PHC 539 Immunogenicity Measurement & Prediction Lecture PDF

Immunogenicity III: Detection and Prediction of Immunogenicity PHC 539 10/25/2024...

Immunogenicity III: Detection and Prediction of Immunogenicity PHC 539 10/25/2024 Nicole Jarvi, Ph.D. Senior Scientist, Biologics & Biopharmaceutics, Merck 1 The views and opinions expressed in this presentation are my own and do not represent the views and opinions of my employer Merck Sharp & Dohme LLC, a Lecture Outline Mechanisms of immunogenicity Review immunogenicity basics Mechanisms of ADA development Clinical immunogenicity analysis and reporting Package insert ADA/neutralizing ADA assays Immunogenicity risk assessment in preclinical setting In vitro prediction methods Mechanism-based prediction methods Animal models 2 Learning objectives Understand mechanisms of anti-drug antibody development Recognize the impact of immunogenicity on protein safety and efficacy Learn how immunogenicity data is reported and appreciate the lack of standardization in anti-drug antibody assays Discover preclinical methods to predict risk of immunogenicity using in vitro and in vivo methods 3 Review What is immunogenicity? What are possible reasons why therapeutic proteins are immunogenic? 4  Immunogenicity “The propensity of the therapeutic protein product to generate immune responses to itself and to related proteins or to induce immunologically related adverse clinical events” Immunologically based adverse events have caused sponsors to terminate development of otherwise efficacious therapeutic protein products For example: cytokine release syndrome, anaphylaxis, or cross-reactive neutralization of endogenous proteins Immune responses to protein drugs vary in clinical relevance based on the impact of anti-drug antibody development on safety and efficacy Why do human therapeutic proteins induce an immune response in some patients? U.S. Food and Drug Administration. Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein 5 Products. https://www.fda.gov/downloads/drugs/guidances/ucm338856.pdf  Unwanted immune responses – two pathways Classical Response Immune response occurring after administration of foreign proteins Within days to weeks, often occurs even after a single injection, neutralizing in nature and persist for long time Replacement therapies such as Factor VIII, CDR region of human Mab Breaking of Immune tolerance Tolerance: immune system recognizes and is tolerant to self proteins Recombinant proteins that are homologous to endogenous proteins should be viewed as “self” Antibody response against self proteins is considered breaking of tolerance Occurs upon slow, chronic administration, and can disappear after treatment stops Cytokines and hormones like EPO or insulin Crosslinking of B cell receptors by multimeric antigen could break tolerance 6 De Groot, A.S. and D.W. Scott, Immunogenicity of protein therapeutics. Trends in Immunology, 2007. 28(11): p. 482-490.  Immunogenicity is observed across all types of protein drugs that vary in target, structure, and indication Immunogenicity (IMG) Rate: Percentage of treated patients that tested positive for anti-drug antibodies in a clinical trial 7 See full table in Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654.  Immunogenicity is observed across all types of protein drugs that vary in target, structure, and indication Immunogenicity (IMG) Rate: Percentage of treated patients that tested positive for anti-drug antibodies in a clinical trial 8 See full table in Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654. Factors influencing immunogenicity Many factors influence immune response  Product, patient, and treatment characteristics Treatment characteristics: SC route can be more immunogenic than IV Frequency of administration (chronic) dosing increases immunogenicity Patient characteristics: Human leukocyte antigen (HLA) is a highly polymorphic protein, with several identified alleles The genes encoding MHC II (HLA DR, DQ, DR, and more) have alleles varying in their peptide binding affinity, leading to interindividual variability in immune response Autoimmunity, immunosuppressive treatment, and pre- existing ADA responses Product characteristics: T cell and B cell epitopes, humanization, chemical degradation, aggregation, and more 9  Donor variability – HLA alleles Variability in HLA alleles or haplotype will likely influence inter-individual variability in immunogenicity  should be considered when designing in vitro risk assessment experiments with human donors http://www.allelefrequencies.net/top10dist.asp 10  11 U.S. Food and Drug Administration. Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products. https://www.fda.gov/downloads/drugs/guidances/ucm338856.pdf  Humanization of monoclonal antibodies Red = mouse Blue = human Strategy to minimize immunogenicity: Replacing drugs from animal to human sources Increasing human sequence content Changing monoclonal antibodies from murine or chimera to humanized and human sequence Due to high immunogenic response of murine (e.g., OKT3), chimeric mAbs were developed Mouse variable region and human constant region (e.g., Remicade, Simulect) Chimeric mAbs are also immunogenic, leading to humanized antibody, where only 12 the CDR region has murine sequences Image: https://absoluteantibody.com/antibody-resources/antibody-engineering/ (e.g., Avastin) Make a prediction You are setting up a clinical trial for a new therapeutic protein. How will you measure immunogenicity of the protein in patients? What types of samples will you need? When should you take the samples? What type of assay would you use? How will you report immunogenicity? 13  Clinical immunogenicity reporting Immunogenicity Information in Human Prescription Therapeutic Protein and Select Drug Product Labeling — Content and Format Guidance for Industry 14 Wang, Y.M., et al., Evaluating and Reporting the Immunogenicity Impacts for Biological Products--a Clinical Pharmacology Perspective. AAPS J,  Immunogenicity sampling in clinical trials Example: Pfizer clinical trial for bococizumab (anti-PCSK9), PF- 04950615 15 https://clinicaltrials.gov/ct2/show/NCT01968954  16 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-  Anti-drug antibody assays Screening assay: detects low and high affinity ADA Broadly detect presence of antibodies in serum samples that bind therapeutic protein Confirmatory assay: confirm the binding of antibodies that are specific to the therapeutic protein product Should have higher specificity than the screening assay Quantitative assay: determine antibody levels of subjects who were confirmed to be ADA positive Express antibody levels using a “titer” or the reciprocal of the highest dilution that gives a value at or just above the assay cutpoint ADA concentration (e.g., ng/ml) can be quantified if using a standard curve of an anti-drug antibody of known concentration 17 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-  Bridging ADA Assay + Color (or substrate fluorescence) HRP conjugated drug Patient ADA Drug 96-well plate Harth, S. and C. Frisch, Recombinant Anti-idiotypic Antibodies in Ligand Binding Assays for Antibody Drug Development, in Proteomic 18 Profiling: Methods and Protocols, A. Posch, Editor. 2021, Springer US: New York, NY. p. 291-306.  Neutralizing ADA (NAb) Assays Cell-based/biological neutralizing assays Use cell lines that express the target and mimic the therapeutic effects of the drug Detecting a decrease in drug activity in the presence of patient ADA indicates neutralizing ADA Sensitive to matrix effects, difficult assay validation, time-consuming Non-cell based assays: Enzymatic activity neutralizing assay If the therapeutic protein has enzymatic activity, the ability of patient antibodies to reduce/block activity of the protein can distinguish neutralizing antibodies For example: FVIII inhibitors are measured in a modified clotting time assay, where inhibitors block FVIII activity and extend the blood clotting time Competitive ligand binding assay Quantify NAbs based on their ability to prevent the therapeutic mAb binding to its target Direct and indirect formats May not correlate with clinical response https://www.medpace.com/neutralizing-antibody-assays/ Image: Indirect competitive ligand binding https://www.creative-biolabs.com/drug-discovery/therapeutics/non-cell-based-competitive-lig 19  Measuring the humoral response with ELISpot 1. Coat PVDF-backed microplate with protein antigen 2. Add PBMCs or B cells to each well 3. Incubate in a humidified 37°C CO2 incubator to allow production of antigen-specific antibodies (that bind to antigen coated on plate) 4. Wash away any cells and unbound substances 5. Add a biotinylated polyclonal detection antibody specific for the secreted antibody 6. Wash away any unbound detection antibody 7. Add alkaline phosphatase-conjugated streptavidin 8. Wash away any unbound substrate 9. Add the BCIP/NBT substrate 10.Incubate until a blue-black precipitate forms 20 https://www.rndsystems.com/products/elispot-serological-assays-coronavirus-research Issues with measuring immunogenicity Lack of assay standardization Antibody titers measured in different clinical trials are difficult to compare, unless the antibody assays were performed in the same laboratory Wide variation in reported incidence of immunogenicity associated with a single product Biological assays or bioassays used to determine neutralizing activity are usually technically difficult and time-consuming Need appropriate cell lines The immune response is dynamic and the kinetics of antibody development must be evaluated as the response evolves over time Antibodies can be transient (i.e. last only for a short time) 21 How do anti-drug antibodies develop? 