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.

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.

Figure 1.

Generation of a monoclonal antibody specific for IdeS cleavage site in the lower hinge of human IgG1. (A) The fully rabbit anti-hinge mAb 91–2 bound to IdeS-generated F(ab’)₂ fragments, but not to intact IgG1 and F(ab’)₂ fragments generated with MMP-3 or GluV8 in a plate-based ELISA assay. Data are representative of two independent experiments (duplicate measurements per experiment). (B) CDC activity was measured using rituximab IgG1 (black circles), rituximab IgG1 F(ab’)_{2 IdeS} (solid gray up triangles), and a fixed concentration of rituximab IgG1 F(ab’)_{2 IdeS} (1 μg/ml) in the presence of serially-diluted 91–2 (solid red down triangles). Data are representative of two independent experiments (triplicate measurements per experiment). (C) The rabbit/human chimeric anti-hinge mAb 2095–2 displayed the same specificity as the fully rabbit mAb 91–2. Data are representative of two independent experiments (duplicate measurements per experiment).

Figure 2.

2095–2 specificity on cell-bound F(ab’)₂ fragments and ADCC/CDC restoration of function. (A) F(ab’)₂ fragments of rituximab generated with IdeS (solid black circles), GluV8 (solid black up triangles), and MMP-3 (solid black squares) displayed comparable binding to WIL2-S cells. (B) The anti-hinge mAb 2095–2 bound to cells opsonized with IdeS-generated F(ab’)₂ fragments (solid black circles), but not to GluV8-generated F(ab’)₂ fragments (solid black up triangles) or MMP-3-generated (solid black squares). Data from (A) and (B) are representative of two independent experiments. (C) The anti-hinge mAb 2095–2 was capable of restoring ADCC function to IdeS-generated F(ab’)₂ fragments (open up red triangles), but not to GluV8-generated F(ab’)₂ fragments (open green diamonds) or MMP-3-generated F(ab’)₂ fragments (open down blue triangles). Addition of 2095–2 alone (solid gray squares) or F(ab’)_{2 IdeS} fragments (solid black up triangles), F(ab’)_{2 GluV8} fragments (solid black diamonds), or F(ab’)_{2 MMP-3} fragments (solid black down triangles) alone did not result in appreciable ADCC activity. Data are representative of 2 independent experiments (duplicate measurements per experiment). (D) The anti-hinge mAb 2095–2 was capable of restoring CDC function to anti-CD20 IdeS-generated F(ab’)₂ fragments (open up red triangles), but not to GluV8-generated F(ab’)₂ fragments (open green diamonds) or MMP-3-generated F(ab’)₂ fragments (open down blue triangles). Addition of 2095–2 alone (solid gray squares) or F(ab’)_{2 IdeS} fragments (solid black up triangles), F(ab’)_{2 GluV8} fragments (solid black diamonds), or F(ab’)_{2 MMP-3} fragments (solid black down triangles) alone did not result in appreciable CDC activity. Data are representative of 2 independent experiments (triplicate measurements per experiment).

Figure 3.

2095–2 restores ADCC and CDC function to both IdeS-generated IgG1 F(ab’)₂ fragments and IgG4 F(ab’)₂ fragments, but not to IgG2 F(ab’)₂ fragments. (A) The intact anti-CD142 IgG1 mAb elicited ADCC against MDA-MB-231 cells (solid black circles), whereas anti-CD142 IgG2 (solid green squares), anti-CD142 IgG4 (solid blue diamonds), and 2095–2 alone (solid gray up diamonds) showed minimal ADCC activity. (B) Anti-CD142 IdeS-generated F(ab’)₂ fragments of IgG1 (solid black circles), IgG2 (solid green squares), and IgG4 (solid blue diamonds) did not elicit detectable ADCC. A fixed concentration of 3 μg/ml of 2095–2 restored ADCC to anti-CD142 IdeS-generated F(ab’)₂ fragments of IgG1 (open red circles) and IgG4 (open blue diamonds). Data in (A) and (B) are representative of three independent experiments (duplicate measurements per experiment). (C) The intact anti-CD20 IgG1 mAb elicited CDC against WIL2-S cells (solid black circles), whereas anti-CD20 IgG2 (solid green squares), anti-CD20 IgG4 (solid blue diamonds), and 2095–2 alone (solid gray up diamonds) displayed minimal CDC activity. (D) Anti-CD20 IdeS-generated F(ab’)₂ fragments of IgG1 (solid black circles), IgG2 (solid green squares), and IgG4 (solid blue diamonds) did not elicit detectable CDC. A fixed concentration of 5 μg/ml of 2095–2 restored CDC to anti-CD20 IdeS-generated F(ab’)₂ fragments of IgG1 (open red circles) and IgG4 (open blue diamonds). Data in (C) and (D) are representative of two independent experiments (triplicate measurements per experiment).

Figure 4.

2095–2 restores in vivo platelet depletion to IdeS-generated F(ab’)₂ fragments of c7E3 in canines. Treatment of canines with saline (open black diamonds), 0.05 mg/kg of 2095–2 alone (gray up triangles), and 0.05 mg/kg of IdeS-generated F(ab’)₂ fragments of c7E3 alone (solid brown squares) did not result in platelet depletion, whereas treatment with 0.05 mg/kg of intact c7E3 (solid black circles) resulted in platelet depletion. The combined treatment of 0.05 mg/kg of c7E3 F(ab’)_{2 IdeS} and 0.5 mg/kg of 2095–2 (open red circles) resulted in rapid platelet clearance. Data are representative of the average +/− SEM of three animals per group.

Figure 5.

2095–2 amplifies in vivo platelet depletion to IdeS-generated F(ab’)₂ fragments of c7E3 in rats compared with intact c7E3. (A) Treatment of rats with PBS (solid gray squares), 1 mg/kg of c7E3 (solid black circles), or 3 mg/kg of 2095–2 (solid red down triangles) did not result in platelet depletion, whereas treatment with 3 mg/kg of c7E3 (solid blue up triangles) displayed detectable platelet depletion. (B) Treatment of rats with 1 mg/kg of c7E3 F(ab’)₂ fragment did not result in platelet depletion (solid gray squares), whereas the combination of 1 mg/kg of c7E3 F(ab’)₂ fragment and 0.1 mg/kg of 2095–2 (solid black diamonds), 0.33 mg/kg of 2095–2 (solid red down triangles), 1 mg/kg of 2095–2 (solid blue up triangles), or 3 mg/kg of 2095–2 (solid black circles) resulted in a dose-dependent increase in platelet depletion. Data are representative of the average +/− SEM of three animals per group.