Background: Adoptive cell therapy is the most promising approach for battling cancer, with T cell receptor-engineered T (TCR-T) cell therapy emerging as the most viable option for treating solid tumors. Current techniques for preparing TCR-T cell therapy provide a limited number of candidates TCRs, missing the comprehensive view of the repertoire, which may hinder the identification of the most effective TCRs.Methods: Dendritic cells were primed with immunogenic peptides of the antigen of interest to expand antigen-specific CD8 T lymphocytes from peripheral blood. Following that, the entire repertoire of naturally occurring antigen-specific TCRs was analyzed using single-cell RNA sequencing, alongside the assessment of the dominancy, transcriptome, and binding specificity of the obtained clonotypes, utilizing the TCRscape tool and ERGO-II neural network to identify the most effective candidate for TCR-T cell therapy development. Finally, TCR-T cells with the candidate TCR were obtained, followed by assessing their functionality and selectivity.Results: The developed protocol achieved a remarkable increase in the percentage of antigen-specific T cells by more than 200-fold, with more than 100 antigen-specific TCR clonotypes identified. The resulting TCR-T cells demonstrated high cytotoxicity and selectivity for the targeted antigen, indicating their potential to preferentially target tumor cells.Conclusions: This study offers a comprehensive approach for the discovery and analysis of not only few, but the entire repertoire of naturally occurring antigen-specific TCRs for TCR-T cell therapy development. Additionally, the proposed approach can be tailored to accommodate different types of antigens and MHC variants, making it a highly versatile tool for both research and clinical applications.
背景：过继性细胞疗法是对抗癌症最具前景的策略，其中T细胞受体工程化T细胞（TCR-T）疗法已成为治疗实体瘤最可行的选择。目前制备TCR-T疗法的技术仅能提供有限数量的候选TCR，缺乏对TCR库的整体性观察，这可能阻碍最有效TCR的识别。 方法：通过用目标抗原的免疫原性肽段刺激树突状细胞，从外周血扩增抗原特异性CD8 T淋巴细胞。随后，利用单细胞RNA测序技术分析天然存在的抗原特异性TCR完整谱系，并结合TCRscape工具与ERGO-II神经网络评估所得克隆型的优势度、转录组特征及结合特异性，从而筛选出用于TCR-T细胞疗法开发的最有效候选TCR。最终获得携带候选TCR的TCR-T细胞，并评估其功能活性与选择性。 结果：本研究建立的方案使抗原特异性T细胞比例实现超过200倍的显著提升，成功鉴定出100余种抗原特异性TCR克隆型。所得TCR-T细胞对靶抗原展现出高效细胞毒性及优异选择性，表明其具备特异性靶向肿瘤细胞的潜力。 结论：本研究提供了一套综合性策略，不仅能够发现少量TCR，更能系统分析天然存在的抗原特异性TCR完整谱系，为TCR-T细胞疗法开发奠定基础。此外，该策略可根据不同抗原类型及MHC变异体进行定制化调整，使其成为兼具科研与临床应用价值的高度通用型工具。
肿瘤(癌症)患者之家