Mechanistic Basis for the Selective Recognition of the Fcγ Receptor IIa by Monoclonal Antibody IV.3

The monoclonal antibody IV.3 selectively binds the platelet Fcγ receptor IIa (FcγRIIa), potently blocking immune complex engagement without cross-reacting with the closely-related FcγRIIb. This specificity has made IV.3 invaluable for dissecting FcγRIIa-mediated activation in diverse conditions, including infection, autoimmunity, thromboinflammation, and platelet-mediated thrombosis. We combined cryogenic electron microscopy, surface plasmon resonance, alchemical free energy calculations, and molecular dynamics simulations to elucidate IV.3's binding sites on FcγRIIa and the mechanistic basis of IV.3 specificity. We find that IV.3 engages a broader FcγRIIa epitope than previously recognized, extending beyond residues H/R134 and L135 (R and S in FcγRIIb). Simulations of FcγIIa-R134 variants bearing either L135 or S135 reveal that IV.3 specificity arises from hydrophobic stabilization mediated by L135 and disruption of an R134-specific interaction network in the presence of S135. These findings provide a mechanistic framework for rational design of FcγRIIa-targeted therapeutics.