Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fc gamma receptors (Fc gamma R). Although an early goal has been enhanced Fc gamma RIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as Fc gamma RIIa. Here, we describe a set of engineered Fc variants with diverse Fc gamma R affinities, including a novel substitution G236A that provides selectively enhanced binding to Fc gamma RIIa relative to FcyRIIb. Variants containing this substitution have up to 70-fold greater Fc gamma RIIa affinity and 15-fold improvement in Fc gamma RIIa/Fc gamma RIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual Fc gamma R to effector functions mediated by NK cells and macrophages. These experiments show that Fc gamma RIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor Fc gamma RIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.