Fc Gamma Receptor Glycosylation Modulates the Binding of IgG Glycoforms: A Requirement for Stable Antibody Interactions

Fc gamma Rs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of Fc gamma R glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant Fc gamma Rs (Fc gamma RIa, Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIIaPhe158/Val158, and Fc gamma RIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for Fc gamma RIa and Fc gamma RIIIa. The IgG-Fc gamma R interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated Fc gamma RIa and Fc gamma RIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated Fc gamma RIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to Fc gamma RIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-Fc gamma R interaction and the influence of Fc gamma R glycosylation.