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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, GeneCell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay Comparabil ityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

BackgroundPD-1/PD-L1 immune checkpoint inhibitors have transformed oncology, but a significant unmet need persists for patients with relapsed/refractory tumors following PD-1/PD-L1 treatment. PD-L1 is expressed in patients across a broad spectrum of tumor types and displays limited normal tissue expression, highlighting the potential of PD-L1 as a target for antibody-drug conjugates (ADCs) in addition to its role as an immune checkpoint. SGN-PDL1V is a PD-L1-directed ADC currently under preclinical investigation, which is comprised of an anti-PD-L1 antibody conjugated to the vedotin drug-linker. The vedotin drug-linker, consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), and a protease-cleavable peptide linker, which has been clinically validated in multiple ADC programs including brentuximab vedotin, enfortumab vedotin and polatuzumab vedotin.1–3 The proposed SGN-PDL1V primary mechanism of action is direct cytotoxicity against PD-L1-expressing malignant cells through delivery of the MMAE payload. Additionally, MMAE induces immunogenic cell death, leading to subsequent immune activation in the tumor microenvironment.4 Here, we characterize the preclinical activity and tolerability of SGN-PDL1V.MethodsSGN-PDL1V cytotoxicity was evaluated using PD-L1 expressing tumor cell lines in vitro and xenograft tumor models in vivo. Inhibition of the PD-1/PD-L1 immune checkpoint was assessed in a luminescent reporter system in vitro and a syngeneic tumor model in vivo. The tolerability and safety profile of SGN-PDL1V was determined in a non-human primate study.ResultsIn vitro, SGN-PDL1V demonstrated internalization and potent cytotoxic activity against PD-L1 expressing tumor cells. In vivo, SGN-PDL1V achieved tumor regressions in multiple tumor xenograft models at doses as low as 1 mg/kg when dosed weekly for a total of three doses. This activity was observed in immunocompromised mice, which lack responses to PD-1/PD-L1 inhibition. Notably, activity was observed even in xenograft models with low, heterogeneous PD-L1 expression, supporting the possibility to treat patients across a wide range of PD-L1 expression levels. Additionally, SGN-PDL1V exhibited potential to inhibit the PD-1/PD-L1 checkpoint in vitro and in vivo. The tolerability and safety profile of SGN-PDL1V were assessed in a non-human primate study and found to be comparable to other FDA-approved vedotin ADCs.ConclusionsSGN-PDL1V is a promising PD-L1 directed ADC with a unique cytotoxic mechanism of action among other PD-L1-targeted therapeutics. SGN-PDL1V demonstrated robust activity in multiple preclinical models and comparable tolerability and safety profile to other vedotin ADCs in non-human primates. Collectively, these data support further evaluation of SGN-PDL1V in a planned, first-in-human Phase 1 study.AcknowledgementsWe would like to thank Kerry Klussman for assay support and Jamie Mitchell for conjugation support.Trial RegistrationN/AReferencesSenter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol 2012;30(7):631–7. Epub 2012/07/12. doi: 10.1038/nbt.2289. PubMed PMID: 22781692.Rosenberg JE, O'Donnell PH, Balar AV, McGregor BA, Heath EI, Yu EY, et al. Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/Programmed death ligand 1 therapy. J Clin Oncol 2019;37(29):2592–600. Epub 2019/07/30. doi: 10.1200/JCO.19.01140. PubMed PMID: 31356140; PubMed Central PMCID: PMC6784850.Tilly H, Morschhauser F, Bartlett NL, Mehta A, Salles G, Haioun C, et al. Polatuzumab vedotin in combination with immunochemotherapy in patients with previously untreated diffuse large B-cell lymphoma: an open-label, non-randomised, phase 1b-2 study. Lancet Oncol 2019;20(7):998–1010. Epub 2019/05/19. doi: 10.1016/S1470-2045(19)30091-9. PubMed PMID: 31101489.Klussman K, Tenn E, Higgins S, Mazahreh R, Snead K, Hamilton J, Grogan B, Sigurjonsson J, Cao A, Gardai S, Liu B. 618 Vedotin ADCs induce ER stress and elicit hallmarks of ICD across multiple cancer indications. J Immunother Cancer 2020;8(Suppl 3):A372. DOI:10.1136/jitc-2020-SITC2020.0618.Ethics ApprovalAll animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

