The T cell receptor (TCR) provides the fine specificity of T cells to recognize mutations in cancer cells ^1-3. We developed a clinical-grade approach based on CRISPR/Cas9 non-viral precision genome editing to simultaneously knock-out the two endogenous TCR genes, TCRα (TRAC) and TCRβ (TRBC), and insert in the TRAC locus the two chains of a neoantigen-specific TCR (neoTCR), isolated from the patient’s own circulating T cells using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with refractory solid cancers received up to three distinct neoTCR-transgenic cell products, each expressing a patient-specific neoTCR, in a cell dose-escalation, first-in-human phase 1 clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome, and one grade 3 encephalitis. All had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease, and the other 11 had disease progression as best response on therapy. NeoTCR-transgenic T cells were detected in tumour biopsies post-infusion at frequencies higher than the native TCRs pre-infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs recognizing mutational neoantigens, the simultaneous knock-out of the endogenous TCR and knock-in of the neoTCRs using single-step, non-viral precision genome editing, the manufacturing of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene edited neoTCR T cell products, and the ability of the transgenic T cells to traffic to the patients’ tumours.

T 细胞受体 (TCR) 提供 T 细胞的精细特异性来识别癌细胞中的突变^1-3。我们开发了一种基于 CRISPR/Cas9 非病毒精准基因组编辑的临床级方法，可同时敲除两个内源 TCR 基因 TCRα ( TRAC ) 和 TCRβ ( TRBC )，并在TRAC基因座中插入 A 的两条链。新抗原特异性 TCR (neoTCR)，使用可溶性预测新抗原-HLA 捕获试剂的个性化库从患者自身的循环 T 细胞中分离出来。在一项细胞剂量递增、首次人体 1 期临床试验 (NCT03970382) 中，16 名患有难治性实体癌的患者接受了多达三种不同的 neoTCR 转基因细胞产品，每种产品都表达患者特异性的 neoTCR。一名患者患有 1 级细胞因子释放综合征，一名患者患有 3 级脑炎。所有患者都出现了淋巴清除化疗带来的预期副作用。5 名患者病情稳定，另外 11 名患者病情进展，这是治疗的最佳反应。输注后的肿瘤活检中检测到的 NeoTCR 转基因 T 细胞的频率高于输注前的天然 TCR。这项研究证明了分离和克隆多个识别突变新抗原的TCR、使用单步非病毒精确基因组编辑同时敲除内源TCR和敲入neoTCR、制造neoTCR工程化T细胞的可行性在临床级别，输注多达三种基因编辑的 neoTCR T 细胞产品的安全性，以及转基因 T 细胞运输至患者肿瘤的能力。