In situ generation of proinflammatory CAR macrophages via mRNA-TLR agonist co-delivery for triple-negative breast cancer immunotherapy

BACKGROUND: Chimeric antigen receptor (CAR) macrophage therapy shows significant potential for solid tumors owing to the intrinsic tumor infiltration and phagocytic capacity of macrophages. However, its clinical translation is limited by macrophage phenotypic plasticity within the immunosuppressive tumor microenvironment and the complexity of ex vivo cell manufacturing. It is essential to develop techniques that enable macrophages to be activated specifically by antigens while sustaining their proinflammatory activity in vivo. METHODS: Here, we report a mannose-modified lipid nanoparticle (LNP) platform for the co-delivery of CAR-encoding messenger RNA (mRNA) and the Toll-like receptor (TLR) 7/8 agonist resiquimod (R848), enabling in situ generation of proinflammatory CAR macrophages. In vitro, we assessed macrophage-preferential uptake, CAR expression efficiency, TLR7/8 agonist-mediated macrophage polarization, and immune activation. In vivo efficacy was assessed in syngeneic and humanized mouse models of triple-negative breast cancer, including postoperative recurrence and lung metastasis models. RESULTS: Systemic administration of M-LNP/CAR+R848 induced robust CAR expression in tumor-associated macrophages and promoted sustained M1 polarization. Engineered macrophages exhibited enhanced antigen-specific phagocytic activity and tumor cell clearance, and promoted CD8+ T cell proliferation and NK cell infiltration, thus coordinating innate and adaptive immune responses. Functional macrophage depletion experiments demonstrated that tumor control was dependent on macrophages. In vivo treatment significantly reduced the growth of primary tumors, prevented postoperative recurrence, and prolonged survival in mice with lung metastases in both syngeneic and humanized models. CONCLUSIONS: Our findings demonstrate that M-LNPs enabling co-delivery of mRNA and an innate immune agonist enable in situ generation of proinflammatory CAR macrophages and induce durable antitumor immunity. This controllable and non-integrative strategy allows tunable immune activation, provides a flexible platform for CAR macrophage-based immunotherapy in triple-negative breast cancer.