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.

从人多能干细胞（hPSC）生成嵌合抗原受体巨噬细胞（CAR-Ms）为癌症免疫治疗提供了新的前景，但目前面临着分化效率低和功能有限的挑战。在这里，我们开发了一种高效的单层系统，可以在 3 周内从单个 hPSC 产生约 6,000 个巨噬细胞。基于CAR结构筛选，我们生成了具有稳定CAR表达和有效体外杀肿瘤活性的hPSC-CAR-Ms 。为了克服 hPSC-CAR-Ms体内抗肿瘤活性的丧失，我们使用干扰素-γ和单磷酰脂质 A 来激活先天免疫反应，使 hPSC-CAR-Ms 重新极化为抗肿瘤巨噬细胞。此外，通过hPSC-CAR-Ms联合激活T细胞，我们证明激活协同先天适应性免疫反应可以进一步增强hPSC-CAR-Ms在体内的抗肿瘤作用。总的来说，我们的研究提供了可行的方法，可显着改善 hPSC-CAR-M 的生产和功能，以支持其转化为临床应用。