固体肿瘤对嵌合抗原受体（CAR）T细胞治疗具有独特的阻碍作用，包括T细胞存留时间有限、肿瘤浸润效率低、以及免疫抑制性肿瘤微环境。迄今为止，克服这些障碍的尝试并不令人满意。本文报道一种策略，即将Runx3（编码RUNX家族转录因子3）过表达与外体蛋白激酶B（AKT）抑制相结合，以生成既具有中央记忆又具有组织驻留记忆特性的CAR-T细胞以克服这些障碍。我们生成了表达针对人类碳酸酐酶9的二代小鼠CAR-T细胞，配合Runx3过表达并在AKTi-1/2的存在下扩增，这是AKT1/AKT2的选择性和可逆抑制剂。我们利用流式细胞术、转录组学分析和质谱细胞术探索了AKT抑制剂（AKTi）、Runx3过表达及其组合对CAR-T细胞表型的影响。在皮下胰腺导管腺癌（PDAC）肿瘤模型中评估了CAR-T细胞的存留、肿瘤浸润和抗肿瘤功效。AKTi生成了CD62L+中央记忆型CAR-T细胞群体，具有增强的存活时间，但也可促进其细胞毒性潜力。Runx3过表达与AKTi相配合，生成了既具有中央记忆又具有组织驻留记忆特性的CAR-T细胞。Runx3过表达加强了CD4+CAR T细胞的潜力，并与AKTi相配合，抑制了由调节信号引起的CD8+CAR T细胞的末级分化。虽然AKTi促进了CAR-T细胞的中央记忆表型，但扩张能力显著增强；Runx3过表达促进了CAR-T细胞的组织驻留记忆表型，并进一步增强了细胞的存留力、效应器功能和肿瘤驻留。这些新型AKTi生成的Runx3过表达CAR-T细胞在皮下PDAC肿瘤模型中表现出强大的抗肿瘤活性，并对程序性细胞死亡1阻滞有良好的响应。Runx3过表达与外体AKTi相配合，生成了具有组织驻留和中央记忆特性的CAR-T细胞，这装备了CAR-T细胞更好的存活时间、细胞毒性潜力和肿瘤驻留能力，以克服固体肿瘤治疗中的障碍。 ©作者（或其雇主（S））2023年允许重用CC BY-NC。不允许商业再利用。由BMJ发布。

Solid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to overcome these roadblocks have been unsatisfactory. Herein, we reported a strategy of combining Runx3 (encoding RUNX family transcription factor 3)-overexpression with ex vivo protein kinase B (AKT) inhibition to generate CAR-T cells with both central memory and tissue-resident memory characteristics to overcome these roadblocks.We generated second-generation murine CAR-T cells expressing a CAR against human carbonic anhydrase 9 together with Runx3-overexpression and expanded them in the presence of AKTi-1/2, a selective and reversible inhibitor of AKT1/AKT2. We explored the influence of AKT inhibition (AKTi), Runx3-overexpression, and their combination on CAR-T cell phenotypes using flow cytometry, transcriptome profiling, and mass cytometry. The persistence, tumor-infiltration, and antitumor efficacy of CAR-T cells were evaluated in subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor models.AKTi generated a CD62L+central memory-like CAR-T cell population with enhanced persistence, but promotable cytotoxic potential. Runx3-overexpression cooperated with AKTi to generate CAR-T cells with both central memory and tissue-resident memory characteristics. Runx3-overexpression enhanced the potential of CD4+CAR T cells and cooperated with AKTi to inhibit the terminal differentiation of CD8+CAR T cells induced by tonic signaling. While AKTi promoted CAR-T cell central memory phenotype with prominently enhanced expansion ability, Runx3-overexpression promoted the CAR-T cell tissue-resident memory phenotype and further enhanced persistence, effector function, and tumor-residency. These novel AKTi-generated Runx3-overexpressing CAR-T cells exhibited robust antitumor activity and responded well to programmed cell death 1 blockade in subcutaneous PDAC tumor models.Runx3-overexpression cooperated with ex vivo AKTi to generate CAR-T cells with both tissue-resident and central memory characteristics, which equipped CAR-T cells with better persistence, cytotoxic potential, and tumor-residency ability to overcome roadblocks in the treatment of solid tumors.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.