Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab

Fc gamma receptors (Fc gamma R) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of Fc gamma Rs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the Fc gamma R glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fc gamma receptors RI (CD64), RIIa(Arg131/ His131) (CD32a), RIIb (CD32b) and RIIIa(Phe158/Val158) (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each Fc gamma R were characterized and we discuss the CHO-Fc gamma RIIIaPhe158/Val158 and CHO-Fc gamma RI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent Fc gamma R glycosylation. The glycan profiles of CHO expressed Fc gamma RI and Fc gamma RIIIa Phe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective Fc gamma R binding patterns with rituximab. Oligomannose structures are prevalent on Fc gamma RI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On Fc gamma RI and Fc gamma RIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-Fc gamma R binding kinetics differ depending on the glycosylation of the Fc gamma R and further support a stabilizing role of Fc gamma R glycans in the antibody binding interaction.