Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

Abstract

Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fc gamma receptors or blocking antibodies that disrupt inhibitory SIRP-CD47 engagement. Yet how these competing signals are integrated is poorly understood mechanistically, raising questions about how to effectively titrate immune responses. Here we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand on targets over a broad range of absolute molecular densities. Using endogenous as well as chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering this ratio reduces Fc{gamma}R phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRP. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRP in vitro, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.