Glycosylation of Fcγ receptors influences their interaction with various IgG1 glycoforms.

Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIa/b and FcγRIIIa) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.