Motte-Mohs (lamottemohsr@macrogenics.com) Background Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P336 Monoclonal antibodies (mAbs) that target the immune checkpoints, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and pro- Background grammed cell death protein-1 (PD-1), have shown enhanced clinical Monoclonal antibodies (mAbs) that target immune checkpoint path- anti-tumor activity when given in combination, triggering interest in ways, such as the cytotoxic T lymphocyte-associated antigen-4 and determining whether additional checkpoint inhibitor combinations the programmed cell death protein 1 (PD-1) pathways, have demon- may afford enhanced clinical benefit. Lymphocyte activation strated broad clinical efficacy against a variety of malignancies as gene-3 (LAG-3) is another immune checkpoint expressed on acti- monotherapy or in a combination. MGA012 is a novel anti-PD-1 mAb vated T cells and tumor infiltrating lymphocytes. Recognizing the developed to disrupt the PD-1 interaction with PD-L1/PD-L2 to re- therapeutic potential of dual checkpoint blockade, we have engi- store or improve T-cell function as stand-alone therapy or in combin- neered MGD013, a IgG4κ bispecific DART molecule to bind PD-1 ation with other immune approaches. and LAG-3 concomitantly or independently and disrupt these Methods non-redundant inhibitory pathways to further restore exhausted Murine PD-1 mAbs were generated and benchmarked against replicas T-cell function. of the approved mAbs, nivolumab and pembrolizumab. Several mAbs Methods with favorable characteristics were further chimerized or humanized. Proprietary PD-1 and LAG-3 mAbs were generated and selected MGA012, a humanized, hinge-stabilized IgG4κ mAb, was selected based on binding characteristics, biophysical properties, the ability to based on binding and biophysical properties as well as a functional block their respective receptor/ligand axes and to synergize in T-cell characterization inclusive of enhanced T-cell activation following super- stimulation assays. Humanized sequences were incorporated into a antigen re-stimulation. tetravalent bispecific DART format and benchmarked against Results combinations of replicas of the approved PD-1 mAb, nivolumab, MGA012 bound human PD-1 with an affinity equal to or exceeding and BMS-986016 anti-LAG-3 mAb, which is currently under clinical those of replicas of nivolumab or pembrolizumab. MGA012 bound evaluation. MGD013 biological activity was evaluated in various PD-1-expressing cell lines and chronically-activated T cells, blocked primary cell-based immune assays. Safety was assessed in cyno- PD-1 interactions with PD-L1/PD-L2, resulting in inhibition of PD-1 molgus monkey toxicology studies performed at MPI (Mattawan, signaling and enhanced antigen-driven cytokine secretion to levels MI) under Institutional Animal Care and Use Committee-approved comparable to those observed with nivolumab or pembrolizumab protocols. replicas. Furthermore, characterization of MGA012 in ex-vivo tumor Results microenvironment immune models showed activation profiles recap- MGD013 bound with high affinity to human and cynomolgus mon- itulating the benchmark PD-1 mAbs. MGA012 showed combinatorial key PD-1- and LAG-3-expressing cells and blocked PD-1/PD-L1, PD-1/ activity in vitro when added to anti-CTLA-4 or anti-LAG-3 mAbs and PD-L2 and LAG-3/HLA (MHC-II) interactions, with resultant signaling enhanced the activity of a T-cell redirecting molecule in a mouse blockade. Functional characterization revealed enhanced cytokine tumor model. MGA012 showed no unexpected cross-reactivity in secretion in response to antigen stimulation that was greater than human tissues, with staining observed primarily in lymphocytes and that of the combination of individual equimolar amounts of PD-1 lymphoid organs. In a repeat-dose (10-150 mg/kg QWx4) study in cy- and LAG-3 mAbs. MGD013 was well-tolerated in a repeated-dose nomolgus monkeys, PK was linear with a beta half-life of 11.2 days (Q1Wx4) cynomolgus monkey toxicology study. Except for the occur- (±4.6 SD) and full PD-1 occupancy on circulating T cells at all doses rence of watery feces in a few animals, no MGD013-related adverse tested. Occupancy of ≥80%, persisting for 4-7 weeks, was also ob- findings were noted, including hematological or clinical chemistry served in monkeys receiving a single 10 mg/kg dose. MGA012 was changes, serum cytokine levels or gross and microscopic histological well tolerated in cynomolgus monkeys and demonstrated a favorable findings, establishing a no-observed adverse-effect level (NOAEL) of safety profile with a no-adverse effect level (NOAEL) of 150 mg/kg. 100 mg/kg. Conclusions Conclusions MGA012 is a novel anti-PD-1 mAb with favorable preclinical charac- MGD013 is a bispecific DART molecule capable of simultaneously teristics, including PD-1 binding and biophysical properties, PD-1 blocking the PD-1 and LAG-3 pathways, resulting in enhanced T- pathway blockade, the ability to enhance T-cell responses in vitro cell activation compared to single or combination mAb blockade. and in vivo, and a favorable PK and safety profile in cynomolgus MGD013 has demonstrated a favorable pre-clinical safety and monkeys. Clinical trials are ongoing [NCT03059823] or in planning toxicological profile and is currently initiating clinical testing stage to ascertain the safety and preliminary activity of MGA012 [NCT03219268]. alone or in combination with other immune oncology agents, includ- Trial Registration ing T-cell redirecting bispecific DART® molecules. ClinicalTrials.gov NCT03219268 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 169 of 244 P338 Background Combined IL-6 and CTLA4 blockade enhances CD8+ T cell Hematopoietic progenitor kinase 1 (HPK1) was previously reported as infiltration via CXCR3 and limits growth of pancreatic cancer in a negative regulator of T cell responses due to its ability to attenuate murine models T cell receptor (TCR) signaling. T cells from HPK1 knockout (HPK1-/-) 1 2 1 3 Christopher McQuinn , Matthew Farren , Thomas Mace , Jacob Bowers , mice demonstrated elevated proliferation and cytokine production in 1 2 1 1 Reena Shakya , A. Brad Ferris , Gregory Young , William Carson, III , response to TCR engagement and are resistant to prostaglandin E2 3 2 2 Chrystal Paulos , Bassel El-Rayes , Gregory Lesinski (PGE2)-mediated suppression. Additionally, HPK1-/- bone marrow 1 2 The Ohio State University, Columbus, OH, USA; Winship Cancer derived dendritic cells (BMDCs) eliminate established Lewis Lung Institute of Emory University, Atlanta, GA, USA; Medical University of Carcinoma more effectively compared with HPK1 +/+ BMDCs, sug- South Carolina, Charleston, SC, USA gesting HPK1 is a critical negative regulatory component in key im- Correspondence: Gregory Lesinski (gregory.b.lesinski@emory.edu) mune cell types involved in anti-tumor immunity. To further Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P338 investigate the potential value of targeting HPK1 pharmacologically in cancer immunotherapy, we generated HPK1 kinase dead mice Background (HPK1 KD). Extensive characterization of these mice was conducted Immune checkpoint blockade has not shown efficacy in pancreatic via employing syngeneic tumor models and ex vivo studies. We re- cancer. We hypothesized the cytokine interleukin-6 (IL-6) enhances port here our findings on the blockade of HPK1 kinase activity in immune suppression in this disease, and represents a logical target modulating anti-tumor immune responses. to augment immunotherapy. Prior publications from our group and Methods others demonstrate IL-6 is derived from pancreatic stroma, is in- HPK1 KD mice were generated and characterized, including immune versely associated with overall survival in metastatic patients and cell phenotyping in peripheral blood and secondary lymphoid organs. enhances efficacy of anti-PD-L1 antibodies in pre-clinical models. This These mice were then subjected to in vivo stimulation with agonistic report addresses the efficacy of combined IL-6 and CTLA4 blockade, anti-CD3 followed by measurement of plasma cytokines. HPK1 WT and and identifies a mechanism by which this combination enhances KD mice were further interrogated in syngeneic tumor models in com- effector T cell infiltration into tumors. bination with a cyclooxygenase inhibitor (celecoxib) or anti-PD1. Anti- Methods tumor efficacy was monitored, immune phenotyping was conducted In vivo efficacy of Ab targeting IL-6 and CTLA4 was tested in mice for draining lymph nodes and tumor tissues, and ex vivo studies were bearing subcutaneous MT-5 murine pancreatic tumor cells with performed to evaluate the enhancement of immune responses. G12D R172H Kras and TP53 mutations. CXCR3 blocking antibodies were Results used for in vivo mechanistic studies, while immune biomarkers were HPK1 KD mice were grossly normal and immune phenotyping re- analyzed using flow cytometry or immunohistochemistry. vealed no difference compared with WT mice. HPK1 KD mice demon- Results strated superior anti-tumor efficacy in a sarcoma model. Significantly Combined blockade of IL-6 and CTLA4 inhibited tumor growth rate better anti-tumor efficacy with 50-70% tumor free was observed in as compared to controls (p’s <0.05). IHC analysis revealed increased T HPK1 KD mice compared to WT mice treated with either celecoxib or cells in tumors from combination treated mice (p=0.035 vs. anti-IL-6; anti-PD1, in sarcoma and colorectal tumor models, respectively. Fur- p=0.038 vs. anti-CTLA4; p<0.0001 vs. iso control). Analysis of systemic ther immune phenotyping and ex vivo studies indicated enhanced + + immune biomarkers was conducted via flow cytometric analysis of CD4 and CD8 T cell populations as well as IFNγ and TNFα secretion splenocytes. Despite the role for IL-6 in expanding MDSC, no change along with reduced ratios of CD8/Tregs. Cytolytic activities against + - + + + in monocytic (CD11b Ly6G Ly6C ) or granulocytic (CD11b Ly6G syngeneic tumor cells were also augmented with HPK1 KD CD8 T + low + Ly6C ) cells were observed. However, analysis of T cell subsets re- cells compared with WT CD8 cells. vealed both anti-CTLA4 alone or the combination increased cells with Conclusions Th1 or Th17, but not Th2 phenotypes (p<0.05). Cells with a CD4 Genetic blockade of HPK1 kinase activity revealed a significant en- + + + CD25 FoxP3 phenotype were increased in combination treated hancement of immune responses and resulted in improved anti-tumor mice (p<0.05). We are studying if these cells are suppressive or a efficacy in combination with inhibition of PGE2 or PD-1 pathways. byproduct of enhanced activation marker expression. Blockade of IL-6, CTLA4 or the combination altered circulating cells with phenotypic properties of naïve, effector and central memory T cells (based on P340 + + + CD44/CD62L). Notably, circulating CD4 CD44 CD62L T cells were sig- In vitro effects and mechanism of hu14.18-IL2 immunocytokine nificantly higher in the combination (p<0.05). Consistent with T cell in- against GD positive pediatric malignancies 1 1 1 1 filtration data, sequencing of TCRβ within n=3 representative tumors Holger Lode , Maxi Zumpe , Madlen Juettner , Sascha Troschke-Meurer , 2 2 2 1 from each group of mice revealed a trend toward increased number of Evelyne Janzek , Romana Schaefer , Bettina Neuchrist , Nikolai Siebert , both productive templates and rearrangements. Finally, in vivo adminis- Hans Loibner 1 2 tration of Ab targeting CXCR3 limited the efficacy of the treatment University Medicine Greifswald, Greifswald, Germany; Apeiron Biologics, combination, and reduced CD8 T cell infiltration into tumors. Vienna, Austria Conclusions Correspondence: Holger Lode (lode@uni-greifswald.de) IL-6 blockade enhances efficacy of antibodies targeting CTLA4 in pre- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P340 clinical models of pancreatic cancer. Mechanistically, this involves CXCR3- mediated intratumoral infiltration of CD8 T cells. These data suggest that Background clinically-available IL-6/IL-6R targeted agents could be repurposed to en- Passive immunotherapy with anti-GD antibody (Ab) ch14.18/CHO hance immune checkpoint blockade as a novel combination therapy. (dinutuximab beta) showed activity for the treatment high-risk neuroblastoma (NB) patients and received recently marketing ap- proval in the EU. Immunocytokines are antibody-cytokine fusion P339 proteins that combine the targeting properties of monoclonal Critical role of hematopoietic progenitor kinase 1 in anti-tumor antibodies with the immune stimulatory function of cytokines. immune responses Here we demonstrate activity and mechanism of hu14.18-IL2 1 1 1 1 Dan You , Stephen Hillerman , Jesse Swanson , Ching-Ping Ho , Anwar against a variety of GD positive pediatric tumor cell lines (neuro- 1 1 1 1 Murtaza , Rukiye Eraslan , Miguel Sanjuan , John Hunt , blastoma, osteosarcoma, Ewing’s sarcoma, retinoblastoma) in pre- 1 2 Luisa Salter-Cid , Joshua Curtin clinical models. 1 2 Bristol-Myers Squibb, Lawrence Township, NJ, USA; Johnson & Johnson Methods Pharmaceutical, Spring House, PA, USA Expression of the target antigen GD on LAN-1 (neuroblastoma), MG63 Correspondence: Jinqi Liu (jinqi.liu1@gmail.com) (osteosarcoma), TC-71 (Ewing’s sarcoma) and Y79 (retinoblastoma), and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P339 PD-1/PD-L1 checkpoint on LAN-1 was analyzed by flow cytometry. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 170 of 244 Hu14.18-IL2 mediated ADCC, CDC and whole blood cytotoxicity (WBT) reflecting temporal and spatial heterogeneity during immune system was determined by Cr release assays. modulation by immunotherapies. Differential baseline levels of cyto- Results kines and immune checkpoint expression in the R group may be We found expression of antigen GD on all cell lines derived of major used as a predictive biomarker for nivolumab response if confirmed neuro-ectodermal malignancies in childhood. The highest level was in larger series. found in neuroblastoma but all other cell lines were clearly GD anti- gen positive. Importantly, hu14.18-IL2 was effective in mediating References ADCC, CDC and WBT against all cell lines in vitro, and potency was 1. Choueiri, T. K., et al. Immunomodulatory Activity of Nivolumab in Metastatic found higher than that of ch14.18/CHO in the osteosarcoma and ret- Renal Cell Carcinoma. Clinical Cancer Research.2016;22(22): 546. inoblastoma models. The effects were antigen specific as addition of 2. Chalmin F, et al. Membrane-associated Hsp72 from tumor-derived an anti-idiotypic antibody abrogated the cytolytic activity. exosomes mediates STAT3-dependent immunosuppressive function of Interestingly, tumor specific ADCC in the presence of LA-N-1 neuro- mouse and human MDSC. J Clin Invest. 2010;120:457-71. blastoma cells, leukocytes and sub-therapeutic hu14.18-IL2 concentra- 3. Gao, J., et al. VISTA is an inhibitory immune checkpoint that is increased tions (10-100 ng/ml) resulted in a strong increase of PD-L1 expression after ipilimumab therapy in patients with prostate cancer. Nat both on effector and target cells. This effect required cell-cell contact, Med.2017;23(5): 551-555. since separation of effector cells from target cells using a membrane abrogated the activity. This provides a rationale to explore combinator- P342 ial approaches with agents that inhibit the PD-1/PD-L1 checkpoint. Blood in circulation, with intact cascade systems, as a tool to Conclusions predict first-infusion reactions and mechanism-of-action of Immunocytokine hu14.18-IL2 is effective against GD positive pediatric immunotherapeutics malignancies derived of neuroectodermal origin. Engaged immune ef- 1 1 2 1 Erika Fletcher , Mohamed Elthahir , Frida Lindqvist , Jonas Rieth , fector functions by hu14.18-IL2 result in a concomitant upregulation of 2 1,2 1,2 Gunilla Törnqvist , Justyna Leja-Jarblad , Sara Mangsbo immune checkpoints that suggests a combinatorial approach with 1 2 Uppsala University, Uppsala, Sweden; Immuneed AB, Uppsala, Sweden checkpoint inhibitors early during clinical development. Correspondence: Sara Mangsbo (sara.mangsbo@farmbio.uu.se) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P342 P341 Peripheral immunomodulatory changes in metastatic renal cell Background carcinoma (mRCC) patients treated with nivolumab Since the TGN1412 disaster in London, where an anti-CD28 supera- Lorena Almeida, Manuel Maia, Haejung Won, Dayson Moreira, gonistic antibody induced a life-threatening cytokine storm in six Paulo Bergerot, Marcin Kortylewski, Sumanta Pal healthy men during a clinical trial [1], risks of first-infusion reactions City of Hope Comprehensive Cancer Center, Duarte, CA, USA are important to study prior clinical trial initiation. These assays can Correspondence: Manuel Maia (mcaitano@coh.org) constructed in different ways and depending on the setup, the sensi- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P341 tivity of the assay depends on the antibody tested. It is therefore of great need to validate a cytokine release assay with the potential to Background detect first-infusion reactions regardless of the nature of the anti- CheckMate-009 was a biomarker-based study in which serial biopsies body tested. were conducted for patients receiving nivolumab [1]. Lesser data is Methods available for fluxes in circulating biomarkers during the course of As the assays used today to predict cytokine release syndrome (CRS) treatment. We hypothesized that immunomodulatory changes in are devoid of one or several blood components we setout to investi- blood would mirror previous observations in tissue. gate if a modified chandler loop model could be used to predict Methods CRS. The assay makes use of extracorporeal fresh whole blood in cir- Patients with mRCC receiving nivolumab as standard of care were culation, thus it contains blood immune cells, immunoglobulins prospectively enrolled. Blood samples were drawn at baseline, week along with intact cascade systems [2]. The challenge with such sys- 4 and week 12 during treatment. Analysis of samples for relevant tems is to avoid clotting during assay performance, but correctly per- white blood subsets was conducted by immersing peripheral blood formed, will allow for both monitoring of cytokine release along with mononuclear cells in a mixture of PBS, 2% FCS and 0.1% sodium studies of ADCC and CDC. azide with FcIII/IIR-specific antibody to block nonspecific binding and Results stain the cells with different combinations of fluorochrome-coupled Uniquely the assessed agonistic antibodies were scored to induce antibodies. We further collected fluorescence data on Fortessa (Beckton CRS in blood from all tested donors (n=22) after only 4 hours of incu- Dickinson) and analyzed them using FlowJo software (Tree Star) [2]. bation in the loop assay, whereas non-agonistic antibodies associ- Plasmas were analyzed for human cytokines using Luminex. Changes ated with no or low infusion reactions in the clinic neither induce in cells and cytokines overtime in the overall population were inter- cytokine release nor cause false positive responses. Additionally, the rogated. We also compared baseline levels of cytokines in responders value of an intact complement system in the assay was highlighted (R; including partial/complete response and stable disease) and non- by the possibility to dissect out the mechanism-of-action of alemtu- responders (P; primary progression). zumab and rituximab. Results Conclusions A total of 10 patients were included, with a total of 29 samples col- A modified chandler loop system can complement lymph node-like lected. During treatment, we observed a significant decrease in the pro- assays or be used as an excellent stand-alone test to investigate portion of CD3+CD4+ T cells and an increase in the CD3+CD8+ T cells drug/blood interactions during preclinical development, or for indi- in the overall population (p<0.05). In addition, the expression of PD-1 vidual safety screening prior a first-in-man clinical trial. was significantly decreased in both CD4+ and CD8+ T cells overtime, while serum level of IL2R and CXCL9 significantly increased (p<0.05). References Compared to P, R group showed a trend towards higher baseline levels 1. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner of IFN-alpha and IFN-gamma (p=0.05 and 0.11, respectively) as well as MD, Panoskaltsis N. Cytokine storm in a phase 1 trial of the anti-CD28 VISTA expression on CD4+ T cells (p=0.15) monoclonal antibody TGN1412. N Engl J Med. 2006;355(10):1018-28. Conclusions 2. Mangsbo SM, Sanchez J, Anger K, Lambris JD, Ekdahl KN, Loskog AS, Our results emphasize the immunomodulatory changes in the per- Nilsson B, Totterman TH. Complement activation by CpG in a human ipheral blood occurring during treatment with nivolumab in mRCC whole blood loop system: mechanisms and immunomodulatory effects. pts. Some dynamics contrast with previous observations in tissue [3], J Immunol. 2009;183(10):6724-32. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 171 of 244 P343 Background Novel immuno-oncology biologics derived via directed evolution TIGIT is a coinhibitory receptor that is expressed on lymphocytes, + + of IgSF domains including effector and regulatory CD4 T cells (Tregs), effector CD8 Mark Maurer, Ryan Swanson, Michael Kornacker, Steve Levin, Chris Navas, T cells, and NK cells, that infiltrate different types of tumors. Engage- Chelsea Gudgeon, Lawrence Evans, Martin Wolfson, Dan Ardourel, ment of TIGIT with its reported ligands, poliovirus receptor (PVR) and Daniel DeMonte, Joseph Kuijper, Sean MacNeil, Janhavi Bhandari, Mark PVR-like proteins (PVRL2 and PVRL3) directly suppresses lymphocyte Rixon, Stanford Peng activation. PVR is also broadly expressed in tumors, suggesting that Alpine Immune Sciences, Inc., Seattle, WA, USA the TIGIT-PVR signaling axis may be a dominant immune escape Correspondence: Mark Maurer mechanism for cancer. We report here the biophysical and functional (mark.maurer@alpineimmunesciences.com) characterization of COM902, a therapeutic antibody targeting TIGIT. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P343 We demonstrate that co-blockade of TIGIT and a new checkpoint in- hibitor, PVRIG, augments T cell responses. Background Methods Our variant Ig domain (vIgD™) platform creates novel, therapeutically- Human phage display and mouse hybridoma antibody discovery cam- applicable protein domains with tailored specificity and affinity. These paigns were conducted to generate therapeutic anti-TIGIT antibodies. vIgDs are created through directed evolution of immunoglobulin super- The resulting antibodies were evaluated for their ability to bind to re- family (IgSF) proteins and have unique biochemical properties includ- combinant and cell surface-expressed human TIGIT with high affinity. ing small size, single domain structure, and the capacity to interact Cross-reactivity of the antibodies to cynomolgus and mouse TIGIT was with multiple counter-structures. Because many IgSF family members also examined. A subset of antibodies that bound with high affinity to and their counter-structures are widely expressed on immune cells and human TIGIT, and cross-reactive to cynomolgus TIGIT were further char- tumors, the vIgD platform is well positioned for the development of acterized for their ability to block the interaction between TIGIT and immuno-oncology biologics with potential first-in-class mechanisms of PVR. Blocking antibodies were screened for their ability to enhance action. Here, multiple therapeutic formats for vIgDs were developed antigen-specific T cell activation in vitro either alone, or in combination and characterized. with an anti-PVRIG antibody, COM701. Methods Results Novel vIgDs were created with tailored affinities and modulatory We identified a lead antibody, COM902, that binds to human activities against PD-1, TIGIT, PD-L1, CTLA-4, CD28, and/or ICOS. TIGIT with high femtomolar affinity. This antibody bound to TIGIT These domains were successfully developed into multiple therapeutic endogenously expressed on human CD8 T cells with higher af- formats, including single and multiple domain Fc fusion proteins and finity than tested benchmark antibodies, and was also cross- vIgD-monoclonal antibody (V-mAb) fusion proteins. In addition to reactive to both cynomolgus and mouse TIGIT. When tested for ligand binding and specificity assays, in vitro functional activity was in vitro activity, COM902 augmented cytokine secretion and characterized in several T cell-based assays including cell-based re- tumor cell killing by CMV-specific CD8 T cells with superior or porter systems for pathway agonism or antagonism, primary human equivalent potency to the tested benchmark antibodies. Combin- mixed lymphocyte reactions (MLRs), and costimulation assays utiliz- ation of COM902 with an anti-PD1 antibody or COM701 resulted ing artificial APCs (assessed by proliferation and IFNg production). in enhanced CMV-specific CD8 T cell activity. Furthermore, we Results demonstrated that TIGIT is predominantly expressed on Tregs Several functionally active therapeutic vIgD-based molecules were and effector CD8 T cells from solid tumors compared to periph- created successfully. (1) Single-domain vIgD-Fc fusion proteins with eral blood, suggesting that these populations will likely be prefer- tailored binding to CD28, CTLA-4, and PD-L1 demonstrated differen- entially targeted by COM902. tial activity in T cell activation assays and, depending on their ligand Conclusions binding profile, resulted in greater or reduced IFNγ production and T We describe the development of a high affinity antagonistic TIGIT anti- cell proliferation in human T cell activation assays. (2) Multi-domain body, COM902, that is currently in preclinical development. We postu- vIgD-Fc fusion proteins demonstrated promising targeting of immuno- late that the femtomolar affinity of COM902 could result in lower and modulatory pathways in cell-based reporter assays and MLRs as less frequent dosing in patients. COM902 can enhance human T cell assessed by IL-2 signaling and IFNγ production. Efficacy was compar- activation either alone or in combination with other checkpoint anti- able to monoclonal antibodies against the individual vIgD targets. (3) bodies. Thus, our data demonstrates the utility of targeting TIGIT, PD1, V-mAbs demonstrated target-specific T cell proliferation and IFNγ pro- and PVRIG for the treatment of cancer. duction in vitro, using both recombinant target proteins or target- specific cell lines. P345 Conclusions Tumor protective effect of anti-MUC1.IgE in pancreatic cancer The vIgD platform has successfully generated multiple immuno-oncology 1 1 2 Kamiya Mehla , James A Grunkemeyer , Ragupathy Madiyalakan , therapeutic candidates, in various formats including single- and multiple- 3 1 Christopher F Nicodemus , Michael A Hollingsworth domain Fc fusion proteins as well as V-mAbs. These varied formats con- 1 2 University of Nebraska Medical Centre, Omaha, NE, USA; Oncoquest, fer, from a single molecule, multiple advantages including the multi- Edmonton,, AB, Canada; AIT Strategies, Franconia, NH, USA target modulation capability of evolved IgSFs, and, where applicable, Correspondence: Michael A Hollingswort (kamiya.mehla@unmc.edu) tumor localizing capability of partner molecules or domains. This platform Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P345 may contribute to the next generation of immunotherapeutic proteins in an oncology setting and efforts are ongoing to develop these candidates Background for human therapeutic use. Pancreatic adenocarcinoma remains a highly lethal disease, that has thus far proven to be highly refractory to immunotherapeutic strat- P344 egies. Hence, new approaches are needed. There is epidemiological Development and functional characterization of COM902, a novel evidence that individuals with allergies have a lower incidence of therapeutic antibody targeting the immune checkpoint TIGIT pancreatic cancer [1]. This suggests that distinct immune surveillance 1 1 1 1 Sandeep Kumar , Radhika Desai , Hsin-Yuan Cheng , Kyle Hansen , (Th2 based) might underlie protection against pancreatic cancer ob- 1 1 1 2 Andy Drake , Patrick Wall , Kathryn Logronio , Gady Cojocaru , served in allergic individuals. We hypothesized that therapeutic tar- 1 1 1 1 John Hunter , Mark White , Spencer Liang , Maya Kotturi geting with IgE to enhance FcεRI signaling would trigger an effective 1 2 Compugen USA, Inc., South San Francisco, CA, USA; Compugen Ltd., anti-tumor immune response against pancreatic cancer. We posited Holon, Israel that tumor antigen directed IgE/FcεRI cross-linking on mast cell and Correspondence: Maya Kotturi (mayak@cgen.com) controlled release of histamine near intra-tumor blood vessels could Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P344 be exploited to improve drug delivery in pancreatic cancer. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 172 of 244 Methods Response, FDR p<0.001). Treatment with anti-MMP9 and anti-PDL1 anti- We therefore explored the efficacy of human tumor antigen targeted bodies decreased TCR clonality, with evidence of a more diverse TCR IgE antibody (humanized anti-MUC1.IgE) in combination of anti-PD-L1 repertoire (p=0.005). Immunophenotyping of tumor-associated T cells (for relieving T cell exhaustion [2]) and PolyICLC (for effective dendritic by flow cytometry showed that anti-MMP9 and anti-PDL1 co-treatment cell maturation [2]) in a pre-clinical model of pancreatic cancer using promoted a 2.8-fold increase in CD3+ cells in tumors (p=0.01), which mice transgenic for human MUC1 and FcεRI (hMUC1/hFcεRI). was associated with an increase in CD4+ T cells (3.2-fold increase; Results p=0.006) and CD8+ T cells (2.8-fold increase; p=0.013). In contrast, anti- We observed that a combination of anti-MUC1.IgE+anti-PD-L1 MMP9 and combination treatment resulted in a decrease in tumor- +PolyICLC induced antigen specific rejection of two different MUC1 associated regulatory T cells (CD25+ FoxP3+ cells, p = 0.04). MMP9 expressing pancreatic tumors cell lines (Panc02.MUC1, KPC.MUC1) cleavage of T cell chemoattractant ligands in vitro rendered them func- and prolonged the overall survival of mice challenged with sub- tionally inactive for recruitment of activated primary human effector T cutaneous and orthotopic tumors as compared to mice treated with cells. Luminex analysis of tumor protein lysates showed elevated levels an isotype control antibody (anti-PSA.IgE). Furthermore, anti-MUC1.IgE of key immune cytokines IL-12p70, IL-18, and CXCL10. +anti-PD-L1+PolyICLC combination induced MUC1 specific memory re- Conclusions sponses as evidenced by antigen specific rejection/delays of tumors in Inhibition of MMP9 reduces tumor burden and promotes cytotoxic T mice re-challenged with Panc02.MUC1 tumors. Importantly, we observed cell infiltration in a PD1-axis refractory mouse model. The combin- that NK and CD8 T cells were required for the cell mediated anti-tumor ation of nivolumab and GS-5745, a humanized anti-MMP9 inhibitory responses, as in vivo depletion of these subtypes but not CD4 abrogated antibody, is being evaluated in gastric cancer (NCT02864381). the tumor protective in mice bearing orthotopic tumors. Additionally, our Trial Registration study demonstrated a time dependent increase in intra-tumor vascular ClinicalTrials.gov: NCT02864381. permeability (increased dextran-647 influx) post anti-MUC1.IgE treatment (iv) in subcutaneous tumor (Panc02.MUC1) bearing dTg mice. P347 Conclusions Anti-PD1 x anti-ICOS bispecific antibody XmAb23104 brings Taken together, this is the first study to show that cellular immune re- together PD1 blockade and ICOS costimulation to promote human sponses induced by antigen specific stimulation of the IgE/FcεRI axis in T cell activation and proliferation combination with PolyICLC and anti-PD-L1provided tumor protective Gregory Moore, Michael Hedvat, Matthew Bernett, Christine Bonzon, benefits against pancreatic cancer. Our study provides evidence for the Rajat Varma, Suzanne Schubbert, Sung-Hyung Lee, Kendra Avery, clinical applicability and rapid translation of anti-MUC1.IgE based com- Rumana Rashid, Alex Nisthal, Liz Bogaert, Irene Leung, Seung Chu, bination therapy against pancreatic tumors. Umesh Muchhal, John Desjarlais Xencor, Inc., Monrovia, CA, USA References Correspondence: John Desjarlais (gmoore@xencor.com) 1. Gandini S, Lowenfel AB, Jaffee EM, Armstrong TD. Allergies and risk of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P347 pancreatic cancer: a meta-analysis with review of epidemiology and bio- logical mechanisms. Cancer Epidemio Biomarker Prev. 2005;14(8):1908-16. Background 2. Nagato T, Celis E. A novel combinatorial cancer immunotherapy:poly-IC Tumor infiltrating lymphocytes (TILs) often express multiple immune and blockade of the PD-1/PD-L1 pathway. Oncoimmunology. checkpoints and costimulatory receptors. Checkpoint blockade has 2014;15:3:e28440. demonstrated increased clinical response rates relative to other treat- ment options; however, many patients fail to achieve a response to P346 checkpoint blockade. We sought to identify an additional therapeutic Inhibition of MMP9 yields improved effector T cell responses in a modality to stack with checkpoint blockade that could increase patient PD1-Axis refractory model response rate. We hypothesized that engagement of T cell costimula- Amanda Mikels-Vigdal, Vladi Juric, Chris O'Sullivan, Erin Stefanutti, tory receptors in concert with checkpoint blockade could further Andrew Greenstein, Maria Kovalenko, Jeremiah Degenhardt, Peng Yue, increase T cell activation and proliferation. The combination of check- Victoria Smith point blockade with costimulation could be accomplished using a bis- Gilead Sciences, Inc, Foster City, CA, USA pecific antibody format, with the potential benefits of reduced cost and Correspondence: Amanda Mikels-Vigdal (amanda.mikels- more selective targeting of TILs to improve safety. vigdal@gilead.com) Methods Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P346 XmAb23104, which binds to immune checkpoint PD1 and T cell cost- imulatory receptor ICOS, was assembled in a bispecific antibody plat- Background form with substitutions in the Fc domain to suppress effector Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to function. XmAb23104 was evaluated in vitro by measuring antibody promote tumor growth and metastasis, and is a key component of binding to and cytokine release from Staphylococcal enterotoxin B the immune-suppressive myeloid inflammatory milieu. We developed (SEB) stimulated PBMCs. In vivo activity was evaluated using a mouse a monoclonal antibody (AB0046) that inhibits murine MMP9 and model in which human PBMCs are engrafted into NSG mice assessed its mechanism of action in immunocompetent mice as a (huPBMC-NSG) and the extent of T cell engraftment monitored by single agent or in combination with a murine anti-PDL1 antibody. flow cytometry. Methods Results An orthotopic, syngeneic tumor model of Her2-driven breast cancer We produced XmAb23104, a PD1 x ICOS bispecific antibody that binds was utilized for both efficacy and pharmacodynamic studies involv- both PD1 and ICOS monovalently. XmAb23104 selectively targeted + + ing RNA and T cell receptor (TCR) sequencing, flow cytometry, and PD1 ICOS T cells more than monovalent controls, indicating that a sin- protein analysis. Enzymatic analyses were performed on T cell gle bispecific molecule was capable of avid co-engagement of both chemoattractant CXCR3 ligands (CXCL9, CXCL10, and CXCL11) which PD1 and ICOS. Surprisingly, despite monovalent engagement of ICOS, were subsequently evaluated in chemotaxis assays. XmAb23104 promoted strong T cell activation above that expected of Results PD1 blockade alone, both in vitro and in vivo. In vitro, XmAb23104 en- Anti-MMP9 treatment alone or in combination with an anti-PDL1 anti- hanced IL2 production in an SEB stimulation assay (n = 16 donors) rela- body decreased primary tumor growth as compared to IgG control- tive to control, anti-PD1 alone, and anti-ICOS alone (p < 0.001 for all treated animals (56% vs 335% tumor growth increase, p=0.0005) or comparisons), indicating productive combination of PD1 blockade plus anti-PDL1 alone. Profiling of tumors by RNA sequencing revealed that ICOS costimulation. Analysis of XmAb23104-treated cells revealed hall- inhibition of MMP9 resulted in elevated expression of genes associated marks of ICOS signaling, including AKT phosphorylation and a multi- with immune cell activation pathways (Hallmark Interferon Gamma gene expression signature consistent with ICOS costimulation. In vivo, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 173 of 244 treatment of huPBMC-NSG mice with XmAb23104 promoted superior T P349 cell engraftment to that found for anti-PD1 treatment alone. In a repre- Molecular-targeted radiotherapy with an alkyl-phosphocholine sentative study, XmAb23104 induced a 14-fold increase in human CD45 analog leads toimmunomodulation in a syngeneic murine + cells, while anti-PD1 treatment alone only promoted a 2-fold increase. melanoma model Conclusions Suresh Veerankutty, Peter Carlson, Dana Baiu, Clinton Heinze, XmAb23104, a PD1 x ICOS bispecific antibody, brings together PD1 Alexander Boruch, Amy Erbe, Joseph Grudzinski, Reinier Hernandez, blockade and ICOS costimulation and promotes strong T cell activa- Bryan Bednarz, Jamey Weichert, Paul Sondel, Zachary Morris, tion in vitro and in vivo. Compelling activity suggests clinical develop- Mario Otto ment is warranted for the treatment of human malignancies. University of Wisconsin, Madison, WI, USA Correspondence: Mario Otto (motto@pediatrics.wisc.edu); Zachary Morris P348 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P349 Dual blockade of PD-L1 and LAG-3 with FS118, a unique bispecific antibody, induces T-cell activation with the potential to drive Background potent anti-tumour immune responses Ionizing radiation can influence the immune response to tumors, and Matthew Kraman, Natalie Fosh, Katarzyna Kmiecik, Katy Everett, Carlo the combination of external beam radiotherapy with immunotherapy Zimarino, Mustapha Faroudi, Mateusz Wydro, Alexander Koers, Lesley has increasingly garnered attention in clinical trials. Little is known Young, Michelle Morrow, Jacqueline Doody, Mihriban Tuna, Neil Brewis about the immunomodulatory effects of molecular-targeted radiophar- F-star, Cambridge, United Kingdom maceuticals. 18-(p-iodophenyl) octadecyl phosphocholine (NM404) is a Correspondence: Michelle Morrow (michelle.morrow@f-star.com) phospholipid ether analog with selective sequestration in cancer cells Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P348 [1]. Here, we report on the immunomodulatory properties of the radio- active isostere, I-NM404 in a syngeneic murine melanoma model. Background Methods Despite advances with therapies targeting the PD-1/PD-L1 pathway, In vitro cell uptake of NM404 was evaluated by flow cytometry using many patients are refractory or relapse following treatment. LAG-3 a fluorescent-labeled NM404 analog. Mice, bearing syngeneic B78 expression on exhausted T cells and T-regulatory cells (Tregs) in the melanoma tumors, were injected with a subtherapeutic dose of 2.2 tumour may be responsible for this resistance and provides a ration- MBq I-NM404 via lateral tail vein. Mice were euthanized and tumor ale for co-treatment with antibodies targeting LAG-3 and PD-L1. An tissue was harvested at 4 consecutive 1-week intervals post injection; alternative approach is the development of a bispecific antibody the half-life of iodine-131 is ~1 week. To evaluate markers of immu- encompassing binding sites for two antigens. FS118 is a bispecific nomodulation over time, quantitative PCR and immunohistochemis- antibody targeting LAG-3 and PD-L1 that provides dual pathway try (IHC) were performed on the serially collected tumor tissue blockade with the potential to drive unique biology via co-binding of samples. PD-L1 and LAG-3. Results Methods In vitro, murine B78 melanoma cells sequestered 3-4 times more A LAG-3/PD-L1 mAb bispecific antibody, termed FS118, was engi- NM404 than normal murine splenocytes, and 6-10 times more than pri- neered by introducing a distinct LAG-3 binding capability into the mary human fibroblasts over an 18 hour incubation period. The single, constant region of a human IgG1 molecule and assembled into a bis- sub-therapeutic dose of I-NM404 did not significantly reduce the pecific format with anti-PD-L1 Fabs. Additional mutations introduced growth rate of B78 tumors in the treated mice compared to a control into the Fc region suppress effector function. FS118 was evaluated cohort injected with equivalent mass dose of nonradioactive NM404 in vitro for antigen binding and de-repression of LAG-3 and PD-L1 (excipient). Gene expression analysis revealed a marked modulation of function in both a D011.10 T-cell activation system and a super- various immune and tumor-associated markers during the course of antigen stimulated peripheral blood mononuclear cells (PBMC) assay. radiotherapy, such as IL-1β,IFNγ, CXCL1,IL-18,TGF-β2, B7-H3, PD-L1 Anti-tumour activity of a murine-specific molecule, mLAG-3/PD-L1 and PD-L2. Our preliminary data suggest time-dependent patterns. IHC mAb , was assessed in vivo in the MC38 mouse tumour model and demonstrated that the number of tumor-infiltrating CD4 and CD8 T associated immunophenotypic changes were evaluated using flow cells did not change significantly over time. cytometry. Conclusions Results Our preliminary results suggest time-dependent immunomodulation FS118 is bivalent for both LAG-3 and PD-L1. It is capable of binding induced by I-NM404 in B78 flank tumors. Further research should to both targets simultaneously and can de-repress the inhibitory address the influence of variables such as radioactive dose, choice of function of human PD-L1 and human LAG-3 in an engineered murine radionuclide, tumor type and location on immunomodulation. This T-cell system. FS118 is a potent activator of immune cell function information has the potential to provide guidance in the design of generating sub-nanomolar EC values in a human PBMC assay as more effective therapeutic strategies combining molecular-targeted measured by cytokine with at least equivalent activity to a combin- radiotherapy and immunotherapy for cancer patients. ation of anti-LAG-3 and anti-PD-L1. In murine in vitro assay systems, mLAG3/PD-L1 mAb recapitulates References the function of FS118 in human systems. MC38 tumour growth stud- 1. Pinchuk AN et al. Synthesis and structure-activity relationship effects on ies indicated that mLAG-3/PD-L1 could result in significant anti- the tumor avidity of radioiodinated phospholipid ether analogues. J Med tumour activity equivalent to a combination of antibodies targeting Chem. 2006;49(7):2155-65. LAG-3 and PD-L1. Pharmacodynamic assessment demonstrated changes in the immunophenotype of tumour-infiltrating lymphocytes in the tumour of mLAG3/PD-L1 mAb treated mice. P350 Conclusions Breast tumor suppression mediated by therapeutic expression of Dual blockade of LAG-3 and PD-L1 with a bispecific antibody results chemerin, an innate leukocyte chemoattractant 1 1 2 2 in T-cell activation at least comparable to a combination of anti- Russell Pachynski , Ping Wang , Brian Zabel , Eugene Butcher 1 2 bodies targeting LAG-3 and PD-L1 in primary T-cell assays and mur- Washington University School of Medicine, St Louis, MO, USA; Stanford ine tumour models. These data provide evidence to support the University, Palo Alto, CA, USA rationale for clinical development of FS118, a LAG-3/PD-L1 mAb , for Correspondence: Russell Pachynski (rkpachynski@wustl.edu) the treatment of human cancer. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P350 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 174 of 244 null Background xenograft tumor models were established in NOD-scid IL2Rgamma The infiltration of immune cells into the tumor microenvironment mice (NSG) using tumor cells derived from metastatic melanoma pa- can regulate growth and survival of neoplastic cells. Several studies tients following surgical resection. Upon tumor implantation and have shown a correlation between increases in the number of ef- palpable growth, mice received an adoptive transfer of autologous, fector immune cells present in a tumor and clinical outcomes in unexpanded PBMC from the same patient and treatment with ALKS many human tumors, including breast. Current strategies employing 4230. checkpoint inhibitors aim to stimulate effector immune cells, how- Results ever lack of adequate effector cell numbers within the tumor micro- We found that autologous T cells successfully engrafted NSG recipi- environment can result in suboptimal responses to these agents. ent mice after ALKS 4230 treatment similar to IL-2 and that both Here, we describe a novel strategy employing therapeutic overex- treatments induced cellular expansion over vehicle controls. Follow- pression of chemerin to recruit immune cells into the tumor micro- ing treatment with ALKS 4230 and adoptive transfer of autologous environment (TME) and suppress tumor growth. PBMCs, PDX tumor-bearing mice consistently displayed increased Methods numbers of both CD8 and CD4 T cells migrating into tumor tissue, Publically available whole genome expression datasets were analyzed preferential expansion of non-regulatory T cell subsets, and signifi- for RARRES2 expression, comparing normal breast tissue to invasive cant delays in tumor growth as compared to vehicle-treated controls. breast cancers. Both the JC and EMT6 tumor lines were used in fully im- Conclusions mune competent BALB/c mice. Mammary fat pads were used for inocu- Together these data support the rationale for ALKS 4230 as a novel lation, and chemerin-expressing tumors were compared to control immunotherapeutic for the treatment of melanoma and potentially tumors. Tumors were measured using standard caliper measurements, other solid cancers, as well as the strategy of screening individual, and infiltrating leukocytes measured by flow cytometry. Leukocyte de- patient-specific xenograft models to assess potential treatment pletion studies were performed using specific depleting antibodies. efficacy. Results Our analyses of both TCGA and other public whole genome expres- sion datasets show that RARRES2 (the gene for chemerin), a widely P352 expressed endogenous chemoattractant protein for innate immune A novel CD73 blocking antibody restores T cell function and cells, is downregulated in several studies of human breast cancer. augments efficacy of Adenosine 2A Receptor (A2AR) inhibitor Significant downregulation of RARRES2 is seen in both invasive ductal CPI-444 and lobular breast cancers, compared to normal breast tissue. In Emily Piccione, Jenny Rudnick, Barbara Daine-Matsuoka, Glen Mikesell, mouse models using both the JC and EMT6 tumor lines, we have Richard Miller, Ian McCaffery found that forced overexpression of chemerin within the TME signifi- Corvus Pharmaceuticals, Burlingame, CA, USA cantly suppressed tumor growth with increased numbers of infiltrat- Correspondence: Emily Piccione (epiccione@corvuspharma.com) ing leukocytes compared to controls. Systemic depletion of NK cells Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P352 using anti-GM1 antibody treatment resulted in significant abrogation of chemerin’s anti-tumor effect, suggesting – at least in part – a Background reliance on the innate response. Adenosine accumulates in the tumor microenvironment (TME) Conclusions through degradation of extracellular ATP by ectonucleotidases CD39 We have shown, for the first time, the use of therapeutic overexpres- and CD73 and suppresses T cell activity through activation of A2AR. sion of chemerin is effective at suppressing breast tumor growth in CD73 levels are elevated and are prognostic in certain tumors sug- in vivo mouse models. This approach has been used successfully in gesting CD73 is an important immune-suppressive mechanism. Mul- our melanoma models, and may be a broadly applicable approach to tiple CD73 antibodies are in clinical development that are allosteric increase the number of immune effector cells within the TME. CD73 inhibitors that stimulate internalization (type 2 mechanism), Ongoing studies are looking at the combination of therapeutic che- leading to incomplete inhibition of cell surface CD73 activity. We de- merin modulation in combination with available checkpoint inhibi- scribe CPI-006, a novel CD73 antibody that directly inhibits CD73 en- tors for determination of additive or synergistic effects. zymatic activity (type 1 mechanism). Methods CD73 activity was assayed by Malachite Green. Internalization was P351 measured by flow cytometry. T cell proliferation was assayed by flow A novel, individualized xenograft model of cancer immunotherapy cytometry and cytokine levels by ELISA. Tumor CD73 was assessed and tumor growth inhibition by ALKS 4230 by immunohistochemistry. Gene expression was determined by 1 2 2 2 Lukas Pfannenstiel , Jared Lopes , Heather Losey , Juan Alvarez , Nanostring. Brian Gastman Results 1 2 Cleveland Clinic, Cleveland, OH, USA; Alkermes, Inc., Waltham, MA, USA CPI-006 (nM affinity) was compared to CPX-016 (pM affinity), a potent Correspondence: Lukas Pfannenstiel (pfannel@ccf.org) type 2 CD73 antibody. CPI-006 completely inhibited CD73 catalytic Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P351 activity across a broad range of CD73 expression. In contrast, CPX- 016 incompletely inhibited CD73 activity (~60%) and was not effect- Background ive towards cell lines with high CD73 expression. Recent successes in tumor immunotherapy highlight the curative po- CPI-006 was significantly more effective than CPX-016 at blocking tential of modulating patient anti-tumor immune responses. How- suppression of T cell proliferation and cytokine secretion by adeno- ever, preclinical in vivo modeling of immune/tumor interactions often sine (80% restoration compared to 45%). CPI-006 was effective across depend on a limited number of well-established cell lines. Of avail- a broad range of CD73 expression while CPX-016 efficacy was re- able treatment strategies, the use of cytokine therapy offers the ad- stricted to samples with lower CD73 levels. vantage of using the patient’s own immune cells as anti-tumor CD73 expression was evaluated by IHC in renal cell, melanoma, non- effectors. ALKS 4230 is a fusion protein of circularly permuted IL-2 small cell lung and breast cancers (N=70 each). CD73 expression was and IL-2 receptor α that is selective for the intermediate-affinity IL-2 heterogeneous in the tumor and stromal compartments; however, receptor expressed on NK cells and subsets of memory and effector when expressed, CD73 levels were high, exceeding expression in the T cells. ALKS 4230 is currently in a phase 1 trial to evaluate safety high expressing CD73 cell lines that were incompletely inhibited by and tolerability in the treatment of patients with refractory solid CPX-016. These data suggest that type 2 antibodies will have limited tumors. efficacy in CD73 high expressing tumors. Methods Tumors with high CD73 expression are more responsive to A2AR in- In order to evaluate the ability of ALKS 4230 to promote and en- hibition [1] therefore we investigated the combined effect of CPI-006 hance anti-human tumor immune responses preclinically, individual with A2AR inhibitor, CPI-444. The combination significantly restored Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 175 of 244 T cell proliferation and interferon-gamma production and suppressed activity in cellular binding assay and phagocytosis assay. The lead adenosine responsive gene expression to levels greater than either compound exhibited desirable metabolic stability, solubility and oral agent alone. bioavailability. In in-vivo studies, lead compound demonstrated sig- Conclusions nificant anti-tumor efficacy in A20 B-cell lymphoma tumor model CPI-006 is a novel CD73 blocking antibody that directly and completely upon oral dosing. The above findings support further development inhibits CD73 activity, in contrast to type 2 antibodies in development. of these orally bioavailable agents for use in the clinic. CPI-006 blocks CD73 activity and improves T cell function independent of CD73 expression levels and augments the effect of CPI-444. P354 An orally bioavailable small molecule antagonist of VISTA and References VSIG8 signaling pathways shows potent anti-tumor activity 1. Beavis, P.A., et al., Blockade of A2A receptors potently suppresses the Pottayil Sasikumar, Sudarshan Naremaddepalli, Raghuveer Ramachandra, metastasis of CD73+ tumors. PNAS. 2013;110(36):14711-6. Nagesh Gowda, Govardhan Alluri, Manikyalarao Yerramsetti, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Amit Dhudashiya, P353 Samiulla- Dodheri, Nagaraj Gowda, Murali Ramachandra Discovery and pre-clinical development of an orally available small Aurigene Discovery Technologies Limited, Bangalore, India molecule antagonist targeting the CD47/SIRPα pathway for cancer Correspondence: Murali Ramachandra (sasikumar_g@aurigene.com) immunotherapy Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P354 Pottayil Sasikumar, Chennakrishna Gundala, Wesley Balasubramanian, Sudarshan Naremaddepalli, Archana Bhumireddy, Sandeep Patil, Amit Background Dhudashiya, Vijaysai Rayavarapu, Anirudha Lakshminarasimhan, Samiulla Antibody-mediated blockade of immune checkpoint pathways have Dodheri, Kiran Aithal, Girish Daginakatte, Murali Ramachandra transformed the outlook for cancer therapy. While PD-1/PD-L1 anti- Aurigene Discovery Technologies Limited, Bangalore, India bodies primarily focus on T-cells to achieve anti-tumor efficacy, other Correspondence: Murali Ramachandra (sasikumar_g@aurigene.com) cells in the tumor microenvironment such as myeloid cells including Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P353 MDSCs also play a role in immune evasion. To overcome the immune resistance induced by MDSCs, V-domain Ig suppressor of T-cell activa- Background tion (VISTA) expressed predominantly on myeloid cells and tumor- The CD47/signal regulatory protein alpha (SIRPα) axis is a critical infiltrating lymphocytes is considered as an ideal target. Recent findings regulator of myeloid cell activation and serves as an immune check- also support the role of VISTA pathway in clearance of apoptotic bodies point for macrophage mediated phagocytosis. Because of its fre- and prevention of autoimmunity. VISTA is reported to mediate immune quent upregualtion in several cancers, CD47 contributes to immune suppression through homophilic interaction as well as interaction with evasion and cancer progression. Most of the current approaches in V-Set and immunoglobulin domain containing 8 (VSIG8). We sought to the immunotherapy focus on T-cell axis. Macrophages and other discover and develop an orally available small molecule VISTA antagon- myeloid immune cells offer much promise as effectors of cancer im- ist targeting both VISTA and VSIG8 pathways. Unlike antibodies an oral munotherapy, hence efforts to modulate them for therapeutic bene- gent potentially offers the convenience, flexibility to adjust dose and fit are gaining momentum. In this regard disruption of CD47-SIRPα schedule to address any emergent adverse events and ease of combin- interaction is now being evaluated as a therapeutic strategy for can- ation therapy. cer to stimulating macrophage mediated anti-tumor immune re- Methods sponse. In view of the requirement for the intravenous dosing for all VISTA belongs to B7 family and shares sequence homology with the B7 reported CD47 targeting agents, we sought to discover and develop family ligands PD-L1, a focused library of compounds mimicking the an orally available small molecule CD47 antagonist. An oral CD47 interaction of checkpoint proteins of B7 family was designed and syn- agent potentially offers the convenience, flexibility to adjust the dose thesized. These compounds were tested in VISTA and VSIG8 specific and schedule to address any emergent adverse events and ease of functional assays. In-vivo efficacy was evaluated in mouse syngeneic combination therapy. B16F10 melanoma and CT26 colon carcinoma models. Impact of test Methods agents on various immune populations was measured by flow cytomet- Series of peptide disrupting CD47-SIRPα interactions were identified by ric analysis in tumor–bearing animals upon repeated dosing. rational design strategy based on CD47/SIRPα interacting interface. An Results FITC probe based cellular binding assay was established to identify high Screening and analysis of the focused library of compounds led to peptide fragments. The elements of this pharmacophore were incorpo- the identification of hits capable of functional disruption of the rated into non-peptidic small molecule scaffolds resulting in lead com- checkpoint protein(s) signaling. Further optimization resulted in lead pounds disrupting CD47/SIRPα interaction. The shortlisted compounds compounds targeting both VISTA and VSIG8 signaling pathways with were further screened in a phagocytosis assay. In vivo efficacy was eval- desirable drug-like properties. Potent functional activity comparable uated in A20 B Cell lymphoma syngeneic tumor model. to that obtained with an anti-VISTA or anti-VSIG8 antibody in rescu- Results ing effector functions was observed with the lead compound along Initial hits identified in FITC probe based cellular binding assay was with selectivity against other immune checkpoint proteins. An ad- further optimised for their activity in binding assay. Lead CD47 an- vanced lead compound exhibited sustained immune PD in tumor- tagonists induced phagocytototic activity of human macrophages to bearing animals including desirable impact on myeloid and T-cells in a similar extent as commercially available anti-CD47 antibodies. Fur- both circulation and tumor. The advanced lead compound also ther optimization of these leads resulted in compounds with desir- exhibited significant efficacy in syngeneic pre-clinical tumor models able physico-chemical properties and good oral bioavailability. An of melanoma and colon cancers upon once a day oral dosing with advanced lead CD47 antagonist inhibited primary tumor growth excellent tolerability. (~90%TGI) in a mouse syngeneic model of B-cell lymphoma upon Conclusions twice a day oral dosing. Biomarker characterization and efficacy stud- Our efforts have resulted in the identification of novel oral antago- ies in additional tumor models are ongoing. nists of VISTA and VSIG8 signaling. Desirable drug-like properties and Conclusions anti-tumor efficacy at well-tolerated doses by the advanced lead Rational design based on CD47/SIRPα interacting interface led to the compound in pre-clinical models supports its further development identification of a novel and selective CD-47 antagonist with potent towards advancing to the clinic. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 176 of 244 P355 Background Epigenetic modulation promotes tumor suppression and improves In our previous studies, we demonstrated that combination of a CXCR4 survival when combined with checkpoint inhibition in murine antagonist with an anti-angiogenesis agent axitinib relieved myeloid- models of breast cancer derived suppressor cells (MDSCs) mediated immunosuppression and Evanthia Roussos Torres, Hayley Ma, Christine Rafie, Brian Christmas, suppressed HIF-2a expression, which resulted in synergistic antitumor Todd Armstrong, Elizabeth Jaffee effects in 786-0 and A498 RCC xenografts. To further study the CXCR4 Johns Hopkins University, Baltimore, MD, USA mechanism of action in an immune-proficient background, we investi- Correspondence: Evanthia Roussos Torres (ertorres@jhu.edu); Elizabeth gated the activity of a CXCR4 antagonist in a syngeneic mouse tumor Jaffee model. As the current murine models for RCC do not share the same Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P355 genetic alterations with the human disease, the B16-OVA model was selected for this study. The CXCR4 antagonist was tested in combin- Background ation with axitinib or with checkpoint inhibitors which are part of the Checkpoint inhibition is a very successful treatment strategy in can- SOC in managing melanoma in clinic. cers that are naturally immunogenic by attracting T cells into the Methods tumor microenvironment (TME) and promoting cytotoxic signaling B16-OVA cells were implanted into C57BL/6 mice. Seven-days post pathways. Most breast cancers are not highly immunogenic likely implantation, mice were treated with axitinib, X4-136 (CXCR4 inhibi- due to an immunosuppressive microenvironment. One strategy to tor) or anti-PD-L1/anti-CTLA4, or the combinations for 16 days. At transform this TME is to use epigenetic modulation to affect activa- sacrifice, tumors were excised and flash frozen in liquid nitrogen for tion and trafficking of myeloid derived suppressor cells (MDSCs), Western Blot analysis or treated with collagenase for the analysis of known to alter the immunogenicity of the TME and sensitize tumors subsets of T-cells by FACS. to checkpoint modulation. We hypothesize that combinatorial ther- Results apy primes the TME by altering infiltration and function of MDSCs Axitinib alone modestly inhibited growth of B16-OVA tumors. X4-136, leading to a more robust T cell response. the CXCR4 inhibitor, alone demonstrated more robust activity than Methods the combination treatment of anti-PD-L1 and anti-CTLA4. The anti- We use the HER-2/neu transgenic mouse model with challenge of syn- tumor activity of the anti-PD-L1 and anti-CTLA4 regimen was further geneic cell lines. This model enables us to study the efficacy of different enhanced in combination with X4-136. combinations of an anti-HER2 antibody (a-Her2), an epigenetic agent, Preliminary analysis of infiltrating immune cells by flow cytometry the histone deacetylase inhibitor entinostat, and checkpoint inhibitors, showed that axitinib treatment led to an increase in immunosuppres- anti -programmed cell death protein (PD-1) and anti-cytotoxic T- sive Treg cell population in tumors, while treatment with X4-136 lymphocyte-associated protein 4 (CTLA-4) antibodies, on tumor growth alone or the anti-PD-L1/anti-CTLA4 combination led to a decrease in and to help identify co-stimulatory and inhibitory factors regulating T Tregs. The decrease was more pronounced when X4-136 was used in cell and MDSC responses. Characterization of tumor infiltrating lympho- combination with axitinib. Additional analysis of other immune cell cytes (TIL) and their functional capabilities are being investigated in pri- subsets will also be presented. mary tumors using fluorescence-activated cell sorting, nanostring gene This combination also led to a decrease in the expression of HIF-2α expression profiling, and immunohistochemistry. and cyclin D1 in comparison to vehicle treated mice as demonstrated Results by western blot analysis of tumors, suggesting an anti-proliferative We found significant improvement in survival and delay in tumor effect. PARP cleavage was apparent when X4-136 was used in com- growth in tumor bearing mice- treated with entinostat, anti-PD1, and bination with axitinib indicating the role of apoptosis in anti-tumor anti-CTLA-4 and with a-Her2, entinostat and anti-PD1 or anti-CTLA4. effect. Additional analysis of tumors after treatment in earlier time We show that addition of entinostat to checkpoint inhibition leads to points will also be presented. significantly increased infiltration of granulocytic-MDSCs and CD8 T Conclusions effector cells into the TME. Flow cytometric evaluation suggests in- X4-136 alone exhibited potent anti-tumor activity in the B16-OVA creased T cell activation, exhaustion, and altered MDSC function. murine melanoma model. Added benefit was observed when X4-136 Functional assays of isolated MDSCs from mice treated with combin- was added to either axitinib or to anti-PD-L1/anti-CTLA-4 treatments. ation therapy demonstrates reduced suppressive ability of these cells. Treatment benefits were associated with the reduction of Treg popu- Gene expression profiling of isolated MDSCs and TIL is underway to lation in the tumor microenvironment. Induction of apoptosis was help determine significant changes in immune related pathways that observed in combination treatment of axitinib and X4-136. have lead to our observed outcomes. Conclusions P357 Addition of entinostat and checkpoint inhibition to a-Her2 therapy The Tt-cell growth factor cocktail IL-2/IL-15/IL-21 enhances significantly increases infiltration of innate and adaptive immune expansion and effector function of tumor-infiltrating T cells in a cells into the highly tolerant breast tumors and leads to improved novel process developed by iovance survival and decreased tumor burden. Results suggest that this com- Ian Frank, Michelle Simpson-Abelson, Michael Lotze, Krit Ritthipichai, binatorial treatment alters the function of the infiltrating cell types Christopher Mosychuk that lead to the observed phenotype. We aim to delineate genetic al- Iovance Biotherapeutics, Tampa Bay, FL, USA terations responsible for these observations. It is our hope that our Correspondence: Michael Lotze (michelle.abelson@iovance.com) novel findings will provide further rationale for combination therapy Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P357 and improve the response rate to immunotherapy in patients with breast cancer. Background Adoptive T cell therapy with autologous tumor infiltrating lymphocytes P356 (TIL) has demonstrated clinical efficacy in patients with metastatic mel- Efficacy and Mechanism of Action of CXCR4 Inhibition in B16-OVA anoma and cervical carcinoma. In some studies, clinical outcomes have Melanoma Model positively correlated with the total number of cells infused and/or per- 1 2 1 Ruchi Saxena , Yan Wang , James Mier centage of CD8+ T cells. Most current production regimens solely utilize 1 2 Beth Israel Deaconess Medical Center, Boston, MA, USA; X4 IL-2 to promote TIL growth, although enhanced lymphocyte expansion Pharmaceuticals, Cambridge, MA, USA has been reported using IL-15 and IL-21-containing regimens. This Correspondence: James Mier (rsaxena@bidmc.harvard.edu) study describes the positive effects of adding IL-15 and IL-21 to the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P356 standard IL-2-alone TIL generation protocol. