Reduction of disulfide bonds within anti‐D results in enhanced Fcγ receptor blockade

Abstract

We have investigated whether chemicals known to disrupt disulfide bonds are capable of altering immunoglobulin anti-D structure resulting in an increased efficacy of the chemically modified anti-D to inhibit Fcgamma receptor (FcgammaR)-mediated phagocytosis. If successful, this would provide a rationale to explore this mechanism of enhancing FcgammaR blockade for future use in immunoglobulin therapies for immune cytopenias. Anti-D that was shown to block 50 percent of the FcgammaR-mediated phagocytosis of opsonized red blood cells (RBCs) using a monocyte monolayer assay (MMA) was combined with two different thiol-containing compounds, dithiothreitol (DTT) or p-toluenesulfonylmethyl mercaptan, with or without treatment with iodoacetamide, and allowed to react. Excess chemical was removed by extensive dialysis. FcgammaR blockade was assessed by MMA with dialyzed, untreated, or chemically treated anti-D using both D+ and D- opsonized target RBCs. Toxicity was determined by fluorescence-activated cell sorting. Aggregates and oligomerization of chemically treated anti-D were examined using gel filtration-high pressure liquid chromatography. Using disulfide-reducing compounds to chemically modify anti-D significantly increases the efficacy of the anti-D to induce an FcgammaR blockade and decrease phagocytosis in vitro of opsonized D+ or D- RBCs. This effect was shown not due to unbound residual chemical, toxicity, or formation of immunoglobulin G oligomers. S-alkylation was required when using low concentrations of reducing compound. Our results demonstrate that irreversible reduction of interchain disulfide bonds within the immunoglobulin anti-D results in a significantly increased efficacy to inhibit FcgammaR-mediated phagocytosis regardless of opsonized target cell. With the use of this strategy, more effective and less expensive immunoglobulin treatment for immune cytopenias such as immune thrombocytopenic purpura or autoimmune hemolytic anemia may be developed.