Antibody binding geometry and affinity control inhibitory hFcγRIIB receptor signaling.

The inhibitory human Fc gamma receptor, hFcγRIIB, is a key mediator of humoral immunity and regulator of antibody-mediated effector function. hFcγRIIB function can be modulated by anti-hFcγRIIB antibodies. We demonstrate that agonistic, but not antagonistic, antibodies reduce hFcγRIIB mobility in the plasma membrane, associated with receptor clustering and redistribution into lipid rafts. Agonists display lower affinity binding with higher off rates compared with antagonists. Using crystallographic structure determination and alanine-scanning mutagenesis, we show that epitopes targeted by agonistic and antagonistic antibodies are overlapping but distinct. Using small-angle X-ray scattering (SAXS) and molecular dynamics simulations, we demonstrate that agonists nucleate more compact receptor complexes. Through their high off rates, we propose that agonists facilitate a catch-and-release mechanism that promotes receptor clustering and subsequent activation. By contrast, antagonists adopt a binding geometry that prevents effective clustering, with their low off rate reducing receptor disengagement and subsequent clustering. These findings provide key principles underpinning agonism versus antagonism of immunomodulatory receptors.