Engineering TME-activated CD47-specific CAR macrophage via Arg1 promoter for safe and effective solid tumor immunotherapy.
BACKGROUND: Chimeric antigen receptor macrophage (CAR-Mφ) therapy has promising therapeutic potential in solid tumors, yet challenges remain in target compatibility and systemic toxicity. METHODS: In this study, we screened the CD47-scFv sequence of CAR-Mφ as the extracellular structure. We then constructed a classical CD47 CAR-Mφ incorporated the costimulatory domain of the α1β1 integrin-mediated Fc-gamma receptor I (FcγRI) signaling component. Subsequently, we developed a tumor microenvironment (TME)-responsive CAR macrophage platform by the arginase 1 (Arg1) promoter to target CD47, a highly expressed but clinically challenging immune checkpoint in solid tumors. RESULTS: We found that anti-CD47-scFv-mediated macrophages can effectively kill tumor cells both in vivo and in vitro. Furthermore, by integrating an α1β1 integrin-mediated FcγRI signaling domain, CD47 CAR-Mφ exhibited superior antitumor activity in hCD47+4T1 and SGC-7901 cells in vitro, which demonstrated that the CD47 CAR-Mφ was effective against solid tumors. Subsequently, Arg1-mediated activated pArg1 CD47 CAR-Mφ exhibited strong cytotoxicity against target cancer cells. We further demonstrated TME-controllable CAR gene expression in situ and induced a significant regression of established tumors in vivo. Besides, TME-dependent activation of CD47 CAR Mφ reduced the cytotoxic killing effect on erythrocytes. CONCLUSIONS: Our findings confirmed that the TME-specific activation mechanism of pArg1 CD47 CAR-Mφ based on intrinsic Arg1 promoter reprogramming endowed CAR-Mφ to effectively mitigate erythrocyte toxicity while enabling safe multidose administration regimens. This Trojan horse-like CAR-Mφ system achieves tumor-specific activation while minimizing systemic toxicity, offering a novel strategy to expand CAR-Mφ applications for solid tumors.