Exploitable mechanisms of antibody and CAR mediated macrophage cytotoxicity.

Macrophages infiltrate solid tumors and either support survival or induce cancer cell death through phagocytosis or cytotoxicity. To uncover regulators of macrophage cytotoxicity towards cancer cells, we perform two co-culture CRISPR screens using CAR-macrophages targeting different tumor associated antigens. Both identify ATG9A as an important regulator of this cytotoxic activity. In vitro and in vivo, ATG9A depletion in cancer cells sensitizes them to macrophage-mediated killing. Proteomic and lipidomic analyses reveal that ATG9A deficiency impairs the cancer cell response to macrophage-induced plasma membrane damage through defective lysosomal exocytosis, reduced ceramide production, and disrupted caveolar endocytosis. Depleting non-cytotoxic macrophages using CSF1R inhibition while preventing ATG9A-mediated tumor membrane repair enhances the anti-tumor activity of therapeutic antibodies in mice. Thus, macrophage cytotoxicity plays an important role in tumor elimination during antibody or CAR-macrophage treatment, and inhibiting tumor membrane repair via ATG9A, particularly in combination with cytotoxic macrophage enrichment through CSF1R inhibition, improves tumor-targeting macrophage efficacy.