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

Brezski RJ, Kinder M, Grugan KD, Soring KL, Carton J, Greenplate AR, Petley T, Capaldi D, Brosnan K, Emmell E, Watson S, Jordan RE
MAbs. 2014 6 (5): 1265-73

PMID: 25517311 · PMCID: PMC4623506 · DOI:10.4161/mabs.29825

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.

MeSH Terms (20)

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

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