Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, Macaca mulatta (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile158 and Val158 variants. Here we describe structure-function relationships of the Ile/Val158 polymorphism in Mm FcγRIII. Our data indicate that the affinity of the allelic variants of Mm FcγRIII for the macaque IgG subclasses vary greatly with changes in glycan composition both on the Fc and the receptor. However, unlike the human Phe/Val158 polymorphism in FcγRIIIa, the higher affinity variant corresponds to the larger, more hydrophobic side chain, Ile, even though it is not directly involved in the binding interface. Instead, this side chain appears to modulate glycan-glycan interactions at the Fc/FcγRIII interface. Furthermore, changes in glycan composition on the receptor have a greater effect for the Val158 variant such that with oligomannose type glycans and with glycans only on Asn45 and Asn162, Val158 becomes the variant with higher affinity to Fc. These results have implications not only for the better interpretation of nonhuman primate studies but also for studies performed with human effector cells carrying different FcγRIIIa alleles.

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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.

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