A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

MAbs. 2014;6(5):1265-73. doi: 10.4161/mabs.29825. Epub 2014 Oct 30.

Abstract

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')₂ fragments, but not for full-length IgG or for closely-related F(ab')₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')₂ fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')₂ fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; CDC, complement-dependent cytotoxicity; FACS, fluorescence-activated cell sorter; GluV8, glutamyl endopeptidase V8; IdeS, Immunoglobulin G-degrading enzyme of Streptococcus pyogenes; IgG fragments; MMP, matrix metalloproteinase; PBMC, peripheral blood mononuclear cell; antibody-dependent cell-mediated cytotoxicity; chimeric antibody; complement-dependent cytotoxicity; hinge region; mAb, monoclonal antibody.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / pharmacology
  • Antibodies, Monoclonal, Murine-Derived / immunology
  • Antibodies, Monoclonal, Murine-Derived / metabolism
  • Antibodies, Monoclonal, Murine-Derived / pharmacology
  • Antibody-Dependent Cell Cytotoxicity / drug effects
  • Antibody-Dependent Cell Cytotoxicity / immunology
  • Bacterial Proteins / metabolism
  • Blood Platelets / immunology
  • Blood Platelets / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Cysteine Endopeptidases / metabolism
  • Dogs
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / immunology
  • Epitopes / metabolism
  • Humans
  • Immunoglobulin Fab Fragments / immunology*
  • Immunoglobulin Fab Fragments / metabolism
  • Immunoglobulin Fab Fragments / pharmacology
  • Immunoglobulin G / immunology*
  • Immunoglobulin G / metabolism
  • Matrix Metalloproteinase 3 / metabolism
  • Platelet Count
  • Proteolysis
  • Rats
  • Rituximab

Substances

  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Murine-Derived
  • Bacterial Proteins
  • Epitopes
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • Mac-1-like protein, Streptococcus
  • Rituximab
  • Cysteine Endopeptidases
  • Matrix Metalloproteinase 3