GE2, a human bifunctional Fcγ-Fcɛ fusion protein cross-links FcγRIIb and FcɛRI on human mast cells and basophils and results in inhibition of FcɛRI-mediated functions.Three modified Fcγ-Fcɛ (GE) proteins were compared with GE2 for their effect on inhibition of FcɛRI-mediated cellular responses.GE2 was modified to potentially improve its therapeutic efficacy by increasing binding to FcγRIIb (GE S mutant) and decreasing binding to FcγRIII (GE H mutant) or reversing the Fcγ and Fcɛ domains and removing nonhuman linker sequences (E2G). These proteins were tested for their ability to bind a basophil-like cell line, block FcɛRI-mediated degranulation in human basophils, and inhibit passive cutaneous anaphylaxis in human FcɛRIα-transgenic mice.All 4 GE proteins bound cells that express FcɛRI and FcγRIIb, 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.Optimization of GE2 as an inhibitor of FcɛRI-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 C H 2 domain aimed at enhancing GE2's inhibitory function by increasing FcγRII binding or additionally decreasing FcγRIII binding were not beneficial.GE2 and E2G molecules are effective inhibitors of FcɛRI-mediated degranulation and are of interest as potential therapeutics for IgE-mediated allergic reactions.