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

Fc gamma receptors (FcR) 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 FcRs 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 FcR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fc 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 FcR were characterized and we discuss the CHO-FcRIIIa_{Phe158/Val158} and CHO-FcRI 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 FcR glycosylation. The glycan profiles of CHO expressed FcRI and FcRIIIa_{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 FcR binding patterns with rituximab. Oligomannose structures are prevalent on FcRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcRI and FcRIIIa 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-FcR binding kinetics differ depending on the glycosylation of the FcR and further support a stabilizing role of FcR glycans in the antibody binding interaction.

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