IMMUNOGENICITY ASSESSMENT FOR ANTIBODY DRUG CONJUGATES AND CONSIDERATIONS FOR ANALYTICAL ASSAYS

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1 IMMUNOGENICITY ASSESSMENT FOR ANTIBODY DRUG CONJUGATES AND CONSIDERATIONS FOR ANALYTICAL ASSAYS James Munday Senior Study Director Copyright 2016 Covance. All Rights Reserved

2 Agenda Background for immunogenicity considerations Impact of Immunogenicity on ADCs Additional analytical considerations for ADCs Immunogenic epitope mapping for understanding ADC mode of action Utilising a broad array of bioanalytical assays to derisk your ADC development program 2 6th World ADC Summit, Berlin February 9th, 2016

3 Background For Immunogenicity Considerations 3 6th World ADC Summit, Berlin February 9th, 2016

4 Immunogenicity Of Therapeutic Proteins It has been found that nearly all recombinant therapeutic proteins (or biotherapeutic) are immunogenic and can lead to the production of antibodies (Abs) Immunogenicity of a biotherapeutic is a function of it s foreigness to the recipient Immunogenicity can be either desired or undesired Immunogenicity is desired in the case of vaccine administration Immunogenicity is a major safety concern in the case of largemolecule therapeutic administration (Safety Immunogenicity) Testing during pre-clinical and clinical programs is required 4 6th World ADC Summit, Berlin February 9th, 2016

5 Immunogenicity Overview For the humoral response > the ability of an antigen to induce antibodies that are specific to an antigen Antigen Presenting Cell Antigen T-Helper Cell Plasma B-Cell Naive B-Cell Memory B-Cell ADC can influence immunogenicity by being a target antigen or by targeting immune effector cells 5 6th World ADC Summit, Berlin February 9th, 2016

6 Immunogenicity Considerations For Biotherapeutics Biotherapeutics (biologics) are typically large complex molecules generated by living organisms and therefore are sensitive to multiple factors and changes in the manufacturing process Factors Influencing Immunogenicity Product related Factors Glycosylation Aggregation / protein folding Level of impurities Formulation Potency Patient related Factors Route of Administration Dose and length of treatment Patient factors 6 6th World ADC Summit, Berlin February 9th, 2016

7 Impact Of Immunogenicity On ADCs 7 6th World ADC Summit, Berlin February 9th, 2016

8 Specific ADC Components That Can Be Affected By Immunogenicity Monoclonal Antibody Immunogenic responses that have biological impact: ADA to CDR > Neutralise antigen binding ADA to FcgammaR > Inhibit ADCC or CDC ADA to FcRn > Inhibit exposure Cytotoxic Agent Immunogenic responses that have biological impact: ADC reduce immune response by killing antigen presenting cells ADA may reduce efficacy ADA may result in immune complex Linker Immunogenic responses that have biological impact: ADA may reduce efficacy May lead to epitope spreading ADA may result in immune complex 8 6th World ADC Summit, Berlin February 9th, 2016

9 What Influence Do ADAs Have On ADC Mode Of Action? 9 6th World ADC Summit, Berlin February 9th, 2016 ADC Mode of Action 1. Cell binding 2. Internalisation 3. Toxin release 4. Cell death

10 Interpretation Of Immunogenicity Immunogenicity must be considered with other endpoints ADA onset of immune response & kinetics PK.pharmacokinetics (drug exposure) PD.pharmacodynamics (drug activity/efficacy) AE.adverse events/clinical observations (drug toxicity).all are important when interpreting study results! ADA + PK + PD + AE = Immunogenicity Assessment 10 6th World ADC Summit, Berlin February 9th, 2016

11 Animal Data Do Not Always Predict Clinical Outcomes, But Is Still Of Value For immunogenicity assessment in pre-clinical, the main purpose is to confirm appropriate interpretation of PK and PD In general the greater the difference between the foreign protein and the human protein sequence, the more immunogenic the foreign protein will be ADC reagents can introduce neo-epitopes ADC regents can introduce immune complex formation Work done in Pre-clinical to develop bioanalytical assays can be helpful for clinical program 11 6th World ADC Summit, Berlin February 9th, 2016

12 Additional Analytical Considerations For ADCs 12 6th World ADC Summit, Berlin February 9th, 2016

13 Additional Analytical Considerations For Nonclinical Safety Assessment Of ADCs Analytical consideration Small molecules Large Molecules Manufacturing Chemical synthesis Biologically-derived Toxicity On- and off-target Generally low toxicity due to selectivity; Pharmacology can be more complex ADCs (Combination of Large and Small) Biologically derived + conjugation from chemical synthesis Ag-independent, on- and off- target Pharmacokinetics Short half-life High Volume of distribution Long half-life Lower Volume of distribution as target dependent Long Ab half-life, toxin: rapid clearance Lower Volume of distribution due to target dependence PK assays Total drug product and metabolites Total Ab Conjugated Drug (ADC), Total Antibody DAR=0, unconjugated toxin Immunogenicity assays No Yes Yes 13 6th World ADC Summit, Berlin February 9th, 2016

