Shao Hongwei, Lin Yanmei, Wang Teng, Ou Yusheng, Shen Han, Tao Changli, Wu Fenglin, Zhang Wenfeng, Bo Huaben, Wang Hui, Huang Shulin
School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Southern Medical University, Guangzhou, China.
Cancer Lett. 2015 Jul 10;363(1):83-91. doi: 10.1016/j.canlet.2015.04.001. Epub 2015 Apr 15.
Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells.
鉴定肿瘤相关抗原（TAA）特异性的TCR基因对于T细胞的TCR基因修饰是必要的，这应用于抗肿瘤过继性T细胞疗法（ACT）。通常的鉴定方法是基于分离单个对肽有反应的T细胞，并通过体外扩增或单细胞RT-PCR克隆TCR基因。然而，漫长且严格的体外培养周期和苛刻的操作要求限制了这些方法的应用。免疫分析仪是一种有效的工具，它可以分析TCR库，并通过CDR3长度分析识别显著扩增的克隆。在本研究中，选择了一种针对HLA-A2结合进行优化的生存素衍生突变肽来负载树突状细胞（DC）并激活T细胞。通过免疫分析仪分析分别鉴定TCRA和TCRB基因的单克隆扩增，在序列鉴定之后，将正确配对的TCR基因转入T细胞。肽识别和细胞毒性试验表明，经TCR修饰的外周血单核细胞（PBMC）可以对突变肽和野生型肽作出反应并裂解靶细胞。这些结果表明，将免疫分析仪与体外肽刺激相结合为鉴定特异性TCR基因提供了一种替代且优越的方法，这代表了TCR基因修饰T细胞应用方面的一项重大进展。
