Engineered macrophages with IL-10-TLR9 signal switch receptors for reprogramming tumor microenvironment and enhancing antitumor immunity.

Chimeric antigen receptor (CAR) T cell immunotherapy has achieved remarkable success in hematologic malignancies, prompting the exploration of CAR strategies in solid tumors. Here, we developed a CAR-like signal-switching receptor-macrophage (SR CAR-M) that recognizes IL-10, a major immunosuppressive cytokine enriched in solid tumors, and converts this inhibitory signal into a pro-inflammatory activation through the Toll-like receptor 9 (TLR9) intracellular signaling pathway. SR CAR-Ms effectively blocked IL-10-mediated STAT3 phosphorylation while activating TLR9 downstream signaling, including nuclear translocation of nuclear factor-κB and upregulation of IRF1, thereby adopting an M1-like phenotype with enhanced phagocytic capacity and tumor cytotoxicity. Domain-deletion controls demonstrated that both IL-10 binding and TLR9 signaling are indispensable. In addition, SR CAR-Ms promoted dendritic cell maturation, enhanced T cell proliferation and effector function, and prevented T cell exhaustion. In an orthotopic 4T1 breast cancer model, infused SR CAR-Ms selectively accumulated in tumors, depleted local IL-10 while inducing inflammatory cytokine production, suppressed tumor growth, and prolonged survival without systemic toxicity. The signal-switching mechanism was validated in primary bone marrow-derived macrophages and human monocyte-derived macrophages, supporting the translational applicability. Further enhancement was achieved by engineering dual-function SRPα CAR-Ms secreting anti-PD-L1 antibodies, which outperformed either SR CAR-M or anti-PD-L1 monotherapy. The SR CAR-M platform transforms an immunosuppressive cytokine into a location-specific activation trigger, simultaneously depleting the inhibitory signal and remodeling the tumor microenvironment. This signal-switching paradigm offers a versatile approach for treating solid malignancies that are resistant to conventional immunotherapies.