Mapping affinity and allostery in human IgG antibody Fc region-Fc γ receptor interactions

IgG antibodies, required for a functional immune system, recognize antigens and neutralize pathogens using their Fab regions, while signaling to the immune system by binding to host Fc γ receptors (FcγRs) through their Fc regions. These FcγR interactions initiate and modulate antibody-mediated effector functions that are essential for host immunity, therapeutic monoclonal antibody effectiveness and IgG-mediated pathologies. FcγRs include both activating and inhibitory receptors and the relative binding affinities of the IgG Fc region to FcγRs that generate opposing signals is a key determinant of the immune response. Substantial research effort has been devoted to understanding and manipulating FcγR interactions to decipher their fundamental biological activities and to develop therapeutic monoclonal antibodies with tailored effector functions. However, a common Fc-FcγR binding interface, the high sequence identity of FcγRs, and the inherent conformational dynamics of the IgG Fc region, have prohibited a full understanding of these interactions, even when employing state-of-the-art biophysical and biological methods. Here, we used site-saturation libraries of the human IgG1 Fc region to determine the effective affinities of more than 98% of all possible single-site amino acid substitutions in the Fc to all human FcγRs, as well as the most common FcγR polymorphisms. We provide a comprehensive analysis of Fc amino acid variations that determine Fc stability, orthosteric control of FcγR binding, and short- and long-range allosteric control of FcγR binding. We also predict the relative activating versus inhibitory effector function capacity of nearly every possible single-site Fc mutation.