Abstract

BACKGROUND Chimeric antigen receptor T-cell (CAR-T) therapy shows limited efficacy against solid tumors due to the immunosuppressive tumor microenvironment (TME). Macrophages possess superior infiltration capabilities, yet their therapeutic potential remains under-realized. METHODS We engineered a synNotch-iNOS CAR-macrophage (CAR iNOS-M) that releases nitric oxide (NO) upon CD19 recognition. Its efficacy was evaluated in syngeneic, immunocompetent murine models of metastatic melanoma. RESULTS CAR iNOS-M therapy effectively reprogrammed the pulmonary tumor immune microenvironment (TIME), inducing potent antitumor responses independent of CD8+ T cells but strictly dependent on CD4+ T cells. Mechanistically, CAR iNOS-M treatment led to a significant reduction in pro-tumorigenic lung interstitial macrophages (IMs), subsequently decreasing platelet factor 4 (PF4) levels. This disruption of the PF4 signaling axis inhibited the polarization of immunosuppressive Th1-Tregs, alleviating T-cell exhaustion. CONCLUSIONS This study delineates a novel indirect mechanism for CAR-M, shifting the focus from direct phagocytosis to strategic TIME remodeling, providing a foundation for treating solid tumors.