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Antibody-drug conjugates: integrated bioanalytical and biodisposition assessments in lead optimization and selection

Antibody-drug conjugates: integrated bioanalytical and biodisposition assessments in lead... Therapies based on monoclonal antibodies (mAbs) have delivered an impressive success in the clinics due to their exquisite specificity, potential for agonistic or antagonistic responses, tunable effector function, and optimal pharmacokinetic properties. Building on these inherent antibody properties, the design and development of antibody-drug conjugates (ADCs) with improved or gained therapeutic activity and safety has been successfully demonstrated in oncological applications. There is enormous potential for this new type of hybrid biologics but there are also significant engineering, manufacturing and bioanalytical challenges. In this manuscript, we highlight the range and diversity of assays that are critical to characterize the individual components of ADCs-linker, carrier, and payload. We discuss a series of in vitro and in vivo preclinical experimental approaches we implemented to characterize two anti-inflammatory steroid bearing ADCs, and an ADC bearing a modified glucagon-like peptide 1 receptor/glucagon receptor co-agonist peptide. Keywords: Antibody-drug conjugates, Immunoassay, LC-MS, Biodisposition, CD74, CD25, GLP1R, GCGR Background Antibody-drug conjugates represent a new kind of Antibodies and antibody-based molecules constitute one therapeutic agents which are gaining importance espe- of the most efficacious classes of therapeutic products in cially in the oncology field (Sievers and Senter, 2013). the biotechnology industry. Due to their generally long They are the product of covalently linking a monoclonal half-life and specificity encoded in discrete complemen- antibody with a small molecule or peptide drug (pay- tary determining regions, it was originally believed that, load). Typically, the mAb will enable a targeted delivery in addition to an excellent therapeutic window, the phar- of the payload to a defined cell population of choice. macokinetic properties of recombinant antibodies will The increase in total mass of ADC compared to payload be both similar and predictable. However, it is now well alone improves the payload exposure due to diminished understood that many factors, depending on the specific renal uptake and clearance of ADC. In addition, ADCs antibody and intended target, may significantly affect the are expected to retain binding to the neonatal Fc recep- drug pharmacokinetic behavior (Tabrizi et al., 2006). tor (FcRn), which is responsible for salvaging both im- munoglobulin G (IgG) and albumin from cellular catabolism in a pH-dependent recycling and transcytosis * Correspondence: Enrique.escandon@merck.com 2 mechanism (Andersen and Sandlie, 2009; Roopenian Biologics DMPK and Disposition, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, CA 94304-1104, USA and Akilesh, 2007). Full list of author information is available at the end of the article © The Author(s). 2018 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. Beaumont et al. AAPS Open (2018) 4:6 Page 2 of 17 For ADCs with a targeted intracellular payload, the al., 2014). Descriptions of bioanalytical techniques have challenge is to successfully design ADCs with a balanced been recently published (Gorovits et al., 2013; Kaur et chemical linker strong enough to be stable in blood al., 2013; Wakankar et al., 2011). In this manuscript, we while efficiently cleaved once inside the cell lysosomal describe several specific experimental examples of differ- compartment to release its active payload. These proper- ent site-specific conjugated ADC case-studies in order ties would reduce systemic pharmacological exposure to highlight the complexity and range of bioanalytical and non-specific effects associated with the free drug. methods required to characterize their properties in Alternatively, for extracellular targets the mAb portion vitro and their pharmacokinetic and disposition behavior can serve as a carrier to extend the half-life of the pay- in vivo (Outlined in Fig. 1). Two cases presented here load from minutes to days and systemic exposure by are: 1) Anti-inflammatory steroid-ADCs directed to several folds compared to when dosed as a small mol- specific cell surface receptors (CD25 and CD74) for tar- ecule drug. In this scenario it is critical that the stability geted internalization; 2) A modified Glucagon-like of the linker and payload in the circulation are engi- Peptide 1 receptor/Glucagon receptor (GLP1R/GCGR) neered to match appropriately the expected increase in co-agonist-bearing ADC (Pep-ADC) designed for im- exposure and half-life of the immunoconjugate. This is proved systemic exposure and prolonged pharmaco- of particular importance for peptide payloads as they are logical activity via peptide cell surface receptor binding more likely to be targeted by enzymatic catabolism in and activation. the systemic circulation. Generally, conjugation of the drug to the antibody has Methods been achieved through lysine or existing cysteine resi- All animal procedures described here were performed in dues resulting in heterogeneous conjugations (Casi and accordance with the established guidelines and regula- Neri, 2012). However, there is a tremendous interest and tions by our Institutional Animal Care and Use Commit- research to produce more homogeneous ADCs through tee (IACUC). Animals were housed and maintained in site specific conjugations using either engineered cyst- facilities under a protocol approved by and in accord- eine residues (Junutula et al., 2010) or through the ance with the guidelines of the American Association for site-specific incorporation of non-natural amino acids Assessment and Accreditation of Laboratory Animal with unique reactive groups while minimizing antibody Care. scaffold modifications (Axup et al., 2012; Kern et al., 2016b; Tian et al., 2014; Zimmerman et al., 2014). Re- Monoclonal antibodies and ADC compounds gardless of the methods used for generating ADCs, a Monoclonal rat-mouse chimeric anti- mouse IL-2Rα thorough characterization of the effects that the conju- (CD25) IgG1 was coupled to dexamethasone (Dex) with gated products and procedures may have on the either a novel proprietary phosphate based linker (anti-C- functional properties of the antibody moiety itself is crit- D25-Phos-Dex) (Kern et al., 2016a) or with a cathepsin ical for lead selection and development; including bind- based linker (anti-CD25-Cat-Dex) (Kern et al., 2016b), as ing properties, affinity and specificity, Fc effector described in the respective references. These ADCs were function, off-target disposition, aggregation and novel generated in partnership with Ambrx Inc. (La Jolla, CA). protein interactions. In addition, because of their Humanized anti-human CD74 IgG1 either unconjugated multi-component nature (mAb, linker and payload), (unconjugated anti-CD74) or conjugated to budesonide these hybrid entities are more complex than a “simple” using phosphate linker (anti-CD74-Phos-Bud), humanized mAb or separate payload. For any given combination of anti-human CD74 IgG4 (anti-CD74–011), human mAb, linker, payload, site, type of conjugation, and the anti-viral IgG4 (mAb3), and mAb3 conjugated to modified achieved drug-antibody ratio (DAR), the resulting prod- Glucagon-like Peptide 1 receptor/Glucagon receptor ucts will consist of a mixture of ADC variants with dif- (GLP1R/GCGR) co-agonist peptide (Pep-ADC), were all ferent physico-chemical properties. This in turn, generated in partnership with Ambrx. Mouse depending on the resulting heterogeneity, may affect its anti-Dexamethasone IgG1 (MEB113.53F3.104A) and stability, pharmacokinetics and disposition, adversely mouse anti-Dexamethasone IgG2a (MEB113.8D1.7F) were changing the overall ADC safety and efficacy profile. generated at Merck & Co., Inc. (MRL, Palo Alto, CA). Therefore, each experimental conjugate requires the de- velopment of a combination of various analytical proce- In vitro stability of anti-CD25-Phos-Dex and anti- CD25- dures for a thorough physico-chemical monitoring, from Cat-Dex antibody and payload conjugation, immunoassay, and Anti -CD25-Phos-Dex or anti-CD25-Cat-Dex (15 μg/ cell based procedures to standard and innovative 150 μL plasma) was spiked in normal male DBA1 mouse fit-for-purpose LC-MS methods. There are excellent re- plasma for up to 18 days at 37 °C. After incubation, sam- views that cover several aspects of this process(Perez et ples at selected time points were analyzed by total Beaumont et al. AAPS Open (2018) 4:6 Page 3 of 17 Fig. 1 Schematic illustrating the range of bioanalytical methods used in the studies described in this paper. These methods can be applied to understand concentration, activity, and intactness of antibody-drug conjugates in vitro and in vivo antibody immunoassay (Total IA), measuring anti-CD25 Buffer B (Buffer A: 0.1% formic acid aqueous solution; antibody still capable of binding the target regardless of Buffer B: 0.1% formic acid in acetonitrile) at 0.4 mL/min. the presence or absence of payload, and ADC specific Dexamethasone and dexamethasone-d4 were detected immunoassay (ADC IA) using Mesoscale Discovery using the transitions 437.0 > 361.3, and 441.0 > 363.3, re- (MSD) sandwich immunoassay (Meso Scale Discovery, spectively. Method showed linearity from 0.1 ng/mL to Rockville, MD), and free dexamethasone was determined 2000 ng/mL. by LC-MS (Free Dex). For Total IA, the plasma samples spiked with each ADC were added to MSD MA6000 In vivo stability of anti-CD74-Phos-Bud in human CD74 96-Small Spot plate coated with recombinant mouse transgenic mice IL-2 Rα (R&D System, Minneapolis, MN) as a capture Human CD74 expressing transgenic (Tg) mice molecule. After incubation and washes, wells were incu- (B6.Cg-Tg(CD74)AR194Ayr/j) were generated and char- bated in goat anti-rat IgG (Fc), F (ab’)2-Biotin (Sig- acterized previously (Honey et al., 2004). These Tg mice ma-Aldrich, St. Louis, MO). Finally for detection, (n = 2/group) were dosed intravenously (IV) with either SULTO-TAG labeled Streptavidin was added to the 3, 10 or 30 mg/kg of anti-CD74-Phos-Bud and plasma wells, signal was read on the MESO SECTOR S 600 samples were collected at designated time points. reader after adding MSD read Buffer T. For Anti-CD25 Samples were subjected to MSD immunoassays. For conjugate ADC specific immunoassay, similar procedure total anti-CD74 immunoassay (Total IA), measuring was followed, however, mouse anti-dexamethasone anti-CD74 antibody still capable of binding the target IgG2a (MEB113.8D1.7F)–Biotin (Merck) was used to de- regardless of the presence or absence of payload, tect dexamethasone containing intact ADC in spiked recombinant human CD74 (R&D System) was plated on plasma. MSD MA6000 96-Small Spot plate as a target capture For LC-MS detection of Dexamethasone, plasma pro- and mouse anti-human IgG-SULTO-TAG (SouthernBio- tein was precipitated by addition of 400 μL of internal tech, Birmingham, AL) was used as detection standard solution, consisting of 20 ng/mL of reagent. For anti-CD74–Phos-Bud ADC specific im- Dexamethasone-d4 (TLC PharmaChem, Vaughan, ON) munoassay (ADC IA), mouse anti-dexamethasone IgG1 in acetonitrile to 40 μL of plasma. Samples were vor- (MEB113.53F3.104A)–Biotin (Merck) was coated on to texed for 30 s and centrifuged at 14,000 rpm for 10 min MSD Streptavidin multi-array 96-well plate, and at 4 °C. Supernatants were transferred to a clean tube MEB113.53F3.104A-SULFO-TAG was used as the detec- and dried in Centrivap vacuum concentrator (Labconco, tion reagent. Kansas City, MO). Samples were reconstituted with 40 μL of 50% methanol /aqueous solution and 5 μLwas Anti-CD74-Phos-Bud interacting protein in C57BL/6 J injected into the ESI(−)LC-MS/MS (Acquity Waters, mouse plasma Milford, MA; TSQ Vantagem, ThermoScientific, Wal- Anti-CD74-Phos-Bud was immune-purified from spiked tham MA). Analysis was performed on a Ascentis Ex- C57BL/6 J mouse plasma using NanoLink Streptavidin press C18 (50 mm x3mm × 2.7 μm) (Sigma-Aldrich) Magnetic beads (Solulink, San Diego, CA) immobilized column with a 3 min linear gradient from 50 to 95% with biotin-SP-conjugated AffiniPure F(ab’)2 fragment Beaumont et al. AAPS Open (2018) 4:6 Page 4 of 17 goat anti-human IgG (H + L) (Jackson ImmunoResearch) Biodistribution of DyLight 650 labeled anti-CD74-Phos- with 3 h incubation at 4 °C. After capture, beads were Bud and unconjugated anti-CD74–011 mAb in mice separated from plasma using a magnetic rack and Transgenic human CD74 mice (B6.Cg-Tg(CD74)AR194- washed five times with 0.1% RapiGest (Waters) in TBS Ayr/j) and normal wild type C57BL/6 J female littermates 1× solution. Samples were eluted with 30 μL of 0.25% (WT) (n = 2/group/time point) were given 5 mg/kg IV formic acid solution and neutralized by the addition of bolus of DyLight 650 labeled-anti-CD74-Phos-Bud. A sep- TRIS HCl (1 M). Samples were heated for 3 min at 80 ° arate group of wild type littermates (n = 2/time point) C for denaturation, reduced with DDT (final concentra- were administered with 5 mg/kg of DyLight 650 tion of 5 mM) for 30 min at 60 °C, and alkylated with labeled-anti-CD74–011. Animals were euthanized with iodoacetamide (final concentration of 15 mM) for carbon dioxide followed by terminal cardiac puncture. 30 min at room temperature in dark. Digestion was per- Blood, plasma, and selected tissues were harvested to formed by addition of 2.5 μg of trypsin (Promega, Madi- measure drug concentration. Whole blood samples were son, WI) to each sample, and incubation for 4 h at 37 ° collected into K2-EDTA CapiJect microcollection tubes C. Digestion was quenched by addition of HCl (final (Terumo Medical Corporation, Somerset, NJ) and placed concentration of 200 mM) and samples were further in- on ice. For drug analysis in whole blood, samples were ali- cubated at 37 °C for 45 min for hydrolysis of remaining quoted into polypropylene vials (Corning Glassworks, RapiGest. Samples were centrifuged at 14,000 rpm for Corning, NY) and stored at − 80 °C. Plasma was separated 10 min at 4 °C, and 4 μL of digests were injected in a from whole blood via centrifugation for 6 min at 6000×g LC-MS/MS system NanoAcquity (Waters)/ at 4 °C, and stored at − 80 °C. For tissue lysates, organ LTQ-Orbritrap XL (ThermoScientific) with a 60 min lin- samples were collected and immediately placed into 2-ml ear gradient from 5 to 56% Buffer B (Buffer A: 0.1% for- Precellys Lysing Tubes (Bertin Technologies, Rockville, mic acid aqueous solution; Buffer B: 0.1% formic acid in MD), weighed, and frozen by placing on dry ice. To pre- acetonitrile) at 0.4 μL/min. Search was performed using pare lysates, upon partial thawing, a 1:5 dilution of Dul- the Proteome Discoverer Software (ThermoScientific). becco’s PBS containing 1% Triton X-100 (MP Biomedicals, Solon, OH) and 1× Halt Protease Inhibitor single use cocktail (ThermoFisher Scientific) was added to DyLight™ 650 labeling of antibodies the samples. Then, tissue slurries were prepared using a DyLight™ 650 labeling kits (ThermoScientific) were used Precellys Evolution Homogenizer (Bertin Technologies). to conjugate an N-hydroxysuccinimide ester fluores- The slurry was centrifuged (10,000×g at 4 °C for 10 min) cence dye (excitation at 652 nm and emission at and the tissue lysate supernatants were collected and ei- 672 nm) to anti-CD74, anti-CD74-Phos-Bud, and isotype ther processed immediately or stored at − 80 °C until matched control antibody (mAb-C), Pep-ADC, and analysis. mAb3, using the method described in previous publica- tion from our group (Brunn et al., 2016). Determination of DyLight 650 labeled -anti-CD74-Phos- Bud and anti-CD74–011 mAb concentration in tissue In vitro stability assessment of DyLight 650 labeled anti- samples by fluorescence emission–linked analysis (FELA) CD74, anti-CD74-Phos-Bud, and isotype control antibody For assessment of tissue drug concentration, the col- (mAb-C) by SEC-HPLC lected blood, plasma, and tissue lysate samples or a set For assessing stability in mouse plasma in vitro, DyLight of corressponding blank tissue samples were diluted 1:10 650 labeled anti-CD74, anti-CD74-Phos-Bud and isotype (final dilution) with tissue lysis buffer (1× DPBS with 1% matched control antibody (mAb-C) were incubated at Triton X-100). The diluted samples (150 μL) were trans- 37 °C in C57BL/6 J mouse plasma (100 μL) at a final ferred to 96-well polystyrene plates with low fluores- concentration of 80 μg/mL, samples were taken out and cence background for analysis in the Glomax® snap frozen at designated time points, and stored at − Multi-Detection system microplate reader (Promega). 