Development and Characterization of Multi-Platform Antibody Drug Conjugates

Transcription

1 Development and Characterization of Multi-Platform Antibody Drug Conjugates Jason A. Starkey Senior Principal Scientist - BioTherapeutics Pharmaceutical Sciences Pfizer, Inc. This document provides an outline of a presentation and is incomplete without the accompanying oral commentary and discussion. Conclusions and/ or potential strategies contained herein are NOT necessarily endorsed by Pfizer management. Any implied strategy herein would be subject to management, regulatory and legal review and approval before implementation.

2 ADCs Approved and in Clinical Development FDA Approvals Tomorrow Seattle Genetics Anti-CD30 mab conjugated with vcmmae Pfizer Approved in 2000 for AML Anti-CD33 conjugated to calicheamicin Subsequently withdrawn from US in 2010 Available in Japan C&E News 92:3, 13-21(2014) 2

3 What is an Antibody Drug Conjugate? Antibody: Drug: Linker: Monoclonal antibody (mab) or related molecule (e.g. Fc fusion protein) specific to a cell surface tumor antigen/protein Abundant target expression and internalization Often highly potent small molecule drug/toxin with validated antitumor/cytotoxic mechanism of action (e.g. microtubule inhibition, DNA damage) Tethers the drug/toxin to the antibody Clin Cancer Res 17, (2011) Stable in plasma, labile upon internalization to release drug 3 3

4 Antibody-Drug Conjugate Concept Goals Improve selectivity Improve efficacy Decrease systemic toxicity Improve therapeutic index 4

5 Examples of ADC Structures (Shaded: bonds lending to drug release) 5 Wu and Senter Nature Biotech. 23:9, (2005)

6 Common ADC Conjugation Chemistries Lysine conjugation Cysteine conjugation Site-specific conjugation Potential impact of new technologies (e.g site specific conjugation) Controlled drug loading, eliminate mixtures May improve in pharmaceutical properties May simplify analytics and development, and present newer challenges Junutula, J. R et al Nat Biotechnol, 26, (2008) Strop, P, et al Chem Biol, 20, (2013) 6

7 Unique Regulatory Space of ADCs EMA: EMEA/CHMP/BWP/157653/2007 Guideline on the Development, Production Characterization and Specifications for Monoclonal Antibodies and Related Products FDA: Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use, 1997 Unique aspects of ADCs include: Characterization of each individual component (mab, linker, drug) Understanding of potency pre- and post-conjugation Understanding of product-related impurities pre- and post-conjugation Understanding of the amount of free vs conjugated mab Understanding of the amount of free drug and related species Number LP attached to mab and sites of conjugation 7

8 Early Stage Testing Strategy for mab and ADC DS/DP - Release/Stability Test Methods Test Relevant To Test Relevant To Appearance and Description mab/ds/dp Safety (Bioburden or Sterility) mab/ds/dp ph Protein Content (UV) Drug loading and drug profile (HIC, UV, ice or RP HPLC) mab/ds/dp mab/ds/dp DS/DP Charge profiles 1 mab/ds 1 /DP 1 Identity (peptide mapping) Purity/Impurity (SEC-HPLC) Purity/Impurity (CGE or SDS-CE) Impurity: free drug & related/ free antibody mab/ds/dp mab/ds/dp mab/ds/dp DS/DP Biological Activity (Binding ELISA) mab/ds 2 /DP 2 Safety (Endotoxin) Uniformity of Dosage Units Reconstitution Time (for Lyo) Residual Moisture (for Lyo) Particulate Matter (Subvisible particles) Impurities: Residual Host Cell Protein, rprotein A (ELISAs) & DNA N-linked Oligosaccharide profile (Glycan fingerprint) Osmolality mab/ds/dp DP DP DP DP mab mab DS/DP Biological Activity (Cytotoxicity) DS/DP 1 charge profiles may not be as meaningful for Lys conjugates 2 may be replaced by cytotoxicity 8

9 Development Considerations - Unconjugated Antibody Unconjugated antibody is a recognized attribute that reflects process consistency capabilities 25% 25% 20% 20% 15% 15% 10% 10% 5% 5% 0% % Drug Load Analytical methods for unconjugated antibody have evolved significantly since the first ADC licensure Capillary electrophoresis for charge distribution of lysine-based conjugates HIC for cysteine-based conjugates Drug Load Mass Spectrometry as an orthogonal characterization technique 9

10 AU Drug Load Profile by HIC - Cysteine Conjugates DL0 DL2 DL4a DL4b DL6 Cysteine Chemistry Minutes DL2 Site Specific Conjugation DL0 DL1 HIC is a powerful tool for determining drug load profiles in ADCs 10

11 low conjugated fractions Drug Load Profile by HIC - Lysine Conjugates Lysine Chemistry Heterogenous conjugation at various sites and not in sets, like cysteine conjugation 11

12 Absorbance Absorbance Absorbance Absorbance Absorbance Absorbance pi Characterization of Drug Load Distribution - Lysine Conjugation HIC and ice HIC D5 D ADC + mab ice D0 D4 D pi D pi 0.20 D ice D7 D5 D pi pi pi D8 D7

