用嵌合抗原受体（CAR）改造的巨噬细胞因其免疫调节活性和浸润实体瘤的能力而适用于免疫治疗。然而，基因编辑、高效、大规模生产的CAR修饰巨噬细胞（CAR-Ms）的生产和应用具有挑战性。在这里，我们使用同源独立靶向插入（HITI）将CAR定点整合到安全的载体中。 -人类多能干细胞（hPSC）的港口区域。这种方法与简单的分化方案相结合，产生了稳定且高效且无异质性的 CAR-M。这些工程细胞吞噬癌细胞，从而在体外和体内显着抑制癌细胞增殖。此外，工程化的CAR结合了CD3ze和Megf10（称为FRP5Mze），通过促进M1而非M2极化，显着增强了CAR-M的抗肿瘤作用。 FRP5Mze 通过核因子 kappa B (NF-κB)、ERK 和 STAT1 信号传导促进 M1 极化，并同时抑制 STAT3 信号传导，即使在 M2 条件下也是如此。 CAR-Ms 的这些特性通过激活炎症信号传导、诱导旁观者非 CAR 巨噬细胞的 M1 极化以及增强癌症球体中 T 细胞的浸润来调节肿瘤微环境。我们的研究结果表明 CAR-Ms 作为免疫治疗药物具有前景。总之，引导插入包含 CD3z 和 Megf10 结构域的 CAR 是实体瘤免疫治疗的有效策略。这项工作得到了 KRIBB 研究计划资助 (KGM4562431、KGM5282423) 和韩国再生医学基金会 (KFRM) 资助的支持由韩国政府（科学和信息通信技术部、卫生和福利部）资助（22A0304L1-01）。版权所有 © 2024 作者。由 Elsevier B.V. 出版。保留所有权利。

Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging.Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity.These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids.Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours.This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.