肿瘤微环境（TME）对嵌合抗原受体（CAR）T细胞的抑制作用是T细胞治疗实体瘤的重要障碍。额外结构域B（EDB）-纤维连接蛋白是新生血管内皮层和癌细胞上表达的胎儿型抗原。尽管被认为是T细胞治疗的靶点，但迄今为止，经过工程改造的CAR T细胞在体内效果并不理想。本研究报告了通过以EDB-纤维连接蛋白为靶点的重定向TCR-CAR T细胞（rTCR-CAR）绕过抑制性TME以达到治疗实体瘤和有效抑制肿瘤生长的结果。我们通过将单链可变片段融合到CD3ε上生成EDB-靶向CAR，从而产生了rTCR-CAR。人类原代T细胞和Jurkat细胞被用于研究EDB-靶向T细胞。我们对其在信号传导、免疫突触形成和T细胞耗竭方面与传统的第二代CAR T细胞的不同进行了表征。我们在体外测试了rTCR-CAR T细胞的细胞毒性，并使用异种移植模型证明了其治疗有效性。结果显示，在异种移植模型中，rTCR-CAR T细胞在体内显示出优于基于传统CAR设计的治疗效果。与肿瘤生长抑制一起观察到肿瘤血管密度的显著减少，甚至在建立了EDB阴性癌细胞的肿瘤模型中也存在这种现象。rTCR-CAR与固定的EDB结合，该结合导致了与第二代CAR形成的免疫突触结构相比更好的免疫突触结构。与常规TCR复合物类似的机制，EDB-纤维连接蛋白激活rTCR-CAR，导致基础激活水平低且体内扩增增加的rTCR-CAR T细胞。我们的研究证明了以EDB-纤维连接蛋白为靶点的rTCR-CAR T细胞具有抗癌潜力。rTCR-CAR T细胞经过工程改造以具备抗血管生成和细胞毒性活性，显示出治疗有效性不受抑制性TME影响。这种单一治疗剂的综合特性指向其达到持续控制实体瘤的潜力。©作者（或其雇主）2023。在CC BY-NC下允许再使用。不得进行商业再利用。由BMJ出版。

The suppression of chimeric antigen receptor (CAR) T cells by the tumor microenvironment (TME) is a crucial obstacle in the T-cell-based treatment of solid tumors. Extra domain B (EDB)-fibronectin is an oncofetal antigen expressed on the endothelium layer of the neovasculature and cancer cells. Though recognized as a T cell therapy target, engineered CAR T cells thus far have failed to demonstrate satisfactory in vivo efficacy. In this study, we report that targeting EDB-fibronectin by redirected TCR-CAR T cells (rTCR-CAR) bypasses the suppressive TME for solid tumor treatment and sufficiently suppressed tumor growth.We generated EDB-targeting CAR by fusing single-chain variable fragment to CD3ε, resulting in rTCR-CAR. Human primary T cells and Jurkat cells were used to study the EDB-targeting T cells. Differences to the traditional second-generation CAR T cell in signaling, immune synapse formation, and T cell exhaustion were characterized. Cytotoxicity of the rTCR-CAR T cells was tested in vitro, and therapeutic efficacies were demonstrated using xenograft models.RESULTS: In the xenograft models, the rTCR-CAR T cells demonstrated in vivo efficacies superior to that based on traditional CAR design. A significant reduction in tumor vessel density was observed alongside tumor growth inhibition, extending even to tumor models established with EDB-negative cancer cells. The rTCR-CAR bound to immobilized EDB, and the binding led to immune synapse structures superior to that formed by second-generation CARs. By a mechanism similar to that for the conventional TCR complex, EDB-fibronectin activated the rTCR-CAR, resulting in rTCR-CAR T cells with low basal activation levels and increased in vivo expansion.Our study has demonstrated the potential of rTCR-CAR T cells targeting the EDB-fibronectin as an anticancer therapeutic. Engineered to possess antiangiogenic and cytotoxic activities, the rTCR-CAR T cells showed therapeutic efficacies not impacted by the suppressive TMEs. These combined characteristics of a single therapeutic agent point to its potential to achieve sustained control 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.