Programmed cell death protein-1 (PD-1)-mediated immunosuppression has been proposed to contribute to the limited clinical efficacy of chimeric antigen receptor T (CAR-T) cells in solid tumors. We generated PD-1 and T cell receptor (TCR) deficient mesothelin-specific CAR-T (MPTK-CAR-T) cells using CRISPR-Cas9 technology and evaluated them in a dose-escalation study. A total of 15 patients received one or more infusions of MPTK-CAR-T cells without prior lymphodepletion. No dose-limiting toxicity or unexpected adverse events were observed in any of the 15 patients. The best overall response was stable disease (2/15 patients). Circulating MPTK-CAR-T cells peaked at days 7–14 and became undetectable beyond 1 month. TCR-positive CAR-T cells rather than TCR-negative CAR-T cells were predominantly detected in effusion or peripheral blood from three patients after infusion. We further confirmed the reduced persistence of TCR-deficient CAR-T cells in animal models. Our results establish the preliminary feasibility and safety of CRISPR-engineered CAR-T cells with PD-1 disruption and suggest that the natural TCR plays an important role in the persistence of CAR-T cells when treating solid tumors.

已提出程序性细胞死亡蛋白-1 (PD-1) 介导的免疫抑制有助于嵌合抗原受体 T (CAR-T) 细胞在实体瘤中的有限临床疗效。我们使用 CRISPR-Cas9 技术生成了 PD-1 和 T 细胞受体 (TCR) 缺陷型间皮素特异性 CAR-T (MPTK-CAR-T) 细胞，并在剂量递增研究中对其进行了评估。共有 15 名患者接受了一次或多次 MPTK-CAR-T 细胞输注，而没有事先进行淋巴清除。15 名患者均未观察到剂量限制性毒性或意外不良事件。最佳的总体反应是疾病稳定（2/15 患者）。循环中的 MPTK-CAR-T 细胞在第 7-14 天达到峰值，并且在 1 个月后变得无法检测到。输注后，三名患者的积液或外周血中主要检测到 TCR 阳性 CAR-T 细胞而不是 TCR 阴性 CAR-T 细胞。我们进一步证实了动物模型中 TCR 缺陷型 CAR-T 细胞的持久性降低。我们的结果确立了 CRISPR 工程 CAR-T 细胞与 PD-1 破坏的初步可行性和安全性，并表明天然 TCR 在治疗实体瘤时在 CAR-T 细胞的持久性中起重要作用。