Wang Xianyao, Song Xuelian, Li Yi, Ding Yanshu, Yin Chenfei, Ren Tianyuan, Zhang Weiguo
National Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, China.
Department of Immunology, Zunyi Medical University, Zunyi, Guizhou, China.
J Immunother Cancer. 2025 Jul 31;13(7):e012029. doi: 10.1136/jitc-2025-012029.
T-cell receptor (TCR)-T immunotherapy has emerged as a promising strategy for cancer treatment. However, identifying TCRs that can be used to generate TCR-T cells remains challenging due to tumor heterogeneity, the scarcity of tumor-specific T cells, and the diversity of human leukocyte antigens (HLA). To advance TCR-T immunotherapy, it is crucial to develop an efficient and scalable method to identify tumor-specific TCRs.
To identify tumor-specific TCRs, epitopes, and their corresponding HLA subtypes, we developed a method for rapidly assembling TCRs identified through the single-cell analysis of T cells from various tumors. For each TCR, only two pairs of oligonucleotides corresponding to the CDR3 regions of TCR-α and β chains needed to be synthesized, enabling the construction of a TCR library quickly in a cost-effective manner. Additionally, we engineered HLA-knockout HEK-293T cells as antigen-presenting cells to express patient-specific HLA-I and neoantigens, and a Jurkat NFAT-GFP reporter cell line for screening antigen-reactive TCRs. The efficacy of our TCR-screening system was validated through a small-scale screening of HPV16-specific TCRs from patients with cervical cancer.
We successfully developed a TCR assembly method that enables the rapid cloning and construction of TCR libraries within 2 days, significantly accelerating the process and reducing costs. Our antigen-presenting system also allows for flexible expression of patient-specific HLA-I molecules, facilitating personalized screening. The Jurkat reporter cells demonstrated high sensitivity for screening functional TCRs. Using published datasets from patients with HPV16-positive cervical cancer, we successfully used our system to isolate a human papillomavirus (HPV)-specific TCR. Through deletion, alanine scanning, and mass spectrometry analysis, we determined that this TCR specifically recognized an 8-mer peptide (MHGDTPTL) from HPV-E7 presented by HLA-B*15:18. Moreover, T cells expressing this TCR were able to effectively kill HPV-positive cells.
We developed an integrated antigen-presenting, TCR assembly, and TCR reporter system for screening tumor-specific TCRs using single-cell sequencing datasets. By using this system, we have successfully identified a functional, HPV-specific TCR, demonstrating the potential of our approach for the efficient screening of tumor-specific TCRs to advance TCR-T immunotherapy.
T细胞受体（TCR）-T免疫疗法已成为一种有前景的癌症治疗策略。然而，由于肿瘤异质性、肿瘤特异性T细胞稀缺以及人类白细胞抗原（HLA）的多样性，识别可用于产生TCR-T细胞的TCR仍然具有挑战性。为了推进TCR-T免疫疗法，开发一种高效且可扩展的方法来识别肿瘤特异性TCR至关重要。
为了识别肿瘤特异性TCR、表位及其相应的HLA亚型，我们开发了一种方法，用于快速组装通过对来自各种肿瘤的T细胞进行单细胞分析而鉴定出的TCR。对于每个TCR，只需合成两对分别对应于TCR-α和β链CDR3区域的寡核苷酸，就能以经济高效的方式快速构建TCR文库。此外，我们构建了HLA敲除的HEK-293T细胞作为抗原呈递细胞，以表达患者特异性HLA-I和新抗原，并构建了一个Jurkat NFAT-GFP报告细胞系用于筛选抗原反应性TCR。我们通过对宫颈癌患者的HPV16特异性TCR进行小规模筛选，验证了我们的TCR筛选系统的有效性。
我们成功开发了一种TCR组装方法，能够在2天内快速克隆和构建TCR文库，显著加快了这一过程并降低了成本。我们的抗原呈递系统还允许灵活表达患者特异性HLA-I分子，便于进行个性化筛选。Jurkat报告细胞对筛选功能性TCR表现出高灵敏度。利用已发表的HPV16阳性宫颈癌患者数据集，我们成功地使用我们的系统分离出一种人乳头瘤病毒（HPV）特异性TCR。通过缺失、丙氨酸扫描和质谱分析，我们确定该TCR特异性识别由HLA-B*15:18呈递的来自HPV-E7的一个8肽（MHGDTPTL）。此外，表达该TCR的T细胞能够有效杀伤HPV阳性细胞。
我们开发了一种整合的抗原呈递、TCR组装和TCR报告系统，用于使用单细胞测序数据集筛选肿瘤特异性TCR。通过使用该系统，我们成功鉴定出一种功能性的HPV特异性TCR，证明了我们的方法在高效筛选肿瘤特异性TCR以推进TCR-T免疫疗法方面的潜力。
