Supplementary Fig. S1 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Table S4 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Fig. S2 from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Supplementary Table S3 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Data from Intracellular Activation of SGN-35, a Potent Anti-CD30 Antibody-Drug Conjugate

Supplementary Fig. S2 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Material from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Fig. S1 from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Supplementary Material from Anti-CD30 diabody-drug conjugates with potent antitumor activity

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 14 th edition of the Workshop on Recent Issues in Bioanalysis (14 th WRIB) was held virtually on June 15–29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

Tisotumab vedotin is an antibody-drug conjugate composed of a human IgG targeting tissue factor (TF), a valine citruline linker, and the microtubule disrupting agent monomethyl auristatin E (MMAE). TF is aberrantly expressed in solid tumors and is thought to contribute to tumor progression by enhancing tumor growth, neo-angiogenesis and metastatic potential through local activation of coagulation and protease-activated receptor-2 (PAR-2) signaling. Tisotumab vedotin was previously shown to induce cytotoxicity through MMAE- and Fc-dependent mechanisms. In addition, tisotumab vedotin inhibited PAR-2 signaling in TF-positive tumor cells. Here we aimed to elucidate additional effector mechanisms by assessing the capacity of tisotumab vedotin to induce immunogenic cell death (ICD), bystander cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP) in vitro. ICD was assessed by incubating A431, MDA-MB-231, and HPAFII cells with tisotumab vedotin, and measuring ER stress by western blot, ATP secretion by reporter assay, and HMGB1 release by ELISA. Bystander cytotoxicity was assessed by incubating mixed cultures of TF+ (MDA-MB-231) and TF- (A549) tumor cells with tisotumab vedotin and quantifying viable cells by flow cytometry. To assess ADCP, PKH26-labeled BxPC-3 or A431 cells were incubated with human monocyte derived macrophages in presence of tisotumab vedotin. ADCP activity was analyzed by measuring the fraction of macrophages that double stained with PKH26 and anti-CD11c antibody using flow cytometry. Tisotumab vedotin induced key hallmarks of ICD including induction of ER stress, ATP secretion, and release of HMGB1, which have been shown to facilitate antitumor immunity. In co-cultures of TF+ and TF- tumor cells, tisotumab vedotin induced cytotoxicity in both cell types, even at a target-positive to target-negative cellular ratio of 1:15. This was mediated through bystander cytotoxicity, as demonstrated by the lack of cytotoxicity in monocultures of TF- cells. Finally, ADCP was observed with macrophages derived from multiple donors, with double-positive macrophages comprising up to 50% of total macrophages. In summary, ICD, bystander cytotoxicity, and ADCP were identified as novel effector mechanisms of tisotumab vedotin in vitro. Combining the present study with previous results, tisotumab vedotin induces TF-dependent anti-tumor activity through 1) MMAE-mediated effector mechanisms, including MMAE-mediated direct and bystander cytotoxicity, and induction of ICD; 2) Fc-mediated effector mechanisms, including ADCC and ADCP; and 3) Fab-mediated inhibition of PAR-2 dependent signaling. Tisotumab vedotin is currently being investigated in a variety of solid tumors (NCT03245736, NCT03485209, NCT03438396, NCT03657043). Citation Format: Stephen C. Alley, Jeffrey R. Harris, Anthony Cao, Elke Gresnigt-van den Heuvel, Jyoti Velayudhan, David Satijn, Sandra Verploegen, Teresa Dominguez, Esther C. Breij. Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 221.

The 2018 12(th) Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 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 and immunogenicity. 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 and LBA/cell-based assays approaches. This 2018 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 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

The antibody-drug conjugate field has made significant progress recently owing to careful optimization of several parameters, including mAb specificity, drug potency, linker technology, and the stoichiometry and placement of conjugated drugs. The underlying reason for this has been obtained in pre-clinical biodistribution and pharmacokinetics studies showing that targeted delivery leads to high intratumoral free drug concentrations, while non-target tissues are largely spared from chemotherapeutic exposure. Recent developments in the field have led to an increase in the number of ADCs being tested clinically, with 3 in late stage clinical trials: brentuximab vedotin (also referred to as SGN-35) for Hodgkin lymphoma; Trastuzumab-DM1 for breast cancer; and Inotuzumab ozogamicin for non-Hodgkin lymphoma. This review highlights the recent pre-clinical and clinical advances that have been made.

