title: A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo.
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.
authors.affiliation: a Biologics Research, Janssen Research & Development, LLC , Spring House , PA , USA.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Brezski
authors.forename: Michelle
authors.initials: M
authors.lastname: Kinder
authors.forename: Katharine D
authors.initials: KD
authors.lastname: Grugan
authors.forename: Keri L
authors.initials: KL
authors.lastname: Soring
authors.forename: Jill
authors.initials: J
authors.lastname: Carton
authors.forename: Allison R
authors.initials: AR
authors.lastname: Greenplate
authors.forename: Theodore
authors.initials: T
authors.lastname: Petley
authors.forename: Dorie
authors.initials: D
authors.lastname: Capaldi
authors.forename: Kerry
authors.initials: K
authors.lastname: Brosnan
authors.forename: Eva
authors.initials: E
authors.lastname: Emmell
authors.forename: Sharon
authors.initials: S
authors.lastname: Watson
authors.forename: Robert E
authors.initials: RE
authors.lastname: Jordan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology; D001426:Bacterial Proteins / Q000378:metabolism; D001792:Blood Platelets / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D010976:Platelet Count; D059748:Proteolysis; D051381:Rats; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D001426:Bacterial Proteins; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; C442815:Mac-1-like protein, Streptococcus; D000069283:Rituximab; D003546:Cysteine Endopeptidases; D019278:Matrix Metalloproteinase 3
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