Background: Tucatinib is an orally administered, reversible HER2-targeted small molecule tyrosine kinase inhibitor that potently and selectively inhibits HER2 relative to the closely related kinase EGFR. In a phase Ib clinical trial in HER2+ metastatic breast cancer, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in pretreated patients with HER2-positive metastatic breast cancer (Borges VF et al., 2018). Here, we present preclinical data demonstrating tucatinib potentiates the activity of T-DM1 in HER2+ breast cancer models in vitro, and in vivo. In addition, tucatinib also enhanced the activity of a camptothecin-based HER2 ADC comprising trastuzumab conjugated with 8 exatecan moieties (T-Ex). Methods: In vitro assays were conducted to evaluate the potency of tucatinib, T-DM1 and T-Ex as single agents, and in combination, using a panel of breast cancer cell lines expressing various levels of HER2. The combinatorial effects of tucatinib with T-DM1 or T-Ex was assessed by isobologram analysis to determine additivity, synergy or antagonistic properties of the drug combinations. The activity of tucatinib either alone (50 mg/kg BID) or in combination with T-DM1 (10 mg/kg single dose IV) was evaluated in vivo using the HER2+ breast cancer cell line BT-474, and in 3 patient-derived xenograft (PDX) models of HER2+ breast cancer. Results: Tucatinib demonstrated potent anti-tumor activity in HER2 overexpressing cell lines in vitro with a similar selectivity profile as T-DM1 or T-Ex. When co-administered in vitro and subjected to isobologram analysis, tucatinib and T-DM1 or T-Ex combinations produced additive or synergistic effects. Moreover, tucatinib potently inhibited a subset of cell lines that showed reduced sensitivity to either T-DM1 or T-Ex. In BT-474 cells, the co-administration of tucatinib with T-DM1 in vitro was synergistic and resulted in an increased intracellular concentration of the TDM-1 catabolite Lys-MCC-DM1. The combination of tucatinib with T-DM1 was also more effective than either single agent alone in BT-474 xenografts in vivo and increased the number of complete tumor regressions. In 2 of 3 PDX models tested, the combination of tucatinib with T-DM1 was significantly more active than T-DM1 or tucatinib alone and produced a higher proportion of partial or complete tumor regressions compared with the single agent treatments. Conclusions: These data demonstrate tucatinib results in selective and potent anti-tumor activity in HER2+ tumor derived cell lines, including cell lines that show reduced sensitivity to T-DM1 or T-Ex in vitro. The results also demonstrate that tucatinib is either additive or synergistic when combined with T-DM1 or T-Ex in vitro. In addition, tucatinib in combination with T-DM1 showed enhanced anti-tumor activity in vivo in models of HER2+ breast cancer when compared to T-DM1 as a single agent. These results, taken together with the early clinical data demonstrating preliminary safety and activity of tucatinib with T-DM1, support continued assessment of tucatinib in combination with T-DM1, as well as other HER2 targeted ADCs, in HER2+ metastatic breast cancer patients. Borges VF, Ferrario C, Aucoin N, Falkson C, Khan Q, Krop I, Welch S, Conlin A, Chaves J, Bedard PL, Chamberlain M, Gray T, Vo A, Hamilton E. JAMA Oncol. 2018;4(9):1214-1220. Citation Format: Anita Kulukian, Janelle Taylor, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Tim S Lewis, Scott R Peterson. Tucatinib, a HER2-selective tyrosine kinase inhibitor, increases the anti-tumor activity of trastuzumab antibody-drug conjugates in preclinical models of HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-09.