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 177 of 244 Methods CD40L. CDX-1140 also activates B cells, and has direct anti-lymphoma The process for generating TIL includes a pre-Rapid Expansion Proto- activity in xenograft models. However, the antibody does not promote col (pre-REP), in which TIL emigrate out of tumor fragments of 1-3 cytokine production in whole blood assays. Importantly, CDX-1140 has mm in media containing IL-2. To further stimulate TIL growth, TIL shown good pharmacodynamic and safety profiles in a cynomolgus are expanded using a secondary culture period termed the Rapid Ex- macaque study investigating doses from 0.01 to 10 mg/kg. This study pansion Protocol (REP) that includes irradiated PBMC feeders, IL-2 showed a clear dose response with respect to changes in hematologic and anti-CD3. In this study, a shortened pre-REP and REP expansion and circulating cytokine values (IL-12p40) that were expected to result protocol was developed to expand TIL while maintaining the pheno- from CD40 activation. typic and functional attributes of the final TIL product. This shortened Conclusions TIL-generation protocol was used to assess the impact of IL-2 alone These data support the potential of CDX-1140 as part of a cancer versus a combination of IL-2/IL-15/IL-21. These two culture regimens therapy regimen, and the phase 1 dose-escalation clinical study is an- were compared for the generation of TIL grown from colorectal, mel- ticipated to begin this year. anoma, cervical, triple negative breast, lung and renal tumors. At the completion of the pre-REP, cultured TIL were assessed for expansion, P359 phenotype, function (CD107a+ expression and IFNg release) and TCR Effect of targeted anti-GD2/-CD16 Bispecific NK cell Engager (BiKE) Vβ repertoire. with or without IL-15 super agonist ALT803 against GD2+ Results Neuroblastoma (NB) and Ewing Sarcoma (ES) Enhanced TIL expansion (>20%), in both CD4+ and CD8+ cells in the 1 1 1 2 Aradhana Tiwari , Dina Edani , Janet Ayello , Hing C. Wong , IL-2/IL-15/IL-21 culture cocktail was observed across multiple tumor Mitchell S Cairo histologies. Preliminary analysis demonstrated a shift towards a pre- 1 2 New York Medical College, Valhalla, NY, USA; Altor dominantly CD8+ TIL population with a skewed TCR Vβ repertoire in BioscienceWebsiteDirections, Miramar, FL, USA TIL cultured with the IL-2/IL-15/IL-21, versus IL-2 alone. IFNg release Correspondence: Aradhana Tiwari (aradhana_tiwari@nymc.edu) and CD107a expression were also elevated in TIL cultured in the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P359 presence of IL-2/IL-15/IL-21 versus those cultured using IL-2 alone. Conclusions Background Rapidly expanding TIL ex vivo for adoptive cell therapy is essential in GD2 is a surface disialoganglioside that is a well-characterized immu- treating patients with cancer. We report an increased TIL-product notherapeutic target in NB and Sarcomas [1]. The efficacy of anti- yield, in addition to potentially beneficial phenotypic and functional GD2 antibodies depends on engaging functional NK cells to kill GD2- differences, when TIL are cultured with IL-2/IL15/IL-21 as compared positive targets through antibody dependent cellular cytotoxicity to IL-2 alone. We suggest a more robust process encompassing the (ADCC). However, NK cell number and function are decreased in use of IL-2/IL-15/IL-21 in TIL culture may provide a means to promote most cancer patients at diagnosis, are further reduced by radiation TIL expansion particularly in tumors with poor T cell infiltration. chemotherapy, antigen loss and immunosuppressive tumor micro- environment (TME) which contribute to treatment failure. One of the P358 new approaches to overcome TME resistance and improve NK cell Development of CDX-1140, an agonist CD40 antibody for cancer mediated ADCC against tumor cells is using a BiKE [2]. ALT-803 is a immunotherapy superagonist of an IL-15 variant bound to an IL-15RaSu-Fc fusion 1 2 2 2 Lawrence Thomas ,Laura Vitale , Thomas O'Neill , Jenifer Widger , with enhanced IL-15 biological activity. 2 2 2 2 Andrea Crocker , Li-Zhen He , Jeffrey Weidlick , Karuna Sundarapandiyan , To investigate the in-vitro activity of hu-anti-GD2/-CD16 BiKE with or 1 1 1 1 James Storey ,Eric Forsberg , Catherine Pilsmaker , Lauren Gergel , without ALT803 against GD2 expressing NB/ES 1 2 1 2 Elizabeth Do ,Joel Goldstein , Henry Marsh ,Tibor Keler Methods 1 2 Celldex Therapeutics, Needham, MA, USA; Celldex Therapeutics, Anti-GD2/-CD16 BiKE was constructed in pBudCE4.1 mammalian ex- Hampton, NJ, USA pression vector and transfected in to HEK293-EBNA Cells. Stable Correspondence: Lawrence Thomas (Lthomas@celldex.com) clone were selected by Zeocin for the secretion and purification of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P358 anti-GD2/-CD16 plasmid by ProBond™ Ni column and validated by Western blot. Cytotoxicity was examined against NB/ES cells with or Background without ALT803 (generously supplied by Altor Biosciences) with Current limitations for immunotherapy approaches include poorly func- K562-mbIL21-41BBL expanded NK cells by DELFIA cytotoxicity assay tioning events early in the immune response cycle, such as efficient at 10:1 E:T ratio. antigen presentation and T cell priming. CD40 signaling in dendritic Results cells (DCs) leads to upregulation of cell surface costimulatory and MHC GD2 expressing NB (54.4± 10.61%) and ES (71.93±8.33%) cells were molecules and the generation of cytokines, which promotes effective used for functional assays. Anti-GD2/-CD16 BiKE was purified from the priming of CD8+ effector T cells while minimizing T cell anergy and the transfected culture supernatant and validated by western blot (Fig. 1A). generation of regulatory T cells. This naturally occurs through inter- BiKE+NK compared to Medium+NK significantly increased NK mediated action with CD40 ligand (CD40L) expressed on CD4+ T-helper cells. cytotoxicity against NB: SKNF1 (64.5±5.9% vs. 20.1±1.3%, p=0.002), CD40 signaling can also be achieved using specific monoclonal anti- SKNBE2 (67.4±4.02% vs. 15.1±0.9%, p=0.004), SHS5Y5 (68.9±0.9% vs. bodies (mAbs), leading to the development of several agonist CD40 30.2±0.85%, p=0.005) and ES: EWS502 (68.05±3.06% vs. 28.1±4.05%, antibodies that have initiated clinical development. p=0.004), A673 (66.02±4.05% vs. 20.3±0.8%, p=0.005) respectively. Methods Further, BiKE+NK+ALT803 compared to BiKE+NK+rhuIL15 improved NK Our approach for the development of a CD40 agonist antibody was mediated cytotoxicity against NB: SKNF1 (73.9±9.3% vs. 56.5±8.5%, to define a balanced profile between sufficiently strong immune p=0.007) and SKNBE2 (63.9±0.6% vs. 40.5±0.49%, p=0.002) respectively, stimulation and the untoward effects of systemic immune activation. against NB/ES cell lines (Fig. 1B). Results Conclusions CDX-1140 is a human IgG2 antibody derived from human Ig transgenic Our preliminary results demonstrated that the BiKE with ALT803 sig- mice, that activates DCs as demonstrated by upregulation of costimula- nificantly enhanced NK cytotoxicity against NB and ES. Future studies tory molecules and increased production of cytokines. This activity is Fc will investigate the efficacy of this BiKE with ALT803 against GD2 ex- independent as it is maintained using an F(ab’) fragment of the anti- pressing solid tumor in humanized NSG xenografted mouse model. body. CDX-1140 does not block the binding of CD40L and the addition of soluble CD40L greatly enhances DC activation by CDX-1140, suggest- References ing that CDX-1140 may act synergistically with naturally expressed 1. Perkins S M, Shinohara ET, DeWees T, Frangoul H. Outcome for children Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 178 of 244 with metastatic solid tumors over the last four decades. PLoS One. 2014;9. safety profile. Combination of APX005M with ICI enhances T-cell re- 2. Gleason MK, et al. Bispecific and trispecific killer cell engagers directly sponses providing a rationale for the ongoing clinical studies com- activate human NK cells through CD16 signaling and induce cytotoxicity bining APX005M with anti-PD-1 antibodies in NSCLC and melanoma and cytokine production. Mol Cancer Ther. 2012;11:2674-2684. patients (trials NCT03123783 and NCT02706353). P361 A novel T-cell engaging bispecific antibody platform: Efficient In vivo tumor clearance with minimal cytokine release Nathan Trinklein, Andrew Boudreau, Ben Buelow, Starlynn Clarke, Kevin Dang, Laura Davison, Shelley Force Aldred, Katherine Harris, Suhasini Iyer, Brett Jorgensen, Heather Ogana, Duy Pham, Payal Pratap, Udaya Rangaswamy, Ute Schellenberger, Harshad Ugamraj, Omid Vafa, Wim van Schooten Teneobio, Menlo Park, CA, USA Correspondence: Nathan Trinklein (ntrinklein@teneobio.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P361 Background Fig. 1 (abstract P359). See text for description Using transgenic rats and a unique sequence-based discovery ap- proach, we have created a large collection of fully human anti-CD3 antibodies with diverse T-cell agonist activities. We have used these anti-CD3 antibodies to create a unique multi-specific platform for T- cell redirected tumor cell killing. P360 Methods APX005M is a potent CD40 agonistic antibody capable of stimulating Our novel discovery platform combines antibody repertoire deep se- both innate and adaptive immune responses against cancer quencing, high-throughput gene assembly, and recombinant expres- Pia Björck, Erin Filbert, Ovid Trifan, Xiaodong Yang sion and generates a much larger diversity of antibodies than Apexigen Inc., San Carlos, CA, USA traditional approaches. The CD3 antibodies identified by our platform Correspondence: Xiaodong Yang (otrifan@apexigen.com) show diverse in vitro T-cell activation profiles measured by CD69 up- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P360 regulation, IL2, and IFNg production. Using our discovery platform, we have also generated human domain antibodies targeting tumor Background antigens that may be combined with our unique CD3 antibodies to CD40 plays an important role in activation and regulation of both in- create multi-specific antibodies. nate and adaptive immunity. Using the proprietary APXiMAB™ dis- Results covery platform, Apexigen discovered APX005M, a potent immune As one example, we have created a CD3xBCMA bispecific antibody stimulatory CD40 agonistic antibody currently being developed for (TNB-383B) for the treatment of multiple myeloma. TNB-383B kills mul- cancer immunotherapy. APX005M is a high-affinity humanized IgG1 tiple myeloma cells in vitro and in vivo in a BCMA-dependent manner, antibody targeting human CD40. APX005M was engineered to carry and kills primary patient myeloma cells ex vivo. The EC50 for cytotox- an S267E mutation in its Fc region to enhance CD40 agonistic activity icity was in the single-digit nanomolar range for TNB-383B against MM via cross-linking to the FcRγIIb. cell lines in vitro. TNB-383B showed much reduced (ie IFN-ɣ)or absent Methods (ie IL-2) cytokine release compared to other anti-CD3 antibodies. In vitro PBMCs were obtained from healthy human subjects. Isolated cell results were consistent across 10 healthy huPBMC donors. Ex vivo, TNB- subsets were cultured in various conditions with APX005M. The po- 383B efficiently lysed primary MM cells in the presence and absence of tential synergistic effect of APX005M with immune checkpoint inhibi- supplementary T-cells. In vivo, TNB-383B mediated clearance of MM tu- tors (ICI) was assessed by in vitro culture of human dendritic cells mors from NSG mice at doses as low as 10ng of bispecific antibody. (DC) and allogeneic T cells with APX005M and anti-PD-1 or anti-PD- Conclusions L1 antibodies. The duration of APC activation following exposure to In summary, we have created a T-cell engaging bispecific antibody APX005M was determined by culturing PBMCs with APX005M for 24 platform with tunable T-cell agonism that can be used to optimize hours. Cell activation was measured at various times post washout. the therapeutic index for a variety of tumor antigens. Results APX005M binds with high-affinity to human CD40 (Kd=0.12 nM) and recognizes a unique epitope that overlaps with the CD40 ligand (CD40L) binding domain thus mimicking CD40L-induced activation. APX005M is P362 capable of activating human B cells (EC50=12 pM) and DC (EC50=0.49 A novel assay using RNA aptamers to quantitate the fraction of nM) and also promotes proliferative responses of tumor-infiltrating T IL2Ra (CD25) receptors occupied by IL2 cells. APX005M’s CD40 agonistic activity depends on cross-linking of Fc- Suresh Veeramani, Sue Blackwell, William Thiel, Paloma Giangrande, gamma receptors. Receptor occupancy studies revealed that 10% of George Weiner CD40 receptor occupancy is sufficient to produce maximum APC and T University of Iowa, Iowa City, IA, USA cell activation. Short-term exposure (24 hours) of PBMC to APX005M in- Correspondence: George Weiner (suresh-veeramani@uiowa.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P362 duced long-lasting activation of B cells, monocytes and T cells that remained activated 2 weeks after removal of APX005M. In vitro co- Background culture of human DC and allogeneic T cells showed that APX005M in- RNA aptamers bind to antigens in a manner analogous to antibodies duces a dose-dependent increase of CD4 and CD8 T cell proliferation and their binding can be quantitated using simple RT-PCR. Using IL2- and IFN-γ secretion, and the T-cell responses were further enhanced by IL2Ra as a model, we report a novel assay using IL2-IL2Ra-binding anti-PD-1 or anti-PD-L1 antibodies suggesting that APX005M combined RNA aptamers that allows for quantitation of receptor occupancy by with ICI can synergistically stimulate T-cell responses. its ligand. Conclusions Methods The immune stimulatory activity of APX005M results from its unique epitope and is dependent on cross-linking by Fc-gamma receptors. A modification of whole cell Systematic Evolution of Ligands by EX- ponential enrichment (SELEX) was used to select T regulatory (Treg) The properties of APX005M make it an optimal CD40 agonist for cell-specific RNA aptamers. Aptamers specific for common T cell stimulating anti-tumor immune responses while maintaining a good Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 179 of 244 + - antigens were precleared using normal donor CD4 IL2Ra T cells, P363 + + followed by enrichment for aptamers specific for CD4 IL2Ra Tregs The OX40-CTLA-4 bispecific antibody, ATOR-1015, induces immune obtained from the same donor. The process was repeated for eight activation and anti-tumor effect rounds with each round using T cells from a different normal donor. Niina Veitonmäki, Karin Hägerbrand, Mia Thagesson, Doreen Werchau, High-throughput sequencing and bioinformatics analysis was used to Karin Enell-Smith, Anna Rosén, Sara Frizell, Anne Månsson-Kvarhammar, select top Treg-binding aptamers. Binding of aptamers to unoccu- Per Norlén, Furebring Tina, Peter Ellmark pied IL2Ra and IL2Ra occupied by IL2 was determined by RT-qPCR. Alligator Bioscience, Lund, Sweden Some aptamers bound preferentially to unoccupied IL2Ra while Correspondence: Niina Veitonmäki (niv@alligatorbioscience.com) others bound preferentially to IL2Ra occupied by IL2. To determine Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P363 the fraction of IL2Ra occupied by IL2, aptamers that bound preferen- tially to unoccupied receptor or to IL2-occupied receptor were mixed Background in equimolar quantities. Aptamer mix was added to IL2Ra-coated ATOR-1015 is an OX40-CTLA-4 bispecific immune activating antibody Dynabeads that were pre-incubated with various concentrations of developed for tumor-directed immunotherapy. The compound was IL2 to create various IL2-occupied receptor fractions. Aptamer bind- generated by fusing a high affinity CTLA-4 binder, derived by FIND® ing was quantified by RT-qPCR using a set of primers specific for optimization of CD86, to an agonistic OX40 antibody derived from both aptamers. Fluorescent probes specific for the variable region for the human antibody library ALLIGATOR-GOLD®. each aptamer were used to quantify each of the aptamers. ATOR-1015 binds both targets simultaneously resulting in cell-cell in- Results teractions expected to enhance the immuno-stimulating effect of the Five of the top 12 Treg-binding aptamers recognized IL2Ra. Some compound. The mode of action of ATOR-1015 is thought to be a IL2Ra-specific aptamers (e.g. Tr-8) bound preferentially to unoccupied combination of effector T cell activation and regulatory T cell (Treg) IL2Ra and others (e.g. Tr-7) to the IL2-IL2Ra complex (Fig. 1). Standard depletion. curves were developed which allowed for determination of the frac- Methods tion of IL2Ra occupied by IL2 (Fig. 2). Human primary cells used in in vitro assays were isolated from Conclusions leukocyte concentrates from healthy donors. The in vitro effects were A Treg cell-based SELEX was used to select Treg-binding aptamers measured by using either standard cytokine release assays or Pro- specific for IL2Ra. Aptamer pairs showing differential binding prefer- mega reporter assays. ences towards unoccupied IL2Ra versus the IL2-IL2Ra complex were Human OX40 transgenic (knock-in) mice were generated by genO- identified. Because the selected aptamer pairs share the same way SA and female mice were used for the anti-tumor effect studies primers, RT-qPCR amplification using a single set of primers allowed using syngeneic mouse models. Tumors and spleens from treated for quantitation of the ratio of bound aptamers. The resulting ratio mice were analyzed for Treg and T cell populations by flow reflects the occupancy of the IL2Ra receptor by its ligand IL2. This cytometry. technique could be valuable for studying the role of IL2-IL2Ra ligand- Results receptor complex in anti-tumor immunity. Importantly, modifications ATOR-1015 dependent T cell activation and Treg depletion is supported of this assay could be used to quantitate other receptor-ligand com- by cell-based in vitro studies and in vivo syngeneic tumor models. plexes relevant in the field of cancer immunotherapy. The ability to induce ADCC of human Treg was investigated using a FcγR expressing reporter assay demonstrating superior effect of ATOR-1015 compared to the monospecific antibodies. Further, ATOR- 1015 has been shown to induce activation of T cells in the presence of CTLA-4 expressing cells. Treatment with ATOR-1015 reduces tumor growth and prolongs survival in syngeneic tumor models in vivo using human OX40 transgenic mice. Further, ATOR-1015 treatment demonstrates an increase in the intratumoral CD8 T cell/Treg ratio which is super- ior compared to the monospecific counterparts, without affecting systemic T cells. Conclusions ATOR-1015 suppresses/depletes Tregs and activates CD8 Tcells in vivo and is currently in the second phase of production, and process development. ATOR-1015 is planned to enter clinical trials in 2018. P364 Discovery and pharmacological characterization of JNJ-63723283, an anti-programmed cell death protein-1 (PD-1) antibody that blocks PD-1 function Fig. 1 (abstract P362) See text for description 1 1 1 1 Catherine Ferrante , Nikki DeAngelis , Gordon Powers ,Jocelyn Sendecki , 2 1 1 1 Hillary Millar , Bethany Mattson , Darlene Pizutti , Christina Lourdes Mayer , 1 1 1 1 Weirong Wang , Raymond Brittingham , John Wheeler ,Linda Barone , 1 1 1 3 Rupesh Nanjunda , Eilyn R. Lacy ,Sheng-Jiun Wu ,Jinquan Luo , 1 1 1 1 Enrique Zudaire , Kathryn Packman , Brian Geist , Kevin Trouba , 1 1 Matthew V. Lorenzi , Raluca I. Verona 1 2 Janssen Research & Development, Spring House, PA, USA; Janssen Research & Development, Beerse, Belgium; Janssen Resaerch & Development, Spring House, PA, USA Correspondence: Raluca I. Verona (RVerona@its.jnj.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P364 Background Programmed cell death protein-1 (PD-1), a negative immune check- Fig. 2 (abstract P362) See text for description point receptor, suppresses T cell function when bound to its ligands Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 180 of 244 PD-L1 and PD-L2. Tumor cells exploit this pathway to evade immune P365 surveillance; therefore, inhibiting the PD-1 pathway can restore T cell Development of JNJ-64158120, an anti-TIM-3 antibody to overcome function, stimulating their anti-tumor response. Here we report the innate and acquired mechanisms of resistance to PD-1 therapy 1 1 1 1 characterization of JNJ-63723283 (JNJ-283) to support new combin- Nikki DeAngelis , Gordon Powers , Keri Dorn , Matthew Chomo , 1 1 1 1 ation approaches in the future. Douglas Dolfi , Karla Wiehagen , Douglas Marvel , Bethany Mattson , 1 1 1 1 Methods Darlene Pizutti , Liam Campion , Caitlin Morgan , Brian Tomkowicz , 1 2 1 1 JNJ-283 was generated by phage panning against human and cy- Nina Sabins , Sandra Santulli-Marotto , Jocelyn Sendecki , John Wheeler , 1 1 1 1 nomolgus monkey (cyno) PD-1 extracellular domain followed by Brian Whitaker , Annmarie Winkis ,Linda Barone , Deborah Kwok , 1 3 3 1 affinity maturation. In vitro activity was evaluated using cyto- Eilyn R. Lacy ,Edmund Moon ,Steve Albelda , Kathryn Packman , 1 1 1 megalovirus (CMV) recall, mixed lymphocyte reaction (MLR), and Enrique Zudaire , Matthew V. Lorenzi , Raluca I. Verona 1 2 Jurkat-PD-1 nuclear factor of activated T cells (NFAT) reporter as- Janssen Research & Development, Spring House, PA, USA; Alios says. In vivo activity was assessed using human PD-1 knock-in BioPharma/Janssen Research & Development, South San Francisco, PA, mice implanted with MC38 tumors and a lung patient-derived USA; University of Pennsylvania, Philadelphia, PA, USA xenograft (PDX) model (LG1306) using CD34 cord blood human- Correspondence: Raluca I. Verona (RVerona@its.jnj.com) ized NSG mice. Pharmacodynamic, toxicokinetic, and safety as- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P365 sessments were performed in cyno following single (0.1, 1, 10 mg/kg) and/or repeat (10, 30, 100 mg/kg/wk up to 5 weeks) Background intravenous (IV) dosing. Programmed cell death protein-1 (PD-1) pathway blockade has revolu- Results tionized the treatment of a variety of malignancies but has only dem- JNJ-283 displayed high affinity for human (1.72 ± 0.99 nM) and cyno onstrated clinical benefit in a subset of patients. Emerging preclinical (0.90 ± 0.08 nM) PD-1, blocked PD-1 binding to PD-L1 (IC =111.7 ± and clinical data have proposed T cell immunoglobulin and mucin 22.0 ng/mL) and PD-L2 (IC =138.6 ± 12.4 ng/mL), and cross- domain-3 (TIM-3) as an inhibitory mechanism that is co-regulated with competed with nivolumab and pembrolizumab analogs. PD-1 to restrict T cell-mediated immune responses. Dynamic upregula- JNJ-283 dose-dependently increased T cell-mediated cytokine pro- tion of TIM-3 has been observed in patients in response to anti-PD-1/ duction in CMV recall and MLR assays and enhanced NFAT activity in PD-L1 therapy, implicating TIM-3 as a potential mechanism of immune a Jurkat-PD-1 reporter assay, indicating functional disruption of the resistance. These observations suggest that dual blockade of the TIM-3 PD-1/PD-L1 interaction. JNJ-283 in vitro activity was comparable to and PD-1 pathways can broaden the effectiveness of anti-PD-1/PD-L1 that of competitor molecule analogs. therapy as a new immunotherapy for PD-1 responsive malignancies. Following intraperitoneal injection with 10 mg/kg JNJ-283 or nivolu- Here we describe the characterization and functional activity of JNJ- mab analog (2x weekly; day 7 after tumor implantation), mean MC38 64158120 (JNJ-120), an antibody targeting human TIM-3. tumor volume in PD-1 knock-in mice was significantly lower at days Methods 17 (p<0.001) and 21 (p<0.0001) compared to control (Fig. 1). In the lung JNJ-120 in vitro activity was assessed by CMV, MART-1, and NK cell PDX model, 10 mg/kg JNJ-283 or pembrolizumab (q5d x 6) reduced activation assays; in vivo activity was evaluated using a NY-ESO TCR (p<0.01) mean tumor volume compared to control. transgenic model of T cell exhaustion and a human TIM-3 knock-in JNJ-283 was tolerated in cyno following single and/or repeat dosing; mouse model bearing MB49 murine bladder carcinoma tumors. mean drug exposures increased in a dose dependent manner. Primary Results findings attributed to JNJ-283 included proliferation of T-lymphocyte JNJ-120 is a fully human, phage-derived, IgG2σ monoclonal antibody subsets, stimulation-related cytokine expression (in vitro), increased IgM that binds to human (K =2.5 nM) and cynomolgus monkey (K =0.75 d d and IgG titer secondary to antigen challenge, and decreased cellularity nM) TIM-3. JNJ-120 inhibited the binding of TIM-3 to two putative TIM- (lymphocytes) in the thymus. 3 ligands, phosphatidyl serine and galectin-9 and enhanced T cell func- Conclusions tion in primary antigen-specific T cell assays (CMV and MART-1). In CMV JNJ-283, a high affinity anti-PD-1 antibody, was well-tolerated in toxi- stimulated PBMCs, JNJ-120 triggered expression of IFN-γ and TNF-α + + cology studies and demonstrated robust activity in vitro as well as and enhanced CD137 expression on CD8 and CD4 T cells. Further- displayed anti-tumor efficacy in vivo comparable to that observed more, in MART-1 assays, JNJ-120 enhanced IFN-γ,TNF-α, IL-2, and IL-8 with other agents targeting PD-1. These data support the ongoing production by CD8 T cells. JNJ-120 also enhanced T cell function in clinical study of JNJ-283 and combination potential with other combination with anti-PD-1 antibodies in peripheral blood samples de- modalities. rived from melanoma patients. JNJ-120 led to higher NK cell activation, as measured by CD69 expression and cytokine secretion, in IL-2- stimulated PBMCs derived from normal donors and cancer patients. In vivo, JNJ-120 treatment enhanced the anti-tumor activity of PD-1 blockade in tumor-bearing TIM-3 knock-in mice and in a NY-ESO TCR transgenic model of T cell exhaustion. In the NY-ESO TCR model, JNJ- 120 increased T cell infiltration in NY-ESO-expressing A549 tumors and, in combination with anti-PD-1 antibodies, enhanced the ability of infiltrating T cells to kill tumor cells ex-vivo. Conclusions These data demonstrated that targeting TIM-3 in combination with PD-1 leads to increased tumor T cell infiltration and superior anti- tumor efficacy compared to PD-1 blockade alone. These data support the clinical testing of PD-1 and TIM-3 in tumor types not broadly re- sponsive to current immunotherapy regimens. P366 Anti–PD-L1 mAb treatment combined with cisplatin modulates intratumoral immune responses and promotes antitumor effects Daiko Wakita, Toshiki Iwai, Masamichi Sugimoto, Suguru Harada, Miho Suzuki, Kaname Yamamoto Chugai pharmaceutical CO., LTD., Kamakura, Japan Fig. 1 (abstract P364). In vivo anti-tumor activity of JNJ-283 in hu- Correspondence: Kaname Yamamoto (wakita.daiko59@chugai-pharm.co.jp) man PD-1 knock-in mice implanted with MC38 tumors Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P366 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 181 of 244 Background Methods Immune checkpoint inhibitors such as anti–PD-L1/PD-1 agents have The primary objectives for this ongoing biomarker-driven Phase Ib trial shown marked antitumor effect in a range of cancer types, and to fur- of X4P-001 alone and with pembrolizumab are to evaluate the safety ther expand their effects, combination treatments with chemotherapeu- and tolerability in patients (pts) with metastatic melanoma, and to tic drugs have been actively investigated. Although cisplatin is widely characterize the effects of X4P-001 alone and with pembrolizumab on used as a standard chemotherapy, the antitumor activity of cisplatin tumor immune cell infiltrates. Serial biopsies of cutaneous or subcuta- combined with immune checkpoint inhibitors remains largely unknown. neous metastatic melanoma lesions and peripheral blood mononuclear Here, we investigated if anti–PD-L1 plus cisplatin combination can aug- cells (PBMCs) are collected at pre-dose, after 3 weeks of X4P-001 treat- ment antitumor immunity in a syngeneic mouse tumor model. ment and after 6 weeks of combination treatment. Biopsies are Methods assessed by immunohistochemistry (IHC) for multiple markers including E.G7-OVA cells, expressing ovalbumin (OVA) gene as a model tumor CD3, CD8, FoxP3 and CXCL12, and PBMCs are analyzed by flow cytome- antigen, were subcutaneously inoculated into syngeneic C57BL/6 try for both lymphoid and myeloid-cells. mouse. After tumor mass was established, anti-mouse PD-L1 mAb Results (anti–PD-L1; 10 mg/kg, three times a week) and cisplatin (1 mg/kg, As of June 30, 2017, 13 pts have been enrolled and 4 of the 13 pts have once) were administered intraperitoneally on the day of treatment completed the study. The median age was 74 years (range 53-91). Most initiation (Day1). The frequency and activation status of immune cells pts (12/13) had not received prior systemic therapy. X4P-001 was well in tumor tissues were evaluated by flow cytometry. For detection of tolerated. Treatment related AEs (related to either X4P-001 or pembroli- IFNg-producing cells, the immune cells were stimulated with anti- zumab; >5%) of any grade were: diarrhea (15%), chill, fatigue, head- CD3/anti-CD28 mAbs in vitro before staining. ache, ocular hyperemia, photophobia, pruritus, rash, and vomiting Results (7.7%). Two treatment related G3 SAEs were reported: acute diarrhea We found that anti–PD-L1 plus cisplatin combination resulted in pro- and immune-mediated drug-reaction. Three of the 4 pts who com- found effect leading to tumor shrinkage vs anti–PD-L1 alone or cis- pleted the study had IHC evaluable tumor samples. All showed an platin alone even though each monotherapy showed significant increase in T-cell infiltration in the central region of the tumors follow- inhibition of the tumor growth than the control group at Day17. In ing both single agent and combination treatment. The percentage of parallel with the clinical effect, the combination therapy significantly Ki67 positive CD8+ T cells in PBMC were increased post treatment in all increased tumor-infiltrating CD8+ T cells vs each monotherapy at 4 pts. These data along with additional biomarker readouts in all en- Day7 even though more tumor-infiltrating CD8+ T cells were ob- rolled patients will be presented. served in mice treated with anti–PD-L1 or cisplatin alone than Conclusions control. Further, anti–PD-L1 plus cisplatin combination activated Treatment with X4P-001 as a single agent, and in combination tumor-infiltrating CD8+ T cells, characterized by higher frequency of with pembrolizumab, is well tolerated with preliminary evidence granzyme B-expressing cells than each monotherapy. We also ob- of enhanced immune cell infiltration and activation. The enroll- served increased OVA-specific CD8+ T cells with combination vs ment of the study is near completion and further biomarker ana- monotherapies. Additional analysis of CD4+ T cells showed that anti– lysis is on-going. PD-L1 plus cisplatin combination or anti–PD-L1 alone, but not cis- platin alone, induced IFNg-producing CD4+ T cells in the tumor P368 tissues. eFT508, a potent and highly selective inhibitor of MNK1 and Conclusions MNK2, is an activator of anti-tumor immune response Anti–PD-L1 plus cisplatin combination therapy demonstrated marked 1 1 1 1 Kevin Webster , Rajesh Sharma , Vikas Goel , Craig Stumpf , Jocelyn antitumor effect in this mouse tumor model. This robust therapeutic 1 1 1 2 2 Stauton , Peggy Thompson , Gary Chiang , Yichen Xu , Hyun Yong Jin , effects may at least partly be due to the increased tumor-specific CD8+ Davide Ruggero T cells in tumor tissues. Cisplatin or anti–PD-L1 alone increased tumor- 1 2 eFFECTOR Therapeutics, San Diego, CA, USA; Helen Diller Family infiltrating CD8+ T cells, however anti–PD-L1 but not cisplatin induced Comprehensive Cancer Center, San Francisco, CA, USA IFNg+ CD4+ T cells at tumor sites. These differences in immune status Correspondence: Kevin Webster (kwebster@effector.com) in response to anti–PD-L1 or cisplatin may lead to the combined thera- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P368 peutic effect and provide a rationale for the combination. Background P367 eFT508 is a potent and highly selective inhibitor of MNK1 and X4P-001, an orally bioavailable CXCR4 antagonist, increases T cell MNK2, kinases that function to mediate tumor immune evasion infiltration in human metastatic melanoma downstream of MEK and MAPK signaling. eFT508 treatment es- 1 2 3 1 Robert Andtbacka , Melinda Yushak , Merrick Ross , Kenneth Grossman , tablishes a regulatory program that promotes multiple steps in 4 4 4 4 Eleni Tsiroyanni , Sarah Blanchette , Lu Gan , Yan Wang , the cancer immunity cycle including antigen presentation and T Mohammed Milhem cell priming, expansion of memory T cells, and prevention of T 1 2 Huntsman Cancer Institute, Salt Lake City, UT, USA; Winship Cancer cell exhaustion. Institute, Atlanta, GA, USA; MD Anderson Cancer Center, Houston, TX, Methods 4 5 USA; X4 Pharmaceuticals, Cambridge, MA, USA; University of Iowa, The immunological effects of eFT508 have been evaluated in the Iowa City, IA, USA context of normal human immune cells in vitro and in immuno- Correspondence: Robert Andtbacka (jennifer@theyatesnetwork.com) competent syngeneic and genetically engineered mouse models Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P367 in vivo. Results Background eFT508 treatment of normal donor T cells has no deleterious effect The CXCR4/CXCL12 axis plays a central role in the trafficking of key im- on αCD3/αCD28 stimulated T cell proliferation or T cell viability in mune cells in the tumor microenvironment. Enhanced survival is re- contrast to inhibitors acting upstream of MAPK signaling. eFT508 ported in multiple syngeneic mouse models when a CXCR4 antagonist selectively down regulates key immune checkpoint proteins and is combined with a check-point inhibitor. X4P-001 is an oral, selective, the production of a subset of proinflammatory and immunosup- allosteric inhibitor of CXCR4, and X4P-001 alone demonstrated robust pressive cytokines. In vitro mechanism of action studies have dem- inhibition of murine B16-OVA melanoma growth. We hypothesize that onstrated that MNK selectively regulates gene expression at the disruption of CXCR4/CXCL12 signaling will result in modulation of the level of mRNA translation via specific sequence elements in the 5’- immune cell profile within the tumor microenvironment and ultimately and 3’-untranslated regions. In addition, eFT508 activated antigen lead to increased CD8+ T-cell infiltration, favoring an improved re- presenting cells leading to more effective T cell priming. eFT508 sponse to checkpoint inhibitors in metastatic melanoma. also affected T cell memory formation, both in the context of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 182 of 244 specific peptide antigen stimulation and in a mixed lymphocyte re- COM701 as a cancer therapeutic, either as a monotherapy or as a + + action, shifting the distribution of T cells towards a CD62L CD44 dual- or triple-combination therapy with antibodies targeting TIGIT, central memory T cell population. eFT508 also enhanced the cyto- and PD-1. These data highlight the potential of this combination ap- toxic function of T cells from OT-I mice stimulated with SIINFEKL proach to expand the immune checkpoint inhibitor responsive can- peptide demonstrating a dose-dependent increase of cell killing. cer patient population, including those who are non-responsive to Consistent with the mechanisms elaborated upon in vitro, eFT508 PD-1 inhibitors. showed significant anti-tumor activity mediated through tumor in- filtrating lymphocytes in the CT26 syngeneic tumor model as well P370 as genetically engineered mouse models of NSCLC and HCC. Enhancement of tumor specific immunity by activation of CD40 Conclusions through a bispecific molecule targeting CD40 and a tumor surface eFT508 treatment establishes a regulatory program that promotes antigen anti-tumor immunity. eFT508 is currently under evaluation as a single 1 1 1 1 1 Shiming Ye , Diane Yu , Nicole Belmar , Donghee Choi , Debra Chao , agent in two phase 1/2 clinical trials for patients with advanced solid 1 1 2 2 2 Mien Sho , Han Kim , Jean Cabel , Danying Song , Kelley Hiser , tumors and patients with advanced lymphoma. A biomarker driven 2 1 1 1 3 John Harlan , Catherine Zhang , Yuni Fang , Steve Keller , Alan Wahl , proof of concept study, including mandatory pre- and on-treatment 4 1 Patricia Culp , Diane Hollenbaugh biopsies, to evaluate the immunological mechanism of action of the 1 2 AbbVie Biotherapeutics Inc., Redwood City, CA, USA; AbbVie Inc., North drug is planned to be initiated later this year. In addition, a phase 2 3 4 Chicago, IL, USA; Former AbbVie Employee, Del Mar, CA, USA; Former study evaluating eFT508, alone or in combination with avelumab, a AbbVie Employee (currently working at Alector Inc.), South San PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed Francisco, CA, USA or refractory CRC patients is planned. Correspondence: Shiming Ye (shiming.ye@abbvie.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P370 P369 Analysis of the TIGIT/PVRIG axis in human cancers to support Background indication selection and biomarkers for COM701 and COM902 CD40 activation can bridge the innate and adaptive immune sys- Sarah Whelan, Ling Leung, David Bernardos, John Hunter, Mark White, tems during immune activation. Agonistic anti-CD40 monoclonal Spencer Liang antibodies (mAbs) have demonstrated anti-tumor activity in clin- Compugen USA, Inc., South San Francisco, CA, USA ical studies. Due to the dose-limiting toxicity observed, an alter- Correspondence: Sarah Whelan (sarahw@cgen.com) native approach to developing an anti-CD40 therapy, and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P369 potentially reducing systemic toxicity, is to achieve immune acti- vation via a bispecific molecule that is maximally active in the Background presence of a tumor antigen (TA), but with limited activity in its PVRIG and TIGIT were identified by Compugen’s Predictive Discov- absence. ery Platform as immune inhibitory receptors and have been re- Methods ported to inhibit anti-tumor activity. We are pursuing clinical Different bispecific formats targeting both CD40 and a TA were development of antagonistic antibodies to PVRIG (COM701) and to designed and constructed. Bispecific molecules with desired TIGIT (COM902). Here, we analyzed primary human cancer tissues properties were screened and assayed for activation of primary and immune cells to characterize expression in the TIGIT/PVRIG axis B cells or monocyte-derived dendritic cells (moDC) co-cultured to support indication selection and combination strategies for with TA positive or negative cells. Selected bispecific molecules COM701 and COM902. were tested for anti-tumor activity using PC3 tumor cells with Methods or without TA expression mixed with autologous moDC and T COM701 and COM902 were identified based on ability to block the cells, cultured in vitro or inoculated in NSG mice. Syngeneic interaction of PVRIG and TIGIT with their cognate ligands (PVRL2 mouse models of TA positive tumors were used to test bispeci- and PVR respectively) and were screened for their ability to en- fic molecules for immune activation, anti-tumor potency, and hance antigen-specific CD8 T cell activation in a co-culture with tolerability. tumor cell lines. Immunohistochemistry and Flow cytometry were Results performed to assess receptor/ligand expression in dissociated blad- ABBV-428 is a bispecific molecule with single chain Fv (scFv) do- der, breast, colorectal, head and neck, lung, kidney, ovarian, pros- mains targeting human CD40 and a TA. ABBV-428 was less potent tate, and stomach tumors. than a CD40 mAb in stimulating B cells and moDCs when cultured Results alone or in the presence of cells that do not express TA. However, Among the cancers examined, PVRIG and PVRL2 expression was high- ABBV-428 exhibited enhanced B cell and moDC activation when est in endometrial, lung, kidney, ovarian, and head and neck cancers cultured with cells expressing TA. T cells were also activated by compared to normal adjacent tissue. From dissociated tumors, PVRIG ABBV-428 when mixed with moDC in the presence of cell-surface expression was detected on T and NK TILs whereas PVRL2 expression TA, as evidenced by a reduction in growth of PC3 cells, both was detected on CD45 cells and myeloid cells. A co-expression analysis in vitro and in NSG mice. Although expression of the cell surface TA of PVRIG, TIGIT, and PD1 demonstrated that PVRIG was co-expressed is necessary for immune-mediated anti-tumor activity, ABBV-428 + + + with both TIGIT and PD1 and that PVRIG TIGIT PD1 cells comprised a inhibited the growth of both TA-positive and TA-negative PC3 cells major proportion of CD8 TILs. In comparison to PD-L1, PVRL2 expres- as long as the TA was expressed within the tumor environment. sion was more prevalent across several cancer types and expression of This phenomenon was confirmed in a mouse model carrying PVRL2 was detected in PD-L1 negative samples. In vitro, combination syngeneic 4T1 tumors expressing the cell surface TA. A surrogate of COM701 with PD1 inhibitors or COM902 enhanced CD8 cytokine molecule of ABBV-428 elicited T cell responses against both TA- production and cytotoxic activity, with the triple combination of expressing and non-expressing 4T1 cells, where the anti-tumor COM701, COM902, and PD-1 antibody yielding the greatest increase in activity was similar to anti-CD40 mAb, but did not elicit increases in functional activity. Several immune receptors were induced in response serum cytokines and liver enzyme levels observed in anti-CD40 of PVRIG blockade by COM701 on CD8 T cells. Taken together, these mAb treated mice. data support indication selection and combination strategies for Conclusions COM701 and COM902 and potential biomarkers that could be indica- ABBV-428 exhibits enhanced CD40 activation upon binding to TA- tors of response. expressing cells, and provides tumor-specific immune stimulation Conclusions with systemic administration. The enhanced tumor-specific immune In summary, we demonstrate that PVRIG and PVRL2 are induced in activation is hypothesized to maximize anti-tumor potency while lim- the tumor microenvironment of human cancers, and the potential of iting systemic toxicity in clinical studies. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 183 of 244 Impact of Diet, Exercise, and/or Stress on may lead to severe neurotoxicity. We have developed a novel model system where conditioning immune-competent mice with Antitumor Immunity cyclophosphamide (CPA) followed by transfer of murine CD19- directed CAR-T cells induces acute symptoms including systemic P371 cytokine release in concordance with what is observed in patients Featuring adipocytes secretion as pharmacological target for that develop CRS and/or neurotoxicity. This systemic toxicity is adjuvant immunotherapy against breast cancer accompanied by the alteration of gene expression levels in the Luis Henrique Correa, LÍVIA PIMENTEL SANT’ANA DOURADO, Kelly Grace mouse brain indicating a possible neuro-inflammatory response. Magalhaes Methods University of Brasilia, Brasilia, Brazil We examined the effects of murine CD19-directed CAR-T cell therapy Correspondence: Kelly Grace Magalhaes (kellymagalhaes@gmail.com) in a syngeneic BALB/c model. Following dose optimization for both Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P371 CPA conditioning and the intravenous transfer of CAR-T cells, mice were evaluated for acute toxicity and systemic and brain-related Background pathologies including serum cytokine levels, brain and peripheral Obesity and adipose tissue have been shown to be associated with organ histopathology by microscopy of hematoxylin and eosin- low grade inflammation resulting in cellular and humoral inflamma- stained sections, evaluation of blood brain barrier (BBB) integrity by tory factors that contribute to carcinogenesis . It has been known extravasation of fluorescently-labeled low-molecular weight dextrans, that inflammation exerts an important role in carcinogenesis and wet-dry brain weights indicative of cerebral edema, and flow cytom- tumor progression. Inflammatory molecules can potentially be used etry and gene expression analysis of transcardially-perfused brain as adjuvant in immunotherapy against cancer. However, little is tissues. known about the differential role of brown and white adipocytes Results against breast cancer. In the present work, we aimed to Conditioning with CPA, followed by administration of murine characterize the differential function of brown and white adipo- CD19-directed CAR T cells, but not control CAR-T cells, induced cytes in breast cancer cell death in vitro and investigate the role of rapid weight loss, peripheral organ pathologies and elevated NLRP3 and caspase-1/11 in this process. serum cytokine levels (including a spectrum of cytokines similar Methods to those observed in clinical CRS). In the brain, we observed sig- Brown and white adipose tissue were isolated from wild type and nificant changes in gene expression indicative of neuro- caspase-1/11 and NLRP3 knockout mice. Conditioned medium from inflammation including genes associated with interferon response these brown and white adipocytes were used to stimulate breast pathways, vascular endothelial activation and oxidative stress, ac- cancer cells 4T1. Breast cancer cells (I) viability was analyzed by companied by CAR-T cell infiltrate into the brain. No evidence for MTT assay; (II) cell death was investigated by annexin V/PI staining overt brain histopathology was observed, nor increased BBB per- and flow cytometry analysis (III) membrane pore formation was ob- meability or cerebral edema. served by PI staining and spectrophotometry analysis and (IV) lipid Conclusions droplet biogenesis was analyzed by Bodipy staining and flow cy- These findings describe a new animal model and highlight its poten- tometry analysis. tial use to elucidate the mechanisms underlying CAR-T-cell mediated Results toxicities and test proposed interventions to reduce neuro- Our data showed that brown adipocytes conditioned medium trig- inflammation that may arise from CD19-directed CAR-T cell therapies. gered significant higher levels of breast cancer cell death and pore Ongoing work seeks to identify and evaluate various pharmaco- membrane formation and lower levels of lipid droplet biogenesis logical interventions with the potential to ameliorate systemic and and cell viability compared to white adipocytes conditioned medium. neuro-inflammation in this model, with the goal to translate these Conclusions learnings to the clinic. The absence of caspase-1/11 in brown adipocytes, but not NLRP3, enhanced these cell death and carcinogenic parameters in breast cancer cells. Identification of molecules from these adipocytes secre- tion is ongoing and can be potentially used as adjuvant immunother- P373 apy agains breast cancer. Optimizing anti-OX40 mediated immunotherapy: preclinical exploration of the relationship between antitumor activity References and isotype choice, ligand blocking capacity, dose and 1. Alderton G. The supersized tumour microenvironment. Nature Reviews schedule 1 2 1 1 Cancer. 2015;15:575. Chan Gao , John Engelhardt , Gennaro Dito , Susan Glick , 1 1 2 Megan Raymond , Marie-Claude Gaudreau , Alan Korman , Michael Quigley Mechanisms of Efficacy or Toxicity 1 2 Bristol-Myers Squibb, Princeton, NJ, USA; Bristol-Myers Squibb, P372 Redwood City, CA, USA A syngeneic mouse model of CAR-T mediated toxicity and Correspondence: Michael Quigley (gyrase@gmail.com); Alan Korman neuroinflammation Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P373 Eric Chadwick, Alyssa Noll, Yue Jiang, Ronald Hause, Rafael Ponce, Hyam Levitsky, Ruth Salmon Background Juno Therapeutics, Seattle, WA, USA Following the clinical success of checkpoint blockade, the field of Correspondence: Ruth Salmon (eric.chadwick@junotherapeutics.com) cancer immunotherapy is rapidly expanding. Extensive preclinical Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P372 data have demonstrated that treatment with an agonist OX40 anti- body can result in anti-tumor immune responses both alone and in Background combination with other immune-targeting agents including CTLA-4 Interrogating the spectrum of systemic effects of CAR-T cell ther- and PD-1 blockers. The reported mechanism of action for OX40 in- apy, especially in the context of adverse events (i.e. systemic cludes costimulation of effector T cells as well as reduction in regula- cytokine release syndrome (sCRS) and neurotoxicity) that have oc- tory T cell (T ) suppression either through depletion or receptor reg curred in clinical trials, may provide important insights into fac- engagement. However, the vast majority of published work utilizes a tors contributing to the cause and/or progression of such events single, ligand non-blocking antibody to define the role of anti-OX40 and potential interventions. Preclinical CAR-T cell efficacy models in enhancing tumor immunity in vivo. We generated an alternative in immune-compromised mice do not capture the potential role agonistic, ligand-blocking mouse OX40 antibody as well as series of of the host immune system in sCRS and central nervous system isotype variants in order to better define the role of isotype, ligand (CNS) pathologies that, in combination with other patient factors, blocking vs non-blocking epitopes, dose and schedule on the anti- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 184 of 244 tumor activity of anti-OX40 alone and in combination with PD-1 exacerbates 4-1BB agonist hepatitis. Co-administration of CTLA-4 blockade in vivo. blockade ameliorates transaminase elevation, whereas PD-1 block- Methods ade exacerbates it. Loss of the chemokine receptor CCR2 blocks The activity of OX40 agonist antibodies, OX86 (ligand non-blocker) and 4-1BB agonist hepatitis without diminishing tumor-specific im- OX40.23 (ligand blocker), were tested across a dose range from 0.03 munity against B16 melanoma. mg/kg to 10mg/kg in the CT26 tumor model. Anti-PD-1 (10 mg/kg) was Conclusions dosed either concurrently or 5 days after initial OX40 treatment. On- 4-1BB agonist antibodies trigger hepatitis via activation of myeloid treatment immune monitoring of peripheral and tumor-infiltrating lym- cells to produce Interleukin-27. Co-administration of CTLA-4 and/or phocytes was analyzed by flow cytometry. CCR2 blockade may minimizing hepatitis but yield equal or greater Results antitumor immunity. Fc-competent agonist OX86 antibodies display potent in vivo anti- tumor activity at 10 mg/kg, whereas an Fc inert antibody at the same P375 dose had no effect on CT26 tumor growth. Relative anti-tumor activ- Evaluation of surrogate endpoints for overall survival in patients ity was related to their ability to preferentially bind activating FcγR treated with immunotherapies and deplete intratumoral T s. Maximal activity of OX40.23 was reg 1 2 3 4 Howard Kaufman , Lawrence Schwartz , William William , Mario Sznol , achieved at 3 mg/kg and 0.3 mg/kg as monotherapy and in combin- 5 5 6 6 Michael del Aguila , Craig Whittington , Kyle Fahrbach , Yingxin Xu , ation with anti-PD-1, respectively. Interestingly, administration of 7 8 Eric Masson , Andrea Vergara-Silva OX40.23 at 10 mg/kg in both monotherapy and combination dis- Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; played diminished activity, accompanied by a reduction in peripheral 2 3 Columbia University Medical Center, New York, NY, USA; MD Anderson T cell activation. Evaluation of OX40 RO demonstrated that peripheral Cancer Center, Houston, TX, USA; Yale School of Medicine, New Haven, and intratumoral RO was similar as was RO between monotherapy 5 6 CT, USA; Doctor Evidence, Santa Monica, CA, USA; Evidera, Bethesda, and combination treatment. Maximal anti-tumor activity of the com- 7 8 MD, USA; AstraZeneca, Waltham, MA, USA; AstraZeneca, Gaithersburg, bination was achieved well below 100% OX40 RO. Furthermore, for MD, USA combination treatment, concurrent dosing resulted in greater anti- Correspondence: Howard Kaufman (hk553@cinj.rutgers.edu) tumor activity than a staggered regimen. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P375 Conclusions Our results demonstrate the importance of isotype choice, lig- Background and blockade capacity, dose and schedule on the in vivo anti- Surrogate endpoints have not been clearly determined in oncology tumor activity of mouse OX40 agonist antibodies alone and in immunotherapy (IT). Using both arm- and comparison-level data, this combination with PD-1 blockade. These data provide valuable study explored the relationship between overall survival (OS) and insight relevant in biomarker, dose and schedule selection for clinical endpoints (objective response rate [ORR], disease control rate agonist OX40 antibody-containing regimens currently in clinical [DCR], and progression-free survival [PFS]) in patients treated with IT. development. The aim was to assess whether any of these endpoints could func- tion as surrogates for OS. P374 Methods Activation of 4-1BB on liver myeloid cells triggers hepatitis via an A systematic review was conducted in MEDLINE™ and Embase interleukin-27 dependent pathway (January 2005–March 2017), and supplemented with conference 1 1 1 1 Ashvin R. Jaiswal , Todd Bartkowiak , Casey R. Ager , Renee Chin , proceedings (2014–2017). Eligible studies were randomized con- 1 1 1 1 Chao-Hsien Chen , Pratha Budhani , Midan Ai , Matthew J. Reilley , trolled trials (RCTs) that investigated blocking antibodies target- 2 1 1 Manu M. Sebastian , David S Hong , Michael A Curran ingprogrammedcelldeath-1 (PD-1)/programmedcelldeath The University of Texas MD Anderson Cancer Center, Houston, TX, USA; ligand-1 (PD-L1) or cytotoxic T-lymphocyte-associated antigen-4 The University of Texas MD Anderson Cancer Center, Smithville, TX, USA (CTLA-4). Studies were included in the arm-level analyses if the Correspondence: Ashvin R. Jaiswal (arjaiswal@mdanderson.org); treatment arm’s absolute effects could be obtained for ORR, Michael A Curran DCR, 6- and 9-month PFS, median PFS, median OS, or OS at 12 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P374 or 18 months. They were included in comparison-level analyses if the treatment’s relative effects (odds ratios [ORs] on ORR and Background DCR or hazard ratios [HR] on PFS and OS) were reported/could Agonist antibodies targeting the T cell co-stimulatory receptor 4-1BB be derived. (CD137) are among the most effective immunotherapeutic agents Weighted linear regression models were fitted and adjusted R values across multiple pre-clinical models of cancer. In the clinic, however, de- estimated, with analyses stratified by treatment regimen (IT mono or IT velopment of these agents has been stymied by dose-limiting liver tox- plus chemotherapy), by type of IT (PD-1/PD-L1 or CTLA-4), and by indi- icity. Lack of knowledge of the mechanisms underlying this toxicity has cation, as data permitted. limited the potential to separate 4-1BB agonist driven anti-tumor im- Results munity from hepatotoxicity. Twenty-nine RCTs involving 66 treatment arms (11,797 patients) were Methods included. In the arm-level analyses, higher PFS rates at 6 months pre- The capacity of 4-1BB agonist antibodies to induce liver toxicity dicted better OS rates at 12 and 18 months, with similar trends was investigated in immune competent mice, with or without co- across subgroups (Fig. 1A–D); results by type of IT revealed stronger administration of checkpoint blockade, via measurement of serum correlations for PD-1/PD-L1 than CTLA-4 as potential surrogates for transaminase levels, through imaging of liver immune infiltrates, OS (Fig. 1A, Fig. 1B). Similarly, the comparison-level analyses only and via qualitative and quantitative assessment of liver myeloid found the PFS HR to be moderately correlated with the OS HR and T cells via flow cytometry. Knockout mice were used to clar- (R =0.372; P=0.002; Fig. 1e); this finding remained consistent when ify the contribution of specific cell subsets, cytokines and restricted to studies assessing IT monotherapy only (R =0.460; chemokines. P=0.002; Fig. 1F) and in subgroups stratified by treatment type or Results indication. We find that activation of 4-1BB on liver myeloid cells is essential Conclusions to initiate hepatitis. Once activated, these cells produce Among anti–PD-1/PD-L1 studies, PFS was an imperfect surrogate interleukin-27 which is required for liver toxicity. CD8 T cells infil- (low to moderate correlation) for OS, whereas other surrogate trate the liver in response to this myeloid activation and mediate clinical endpoints were not correlated with OS. Thus, a new sur- tissue damage triggering transaminase elevation. FoxP3+ regula- rogate, such as a biomarker, is needed to better predict the OS tory T cells limit liver damage and their removal dramatically benefit for IT. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 185 of 244 myeloma cell line (MM.1R; 75,590 average receptors/cell). Detectable CD38 receptor expression was observed on tumor cells from 5/21 pri- mary NSCLC tumor samples and was highly expressed on intratumoral immunosuppressive cells including: monocytic MDSC, tumor-associated macrophages and T . In the PB of lung cancer patients, CD38 receptor reg was expressed on >50% of NK cells, B-cells, monocytic MDSC, immature MDSC, and monocytes. Significantly, DARA induced cell-mediated kill- ing of CD38 lung cancer cell lines in vitro, and depleted monocytic MDSC from the PB of lung cancer patients ex vivo. Conclusions Tumor cell CD38 expression is observed in a subset of lung cancers and is highly expressed on patient tumor-infiltrating immune cells and immunosuppressive cells in PB samples. Because DARA has demonstrated in vitro activity in depleting these cells, DARA may represent a novel therapeutic approach to targeting the immune microenvironment in solid tumors. Clinical trials in NSCLC are un- derway (NCT03023423) and/or planned. P377 Cisplatin induces immunogenic cell death in preclinical models of head and neck squamous cell carcinoma 1 1 1,2 1 1,3 So-Jin Park , Linda Tran , Roy Xiao , Carter Van Waes , Nicole Schmitt 1 2 NIDCD, NIH, Bethesda, MD, USA; Cleveland Clinic Lerner College of Medicine, Bethesda, MD, USA; Johns Hopkins University, Bethesda, MD, USA Correspondence: Nicole Schmitt (nicole.schmitt@nih.gov) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P377 Fig. 1 (abstract P375). Abstract Summary Background Immunogenic cell death (ICD) is the process by which stressed cells exhibit translocation of markers including calreticulin and heat shock proteins (HSPs) to the cell surface along with release of HMGB1 and P376 ATP. This activates toll-like receptors and adaptive immunity. Studies Evaluating CD38 as a therapeutic target in non-small cell lung in several cancer types suggest that oxaliplatin induces superior ICD cancer (NSCLC) compared to cisplatin. This has never been studied in head and neck Michelle Kinder, Mark Mendonca, Khaja Syed, Melissa Parker, squamous cell carcinoma (HNSCC). Patrick Wilkinson, Edward Thompson, Gerald Chu, Christopher Chiu, Methods Natalie Hutnick Three HNSCC cell lines were treated with sublethal doses of the platinum Janssen Research and Development, LLC, Spring House, PA, USA chemotherapy drugs cisplatin, oxaliplatin, and carboplatin for 48 hours. Correspondence: Michelle Kinder (mkinder@its.jnj.com) Cell surface levels of HSP70 and calreticulin were then assayed by flow- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P376 cytometry. Intracellular HMGB1, an indirect measurement of HMGB1 re- lease, was also quantified. Release of HMGB1 was measured in the cell Background culture supernatants. A syngeneic mouse model was then used to com- Daratumumab (DARA), a human CD38-targeted monoclonal antibody, pare the effects of cisplatin vs. oxaliplatin, alone or in combination with is approved as monotherapy and in combination with standard of care anti-PD-1 immunotherapy, on tumor growth and survival. A subset of tu- regimens for patients with relapsed or refractory multiple myeloma. mors were analyzed for immune cell infiltrates by flow cytometry. DARA demonstrates direct on-tumor and immunomodulatory mecha- Results nisms of action. In multiple myeloma patients treated with DARA, CD38 All three platinum drugs induced translocation of HSP70 and calreticu- immunosuppressive cells including regulatory T-cells (T ), myeloid reg lin to the cell surface, as well as release of HMGB1 in multiple cell lines. derived suppressor cells (MDSC) and regulatory B-cells (B )are de- reg Cisplatin was the superior ICD inducer in these cell lines. Cisplatin and pleted. DARA also results in increased CD8 T-cells and T-cell clonality. oxaliplatin induced similar tumor growth delay when combined with We hypothesized that DARA could be effective in solid tumors that anti-PD-1 immunotherapy in tumor-bearing, immunocompetent mice. have elevated levels of CD38 expression on the surface of tumor and/ Conclusions or immunosuppressive cells. Here, we investigated CD38 expression on Treatment of HNSCC cells with platinum chemotherapy drugs appears lung cancer and intra-tumoral immune cells to determine whether to induce ICD, which may enhance anti-tumor immunity. Cisplatin, NSCLC may be susceptible to the immunomodulatory effects of DARA. which is the standard chemotherapy drug for treatment of HNSCC, ap- Methods pears to be at least as effective as oxaliplatin as an ICD inducer in these CD38 RNA expression in lung cancer cell lines and tumor sections from preclinical models. The Cancer Genome Atlas (TCGA) database was investigated using RNA-sequencing. Protein expression on lung cancer cell lines, tumor- infiltrating immune cells, and peripheral blood (PB) immune cells was analyzed by flow cytometry. Primary tumor samples were evaluated by P378 immunohistochemistry. The effect of DARA on lung cancer cell viability Improving the efficacy of cancer immunotherapy: An intimate play was measured in an in vitro assay. DARA-mediated depletion of im- of CD8 T and NK cells 1 1 2 , J Shawn Goodwin , William Hofmeister , mune cells in the PB of lung cancer patients was assessed ex vivo. Roman Uzhachenko 1,3 Anil Shanker Results Meharry Medical College School of Medicine, Nashville, TN, USA; CD38 RNA was highly expressed in lung tumor specimens from the University of Tennessee Space Institute, Tullahoma, TN, USA; TCGA database. Of the lung cancer cell lines investigated, 3/12 (A549, Vanderbilt-Ingram Cancer Center, Nashville, TN, USA NCI-H2023, and NCI-H2073) showed detectable CD38 receptor. These Correspondence: Anil Shanker (ashanker@mmc.edu) cell lines demonstrated moderate receptor density (8,830, 20,900, and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P378 58,100 average receptors/cell, respectively) compared to a multiple Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 186 of 244 Background also performed similar analyses of surgically resected melanomas from The interaction between the innate and adaptive immune compo- patients being treated with checkpoint blockade therapy. nents is fundamental for an effective antitumor immunity. Our stud- Results ies in murine models of mastocytoma, kidney, breast and lung solid In both tumor models, more than 13 distinct tumor infiltrating T cell tumors showed that productive antitumor effector response relies on populations were identified. Both anti-CTLA-4 and anti-PD-1 checkpoint functional crosstalk between innate immune effectors—natural killer blockade modulated the frequencies of only a subset of these tumor (NK) cells, and adaptive immune effectors—cytolytic CD8 T lympho- infiltrating T cell populations. Furthermore, of multiple exhausted-like cytes. We found that this lymphocyte cooperativity between CD8 T CD8 T cell populations identified, the frequencies of only two subsets and NK cells can prevent the development of antigen-escape tumor correlate with outcome, suggestive of functional heterogeneity within variants. phenotypically exhausted T cells. Most notably, we find that anti-CTLA- Methods 4, but not anti-PD-1, modulates the CD4 effector compartment by Using a nanofiber matrix engineered to provide lymphocytes a controlled inducing the expansion of Th1-like CD4 effector T cells. Observations + high high 3D interaction, we found that activated CD8 T cells (CD69 CD25 ) from mass cytometry analyses of surgically resected melanoma tumors formed multiple intercellular contacts with several naïve NK cells, while from patients being treated with anti-CTLA-4 or anti-PD-1 therapy were naïve CD8 T cells made single or no contact with NK cells. consistent with these preclinical findings. Results Next we utilized similar methodologies to investigate the cellular In lymphocyte coculture (physical contact possible), activated CD8 T mechanism of combination anti-CTLA-4 and anti-PD-1 therapy. Al- and NK cells cross-regulated each other’s phenotype wherein NK cells though combination therapy largely enhanced effects observed in polarized activated CD8 T cells towards T central memory phenotype monotherapies, combination therapy differentially modulated spe- and activated CD8 T lymphocytes induced acquisition of effector/ cific exhausted-like CD8 T cell populations. These data suggest that regulatory phenotype by naïve NK cells. This cross-regulation of lym- combination therapy modulates T cell function and mediates tumor phocytes disappeared in a trans-well system (no physical contact) in- rejection through mechanisms that are in part distinct from either dicating the necessity of cell-to-cell physical interaction during CD8 monotherapy. T—NK crosstalk. Notably, intercellular physical interaction led to Conclusions 2+ + cross-regulation of mitoCa oscillations in both activated CD8 T and Our findings indicate that anti-CTLA-4 and anti-PD-1 modulate spe- 2+ + 2+ NK cells. Inhibition of mitochondrial Ca uptake or Na /Ca exchan- cific tumor infiltrating T cell populations and utilize distinct cellular ger with Ru360 and CGP37157, respectively, mimicked observed mechanisms to induce tumor rejection. Anti-CTLA-4, but not anti-PD- alterations in both lymphocytes. Further, NK cells displayed increased 1, induces expansion of CD4 effector T cells. Furthermore, these data oxidative signaling, Tyk2, Jak 1 and 3, Stat2 and Stat6 phosphoryl- suggest that combination therapy modulates T cell function differ- ation while inhibiting TCR- and various cytokine receptor-mediated ently than monotherapies. These findings have implications for the signaling. In turn, NK cells selectively restrained IL-2 signaling in CD8 rational design of combinatorial therapeutic approaches and expand T cells by dampening activation-induced up-regulation of CD25, our understanding of the mechanisms that regulate T cell activity. Stat5 phosphorylation, IL-2 synthesis and elevation in IL-2 uptake. Conclusions 2+ P380 These data suggest a model, where mitochondrial Ca flux acts as a The role of radiotherapy in driving anti-tumor CD8 T cell key biological controller to guide cellular crosstalk allowing acquisition responses of NK cell effector/regulatory and T cell central-memory phenotypes 1 2 2 Lauren Zebertavage , Alejandro Alice , Marka Crittenden , upon their interaction. Further understanding of the characteristics and + 2 Michael Gough regulatory factors involved in this NK—CD8 T cell physical play in the 1 2 Oregon Health and Science University, Portland, OR, USA; Earle A tumor microenvironment will provide new insights on controlling im- Chiles Research Institute, Portland, OR, USA mune escape variants of tumor. This could lead to novel strategies for Correspondence: Michael Gough (uhde@ohsu.edu) effective cancer immunotherapies, with a potential of relapse-free sur- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P380 vival in cancer patients. Background P379 Radiotherapy (RT) is one of the three main arms of traditional cancer Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 therapy, and has been show to synergize well with immunotherapies in checkpoint blockade preclinical models. Consistent with the findings of other investigators, in 1 2 1 1 Spencer Wei , Jacob Levine , Alexandria Cogdill , Yang Zhao , immunogenic murine models of radiation therapy and immunotherapy 1 1 1 1 Nana-Ama Anang , Miles Andrews , Padmanee Sharma , Jing Wang , we have observed CD8+ T cell-dependent clearance of tumors given sin- 1 2 1 Jennifer Wargo , Dana Pe'er , James Allison gle, high-dose radiation (20Gy) and anti-PD1 checkpoint blockade. In this The University of Texas MD Anderson Cancer Center, Houston, TX, USA; setting, RT has been proposed to generate tumor antigen-specific T cells Sloan Kettering Institute, New York, NY, USA following the release of antigens by dying cancer cells, and PD1 block- Correspondence: Spencer Wei (scwei@mdanderson.org); James Allison ade allows T cell control of residual cancer cells in the suppressive tumor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P379 environment. We hypothesized that the role of radiation is to boost the number of existing tumor antigen-specific CD8 T cells and drive an influx Background of tumor-antigen reactive cytotoxic T cells into the tumor. If this hypoth- Immune checkpoint blockade is able to achieve durable responses in a esis is true, then providing T cells through other means should be suffi- subset of patients, however despite such significant clinical progress cient to replace radiation therapy in this therapeutic combination. we still lack a fundamental understanding of the mechanisms of anti- Methods CTLA-4 and anti-PD-1 induced tumor immune rejection. CTLA-4 and In order to deliver targeted beams of radiation, we use the Xstrahl PD-1 regulate T cell activation through different molecular and cellular Small Animal Radiation Research Platform (SARRP). Tumor-reactive mechanisms and act at different stages of T cell activation. As such, we CD8 T cell numbers were boosted using live-attenuated Listeria hypothesized that anti-CTLA-4 and anti-PD-1 checkpoint blockade in- monocytogenes vaccines against potent tumor-specific CD8 model duce tumor rejection through distinct cellular mechanisms. antigens. Nur77-GFP transgenic mice were used to measure antigen Methods recognition by T cells in vitro and in vivo. To address this hypothesis, we utilized mass cytometry which enables Results characterization of more than 40 parameters at single cell resolution and We demonstrate that RT generates marginal increases in tumor-antigen unsupervised cellular classification. Using this approach, we comprehen- specific T cells in the peripheral circulation. In order to determine sively profiled the immune infiltrates of MC38 and B16BL6 murine tumors whether higher numbers of tumor-responsive CD8s were driving RT from mice treated with anti-CTLA-4, anti-PD-1, or control antibodies. We plus checkpoint blockade efficacy, we vaccinated mice with attenuated Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 187 of 244 Listeria expressing tumor-specific antigens. Using immunohistology and modulate PD-L1 and other relevant immuno-oncology targets in pa- flow cytometry we demonstrate that these antigen-specific cells were tients at therapeutically well-tolerated doses. Follow up studies to present at high levels in the tumor. We demonstrate that antigen- correlate these changes with protein expression, immune cell activa- specific T cells generated by vaccination kill antigen-pulsed targets in tion, and tumor-specific targets in patients are underway. in vivo cytotoxicity assays, recognize cancer cells ex vivo, and respond to tumor-associated antigen within the tumor. However, mice treated P382 with Listeria vaccination and PD1 blockade showed no tumor growth ATR inhibition sequenced with radiation therapy abrogates control advantage when compared to controls, despite the high level immune exhaustion of antigen-specific T cells in the tumor. 