National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, 28116, Republic of Korea; Department of Nanoscience and Nanotechnology, Graduate School, Kyungpook National University, Daegu, 41566, Republic of Korea.
National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, 28116, Republic of Korea; Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, 06591, Republic of Korea; Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul, 06591, Republic of Korea.
EBioMedicine. 2024 Nov;109:105390. doi: 10.1016/j.ebiom.2024.105390. Epub 2024 Oct 9.
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 FRP5), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5 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).
嵌合抗原受体 (CAR) 修饰的巨噬细胞具有免疫调节活性和浸润实体瘤的能力，适合基于免疫的治疗。然而，生产和应用遗传编辑的、高效的、大规模生产的 CAR 修饰巨噬细胞 (CAR-M) 具有挑战性。
在这里，我们使用同源独立靶向插入 (HITI) 将 CAR 定点整合到人多能干细胞 (hPSC) 的安全港区域。这种方法与简单的分化方案相结合，产生了稳定且高效的 CAR-M，没有异质性。
这些工程化细胞吞噬癌细胞，导致体外和体内癌细胞增殖的显著抑制。此外，工程化的 CAR 包含 CD3ζ 和 Megf10 的组合（称为 FRP5），通过促进 M1，而不是 M2，极化，显著增强了 CAR-M 的抗肿瘤作用。FRP5 通过核因子 kappa B (NF-κB)、ERK 和 STAT1 信号促进 M1 极化，同时即使在 M2 条件下也抑制 STAT3 信号。CAR-M 的这些特征通过激活炎症信号、诱导旁观者非 CAR 巨噬细胞的 M1 极化以及增强 T 细胞在癌症球体中的浸润来调节肿瘤微环境。
我们的研究结果表明，CAR-M 具有作为免疫疗法的潜力。总之，CAR 包含 CD3ζ 和 Megf10 结构域的引导插入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（KFRM）资助，该基金由韩国政府（科学和信息通信技术部、卫生部和福利部）资助（22A0304L1-01）。
