Immunogenicity 1 Measurement and Prediction Lecture (PHC 539)

Immunogenicity 1: Introduction to detection and prediction of immunogenicity PHC 539 10/17/2022 Nicole Jarvi 1 Lecture Outline Introduction to immunogenicity Anti-drug antibodies: neutralizing vs binding Factors that co...

Immunogenicity 1: Introduction to detection and prediction of immunogenicity PHC 539 10/17/2022 Nicole Jarvi 1 Lecture Outline Introduction to immunogenicity Anti-drug antibodies: neutralizing vs binding Factors that contribute to immunogenicity Mechanism of immune response Clinical immunogenicity analysis and reporting ADA/neutralizing ADA assays Immunogenicity risk assessment in preclinical setting In vitro prediction methods Mechanism-based prediction methods Animal models 2 Learning objectives Define immunogenicity of biologics Name factors that contribute to immunogenicity of proteins and monoclonal antibodies 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 Introduction 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 Products. 5 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. Anti-drug antibodies Anti-drug antibodies can have binding (non-neutralizing) or neutralizing activity Neutralizing/inhibitory antibodies leads to loss of biological activity of the therapeutic proteins In some instances, Nab can neutralize endogenous protein leading to a new clinical syndrome EPO – red cell aplasia Binding antibodies can alter the pharmacokinetics of the therapeutic protein, e.g., by increasing clearance ADA binding can introduce a new clearance pathway, such as clearance of immune complexes by phagocytes 7  Anti-drug antibodies Left: https://absoluteantibody.com/antibody-resources/antibody-overview/antibody-isotypes-subtypes/ 8 Right: https://www.antibody-creativebiolabs.com/antibody-structure-isotypes.htm  Neutralizing anti-drug antibodies (NAb) Example: factor VIII inhibitors Anti-factor VIII antibodies that block functional binding sites are neutralizing Prevent factor VIII from performing its blood clotting activity Occur in approx. 30% of severe hemophilia A patients receiving FVIII Anti-idiotypic antibodies Neutralizing ADA prevent the antibody drug from binding to its target antigen Left: https://www.rndsystems.com/products/anti-idiotype-antibodies 9 Right: https://teens.aboutkidshealth.ca/Article?contentid=3238&language=English Hemophilia and inhibitors  Other immunological adverse events mediated by hypersensitivity responses Immediate hypersensitivity clinically consistent with type I hypersensitivity, IgE cross linking antigen to mast cells leading to secretion of vasoactive mediators, occurs within 48 hrs Handled with improved purity, antihistamines, Black box warning: Type III: immune complex mediated hypersensitivity, protein-antibody complex deposited in various tissues induce complement activation and inflammatory response, typical manifestations include serum sickness Treatment: NSAIDs, anti-histamines, and corticosteroids Left: https://webpath.med.utah.edu/IMMHTML/IMM101.html 10  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 11 Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654. See full table in reference  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 12 Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654. See full table in reference Factors influencing immunogenicity Many factors influence immune response à 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 13  Donor variability – HLA alleles Variability in HLA alleles or haplotype will likely influence inter-individual variability in immunogenicity à should be considered when designing in vitro risk assessment experiments with human donors 14 http://www.allelefrequencies.net/top10dist.asp  15 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 the CDR region has murine sequences (e.g., Avastin) Fully human antibodies (e.g., Humira) also show clinical immunogenicity, albeit low 16 Image: https://absoluteantibody.com/antibody-resources/antibody-engineering/humanisation/ 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? 17  Clinical immunogenicity reporting Immunogenicity Information in Human Prescription Therapeutic Protein and Select Drug Product Labeling — Content and Format Guidance for Industry 18 Wang, Y.M., et al., Evaluating and Reporting the Immunogenicity Impacts for Biological Products--a Clinical Pharmacology Perspective. AAPS J, 2016. 18(2): p. 395-403.  Immunogenicity sampling in clinical trials Example: Pfizer clinical trial for bococizumab (anti-PCSK9), PF-04950615 19 https://clinicaltrials.gov/ct2/show/NCT01968954  20 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-and-validating-assays-anti-drug  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 21 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-testing-therapeutic-protein-products-developing-and-validating-assays-anti-drug  Bridging ADA Assay + substrate Color (or 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 Profiling: Methods and Protocols, 22 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: https://www.creative-biolabs.com/drug-discovery/therapeutics/non-cell-based-competitive-ligand- binding-assay.htm Indirect competitive ligand binding assay 23  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 11. Identify and count the spots with an automated ELISpot reader 24 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 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) 25 How do anti-drug antibodies develop? 26  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 other immune cells, through TCR interactions or cytokine production B cells recognize antigen directly and produce antibodies against that antigen Marshall, J.S., et al., An introduction to immunology and immunopathology. Allergy, Asthma & Clinical Immunology, 2018. 14(2): p. 49. 27 Image: https://blog.cellsignal.com/immunology-overview-how-does-our-immune-system-protect-us  Lymphocytes (T and B cells) develop in different compartments before entering the periphery Naïve T and B cells reside in secondary lymphoid organs T cells develop in (lymph nodes) the thymus B cells develop in the bone marrow There are 1,000,000,000,000,000 (quadrillion) potential different T cell receptors for all 𝜶β T cells !! 