Unlike with new chemical entities, the biotransformation of therapeutic proteins (TPs) has not been routinely investigated or included in regulatory filings. Nevertheless, there is an expanding pool of evidence suggesting that a more in-depth understanding of biotransformation could better aid the discovery and development of increasingly diverse modalities. For instance, such biotransformation analysis of TPs affords important information on molecular stability, which in turn may shed light on any potential impact on binding affinity, potency, pharmacokinetics, efficacy, safety, or bioanalysis. This perspective summarizes the current practices in studying biotransformation of TPs and related findings in the biopharmaceutical industry. Various TP case studies are discussed, and a fit-for-purpose approach is recommended when investigating their biotransformation. In addition, we provide a decision tree to guide the biotransformation characterization for selected modalities. By raising the awareness of this important topic, which remains relatively underexplored in the development of TPs (Bolleddula et al., 2022), we hope that current and developing practices can pave the way for establishing a consensus on the biotransformation assessment of TPs. SIGNIFICANCE STATEMENT: This article provides a comprehensive perspective of the current practices for exploring the biotransformation of therapeutic proteins across the drug development industry. We, the participants of the Innovation and Quality therapeutic protein absorption distribution metabolism excretion working group, recommend and summarize appropriate approaches for conducting biotransformation studies to support internal decision making based on the data generated in discovery and development.

Lessons Learned Background We conducted a first-in-human, first-in-class, phase I study of SGN-2FF, a potent small-molecule inhibitor of glycoprotein fucosylation, in patients with advanced solid tumors. Methods The study consisted of four parts: SGN-2FF monotherapy dose-escalation (part A) and expansion (part B), and SGN-2FF + pembrolizumab dose-escalation (part C) and expansion (part D). The objectives were to evaluate safety and tolerability, maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of SGN-2FF monotherapy and SGN-2FF + pembrolizumab. Results Forty-six patients were enrolled (part A, n = 33; part B, n = 6; part C, n = 7; part D did not enroll any patients). During part A (n = 32) exploring 1–15 g once daily (QD) and 2–5 g twice daily (b.i.d.), grade 3 dose-limiting toxicities were diarrhea (2 g and 15 g QD) and increased lipase (2 g QD). The MTD was 10 g daily. In part A, common toxicities were grades 1–2 diarrhea, fatigue, and nausea (each 47%); thromboembolic events (grades 2–5) occurred in 5 of 32 patients (16%). Safety measures included concurrent prophylactic anticoagulation with low-molecular weight heparin (LMWH). In part C, despite the safety measures implemented, a thromboembolic event occurred in one of seven patients (14%) during the SGN-2FF lead-in period. Of 28 evaluable patients in part A, 1 patient with advanced head and neck squamous cell carcinoma achieved Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 complete response (CR) and 10 (36%) had RECIST v1.1 stable disease, including 1 patient with advanced triple-negative breast cancer with 51% tumor burden reduction. SGN-2FF administration led to dose-proportional increases in exposure and PD reduction in protein fucosylation. Conclusion SGN-2FF demonstrated proof-of-mechanism and preliminary antitumor activity but was associated with thromboembolic events leading to study termination.

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1–5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations on Innovation in Small Molecules and Oligonucleotides & Mass Spec Method Development Strategies for Large Molecules Bioanalysis. Part 2 (2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) and Part 3 (New Insights in Biomarkers Assays Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in drug discovery & development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and The Gene Therapy Bioanalytical Challenges) are published in volume 11 of Bioanalysis, issues 23 and 24 (2019), respectively.

