Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel monoclonal antibody (mAb) biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated mAbs demonstrate increased affinity towards the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity (ADCC). Crystal structure analysis has led to the hypothesis that afucosylation in the Fc-region results in reduced steric hindrance between antibody-receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data has yet to corroborate this hypothesis. In addition, recent work has shown that the antigen-binding region (Fab) may directly interact with Fc receptors; however, the biological consequences of these interactions remains unclear. By probing differences in solvent accessibility between native and afucoslyated IgG1 using hydroxyl radical footprinting (HRF)-mass spectrometry, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc-region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamic (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc-fucose in IgG1, and corroborates previously proposed Fab-receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab-receptor interactions directly contribute to the modulation of ADCC activity. This work demonstrates that in addition to Fc-polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG-Fc receptor biology.

Keywords: Antibody-Dependent Cellular Cytotoxicity; Fab-Fc Receptor Interactions; Fast Photochemical Oxidation of Proteins (FPOP); FcγRIIIa; Hydroxyl Radical Footprinting-Mass Spectrometry; IgG1; Molecular Dynamics; Monoclonal Antibody; afucosylation.