28 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. Top right: Janeway Immunobiology 9th edition  Immune cells involved in immunogenicity 29 Prezotti, A.N.L., et al., Immunogenicity of Current and New Therapies for Hemophilia A. Pharmaceuticals, 2022. 15(8): p. 911. 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 presentation. Nature reviews. 30 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) 2. Verification /survival signals provided by co-stimulatory Peptide 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 31 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) 32 De Silva, N.S. and U. Klein, Dynamics of B cells in germinal centres. Nature Reviews Immunology, 2015. 15(3): p. 137-148.  Humoral immune response generates anti-drug antibodies B cells play a major role in the 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 higher affinity for antigen Due to affinity maturation and selective proliferation Time course of the antibody response of the highest affinity B cell clones 33 Janeway Immunobiology. 9th Edition. How do you predict whether a therapeutic protein will be immunogenic in a patient? 34  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 à 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 prediction In vitro prediction In vivo prediction Clinical immunogenicity 35 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).  In silico screening 36 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? 37  38 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).  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-alpha 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 39 Walsh, R.E., et al., Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms. mAbs, 2020. 12(1): p. 1764829-1764829.  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). Walsh, R.E., et al., Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms. mAbs, 2020. 12(1): p. 1764829-1764829. 40 Image: flowmetric.com/cytometry-blog/optimization-cell-proliferation-flow-cytometry  MHC II associated peptide proteomics (MAPPS) DC uptake of antigen is also a measure of immunogenic risk (in a separate assay) FIGURE 5 | MAPPs assay design. 1. Monocytes are isolated from whole PBMCs and differentiated into Dendritic Cells (DCs) in the presence of IL-4 and GMCSF 2. Immature DCs are matured by incubating cells with LPS and antigen. 3. Mature DCs are lysed releasing peptide- loaded HLA molecules from the plasma membrane which are collected by immunoprecipitation. 4. Next peptides are eluted from the HLA molecules and analyzed by Mass Spec. 5. Peptides are identified by screening them against a database of known antigens. 41 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).  3D human artificial lymph node (HuALN) model A photograph of the bioreactor is shown in the upper right corner of b). Follicle-like spheroid formation and proliferation (image c; Ki67; red staining), plasma cell differentiation (image d; CD138; red staining) and antigen-specific binding on plasma cells (image e; biotynilated CMV-lysate; red staining) was confirmed histologically on tissue slices upon tissue culture completion. Kraus, T., et al., Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates. Journal of Pharmaceutical Sciences, 2019. 108(7): p. 2358-2366. 42 Giese, C., et al., Immunological substance testing on human lymphatic micro-organoids in vitro. J Biotechnol, 2010. 148(1): p. 38-45.  Human skin model In vitro human skin model Skin cells only No leukocytes Innate immune response Response to process-related impurities (human TLR1-9 agonist kit) (top left) or mAb aggregates from stirring (top right) or pH stress (bottom) Tokuda, J.M., et al., Use of in vitro human skin models to assess potential immune activation in response to biotherapeutic attributes and process-related impurities. Journal of Pharmaceutical Sciences, 2022. 43 Image: https://www.mattek.com/products/epidermft/  Mechanism based model for SC delivery Mechanism-based risk markers on dendritic cells are upregulated by immunogenic proteins Migratory Phenotype Activation Phenotype Percent of IL-12-producing Expression level of dendritic cells chemokine receptor Expression level of co- stimulatory markers Dendritic cell Direct Migratory Potential Number of dendritic cells migrating toward therapeutic protein in the Dendritic cell migration from the subcutaneous injection site to presence of chemokines in a draining lymph nodes is a driving force for immunogenicity Transwell assay 44 Jarvi and Balu-Iyer, unpublished data  In vivo models – use of animals Animal models appear to have predictive potential when assessing relative immunogenicity, neo-epitopes, or breaking of tolerance, but results should be interpreted with caution May be useful and somewhat predictive if sequence homology is high Mechanisms underlying immunogenicity are not fully understood and there may be important differences between the human and animal response Transgenic mice can be developed to be immune tolerant to some human proteins to predict immunogenicity Constriction in genetic variability in animal models can limit immunogenicity prediction for diverse human populations Although non-human primates are tolerant to many human proteins, they are not always able to predict incidence of immunogenicity 45 Brinks, V., W. Jiskoot, and H. Schellekens, Immunogenicity of therapeutic proteins: the use of animal models. Pharm Res, 2011. 28(10): p. 2379-85. Recommended reading Dingman, R. and S.V. Balu-Iyer, Immunogenicity of Protein Pharmaceuticals. J Pharm Sci, 2019. 108(5): p. 1637-1654. Jarvi, N.L. and S.V. Balu-Iyer, Immunogenicity Challenges Associated with Subcutaneous Delivery of Therapeutic Proteins. BioDrugs, 2021. 35(2): p. 125-146. 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). De Groot, A.S. and D.W. Scott, Immunogenicity of protein therapeutics. Trends in Immunology, 2007. 28(11): p. 482-490. Hamuro, L., et al., Perspectives on Subcutaneous Route of Administration as an Immunogenicity Risk Factor for Therapeutic Proteins. J Pharm Sci, 2017. 106(10): p. 2946-2954. 46 Questions? My email: [email protected] My location: 358 Pharmacy Building 47