22  Overview of the immune response: innate vs adaptive Innate immunity: First line of defense for fighting an intruding pathogen Rapid, initiated within minutes or hours No immunologic memory (not pathogen specific) Defensive barriers: anatomic, physiologic, endocytic and phagocytic, and inflammatory Adaptive immunity: Antigen-dependent and antigen-specific immune responses Delayed Has memory – so the response to re-infection is stronger and faster Dendritic cells phagocytose antigens and initiate the acquired immune response, activate antigen- specific T cells CD4+ T cells mediate the immune response by directing Marshall, J.S., other toimmune et al., An introduction cells, immunology and throughAllergy, immunopathology. TCR Asthma & Clinical Immunology, 2018. 14(2): p. 49. 23 interactions or cytokine production Image: https://blog.cellsignal.com/immunology-overview-how-does-our-immune-system-protect-us  Innate Immune Activation Pattern recognition receptor-mediated NF-κB Innate immune cells are signaling capable of distinguishing “self” and “non-self” using a host of pattern-recognition receptors Innate immune cells: Monocytes, neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, eosinophils, and basophils PRR signaling by pathogen- associated molecular patterns (PAMPs) or damage- associated molecular patterns (DAMPs) induces proinflammatory cytokine production PAMPs: nucleic acids, Proinflammatory lipids,or proteins conserved cytokine production across pathogenic microorganisms 24 Li D, Wu M. Pattern recognition receptors in health and diseases. Signal Transduct Target Ther. 2021 Aug  Immune cells involved in immunogenicity 25 Prezotti, A.N.L., et al., Immunogenicity of Current and New Therapies for Hemophilia A. Pharmaceuticals, 2022.  Lymphocytes (T and B cells) develop in different compartments before entering the periphery Naïve T and B cells reside in secondary T cells lymphoid organs develop in (lymph nodes) the thymus B cells develop in the bone marrow potential different T cell receptors for all 𝜶β T There are 1,000,000,000,000,000 (quadrillion) 26 cells !! Left: https://step1.medbullets.com/immunology/105063/lymphocyte-development-and-structure Right: Edwards, J.C.W. and G. Cambridge, B-cell targeting in rheumatoid arthritis and other autoimmune diseases. Nature Reviews Immunology, 2006. 6(5): p. 394-403. Antigen-presenting cells (macrophages, DCs, and B cells) engulf protein antigen and present peptide-MHC-II complexes In immature DCs, MHC II was predominantly in late endosomes and lysosomes, whereas in resting B cells it was primarily on the cell surface. Ma, J.K., et al., MHC class II distribution in dendritic cells and B cells is determined by ubiquitin chain length. Proceedings of the National Academy of Sciences, 2012. 109(23): p. 8820-8827. Roche, P.A. and K. Furuta, The ins and outs of MHC class II-mediated antigen processing and 27 presentation. Nature reviews. Immunology, 2015. 15(4): p. 203-216.  T cells are activated by antigen-presenting cells by a combination of signals T cells need 3 signals from APCs in order to become activated: 1. Recognition of peptide-MHC-II complex by the T cell receptor (TCR) Peptide 2. Verification /survival signals provided by co- stimulatory molecules (CD86 and CD40) 3. Proliferation and differentiation induced by cytokine secretion Activated T cells will go on to direct the immune response and activate antigen-specific B cells 28 Janeway Immunobiology. 9th Edition.  Germinal centers in lymph nodes are the main site of B cell differentiation and proliferation Full activation of B cells by cognate T cells Early activation of B cells by antigen Activation of T cells by DCs in antigen- presenting mode Proliferating B cell clones Formation of the germinal center (GC) 29 De Silva, N.S. and U. Klein, Dynamics of B cells in germinal centres. Nature Reviews Immunology, 2015.  