The antibody-binding Fc gamma receptor IIIa / CD16a is N-glycosylated with high occupancy at all five sites.

The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40-70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65-100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site.