13 Cytotoxicity Assay for ADC Drug Substance and Drug Product Assay format Plate tumor cells in assay plate Serially dilute ADC in dilution plate Transfer from dilution plate to assay plate Incubate ~72 hrs in 37 C in 5% CO 2 incubator Add Cell Titer-Glo, and lyse cells. Cell Titer-Glo generates a luminescent response directly proportional to the amount of ATP present. mab target antigen payload Qualification parameters evaluated: Plate bias Precision Accuracy Range Linearity Specificity/selectivity Representative Standard Curve ADC Tumor cell tubulin Payload inhibits tubulin polymerization, inhibiting cell division and results in decreased ATP levels. ADC concentration Cell number ATP Luminescence ADC concentration Cell number ATP Luminescence 13

14 Cytotoxicity Assay Specifications Strategy Specification ranges are initially established based on bioassay variability and not process variability. Cytotoxicity assay is one of many analytical release and/or characterization tools used to define a product. Understand clinical dosing escalation plans in order to determine what range cytotoxicity specification needs to be within. Initially set assay specification wide with tighter limits to allow investigation into out-of-limit results and further understand assay variability Based on assay and process variability, design assay to support tighter specification if able Clinical development timelines and assay development timelines are challenging to align. Requires early discussions on clinical plan, early start of cytotoxicity assay development & more resources to develop and characterize assay. 14

15 Characterization Strategy for mab and ADC DS/DP Roadmap for ADCs in Early Development "initial characterization and process development support: MS analyses: Intact protein mass Subunit/domain isoforms Peptide map sites Biochemical analyses: Drug loading profile (HIC) Drug-mAb ratio (DAR) Free drug Size heterogeneity Charge heterogeneity N-glycan profiling (for mab DSI only) Reference Material Characterization MS analyses: Intact protein mass Subunit/domain isoforms Peptide map sites Biochemical analyses: Drug loading profile (HIC) Drug-mAb ratio (DAR) Free drug Size heterogeneity Charge heterogeneity Free sulfhydryl analysis Biophysical analyses: 2º and 3º structure 15

16 AU AU AU Fd (1) Fd (2) Fd (2) Fd (3) AU AU Summary of LC/MS Subunit Analyses for ADCs Comparison of Characterization Methods Cys Chemistry: 1. ADC is IdeS digested & reduced 2. Chromatographic profile shows extent of conjugation in L chain and Fd Lys Chemistry: scfc L Chain Fd Minutes 1. ADC is reduced & alkylated L Chain 2/3 ADC 3 2. Acidic ph quantitatively cleaves /4 hydrazone bond in linker 1 3. H chain is fully conjugated at known 0 H Chain 4/5 Fc sites; trace amts. in L chain Site-specific Chemistry: 1. ADC is IdeS digested & reduced 2. Fc region is essentially fully conjugated via quantitative chromatographic shift Intens. [mau] Intens. [mau] scfc scfc D/P scfc L Chain L Chain(1) Fd Minutes LC LC Fd (1) scfc D/P +vc0101 XXXX scfc+vc0101 pq-fd Fd pq-fd mab ADC mab mab ADC ADC 0 LC- H20 Fd Time [min]

17 LC/MS Peptide Mapping (Reduced / Alkylated / Lys-C) Identify Lys Conjugation Sites, Molecular Integrity & Conjugation Extent mab-calicheamicin = conjugated peptide mab Identify the sites of conjugation Qualitatively determine the relative abundance of conjugation at each site 17

18 AU H12 (1) L15 (1) H13H14 (1) H13H14 (1) H13H14 (2) L7 H13H14 H15 L8 H4 H28 H1 AU H27 H13H14 G238R H13H14 H7 LC/MS Peptide Mapping (Reduced / Alkylated / Lys-C) Identify Cys Conjugation Sites, Molecular Integrity & Conjugation Extent mab L5 H7H H17H18 (Nglycan), H H27 D H15 Ox H1 +VHS Cys Chemistry ADC Minutes L15: SFNRGEC H12: SCDK H13H14: THTCPPCPAPELLGGPSVFLFPPKPK All peptide mass measurements are less than 2 ppm. 18 Numbers in ( ) indicate number of linker-payloads conjugated

19 Summary Pfizer has significant experience with various Antibody Drug Conjugate (ADC) technologies, and a portfolio that has evolved over the years Analytics are a key component to the advancement of an ADC development program. The evolution over time of analytical procedures, approaches, and technologies has greatly increased our capabilities Our ability to better characterize ADCs enhances our capability to effectively design compounds and develop robust manufacturing processes 19

20 Acknowledgements Pfizer BioTherapeutics Pharm Sci ARD Rob Morrison, Heyi Li, April Xu, Debbie Meyer, Jeff Borgmeyer, Jim Mo, Meg Ruesch, Jason Rouse, Tom Porter, Marta Czupryn, Ned Mozier, Steve Max, Olga Friese, Laura Bass Pfizer GCMC Leslie Bloom, Jackie Moxham, Kristin Murray, Stephanie Pluschkell, Mark Tardie, Ann Subashi, Tish Webber Pfizer PPMT Steve Max Pfizer Oncology Research Unit Puja Supra 20