Effective production of highly functional car-macrophages from human pluripotent stem cells for cancer immunotherapy

Generation of chimeric antigen receptor macrophages (CAR-M) from human pluripotent stem cells (hPSCs) holds new prospects for tumor immunotherapy, but currently is challenged by low differentiation efficiency and limited function. Here, we develop a highly efficient monolayer system that increases the production of CAR-M by more than 100 times, enabling a single hPSC to produce up to 6000 CAR-M within 3 weeks. By optimizing CAR structure, we generate hPSC-CAR-M with stable CAR expression and potent tumoricidal activity in vitro. To overcome the lost tumoricidal activity of hPSC-CAR-M in vivo, we use interferon-γ and toll-like receptor 4 agonist to activate innate immunity in vivo that improves the antitumor potential of hPSC-CAR-M. Moreover, we demonstrate that activating a collaborative innate-adaptive immune response can further enhance the anti-tumor effect of hPSC-CAR-M in vivo. Collectively, our study provides feasible methodologies that significantly improve the production and antitumor activity of hPSC-CAR-M to support the translation of hPSC-based immunotherapies to clinical applications. Generation of chimeric antigen receptor macrophages (CAR-M) from human pluripotent stem cells (hPSCs) holds new prospects for tumor immunotherapy, but currently is challenged by low differentiation efficiency and limited function. Here, we develop a highly efficient monolayer system that increases the production of CAR-M by more than 100 times, enabling a single hPSC to produce up to 6000 CAR-M within 3 weeks. By optimizing CAR structure, we generate hPSC-CAR-M with stable CAR expression and potent tumoricidal activity in vitro. To overcome the lost tumoricidal activity of hPSC-CAR-M in vivo, we use interferon-γ and toll-like receptor 4 agonist to activate innate immunity in vivo that improves the antitumor potential of hPSC-CAR-M. Moreover, we demonstrate that activating a collaborative innate-adaptive immune response can further enhance the anti-tumor effect of hPSC-CAR-M in vivo. Collectively, our study provides feasible methodologies that significantly improve the production and antitumor activity of hPSC-CAR-M to support the translation of hPSC-based immunotherapies to clinical applications.