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Séminaire de Communication Scientifique Faculté de Pharmacie de Lille 20 février 2014 DESPRES Chloé NOURRY Sandra OLIVIER Agathe IMMUNOGENICITY OF BIOLOGICS Prediction tools & Risks assessment CSA Biopharm 1 CSA Biopharm CEO Chief Medical Officer Chief Scientic Officer Goal : to develop the pipeline in biotechnologies Just discovered a new mouse mAb for Alzheimer’s disease 2 CSA Biopharm CSA Biopharm CEO Chief Medical Officer Chief Scientic Officer We want to be sure there will be no immunogenicity problems that could stop the development during the clinical trials and later. 3 CSA Biopharm Humanization 4 CSA Biopharm Humanization : HOW ? Goal : Human antibody (80%) : reduce immunogenicity • • • AHAM : anticorps humains anti-anticorps murins AHAC : anti-corps humains anti-anticorps chimériques AHAH : anticorps humains anti-anticorps humanisés 5 CSA Biopharm Examples Antibody name Company Type Target Indication(s) Reported immunogenicity Infliximab (REMICADE) Centocor (J&J) Chimeric TNFα RA/Crohn 10-15% Panitumumab (VECTIBIX) Amgen Human EGFR Colorectal Cancer 4,6% Ustekinumab (STELARA) Centocor (J&J) Human IL12-IL23 Plaque Psoriasis 3-5% TNFα RA/Crohn/ Ankylosing spondylitis/ plaque psoriasis… 2.6%–26% Adalimumab (HUMIRA) Abbott Human 6 Matthew P Baker et Al. Immunogenicity of protein therapeutics:The key causes, consequences and challenges. Self/Nonself 1:4, 314-322; October/November/December 2010 © 2010 Landes Bioscience Humanization : HOW ? Goal : - Murines CDR (20%) : responsible for the desired binding properties Risk : - Decrease of affinity and activity. - Creation of new epitope T at junctions and induced immunogenicity and side effects. 7 CSA Biopharm Humanization : illustrating Not just insert murines CDR 8 CSA Biopharm Examples Antibody name Company Type Target Indication(s) Reported immunogenicity Infliximab (REMICADE) Centocor (J&J) Chimeric TNFα RA/Crohn 10-15% Panitumumab (VECTIBIX) Amgen Human EGFR Colorectal Cancer 4,6% Ustekinumab (STELARA) Centocor (J&J) Human IL12-IL23 Plaque Psoriasis 3-5% TNFα RA/Crohn/ Ankylosing spondylitis/ plaque psoriasis… 2.6%–26% Adalimumab (HUMIRA) Abbott Human 9 Matthew P Baker et Al. Immunogenicity of protein therapeutics:The key causes, consequences and challenges. Self/Nonself 1:4, 314-322; October/November/December 2010 © 2010 Landes Bioscience Immune Response to Therapeutic Protein : Why is it important ? Development of Anti-Drug Antibody (ADA) Affect both efficacy and safety of the product 1. Result in reduced efficacy and sometimes of a complete lack of a clinical response 2. Result in adverse events (benign to life-threatening) Hypersensibility… 10 CSA Biopharm And for the regulatory agencies ? Widespread use therapeutic proteins has demonstrated that nearly all biologicals can elicit antibody responses FDA & EMA demand : No production of ADA causing adverse effect Animal models are not predictive for immunogenicity : development of anti human protein antibody 11 CSA Biopharm Immunogenicity : Predictive Tools 12 CSA Biopharm But before… … The Immunogenicity mechanism 13 CSA Biopharm How does it work ? CD/LcB http://www.youtube.com/watch?v=Mn9_sgUnemk 14 CSA Biopharm Immunogenicity tests 16 CSA Biopharm T Cell Epitope Antigenicity Profiling In silico assays In vitro assays Class II HLA binding assays Antigen presentation assays T cell proliferation assays Cytokines release assays 17 CSA Biopharm Immunogenicity tests In silico 18 CSA Biopharm In silico binding prediction 19 CSA Biopharm In silico binding prediction Macromolecular Modeling Blog ™ Zavala-Ruiz Z et al. PNAS 2004;101:13279-13284 20 CSA Biopharm In silico binding prediction Goal : prediction of MHC II/ peptide interaction Ab is virtually splited peptides Peptides created are shifted by 1 amino acid AEYILPMKFDVSKLGFQ AEYILPMKFDVS EYILPMKFDVSK YILPMKFDVSKL ILPMKFDVSKLG LPMKFDVSKLGF PMKFDVSKLGFQ 21 CSA Biopharm In silico – Results L. Moise et Al. Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo.ClinicalImmunology (2012) 142, 320–331 22 CSA Biopharm Deimmunization YILPMKFDVSKL YGLPMKFDVSGL Identify and eliminate T-cell epitopes from the variable region sequences of antibodies and proteins. 23 CSA Biopharm De-immunization : it is working! 24 CSA Biopharm L. Moise et Al. Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo.ClinicalImmunology (2012) In Silico Strengths and Limitations High throughput No proof of TCR interaction Low cost Consequences of this interaction? (production of Ab or T rég, affinity…) Reduce downstream in vitro testing No proof of real interaction Opportunity in early development to modify a protein to decrease its immunogenic potential 25 Immunogenicity tests In vitro 26 Class II HLA-peptide Binding Assay 27 Class II HLA-peptide Binding Assay Goal : Determines the ability of each candidate peptide to bind class II HLA alleles and to stabilize the HLA-peptide complex 28 HLA Binding Assay Strengths and Limitations Straight forward & Easier No proof of TCR interaction Improve accuracy of immunogenicity predictions before using biological assay Artificial processing 29 Antigen Presentation Assays 30 How does it work ? Culture of the protein of interest with DCs.DCs take up the protein and process it HLA molecules present epitopes from the protein on the DC surface. Cells are lyzedand HLApeptide molecules are then recovered in an immune affinity step and analyzed by sequencing mass spectrometry. Peptides are recovered from the HLA CSABiopharm Peptides identified by mass spectrometry are subjected to analysis to identify true positive peptides with high 32 confidence. Antigen Presentation Assay Strengths and limitations Inform about natural antigen processing. No proof of TCR interaction and T cell proliferation Assessment of MHC peptide binding Rapid way to identify sequences of key relevance for the immunogenicity. 36 T-Cell Activation 37 T-cell proliferation – Fluorescent marker dilution Using whole protein or synthesized peptide Flow Cytometry 39 T-cell proliferation – Fluorescent marker dilution 40 Cytokine Release Assay PRINCIPLE : ELISA Sandwich 41 X8 X9 X4 X5 X6 X7 PBS SEB X1 X2 X3 Results 42 T-cell Activation Assay Strengths and limitations Brings together all the partners of immunogenicity Risk of variability Difficulties of reproductibility Incorporate Ag natural processing and presentation pathways Overestimation of the frequency of activated T cells Number of individual blood (PBMC) is quite large (> 40) HLA diversity of a patient population 43 TESTS - summary In silico Algorithm to screen for potential T-cell epitopes Identify linear motifs of 9-10 amino-acids that bind to MHC Class II molecules Fast, extensive databases exist Tend to over-predict potential for immune response relative to in vitromethods Typically used as part of lead selection/optimization In vitro HLA binding assays Antigen Presentation Assay T cells assays Peptides or proteins Typically used as part of lead selection/optimization 44 Decision Tree + affinity assays efficacy Humanized mAB De-immunization beginning modifications immunogenic sequences ? (In silico tests) « in silico » High immunogenicity « in silico » Low immunogenicity Overlapping peptides production HLA-binding test Ag Presentation test High immunogenicity T-cell activation test Low immunogenicity mAB candidates Choice of the best mAB (Safety/Efficacy) Olinoudezumab Mouse model 46 Thank you for your attention ! Question ? 47