1 1 1 2 David Clump , Christopher Bakkenist , Frank Vindetti , Greg Delgoffe , Conclusions Robert Ferris These data demonstrate that while radiotherapy generates marginal 1 2 UPMC Hillman Cancer Center, Pittsburgh, PA, USA; University of increases in the number of antigen-specific T cells, which are log-fold Pittsburgh School of Medicine, Pittsburgh, PA, USA fewer than generated by Listeria vaccination, RT is a superior partner Correspondence: David Clump (clumpda2@upmc.edu) for combination with checkpoint blockade. We find that generating Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P382 large numbers of tumor antigen-specific T cells cannot substitute for the efficacy of radiotherapy in combination with anti-PD1 checkpoint Background blockade, and question whether the main driver of radiotherapeutic Immunotherapy that targets the immunosuppressive interaction be- efficacy is augmenting CD8 T cell numbers. tween the T cell activation-induced programmed cell death receptor 1 (PD-1) and its ligand PD-L1 and radiation therapy are used in the Mechanisms of Resistance to Immunotherapy management of a majority of metastatic non-small cell lung cancer (NSCLC) patients. ATR is a DNA damage signaling kinase activated at P381 damaged replication forks and ATR kinase inhibitors potentiate the The BET bromodomain inhibitor ZEN-3694 modulates the cytotoxicity of DNA damaging chemotherapies and radiation expression of checkpoint receptors and immune suppressive Methods factors in the blood of mCRPC patients 1 1 2 1 Here we show that ATR kinase inhibitors potentiate radiation-induced Eric Campeau , Henrik Hansen , Sanjay Lakhotia , Karen Norek , 1 1 tumor immune responses in genetically engineered and syngeneic Laura Tsujikawa , Sarah Attwell 1 2 mouse models of Kras-mutant lung adenocarcinoma and colorectal car- Zenith Epigenetics, Calgary, AB, Canada; Zenith Epigenetics, San cinoma, respectively. Francisco, CA, USA Results Correspondence: Eric Campeau (sattwell@zenithepigenetics.com) ATR kinase inhibitors attenuate radiation-induced PD-L1 upregulation on Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P381 tumor cells and dramatically decrease the number of tumor infiltrating Tregs. ATR kinase inhibitors further attenuate radiation-induced T cell ex- Background haustion and increase T cell activity in the tumor microenvironment. Epigenetic regulation of the immune system plays a significant role in Conclusions the response to immunotherapies. The potential for epigenetic modula- Our work raises the exciting possibility that a single pharmacologic tors to prime the immune system and increase the duration and fre- agent may enhance the cytotoxic effects of DNA damaging chemother- quency of response to checkpoint inhibitors has been supported by both apy and radiation while concurrently potentiating radiation-induced pre-clinical and clinical evidence. ZEN-3694 is an orally available inhibitor tumor immunity. of the bromodomain and extra-terminal (BET) domain family of proteins currently in phase I clinical trials in metastatic castration-resistant prostate cancer (mCRPC) (NCT02705469 and NCT02711956). Previously, we have shown that ZEN-3694 modulates multiple checkpoint receptors, immune P383 suppressive factors and cytokines in vitro, and acts synergistically with a Molecular and immune characterization of melanoma metastases PD-1 mAb to inhibit tumor growth in a MC-38 syngeneic mouse model. with heterogeneous PTEN expression 1 2 3 To follow up with these findings, we examined the ability of ZEN-3694 to Mariana Petaccia de Macedo , Feng Wang , Diego Marzese , 2 2 2 2 modulate the expression of Immuno-Oncology target genes in mCRPC Courtney Hudgens , Meredith McKean , Khalida Wani , Lauren Haydu , 4 4 4 4 patients at multiple doses in our current phase 1 clinical trial. Patrick Danaher , Christopher Merritt , Giang Ong , Sarah Warren , 4 3 2 2 Methods Joseph Beechem , Dave Hoon , Weiyi Peng , Lawrence Kwong , 2 2 2 The Nanostring nCounter® PanCancer Immune Profiling Panel was Michael Tetzlaff , Alexander Lazar , Michael Davies 1 2 used to measure immune marker expression in patient whole blood AC Camargo Cancer Center, São Paulo, Brazil; MD Anderson Cancer RNA taken at 0, 4 and 24 h post dosing with ZEN-3694. Center, Houston, TX, USA; John Wayne Cancer Institute, Santa Monica, Results CA, USA; NanoString Technologies, Seattle, WA, USA ZEN-3694 modulates multiple checkpoints, suppressive factors and cyto- Correspondence: Michael Davies (mdavies@mdanderson.org) kines in peripheral immune cells 4 hours after a single dose. Most of these Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P383 changes return to baseline at 24 hours, following clearance of the drug. Significant effects were detected at all doses tested, including at well- Background tolerated doses below the maximum tolerated dose. Multiple checkpoint There is growing evidence that oncogenic pathways in tumor cells can receptors, including TIM3 and PD-L1, chemokines CCL2/CCR2 and IL-8, impact the anti-tumor immune response and the efficacy of immuno- and the suppressive factors IDO1 and ARG1, were significantly inhibited 4 therapy. Previously we showed that loss of PTEN inhibits immune infil- h post-dose across 16 patients. These results were also confirmed by real- tration and promotes resistance to immune-mediated tumor killing in time PCR for several of these markers. Furthermore, mRNA levels of mul- melanoma [1]. Although PTEN expression is generally uniform in mel- tiple co-stimulatory markers, including ICOSLG and CD28, were maintained anoma, we identified a cohort of melanoma metastases with distinct or induced. Several of these markers show a strong dose/exposure re- areas with (+) and without (-) PTEN protein expression. Comparative sponse, and Ingenuity® Pathway Analysis suggests that lower doses may molecular and immune analyses were performed on the PTEN (+) and be superior to higher doses for cancer immune response modulation. (-) regions of these tumors to improve our understanding of the mech- Conclusions anisms, pathogenesis, and significance of PTEN loss. Taken together, these data suggest that ZEN-3694 has the potential Methods to modulate multiple factors involved in adaptive resistance to thera- Core biopsies were performed on formalin-fixed paraffin-embedded peutic PD-1 blockade, and therefore may improve response rate and FFPE blocks to isolate analytes from tumor-matched regions with (+) duration in combination with a checkpoint inhibitor. This is the first and (-) PTEN expression by immunohistochemistry (IHC). DNA was presented clinical evidence that a BET bromodomain inhibitor can analyzed for somatic mutations and copy number variations (CNVs) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 188 of 244 in 200 cancer-related genes by next generation sequencing (NGS), increases in B regulatory populations and IL-10 expression in CT26 tumor and globally for DNA methylation patterns by Illumina Infinium draining lymph nodes from treated animals. Human Methylation450 arrays. Expression of proteins involved in sig- Conclusions naling pathways (n=42) and immune populations/regulators (n=40) As B cell subsets express apoptotic cellular receptors similar to mac- in distinct PTEN (+) and (-) regions within the tumors was quantified rophages, we propose B cells may be one mechanism of resistance by Nanostring Digital Spatial Profiling. to this combination therapy and the direct blockade of their activity Results may optimize T cell effectiveness for patients with rectal cancer. NGS showed that PTEN (+) and (-) regionsdid notdifferinoverall muta- tion patterns. PTEN (-) regions had increased rates of chromosomal loss P385 affecting PTEN, EMB and ZNF567; gain of LETM1, WDR17 and SPATA4 was A HSP-TLR-Wnt5a paracrine signaling axis drives CXCR2 ligand detected in PTEN (+) regions. Hierarchical clustering of DNA methylation recruitment of myeloid-derived suppressor cells and represents a patterns demonstrated overall clustering of samples by tumor. Loss of novel adaptive resistance mechanism to anti-PD-1 antibody therapy PTEN expression did not correlate with increased methylation of the 1 1 1 1 Bala Theivanthiran , Nicholas C. DeVito , Kathy Evans , Fei Zhao , Pten locus, but differential methylation of several potential regulatory 1 2 2 3 Christine Xiao , Benjamin S. Goldschmidt ,Rob Edgar , Alisha H. Holtzhausen , transcription factors (Pax3, Gata2, Ikzf1, Pax5, and Gata5) was detected. 1 4 1 2 5 April K.S. Salama , John Lewicki ,JohnH.Strickler , John A. Viator , Brent A. Hanks Proteomic analysis identified multiple signaling and immune related pro- 1 2 Duke University, Durham, NC, USA; Duquesne University, Pittsburgh, teins with significantly different expression between PTEN (+) and (-) re- 3 4 PA, USA; University of North Carolina, Chapel Hill, NC, USA; OncoMed gions. Loss of PTEN correlated with significantly increased P-AKT and P- Pharmaceuticals, Inc., Redwood City, CA, USA; Duke University, Durham, P70S6K, consistent with increased activation of PI3K-AKT-mTOR signal- NC, USA ing, and significantly decreased VISTA, CD8A, and CD3. PD-L1 expression Correspondence: Brent A. Hanks (hanks004@mc.duke.edu) did not correspond with PTEN status. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P385 Conclusions Intratumoral loss of PTEN in melanoma metastases corresponded Background with genetic alterations in the Pten gene but not its methylation sta- Despite the impressive impact generated by the anti-PD-1/PD-L1 anti- tus. Proteomic analysis supports that PTEN status corresponds locally body (ab) therapies in oncology, a significant percentage of patients with PI3K-AKT pathway activation and with heterogeneity of compo- exhibit either primary or secondary resistance to these treatment mo- nents of the anti-tumor immune response. Together the findings fur- dalities. The evolution of these resistance mechanisms to cancer im- ther support the biological significance of PTEN loss in melanoma. munotherapy remains poorly understood. Myeloid-derived suppressor Consent to publish cells (MDSCs) have been demonstrated to suppress anti-tumor immun- All patients provided informed consent. ity and previous work has shown the granulocytic subset of MDSCs (Gr- MDSDCs) to migrate in response to CXCR2 chemokine gradients. References Methods 1.Peng We utilized RNAseq differential gene expression analysis, serial tissue bi- W, et al. Loss of PTEN promotes resistance to T-cell mediated immunother- opsy qrt-PCR, and SILAC-AHA metabolic labeling mass spectrometry-based apy. Cancer Discovery. 2016; 6 (2):201-216. differential secretome analysis to identify those genes and secreted pro- teins upregulated in melanomas escaping anti-PD-1 ab therapy in an au- V600E -/- P384 tochthonous BRAF PTEN melanoma model. Multi-parameter flow Mechanisms of efficacy during TGFbR1 inhibition/cytotoxic cytometry and immunohistochemistry were utilized to investigate MDSC combination therapy for rectal cancer recruitment to tumor tissues. Lentiviral shRNA gene silencing and in vivo Andrew Gunderson, Kayla McCarty, Michaela Phillips, Emily Hodel, CD8 T cell ablation assays were conducted to define the underlying sig- Michael Gough, Marka Crittenden, Kristina Young naling mechanisms driving MDSC recruitment to anti-PD-1 ab-treated Earle A. Chiles Cancer Research Institute, Portland, OR, USA melanomas. Circulating melanoma cells (CMCs) were derived from patients Correspondence: Andrew Gunderson (agun1107@gmail.com); Kristina undergoing active anti-PD-1/PD-L1 ab therapy using a photoacoustic cy- Young tometry technique for single cell qrt-PCR validation of pre-clinical findings. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P384 Results V600E -/- Autochthonous BRAF PTEN melanomas upregulate the expres- Background sion of several CXCR2 ligands, including CXCL5, following escape Neoadjuvant chemoradiation is the standard of care for patients with lo- from anti-PD-1 ab therapy (Fig. 1A-B). This is associated with the cally advanced rectal cancer with the intention of downstaging and rapid recruitment of Gr-MDSCs and an inhibition of local CD8 T cell sphincter preservation prior to surgical intervention. Responses may be responses (Fig. 1C-D). We determined that melanoma CXCL5 expres- limited, however, in part due to TGFb1 regulated resistance to these cyto- sion is induced by a Wnt5a-YAP pathway and that Wnt5a is, in turn, toxic modalities. We have shown previously that blocking ALK5 signaling regulated by local CD8 T cell killing and paracrine heat shock pro- through use of a small molecule inhibitor sensitizes CT26 tumor-bearing tein (HSP)-TLR2 signaling (Fig. 2). The individual genetic silencing of mice to the effects of radiation in part by boosting CD8 T cell mediated Wnt5a and CXCL5 reverses Gr-MDSC recruitment in response to anti- V600E -/- cytotoxicity. The mechanism for this efficacy is currently unknown. PD-1 ab therapy and sensitizes these BRAF PTEN melanomas to Methods checkpoint inhibition (Fig. 3). Indeed, blockade of Wnt-mediated signal- By using LY2157299 prior to 5-FU and hypofractionated radiation, tumor- ing with a OMP-54F28 'Wnt trap' diminishes Gr-MDSC recruitment and bearing animals elicit suboptimal T cell responses capable of controlling augments the efficacy of anti-PD-1 ab therapy in this autochthonous tumor growth in a significant percentage of animals but fail to cure. melanoma model (Fig. 4). Plasma levels of CXCL5 correlate with pro- Results gression and anti-PD-1 ab escape in the transgenic melanoma model Early results from a clinical trial of neoadjuvant TGFbR1 inhibition with while patient-derived CMCs also demonstrate CXCR2 ligand upregula- chemoradiation in 3 rectal cancer patients demonstrate objective clinical tion following progression on anti-PD-1/PD-L1 ab therapy (Fig. 5). responses and markers of enhanced anti-tumor immunity but indicators Conclusions of compensatory immunosuppression are also observed in surgically ex- A HSP-TLR-Wnt5a paracrine signaling axis mediates the recruitment of cised tissue. One visually prominent aspect in a proportion of these pa- Gr-MDSCs in response to anti-PD-1 ab therapy-mediated CD8 T cell tients is the infiltration of plasma cells into tumor parenchyma. Typically killing and represents a novel adaptive resistance mechanism to cancer these fully differentiated B cells would be a hallmark of immune medi- immunotherapy. Additional studies are ongoing to exploit this pathway ated rejection through secretion of high affinity immunoglobulin, but as a strategy for augmenting the therapeutic efficacy of anti-PD-1 ab previous reports suggest they may also function to suppress cytotoxic T therapy and for monitoring the development of immunotherapy resist- cells through TGFb1, IL-10 and PDL1. Correspondingly, we also observe ance in advanced melanoma patients. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 189 of 244 Fig. 1 (abstract P385). Escape from anti-PD-1 Ab Immunotherapy in a Fig. 3 (abstract P385). The Wnt5a-CXCL5 Pathway Drives Gr-MDSC Transgenic Melanoma Model Involves the Recruitment of Gr-MDSCs. A. Recruitment in Response to anti-PD-1 Ab Therapy and Promotes RNAseq differential gene expression analysis showing an upregulation of the Development of anti-PD-1 Ab Adaptive Resistance. A. Flow cy- several CXCR2 ligands following anti-PD-1 ab escape in a transgenic tometry analysis of Gr-MDSCs in Wnt5a-silenced BRAF(V600E)- BRAF(V600)-PTEN-/- melanoma model. B. RNAseq differential gene expres- PTEN-/- melanomas. B. Immunohistochemistry of Gr-MDSCS in sion analysis demonstrating increased MDSC markers in BRAF(V600E)- Wnt5a-silenced BRAF(V600E)-PTEN-/- melanomas. C. Flow cytome- PTEN-/- melanomas following escape from anti-PD-1 ab therapy. C. Immu- try analysis demonstrates that Gr-MDSC recruitment in response to nohistochemistry and flow cytometry analysis demonstrating increased anti-PD-1 ab therapy is ablated in Wnt5a-silenced BRAF(V600E)- levels of Gr-MDSCs in anti-PD-1 ab-treated versus IgG isotype control- PTEN-/- melanomas. Representative flow cytometry dot plots treated BRAF(V600E)-PTEN-/- melanomas. Representative IHC images and provided. D. IFNg ELISPOT and tissue immunofluorescence show flow cytometry dot plots are provided. D. Serial tissue biopsy and qrt-PCR that melanoma Wnt5a silencing enhances the activation of TRP2- analysis of anti-PD-1 ab-treated versus IgG isotype control-treated specific CD8+ T cells and promotes the expression of PD-L1. BRAF(V600E)-PTEN-/- melanomas. All data is mean +/- SEM. *P<0.05 Representative images provided on the left. E. Wnt5a silencing sensitizes BRAF(V600E)-PTEN-/- melanomas to anti-PD-1 ab therapy. F. CXCL5 silencing suppresses Gr-MDSC recruitment and sensitizes BRAF(V600E)-PTEN-/- melanomas to anti-PD-1 ab therapy. Representative flow cytometry dot plots shown. All data is mean +/- SEM. *P<0.05 Fig. 2 (abstract P385). Induction of CXCL5 in Melanomas Undergoing Treatment with Anti-PD-1 Ab Therapy is Dependent Upon a Paracrine HSP-TLR-Wnt5a Signaling Pathway. A. Qrt-PCR analysis shows that Wnt5a stimulates the expression of several CXCR2 ligands in a BRAF(V600E)- PTEN-/- melanoma cell line. B. Western blot analysis of nuclear YAP1 levels in response to Wnt5a stimulation in a BRAF(V600E)-PTEN-/- melan- oma cell line. C. Immunohistochemistry studies show that CXCL5 expres- sion is significantly suppressed in Wnt5a-silenced BRAF(V600E)-PTEN-/- melanomas. D. SILAC-AHA metabolic labeling mass spectrometry-based differential secretome analysis and RNAseq differential gene expression analysis of BRAF(V600E)-PTEN-/- melanomas following treatment with Fig. 4 (abstract P385). Inhibition of Wnt Signaling Suppresses anti-PD-1 ab vs IgG isotype control ab. Several cellular stress pathways Gr-MDSC Recruitment in Response to anti-PD-1 Ab Therapy and identified as being upregulated in those melanomas undergoing anti- Augments the Efficacy of this Checkpoint Inhibitor in a Transgenic PD-1 ab therapy. Several heat shock proteins including HSP70 noted to BRAF(V600E)-PTEN-/- Melanoma Model. A. Immunohistochemistry be induced in anti-PD-1 ab-treated melanomas. E. BRAF(V600E)-PTEN-/- of CXCL5 and Gr-1 in BRAF(V600E)-PTEN-/- melanoma tissue melanomas express elevated levels of TLR2 and TLR4 based on qrt-PCR. undergoing treatment with anti-PD-1 ab therapy in the absence Western blot demonstrates that HSP70 and agonistic anti-TLR2 ab and presence of OMP-18R5 (vantictumab, anti-Fzd7 ab) or stimulates Wnt5a expression in the BRAF(V600E)-PTEN-/- melanoma cell OMP-54F28 (ipafricept, Fzd8-Fc). B. Histologic analysis of resected line. F. top, CD8+ T cell levels and Gr-MDSC levels correlate significantly lung tissues to enumerate lung metastases in the transgenic in BRAF(V600E)-PTEN-/- melanomas only when treated with anti-PD-1 ab BRAF(V600E)-PTEN-/- melanoma model following the indicated therapy (p = 0.044). bottom, Ablation of CD8+ T cells diminishes the treatment regimens. C. Flow cytometry analysis of BRAF(V600E)- recruitment of Gr-MDSCs in response to anti-PD-1 ab therapy. G. top, PTEN-/- melanoma tissue following the indicated treatment Whole tissue Western blot analysis shows that anti-PD-1 ab therapy regimens. CD45+CD3+CD8+ T cells and CD45+CD4+FoxP3+ induces the upregulation of Wnt5a and YAP1 in BRAF(V600E)-PTEN-/- regulatory T cell (Treg) populations quantitated and expressed as a melanomas. bottom, Dot blot analysis shows that CXCL5 expression is CD8+ T cell : Treg ratio. D. TRP2 IFNg ELISPOT analysis performed upregulated in the serum of transgenic BRAF(V600E)-PTEN-/- mice using harvested splenocytes from transgenic BRAF(V600E)-PTEN-/- undergoing treatment with anti-PD-1 ab therapy (red, blot density mice after undergoing the indicated therapy. Representative wells analysis). Representative blots shown. All data is mean +/- SEM. *P<0.05 shown on the right. All data is mean +/- SEM. *P<0.05 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 190 of 244 tumor microenvironment were functionally suppressed. Tumor tran- scriptional profiling revealed that prime/boost vaccination led to induc- tion of numerous inflammatory processes, with gene signatures consistent with CD8+ T cell infiltration, upregulation of multiple co- stimulatory/ immune checkpoint receptors, and induction of chemo- kine networks associated with lymphoid and myeloid cell trafficking. Based on these signatures, we tested candidate combination therapies to further enhance the impact of prime/boost vaccination, of which checkpoint blockade using αPD-1 produced the greatest benefit. Analysis of the response to prime/boost vaccination + αPD-1 by MRI revealed significant intratumoral inflammation after Maraba boosting followed by dramatic tumor regression, consist- ent with pseudo-progression. Tumors were effectively controlled, Fig. 5 (abstract P385). Circulating Melanoma Cell (CMC) Qrt-PCR but ultimately relapsed. Using MRI guided assessment, treated tu- Analysis Demonstrates Enhanced CXCR2 Ligand Expression in mors were subsequently isolated during all phases of this re- Progressing Melanoma Patients Following anti-PD-1/anti-PD-L1 Ab sponse and subjected to RNAseq analysis. The findings from Therapy. A. Schematic showing clinical specimen acquisition and these studies will be presented. analysis work flow. Samples are harvested from patients before and Conclusions during checkpoint inhibitor immunotherapy. CMCs are enriched Our data suggest that interrogating the tumor microenvironment through centrifugation, analyzed by the photoacoustic flow cytometer, during the course of vaccine prime/oncolytic boost immunotherapy and subjected to integrated fluidic chip single cell qrt-PCR analysis of can identify candidate therapeutic targets, as well as key biomarkers select target genes. Samples positive for CD45 or negative for both of therapeutic response and/or intratumoral changes leading to MART1 and S100 are excluded from analysis. Target gene expression tumor progression. normalized to Ubb expression levels. 6 CMCs analyzed per patient time point. B. Spider plot of target lesion tumor volume versus time for an advanced melanoma patient undergoing anti-PD-L1 ab therapy. Blood P387 samples taken at week 0 prior to therapy and week 24. C. MART1 IFNγ signaling in T acts as a barrier to immunotherapeutic response regs immunofluoresence analysis of captured CMCs. Brightfield images 1 1 1 Abigail Overacre-Delgoffe , Maria Chikina , Rebecca Dadey , shown on left. D. CMC single-cell qrt-PCR analysis and quantitation of 1 1 2 1 Hiroshi Yano , Erin Brunazzi , Gulidanna Shayan , William Horne , CXCR2 ligands and other target genes of interest in the same patient 2 1 2 1 Jessica Moskovitz , Jay Kolls , Cindy Sander , Yongli Shuai , described in B. Data has been normalized to week 0 expression levels 1 2 2 2 Daniel Normolle , John Kirkwood , Robert Ferris , Greg Delgoffe , of respective target genes 2 1 1 Tullia Bruno , Creg Workman , Dario Vignali 1 2 University of Pittsburgh, Pittsburgh, PA, USA; University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA P386 Correspondence: Abigail Overacre-Delgoffe (overacre@pitt.edu); Dario Vignali Tracking the dynamic response to vaccine prime/oncolytic boost Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P387 immunotherapy identifies key mechanisms of immune resistance in metastatic ovarian cancer Background 1 1 1 1 AJ Robert McGray , Raya Huang , Sebastiano Battaglia , Cheryl Eppolito , Checkpoint blockade (anti-PD1/PDL1) has displayed striking, durable re- 1 1 2 2 Anthony Miliotto , Mukund Seshadri , Kyle Stephenson , Brian Lichty , sponses in cancer patients; however, many still succumb to disease. The Kunle Odunsi underlying reasons for this lack of response remain obscure. Therefore, 1 2 Roswell Park Cancer Institute, Buffalo, NY, USA; McMaster University/ identifying resistance mechanisms to current immunotherapies is critical. Turnstone Biologics, Hamilton & Ottawa, Canada While regulatory T cells (T ) are required to maintain immune homeo- regs Correspondence: Kunle Odunsi (bobmcgray@gmail.com) stasis, they are also a major barrier to the anti-tumor immune response, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P386 and as such, are an attractive therapeutic target. We have previously shown that intratumoral T stability is reliant on the Neuropilin-1 reg Background (Nrp1):Semaphorin-4a axis. In its absence, T become fragile and se- regs While spectacular responses to cancer immunotherapy have been ob- crete IFNγ while maintaining Foxp3 expression [1,2]. However, 1) served, most responses are short-lived with ultimate tumor relapse. whether T fragility is required for response to immunotherapy, and 2) reg Using a vaccine prime/oncolytic boost immunotherapy, we sought to the underlying T -mediated mechanisms of immunotherapeutic resist- reg define robust biomarkers indicative of therapeutic response, response ance in mice and patients remain unclear. durability, as well as changes leading to tumor relapse in a pre-clinical Methods mouse model of intraperitoneal metastatic ovarian cancer. We utilized syngeneic tumor models, including MC38 (anti-PD-1 Cre-YFP Methods sensitive) and B16.F10 (anti-PD-1 resistant) in both Foxp3 and L/L Cre-YFP We utilized a heterologous prime/boost vaccine to treat ovarian can- Ifngr1 Foxp3 mice. Mice were treated with various immunother- cer, combining an adjuvant-based vaccine with a novel oncolytic apies to assess the role of IFNγ signaling by T in mediating resistance. regs Maraba viral vector to generate robust anti-tumor T cell responses. In addition, we are assessing the direct effect of IFNγ in vivo through the To investigate treatment impact on both anti-tumor immunity and use of novel delivery mechanisms. Lastly, we are determining the impact local events within the tumor, we combined analyses of immune of T fragility on patient response rate after PD-1 blockade in meta- reg cells within the tumor microenvironment with transcriptional profil- static melanoma and head and neck squamous cell carcinoma. ing of solid tumors, and pre-clinical MRI to directly monitor changes Results Cre-YFP L/L Cre-YFP in intraperitoneal tumors in response to therapy. This approach led Using Foxp3 and Ifngr1 Foxp3 mice, we found that re- to the identification/testing of candidate combination treatments sponse to PD-1 blockade required IFNγ-induced T fragility, as reg L/L Cre-YFP with the potential to improve prime/boost vaccination. Ifngr1 Foxp3 mice treated with anti-PD1 were completely re- Results sistant to therapy. In addition, PD-1 blockade led to increased IFNγ L/L Cre-YFP Heterologous prime/boost vaccination elicited robust tumor-specific secretion by T , an effect that was absent in Ifngr1 Foxp3 regs CD8+ T cell responses, with high numbers of therapy-induced CD8+ T mice. Furthermore, pre-treatment of tumor-derived T with IFNγ regs cells effectively trafficking to the tumor microenvironment. This led to significantly reduced suppressive function in both mouse and approach greatly improved tumor control and long-term survival com- human T . We are currently extending our studies to patients regs pared to vaccine priming alone, however the combination therapy was treated with anti-PD1 in order to determine whether T fragility is reg not curative and cellular analysis suggested that T cells within the associated with immunotherapeutic response. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 191 of 244 Conclusions References Overall, we have shown that IFNγ-induced T fragility is required for 1. Fridlender ZG, Sun J, Kim S, et al. Polarization of tumor-associated neutrophil reg anti-PD1 response and that IFNγ leads to reduced T suppressive phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer cell. 2009;16:183-194. reg capacity while maintaining Foxp3 expression. These studies uncover a novel potential resistance mechanism to immunotherapy, and pro- P389 vides an avenue to target T selectively in the tumor microenviron- regs Aurora kinase inhibition enhances the efficacy of ment to bolster current immunotherapies. immunotherapy 1 2 1 1 3 Simone Punt , Shruti Malu , Rina Mbofung , Jodi McKenzie , Zhe Wang , References 1 1 1 1 Leila Williams , Jie Qing Chen , Sourindra Maiti , Trang Tieu , 1. Delgoffe GM, et al. Stability and function of regulatory T cells is maintained 1 1 1 1 Weiyi Peng , Chengwen Liu , Chunyu Xu , Marie-Andrée Forget , by a neuropilin-1-semaphorin-4a axis. Nature. 2013; 501: 252-256. 1 4 1 1 Cara Haymaker , Jahan Khalili , Nikunj Satani , Florian Muller , 2. Overacre-Delgoffe AE, et al. Interferon-γ Drives Treg Fragility to Promote 5 1 1 1 Laurence Cooper , Jason Roszik , Rodabe Amaria , Chantale Bernatchez , Anti-tumor Immunity. Cell. 2017. 169. 1130-1141. 1 1 Timothy Heffernan , Patrick Hwu The University of Texas MD Anderson Cancer Center, Houston, TX, USA; The University of Texas MD Anderson Cancer Center (current: Eli Lilly P388 and Company), Indianapolis, IN, USA; The University of Texas MD Neutrophil-rich inflammation and PD1/PD-L1 inhibitor resistance in Anderson Cancer Center (current: The Journal of Experimental Medicine), advanced non-small cell lung cancer patients New York, NY, USA; The University of MD Anderson Cancer Center 1 2 1 1 Wungki Park , Sandra Algaze , Vaia Florou , Diana Saravia , (current: Personal Peptides LLC), Seattle, WA, USA; The University of 1 1 Deukwoo Kwon , Gilberto Lopes Texas MD Anderson Cancer Center (current: Ziopharm Oncology, Inc.), University of Miami, Miller School of Medicine, Sylvester Comprehensive Houston, TX, USA Cancer Center, Miami, FL, USA; University of Miami, Miller School of Correspondence: Simone Punt (s.vd.garde@outlook.com) Medicine, Miami, FL, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P389 Correspondence: Gilberto Lopes (wungkipark@gmail.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P388 Background Immunotherapy has improved clinical outcomes for a subpopula- Background tion of melanoma patients, but over half of treated patients The pro-tumoral role of neutrophils in chronic inflammation in car- develop primary or secondary resistance. Current efforts are thus cinogenesis and metastasis has been well-reported in the animal focused on developing combinatorial strategies involving im- models in the literature.[1] However, the role of neutrophil-to- munotherapy and other treatment modalities, such as targeted lymphocyte ratio (NLR) and the acquired resistance to PD1/PD-L1 in- therapy, to improve clinical outcomes. We aimed to identify fac- hibitors (PD1/PD-L1i) in advanced non-small cell lung cancer patients tors that regulate the tumor cell response to T cell mediated (NSCLC) has not been described. cytotoxicity, with the goal to develop more efficacious treatment Methods combinations. We retrospectively evaluated 115 advanced NSCLC patients who Methods received PD-1 and PD-L1 inhibitors as their second-line therapy after A high-throughput screen of 850 bioactive compounds was used to platinum-based chemotherapy. Two groups were defined: Progres- identify proteins/pathways that confer resistance to T cell mediated sors (P) as patients who had radiographic and clinical progression cytotoxicity (Fig. 1A). A comboscore was calculated for apoptosis after at least 2 months of treatment and Clinical Durable Responders (cleaved caspase-3) induced by combined compound and T cell (CDR) as patients with at least treatment duration of 6 months and treatment compared to either treatment alone (Fig. 1B). A library of ongoing response. Patients with baseline NLR > 10 were excluded. 576 ORF was subsequently used to identify proteins that induce resist- We performed paired-samples T test to analyze the difference of ance to T cell mediated cytotoxicity. The genes with the lowest combo- each patient’s NLR at baseline and at progression in P group. In CDR score in the ORF screen were analyzed using Nanostring on 23 samples group, the difference of each patient’s NLR at baseline and at cen- from adoptive cell therapy (ACT) treated melanoma patients. One of sored time while receiving ongoing PD-1/PD-L1i above 6 months the hits was further validated using in vitro and in vivo assays. were compared. Mann-Whitney test was performed to compare indi- Results vidual patient’s NLR at baseline and at progression with the presence After challenging melanoma cell lines with the compounds and of bone metastasis. autologous TILs, Aurora kinase inhibitors (AURKi) obtained a high Results comboscore. Two Aurora kinase inhibitors were validated to There were 72 patients in P group and 43 patients in the CDR group. synergize with T cell mediated cytotoxicity in multiple melanoma Total 82 patients received nivolumab, 25 patients received pembroli- cell lines (example of enhanced cytotoxicity in one cell line zumab, and 8 patients received atezolizumab. The duration of clinical shown in Fig. 1C). Complementary to this finding, Aurora kinase response in P ranged from 2 to 17.4 months and 6.5 to 23.8 months A (AURKA) and AURKB overexpression induced resistance to T cell in CDR group. The mean NLR at baseline in P group was 4.89 and cytotoxicity. Additionally, patients who did not respond to ACT 6.59 at progression. Patients in P group had statistically significant in- expressed higher levels of AURKA compared to responding pa- creases of NLR at progression with mean increase of 1.69. The 95% tients (p=0.04), further demonstrating the relevance of Aurora confidence interval was 3.04 to 0.35 (p-value = 0.01). However, there kinase in immune suppression. The combination of AURKi was no statistical difference between the NLR at baseline and AZD1152 and anti-CTLA4 significantly reduced tumor growth censored time while receiving ongoing treatment in CDR group (p<0.05) and enhanced survival (p<0.01) as compared to either (95% C.I.: -1.90 - 0.822, p-value=0.428). Bone metastasis did not therapy alone in an MC38/gp100 in vivo model. Ongoing studies correlate with the degree of NLR. (p=0.56) Patients who received are now aimed at identifying the molecular mechanisms by corticosteroid at the time of progression were not included in which AURKi enhance T cell mediated cytotoxicity. the study. Conclusions Conclusions Aurora kinase inhibition may not be cytotoxic per se, but we Persistent increase of NLR may represent unfavorable pro-tumoral in- showed that AURKi treatment sensitizes tumor cells to T cell me- flammation and can represent unfavorable peripheral balance of PD- diated cytotoxicity and enhances the efficacy of immunotherapy. 1 and PD-L1 interplay in myeloid-effector T cell immune synapse. The combination of AURKi treatment and immunotherapy may This may serve as acquired resistance mechanism of PD-1/PD-L1i and thus induce an effective and durable response in melanoma pa- warrants further investigation. tients, making Aurora kinase inhibitors a promising combination Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 192 of 244 strategy with immunotherapy, especially for patients who do not colon carcinoma bearing BALB/c mice were treated with IT, RT, or respond to immunotherapy alone. combined IT/RT either 10 or 17 days after tumor implantation. The fre- quency/function of tumor-specific (OT-I or AH1) CD8 T cells was moni- tored in blood, lymph node (LN), and tumor infiltrating lymphocytes (TIL). Reporter Nur77-GFP-OT-I CD8 T cells expressing green fluorescent protein in response to TCR ligation were used to measure TCR signaling following IT/RT. Results We demonstrate OT-I cells become anergic/exhausted and produce few effector cytokines 21 days after adoptive transfer. Combination therapy with IT/RT resulted in increased TCR stimulation (measured by GFP expression) and reversed anergy (measured by proliferation) within OT-I T cells in blood, LN, and TIL compared to IT or RT alone. Combin- ation therapy was able to reverse functional exhaustion (measured by cytokine production) within LN but not TIL in this model. In CT26 tumor bearing mice combination IT/RT significantly increased (up to 4-fold) the frequency of tumor-specific endogenous AH1 CD8 TIL and pre- vented development of functional exhaustion within TIL compared to IT or RT alone. Conclusions Combined ablative RT and IT with αOX40/αCTLA-4 results in increased CD8 TCR signaling, reversal of T cell anergy, and can prevent or reverse functional exhaustion in some cases. These novel results suggest the addition of RT to IT can effectively reverse tumor-associated T cell dys- function where IT alone is insufficient and provide rationale for early Fig. 1 (abstract P389). Aurora kinase inhibitors were identified to phase clinical trial design. enhance T cell mediated cytotoxicity. (A) Patient derived melanoma tumor cells were incubated with one of 850 different compounds per well in triplicate in a high-throughput screen. After 24 hours, the P391 compound was washed off and the tumor cells were co-cultured + - hi Non-conventional inhibitory CD4 Foxp3 PD-1 T cells as a with autologous TILs for three hours, followed by intracellular biomarker of immune checkpoint blockade activity staining for cleaved caspase-3. The percentage caspase-3 positive Roberta Zappasodi, Sadna Budhu, Matthew Hellmann, Michael Postow, tumor cells is used as a read-out for the amount of apoptosis among Yasin Senbabaoglu, Yanyun Li, Cailian Liu, Hong Zhong, Billel Gasmi, DDAO stained tumor cells. (B) The comboscore is calculated to Daniel Hirschhorn-Cymerman, Katherine Panageas, Taha Merghoub, assess the enhanced cytotoxicity of combined compound and Jedd Wolchok TIL treatment over either treatment alone. (C) Mel2549 cells were MSKCC, New York, NY, USA treated with 2uM AZD1152 or DMSO, TIL at a 3:1 E:T ratio or the Correspondence: Jedd Wolchok (zappasor@mskcc.org) combination of AZD1152 and TIL. Four other patient derived Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P391 melanoma cell lines showed similar enhanced cytotoxicity for the combination treatment Background A significant proportion of cancer patients do not respond to im- mune checkpoint blockade therapy. To deepen our understanding of P390 the mechanisms of action of this strategy, we explored the role of a Combination radiotherapy and αOX40/αCTLA-4 immunotherapy subset of CD4+Foxp3-negative T cells expressing PD-1 (4PD1hi), reverses anergy and prevents development of functional exhaustion which we found to be up-regulated after CTLA-4 blockade in associ- within tumor-specific CD8 T cells ation with limited response to treatment. 1 2 2 Joshua Walker , Melissa Kasiewicz , William Redmond Methods 1 2 Oregon Health & Science University, Portland, OR, USA; Providence 4PD1hi frequency was monitored by flow cytometry. Their function Cancer Center, Portland, OR, USA was tested in standard in vitro suppression assays and 3D collagen- Correspondence: William Redmond (walkejos@gmail.com) fibrin gel killing assays. RNAseq gene expression analyses were per- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P390 formed on a Proton sequencing system at the MSK Genomics Core Facility. Background Results The immunosuppressive tumor microenvironment and chronic T cell We observed that 4PD1hi accumulate intratumorally as a function stimulation that occurs in the presence of cancer results in CD8 T cell of tumor burden in untreated tumor-bearing hosts. In addition, dysfunction that has been difficult to reverse with immunotherapy (IT) mouse and human circulating and intra-tumor 4PD1hi inhibit T- alone. This dysfunction can be separated into two categories of anergy cell functions in a PD-1/PD-L1 dependent fashion. Interestingly, and exhaustion. Anergy being a poor proliferative response to stimula- CTLA-4 blockade promotes intratumoral and peripheral 4PD1hi tion after initial priming with inadequate costimulation or suboptimal T increases in a dose-dependent manner, while combination with cell receptor ligation, and functional exhaustion being the inability of T PD-1 blockade mitigates this effect and significantly improves cells to kill or produce effector cytokines in response to stimulation. We anti-tumor activity. In keeping with these observations, we found hypothesized the addition of ionizing radiotherapy (RT) to IT with agon- that patients have a significantly higher risk of death if high ist αOX40 & blocking αCTLA-4 antibodies would reverse tumor associ- 4PD1hi levels persist after PD-1 blockade. Mechanistically, we pro- ated CD8 T cell anergy and exhaustion. vide evidence that mouse and human 4PD1hi resemble follicular- Methods helper-T-cell(TFH)-like cells and that CTLA-4 blockade increases B- We utilized a model of CD8 T cell anergy in which transgenic, OVA- cell co-stimulatory potential in vivo and in vitro, pointing to specific, OT-I CD8 T cells are adoptively transferred into centrally toler- 4PD1hi as a possible downstream effect of enhanced T-cell prim- ant POET-1 mice bearing OVA expressing tumors. After development of ing during CTLA-4 blockade. anergy/exhaustion, 21 days after transfer, animals were treated with a Conclusions single 20 Gy fraction of RT, αOX40/αCTLA-4, or combined IT/RT. To as- These findings broaden our understanding of the incremental activ- sess anergy/exhaustion in endogenous CD8 T cell responses, CT26 ity of combination checkpoint blockade. They also provide a new Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 193 of 244 pharmacodynamic and prognostic biomarker that may inform the design of optimal combination schedules and checkpoint blockade dosage. Microbiome P392 Microbiome analyses in patients with previously treated, deficient DNA mismatch repair/microsatellite instability-high metastatic colorectal cancer treated with nivolumab ± ipilimumab: CheckMate 1 2 1 3 Scott Kopetz , Vittorina Zagonel , Michael J. Overman , Ray McDermott , 4 5 6 7 Michael A. Morse , Franklin L. Chen , James Lee , Rebecca A. Moss , 8 7 7 7 Lilan Ling , Alexander Greenfield , Danielle Greenawalt , Z. Alexander Cao 1 2 MD Anderson Cancer Center, Houston, TX, USA; Istituto Oncologico Veneto IOV-IRCSS, Padova, Italy; St. Vincent's University, Dublin, Ireland; 4 5 Duke University Medical Center, Durham, NC, USA; Novant Health Oncology Specialists, Winston-Salem, NC, USA; University of Pittsburgh School of Medicine UPMC, Pittsburgh, PA, USA; Bristol-Myers Squibb, Fig. 1 (abstract P392). See text for description Princeton, NJ, USA; Memorial Sloan-Kettering Cancer Center, New York, NY, USA Correspondence: Scott Kopetz (chris.geraci@chrysalismedical.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P392 P393 Association of microbiome to nivolumab response in metastatic Background renal cell carcinoma (mRCC) 1 1 2 1 Microbiota may have a role in the carcinogenesis of colorectal cancer Manuel Maia , Valeriy Poroyko , Hae Won , Paulo Bergerot , 1 1 1 2 (CRC) through chronic low-grade inflammation and the influence of Lorena Almeida , Nazli Dizman , JoAnn Hsu , Jeremy Jones , 1 1 microbiota-derived metabolites on host metabolism and immune Ravi Salgia , Sumanta Pal function.[1,2] Microbiome diversity was significantly different be- City of Hope Comprehensive Cancer Center, Duarte, CA, USA; tween patients with melanoma who responded to anti–PD-1 therapy Beckman Research Institute, City of Hope Comprehensive Cancer and those who did not respond.[3] Here, we explore the relationship Center, Duarte, CA, USA between microbiota and response to nivolumab ± ipilimumab in pa- Correspondence: Manuel Maia (mcaitano@coh.org) tients with deficient DNA mismatch repair/microsatellite instability- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P393 high (dMMR/MSI-H) CRC enrolled in CheckMate 142 (NCT02060188). Methods Background Patients received nivolumab 3 mg/kg Q2W or nivolumab 3 mg/kg + Checkpoint inhibitors (CPI) have improved clinical outcomes in vari- ipilimumab 1 mg/kg Q3W × 4 doses followed by nivolumab 3 mg/kg ous cancer types, including mRCC [1]. Predictive factors of benefit Q2W until discontinuation. 16S ribosomal RNA sequencing was per- from CPIs are not well defined. Relationship of the microbiome to formed on baseline stool samples. Alpha diversity and differential benefit from CPIs has been suggested in preclinical studies [2]. Here measures of operational taxonomic units were evaluated at each taxa we provide the first evidence of association between microbiome level. Microbiota differences were compared between patients with composition and response to nivolumab in mRCC patients (pts). an investigator-assessed best overall response (BOR) of partial re- Methods sponse (PR) vs those with a BOR of progressive disease (PD). Due to Stool samples were prospectively collected from pts with mRCC at small patient numbers, those treated with nivolumab and nivolumab 3 time points relative to nivolumab treatment (baseline, week 4 + ipilimumab were combined for the analysis. and week 12) and used to assess gut microbiota composition in Results two different groups: responders (R; including complete/partial re- Baseline stool samples were collected from 72 patients. Among these sponse and stable disease) and non-responders (P; primary progres- patients, BORs of PR were achieved in 25, stable disease in 31, and PD sion). In short, microbial DNA was extracted, 16s rRNA gene tags in 14; 2 were not evaluable. Alpha diversity was not statistically differ- (v4) were generated by PCR amplification and sequenced using ent between patients with a BOR of PR vs those with PD. The abun- MiSeq (Illumina). Sequence reads were processed by Mothur soft- dance of Actinobacteria Micrococcaceae was 2.7-fold lower in patients ware, assembled in operational taxonomic units (OTUs), taxonomic- with a BOR of PR compared with patients with a BOR of PD (P=0.0009; ally annotated to the level of genus and used to construct Bray- Fig. 1); significant differences were also observed in the genus Rothia Curtis dissimilarity matrices. The similarity of samples was visualized (2.7-fold lower in patients with a PR; P=0.0007). These results remained by principal coordinates analysis (PCoA) and further confirmed by significantly different (P=0.04) after corrections for false discovery rate. k-means clustering (k=2) and ANOSIM tests. Differentially abundant Analyses on additional microbiota will be presented. taxa were determined by METASTATS and compared between R Conclusions and P. Patients with dMMR/MSI-H CRC who achieved a BOR of PR with nivo- Results lumab ± ipilimumab treatment were found to have a lower abun- Of 11 pts, 9 were evaluable for response. In this study, 25,304 OTUs dance of Actinobacteria Micrococcaceae, and in particular, the genus were attributed to 165 genera from 8 phyla. PCoA analysis reveals Rothia, compared with those with a BOR of PD. These results suggest that the first two principal coordinates can explain 49.2% of data set that there may be differences in the gut microbiota of patients with variation. Subsequent k-means clustering shows an almost complete dMMR/MSI-H CRC who respond to treatment with checkpoint inhibi- separation of microbiota in R and P groups. The produced clusters al- tors vs those who progress. Additional analyses investigating correla- most perfectly aligned with R and P groups, although ANOSIM of this tions with tumor immune phenotype and microenvironment, and separation was not significant (p=0.07). The analysis of microbiota other clinical parameters, are warranted. membership in P and R groups revealed 4 differentially abundant taxonomic units at the genus level (Fig. 1), with 2 present above 1% References abundance. Namely, Roseburia spp and Faecalibacterium spp were 1. Oke S, Martin A. Therap Adv Gastroenterol. 2017;10:417-428. significantly elevated in R as compared to P (p<0.05). Notably, no sig- 2. Gao R et al. Eur J Clin Microbiol Infect Dis. 2017; 36 :757-769. nificant differences were noted in Bifidobacterium spp or Bacteriodes 3. Sivan A et al. Science. 2015; 350:1084-1089. spp in R and P (P=0.15 for both). Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 194 of 244 Conclusions differentially abundant in responders relative to non-responders. Our results are the first to associate specific microbial genera to nivo- Forty-three of these OTUs were assigned potential species-level iden- lumab response in mRCC and warrant confirmation in larger series. tities, based on metagenomics sequencing of the same stool sam- Furthermore, manipulation of the stool microbiome as a means of ples. Selected responder-associated species, including Bifidobacterium modulating nivolumab response should be investigated. longum, Akkermansia muciniphila, Collinsella aerofaciens, and Entero- coccus faecium were also confirmed by quantitative PCR. Reconstitu- References tion of germ-free mice with fecal material from a responding patient 1. Motzer RJ, et al. Nivolumab versus Everolimus in Advanced Renal-Cell led to improved tumor control, augmented T cell responses, and a Carcinoma. N Engl J Med.2015: 1803-1813. distinct microbiota compared to a non-responding patient. 2. Vetizou M,et al. Anticancer immunotherapy by CTLA-4 blockade relies on Conclusions the gut microbiota. Science. 2015; 350: 1079-84. Our results indicate a correlation between the abundance of certain gut bacteria and clinical response to anti-PD-1 immunotherapy in cancer patients, and suggest a mechanistic impact of the commensal microbiota on anti-tumor immunity References Sivan A, Corrales L, Hubert N, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 2015; 350: 1084-1089. 2. Daillere R, Vétizou M, Waldschmitt N, et al. Enterococcus hirae and Barnesiella intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects. Immunity. 2016; 45: 931-943. 3. Iida N, Dzutsev A, Stewart CA, et al. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment. Science. 2013; 342 : 967-970. P395 DNA repair genetic aberrations and tumor mutation burden in biliary tract cancers 1,2 1 1 Fig. 1 (abstract P393). Relative abundance of selected genera Reham Abdel-Wahab , Rachna Shroff , Jean Nicolas Vauthey , 1 3 1 1 demonstrating significant differences between responders (R) and Eugene Jon Koay , Siraj Ali , Lawrence Kwong , Mingxin Zuo , 1 1 progressors (P). P: Primary progression group. R: Responders group Asif Rashid , Milind Javle 1 2 MD Anderson Cancer Center, Houston, TX, USA; Assiut University Hospital, Assiut Faculty of Medicine, Assiut, Egypt; Foundation Medicine, Cambridge, MA, USA Correspondence: Milind Javle (raali@mdanderson.org) P394 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P395 Commensal microbiota associated with anti-PD-1 efficacy in metastatic melanoma patients Background Vyara Matson, Jessica Fessler, Riyue Bao, Tara Chongsuwat, Yuanyuan Zha, Immune checkpoint inhibitors (ICPI) has been approved for several Maria-Luisa Alegre, Jason Luke, Thomas Gajewski cancers. The association between tumor mutational burden (TMB) University of Chicago, Chicago, IL, USA and ICPI has been previously studied and high TMB is associated Correspondence: Thomas Gajewski (vmatson@uchicago.edu) with prolonged progression free survival (PFS) after ICPI. DNA repair Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P394 gene aberrations (GAs) including those leading to microsatellite in- stability (MSI) is associated with response to ICPI. Data regarding this Background in biliary tract cancers (BTC) are limited.[1] Anti-PD-1-based immunotherapy is having a major impact on cancer Methods outcomes, but only a subset of patients respond favorably. To predict A comprehensive genomic profiling of 422 formalin-fixed, paraffin- response and ultimately improve therapeutic efficacy, it is of interest embedded tumor tissue from BTC patients, including 92 gall bladder to identify variables that cause this inter-patient heterogeneity. Pre- cancer and 330 cholangiocarcinoma, was performed using next gener- clinically, commensal bacterial species have been demonstrated to ation sequencing. We included 20 DNA repair genes that were previ- modulate spontaneous anti-tumor immunity and the outcome of im- ously described.[1] These included direct DNA repair genes (ATM, ATR, munotherapy and chemotherapy in mice (1-3). Here, we examine the BRCA1, BRCA2, FANCA, FANCD2, MLH1, MSH2, MSH6, PALB2, POLD1, POLE, association between gut microbiota composition and response to PRKDC, RAD50, SLX4) and caretaker genes that induce genomic instability anti-PD-1 immunotherapy in patients with melanoma. (BAP1, CDK12, MLL3, TP53, BLM). TMB was calculated in 205 samples by Methods counting the number of mutations across a 1.25Mb region and classified Stool samples were collected from 42 metastatic melanoma patients into three groups; low (< 6 mutation/Mb), intermediate (6 – 19 muta- prior to immunotherapy and microbial composition was analyzed tion/Mb), and high (≥20 mutation/Mb). We assessed MSI status by a through an integration of 16S rRNA gene sequencing, metagenomic computational algorithm examining 114 intronic homopolymer loci. shotgun sequencing, and quantitative PCR for selected bacteria. Clin- Results ical response was determined using Response Evaluation Criteria In Direct DNA repair genes were identified in 61 BTC tumors (14.5%) in- Solid Tumors (RECIST) version 1.1. Germ-free mice were colonized cluding GAs in ATM (5.5%) or BRCA2 (2.1%). Majority of the tumors with stool from responding and non-responding patients, and tumor had GAs in caretaker genes (42.9%), predominately in TP53 (33.4%), growth kinetics and anti-tumor immune responses were evaluated. and BAP1 (9.5%). However, FANCD2, POLD1, POLE, PRKDC, RAD50, Results SLX4, BLM mutations were not identified in our cohort. TMB was eval- There were 16 responders and 26 non-responders. There was a sig- uated in 205 patients. Among patients with direct DNA repair GAs, 20 nificant association between the commensal microbiota composition out of 35 (57.2%) had intermediate or high TMB as compared with and clinical response. Sixty-two operational taxonomic units (OTUs) 47 out of 170 (27.7%) patients without direct DNA repair GAs (P < assigned from the 16S rRNA gene sequencing dataset were .0001). Patients with caretaker GAs also had significantly higher TMB Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 195 of 244 [intermediate and high (48.4%)] than patients without these muta- P398 tions (P < .0001). MSI was determined in 88 patients and classified as Tumor mutation load assessment of FFPE samples using an NGS stable, ambiguous, and high. All patients with MSI high had direct based assay 1 1 1 1 2 DNA repair GAs (P < .0001). Ruchi Chaudhary ,AshaKamat ,WarrenTom ,IrisCasuga ,DineshCyanam , 1 1 1 Conclusions Dumitru Brinza , Janice Au-Young , Fiona Hyland 1 2 DNA repair GAs occur at a relatively high frequency in BTC and are Thermofisher Scientific, South San Francisco, CA, USA; Thermofisher associated with higher TMB and MSI. Future clinical trials targeting Scientific, Ann Arbor, MI, USA this subgroup with ICPI are warranted. Correspondence: Asha Kamat (asha.kamat@thermofisher.com); Ruchi Chaudhary References Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P398 1. Chae YK, Anker JF, Carneiro BA, Chandra S, Kaplan J, Kalyan A, Santa- Maria CA, Platanias LC, Giles FJ. Genomic landscape of DNA repair genes Background in cancer. Oncotarget. 2016;7(17):23312-21. Background – Tumors with high mutation load respond to therapy with immune checkpoint inhibitors. Hence high mutation load of a tumor can act as a predictive biomarker to immuno-therapy. We P396 developed an Ion S5 Sequencing System assay to determine the mu- Evolving immune response in lung squamous carcinogenesis tation status of solid tumors. 1 2 2 3 Celine Mascaux , Mihaela Angelova , Angela Vasaturo , Jennifer Beane , Methods 4 5 5 Kahkeshan Hijazi , Geraldine Anthoine , Karen Willard-Gallo , Methods: Our assay uses an FFPE sample, utilizing an Ampliseq 6 7 5 8 Annick Haller , Vincent Ninane , Arsène Burny , Jean-Paul Sculier , based target panel that covers 1.7 Mb of genomic DNA including 9 2 Avrum Spira , Jérôme Galon 409 genes. The assay does not require matched normal sample to es- Marseille Early Phases Cancer Trials Center CLIP, Marseille, France; timate the mutation status of a tumor. 2 3 Laboratory of Integrative Cancer Immunology, Paris, France; Boston Results Univ. School of Medicine, Boston, MA, USA; Research Center for Results: Our panel exhibits >90% sensitivity and >95% specificity in dis- Modeling and Simulation, Islamabad, Pakistan; Institut Jules Bordet, tinguishing between high and low mutation burden samples. Using a Université Libre de Bruxelles, Brussels, Belgium; University Chidren's variety of public annotation sources, we have successfully eliminated Hospital Queen Fabiola, Brussels, Belgium; CHU Saint-Pierre - Service de approximately 98% of germline variants. Matched tumor-normal ana- Pneumologie, Brussels, Belgium; Institut Jules Bordet, Centre des lyses on 9 lung and colorectal samples suggested that our single sam- Tumeurs de l'Université Libre de Bruxelles, Brussels, Belgium; Boston ple analysis on tumor samples detects mutation load with strong University Medical Center, Boston, MA, USA correlation (r=0.9358) with tumor-normal analysis. On engineered con- Correspondence: Jérôme Galon (mihaela.angelova@crc.jussieu.fr) trol from Acrometix, mutation load count consistently comprise true Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P396 mutations with positive predictive value of 89%. To assess reproducibil- ity, we compared the mutation load in library replicates for a cohort of Background 10 samples (FFPE and fresh frozen tumors, Acrometrix control, and NIST Molecular characterization of pre-invasive and invasive bronchial cell-lines) and observed high correlation (r=0.9948). lesions can provide insights into the mechanisms behind genesis and Conclusions development of squamous cell carcinoma, revealing potentially promis- We have developed a single tumor-only assay for accurate estimation ing new biomarkers for early detection and treatment of lung cancer. of tumor mutation burden, useful for cancer immuno-therapy re- Methods search and development. At 8 different morphological stages of lung squamous carcinogen- esis, from normal, to low grades dysplasia, high grades dysplasia, to carcinoma, fresh frozen human bronchial biopsies (n=122) were P399 subjected to gene expression profiling (Agilent microarrays) from 77 Rationally improving T cell-mediated cancer immunotherapy patients. A linear mixed-effects model was applied to infer modules using Sleeping Beauty mutagenesis to identify novel drug of gene co-expression, adjusted for smoking status, gender, and can- targets cer history as fixed effects, and patient as a random effect. Immune Laura Rogers, Adam Dupuy, George Weiner cell-specific gene signatures were used to characterize the immune University of Iowa, Iowa City, IA, USA response through the different development stages. Correspondence: Laura Rogers (laura-rogers@uiowa.edu) Results Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P399 The gene expression alterations could distinguish four groups of succes- sive stages of development. There were eight modules of co-expressed Background genes with different expression patternsacrossthe stages:up- or down- T cells have amazing potential to eliminate tumor cells throughout the regulation among the early or late steps,linearincreaseordecreasein body, evidenced by the clinical success of immunotherapies designed expression, and a biphasic pattern. Transitioning to early grade, there to enhance T cell function like immune checkpoint blockade and was a depletion of immune gene expression.However,atthe transition chimeric antigen receptor T cell (CAR-T) therapies. Expanding success to high-grade dysplasia, there was a significant up-regulation of im- to a broader number of patients is a top priority in the field. T cell mune/inflammatory response genes, representing T helper 1, myeloid infiltration into tumors appears to be an important prerequisite for the cells, as well as co-inhibitory and co-stimulatory molecules. success of both immune checkpoint blockade and CAR-T therapies. Conclusions Thus, increasing T cell infiltration into tumors could have beneficial At a critical stage of carcinogenesis there is a significant modification therapeutic impact. of the immune response. The up-regulation of immune genes in Methods high-grade suggests a major role of the surrounding microenviron- We have designed a forward genetic screen to identify T cell genes that ment thorough adaptive and innate immune response, but also a contribute to intratumoral T cell accumulation using Sleeping Beauty tumor versus host immunosuppressive response before tumor inva- (SB) transposon insertional mutagenesis in T cells. Our systematic sion across the basal membrane. This analysis may identify promising screen approach allows us to evaluate both loss- and gain-of-function new immune signatures of carcinogenesis, and may provide novel mutations across the entire genome in immunocompetent melanoma therapeutic targets for lung cancer. and lymphoma models that preserve the complexity of the tumor microenvironment. Importantly, the vast majority of genes we identi- fied were not previously known to be involved in tumor-associated T cell biology, and likely would not have been considered as drug targets P397 to enhance immunotherapy. These genes have the potential to modify Withdrawn Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 196 of 244 important T cell functions including trafficking to the tumor, clonal ex- References pansion, and sustained viability once inside the tumor. 