80 °C until analysis. For SEC-HPLC analysis, 25 μLof The microplate reader was equipped with a fluorescence plasma samples from time points were applied onto a optical filter featuring excitation and emission wave- BioSep-SEC-S3000 column (Phenomenex, Torrance, lengths of 625 nm and 660 to 720 nm, respectively. CA) at 1 mL/min elution rate with PBS as a mobile For each DyLight 650 labeled antibody, fluorescence phase for 15 min, and analyzed using an Agilent 1200 intensity calibration curves were established by serially HPLC system (Agilent Technologies, Santa Clara, CA). diluting spiked blood and liver lysate. A linear correl- The effluent was monitored continuously at 280 nm and ation function was fitted to the data using best-fit pa- by the net fluorescence intensity at an excitation wave- rameters on Microsoft Excel. The resulting blood length of 646 nm and emission of 674 nm, as described calibration curve equation was used to fit unknown previously (Brunn et al., 2016). blood and spleen lysate samples, and the liver calibration Beaumont et al. AAPS Open (2018) 4:6 Page 5 of 17 curve equation was used to fit unknown liver lysate sam- (2.1 × 30 mm) (Applied Biosystems, Waltham, MA) with ples. Concentrations of DyLight-650 labeled antibodies flow rate 0.1 ml/min, 7 min gradient 30% to 58% B, 65 ° were calculated as microgram equivalents per gram of C. Buffer A was water, 0.1% FA, buffer B was 90% Aceto- wet tissue or as microgram equivalents per mL of blood. nitrile, 0.1% FA. Mass Spectrometry conditions were: ca- Subsequently, tissues to blood ratios were calculated by pillary voltage of 3.2 kV, cone voltage of 80 V, dividing concentration in respective tissue by concentra- desolvation temperature 450 °C. Mass data was acquired tion in blood at the designated time point. in m/z mass range of 500–4000. The mass spectrum was deconvoluted and integrated using MassLynx software Assessment of bioactive and total Pep-ADC in mice (Waters). In order to measure the bioactivity of Pep-ADC in plasma (Bioassay), plasma samples from C57BL/6 J male Pep-ADC quantitative mass spectrometry based assay mice with diet induced obesity (DIO) dosed with 10 mg/ (LC-MRM) kg IV bolus of Pep-ADC were incubated with GLP1R 50 μL of solulink magnetic beads were coated by rota- transfected Chinese hamster ovarian (CHO) cells for tion with 250 μL of goat anti-human IgG (monkey 1 h. The reactions were stopped by adding cell-lysis buf- absorbed)-Biotin (SouthernBiotech) for 30 min at room fer from cAMP CISBIO kit (Cisbio, Bedford, MA). A temperature. The beads were washed 3 times with TBS/ calibration curve was established using different amount 0.02% RapiGest and resuspended in 50 μL of TBS/0.02% of Pep-ADC spiked into plasma to stimulate RapiGest. For affinity enrichment, 5 μL of bead was in- GLP1R-CHO cells and quantify cAMP release in a cubated with 30 μL of plasma and TBS/0.02% RapiGest 96-well corning white plate using cAMP CISBIO kit. up to 50 μl for 30 min on a rotator. The beads were Bioactivity of Pep-ADC was back-calculated and re- washed 3 times with TBS/0.02% RapiGest and once with ported as μg/mL. TBS (pH 7.4), eluted with 30 μL of 0.1% formic acid, To determine the total antibody level (Total mAb3 and neutralized with 2.5 μL of 1 M ammonium assay), MSD immunoassay was used. Plasma samples bicarbonate. from Pep-ADC dosed animals were applied to Streptavi- 50 mM TCEP was added for reduction and the mix- din Gold Multi-Array 96-well Plate (MSD) coated with ture incubated for 30 min at 60 °C. Digestion was done mouse anti-Human IgG4-Biotin (SouthernBiotech) as a with 0.25 μg of trypsin for 5 h at 37 °C. 1 pmol of la- capture reagent, and SULFO-TAG-mouse anti-Human beled peptides spanning residue 1 to 12 (representing in- IgG (Southern Biotech) was used for detection. tact molecule) and residue 24 to 30 (representing M1) was added as internal standard. 5 μL was injected into Pep-ADC intact mass analysis by AP-LC-MS an Acquity TSQ Vantage for multiple reactions monitor- Mouse study: C57BL/6 J male mice (n = 3) with diet in- ing of R1-R12 and R24–30 peptides. duced obesity were dosed with 3 mg/kg IV bolus of Pep-ADC. Plasma samples collected at day 1, 3, 7, and Biodisposition of Pep-ADC in mice 10 were processed for intact mass analysis. Normal C57BL/6 J mice (n = 2/group/time point) were Non-human primates study: Treatment naïve male administered a single 3 mg/kg IV dose of either DyLight rhesus monkeys (n = 3) weighing 6–7.1 kg were fasted 650 labeled Pep-ADC or unconjugated mAb3. Plasma overnight and dosed with 3 mg/kg IV bolus of and tissue samples were collected at 1 h, day 1, 2, 3, 5, 7, Pep-ADC. Plasma samples collected at 5 min, day 1, day 9, and day 14 post dose. 3, day 7, day 10, and day 14 were processed for intact To generate plasma concentration vs time profiles, mass analysis. DyLight 650 labeled drug concentration in plasma was Pep-ADC was retrieved from plasma samples from determined by FELA as described previously for both studies by affinity purification for LC-MS as fol- anti-CD74 studies. lows. Nanolink Streptavidin Magnetic beads (Solulink) To determine in vivo stability of DyLight 650 labeled were preloaded with biotinylated polyclonal goat anti Pep-ADC and unconjugated mAb3 by SEC-HPLC, un- human IgG (SouthernBiotech). Preloaded magnetic diluted plasma samples collected at 1 h, day 2, 5, 9 and beads (20 μl) at 2.5 mg/ml in TBS pH 7.4, 0.02% Rapi- day 14 from each group were applied onto a gest SF Surfactant (Waters) was added to 30 μlof BioSep-SEC-S3000 column (Phenomenex) at 1 mL/min K2-EDTA plasma sample. Captured Pep-ADC was elution rate with PBS as a mobile phase for 15 min, and eluted in 30 μl of 0.25% formic acid (FA), reduced with analyzed using an Agilent 1200 HPLC system (Agilent 5 mM TCEP for 30 min at 60 °C, and 10 μl was injected Technologies). The effluent was monitored continuously into Waters Acquity UPLC I class on line with Waters at 280 nm and by the net fluorescence intensity at an ex- Synapt G2S QTOF mass-spectrometer. Reverse phase citation wavelength of 646 nm and emission of 674 nm, chromatography was used on the Poros R2/10 column as described previously (Brunn et al., 2016). Beaumont et al. AAPS Open (2018) 4:6 Page 6 of 17 To determine cellular disposition of the labeled anti- Anti-CD25 mAb conjugated to dexamethasone with bodies, 1 h and day 1 post dose pancreas sections cut cathepsin or phosphate linker: Importance of linker from formalin fixed paraffin embedded (FFPE) tissue stability blocks were deparaffinized and rehydrated with serial A monoclonal antibody against CD25 or Interleukin −2re- passage through changes of xylene and graded ethanols ceptor alpha chain (IL-2Rα), expressed in a subset of im- for DyLight 650 immunohistochemistry (IHC). All slides mune cells (Triplett et al., 2012), was coupled to were subjected to heat induced epitope retrieval in TRS dexamethasone (Dex) using either a novel proprietary solution (pH 6.1) (Dako, Carpineteria, CA). Endogenous phosphate based linker (anti-CD25-Phos-Dex) (Kern et al., peroxidase in tissues was blocked by incubation of slides 2016a) or a cathepsin based linker (anti-CD25-Cat-Dex) in 3% hydrogen peroxide solution prior to incubation (Kern et al., 2016b). In order to fully characterize the ADC with primary antibody, FITC-conjugated anti-DyLight stability in mouse plasma, several assays were developed. 650 clone 12A3 (Merck) for 60 min. Antigen-antibody Initially these included: A total antibody immunoassay binding was visualized via application of rabbit measuring anti-CD25 antibody still capable of binding the anti-FITC antibody (Invitrogen, Carlsbad, CA) then En- target regardless of presence or absence of payload (target-- vision Rabbit HRP (Dako) and application of 3, 3′ diami- capture and anti-Fc detection); A conjugate specific ADC nobenzidine (DAB) chromogen (Dako). Stained slides immunoassay (target capture and anti-Dex detection); And were counterstained with hematoxylin and coverslipped aLC-MS assay tomeasure free Dex. for review. Mouse neat plasma spiked with anti-CD25-Phos-Dex was incubated over the course of 18 days at 37 °C. The Pharmacokinetics total antibody and ADC specific immunoassays showed a All pharmacokinetic parameters were estimated or cal- constant concentration across all time points (Fig. 2a). culated by non-compartmental analysis (NCA) using Likewise, the LC-MS assay for free Dex showed no ap- Phoenix® WinNonlin® software (Certara, Princeton, NJ). pearance of unconjugated payload at any time-point con- Model 201 (IV input bolus) was used for the NCA. sistent with the intactness of this construct in plasma. In contrast, the same ADC but with the cathepsin linker Results (anti-CD25-Cat-Dex conjugate) showed a very different Case-study 1: Anti-inflammatory steroid-ADCs stability-time profile. Total antibody levels were unchanged Systemic anti-inflammatory drugs are often used in the and no free Dex was detected (Fig. 2b). However, the ADC treatment of chronic conditions such as inflammatory proved to be highly unstable as illustrated by a dramatic de- bowel disease (Baumgart and Sandborn, 2007) and crease in concentration over time measured by the asthma (Lipworth, 1999). As a class, steroids are largely ADC-specific immunoassay (Fig. 2b). To investigate the un- not cell or tissue specific and their systemic use is expected lack of stability of anti-CD25-Cat-Dex in plasma, dose-limited due to harmful side effects (Brunton, 2011). we developed a method based on affinity purification Developing a targeted delivery of the most potent im- followed by intact mass measurement by LC-MS mune system repressors through an antibody-drug con- (AP-LC-MS). Anti-CD25-Cat-Dex spiked in plasma was re- jugate could provide a more effective dose and dosing trieved using antigen-bound beads and its mass was mea- rational for enhanced efficacy without triggering nonspe- sured on a Q-TOF mass spectrometer Synapt G2S. The cific detrimental side effects (Kern et al., 2016a). analysis was consistent with a clipping site in the linker Through site specific conjugation in the CH1 domain moiety at a C-O bond, between the linker and the drug (Axup et al., 2012), we coupled potent anti-inflammatory moieties, which was not the part of the predicted cathepsin molecules, dexamethasone and budesonide, to different cleavage site (Fig. 2c). Linker cleavage at this novel site ex- monoclonal antibodies aimed at surface receptors broadly plains the decrease in intact ADC in the immunoassay and expressed on immune cells involved in inflammation. In the absence of free drug by LC-MS as the drug remains at- this modality, the ADC should remain intact in the sys- tached to a part of the broken linker. The same clipping temic circulation, bind to its intended target on the sur- was also observed when anti-CD25-Cat-Dex was incubated face of immune cells followed by target-mediated in phosphate buffered saline (PBS). Regardless of other ap- internalization and drug release in the lysosomal compart- proaches, the implementation of an AP-LC-MS intact mass ment. The active drug is then released to engage gluco- method should become an essential tool to monitor the in- corticoid receptors inside the cell. In addition to their tegrity of the ADCs. affinity and specificity, it is expected that these antibody Contrary to the cathepsin sensitive linker ADC conjugates should retain ideal pharmacokinetic properties (anti-CD25-Cat-Dex) instability, the AP-LC-MS intact mainly affected by target-mediated clearance and display a mass analysis of anti-CD25-Phos-Dex showed no evi- pattern of organ disposition similar to normal endogenous dence of drug release or linker instability resulting in a IgGs. constant drug to antibody ratio with molecular weights Beaumont et al. AAPS Open (2018) 4:6 Page 7 of 17 AC Fig. 2 In vitro stability of anti-CD25-Phos-Dex and anti-CD25-Cat-Dex in mouse plasma. To assess the trend in stability of these antibodies, Anti- CD25-Phos-Dex (a) or anti-CD25-Cat-Dex (b) was spiked in normal male DBA1 mouse plasma for up to 18 days at 37 °C. After incubation, the levels of antibody was determined by total antibody immunoassay (Total IA) and the levels of intact ADC was determined by ADC specific immunoassay (ADC IA) on plasma samples using Mesoscale Discovery (MSD) sandwich immunoassay, and free dexamethasone was determined by LC-MS (Free Dex). Amounts in picomole (pmol) versus time are plotted to compare across different methods of analysis. Values represent the results of a single incubation at each time point. Panel c shows the unexpected site of cleavage in the cathepsin linker of anti-CD25-Cat-Dex comparable to the stock solution at all-time points (un- known to associate with MHC class II molecule published observations). This result was consistent with (Borghese and Clanchy, 2011), a more potent ADC-specific immunoassay results (Fig. 2a). anti-inflammatory steroid, budesonide (Bud), was conju- The potential relevance of in vitro ADC analysis de- gated using an identical phosphate linker at the same pends largely on their translatability to appropriate pre- amino acid residue position in the CH1 domain. The clinical models for safety and efficacy. These in turn systemic stability of the resulting ADC (anti-C- should be selected based on their relevance to the human D74-Phos-Bud) was characterized following IV dosing in physiology and expected mechanism of action (MoA) of mice at 3, 10, and 30 mg/kg. The plasma levels of total the ADC and free drug. Therefore, the stability of mAb versus conjugate-specific immunoassay results anti-CD25-Phos-Dex was further tested in a preclinical showed an apparent linker and/or payload instability PK study. Mice were administered a single intravenous in- (Fig. 3), contrary to the first example of jection of 4 mg/kg of anti-CD25-Phos-Dex. Both immu- anti-CD25-Phos-Dex. However, the AP-LC-MS analysis noassays (total and ADC) showed overlapping indicated a constant peak, identical to the original dos- concentrations with a typical IgG PK profile (unpublished ing material demonstrating the intactness of the ADC observations). Likewise, the total mass spectrum for the construct up to 6 h, after which strong peak broadening ADC showed an identical profile to the stock solution and was observed and the total ion chromatograms could unchanged drug to antibody ratio of 2 at all time points. not be deconvoluted, suggesting heterogeneity. This These results are consistent with the overall good stability could be due to severe conjugate modifications, aggrega- of the anti-CD25-Phos-Dex including the drug, linker, and tion, or protein interactions. In addition, no free Bud antibody components both in vitro and in vivo. could be detected by LC-MS at any time point. There- fore, additional studies were implemented to better Anti-CD74 mAb conjugated to budesonide with understand these discrepancies. Anti-CD74-Phos-Bud phosphate linker: Importance of the targeting antibody was incubated in mouse plasma for 14 days at 37 °C and properties retrieved by pull-down for tryptic digestion and analysis In a different ADC construct with a monoclonal anti- by LC-MS for identification of ADC associated plasma body targeting CD74, a cell surface receptor which is proteins. Analysis of these samples and a control sample Beaumont et al. AAPS Open (2018) 4:6 Page 8 of 17 A B Fig. 3 In vivo stability of anti-CD74-Phos-Bud. To assess the stability of anti-CD74-Phos Bud in mice, the levels of antibody was determined by total antibody immunoassay (Total IA) and levels of intact ADC was determined by ADC specific immunoassay (ADC IA) on plasma samples from human CD74+ transgenic (Tg) mice IV dosed with 30 mg/kg (a), 10 mg/kg (b) and 3 mg/kg (c). Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not visible with no ADC spiked in the original mouse plasma re- (HPLC) system equipped with an in-line fluorescent de- vealed the presence of several unexpected, off-target tector. The resulting chromatographic profiles (Fig. 5a ADC-associated proteins (Fig. 4). These included: and b) indicated antibody instability as shown by the for- alpha-2 macroglobulin, ceruloplasmin, complement C3, mation of high molecular weight (HMW) complexes for albumin, fibrinogen beta, and murinoglobulin 1. Using a both constructs. By day 14 of incubation, only 53% of different approach, anti-CD74-Phos-Bud and unconju- anti-CD74-Phos-Bud and 60% of drug unconjugated gated (naked) anti-CD74 were labeled using a anti-CD74 antibody remained as a single intact mono- DyLight-650 fluorescent dye to follow their size exclu- meric IgG peak. A control antibody (Fig. 5c) remained at sion chromatography (SEC) profile over time upon incu- 96% in the same conditions. The immunoassay discrep- bation in mouse plasma at 37 °C. Samples were analyzed ancies between total anti-CD74 versus intact ADC on an Agilent 1100 high pressure liquid chromatography values may be attributed to assay interferences due to antibody aggregation or off-target association with plasma proteins preventing payload detection. The iden- tification of anti-CD74-Phos-Bud HMW complex forma- tion in plasma was consistent with the off-target interactions identified by LC-MS, and pointed to a po- tential flaw in the properties of the anti-CD74 mAb moi- ety but not in the linker or the payload or the conjugation process. Unconjugated anti-CD74 mAb shows off-target clearance mechanism in mice without CD74 target Clearance of free IgGs and small, non-precipitating IgG complexes happen primarily by interactions with Fc receptors (FcRs) and non-specific protein clearance via fluid-phase endocytosis (Nash et al., 2001;Rojas et al., 2005; Takai, 2002). These pathways are ex- pected to drive the elimination of anti-CD74 in spe- cies without the target expression or binding. To Fig. 4 Anti-CD74-Phos-Bud-interacting proteins in C57BL/6 J plasma. determine the PK properties of this anti-CD74 anti- To determine any off-target protein binding, Anti-CD74-Phos-Bud body, wild C57BL/6 J mice were administered a single was incubated in C57BL/6 J mouse plasma for 14 days at 37 °C and retrieved by pull-down. Pulled-down products were digested with intravenous dose of unconjugated anti-CD74. As show trypsin and analyzed by LC-MS/MS for peptide mapping. Data in Table 1, non-linear PK properties were observed represent the results of a single incubation at each time point for this antibody (Table 1). Concentration ( g/mL) Concentration ( g/mL) Concentration ( g/mL) Beaumont et al. AAPS Open (2018) 4:6 Page 9 of 17 Table 1 PK parameters following IV administration of anti-CD74 to C57BL/6 J mice Dose t C CL V AUC 1/2 max ss 0-INF (mg/kg) (day) (μg/mL) (mL/day/kg) (mL/kg) (day*μg/mL) 10 3.75 90.0 195 589 51.4 3 3.42 27.8 70.3 295 42.7 1 3.90 12.0 39.8 197 25.2 0.3 3.97 2.72 31.5 170 9.52 Here, observed increases in clearance at higher doses are consistent with the presence of a low affinity, high cap- acity off-target antibody sink (i.e., a non-specific clear- ance mechanism). This is, in general, an undesirable property for a targeting antibody that is characterized for its specificity and extended systemic half-life. Anti-CD74-Phos-Bud tissue disposition pattern in mice with and without human CD74 target confirms off target effect with anti-CD74 mAb To gain a better understanding of the DMPK properties of Anti-CD74-Phos-Bud, and the relevance of target ver- sus off-target interactions in the fate of anti-CD74-Phos-Bud in vivo, we utilized a transgenic (Tg) mouse strain that expresses the human CD74 re- ceptor (Honey et al., 2004). The expression of this hu- man receptor is driven by the endogenous analog murine promoter; therefore, these transgenic mice dis- play abundant receptor expression in spleen and to a lesser degree in hepatic tissues. Since anti-CD74-Phos-Bud does not recognize murine CD74, wild type (WT) littermates were used to evaluate the ef- fect of target versus off-target interactions in the distri- bution and elimination of this antibody-drug-conjugate. Fig. 5 In vitro stability of unconjugated anti-CD74 (a), anti-CD74- In addition, a different, unconjugated anti-CD74 mono- Phos-Bud (b), and isotype matched control antibody, mAb-C (c). clonal antibody (referred to as anti-CD74–011 mAb Shown are the fluorescence SEC-HPLC profiles of DyLight 650- labeled antibodies in undiluted C57BL/6 J mouse plasma following hereafter) was used as a control for background tissue incubation at 37 °C for 5 min, 7 days, and 14 days. The plasma uptake characteristic of a non-specific IgG clearance samples were run on a BioSep-SEC-S3000 column at 1 mL/min mechanism, with known negative organ accumulation elution rate with PBS as a mobile phase for 15 min. Black arrows and tissue to blood ratios below 1. Mice were given an indicate the percent of intact, monomeric IgG at day 14 IV dose of 5 mg/kg of DyLight-650-labeled anti-CD74-Phos-Bud and anti-CD74–011 mAb. Figure 6 AUC : Area under the curve from zero to infinity; CL: shows the μg-equivalents/gram (μg/g) of wet tissue or 0-INF Clearance; C : Observed maximum concentration; t1 : μg/ml of blood in Tg and WT mice. In concordance max /2 Terminal half-life; V : Volume of distribution at steady with the organ receptor expression levels, ss state anti-CD74-Phos-Bud distributed extensively to the Frequently, non-linear PK of mAbs may be explained by spleen and liver in the Tg mice with peak concentrations target mediated disposition (TMD). TMD is a saturable of 87 μg/g at 2 h in spleen and 43.5 μg/g at 6 h in liver. process and it is characterized by decreasing clearance However, disposition to spleen and liver in the wild type with increasing doses. On the contrary, clearance of un- littermates was also unusually high (34.1 and 33.7 μg/g) conjugated anti-CD74 appeared to be higher when six hours post IV dosing. In contrast, the spleen and liver higher doses were administered. In addition, since this drug levels in the anti-CD74–011 mAb clone group using anti-CD74 antibody does not recognize the murine tar- wild type littermates were below 11 μg/g at all time points get receptor, its PK was not expected to reflect TMD. in both organs. Accordingly, anti-CD74-Phos-Bud tissue Beaumont et al. AAPS Open (2018) 4:6 Page 10 of 17 A B Fig. 6 Tissue distribution of anti-CD74–011 and anti-CD74-Phos-Bud. Microgram equivalent per gram concentration of antibodies in spleen (a) and liver (b) of WT or human CD74 transgenic (Tg) mice were determined by fluorescence emission-linked assay at multiple time points over 2 days after 5 mg/kg IV dosing. An additional 15 min time-point was collected for anti-CD74-Phos-Bud in Tg mice. Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not plotted Tg Spleen Tg Liver WT Liver WT Spleen Fig. 7 Spleen and liver tissue to blood ratios of anti-CD74-Phos-Bud (a) and an unconjugated anti-CD74–011 (b) in wild type (WT) and human CD74 transgenic (Tg) mice. Tissue/blood ratios were calculated from drug concentration measured by fluorescence emission-linked assay in blood and the tissues at 2 h, 6 h, 1 and 2 days after 5 mg/kg IV dosing. An additional 15 min time-point was collected for anti-CD74-Phos-Bud in Tg mice. Tissue/blood ratios greater than 1 are indicative of tissue uptake. Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not plotted Beaumont et al. AAPS Open (2018) 4:6 Page 11 of 17 to blood ratios were as high as 99 for the spleen at day 1 with the required stability to match the enhanced pharma- and 66.9 for liver at day 2 in Tg mice (Fig. 7a). However, cokinetic properties conferred by the antibody conjuga- tissue to blood ratios higher than 1 were also found in tion. For measurements of total versus intact ADC spleen and liver from wild type littermates consistent with concentrations, an immunoassay (Fc capture and Fc de- off-target interactions. Overall these results demonstrated tection) and a cell based bioactivity (peptide-induced re- the presence of an antibody driven, off-target mechanism ceptor activation) procedures were implemented. of tissue uptake and disposition, and were consistent with the identification of non-specific protein interactions in Immunoassay, cell based bioactivity, and mass the circulation and undesirable PK for this construct. spectrometric characterization helped to determine the Based on these results, this anti-CD74 mAb was therefore pep-ADC stability issue in mice not further considered as a lead therapeutic candidate. In To characterize the metabolic fate of Pep-ADC in vivo, this case, the intrinsic unfavorable properties of the anti- Pep-ADC was administered to mice at 1 and 10 mg/kg body alone determined the overall poor fate of this ADC as a single intravenous injection. Plasma was collected in vivo. This conclusion was further supported by obser- over time and analyzed for both total ADC (mAb3 vations with the anti-CD74–011 mAb, which demon- levels) and cell-based bioactivity (assessment of free and/ strated excellent stability in biomatrices. In a follow up or conjugated active peptide). Previous in vitro work disposition study, anti-CD74–011 mAb exhibited higher with the free peptide (Pep) had demonstrated that only tissue to blood ratios in spleen compared to liver in hu- the intact moiety retained receptor binding and signaling man CD74 expressing transgenic mice at any evaluated in this cell-based assay. Pharmacokinetic results (Table 2 time points, with ratio in spleen reaching as high as 224 at and Fig. 8) demonstrated more than 2-fold differences in day 2 compared to 48.7 in liver (Fig. 7b). Furthermore, exposure and clearance between total mAb3 and bio- tissue-to-blood ratios in wild type mice for both tissues active measurements in a cell based assay. Therefore, were lower than 0.5 at any evaluated time point (Fig. 7b). the smaller exposure and increased clearance for the These observations were consistent with anti-CD74–011 bioactive ADC compared to the total ADC -suggested a mAb target-mediated uptake following administration to potential linker instability and/or peptide catabolism. the transgenic mouse strain that expresses the human To further understand the molecular nature of form of CD74 (Fig. 7b). Pep-ADC potency loss, we conducted an AP-LC-MS analysis. In agreement with the bioactivity assay data, Case-study 2: GLP1R/GCGR peptide-ADC (Pep-ADC): the results showed that while the intact conjugated Importance of payload stability Pep-ADC decreased with time and eventually became Due to their precise specificity, peptide drugs are gaining undetectable by day 5, several clipped variants (M1, M2, importance as therapeutic agents. However, their small M3 and M4) representing degradation of the conjugated size, rapid clearance rates, and systemic catabolism have peptide at various positions became the predominant limited their clinical potential and applications (McGregor, ADC species (Fig. 9a and b). A control group of mice 2008;Sato et al., 2006). The use of balanced Glucagon dosed with the mAb3 only, without the conjugated -like peptide 1 receptor/Glucagon receptor (GLP1R/ linker-peptide, showed a constant peak (intact mAb) at GCGR) co-agonist has shown enhanced efficacy and safety the same molecular weight at all tested time points (un- relative to pure GLP1R agonists in the treatment of rodent published observations) indicating no catabolism of the obesity, with simultaneous improvement in glycemic con- antibody moiety itself. Mass difference calculations iden- trol (Day et al., 2012; Day et al., 2009; Pocai et al., 2009). tified that the conjugated peptide clipping between resi- Using site specific conjugation, we coupled a dues 2/3 (M4), 25/26 (M1), 27/28 (M2) and 30/31 (M3) modified-for-stability GLP1R/GCGR co-agonist peptide of the peptide sequence (Fig. 9c). All clipped M-variants (payload) to a residue in the CDR H1 of a non-targeting are inactive in bioassay, as it is known that the removal human mAb (mAb3) via a non-cleavable linker. The Table 2 Pharmacokinetic parameters following IV linked peptide-mAb3 immunoconjugate is referred to as administration of Pep-ADC to C57BL/6 J mice Pep-ADC hereafter. In this modality, it is expected that Assay Dose t AUC CL V the antibody moiety will provide a stable carrier for pro- 1/2 0–14 ss (mg/kg) (day) (day*μg/mL) (mL/day/kg) (mL/kg) longed exposure in the circulation of the linked peptide Total mAb3 1 5.12 56.9 15.5 89.1 due to reduced renal clearance and engagement of the 10 4.76 657 13.9 69.0 FcRn recycle mechanism. In turn, this should result in a substantial improvement in the biological outcome com- Bioassay 1 2.55 28.5 34.9 49.3 pared to the co-agonist peptide dosed alone (Kompella 10 2.78 264 37.5 64.7 and Lee, 1991). In order for this to occur, it is essential AUC : Area under the curve from zero to 14 days; CL: Clearance; t : 0–14 1/2 that both the linker and the bioactive peptide are designed Terminal half-life; V : Volume of distribution at steady state ss Beaumont et al. AAPS Open (2018) 4:6 Page 12 of 17 receptor (GLP1R/GCGR) co-agonist-bearing ADC. Fi- nally, a mass spec-based quantitative assay was developed to measure the disappearance of the intact form and ap- pearance of the main peptide catabolite (Fig. 10a). It con- firmed that intact Pep-ADC disappeared fast and by day 2, only about 10% of intact Pep-ADC remained (Fig. 10b). Conversely, M1 increased for the first two days and then declined over the remaining 5 days as it was cleared and/ or transformed into M2 and M3 (Fig. 10b). Overall, these data were consistent with the bioactivity assay and intact mAb measurements, and confirmed M1 as one of the most abundant metabolites in the circulation. These re- sults indicated a path forward in the rational design of im- Fig. 8 The concentration time profile of Pep-ADC measured by two proved ADC versions focused on strengthening the different assays. Plasma samples were collected at multiple time stability of the peptide moiety. It is important to note that points over 14 days from mice IV dosed with 10 mg/kg of Pep-ADC, although bioactive drug exposure can be further increased and measured for intact antibody concentrations by MSD assay with potentially more stable peptide properties in the cir- (Total mAb3 assay) as well as bioactive ADC using a cell-based assay culation, the current exposure to active drug in this ADC (Bioassay). Data plotted are mean from 3 mice. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter was considerably better than those of similar peptides than the symbols are not plotted when dosed alone with half-lives ranging in the order of minutes and elimination rates closer to glomerular filtra- of the first two amino acids of Glucagon-like peptide-1 tion rates (Galaske et al., 1979). For free peptides in the (GLP-1) by dipeptidyl peptidase 4 (DPP4) kills the activ- circulation at pharmacological doses, two powerful mech- ity of GLP-1 through GLP-1 receptor (GLP1R) (Knudsen anisms drive the fast elimination of these molecules and Pridal, 1996). This is also true for Pep-ADC, which mainly by renal clearance followed by systemic enzymatic is a modified Glucagon-like Peptide 1 receptor/Glucagon degradation (Rabkin and Dahl, 1993). Peptide clearance by AB Fig. 9 Intact mass analysis of Pep-ADC in mice by AP-LC-MS. Pep-ADC (3 mg/kg) administered to C57BL/6 J diet induced obese (DIO) mice by IV was retrieved by pull-down for intact mass analysis on a Synapt G2S Q-TOF. Intact mass profiles show the appearance of several metabolites named M1, M2, and M4 (Panel a). Panel b shows the relative abundance of each species up to 10 days post-dosing. Data plotted are mean peak intensity from four mice per time point. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted. Panel c indicates the sites of cleavages on the peptide that matches with metabolites Beaumont et al. AAPS Open (2018) 4:6 Page 13 of 17 Fig. 10 Quantitative mass spectrometry based assay (LC-MRM) of intact Pep-ADC. This assay measures the concentration of the intact Pep-ADC by monitoring peptide R1–12 which covers the first 12 amino-acids of Pep-ADC (a). The relative concentration (compared to immediately after dosing) of intact Pep-ADC and main metabolite (M1) in plasma samples from C57BL/6 J normal mice IV dosed with 3 mg/kg of Pep-ADC (b). Data plotted are mean from three mice per time point (Exception: Day 3 has n = 2). Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted Fig. 11 Intact mass analysis of Pep-ADC by AP-LC-MS in non-human primates. Rhesus monkeys (n = 3) were dosed IV with a single 3 mg/kg Pep- ADC dose. Pep-ADC in dosed plasma samples at indicated time points was retrieved by pull-down for intact mass analysis on a Synapt G2S Q- TOF. Relative abundance of intact Pep-ADC and main metabolites (M1 and M2) is depicted by plotting average peak intensity for up to 14 days after dosing. Data plotted are mean peak intensity from three animals per time point. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted Beaumont et al. AAPS Open (2018) 4:6 Page 14 of 17 target mediated uptake and cellular internalization plays a analysis, with values below 0.6 for all the organs tested more modest role (McMartin, 1992). over the course of the study (unpublished observations). Selected plasma samples were further characterized by Pep-ADC stability issue was also observed in non-human SEC-HPLC. As expected, the total amount of the main primates monomeric peak representing intact IgG decreased with To determine whether the Pep-ADC metabolic profile time due to antibody clearance (Fig. 13). However, observed in mice was translatable to higher order spe- SEC-HPLC profile showed that both conjugated and un- cies, rhesus monkeys were administered intravenously conjugated constructs were stable in the circulation dur- with a single dose of Pep-ADC at 3 mg/kg. Plasma was ing this time with no evidence of formation of HMW collected at 5 min, day 1, day 3, day 7, day 10, and day complexes, aggregates, or major catabolic species. More 14. Intact mass analysis by AP-LC-MS showed that the than 90% of the labeled test materials remained as a single intact Pep-ADC was undetectable by day 7 (Fig. 11), intact IgG peak by day 14 (Fig. 13). Importantly, immuno- similar to what had been observed in mice by day 2. Fur- histochemical analysis clearly demonstrated that thermore, M1 and M2 were the most abundant metabo- Pep-ADC but not unconjugated mAb3 efficiently localized lites as early as day 3 with M1 almost completely to pancreatic Islets of Langerhans consistent with receptor converted to M2 by day 7. All three individual animals mediated Pep-ADC uptake via specific peptide/receptor exhibited similar degradation profile of the Pep-ADC. binding interactions (Fig. 14). Pep-ADC exhibited normal biodisposition pattern in mice To complete the characterization of Pep-ADC in vivo and the potential role of the conjugation and the target in the tissue distribution and catabolism of this ADC, fluoro- phore labeled Pep-ADC and unconjugated mAb3 were intravenously administered to normal C57BL/6 J mice at 3 mg/kg. Selected organs including plasma and whole blood were collected for up to 14 days after IV administra- tion. The concentration versus time values (based on mea- surements of fluorescent tag on the antibody moiety) for the Pep-ADC and unconjugated mAb3 were similar, sug- gesting minimal effects of both the conjugation and the presence of the target in vivo at this dose (Fig. 12). The lack of noticeable gross organ uptake of both compounds was supported by the results of the tissue to blood ratios Fig. 12 The concentration time profiles of Pep-ADC and unconjugated mAb3. Plasma was collected at indicated time points over 14 days following a single 3 mg/kg IV administration of DyLight Fig. 13 In vivo stability of Pep-ADC (a) and unconjugated mAb3 (b). 650-labeled Pep-ADC or DyLight 650-labeled unconjugated mAb3 in Shown are the fluorescence SEC-HPLC profiles of the DyLight650 normal C57BL/6 J mice, and the concentrations were determined by labeled antibodies at indicated time points in plasma of C57BL/6 J fluorescence emission-linked assays. Data plotted are mean from mice after a single 3 mg/kg IV administration. The plasma samples two mice per time point. Error bars correspond to the range. Error were run on a BioSep-SEC-S3000 column at 1 mL/min elution rate bars smaller than the symbols are not plotted with PBS as a mobile phase for 15 min Beaumont et al. AAPS Open (2018) 4:6 Page 15 of 17 AB CD Fig. 14 Immunohistochemical analysis of cellular disposition of DyLight 650-labeled Pep-ADC (a and b) and Dylight 650-labeled unconjugated mAb3 (c and d). Tissue sections were stained with anti-DyLight 650 dye specific antibody. Asterisk (*) represents Pep-ADC localized to pancreatic Islets of Langerhans at Day 1. Arrowheads show labeling of capillaries with both antibodies Discussion lysosomal compartment for efficient drug release di- ADCs or armed antibodies represent one of the next lo- rected towards intracellular targets. gical steps in the implementation of smarter, more effi- In a different modality, ADCs can also be used to ex- cient targeted drugs. Specific delivery of highly bioactive ploit the excellent pharmacokinetic properties of anti- compounds to intracellular targets via antibody-receptor bodies to extend the exposure and half-life of conjugated binding and internalization holds promise for the arrival bioactive compounds (small molecules, ligands, or pep- of novel hybrid biologics with improved therapeutic win- tides). It has been our experience that in general, ADME dows. There are currently 2 ADCs approved in the USA information collected from one ADC is not directly (Garnock-Jones, 2013; Lambert and Chari, 2014) for translatable to another immunoconjugate even when oncological applications and more than 40 ADCs cur- using the same drug-linker combination in a different rently in various stages of clinical trials (Polakis, 2016; antibody and against the same target. It has also been Sievers and Senter, 2013). It is recognized that there are shown that even in the same ADC construct, change in additional challenges and complexities associated with drug to antibody ratios or drug load is sufficient to dra- the design and manufacturing of bio-conjugates versus matically change the pharmacokinetic properties of the standard antibodies or small molecules production. The resulting conjugates (Kamath and Iyer, 2015,; Lyon et al., CMC development that made possible the production of 2015). This is more evident when the new ADC is di- heterogeneous ADCs for successful clinical applications rected to a different target. In non-oncological applica- marks a cornerstone in biotechnology applications (Beck tions, the need for early ADME characterization of each and Reichert, 2014). Furthermore, as improvements in lead ADC-candidate is a critical step in molecule selec- the technology or novel approaches become available, tion and rational drug design. For this purpose, the fol- we can expect additional ADCs with better overall lowing issues need to be addressed properly: 1) Is the pharmacological properties and safer profiles. The right linker stable? 2) Is the drug stable? 3) And is the entire balance needs to be engineered in the ADC so that the ADC stable? The several approaches to address these linker and drug are stable in blood but are promptly questions are also discussed in an industry white paper sorted via antibody binding and internalization to the (Kraynov et al., 2016). Beaumont et al. AAPS Open (2018) 4:6 Page 16 of 17 Conclusion Author details Bioanalytics, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, In this paper, we have shown several experimental exam- California 94304-1104, USA. Biologics DMPK and Disposition, MRL, Merck & ples of two different ADC modalities (targeted internal- 3 Co., Inc., 901 S. California Avenue, Palo Alto, CA 94304-1104, USA. Profiling ization vs. half-life extension) and the corresponding and Expression Departments, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, California 94304-1104, USA. Ambrx, Inc., 10975 North Torrey Pines complexities of the bioanalytical and preclinical ADME Road, La Jolla, California 92037, USA. work necessary to properly characterize these entities both in vitro and in vivo. An early understanding of the Received: 27 February 2018 Accepted: 10 July 2018 critical physico-chemical components responsible for non-favorable or unexpected PK and ADME properties References of experimental conjugates provides critical experimen- Andersen JT, Sandlie I (2009) The versatile MHC class I-related FcRn protects IgG tal feedback for a rational design, optimization, and se- and albumin from degradation: implications for development of new lection of successful lead candidates. diagnostics and therapeutics. Drug metab pharmacokinet 24(4):318–332 Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth Abbreviations JS, Santidrian AF, Stafin K, Lu Y, Tran H, Seller AJ, Biroc SL, Szydlik A, Pinkstaff (D)PBS: (Dulbecco’s) Phosphate buffered saline; ADC (s): Antibody-drug JK, Tian F, Sinha SC, Felding-Habermann B, Smider VV, Schultz PG (2012) conjugate (s); AP-LC-MS: Affinity purified LC-MS; AUC : Area under the Synthesis of site-specific antibody-drug conjugates using unnatural amino 0–14 curve from zero to 14 days; AUC : Area under the curve from zero to acids. Proc Natl Acad Sci U S A 109(40):16101–16106 0-INF infinity; Bud: Budenoside; cAMP: Cyclic adenosine monophosphate; Baumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects Cat: Cathepsin based linker; CH1: Heavy chain constant domain 1; and established and evolving therapies. Lancet (London, England) 369(9573): CHO: Chinese hamster ovary; CL: Clearance; C : Observed maximum 1641–1657 max concentration; DAB: 3,3′ Diaminobenzidine; DAR: Drug to antibody ratio; Beck A, Reichert JM (2014) Antibody-drug conjugates: present and future. mAbs Dex: Dexamethasone; DIO: Diet induced obesity; FcRn: Neonatal Fc receptor; 6(1):15–17 FELA: Fluorescence emission-linked analysis; FFPE: Formalin fixed paraffin Borghese F, Clanchy FI (2011) CD74: an emerging opportunity as a therapeutic embedded; GCGR: Glucagon receptor; GLP1R: Glucagon-like peptide 1 target in cancer and autoimmune disease. Expert Opin Ther Targets 15(3): receptor; HMW: High molecular weight; HRP: Horseradish peroxidase; 237–251 IA: Immunoassay; IACUC: Institutional animal care and use committee; Brunn ND, Mauze S, Gu D, Wiswell D, Ueda R, Hodges D, Beebe AM, Zhang S, IgG: Immunoglobulin; IHC: Immunohistochemistry; IL-2Rα: Interleukin-2 Escandon E (2016) The role of anti-drug antibodies in the pharmacokinetics, receptor alpha; IV: Intravenous (−ly); K2-EDTA: Ethylenediaminetetraacetic disposition, target engagement, and efficacy of a GITR agonist monoclonal acid-dipotassium; LC-MS: Liquid chromatography-mass spectrometry; mAb antibody in mice. J Pharmacol Exp Ther 356(3):574–586 (s): Monoclonal antibody (−ies); MHC: Major histocompatibility complex; Brunton LL (2011) Goodman & Gilman's the pharmacological basis of Min: Minute (s); MSD: Mesoscale discovery; NCA: Non-compartmental therapeutics. McGraw-Hill Education, New York analysis; nm: Nanometer; Pep-ADC: GLP1R/GCGR co-agonist bearing antibody Casi G, Neri D (2012) Antibody-drug conjugates: basic concepts, examples and drug conjugate; Phos: Phosphate based linker; PK: Pharmacokinetic; future perspectives. J Control Release 161(2):422–428 S.D.: Standard Deviation; SEC-HPLC: Size exclusion-high pressure liquid chro- Day JW, Gelfanov V, Smiley D, Carrington PE, Eiermann G, Chicchi G, Erion MD, matography; t : Terminal half life; TBS: Tris buffered saline; TCEP: Tris (2- Gidda J, Thornberry NA, Tschop MH, Marsh DJ, SinhaRoy R, DiMarchi R, Pocai 1/2 carboxyethyl)phosphine; Tg: Transgenic; TMD: Target mediated disposition; A (2012) Optimization of co-agonism at GLP-1 and glucagon receptors to Vss: Volume of distribution at steady state safely maximize weight reduction in DIO-rodents. Biopolymers 98(5):443–450 Day JW, Ottaway N, Patterson JT, Gelfanov V, Smiley D, Gidda J, Findeisen H, Bruemmer D, Drucker DJ, Chaudhary N, Holland J, Hembree J, Abplanalp W, Acknowledgments Grant E, Ruehl J, Wilson H, Kirchner H, Lockie SH, Hofmann S, Woods SC, The authors acknowledge the contributions of Dr. Laurence Fayadat-Dilman Nogueiras R, Pfluger PT, Perez-Tilve D, DiMarchi R, Tschop MH (2009) A new at the Department of Protein Sciences; Dr. Wolfgang Seghezzi at the Depart- glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nat Chem Biol ment of Bioanalytics; Phillip E. Brandish and Paul E. Carrington at the Biology- 5(10):749–757 Discovery Department; Robert M. Garbaccio at the Chemistry-Discovery De- Galaske RG, Van Liew JB, Feld LG (1979) Filtration and reabsorption of partment; And Ambrx, Inc. endogenous low-molecular-weight protein in the rat kidney. Kidney Int 16(3): 394–403 Funding Garnock-Jones KP (2013) Brentuximab vedotin: a review of its use in patients This work was supported by Merck Research Laboratories (MRL), Merck & Co. with hodgkin lymphoma and systemic anaplastic large cell lymphoma Inc. following previous treatment failure. Drugs 73(4):371–381 Gorovits B, Alley SC, Bilic S, Booth B, Kaur S, Oldfield P, Purushothama S, Rao C, Availability of data and materials Shord S, Siguenza P (2013) Bioanalysis of antibody-drug conjugates: The datasets supporting the conclusions of this article are included within American Association of Pharmaceutical Scientists antibody-drug conjugate the article. working group position paper. Bioanalysis 5(9):997–1006 Honey K, Forbush K, Jensen PE, Rudensky AY (2004) Effect of decreasing the Authors’ contributions affinity of the class II-associated invariant chain peptide on the MHC class II Participated in research design: MB, GA, SZ, JHY, EE Conducted experiments: DT, peptide repertoire in the presence or absence of H-2M. J Immunol 172(7): DH, GE, EH, O-YS, YS, HM, SA, WM, YZ, SH, XD, ER, MJ, FV, CM. Contributed re- 4142–4150 agents: AM, NK, AB, DB, DG Performed data analysis: MB, IF, DT, DH, GE, EH, Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen O-YS, YS, SCH, XD, ER, MJ, DN Wrote or contributed to the writing of the manu- T, Dugger DL, Li G, Mai E, Lewis Phillips GD, Hiraragi H, Fuji RN, Tibbitts J, script: MB, DN, EE. All authors read and approved the final manuscript. 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Antibody-drug conjugates: integrated bioanalytical and biodisposition assessments in lead optimization and selection