Camptothecins (CPT) are an important class of molecules that have been employed in cancer therapy for the past 20 years. These molecules interact with the DNA-topoisomerase I complex leading to double-strand breaks and cell death. While these molecules are important cancer therapies, they also induce toxicities including diarrhea and myelosuppression. Antibody-drug conjugates (ADCs) have emerged as a method to help mitigate small molecule toxicities by targeting them to tumors via conjugation to tumor-specific antibodies. Recently, encouraging clinical results have been observed with ADCs bearing CPT payloads, demonstrating a potential new utility for this class. The linker that is used to conjugate a payload to an ADC influences its activity, by controlling the identity of the molecular species that can be released, as well as efficiency of drug release. When employed as an ADC, maleimidopropionyl-PEG7-valine-lysine-glycine-7-aminomethyl-10,11-methylenedioxycamptothecin (VKG-AMDCPT), a novel, highly active and enzyme-cleavable tripeptide CPT drug-linker, releases two CPT payloads in cancer cells, as demonstrated both in vitro and in vivo. Using tandem mass spectrometry (LC-MS/MS), the two CPTs have been identified as 7-amino methyl 10,11-methylenedioxy CPT (AMDCPT) and the corresponding glycine adduct of AMDCPT (G-AMDCPT). We have characterized the efficiency of drug release and intracellular retention of the small molecules released from VKG-AMDCPT-conjugated ADCs. This was accomplished through quantification of each species released in vitro and in vivo using multiple cancer cell lines and xenografts. We report here characterization of the biochemical, cellular and bystander killing potencies of each released drug species to further understand their contributions to ADC activity. Citation Format: Julia H. Cochran, Lauren Farr, Ryan Lyski, Peter D. Senter, Scott C. Jeffrey, Shawna M. Hengel, Nicole M. Okeley. Characterization of payload release from a novel camptothecin drug-linker [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2895.

Background LIV-1, a multispan transmembrane protein and downstream target of STAT3, is highly expressed in breast cancer cells (Sussman 2014). It has been associated with lymph node involvement and linked with malignant progression to metastasis (Manning 1994). SGN-LIV1A is an anti-LIV-1 antibody conjugated via a protease-cleavable linker to monomethyl auristatin E (MMAE). Upon binding to cell-surface LIV-1, SGN-LIV1A is internalized and releases MMAE, which binds to tubulin and induces G2/M arrest and apoptosis. Methods A phase 1, open-label, dose-escalation study is ongoing to evaluate the safety, tolerability, antitumor activity, pharmacokinetics (PK), and the maximum tolerated dose (MTD) of SGN-LIV1A monotherapy (q3 wks IV) in women with LIV-1-positive, metastatic breast cancer (MBC) (NCT01969643). Patients (pts) with either hormone receptor-positive/HER2-negative (HR+/HER2–) or triple-negative (TN) disease were eligible if they had measurable disease and received ≥2 prior cytotoxic regimens in the metastatic setting. Pts with ≥ Grade 2 neuropathy were excluded. Response was assessed per RECIST v1.1, and pts with stable disease (SD) or better were eligible to continue treatment until disease progression or intolerable toxicity. Fresh tumor biopsies were obtained pre- and post-treatment to evaluate the LIV-1 expression-response relationship, mechanism of action, and tumor sensitivity to SGN-LIV1A. Results To date, 21 pts (17 HR+/HER2–, 4 TN) have received a median of 3 cycles (range, 1–7) of SGN-LIV1A at doses of 0.5–2.8 mg/kg. Median age was 58 yrs (range, 34–73), and pts had a median of 8 prior systemic metastatic therapies (range, 2–15). At baseline, 19 pts had visceral disease and 15 had bone involvement. No dose-limiting toxicities (DLTs) in Cycle 1 were observed in the 18 DLT-evaluable pts; MTD has not yet been identified. Treatment-emergent adverse events (AEs) reported in ≥30% of pts were primarily Grade 1/2 in severity and were: nausea (57%), fatigue and peripheral neuropathy (43% each), alopecia (38%), and vomiting (33%). Two pts discontinued treatment due to AEs (1 nausea, 1 tachycardia). Preliminary PK data suggest a linear increase in antibody-drug conjugate (ADC) exposure with increasing dose. In the 19 efficacy-evaluable pts, the overall response rate (ORR) was 11% (2 partial responses [PRs]) with clinical benefit of SD or better achieved in 63% (2 PR, 10 SD) of pts. Of note, all 4 pts with TN MBC achieved clinical benefit: 2 PR and 2 SD. Currently, the median duration of clinical benefit is 12.7 wks (range, 6.1–26.3). Four pts remain on treatment. To date, of the 179 MBC tumor specimens evaluated for LIV-1, 156 (87%) were LIV-1-positive; moderate-to-high LIV-1 expression (H-score ≥100) was present in 91% (94 of 103) of HR+/HER2– and 81% (47 of 58) of TN samples. Conclusions LIV-1 is expressed in the majority of metastatic breast cancer tissue samples, with moderate-to-high expression in both HR+/HER2- and TN disease. To date, SGN-LIV1A monotherapy has been generally well tolerated and resulted in a clinical benefit of SD or better in 63% of these heavily pre-treated pts, including 2 PRs in the 4 TN MBC pts. Response duration data continue to evolve. Clinical subtype-specific expansion cohorts are planned. Citation Format: Forero A, Burris III H, Mita M, Specht J, Weise A, Liu MC, Modi S, Pusztai L, Kostic A, Yang J, Li M, Hengel S, Miller K. Interim analysis of a phase 1 study of the antibody-drug conjugate SGN-LIV1A in patients with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-05.

