Site-specific N-glycan Analysis of Antibody-binding Fc γ Receptors from Primary Human Monocytes

Abstract

FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ∼20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145).

Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity

“…By far the most important contributor to antibody ADCC activity studied so far is antibody subclass and glycosylation. While the basis of subclass remains somewhat of a mystery, we do have substantial evidence as to the importance of Fc glycosylation (43,47,49,105,107,108). Specifically, if the glycan at Asn 297 is fucosylated, then the binding to FcγRIIIa is impaired (43,(47)(48)(49)109).…”

“…With removal of this core fucose, however, affinity is bolstered to the low nanomolar level. Further affinity can be gained from di-sialylated, complex glycans lacking core fucose, which also have strong anti-inflammatory properties (107,108,110). Nuclear magnetic resonance (NMR) studies suggest that the type of glycan attached to the Fc modulates Fc dynamics as well through the C'E loop that contains Asn 297 (111,112).…”

“…Nuclear magnetic resonance (NMR) studies suggest that the type of glycan attached to the Fc modulates Fc dynamics as well through the C'E loop that contains Asn 297 (111,112). Conversely, FcγRIIIa glycosylation itself can also influence binding of IgG, hinging primarily upon a single glycan at Asn 162 (107,108,110,113). Indeed, there are vast donor-specific differences in monocyte derived FcγR glycoforms that could influence the effectiveness of antibody therapies as well as donor-derived cell therapies (110).…”

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development

“…N38 and N74 -Two N-glycosylated loops opposite the antibody-binding site display the most highly processed sites on CD16a expressed by primary NK cells and monocytes (33,41). These are predominantly tetraantennary complex-type N-glycans (containing four branches) with four sialic acid residues plus the addition of N-acetyllactosamine (LacNAc) repeats that extend the complex-type branches (Figure 2).…”

Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors

“…These recent glycoproteomics studies focused on healthy volunteers. They revealed FcγRIIIa glycosylation of NK cells 29 and monocytes 30 and FcγRIIIb glycosylation of neutrophils 31 . The basis was purification of the cells by negative selection with magnetic beads, followed by immunoprecipitation of FcγR.…”