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Springer Journals
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Copyright © 2018 by The Author(s)
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Biomedicine; Pharmaceutical Sciences/Technology; Pharmacology/Toxicology
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10.1186/s41120-018-0026-0
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Abstract

Therapies based on monoclonal antibodies (mAbs) have delivered an impressive success in the clinics due to their exquisite specificity, potential for agonistic or antagonistic responses, tunable effector function, and optimal pharmacokinetic properties. Building on these inherent antibody properties, the design and development of antibody-drug conjugates (ADCs) with improved or gained therapeutic activity and safety has been successfully demonstrated in oncological applications. There is enormous potential for this new type of hybrid biologics but there are also significant engineering, manufacturing and bioanalytical challenges. In this manuscript, we highlight the range and diversity of assays that are critical to characterize the individual components of ADCs-linker, carrier, and payload. We discuss a series of in vitro and in vivo preclinical experimental approaches we implemented to characterize two anti-inflammatory steroid bearing ADCs, and an ADC bearing a modified glucagon-like peptide 1 receptor/glucagon receptor co-agonist peptide. Keywords: Antibody-drug conjugates, Immunoassay, LC-MS, Biodisposition, CD74, CD25, GLP1R, GCGR Background Antibody-drug conjugates represent a new kind of Antibodies and antibody-based molecules constitute one therapeutic agents which are gaining importance espe- of the most efficacious classes of therapeutic products in cially in the oncology field (Sievers and Senter, 2013). the biotechnology industry. Due to their generally long They are the product of covalently linking a monoclonal half-life and specificity encoded in discrete complemen- antibody with a small molecule or peptide drug (pay- tary determining regions, it was originally believed that, load). Typically, the mAb will enable a targeted delivery in addition to an excellent therapeutic window, the phar- of the payload to a defined cell population of choice. macokinetic properties of recombinant antibodies will The increase in total mass of ADC compared to payload be both similar and predictable. However, it is now well alone improves the payload exposure due to diminished understood that many factors, depending on the specific renal uptake and clearance of ADC. In addition, ADCs antibody and intended target, may significantly affect the are expected to retain binding to the neonatal Fc recep- drug pharmacokinetic behavior (Tabrizi et al., 2006). tor (FcRn), which is responsible for salvaging both im- munoglobulin G (IgG) and albumin from cellular catabolism in a pH-dependent recycling and transcytosis * Correspondence: Enrique.escandon@merck.com 2 mechanism (Andersen and Sandlie, 2009; Roopenian Biologics DMPK and Disposition, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, CA 94304-1104, USA and Akilesh, 2007). Full list of author information is available at the end of the article © The Author(s). 2018 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. Beaumont et al. AAPS Open (2018) 4:6 Page 2 of 17 For ADCs with a targeted intracellular payload, the al., 2014). Descriptions of bioanalytical techniques have challenge is to successfully design ADCs with a balanced been recently published (Gorovits et al., 2013; Kaur et chemical linker strong enough to be stable in blood al., 2013; Wakankar et al., 2011). In this manuscript, we while efficiently cleaved once inside the cell lysosomal describe several specific experimental examples of differ- compartment to release its active payload. These proper- ent site-specific conjugated ADC case-studies in order ties would reduce systemic pharmacological exposure to highlight the complexity and range of bioanalytical and non-specific effects associated with the free drug. methods required to characterize their properties in Alternatively, for extracellular targets the mAb portion vitro and their pharmacokinetic and disposition behavior can serve as a carrier to extend the half-life of the pay- in vivo (Outlined in Fig. 1). Two cases presented here load from minutes to days and systemic exposure by are: 1) Anti-inflammatory steroid-ADCs directed to several folds compared to when dosed as a small mol- specific cell surface receptors (CD25 and CD74) for tar- ecule drug. In this scenario it is critical that the stability geted internalization; 2) A modified Glucagon-like of the linker and payload in the circulation are engi- Peptide 1 receptor/Glucagon receptor (GLP1R/GCGR) neered to match appropriately the expected increase in co-agonist-bearing ADC (Pep-ADC) designed for im- exposure and half-life of the immunoconjugate. This is proved systemic exposure and prolonged pharmaco- of particular importance for peptide payloads as they are logical activity via peptide cell surface receptor binding more likely to be targeted by enzymatic catabolism in and activation. the systemic circulation. Generally, conjugation of the drug to the antibody has Methods been achieved through lysine or existing cysteine resi- All animal procedures described here were performed in dues resulting in heterogeneous conjugations (Casi and accordance with the established guidelines and regula- Neri, 2012). However, there is a tremendous interest and tions by our Institutional Animal Care and Use Commit- research to produce more homogeneous ADCs through tee (IACUC). Animals were housed and maintained in site specific conjugations using either engineered cyst- facilities under a protocol approved by and in accord- eine residues (Junutula et al., 2010) or through the ance with the guidelines of the American Association for site-specific incorporation of non-natural amino acids Assessment and Accreditation of Laboratory Animal with unique reactive groups while minimizing antibody Care. scaffold modifications (Axup et al., 2012; Kern et al., 2016b; Tian et al., 2014; Zimmerman et al., 2014). Re- Monoclonal antibodies and ADC compounds gardless of the methods used for generating ADCs, a Monoclonal rat-mouse chimeric anti- mouse IL-2Rα thorough characterization of the effects that the conju- (CD25) IgG1 was coupled to dexamethasone (Dex) with gated products and procedures may have on the either a novel proprietary phosphate based linker (anti-C- functional properties of the antibody moiety itself is crit- D25-Phos-Dex) (Kern et al., 2016a) or with a cathepsin ical for lead selection and development; including bind- based linker (anti-CD25-Cat-Dex) (Kern et al., 2016b), as ing properties, affinity and specificity, Fc effector described in the respective references. These ADCs were function, off-target disposition, aggregation and novel generated in partnership with Ambrx Inc. (La Jolla, CA). protein interactions. In addition, because of their Humanized anti-human CD74 IgG1 either unconjugated multi-component nature (mAb, linker and payload), (unconjugated anti-CD74) or conjugated to budesonide these hybrid entities are more complex than a “simple” using phosphate linker (anti-CD74-Phos-Bud), humanized mAb or separate payload. For any given combination of anti-human CD74 IgG4 (anti-CD74–011), human mAb, linker, payload, site, type of conjugation, and the anti-viral IgG4 (mAb3), and mAb3 conjugated to modified achieved drug-antibody ratio (DAR), the resulting prod- Glucagon-like Peptide 1 receptor/Glucagon receptor ucts will consist of a mixture of ADC variants with dif- (GLP1R/GCGR) co-agonist peptide (Pep-ADC), were all ferent physico-chemical properties. This in turn, generated in partnership with Ambrx. Mouse depending on the resulting heterogeneity, may affect its anti-Dexamethasone IgG1 (MEB113.53F3.104A) and stability, pharmacokinetics and disposition, adversely mouse anti-Dexamethasone IgG2a (MEB113.8D1.7F) were changing the overall ADC safety and efficacy profile. generated at Merck & Co., Inc. (MRL, Palo Alto, CA). Therefore, each experimental conjugate requires the de- velopment of a combination of various analytical proce- In vitro stability of anti-CD25-Phos-Dex and anti- CD25- dures for a thorough physico-chemical monitoring, from Cat-Dex antibody and payload conjugation, immunoassay, and Anti -CD25-Phos-Dex or anti-CD25-Cat-Dex (15 μg/ cell based procedures to standard and innovative 150 μL plasma) was spiked in normal male DBA1 mouse fit-for-purpose LC-MS methods. There are excellent re- plasma for up to 18 days at 37 °C. After incubation, sam- views that cover several aspects of this process(Perez et ples at selected time points were analyzed by total Beaumont et al. AAPS Open (2018) 4:6 Page 3 of 17 Fig. 1 Schematic illustrating the range of bioanalytical methods used in the studies described in this paper. These methods can be applied to understand concentration, activity, and intactness of antibody-drug conjugates in vitro and in vivo antibody immunoassay (Total IA), measuring anti-CD25 Buffer B (Buffer A: 0.1% formic acid aqueous solution; antibody still capable of binding the target regardless of Buffer B: 0.1% formic acid in acetonitrile) at 0.4 mL/min. the presence or absence of payload, and ADC specific Dexamethasone and dexamethasone-d4 were detected immunoassay (ADC IA) using Mesoscale Discovery using the transitions 437.0 > 361.3, and 441.0 > 363.3, re- (MSD) sandwich immunoassay (Meso Scale Discovery, spectively. Method showed linearity from 0.1 ng/mL to Rockville, MD), and free dexamethasone was determined 2000 ng/mL. by LC-MS (Free Dex). For Total IA, the plasma samples spiked with each ADC were added to MSD MA6000 In vivo stability of anti-CD74-Phos-Bud in human CD74 96-Small Spot plate coated with recombinant mouse transgenic mice IL-2 Rα (R&D System, Minneapolis, MN) as a capture Human CD74 expressing transgenic (Tg) mice molecule. After incubation and washes, wells were incu- (B6.Cg-Tg(CD74)AR194Ayr/j) were generated and char- bated in goat anti-rat IgG (Fc), F (ab’)2-Biotin (Sig- acterized previously (Honey et al., 2004). These Tg mice ma-Aldrich, St. Louis, MO). Finally for detection, (n = 2/group) were dosed intravenously (IV) with either SULTO-TAG labeled Streptavidin was added to the 3, 10 or 30 mg/kg of anti-CD74-Phos-Bud and plasma wells, signal was read on the MESO SECTOR S 600 samples were collected at designated time points. reader after adding MSD read Buffer T. For Anti-CD25 Samples were subjected to MSD immunoassays. For conjugate ADC specific immunoassay, similar procedure total anti-CD74 immunoassay (Total IA), measuring was followed, however, mouse anti-dexamethasone anti-CD74 antibody still capable of binding the target IgG2a (MEB113.8D1.7F)–Biotin (Merck) was used to de- regardless of the presence or absence of payload, tect dexamethasone containing intact ADC in spiked recombinant human CD74 (R&D System) was plated on plasma. MSD MA6000 96-Small Spot plate as a target capture For LC-MS detection of Dexamethasone, plasma pro- and mouse anti-human IgG-SULTO-TAG (SouthernBio- tein was precipitated by addition of 400 μL of internal tech, Birmingham, AL) was used as detection standard solution, consisting of 20 ng/mL of reagent. For anti-CD74–Phos-Bud ADC specific im- Dexamethasone-d4 (TLC PharmaChem, Vaughan, ON) munoassay (ADC IA), mouse anti-dexamethasone IgG1 in acetonitrile to 40 μL of plasma. Samples were vor- (MEB113.53F3.104A)–Biotin (Merck) was coated on to texed for 30 s and centrifuged at 14,000 rpm for 10 min MSD Streptavidin multi-array 96-well plate, and at 4 °C. Supernatants were transferred to a clean tube MEB113.53F3.104A-SULFO-TAG was used as the detec- and dried in Centrivap vacuum concentrator (Labconco, tion reagent. Kansas City, MO). Samples were reconstituted with 40 μL of 50% methanol /aqueous solution and 5 μLwas Anti-CD74-Phos-Bud interacting protein in C57BL/6 J injected into the ESI(−)LC-MS/MS (Acquity Waters, mouse plasma Milford, MA; TSQ Vantagem, ThermoScientific, Wal- Anti-CD74-Phos-Bud was immune-purified from spiked tham MA). Analysis was performed on a Ascentis Ex- C57BL/6 J mouse plasma using NanoLink Streptavidin press C18 (50 mm x3mm × 2.7 μm) (Sigma-Aldrich) Magnetic beads (Solulink, San Diego, CA) immobilized column with a 3 min linear gradient from 50 to 95% with biotin-SP-conjugated AffiniPure F(ab’)2 fragment Beaumont et al. AAPS Open (2018) 4:6 Page 4 of 17 goat anti-human IgG (H + L) (Jackson ImmunoResearch) Biodistribution of DyLight 650 labeled anti-CD74-Phos- with 3 h incubation at 4 °C. After capture, beads were Bud and unconjugated anti-CD74–011 mAb in mice separated from plasma using a magnetic rack and Transgenic human CD74 mice (B6.Cg-Tg(CD74)AR194- washed five times with 0.1% RapiGest (Waters) in TBS Ayr/j) and normal wild type C57BL/6 J female littermates 1× solution. Samples were eluted with 30 μL of 0.25% (WT) (n = 2/group/time point) were given 5 mg/kg IV formic acid solution and neutralized by the addition of bolus of DyLight 650 labeled-anti-CD74-Phos-Bud. A sep- TRIS HCl (1 M). Samples were heated for 3 min at 80 ° arate group of wild type littermates (n = 2/time point) C for denaturation, reduced with DDT (final concentra- were administered with 5 mg/kg of DyLight 650 tion of 5 mM) for 30 min at 60 °C, and alkylated with labeled-anti-CD74–011. Animals were euthanized with iodoacetamide (final concentration of 15 mM) for carbon dioxide followed by terminal cardiac puncture. 