Inhibiting N-glycan processing increases the antibody binding affinity and effector function of human natural killer cells.

Novel approaches are required to improve the efficacy of immunotherapies and increase the proportion of patients who experience a benefit. Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to the efficacy of many monoclonal antibodies therapies. Natural killer (NK) cells mediate ADCC, though responses are highly variable and depend on prior treatment as well as other factors. Thus, strategies to increase NK cell activity are expected to improve multiple therapies. Both cytokine treatment and NK cell receptor engineering are being explored to increase ADCC. Post-translational modifications, including glycosylation, are widely recognized as mediators of cellular processes but minimally explored as an alternative strategy to increase ADCC. We evaluated the impact of treatment with kifunensine, an inhibitor of asparagine-linked (N-)glycan processing, on ADCC using primary and cultured human NK cells. We also probed affinity using binding assays and CD16a structure with nuclear magnetic resonance spectroscopy. Treating primary human NK cells and cultured YTS-CD16a cells with kifunensine doubled ADCC in a CD16a-dependent manner. Kifunensine treatment also increased the antibody-binding affinity of CD16a on the NK cell surface. Structural interrogation identified a single CD16a region, proximal to the N162 glycan and the antibody-binding interface, perturbed by the N-glycan composition. The observed increase in NK cell activity following kifunensine treatment synergized with afucosylated antibodies, further increasing ADCC by an additional 33%. These results demonstrate native N-glycan processing is an important factor that limits NK cell ADCC. Furthermore, optimal antibody and CD16a glycoforms are defined that provide the greatest ADCC activity.

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.