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

Abstract

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 {gamma} receptors (Fc{gamma}Rs) through their Fc regions. These Fc{gamma}R interactions initiate and modulate antibody-mediated effector functions that are essential for host immunity, therapeutic monoclonal antibody effectiveness and IgG-mediated pathologies. Fc{gamma}Rs include both activating and inhibitory receptors and the relative binding affinities of the IgG Fc region to Fc{gamma}Rs that generate opposing signals is a key determinant of the immune response. Substantial research effort has been devoted to understanding and manipulating Fc{gamma}R interactions to decipher their fundamental biological activities and to develop therapeutic monoclonal antibodies with tailored effector functions. However, a common Fc-Fc{gamma}R binding interface, the high sequence identity of Fc{gamma}Rs, 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 Fc{gamma}Rs, as well as the most common Fc{gamma}R polymorphisms. We provide a comprehensive analysis of Fc amino acid variations that determine Fc stability, orthosteric control of Fc{gamma}R binding, and short- and long-range allosteric control of Fc{gamma}R binding. We also predict the relative activating versus inhibitory effector function capacity of nearly every possible single-site Fc mutation.