Background: Antibody–drug conjugates (ADCs) require multiple assays to characterize their PK. These assays can separately evaluate the ADC by quantifying the antibody or the conjugated drug and may give different answers due to assay measurement differences, heterogeneous nature of ADCs and potential biotransformations that occur in vivo. Results: We present a new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs. A stable isotope-labeled internal standard, protein A affinity capture and solid-phase cleavage of MMAE using papain was used prior to LC–MS/MS analysis. Conclusion: The assay was used to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native-cysteine ADC in vivo.

Therapeutic antibody-drug conjugates (ADCs) harness the cell-killing potential of cytotoxic agents and the tumor targeting specificity of monoclonal antibodies to selectively kill tumor cells. Recent years have witnessed the development of several promising modalities that follow the same basic principles of ADC based therapies but which employ unique cytotoxic agents and conjugation strategies in order to realize therapeutic benefit. The complexity and heterogeneity of ADCs present a challenge to some of the conventional analytical methods that industry has relied upon for biologics characterization. This current review will highlight some of the more recent methodological approaches in mass spectrometry that have bridged the gap that is created when conventional analytical techniques provide an incomplete picture of ADC product quality. Specifically, we will discuss mass spectrometric approaches that preserve and/or capture information about the native structure of ADCs and provide unique insights into the higher order structure (HOS) of these therapeutic molecules.

Analysis of samples containing intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.