30 min at room temperature in dark. Digestion was per- Blood, plasma, and selected tissues were harvested to formed by addition of 2.5 μg of trypsin (Promega, Madi- measure drug concentration. Whole blood samples were son, WI) to each sample, and incubation for 4 h at 37 ° collected into K2-EDTA CapiJect microcollection tubes C. Digestion was quenched by addition of HCl (final (Terumo Medical Corporation, Somerset, NJ) and placed concentration of 200 mM) and samples were further in- on ice. For drug analysis in whole blood, samples were ali- cubated at 37 °C for 45 min for hydrolysis of remaining quoted into polypropylene vials (Corning Glassworks, RapiGest. Samples were centrifuged at 14,000 rpm for Corning, NY) and stored at − 80 °C. Plasma was separated 10 min at 4 °C, and 4 μL of digests were injected in a from whole blood via centrifugation for 6 min at 6000×g LC-MS/MS system NanoAcquity (Waters)/ at 4 °C, and stored at − 80 °C. For tissue lysates, organ LTQ-Orbritrap XL (ThermoScientific) with a 60 min lin- samples were collected and immediately placed into 2-ml ear gradient from 5 to 56% Buffer B (Buffer A: 0.1% for- Precellys Lysing Tubes (Bertin Technologies, Rockville, mic acid aqueous solution; Buffer B: 0.1% formic acid in MD), weighed, and frozen by placing on dry ice. To pre- acetonitrile) at 0.4 μL/min. Search was performed using pare lysates, upon partial thawing, a 1:5 dilution of Dul- the Proteome Discoverer Software (ThermoScientific). becco’s PBS containing 1% Triton X-100 (MP Biomedicals, Solon, OH) and 1× Halt Protease Inhibitor single use cocktail (ThermoFisher Scientific) was added to DyLight™ 650 labeling of antibodies the samples. Then, tissue slurries were prepared using a DyLight™ 650 labeling kits (ThermoScientific) were used Precellys Evolution Homogenizer (Bertin Technologies). to conjugate an N-hydroxysuccinimide ester fluores- The slurry was centrifuged (10,000×g at 4 °C for 10 min) cence dye (excitation at 652 nm and emission at and the tissue lysate supernatants were collected and ei- 672 nm) to anti-CD74, anti-CD74-Phos-Bud, and isotype ther processed immediately or stored at − 80 °C until matched control antibody (mAb-C), Pep-ADC, and analysis. mAb3, using the method described in previous publica- tion from our group (Brunn et al., 2016). Determination of DyLight 650 labeled -anti-CD74-Phos- Bud and anti-CD74–011 mAb concentration in tissue In vitro stability assessment of DyLight 650 labeled anti- samples by fluorescence emission–linked analysis (FELA) CD74, anti-CD74-Phos-Bud, and isotype control antibody For assessment of tissue drug concentration, the col- (mAb-C) by SEC-HPLC lected blood, plasma, and tissue lysate samples or a set For assessing stability in mouse plasma in vitro, DyLight of corressponding blank tissue samples were diluted 1:10 650 labeled anti-CD74, anti-CD74-Phos-Bud and isotype (final dilution) with tissue lysis buffer (1× DPBS with 1% matched control antibody (mAb-C) were incubated at Triton X-100). The diluted samples (150 μL) were trans- 37 °C in C57BL/6 J mouse plasma (100 μL) at a final ferred to 96-well polystyrene plates with low fluores- concentration of 80 μg/mL, samples were taken out and cence background for analysis in the Glomax® snap frozen at designated time points, and stored at − Multi-Detection system microplate reader (Promega). 80 °C until analysis. For SEC-HPLC analysis, 25 μLof The microplate reader was equipped with a fluorescence plasma samples from time points were applied onto a optical filter featuring excitation and emission wave- BioSep-SEC-S3000 column (Phenomenex, Torrance, lengths of 625 nm and 660 to 720 nm, respectively. CA) at 1 mL/min elution rate with PBS as a mobile For each DyLight 650 labeled antibody, fluorescence phase for 15 min, and analyzed using an Agilent 1200 intensity calibration curves were established by serially HPLC system (Agilent Technologies, Santa Clara, CA). diluting spiked blood and liver lysate. A linear correl- The effluent was monitored continuously at 280 nm and ation function was fitted to the data using best-fit pa- by the net fluorescence intensity at an excitation wave- rameters on Microsoft Excel. The resulting blood length of 646 nm and emission of 674 nm, as described calibration curve equation was used to fit unknown previously (Brunn et al., 2016). blood and spleen lysate samples, and the liver calibration Beaumont et al. AAPS Open (2018) 4:6 Page 5 of 17 curve equation was used to fit unknown liver lysate sam- (2.1 × 30 mm) (Applied Biosystems, Waltham, MA) with ples. Concentrations of DyLight-650 labeled antibodies flow rate 0.1 ml/min, 7 min gradient 30% to 58% B, 65 ° were calculated as microgram equivalents per gram of C. Buffer A was water, 0.1% FA, buffer B was 90% Aceto- wet tissue or as microgram equivalents per mL of blood. nitrile, 0.1% FA. Mass Spectrometry conditions were: ca- Subsequently, tissues to blood ratios were calculated by pillary voltage of 3.2 kV, cone voltage of 80 V, dividing concentration in respective tissue by concentra- desolvation temperature 450 °C. Mass data was acquired tion in blood at the designated time point. in m/z mass range of 500–4000. The mass spectrum was deconvoluted and integrated using MassLynx software Assessment of bioactive and total Pep-ADC in mice (Waters). In order to measure the bioactivity of Pep-ADC in plasma (Bioassay), plasma samples from C57BL/6 J male Pep-ADC quantitative mass spectrometry based assay mice with diet induced obesity (DIO) dosed with 10 mg/ (LC-MRM) kg IV bolus of Pep-ADC were incubated with GLP1R 50 μL of solulink magnetic beads were coated by rota- transfected Chinese hamster ovarian (CHO) cells for tion with 250 μL of goat anti-human IgG (monkey 1 h. The reactions were stopped by adding cell-lysis buf- absorbed)-Biotin (SouthernBiotech) for 30 min at room fer from cAMP CISBIO kit (Cisbio, Bedford, MA). A temperature. The beads were washed 3 times with TBS/ calibration curve was established using different amount 0.02% RapiGest and resuspended in 50 μL of TBS/0.02% of Pep-ADC spiked into plasma to stimulate RapiGest. For affinity enrichment, 5 μL of bead was in- GLP1R-CHO cells and quantify cAMP release in a cubated with 30 μL of plasma and TBS/0.02% RapiGest 96-well corning white plate using cAMP CISBIO kit. up to 50 μl for 30 min on a rotator. The beads were Bioactivity of Pep-ADC was back-calculated and re- washed 3 times with TBS/0.02% RapiGest and once with ported as μg/mL. TBS (pH 7.4), eluted with 30 μL of 0.1% formic acid, To determine the total antibody level (Total mAb3 and neutralized with 2.5 μL of 1 M ammonium assay), MSD immunoassay was used. Plasma samples bicarbonate. from Pep-ADC dosed animals were applied to Streptavi- 50 mM TCEP was added for reduction and the mix- din Gold Multi-Array 96-well Plate (MSD) coated with ture incubated for 30 min at 60 °C. Digestion was done mouse anti-Human IgG4-Biotin (SouthernBiotech) as a with 0.25 μg of trypsin for 5 h at 37 °C. 1 pmol of la- capture reagent, and SULFO-TAG-mouse anti-Human beled peptides spanning residue 1 to 12 (representing in- IgG (Southern Biotech) was used for detection. tact molecule) and residue 24 to 30 (representing M1) was added as internal standard. 5 μL was injected into Pep-ADC intact mass analysis by AP-LC-MS an Acquity TSQ Vantage for multiple reactions monitor- Mouse study: C57BL/6 J male mice (n = 3) with diet in- ing of R1-R12 and R24–30 peptides. duced obesity were dosed with 3 mg/kg IV bolus of Pep-ADC. Plasma samples collected at day 1, 3, 7, and Biodisposition of Pep-ADC in mice 10 were processed for intact mass analysis. Normal C57BL/6 J mice (n = 2/group/time point) were Non-human primates study: Treatment naïve male administered a single 3 mg/kg IV dose of either DyLight rhesus monkeys (n = 3) weighing 6–7.1 kg were fasted 650 labeled Pep-ADC or unconjugated mAb3. Plasma overnight and dosed with 3 mg/kg IV bolus of and tissue samples were collected at 1 h, day 1, 2, 3, 5, 7, Pep-ADC. Plasma samples collected at 5 min, day 1, day 9, and day 14 post dose. 3, day 7, day 10, and day 14 were processed for intact To generate plasma concentration vs time profiles, mass analysis. DyLight 650 labeled drug concentration in plasma was Pep-ADC was retrieved from plasma samples from determined by FELA as described previously for both studies by affinity purification for LC-MS as fol- anti-CD74 studies. lows. Nanolink Streptavidin Magnetic beads (Solulink) To determine in vivo stability of DyLight 650 labeled were preloaded with biotinylated polyclonal goat anti Pep-ADC and unconjugated mAb3 by SEC-HPLC, un- human IgG (SouthernBiotech). Preloaded magnetic diluted plasma samples collected at 1 h, day 2, 5, 9 and beads (20 μl) at 2.5 mg/ml in TBS pH 7.4, 0.02% Rapi- day 14 from each group were applied onto a gest SF Surfactant (Waters) was added to 30 μlof BioSep-SEC-S3000 column (Phenomenex) at 1 mL/min K2-EDTA plasma sample. Captured Pep-ADC was elution rate with PBS as a mobile phase for 15 min, and eluted in 30 μl of 0.25% formic acid (FA), reduced with analyzed using an Agilent 1200 HPLC system (Agilent 5 mM TCEP for 30 min at 60 °C, and 10 μl was injected Technologies). The effluent was monitored continuously into Waters Acquity UPLC I class on line with Waters at 280 nm and by the net fluorescence intensity at an ex- Synapt G2S QTOF mass-spectrometer. Reverse phase citation wavelength of 646 nm and emission of 674 nm, chromatography was used on the Poros R2/10 column as described previously (Brunn et al., 2016). Beaumont et al. AAPS Open (2018) 4:6 Page 6 of 17 To determine cellular disposition of the labeled anti- Anti-CD25 mAb conjugated to dexamethasone with bodies, 1 h and day 1 post dose pancreas sections cut cathepsin or phosphate linker: Importance of linker from formalin fixed paraffin embedded (FFPE) tissue stability blocks were deparaffinized and rehydrated with serial A monoclonal antibody against CD25 or Interleukin −2re- passage through changes of xylene and graded ethanols ceptor alpha chain (IL-2Rα), expressed in a subset of im- for DyLight 650 immunohistochemistry (IHC). All slides mune cells (Triplett et al., 2012), was coupled to were subjected to heat induced epitope retrieval in TRS dexamethasone (Dex) using either a novel proprietary solution (pH 6.1) (Dako, Carpineteria, CA). Endogenous phosphate based linker (anti-CD25-Phos-Dex) (Kern et al., peroxidase in tissues was blocked by incubation of slides 2016a) or a cathepsin based linker (anti-CD25-Cat-Dex) in 3% hydrogen peroxide solution prior to incubation (Kern et al., 2016b). In order to fully characterize the ADC with primary antibody, FITC-conjugated anti-DyLight stability in mouse plasma, several assays were developed. 650 clone 12A3 (Merck) for 60 min. Antigen-antibody Initially these included: A total antibody immunoassay binding was visualized via application of rabbit measuring anti-CD25 antibody still capable of binding the anti-FITC antibody (Invitrogen, Carlsbad, CA) then En- target regardless of presence or absence of payload (target-- vision Rabbit HRP (Dako) and application of 3, 3′ diami- capture and anti-Fc detection); A conjugate specific ADC nobenzidine (DAB) chromogen (Dako). Stained slides immunoassay (target capture and anti-Dex detection); And were counterstained with hematoxylin and coverslipped aLC-MS assay tomeasure free Dex. for review. Mouse neat plasma spiked with anti-CD25-Phos-Dex was incubated over the course of 18 days at 37 °C. The Pharmacokinetics total antibody and ADC specific immunoassays showed a All pharmacokinetic parameters were estimated or cal- constant concentration across all time points (Fig. 2a). culated by non-compartmental analysis (NCA) using Likewise, the LC-MS assay for free Dex showed no ap- Phoenix® WinNonlin® software (Certara, Princeton, NJ). pearance of unconjugated payload at any time-point con- Model 201 (IV input bolus) was used for the NCA. sistent with the intactness of this construct in plasma. In contrast, the same ADC but with the cathepsin linker Results (anti-CD25-Cat-Dex conjugate) showed a very different Case-study 1: Anti-inflammatory steroid-ADCs stability-time profile. Total antibody levels were unchanged Systemic anti-inflammatory drugs are often used in the and no free Dex was detected (Fig. 2b). However, the ADC treatment of chronic conditions such as inflammatory proved to be highly unstable as illustrated by a dramatic de- bowel disease (Baumgart and Sandborn, 2007) and crease in concentration over time measured by the asthma (Lipworth, 1999). As a class, steroids are largely ADC-specific immunoassay (Fig. 2b). To investigate the un- not cell or tissue specific and their systemic use is expected lack of stability of anti-CD25-Cat-Dex in plasma, dose-limited due to harmful side effects (Brunton, 2011). we developed a method based on affinity purification Developing a targeted delivery of the most potent im- followed by intact mass measurement by LC-MS mune system repressors through an antibody-drug con- (AP-LC-MS). Anti-CD25-Cat-Dex spiked in plasma was re- jugate could provide a more effective dose and dosing trieved using antigen-bound beads and its mass was mea- rational for enhanced efficacy without triggering nonspe- sured on a Q-TOF mass spectrometer Synapt G2S. The cific detrimental side effects (Kern et al., 2016a). analysis was consistent with a clipping site in the linker Through site specific conjugation in the CH1 domain moiety at a C-O bond, between the linker and the drug (Axup et al., 2012), we coupled potent anti-inflammatory moieties, which was not the part of the predicted cathepsin molecules, dexamethasone and budesonide, to different cleavage site (Fig. 2c). Linker cleavage at this novel site ex- monoclonal antibodies aimed at surface receptors broadly plains the decrease in intact ADC in the immunoassay and expressed on immune cells involved in inflammation. In the absence of free drug by LC-MS as the drug remains at- this modality, the ADC should remain intact in the sys- tached to a part of the broken linker. The same clipping temic circulation, bind to its intended target on the sur- was also observed when anti-CD25-Cat-Dex was incubated face of immune cells followed by target-mediated in phosphate buffered saline (PBS). Regardless of other ap- internalization and drug release in the lysosomal compart- proaches, the implementation of an AP-LC-MS intact mass ment. The active drug is then released to engage gluco- method should become an essential tool to monitor the in- corticoid receptors inside the cell. In addition to their tegrity of the ADCs. affinity and specificity, it is expected that these antibody Contrary to the cathepsin sensitive linker ADC conjugates should retain ideal pharmacokinetic properties (anti-CD25-Cat-Dex) instability, the AP-LC-MS intact mainly affected by target-mediated clearance and display a mass analysis of anti-CD25-Phos-Dex showed no evi- pattern of organ disposition similar to normal endogenous dence of drug release or linker instability resulting in a IgGs. constant drug to antibody ratio with molecular weights Beaumont et al. AAPS Open (2018) 4:6 Page 7 of 17 AC Fig. 2 In vitro stability of anti-CD25-Phos-Dex and anti-CD25-Cat-Dex in mouse plasma. To assess the trend in stability of these antibodies, Anti- CD25-Phos-Dex (a) or anti-CD25-Cat-Dex (b) was spiked in normal male DBA1 mouse plasma for up to 18 days at 37 °C. After incubation, the levels of antibody was determined by total antibody immunoassay (Total IA) and the levels of intact ADC was determined by ADC specific immunoassay (ADC IA) on plasma samples using Mesoscale Discovery (MSD) sandwich immunoassay, and free dexamethasone was determined by LC-MS (Free Dex). Amounts in picomole (pmol) versus time are plotted to compare across different methods of analysis. Values represent the results of a single incubation at each time point. Panel c shows the unexpected site of cleavage in the cathepsin linker of anti-CD25-Cat-Dex comparable to the stock solution at all-time points (un- known to associate with MHC class II molecule published observations). This result was consistent with (Borghese and Clanchy, 2011), a more potent ADC-specific immunoassay results (Fig. 2a). anti-inflammatory steroid, budesonide (Bud), was conju- The potential relevance of in vitro ADC analysis de- gated using an identical phosphate linker at the same pends largely on their translatability to appropriate pre- amino acid residue position in the CH1 domain. The clinical models for safety and efficacy. These in turn systemic stability of the resulting ADC (anti-C- should be selected based on their relevance to the human D74-Phos-Bud) was characterized following IV dosing in physiology and expected mechanism of action (MoA) of mice at 3, 10, and 30 mg/kg. The plasma levels of total the ADC and free drug. Therefore, the stability of mAb versus conjugate-specific immunoassay results anti-CD25-Phos-Dex was further tested in a preclinical showed an apparent linker and/or payload instability PK study. Mice were administered a single intravenous in- (Fig. 3), contrary to the first example of jection of 4 mg/kg of anti-CD25-Phos-Dex. Both immu- anti-CD25-Phos-Dex. However, the AP-LC-MS analysis noassays (total and ADC) showed overlapping indicated a constant peak, identical to the original dos- concentrations with a typical IgG PK profile (unpublished ing material demonstrating the intactness of the ADC observations). Likewise, the total mass spectrum for the construct up to 6 h, after which strong peak broadening ADC showed an identical profile to the stock solution and was observed and the total ion chromatograms could unchanged drug to antibody ratio of 2 at all time points. not be deconvoluted, suggesting heterogeneity. This These results are consistent with the overall good stability could be due to severe conjugate modifications, aggrega- of the anti-CD25-Phos-Dex including the drug, linker, and tion, or protein interactions. In addition, no free Bud antibody components both in vitro and in vivo. could be detected by LC-MS at any time point. There- fore, additional studies were implemented to better Anti-CD74 mAb conjugated to budesonide with understand these discrepancies. Anti-CD74-Phos-Bud phosphate linker: Importance of the targeting antibody was incubated in mouse plasma for 14 days at 37 °C and properties retrieved by pull-down for tryptic digestion and analysis In a different ADC construct with a monoclonal anti- by LC-MS for identification of ADC associated plasma body targeting CD74, a cell surface receptor which is proteins. Analysis of these samples and a control sample Beaumont et al. AAPS Open (2018) 4:6 Page 8 of 17 A B Fig. 3 In vivo stability of anti-CD74-Phos-Bud. To assess the stability of anti-CD74-Phos Bud in mice, the levels of antibody was determined by total antibody immunoassay (Total IA) and levels of intact ADC was determined by ADC specific immunoassay (ADC IA) on plasma samples from human CD74+ transgenic (Tg) mice IV dosed with 30 mg/kg (a), 10 mg/kg (b) and 3 mg/kg (c). Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not visible with no ADC spiked in the original mouse plasma re- (HPLC) system equipped with an in-line fluorescent de- vealed the presence of several unexpected, off-target tector. The resulting chromatographic profiles (Fig. 5a ADC-associated proteins (Fig. 4). These included: and b) indicated antibody instability as shown by the for- alpha-2 macroglobulin, ceruloplasmin, complement C3, mation of high molecular weight (HMW) complexes for albumin, fibrinogen beta, and murinoglobulin 1. Using a both constructs. By day 14 of incubation, only 53% of different approach, anti-CD74-Phos-Bud and unconju- anti-CD74-Phos-Bud and 60% of drug unconjugated gated (naked) anti-CD74 were labeled using a anti-CD74 antibody remained as a single intact mono- DyLight-650 fluorescent dye to follow their size exclu- meric IgG peak. A control antibody (Fig. 5c) remained at sion chromatography (SEC) profile over time upon incu- 96% in the same conditions. The immunoassay discrep- bation in mouse plasma at 37 °C. Samples were analyzed ancies between total anti-CD74 versus intact ADC on an Agilent 1100 high pressure liquid chromatography values may be attributed to assay interferences due to antibody aggregation or off-target association with plasma proteins preventing payload detection. The iden- tification of anti-CD74-Phos-Bud HMW complex forma- tion in plasma was consistent with the off-target interactions identified by LC-MS, and pointed to a po- tential flaw in the properties of the anti-CD74 mAb moi- ety but not in the linker or the payload or the conjugation process. Unconjugated anti-CD74 mAb shows off-target clearance mechanism in mice without CD74 target Clearance of free IgGs and small, non-precipitating IgG complexes happen primarily by interactions with Fc receptors (FcRs) and non-specific protein clearance via fluid-phase endocytosis (Nash et al., 2001;Rojas et al., 2005; Takai, 2002). These pathways are ex- pected to drive the elimination of anti-CD74 in spe- cies without the target expression or binding. To Fig. 4 Anti-CD74-Phos-Bud-interacting proteins in C57BL/6 J plasma. determine the PK properties of this anti-CD74 anti- To determine any off-target protein binding, Anti-CD74-Phos-Bud body, wild C57BL/6 J mice were administered a single was incubated in C57BL/6 J mouse plasma for 14 days at 37 °C and retrieved by pull-down. Pulled-down products were digested with intravenous dose of unconjugated anti-CD74. As show trypsin and analyzed by LC-MS/MS for peptide mapping. Data in Table 1, non-linear PK properties were observed represent the results of a single incubation at each time point for this antibody (Table 1). Concentration ( g/mL) Concentration ( g/mL) Concentration ( g/mL) Beaumont et al. AAPS Open (2018) 4:6 Page 9 of 17 Table 1 PK parameters following IV administration of anti-CD74 to C57BL/6 J mice Dose t C CL V AUC 1/2 max ss 0-INF (mg/kg) (day) (μg/mL) (mL/day/kg) (mL/kg) (day*μg/mL) 10 3.75 90.0 195 589 51.4 3 3.42 27.8 70.3 295 42.7 1 3.90 12.0 39.8 197 25.2 0.3 3.97 2.72 31.5 170 9.52 Here, observed increases in clearance at higher doses are consistent with the presence of a low affinity, high cap- acity off-target antibody sink (i.e., a non-specific clear- ance mechanism). This is, in general, an undesirable property for a targeting antibody that is characterized for its specificity and extended systemic half-life. Anti-CD74-Phos-Bud tissue disposition pattern in mice with and without human CD74 target confirms off target effect with anti-CD74 mAb To gain a better understanding of the DMPK properties of Anti-CD74-Phos-Bud, and the relevance of target ver- sus off-target interactions in the fate of anti-CD74-Phos-Bud in vivo, we utilized a transgenic (Tg) mouse strain that expresses the human CD74 re- ceptor (Honey et al., 2004). The expression of this hu- man receptor is driven by the endogenous analog murine promoter; therefore, these transgenic mice dis- play abundant receptor expression in spleen and to a lesser degree in hepatic tissues. Since anti-CD74-Phos-Bud does not recognize murine CD74, wild type (WT) littermates were used to evaluate the ef- fect of target versus off-target interactions in the distri- bution and elimination of this antibody-drug-conjugate. Fig. 5 In vitro stability of unconjugated anti-CD74 (a), anti-CD74- In addition, a different, unconjugated anti-CD74 mono- Phos-Bud (b), and isotype matched control antibody, mAb-C (c). clonal antibody (referred to as anti-CD74–011 mAb Shown are the fluorescence SEC-HPLC profiles of DyLight 650- labeled antibodies in undiluted C57BL/6 J mouse plasma following hereafter) was used as a control for background tissue incubation at 37 °C for 5 min, 7 days, and 14 days. The plasma uptake characteristic of a non-specific IgG clearance samples were run on a BioSep-SEC-S3000 column at 1 mL/min mechanism, with known negative organ accumulation elution rate with PBS as a mobile phase for 15 min. Black arrows and tissue to blood ratios below 1. Mice were given an indicate the percent of intact, monomeric IgG at day 14 IV dose of 5 mg/kg of DyLight-650-labeled anti-CD74-Phos-Bud and anti-CD74–011 mAb. Figure 6 AUC : Area under the curve from zero to infinity; CL: shows the μg-equivalents/gram (μg/g) of wet tissue or 0-INF Clearance; C : Observed maximum concentration; t1 : μg/ml of blood in Tg and WT mice. In concordance max /2 Terminal half-life; V : Volume of distribution at steady with the organ receptor expression levels, ss state anti-CD74-Phos-Bud distributed extensively to the Frequently, non-linear PK of mAbs may be explained by spleen and liver in the Tg mice with peak concentrations target mediated disposition (TMD). TMD is a saturable of 87 μg/g at 2 h in spleen and 43.5 μg/g at 6 h in liver. process and it is characterized by decreasing clearance However, disposition to spleen and liver in the wild type with increasing doses. On the contrary, clearance of un- littermates was also unusually high (34.1 and 33.7 μg/g) conjugated anti-CD74 appeared to be higher when six hours post IV dosing. In contrast, the spleen and liver higher doses were administered. In addition, since this drug levels in the anti-CD74–011 mAb clone group using anti-CD74 antibody does not recognize the murine tar- wild type littermates were below 11 μg/g at all time points get receptor, its PK was not expected to reflect TMD. in both organs. Accordingly, anti-CD74-Phos-Bud tissue Beaumont et al. AAPS Open (2018) 4:6 Page 10 of 17 A B Fig. 6 Tissue distribution of anti-CD74–011 and anti-CD74-Phos-Bud. Microgram equivalent per gram concentration of antibodies in spleen (a) and liver (b) of WT or human CD74 transgenic (Tg) mice were determined by fluorescence emission-linked assay at multiple time points over 2 days after 5 mg/kg IV dosing. An additional 15 min time-point was collected for anti-CD74-Phos-Bud in Tg mice. Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not plotted Tg Spleen Tg Liver WT Liver WT Spleen Fig. 7 Spleen and liver tissue to blood ratios of anti-CD74-Phos-Bud (a) and an unconjugated anti-CD74–011 (b) in wild type (WT) and human CD74 transgenic (Tg) mice. Tissue/blood ratios were calculated from drug concentration measured by fluorescence emission-linked assay in blood and the tissues at 2 h, 6 h, 1 and 2 days after 5 mg/kg IV dosing. An additional 15 min time-point was collected for anti-CD74-Phos-Bud in Tg mice. Tissue/blood ratios greater than 1 are indicative of tissue uptake. Data plotted are mean from two mice per time point. Error bars correspond to the range. Error bars smaller than the symbol are not plotted Beaumont et al. AAPS Open (2018) 4:6 Page 11 of 17 to blood ratios were as high as 99 for the spleen at day 1 with the required stability to match the enhanced pharma- and 66.9 for liver at day 2 in Tg mice (Fig. 7a). However, cokinetic properties conferred by the antibody conjuga- tissue to blood ratios higher than 1 were also found in tion. For measurements of total versus intact ADC spleen and liver from wild type littermates consistent with concentrations, an immunoassay (Fc capture and Fc de- off-target interactions. Overall these results demonstrated tection) and a cell based bioactivity (peptide-induced re- the presence of an antibody driven, off-target mechanism ceptor activation) procedures were implemented. of tissue uptake and disposition, and were consistent with the identification of non-specific protein interactions in Immunoassay, cell based bioactivity, and mass the circulation and undesirable PK for this construct. spectrometric characterization helped to determine the Based on these results, this anti-CD74 mAb was therefore pep-ADC stability issue in mice not further considered as a lead therapeutic candidate. In To characterize the metabolic fate of Pep-ADC in vivo, this case, the intrinsic unfavorable properties of the anti- Pep-ADC was administered to mice at 1 and 10 mg/kg body alone determined the overall poor fate of this ADC as a single intravenous injection. Plasma was collected in vivo. This conclusion was further supported by obser- over time and analyzed for both total ADC (mAb3 vations with the anti-CD74–011 mAb, which demon- levels) and cell-based bioactivity (assessment of free and/ strated excellent stability in biomatrices. In a follow up or conjugated active peptide). Previous in vitro work disposition study, anti-CD74–011 mAb exhibited higher with the free peptide (Pep) had demonstrated that only tissue to blood ratios in spleen compared to liver in hu- the intact moiety retained receptor binding and signaling man CD74 expressing transgenic mice at any evaluated in this cell-based assay. Pharmacokinetic results (Table 2 time points, with ratio in spleen reaching as high as 224 at and Fig. 8) demonstrated more than 2-fold differences in day 2 compared to 48.7 in liver (Fig. 7b). Furthermore, exposure and clearance between total mAb3 and bio- tissue-to-blood ratios in wild type mice for both tissues active measurements in a cell based assay. Therefore, were lower than 0.5 at any evaluated time point (Fig. 7b). the smaller exposure and increased clearance for the These observations were consistent with anti-CD74–011 bioactive ADC compared to the total ADC -suggested a mAb target-mediated uptake following administration to potential linker instability and/or peptide catabolism. the transgenic mouse strain that expresses the human To further understand the molecular nature of form of CD74 (Fig. 7b). Pep-ADC potency loss, we conducted an AP-LC-MS analysis. In agreement with the bioactivity assay data, Case-study 2: GLP1R/GCGR peptide-ADC (Pep-ADC): the results showed that while the intact conjugated Importance of payload stability Pep-ADC decreased with time and eventually became Due to their precise specificity, peptide drugs are gaining undetectable by day 5, several clipped variants (M1, M2, importance as therapeutic agents. However, their small M3 and M4) representing degradation of the conjugated size, rapid clearance rates, and systemic catabolism have peptide at various positions became the predominant limited their clinical potential and applications (McGregor, ADC species (Fig. 9a and b). A control group of mice 2008;Sato et al., 2006). The use of balanced Glucagon dosed with the mAb3 only, without the conjugated -like peptide 1 receptor/Glucagon receptor (GLP1R/ linker-peptide, showed a constant peak (intact mAb) at GCGR) co-agonist has shown enhanced efficacy and safety the same molecular weight at all tested time points (un- relative to pure GLP1R agonists in the treatment of rodent published observations) indicating no catabolism of the obesity, with simultaneous improvement in glycemic con- antibody moiety itself. Mass difference calculations iden- trol (Day et al., 2012; Day et al., 2009; Pocai et al., 2009). tified that the conjugated peptide clipping between resi- Using site specific conjugation, we coupled a dues 2/3 (M4), 25/26 (M1), 27/28 (M2) and 30/31 (M3) modified-for-stability GLP1R/GCGR co-agonist peptide of the peptide sequence (Fig. 9c). All clipped M-variants (payload) to a residue in the CDR H1 of a non-targeting are inactive in bioassay, as it is known that the removal human mAb (mAb3) via a non-cleavable linker. The Table 2 Pharmacokinetic parameters following IV linked peptide-mAb3 immunoconjugate is referred to as administration of Pep-ADC to C57BL/6 J mice Pep-ADC hereafter. In this modality, it is expected that Assay Dose t AUC CL V the antibody moiety will provide a stable carrier for pro- 1/2 0–14 ss (mg/kg) (day) (day*μg/mL) (mL/day/kg) (mL/kg) longed exposure in the circulation of the linked peptide Total mAb3 1 5.12 56.9 15.5 89.1 due to reduced renal clearance and engagement of the 10 4.76 657 13.9 69.0 FcRn recycle mechanism. In turn, this should result in a substantial improvement in the biological outcome com- Bioassay 1 2.55 28.5 34.9 49.3 pared to the co-agonist peptide dosed alone (Kompella 10 2.78 264 37.5 64.7 and Lee, 1991). In order for this to occur, it is essential AUC : Area under the curve from zero to 14 days; CL: Clearance; t : 0–14 1/2 that both the linker and the bioactive peptide are designed Terminal half-life; V : Volume of distribution at steady state ss Beaumont et al. AAPS Open (2018) 4:6 Page 12 of 17 receptor (GLP1R/GCGR) co-agonist-bearing ADC. Fi- nally, a mass spec-based quantitative assay was developed to measure the disappearance of the intact form and ap- pearance of the main peptide catabolite (Fig. 10a). It con- firmed that intact Pep-ADC disappeared fast and by day 2, only about 10% of intact Pep-ADC remained (Fig. 10b). Conversely, M1 increased for the first two days and then declined over the remaining 5 days as it was cleared and/ or transformed into M2 and M3 (Fig. 10b). Overall, these data were consistent with the bioactivity assay and intact mAb measurements, and confirmed M1 as one of the most abundant metabolites in the circulation. These re- sults indicated a path forward in the rational design of im- Fig. 8 The concentration time profile of Pep-ADC measured by two proved ADC versions focused on strengthening the different assays. Plasma samples were collected at multiple time stability of the peptide moiety. It is important to note that points over 14 days from mice IV dosed with 10 mg/kg of Pep-ADC, although bioactive drug exposure can be further increased and measured for intact antibody concentrations by MSD assay with potentially more stable peptide properties in the cir- (Total mAb3 assay) as well as bioactive ADC using a cell-based assay culation, the current exposure to active drug in this ADC (Bioassay). Data plotted are mean from 3 mice. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter was considerably better than those of similar peptides than the symbols are not plotted when dosed alone with half-lives ranging in the order of minutes and elimination rates closer to glomerular filtra- of the first two amino acids of Glucagon-like peptide-1 tion rates (Galaske et al., 1979). For free peptides in the (GLP-1) by dipeptidyl peptidase 4 (DPP4) kills the activ- circulation at pharmacological doses, two powerful mech- ity of GLP-1 through GLP-1 receptor (GLP1R) (Knudsen anisms drive the fast elimination of these molecules and Pridal, 1996). This is also true for Pep-ADC, which mainly by renal clearance followed by systemic enzymatic is a modified Glucagon-like Peptide 1 receptor/Glucagon degradation (Rabkin and Dahl, 1993). Peptide clearance by AB Fig. 9 Intact mass analysis of Pep-ADC in mice by AP-LC-MS. Pep-ADC (3 mg/kg) administered to C57BL/6 J diet induced obese (DIO) mice by IV was retrieved by pull-down for intact mass analysis on a Synapt G2S Q-TOF. Intact mass profiles show the appearance of several metabolites named M1, M2, and M4 (Panel a). Panel b shows the relative abundance of each species up to 10 days post-dosing. Data plotted are mean peak intensity from four mice per time point. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted. Panel c indicates the sites of cleavages on the peptide that matches with metabolites Beaumont et al. AAPS Open (2018) 4:6 Page 13 of 17 Fig. 10 Quantitative mass spectrometry based assay (LC-MRM) of intact Pep-ADC. This assay measures the concentration of the intact Pep-ADC by monitoring peptide R1–12 which covers the first 12 amino-acids of Pep-ADC (a). The relative concentration (compared to immediately after dosing) of intact Pep-ADC and main metabolite (M1) in plasma samples from C57BL/6 J normal mice IV dosed with 3 mg/kg of Pep-ADC (b). Data plotted are mean from three mice per time point (Exception: Day 3 has n = 2). Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted Fig. 11 Intact mass analysis of Pep-ADC by AP-LC-MS in non-human primates. Rhesus monkeys (n = 3) were dosed IV with a single 3 mg/kg Pep- ADC dose. Pep-ADC in dosed plasma samples at indicated time points was retrieved by pull-down for intact mass analysis on a Synapt G2S Q- TOF. Relative abundance of intact Pep-ADC and main metabolites (M1 and M2) is depicted by plotting average peak intensity for up to 14 days after dosing. Data plotted are mean peak intensity from three animals per time point. Error bars correspond to the S.D. calculated using GraphPad Prism 7. Error bars shorter than the symbols are not plotted Beaumont et al. AAPS Open (2018) 4:6 Page 14 of 17 target mediated uptake and cellular internalization plays a analysis, with values below 0.6 for all the organs tested more modest role (McMartin, 1992). over the course of the study (unpublished observations). Selected plasma samples were further characterized by Pep-ADC stability issue was also observed in non-human SEC-HPLC. As expected, the total amount of the main primates monomeric peak representing intact IgG decreased with To determine whether the Pep-ADC metabolic profile time due to antibody clearance (Fig. 13). However, observed in mice was translatable to higher order spe- SEC-HPLC profile showed that both conjugated and un- cies, rhesus monkeys were administered intravenously conjugated constructs were stable in the circulation dur- with a single dose of Pep-ADC at 3 mg/kg. Plasma was ing this time with no evidence of formation of HMW collected at 5 min, day 1, day 3, day 7, day 10, and day complexes, aggregates, or major catabolic species. More 14. Intact mass analysis by AP-LC-MS showed that the than 90% of the labeled test materials remained as a single intact Pep-ADC was undetectable by day 7 (Fig. 11), intact IgG peak by day 14 (Fig. 13). Importantly, immuno- similar to what had been observed in mice by day 2. Fur- histochemical analysis clearly demonstrated that thermore, M1 and M2 were the most abundant metabo- Pep-ADC but not unconjugated mAb3 efficiently localized lites as early as day 3 with M1 almost completely to pancreatic Islets of Langerhans consistent with receptor converted to M2 by day 7. All three individual animals mediated Pep-ADC uptake via specific peptide/receptor exhibited similar degradation profile of the Pep-ADC. binding interactions (Fig. 14). Pep-ADC exhibited normal biodisposition pattern in mice To complete the characterization of Pep-ADC in vivo and the potential role of the conjugation and the target in the tissue distribution and catabolism of this ADC, fluoro- phore labeled Pep-ADC and unconjugated mAb3 were intravenously administered to normal C57BL/6 J mice at 3 mg/kg. Selected organs including plasma and whole blood were collected for up to 14 days after IV administra- tion. The concentration versus time values (based on mea- surements of fluorescent tag on the antibody moiety) for the Pep-ADC and unconjugated mAb3 were similar, sug- gesting minimal effects of both the conjugation and the presence of the target in vivo at this dose (Fig. 12). The lack of noticeable gross organ uptake of both compounds was supported by the results of the tissue to blood ratios Fig. 12 The concentration time profiles of Pep-ADC and unconjugated mAb3. Plasma was collected at indicated time points over 14 days following a single 3 mg/kg IV administration of DyLight Fig. 13 In vivo stability of Pep-ADC (a) and unconjugated mAb3 (b). 650-labeled Pep-ADC or DyLight 650-labeled unconjugated mAb3 in Shown are the fluorescence SEC-HPLC profiles of the DyLight650 normal C57BL/6 J mice, and the concentrations were determined by labeled antibodies at indicated time points in plasma of C57BL/6 J fluorescence emission-linked assays. Data plotted are mean from mice after a single 3 mg/kg IV administration. The plasma samples two mice per time point. Error bars correspond to the range. Error were run on a BioSep-SEC-S3000 column at 1 mL/min elution rate bars smaller than the symbols are not plotted with PBS as a mobile phase for 15 min Beaumont et al. AAPS Open (2018) 4:6 Page 15 of 17 AB CD Fig. 14 Immunohistochemical analysis of cellular disposition of DyLight 650-labeled Pep-ADC (a and b) and Dylight 650-labeled unconjugated mAb3 (c and d). Tissue sections were stained with anti-DyLight 650 dye specific antibody. Asterisk (*) represents Pep-ADC localized to pancreatic Islets of Langerhans at Day 1. Arrowheads show labeling of capillaries with both antibodies Discussion lysosomal compartment for efficient drug release di- ADCs or armed antibodies represent one of the next lo- rected towards intracellular targets. gical steps in the implementation of smarter, more effi- In a different modality, ADCs can also be used to ex- cient targeted drugs. Specific delivery of highly bioactive ploit the excellent pharmacokinetic properties of anti- compounds to intracellular targets via antibody-receptor bodies to extend the exposure and half-life of conjugated binding and internalization holds promise for the arrival bioactive compounds (small molecules, ligands, or pep- of novel hybrid biologics with improved therapeutic win- tides). It has been our experience that in general, ADME dows. There are currently 2 ADCs approved in the USA information collected from one ADC is not directly (Garnock-Jones, 2013; Lambert and Chari, 2014) for translatable to another immunoconjugate even when oncological applications and more than 40 ADCs cur- using the same drug-linker combination in a different rently in various stages of clinical trials (Polakis, 2016; antibody and against the same target. It has also been Sievers and Senter, 2013). It is recognized that there are shown that even in the same ADC construct, change in additional challenges and complexities associated with drug to antibody ratios or drug load is sufficient to dra- the design and manufacturing of bio-conjugates versus matically change the pharmacokinetic properties of the standard antibodies or small molecules production. The resulting conjugates (Kamath and Iyer, 2015,; Lyon et al., CMC development that made possible the production of 2015). This is more evident when the new ADC is di- heterogeneous ADCs for successful clinical applications rected to a different target. In non-oncological applica- marks a cornerstone in biotechnology applications (Beck tions, the need for early ADME characterization of each and Reichert, 2014). Furthermore, as improvements in lead ADC-candidate is a critical step in molecule selec- the technology or novel approaches become available, tion and rational drug design. For this purpose, the fol- we can expect additional ADCs with better overall lowing issues need to be addressed properly: 1) Is the pharmacological properties and safer profiles. The right linker stable? 2) Is the drug stable? 3) And is the entire balance needs to be engineered in the ADC so that the ADC stable? The several approaches to address these linker and drug are stable in blood but are promptly questions are also discussed in an industry white paper sorted via antibody binding and internalization to the (Kraynov et al., 2016). Beaumont et al. AAPS Open (2018) 4:6 Page 16 of 17 Conclusion Author details Bioanalytics, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, In this paper, we have shown several experimental exam- California 94304-1104, USA. Biologics DMPK and Disposition, MRL, Merck & ples of two different ADC modalities (targeted internal- 3 Co., Inc., 901 S. California Avenue, Palo Alto, CA 94304-1104, USA. Profiling ization vs. half-life extension) and the corresponding and Expression Departments, MRL, Merck & Co., Inc., 901 S. California Avenue, Palo Alto, California 94304-1104, USA. Ambrx, Inc., 10975 North Torrey Pines complexities of the bioanalytical and preclinical ADME Road, La Jolla, California 92037, USA. work necessary to properly characterize these entities both in vitro and in vivo. An early understanding of the Received: 27 February 2018 Accepted: 10 July 2018 critical physico-chemical components responsible for non-favorable or unexpected PK and ADME properties References of experimental conjugates provides critical experimen- Andersen JT, Sandlie I (2009) The versatile MHC class I-related FcRn protects IgG tal feedback for a rational design, optimization, and se- and albumin from degradation: implications for development of new lection of successful lead candidates. diagnostics and therapeutics. Drug metab pharmacokinet 24(4):318–332 Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth Abbreviations JS, Santidrian AF, Stafin K, Lu Y, Tran H, Seller AJ, Biroc SL, Szydlik A, Pinkstaff (D)PBS: (Dulbecco’s) Phosphate buffered saline; ADC (s): Antibody-drug JK, Tian F, Sinha SC, Felding-Habermann B, Smider VV, Schultz PG (2012) conjugate (s); AP-LC-MS: Affinity purified LC-MS; AUC : Area under the Synthesis of site-specific antibody-drug conjugates using unnatural amino 0–14 curve from zero to 14 days; AUC : Area under the curve from zero to acids. Proc Natl Acad Sci U S A 109(40):16101–16106 0-INF infinity; Bud: Budenoside; cAMP: Cyclic adenosine monophosphate; Baumgart DC, Sandborn WJ (2007) Inflammatory bowel disease: clinical aspects Cat: Cathepsin based linker; CH1: Heavy chain constant domain 1; and established and evolving therapies. Lancet (London, England) 369(9573): CHO: Chinese hamster ovary; CL: Clearance; C : Observed maximum 1641–1657 max concentration; DAB: 3,3′ Diaminobenzidine; DAR: Drug to antibody ratio; Beck A, Reichert JM (2014) Antibody-drug conjugates: present and future. mAbs Dex: Dexamethasone; DIO: Diet induced obesity; FcRn: Neonatal Fc receptor; 6(1):15–17 FELA: Fluorescence emission-linked analysis; FFPE: Formalin fixed paraffin Borghese F, Clanchy FI (2011) CD74: an emerging opportunity as a therapeutic embedded; GCGR: Glucagon receptor; GLP1R: Glucagon-like peptide 1 target in cancer and autoimmune disease. 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McGraw-Hill Education, New York analysis; nm: Nanometer; Pep-ADC: GLP1R/GCGR co-agonist bearing antibody Casi G, Neri D (2012) Antibody-drug conjugates: basic concepts, examples and drug conjugate; Phos: Phosphate based linker; PK: Pharmacokinetic; future perspectives. J Control Release 161(2):422–428 S.D.: Standard Deviation; SEC-HPLC: Size exclusion-high pressure liquid chro- Day JW, Gelfanov V, Smiley D, Carrington PE, Eiermann G, Chicchi G, Erion MD, matography; t : Terminal half life; TBS: Tris buffered saline; TCEP: Tris (2- Gidda J, Thornberry NA, Tschop MH, Marsh DJ, SinhaRoy R, DiMarchi R, Pocai 1/2 carboxyethyl)phosphine; Tg: Transgenic; TMD: Target mediated disposition; A (2012) Optimization of co-agonism at GLP-1 and glucagon receptors to Vss: Volume of distribution at steady state safely maximize weight reduction in DIO-rodents. 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Kidney Int 16(3): 394–403 Funding Garnock-Jones KP (2013) Brentuximab vedotin: a review of its use in patients This work was supported by Merck Research Laboratories (MRL), Merck & Co. with hodgkin lymphoma and systemic anaplastic large cell lymphoma Inc. following previous treatment failure. Drugs 73(4):371–381 Gorovits B, Alley SC, Bilic S, Booth B, Kaur S, Oldfield P, Purushothama S, Rao C, Availability of data and materials Shord S, Siguenza P (2013) Bioanalysis of antibody-drug conjugates: The datasets supporting the conclusions of this article are included within American Association of Pharmaceutical Scientists antibody-drug conjugate the article. working group position paper. Bioanalysis 5(9):997–1006 Honey K, Forbush K, Jensen PE, Rudensky AY (2004) Effect of decreasing the Authors’ contributions affinity of the class II-associated invariant chain peptide on the MHC class II Participated in research design: MB, GA, SZ, JHY, EE Conducted experiments: DT, peptide repertoire in the presence or absence of H-2M. J Immunol 172(7): DH, GE, EH, O-YS, YS, HM, SA, WM, YZ, SH, XD, ER, MJ, FV, CM. Contributed re- 4142–4150 agents: AM, NK, AB, DB, DG Performed data analysis: MB, IF, DT, DH, GE, EH, Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen O-YS, YS, SCH, XD, ER, MJ, DN Wrote or contributed to the writing of the manu- T, Dugger DL, Li G, Mai E, Lewis Phillips GD, Hiraragi H, Fuji RN, Tibbitts J, script: MB, DN, EE. All authors read and approved the final manuscript. 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Published: Aug 8, 2018

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