1. Rizvi, NA, et al. Mutational landscape determines sensitivity to PD-1 blockade Results in non-small cell lung cancer. Science. 2016;348:1-13. We identified 312 tumor-enriched genes that were mutated in tu- mors, but not the spleen, of individual mice. Twenty of these were Oncolytic Viruses and Intratumoral Therapies detected in more than one mouse, representing strong gene candi- dates for validation. We demonstrate that one gene candidate is P401 functionally associated with T cell response to activation signals. Intratumoral expression of IL12 using the ZVex® dendritic cell- Specifically, CRISPR-mediated knockout of this gene in a murine T cell targeting lentiviral vector exerts potent anti-tumor effects via line resulted in cytokine production defects after T cell receptor induction of multiple immune effectors, including CD8 T cell stimulation. Additional screening has begun using other tumor responses models and anti-PD-1 therapy. Preliminary data from these indicate Tina C. Albershardt, Jardin Leleux, Rebecca S. Reeves, Jan ter Meulen, that few genes are conserved across tumor models and treatment Peter Berglund groups. Immune Design, Seattle, WA, USA Conclusions Correspondence: Tina C. Albershardt We are currently investigating the role of candidate genes in intratu- (tina.albershardt@immunedesign.com) moral T cell accumulation and immunotherapy enhancement using Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P401 an in vivo approach. Together, these experiments have the potential to expand our understanding of T cell infiltration into tumors and Background may provide previously unexplored strategies to rationally enhance Interleukin 12 (IL12), produced by antigen-presenting cells, plays a immunotherapy efficacy. pivotal role in the interplay between the innate and adaptive arms of the immune system. IL12 treatment has been shown to augment cytotoxic T lymphocyte and T 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to P400 toxicity. Intratumoral administration of IL12 is therefore being ex- Evaluation of tumor genetics and microenvironment through plored as an alternative route. We have previously reported on the next-generation sequencing (NGS) strong anti-tumor effects of IL12 delivered intratumorally using Im- Alex So, Shannon Kaplan, Joyee Yao, Shile Zhang, Aaron Wise, mune Design’s dendritic cell-targeting lentiviral platform ZVex in vari- Kristina Kruglyak, Naomi O'Grady, Jeremiah Mcdole, Marina Bibikova ous murine tumor models. Here, we evaluated the mechanism of Illumina, San Diego, CA, USA action mediating the local and systemic immune responses induced Correspondence: Alex So (alexsoas@gmail.com) by intratumoral ZVex/IL12 in different animal models. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P400 Methods Tumor cell lines (B16 melanoma, CT26 colon carcinoma, 4T1 breast Background cancer, A20 lymphoma, P815 mastocytoma, GL261 glioma) were im- The effectiveness of cancer immunotherapy is determined by the planted subcutaneously into mice. ZVex was engineered to express genetic basis of the patient’s tumor as well as the state of the tumor murine IL12 and administered as a single intratumoral injection into microenvironment. For instance, tumor mutation burden (TMB) and palpable tumors (50-100 mm ), typically at 7 days post-tumor inocu- microsatellite instability (MSI) status correlate with patient response to lation. Mice were monitored twice weekly for tumor growth and checkpoint inhibitors [1]. Also, the presence of immune cells in the survival. In lymphocyte depletion studies, anti-CD8, anti-CD4 or anti- tumor microenvironment correlates with immunotherapy responsive- NK1.1 antibodies were administered twice weekly, and their effect on ness. Thus, this project aims to develop a next-generation sequencing immediate and long-term anti-tumor response was studied. (NGS) workflow that reports various biomarkers that can be used to Results guide cancer immunotherapy selection. Here we present our data on A single intratumoral injection of ZVex/mIL12 induced complete tumor using whole exome sequencing (WES) and whole transcriptome se- regression in B16, CT26,A20,GL261,and P815 models,delayed tumor quencing (WTS) to report human leukocyte antigen (HLA) sequence, growth in the 4T1 model. In mice depleted of either CD8 T, CD4 T, or NK TMB load, prioritized neoantigens, and presence of immune cells. cells, complete tumor regression was still observed in 13-40% of mice. Methods However, only depletion of CD8 T cells together with CD4 T and/or NK WES and WTS libraries were generated using Illumina’s TruSeq® cells completely abrogated the anti-tumor response, suggesting that CD8 Exome and RNA Access library preparation kits, respectively. The T cells are required but not sufficient effectors in ZVex/mIL12-mediated samples were paired-end sequenced (2x75bp) using HiSeq® 2000 anti-tumor control. Furthermore, depletion of CD4 T cells resulted in very and 2500 instruments. pronounced vitiligo, demonstrating that intratumoral ZVex/mIL12 pro- Results foundly breaks self-tolerance, likely in the absence of regulatory T cells. Here we demonstrate a workflow using tumor/normal WES and tumor- Conclusions only WTS to determine MSI status and expressed TMB load. We also Taken together, the results demonstrate that intratumoral administra- show data that WES can be used to haplotype HLA Class I genes with tion of IL12 induces complex protective cellular immune responses, 97% accuracy. Using the HLA haplotype sequence, we computationally requiring CD8 effector T cells for maximum efficacy. predicted putative neoantigens and experimentally validated a subset, as true binders to their respective major histocompatibility com- plex. In addition, we applied FRICTION, a novel algorithm for cell deconvolution, to WTS data extracted from bulk tumor to quantify P402 the fraction of immune cells present in the tumor microenviron- Chemotherapy enhances oncolytic recombinant poliovirus efficacy 1 2 2 ment. Our titration experiments demonstrated our method’slinear- Jeffrey Bryant , Michael Brown , Matthias Gromeier + + + 1 2 ity in quantifying CD4 T cells, CD8 T cells, and CD19 B cells in a Duke University, Durham, NC, USA; Duke University Medical Center, variety of tissue backgrounds (median R > 0.97). Finally, we ap- Durham, NC, USA plied FRICTION to WTS data from melanoma samples extracted Correspondence: Jeffrey Bryant (jeffrey.bryant@duke.edu); Matthias from different subjects and showed that our workflow could also Gromeier quantify the fraction of immune cells in these samples when com- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P402 pared to quantitation by flow cytometry. Conclusions Background In summary, we demonstrate the utility of a NGS workflow that uses Oncolytic viruses are capable of direct tumor lysis along with the WES and WTS to report biomarkers relevant to immunotherapy and ability to activated pro-inflammatory programs that may contribute characterize the inflammation status of the tumor microenvironment. to the recruitment of adaptive immune effector responses. We use Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 197 of 244 a recombinant poliovirus:rhinovirus hybrid, PVSRIPO, which has be correlated to patient outcomes. Here we demonstrate the first func- tropism for neoplastic cells, due to the ectopic expression of the tional characterization of PIIM. poliovirus receptor, CD155, and a reliance on oncogenic signaling Methods to initiate viral protein translation. PVSRIPO is currently in Phase-I/II Two PIIM constructs were created for functional characterization: PIIM- clinical trials against recurrent glioblastoma multiforme (GBM). The OVA (IL12~Flt3L-OVA) for mouse experiments, and PIIM-NYESO1 first patients treated with PVSRIPO are radiographically tumor-free (IL12~Flt3L-NYESO1) for testing in human cells. The expression and and clinically normal >52 months after single intratumoral infusion functional activity of PIIM components were determined. Treated tu- th of PVSRIPO. On May 10 , 2016, the FDA/CBER granted Break- mors and spleens were assessed for transcriptional changes by Nano- through Therapy Designation to PVSRIPO. A co-incidental finding String® and phenotypic changes by flow cytometry. Systemic effects of emerging from our ongoing trial is remarkable durable, complete PIIM were assessed using a syngeneic two-tumor model of B16.F10 in responses in patients upon treatment with chemotherapy following which only one tumor received IT-PIIM-EP while the other contralateral tumor progression after PVSRIPO administration. lesion remained untreated. Methods Results This study used syngeneic mouse tumor models (TRAMPC2 and PIIM-OVA and PIIM-NYESO1 secrete functional IL-12p70, Flt3L-OVA and CT2A) to test the same chemotherapy combination used in the clinic. Flt3L-NYESO1 fusion proteins as assessed by ELISA, flow and cell-based Both the clinical trial and mouse study use chemotherapy doses that assays. PIIM promotes DC maturation and antigen-specific T cell prolif- induce severe lymphotoxic side effects which have been suggested eration both ex vivo and in vivo. Hydrodynamic-based gene delivery of to augment antitumor immunity when combined with an immuno- PIIM-OVA lead to splenomegaly and significantly increased splenic therapy regimen. To mirror what is done in the clinic the established CD11c+ DCs. Furthermore, IT-PIIM-EP lead to generation of splenic mouse tumors receive a single injection of PVSRIPO followed a week OVA-specific CD8s and increased APM gene expression. When intro- later by a single round of the alkylating chemotherapeutic drug Tem- duced intratumorally in a mouse two-tumor model, IT-PIIM-EP delays ozolomide (TMZ). Tumor growth and end point immune cells popula- B16.F10 tumor growth in both treated and contralateral tumors com- tions were monitored. pared to untreated controls resulting in increased overall survival. Results Conclusions This study demonstrates that this PVSRIPO TMZ combination can PIIM represents a novel approach to cancer immunotherapy. A be tested in syngeneic mouse models resulting in both an in- combination of functional immune modulators can be expressed lo- crease in survival and a delay in tumor growth over both cally in the tumor microenvironment that increase inflammatory in- PVSRIPO and TMZ treatment alone. Our data also suggests that filtrate, enhances antigen presentation and produces a systemic T this combination induces an increase in memory and infiltrating cell response specific to the antigen encoded on the plasmid. This Treg cell populations. customizable approach has the potential to improve therapeutic Conclusions outcome by enhancing adaptive-immunity and addressing patient- Ongoing work is being done to further validate these finding in mul- specific neoantigens needs. tiple mouse tumor models including intra-cerebral brain tumors models and to determine the mechanisms contributing to the syn- P404 ergy seen resulting from the PVSRIPO and chemotherapy combin- Targeting T-cells to human cancer associated fibroblasts using an ation. These intriguing results suggest that the memory T cells seen oncolytic virus expressing a FAP-specific T-cell engager after PVSRIPO chemotherapy combination may be responsible for 1 2 2 2 Brian Champion , Joshua Freedman , Rebecca Ashfield , Margaret Duffy , the new anti-tumor immune response. However, chemotherapy also 1 2 2 Alice Brown , Leonard Seymour , Kerry Fisher induced an increased tumor infiltrating inhibitory Treg cells. Future 1 2 PsiOxus Therapeutics Ltd, Abingdon, United Kingdom; Oxford work will look at ways to enhance this positive memory T cell popu- University, Oxford, United Kingdom lation while preventing intratumoral Treg cell accumulation. Correspondence: Brian Champion (brian.champion@psioxus.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P404 P403 Intratumoral administration of a multigene construct by Background electroporation can effectively modulate anti-tumor response in a Enadenotucirev (EnAd) is a chimeric oncolytic group B adenovirus with murine B16.F10 model potent and selective anti-tumor activity against a range of epithelial 1 1 1 Shawna Shirley , Anandaroop Mukhopadhyay , Christoph Burkart , cancer cells, with a blood stability profile that enables systemic dosing 1 2 2 1 Jocelyn Wright , Robert Pierce , Jean Campbell , David Canton and has been administered intravenously to over 100 cancer patients. 1 2 Oncosec Medical Incorporated, San Diego, CA, USA; Fred Hutchinson As an approach to immunogene therapy targeting stromal rich tumors, Cancer Research Center, Seattle, WA, USA we have created transgene-modified variants of EnAd expressing a bi- Correspondence: David Canton (dcanton@oncosec.com) specific T-cell engager (BiTE) molecule recognizing human fibroblast ac- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P403 tivating protein (FAP) on cancer associated fibroblasts (CAFs) and CD3 on T-cells. CAFs play a pivotal role in the development of solid carcin- Background omas by facilitating invasion, coordinating angiogenesis and establish- Immunomodulatory cytokines, such as interleukin-12 (IL-12) are attract- ing and maintaining an immune suppressive microenvironment in the ive candidates for cancer immunotherapy. IL-12 is a pro-inflammatory tumor stromal tissue. We hypothesize that production of BiTE proteins cytokine with potent anti-tumor effects; however, systemic administra- by virus infected tumor cells could be an effective way of modifying tion of IL-12 shows limited clinical efficacy and dose-associated toxicity. the stromal microenvironment to drive effective anti-tumor immunity In preclinical and clinical studies, intratumoral (IT) delivery of IL-12 plas- and would bypass delivery and safety issues related to systemic dosing mid DNA by electroporation (EP) can provide a safe and effective alter- of BiTE proteins. native for efficacious dosing. To augment the effects of IL-12, we Methods developed a DNA plasmid platform that allows for delivery of agents Bi-specific T-cell engager constructs comprising linked ScFv anti- that modulate multiple immune pathways as well as tumor- or patient- bodies specific for human FAP and CD3 were designed and used to specific neoantigens. Polycistronic IL-12 Immune Modulator plasmid generate EnAd viruses expressing the BiTE such that transgene ex- (PIIM) is a single plasmid encoding IL-12, and a fusion of Flt3L to an pression was under the control of either a CMV promoter or the virus antigen. Flt3L is a ligand that stimulates dendritic cell (DC) maturation major late promoter to allow broad or tumor-selective expression, re- and enhances antigen processing and presentation. The encoded anti- spectively. Tumor and fibroblast cell lines, together with control BiTE gen can be a viral or shared antigen, or a patient-specific neoantigen, proteins and BiTE-encoding viruses, were used to evaluate initial ac- which enables customization to patient populations, as well as provid- tivities of the FAP-BiTE viruses prior to testing with freshly isolated ing an aid to monitoring antigen-specific immune response(s) that can malignant peritoneal ascites from patients with advanced cancer. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 198 of 244 Results approach change the tumor immune microenvironment, induce We have shown that the viruses selectively express and secrete epitope spreading, and confer long term anti-tumor immunity. These functional FAP-specific BiTEs following infection of tumor cell lines. In findings prompt further evaluation in other spontaneous tumor co-culture with FAP+ fibroblasts and PBMC-derived T cells this leads models and provide a model to decipher the mechanisms of epitope to T cell-activation and cytotoxicity towards the fibroblasts. Similarly, spreading and the role of CD4 help. infection of primary malignant cancer ascites with FAP-BiTE-encoding viruses led to polyclonal activation of endogenous T-cells and deple- References tion of FAP+ cells from the cultures. Unlike activation of T-cells with 1. Gulley JL, et al. Immune impact induced by PROSTVAC (PSA-TRICOM), anti-CD3/CD28, FAP-BiTE activation was also shown to be effective a therapeutic vaccine for prostate cancer. Cancer Immunol Res. even in the presence of immunosuppressive (cell-free) ascitic fluid 2014;2(2):133-41. samples added to the cultures. 2. O'Hara GA, et al. Memory T cell inflation: understanding cause and effect. Conclusions Trends Immunol. 2012;33(2):84-90. In this study, we have shown that CAFs can be effectively targeted for T-cell mediated destruction by a FAP-BiTE transgene-bearing P406 oncolytic virus, with an associated strong activation of endogenous Dissecting the mechanisms underlying antitumor effects of live T-cells to kill endogenous CAFs even in the presence of an immuno- oncolytic vaccinia and heat-inactivated vaccinia suppressive microenvironment. Systemic dosing of such a virus to Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Taha Merghoub, patients with stromal rich tumors may provide an effective approach Jedd Wolchok, Liang Deng for driving effective anti-tumor immunity. Memorial Sloan Kettering Cancer Center, New York, NY, USA Correspondence: Liang Deng (dengl@mskcc.org) P405 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P406 Harnessing pre-existing antiviral immunity to treat solid tumors 1 1 2 1 Nicolas Cuburu , Rina Kim , Sergio Pontejo , Cynthia Thompson , Background 1 1,3 Douglas Lowy , John Schiller Preclinical and clinical studies have shown that viral-based immuno- 1 2 National Cancer Institute, NIH, Bethesda, MD, USA; National Institute of therapy has the potential to overcome resistance to immune check- Allergy and Infectious Diseases, NIH, Bethesda, MD, USA; NCI/NIH, point blockade and to fill the unmet needs of many cancer patients. Bethesda, MD, USA Oncolytic viruses are defined as engineered or naturally occurring vi- Correspondence: Nicolas Cuburu (cuburun@mail.nih.gov); John Schiller ruses that selectively replicate in and kill cancer cells. Oncolytic vi- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P405 ruses also induce antitumor immunity. We recently showed that intratumoral (IT) delivery of inactivated modified vaccinia virus Background Ankara (iMVA) induces antitumor systemic immunity via the STING- Cancer immunotherapy rely on adaptive immune responses against mediated cytosolic DNA-sensing pathway and Batf3-dependent + + tumor-associated antigens. The diversity of tumor antigens makes CD103 /CD8α dendritic cells (DCs). The combination of IT iMVA and the development of cancer vaccines a highly personalized endeavor. systemic delivery of immune checkpoint blockade is highly effective Epitope spreading results from the induction of de novo responses in treating large established tumors and distant metastasis [1]. MVA against tumor antigens not initially included in vaccines [[1]. Intratu- is non-replicative in tumor cells, and therefore whether or not viral moral therapies also require to induce de novo responses against replication is necessary for antitumor effects of IT oncolytic DNA virus tumor antigen for abscopal therapeutic effect. is unknown. Human cytomegalovirus is highly prevalent in humans with polyfunc- Methods tional T cell responses expanding with age [2]. We questioned In the study, we engineered a replication-competent, attenuated whether redirecting pre-existing anti-cytomegalovirus T cells into vaccinia virus (VC) by removing the Z-DNA-binding domain of solid tumor could arrest tumor growth, induce epitope spreading, vaccinia virulence gene E3 and inserting murine GM-CSF gene and confer long-term anti-tumor immunity? into the thymidine kinase (TK) locus (E3LΔ83N-TK -mGM-CSF). We Methods compared antitumor effects of live VC and iVC in murine tumor Persistently infected mice with mouse cytomegalovirus (MCMV) were models. challenged with TC-1 tumor cells expressing human papillomavirus Results (HPV) E6 and E7 oncogenes. We generated HPV pseudovirons express- We found that IT Heat-inactivated VC (iVC; by heating the live ing MCMV antigens or peptides derived from minimal MCMV epitopes. virus at 55°C for 1 h) is more effective in generating systemic an- These reagents were injected intratumorally together with the poly(I:C), titumor immunity than live VC murine tumor models. The antitu- as adjuvant. We monitored tumor growth, survival and measured anti- mor effects of live VC also requires Batf3-dependent DCs. IT Heat- + + tumor CD8 T cell responses against an E7 immunodominant epitope iVC induces higher numbers of infiltrating activated CD8 and using MHC-I tetramer and intracellular cytokine stainings. In rechallenge CD4 T cells in the non-injected tumors than live VC and is more experiments, we evaluated the induction of long-term anti-tumor im- potent in depleting tumor-associated macrophages than live VC munity. Using PanCancerImmune panel (Nanostring), we analyzed the in both injected and non-injected distant tumors. The surviving tumor immune microenvironment after treatment. mice treated with Heat-iVC are more effective in rejecting tumor Results rechallenge than those treated with live-VC, indicating that IT Transduction of TC-1 tumors with HPV pseudovirions expressing MCMV Heat-iVC generates stronger antitumor memory T cell responses antigens or peptidic MCMV epitopes caused the expansion of MCMV- than live VC. + + specific CD4 and CD8 T cells. These treatments also caused broad Conclusions modifications of the tumor immune environement. Intratumoral In conclusion, our results support the use the IT delivery of inacti- injection of MCMV CD8 epitopes provoked the arrest of tumor growth. vated vaccinia virus as a safe and effective cancer immunotherapy ei- Intratumoral injection of MCMV CD4 epitopes with poly(I:C) promoted ther alone or in combination with immune checkpoint blockade induction of E7-specific CD8 T cells. Sequential administration of CD4 antibodies. and CD8 MCMV epitopes together with poly(I:C) was the best protocol to eradicate pre-existing tumor of 5 to 10mm , and rechallenge experi- References ments showed anti-tumor immunity up to 4 months after the last 1. Dai P, Wang W, Serna-Tamayo C, Ricca JM, Zamarin D, Shuman S., treatment. Merghoub T, Wolchok JD, and Deng L. Intratumoral delivery of inacti- Conclusions vated modified vaccinia virus Ankara (iMVA) induces systemic antitumor Our results provide a proof of concept to design “antigen-agnostic” immunity via STING and Batf3-dependent DC. Science Immunology. intratumoral therapies based on pre-existing antiviral T cells. Such 2017;2: eaal1713. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 199 of 244 P407 Background In situ vaccination for cancer immunotherapy: treat locally, Our laboratory has previously shown that immunogenic tumors respond systemically spontaneously activate the innate immune system through the 1 2 2 2 Nicole Steinmetz , P. Jack Hoopes , Mee Ree Sheen , Chenkai Mao , STING pathway. The STING pathway senses cytosolic DNA, which acti- 1 2 2 3 Scott Sieg , Steven Fiering , Patrick :Lizotte , Amy Wen vates a signal transduction pathway culminating in phosphorylated 1 2 Case Western Reserve University, Cleveland, NH, USA; Geisel School of IRF3 that translocates to the nucleus where it acts as a transcription Medicine at Dartmouth, Lebanon, NH, USA; Case Western Reserve factor to induce several genes including IFN-β. STING signaling and University, Cleveland, OH, USA IFN-β receptor signaling in tumor-infiltrating immune cells, in turn, Correspondence: Steven Fiering (fiering@dartmouth.edu) are required for optimal priming of CD8+ T cells against tumor anti- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P407 gens. Based on this notion, STING agonists were developed and tested as a pharmacologic approach to activate the pathway. Background Methods Immunotherapy for cancer is making impressive impacts in the clinic. We stimulated various cell populations present in the tumor micro- One strategy dating back to William Coley in 1900 is in situ vaccin- environment as well as several tumor cell lines with STING agonists ation. This approach puts immunostimulatory reagents into an identi- to test their ability produce IFN-β and analyze each step in STING fied tumor to break the local immunosuppression, stimulate a local pathway signaling. anti-tumor response and most importantly stimulate systemic antitu- Results mor immune responses to eliminate metastatic disease. This is essen- We observe that tumor cells themselves frequently fail to produce tially an antitumor therapeutic vaccination, because the tumor IFN-β in response to STING agonists or cytoplasmic DNA, arguing provides the antigens and the adjuvants are the immunostimulatory that loss of activation of this pathway might occur regularly as a reagents, thus “in situ vaccination”. The approach has been part of component of oncogenesis. Surprisingly, we find that tumor cells re- standard of care for superficial bladder cancer using BCG bacteria. tain expression of each gene in the STING pathway and STING signal There are many immunostimulatory reagents that can be used and transduction is intact up to and including nuclear translocation of each has different capabilities. We have done previous studies with IRF3. ChIP assays demonstrate IRF3 is unable to bind the IFN-β pro- attenuated microorganisms including Toxoplasma gondii, and Listeria moter but can still bind the promoters of other genes. monocytogenes. Conclusions Methods These data indicate tumor cells fail to express IFN-β following STING Our recent focus has been on a plant virus that cannot infect ani- pathway activation due to a defect in transcription factor binding to mals, compea mosaic virus (CPMV). Complete CPMV or CPMV lacking the IFN locus. Based on ChIP experiments the defect in IRF3 DNA bind- nucleic acids (eCPMV) are grown in plants and biochemically ing may be specific to the IFN-β locus. Sequencing of the locus shows purified. no mutations in or around the binding site so we are currently investi- Results gating epigenetic mechanisms regulating IFN-β gene expression. CPMV nanoparticles were used in mouse cancer models and commu- nity dogs with oral melanoma and other tumors. These particles are P409 composed of assembled viral capsid proteins. eCPMV has no nucleic Efficacious anti-melanoma immunity induced by OX40 ligand- acids and no recognized immunostimulatory reagents. However, expressing oncolytic adenovirus Delta-24-RGDOX eCPMV is strongly immunostimulatory through unknown pathways Hong Jiang, Andrew Dong, Caroline Carrillo, Verlene Henry, Xuejun Fan, and causes changes in the tumor microenvironment that lead to pri- Yisel Rivera-Molina, Francisco Puerta-Martinez, Teresa Nguyen, Karen mary tumor reduction or elimination and potent resistance to meta- Clise-Dwyer, Frederick Lang, Candelaria Gomez-Manzano, Juan Fueyo static tumors (1). The treatment is immune-mediated but response in The University of Texas MD Anderson Cancer Center, Houston, TX, USA the lungs requires different immune components than response in Correspondence: Hong Jiang (hjiang@mdanderson.org) flank tumors of the same B16F10 melanoma cell line. Tumor reduc- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P409 tion or elimination occurs in many anatomic locations with multiple tumor types and in multiple strains of inbred mice. Treatment of pri- Background mary tumors by direct intratumoral injection mediates robust rejec- Oncolytic adenoviruses are highly immunogenic and cancer-selective tion of a rechallenge with the same tumor. Response in community but show limited efficacy as a single agent. Immune checkpoint modu- dogs was highly effective at both eliminating the local tumor and lation has shown efficacy in a variety of cancers but is associated with resisting metastatic disease. The mechanisms and pathways of immu- nonspecific T-cell activation, and has had a limited effect in tumors with nostimulation are under investigation. a nonimmunogenic microenvironment.Thus, combining these two Conclusions strategies likely resulted in both efficacious and specific cancer therapy. In situ vaccination has considerable potential for clinical use. In To this end, we constructed the oncolytic adenovirus Delta-24-RGDOX addition to the inherent immunostimulatory adjuvant properties of which expressed the immune co-stimulator OX40L. This new virus in- CPMV, they are a versatile platform to which other reagents for im- duces a superior immunotherapeutic effect in immunocompetent mune modulation can be attached. This demonstration of the value mouse glioma models than its predecessor Delta-24-RGDOX. of select viral-like nanoparticles for treatment of cancer opens a new Methods avenue of cancer immunotherapy. In a s.c./s.c. melanoma mouse model with B16F10-Red-FLuc cells, Delta-24-RGDOX and/or anti-CD47 antibody were injected into the References first implanted tumor. The tumor growth of the first and second im- 1. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz planted tumors was monitored with bioluninescence. The survival NF, Fiering S. In situ vaccination with cowpea mosaic virus nanoparticles curves were plotted according to the Kaplan–Meier method. Survival suppresses metastatic cancer. Nat Nanotechnol. 2016;11:295-303. rates in the different treatment groups were compared using the log-rank test. The immune cells in the tumor were isolated and ana- lyzed with flow cytometry. P408 Results Tumor cell-intrinsic STING signaling and regulation of IFN-β gene Delta-24-RGDOX expressed OX40L effectively in mouse melanoma cells. expression Compared to treatment with PBS, four doses of intratumoral injection 1 2 1 Blake Flood , Leticia Corrales , Thomas Gajewski of the virus significantly inhibited the growth of both the injected tu- 1 2 University of Chicago, Chicago, IL, USA; Aduro Biotech, Chicago, IL, USA mors and the untreated distant tumors, resulting in prolonged survival Correspondence: Blake Flood (blakeflood@uchicago.edu) of the mice with 40% long-term survival (P = 0.01). The surviving mice Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P408 is resistant to rechallenging with the same tumor cells but is Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 200 of 244 susceptible to lung cancer cells, suggesting the development of im- Conclusions mune memory specific to the virus-injected tumor type. Through flow ICR2 and ICR4 are potent PRR-stimulating cytotoxic agents against cytometry analysis of the tumor-infiltrating lymphocytes, we found the human cancers. ICR2 and ICR4 have distinctive immune stimulatory virus injection increased the presence of CD3+ T lymphocytes, CD3 and coagulating activities. Combination of dual checkpoint inhibitors +CD4+ helper T cells and CD3+CD8+ cytotoxic T cells in the tumor, and ICR4 and/or ICR2 would be a potent and effective anti-cancer causing a decreased ratio of CD4+/CD8+ cells. To further increase the therapies against advanced cancers. efficacy of Delta-24-RGDOX, we combined the virus with intratumoral injection of an antibody against CD47, a “don’teatme” signal overex- P411 pressed on tumor cell surface to protect them from phagocytosis. We Immunotherapy with BO-112, a novel double-stranded RNA-based found high expression levels of CD47 in cultured B16 cells and the agent, promotes tumor cell death and boosts T cell immunity in tumor cells from the xenografts. Intratumoral injection of Delta-24- preclinical mouse models RGDOX caused much more phagocytes presented at the tumor site 1 2 1 Lourdes Planelles , Angela Aznar , MªJosé Gómez-Sánchez , than PBS treatment. Combination of anti-CD47 antibody with Delta-24- 1 2 1 Mercedes Pérez-Olivares , Saray Garasa , Mercedes Pozuelo , RGDOX mediated longer survival time than the virus alone (median sur- 3 1 2 Ivan Márquez-Rodas , Marisol Quintero , Ignacio Melero vival: 56 days vs. 26 days). 1 2 BIoncotech Therapeutics, Valencia, Spain; Center for Applied Medical Conclusions Research (CIMA) & University of Navarra, Pamplona, Spain; Hospital Delta-24-RGDOX induced efficacious local and systemic anti-melanoma General Universitario Gregorio Marañón, Madrid, Spain immunity in B16-C57BL/6 mouse model and combination of the virus Correspondence: Lourdes Planelles (lplanelles@bioncotech.com) with anti-CD47 antibody further increased the therapeutic efficacy. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P411 P410 Background Generation of therapeutic RNAs to induce immunogenic cell death Unresponsive or refractory tumors are the main research objective in and interferon expression in cancer cells cancer immunotherapy. Strategies that promote immunogenic tumor 1 2 Jaewoo Lee , Youngju Lee cell death in the context of type I interferon (IFN) signaling can be 1 2 Duke University Medical Center, Durham, NC, USA; Duke University, combined effectively with existing therapies that target checkpoint Durham, NC, USA inhibitors to boost anti-tumor immunity. BO-112 is a double-stranded Correspondence: Jaewoo Lee (jaewoo.lee@duke.edu) synthetic RNA currently undergoing phase I clinical trials for intratu- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P410 mor injection. To enhance local antitumor immunity, BO-112 bears poly I:C chains that trigger endosomal Toll-like receptor 3 and cyto- Background solic helicases. Pattern-recognition receptors (PRRs) are immunological sensors that ini- Methods tiate the host defense response against infections. They are located at B16F10 and B16F10-OVA melanoma, MC38 colon carcinoma or the cell surface, within endosomal compartments and in the cytoplasm, 4T1 breast carcinoma cells (5 x 10 ) were implanted subcutane- where they are poised to recognize different molecular signatures asso- ously in the right flank of C57BL/6J and BALB/c mice; a second ciated with invading pathogens. In addition to anti-infectious immunity, tumor was engrafted in the left flank to evaluate distant antitu- the activation of RNA-sensing PRRs can mediate programmed cell mor effects. Beginning 7-10 days post-implant, mice were treated death of infected cells, which allows the host to efficiently block viral intratumorally with BO-112 only in the primary lesion. Therapeutic replication by sacrificing infected cells. Transfection with certain types efficacy was evaluated by monitoring tumor growth and survival, of RNA ligands that stimulate RIG-I-like receptor (RLR) family can induce and cellular responses were analyzed by multiparametric flow cy- type I interferon (IFN) and immunogenic cell death (ICD) in cancer cells, tometry. Antigen-specific CD8+ T cell in vivo priming was ad- which together orchestrate anti-tumor immune responses that can pre- dressed by H-2K tetramer analysis in B16F10-OVA tumors. The vent tumor recurrence and metastasis. Currently RNA-sensing PRR ago- role of CD4+ or CD8+ T cells was studied by depletion and de- nists have demonstrated little or no overall benefit to patients with pletion/rechallenge experiments. Combined BO-112 and anti-PD- cancers because of, mostly but not exclusively, toxicity driven by non- L1 (programmed death-ligand 1) antibody therapy was evaluated specific induction of immune reactions. In this study, we generate and in established B16F10 melanomas. screen multiple nuclease-resistant RNA molecules that can differentially Results induce immunogenic cancer cell death with or without concomitant In vitro and in vivo studies showed that BO-112 triggered cell death expression of IFN-β, pro-inflammatory cytokines and pro-coagulation in tumor cells of distinct tissue origins (melanoma, colon or breast mediators. carcinoma). The cytotoxic effect was also detected in engineered B16 Methods cells that do not respond to type I IFN. Intratumor BO-112 adminis- Therapeutic RNAs are designed using mFold RNA structure prediction tration in all three mouse tumor models showed therapeutic effects, program and generated by in vitro transcription. All RNAs contain with complete tumor regression in up to 30% of mice. FACS analyses 5’triphosphate and 2’fluoro pyrimidine that are recognized by RLRs of tumor-derived cell suspensions showed that BO-112 increased the and resistant to nucleases. Immunogenic cell death in human melan- tumor immune infiltrate and CD8+ T cell frequency. Moreover, the oma, pancreatic cancer and prostate cancer cells and PBMCs was an- receptors PD1 and CD137, which denote activation, were upregu- alyzed by western blot, ELISA and Flow cytometry. In vivo anti-tumor lated on CD8+ T cells. BO-112 treatment enhanced intratumor OVA- effects of the RNAs were induced by intratumoral transfection with specific CD8+ T cells, and depletion and rechallenge experiments RNAs into immunocompromised mice bearing human melanoma. confirmed CD8+ T cell involvement in its immunotherapeutic effects. Results A combination of BO-112 and anti-PD-L1 antibody significantly im- We generated two novel and distinct ssRNA molecules (Immuno- proved the poor response to PD-L1 blockade in B16F10 and B16F10- genic Cell- killing RNA (ICR)2 and ICR4). ICR2 and ICR4 differentially OVA tumor-bearing mice. Responses were observed in treated and in stimulated cell death and PRR signaling pathways and induced differ- untreated distant tumors, which provides a rationale for clinical test- ent patterns of cytokine expression in cancer and innate immune ing of this combination. cells. Interestingly, damage-associated molecular patterns (DAMPs) Conclusions released from ICR2- and ICR4-treated cancer cells had distinct pat- These data identify local BO-112 release as a new immunotherapy terns of stimulation of innate immune receptors and coagulation. agent beneficial in the treatment of refractory and recurrent tumors, Finally, ICR2 and ICR4 inhibited in vivo tumor growth as effectively as especially when combined with checkpoint inhibitors. A phase Ib polyI:C. ICR2 and ICR4 are potential therapeutic agents that differen- clinical trial is being designed to address this question. tially induce cell death, immune stimulation and coagulation when Trial Registration introduced into tumors. http://ClinicalTrials.gov identifier, NCT02828098. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 201 of 244 P412 Background Antitumor immunity in patients with locally advanced soft tissue Hafnium oxide, an electron-dense material, was designed at the sarcoma treated with hafnium oxide nanoparticles and radiation nanoscale to increase the radiation dose deposited from within the therapy cancer cells: “Hot spot” of energy deposit where the nanoparticles 1 2 3 4 Jérôme Galon , Marick Laé , Juliette Thariat , Sébastien Carrere , are when exposed to radiation therapy (RT). Preclinical studies have 5 6 6 7 Zsuzsanna Papai , Martine Delannes , Philippe Rochaix , László Mangel , demonstrated increase of cancer cells killing in vitro and marked anti- 8 9 7 2 Fabienne Hermitte , Zoltán Sapi , Tamas Tornoczky , Vincent Servois , tumor efficacy in vivo with presence of these nanoparticles (HfO -NP) 3 4 4 Isabelle Birtwisle Peyrottes , Raphaël Tetreau , Marie-Christine Château , exposed to RT, when compared to RT alone. HfO -NP is intended for 10 2 2 Sébastien Paris , Hervé Brisse , Sylvie Bonvalot a single intratumor injection and is currently evaluated in clinical tri- 1 2 3 INSERM, Paris, France; Institut Curie, Paris, France; Centre Antoine als including soft tissue sarcoma, head and neck, prostate, liver and Lacassagne, Nice, France; Centre Régional De Lutte Contre Le Cancer rectum cancers. Paul Lamarque, Montpellier, France; Medical Centre, Hungarian Defence Here, we explore the ability of nanosized hafnium oxide exposed to Forces, Budapest, Hungary; Institut Universitaire du Cancer Toulouse - RT to bring substantial immune cells infiltrations in the tumors and 7 8 Oncopole, Toulouse, France; Pecs University, Pecs, Hungary; HalioDX, convert immunologically “cold” tumor into “hot” tumor. Marseille, France; Semmelweis University, Budapest, Hungary; Methods Nanobiotix, Paris, France CT26 (murine colorectal cancer cells) were subcutaneously injected in Correspondence: Sébastien Paris (agnes.pottier@nanobiotix.com) the flank of BALB/c mice. Once the mean tumors volume reached 115 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P412 ±30 mm , tumors were intratumor injected with HfO -NP and irradiated with 2Gyx3 or 4Gyx3, or irradiated only. Tumors were collected 5 days Background after the last RT fraction and analyzed for immune cell infiltrates by im- Soft tissue sarcoma (STS) is a large and heterogeneous group of munohistochemistry (2Gyx3 and 4Gyx3) and cytokines content by flow malignant mesenchymal neoplasms characterized by a strong ten- cytometry (2Gyx3). dency toward local recurrence and metastatic spreading. Consistently, A second study evaluated HfO -NP exposed to RT vs RT alone using the immune microenvironment in sarcomas is highly variable. A new the 4T1 murine breast cancer model. Cells treated or not with class of material with high electron density, hafnium oxide, was de- HfO -NP were exposed to irradiation (40Gy). Irradiated cells (1.10 ) signed at the nanoscale to efficiently absorb ionizing radiation from (or phosphate-buffered saline as control) were inoculated subcuta- within the tumor cells and augment the dose deposited to a tumor. neously into the flank of BALB/c mice (vaccination phase). Seven These nanoparticles (HfO -NP) administered in a single intratumor in- days after, mice were challenged with untreated 4T1 cells (1.10 ) jection and activated by fractionated radiotherapy are evaluated in a (challenge phase). Grown tumors (challenge site) were collected 19 phase II/III trial in patients with locally advanced STS as neoadjuvant days after the challenge phase and analyzed for immune cell infil- treatment. Besides, beyond the broadly cytotoxic effect of radiation trates by immunohistochemistry. therapy (RT), RT may promote the release of tumor neoantigens during Results cancer cell death and stimulate local immunological effects. In mice bearing CT26 tumors, a marked increase of cytokines content Here, we explore the effects of nanosized hafnium oxide exposed to and immune cell infiltrates was observed with HfO -NP + 2Gyx3 RT in terms of tumor immune profile changes in patients with STS when compared to RT alone. The tumor immune cell infiltrates were when compared to RT alone. further enhanced with HfO -NP + 4Gyx3. Methods In mice inoculated with 4T1 cells treated with HfO -NP + 40Gy, a Tumor tissues pre- (biopsy) and post-treatment (resection) are collected marked increase of immune cell infiltrate (CD8+) was observed in tu- from patients with locally advanced STS (NCT02379845), who received mors when compared to tumors in mice inoculated with 4T1 cells either HfO -NP activated by RT or RT alone. Immunohistochemistry and treated with 40Gy and control. Digital Pathology for immune biomarkers and Pan-Immune gene Conclusions expression profiling are analyzed. These in vivo data generated from CT26 and 4T1 tumor models suggest Results that HfO -NP + RT triggers immunogenic conversion of the tumor A significant increase of CD8+ T cells and a marked increase of CD3+ microenvironement when compared to RT alone. HfO -NP treatment and PD-1 T cells and CD103+ immune cell infiltration post- vs pre- may represent a therapeutical approach for broad applications since it treatment are observed for HfO -NP + RT while no differences are seen does not rely on any molecular characteristics of the tumor. for RT alone (more than 10 patients analyzed in each arm). Functional analysis of genes expression up-regulated in HfO -NP + RT post- vs pre- P414 treatment shows an enrichment of cytokine activity (IL7, IFNA, IL11, PVSRIPO is an interferon-resistant, immunotherapeutic oncolytic IFNG), adaptive immunity (RAG1, TAP1, TAP2, TBX21, IFNG, LTK, CD37, virus CD22) and T cell receptor signaling pathway (CD28, CTLA4, CD274, Ross Walton, Michael Brown, Eda Holl, David Boczkowski, Vidya BTLA,TIGIT, CD5,ZAP70)when compared to RT. Chandramohan, Smita Nair, Matthias Gromeier Conclusions Duke University, Durham, NC, USA Promising results are observed in patients who received HfO -NP + Correspondence: Ross Walton (rww7@duke.edu) RT in terms of immune cells infiltration post- vs pre-treatment when Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P414 compared to RT. Moreover, HfO -NP + RT induces a specific adaptive immune pattern. So far, nanosized hafnium oxide exposed to RT Background bring substantial changes to the tumor immune profile in patients Oncolytic viruses are attractive cancer therapies because they select- with STS when compared to RT. As such, it may convert immuno- ively lyse cancer cells, but show attenuation in normal cells. Many logically “cold” tumor into “hot” tumor and be effectively combined oncolytic virus are attenuated through susceptibility to innate anti- with immunotherapeutic agents across oncology. More tissue sam- viral immune responses, like Type I Interferon (IFN) responses. Until ples are under evaluation to reinforce these findings. recently it was believed that cancer cells lack these responses. How- ever, our lab and others have shown cancer cells can have intact IFN P413 responses which inhibit IFN-susceptible viruses, but may activate po- Transforming immunologically “cold” tumor into “hot” tumor with tent anti-cancer immunity. Innate immunity in cancer is thus an area hafnium oxide nanoparticles and radiation therapy of extreme interest. Sébastien Paris, Audrey Darmon, Ping Zhang, Maxime Bergère, Laurent Levy Our lab developed an oncolytic human rhinovirus:poliovirus chimera, Nanobiotix, Paris, France PVSRIPO, which is in clinical trial against glioblastoma multiforme, an Correspondence: Sébastien Paris (agnes.pottier@nanobiotix.com) aggressive form for brain cancer. Using PVSRIPO, and the related pi- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P413 cornavirus encephalomyocarditis virus (EMCV), we investigated MDA5 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 202 of 244 and IFNs in infection of cancer cells. MDA5 is a pattern recognition re- upregulation is likely a reflection of adaptive immune resistance to ceptor, targeted to viral double-stranded RNA produced by picornavi- the increased tumor infiltrating lymphocytes. Systemic therapeutic ruses. We also investigated PVSRIPO’s role in anti-tumor immune targeting of PD-1 or PD-L1 in combination with intratumoral NDV responses after treatment of cancer cells. resulted in rejection of both treated and distant tumors. Anti-tumor Methods efficacy was fully dependent on CD8 cells and on early presence of DM440, a human melanoma cell line, was engineered with stable NK cells. MDA5 knock-down by lentivirus transduction containing MDA5- Conclusions targeting small-hairpin RNA. PVSRIPO or EMCV was added at a multi- These findings provide implications for timing of PD-1/PD-L1 block- plicity of infection (MOI) of 0.1 or 0.01 and samples collected for im- ade in conjunction with OV therapy and highlight that understand- munoblot, ELISA, or viral titer. Cells were also incubated with IFN-α2 ing of adaptive mechanisms of immune resistance to specific OV will for 24 hours, infected, and assayed as above. Human dendritic cells be important for rational design of appropriate combinatorial ap- (DCs) were infected at 1, 10, 50, or 100 MOI and collected as above. proaches with these agents. DCs were also treated with oncolysate from PVSRIPO-infected SUM149 human breast cancer cells, non-infectious lysate, or virus- Other filtered lysate. DCs were analyzed by flow cytometry or used for a cytotoxic HLA-matched T-cell assay using europium-labeled target P416 cells, including cancer cells, tumor antigen transfected DCs, and con- Anti-tumor effects and immunological response following immune trol antigen expressing DCs. stimulating interstitial laser thermotherapy Results Jakob Axelsson, Cristina Pantaleone DM440 cells produce and respond to IFNs in an MDA5-dependent Clinical Laserthermia Systems AB, Lund, Sweden manner after PVSRIPO and EMCV infection. MDA5 activation and IFN Correspondence: Jakob Axelsson (jakob@clinicallaser.se) treatment inhibits EMCV replication significantly more than PVSRIPO Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P416 replication. Human DCs can be productively infected and activated with PVSRIPO without toxicity. Oncolysate activates human DCs in a Background virus dependent manner, and DCs activated in this way promote Immune stimulating interstitial laser thermotherapy (imILT) is a local cytotoxic T-cell activation against tumor-specific antigen. ablation modality designed for local destruction of tumor tissue as Conclusions well as to optimize systemic immunologically mediated anti-cancer PVSRIPO is an IFN-resistant oncolytic virus that provokes MDA5- effects. Safety and feasibility in the clinic has been studied and previ- dependent IFN responses in cancer cells, without significant viral in- ous rodent data show absopal effects. This study expands on the hibition. Further, PVSRIPO infects DCs, activating them without sig- mechanisms behind these immunological effects. nificant cytotoxicity, which can lead to tumor-specific cytotoxic T-cell Methods responses, and may lead to antigen spreading. It is possible this po- An imILT treatment protocol was developed and a syngenic mouse tent DC activation phenotype is due to PVSRIPO’s resistance to IFNs. tumor model was established. Mice were inoculated subcutaneously with tumor cells and tumor growth was monitored. When tumors reached adequate size imILT treatment was performed and subse- P415 quently another tumor was inoculated and tumor progression was Upregulation of PD-L1 in tumor microenvironment is a resistance measured. In another set of experiments, two tumors were inoculated mechanism for oncolytic virus immunotherapy simultaneously (twin tumor model) and one of them was treated at ap- Dmitriy Zamarin, Jacob Ricca, Anton Oseledchyk, Mathieu Gigoux, propriate size. Tumor progression of both tumors were monitored. Im- Taha Merghoub, Jedd Wolchok mune infiltrate and morphology of all tumors were studied. Memorial Sloan Kettering Cancer Center, New York, NY, USA Results Correspondence: Dmitriy Zamarin (zamarind@mskcc.org) Preliminary data show both abscopal effects in the twin tumor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P415 model as indicated as reduced growth of the non-treated tumor of the imILT treated mice as compared to the control mice. Memory Background effects can also be demonstrated in the first set of experiment as Intralesional therapy with oncolytic viruses (OV) leads to activation of seen as reduced growth and take of tumor inoculation in mice multiple immune pathways such as type I IFN. Both OV and OV- which have previously been receiving imILT as compared to control activated type I IFN pathway can exert a variety of pleiotropic effects on mice. both immune and non-immune cells, activating resistance to the im- Conclusions mune system on both local and abscopal level. Identification of such Taken together these data suggest immunologically mediated effects pathways could provide insights into the mechanisms of OV-mediated T are evoked following imILT treatment. This new information will be cell activation as well as generate rationale for combinatorial strategies. useful for deducing the mechanisms of action involved in these Methods effects. Using Newcastle Disease Virus (NDV) as a model OV, we explored the effect of intratumoral OV therapy on the microenvironment of the treated and distant tumors in syngeneic bilateral flank tumor models, P417 and explored combination therapies using intratumoral NDV with Post-marketing safety of checkpoint inhibitors: analysis of the FDA systemic immune checkpoint blockade. adverse event reporting system 1 2 3 Results Rawad Elias , Jennifer Rider , Osama Rahma 1 2 Intratumoral therapy with NDV led to upregulation of a range of ac- Boston University Medical Center, Boston, MA, USA; Boston University tivating and inhibitory immune targets in the treated and distant School of Public Health, Boston, MA, USA; Dana-Farber Cancer Institute, tumors. While NDV therapy shifted the balance from exhausted to Boston, MA, USA effector T cell phenotype in distant tumors, it was not sufficient for Correspondence: Rawad Elias (rawadelias@hotmail.com) complete tumor rejection. We further demonstrate that infection Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P417 with NDV leads to upregulation of PD-L1, an effect that is mediated early through paracrine action of type I IFN. Consistent with these Background findings, intratumoral therapy with NDV or IFNα results in upregula- Information about the toxicity profile of immune checkpoint Inhibi- tion of PD-L1 on both tumor cells and tumor-infiltrating lympho- tors (ICIs) is limited. Available data primarily comes from clinical trials. cytes in the treated tumors. In contrast to NDV, intratumoral IFNα However, subjects enrolled in clinical trials are usually fitter than pa- therapy had no effect on late PD-L1 expression, immune cell infil- tients typically seen in clinical practice and therefore may have a dif- tration, or growth of distant tumors, highlighting that late PD-L1 ferent toxicity profile. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 203 of 244 Methods P418 We reviewed data from the FDA Adverse Event Reporting System Bromodomain and extraterminal region inhibitors slow melanoma (FAERS) summarizing adverse events (AEs) associated with the tumor growth by altering the tumor microenvironment 1 1 1 1 PD-1 inhibitors (nivolumab and pembrolizumab); PD-L1 inhibitor Dan A. Erkes , Claudia Capparelli , Shea A. Heilman , Timothy J. Purwin , 1 2 1 (atezolizumab); and CTLA-4 inhibitor (ipilimumab). We restricted Adam C. Berger , Michael A. Davies , Andrew E. Aplin 1 2 our analysis to reports that included only an ICI as suspect agent. Thomas Jefferson University, philadephia, PA, USA; MD Anderson For each agent, we performed a descriptive analysis of Cancer Center, houston, TX, USA hospitalization (HO) and death (DE) outcomes, as well as AEs of Correspondence: Andrew E. Aplin (dan.erkes@jefferson.edu) special interest (AESI). We compared the distribution of each out- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P418 come within age groups (<65 years; 67-75; >75) using Mantel- Haenszel chi -quare test for trend. Background Results Melanoma is the deadliest form of skin cancer, yielding a 2-16% sur- Our analysis included a total of 21,588 safety reports. 415 for ate- vival rate for metastatic patients. Despite the onset of new successful zolizumab, 10,026 for nivolumab, 4,808 for pembrolizumab, and therapies, i.e. targeted inhibitors and immunotherapy, there is a large 6,339 for ipilimumab (Table 1). There was a statistically significant need for drugs that alter the entire tumor microenvironment. To this trend of more hospitalization with increasing age for all drugs. end, we aim to study drugs that have widespread tumor microenviron- Prevalence of any of the AESI was higher as age increased for all ment effects, such as epigenetic modifiers, i.e. bromodomain and extra drugs (p<0.0001) except for atezolizumab (p 0.12). However, no terminal domain inhibitors (BETi), which slow tumor growth in a variety statistically significant trend by age was found for any individual of cancers and effect inflammatory responses. Thus, BETis potentially AESI (data not shown). Proportion of older patients who experi- represent a novel treatment for metastatic melanoma, as they can tar- enced death was higher for pembrolizumab (p<0.001) and ipili- get tumor cell growth and anti-tumor immune mechanisms. mumab (p 0.002). Methods V600E Conclusions Several mouse and human BRAF melanoma cell lines, im- Our analysis suggests that older patients receiving pembrolizumab, mune competent mouse models, and a patient-dreived xenograft Nivolumab or ipilimumab are more likely to develop immune related model were used to uncover the effects of BETi treatment on AEs, and to be hospitalized. This resulted in higher rate of deaths only melanoma. in pembrolizumab and ipilimumab. The smaller sample size of the pa- Results tients receiving atezolizumab may have contributed to the lack of stat- Here we show that the BETi PLX51107 was able to differentially impact V600E istical significance. Older patients treated with ICIs should be BRAF melanoma tumor growth in a number of mouse and human monitored carefully for treatment-related AEs. Toxicity of ICIs should be melanoma cell lines in vitro and in vivo. PLX51107 induced cell cycle further evaluated prospectively in the setting of trials in older adults arrest and cell death of melanoma cell lines by increasing Bim and that would take in consideration impact of physiologic and immune decreasing Cyclin B1 in mouse and human lines. Using immune com- aging. petent mouse models, BETi treatment altered the immunogenicity of the microenvironment of responsive melanoma tumors by lowering the expression of anti-inflammatory PD-L1 and FasL, while increasing pro-inflammatory MHC-I. This was also seen in mouse and human mel- anoma cell lines and cancer-associated fibroblasts. PLX51107 treatment Table 1 (abstract P417). See text for description differentially altered tumor infiltrating lymphocyte (TIL) populations in mouse melanoma models, increasing activated, proliferating, and functional intratumoral CD8+ T cells and leading to a CD8+ T cell mediated tumor growth delay in highly responsive tumors. How- ever, in moderately or poorly responsive tumors, these CD8+ T cell populations were unchanged or decreased after BETi treatment. As BETi treatment was a successful primary therapy, it was prudent to determine its efficacy as a second line therapy. In this vein, an im- mune compotent mouse model and patient-derived xenograft model were used to demonstrate BETi efficacy on anti-PD-1 resist- ant tumors. BETi treatment was able to delay the growth of anti- PD-1 resistant tumors, by increasing intratumoral CD8+ T cells and pushing tumors to a more responsive IPRES signature. Conclusions As hypothesized, BETi treatment had wide-ranging effects on melan- oma tumors by altering the tumor cells themselves, the microenviron- ment, and anti-tumor immunity to induce robust tumor growth delay. From these findings, BETis represent a potential primary or secondary treatment for metastatic melanoma patients. P419 Tobacco use, awareness and cessation among Malayali tribes, Yelagiri Hills, Tamil nadu, India Delfin lovelina Francis (delfin_lovelina@yahoo.co.in) Dr MGR Medical University, Chennai, India Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P419 Background Health is a state of complete wellbeing free from any discomfort and pain. Despite remarkable world-wide progress in the field of diagnostic, curative and preventive medicine, still there are large populations of people living in isolation in natural and unpolluted surroundings far away from civilisation, maintaining their traditional Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 204 of 244 values, customs, beliefs and myths. India has the second largest tribal Targeting the same AUC and C in mice into a human PK/PD predic- max population of the world next to the African countries. About half of the tion model, the human efficacious dose was projected to be 1 mg/kg. world’s autochthonous people live in India, thus making India home to The proposed FIH starting dose was then selected to be 0.25 mg/kg, 4- many tribes which have an interesting and varied history of origins, fold below the projected efficacious dose. First, using preclinical GLP customs and social practices. The present study was conducted to as- safety assessments, the proposed starting dose was 68- to 80-fold lower sess the tobacco use, awarness and its effect on health among Malayali than 1/6th of the highest non-severely toxic dose (120 mg/kg/week) in tribes, Yelagiri Hills, Tamil nadu, India. monkeys. Second, the C (6.25 μg/mL) at the starting dose was 5-fold max Methods below the highest no-effect concentration (33 μg/mL) evaluated in the The inhabitants of the 14 villages of the Yelagiri hills, who have com- in vitro cytokine release assay. Third, there was a >10-fold exposure mar- pleted 18years and residing for more than 15years present on the gin between the minimal pharmacologic effect (antigen-specific T-cell or day of examination and who were willing to participate in the study antibody responses) observed in monkeys at doses of 2-4 mg/kg and were included. the FIH starting dose selected. Data was collected from a cross-sectional survey, using a Survey Pro- The FIH trial was successfully initiated at 20 mg (0.25 mg/kg with a forma, clinical examinationand a pre-tested questionnaire which in- BW of 80 kg). BMS-986178 was well tolerated without any adverse cluded Demographic data, tobacco habits. An intra-oral examination event at this dose. OX40 receptor occupancy observed in peripheral was carried out by a single examiner to assess the Oral Health Status blood at the starting dose maintained ~80% in line with the predic- using WHO Oral Health Surveys – Basic Methods Proforma (1997).SPSS tion made from in vitro cellular binding affinity and clinical informa- version15 was used for statistical analysis. tion observed from other anti-OX40 antibodies. Results Conclusions Results showed that among 660 study population, 381(57.7%) This PK/PD-based approach was successfully applied to select the FIH had no formal education. Among the study population 75%) had starting dose of BMS-986178. This strategy appropriately balanced the habit of alcohol consumption. Of those who had the habit of the selection of a safe dose, while minimizing the number of cancer smoking, 26% smoked beedi, 10.9% smoked cigarette, 65% patients receiving sub-therapeutic doses. chewed raw tobacco, 18% chewed Hans and 28% had a combin- ation of smoking and smokeless tobacco usage. The reason for P421 practicing these habits were as a measure to combat the cold, Co-administration of dexamethasone with checkpoint blockade relieving stress and body pain after work, and the lack of aware- therapy increases survival in brain tumor model ness of the hazards of the materials used. Prevalence of oral mu- Marsha-Kay Hutchinson, Amber Giles, Heather Sonnemann, Jinkyu Jung, cosal lesions in the study population was due to tobacco usage Caitlin Reid, Deric Park, Mark Gilbert and alcohol consumption and lack of awareness regarding the National Institutes of Health, Bethesda, MD, USA deleterious effects of the products used. Correspondence: Amber Giles (hutchinsonmn@mail.nih.gov); Deric Conclusions Park; Mark Gilbert From the results of this study it may be concluded that the Malayali Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P421 tribes were characterized by a lack of awareness about oral health, deep rooted dental beliefs, high prevalence of tobacco use and lim- Background ited access to health services. Theuse of corticosteroids fortherapeutic benefitmustbeweighed against risks of adverse consequences associated with these drugs. Brain P420 tumor patients are routinely prescribed dexamethasone to reduce Selection of first-in-human starting dose of anti-OX40 agonist tumor-associated edema. Checkpoint blockade, a type of immune ther- monoclonal antibody BMS-986178 using a pharmacokinetic/ apy, is currently being investigated as a potential treatment for brain tu- pharmacodynamic-based approach mors. Although glucocorticoid signaling has been shown to attenuate 1 1 2 1 Christine Huang , Yan Feng , Bryan Barnhart , Michael Quigley , theimmuneresponse, theeffect of glucocorticoids on the anti-tumor 1 1 1 1 John Huber , Akintunde Bello , Punit Marathe , Praveen Aanur , immune response during checkpoint blockade remains unclear. Here, 1 1 Timothy Reilly , Zheng Yang we propose that dexamethasone’s ability to upregulate T-cell checkpoint 1 2 Bristol-Myers Squibb, Princeton, NJ, USA; Bristol-Myers Squibb, molecules such as CTLA-4 might be an immunosuppressive mechanism, Redwood City, CA, USA but can be countered in unison with checkpoint blockade therapy. Correspondence: Christine Huang (christine.huang@bms.com) Methods Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P420 Healthy donor T cells were tested for response to dexamethasone. T cell proliferation, cell cycle analysis, apoptosis, glucose uptake, and Background protein expression were assessed with flow cytometry and Western Following the clinical success of checkpoint blockade, the field of can- blots. Transcriptional changes were assessed with qPCR. CTLA-4 was cer immunotherapy rapidly expanded. Co-stimulatory molecules from blocked using monoclonal antibodies in vitro on human PBMCs and the TNF receptor superfamily, including OX40, may be a promising ap- in vivo with the GL261 syngeneic glioblastoma model. proach to enhance the benefits of immunotherapy. BMS-986178 is a Results fully human agonist antibody of the immunoglobulin G1 isotype that Checkpoint molecule CTLA-4 was increased by dexamethasone treat- binds with high affinity to the human OX40 receptor, currently being ment upon stimulation. Unexpectedly, dexamethasone did not elicit a developed for the treatment of advanced solid tumors. direct lymphotoxic effect on T cells but significantly reduced T cell Methods entry into cell cycle. Dexamethasone also abated CD28 signaling as BMS-986178 does not bind to mouse OX40, necessitating antibody shown by reduced AKT phosphorylation and reduced glucose uptake. surrogates to assess in vivo activity of OX40 agonism. Two hamster Blockade of CTLA-4 resulted in a substantial reversal of these effects. In anti-mouse OX40 agonist mAbs (reformatted as mouse antibodies: addition, we discovered that antigen-experienced memory T cells were mIgG1 and mIgG2a) were studied in the mouse MC38 colon adeno- invulnerable to dexamethasone treatment. In vivo, dexamethasone and carcinoma model. A pharmacokinetic/pharmacodynamic (PK/PD)- CTLA-4 blockade provided a survival benefit to tumor bearing mice. based approach was employed to integrate the preclinical data and Conclusions guide the first-in-human (FIH) starting dose selection. These results suggest that mature T cells, which predominate in the Results tumor microenvironment, are resilient to the negative effects of corti- In vitro, binding EC of mIgG1 and mIgG2a mAbs in activated mouse costeroids. Thus, corticosteroids are unlikely to destroy anti-tumor T T-cells was similar to in vitro binding EC of BMS-986178 in activated cells. For patients who have not responded to immune therapy, human T-cells. PK/PD analysis of the anti-tumor efficacy data showed CTLA-4 blockade may provide protection from corticosteroids to the maximum efficacy at an AUC of 100 μg*h/mL with a C of 25 μg/mL. naïve T cell pool. max Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 205 of 244 P422 P423 Overall survival and treatment patterns among real-world patients Overall survival and treatment patterns among real-world patients with metastatic non-small cell lung cancer not previously treated with stage IIIB non-small cell lung cancer treated with platinum- with systemic therapy for advanced cancer based chemotherapy 1 1 2 3 1 1 2 3 Jason C. Simeone , Beth Nordstrom , Ketan Patel , Alyssa B. Klein Jason C. Simeone , Beth Nordstrom , Ketan Patel , Alyssa B. Klein 1 2 1 2 Evidera, Waltham, MA, USA; AstraZeneca, Milton, United Kingdom; Evidera, Waltham, MA, USA; AstraZeneca, Milton, United Kingdom; 3 3 AstraZeneca, Gaithersburg, MD, USA AstraZeneca, Gaithersburg, MD, USA Correspondence: Alyssa B. Klein (caroline.moreau@evidera.com) Correspondence: Alyssa B. Klein (caroline.moreau@evidera.