Abstract 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.

Abstract Increased fucosylation is associated with tumor progression and metastasis, and targeting fucosylation is a novel strategy in cancer therapy. 2-Fluorofucose (SGN-2FF) has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate GDP-fucose, as well as by direct inhibition of fucosyltransferases, leading to the production of afucosylated glycoproteins including antibodies. SGN-2FF has antitumor activity in multiple mouse tumor models, showing substantial tumor growth delay. SGN-2FF also enhanced the protective effect of a lymphoma vaccine in a syngeneic mouse model (1). This protection was determined to be immune dependent since depletion of CD4 and CD8 T cells reduced the SGN-2FF/vaccine activity. In vitro, SGN-2FF has been shown to activate human T cells in an antigen-dependent manner. Therefore, by inhibiting fucosylation SGN-2FF can potentially work through multiple mechanisms, including but not limited to effects on immune cells, tumor cells, and the tumor microenvironment. A first-in-human, phase 1, multicenter dose-escalation study of SGN-2FF is ongoing to investigate the safety, pharmacokinetics, and antitumor activity of SGN-2FF given orally to patients with advanced solid tumors (NCT# 02952989). Pharmacodynamic effects, including markers of fucosylation status, will also be evaluated to help determine the optimal biologic dose. This presentation reviews the preclinical activity data of SGN-2FF and describes the design of the phase 1 study, showing how demonstrated preclinical pharmacodynamic effects on fucosylation status informed how activity and pharmacodynamics will be monitored and evaluated in the phase 1 study. Reference 1. Okeley NM, Alley SC, Anderson ME, Boursalian TE, Burke PJ, Emmerton KM, et al. Development of orally active inhibitors of protein and cellular fucosylation. Proc Natl Acad Sci U S A 2013;110:5404-9. Citation Format: Stephen C. Alley, Megan O'Meara, Shyra J. Gardai, Nicole M. Okeley. SGN-2FF: A novel small molecule inhibitor of fucosylation with preclinical antitumor activity through multiple immune mechanisms [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 DDT02-02. doi:10.1158/1538-7445.AM2017-DDT02-02

Increased fucosylation is associated with tumor progression and metastasis, and targeting fucosylation is a novel strategy in cancer therapy. 2-Fluorofucose (SGN-2FF) has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate GDP-fucose, as well as by direct inhibition of fucosyltransferases, leading to the production of afucosylated glycoproteins including antibodies. SGN-2FF has antitumor activity in multiple mouse tumor models, showing substantial tumor growth delay. SGN-2FF also enhanced the protective effect of a lymphoma vaccine in a syngeneic mouse model (1). This protection was determined to be immune dependent since depletion of CD4 and CD8 T cells reduced the SGN-2FF/vaccine activity. In vitro, SGN-2FF has been shown to activate human T cells in an antigen-dependent manner. Therefore, by inhibiting fucosylation SGN-2FF can potentially work through multiple mechanisms, including but not limited to effects on immune cells, tumor cells, and the tumor microenvironment. A first-in-human, phase 1, multicenter dose-escalation study of SGN-2FF is ongoing to investigate the safety, pharmacokinetics, and antitumor activity of SGN-2FF given orally to patients with advanced solid tumors (NCT# 02952989). Pharmacodynamic effects, including markers of fucosylation status, will also be evaluated to help determine the optimal biologic dose. This presentation reviews the preclinical activity data of SGN-2FF and describes the design of the phase 1 study, showing how demonstrated preclinical pharmacodynamic effects on fucosylation status informed how activity and pharmacodynamics will be monitored and evaluated in the phase 1 study. Reference 1. Okeley NM, Alley SC, Anderson ME, Boursalian TE, Burke PJ, Emmerton KM, et al. Development of orally active inhibitors of protein and cellular fucosylation. Proc Natl Acad Sci U S A 2013;110:5404-9. Citation Format: Stephen C. Alley, Megan O9Meara, Shyra J. Gardai, Nicole M. Okeley. SGN-2FF: A novel small molecule inhibitor of fucosylation with preclinical antitumor activity through multiple immune mechanisms [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 DDT02-02. doi:10.1158/1538-7445.AM2017-DDT02-02

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. 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 analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies' Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.

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.