SGN-2FF, an orally bioavailable small molecule inhibitor of glycoprotein fucosylation, demonstrates encouraging preclinical antitumor activity in mouse models with suggested multiple mechanisms of action, including direct and indirect effects on immune cells, tumor cells, and the tumor microenvironment. The effects of SGN-2FF were evaluated on tumors implanted in multiple strains of mice to determine how differences in the immune repertoire affect the antitumor activity. SGN-2FF treatment of nude mice, which maintain functional B cells and antibody production, resulted in a delay in LS174T tumor growth compared with untreated mice, while LS174T tumors in SCID mice, which lack B cells, were unaffected by SGN-2FF. These data suggest that activity of SGN-2FF in nude mice may be dependent on residual B cells and circulating antibodies. The antitumor effect of SGN-2FF in syngeneic mouse models with intact immune systems also appears to be dependent on T cell activity. Transfer of T cells isolated from SGN-2FF-treated tumor-bearing mice to naïve tumor-bearing mice was sufficient to delay tumor growth. T cells isolated from untreated tumor-bearing mice did not have the same effect. These results demonstrate that afucosylated immune cells play a key role in the preclinical activity of SGN-2FF. Various preclinical assays were used to detect SGN-2FF-mediated changes in cellular and IgG fucosylation important for biological activity. These assays are being applied in evaluating patient samples in the ongoing phase 1, multicenter, dose-escalation study investigating the safety, tolerability, PK, and biomarkers of antitumor activity of SGN-2FF administered orally to adult patients with advanced solid tumors (NCT# 02952989). Changes in peripheral IgG fucosylation, absolute neutrophil count, and immune cell surface fucosylation were identified as initial biomarkers for proof of pharmacodynamic activity. Preliminary data following daily doses of SGN-2FF demonstrate that cell surface fucosylation on granulocytes was significantly reduced and neutrophil count was significantly increased in 6 of 7 treated subjects; additionally, IgG fucosylation was significantly decreased in 7 of 7 subjects. PK have been characterized, and preliminary results are within the expected range as predicted from preclinical studies. Following daily administration of SGN-2FF, accumulation of the active metabolite, GDP-2FF, was observed intracellularly, while no accumulation of SGN-2FF was observed in plasma. Collectively, these data demonstrate robust biological effects of SGN-2FF. The pharmacodynamic biomarkers and PK analysis are informing next steps in identifying an optimal dose and dosing schedule for SGN-2FF. Citation Format: Nicole M. Okeley, Ryan A. Heiser, Weiping Zeng, Shawna Mae Hengel, Jason Wall, Peter C. Haughney, Timothy Anthony Yap, Francisco Robert, Rachel E. Sanborn, Howard Burris, Laura Q. Chow, Khanh T. Do, Martin Gutierrez, Karen Reckamp, Amy Weise, D Ross Camidge, John Strickler, Conor Steuer, Zejing Wang, Megan M. O'Meara, Stephen C. Alley, Shyra J. Gardai. SGN-2FF: A small-molecule inhibitor of fucosylation modulates immune cell activity in preclinical models and demonstrates pharmacodynamic activity in early phase 1 analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5551.

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications, with two agents currently approved and many more in late-stage clinical trials. These ADCs employ drug-linkers that were developed many years ago, and are now recognized to have properties that may adversely impact the activity and toxicology of the ADCs prepared with them. Two such properties that are now well appreciated are the reversibility of maleimide-based drug conjugation, and the impact of drug conjugation on the pharmacokinetics of the ADC. We recently reported advances in drug-linker design that independently address both of these properties, resulting in the irreversible conjugation of drugs which have minimal impact on antibody pharmacokinetics, even at high levels of drug loading (Nature Biotechnology 32, 1059-1062 (2014), Nature Biotechnology 33, 733-735 (2015)). We have now prepared drug-linkers of monomethylauristatin E (MMAE) that orthogonally employ these features to enable a systematic evaluation of the relative contributions of maleimide instability and accelerated plasma clearance on the in vivo behavior of MMAE ADCs. Biodistribution studies with these molecules have revealed that the concentration of released MMAE in normal tissues is greatly impacted by the rate of ADC clearance (fast clearance results in greater Cmax of free drug), while stabilization of the maleimide has a relatively small effect. These differences in observed free drug concentrations were paralleled in tolerability studies, with ADC clearance rates exerting a greater impact on hematology parameters than maleimide stability. Collectively, these results suggest that ADC pharmacokinetics dominate the biodistribution and toxicology profiles for a given drug payload, with conjugate stability playing a relatively minor role. Citation Format: Haley Neff-LaFord, Franciso Zapata, Wendi Schultz, Cindy Balasubramanian, Paul Pittmen, Shawna Hengel, Russell Sanderson, Nagendra Chemuturi, Jocelyn Setter, Robert P. Lyon. Elucidating the roles of antibody pharmacokinetics and maleimide stability in the toxicology of antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 70. doi:10.1158/1538-7445.AM2017-70