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P422 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P423 Background Background The majority of patients with non-small cell lung cancer (NSCLC) are di- Platinum-based chemotherapy has been the standard of care in the agnosed at an advanced stage of disease, typically with a poor progno- management of patients with advanced non-small cell lung cancer sis. While treatment of metastatic NSCLC is focused on extending (NSCLC), but evidence of effectiveness and patterns of treatment in a survival, real-world data on the effectiveness of existing therapies and real-world setting is needed. patterns of treatment is limited. Methods Methods Electronic medical record data of patients from the Flatiron On- Patients with metastatic NSCLC, that are EGFR and ALK WT and cology database with Stage IIIB NSCLC treated with at least two no prior systemic therapy for advanced disease were identified cycles of platinum-based chemotherapy during 2013–2016 were from the Flatiron Oncology electronic medical record database identified; patients with prior evidence of metastatic disease were from 2013–2016. Treatment patterns, including regimens adminis- excluded. Treatment patterns were examined, including the dur- tered and time to treatment, were summarized. The median over- ation and number of lines received. The median overall survival all survival (OS) from the initial diagnosis of metastatic NSCLC (OS) from the start date of the third cycle of chemotherapy was was calculated from Kaplan-Meier curves, with associated 95% calculated from Kaplan-Meier curves, with 95% confidence inter- confidence intervals (CI). vals (CI). Results Results A total of 10,123 patients were eligible for analysis. The mean age of A total of 1,226 patients met all criteria for study inclusion. The patients was 68.0 ± 10.0 years, and 54.0% were male. The most com- mean age of patients was 68.5 ± 9.0 years, and 51.9% were male. monly observed regimens after the diagnosis of metastatic disease The most common regimens observed at any time during follow- were nivolumab (17.4%), carboplatin + pemetrexed (16.0%), and car- up were carboplatin + paclitaxel (43.9%), nivolumab (23.7%), and boplatin + paclitaxel (15.0%), while 25.4% did not receive treatment. carboplatin + pemetrexed (21.0%); patients received a median of The median time from diagnosis of metastatic disease to start of 2 lines of therapy (range: 1–7). The median OS was 19.4 months first-line therapy was 36 days (range: 1–1,770) and the median num- (95% CI: 17.4–21.6) from the start of the third cycle of chemo- ber of therapy lines was 1 (range: 1–8). The median OS was 11.2 therapy (Fig. 1). months (95% CI: 10.9–11.6) from the initial diagnosis of metastatic Conclusions disease (Fig. 1). Overall survival among Stage IIIB NSCLC patients who were Conclusions treated with platinum-based chemotherapy in a real-world setting In this real-world study, overall survival of metastatic patients was was less than 20 months. Previous literature indicates survival of less than 12 months, and one quarter of patients did not receive 7-13 months among advanced NSCLC patients treated with treatment after diagnosis. This evidence indicates a potential unmet chemotherapy. However, the present study excludes patients with need for additional treatment options, as more patient information metastatic disease. The difference in OS may also be related to such as biomarkers become available to physicians at initial the use of newer, more effective treatments that were not avail- diagnosis. able in earlier studies. nt–> Fig. 1 (abstract P422). Overall Survival for Patients with Stage IV Fig. 1 (abstract P423). Overall Survival of Patients with Stage IIIB NSCLC, From Initial Diagnosis of Advanced or Metastatic NSCLC NSCLC, From Start of Third Cycle of Chemotherapy Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 206 of 244 P424 UV radiation suggest that cSCC is appropriate for the clinical study Efficacy of pembrolizumab (MK-3475) in patients with of checkpoint inhibitors. adrenocortical carcinoma Methods Mohammed Amir Habra, Matthew Campbell, Camilo Jimenez, This is a phase II study of pembrolizumab in patients with rare tu- Daniel Karp, David Hong, Vivek Subbiah, Shubham Pant, Jeane Painter, mors including a pre-specified cutaneous squamous cell carcinoma Saria Khan, Chantale Bernatchez, Bettzy Stephen, Anas Alshawa, cohort (http://ClinicalTrials.gov identifier, NCT02721732). Patients re- Coya Tapia, Tito Mendoza, Rivka Colen, Kenneth Hess, ceived pembrolizumab 200 mg intravenously once every three Funda Meric-Bernstam, Aung Naing weeks. The response was assessed every nine weeks using RECIST1.1. MD Anderson Cancer Center, Houston, TX, USA The primary end point was progression-free survival at 27 weeks Correspondence: Aung Naing (anaing@mdanderson.org) (PFS27wk). Mandatory biopsies are taken at baseline, on cycle 1 day Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P424 15 -21, and at the time of progression. Results Background A total of 11 patients were enrolled and treated. At the date of analysis, Adrenocortical carcinoma (ACC) is an orphan endocrine malignancy patients received a median of 5.5 treatment cycles (range 1-15). Three with poor prognosis and limited response to chemotherapy. ACC can out of eleven patients have been treated beyond 27 weeks. Four patients express immune markers which could make it susceptible to im- had a partial response and the tumor reduction from the baseline have munotherapy. So far, there is no published clinical trial about using reached 33%, 55%, 63%, and 70%. The fifth patient had stable disease immunotherapy in ACC. while the other five patients had a progressive disease within 27 weeks Methods of starting the study. The last patient has not had his first restaging scans This is a phase II study of pembrolizumab in patients with rare tu- yet. The safety profile of pembrolizumab was very favorable. mors including a pre-specified ACC cohort (http://ClinicalTrials.- Conclusions gov identifier, NCT02721732). Patients received pembrolizumab Single agent pembrolizumab may be effective in a patient with ad- 200 mg intravenously once every 3 weeks. Response was assessed vanced cutaneous squamous cell carcinoma. Expanding the study is every 9 weeks using RECIST1.1. The primary end point was needed to verify the initial observation. Translational data will be progression-free survival at 27 weeks (PFS27wk). Mandatory biop- presented at this meeting. sies are taken at baseline, on cycle 1 day 15 -21, and at the time Trial Registration of progression. http://ClinicalTrials.gov identifier, NCT02721732. Results A total of 11 patients were enrolled and treated. At the time of ana- P426 lysis, patients received a median of 6 treatment cycles (range 2-17). Antibody-drug conjugate induced cytotoxicity of tumor cell lines A PFS of greater than 27 weeks was seen in 3/11 patients (27%). The by targeting the SAS1B N-terminus first patient reached 37% tumor reduction (partial response) while 1 2 1 1 Arabinda Mandal , Mriganka Mandal , Walter Olson , Jagathpala Shetty , the second patient had a response of 41% tumor reduction (partial 3 1 1 4 4 Kiley Knapp , Eusebio Pires , Todd Bauer , Timothy Bullock , John Herr , response) and the third patient had stable disease. The remaining 8 Craig Slingluff patients had evidence of disease progression within 27 weeks of 1 2 University of Virginia, Charlottesville, VA, USA; Albemarle High School, starting the study (median time 18 weeks, range 6-27 weeks). Pro- 3 4 Charlottesville, VA, USA; University, Charlottesville, VA, USA; University gression was seen in most patients with hormonally active ACC (6/7) of Virginia.edu, Charlottesville, VA, USA and only in 2/4 patients without evidence of hormonal excess at the Correspondence: Craig Slingluff (wco3j@virginia.edu) time of their initial diagnosis. The safety profile of pembrolizumab Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P426 was very favorable and none of these patients had severe grade 3 or grade 4 adverse effects. Background Conclusions SAS1B (ASTL, Ovastacin) is a zinc metalloproteinase found normally Single agent pembrolizumab may be an effective option in subset of in growing oocytes that has been detected in uterine cancer cells, ACCs without hormonal overproduction while it is associated with and its expression is restricted to oocytes, among adult tissues. Thus, high failure rate among patients with hormonally active ACCs. it is classified as a cancer-oocyte neoantigen [1]. Treatment of uterine Expanding the study to hormonally silent ACCs is needed to verify tumor cells with anti-SAS1B polyclonal antibodies and complement the initial observation from limited number of patients treated in this arrested growth, and a saporin-immunotoxin directed to SAS1B study. Translational data will be presented at this meeting. caused cell death. The goal of the present study was to develop im- Trial Registration munotherapeutic monoclonal antibodies against the human SAS1B http://ClinicalTrials.gov identifier, NCT02721732. target, to reduce risks of off-target effects during therapy. Methods P425 Monoclonal antibodies (mAbs) were generated to recombinant (r) Efficacy of pembrolizumab in patients with cutaneous aquamous human (h) SAS1B immunogen without its signal peptide. cell carcinoma Results Renata Ferrarotto, Bonnie Glisson, George Blumenschein, David Hong, SAS1B transcript in human uterine and lung cancer cell lines was Sarina Piha-Paul, Dipti Jain, Anas Alshawa, Jeane Painter, Kenneth Hess, confirmedbyPCR andsequencing. SB2 and SB5 mAbs demon- Rivka Colen, Chantale Bernatchez, Charles Lu, Bettzy Stephen, strated Western immunoreactivity to rhSAS1B expressed in E. coli Coya Tapia, Tito Mendoza, Funda Meric-Bernstam, Aung Naing or HEK293T cells, and recognition of SAS1B was confirmed by The University of Texas MD Anderson Cancer Center, Houston, TX, USA mass spectrometry on immunoprecipitated antigen. SAS1B dele- Correspondence: Aung Naing (anaing@mdanderson.org) tion constructs mapped the epitopes recognized by the SB2 and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P425 SB5 mAbs to the N-terminal domain between residues 32-40. Sur- face expression of SAS1B in live uterine, lung and pancreatic Background tumor cell lines was detected by both mAbs. The expression of nd Cutaneous squamous cell carcinoma (cSCC) is the 2 most com- SAS1B N-terminus on the surface of live tumor cells and its cyto- mon malignancy in the US. While the majority of patients are cured plasmic expression from uterus (SNU539, Fig. 1B), ovary, breast, with surgery, approximately 6,400 die annually as a consequence of melanoma (Malme3M,Fig.1C) lung (H226,Fig. 1D),and were con- disease. There is limited data on the role of chemotherapy in the firmed by flow cytometry and SAS1B peptide blocking. SAS1B ex- treatment of metastatic cSCC. The high-mutational burden, the pression was not evident on the surface of normal peripheral presence of tumor-infiltrating lymphocytes, immunosuppression as blood mononuclear cells. Monoclonal antibodies SB2 and SB5, a risk factor, and evidence of direct immunosuppressive effects of complexed with a Fab-Duocarmycin DM, that incorporated a Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 207 of 244 cathepsin-cleavable linker arm, were cytotoxic to SAS1B positive P428 uterine (SNU539, Fig. 1A), pancreatic and lung tumor cells at 10 - PD-1 blockade activates CD4 T cells and the innate immune 100 picomolar concentrations. response for glioblastoma eradication Conclusions Sarah R. Klein, Maria C. Speranza, Prafulla C. Gokhale, Margaret K. Wilkens, The SB2 and SB5 mAbs offer promise as candidate immunotherapeutic Kristen L. Jones, Apoorvi Chaudhri, Paul T. Kirschmeier, David A. Reardon, entities for targeting SAS1B positive cancer cells with defined epitopes Gordon J. Freeman located in the SAS1B N-terminal domain suitable for immunotherapeu- Dana-Farber Cancer Institute, Boston, MA, USA tic targeting. Correspondence: Gordon J. Freeman (maria_speranza@dfci.harvard.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P428 References 1. Pires ES, et al. Membrane Associated Cancer-Oocyte Neoantigen SAS1B/ Background Ovastacin is a Candidate Immunotherapeutic Target for Uterine Tumors. Cancer immunotherapy has come of age as a result of substantive Oncotarget. 2015; 6:30194-30211. advances in basic and translational tumor immunology [1]. In fact, multiple advanced clinical trials with human anti-PD-1 antibodies (i.e. nivolumab and pembrolizumab) have convincingly established the ability of anti-PD-1 antibodies to trigger clinically significant tumor destruction in multiple tumor types, reinforced by dramatic anti- cancer responses in animal models and encouraging durability in early clinical trials [2]. Methods Through flow cytometry and in in vivo intracranial injection of mouse gli- oma tumor cells we recently demonstrated that PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice, despite the absence of accumulat- ing CD8+ cytotoxic T cells in the tumor or draining lymph nodes [3]. Results In this investigation, we provide evidence for the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti- glioma immunity in this model. In response to anti-PD-1 monotherapy, intratumoral CD4+ T cells, but not CD8+ T cells, expressed significantly el- evated levels of IFN-γ and TNF-α pro-inflammatory cytokines and the cytotoxic enzyme, granzyme B. Tbet, GATA3, and EOMES, transcription factors required for T cell proliferation, activation, and effector function, were also up-regulated in CD4+, but not CD8+ T cells in the brains of Fig. 1 (abstract P426). Profile of SAS1B expression on tumor cell mice treated with PD-1 mAbs when compared to controls. We lines by ADC and flow cytometry demonstrated that depletion of CD4+ or CD8+ T cells, but not NK, was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. CD4+ T cell activation was ac- companied by the classical activation and M1 polarization of resi- P427 dent microglia and tumor-infiltrating macrophages. Moreover, Democratizing analysis of cancer data using the cancer genomics CSF-1 inhibition with PLX3397 to deplete microglia and macro- cloud phages did not affect tumor growth, but did lead to a significant Anurag Sethi (anurag.sethi@sbgenomics.com) survival advantage when combined with anti-PD-1 mAb. Seven Bridges Genomics, Inc, Boston, MA, USA Conclusions Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P427 Together, these studies demonstrate for the first time a role for CD4 + T cells and the innate immune response in the eradication of glio- Background blastoma by PD-1 blockade. The advent of next generation sequencing has resulted in the generation of petabytes of multi-dimensional information, but the References access and analyses of this data remains challenging. This difficulty 1. Mittal D, Gubin MM, Schreiber RD et al. New insights into cancer is exemplified when we consider data generated by the efforts of immunoediting and its three component phases–elimination, The Cancer Genomics Atlas (TCGA) network. Historically, the only equilibrium and escape. Current opinion in immunology. 2014;27:16-25. means for gaining insight from the TCGA data was to download 2. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibi- the complete TCGA repository, which can require several weeks tory receptor by a novel B7 family member leads to negative regulation of with a highly optimized network connection, followed by analyses lymphocyte activation. J Exp Med. 2000;192:1027-1034. in a very expensive high performance computational cluster. 3. Checkpoint Blockade in an Orthotopic, Immunocompetent Model. Cancer Methods Immunol Res. 2015;4(2): 124–35. The Cancer Genomics Cloud Pilots project seeks to democratize can- cer data analysis by co-localizing data with the computational re- sources. Funded by the National Cancer Institute, the Cancer Personalized Vaccines and Technologies/ Genomics Cloud Pilot (www.cancergenomicscloud.org) project en- ables researchers to leverage the power of cloud computing to gain Personalized Medicine actionable insights on cancer biology and human genetics from massive public datasets including TCGA. P429 Results Nanoparticle based enrichment and expansion of self and neo- The CGC removes the need to download data, enables easy query- epitope specific CD8 T cells in murine melanoma ing, and much more. Catherine A Bessell, Joan Glick Bieler, John-William Sidhom, Jonathan P Conclusions Schneck We will highlight our approach to optimized computation, data min- Johns Hopkins University, Baltimore, MD, USA ing, and visualization solutions that address the challenges associ- Correspondence: Jonathan P Schneck (cbessel1@jhmi.edu) ated with analyses of petabyte-scale datasets and beyond. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P429 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 208 of 244 Background Methods T cell responses against neo-antigens has created a new avenue to tar- Multiple patients with solid tumors were analyzed. CD14 monocytes get and kill patients’ tumors while decreasing autoimmune side effects. and T cell subsets were isolated from patient peripheral blood mono- Neo-antigen-specific CD8+ T cells have the potential benefit to be a nuclear cells. Monocytes were differentiated into dendritic cells (MDDC), source of T cells that are not limited by peripheral tolerance or deleted and T cells were non-specifically expanded. Whole exome sequencing from the T cell repertoire by central tolerance. To understand the differ- was performed on tumor biopsies and matched normal genomic DNA, ences between T cell responses against neo- and self-antigens, these and tumor specific changes (single nucleotide variants and insertion/ two types of T cell populations must be compared side-by-side for anti- deletions) were identified and cloned into E. coli expression vectors tumor responses and phenotypic differences. with and without co-expressed listeriolysin O to enable presentation Methods via MHC class I or class II, respectively. For each patient, their unique Following another group that identified mutated proteins in B16 clones were co-cultured with autologous MDDCs in an ordered array, + + murine melanoma [1], our current work has identified the short pep- then their CD4 or CD8 T cells were added and incubated overnight. T tide epitopes that can stimulate neo-antigen T cell responses. Artifi- cell activation was determined by measurement of TNF-α and IFN-γ in cial antigen presenting cells (aAPCs) were used to access neo- and the supernatants by meso-scale discovery. Cytokine concentrations self-antigen tumor specific responses in the endogenous T cell popu- were normalized against responses to negative control bacteria and lation of naïve and B16 tumor bearing animals. In a B16-SIY melan- neoantigens were defined as clones that elicited responses >3 median oma model, neo-epitope Kb-SIY serves as a model to collect T cells absolute deviations of the median of negative controls. for self and neo-epitopes and analyze the T cell receptor (TCR) reper- Results TM + + toire of the separate populations. ATLAS identified CD4 and CD8 T cells responses to up to Results 15% of mutant polypeptide sequences, across a broad cohort of After exposure to tumor, neo-epitope T cell responses showed larger patients with different tumor types, including tumors with either fold expansion, while T cell expansions for self-antigen Kb-TRP2 ex- low or high mutational burden. “Inhibitory” neoantigens, which panded similar in the naïve animals. TCR repertoire analysis of neo- and shut off all cytokine production, were also identified in each sub- self-antigen reactive T cells showed a highly conserved V beta gene ject. Many neoantigens were not predicted by algorithms. When usage in the naive Kb-SIY TCR repertoire, while the naïve Kb-TRP2 TCR exploring neoantigens by tumor type, no patterns in overall mu- repertoire had a more broad V beta selection. The tumor did not alter tational burden, RNA expression level, or DNA mutant allele fre- the V beta usage for Kb-SIY repertoire, however the Kb-TRP2 repertoire quency have yet been identified. changed to different V beta genes after tumor exposure. Conclusions TM Conclusions The ATLAS platform empirically defines which potential neoan- The increased expansion ability of neo-antigens after tumor exposure tigens created by somatic mutations elicit immune responses in demonstrates the potential anti-tumor ability of neo-epitope responses individual patients independently of a patient’sHLA typeandT compared to self-epitope T cell responses. TCR repertoire analysis of cell receptor repertoire. The identification of activating and inhibi- + + neo- and self-antigen reactive T cells shows that the tumor shifts the tory neoantigens for CD8 T cells, as well as for CD4 T cells for TCR clonal diversity of self-antigen specific populations to but leaves which the algorithmic approaches do not perform nearly as well, the neo-epitope repertoire intact and similar to the naïve repertoire. provides the opportunity to identify better targets to include in a From this work, we show proof of concept of a streamlined method to vaccine formulation. Genocea is currently developing personalized TM screen candidate neo-epitopes, identify rare endogenous T cell popula- cancer vaccines with neoantigens prioritized by ATLAS which tions, and assess the benefit of T cell directed immunotherapies on dif- we hypothesize will provide better therapeutic impact than algo- ferent T cell populations. rithmic approaches. References P431 1. Castle JC, Kreiter S, Diekmann J, Lower M, van de Roemer N, de Graaf J, Identification of immunogenic breast cancer neoantigens exposed Selmi A, Diken M, Boegel S, Paret C, Koslowski M, Kuhn AN, Britten CM, by radiation therapy Huber C, Tureci O, Sahin U. Exploiting the Mutanome for Tumor Claire Lhuillier, Nils Rudqvist, Takahiro Yamazaki, Tuo Zhang, Vaccination. Cancer Res. 2012; 72:1081-1091. Lorenzo Galluzzi, Sandra Demaria Weill Cornell Medical College, New-York, NY, USA P430 Correspondence: Sandra Demaria (cfl2002@med.cornell.edu) TM Neoantigen identification using ATLAS across multiple tumor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P431 types highlights limitations of prediction algorithms Jason Dobson, Huilei Xu, Kyle Ferber, Johanna Kaufmann, Judy Jacques, Background Christine McCoy, Michael O'Keeffe, Yana Ostrovsky, Crystal Cabral, Recent studies have highlighted the key role of mutation-generated Pamela Carroll, Theresa Zhang, Jessica Flechtner, Wendy Broom neoantigens in tumor response to immunotherapy [1]. We have previ- Genocea Biosciences, Cambridge, MA, USA ously shown in the 4T1 mouse model (syngeneic with BALB/c mice) of Correspondence: Wendy Broom (jason.dobson@genocea.com) immune-checkpoint blockade-resistant metastatic breast cancer that local Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P430 radiation therapy (RT) combined with CTLA-4 blockade induces the CD8 T cell-mediated regression of irradiated tumors and limits metastatic lung Background colonization [2]. Preliminary analysis of the T-cell receptor repertoire indi- Neoantigens arise from tumor-specific, somatic mutations and have the cated that unique clonotypes are expanded in treated tumors, suggest- potential to be recognized by T cells that are associated with anti-tumor ing that tumor rejection involves T cells reactive to a set of tumor immune responses. Since they are non-self, they are hypothesized to pro- antigens that are made available to the immune system by RT. Therefore, vide an attractive therapeutic modality since T cells that can respond to we hypothesize that RT, by changing the transcriptional profile of cancer TM those sequences have not undergone thymic selection. The ATLAS cells, may expose antigenic mutations not transcribed at sufficient levels + + platform enables identification of biologically relevant CD4 and CD8 T in untreated tumors and hence promote priming of T cells to these cell neoantigens in any subject in an unbiased manner. unique mutated antigens. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 209 of 244 Methods coverage, maximising individual protein coverage, and focusing on We performed whole-exome sequencing and RNA sequencing (RNA-seq) dominant HLA restrictions. By integrating novel cell biology, mass of 4T1 cells irradiated (8GyX3) or not (0Gy) in vitro to identify tumor- spectrometry, and bioinformatic technologies in over 1000 individual specific mutations. We used several algorithms to predict MHC-I (H2 )- experiments we have dramatically increased the depth of the HLA binding epitopes from these mutated genes, and we selected those with ligandome captured and achieved near total coverage of the a predicted affinity <500 nM. Based on the RNA-seq data, we prioritized protein-coding genome, with 90% of the proteome captured for variants that were upregulated by radiation. These candidate peptides HLA-A*02:01. d d were synthesized and tested in vitro for binding to H2-L or H2-K in a Our comprehensive genome coverage has enabled us to probe both MHC stabilization assay using RMA-S cells. Finally, the mutated peptides directly and indirectly for the presence of neoantigens. Known som- with the highest affinity were used to vaccinate BALB/c mice, followed atic mutations within immortalised lines were used to generate be- by challenge with 4T1 cells to test for the induction of protective anti- spoke reference databases that has led to direct identification of tumor immunity. many hundreds of neoantigens. Results Results Out of 309 total mutations initially identified in the 4T1 cancer Proteins that were found to contain neoantigens appeared to follow cells, we selected and tested in vitro 17 candidate neo-epitopes. the processing and presentation behaviours of their unmutated Two of them bound H2-L with a high affinity and were used equivalent. We have therefore found our HLA peptide dataset is able alone or in combination to vaccinate mice. Our preliminary data to offer significant value in predicting the likelihood of a somatic mu- indicate that the combination of these two neoepitopes is im- tation creating a neoantigen. munogenic and delays the tumor growth after challenge with To test this, somatic mutations reported in 980 cell lines were 4T1 cells. probed against the database of HLA peptides. On average we Conclusions find one peptide containing the mutated amino acid for every 5 In conclusion, these data provide initial proof-of-principle evidence somatic mutations reported. By incorporating the HLA back- that RT can expose existing neoantigens to the immune system. ground of the cell carrying the mutation we narrow this predic- tion to one high affinity HLA peptide for every 14 somatic References mutations reported. Comparing the peptides predicted in this 1. Schumacher TN, and Schreiber RD. Neoantigens in cancer immunotherapy. analysis with those directly identified by mass spectrometry, we Science. 2015; 348: 69-74. are able to show that we can prioritise mutation data by accur- 2. Demaria S, Kawashima N, Yang AM, Devitt ML, Babb JS, Allison JP, and ately predicting the presence and relative abundance of Formenti SC Immune-mediated inhibition of metastases after treatment neoantigens. with local radiation and CTLA-4 blockade in a mouse model of breast Conclusions cancer. Clinical cancer research: an official journal of the American An integrative approach to HLA peptidomics has delivered a power- Association for Cancer Research. 2005;11:728-734. ful reference database that can be used as a source for developing novel immunotherapies. P432 Exploiting large scale HLA peptidomics to generate novel P433 Immunotherapies; A data-driven approach to neoantigen Computational pipeline for the PGV-001 (personalized genomic prioritization vaccine) clinical trial 1 2 1 Alex S. Powlesland, Geert P.M. Mommen, Ricardo J. Carreira, David Lowne, Alexander Rubinsteyn , Julia Kodysh , Nina Bhardwaj , Michael J. Cundell, Floriana Capuano, Bent K. Jakobsen Jeffrey Hammerbacher 1 2 Immunocore Ltd, Abingdon, United Kingdom Icahn School of Medicine at Mount Sinai, New York, NY, USA; Icahn Correspondence: Alex S. Powlesland (alex.powlesland@gmail.com) School of Medicine, New York, NY, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P432 Correspondence: Alexander Rubinsteyn (alex.rubinsteyn@gmail.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P433 Background Peptides presented to the immune system on HLA complexes are Background valuable targets for immunotherapeutic treatments. Identifying PGV-001 (NCT02721043) is a phase I clinical trial at Mount Sinai the full complement of peptides derived from a particular protein Hospital, studying the safety and immunogenicity of a multi- that are presented on major class I HLA restrictions will provide a peptide personalized genomic vaccine for treatment of several dif- vital step toward increasing the speed and viability of many im- ferent malignancies. Patients are treated with a TLR3 agonist (Poly- munotherapeutic strategies. Advances in next generation sequen- ICLC) and ten synthetic long peptides containing tumor mutations cing and single cell technologies have enabled the accurate that are predicted to be abundantly expressed and to form mu- capture of somatic mutations accumulated by a tumour, yet a tated MHC ligands. The personalized vaccine is administered as an significant hurdle remains how this information can be utilised intramuscular injection and is given to each patient 10 times over a for immunotherapeutic benefit. In particular, identifying which span of 6 months. Thus far, two patients (of an eventual twenty) somatic mutations produce neoantigens, (peptides that contain a have enrolled in the trial and one has been treated. The vaccine is somatic mutation and are presented to the immune system in administered in the adjuvant setting for patients who undergo a complex with HLA), is crucial to linking genetic changes with im- complete resection and have no evidence of residual disease. munological impact. Methods Methods Tumor DNA and RNA are extracted from fresh frozen tissue immedi- Our approach to understanding the targetable human HLA pepti- ately following surgery. Normal DNA is extracted from a patient dome is based on three key principles; achieving full proteome blood sample. Both DNA samples are enriched for coding regions Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 210 of 244 using an exon capture kit. Normal DNA is sequenced to mean on- Results target coverage of 150x, whereas tumor DNA is sequenced to 300x Combination treatment of a GAd-CT26 neoantigens vaccine with coverage. RNA is enriched for mRNA using poly-A capture and then NKTR-214 results in tumor regression in 90% of mice. Single agent >100M paired-end reads are sequenced. Somatic variants are de- NKTR-214 treatment led to tumor regression in < 40% of mice. tected from the tumor and normal DNA sequencing data. The RNA Interestingly, all animals cured by the combo were resistant to a sequencing data is then used to assemble the most abundant coding second tumor challenge. NKTR-214 treatment rescued CD4 T cell re- sequence for each variant, which naturally phases adjacent variants activity against at least one of the predicted neoantigens, likely in- and allows for an allele-specific estimate of expression. Each coding duced by the tumor itself. GAd vaccination in the presence of sequence is then translated to a mutated protein fragment, which is NKTR-214 induced a greater breadth and depth of immune re- ranked according to (1) expression and (2) predicted binding affinity sponse. When compared to Gad vaccine alone, the Gad + NKTR-214 to patient MHC I molecules. The window of wildtype amino acids combination had immune reactivity detected against a larger number around a mutation in each peptide is optimized to improve the odds of vaccine-encoded neoantigens and there was a greater proportion of of successful solid phase synthesis. IFN-gamma+ CD4 and CD8 T cells against each vaccine-encoded Results neoantigens. Tumors in regression of mice treated by the combo were Though only two patients have enrolled in PGV-001 so far, we have highly enriched in T cells reactive to vaccine-encoded neoantigens. evaluated the pipeline on a variety of patient (and mouse) samples Conclusions across different kinds of cancers. We demonstrate that even cancers Vaccination with GAd encoding neoantigens is a very effective and safe with intermediate mutational burden still yield sufficient predicted approach to strengthen and broaden tumor-killing T cells. Therapeutic neoantigens to potentially benefit from vaccination. Furthermore, cancer vaccines have been unsuccessful in the past likely because T cell even with our existing attempt to optimize peptide sequences for growing capacity in the tumor is suppressed by the tumor itself. Vigor- manufacturability, we have still found many peptide sequences can- ous growth of vaccine-induced T cells in the tumour can be successfully not be synthesized or purified. Analysis of these peptide sequences achieved by the cytokine NKTR-214, overcoming the tumor inhibitory hint at additional rules which may improve synthetic yield for activity. NKTR-214 was shown to be safe and well tolerated in cancer peptide-based neoantigen vaccines. patients and GAd vaccines have been safely used in many healthy vol- Conclusions unteers, opening up the opportunity for a fast evaluation of a combo We hope that discussion of the design and rationale for PGV-001's treatment in the clinic. computational pipeline will help other groups start their own person- alized vaccine programs. P435 Trial Registration Immunogenomics for the development of personalized T cell NCT02721043. therapy for ovarian cancer Muzamil Want, Sebastiano Battaglia, Takemasa Tsuji, Richard Koya P434 Roswell Park Cancer Institute, Buffalo, NY, USA Great Apes Adenoviral vaccine encoding neoantigens synergizes Correspondence: Sebastiano Battaglia (muzamil.want@roswellpark.org) with immunomodulators to cure established tumors in mice Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P435 1 1 1 Anna Morena D'Alise , Gabriella Cotugno , Guido Leoni , Francesca 1 1 2 1 1 Langone , Imma Fichera , Maria De Lucia , Rosa Vitale , Adriano Leuzzi , Background 1 1 1 1,2 Elena Di Matteo , Antonella Folgori , Stefano Colloca , Alfredo Nicosia , Ovarian cancer (OC) is the fifth leading cause of cancer death in the 3 1 Jonathan Zalevsky , Elisa Scarselli United States with approximately 20,000 women being diagnosed 1 2 Nouscom srl, Rome, Italy; University of Naples Federico II, Naples, Italy; every year. Since OC development is mainly asymptomatic, patients Nektar Therapeutics, San Francisco, CA, USA are often diagnosed at late stage and present local and distal metas- Correspondence: Elisa Scarselli (g.napolitano@nouscom.com) tases. This offers a clinical challenge, as roughly 70% of the patients Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P434 develop chemoresistant disease. With the advent of cancer immuno- genomics it is now possible to identify tumor specific mutations, or Background neoantigens/neoepitopes, that can be exploited for the development Current immunotherapies based on checkpoint inhibitors (CPI) re- of personalized T cell therapies via DC vaccines or adoptive cell ther- vealed the importance of neoantigens-specific T cells for tumor con- apy (ACT). We therefore hypothesize that tumor cells isolated from trol. Here, we developed a novel neoantigens vaccine approach patients with ovarian cancer offer a unique opportunity to identify based on the use of a viral vector, Great Apes Adenovirus (GAd), en- clinically actionable tumor neoantigens. coding multiple cancer neoantigens in tandem. NKTR-214 is a Methods CD122-biased cytokine agonist currently in clinical trials. NKTR-214 is Somatic mutations were identified by whole exome sequencing (WES) designed to expand a specific population of cancer-killing T cells, and RNASeq analysis in tumor samples, using PBMCs as germline con- known as tumor-infiltrating lymphocytes. Synergy between the two trol. In parallel ovarian cancer PDXs were established in NSG mice drugs was investigated. by injecting 2×106 patient tumor cells/mouse subcutaneously (SQ, Methods n=20). Tumor growth was monitored weekly via caliper measure- Balb/c mice were implanted subcutaneously with CT26 colon carcin- ment. Mutational analysis and immunogenicity prediction for the oma cells. Once tumors were established, mice were randomized and mutated peptides was done using a combination of home- vaccinated with a single intramuscular injection of GAd encoding up developed bioinformatic approaches and NetMHC. Immunogenicity to 20 different CT-26 neoantigens identified by NGS and selected predictions were validated in-vitro by pulsing wild type and mu- with a NousCom proprietary pipeline. NKTR-214 was administered i.v. tated peptides into CD8+ depleted PBMCs and co-cultured with on q9dx3 schedule. Tumor growth in treated animals was monitored CD8+ T cells for up to 20 days. T cell activation was quantitated via and neoantigen-specific T cells were measured both in the periphery ELISA measuring IFN-g release and via flow cytometry by staining and in tumors. for CD137 expression.Lastly, the therapeutic potential of ACT Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 211 of 244 targeting neoantiges was tested in-vivo in ovarian cancer PDXs and dramatically regressed or inhibited the progression of established pri- tumor size is being monitored weekly. mary or lung metastatic EG7.OVA (Fig. 1G), B16F10 (Fig. 1H), and Results MC38 tumors (Fig. 1I). Remarkably, combining AlbiVax with immune We have established patient-derived xenografts (PDX) mice and interro- checkpoint inhibitor anti-PD-1 further improved the therapeutic effi- gated the tumor mutational landscape. Targeted mutations were con- cacy, and led to the complete regression of 60% MC38 tumors for at firmed in the following PDX passages by NGS and Sanger sequencing. least 5 months, and triple combination of AlbiVax, anti-PD-1, and Out of 168 mutated peptides predicted from the patient tumor, 13 Abraxane exhibited synergistic therapeutic efficacy (Fig. 1H, Fig. 1I). were found to have high affinity for different HLAs compared to the AlbiVax showed excellent safety profile throughout the study. wild type peptide. In vitro screening of high affinity peptides showed Conclusions that few of the mutated peptides can activate the T cells from patient AlbiVax represents a promising safe, widely applicable, and robust T PBMCs and tumor infiltrating lymphocytes thereby confirming the pres- cell vaccine for combination cancer immunotherapy. ence of neoantigen reactive T cells. Conclusions In this study, we demonstrate that the patient’s own T lymphocytes can recognize tumor neoantigens and could be used as personalized therapy in ovarian cancer patients P436 In vivo self-assembled albumin/vaccine nanocomplexes for lymph- node-targeted vaccine delivery in combination cancer immunotherapy Guizhi Zhu, Xiaoyuan Chen National Institutes of Health, Bethesda, MD, USA Correspondence: Guizhi Zhu (zhu.julian21@gmail.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P436 Background Cancer immunotherapy aims to normalize the immune system in pa- tients to harness the power of the immune system to systemically treat cancer. Molecular vaccines hold tremendous potential for tumor immunotherapy, but its clinic outcome has yet been optimal, largely due to inefficient co-delivery of heterogeneous peptide antigens and adjuvants to secondary lymphoid organs such as lymph nodes (LNs). Nanovaccines were attempted to improve vaccine delivery, however, clinical translation of most of them have been impeded by compli- cated large-scale manufacturing and safety concerns. Methods We developed an albumin-binding Evans blue derivative as LN- homing moieties and cell uptake mediators for cancer vaccine de- livery. The safety of this derivative has been validated in 2 ongoing clinical trials involving more than 200 patients and healthy volun- teers. By conjugating this derivative with molecular vaccines (CpG adjuvant, B16F10 melanoma-associated antigen Trp2, and MC38 colon adenocarcinoma-specific neoantigen Adpgk), we developed albumin-binding vaccine (AlbiVax) (Fig. 1A) to efficiently co-deliver molecular adjuvants and antigens to LNs. By PET imaging of radi- olabeled AlbiVax in mice, we systematically optimized AlbiVax for LN-targeted delivery, and light-sheet fluorescence imaging and super-resolution confocal microscopy were employed to study vac- Fig. 1 (abstract P436). In vivo self-assembled albumin/vaccine cine behaviors in transparent LNs and single antigen-presenting nanocomplexes for lymph-node-targeted vaccine delivery in cells (APCs). In three tumor models, we studied the antigen-specific combination cancer immunotherapy. (a) Schematics of albumin- immunogenicity and tumor therapy efficacy of AlbiVax, either alone binding vaccine (AlbiVax) for LN-targeted vaccine delivery for cancer or in combination with anti-PD-1 or Abraxane. immunotherapy. (b, f) by PET imaging of radiolabeled AlbiVax in Results small animals, AlbiVax was systematically optimized for optimal Quantitative PET imaging revealed that AlbiVax was delivered to LNs LN-targeted delivery. (c, d) By light-sheet fluorescence imaging, more efficiently (20-fold for CpG, up to 91-fold for antigens) than un- we elucidated the distribution of AlbiVax in draining LNs that were conjugated vaccines or vaccines emulsified in Incomplete Freud’s Ad- cleared to be transparent. (e) By super-resolution imaging, we juvant (IFA) (Fig. 1B, Fig. 1F). Light-sheet fluorescence imaging discovered efficient intracellular co-delivery of AlbiVax via mouse elucidated the distribution of AlbiVax in transparent draining LNs serum albumin (MSA) into endolysosome of bone marrow derived (Fig. 1, Fig. 1D); and super-resolution imaging revealed efficient intra- dendritic cells (BMDCs). (g) AlbiVax elicited 21-fold higher frequency cellular co-delivery of AlbiVax via albumin into endolysosome of of Antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) than IFA- APCs (Fig. 1E). AlbiVax elicited >21-fold higher frequency of Antigen- emulsified vaccine. (h-j) AlbiVax dramatically regressed or inhibited specific CD8 cytotoxic T lymphocytes (CTLs) than IFA-emulsified vac- the progression of EG7.OVA (g), B16F10 (h), and MC38 tumors (i) cines and induced immune memory for > 5 months (Fig. g). AlbiVax Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 212 of 244 PHYSICIAN/NURSE/PHARM: Best Practices for References 1. Nathan P, Cohen V, Coupland S, Curtis K, Damato B, Evans J, Fenwick S, Improving Cancer Immunotherapy Kirkpatrick L, Li O, Marshall E, McGuirk K, Ottensmeier C, Pearce N, Salvi S, Stedman B, Szlosarek P, Turnbull N. United Kingdom Uveal Melanoma P437 Guideline Development Working Group, Uveal Melanoma UK National Mapping the treatment pathway for metastatic uveal melanoma Guidelines. European Journal of Cancer. 2015; 51: 2404– 2412. (mUM) patients in England: A qualitative pilot study 1 1 2 3 Elisabeth Adams , Chih-Yuan Cheng , Joseph Sacco , Sarah Danson , 4 5 6 2 Pippa Corrie , Paul Nathan , Peter Szlosarek , Joanne Upton , 6 7 Abolore Amuludun , Toby Toward 1 2 P438 Aquarius Population Health Limited, London, United Kingdom; The 3 Continued challenges and opportunities in oncologists’ Clatterbridge Cancer Centre, Wirral, United Kingdom; Weston Park 4 COMPREHension of clinical immunology and its relationship with Hospital, Sheffield, United Kingdom; Addenbrooke's Hospital, 5 cancer immunotherapy Cambridge, United Kingdom; Mount Vernon Cancer Centre, London, 1 1 2 3 6 Tara Herrmann , Charlotte Warren , Haleh Kadkhoda , Lianne Wiggins United Kingdom; Barts Cancer Institute, London, United Kingdom; 1 2 7 Medscape Education Oncology, New York, NY, USA; Medscape Immunocore Limited, Abingdon, United Kingdom Education, New York, NY, USA; SITC, Milwaukee, WI, USA Correspondence: Toby Toward (chihyuan.cheng@aquariusph.com) Correspondence: Tara Herrmann (therrmann@medscape.net) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P437 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P438 Background Background mUM is a rare disease (annual UK incidence <150 patients), with a re- Cancer immunotherapy (CIT) is revolutionizing the treatment of can- ported median overall survival of 7-12 months. In 2015, the UK published cer. As part of an ongoing educational curriculum, assessment of on- their first national uveal melanoma guidelines [1]. However, with few cologists' knowledge, competence, and performance in the use of therapeutically effective options available for metastatic patients, it is un- CIT identified multiple challenges and barriers. This study’s objective clear what patients actually receive as standard of care (SoC). This pilot was to assess and compare practicing oncologists’ comprehension of, study aimed to map the real-world pathway for recently diagnosed mUM and confidence in, the interplay between the immune system and patients, through qualitatively evaluating their clinical management and cancer tumor development and metastasis. treatments received within SoC. Methods Methods An expert panel was convened to identify knowledge, competence, Based on treatment recommendations within national guidelines, an out- and performance gaps in the area of CIT. Educational curricula con- line decision-tree for mUM patients was developed. On presenting the sisting of 8 activities that focused on foundational education were decision-tree to 5 specialist centres across England (January-June, 2017), developed in 2014-2016, and posted online. Intra-activity questions 6 senior clinicians were interviewed about their typical: clinical manage- embedded in each activity allowed learners to self-report their confi- ment; medical resource use; treatment decision-making and patient dence with CIT concepts, while matching participants’ responses to flow(s) between local and regional facilities, when treating mUM patients. knowledge-based questions presented before and after education The interview data were analysed to populate centre-level decision-trees, were collected to measure reinforced learning and gains in compre- and consolidated to inform a consensus SoC pathway. hension. Confidentiality of respondents was maintained. Responses Results were collected from 5/2014-10/2016. Interviews revealed considerable variation in first-line treatments of- Oncologists (N=4,684) who participated in the activities demon- fered at specialist centres: strated on average a 20% improvement from initial baseline be- tween years 1 and 2. Additional education in year 3 was provided hepatic resection for liver metastases [1-25%]; to help prevent decay in oncologists’ comprehension of immune loco-regional therapies for liver metastases (including percutaneous system functions. However, comparison of oncologists' knowledge hepatic perfusion and radiofrequency ablation) [0-20%]; of the role of immune checkpoints indicates that while gains ob- new treatments in clinical trials (TRAP, SelPac or IMCgp100) served in year 1 were maintained, a drop off was observed between [10-80%]; years 2 and 3. This is likely a result of more data becoming avail- immunotherapies (Ipilimumab, Pembrolizumab or Nivolumab) able on alternative CITs while simultaneously less foundational edu- [0-54%]; and cation was available. chemotherapies (Dacarbazine or Temozolomide) [0-1%]. Results Oncologists continued to report a lack of confidence with the con- There was, however, consensus on treatment priorities - if liver metastases cepts of clinical immunology. Only 2%-11% of oncologists were very were operable then surgical options were considered first. If non-operable, confident in their comprehension of the relationship between the clinical trials were subsequently considered, before other therapeutic op- immune system and tumor development. In addition, fewer than tions. Whilst centres agreed that treatment should be initiated at a special- 50% of participants post-activity could recognize common T cell co- ist (supra-regional) mUM centre, there were mixed attitudes on “if” and receptors that could be targeted as part of CIT. One quarter did not “how” patients could receive ongoing treatments at more local centres. realize that tumors interfere with signal 1 by blocking MHC antigen Conclusions processing. The main reason suggested for treatment pathway variations amongst Conclusions centres, was the absence of effective treatments. Introducing an effective This study demonstrates the value of and continued need for educa- therapeutic option at a defined optimum point in the pathway would be tional interventions on CIT. Retention of CIT concepts and improve- considered a “catalyst to transform the management of care”.This inturn ments in knowledge of clinical immunology should facilitate would lend itself to greater consistency of practice, harmonise care path- competence among oncologists, which has valuable implications for ways and improve overall outcomes for mUM patients. use of CIT and for patient outcomes. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 213 of 244 P439 PDL-1 status and value for Extrapulmonary small cell carcinomas Mohammed Salhab, Yiqin Xiong, Karen Dresser, Benjamin Chen, James Liebmann University of Massachusetts, worcester, MA, USA Correspondence: Mohammed Salhab (mohammed.salhab@umassmemorial.org) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P439 Background Extrapulmonary small cell carcinomas (ESCCs) are rare but aggressive tumors with an overall 5-year survival rate of less than 15%. Relapse is common despite rigorous chemotherapy and radiotherapy. Cancer im- munotherapy targeting PD-1/PD-L1 pathway emerges as an increas- ingly appealing strategy [1, 2, 3]. Methods We investigated PD-L1 expression by immunochemistry (IHC) in ESCCs diagnosed at our institution from 1999-2016. 34 cases with sufficient material were selected for PD-L1 IHC analysis using clone E1L3N. Sites of origin include bladder (9), cervix (3), prostate (2), ovary (2), gallbladder (2), duodenum (1), esophagus (1), biliary tract Fig. 1 (abstract P439). See text for description (1), vagina (1), palate (1), ureter (1), and unknown primary (10). Results Two cases showed diffuse PD-L1 expression in both tumor and stro- mal cells, one from ureter and one from a gallbladder in origin. 10 PHYSICIAN/NURSE/PHARM: Case Studies cases (29%) showed at least patchy PD-L1 expression in stromal cells. demonstrating exceptional responses 22 cases (65%) were completely negative for PD-L1 expression. Pa- tients of both groups who was treated, did received chemotherapy P440 +/- surgery or radiation. The overall response rate for PDL positive Durable complete response with PANVAC and Trastuzumab in group was 80% while it was 67% for PDL-1 negative group (Table 1). metastatic triple positive breast cancer (mTPBC) The median overall survival for PD-L1 positive staining group, regard- 1 2 1 1 Margaret Gatti-Mays , Mahsa Mohebtash , Ravi Madan , Julius Strauss , less of stage was 11.5 months’ vs 7 months for PDL-1 negative group 1 1 1 3 Marijo Bilusic , Caroline Jochems , Myrna Rauckhorst , Elizabeth Jones , (Fig. 1). Patients with limited stage disease with positive PDL-1 1 1 1 William Dahut , Jeffrey Schlom , James Gulley showed an impressive 53 months’ survival compared to PDL-1 nega- 1 2 National Cancer Institute, NIH, Bethesda, MD, USA; Medstar Union tive limited disease with 15 months. Memorial Hospital, Baltimore, MD, USA; National Institutes of Health, Conclusions Bethesda, MD, USA This study suggests that PD-L1 staining in extrapulmonary small cell Correspondence: Margaret Gatti-Mays (margaret.gatti-mays@nih.gov) carcinoma may be of prognostic relevance and further studies are re- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P440 quired to understand the implications of immune dysregulation in these aggressive tumors. PD-1 inhibitors should be investigated in Background this group of patients. First line treatment of metastatic HER2+ breast cancer with Docetaxel+Trastuzumab(TH) carries a median PFS of 12months References and OS of 40months. The addition of Trastuzumab+/-Pertuzumab 1. Annemiek ME, Walenkamp A, Sonke G, Sleijfer D. Clinical and therapeutic to chemotherapy prolongs survival in the metastatic setting but aspects of extrapulmonary small cell carcinoma. Cancer Treatment no therapies are curative. Trastuzumab has immune-related ef- Reviews. 2009; 35 :228–236. fects including antibody-dependent cell-mediated cytotoxicity and 2. Wong YN, Jack R, Mak V, Henrik M, Davies E. The epidemiology and cross-presentation of tumor antigens. PANVAC is a recombinant survival of extrapulmonary small cell carcinoma in South East England, poxviral vaccine that contains transgenes for MUC-1, CEA and T- 1970–2004. BMC Cancer. 2009; 9:209. cell costimulatory molecules (B7.1, ICAM-1 and LFA-3). For optimal 3. Brennan S, Gregory D, Stillie A, Herschtal A, Manus M, Ball D. Should response, PANVAC employs a prime-boost regimen, using vaccinia Extrapulmonary Small Cell Cancer BeManaged Like Small Cell Lung (PANVAC-V) as the primer and fowlpox (PANVAC-F) as the boost. Cancer? Cancer.2010. We report an exceptional response of mTPBC to PANVAC+Trastu- zumab after progression on Letrozole+Trastuzumab. Table 1 (abstract P439). See text for description Methods A pilot study of PANVAC+Sargramostim(NCT00088413) demonstrated minimal side effects and suggested clinical benefit in low disease burden. On Day 1(D1), patients received PANVAC-V 2x10e8pfu SC followed by PANVAC-F 1x10e9pfu SC every 2weeks x 3, then monthly x 12 followed by maintenance every 3months until progression. Sar- gramostim 100μg SC was given on D1-D4 of each PANVAC injection. Scans were performed every 2-3months. Results A 32-year-old female, BRCA wildtype, with poorly differentiated, inva- sive ductal carcinoma, ER60%, PR70%, HER2+(IHC3+) had biopsy- confirmed metastasis to an axillary node and liver shortly after diagno- sis. After bilateral oophorectomy, she received THx6cycles followed by a partial hepatectomy, which demonstrated a pathologic complete Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 214 of 244 response. She received radiation to supraclavicular lymph nodes with a for a period of 15 months spanning 27 cycles of Nivolumab without th tumor bed boost. After 12months on Letrozole+Trastuzumab she re- any significant toxicity. Unfortunately, by the 27 cycles the patient’s curred. While on Letrozole she completed 6injections for a HER2/neu lung cancer progressed and he decided to stop all treatment and en- peptide vaccine trial(NCT00194714). Due to progression in bilateral hilar roll in hospice where he eventually expired due to his progressive and subcarinal(2.8cm) lymph nodes determined by PET, she enrolled lung cancer. on trial at NCI in January 2008. Per protocol above, she received Conclusions PANVAC-V followed by PANVAC-F. Restaging at 10months showed a This case demonstrates for the first time the notable activity and clinical partial response(0.8cm) and at 18months showed a complete respon- benefit of Nivolumab as a single agent in PTCL-NOS. Further evaluation se(CR). She has remained on-study and receives PANVAC-F+Sargramos- of Nivolumab’s activity in PTCL as well its incorporation into PTCL treat- tim every 90days and Trastuzumab every 3weeks. Restaging in May ment approaches need additional study and validation. 2017 shows a continued CR at 113months. Conclusions This case report highlights the potential of immunotherapy and pos- P442 sibly multiple immunotherapies in a patient with breast cancer. Fur- Pseudoprogression manifesting as recurrent ascities with anti-PD-1 ther study is required to understand the mechanism of response in therapy this patient and if it can be applied on a broader scale. Randy Sweis, Yuanyuan Zha, Russell Szmulewitz, Lomax Pass, Trial Registration Tara Chongsuwat, Jason Luke, Thomas Gajewski NCT00088413 University of Chicago, Chicago, IL, USA Correspondence: Randy Sweis (rsweis@uchicago.edu) References Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P442 1. Swain SM, Baselga J, Kim SB, et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med. Background 2015;372(8):724-734. Pseudoprogression occurs when patients treated with immunother- 2. Mohebtash M, Tsang KY, Madan RA, et al. A pilot study of MUC-1/CEA/ apy have an initial increase in apparent tumor size prior to regres- TRICOM poxviral-based vaccine in patients with metastatic breast and sion. This phenomenon results from an influx of activated immune ovarian cancer. Clin Cancer Res. 2011;17(22):7164-7173. cells, occurring in up to 7% of patients [1]. Ascites is also a common manifestation of progressive malignancy from peritoneal metastases. However, ascites has not been previously reported as a manifestation P441 of pseudoprogression. We describe a metastatic urothelial cancer pa- Activity of Nivolumab (Opdivo®) in relapsed progressive peripheral tient with progressive ascites and concurrent reduction in tumor bur- T-cell Lymphoma-NOS (PTCL-NOS) den on anti-PD-1 therapy. 1 2 Philip A Haddad , David Sommerhalder Methods 1 2 Overton Brooks VAMC, Shreveport, LA, USA; LSUHSC-S, Shreveport, LA, Pembrolizumab was given 200 mg IV every 3 weeks. Computed tomog- USA raphy scans were obtained and tumor burden assessed by RECIST v1.1. Correspondence: Philip A Haddad (haddad8838@msn.com) Peritoneal fluid was analyzed for cytology and fluorescence-activated Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P441 cell sorting (FACS) using FlowJo10.1r5. Multiplex cytokine assay was performed on peritoneal fluid. Normal donor PBMCs stimulated with Background phorbol myristate acetate/ionomycin was used as a positive control. Nivolumab is a member of the family of check-point inhibitors. It is a Results PD-L1 inhibitor that has been approved by the FDA as treatment for sev- A 62-year-old female with urothelial bladder cancer previously treated eral solid tumors including non-smallcelllungcancer. It’salsoapproved with chemotherapy and radiation presented with recurrent metastatic to treat Hodgkin’s Lymphoma after failure of several lines of therapy. disease including two peritoneal nodules and an enlarged para-aortic PTCL is often aggressive and incurable. Compared to B-cell lymphomas, lymph node. Pembrolizumab was started and she had a significant par- they tend to present at more advanced stages and respond less fre- tial response with a 49% reduction in target lesions. During therapy, quently and with shorter duration to the same chemotherapy regimens. she developed large-volume ascites. Initial paracentesis showed a Nevertheless, PCTL is still approached with suboptimal B-cell therapeutic serum-ascites albumin gradient >1.5, total protein of 1.2 g/dL, and strategies. As to the recently approved PTCL specific therapies, they are leukocyte count of 231/μL including 45% lymphocytes. Cytology from palliative in nature. An urgent need for better therapeutic agents in this four separate paracenteses over 8 weeks showed reactive cellular desperate lymphoma continues to exist. changes/inflammation, but no malignant cells were identified. Methods FACS analysis on peritoneal fluid showed CD8 lymphocytes com- th + + + We report on the activity of Nivolumab as a 4 line treatment in a prised 53% of CD45 CD3 cells. The majority of CD8 lymphocytes case of relapsed and rapidly progressive PTCL-NOS. were PD-1 (78%). Class II-MHC (HLA-DR) and CD40 ligand (CD154) Results were expressed on 43% and 20% of CD8 lymphocytes, respectively. Our patient was a 62 y.o. white man who was diagnosed with poor All three markers were increased relative to normal donor PBMC con- risk stage IVA PTCL and Stage IA lung squamous cell carcinoma in trols, suggesting a locally high number of antigen-experienced, + + 2014. Due to his borderline functional performance status and de- activated T cells. FoxP3 CD25 Tregs were also detected in 17% of + + + creased left ventricular ejection fraction, he was treated with Prala- the CD45 CD3 CD4 compartment. Multiplex cytokine assay for 38 trexate that is approved for PTCL and has activity in lung cancer. He analytes was performed. The most significantly elevated analytes received 2 cycles of Pralatrexate which stabilized both malignancies were IL-6, IL-15, G-GSF and FGF-2 (each >2-fold above control). for 3 months after which they progressed. Subsequently, he was Conclusions given Carboplatin and Gemcitabine combination that has activity in Pseudoprogression in cancer patients treated with immunotherapy both malignancies. This combination seemed to stabilize his lung can manifest as recurrent ascites due to a rapid influx of activated T th cancer and PTCL for 4 months. After the 5 cycle, the patient’s lymphocytes into the peritoneum. Since ascites can also be caused lymphoma progressed. At this time, the patient was started on Beli- by disease progression, it is important to clinically distinguish the nostat to address his PTCL. Initially, PTCL responded to Belinostat two, so that treatment may be appropriately continued or stopped. and then stabilized for 5 months. After the fifth cycle, his lung cancer started progressing requiring lung cancer directed therapy. He was References th started on Nivolumab which stabilized his lung cancer. After the 4 1.Blumenthal, dose of Nivolumab further unexpected PTCL response was noted et al. Treatment beyond progression with immune checkpoint clinically and on follow up imaging. This response was maintained inhibitors—known unknowns. JAMA Oncology. 2017. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 215 of 244 P443 (irAEs), affecting any organ, with signs and symptoms usually seen in Malignant melanoma metastatic to the heart: exploring patient inflammatory and autoimmune diseases. There is paucity of data on outcomes and cardiovascular complications in the era of CPI induced arthritis, primarily from case reports or small series. immunotherapy Methods Laura Talamo, William Grosh Patients developed arthritis while using CPI were identified from the University of Virginia, Charlottesville, VA, USA Rheumatology Clinic at UT MD Anderson Cancer Center. We diagnosed Correspondence: William Grosh (lt3j@virginia.edu) arthritis if patients developed joint swelling after receiving any FDA ap- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P443 proved CPI, after ruling out other potential underlying causes or path- ologies. Here we report 13 pts. Cancer types included melanoma (n=5), Background non-small cell lung cancer (n=4), renal cell carcinoma (n=2), acute mye- Cardiac metastasis (CM) from melanoma is being recognized more loid leukemia, and cancer colon (1 each). CPI included nivolumab (n=8), frequently, but often presents without signal symptoms. A rate of CM pembrolizumab (n=3), and ipilimumab and atezolizumab (1 each). of 64% has been quoted in one autopsy series [1], though the clinical Results diagnosis rate is considerably lower [2]. We evaluated patients with Patients age ranged from 47-74 years and 62% were male. CPI induced known CM for cardiac complications and impact of therapy, and de- arthritis was the sole irAE in eight pts (62%), and was associated with fined survival among these patients. other irAEs including sicca symptoms (xerostomia, dry eyes), colitis, Methods pancreatitis, or dermatitis in the remaining five (38%). Overall, median This retrospective review identified patients with CM seen in the Univer- time to onset of arthritis after the initiation of CPI was 6 (range 1 to 15) sity of Virginia Oncology clinic between 2010 and 2017. Eligible patients months. When occurred with other irAEs, arthritis always manifested up were at least 18 years old with metastatic melanoma (MM) and CM. to 4 months after the first occurring irAE despite sufficient immunosup- Our primary outcome metrics were 2 year survival from time of diag- pressant treatment (infliximab and/or high dose steroid) and symptoms nosis of melanoma (M) and MM. We recorded cardiac complications, control, with the exception of one patient with sicca symptoms. Initial diagnostic procedures, patient outcomes, and treatment modality. symptoms varied widely between small and large joints of upper and Ten patients were identified with the demographics outlined below. lower extremities. Over time, symmetrical polyarthritis resembling Longest follow up was 21 months from diagnosis of CM (DCM). rheumatoid arthritis, predominantly involving small joints of the hands Results and wrists, along with larger joints was reported in six patients. Two Two year survival from M diagnosis was 70%. Two year survival from had oligoarthritis resembling seronegative spondyloarthropathy with DCM could not be calculated (two patients are still living at 9 and 21 axial pain and enthesopathy. Four had asymmetrical inflammatory poly- months from DCM). Figure 1 shows Kaplan-Meier survival analysis. Of arthritis, and one had oligoarthritis. Rheumatoid factor and/or anti- the eight patients who died, all died within one year of DCM. citrullinated peptide antibodies were positive in two patients, and anti- Responses to immunotherapy (IO) are shown in Table 2. nuclear antibodies in two others. Eleven patients were treated with ste- Conclusions roids, six were controlled, while five needed additional biological The rate of cardiac complications was low. Only one death was a agents (4 tocilizumab, 1 infliximab) to achieve appropriate steroid taper direct result of CM (patient 10). (< 7.5 mg). One patient improved with intra-articular steroid injection, 90% of patients received IO. At the time of data collection, two patients re- and one received tocilizumab as first line. main (2 and 3) alive nine and 21 months since DCM; both received >20 Conclusions cycles of P. Patients 1, 2, 3, 8, 9, and 10 received IO after DCM, but only pa- Our data suggest that arthritis is a late occurring irAE that may be tient 3’s CM responded to IO (IN induction, P maintenance). Patient 1 did due to a different immunopathology. A multi-institutional collabora- not respond to I; patient 2 was lost to follow up on IL-2; patient 8’sre- tive work including tissue analysis is needed to enhance our under- sponse cannot be determined due to death from septic shock; patient 9 standing of the pathogenesis and design optiomal therapy without progressed through P; and patient 10 did not respond after one cycle of I. dampening antitumor immunity. This study is limited by small patient numbers, but supports a poor prognosis for patients with CM. Responses can be seen with IO, spe- P445 cifically PD-L1 inhibitors, which may improve survival. Other treat- Clinical features of immune checkpoint inhibitor-induced ments cannot be assessed. The only patient who survived one year inflammatory arthritis differ in those treated with combination after DCM has had an ongoing response to P. versus single agent therapy Laura Cappelli, Clifton Bingham, Julie Brahmer, Patrick Forde, Dung Le, References Evan Lipson, Jarushka Naidoo, Lei Zhang, Ami Shah 1. Glancy DL, Roberts WC. The heart in malignant melanoma. A study of 70 Johns Hopkins, Baltimore, MD, USA autopsy cases. Am J Cardiol. 1968;21:555–71. Correspondence: Laura Cappelli (lcappel1@jhmi.edu) 2. Villa et al. Cardiac metastases of melanoma as first manifestation of the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P445 disease. J Radiol Case Rep. 2014;1;8(4):8-15. Background PHYSICIAN/NURSE/PHARM: irAE Management: Multi-system immune-related adverse events (iRAE) can occur as a re- sult of immune checkpoint inhibitors (ICIs) targeting PD-1, PD-L1, Clinical Care and Best Practices and CTLA-4, with variations depending on tumor type and agent P444 used. Inflammatory arthritis (IA) has been increasingly recognized as Inflammatory arthritis induced by the use of checkpoint inhibitors an iRAE that may occur as a result of anti-PD-1/PD-L1/CTLA-4 therapy for immunotherapy of cancer used in the treatment of malignancy [1, 2]. We evaluated associations 1,2 1 1 1 Noha Abdel-Wahab , Jean H. Tayar , Adi Diab , Sang T. Kim , between ICI regimen and clinical presentation of IA in a single 1 1 Huifang Lu , Maria E. Suarez-Almazor institution. The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Methods Assiut University Hospitals, Faculty of Medicine, Assiut, Egypt Patients referred to rheumatology for suspected ICI-induced IA, were Correspondence: Maria E. Suarez-Almazor (nahassan@mdanderson.org) identified January 2013- July 2017. Patients were included if they de- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P444 veloped de novo IA during or after ICI therapy and had no history of pre-existing autoimmune disease. Patients were included for the sin- Background gle agent group if they were on a PD-1 or PD-L1 inhibitor without Checkpoint inhibitors (CPI) have revolutionized cancer treatment with CTLA-4 inhibition. Those in the combination group were treated with remarkable survival benefits in multiple cancer patients. Yet, their PD-1 and CTLA-4 inhibition. Clinical, demographic and laboratory use can be hampered by frequent immune-related adverse events data were obtained from the medical records. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 216 of 244 Results P446 Tumor types for included patients were melanoma, non-small cell lung Immunosuppression use and clinical outcomes in patients with cancer, small cell lung cancer, colon cancer, Hodgkin or cutaneous lymph- immune checkpoint inhibitor -induced inflammatory arthritis oma, renal cell carcinoma, endometrial carcinoma, duodenal carcinoma, Laura Cappelli, Clifton Bingham, Julie Brahmer, Patrick Forde, Dung Le, merkel cell carcinoma, basal cell carcinoma, or squamous cell carcinoma. Evan Lipson, Jarushka Naidoo, Lei Zheng, Ami Shah The group treated with combination CTLA-4/PD-1 inhibition was signifi- Johns Hopkins, Baltimore, MD, USA cantly younger than the monotherapy group; those in the combination Correspondence: Laura Cappelli (lcappel1@jhmi.edu) groupwerealsomorelikelyto bemaleand tohave melanoma (Table 1). Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P446 Combination therapy was associated with higher levels of C-reactive pro- tein (4 mg/dl vs. 0.7 mg/dL) and a higher likelihood of having a large joint Background affected first in the arthritis course (table 1). All instances of reactive Inflammatory arthritis (IA) resulting from immune checkpoint in- arthritis-like presentation (arthritis + sterile urethritis, conjunctivitis) were hibitors (ICIs) is an increasingly recognized immune-related ad- in the combination therapy group. Patients who received PD-1/PD-L1 verse event. The requirement for immunosuppression to treat IA, monotherapy were more likely to have IA as their first IRAE. No patients including TNF-inhibitors (TNF-I), has been documented in previ- had CCP antibodies; two patients were positive for rheumatoid factor. ous case series [1, 2]. How often immunosuppression is required Conclusions to treat ICI-induced IA is unclear. The consequences of long- This study illustrates the diversity in clinical phenotypes of patients term immunosuppression on tumor response from ICIs is also with ICI-induced IA. Differences in presentation may be explained by uncertain. the regimen used to treat a patient’s malignancy. Understanding the Methods diversity of clinical presentations will help oncologists identify and Patients treated at Johns Hopkins Rheumatology for IA due to ICIs treat this increasingly important iRAE. were included if they received anti-CTLA-4, anti-PD-1, and/or anti-PD- L1 therapy and developed IA during or after therapy. Patients with References pre-existing autoimmune disease were excluded. Patients were man- 1. Cappelli LC, Gutierrez AK, Baer AN, et al. Inflammatory arthritis and sicca aged by rheumatology and oncology based on preliminary recom- syndrome induced by nivolumab and ipilimumab. Ann Rheum Dis 2017; mendations for ICI-induced IA treatment [3]. 761):43-50. Results 2. Calabrese C, Kirchner E, Kontzias K, et al. Rheumatic immune-related ad- Included patients (n=32) had melanoma, non-small cell lung can- verse events of checkpoint therapy for cancer: case series of a new cer (NSCLC), small cell lung cancer, colon cancer, Hodgkin or cu- nosological entity. RMD open 2017;3(1):e000412. taneous lymphoma, renal cell carcinoma, endometrial carcinoma, duodenal carcinoma, merkel cell carcinoma, basal cell carcinoma, or squamous cell carcinoma. Of these patients, 20 (63%) had a partial or complete tumor response to ICIs clinically or by RECIST Table 1 (abstract P445). See text for description criteria. Twenty six (81.3%) required corticosteroids for manage- ment of their IA. Eleven patients (34.3%) required additional im- munosuppression. TNF-Is were used in 7 patients (tumor types: melanoma n=6, RCC n=1). Methotrexate or leflunomide was used in 4 patients (tumor types: NSCLC, small cell lung cancer, duo- denal carcinoma, endometrial carcinoma). Duration of TNF-I use ranged from 2-16 months. Four patients treated with TNF-I, all with melanoma, had complete response by RECIST criteria or no evidence of disease at ICI start. None of these patients had tumor progression while on TNF-I (duration: 3-16 months). Of the pa- tients treated with methotrexate or leflunomide, 3 had a complete or partial response to ICIs, and none had tumor pro- gression during 2-12 months of IA management. Conclusions In a cohort of patients with ICI-induced IA, the majority of patients required corticosteroids to control IA symptoms. In this series, TNF- inhibitors and other forms of non-corticosteroid immunosuppression did not affect tumor response in those who had an initial response to ICIs for malignancy. References 1. Cappelli LC, Gutierrez AK, Baer AN, et al. Inflammatory arthritis and sicca syndrome induced by nivolumab and ipilimumab. Ann Rheum Dis 2017;76(1):43-50. 2. Calabrese C, Kirchner E, Kontzias K, et al. Rheumatic immune-related adverse events of checkpoint therapy for cancer: case series of a new nosological entity. RMD open 2017;3(1):e000412. 3. Naidoo J, Cappelli LC, Forde PM, et al. Inflammatory Arthritis: A Newly Recognized Adverse Event of Immune Checkpoint Blockade. Oncologist Demographic and clinical variables at initial evaluation for inflammatory arthritis 2017;22(6):627-30. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 217 of 244 P447 P448 Infliximab associated with faster symptom resolution compared to Incidence of thyroid test function abnormalities in patients corticosteroids alone for management of immune related receiving immune-checkpoint inhibitors for cancer treatment enterocolitis Nisha Patel, Anais Oury, Gregory Daniels, Lyudmila Bazhenova, 1 2 2 2 Daniel H Johnson , Chrystia Zobniw , Van A Trinh , Roland Bassett , Sandip Patel 2 2 2 2 Junsheng Ma , Jamie Anderson , Jennifer Davis , Jocelyn Joseph , University of California San Diego, La Jolla, CA, USA 2 2 2 2 Marc Uemura , Cassian Yee , Rodabe Amaria , Sapna Patel , Correspondence: Nisha Patel (nip011@ucsd.edu) 2 2 2 2 Hussein Tawbi , Isabella Glitza , Michael A Davies , Michael Wong , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P448 2 2 2 2 Scott Woodman , Patrick Hwu , Wen-Jen Hwu , Yinghong Wang , Adi Diab Background University of Texas MD Anderson Cancer Center, Houston, TX, USA; With the advent of immune-checkpoint inhibitor (ICI) therapy (anti- UT-MD Anderson Cancer Center, Houston, TX, USA CTLA-4, anti-PD-1), immune related adverse events (irAE) such as thy- Correspondence: Daniel H Johnson (dhjohnson@mdanderson.org); Adi Diab roid function test abnormalities (TFTA) are common with a reported Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P447 incidence range of 2-15% depending upon the ICI utilized [1,2]. The aim of this study was to describe the incidence of TFTAs retrospect- Background ively in patients who received ICI therapy. Immune-related enterocolitis (irEC) is the most common serious compli- Methods cation from checkpoint inhibitors (CPIs) that can lead to bowel perfor- A total of 285 patients were reviewed (178 male, 107 female; ages ation, sepsis, and death. The current front-line treatment for irEC is 16-94) of which 218 had no baseline TFTA, 61 had baseline TFTAs, high-dose corticosteroids (CS). However, prolonged high-dose CS ther- and 6 had history of thyroidectomy and were excluded. Patients re- apy has significant toxicities and side effects, and prolonged therapy ceived at least one dose of ipilimumab and/or nivolumab or pembro- may reduce CPI anti-tumor activity. Early addition of TNF-alpha inhibi- lizumab. Post-ICI therapy TFTA was classified according to definitions tors such as infliximab may expedite symptom resolution and shorten of thyroid abnormalities when possible [3]. CS duration. The only prospective trial evaluating infliximab with CS Results versus CS alone for irEC (NCT02763761) has recently been terminated. A total of 35% (76/218) patients had new onset TFTAs on ICI therapy. Of Thus we conducted a retrospective study to evaluate symptom reso- note, 70.5% (43/61) had baseline TFTA that were exacerbated by ICI ther- lution in patients with irEC treated with infliximab + CS versus CS alone. apy. Median time to new onset or exacerbated baseline TFTA were 46 & Methods 33 days respectively. Of note, 65% (20/31) of patients on both ipilimumab The medical records of patients with irEC between January 1, 2012 and and nivolumab had new onset TFTA, compared to 31.3% (15/48) with ipili- June 30, 2017 were reviewed under an institution review board- mumab, 31.5% (28/89) nivolumab, 26% (13/50) pembrolizumab (Table 1). approved protocol. Patient demographics, CPI regimen (aCTLA-4, aPD- Conclusions 1/L-1, alone or in combination), colitis grade, colitis treatment, The incidence of TFTAs with ICI therapy was higher than previously treatment-related toxicities, time to symptom resolution, and CS dur- reported. Patients with baseline TFTA and/or receiving ipilimumab ation were collected. The primary-endpoint was time to symptom reso- and nivolumab combination therapy had a higher incidence of TFTA lution for irEC (times to diarrhea resolution and to initiation of steroid than one agent ICI therapy. In conclusion, we recommend more fre- titration) for patients managed with versus without infliximab. Duration quent evaluation of TFT in the first two months, especially in those of CS and rate of CS-associated toxicities were secondary-endpoints. with baseline TFTA. Categorical and continuous variables were assessed between groups by Fisher's exact test and Kruskal-Wallis test (or two-sample t-test). References Results 1. Corsello S, Barnabei A, Marchetti P, et al. Endocrine Side Effects Induced Among 75 patients with irEC, 39 (52%) received CS alone and 36 by Immune Checkpoint Inhibitors.J Clin Endocrinol Metab. (48%) received infliximab within a median of 8 days after CS initi- 2013;98(4):1361-1375. ation. Patient characteristics (age, comorbidities, cancer type, im- 2. Morganstein D.L, Lai Z, Spain L, et al. Thyroid abnormalities following the munotherapy regimen, and initial steroid dose), were similar use of cytotoxic T-lymphocyte antigen-4 and programmed death recep- between the two arms. The incidence of grade 3+ colitis was higher tor protein -1 inhibitors in the treatment of melanoma. Clinical Endocrin- in the infliximab + CS arm (86.1% vs. 34.2%, p<0.001). Despite this, ology. 2017;(0) 1-7. times to diarrhea resolution (median 3 vs. 9 days, p<0.001) and to 3. Rossi E, Sgambato A, De Chiara G, et al. Thyroid-Induced Toxicity of steroid titration (median 4 vs. 13 days, p<0.001) were shorter in pa- Check-Point Inhibitors Immunotherapy in the Treatment of Advanced tients treated with infliximab + CS versus CS alone. Rates of CS- Non-Small Cell Lung Cancer. J Endocrinol Diabetes. 2016; 3 (1):1-10. associated toxicities (39% vs. 54%; p= 0.249) and total steroid dur- ation (median 35 days vs. 51 days; p= 0.241) were numerically lower in the infliximab-treated patients (Table 1). Table 1 (abstract P448). See text for description Conclusions To our knowledge this is the first study to compare time to symptom resolution for irEC with infliximab + CS versus CS alone. Despite higher incidence of grade 3+ colitis, infliximab treatment was associ- ated with a statistically significant shorter time to symptom reso- lution without additional toxicities. The data suggest that early introduction of infliximab should be considered for patients with irEC until definitive prospective clinical trials. Table 1 (abstract P447). See text for description Summary of new onset TFTA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 218 of 244 P449 Background Neurotoxicity in patients with metastatic solid tumors treated with Immunotherapy continues to offer promising treatments for many immune checkpoint inhibitors: a single institution retrospective tumor types. Unfortunately, patients potentially eligible for immunother- analysis apy are often not aware of options. According to a 2014 Cancer Support Bianca Santomasso, Rachna Malani, Aya Haggiagi, Jenessa Holder, Yelena Community (CSC) online survey, only 34.8% of cancer survivors knew Shames, Samuel Briggs, Margaret Callahan the term “immuno-oncology” and 64.9% had heard of “immunotherapy.” Memorial Sloan Kettering Cancer Center, New York, NY, USA Despite low awareness, most (84%) wanted to know more about these Correspondence: Bianca Santomasso (Santomab@mskcc.org); Margaret topics. Recognizing this need, the CSC developed a national psychoedu- Callahan cational immunotherapy workshop. The current analyses investigate the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P449 program’s informational and empowerment outcomes among attendees with cancer. Background Methods Immune checkpoint inhibitors (ICIs) have dramatically improved From 2014-16, 706 program attendees of the CSC’s Frankly Speaking survival for patients with melanoma and are playing an increasingly About Cancer: Your Immune System & Cancer Treatment workshop com- important role in the management of other tumor types. Anti-CTLA4, pleted a post-program evaluation assessing knowledge (on 5-point anti-PD1, and anti-PDL1 antibodies have been approved for multiple scale) and program outcomes, including program satisfaction. Individ- tumor types based on their clinical benefit, however they can be as- uals with cancer (n=478) comprised 68.0% of participants in the work- sociated with a unique set of toxicities termed immune related ad- shop. These attendees were White (81.7%), female (79.6%), and verse events (irAEs). We aimed to identify the incidence and clinical averaged 62.6 years old [1]. Workshop outcomes were ascertained by manifestations of patients who developed neurologic irAEs with ICIs descriptive analyses and ANOVAs. at our institution in order to inform investigation and management Results guidelines. Over half of workshop attendees with cancer (53.0%) reported generally Methods being very involved in their overall treatment decision-making, yet 47.2% An IRB approved retrospective study involved review of charts and had been unsure whether immunotherapy was a treatment option for institutional databases at MSKCC. We identified patients who devel- them[m1]. [AKZ2] About one-third (36.8%) had spoken with their doctor oped neurotoxicity while on at least one of the following ICIs: anti- about immunotherapy and 26.2% inquired about potential side effects of CTLA4, anti-PD1 or anti-PD-L1 over a 6 year period (1/1/2010-11/16/ immunotherapy; 44.1% had searched for information about immunother- 2016). apy on their own before the workshop. The majority (74.6%) reported Results low knowledge about immunotherapy before the workshop. We identified a total of 3,804 patients who were treated with one or After the workshop, most attendees with cancer reported a high level of more ICI during the period of review. Neurotoxicity was observed in knowledge of immunotherapy (57.8%), representing a significant gain 81 patients (2.1%) and affected both central and peripheral nervous from attending the program (F (4,416) = 39.2, p <.01). Post-workshop, systems. 31 patients (38.2%) received more than one ICI. Median most (86.5%) felt confident speaking about immunotherapy with their number of cycles prior to developing toxicity was 3 (1-29). Ten doctors. Furthermore, a majority felt “more confident” discussing potential patients had more than one neurotoxicity. The various neurologic side effects (86.3%), asking about treatment options generally (90.3%), phenotypes observed in patients included: sensory neuropathy, en- and making decisions with their doctors (84.8%) post-workshop. Nearly cephalopathy, headache and aseptic meningitis, myasthenia gravis- all (92.5%) strongly or moderately recommend attending the workshop. like syndrome, myopathy, autonomic neuropathy, radiculopathy, Conclusions brachial plexitis, mononeuritis multiplex, Guillain-Barre syndrome or Results from the attendee evaluations demonstrate significant informa- chronic inflammatory demyelinating polyneuropathy, paraneoplastic tional and confidence gains from attending a two-hour psychoeducational neurologic disorder, and PRES. 43 patients required hospital admis- workshop. Attendees reported increased awareness of options, knowledge, sion and 2 required ICU level of care. A diagnosis of neurotoxicity and efficacy in discussing potential options for them. Results suggest that was based upon the temporal association with ICIs and appropriate improving access to comprehensive information about immunotherapy workup including but not limited to neurologic consultation, lumbar and promoting communication between the patient and the healthcare puncture, and neuroimaging. Patients were treated with drug holi- team about treatment options and decision-making via an educational day, observation, corticosteroids, plasma exchange and/or IVIG. workshop is viable and can be implemented nationally to reach many. Conclusions As ICIs are being used with increased frequency, oncologists and References other health care providers should be aware of the varied manifesta- 1. Cancer Support Community. CSC Immuno-Oncology online survey. 2014. tions of neurotoxicity in order to ensure appropriate diagnosis, work up and treatment. Prompt treatment may relay a benefit in regards to outcome. Tumor Microenvironment (Mechanisms and Therapies) PHYSICIAN/NURSE/PHARM: Patient Experience: P451 Alpha-TEA demonstrates antitumor activity against trastuzumab Patient Education Regarding I sensitive and resistant breast cancer 1 2 2 1 P450 Emmanuel Akporiaye , Mary Disis , Ekram Gad , Tobias Hahn , 3 1 4 5 Educating cancer patients about immunotherapy: gains from Brook Cole , Michael Gough , Talicia Savage , Hailing Lu 1 2 attending a national educational immunotherapy workshop Providence Portland Medical Center, Portland, OR, USA; University of Washington School of Medicine, Seattle, WA, USA; University of Claire Saxton, Heather Hollen, Marni Amsellem Cancer Support Community, Washington, DC, USA Minnesota, Minneapolis, MN, USA; Albert Einstein College of Medicine, Correspondence: Claire Saxton Bronx, NY, USA; Immune Design, Seattle, WA, USA (hhollen@cancersupportcommunity.org) Correspondence: Emmanuel Akporiaye (etakporiaye@gmail.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P451 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P450 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 219 of 244 Background adenocarcinoma cell line. After two weeks tumors were stained for Alpha-tocopheryloxyacetic acid (α-TEA) is an orally administered, small myeloid cell surface markers and analyzed via flow cytometry. Distribu- molecule, multi-pathway cancer therapeutic agent that drives tumor tion of myeloid cell populations in tumors were also monitored via con- + + cell apoptosis and autophagy to stimulate antigen cross-presentation. focal laser-scanning microscopy of IL-15 reporter mice. CCR2 IL-15 Anti-tumor activity of α-TEA in combination with trastuzumab was eval- double reporter mice were used to further characterize myeloid cells. uated in vivo against established Her2/neu-expressing mouse tumors To investigate the cellular source of sIL-15 complexes, we used mice (MMC) and in vitro against HER2 positive, human trastuzumab-sensitive with a conditional deletion of IL-15Rα from DCs (CD11c-Cre x IL-15Rαfl/ (BT-474) and trastuzumab-resistant (HR6) tumor cells. fl) or macrophages (LysM-Cre x IL-15Rαfl/fl); or mice were treated with Methods anti-Ly6G depleting antibodies. In vivo studies: Alpha-TEA was formulated in mouse chow and adminis- Results tered to mice ad libitum. FVB/N-TgN (MMTVneu) on nutrient-matched In both IL-15 reporter lines, we found that the EGFP expressing cells in control chow mice received s.c. implant of 1 million Her2/neu positive the tumor microenvironment are composed of three main populations: + hi - + + + + -/lo - murine mammary cancer (MMC) cells. On day 25, after tumors were CD11b Ly6C Ly6G,CD11b Ly6C Ly6G and CD11b Ly6C Ly6G cells + hi + established, mice were transferred to α-TEA diet, and treatment with with the CD11b Ly6C CCR2 cells being the most abundant. Similar anti-Her2/neu monoclonal antibody (7.16.4) was initiated. Mice received composition of EGFP myeloid populations was also seen in MCA-205 3 injections of 7.16.4 antibody 3 times a week for 1 month and tumor and MC-38 tumors. Imaging studies of MCA-205 tumors showed that + + + growth was monitored until day 60. Mice demonstrating complete the majority of the F4/80 or CCR2 cells were also EGFP . In MCA-205 + + + tumor regression were challenged with viable tumor cells to assess the tumors, EGFP expression was decreased in CD11b Ly6C Ly6G cells establishment of a memory response. Spleen cells were removed from compared to the spleen. Analyses with conditional knockout mice or treated mice and assessed for cytokine production by ELISPOT assay. depleting antibodies showed that the monocytic cells and macro- In vitro studies: Human trastuzumab-sensitive (BT-474) and a resist- phages are major sources of sIL-15 complexes in early B16 tumors. ant sub-clone (HR-6) were treated continuously with α-TEA alone or Conclusions in combination with trastuzumab, both at various doses and evalu- In the tumor microenvironment, IL-15 is primarily produced by in- ated for clonogenic potential and activation of ERK and AKT signaling flammatory monocytes and macrophages in early tumors and is pathways. down regulated with tumor growth. Results Treatment of mice harboring established MMC tumors with α-TEA References plus trastuzumab caused complete tumor regression, and increased 1. Mlecnik B, et al. Functional Network Pipeline Reveals Genetic Determinants cytokine production by splenic lymphocytes. Individually, α-TEA and Associated with in Situ Lymphocyte Proliferation and Survival of Cancer trastuzumab caused cell death and reduced colony formation of BT- Patients. Sci Transl Med. 2014; 6228:228ra37. 474 tumor cells in vitro; however only α-TEA was able to kill and re- duce the clonogenicity of the trastuzumab-resistant HR6 cell line. Furthermore, α-TEA-mediated cell death of HR6 tumor cells involved inhibition of AKT and ERK phosphorylation. Treatment with α-TEA did P453 not affect the expression of HER2/neu by both cell lines. Targeted tumor stroma depletion in combination with a whole cell Conclusions tumor vaccine enhances effector T cell infiltration in a mouse model of pancreatic cancer 1 1 1 1 Alex Blair , Victoria Kim , Stephen Muth , Todd Armstrong , Alpha-TEA synergizes with trastuzumab to cause regression of 2 1 Sanna Rosengren , Lei Zheng established Her2/neu-expressing tumors. 1 2 Johns Hopkins Medical Institution, Baltimore, MD, USA; Halozyme Anti tumor activity of α-TEA is due to apoptosis induction and Therapeutics, San Diego, CA, USA suppression of AKT and ERK signaling pathways. Correspondence: Lei Zheng (ablair9@jhmi.edu) Alpha-TEA represents a potential opportunity for combination Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P453 treatment with Herceptin in HER2 positive breast cancer. Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with P452 dismal prognosis due to conventional therapy resistance. The tumor Defining IL-15 expressing myeloid cells in the tumor microenvironment (TME) of PDAC is characterized by a desmoplastic re- microenvironment action that excludes effector immune cell infiltration, thus preventing Figen Beceren-Braun, Rosa M. Santana Carrero, Sarai C. Rivas, Kimberly S. optimal efficacy of immunotherapy. A human recombinant hyaluroni- Schluns dase (PEGPH20) has been clinically formulated to enzymatically deplete University of Texas MD Anderson Cancer Center, Houston, TX, USA hyaluronan (HA) in the tumor extracellular matrix. We tested the hypoth- Correspondence: Figen Beceren-Braun (fbeceren@mdanderson.org) esis that combining the stromal targeting agent PEGPH20 with a GM- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P452 CSF secreting allogenic pancreatic tumor vaccine (GVAX) would increase immune cell infiltration and improve survival of PDAC bearing mice. Background Methods Loss of IL-15 within colorectal tumors correlates with higher risk of C57BL/6 mice were orthotopically transplanted with 2 x 10 murine relapse, decreased survival, lower T cell density and decreased T KPC pancreatic tumor cells to form liver metastases by a hemisple- cell proliferation [1] suggesting IL-15 expression within the tumor is nectomy technique on day 0. Following tumor transplantation, mice important for anti-tumor responses. Furthermore, we have found were treated subcutaneously with GVAX in combination with intra- that soluble IL-15 (sIL-15) complexes are produced in early tumors venous PEGPH20 or the appropriate control. PEGPH20 was given on of mice implanted with tumor cell lines. However, the cell types day 6 and GVAX on post-operative day 7. An immune modulating expressing IL-15 and the mechanisms regulating IL-15 responses in dose of Cyclophosphamide (Cy) was given the day before GVAX in the tumor microenvironment are not known. The goal of this study all groups. Tumor immune lymphocyte infiltration was assessed by was to identify myeloid cell populations expressing IL-15 in the immunohistochemistry and flow cytometry. RT-PCR was performed tumor microenvironment and investigate the mechanisms of on tumor associated macrophages isolated with CD11b beads. Mice expression. were sacrificed on day 16 for immune and gene expression analysis. Methods Additional survival analysis followed mice until death. Both IL-15 transcriptional and translational EGFP (enhanced green Results fluorescent protein) reporter mice were injected subcutaneously Combination therapy with PEGPH20 and GVAX yielded higher infiltra- with 3x10 B16 melanoma, MCA-205 fibrosarcoma or MC-38 murine tion of effector CD8+ and CD4+ tumor infiltrating lymphocytes in the Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 220 of 244 TME compared to untreated and monotherapy groups (all p<0.05) “concomitant immune tolerance” (CIT), which drastically reduces the (Fig. 1). Murine survival was improved in the combination therapy efficacy of in situ vaccination. We have shown that CIT is mediated, group as compared to GVAX therapy alone (Hazard Ratio: 0.286 in part, by tumor-infiltrating regulatory T cells (Tregs) and that CIT [0.10-0.82] p=0.02). Additionally, a larger percent of tumor-infiltrating can be overcome by delivering 12Gy EBRT to both tumors. Clinically, CD8+ cells were noted to express the lymphocyte activation surface it is not feasible to deliver this dose to all sites of metastatic disease. marker CD137 in combination therapy compared to monotherapy Systemically delivered molecular targeted radiotherapy (MTRT) with controls. A trend towards lower CXCR4 expression was noted in the the tumor-selective alkyl phosphocholine analog I-NM404 could combination therapy group, suggesting a possible mechanism to overcome this challenge, and potentially deplete immunosuppressive CD8+CD137+ infiltration beyond decreased vascular impedance. cells responsible for CIT in practically all sites of metastasis. We evalu- Conclusions ated doses of EBRT to distant tumor sites needed to eliminate CIT, Our preclinical murine model of PDAC demonstrates enhanced effi- performed dosimetry to determine the administered activity of I- cacy of GVAX in combination with the stromal targeting agent NM404 that approximates this EBRT absorbed dose, and tested PEGPH20 with increased immune cell infiltration and a survival whether systemic administration of MTRT may overcome CIT in a advantage. This study provides rationale for further study of this syngeneic murine melanoma model. combination for PDAC treatment. Methods Total number of live (A) CD8+ and (B) CD4+ tumor infiltrating lym- We monitored tumor volume, survival, and infiltrating lymphocyte phocytes after KPC hemispleen surgery and indicated therapy. Data populations in primary and secondary tumors treated with EBRT+IT- represent mean ± SEM from one representative experiment with IC+/-intravenous MTRT in C57BL/6 mice bearing one or two three mice per group that was repeated twice. (C) Kaplan-Meier sur- ~250mm syngeneic GD2+ B78 melanoma flank tumors. vival curves of mice that were implanted with PDAC cells and treated Results with Cy/GVAX with and without combination of PEGPH20. * = p. We demonstrate that CIT can be substantially attenuated using low- dose (2Gy) EBRT to the secondary tumor. This lower dose is not ad- equate to generate an in situ vaccine response but appeared com- parable to 12Gy in its capacity to overcome CIT. Using longitudinal I-NM404 PET/CT imaging with Monte Carlo dosimetry, we esti- mated that 60 μCi of I-NM404, a dose which does not destroy B78 tumors, given IV provides approximately 2Gy to both tumors in ani- mals bearing two B78 tumors. Following 60μCi of I-NM404 to mice with two B78 tumors, we observed depletion of tumor-infiltrating Tregs by IHC staining over a 4-week period, without bone marrow suppression (thrombocytopenia/leukopenia/anemia). We observed that pretreatment with systemic I-NM404 one week prior to 12Gy EBRT+IT-IC to a single tumor site may substantially attenuate CIT and rescue a systemic response to in situ tumor vaccination. Conclusions We demonstrate that 2Gy EBRT is sufficient to attenuate CIT, and that this dose can be approximated using systemic MTRT. This work sug- gests that MTRT may enable conditioning of the tumor immune microenvironment to overcome intrinsic immune evasion mecha- nisms and restore efficacy of in situ tumor vaccination. P455 Investigating the specificity and response of a native leukemia- reactive CD8 T cell clone 1 1 1 2 Xiufen Chen , Brendan MacNabb , Emily Curran , Bruce Blazar , Justin Kline 1 2 Fig. 1 (abstract P453). Combination therapy of PEGPH20 with University of Chicago, Chicago, IL, USA; University of Minnesota, Cy/GVAX improves immune cell infiltration and survival in a PDAC Minneapolis, MN, USA mouse model Correspondence: Justin Kline (xchen82@bsd.uchicago.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P455 Background P454 We previously reported that high-affinity, antigen-specific CD8 T Molecular targeted radiotherapy facilitates in situ vaccination in a cells were induced to undergo abortive proliferation and deletion in syngeneic murine melanoma model leukemia-bearing hosts, and have subsequently found that this dele- 1 1 1 1 Peter M Carlson , Clinton Heinze , Joseph Grudzinski , Reinier Hernandez , tional CD8 T cell tolerance requires cross-presentation of the 2 3 1 1 Stephen D Gillies , Hans Loibner , Alexander L Rakhmilevich ,Mario Otto , leukemia antigen by splenic Batf3-lineage dendritic cells. 1 1 1 1 Bryan Bednarz , Jamey Weichert , Paul M Sondel , Zachary S Morris Methods University of Wisconsin School of Medicine and Public Health, Madison, Recently, we generated a T cell receptor (TCR) transgenic mouse 2 3 WI, USA; Provenance Biopharmaceuticals, Carlisle, MA, USA; Apeiron strain (referred to as Tg101) that expresses the TCR-α and -β chains Biologics, Vienna, Austria of a CD8 T cell clone specific for a native leukemia antigen. Correspondence: Zachary S Morris (pcarlson2@wisc.edu) Results Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P454 Having confirmed that Tg101 CD8 T cells specifically recognize C1498 acute myeloid leukemia (AML) cells in the context of the MHC class I Background molecule, H-2D , we investigated the behavior and fate these cells in We reported that 12Gy external beam radiation (EBRT) and intratu- naïve and C1498 leukemia-bearing hosts. In naïve mice, Tg101 thymo- moral (IT) administration of the hu14.18-IL2 immunocytokine (IC) [an cytes predominantly developed along the CD8 lineage, and existed as anti-disialoganglioside (GD2) mAb fused to IL2] five days later elicits naïve CD8 T cells in peripheral lymphoid organs, suggesting that the an in situ vaccination effect in GD2 murine tumors. An established Tg101 antigen is likely leukemia-specific. In leukemia-bearing mice, distant, untreated secondary tumor exerts an antagonistic effect, antigen encounter by Tg101 T cells was delayed (day 10-12), and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 221 of 244 primarily occurred in the liver - a prominent site of leukemia progres- exemplified by up-regulation of CD137, GITR, TIM3, PD-1, PD-L1, and sion. Subsequently, Tg101 cells expanded and upregulated expression TIGIT on the surface of both CD4+ and CD8+ T cells correlating with of co-inhibitory receptors, including PD-1, LAG-3, TIM-3 and TIGIT, gene expression changes associated with T cell activation phenotype which correlated with a progressively dysfunctional phenotype. Surpris- (upregulated Granzyme B, CD137, OX40, TNF-alpha, IL2R, IP10, ICAM- ingly, antigen encounter by Tg101 T cells in leukemia-bearing mice oc- 1) with only a modest concentration-dependent decrease in T cell ex- curred independently of Batf3-lineage DCs, suggesting that the Tg101 pansion. Furthermore, intermittent exposure of T cells to abemaciclib antigen was poorly cross-presented in vivo. Given this finding, we de- during various stages of expansion revealed that T cells were capable leted H-2D in C1498 cells to determine the extent to which direct anti- of recovering from abemaciclib-induced growth inhibition whereas gen presentation by leukemia cells is responsible for the acquisition of expression of costimulatory/coinhibitory molecules was maintained dysfunctional properties in Tg101 T cells in vivo. In vitro expansion was once the drug was removed. 2-fold decreased in Tg101 T cells compared to high-affinity 2C CD8 T Conclusions cells (which recognize the SIY peptide in the context of H-2K )upon Taken together, these results demonstrate that abemaciclib can culture with SIY-expressing C1498 cells (C1498.SIY), indicative of a lower exert T cell-intrinsic effects at concentrations comparable to clin- avidity of Tg101 vs the 2C T cells for antigen. In contrast to Tg101 T ical exposure. This is exemplified by increased T cell activation cells, 2C T cells encountered leukemia antigen rapidly (day 3-4) in the phenotype with only a modest and reversible decrease in T cell context of splenic CD8a DCs, proliferated briefly, and were effectively expansion, providing further rationale for clinical exploration of deleted from the host by day 12. combination studies with immunomodulatory agents such as PD-1 Conclusions pathway inhibitors. Collectively, our data reveal that CD8 T cell tolerance is effectively generated in the context of AML. However, the nature of the P457 leukemia antigen, as well as the context in which it is presented, are Pharmacodynamic activity of MEDI9197, a TLR7/8 agonist, critical determinants in the underlying mechanism of tolerance that administered intratumorally in subjects with solid subcutaneous or ensues. cutaneous tumors 1 1 1 1 Zachary A. Cooper , Christopher A. Morehouse , Yuling Wu , Keith Steele , 2 1 1 1 P456 David Brucker , Leo Tseng , Maria Jure-Kunkel , Nancy Mueller , 1 3 4 5 CDK4 & 6 inhibitor Abemaciclib exerts intrinsic Patricia Ryan , Juneko Grilley-Olson ,David Hong , Aurélien Marabelle , 6 6 5 3 immunomodulatory effects on human T cells in vitro Pamela Munster ,RahulAggarwal , Sandrine Aspeslagh , Robert G. Dixon , 1 1 1 2 7 4 8 7 Darin Chin , Carmine Carpenito , Nelusha Amaladas , Linda Chung , Kirk Manish Patel , Vivek Subbiah ,Joshua Brody ,ShilpaGupta 2 3 2 1 1 2 Staschke , David Schaer , Richard Beckmann , Ruslan Novosiadly , MedImmune, Gaithersburg, MD, USA; Definiens, Cambridge, MA, USA; 1 3 Michael Kalos University of North Carolina Lineberger Comprehensive Cancer Center, 1 2 4 Eli Lilly & Co, New York, NY, USA; Eli Lilly & Co, Indianapolis, IN, USA; Chapel Hill, NC, USA; MD Anderson Cancer Center, Houston, TX, USA; 3 5 6 Eli Lilly & Co, Brooklyn, NY, USA Institut Gustave Roussy, 94800 Villejuif, France; UCSF Helen Diller Correspondence: Carmine Carpenito (chin_darin@lilly.com); Kirk Family Comprehensive Cancer Center, San Francisco, CA, USA; Masonic Staschke, David Schaer, Richard Beckmann, Ruslan Novosiadly, Michael Cancer Center, Minneapolis, MN, USA; Mount Sinai (Hess Center for Kalos Science and Medicine), New York, NY, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P456 Correspondence: Zachary A. Cooper (robert.mulvey@ashfieldhealthcare.com) Background Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P457 Abemaciclib is an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 & 6) that suppresses DNA synthesis and cell proliferation Background as result of cell cycle arrest in G1 phase. Significant clinical activ- MEDI9197 (formerly 3M-052), is a novel TLR7/8 dual agonist formulated ity of abemaciclib has been observed in hormone receptor- for intratumoral (IT) injection and optimized for retention within the positive breast cancer patients highlighting the therapeutic po- tumor after IT injection. IT administration aims to focus antigen presen- tential of this compound in oncology, including combinations tation in the tumor by activation of both plasmacytoid (TLR7 express- with other therapeutic modalities. In preclinical studies, pretreat- ing) and conventional dendritic (TLR8 expressing) cells, thus minimizing ment with abemaciclib followed by PD-L1 checkpoint blockade systemic inflammatory toxicities. We report the pharmacodynamic re- has demonstrated synergistic immune activation and antitumor sults of MEDI9197 from a phase I, first-in-human, dose-escalation study efficacy. This has generated interest in combination studies of in patients with solid tumors (NCT02556463). abemaciclib with immunotherapeutic agents. However, the mech- Methods anisms underlying immunomodulatory activity of abemaciclib The dose-escalation trial of MEDI9197 (0.005 to 0.055 mg) has en- have not been fully elucidated. Consequently, the objective of rolled 24 patients with subcutaneous/cutaneous tumors. As reported this study was to understand the direct effects of abemaciclib on at AACR 2017, the maximum tolerated dose is 0.037 mg and IT ad- primary human T cells. ministration is feasible and safe. Here we present updated tumoral Methods and peripheral pharmacodynamic effects in patients treated with Jurkat and primary human T cells were stimulated in vitro with anti- MEDI9197 0.005, 0.012, and 0.037 mg. CD3/CD28 and treated with increasing concentrations of abemaciclib Results ranging between 0 uM to 1 uM for up to ten days. Cells were har- Local pharmacodynamic effects assessed by immunohistochemis- vested at the end of the experiments and nuclear factor of activated try (IHC) in longitudinal biopsies demonstrated the majority of T cells (NFAT) activation, cell surface immune markers (costimulatory patients treated with 0.037 mg had an increase (≥2fold) inCD8, and coinhibitory receptors and ligands) and expression of immune- CD40, CD56, or PD-L1 (tumor and immune cells) markers 3 weeks related genes were analyzed by signal reporter assay, flow cytometry after a single dose of MEDI9197. RNAseq analysis of paired tumor and Quantigene Plex, respectively. biopsies showed an increase in TLR7/8-downstream regulated Results genes and innate and adaptive immune activation signatures Treatment of Jurkat T cells showed a dose dependent increase in such as Type 1 IFN, IFNγ and T effector signatures (≥1.5 fold) NFAT reporter activation during abemaciclib treatment which was consistent with IHC. replicated in primary human T cells. Additionally, treatment of pri- Increases in peripheral levels of IFNγ, CXCL10, and CXCL11 were mary T cells with abemaciclib resulted in a distinct phenotype observed across all 3 doses (0.037, 0.012, and 0.005 mg). Within Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 222 of 244 24 hours of IT administration of MEDI9197 0.037 mg, median monotherapy and control group. However, anti-PD1 + NOS-inhibition peak values of IFNγ, CXCL10, and CXCL11 observed were 236, resulted in a significant reduction in tumor growth. In immune- 9286, and 558 pg/mL respectively. The fold change increase humanized TNBC PDXs, 40% responded to anti-PD1 monotherapy, ranged from 4.5-43, 30-132, and 3.7-52 vs. baseline for IFNγ, 60% had improved response following combination therapy, and CXCL10, and CXCL11 respectively. Peripheral levels of IFNγ 33% responded only to combination therapy. In total, 66% of PDXs showed a significant trend for a dose response with median peak analyzed benefited from combination therapy. Furthermore, both in- values for those treated with MEDI9197 0.005 mg 4.1-fold less vitro and in-vivo samples showed downregulation of ADAR1 levels than those treated with 0.037 mg. Analysis of whole blood micro- when treated with NOS inhibition, with a subsequent increase in array data across all doses demonstrated increases (≥2fold) in HMGB1, a marker of immunogenic cell death. T 1 and Type 1 IFN gene expression signatures with a transient Conclusions decrease (≥1.5 fold) in CD8A transcript and NK cell signature ex- NOS inhibition upregulates PDL-1 expression in TNBC cell lines. Fur- pression, suggesting trafficking of T and NK cells. thermore, combining NOS inhibition with anti-PD1 therapy improved Conclusions antibody response by activating the immunogenic cell death path- IT administration of MEDI9197 induces Type 1 and 2 IFN as well as way. This combination treatment may benefit cancer patients who T 1 responses, suggesting activation of both plasmacytoid and con- do not respond to anti-PD-1 monotherapy. ventional dendritic cells. A combination trial of MEDI9197 with durva- lumab (anti-PD-L1) and radiation in patients with advanced solid P459 tumors is currently ongoing. Class IIa HDAC inhibition promotes an anti-tumor macrophage Trial Registration phenotype that induces breast tumor regression and inhibits NCT02556463. metastasis 1 1 1 1 Jennifer Guerriero , Alaba Sotayo , Holly Ponichtera , Jessica Castrillon , 1 1 1 1 P458 Alexandra Pourzia , Sara Schad , Shawn Johnson , Ruben Carrasco , 1 2 3 3 Nitric oxide synthase inhibition results in PDL-1 upregulation and Suzan Lazo , Roderick Bronson , Scott Davis , Mercedes Lobera , 3 1 ADAR1 inhibition, triggering immunogenic cell death: an in-vitro Michael Nolan , Anthony Letai 1 2 and in-vivo analysis Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical Daniel Davila-Gonzalez, Roberto Rosato, Wei Qian, Anthony Kozielski, School, Boston, MA, USA; GlaxoSmithKline, Cambridge, MA, USA Wen Chen, Dong Soon Choi, Patrick Tucker, Jenny Chang Correspondence: Jennifer Guerriero Houston Methodist, Houston, TX, USA (Jennifer_Guerriero@dfci.harvard.edu) Correspondence: Jenny Chang Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P459 (ddavilagonzalez@houstonmethodist.org) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P458 Background The breast tumor environment is complex and includes both neoplastic Background and immune cells. Among the most prevalent immune cell population Response failure in checkpoint inhibitors, such as death ligand 1 within breast tumors is tumor associated macrophages (TAMs). Macro- (PD-L1) and its receptor programmed death 1 (PD-1), is associated phages have the ability to polarize into either M1 cells, which have po- with cancer-related immunosuppression. Inducible nitric oxide syn- tent anti-tumor capabilities, or M2 macrophages which promote tumor thase (iNOS) product, nitric oxide, has been implicated in immuno- progression by stimulating tumor vasculature, invasion, metastasis, and suppressive environments. In triple negative breast cancer (TNBC), can enhance tumor resistance to chemotherapy. Generally TAMs in iNOS expression is associated with poor survival and increased breast tumors are considered M2 macrophages and a high tumor dens- tumor aggressiveness. We’ve shown ADAR1 (adenosine deaminase ity of TAMs clinically correlates to both overall worse prognosis and in- acting on RNA 1) is upregulated by iNOS activation in TNBC cell creased metastasis. Several therapeutic strategies exist to modulate lines. ADAR1 RNA editing suppresses inflammation, thereby damp- TAMs clinically, focusing on depleting or inhibiting TAMs. However, ing the immune response. This study aims to evaluate whether macrophage are required for optimal tumor regression in response to NOS inhibition plus anti-PD-1 is a feasible therapeutic combination both chemotherapy and immunotherapy. Their embedded location in TNBC. and their untapped potential provide impetus to the discovery of strat- Methods egies to turn them against tumors and to harness for cancer therapy. TNBC cell lines BT-549, SUM-159, SUM-157, HCC-70, MDA-MB-231 Therefore here, we describe a novel method to polarize pro-tumor and MDA-MD-468 were treated with NOS inhibition therapy (L- macrophages to an anti-tumor phenotype. NMMA, 4mM) + amlodipine (5μM). PDL-1, HMGB1, and ADAR1 ex- Methods pression was assessed via western blot. BALBc mice growing orthoto- We recently reported that a first in class selective class IIa HDAC in- pically injected 4t1 cells were treated weekly with: 1) vehicle (saline, hibitor (TMP195) influenced human monocyte responses to colony oral/Rat-IgG2 i.p.); 2) NOS inhibition [L-NMMA (200mg/kg oral) + stimulating factors CSF-1 and CSF-2 in vitro. Here, we utilize a amlodipine (10mg/kg, i.p.), days 1-5]; 3) anti-PD-1 antibody (10 mg/ macrophage-dependent autochthonous mouse model of breast can- kg i.p.; Bio X Cell Clone: RMP1-14, days 1, 3, 5); or 4) anti-PD-1 + NOS cer to demonstrate that in vivo TMP195 treatment alters the tumor inhibition. Humanized mice were developed by injecting microenvironment and reduces tumor burden and pulmonary metas- hematopoietic stem cells (HSC, CD34+) into the tail vein of irradiated tases through macrophage modulation. NOD-scid IL2Rγnull mice. Mice were sorted and TNBC patient-derived Results xenografts (PDXs), representing 12 different patients, were implanted Here, we demonstrate that a first in class, class IIa histone deacetylase into the mammary fat pad (3 mice/PDX). Mice were sorted and (HDAC) inhibitor, TMP195, can activate tumor macrophages in vivo to treated weekly with: 1) vehicle; 2) pembrolizumab (anti-PD-1, 200μg, induce tumor regression and inhibit pulmonary metastases in a mouse day 1, IV); or 3) pembrolizumab + NOS inhibitor. model of breast cancer. We find that TMP195 induces macrophage re- Results cruitment and differentiation of highly phagocytic cells within the NOS inhibition increased PD-L1 in most cell lines. 4T1 syngeneic tumor, which increases tumor cell death while decreasing angiogenesis. model helped assess the potential benefit of combined therapy. Strikingly, we find that TMP195 enhances chemotherapy and immuno- There were no significant differences in tumor growth between therapy to induce durable tumor reduction. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 223 of 244 Conclusions P461 Our findings reveal an immunostimulatory effect of class IIa HDAC in- Listeria monocytogenes-based immunotherapies alter the hibition that contrasts with strategies of depleting or inhibiting TAMs suppressive phenotype of intratumoral regulatory T cells for cancer therapy. Class IIa HDAC inhibition leverages the effector Rachelle E. Kosoff, Nithya Thambi, Lauren K. Pettit, Daniel O. Villarreal, functions of macrophages, opening the door to clinically-relevant co- Kimberly Ramos, Michael F. Princiotta, Robert G. Petit, Sandra M. Hayes operation of checkpoint blockade, agonistic aCD40 or inhibitory Advaxis, Inc., Princeton, NJ, USA aCD47 therapy, peptide vaccine therapy or the antibody-dependent Correspondence: Sandra M. Hayes (hayes@advaxis.com) cellular phagocytosis (ADCP) associated with mAb therapy. Indeed, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P461 the innate immune system is the natural complement to the adap- tive immune system that surveys and fights tumors, and these stud- Background ies demonstrate a novel approach to harness innate immune cells to Advaxis’ Listeria monocytogenes (Lm)-based immunotherapies are live cooperate with agents that stimulate an adaptive anti-tumor immune attenuated bacterial vectors that elicit tumor-reactive T cells through response in an otherwise resistant cancer. the delivery of tumor-associated antigens directly to dendritic cells. In addition, our Lm-based immunotherapies reduce the frequency and function of intratumoral regulatory T cells (Tregs), thereby coun- P460 teracting the immunosuppressive tumor microenvironment (TME) Selenium, the element of the moon, improves immunotherapies and sustaining the newly generated antitumor immune response. on earth The purpose of this study is to investigate further the mechanism by 1 1 1 2 Claudia Lennicke , Shamim Ahmad , Rajeev Shrimali , Barbara Seliger , which Advaxis’ Lm-based immunotherapies alter Treg function. 3 1 1 1 Youcef Rustum , John Janik , Mikayel Mkrtichyan , Seema Gupta , Methods Samir Khleif C57BL/6 mice were implanted with TC-1 tumor cells, which express the 1 2 Augusta University, Augsuta, GA, USA; Institute of Medical human papillomavirus (HPV)16 E6 and E7 proteins. On day 8, when tu- Immunology, Halle, Germany; University of Buffalo, Buffalo, NY, USA mors were palpable, mice were treated with either one dose of ADXS- Correspondence: Samir Khleif (segupta@augusta.edu) HPV-Quad, an Lm-based vector targeting the E6 and E7 proteins from Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P460 both HPV16 and HPV18, or PBS. Five days post-treatment, tumor infil- trating lymphocytes were phenotyped by flow cytometry and intact tu- Background mors were analyzed by immunofluorescence microscopy. Recent advances in immunotherapy have shown amazing results in Results cancer patients, albeit in small fraction. Attempts in developing im- We reasoned that Lm-based immunotherapies, to be effective at tumor munotherapy to generate effective response against tumors are still control, must reprogram the immunosuppressive TME early during the faced with immune suppressive environment. The abundance of course of treatment in order to induce and sustain infiltration of tumor- tumor-infiltrated T regulatory cells (Tregs) highly is one of the main specific T cell effectors. Accordingly, we analyzed the TME 5 days after mechanism by which tumors overcome immune responsiveness. a single dose of ADXS-HPV-Quad. By flow cytometry, we compared the Tregs also correlates with poor patient prognosis. proliferative status and viability of Tregs in tumors of mice treated with Selenium (Se), an essential trace element named after the goddess of either PBS or ADXS-HPV-Quad. The percentage of proliferating (Ki-67 ) the moon, is described to exhibit anti-carcinogenic potentials [1]. Tregs was comparable between the two treatment groups, but the However, the underlying mechanisms of Se action are insufficiently percentage of Tregs undergoing apoptosis was 2-fold higher in ADXS- understood. Since Se is also known to exhibit immune-modulatory HPV-Quad-treated mice than in PBS-treated mice. Further phenotypic properties [2] we hypothesized that Se in combination with vaccine- analysis revealed that the percentage of Tregs expressing CCR8, a che- based immunotherapy might enhance anti-tumor responses. mokine receptor whose expression is essential for Treg survival and Methods function, was almost 2-fold lower in ADXS-HPV-Quad-treated mice than TC-1 tumor-bearing C57BL/6 mice were treated either with Vaccine (E7, in PBS-treated mice. Expression levels of other Treg phenotypic th 100 μg/mouse; PADRE, 20 μg/mouse; every 7 day), Methylselenocys- markers, namely Foxp3 and CD39, were also reduced in the CCR8- teine, as source of Se (MSC, 100 μg/mouse; daily) or with a combination negative Tregs. The phenotypic changes in intratumoral Tregs were as- of both. In addition, an untreated group served as the control. Spleens sociated with a loss in in vivo suppressive activity, as there was a dra- and tumor tissues were analyzed for T cell subsets via flow cytometry. matic increase in the infiltration of activated cytokine-producing T cell ELISpot assays were performed to determine IFNg production in E7/ effectors into the tumor core of ADXS-HPV-Quad-treated mice but not PADRE restimulated splenocytes. In vitro, CD4+ T cells isolated from into the tumor core of PBS-treated mice. spleens of C57BL/6 mice were cultured under iTreg conditions, treated Conclusions with Se and analyzed via Flow Cytometry and Immunoblotting. Within 5 days of administration, a single dose of ADXS-HPV-Quad al- Results ters the tumor microenvironment by impairing Treg survival and We found that combining Selenium with specific antigen vaccine led function and by promoting effector T cell recruitment and function. to significant enhancement of immune response leading to delay in Changes in the suppressive Treg phenotype, specifically the down- tumor growth thereby prolonging the survival rate of mice. Import- regulation of CCR8 expression, may be a key mechanism by which antly, Se significantly reduced the infiltration of Tregs into the tumor Advaxis’ Lm-based immunotherapies impair Treg function. environment thereby enhancing the CD8+/Treg ratio leading to an increased vaccine-induced immune response. In vitro analysis of iTregs revealed that Se reduces Foxp3 expression and destabilizes P462 Tregs. Ubiquitin-specific protease 6 (USP6) oncogene promotes immune Conclusions cell recruitment in sarcoma 1 2 2 3 1 Here we show for the first time that Se an essential trace element Ian Henrich , Laura Quick , Rob Young , Andre Oliveira , Margaret Chou 1 2 improves vaccine-based immunotherapies thereby opening a new University of Pennsylvania, Philadelphia, PA, USA; Children's Hospital of window for the development of novel strategies to fight against Pennsylvania, Philadelphia, PA, USA; Mayo Clinic, Rochester, MN, USA cancer. Correspondence: Ian Henrich (ihenrich@mail.med.upenn.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P462 References 1. Misra S, Boylan M, Selvam A. Redox-active selenium compounds–from Background toxicity and cell death to cancer treatment. Nutrients. 2015;7:3536–56. The de-ubiquitylating enzyme USP6 is a target of chromosomal trans- 2. Huang Z, Rose AH, Hoffmann PR. The role of selenium in inflammation location and the key etiologic agent in several benign bone and soft and immunity: from molecular mechanisms to therapeutic opportunities. tissue tumors (BSTTs) [1]. We have shown that USP6 drives tumori- Antioxid Redox Signal. 2012;16:705–43. genesis by directly de-ubiquitylating the Jak1 kinase, leading to its Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 224 of 244 stabilization and activation of STAT transcription factors [2]. We re- Using syngeneic murine tumor models implanted into WT and TAM -/- cently found that USP6 is also upregulated in sarcomas, malignancies RTK mice, we FACS isolated MDSCs and analyzed the TAM RTKs’ effect of mesenchymal origin. Activation of the Jak-STAT pathway plays a on MDSC suppressive mechanisms. To complement our genetic key role in modulating the tumor microenvironment, however it is models, we employed a pan-TAM RTK inhibitor, UNC4241, and evalu- not known if or how USP6 affects immune cell infiltration and/or ated MDSC function and tumor growth. composition. We sought to determine USP6 effects on sarcoma Results tumor growth and immune cell migration/infiltration. TAM RTKs and their ligands are significantly (M-MDSCs>20 fold, G- Methods MDSCs>15 fold) upregulated in MDSCs from tumor-bearing mice USP6 was expressed in a doxycycline-inducible manner in the patient- compared to non-tumor bearing controls. MDSCs isolated from -/- derived sarcoma cell lines A673 and RD-ES. USP6 expression levels were tumor bearing TAM RTK mice have significantly diminished argi- confirmed to approximate those in primary patient tumor samples. nase, iNOS and ROS activity compared to WT counterparts. Consist- Results ent with these findings, M-MDSC functional suppression assays Overexpression of USP6 results in the production of several immuno- showed that loss of either mertk or tyro3 yielded tumor-elicited M- modulatory cytokines, most notably CXCL10, CCL5, and CCL20. USP6 MDSCs exhibiting reduced suppressive capabilities in T cell expression led to the hallmarks of a heightened immune response, proliferation and IFN-g ELISPOT assays. We performed tumor and -/- such as enhanced migration, polarization, and activation of several MDSC co-implantation studies and demonstrated that WT and axl + + -/- -/- classes of immune cells, such as CD4 and CD8 T cells, monocytes, MDSCs migrate readily to the TDLN while mertk and tyro3 and macrophages. In murine xenografts, sarcoma cells expressing MDSCs exhibit a migration defect and remain within the tumor, USP6 had attenuated tumor growth and enhanced immune cell infil- suggesting that the TAM RTKs play several roles in MDSC-mediated -/- tration compared to parental lines. Furthermore, analysis of sarcoma inhibition of T cell priming. Adoptive transfer of TAM RTK MDSCs patient datasets revealed that elevated USP6 expression correlated results in diminished B16 melanoma development in syngeneic with an immune cell gene signature, and immunohistochemistry of hosts relative to those receiving WT MDSCs. To evaluate whether + + USP6-translocated tumors revealed a high degree of CD4 , CD8 , inhibition of TAM RTKs has clinical potential, we employed a pan- and CD163 infiltration. TAM inhibitor, UNC4241, and found this agent to suppress MDSC Conclusions arginase, iNOS and ROS activity, all while increasing T cell prolifera- V600E -/- USP6 expression results in the production of several immunomodula- tion and diminishing the growth of a BRAF PTEN melanoma tory cytokines that are known to promote the activation and migra- model. In vivo MDSC ablation reversed the effect of UNC4241 indi- tion of several classes of immune cells such as T cells, monocytes, cating that the mechanism of this agent is MDSC-dependent. and natural killer cells. High USP6 expression correlated with in- Conclusions creased immune cell infiltration in mouse xenografts and patient These results indicate that coordinate TAM RTK action mediates samples. The ability of USP6 to provoke an enhanced immune re- MDSC suppressive capabilities and that their pharmacologic inhib- sponse could potentially serve as a biomarker for susceptibility to ition reverses MDSC-dependent T cell suppression and diminishes immunotherapy. tumor growth. These findings suggest that inhibition of TAM RTKs represents a novel approach for modulating MDSC function and aug- References menting the efficacy of checkpoint inhibitor therapy. 1. Oliveira A, Chou M. The TRE17/USP6 oncogene: a riddle wrapped in a mystery inside an enigma. Front Biosci (Schol Ed).2012;4:321-340.2. P464 2. Quick L, Young R, Henrich I, Wang X, Asmann Y, Oliveira A, Chou M.Jak1- Use of evofosfamide for targeting immune-suppressive hypoxia in STAT3 signals are essential effectors of the USP6/TRE17 oncogene in head and neck squamous cell carcinoma tumorigenesis. Cancer Res. 2016;76(18):5337-47. 1 1 1 2 3 Francis Hunter ,Avik Shome , Way Wong , Pratha Budhani ,Neil Senzer , 4 1 1 1 E. Gabriela Chiorean ,Dan Li , Peter Tsai , Nooriyah Poonawala , 1 5 1 1 P463 Purvi Kakadiya , Maria Kondratyev ,CourtneyLynch , Cho Rong Hong , 1 6 6 6 TAM RTKs facilitate MDSC-mediated tumor immunosuppression Tet-Woo Lee , Dennis Kee , Andrew Macann , Nicholas McIvor , 1 1 1 1 1 1 1 7 7 Alisha Holtzhausen , William Harris , Debra Hunter , Eric Ubil , Kevin Hicks , Stefan Bohlander ,Cristin Print , Stew Kroll , Charles Hart , 1 2 1 1 5 1 2 1 Xiaodong Wang , Douglas Graham , Stephen Frye , H. Shelton Earp Bradly Wouters , William Wilson ,Michael Curran , Stephen Jamieson 1 2 1 2 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Emory University of Auckland, Auckland, New Zealand; MD Anderson Cancer University, Atlanta, GA, USA Center, Houston, TX, USA; Mary Crowley Cancer Research Center, Dallas, Correspondence: H. Shelton Earp (alisha_holtzhausen@unc.edu) TX, USA; Indiana University Melvin and Bren Simon Cancer Center, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P463 Indianapolis, IN, USA; University Health Network, Toronto, ON, Canada; 6 7 Auckland City Hospital, Auckland, New Zealand; Threshold Background Pharmaceuticals, San Francisco, CA, USA The innate immune-regulatory receptor tyrosine kinases Tyro3, Axl Correspondence: Francis Hunter (f.hunter@auckland.ac.nz) and MerTK (TAM RTKs) and their ligands, Gas 6 and Protein S, sup- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P464 press the immune response through various mechanisms, including down regulating M1 cytokines, suppressing antigen presenting cell Background function and decreasing CD8 tumor infiltration. Myeloid-derived Hypoxia is prevalent in head and neck squamous cell carcinoma suppressor cells (MDSCs) are potently immunosuppressive and inhibit (HNSCC), where it limits radiotherapy outcomes and may create an T cell activity via various mechanisms, including increased expression immune-suppressive microenvironment. The hypoxia-activated prodrug of arginase, inducible nitric oxide synthase (iNOS), and the produc- (HAP) evofosfamide (TH-302) targets hypoxia by undergoing oxygen- tion of reactive oxygen species (ROS). sensitive reductive activation (Figure 1). Evofosfamide is being clinically Methods evaluated in combination with ipilimumab in solid tumours, including We studied the expression of TAM RTKs in MDSCs and their respective HNSCC (NCT03098160). The present study investigated the efficacy and roles in MDSC-mediated immune suppression in the setting of cancer. sensitivity determinants of evofosfamide in HNSCC. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 225 of 244 Methods pre-metastatic niche are not well defined. We hypothesize that pri- Case reports were retrieved from an unpublished phase 2 evofosfamide mary tumor growth will alter immune cell infiltration and/or function monotherapy expansion cohort in solid tumours (480 or 575 mg.m on in the pre-metastatic niche. days 1, 8 and 15 of 28-day cycles). A collection of 22 human Methods papillomavirus-negative HNSCC cell lines derived from lesions of vary- Lungs and bone marrow from mice bearing M3-9-M, a metastatic ing TNM stages was assessed for sensitivity to 3 HAPs – evofosfamide, embryonal rhabdomyosarcoma cell line, were harvested at various PR-104A and SN30000 – in addition to cisplatin, 5-fluorouracil and the time points and analyzed by flow cytometry for lymphoid and mye- active metabolite of evofosfamide, bromo-iso-phosphoramide mustard loid populations. (Br-IPM). Reductive activation of evofosfamide in cultured cells was Results measured by LC–MS/MS. The lines were molecularly characterised by Primary tumor progression was correlated with infiltration of immune RNAseq and whole-exome sequencing and their genomic and tran- cells into the lung before detectable metastasis. There was a dramatic + + + + scriptomic features compared to public domain HNSCC clinical samples. increase of CD11b Ly6G and CD11b Ly6C myeloid cells in the lung, Molecular predictors of evofosfamide sensitivity were investigated and a shift in macrophage and dendritic cell populations and pheno- using hierarchical clustering, differential expression and correlation ana- types in the lung and bone marrow. Furthermore, the proportion of -1 + + lyses. The antitumour activity of evofosfamide (50 mg.kg qdx5 for 2-3 both CD4 and CD8 T cells that express PD-1 is significantly enhanced cycles with/without 10 Gy radiation on day 5 of cycle 1) was evaluated in the lung, as is the number of leukocytes expressing PD-L1. in two HNSCC xenografts and two HNSCC PDX models derived in- Conclusions house. Evofosfamide was evaluated in combination with CTLA-4 block- We demonstrate that the immune compartment in the bone marrow ade in the murine SCC-VII model. Hypoxic fraction was assessed using and in pre-metastatic lungs changes significantly in response to pri- pimonidazole. mary tumor growth. The differences in immune populations that were Results observed in the lung and bone marrow were distinct, indicating that Of 5 metastatic or locally-advanced HNSCC patients who received evo- the immune changes occurring at these sites in response to tumor pro- fosfamide after failing standard-of-care, two showed partial responses gression are specific to their unique microenvironment. We postulate and three showed stable disease. Evofosfamide was highly selective for that disrupting the formation of the pre-metastatic niche by targeting hypoxic HNSCC cells and more potent and selective than PR-104A or stromal cell populations combined with immunotherapy could have SN30000. Cell line sensitivity to evofosfamide was correlated with Br- anti-metastatic potential. These studies will provide insight into the im- IPM and cisplatin but not with PR-104A, SN30000 or 5-FU, indicating mune signature of metastatic disease and identify new targets for distinct sensitivity determinants. Evofosfamide sensitivity was associ- therapeutic intervention with the potential to reduce metastasis. ated with the expression of genes relating to proliferation. Accordingly, a proliferation metagene identified subtypes within the cell lines that References were differentially sensitive to evofosfamide. Xenografts chosen on the 1. Rosandra Natasha Kaplan et al. VEGFR1-positive haematopoietic bone basis of putative predictive biomarkers (tumour hypoxia, proliferation marrow progenitors initiate the pre-metastatic niche. Nature. 2005; subtype) showed the expected patterns of response. Two PDX models 438(7069):820-7. were also highly responsive to evofosfamide. SCC-VII was refractory to 2. Amber Jin Giles et al: Activation of Hematopoietic Stem/Progenitor Cells evofosfamide monotherapy but showed increased growth delay when Promotes Immunosuppression Within the Pre–metastatic Niche. Cancer. evofosfamide was combined with CTLA-4 inhibition. Res. 2016;76(6):1335-47. Conclusions 3. Meera Murgai et al. KLF4-dependent perivascular plasticity mediates pre- This study provides a rationale for the clinical evaluation of evofosfa- metastatic niche formation and metastasis. Nat. Med. In Press. mide with immunotherapy and/or radiotherapy in genetically de- fined subsets of HNSCC. P466 Rational design of immuno-oncology biologics with improved P465 safety and efficacy 1 2 2 2 Characterization of immune cells in the pre-metastatic niche in a Margaret Karow , Kurt Jenkins , Miso Park , Parker Johnson , 2 2 2 3 murine model of rhabdomyosarcoma Asaul Gonzalez , Veronica Flesch , Tim Miles , Ulrich Rodeck , Sabina Kaczanowska, Meera Murgai, Daniel Beury, Rosandra Kaplan John Williams 1 2 National Cancer Institute, Bethesda, MD, USA Akriveia Therapeutics, Thousand Oaks, CA, USA; City of Hope, Duarte, Correspondence: Rosandra Kaplan (sabina.kaczanowska@nih.gov) CA, USA; Thomas Jefferson University, Philadelphia, PA, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P465 Correspondence: Margaret Karow (karow@akriveiatx.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P466 Background Tumor metastasis is a critical step in the progression of cancer that is Background associated with patient mortality. Prior to the arrival of tumor cells at The benefit of enhancing anti-tumor immune response has been dem- the metastatic site, hematopoietic stem and progenitor cells expand onstrated by the unprecedented clinical responses of immune check- in the bone marrow and mobilize to the pre-metastatic niche, where point antagonists and cell-based therapies . However, the challenge they differentiate into immunosuppressive myeloid cells [1,2]. Fur- with these therapeutic approaches is that they necessarily involve a sys- thermore, recent data has demonstrated that perivascular cells temic activation of the immune system . This leads to treatment- undergo phenotypic changes in the lung that are dependent on the limiting and often debilitating immune related adverse events (irAEs). transcription factor KLF4 and enhance metastasis in response to Attempts to control irAEs include reducing dosing frequency and dos- tumor-derived factors [3]. Targeting the pre-metastatic niche by ei- ing levels, co-dosing immune suppressants, and drug withdrawal; all of ther antibody-mediated depletion of myeloid cells or inhibition of which may limit the utility of immune therapies . Focusing the activity perivascular cell phenotypic switching significantly decreases metas- of immuno-oncology agents to the tumor microenvironment is ex- tasis [2,3]. However, changes in other immune cell populations in the pected to overcome the limitations posed by irAEs. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 226 of 244 Methods Methods Using anti-mouse CTLA4 antibody 9D9 and anti-mouse PD1 antibody We have developed a structurally unique series of small molecule an- J43 as a proof of concept, we have applied a rational approach to tagonists of CCR4 with cellular potencies in multiple assays (e.g. design molecules that are activated in the tumor microenvironment chemotaxis of primary human T in 100% serum) in the low reg by tumor-associated proteases. In vitro testing using antigen binding double-digit nM range. Moreover, these compounds have excellent by Surface Plasmon Resonance (SPR), antigen-capture ELISA and kin- in vitro and in vivo ADME properties, consistent with convenient oral etic assays provided important information to support the design dosing. These CCR4 antagonists were tested in murine syngeneic process. Candidates were further characterized in vivo in C57Bl/6 tumor models alone and in combination with immunomodulatory mice implanted with syngeneic MC38 tumors using tumor regression agents. During and following treatment, CCR4 ligand levels, tumor in- as a measure of efficacy. The potential for improved safety resulting filtrating lymphocytes, and tumor volumes were evaluated. from inhibition of systemic activity was evaluated using a Fluorescent Results Activated Cell (FAC) analysis of the T cell population in the spleen for Preclinically, these CCR4 antagonists block T migration and support reg the CTLA4 targeting agents. Induction of diabetes in NOD mice was expansion of activated T in the tumor. Our antagonists reduce T in eff reg used to evaluate safety potential for the anti-PD1 agents. the tumor, but not in peripheral tissues such as blood, spleen or skin; Results which presents a potential safety advantage to the non-selective ap- The tumor-activated antibodies exhibit significantly reduced affinity proach of depleting anti-CCR4 antibodies. In preclinical efficacy studies, for their targets without activation, and full binding affinity is re- treatment with various checkpoint inhibitors and immune stimulators stored with proteolytic activation. In vivo testing of the tumor- (e.g., anti-CTLA-4 or anti-CD137) induce the upregulation of CCR4 lig- activated antibodies led to tumor reduction similar to the parental and expression. Combination therapy with CCR4 antagonist and immu- antibody. Critically, the tumor-activated antibodies exhibit reduced nomodulatory agents reduced intratumoral T number and increased reg levels of the systemic activity compared to their parental antibody. number of activated and total T , resulting in an increase in the intra- eff + + Conclusions tumoral ratios of both CD4 and CD8 T to T . The change in these eff reg We have developed and demonstrated a rational approach to effi- T to T ratios is greater for our CCR4 antagonist in combination than eff reg ciently design immunotherapeutics that are locally activated in the with the immunomodulatory agent alone and correlates with en- tumor microenvironment and exhibit reduced systemic activities. Our hanced tumor growth inhibition and increased tumor regression. approach, which we have termed Aklusion™, has the potential to im- Conclusions prove the safety and efficacy of immuno-oncology therapeutics. Combination therapy with CCR4 antagonist and immunomodulatory agents overcome T -mediated suppression in tumors and tips the reg References balance toward tumor rejection. 1. Hoos, A. Development of immuno-oncology drugs — from CTLA4 to PD1 to the next generations. Nat Rev Drug Discov. 2016;4:235-47. P468 2. June CH, Warshauer, JT, Bluestone JA. Is autoimmunity the Achilles’ heel A novel 3D ex vivo platform of fresh patient tumor tissue with of cancer immunotherapy? Nat Med. 2017;23(5):540-547. intact tumor microenvironment for immuno-oncology drug 3. Abdel-Wahab N, Shah M, Suarez-Almazor ME. Adverse Events Associated development and biomarker discovery with Immune Checkpoint Blockade in Patients with Cancer: A Systematic Melanie Mediavilla-Varela, Melba Marie Page, Jenny Kreahling, Review of Case Reports. PLoS ONE. 2016;11 (7): e0160221. Scott Antonia, Soner Altiok Nilogen Oncosystems, Tampa, FL, USA P467 Correspondence: Soner Altiok (jenny@nilogen.com) CCR4 antagonists inhibit regulatory T cell (T ) recruitment and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P468 reg increase effector T cell (T ) numbers in the tumor eff microenvironment potentiating an anti-tumor response Background 1 1 1 1 Oezcan Talay , Lisa Marshall , Cesar Meleza , Maureen Reilly , Omar Cancer immunotherapy delivers treatment of high specificity, low 1 1 1 1 Robles , Mikhail Zibinsky , Abood Okal , Jenny McKinnell , Scott toxicity and prolonged activity in subsets of patients. There is an un- 1 2 1 1 Jacobson , Erin Riegler , Sashie Marubayashi , Emily Karbarz , David met need for clinically relevant preclinical models and translational 1 1 1 2 Chian , Angela Wadsworth , David Wustrow , Jordan Fridman , Paul strategies that recapitulate the complexity of tumor immune micro- Kassner environment to test the therapeutic potential of immuno-oncology 1 2 FLX Bio Inc., South San Francisco, CA, USA; Formerly at FLX Bio Inc., (IO) drugs, identify rational combination therapies and to develop South San Francisco, CA, USA novel predictive biomarkers of clinical response. Here we describe a Correspondence: Paul Kassner (pkassner@flxbio.com) novel 3D ex vivo drug assay utilizing fresh patient tumor samples Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P467 with intact immune microenvironment to facilitate oncology and IO drug development and biomarker discovery. Background Methods Regulatory T cells (T ) are essential for immune tolerance and T -me- 3D exvivo studies were performed with fresh tumor tissue ob- reg reg diated suppression of effector T cells (T ) is important to control in- tained from consented patients with non-small cell lung, renal, eff flammation and prevent autoimmune diseases. However, the presence urothelial, head & neck and breast carcinoma. 3D tumor micro- of T in the tumor microenvironment (TME) has been shown to spheroids (150 μm in size) prepared according to a proprietary reg dampen anti-tumor immune responses. Human T express CCR4, the protocol were treated in their intact immune microenvironment reg receptor for the chemokines CCL17 and CCL22. Preclinical and clinical with PD1 inhibitors pembrolizumab or nivolumab at 10μg/ml for data supports a role for CCR4-mediated recruitment and accumulation 36 hours. Culture media was collected over the course of the ex- of T in the TME which can be associated with poor prognosis. Fur- periments to simultaneously analyze the differential release of reg thermore, patients receiving immunomodulatory agents demonstrate cyto-and chemokines. Treatment-mediated changes in T-cell acti- an influx of T in responding patients which may dampen optimal vation and immune cell populations were monitored by flow cy- reg anti-tumor responses. Therefore, CCR4 is an ideal target to selectively tometry while NanoString PanCancer Immune Profiling platform block T recruitment into the TME. containing probes to quantitate 770 immune function genes was reg Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 227 of 244 used to determine positive and negative associations between cures when treatment is initiated at day 7 in GL261 tumor-bearing expression of immune function genes and TIL activation by mice. Furthermore, the combination of OMX with anti-PD-1 in late- ex vivo treatment. Immunohistochemical studies were performed stage GL261 tumor-bearing mice increases mouse survival by ~80% to identify PD-L1 expression and immune cell composition in and in large PD-1 resistant GL261 tumors, OMX significantly increases tumor samples. the median survival by ~50%. The survival benefit observed with OMX Results could be predicted with an identified circulating chemokine signature 3D ex vivo samples treated with pembrolizumab or nivolumab dem- (post-hoc test). onstrated PD1 occupation in all tumors, while approximately 30% of Conclusions tumors showed increased CD8 T-cell activation that closely correlated By delivering oxygen specifically to the hypoxic tumor microenviron- with proinflammatory cytokine release in the conditioned media. Im- ment, OMX may restore anti-cancer immune responses in cancer pa- mune gene expression profiling studies revealed increased expres- tients through activation of inflammatory cytokine cascades and sion of IFNg inducible genes in tumors showing activated CD8 mobilization and activation of tumor-specific T cells. As a unique, population by flow cytometry. No significant correlation was found pleiotropic immunotherapy, OMX may enhance immune control of between tumor PD-L1 expression and ex vivo response to treatment. tumors to improve patient outcomes. Furthermore, we showed pembrolizumab or nivolumab treatment ex vivo led to changes in tumor immune cell composition in specific- P470 ally including changes in monocyte populations in subset of tumors Tertiary lymphoid organs as a good prognostic indicator following used in the ex vivo experiments. neoadjuvant chemo (radio) therapy for pancreatic cancer Conclusions Shota Kuwabara, Takahiro Tsuchikawa, Yoshitsugu Nakanishi, Toshimichi Our studies showed that this approach can be used to identify ra- Asano, Takehiro Noji, Yo Kurashima, Yuma Ebihara, Soichi Murakami, tionale drug combinations and to develop potential companion diag- Toru Nakamura, Keisuke Okamura, Toshiaki Shichinohe, Satoshi Hirano nostics to facilitate biomarker-driven drug development efforts and Hokkaido University Graduate School of Medicine, Sapporo Hokkaido, Japan personalized medicine in immuno-oncology. Correspondence: Takahiro Tsuchikawa (medicineman424@yahoo.co.jp) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P470 P469 Tumor oxygenator OMX reverses multiple modes of Background immunosuppression, activates anti-tumor immunity and cures as a Pancreatic cancer (pancreatic ductal adenocarcinoma; PDAC) is a single agent, and in combination with anti-PD-1 in resistant highly malignant tumor that frequently develops local recurrence orthotopic tumors and distant metastasis even after a curative resection. Prognosis of Ana Krtolica, Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, PDAC is very poor, and the overall 5-year survival rate for patients Carol Liang, Jonathan Winger, Stephen P. L. Cary with PDAC treated by curative resection is 15-25%. Recently, neoad- Omniox, Inc., San Carlos, CA, USA juvant chemo (radio) therapy had been reported to improve treat- Correspondence: Ana Krtolica (akrtolica@omnioxinc.com) ment outcome. Therefore, it is an urgent need to explore surrogate Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P469 markers predicting patients’ outcome. We herein focused on the tumor microenvironment as one of the candidates for monitoring Background local pathological and immunological reactions, and put a spotlight Hypoxia, a common feature of solid tumors that correlates with poor on tertiary lymphoid organs (TLO, alternatively, ectopic lymphoid tis- patient outcomes, is a central mediator of escape from innate and sue) as a biomarker reflecting the effect of preoperative neoadjuvant adaptive anti-tumor immune responses. Numerous classes of immuno- chemotherapy (NAC). therapy target specific pathways of hypoxia-driven immunosuppres- Methods sion. Reversing tumor hypoxia, however, would work upstream at one In this study, we retrospectively analyzed the area of TLO, dividing of the primary nodes of immunosuppression to stimulate effective re- 140 patients diagnosed with PDAC from January 2009 to December sponses. The hypoxic tumor microenvironment arises due to dysfunc- 2015 into two groups, those who performed upfront surgery (SF tional tumor vasculature, and oxygen diffusion is restricted to within group; n=93) and those who underwent NAC (NAC group; n=47). 2 2 ~80 mm of active capillaries. To reverse tumor hypoxia and create an Then we measured the area (mm ) of TLO, the total area (mm )of immunopermissive microenvironment, we engineered OMX, a stable, the tumor, and calculated the area ratio of total tumor to the total non-vasoactive, high affinity oxygen carrier that preferentially accumu- TLO (TLO/ Tumor ratio) by microscopic observation, and statistically lates in solid tumors and specifically re-oxygenates hypoxic microenvi- analyzed the association between the level of TLO formation and ronments without affecting normoxic tissues. OMX improves anti- prognosis in the tumor microenvironment. All the microscopic im- tumor immune responses. ages were imported as digital photo files using a NanoZoomer Methods Digital Pathology system (Hamamatsu Photonics, Hamamatsu, Japan), We used immunohistochemical, flow cytometric, and multiplex cyto- and imaging analysis were performed using Image J software (NIH, kine analyses to evaluate OMX’s ability to weaken immunosuppres- Bethesda, MD, USA). sion in the tumor microenvironment of orthotopic GL261 brain Results tumors and promote tumor cures. The TLO formation was recognized in 128 patients (91.4%; 128/140). Results There were no significant difference in terms of TLO formation nor A single dose of OMX in GL261 tumor-bearing mice reduces tumor TLO/ Tumor ratio between the two groups (94.6%; 88/93 v.s. 85.1%; hypoxia, enhances T cell accumulation into the tumor, decreases Tregs 40/47, p=0.0575 and 0.66±1.22 v.s. 0.64±8.62, p=0.2342, respectively). and increases activation and proliferation of cytotoxic T lymphocytes The 5-year-overall survival rate of NAC group had been significantly (CTLs). Specifically, OMX increases the Teff/Treg ratio by ~3-fold, indi- better than SF group (41% v.s.16%, p=0.0203). On multivariate ana- cating a switch from an immunosuppressive to an immunopermissive lysis, lymphnode metastaisis (HR 0.047, 95% CI: 0.005-0.265, microenvironment. In addition, OMX stimulates secretion of a major p<0.0001) and high TLO/ Tumor ratio (HR 0.108, 95% CI: 0.010-0.593, chemoattractant for Th1 cells, and increases the accumulation of CD8+ p=0.008) were revealed as independent favorable prognostic factors. tumor-specific T cells in the tumor tissue. When combined with anti- Conclusions PD-1, OMX increases CTL infiltration, proliferation and cytotoxic activity, Our present results indicate that the level of TLO serves as one of and modulates IFNγ and IFNγ-inducible cytokines that may polarize T the valuable independent prognostic markers following neoadjuvant cells towards a Th1 phenotype. OMX alone resulted in ~50% tumor chemo (radio) therapy for PDAC. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 228 of 244 P471 References Patterns of immune cell infiltration in murine models of 1. Erdag G, Schaefer JT, Smolkin ME, et al. Immunotype and melanoma: roles of antigen and tissue site in creating inflamed immunohistologic characteristics of tumor-infiltrating immune cells are tumors associated with clinical outcome in metastatic melanoma. Cancer Res. 1 2 1 1 Katie Leick , Joel Pinczewski , Donna Deacon , Amber Woods , Marcus 2012;72(5):1070. 3 1 1 Bosenberg , Victor Engelhard , Craig Slingluff 1 2 University of Virginia, Charlottesville, VA, USA; Dorevitch Pathology, North Melbourne, Australia; Yale University, New Haven, CT, USA Correspondence: Victor Engelhard, Craig Slingluff (kml2h@virginia.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P471 Background Immune cell infiltration is associated with improved survival in mel- anoma and other cancers. Melanoma metastases in humans may be grouped into three Immunotypes representing patterns of immune cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse) . Prior work suggests that anatomical site and intratumoral vascularity can have dramatic effects on T cell responses and immune infiltra- tion. Murine models provide opportunities to understand factors that control immune infiltrates, but Immunotypes have not been defined for common murine models. We hypothesize that patterns of im- mune cell infiltration can be defined in Braf/Pten and B16 murine models that are similar to Immunotypes observed in human melan- oma metastases , and that AAD and OVA transfectants and intraperi- toneal (i.p.) location will facilitate greater immune infiltration in the B16 model. Methods Fig. 1 (abstract P471). Immunotype and immune cell density of We performed immunohistochemistry for S100, CD31, and CD45, Braf/Pten and B16 murine melanomas with immune cell enumeration, Immunotyping, and scoring of vas- cular density in spontaneous melanoma models and in transplant- able melanoma models (B16-F1, B16-OVA, and B16-AAD). The transplantable tumors were grown either in subcutaneous (s.c.) or i.p. locations. Results P472 The Braf/Pten and Braf/Pten/β-catenin tumors had low immune DARPin®-based fibroblast activation protein-targeted agonists of 4- cell counts in 6/6 tumors that were consistent with Immunotype 1BB and OX40 co-stimulate T-cells in a Fc-receptor-independent, A (Fig. 1A, Fig. 1B), which was also seen in 11/12 B16-F1 tumors tumor-restricted manner (Fig. 1C). In comparison, 9/10 tumors in B16-OVA s.c. and i.p. and Alexander Link, Julia Hepp, Ulrike Fiedler, Christian Reichen, 5/6 B16-AAD s.c. tumors were characterized as Immunotype B. Clara Metz, Alexan der Titz, Ivana Tosevski, Joanna Taylor, Only the B16-AAD i.p. tumors were Immunotype C in 4/6 tumors. Zita Arany, Laurent Juglair, Patricia Schildknecht, Yvonne Kaufmann, The i.p. location was characterized by significantly higher immune Denis Villemagne, Simon Fontane, Ralph Bessey, Christof Zitt, cell counts in B16-OVA tumors (P = 0.0008, Fig. 1D) and higher Keith Dawson, Dan Snell, Daniel Steiner, Michael Stumpp, Victor Levitsky tumor vascularity. Interestingly, mutated cancer antigens in mu- Molecular Partners AG, Schlieren-Zürich, Switzerland tant BRAF models and in B16 melanoma were insufficient to in- Correspondence: Victor Levitsky duce significant immune infiltrates, suggesting the existence of (victor.levitsky@molecularpartners.com) barriers to immune infiltration. Addition of model neoantigens Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P472 (OVA or AAD) overcame existing barriers in a manner that was dependent in part on tumor site. Background Conclusions In addition to check point blockade, immunosuppressive tumor These murine models may be useful for preclinical studies of com- microenvironment can be counteracted by enhanced triggering bination immune therapy, and suggest that both tumor antigen ex- of T-cell co-stimulatory receptors. In vivo agonistic activity of pression and tumor location contribute to the extent of immune cell antibodies against such receptors usually requires Fc-dependent infiltration and Immunotype in murine melanoma. crosslinking and comes at a cost of various systemic, immune Spontaneous Braf/Pten and Braf/Pten/β-catenin dermal melanomas related toxicities. We developed a new class of biologics based lacked significant intratumoral immune cell infiltrates characteristic on the DARPin® technology platform which allow tumor- of Immunotype A (A), and the mean CD45+ cell counts for 3 mice of restricted engagement of co-stimulatory molecules independently each tumor type were all less than 10 per hpf in murine melanoma of Fc-receptors. Our preclinical in vitro and in vivo results indi- (B). Associations of B16 tumor cell type and tumor location with im- cate that DARPin®-based co-stimulatory receptor agonists are mune infiltrate. B16-F1 melanomas (B16-F1), or those transfected comparable to or exceed conventional antibodies in agonistic with OVA or AAD, were grown in from s.c. or i.p. locations and evalu- potency but restrict their activity to the tumor site effectively ated for Immunotype (C) and overall CD45+ cell counts (D). Signifi- widening the therapeutic window for compounds exploiting this cance is noted by asterisks: * < 0.05, ** < 0.001, **** <0.0001. mode of action. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 229 of 244 Methods expression and protein analyses, and Luminex bead-based multi- We generated multi-specific DARPin® therapeutics, which bind to plex assays. human fibroblast activation protein (FAP) and either to 4-1BB or Results OX40 co-stimulatory receptor, preferentially expressed on CD8+ and Here, we demonstrated that tumor growth inhibition in an CD4+ lymphocytes, respectively. The agonistic activity of both drug MC38 model was associated with increase in intra-tumoral M1 hi candidates was analyzed using NFkB-pathway activation reporter macrophages, CD169 antigen presenting macrophages, and cells lines and several types of T-cell activation assays. The 4-1BB- PD-L1 expression. Also observed were a higher ratio of CD8 agonist was tested in a xenotransplantation tumor model established +/CD4+ T cells and an increased expression of CD69 and PD-1 in NOG mice humanized by PBMCs. on CD8+ T cells, indicative of activation of cytotoxic T cells. Results Additionally, an increaseinGranzymeB and IFNγ with a con- Both 4-1BB and OX40-specific FAP-targeted DARPin® drug candidates comitant decrease in VEGF in tumor cell lysates indicted T cell efficiently induced activation of the NFkB pathway in 4-1BB or OX40 activation and M1 polarization of macrophages. Furthermore, in expressing reporter cells but only after crosslinking on FAP, either a CT26 syngeneic model, we demonstrated that RXDX-106 surface immobilized or expressed on cells. The capacity of the mole- inhibited tumor growth as a single agent, and the effect was cules to enhance lymphokine secretion and upregulate expression of further potentiated by combination therapy with immune check- activation markers on T-cells stimulated by polyclonal T-cell receptor point inhibitors, as evidenced by upregulation of anti-tumor crosslinking or specific antigen recognition also strictly required FAP- gene expression patterns, upregulation of anti-tumor cytokines dependent oligomerization. in the tumor cell lysates, and an increase in T cell function. Fi- Urelumab, a clinically tested 4-1BB agonistic antibody associated nally, in an AXL-driven tumor model, we demonstrated that AXL with liver toxicity, led to signs of severe graft-versus-host disease expressing tumors induced a pro-tumorigenic immune environ- (GVHD) and weight loss greater than 20% in the humanized NOG ment, and treatment with RXDX-106 resulted in complete tumor mice transplanted with HT-29 tumor cells at termination of the regression and re-polarization of macrophages towards an M1, experiment. In contrast, mice treated with several doses of the 4- anti-tumor phenotype. 1BB-specific DARPin® drug candidate showed only minor signs of Conclusions GVHD and marginal weight loss. Further analysis indicated that RXDX-106 has the potential to restore and enhance immune function urelumab induced CD8+ T-cell expansion both in the peripheral in macrophages and T cells, resulting in repolarization of the immune blood and tumor tissue of animals while the 4-1BB-specific DAR- response towards an anti-tumor microenvironment. The unique Pin® drug candidate triggered a similar change only in the tumor mechanism of activating both innate and adaptive immunity, plus sites. regulating cross-talk between immune cells and tumor cells by Conclusions RXDX-106 supports clinical development of RXDX-106 to potentially Thus, tumor targeted DARPin® agonists of co-stimulatory molecules treat a wide variety of cancers. can enhance T-cell activity in a manner independent of Fc- crosslinking and limited to the tumor site, thereby avoiding systemic toxicity caused by antibodies against the same immunologic targets. P474 This approach with DARPin® therapeutics can be broadly applied to a Gene expression in MHC II pathway may predict triple negative range of tumor targets and immune receptors. breast cancer prognosis 1 1 2 3 Amy Zhao , Gavin Bond , Katherine Varley , Andres Forero-Torres , 3 3 Albert LoBuglio , Dongquan Chen 1 2 P473 University of Pennsylvania, Philadelphia, PA, USA; University of Utah, Immuno-oncological efficacy of RXDX-106, a novel TAM (TYRO3, Salt Lake City, UT, USA; University of Alabama at Birmingham, AXL, MER) family small molecule kinase inhibitor Birmingham, AL, USA 1 1 1 1 1 Erin Lew , Yumi Yokoyama , Colin Walsh , Jack Lee , Joanne Oh , Correspondence: Amy Zhao (yli@uabmc.edu) 1 1 1 1 Elizabeth Tindall , Robin Navarez , Amy Dilberto , Heather Ely , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P474 2 1 2 1 Ruth Seelige , Amanda Albert , Jack Bui , Gary Li 1 2 Ignyta, Inc, San Diego, CA, USA; University of California, San Diego, San Background Diego, CA, USA Triple negative breast cancer (TNBC) is a subtype with heterogeneous Correspondence: Gary Li (elew@ignyta.com) patient outcomes. It behaves aggressively, and patients are not a Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P473 candidate for ER or HER2/Neu targeted therapy. Although there have been major research efforts directed at establishing genomic signa- Background tures to assess prognosis of breast cancer, prognostic gene expres- The receptor tyrosine kinases (RTKs), TYRO3, AXL, and MER (TAM), sion signatures are not well developed for TNBC. In this study, we are emerging targets for cancer therapy. Particularly, TAM RTKs investigated twenty-four genes [1] related to the MHC II pathway in play a key homeostatic role as negative regulators of immune re- 47 TNBC patients and explored gene(s) that can be used to distin- sponses. RXDX-106 is a potent and selective TAM family inhibitor guish who will relapse. This work may lead to TNBC management in preclinical development. Here, we sought (1) to evaluate the and treatment and could also provide mechanism for the generation efficacy of RXDX-106 as a single agent and in combination with of the anti-tumor immune response. immune checkpoint inhibitors; (2) to identify the immuno- Methods modulatory mechanisms of action; and (3) to decipher the recip- Forty-seven snap frozen primary TNBC tumor specimens were ana- rocal regulation of TAM expression on cancer and immune cells, lyzed using RNA-Seq. Individual gene expressions were transformed and how pharmacological inhibition of TAM signaling would be to fit a normal distribution. The Logistic regression analysis was used beneficial, given their complex interaction and regulation in the to identify genes that can predict no relapse. The optimal cutpoint tumor microenvironment. was determined from the receiver operating characteristic analysis. Methods This study has the approval of UAB’s Institutional Review Board. The anti-tumor efficacy of RXDX-106, as single agent or in com- Results bination with checkpoint inhibitors, was studied in tumors grown CIITA had significantly high expression in TNBC patients who had not either in syngeneic models or in immunocompromised mice. relapsed (P<0.0001). With high CIITA expression, TNBC were 10 times Immuno-phenotyping was performed in tissue and tumor samples more likely to have not relapsed than those with low expression (OR collected during or at the end of studies by flow cytometry, gene =10.8, 95% CI 2.8-41.9, p=0.006). The sensitivity and specificity are Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 230 of 244 75% and 78.3%, respectively. Using multivariable approach with all cells in GBM may negatively impact patient survival, and that these 24-gene panel, we identified three genes, CIITA, CTSH and KRT14, cells may constitute Bregs. which together can predict no relapse with 87.5% sensitivity, and Conclusions 78.3% specificity. Further, at the optimal cut point, we found CIITA To optimize immunotherapy, there is a necessity to understand the alone can predict TNBC no relapse with 84% sensitivity and 77% spe- prevalence and the functional role that B cells play in GBM and their cificity and accuracy of 81%. It predicted 17/ 22 relapse, and 21/25 impact on patient survival. no relapse correctly. These biomarkers were independent predictors of TNBC prognosis regardless of age, race, tumor grade and stage. P476 The findings were confirmed in public microarray data from 199 pa- Targeting the IDO/TDO pathway through degradation of the tients with TNBC confirmed. immunosuppressive metabolite kynurenine and inhibition of the Conclusions downstream AHR pathway High expression of genes in the MHC II pathway, e.g. CIITA, may pre- 1 1 3 3 Karen McGovern , X. Michelle Zhang , Everett Stone , Todd Triplett , dict TNBC prognosis with high sensitivity and specificity regardless of 3 1 2 1 Kendra Garrison , Silvia Coma , Luis Felipe Campesato , Meghan Walsh , age, race, and tumor grade and tumor stage. This work may lead 1 3 3 3 Jeremy Tchaicha , Candice Lamb , Christos Karamitros , John Blazek , TNBC management and treatment. Although we explored the opti- 2 2 3 1 Taha Merghoub , Jedd Wolchok , George Georgiou , Mark Manfredi mal point to distinguish patients’ outcome, it is important to further 1 2 Kyn Therapeutics, Cambridge, MA, USA; Memorial Sloan Kettering establish the threshold in a larger TNBC population. Cancer Center, New York, NY, USA; University of Texas, Austin, TX, USA Correspondence: Karen McGovern (karen@kyntherapeutics.com) References Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P476 1. Forero A, et al. Expression of the MHC Class II Pathway in Triple-Negative Breast Cancer Tumor Cells is Associated with a Good Prognosis and Background Infiltrating Lymphocytes. Cancer Immunol Res. 2016; 4(5): 390–399. Checkpoint inhibitors have become the cornerstone of immune- based oncology therapy. Several orthogonal immune pathways are P475 currently being investigated to relieve suppression or boost activity Profiling Immune Infiltration of Glioblastoma of the innate and adaptive immune system, including the study of Camilo Fadul, Craig Slingluff, Ileana Mauldin the immune-modulating role of metabolites. Both Indoleamine- University of Virginia, Charlottesville, VA, USA pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 Correspondence: Craig Slingluff (ileanasoto@virginia.edu) (TDO2) enzymes metabolize tryptophan forming kynurenine, which Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P475 binds the aryl hydrocarbon receptor (AHR) in multiple innate and adap- tive immune cell types, causing a net immunosuppressive environment. Background Both enzymes are upregulated across many tumor types, and the IDO1 The median survival for patients with glioblastoma (GBM) treated enzyme has been clinically validated with small molecule inhibitors in with standard of care therapies is 15-20 months. The efficacy of combination with checkpoint inhibition, lending credence to the tumor immunotherapy for many types of cancer make this an attractive microenvironment containing small molecule metabolites that induce strategy to improve outcomes for patients with GBM; unfortunately, immune cell tolerance, and leaving the opportunity for broader efficacy thus far these have not had demonstrated efficacy in GBM. In other if both pathways can be targeted. Kynureninase (Kynase), an enzyme cancers, the presence of immune cell infiltration, the pattern of infil- degrading kynurenine generated from IDO1 and TDO2 pathway, as well tration, and the expression of immune markers have been associated as AHR antagonism through small molecule inhibition, can potentially with survival and response to some types of immunotherapy. How- relieve the immunosuppression on multiple cells types and provides ever, the immune infiltrate in GBM has not been well-characterized. novel approaches to inhibiting this pathway in multiple tumor types. Methods Methods Multispectral immunohistochemistry images were obtained from 6 For Kynase, a pegylated enzyme was used for in vitro and in vivo GBM specimens stained with CD8, CD20, FoxP3, PNAD, CD83 and Ki67, studies. Pharmacodynamic (kynurenine depletion) and efficacy stud- and analyzed for tumor infiltrating lymphocytes using digital software. ies were performed in syngeneic tumor models as single agent or in Results combination with checkpoint inhibitors (anti-PD1 or anti-CTLA4). Ex- In preliminary studies, we have observed that GBM can have dense B vivo studies were done to analyze the immune cell composition of cell infiltrates located near blood vessels; however, the functional role tumors post-treatment and the effect on AHR signaling. For AHR an- of B cells in GBM, and the prevalence of these cells has been unstud- tagonists, in vitro and in vivo studies were performed to assess ied. Few studies have been performed evaluating B cells in GBM and kynurenine-dependent signaling through AHR. suggest contradictory roles for B cells in GBM. Candolfi et al. studied Results the role of B cells in a GBM murine model and concluded that B cells Kynase depleted kynurenine produced by IDO1+, TDO2+ and dual posi- act as antigen presenting cells, lending a critical role in clonal expan- tive human cancer cells whereas, the IDO1 inhibitor epacadostat or sion of tumor antigen specific T cells, and brain tumor regression in TDO2 inhibitor 680C91 selectively inhibited Kyn production in IDO1+ or mice. Conversely, Saraswathula et al. showed that patients with GBM TDO2+ cells, respectively. Kynase ameliorated the suppressive effects of have Bregs in peripheral blood. Bregs downregulate effector T cell re- kynurenine on CD4+ and CD8+ T-cells. In B16F10 tumor-bearing mice, a sponses through the secretion of immunosuppressive cytokines such single subcutaneous dose of Kynase depleted kynurenine in the plasma as IL-10 and through cell-cell contact. Thus, these studies present and tumors, leading to an increase in tumor infiltrating CD8+ and CD4+ conflicting roles for B cells in the GBM setting. As a preliminary as- lymphocytes. Kynurenine activation of AHR in cells, measured by gene sessment, we examined the association between survival and overex- expression, is inhibited with AHR antagonists. Kynase demonstrated sig- pression of the MS4A1 gene which encodes for CD20, using RNA-seq nificant tumor growth inhibition and survival benefit either as a single gene expression data obtained from TCGA. The expression data agentorincombination with checkpoint inhibitors in B16F10, CT26 and showed that patients with overexpression of the gene encoding for 4T1 models. Interestingly, Kynase combined with anti-PD1, showed CD20 had decreased overall survival (p = 0.0111). While these data greater efficacy than epacadostat / anti-PD1 combination. provide interesting preliminary findings, they need to be further in- Conclusions vestigated, by evaluating markers of B cell effector functions in GBM Our data support clinical development of human Kynase for the and the impact of B cells on T cells infiltrating tumor. However, con- treatment of cancers where both IDO1/TDO2 pathways play a signifi- sidering these gene expression data, we hypothesize that CD20 B cant immunosuppressive role through kynurenine production. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 231 of 244 P477 Results + neg Novel effector phenotype of Tim3+ regulatory T cells leads to Among CD8 TIL from melanoma metastases, RI , CD49a + neg neg + + neg enhanced suppressive function in head and neck cancer patients CD49b CD103 and CD49a CD49b CD103 subpopulations were 1 2 3 3 Zhuqing Liu , Elizabeth McMichael , Gulidanna Shayan , Jing Li , found in virtually all patients. However, only a subset of patients had 2 2 2 + + neg + + Rghvendra Srivastava , Lawrence Kane , Robert Ferris CD103 subpopulations (CD49a CD49b CD103 and CD49a CD49b 1 2 + + Tongji University, Shanghai, China; University of Pittsburgh, Pittsburgh, CD103 ). Each subset was assessed for cytokine production. A large 3 + neg neg PA, USA; Tsinghua University, Beijing, China fraction of CD49a CD49b CD103 TIL was multifunctional, produ- + neg + Correspondence: Robert Ferris (cmichaele@upmc.edu) cing IFNγ,TNFα and IL-2. In contrast, CD49a CD49b CD103 TIL only + + neg + Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P477 expressed IFNγ, and both CD49a CD49b CD103 and CD49a CD49b + + CD103 subsets were incapable of inducing IFNγ,TNFα or IL-2 at com- Background parable levels (Fig.1). Next, we evaluated RI expression of naïve T cells Regulatory T (Treg) cells are important suppressive cells among in response to different stimuli in vitro. TCR or IL-2 stimulation alone in- tumor infiltrating lymphocytes. Treg express the well-known immune duced two RI cell populations: one that co-expressed CD49a and checkpoint receptor PD-1, which is reported to mark “exhausted” CD49b and another that expressed CD49a alone. Adding TNFα to TCR Treg with lower suppressive function. T cell immuglobulin mucin stimulation further induced these populations, whereas TGFβ +TCR (Tim)-3, a negative regulator of Th1 immunity, is expressed by a siz- stimulation generated two additional populations; CD49a + + neg + + + + able fraction of TIL Tregs, but the functional status of Tim-3 Tregs CD49b CD103 and CD49a CD49b CD103 . Each of these subsets remains unclear. was found among melanoma TIL, suggesting that TCR stimulation, IL-2, Methods TNFα and/or TGFβ may be responsible for the generation of RI subsets + - high CD4 CTLA-4 CD25 Treg were sorted from freshly excised head and in the TME. neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Conclusions + - Functional and phenotypic features of these Tim-3 and Tim-3 TIL These observations suggest that CD49b and/or CD103 expression is Tregs were tested by in vitro suppression assays and multi-color flow upregulated as effector TIL lose the capability of producing cytokines + + cytometry. Gene expression profiling and NanoString analysis of Tim-3 and become more exhausted. Generation of CD103 subsets in the TIL Treg was performed. A murine HNSCC model was used to test the TME may be driven by TCR stimulation and TGFβ and given their effect of anti-PD-1 immunotherapy o nTim-3 Treg. absence in a fraction of tumors, may only be present in tumors pro- Results ducing sufficient TGFβ. The other RI subsets can be induced by IL-2, Despite high PD-1 expression, Tim-3 TIL Treg displayed a greater cap- TCR engagement and/or TNFα, which we hypothesize will be avail- acity to inhibit naïve T cell proliferatin than Tim-3- Treg. Tim-3 Treg able in all tumors. Together, our findings identify opportunities to from human HNSCC TIL also displayed an effector-like phenotype, with modulate RI expression in the TME by cytokine therapies and to aug- more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Ex- ment subsets with specific RI expression in the interest of improving ogenous IFN-γ treatment could partially reverse the suppressive func- immune therapies for cancer or other immune-related diseases. tion of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim- 3 expression on Tregs isolated from murine HNSCC tumors, and this P479 treatment reversed the suppressive function of HNSCC TIL Tregs. MPL-5821, an ESM™-p38 MAPK Inhibitor, modulates macrophage Conclusions plasticity leading to enhanced IL-12p70 and IFN gamma, reduced Tim-3+ Treg are functionally and phenotypically distinct in HNSCC IL-10 and the reversal of macrophage induced T-cell suppression TIL, and are highly effective at inhibiting T cell proliferation despite 1 1 2 2 Martin Perry , David Moffat , Justyna Rzepecka , Lucia Janicova , high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy + 2 2 2 2 Anastasia Nika , Darryl Turner , Clare Doris , Stephen Anderton may be beneficial by reversing Tim-3 Treg suppression. 1 2 Macrophage Pharma Limited, Windsor, United Kingdom; Aquila BioMedical Limited, Edinburgh, United Kingdom P478 Correspondence: David Moffat (david@macrophagepharma.com) Formation and functional associations of CD49a-, CD49b- and Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P479 CD103-expressing CD8 T cell populations in human metastatic melanoma Background 1 1 1 1 Marit Melssen , Walter Olson , Nolan Wages , Ciara Hutchison , Myelomonocytic cells orchestrate both innate and adaptive im- 1 2 1 1 Ileana Mauldin , Cornelis Melief , Timothy Bullock , Victor Engelhard , mune response to tumours but frequently adopt an immuno- Craig Slingluff, Jr suppressive, tumor supportive phenotype (M2-like). However, 1 2 University of Virginia, Charlottesville, VA, USA; ISA Pharmaceuticals, their plasticity offers the opportunity for therapeutic repolariza- Leiden, Netherlands tion to one that is non-immunosuppressive and supportive of Correspondence: Marit Melssen (mm2xz@virginia.edu) an anti-tumor immune response (M1-like). A challenge in identi- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P478 fying such pharmaceuticals is their counter-active functionality in other important immune cell types such as lymphocytes. Background Macrophage Pharma Limited’s Esterase Sensitive Motif (ESM™) Integrins α1β1 (CD49a), α2β1 (CD49b) and αEβ7 (CD103) mediate re- technology predominantly targets myelomonocytic cells [1]. We tention of lymphocytes in peripheral tissues, and their expression is describe the preliminary in vitro characterisation of MPL-5821, upregulated on CD8 tumor infiltrating lymphocytes (TIL) compared an ESM-p38 MAPK inhibitor, contrasting it with conventional in- to circulating lymphocytes. Little is known about what induces ex- hibitors, with reference to its cell type specificity, sparing of pression of these retention integrins (RI). We hypothesized that RI ex- myelomonocytic – lymphocyte communication and ability to re- pression marks functionally distinct T cell subsets in the tumor versemacrophagedrivenlymphocyteimmunosuppression. microenvironment (TME) and that these subsets can be induced by Methods selected cytokines and T cell receptor (TCR) stimulation. Human Macrophage Assays: M2 polarised macrophages (M-CSF, +/- Methods IL-4, IL-10, TGFβ) were stimulated with LPS/IFNγ +/- compounds over- CD8 TIL from 19 human melanoma metastatic lesions were stained night. Cytokine production and macrophage markers were measured for CD49a, CD49b, CD103, and cytokines either directly or after PMA/ by ELISA and flow cytometry respectively. Ionomycin stimulation. For in vitro studies, circulating CD8 T cells Human PBMC Assay: PBMCs were stimulated with either LPS or anti-CD3 from normal donors (n=4-7) were cultured with cytokines and/or +/- compounds for 72 hours and cytokine production measured by ELISA. CD3/CD28 stimulation and evaluated by flow cytometry. CD4 and CD8 T-cell proliferation was measured by flow cytometry. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 232 of 244 Macrophage suppression of Human T-cells: M2-polarised macro- Conclusions phages were incubated with autologous PBMCs +/- compound. T cell Increased inhibitory receptors such as TIGIT and NRP1 on CTLA4+ Tregs stimulation was provided by anti-CD3/anti-CD28. Cytokine produc- combined with increased pro-survival markers in patients that had a tion was analysed by ELISA and expression of CD4, CD8 and Ki67 by minimal response to cetuximab suggests that these Tregs maintain flow cytometry. suppressive function and survival despite cetuximab treatment. Further Results characterization of the immune resistance mechanism to cetuximab Human Macrophage Assays: MPL-5821 and reference p38 MAPK in- will provide the basis for designing knowledgeable combinatorial trials hibitors exhibited a similar profile in inhibiting IL-10 production and with other immunotherapy agents i.e. CTLA-4. enhancing IL-12p70 production. Trial Registration Human PBMC Assay: In comparison with reference p38 MAPK inhibi- clinical trials.gov:NCT01218048,NCT0193592. tors, MPL-5821 was several orders of magnitude more potent in en- hancing IL-12p70 production and was the only inhibitor to enhance References IFNγ production under LPS stimulation. Similarly, MPL-5821 was the 1. Vokes EE, Weichselbaum RR, et al. Head and neck cancer. N Engl J Med only inhibitor to enhance IFNγ production in response to anti-CD3 1993;328:184-194. stimulation. 2. Ang KK, Berkey BA,et al. Impact of epidermal growth factor receptor Macrophage suppression of Human T-cells: MPL-5821 reversed the expression on survival and pattern of relapse in patients with advanced suppressive effects of the M2 macrophages as evidenced by a return head and neck carcinoma. Cancer Res. 2002;62(24):7350-6. of CD4 and CD8 proliferation to control levels accompanied by a 3. Jie HB, Ferris RL, et al. CTLA4+ Regulatory T cells are increased in head concomitant increase in IFNγ and reduction in IL-10. and neck cancer patients, supress NK cell cytotoxicity and correlate with Conclusions poor prognosis. Cancer Res. 2015;75 (11):2200-2210. Hi MPL-5821 modulates the macrophage phenotype to IL-12p70 / IL- Lo 10 and reverses the M2 macrophage immune suppression of T-cell P481 functionality. The ESM™ cell selectivity differentiates MPL-5821 from Non-oncogenic acute viral infection modulates the innate immune other p38 MAPK inhibitors by its sparing of other immune cells such response and reduces tumor growth in hosts with established as lymphocytes. Thus MPL-5821 enables the maintenance of the cancer myelomonocytic/lymphocyte IL-12p70/IFNγ axis, key to an effective Jenna Newman, Charles Brent Chesson, Andrew Zloza anti-tumour immune response. Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA Correspondence: Jenna Newman jhn33@gsbs.rutgers.edu References Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P481 1. Needham LA et al, J Pharmacol Exp Ther. (2011), 339 :132-42. Background P480 The impact of infection on the development and progression of cancer Tumor infiltrating T regulatory cells in head and neck cancer has been a source of debate throughout the past century. Several path- patients treated with cetuximab demonstrate increased inhibitory ogens have been linked to the development of cancer, while con- receptors and survival versely, pathogen components (including Coley’stoxins) andoncolytic Jessica Moskovitz, Tullia Bruno, Robert Ferris, Dario Vignali viruses have demonstrated tumor regression. Since infection and University of Pittsburgh, Pittsburgh, PA, USA cancer are two often-concomitant challenges to the immune system, Correspondence: Jessica Moskovitz (jessiemoskovitz@gmail.com) we sought to understand the impact of acute, non-oncogenic, non- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P480 oncolytic infections – which constitute the majority of infections affect- ing humans – on tumor growth and host immunity. Background Methods Patients with locally advanced head and neck squamous cell carcinoma C57BL/6 (B6) or NOD-scid-gamma (NSG) mice were challenged with (HNSCC) incur significant treatment morbidity and have a poor 5 year 1.2 x 10 cells B16-F10 melanoma intradermally (right flank) on day 0 overall [1]. The idea of treating HNSCC by targeting epidermal growth and influenza A/PR8/H1N1 (10,000 PFU) administered intranasally factor receptor (EGFR) developed from the demonstration of EGFR (prior to tumor challenge, after tumor challenge before clinical ap- overexpression correlating with advanced tumor size and decreased pearance of the tumor, or once the tumor was established). Tumor survival [2]. Clinical anti-tumor activity of cetuximab, a monoclonal anti- growth and influenza infection were monitored via caliper measure- body that prevents ligand binding of EGFR, did not correlate with ex- ment and host weight, respectively, every 2-3 days. Immune cell pop- pected response rates despite high HNSCC tumor cell EGFR expression. ulations within tumors, lungs, and spleens were analyzed by flow Further studies to clarify the mechanism of action of cetuximab in cytometry at day 20. Statistical comparisons between groups were HNSCC revealed an increase in CTLA4+ T regulatory cells (Tregs) that determined using the student’s t test with p<0.05 considered statisti- suppressed NK cell mediated cytotoxicity, which correlated with poor cally significant. response to neoadjuvant cetuximab treatment [3]. Results Methods Mice infected with influenza prior to or during the subclinical phase of We aimed to further measure inhibitory receptor [e.g. PD1, TIM3, TIGIT, melanoma development displayed accelerated tumor growth relative CTLA4, neuropilin-1(NRP1)] expression on T cells in tumor and periph- to influenza-naïve mice (p<0.01). Contrarily, mice with established (~3 x eral blood lymphocytes (TIL/PBL) of cetuximab treated patients. Pa- 3 mm) tumors displayed slower tumor growth after infection compared tients with locally advance HNSCC were treated with neoadjuvant to uninfected controls (p<0.01). For the latter, infected mice were found - + + cetuximab for four weeks prior to definitive surgery with CT scans per- to have a 9-fold reduction of systemic MHC-II Ly6-G Ly6-C myeloid- formed prior to each intervention. Nine samples (TIL and PBL) pre and derived suppressor cells (MDSCs) in the spleen, relative to that ob- post cetuximab were analyzed using flow cytometry, and tumor area served in influenza-naïve counterparts. Furthermore, the result of cur- change was measured using delta CT. tailed tumor growth in concomitantly challenged mice was observed in Results immunocompromised NSG mice as well. Tumor infiltrating CTLA4+ Tregs had increased expression of NRP1 Conclusions and TIGIT after cetuximab therapy, specifically in patients that had a Previously we reported that influenza infection in the lung acceler- poor response to cetuximab. Further, tumor infiltrating CTLA4+TIGIT+ ates tumor growth of subclinical melanoma in the flank. However, Tregs correlated with patients having larger baseline tumors and our recent data demonstrate that the reverse effect is observed if minimal change in tumor area post cetuximab. Finally, Tregs from influenza is administered to hosts with an established tumor in the these patients displayed an increase in mean flouresecence intensity flank. The latter effect is observed also in NSG mice, which lack adap- of pro-survival markers such as Bcl2. tive immunity and maintain limited innate immunity. These findings, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 233 of 244 in conjunction with a decrease in MDSCs observed in immunocom- P483 petent infected B6 mice exhibiting reduced tumor growth, suggest A novel non-competitive and non-brain penetrant adenosine A 2A that the innate immune system may promote anti-tumor immunity receptor antagonist designed to reverse adenosine-mediated in the context of infection. Further research into the mechanisms by suppression of anti-tumor immunity which influenza alters tumor growth are being investigated in prep- Erica Houthuys, Reece Marillier, Théo Deregnaucourt, Margreet Brouwer, aration for clinical trials that will focus on harnessing microbial im- Romain Pirson, Joao Marchante, Paola Basilico, Shruthi Prasad, munity for the treatment of cancer. Julie Preillon, Anne-Catherine Michaux, Kim Frederix, Annelise Hermant, Florence Nyawouame, Charlotte Moulin, Vanesa Bol, Bruno Gomes, Florence Lambolez, Véronique Bodo, Jakub Swiercz, Gregory Driessens, P482 Noémie Wald, Chiara Martinoli, Michel Detheux, Christophe Quéva, Somatic TP53 mutations alter the immune micro-environment after Stefano Crosignani chemotherapy in breast cancer iTeos Therapeutics, Gosselies, Belgium 1 2 1 3 Mellissa Nixon , M. Valeria Estrada , Susan Opalenik , Donna Hicks , Correspondence: Christophe Quéva 1 1 1 4 Michael Korrer , Mark Pilkinton , Melinda Sanders , Roberto Salgado , (christophe.queva@iteostherapeutics.com) 1 1 3 1 Simon Mallal , Young Kim , Rebecca Cook , Carlos Arteaga , Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P483 Justin Balko 1 2 Vanderbilt University Medical Center, Nashville, TN, USA; UC San Diego, Background 3 4 La Jolla, CA, USA; Vanderbilt University, Nashville, TN, USA; Institut Jules High levels of extracellular adenosine in the tumor microenviron- Bordet, Brussels, Belgium ment promote tumor immune evasion by suppressing Th1 cyto- Correspondence: Justin Balko (mellissa.nixon@vanderbilt.edu) kine production and the activation and cytolytic activity of T cells Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P482 and NK cells. Methods Background We measured extracellular adenosine concentration using intratu- Neoadjuvant chemotherapy (NAC), followed by surgery, is the moral microdialysis of 11 patient-derived xenografts from various standard of care for triple negative breast cancer (TNBC) patients. cancer indications. In parallel, we defined the expression of the four Unfortunately, only 30% achieve a pathological complete re- adenosine receptors in primary human immune cells using sponse (pathCR). In patients who do not achieve a pathCR, tumor nanostring. infiltrating lymphocytes (TILS) after NAC correlate with improved Results survival. This suggests there is an immune response after NAC Intratumoral adenosine concentrations reached a range of 1 to 37 which may be augmented by immune checkpoint blockade (ICB). μM (mean, 11 μM), significantly higher than the 0.1-0.2 μM reported Clinical trials are underway to examine the efficacy of ICB, there- in normal brain tissue. fore it is vital to identify biomarkers of response to identify pa- A was the main adenosine receptor expressed by CD4 and CD8 T 2A tients who may benefit from these modalities. As more than 80% lymphocytes and monocytes, and the only one in mature monocyte- of TNBC patients harbor somatic TP53 mutations, we investigated derived dendritic cells and NK cells. A mRNA were not abundant in 2B the role of TP53 mutations on the immune microenvironment of T cells and monocytes, while A and A mRNA were not detected in 1 3 breast cancer after NAC. any of the cell types evaluated. We further studied A functions in 2A Methods primary human T lymphocytes and monocytes and demonstrated We examined matched pre and post NAC primary breast cancer. that selective A agonists such as CGS-21680 suppressed cytokine 2A TILs were scored by a pathologist. RNA and DNA were extracted production by activated T lymphocytes and monocytes, highlighting and analyzed using Nanostring Pan-Cancer Immune panel (>700 the role of A as the main receptor mediating adenosine signaling 2A genes) and ImmunoSeq T-cell Receptor (TCR) sequencing respect- in these cells. ively. CRISPR technology was applied to mouse tumor cell lines We showed that A antagonists initially designed for Parkinson’s dis- 2A to develop Trp53 mutations commonly found in TNBC. Cells were ease but recently repurposed for immuno-oncology dramatically lost treated with chemotherapy in vitro to determine tumor specific potency in a high adenosine environment. We therefore developped changes in cytokines and PD-L1 expression. To determine alter- a novel non-brain penetrant and non-competitive inhibitor of A 2A ations in the immune microenvironment, cell lines were injected with sub-nanomolar Ki and selectivity versus A ,A , and A recep- 1 2B 3 into syngeneic mice or genetically engineered mouse models tors. Our compound potently inhibited A signaling in human T lym- 2A (GEMM) were utilized Expression of antigen presentation by the phocytes independently of adenosine concentrations (IC in T cell tumor as well as immune phenotyping of TIL after NAC was cAMP assay = 2.8 and 5.5 nM in 1 and 100 μM adenosine, respect- performed. ively), and rescued cytokine production in the presence of high con- Results centrations of A agonists. iTeos A antagonist potently rescued 2A 2A TILs in residual disease of patients correlated with improved survival. T cytokine production in human whole blood treated by A ago- H1 2A A third of patients had increased immune gene signatures after NAC nists, and increased CD8 T cell cytotoxicity in a co-culture assay of which correlated with increased TCR clonality and increased overall effector CD8 T cells and target cancer cells. survival. CRISPR induced Trp53 mutant cells were used to elucidate Conclusions the role of p53. Cells in vitro had higher basal levels of immune- iTeos Therapeutics non-competitive A receptor antagonist is 2A recruiting chemokines which were further induced with chemother- uniquely designed to address the challenge of counteracting ele- apy treatment. Trp53 mutant cells also exhibited increased antigen vated adenosine concentrations in tumors in order to restore antitu- presentation (MHC-I/II) and PD-L1 expression after NAC. In a GEMM mor immunity. of breast cancer that had recurred after chemotherapy, TILs had in- creased expression of CXCR3, LAG3 and PD-1 by flow cytometry. Conclusions Our work suggests that NAC induces immune gene signatures in a P484 subset of TNBC which is correlated with a better prognosis. TP53 mu- A novel metabolic therapeutic that harnesses the anti-tumor tations in the tumor may contribute to an increased immune infil- immune response trate through upregulating chemokines, antigen presentation, and Dayana Rivadeneira, Nicole Scharping, Greg Delgoffe increased cytokine receptor CXCR3+ T cells that display increased ex- University of Pittsburgh, Pittsburgh, PA, USA haustion marks PD-1 and LAG3. Ongoing studies with ICB will indi- Correspondence: Greg Delgoffe (rivadeneirad@upmc.edu) cate if p53-mutant tumors have a better response to ICB after NAC. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P484 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 234 of 244 Background Methods In the past decade immunotherapy has shown impressive clinical re- This multi-institutional study was undertaken to collect comprehensive sponses, quickly becoming the first line treatment for cancers such immune profiles across the biological heterogeneity of childhood and as melanoma. Despite these promising results, all patients do not adult AML, with the aim to implement new molecularly targeted agents have the same level of response, and recent efforts have focused on for patients with specific immunologic subtypes of AML. We employed delineating avenues to understand and avoid therapy resistance. Our the nCounter™ system (NanoString Technologies, Seattle, USA) to lab and others have recently proposed that tumor cells may com- characterize bone marrow (BM) specimens from 70 patients with non- promise T cell function by generating a metabolically inhospitable promyelocytic AML (42 children and 28 adults). Ninety BM samples (63 microenvironment. Most importantly immune or tumor metabolism de novo AMLs, 18 AMLs in complete remission [CR] and 9 relapsed can be modified to modulate the T cell response to immunotherapy. AMLs) were analyzed on the nCounter® FLEX platform, using the RNA Methods Pan-Cancer Immune Profiling Panel. Transcriptomic data were normal- We hypothesize that leptin, a hormone that affects a wide range of ized using the nSolver™ software package. metabolic processes, might be utilized as a means to remodel the Results metabolic state of the tumor microenvironment, consequently modu- Hierarchical clustering identified patient subgroups with heightened lating tumor growth and immunotherapy response. Previous studies expression of CD8 T-cell, Th1-cell, B-cell and cytotoxicity-related have shown that leptin can enhance T cell proliferation and activa- genes, including genes related to NK-cell function, in the leukemia tion, but the effects of leptin on tumor-infiltrating T cells have not microenvironment (“immune enriched” patients). Interestingly, a se- been thoroughly investigated. In order to assess the effects of leptin lect group of patients over-expressed genes associated with mono- in the tumor microenvironment, we obtained tumor infiltrating T cyte/macrophage, neutrophil and mast cell functions, with low cells (TIL) from tumor-bearing mice treated with recombinant leptin. expression of T-cell and B-cell genes. AML patients with ‘immune Additionally, we generated PTEN/BRAF melanoma cell lines stably enriched’ gene profiles also expressed CD8A, IFNG, FOXP3, and inhibi- overexpressing leptin. tory molecules including IDO1 and the immune checkpoints LAG3, Results CTLA4 and PD-L1. As we have previously shown, glucose uptake in TIL is decreased Gene set analyses identified differences in immune gene expression compared to ndLN. When mice were treated with recombinant lep- levels between childhood and adult AMLs, as well as patients with tin, we observed an increase in TIL glucose uptake, suggesting meta- newly diagnosed AML, relapsed AML or AML in CR. In particular, bolic remodeling. Furthermore, PTEN/BRAF melanoma cells which genes highly expressed in adult AMLs compared with childhood dis- overexpress leptin have significantly slower growth kinetics com- ease included chemokine genes, dendritic cell and macrophage pared to control tumors and an increase in overall survival. Leptin genes and NF-kB inhibitor-a. In contrast, immune genes down- overexpressing tumors have increased T cell infiltration compared to regulated in adult compared with childhood AMLs included integrin control tumors, and these TIL are metabolically and functionally family members, cytokine receptors, TCR complex components and superior. lipocalin-2, a neutrophil gelatinase-associated molecule implicated in Conclusions suppression of tumor invasiveness. Taken together, these data suggest leptin can have a direct effect on Conclusions tumor infiltrating T cell function and tumor growth. Our goal is to Our analysis has captured the immunological status of the leukemia further characterize how leptin can metabolically remodel the tumor tumor microenvironment in children and adults with AML at different microenvironment and modulate immunotherapeutic response. disease stages. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment. P485 Use of the NanoString gene expression profiling platform to capture the immunological status of the leukemia P486 microenvironment Utilization of dual IHC and quantitative image analysis techniques 1 2 1 Jayakumar Vadakekolathu , Tasleema Patel , Stephen Reeder , to evaluate LAG-3-positive T cells in the tumor microenvironment 3 4 3 5 Heidi Schaarschmidt , Marc Schmitz , Martin Bornhäuser , Sarah Warren , of NSCLC tissue 5 5 5 1 1 1 1 Tressa Hood , Patrick Danaher , Alessandra Cesano , Joseph M. Malik Khenkhar , Philipp Uhlig , Nickels Winkler , Hartmut Juhl , 5 1 2 1 2 2 Beechem , A. Graham Pockley , Sarah Tasian , Sergio Rutella Alison Bigley , Lorcan Sherry 1 2 1 2 Nottingham Trent University, Nottingham, United Kingdom; Children's Indivumed GmbH, Hamburg, Germany; OracleBio Limited, Glasgow, Hospital of Philadelphia, Philadelphia, PA, USA; University Hospital Carl United Kingdom Gustav Carus, Dresden, Germany; Medical Faculty Carl Gustav Carus, Correspondence: Lorcan Sherry lorcan.sherry@oraclebio.com Dresden, Germany; NanoString Technologies, Inc., Seattle, WA, USA Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P486 Correspondence: Sergio Rutella (sergio.rutella@ntu.ac.uk) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P485 Background Non-small cell lung cancer (NSCLC) patients have impaired immune Background responses, where the expression of inhibitory checkpoint molecules Acute myeloid leukemia (AML) is characterized by clonal expansion on T cells may lead to reduced immune cell activity. The presence of of poorly differentiated myeloid precursors and results in impaired lymphocyte activation gene-3(LAG-3)-positive T cells in the tumor hematopoiesis and often bone marrow failure. Immune responses microenvironment and their potential impact on prognosis have are defective in patients with AML due to powerful immune suppres- been investigated over many years. One implication has been that sive circuits that are activated by soluble factors and immune check- the engagement of LAG-3 inhibits T cell proliferation resulting in T point molecules, including PD-L1 and IDO1. The development and cell suppression and poor prognosis [1]. We aimed to quantify CD3 delivery of new therapeutic strategies for high-risk AML, including and LAG-3 immune cell relationships in terms of cell infiltrations and immunotherapy, therefore remains a priority. proportions within the tumor microenvironment of NSCLC tissue. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 235 of 244 Methods classification and analysis and the third tier refined cell relationships Ten individual NSCLC clinical tissue samples were immunohisto- and measured immune cell distances to the nearest tumor cell or be- chemically (IHC) stained by Indivumed using the Ventana DISCOVERY tween defined immune cells. XT automated staining platform. The first section was dual-labeled Results for LAG-3 and CD3 and the second for pan-Cytokeratin (CK). Image Selected ROI displayed a range of tumor and stroma content with analysis was performed by OracleBio using Indica Labs Halo software. varying populations of CD4 and CD8 positive immune cell infiltra- Tumor and stroma regions of interest (ROI) were classified using the tions. The mean number of CD4 and CD8 positive cells in tumor was CK section. ROI classifications were automatically transferred, during 30 and 250 per mm respectively and in stroma was 474 and 915 analysis, from the sample-associated CK section to the co-registered per mm respectively. Evaluation of tumor-immune cell spatial rela- dual IHC section. Cellular analysis was performed on the ROI using tionships within the stroma, at a 20μm distance to the tumor border, threshold established to identify and count CD3, LAG-3 and dual- highlighted that the percentage of CD4 and CD8 positive cells within labeled cells. this stromal border was 33% and 54% respectively and the average Results distance between the 2 immune cell types was 13μm. Evaluation of tumor samples demonstrated a mean number of LAG- Conclusions 3/CD3-dual positive cells within the stroma and tumor of 132 ± 59 The exemplar data generated provides information relating to the and 54 ± 20 per mm ROI area, respectively. Interestingly however, potential influences of immune cell recruitment and activation within when the proportion of LAG-3/CD3-dual positive cells was normal- the tumor microenvironment, correlation of spatial distributions and ized to the total population of CD3-positive cells within these re- the inter-dependencies between immune cells and cancer cells, gions, an opposite relationship was observed where the mean % which may be indicative of response to treatment or predicting pa- LAG-3/CD3-positive cells relative to the total CD3 immune cell popu- tient outcome. lation was 5 ± 2% within the stroma and 12 ± 3% within the tumor. Conclusions References These example data highlight the benefits of utilizing dual IHC 1. Hiraoka K, Miyamoto M, Cho Y: British Journal of Cancer. staining with whole slide image analysis to characterize immune cell 2006;94:275–280. relationships within the tumor microenvironment. These techniques can assist in the assessment of changing relationships between pro- P488 portions of immune cells between tissue compartments and evalu- Single-cell RNA sequencing of CD8+ T cells to prioritize ation of LAG-3 as a potential target for modulating T cell responses immunotherapy combinations in NSCLC. Yashaswi Shrestha, Michael Kuziora, Lydia Greenlees, Brandon Higgs, Katie Streicher, Rajiv Raja, Koustubh Ranade References MedImmune, Gaithersburg, MD, USA 1. He Y, Yu H, Rozeboom L. LAG-3 Protein Expression in Non-Small Cell Correspondence: Yashaswi Shrestha Lung Cancer and Its Relationship with PD-1/PD-L1 and Tumor-Infiltrating (robert.mulvey@ashfieldhealthcare.com) Lymphocytes. Journal of Thoracic Oncology. 2017;12(5):814-823. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P488 P487 Background Multiplex immunofluorescence evaluation of an immune cell Immunotherapy has transformed cancer treatment. To address marker panel in the tumor microenvironment of NSCLC tissue remaining unmet needs, combinations of checkpoint inhibitors and ag- using tailored analysis of multi-spectral image component data onists are under investigation, although prioritization strategies are un- 1 1 1 1 Richard Bystry , Edward Durley , Lee Dawson , Rebecca Houliston , clear. We hypothesized that molecular understanding of T-cell 2 2 Alison Bigley , Lorcan Sherry phenotypes under chronic stimulation that mimics the tumor micro- 1 2 Asterand Bioscience, Royston, United Kingdom; OracleBio Limited, environment could aid in optimizing combinations. Additionally, target Glasgow, United Kingdom co-expression is important for designing bispecific monoclonal anti- Correspondence: Lorcan Sherry lorcan.sherry@oraclebio.com bodies (mAbs). Although aggregate gene expression analysis of CD8+ T Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P487 cells is valuable, it does not show target co-expression at the single-cell level. Hence, we explored single-cell RNAseq to identify targetable CD8 Background + T-cell subpopulations at various dysfunctional states. Multiplex fluorescence immunohistochemistry (MIF), multispectral im- Methods aging and image analysis of immune cells within the tumor micro- CD8+ T cells were negatively selected from PBMCs and treated with IL2 environment has been increasingly applied to profile and correlate (NonStim) or IL2+anti-CD3,antiCD28 antibodies (Stim) for 13 days, in- cell interactions in relation to cancer treatment and predicting pa- cluding two cycles of treatment separated by a two-day rest in IL2 [1]. tient outcome. Tumor-infiltrating T lymphocytes are known to play Over 1500 Stim and NonStim cells were profiled on 10X Genomics plat- an important role in anti-cancer mechanisms where coexisting infil- form and analyzed with corresponding pipeline. Gene expression corre- trations of CD8 and CD4 T cells affords a good prognosis in non- sponded to 2 unique molecular indices. Published consensus gene-sets small-cell lung carcinoma (NSCLC) [1]. Here we use CD8/CD4 for functional states of CD8+ T cells in cancer [2] were used for Gene- evaluation to exemplify the use of MIF combined with multispectral Set Variant Analysis (GSVA) to identify populations with significant dif- imaging and tailored spectral and spatial quantification to examine ferences in the four states – dysfunctional, senescent, terminal-effector, relationships between immune cell populations within tumor and and stem-like memory – between Stim and NonStim. stroma. Results Methods GSVA scores indicated significant upregulation of a dysfunction signature An exemplar NSCLC lung tumor section was MIF labelled by Asterand and downregulation of terminal-effector function in Stim versus NonStim for 5 immune cell markers, including CD4 and CD8, a pan cytokeratin cells (P<0.001). PD-1 was most differentially expressed between top and tumor marker and DAPI nuclear marker. The stained slide was digi- bottom tertiles of Stim cells with dysfunctional signature. Similarly, cells tised using the Vectra Polaris multispectral scanner (Perkin Elmer) with high expression of TIM3, another dysfunction marker, showed and exemplar region of interest (ROI) images exported in component downregulation of cytotoxicity and antigen-presentation associated data format. The component data images were analysed by Oracle- genes, compared to those with low TIM3 expression. Bio using a 3-tiered sequence of tailored applications developed in The percentage of cells expressing dysfunction markers increased Visiopharm Oncotopix Software. The first tier enabled the classification under chronic stimulation, while those producing effector cytokines of tumor and stroma ROI, the second tier facilitated cell detection, decreased. Cells expressing PD-1, CTLA-4 and GATA3 increased from Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 236 of 244 5%, 3% and 16% to 17%, 80% and 21%, respectively, whereas cells Conclusions expressing IFNγ decreased from 16% to 7%. Of the Stim cells, 11%, IMC allows for successful multiplex imagining of the TME in FFPE tissues. 15% and 71% co-expressed PD-1 and TIM3, PD-1 and CTLA-4, and The method does not require special processing and can be applied to CTLA-4 and LAG3, respectively. CD137 and TIM3 were co-expressed archival specimens and tumor microarrays. Methods developed here in >30% of Stim cells, but only 1% of NonStim. should be applicable for the study of the TME in other tumor types and Conclusions could be used to identify additional biomarkers of response to immuno- Our results provide new insights into the heterogeneity of activated/ oncology agents. exhausted/dysfunctional CD8+ T cells at the single-cell level, which could aid in prioritizing immunotherapy combinations and designing new bispecific mAbs. References 1. McKinney E, et al. T-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection. Nature. 2015;523:612–616. 2. Apetoh L, et al.Consensus nomenclature for CD8+ T cell phenotypes in cancer. Oncoimmunology. 2015;4:e998538. P489 Studying the cellular heterogeneity and spatial arrangement of immune cells in FFPE sections of Hodgkin Lymphoma and normal lymph node using a highly multiplexed Imaging Mass Cytometry Fig. 1 (abstract P489) FFPE section of Normal lymph node Mohan Singh, Parvesh Chaudhry, Erik Gerdtsson, Lisa Harton, James Hicks, Peter Kuhn, Wendy Cozen, Imran Siddiqi, Akil Merchant University of Southern California, Los Angeles, Los Angeles, CA, USA Correspondence: Akil Merchant (mohansin@usc.edu) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P489 Background Clinical successes with immuno-oncology agents have demonstrated the potency of the immune system in controlling cancers, most strik- ingly in Hodgkin lymphoma (HL), where response rates to PD1/PDL1 in- hibitors approach 90%. The majority of tumor bulk in HL is made up of non-malignant immune cells. High parameter methods such as gene expression profiling or flow cytometry have been applied to the study of the tumor immune microenvironment (TME) in Hodgkin disease, however due to the inherent limitations of these techniques, several questions remain unanswered. Data on cellular heterogeneity and rare cells is lost with gene expression studies while the spatial relationship between tumor and immune cells is lost with flow cytometry. The Flui- Fig. 2 (abstract P489) FFPE section of Hodgkin lymphoma digm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. Here we describe our efforts to develop and optimize a 26 parameter antibody panel for the P490 characterization of the lymphoma TME on the IMC platform. PSMA-specific CARTyrin T-stem cell memory therapy eliminates Methods solid tumor in subcutaneous prostate cancer model Literature was reviewed to identify markers relevant to HL TME. Anti- Jenessa B. Smith, Rebecca Codde, Yening Tan, Burton E. Barnett, David gen retrieval, blocking, and antibody staining concentrations were Hermanson, Srinivas Rengarajan, Eric M. Ostertag, Devon J. Shedlock optimized to identify universal staining conditions. Slides were im- Poseida Therapeutics, San Diego, CA, USA aged using the Fluidigm Hyperion IMC system and image analysis Correspondence: Devon J. Shedlock (jbsmith0711@gmail.com) was performed with MCD viewer (Fluidigm) or CellProfiler/miCAT Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P490 (https://doi.org/10.1101/109207). Results Background We successfully performed IMC imaging of Hodgkin lymphoma, NK Prostate-specific membrane antigen (PSMA) is a transmembrane lymphoma and normal lymph nodes (LN) using a 26 parameter panel. glycoprotein overexpressed in >80% of metastatic prostate cancer. We were able to clearly identify tissue architecture (Fig. 1, normal LN, Although PSMA is a promising target for peptide and antibody drug germinal center(GC), mantle zone(MZ), cortex/paracortex(PC), Fig. 2A) conjugate therapies, early chimeric antigen receptor (CAR) T-cell ther- and characterize specific cellular populations such as CD8+Tcells, Th / apies lacked clinical efficacy. Here we developed a novel CAR T-cell Th subsets, FoxP3+T , tissue associated macrophages, CD56+NK cells product (P-PSMA-101) via piggyBac™ transposition of a tri-cistronic 2 reg and tumor vasculature. Important immuno-oncology targets such as transgene encoding a safety switch, a PSMA-specific Centyrin-based LAG3, PDL1/PDL2 and PD1 are included. Cellular resolution is similar to CAR (CARTyrin), and a selection gene, features that may improve IHC, allowing for imaging of tumor, effector and regulatory cell safety and efficacy compared with previous anti-PSMA CAR T-cell interactions at a single cell level (Fig. 2B, Reed Sternberg(RS)cells (red). therapies. CD8+(green) adjacent to RS cell flanked by T (FoxP3-teal) and Methods REG Th (CCR4-yellow) CD4+(not shown) cells. Abundant CD68+(magenta) We developed and identified a lead anti-PSMA CARTyrin from over 250 cells which co-express PDL1/PDL2 (not shown) in the background.) available Centryin binders. We also tested the previously clinically- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 237 of 244 tested anti-PSMA J591 scFv-based CAR for comparability. Initial assess- expression was further increased when cells were treated with ment utilized mRNA delivery of candidate CARTyrins to confirm CAR GSK126 and HDAC inhibitors compared to IFNγ alone. Migration as- surface expression and specific T-cell degranulation against PSMA+ says confirmed that tumor cells treated with GSK126 and HDAC in- prostate tumor cells or PSMA-engineered cells. We then used piggyBac hibitors released chemotactic factors that increased T-cell migration. to deliver our tri-cistronic vector system encoding the lead PSMA CAR- Results Tyrin (P-PSMA-101), J591 scFv CAR, or a BCMA-specific CARTyrin to T- Expression of CXCL9 and CXCL10 was induced by IFNγ in brain tumor cells, resulting in >95% CAR+ T-cells after selection and expansion. lines. Chemokine expression was further increased when cells were Importantly, our unique production methodology leads to >60% T- treated with GSK126 and HDAC inhibitors compared to IFNγ alone. Migra- stem cell memory (Tscm) cells, an early memory population that corre- tion assays confirmed that tumor cells treated with GSK126 and HDAC in- lates with complete responses in CD19 CAR T-cell clinical trials. In vitro, hibitors released T-cell chemotactic factors that increased T-cell migration. P-PSMA-101 and J591 CAR T-cells specifically proliferated, lysed, and Conclusions secreted IFN-γ against PSMA+ LNCaP or PSMA-engineered K562s. No These studies demonstrate that brain tumors express T-cell attracting evidence of tonic signaling or exhaustion was detected. chemokines CXCL9 and 10 in response to IFNγ. Further, single agent Results and the combination of GSK126 and HDAC inhibitors enhanced ex- P-PSMA-101 demonstrated significantly enhanced anti-tumor efficacy pression of CXCL9 and CXCL10, resulting in increased T-cell migration and survival in comparison to J591 CAR T-cell-treated mice in a low toward tumor cells. Together, these data provide a potential means “stress test” dose of T-cells against established subcutaneous LNCaP(- to enhance the efficacy of immune therapies for brain tumors by fLuc+) solid tumors in NSG mice. The >60% Tscm P-PSMA-101 ex- promoting T-cell trafficking toward tumor cells. panded in vivo and gave rise to differentiated effector CAR+ T-cells that were detected in the peripheral blood concomitant with a decrease in P492 tumor burden below detectable caliper and bioluminescent imaging Galectin-3 inhibition with GR-MD-02 synergizes with agonist anti- limits. P-PSMA-101 then contracted, yet persisted in the peripheral OX40 mAb therapy leading to reduced immune suppression and blood with >70% of T-cells exhibiting a Tscm phenotype. On the con- improved overall survival trary, J591 CAR T-cells did not significantly control tumor burden. In a Elizabeth Sturgill, Stephanie Linch, Courtney Mick, Melissa Kasiewicz, dose titration study, P-PSMA-101 eliminated established LNCaP tumor William Redmond in 100% of animals for the duration of the studies (42 days post- Providence Portland Medical Center, Portland, OR, USA treatment), while 2/3 low-dose animals remained tumor-free. Correspondence: William Redmond (elizabeth.sturgill@providence.org) Conclusions Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P492 P-PSMA-101 is a first-in-class Centyrin-based CAR T-cell therapeutic that exhibits a persistently high frequency of Tscm and mediates dur- Background able anti-solid tumor efficacy that surpasses previously established Cancer immunotherapy aims to harness the immune system to destroy anti-PSMA CAR T-cell therapy in our in vivo model. Future efforts will tumor cells as well as reverse tumor-induced immune suppression. One continue towards clinical application of P-PSMA-101 in patients with such molecule reported to contribute to immune suppression is metastatic castrate resistance prostate cancer. galectin-3 (Gal3). Indeed, Gal3 is upregulated on a wide variety of tumors and is associated with poor patient prognosis. In addition, in- P491 creased amounts of Gal3 are found in patients with metastatic disease. Alerting the immune system by removing epigenetic silencing of Gal3 has been shown to recruit myeloid derived suppressor cells T 1 chemokines (MDSCs) into the tumor microenvironment, thus aiding in tumor es- Heather Sonnemann, Amber Giles, Jinkyu Jung, Caitlin Reid, Marsha-Kay cape. Therefore, we hypothesized that the addition of a galectin-3 in- Hutchinson, Deric Park, Mark Gilbert hibitor (GR-MD-02) in conjunction with an agonist anti-OX40 antibody NIH, NCI, Bethesda, MD, USA (aOX40) would synergize to promote tumor regression and increased Correspondence: Amber Giles (heather.sonnemann@nih.gov) survival via a reduction in tumor-induced immune suppression. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P491 Methods MCA-205 sarcoma cells or TRAMP-C1 prostate adenocarcinoma cells Background were implanted into the flank of C57Bl/6 mice; 4T-1 mammary carcin- BACKGROUND: Solid tumors employ multiple mechanisms to evade oma cells were implanted orthotopically into the mammary fat pad. immunologic detection and destruction. However, the potential to GR-MD-02 (2.4 mg/dose) and aOX40 (250 μg/dose) were administered enhance the immune response to cancer has been proven in several to mice intraperitoneally (ip) either 3x/week for 2 weeks or 2x/week for malignancies and is under investigation in many others, including 1 week, respectively. Tumor growth (area) was assessed 2x/week and primary CNS tumors. Brain tumors, in particular, lack robust T-cell in- mice were sacrificed when tumors exceeded 150 mm2. Tumors were filtration, suggesting poor immune surveillance. Recent studies have either digested and immune cells were analyzed by flow cytometry or found that tumor cells express T-cell attracting chemokines, CXCL9 and homogenized into tumor lysate was assayed by cytokine bead array. CXCL10, in response to interferon gamma (IFNγ). Chemokine expression Results can be further amplified by drugs that prevent epigenetic silencing, such We observed that combined GR-MD-02/aOX40 therapy significantly im- as histone methyltransferase inhibitors or histone deacetylase (HDAC) in- proved survival of MCA-205, 4T-1, and TRAMP-C1 tumor-bearing mice, hibitors. This approach increases T-cell trafficking to tumors in vivo and and additionally reduced lung metastases in the 4T-1 model. Further enhances the anti-tumor efficacy of checkpoint blockade. We hypothe- analysis revealed that GR-MD-02/aOX40 therapy significantly reduced sized that T-cell trafficking to brain tumors could be increased by epige- the amount of both monocytic (Mo-) and granulocytic (Gr-) MDSCs netically enhancing CXCL9 and CXCL10 expression. within the tumor compared to aOX40 alone (p<0.05). In addition, we Methods observed a significant decrease in PD-L1, iNOS, arginase 1 (p<0.05), and Assays were performed on human glioma brain tumor cell lines. CD206 (p<0.001) expression in both Mo- and Gr-MSDCs. Moreover, GR- CXCL9 and CXCL10 expression were measured by real-time PCR and MD-02/aOX40 therapy led to increased CD8 T cell proliferation in the Western blot. GSK126, a commercially available methyltransferase in- lymph node and increased intratumoral concentrations of IL-17a and hibitor, was utilized to demethylate histone H3 (K9 and K27). LB201 IL-15 (p<0.05), suggesting a potential role in modulating immune infil- (Lixte Biotechnology, Setatuket, NY) and SAHA were used to inhibit trate and/or prolonged T cell survival. class 1 and class 2 HDACs and Entinostat was used to inhibit class I Conclusions and class III HDACs. Histone methylation and acetylation status were In summary, our data suggests that Gal3 inhibition plus agonist aOX40 examined using Western blot analysis. T-cell migration was measured therapy may reduce myeloid recruitment to the tumor microenviron- using transwell migration assays. RESULTS: Expression of CXCL9 and ment, thus reducing immune suppression and subsequently mediating CXCL10 was induced by IFNγ in brain tumor lines. Chemokine tumor regression and increased survival. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 238 of 244 P493 Methods + + + LTX-315: A first-in-class oncolytic peptide that reshapes the tumor Human CD8 Tcells,CD4 Tcells, and CD14 monocytes were isolated microenvironment from buffy coats using various RosetteSep and EasySep Enrichment cock- 1 2 2 3 Baldur Sveinbjørnsson , Ketil Camilio , Meng-Yui Wang ,Janne Nestvold , tails. The ability of AB421 to rescue AMP-mediated inhibition of T cell 2 2 1 Gunhild Mælandsmo ,Gunnar Kvalheim , Øystein Rekdal activation was evaluated using CD3/CD28/CD2 stimulation. Mixed 1 2 Lytix Biopharma, Oslo, Norway; Radiumhospitalet, Oslo, Norway; lymphocyte reactions (MLRs) were established by mixing GM-CSF and IL-4 3 + Rikshospitalet, Oslo, Norway differentiated monocytic DCs with allogeneic CD4 T cells. AMP and ADO Correspondence: Øystein Rekdal levels in C57BL/6J mice were measured by mass spectrometry in plasma (baldur.sveinbjornsson@lytixbiopharma.com) samples isolated at various time points after administration of AB421. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P493 Results We have designed a highly potent and selective small-molecule Background CD73 inhibitor, AB421, which inhibits endogenous CD73 activity in + + The oncolytic peptide LTX-315, which has been de novo designed human CD8 and CD4 T cells with IC values of 4.5 pM and 6.2 pM, based on structure-activity relationship studies of host-defense pep- respectively. AB421 prevents AMP-mediated inhibition of T cell acti- + + tides, has the ability to kill human cancer cells and induce long-lasting vation in CD3/CD28/CD2 activated human CD4 and CD8 T cells, as anticancer immune response when injected locally into tumors estab- well as allogeneic CD4 T cell activation in MLRs. Addition of exogen- lished in immunocompetent murine models. The oncolytic effect of ous AMP abrogated the enhanced allogeneic CD4 T cell activation LTX-315 involves perturbation of the plasma membrane and the mito- and IFN-γ production mediated by α-PD-L1 monoclonal antibody chondria with subsequent release of danger-associated molecular pat- (mAb), an effect that was blocked by AB421. Mechanistically, addition tern molecules (DAMPs) such as ATP, Cytochrome C and HMGB1. of AMP repressed expression of activation markers (CD25) and im- Furthermore, LTX-315 effectively disintegrates the cellular compart- mune checkpoint proteins (CTLA-4, PD-1, TIM-3, LAG-3, ICOS, CD28) ments with subsequent release of tumor antigens as demonstrated by in the MLR assays. This suggests that activation of the adenosinergic a greater T-cell infiltration (TILs), TILs clonality and the number of clones pathway is dominant and may limit the utility of most antibodies tar- with greater abundance in the tumor microenvironment. In experimen- geting immune checkpoint proteins by curtailing their expression tal tumor models, LTX-315 exerts abscopal effects and reshapes the and/or upregulation (as was seen with an α-PD-L1 mAb and AMP co- tumor microenvironment by decreasing the local abundance of im- culture systems). Analysis of TCGA databases and tumor microarrays munosuppressive cells and by increasing the frequency of effector T- showed differential expression of CD73 across tumor types, with high cells. LTX-315’s ability to convert immunogenically “cold” tumors to expression detected in non-small cell lung carcinoma (adenocarcin- “hot” makes it ideal combination partner with other immunotherapies oma), colorectal, head and neck, esophageal, and stomach cancers. or immunochemotherapy. Indeed, in preclinical tumor models, a com- Analysis of dissociated tumor cells by flow cytometry showed CD73 bination of LTX-315 and immune checkpoint inhibitor (anti-CTLA4) expression in both hematopoietic and non-hematopoietic cells. Fi- demonstrates significant synergy. nally, we show that AB421 can elevate AMP-to-ADO ratios in vivo in a Methods dose-dependent manner, reflecting systemic inhibition of CD73. In the present study the antitumor efficacy and potential synergy of Conclusions LTX-315 in combination with low-dose chemotherapy was investigated AB421 represents a class of potent, reversible and selective CD73 in- in experimental mouse models. Subcutaneously established murine hibitors that exhibit picomolar potency in primary human immune A20 lymphoma was treated with LTX-315 alone, cyclophosphamide cells and is currently undergoing preclinical evaluation as a candidate alone or LTX-315 in combination with cyclophosphamide. Similarily, for clinical evaluation. orthotopically established 4T1 murine mammary carcinoma was treated with either LTX-315 alone, liposomal doxorubicin (CAELYX) P495 alone or in combination. Separate molecular pathways mediate anti-tumor versus tumor- Results promoting Aspects of dsRNA signaling in cancer LTX-315 showed significantly enhanced anticancer efficacy against microenvironments A20 lymphomas and 4T1 breast carcinomas when combined with 1 1 1 Marie-Nicole Theodoraki , Saumendra Sarkar , Ravi Muthuswamy , Jamie cyclophosphamide and doxorubicin, respectively. 1 2 1 1 Voyten , Robert Edwards , David Bartlett , Kalinski Pawel Conclusions 1 2 University of Pittsburgh, Pittsburgh, PA, USA; Magee-Womens Hospital The LTX-315 unique “release and reshape” properties make it a of UPMC, Pittsburgh, PA, USA promising candidate for combination with several types of anticancer Correspondence: Marie-Nicole Theodoraki therapies. Phase 1b study combining LTX-315 with ipilimumab (anti- (marie.nicole.theodorakis@gmail.com) CTLA-4) in malignant melanoma patients, as well as LTX-315 with Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P495 Pembrolizumab (anti-PD1) in metastatic breast cancer is ongoing. Background P494 Infiltration of tumors with cytotoxic T cells (CTLs) predicts improved prog- Reversal of adenosine-mediated immune suppression by AB421, a nosis and has been shown critical for antitumor effectiveness of check- potent and selective small-molecule CD73 inhibitor point blockers. In contrast, tumor infiltration with regulatory T cells Joanne BL Tan, Annette Becker, Daniel DiRenzo, Kristen Zhang, Ada (Tregs) and myeloid-derived suppressor cells (MDSCs) typically predicts Chen, Rhiannon Thomas-Tran, Joel Beatty, Debashis Mandal, Guifen Xu, rapid progression and poor outcomes. Poly-I:C, a frequently used adju- Steve Young, Matthew Walters, Ulrike Schindler, Jay Powers vant which induces not only the CTL-attracting chemokines but also Treg Arcus Biosciences, Hayward, CA, USA attractants. Here we evaluated the molecular pathways, which lead to Correspondence: Joanne BL Tan jtan@arcusbio.com the induction of chemokines by dsRNA in TME and different types of Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P494 tumor-associated cells, in order to develop improved adjuvants which se- lectively attract the desirable effector cells rather than suppressive cells. Background Methods Extracellular adenosine-triphosphate (ATP) is efficiently hydrolyzed to Isolated cells or human cancer biopsies were cultured in the absence adenosine by ecto-nucleotidases CD39 and CD73, which converts or presence of one of two synthetic TLR3 ligands Poly-I:C (non-select- adenosine-monophosphate (AMP) into adenosine (ADO). ADO sup- ive activator of TLR3 and helicases) or rintatolimod (selective TLR3 presses immune responses including those of T cells, natural killer (NK) ligand) and in the absence or presence of a COX-1/2 inhibitor, NF-kB- cells, and dendritic cells (DCs) through activation of A Rand A R recep- or TNFa inhibitors. mRNA assays. ELISA, chemotaxis assays and mo- 2a 2b tors. CD73 inhibition is a promising therapeutic approach for preventing lecular biology assays were used to analyze the chemokine produc- ADO-mediated immunosuppression in the tumor microenvironment. tion and tumor-associated suppressive factors. Confocal microscopy Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 239 of 244 of macrophage cultures treated with the TLR3 ligands was performed and Tregs) T cells, as well as natural killer cells, compared to controls. to evaluate the activation of the different signaling pathways by the Among CD45+ cells, tumor-associated macrophages (TAMs) were the TLR3 receptor and cytoplasmic helicases. most abundant cell type; PEGPH20 significantly reduced the fre- Results quency of TAMs, while increasing the frequency of dendritic cells. We observed that poly-I:C induced activation of NF-kB- and COX-2 Conclusions pathways leading to induction of COX2-dependent suppressive factors Collectively, these results suggest that degradation of tumor HA by and Treg- and MDSC-attracting chemokines. These undesirable effects PEGPH20 can facilitate an anti-tumor immune response by promot- were blocked with inhibitors of both NF-kB- or COX-2 pathways. In con- ing effector cell infiltration and skewing the immune microenviron- trast rintatolimod selectively induced the desirable chemokines, which ment toward a more anti-tumor composition, thereby enhancing were associated with lack of direct activation of NF-kB- or COX-2 path- the effect of anti-PD-L1. These findings support the ongoing clinical ways, and strongly suppressed attraction of Tregs and MDSCs, with evaluation of PEGPH20 in combination with checkpoint inhibitors in elevated CTL attraction in ex vivo migration assays. Looking at the up- HA-accumulating solid tumors, and provide rationale for investigat- stream signaling pathways, both TLR3 ligands induced IRF3 (Type-1 ing PEGPH20 in combination with additional immune modulating interferon pathway). However, only Poly-I:C induced activation of TRAF3 cancer therapies. and RIP-1 (TLR3 dependent NF-kB pathway) as well as MAVS (cytoplas- mic helicases). Conclusions P497 Our data implicate an important new role of helicases by the induction Tumor microenvironment immune gene signature asscoiated with of tumor-promoting factors by dsRNA and points out to new targets to axicabtagene ciloleucel (axi-cel, KTE-C19), an anti-CD19 chimeric enhance the immunogenic and antitumor activities of adjuvants. antigen receptor (CAR) T cell, in a multicenter trial (ZUMA-1) 1 2 2 2 3 John Rossi ,JérômeGalon , Sarah Turcan , Corinne Danan , Frederick Locke , 4 5 6 7 Sattva Neelapu ,David Miklos ,Nancy Bartlett , Caron Jacobson , 8 9 10 11 P496 Ira Braunschweig , Olalekan Oluwole , Tanya Siddiqi ,YiLin , 12 13 14 1 Degradation of hyaluronan (HA) by PEGPH20 promotes anti-tumor John Timmerman , Patrick Reagan , Lazaros Lekakis , Sherryonne Unabia , 1 1 1 immunity and enhances the effect of checkpoint blockade in an William Go ,JeffWiezorek , Adrian Bot 1 2 3 HA-accumulating mouse syngeneic tumor model Kite Pharma, Santa Monica, CA, USA; HalioDx, Marseille, France; Moffitt Benjamin J Thompson, Renee Clift, Trevor B Kimbler, Jesse D Bahn, Cancer Center, Tampa, FL, USA; MD Anderson Cancer Center, Houston, Chunmei Zhao, Barbara Blouw, Curtis B Thompson, Sanna Rosengren TX, USA; Stanford University School of Medicine, Stanford, CA, USA; 6 7 Halozyme, San Diego, CA, USA Washington University Medical School, Saint Louis, MO, USA; Dana- Correspondence: Benjamin J Thompson (bthompson@halozyme.com) Farber Cacner Institute, Boston, MA, USA; Montefiore Medical Center, Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P496 Albert Einstein College of Medicine, Bronx, NY, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; City of Hope National Medical 11 12 Background Center, Duarte, CA, USA; Mayo Clinic, Rochester, MN, USA; UCLA The glycosaminoglycan hyaluronan (HA) is abundant in many solid David Geffen School of Medicine, Santa Monica, CA, USA; University of tumors, and its accumulation is often associated with poor patient Rochester Medical Center, Rochester, NY, USA; University of Miami outcomes. Its degradation by intravenously administered PEGylated Health System, Sylvester Comprehensive Care Center, Miami, FL, USA recombinant human hyaluronidase PH20 (PEGPH20) remodels the Correspondence: Adrian Bot (kate.trueblood@nexusggmed.com) tumor stroma, reduces intratumoral pressure, decompresses tumor Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P497 blood vessels, and facilitates drug delivery. Recently, PEGPH20 was shown to enhance tumor growth inhibition induced by the immune Background checkpoint inhibitor anti-PD-L1 in HA-accumulating pancreatic and The efficacy of axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 orthotopic breast syngeneic tumor models, and to enhance tumor T CAR T cell therapy, was evaluated in the multicenter, pivotal ZUMA-1 cell infiltration. Here, the HA content of commonly used syngeneic study in patients with refractory, aggressive non-Hodgkin lymphoma models was systematically studied, and MC38 (Charles River Labora- (NHL). In a pre-specified interim analysis, ZUMA-1 met its primary end- tories) was identified to further evaluate the combinatorial effect of point with an objective response rate of 82% and a complete response PEGPH20 and anti-PD-L1 in HA-accumulating tumors. rate of 54% [1]. Based on an analysis of patients enrolled in ZUMA-1, we Methods describe for the first time a tumor microenvironment immune gene signa- Eight syngeneic tumor models were screened for HA content by ture associated with CAR T cell treatment of patients with aggressive NHL. ELISA, and immune activity by qPCR for a panel of transcripts that in- Methods cluded surface markers, cytokines, chemokines, immune checkpoints, In this post-hoc analysis, paired biopsies were taken before and within and immunomodulatory enzymes. For tumor growth and immuno- 3 weeks of axi-cel treatment from 14 patients enrolled in ZUMA-1. TM phenotyping studies, MC38 tumors were implanted subcutaneously, These biopsies were analyzed by digital gene expression (Nanostring ) and PEGPH20 (37.5 μg/kg) was administered intravenously 24 h prior and a pre-specified bioinformatics algorithm was then applied to to anti-PD-L1 (10F.9G2, 5 mg/kg) using a biweekly dosing regimen. IGES15 and IGES21 genes, which are hypothesized to be involved in Tumor HA was visualized by immunohistochemistry followed by posi- immune-mediated tumor regression (Immunosign®) [2]. The Immuno- tive pixel count. Tumor-infiltrating immune cells were evaluated by sign® profiles evaluated a pre-defined set of genes for effector T cells, flow cytometry following tumor dissociation. Th1 cells, chemokines, and cytokines. Expression analysis and hierarch- Results ical clustering were used to define an axi-cel–related tumor immune Of the eight models tested, MC38 tumors had the highest HA con- gene signature. Wilcoxon signed rank test with multiple test correction tent, and displayed evidence of robust immune activity in the qPCR by false discovery rate (FDR; Benjamini-Hochberg) was used. screen. PEGPH20 reduced MC38 tumor HA by nearly 90% 24 h after Results either a single dose, or the last of three biweekly doses. In two out A comparison of gene expression profiles of biopsies from 14 patients on of three blinded studies, PEGPH20 significantly enhanced inhibition ZUMA-1 taken before and after axi-cel treatment showed profound of MC38 tumor growth induced by anti-PD-L1, and combination changes in gene expression within the tumor environment after infusion. treatment also significantly prolonged the survival of tumor-bearing The most upregulated genes post–axi-cel treatment were CCL5 (RANTES), mice compared to either treatment alone. PEGPH20-treated tumors CTLA4,and GZMA (log2 fold change >2, P<0.05, FDR <0.050). Immune contained significantly higher numbers of CD8+ and CD4+ (both Th checkpoints PD-L1 and LAG3 were also upregulated post–axi-cel treatment Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 240 of 244 (log2 fold change >1.6, P<0.05, FDR <0.055). Other genes associated with P499 T cell proliferation, homing, and effector function that were upregulated Expression pattern and prognostic value of IL-36g in colorectal included IL-15, GZMK, CXC3CL1 (Fractalkine), CD8A,and STAT4 (log2 cancer 1 2 3 fold change >1.6; P< 0.05, FDR <0.074). Additional baseline tumor Aliyah M Weinstein , Nicolas A Giraldo , Catherine Julie , Laetitia 2 2 3 2 characteristics and associative analysis will be presented. Lacroix , Florent Petitprez , Jean-François Emile , Wolf H Fridman , 1 2 Conclusions Walter J Storkus , Catherine Sautès-Fridman 1 2 We define a mechanistic tumor immune gene signature in NHL patients University of Pittsburgh, Pittsburgh, PA, USA; UMR_S1138, Centre de associated with axi-cel treatment. This signature comprises upregulation Recherche des Cordeliers, Paris, France; Hopital Ambroise Paré, of T cell activation, effector, chemokine, and immune checkpoint genes. Boulogne, France These data will potentially lead to rational optimization of T cell interven- Correspondence: Walter J Storkus (alw151@pitt.edu); Catherine Sautès- tions in cancer. Fridman Trial Registration Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P499 NCT02348216. Background References IL-36g/IL-1F9 is a member of the IL-1 cytokine superfamily and a me- 1. Locke FL, Neelapu SS, Bartlett, NL, et al. AACR 2017. 9986. diator of Type 1 immunity. It was recently reported to be a necessary 2. Galon J, Angell HK, Bedognetti D, Farincola FM. Immunity. 2013;39: 11-26. mediator of tertiary lymphoid organ (TLO) formation in a murine model of colon carcinoma [1]. Specifically, when IL-36g was intro- duced therapeutically into the tumor microenvironment of MC38- P498 bearing mice, tumors exhibited delayed progression in correlation AB928, a dual antagonist of the A R and A R adenosine with TLO formation; this was not observed in control-treated mice. 2a 2b receptors for the treatment of cancer Co-treatment with the antagonist to IL-36g, IL-1F5, blocked the effi- Matthew Walters, Daniel DiRenzo, Joanne Tan, Ulrike Schindler, Dana cacy of the therapy. CD11c+ dendritic cells (DC) were observed to be Piovesan, Devika Ashok, Tim Park, Nell NarasappaFangfang Yin, Wan Hsin a major source of IL-36g in the tumor microenvironment (TME), and Lim, Stefan Garrido-Shaqfeh, Jenna Jeffrey, Laurent Debien, Brandon signaling through IL-36R+ host cells (including those of the vascula- Rosen, Ehesan Sharif, Dillon Miles, Manmohan Leleti, Juan Jaen, Jay ture) was necessary for the efficacy of IL-36g-based treatment. In the Powers present study, we sought to identify a role for IL-36g in the de novo Arcus Biosciences, Hayward, CA, USA formation of TLO. Correspondence: Matthew Walters (mwalters@arcusbio.com) Methods Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P498 In a cohort of 33 primary colorectal cancer patients, the histologic localization and magnitude of expression of IL-36g, IL-1F5/IL-36RA, CD20, Background CD138, CD8, CD31, PNAd, DC-LAMP, CD68, and Tbet were determined In the tumor micro-environment (TME), extracellular ATP is sequen- using immunohistochemical and immunofluorescent imaging. Images tially hydrolyzed to adenosine by the ecto-nucleotidases CD39 were analyzed using CaloPix and Visiopharm software packages. (ATP→AMP) and CD73 (AMP→adenosine), where it acts to suppress Results immune cell function. A R is expressed by a variety of lymphocytes, Both IL-36g and IL-1F5 can be expressed by vasculature-associated 2a while myeloid cells express both A R&A R. A R activation results smooth muscle cells/myofibroblasts as well as immune cells. Expres- 2a 2b 2a in decreased T cell activation, while binding of adenosine to A Ron sion of IL-36g by the tumor vasculature positively correlates with 2b myeloid cells is thought to promote a tolerogenic phenotype. To alle- CD20+ B cell density in TLO, but not the number of TLO observed in viate immune suppression within the TME, we have designed AB928, the tumor; neither observation holds for IL-1F5. Some IL-36g+ vessels a potent and selective dual antagonist of A R and A R appear to be high endothelial venules, specialized vessels that aid in 2a 2b Methods recruiting lymphocytes to TLO. Interestingly, while B cells are the pri- Human immune cells were isolated from healthy donor PBMC and mary cellular component of TLO in this model, they do not produce cultured as appropriate for the cell type. pCREB assays were per- IL-36g. Within the immune compartment, CD8+ T cells (including formed using freshly collected whole mouse blood. those expressing Tbet) and CD68+ macrophages are major sources Results of IL-36g in the TME, while DC-LAMP+ DC are less so. AB928 inhibits both A Rand A R with similar potencies (K :1.4 nM and Conclusions 2a 2b B 2 nM, respectively). In PBMC sorted from healthy donors, similar levels of These results support that the pro-inflammatory conditions of colo- A R mRNA were observed in naïve and central memory CD4 and CD8 T rectal cancer can promote a Type 1-polarized immune microenviron- 2a cells, as well as NK and B cells; A R was also expressed in both dendritic ment; that IL-36g is secreted by diverse cell types within the TME; 2a cells and monocytes. In contrast to this, A R was absent from lympho- and that IL-36g expression may play a role in the maintenance of 2b cytes but was present in both dendritic cell and monocyte populations. TLO and in B cell recruitment to these structures, within the immune Adenosine-mediated suppression of CD8 T cell activation was reversed contexture of colorectal cancer. by AB928, which restored IFN-g and granzyme B production from these cells. Adenosine-mediated suppression of NK lytic activity was inhibited References by AB928, resulting in a significant increase in cell killing. Monocyte- 1. Weinstein AM, Chen L, Brzana EA, Patil PR, Taylor JL, Fabian KL, Wallace CT, derived dendritic cells (moDC) express high levels of A R and those cells, Jones SD, Watkins SC, Lu B, Stroncek DF, Denning TL, Fu YX, Cohen PA, 2b when cultured in the presence of adenosine, had lower levels of costimu- Storkus WJ. Tbet and IL-36g cooperate in therapeutic DC-mediated promo- latory molecules upon activation and a reduced capacity to stimulate tion of ectopic lymphoid organogenesis in the tumor microenvironment. CD4 T cells in a mixed-lymphocyte reaction (MLR). Inhibition of OncoImmunology. 2017; 6(6). adenosine-mediated signaling by AB928 resulted in a significant increase in the frequency of activated T cells in the MLR assay. Adenosine receptor activation leads to phosphorylation of CREB, which can be detected in circulating leukocytes, allowing for the establishment of PK/PD relation- P500 ships in mice. AB928 exhibited a dose dependent inhibition of pCREB in CD73 blockade restores the abscopal response to radiation whole blood. therapy and anti-CTLA-4 in cGAS-deficient tumors Conclusions Erik Wennerberg, Claire Vanpouille-Box, Silvia Chiara Formenti, Sandra In conclusion, AB928 is a potent dual inhibitor of A R and A R re- Demaria 2a 2b ceptors with the potential to block all immunosuppressive effects of Weill Cornell Medicine, New York, NY, USA extracellular adenosine in the TME. This molecule is anticipated to Correspondence: Erik Wennerberg (erik.ag.wennerberg@gmail.com) enter clinical development later in 2017. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P500 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 241 of 244 Background associated fibroblasts (CAF) in cancer microenvironments, induction of We have recently shown that in poorly immunogenic tumors resistant tumor antigen specific T cells, and therapeutic effects of PD-1/PD-L1 to immune checkpoint blockers (ICB), radiation therapy (RT) used at immune checkpoint blockade therapies, using murine models bearing 8GyX3 induces cancer cell-intrinsic interferon type I (IFN-I) through tumor cell lines in which RAS was not involved in their proliferation cGAS-activation. This process is required for recruitment of BATF3- and angiogenetic ability. dependent CD103 dendritic cells (DCs) and for priming of tumor- Results specific CD8 T cells that mediated abscopal responses in the presence Administration of ARBs to tumor-bearing mice resulted in significant of ICBs [1]. Importantly, knockdown of cGAS in the cancer cells at the ir- enhancement of tumor antigen specific T cells. The ARB administra- radiated tumor site abrogates the induction of abscopal responses by tion did not change the numbers of CD11b+ myeloid cells in tumors, RT+ICB. Extracellular ATP, which is released from irradiated cancer cells, but significantly reduced their T-cell inhibitory ability along with de- provides an alternative signal for recruitment and activation of DCs. creased production of various immunosuppressive factors including However, rapid conversion of ATP into immunosuppressive adenosine IL-6, IL-10, VEGF, and arginase by CD11b+ cells in tumors. ARB also effectively abrogates this process. Therefore, we hypothesized that anti- decreased expression of immunosuppressive factors such as chemo- body blockade of the adenosine-converting ectoenzyme CD73 could kine ligand 12 and nitric oxide synthase 2 in CAFs. Lastly, combin- restore abscopal responses by RT+ICB in cGAS-deficient tumors by aug- ation of ARB and anti-programmed death-ligand 1 (PD-L1) antibodies menting ATP-mediated DC recruitment. resulted in significant augmentation of anti-tumor effects in a CD8- Methods positive T cell-dependent manner. Mice bearing subcutaneously inoculated flank tumors (cGAS-silenced Conclusions shcGAS shNS shNS TSA or mock-silenced TSA as primary tumors and TSA as These results demonstrated that RAS is involved in generation of im- abscopal tumors) were treated with (1) control antibody; (2) RT; (3) RT munosuppressive tumor microenvironments caused by myeloid cells +anti-CD73; (4) RT+anti-CTLA-4 or (5) RT+anti-CD73+anti-CTLA-4. Anti- and fibroblasts, other than the previously shown proliferative and CD73 was administered on day 11, 14, 17 and 20 and anti-CTLA-4 on day angiogenetic properties of cancer cells and macrophages, and that 14, 17 and 20. RT was administered to the primary tumor in doses of 8 Gy ARB can transform the immunosuppressive properties of MDSCs and on day 12, 13 and 14. Tumors were analyzed for DC infiltration on day 18. CAFs and could be used in combination with PD-1/PD-L1 immune Results checkpoint blockade therapies. shNS In mice bearing primary TSA tumors, RT+anti-CTLA-4 elicited absco- shcGAS pal responses. In mice bearing primary TSA tumors, RT+anti-CTLA- P502 4 did not induce abscopal responses, while RT+anti-CTLA-4+anti-CD73 3 3 IDO1, an immune checkpoint with distinct mechanisms and did (Tumor size day 37: 873±533 mm in control; 827±732 mm in RT prognostic significance in glioblastoma +anti-CTLA-4; 131±169 mm in RT+anti-CTLA-4+anti-CD73, p<0.01). shcGAS 1 1 1 2 Lijie Zhai , Derek Wainwright , Erik Ladomersky , Carlos Dostal , Kristen Treatment of mice bearing TSA tumors with RT+anti-CD73 re- + 1 2 3 1 1 Lauing , Robert McCusker , David Binder , Meijing Wu , Matthew Genet , sulted in significantly increased tumor infiltration of CD103 DCs 1 4 5 1 Galina Gritsina , Balaz Gyorffy , Priscilla Brastinanos , Jeffrey Sosman , among intratumoral leukocytes compared to mice receiving RT alone 1 1 1 1 Craig Horbinski , David James , Roger Stupp , Francis Giles (1.2±0.8% in RT v. 2.5±0.7% in RT+anti-CD73, p<0.05). Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Conclusions 2 3 University of Illinois at Urbana-Champaign, Urbana, IL, USA; University of Our results demonstrate that blocking CD73 recovers the ability of + 4 Colorado School of Medicine, Aurora, CO, USA; Semmelweis University, RT to induce recruitment of CD103 DCs to cGAS-deficient tumors Budapest, Hungary; Massachusetts General Hospital, Boston, MA, USA and restores the abscopal anti-tumor responses elicited by RT+ICB. Correspondence: Derek Wainwright (zhailijie12@gmail.com) Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P502 References 1. Vanpouille-Box C, et al. DNA exonuclease Trex1 regulates radiotherapy- Background induced tumour immunogenicity. Nat Commun. 2017;8:15618. It is commonly accepted that indoleamine 2, 3 dioxygenase 1 (IDO1), a pivotal immune checkpoint, mediates potent immunosuppression P501 in cancers through its tryptophan (Trp)-to-kynurenine (Kyn) cataboliz- Involvement of local renin-angiotensin system in ing activity. However, this enzymatic-dependent mechanism of IDO1 immunosuppression of tumor microenvironment and its has not been established in glioblastoma (GBM), the most common modulation for augmentation of cancer immunotherapy malignant brain tumor in adults. Additionally, the basis for elevated 1 2 1 1 Tomonori Yaguchi , Kenta Nakamura , Kenji Morii , Yutaka Kawakami IDO1 expression in GBM is also poorly understood. The major object- 1 2 Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; Shinshu ive of this study is to address this gap in our understanding of how University School of medicine, Matsumoto, Nagano, Japan IDO1 contributes to the biology of GBM and immunotherapy of this Correspondence: Yutaka Kawakami beatless@rr.iij4u.or.jp deadly malignance, as well as whether its level of expression is a de- Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P501 terminant of GBM patients’ outcome. Methods Background Freshly resected human GBM, human T cell:GBM co-cultures, HPLC -/- To improve the current cancer immunotherapies, strategies to modu- analysis, CTLA-4 and PD-L1 immune checkpoint blockade, IDO1 and +/+ +/- fl/ late various immunosuppressive stromal cells need to be developed. IDO1 C57BL/6 mice, spontaneous GBM [GFAP(ERT2)→Cre ;pTEN fl fl/fl fl/fl fl/fl -/- Local renin–angiotensin system (RAS) in cancer tissues has been re- ;Rb ;p53 ;(+/-IDO1 );(+/-IDO1 )] mice, nu/nu mice, NOD-scid ported to be involved in cellular migration, proliferation, inflamma- and humanized (NSG-SGM3-BLT) mice engrafted human GBM as well tion, and angiogenesis in the tumor and supporting stromal cells, as the cancer genome atlas form the basis of our investigation. few studies have evaluated the effect of local RAS on anti-tumor im- Results mune responses. In this study we have evaluated the role of local (1) Depletion of GBM-derived IDO1 did not affect brain tumor Trp RAS in the tumor immune-microenvironment. catabolism, while systemic knockout of IDO1 resulted in decreased Trp- Methods >Kyn conversion in both naïve and tumor-bearing mice; (2)CTLA-4 and -/- We have evaluated the effect of administration of angiotensin receptor PD-L1-based dual immunotherapy did not alter Trp/Kyn level in IDO1 +/+ blockers (ARBs) on phenotypes of CD11+ myeloid cells and cancer and IDO1 mice, but only reached to maximal treatment effect in IDO1 Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 242 of 244 +/+ mice; (3) In situ hybridization for IDO1 revealed consistent transcript Conclusions expression in all human GBM samples (n=172), whereas immunohisto- Conclusion: Our study represents so far the most comprehensive chemical IDO1 expression was highly variable; (4) Multivariate analysis re- characterization of immune cell subtypes in the tumor microenviron- vealed that higher levels of IDO1 transcript predicted/correlated with ment of cholangiocarcinoma. The results suggest that PD-1/PD-L1 is shorter survival in the TCGA dataset (P=0.0076) and IDO1 mRNA levels not a good target in ECC, but a potentially good target in ICC. An positively correlate with increased gene expression for markers of cyto- ideal macrophage-targeting agent would be the one that depletes lytic and regulatory T cells, but no correlation with IFNs gene expression; the M2 macrophages more specifically. (5) Humanized mice intracranially-engrafted with human GBM revealed an IFNγ-dependent and T cell-mediated increase of intratumoral IDO1 expression. Conclusions Our data indicate that tumor cell-derived IDO1 mediates its immuno- suppression in GBM via a non-enzymatic mechanism, while the non- tumor IDO1 dominantly contributes to its Trp catabolic function and is essential for the immune checkpoint blockade therapy. TCGA analysis also revealed high intratumoral IDO1 mRNA levels correlate with infer- ior GBM patient outcome and infiltrating T cells. Future immunothera- peutic efforts aiming to increase T cell-mediated immune response should consider combinatorial approaches that inhibit potential T cell- mediated IDO1 upregulation during therapy. P503 Comprehensive characterization of immune microenvironment of cholangiocarcinoma 1 1 1 1 1 Tao Xia , Dwayne Thomas , Linda Chen , Qingfeng Zhu , Jin He , Robert 1 2 2 1 1 Anders , Shafat Quadri , Elaine Pinherio , Adrian Murphy , Lei Zheng Johns Hopkins University School of Medicine, Baltimore, MD, USA; Merck, Merck, NJ, USA Correspondence: Lei Zheng lzheng6@jhmi.edu Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P503 Background Fig. 1 (abstract P503) See text for description Background: Biliary tract cancers (BTC) are groups of tumors that in- clude both intrahepatic cholangiocarcinoma (ICC), extrahepatic cholan- giocarcinoma (ECC) and gallbladder carcinoma (GC). BTC comprise an uncommon cancer type that is currently on the rise with a high mortal- ity rate. New modalities of treatment for BTC are highly demanded. Im- munotherapy has emerged as a promising treatment for many malignant diseases. Although many risk factors of BTC are associated with chronic inflammatory conditions, the immune microenvironment of BTC has been examined to a very limited extent. Therefore, we per- fored a comprehensive analysis of tumor microenvironment of BTCs to identify immune mechanistic targets for immunotherapy and identify optimal strategies of combinational immunotherapy. Methods Materials and methods: Surgically resected tumors from 31 cases of ECC and 24 cases of ICC were stained with multiplex immunohisto- chemistry of 14 immune cell markers including CD3, CD4, CD8, CD20, CD45, CD68, Cd163, CSF1R, DC-LAMP, DC-Sign, Foxp3, Ki-67, PD-1, PD-L1 on single slide section and analyzed by the Halo software. Nanostring of immune panels and cytokine arrays PCR were also per- formed by RNA purified from FFPE slides. Results Results: Higher density of CD8 T cells is significantly associated with longer overall survival (p=0.024). Higher densities of PD-1+ T cells and PD-L1+ myeloid cells are significantly associated with longer overall survival in ECC (PD-L1, p=0.019; PD-1, p=0.018) (Fig. 1, Fig. 2), but higher density of PD-L1+ cells are significantly associated with shorter overall survival in ICC (p=0.003) (Fig. 3, Fig. 4). CD68 is not prognostic. However, higher density of CD163+CD68+ M2 macro- phages is associated with poorer survival in ECC (Fig. 5). Nanostring and cytokine array PCR results are being analyzed. More cases are Fig. 2 (abstract P503) See text for description being stained by multiplex immunothistochemistry. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 243 of 244 Fig. 3 (abstract P503) See text for description Fig. 5 (abstract P503) See text for description P504 Enhancement of CAR-T cell tumor tropism through expression of CXCR2 chemokine receptors 1 1 1 1 Ismahene Benzaid , Daniel Santiago , Cecilia Ramello , Mibel Pabon , 1 1 1 1 Wendy Kandell , Theresa Boyle , Anders Berglund , Scott Antonia , Daniel Abate-Daga Moffitt Cancer Center, Tampa, FL, USA Correspondence: Daniel Santiago Daniel.AbateDaga@moffitt.org Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P504 Background Chimeric Antigen Receptor (CAR)-T cell-based immunotherapies are novel approaches to cancer therapy. With the expected FDA approval of an anti-CD19 CAR for B-cell malignancies, adoptive immunotherapies will become the standard of care for a large number of patients. How- ever, the clinical success of CAR-T cells beyond hematological malig- nancies has been scarce. We hypothesize that this is in part due to suboptimal bioavailability of CAR-T cells in solid tumors, and we propose to manipulate the expression of chemokine receptors to en- hance the CAR-T cells homing and increase their therapeutic efficacy against the tumor. Methods We first analyzed the expression of chemokines by human lung tu- mors, using publicly available databases of RNA expression (TCGA). In addition, we measured their expression in the supernatants of short- term cultures of human lung tumors. Retroviral vectors were gener- ated for the expression of chemokine receptors, in combination with an anti-mesothelin (MSLN) second generation CAR. T cell migration and function were assessed using migration transwell and cytotoxic assays. Anti-tumor efficacy was assessed using a xenograft model of Fig. 4 (abstract P503) See text for description lung cancer. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):87 Page 244 of 244 Results antibody with high affinity against CSF-1R and has demonstrated an- IL-8 (CXCL8) was consistently expressed by lung tumors, and secreted titumor activity in preclinical tumor models by enhancing the im- by short-term cultures of surgically-removed lesions. Co-expression mune response. of an anti-MSLN CAR and CXCR2 or CXCR1 increased the migration Methods of human T cells towards medium containing recombinant IL-8, or Study SNDX-6352-0001 is a first-in-human, double-blind, randomized, conditioned media from lung cancer cell cultures, in transwell assays. placebo-controlled, single ascending dose, phase I trial in healthy Importantly, co-expression of chemokine receptors did not com- volunteers. The planned dose levels were 0.15, 1.0, 3.0, 6.0, and 10.0 promise CAR expression or function. Furthermore, expression of mg/kg. All treated subjects were observed for 72 hours post-infusion CXCR2 (but not CXCR1) in CAR T cells increased the number of intra- and underwent follow-up evaluations for 12 weeks. Safety assess- tumoral T cells, and significantly enhanced antitumor efficacy in a ments included adverse event (AE) monitoring, clinical laboratories, xenograft tumor model based on H2110 cells. vital signs, 12-lead electrocardiogram (ECG), eye examination, and Conclusions physical examination. Blood samples were collected to characterize Tumor homing and therapeutic efficacy of CAR-T cells can be im- the pharmacokinetics (PK) and pharmacodynamics (PD; change in proved through co-expression of chemokine receptors. This approach plasma colony stimulating factor-1 [CSF-1] and interleukin-34 [IL-34]), may apply to other types of T cell-based immunotherapies, for the as well as to evaluate changes in circulating classical and non-clas- treatment of lung cancer, and other solid tumors. sical CD16+ monocytes in blood and change in CSF-1 receptor occu- pancy (RO). Results The first three placebo-controlled cohorts were completed with 14 P505 subjects receiving SNDX-6352 (2 at 0.15mg/kg, 6 at 1mg/kg and 6 at First in human, single ascending dose study in healthy volunteers 3mg/kg) and 6 subjects receiving placebo (2 in each cohort). Dosing of SNDX-6352, a humanized IgG4 monoclonal antibody targeting up to 1.0 mg/kg was tolerated well with the most common com- colony stimulating factor-1 receptor (CSF-1R) plaint being mild to moderate itching (lasting 2 or 3 days). At the 3 1 2 2 2 Renger Tiessen , Anniek Visser , Henko Tadema , Caryn Peterson , Helen mg/kg dose, similar itching complaints were observed. At this dose, 3 3 4 3 Pentikis , Lei Wang , Michael L. Meyers , Peter Ordentlich mild to moderate eyelid swelling was observed in all subjects (me- PRA Health Sciences, Groningen, Netherlands; dian duration of 40 days), with no impact on vision. Dose escalation PRA Bioanalytical Laboratory, Assen, Netherlands; was then terminated. In blood, a transient reduction in non-classical Syndax Pharmaceuticals, Inc., Waltham, Massachussets, USA; CD16+ monocytes was observed, and an upward trend in LDH and Syndax Pharmaceuticals, Inc., New York, New York, USA ASAT. Cmax increases corresponded with dose, while AUC increases Correspondence: Renger Tiessen tiessenrenger@prahs.com were greater than dose proportional, indicating non-linear PK. Incr. Journal for ImmunoTherapy of Cancer 2017, 5(Suppl 2):P505 Background CSF-1R is expressed on immunosuppressive tumor associated macro- Publisher’s Note phages (TAMs). High numbers of TAMs in tumors are associated with Springer Nature remains neutral with regard to jurisdictional claims in poor prognosis. SNDX-6352 is a humanized IgG4 monoclonal published maps and institutional affiliations. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

32nd Annual Meeting and Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2017): Part Two

Journal for ImmunoTherapy of Cancer , Volume 5 (2) – Nov 7, 2017