Mastering Immunity. Nikolai Schwabe, CEO. Tools & Technologies for Managing Immunogenicity Risk - where does the rubber meet the road?

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1 Mastering Immunity Tools & Technologies for Managing Immunogenicity Risk - where does the rubber meet the road? Nikolai Schwabe, CEO

2 About ProImmune HQ in Oxford UK since 2000, US office since 2004 Specialist products and services for measuring immune responses Working with most industry participants, academic / govt. institutes Wide exposure to different questions, problems: Vaccines, immune oncology, infections, autoimmunity, allergy; immunogenicity of biologics: (mabs, mab derivatives, new scaffolds, viruses, oligos, peptides cell based therapies, replacement factors, conjugates, hybrids of the foregoing); small molecule immune modulators Often dealing with questions of both wanted and unwanted immune responses at the same time

3 Immunogenicity is it really a problem? (or just something you have to measure in the clinic to tick a box) Points to make: Drugs really do fail in the clinic due to immunogenicity Some marketed drugs show high rates of immunogenicity, cf. Humira, Remicade, FVIII replacements, IFN beta Current sponsor developments often focus on either: aggressive new approaches to overcome hard to treat conditions such as cancer (bi-specifics, novel scaffolds, conjugates, T cell engagers, combination of the foregoing) biosimilars : a marketplace where immunogenicity incidence may be becoming the most important product attribute And in both cases they are more often than not in the field of immune modulation, meaning either the objective is to (a) dampen down hyperimmunity (MS, RA, allergy), or (b) counteracting immune suppression, surveillance failure Observation: the commercial reality on this question is changing fundamentally on this from only a few years ago Comments made by Barbara Rellahan, FDA CDER, at NBC 2013 Aglycosylated humanized mab- 100% (6/6) of the subjects were ADA positive after single dose administration Humanized IgG1 fusion protein- ADA rate after single administration was 85% Humanized IgG2 with two point mutations in Fc-region- ADA rate after single dose administration was 20-25% Humanized IgG4 with three point mutations in Fc-region- ADA rate after single dose administration was 57% Human IgG1 from synthetic phage display library- ADA rate after multiple doses was > 80%

4 Exploring potential adaptive immune responses with in vitro assays at a pre-clinical stage (healthy donor PBMC, purified HLA) DC:T cell assays T cell proliferation assays HLA typing T FH HLA Binding Assays GC (In silico HLA binding prediction) Cytokine release assays MHC II TCR MHC II TCR (Linear) B cell epitope mapping DC uptake / maturation assays Ag. presentation assays FDC AG BCR B Ag. presentation assays Lots of other receptors everywhere, controlling many elements in the response

5 Exploring potential adaptive immune responses with in vitro assays at a pre-clinical stage (healthy donor PBMC, purified HLA) Precursor Frequency TCR recognition Clonal expansion Differentiation Migration MHC II TCR T FH MHC II TCR GC Antigen uptake Ag. processing DC maturation MHC II presentation Migration FDC AG BCR B Lots of other receptors everywhere, controlling many elements in the response Precursor Frequency Clonal BCR recognition Antigen uptake Antigen processing MHC II loading Differentiation Clonal expansion Migration

6 Exploring actual adaptive immune responses with in vitro assays on PBMC samples from treated patients Precursor Frequency TCR recognition Clonal expansion Differentiation Migration T FH MHC II TCR MHC II TCR GC Antigen uptake Ag. processing DC maturation MHC II presentation Migration FDC AG BCR B Lots of other receptors everywhere, controlling many elements in the response Precursor Freq. Clonal BCR recog. Antigen uptake Antigen processing MHC II loading Differentiation Clonal expansion Migration

7 Preliminary summary: We can measure numerous aspects of adaptive immunity at a preclinical stage, but immune regulation factors contributing to differentiation and clonal expansion of B cells in germinal centers (GCs) are still hard to work out in absolute terms, such as: Epitope specific B, T precursor frequencies Migratory and spatial factors in immune response, e.g. GC architecture and patient pathophysiology Building human artificial lymph nodes has been proposed as a solution for some of these issues, but we have yet to see any evidence that they work well However we can work with comparables in terms of mode of action, modality, route administration etc., and combine preclinical assay results with results from analysing actual immune responses in treated patients to make better development decisions going forward

8 ProPresent : measuring antigen presentation directly LC MS MS lvgsdwrflrgyhqya lvgsdwrflrgyhqyaygd L GSDWRFLRGYHQYA

9 Sample results: identification of Humira (adalimumab) peptides in subset of donors

10 Summary of identified adalimumab peptides with allele association Unique Peptides Amino Acid Start/End Protein Domain DRB1* Alleles Present with Detected Peptide APKLLIYAASTLQSGVPS Variable Light *01:01 *03:01 WNSGHIDYADSVEGRFT Variable Heavy *01:01 *04:03 *04:01 *04:03 DNAKNSLYLQMNSLRAEDTA Variable Heavy *01:01 *13:01 *13:02 *04:01 *04:03 *14:54 *15:01 *15:02 *03:01 *08:01 AKVSYLSTASSLDYWGQ Variable Heavy *04:01 10 out of 20 donors analyzed presented a portion of peptide 74-93

