Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation Emily C. Suter, Eva M. Schmid, Erik Voets, Brian Francica, Daniel A. Fletcher Department of Bioengineering, University of California Berkeley, Berkeley CA, USA UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA, USA Aduro Biotech Europe, Oss, Netherlands Aduro Biotech Inc., Emeryville, CA, USA Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Chan Zuckerberg Biohub, San Francisco, CA, USA Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fc gamma receptors or blocking antibodies that disrupt inhibitory SIRPα-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 FcγR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα in vitro, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.