Protein%20Therapeutics%20Immunogenicity

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Protein TherapeuticsImmunogenicity

• Stephen Lynn
• 11-16-11
• CHEM645

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Anti-therapeutic Protein Antibodies

• Known as monoclonal antibodies (mAbs) or
  anti-drug antibodies (ADAs)
• Produced by the B lymphocytes of the immune
  system in response to foreign proteins
• Neutralize or compromise the clinical effect of
  therapeutic proteins
• May cause serious adverse events related to
  cross-reactivity with autologous proteins
• ADAs that interact with a ligandreceptor may
  inhibit the ef?cacy of all ligands in the same
  class.

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Immunogenicity

• Ability of a particular substance, such as an
  antigen or epitope, to provoke an immune response
  in the body of a human or animal
• Strategies for reducing immunogenicity
• Reduce the total number of doses
• Minimize impurities (contaminants/aggregates)
• Diminish T cell activation through sequence
  modifications (must maintain biological function)
• Avoid delivery vehicles that have altering
  effects on other biological agents

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Immune ResponseT-cell Dependent

• B cell activation can be T cell-independent (Ti)
  or T cell-dependent (Td)
• Td activation of B cells results in a more robust
  antibody response, isotype switching, and the
  development of B cell memory
• The induction of IgG class ADAs (measurable in
  immunogenicity assays) through B cell secretion
  implies that the therapeutic protein is a Td
  antigen
• Td responses require T cell recognition of
  epitopes contained in the protein drug, binding
  of peptide epitopes derived from internal
  processing by antigen-presenting cells (APCs) to
  HLAs (human leukocyte antigens) MHC class II
  molecules, and recognition of the epitopeHLA
  complex by activated T helper cells

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Reducing risk, improving outcomes
Bioengineering less immunogenic protein
therapeutics

• Anne S. De Groot a,b , William Martin aa EpiVax,
  Inc., USAb University of Rhode Island
  Biotechnology Program, USA

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In vitro Assays

• Class I epitopes are short and fit tightly in the
  bounded MHC molecule by contrast, Class II (T
  helper) epitopes lie within an open-ended groove
  in the MHC II complex
• Class II epitope can shift within the groove
  thereby accommodating MHC of various haplotypes.
  The only limit on the size of the peptide is its
  ability to remain in a linear conformation in the
  open-ended groove.

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In vitro Assays

• HLA binding assays can be used to assess whether
  peptides derived from protein sequences can bind
  to either MHC class I or class II.
• In vitro evaluation of HLA binding can be
  performed by quantifying the ability of
  exogenously added peptides to compete with a
  fluorescently-labeled MHC ligand and can be
  adapted for high throughput.
• ELISA, ELISpot, Fluorescent labeling (FACS), and
  intracellular cytokine staining (ICS) are also
  available methods for measuring and defining T
  cell response.

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In silico Methods

• A number of in silico tools for predicting T-cell
  epitopes have been developed involving computer
  algorithms that map the locations of MHC class I
  and class II-restricted T-cell epitopes within
  proteins of various origins. The in silico
  methods include frequency analysis,
  support-vector machines, hidden Markov models,
  and neural networks.
• MHC class II prediction methods are more directly
  related to Td immunogenicity assessment, as class
  II restricted T cells provide help to B cells,
  leading to the development of ADA.
• Some epitope-mapping algorithms allow researchers
  to measure the potential immunogenicity by
  epitope density of whole proteins such as
  bioengineered protein therapeutics, monoclonal
  antibodies (mAbs) and their sub-regions.

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Comparison of in silico Algorithms

• The Immune Epitope Database (IEDB) developed a
  gold standard list of T-cell epitopes. Using
  the list as a benchmark, a number of tools have
  been compared by the team at the IEDB. Available
  online epitope-mapping algorithms were compared
  for their ability to correctly predict a set of
  validated class I and class II HLA-restricted
  epitopes competitors.

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ClustiMer Analysis

• The ClustiMer algorithm identifies polypeptides
  predicted to bind to an unusually large number of
  HLA alleles these sequences are then extended at
  the n- and c-terminal flanks until the predicted
  epitope density of the candidate epitope cluster
  falls below a given threshold value. In general,
  T-cell epitope clusters identified by the
  ClustiMer algorithm tend to be promiscuous MHC
  binders and are frequently T-cell epitopes

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EpiBar Analysis

• An EpiBar is a single 9-mer frame that is
  predicted to be reactive to at least four
  different HLA alleles (Fig. 4B) EpiBars may be a
  signature feature of highly immunogenic,
  promiscuous class II epitopes.
• Peptides scoring above 1.64 on the EpiMatrix Z
  scale (typically the top 5 of any given sample)
  are likely to be MHC ligands.
• CLIP (Class II invariant chain peptide) binds MHC
  II groove before receptor

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Role of regulatory T-cell epitopes

• Natural regulatory T cells appear to serve as
  regulators or suppressors of autoimmune,
  auto-reactive immune responses
• They do not promote immune function, but act to
  decrease it instead. Despite their low numbers
  during an infection, these cells are believed to
  play an important role in the self-limitation of
  the immune system they have been shown to
  prevent the development of various auto-immune
  diseases.
• T cells with high affinity for self antigens are
  deleted whereas those with moderate affinity for
  self antigens may escape deletion and may be
  re-programmed to function as natural regulatory
  T cells.
• Further studies must be done to fully understand
  role in immune response and immunogenicity.

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Pros and cons of assays for measuringimmunogenici
ty antibody assays