Activating innate immune responses repolarizes hPSC-derived CAR macrophages to improve anti-tumor activity.

Generation of chimeric antigen receptor macrophages (CAR-Ms) from human pluripotent stem cells (hPSCs) offers new prospects for cancer immunotherapy but is currently challenged by low differentiation efficiency and limited function. Here, we develop a highly efficient monolayer-based system that can produce around 6,000 macrophages from a single hPSC within 3 weeks. Based on CAR structure screening, we generate hPSC-CAR-Ms with stable CAR expression and potent tumoricidal activity in vitro. To overcome the loss of tumoricidal activity of hPSC-CAR-Ms in vivo, we use interferon-γ and monophosphoryl lipid A to activate an innate immune response that repolarizes the hPSC-CAR-Ms to tumoricidal macrophages. Moreover, through combined activation of T cells by hPSC-CAR-Ms, we demonstrate that activating a collaborative innate-adaptive immune response can further enhance the anti-tumor effect of hPSC-CAR-Ms in vivo. Collectively, our study provides feasible methodologies that significantly improve the production and function of hPSC-CAR-Ms to support their translation into clinical applications.