Modifications to an Fc?-Fc? fusion protein alter its effectiveness in the inhibition of Fc?RI-mediated functions

Background GE2, a human bifunctional Fc?-Fc fusion protein cross-links Fc?RIIb and FcRI on human mast cells and basophils and results in inhibition of FcRI-mediated functions. Objective Three modified Fc?-Fc (GE) proteins were compared with GE2 for their effect on inhibition of FcRI-mediated cellular responses. Methods GE2 was modified to potentially improve its therapeutic efficacy by increasing binding to Fc?RIIb (GE S mutant) and decreasing binding to Fc?RIII (GE H mutant) or reversing the Fc? and Fc domains and removing nonhuman linker sequences (E2G). These proteins were tested for their ability to bind a basophil-like cell line, block FcRI-mediated degranulation in human basophils, and inhibit passive cutaneous anaphylaxis in human FcRIa-transgenic mice. Results All 4 GE proteins bound cells that express FcRI and Fc?RIIb, although the original GE2 retained the strongest ability to bind to these cells. E2G was as effective as GE2 in its ability to inhibit anti-Fel d 1 IgE-mediated histamine release from human basophils and block passive cutaneous anaphylaxis reactions. The GE S and GE H mutants were less effective. Conclusion Optimization of GE2 as an inhibitor of FcRI-mediated functions showed that effectiveness was maintained when potentially immunogenic linker sequences were removed and Ig domain positions were reversed, but specific residue changes within the IgG CH2 domain aimed at enhancing GE2's inhibitory function by increasing Fc?RII binding or additionally decreasing Fc?RIII binding were not beneficial. Clinical implications GE2 and E2G molecules are effective inhibitors of FcRI-mediated degranulation and are of interest as potential therapeutics for IgE-mediated allergic reactions