Modifications to an Fcγ-Fcε fusion protein alter its effectiveness in the inhibition of FcεRI-mediated functions

Background: GE2, a human bifunctional Fc gamma-Fc epsilon fusion protein cross-links Fc gamma RIIb and Fc epsilon RI on human mast cells and basophils and results in inhibition of Fc epsilon RI-mediated functions. Objective: Three modified Fc gamma-Fc epsilon: (GE) proteins were compared with GE2 for their effect on inhibition of Fc epsilon RI-mediated cellular responses. Methods: GE2 was modified to potentially improve its therapeutic efficacy by increasing binding to Fc gamma RIIb (GE S mutant) and decreasing binding to Fc gamma RIII (GE H mutant) or reversing the Fc gamma and Fc epsilon domains and removing nonhuman linker sequences (E2G). These proteins were tested for their ability to bind a basophil-like cell line, block Fc epsilon RI-mediated degranulation in human basophils, and inhibit passive cutaneous anaphylaxis in human Fc epsilon RI alpha-transgenic mice. Results: All 4 GE proteins bound cells that express Fc epsilon RI and Fc gamma 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 Fc epsilon RI-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 Fc gamma RII binding or additionally decreasing Fc gamma RIII binding were not beneficial. Clinical implications: GE2 and E2G molecules are effective inhibitors of Fc epsilon RI-mediated degranulation and are of interest.