点突变Survivin抗原肽反应性CTL克隆TCR基因的鉴定及其转染修饰T细胞特异性抗肿瘤作用的研究

Tumor patients always lack reactive CTL clones that could recognize tumor antigens, which could lead to the functional defects for ani-tumor immune response. Some evidence had shown that the effector T cells could be activated with the point-mutated tumor antigen peptides and then cross-recognized the tumor cells expressed wild-type antigens. It had been found that the new specificity of anti-tumor ability could be acquired by TCR gene transfer.In our previous study, we had identified two HLA-A2 restricted Survivin peptides of single point-mutated, Sur79L2 and Sur79M2. It had been confirmed that these two peptides had higher MHC binding rates and could stimulate TILs activation，which were derived from hepatocellular carcinoma ascites. And we had determined that the mutated peptides-induced CTLs could specifically lysis tumor cells. In this study,we would establish CTL clones from TILs using stimilation with two point-mutated peptides in vitro and screen the specific anti-tumor functional TCR gene relying on peptide-MHC tetramer technology and 5′-RACE PCR for single cell. After that, the speicfic TCR gene would been transfected into CD8+ T lymphocytes using retroviral vectors and the we would verify the anti-tumor function against various hepatoma cell lines, which all have high expression of wild-tpye Survivin antigen.

肿瘤患者体内缺乏有效识别肿瘤抗原的反应性CTL克隆，是导致抗肿瘤免疫缺陷的重要原因。研究发现，点突变肿瘤抗原肽能有效活化效应T细胞并能对表达野生型抗原的肿瘤细胞发挥交叉识别作用，同时TCR转基因技术也被证明能够赋予T细胞新的特异性杀伤能力。前期研究中我们鉴定出两组与MHC分子结合率显著提高的HLA-A2限制性Survivin点突变肽：Sur79L2和Sur79M2，并证实它们能有效刺激肝癌腹水TILs活化，发挥特异性杀伤肿瘤细胞作用,本课题中，我们将利用两种点突变肽刺激后获得的反应性CTL克隆，筛选具有显著杀伤能力的T淋巴细胞，结合抗原肽-MHC四聚体分选技术及单细胞5′-RACE PCR技术筛选鉴定出杀伤性CTLs克隆的特异性TCR基因，将该TCR基因稳定转染人CD8+T淋巴细胞，验证TCR基因修饰的T细胞对高表达野生型Survivin肝癌细胞系的特异性识别杀功能。

由于肿瘤是由机体自身细胞发生恶性转化而形成的，肿瘤相关抗原（TAA）绝大部分为自身抗原，由于机体的中枢免疫耐受机制，体内能识别这些TAA的TCR高亲和力T细胞在发育早期即被免疫清除，体内存在的识别这些TAA的成熟T细胞，绝大多数为TCR低亲和力的naive T细胞，难以被TAA肽有效激活。本研究，我们利用生物信息学分析结合试验验证筛选到两个与HLA-A2分子结合能力显著提高的点突变Survivin 表位肽Sur79L2 和Sur79M2，将这两个点突变肽负载DC细胞后体外刺激活化HLA-A2阳性的CD8+T淋巴细胞，建立了具有特异性识别杀伤能力的CTLs克隆，并通过进一步的筛选和鉴定获得了针对Sur79M2的特异性基因TCRAv4j8Bv7j2和针对Sur79L2 的特异性基因TCRAv10j6Bv3.1j2.3，我们将这两组TCR基因分别构建pDC315-TCRA-IRES-TCRB双表达重组质粒，并包装成腺病毒转染HLA-A2阳性的健康人T 细胞，结果显示导入的两组TCR 基因均能在T细胞表面正确组装和表达，进一步用TCR基因修饰的T细胞进行体外细胞学实验，结果证实，两组TCR基因转染的T细胞均能有效识别负载突变肽及相应野生型肽的T2细胞，并对表达Survivin抗原的HLA-A2阳性肿瘤细胞系有显著杀伤作用。目前以CAR-T和TCR-T为代表的新一代肿瘤免疫细胞治疗技术日益成为研究热点，合适的CAR基因及TCR基因的筛选是其中的关键技术之一，因此，本项目所建立的针对人工点突变TAA表位肽特异性TCR基因的筛选技术，在肿瘤免疫治疗中具有一定的应用价值。

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