Engineered antibody Fc variant with selectively enhanced FcRIIb binding over both FcRIIaR131 and FcRIIaH131

Engaging inhibitory FcRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcRIIb, also enhances binding affinity to FcRIIa(R131) allotype to a similar degree because FcRIIb and FcRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcRIIb complex to identify a novel Fc variant with selectively enhanced FcRIIb binding over both FcRIIa(R131) and FcRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcRIIb than wild-type IgG1, while binding affinity to FcRIIa(R131) and FcRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the C(H)2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcRIIb and FcRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.