T cell receptor (TCR) gene therapy is a potent form of cellular immunotherapy in which patient T cells are genetically engineered to express TCRs with defined tumor reactivity. However, the isolation of therapeutic TCRs is complicated by both the general scarcity of tumor-specific T cells among patient T cell repertoires and the patient-specific nature of T cell epitopes expressed on tumors. Here we describe a high-throughput, personalized TCR discovery pipeline that enables the assembly of complex synthetic TCR libraries in a one-pot reaction, followed by pooled expression in reporter T cells and functional genetic screening against patient-derived tumor or antigen-presenting cells. We applied the method to screen thousands of tumor-infiltrating lymphocyte (TIL)-derived TCRs from multiple patients and identified dozens of CD4⁺ and CD8⁺ T-cell-derived TCRs with potent tumor reactivity, including TCRs that recognized patient-specific neoantigens.

T 细胞受体 (TCR) 基因疗法是细胞免疫疗法的一种有效形式，其中患者 T 细胞经过基因工程改造，以表达具有明确肿瘤反应性的 TCR。然而，由于患者 T 细胞库中肿瘤特异性 T 细胞普遍缺乏，以及肿瘤上表达的 T 细胞表位具有患者特异性，因此治疗性 TCR 的分离变得复杂。在这里，我们描述了一种高通量、个性化的 TCR 发现流程，该流程能够在一锅反应中组装复杂的合成 TCR 文库，然后在报告 T 细胞中汇集表达，并对患者来源的肿瘤或抗原呈递细胞进行功能性遗传筛选。我们应用该方法筛选了来自多个患者的数千个肿瘤浸润淋巴细胞 (TIL) 衍生的 TCR，并鉴定了数十种具有强肿瘤反应性的 CD4 ⁺和 CD8 ⁺ T 细胞衍生的 TCR，包括识别患者特异性新抗原的 TCR。