Humoral immune response generates anti-drug antibodies B cells are the major player in humoral or antibody-mediated immune response When activated by antigen-specific T cells, B cells proliferate and differentiate into antibody-secreting plasma cells or memory B cells Some plasma cells become “long-lived” after migrating to the bone marrow, when they continuously produce antibody Memory B cells are long-lived survivors of infection and be called upon quickly to respond to re-exposure of antigen Upon secondary exposure, the antibody response becomes much stronger with Time course of the antibody higher affinity for antigen response Due to affinity maturation and selective proliferation of the highest affinity B cell clones 30 Janeway Immunobiology. 9th Edition. How do you predict whether a therapeutic protein will be immunogenic in a patient? 31  Immunogenicity risk assessment Recent FDA guidelines for new drug products and generic versions of existing products have suggested immunogenicity risk assessment approaches Immunogenicity risk assessment is most cost-effective if performed during preclinical drug development stages In silico sequence analysis and in vitro assays can be performed … Early in development to design and select therapeutic candidates with low predicted immunogenicity Later in development to de-immunize a protein exhibiting high immunogenicity in First in Human studies Retrospectively, after program termination, to decipher the mechanisms and immunogenicity risk factors underlying the high observed clinical immunogenicity Remember from earlier slide  89% of approved biologics reported clinical immunogenicity, suggesting the importance of immunogenicity risk assessment and the failure of current methods to consistently predict clinical immunogenicity potential In silico In vitro prediction In vivo Clinical prediction prediction immunogenicity 32 Jawa, V., et al., T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review 2020. Frontiers in  In silico screening 33 Figure 2. https://epivax.com/deimmunization Make a prediction You are developing a new monoclonal antibody to treat cancer. You have a selection of candidate molecules to assess for immunogenicity potential. How might you go about testing their immunogenicity risk in a cell culture (in vitro) setting? What immune cells would you use in your assay? How will you report the immunogenic potential of each molecule? Review: What factors other than the molecule structure could influence its immunogenic potential in a patient? 34  35 Jawa, V., et al., T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review 2020. Frontiers in  36 Jawa, V., et al., T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review 2020. Frontiers in  Cytokine assays Incubate test article with immune cells and measure cytokine production in the media (by MSD or ELISA) or inside the cell (by intracellular staining) Dendritic cells and macrophages are major producers of cytokines, such as TNF- or IL-6 Proinflammatory cytokines produced by innate immune cells can also be investigated A significant increase in cytokine levels over the baseline induced by the therapeutic protein would indicate immunogenic risk The baseline is usually cells incubated with media only 37 Walsh, R.E., et al., Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms. mAbs, 2020. 12(1): p. 1764829-  T cell assays Because of their essential role in the generation of isotype switched and affinity- matured antibodies, CD4+ T helper cells are the primary drivers of the ADA response to biologics in the clinic and thus the focus of most the preclinical immunogenicity risk assessment. CD4+ T cell proliferation assay: Label PBMCs with proliferation dye (e.g., CellTrace Violet or CSFE) Incubate PBMCs with test article for a prolonged time, such as 7 days Analyze % of proliferated CD4+ T cells by flow cytometry Depletion of CD8+ T cells from PBMCs can increase assay sensitivity Increased immunogenicity risk indicated by upregulation of T cell proliferation Jawa, V., et al., T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review 2020. Frontiers in Immunology, 2020. 11(1301). 38 Walsh, R.E., et al., Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms. mAbs, 2020. 12(1): p. 1764829-  MHC II associated peptide proteomics (MAPPs) DC uptake of antigen is also a measure of immunogenic risk (in a separate assay) 39 Jawa, V., et al., T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review 2020. Frontiers in MHC II associated peptide proteomics (MAPPs) Secukinumab (human IgG1/kappa) and Ixekizumab (humanized IgG4 variant/kappa) both target IL-17A Secukinumab ADA incidence