Anti-idiotype Antibody Generation and Application in Antibody Drug Discovery Liusong Yin, PhD Senior Scientist, Group Leader Antibody Discovery, Antibody Department, GenScript [email protected] Apr 21st, 2016 Presentation Overview Anti-idiotype antibody introduction 1 2 Anti-idiotype antibody application 3 Anti-idiotype antibody development 4 Anti-idiotype antibody case study Make Research Easy 2 Structural overview of antibodies PDB ID: 1HZH Liusong Yin, 2014, A Dissertation Make Research Easy 3 Antibody ‘-types’ Isotype (species specific)– the phenotypic variations in the constant regions of the heavy and light chains Allotype (animal specific)– the genetically determined difference in antibodies between individuals in the same species, mainly a couple AA differences in constant region Idiotype (antigen specific)– the antigen binding specificity defined by the distinctive sequence in the variable region of antibodies Make Research Easy 4 ‘-topes’ in anti-idiotype antibodies (anti-IDs) Idiotope – the antigenic determinants in or close to the complementarity determining region (CDR) in variable region Epitope Paratope Paratope – the part of an Ab that recognizes an antigen, the antigen-binding site of an Ab Epitope – the part of the antigen to which the paratope binds Anti-IDs – anti-idiotype antibodies which recognize the shared feature of idiotopes Make Research Easy 5 Different types of Anti-IDs Antigen-blocking Non-blocking Complex-specific Anti-ID Drug Target Anti-ID Anti-ID Antibody drug Antibody drug Antibody drug • Paratope-specific • Not paratope-specific • Drug-target complex • Inhibitory • Not inhibitory specific • Neutralizing • Detects total drug • Not inhibitory • Detects free drug (free, partially bound, • Detects bound drug fully bound) only Make Research Easy 6 Presentation Overview Anti-idiotype antibody introduction 1 2 Anti-idiotype antibody application 3 Anti-idiotype antibody development 4 Anti-idiotype antibody case study Make Research Easy 7 Applications of Anti-IDs 1 Pharmacokinetic (PK) Assays • Used to measure the antibody drug level in patient samples 2 Immunogenicity (anti-drug antibody) Assays • Used as a positive control or reference standard for ADA measurement Make Research Easy 8 Antibody drug market Gary Walsh, 2014, Nature Biotechnology Make Research Easy 9 Pharmacokinetic (PK) Assays Purpose: Detect and quantitate human Ab drugs in serum Human serum contains 5-9.5 mg/ml IgG1 Required sensitivity for Ab drug is in the ng/ml range Thus, assays must accurately detect Ab drug despite a million-fold excess of similar molecules Possible agents for detection include: • Antigen (i.e. drug target) • Anti-ID antibodies Make Research Easy 10 Anti-IDs for detection of Ab drugs Possible binding modes of anti-IDs: • Complex-specific anti-IDs detect bound Ab drugs directly • Antigen-blocking (neutralizing) anti-IDs detect free drug • Non-blocking (non-inhibitory) anti-IDs detect free and bound drug Antigen-blocking Non-blocking Complex-specific Anti-ID Drug Target Anti-ID Anti-ID Antibody drug Antibody drug Make Research Easy 11 Typical PK Assay Formats 1 Antigen Capture 2 Anti-ID-Bridging Anti-ID (labeled) Anti-Fc (labeled) Antibody drug (in human serum) Antibody drug (in human serum) Anti-ID Antigen 3 Anti-ID Capture Sandwich 4 Anti-ID-Antigen-Bridging Anti-Fc (labeled) Antigen (labeled) Antibody drug (in human serum) Antibody drug (in human serum) Anti-ID Anti-ID Make Research Easy 12 Immunogenicity of antibody drugs Liusong Yin, 2015, J. Immunology Research Make Research Easy 13 Immunogenicity of FDA Approved mAbs Product Form Target Indication Company Approval OKT3 (86%) Murine IgG2a K CD3 Graft reject Ortho Biotech 1986 ReoPro (6-44%) Chimeric Fab GP IIb/IIIaR Angioplasty Centocor 1994 Rituxan (1%, 3/356) Chimeric IgG1 K CD20 NHL Genentech 1997 Zenapax (14-34%) Humanized IgG1 K CD25 Graft reject Roche 1997 Remicade (10%) Chimeric IgG1 K TNF RA,CD Centocor 1998 Simulect (4/339, 2/133) Chimeric IgG1 K CD25 Graft reject Novartis 1998 Herceptin (<1%, 1/903) Humanized IgG1 K HER-2/NEU Breast cancer Genentech 1998 Synagis (1-2%) Humanized IgG1 K RSV RSV Medimmune 1998 Mylotarg (0%, 0/277) Humanized IgG4 K CD33 AML Wyeth 2000 Campath (2%, 4/211) Humanized IgG1 K CD52 CLL ILEX 2001 Zevalin (4%, 8/211) Murine IgG1 K CD20 NHL IDEC 2002 Xolair (<0.1%, 1/1723) Humanized IgG1 K IgE Asthma Genentech 2003 Bexxar (99%, 219/220) Murine IgG2a K CD20 NHL Corixa 2003 Raptiva (6.3%, 67/1063) Humanized IgG1 CD11a Psoriasis Genentech 2003 Erbitux (5%, 28/530) Chimeric IgG1 K EGF-R Colorectal cancer Imclone 2004 Avastin (0/500) Humanized IgG1 K VEGF Colorectal cancer Genentech 2004 Tysabri (10%) Humanized IgG4 K A4-integrin MS Biogen/IDEC 2004 Vectibix (0.3-4%) Human IgG2 K EGF-R Colorectal cancer Amgen 2006 Humira (5%, 58/1062) Human IgG1 K TNF RA Abbott 2002 Make Research Easy 14 Immunogenicity Assay Overview of Design Elements 1. FDA recommends a multi-tiered approach • Tier 1: A rapid, sensitive screening assay • Tier 2: Positive should then be subjected to a confirmatory assay, such as ligand or antigen competition assay • Tier 3: Further characterization: neutralization/class/isotype/titer 2. Aspects of Assay Development: • Highly sensitive • Able to detect all isotypes (IgM, IgE and IgG subtypes); carefully consider the avidity of control used to evaluate the assay; Should conduct assay performance test in the same concentration of matrix as that used to assess patient samples. • Rabbit or monkey will be the recommended host animals. • Detection of anti-idiotype antibodies is the most important assay validation for immunogenicity. • Establish negative control with a pool of sera from 5-10 non- exposed individuals Make Research Easy 15 Most common assay for immunogenicity Direct Binding ELISA Bridging ELISA Antibody drug (labeled) Anti-Hu IgG Fc (labeled) Anti-drug antibody Anti-drug antibody Antibody drug Antibody drug Anti-ID (mAb or pAb) serve as the best positive controls for Immunogenicity assay development Make Research Easy 16 Typical immunogenicity assay formats Direct and Bridging ELISA assays • Advantages: Sensitive, inexpensive, equipment readily available • Disadvantages: May not detect early immune response and may be influenced by high levels of circulating drug; false positives Radio-immuno precipitation assay (RIPA) • Advantages: sensitive, inexpensive, equipment readily available • Disadvantages: May not detect early immune response and may be influenced by high levels of circulating drug Surface Plasmon Resonance (SPR) • Advantages: Method of choice for detecting early immune response and has Ab characterization capabilities • Disadvantages: Expensive equipment, false positives Electrochemiluminescence (ECL) assay • Advantages: Sensitive, can be modified to respond in the presence of high levels of circulating drug • Disadvantages: Equipment can be expensive, may not easily detect rapidly dissociating Abs Make Research Easy 17 Presentation Overview Anti-idiotype antibody introduction 1 2 Anti-idiotype antibody application 3 Anti-idiotype antibody development 4 Anti-idiotype antibody case study Make Research Easy 18 Anti-IDs: pAb or mAb Anti-ID pAb (Immunogenicity studies) • Advantages: a heterogeneous population which mimic the real ADA incidence in human serum; less expensive • Disadvantages: heterogeneous; batch variability Anti-ID mAb (PK studies) • Advantages: homogenous; defined specificity • Disadvantages: May not represent the real ADA incidence in human serum; more expensive Make Research Easy 19 Challenges in anti-IDs development 1) CDR region of Ab may not be very immunogenic • Antigen immunogenicity: The ability of a particular molecule to elicit an immune response determined by whether the immune system can recognize the antigen 2) Percentage in antiserum very low • Most Abs raised target the Fc region • Anti-ID clone may be missed if conventional screening methods are used 3) Must optimize clone selection from numerous anti-IDs Make Research Easy 20 Overcoming the obstacles at GenScript 1) CDR region of Ab may not be very immunogenic Solution Use antibody drug-KLH conjugate as immunogen Use F(ab)2 (fragment, antigen binding) as immunogen • Remove Fc-specific antibodies Use our proprietary immunoadjuvant Adjust immunization schedule Consider different animal host Make Research Easy 21 Overcoming the obstacles at GenScript 2) Low percentage of anti-IDs in serum Solution Use proper secondary Ab (anti-mouse Fc to prevent cross- reaction with human Ig) HTP binding screening Capture ELISA to select natural epitope recognized by Ab Make Research Easy 22 Overcoming the obstacles at GenScript 3) Optimize clone selection from numerous anti-IDs Solution Epitope binning (ELISA or SPR-based) Affinity ranking (ELISA EC50, Koff and Kd ranking) Antibody pairing (for PK study) Antigen blocking (different binding modes) Make Research Easy 23 Flow chart of GenScript anti-IDs packages Services Anti-ID monoclonal antibody Anti-ID polyclonal antibody Starting material Target antibody drug 2-3mg Target antibody drug 20 mg or more Work flow QC Cross-reactivity with control IgG <10% Cross-reactivity with control IgG <10% Hybridoma cell lines, supernatants and Deliverables 0.5-3mg purified anti-ID antibody/rabbit purified anti-ID antibody (optional) Make Research Easy 24 Presentation Overview Anti-idiotype antibody introduction 1 2 Anti-idiotype antibody application 3 Anti-idiotype antibody development 4 Anti-idiotype antibody case study Make Research Easy 25 Case Study 1: Blocking and non-blocking Anti-IDs Antibody drug: Keytruda (commercial anti-PD1 pembrolizumab) Application: Currently in developing ELISA kit for Keytruda PK study 1 2 0 [PD-1, mg/ml] 0 1 0 0 0 .1 1 ) % 1 0 ( 8 0 g n i d n i 6 0 B e v i t a l 4 0 e R 2 0 0 mAb1 mAb2 mAb3 mAb4 Non-blocking Non-blocking Blocking Blocking Make Research Easy 26 Case Study 2: Anti-IDs with high specificity Antibody drug: humanized Ab Application: PK study 3 2.5 No.1 No.2 2 No.3 No.4 1.5 No.5 No.6 No.7 1 No.8 No.9 0.5 No.10 0 Antibody drug Human IgG1 Normal IgG Human serum Final sub-clones with high specificity to humanized Ab when screened against isotype control, normal human IgG and human serum.