EB病毒相关胃癌中EB病毒特异性TCR-T细胞的制备及抗肿瘤效果研究

Epstein-Barr virus is a double-stranded DNA virus and associated with human cancer. EBV associated gastric cancer (EBVaGC) comprises 9% of gastric carcinomas. Because of the high incidence of gastric cancer, EBVaGC has an annual incidence of 75,000-90,000 cases per year, representing the largest subpopulation among EBV-related tumors. Adoptive T-cell therapy for Epstein-Barr virus specific protein has made great progress in the lymphoma and nasopharyngeal cancer, but T cells expressing EBNA1-specific T cell receptor against EBVaGC has not been reported. In our previous study, we reported the first genome-wide view of sequence variation of EBV isolated from 9 primary EBVaGC biopsy specimens, including EBNA1 gene. In this study, synthesis long peptide of EBNA1 antigen on the basis of individual HLAs and EBNA1 sequence, will be loaded with dendritic cells (DC) to form the EBNA1 peptide-DC complexes and to activate specific T cells. Through the functional verification and high-throughput sequencing of TCR-beta chain and single cellular TCR alpha and beta chain sequencing, EBNA1-specific TCR will be cloned into peripheral blood T cells, and TCR-T cell will be generated, and then injected into the EBVaGC PDXs mouse model with the same individuals. Thus, generation of individualized EBNA1-TCR-T cells in the preclinical experimental treatment of EBVaGC is of groundbreaking significance.

EB病毒是致瘤性DNA病毒，胃癌中EB病毒阳性占比为9%。由于胃癌的高发病率，EBVaGC所占的绝对数量在EBV相关肿瘤中最大。迄今，针对EB病毒特异蛋白的过继T细胞治疗，已在鼻咽癌和淋巴瘤等取得较大进展，但靶向EBNA1特异性TCR-T细胞治疗在EBVaGC中未见报道。本课题组前期，已对9例EBVaGC中EBV基因组进行全面研究，并对EBNA1的表位进行详实分析。在此基础上，本项目根据个体不同HLA分型和EBNA1序列，体外合成EBNA1抗原长肽，负荷树突状细胞，形成复合物，激活特异T淋巴细胞，通过功能验证、高通量TCRbeta链测序和单细胞TCRalpha/beta链测序相结合，克隆EBNA1特异TCR导入外周血T细胞，制备TCR-T细胞，再回输同一个体来源的EBVaGC移植瘤模型。因此，本课题在EBVaGC临床前实验性治疗中，制备个体化EBNA1特异TCR-T细胞，具有开创性意义。

EB病毒是致瘤性DNA病毒，EBV相关肿瘤表达的病毒潜伏期蛋白具有较强的抗原性和肿瘤特异性，是研究肿瘤过继细胞治疗的很好切入点。迄今，针对EB病毒特异蛋白的过继细胞治疗，已在鼻咽癌和淋巴瘤等取得较大进展，但靶向EBNA1特异性TCR-T细胞治疗在EBV相关胃癌中未见报道。本课题组前期已完成9例EBV相关胃癌中EBV基因组全面研究且详实分析了EBNA1的表位。该项目在此基础上，体外合成EBNA1抗原长肽，负荷树突状细胞形成复合物以激活EBNA1特异T淋巴细胞，通过特异性识别实验进行诱导效果的评价，结果表明诱导后T细胞IFN-分泌能力显著增强。为了筛选诱导后T细胞中特异性TCRs基因，通过单细胞RNA结合TCR测序方法，评价EBNA1抗原肽诱导前后TCR的动态变化特征，结果表明诱导后存在频数增加的TCR基因，推测这些刺激后显著增加的高频TCRs可能靶向EBNA1，选择其中特异性扩增的高频主克隆TCRs基因进行功能验证。将高通量筛选的TCRs基因克隆入慢病毒并导入外周血T细胞构建TCR-T细胞，通过EBNA1抗原肽特异性识别实验进行鉴定。EBNA1抗原肽诱导联合单细胞RNA结合TCR测序方法能富集EBNA1抗原肽特异性T细胞、筛选功能性TCRs并获得EBNA1特异性TCR-T细胞，该方法能高通量地为EBV阳性肿瘤患者筛选个体化EBV特异性的TCRs，并对已知肿瘤抗原肽特异性TCRs的筛选具有启示作用。