Humanization of Monoclonal Antibodies Quiz

Humanization of Monoclonal Antibodies

• Immunogenicity is the ability of a molecule to trigger an immune response
• Strategies to minimize immunogenicity:
  • Replace animal-derived drugs with human sources
  • Increase human sequence content
  • Change from murine (mouse) to humanized or human antibodies
• Murine antibodies are highly immunogenic
• Chimeric antibodies combine mouse variable region with human constant (e.g., Remicade, Simulect)
• Chimeric antibodies can also be immunogenic, leading to humanized antibody, where only the CDR region has murine sequences (e.g., Avastin)
• Humanization involves replacing mouse amino acids with human counterparts to minimize immunogenicity

Immunogenicity Testing in Clinical Trials

• Samples needed:
  • Serum: For antibody detection
  • Plasma: For cytokine and chemokine measurement
  • PBMCs: For cellular assays
• Analyze samples at different time points:
  • Baseline (before treatment)
  • During treatment (various time points)
  • After treatment
• Assays used:
  • ELISA: To quantify levels of antibodies to the therapeutic protein
  • Flow cytometry: To measure immune cell populations and their activation
  • Cytokine assay: To assess the production of inflammatory cytokines
  • T cell proliferation assay: To determine the activation and proliferation of T cells
• Report immunogenicity:
  • Incidence: The percentage of individuals developing an immune response
  • Titers: The concentration of antibodies in serum
  • Correlation between immunogenicity and outcomes: Relationship between immune response and treatment efficacy, adverse events, and other clinical endpoints

Innate Immune Activation

• Innate immune cells recognize "self" and "non-self" through pattern-recognition receptors (PRRs)
• Examples of innate immune cells:
  • Monocytes, neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, eosinophils, basophils
• PRRs trigger a cascade of signals that lead to the production of proinflammatory cytokines
• PAMPs are pathogen-associated molecular patterns
• DAMPs are damage-associated molecular patterns
• PAMPs and DAMPs are conserved structures that are recognized by PRRs

Immune Cells Involved in Immunogenicity

• B cells: Produce antibodies that target the therapeutic protein
• T cells:
  • CD4+ T helper cells: Amplify the immune response by stimulating B cells and other immune cells
  • CD8+ cytotoxic T cells: Can directly eliminate cells that express the therapeutic protein
• Dendritic cells: Present antigens to T cells, initiating adaptive immune responses
• Macrophages: Engulf and destroy pathogens
• Neutrophils: Combat infections by releasing enzymes and other antimicrobial substances

Immunogenicity Risk Assessment

• FDA guidelines recommend immunogenicity risk assessment during drug development
• Immunogenicity is a significant factor in determining the safety and efficacy of therapeutic proteins
• Immunogenicity risk assessment strategies:
  • In silico: Computational analysis of protein sequence to predict the likelihood of immune recognition
  • In vitro prediction: Cell culture assays using immune cells to assess the interaction of the therapeutic protein with the immune system
  • In vivo: Animal studies to evaluate the immunogenic potential of the protein in a living organism
  • Clinical immunogenicity assessments: Monitoring patients in clinical trials to evaluate the occurrence and severity of immune response
• More than 89% of approved biologics have reported clinical immunogenicity, highlighting the importance of risk assessment

In Silico Screening

• Computational approaches to analyze protein sequences and identify features that might trigger an immune response
• Methods assess:
  • MHC protein binding: Predication of whether protein fragments can bind to major histocompatibility complex (MHC) molecules, a crucial step in antigen presentation to T cells
  • T cell epitope prediction: Identify peptide sequences within the protein that are likely to be recognized by T cell receptors
• Predictive algorithms prioritize molecules based on immune response likelihood
• These tools are valuable in early stage development to select candidates with reduced immunogenicity risk

In Vitro Immunogenicity Testing

• Cell culture methods to study the interaction of the therapeutic protein with immune cells
• Commonly used cells types:
  • PBMCs: Peripherial blood mononuclear cells containing a variety of immune cells
  • Dendritic cells: Present antigens to T cells
  • Macrophages: Phagocytic cells that can engulf and process antigens
• Assays:
  • Cytokine release: Incubate test article with immune cells and measure cytokine production in the media or inside the cell
  • T cell proliferation: Assess the ability of the therapeutic protein to stimulate T cell growth and proliferation
• These assays provide insights into the immune response at the cellular level and help identify potential immunogenic risks

Factors Influencing Immunogenicity

• Molecule structure:
  • Amino acid sequence: Differences from human proteins can trigger immune recognition
  • Post-translational modifications: Changes to the protein after synthesis, like glycosylation
• Dosage and route of administration
• Patient factors:
  • Genetics: MHC genes variability
  • Previous exposure to similar proteins
  • Underlying immune status

Cytokine Assays

• Measure production of cytokines, small signaling molecules that mediate communication between immune system cells
• Common cytokines assessed:
  • TNF-α: Tumor necrosis factor alpha, a proinflammatory cytokine
  • IL-6: Interleukin 6, a proinflammatory cytokine
• Cells often tested include:
  • Dendritic cells
  • Macrophages
• A significant increase in cytokine levels compared to baseline may indicate immunogenic risk

T Cell Assays

• Investigate the activation of T cells using proliferation assay or by measuring cytokine production and cell surface marker expression
• Techniques:
  • Proliferation assay: Quantify the ability of the therapeutic protein to stimulate T cell division
  • Flow cytometry: Analyze T cells surface markers to determine activation status
• Depletion of CD8+ T cells from PBMCs is common to enhance the assay sensitivity

MHC II Associated Peptide Proteomics (MAPPs)

• Identify the peptides that are bound to MHC II molecules, which are expressed on antigen-presenting cells like dendritic cells
• MHC II binding: Determine the peptides from the therapeutic protein that are likely to be presented to T cells, a key step initiating an adaptive immune response.
• MAPPs assess the potential of a protein to interact with MHC II and trigger an immune response
• Predictive data can guide the development of proteins with less immunogenic potential

Immunogenicity Assessment in a Novel Monoclonal Antibody Development

• In vitro testing:
  • Stimulate immune cells (e.g., PBMCs, dendritic cells) with candidate monoclonal antibodies
  • Measure cytokine production (e.g., TNF-α, IL-6) and T cell proliferation
• Report immunogenic potential:
  • Compare cytokine levels and T cell proliferation between control and candidate molecule stimulated cells
  • Evaluate statistically significant differences
  • Calculate immunogenic potential based on established metrics
• Factors besides structure:
  • Dosage
  • Route of administration
  • Patient’s immune status