11 Functional confirmation of putative adalimumab epitopes CD8 + depleted PBMC from ~40 x HLA-typed individuals representing tissue type distribution in the general population: HLA-DR, -DP and DQ; specific HLA distributions can be requested Generate overlapping peptides for protein of interest: 15-mers offset by 3 amino acids; alternatively unique presented peptides identified by ProPresent can be tested Co-culture of PBMC with synthetic peptides T cell proliferationmeasured over 7 day period using CFSE flow assay + CFSE overlapping peptides PBMC

12 T cell proliferation adalimumab variable heavy chain CDR1 CDR2 CDR3 DNAKNSLYLQMNSLRAEDTA

13 ProImmune REVEAL HLA-binding assay Analysis of functional T cell epitope peptide 102 (SLYLQMNSLRAEDTA) from adalimumab Heavy Chain CDR3 Key Unique Peptides Amino Acid Start/End Protein Domain Red No Binding to MHC APKLLIYAASTLQSGVPS Variable Light Chain *01:01 *03:01 Green Stable Binding WNSGHIDYADSVEGRFT Variable Heavy Chain *01:01 *04:03 DRB1* Alleles Present with Detected Peptide *04:01 *04:03 Yellow Weak affinity binding DNAKNSLYLQMNSLRAEDTA Variable Heavy Chain *01:01 *13:01 *13:02 *04:01 *04:03 *14:54 *15:01 *15:02 *03:01 *08:01 Grey Untested / Unavailable AKVSYLSTASSLDYWGQ Variable Heavy Chain *04:01

14 Case study: F-VIII R S T U W X ProImmune REVEAL HLA binding assays used to characterize immunogenic epitopes of recombinant Factor VIII found in DR15 transgenic mouse model Steinitz, pes K. et al Blood 2012 Apr 26;119(17): [PubMedID: ] Watch complete video presentation of case study on our website Y Z

15 Validating antibody humanization with ProMap CFSE PBMC T cell assays Passive immunization anti VEEV antibody O Brien L. et al. Virology (2012); 426: Comparison of chimeric and humanized sequence to assess impact on antigenicity T cell antigenicity significantly reduced from humanized peptides

16 ProScern DC-T cell assays: whole protein analysis x HLA-typed PBMC donors representing general tissue type distribution Drug-loaded DCs are incubated with autologous CFSE-labelled PBMCs; 7d prolif. flow readout, optionally with addl. phenotyping Used as a direct comparator for formulated drugs, incl. PTMs, aggregates, excipients Verdict on total T cell antigenicity, including processing/uptake factors Can and has revealed batch differences that are not picked up by other biophysical QC methods RI = (% donors responding) x (average strength of response) / 100

17 No Tetramer DR7/PDDYSNTHSTRYVYV ProT2 Tetramer ProT2 MHC Class II Tetramers Detect and separate single antigen-specific CD4+ T cells HLA-DRB1*07:01 CMV-positive donor HLA-DRB1*07:01 CMV-negative donor CD4 ProT2 MHC Class II Tetramer CD4 Cells stained ex vivo, gated on live, CD19-CD14-CD3+ lymphocytes Data kindly provided by Prof. Paul Moss, Birmingham, UK

18 EU funded initiative involving major EU pharma, SMEs and academics with total funding of 35M; started in March 2012 to run for 5 years Objectives: several but in one aspect to try and confirm that T cell epitopes predicted and measured at pre-clinical stage correlate with observed T cell responses in patients with ADA. Summary of recent presentation by B Maillere, CEA, France Recall T cell epitopes found for both VH and VL of Remicade and Rituxan ; these bind to relevant HLA alleles in responding donors; and usually show up in antigen presentation assays

19 Binding prediction and biding assays are both over-predictive; many more sequences bind than are relevant T cell epitopes; binding assays are best used to answer HLA restriction questions in sequence regions of particular interest Antigen-presentation assays reveal key sequences (much fewer than binding assays) that may be T cell epitopes and are processed; these sequences can include sequences for which central and peripheral tolerance exists Peptide mapping T cell assays reveal sequences that are recognizable to T cells and cause T cell proliferation; responses may be seen from peptides for which no central tolerance exists because they are not naturally presented Multi-assay strategies are most powerful in pinpointing epitopes that have actual immunological relevance; epitopes that are triangulated with such strategies are likely to be confirmed in patients treated with the biologic who show a class switched antibody response to the biologic (our own experience in this area is still principally in the vaccines field) More ABIRISK data will be published over the next 24 months; our prediction is that this will confirm the trends outlined above further We need to develop a better understanding what the most relevant time window is to measure T cell responses in treated patients; Class II tetramer analysis will be very useful in answering this question parallels between ABIRISK results disclosed to date and ProImmune hands on experience

20 Summary Understanding and managing the mechanisms of molecular immunobiology in your system / technology platform / treatment modality is essential for winning in the drug markets of the future Access to a wide range of specialist assays is necessary to develop an understanding that is adequate to meet this challenge Appropriate data need to be collected across the entire drug development life-cycle (i.e. including measuring cellular responses in the clinic) to optimize the value added from assay work

21 ProImmune event video content (free educational resource on our website, no registration required) Speakers and attending organisations from past events: Arlene Sharpe Harvard Val Quarmby Genentech Larry Steinman Stanford Bonnie Rup Pfizer Strategies for assessing & managing wanted & unwanted immune responses Regulatory guidance

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