14 Technology Platforms Applicable For ADC Bioanalysis Platform Application Assay Photometric or Fluorometric plate reader Standard ELISA PK/ADA MSD Sector Imager ECL, Multiplex PK/ADA Gyrolab Platform Automated LBA PK/ADA LC-MS/MS Small and Large molecule analysis PK 14 6th World ADC Summit, Berlin February 9th, 2016

15 Specific Considerations For Bioanalysis Of ADCs Conjugated Antibody (ADC) DAR 1 Unconjugated Drug Total Antibody DAR 0 Drug Drug exposure exposure Biotherapeutic ADCs ADA Anti-Drug Immunogenicity Antibody Epitope Mapping NAb th World ADC Summit, Berlin February 9th, 2016

16 Immunogenic Epitope Mapping For Understanding ADC Mode of Action 16 6th World ADC Summit, Berlin February 9th, 2016

17 Strategies For Epitope Mapping Competition Assay Ru Ru No signal Biotin Streptavidin Plate Biotin Streptavidin Plate Screen assay Specificity for unconjugated antibody 17 6th World ADC Summit, Berlin February 9th, 2016

18 Strategies For Epitope Mapping Detection Assay Labelled Linker Ru Labelled Unconjugated MAb Ru ADC ADC Biotin Streptavidin Plate Biotin Streptavidin Plate Detection of unconjugated MAb Detection of Linker 18 6th World ADC Summit, Berlin February 9th, 2016

19 Example Triage Of ADA Data For ADC Antibodies Screening tier 1 Identify all Antibody responses Screening assay for Total drug Negative No further analysis Positive Confirmatory tier 2 Identify Antibody drug response for total drug Confirmatory assay for Total drug Negative No further analysis Positive Confirmatory tier 3 Identify ADA epitopes Confirmatory assay for epitopes Negative Linker Positive Unconjugated Mab Negative Positive 19 6th World ADC Summit, Berlin February 9th, 2016

20 Utilising a Broad Array Of Bioanalytical Assays to De-risk Your ADC Development Program 20 6th World ADC Summit, Berlin February 9th, 2016

21 What Can a Broad Array of Analytical Assays Reveal About ADC Mode of Action? Mode of Action Cell binding Internalisation Toxin release Cell death Nab (cell death) +ve result Nab (cell death) -ve result ADA (Total ADC) +ve ADA (Total ADC) -ve Can not differentiate MOA If neg result could still have ADA binding Can not differentiate MOA No ADA Can not differentiate MOA If neg result could still have ADA binding Can not differentiate MOA Can not differentiate MOA If neg result could still have ADA binding Can not differentiate MOA Some aspect of MOA is affected No affect on efficacy but could miss immune complex formation Can not differentiate MOA No ADA No ADA No ADA ADA (Unconjugated) +ve Most likely involve in cell binding No affect on linker No affect on toxin No cell death due to not getting into cell ADA (Unconjugated) - ve Cell binding not affected Possible affect on linker Possible affect on toxin No affect on efficacy if Total ADC also neg ADA (Toxin/Linker) +ve Cell binding affected by complex formation Internalisation affected by complex formation Toxin affected by complex formation No efficacy due to complex formation ADA (Toxin/Linker) -ve If neg result could still have ADA affecting binding No affect on linker/toxin No affect on linker/toxin No affect on efficacy if Unconjugated ADA also neg 21 6th World ADC Summit, Berlin February 9th, 2016

22 Concentration mg/ml ADC Bioanalytical PK Assay Data For Pre-clinical Development Assay Purpose Technology PK Total Antibody, DAR = 0 PK Conjugated Antibody (ADC), DAR 1 PK Unconjugated Drug Assess overall Antibody PK behaviour Assess level of active drug Free drug level Ligand Binding Ligand Binding LC-MS/MS Example data (Covance): Total Antibody ADC Toxin th World ADC Summit, Berlin February 9th, 2016 Days

23 Ways To Mitigate Risks Of ADC Development Program Build a panel of analytical assays to fully understand the mode of action: Full Bio CMC data package (Physiochemical characterisation) Full Pharmacokinetic characterisation (ADC, Unconjugated and toxin) Full immunogenicity package with possible epitope mapping 23 6th World ADC Summit, Berlin February 9th, 2016

24 Conclusion No single package can be applied to all ADCs (diverse molecules: mabs, linkers, payloads) Bespoke approaches necessary to answer specific questions Understanding the impact of immunogenicity on the mode of action can de-risk the development pathway Broad array of assays in the analytical toolbox 24 6th World ADC Summit, Berlin February 9th, 2016

25 Covance Inc., headquartered in Princeton, NJ, USA, is the drug development business of Laboratory Corporation of America Holdings (LabCorp). COVANCE is a registered trademark and the marketing name for Covance Inc. and its subsidiaries around the world. 25 6th World ADC Summit, Berlin February 9th, 2016