HBV相关HCC新抗原特异性TCR介导的杀肿瘤活性研究

我国乙肝相关肝癌（HCC）患者数量多且治疗手段有限，亟需探索新的治疗方法。以肿瘤新抗原为靶点的特异性T细胞具有特异和高效的杀肿瘤活性，临床效果显著，但有关HCC新抗原特异性T细胞受体（TCR）介导的肿瘤杀伤活性研究数据欠缺。我们在前期研究中对慢乙肝、肝硬化以及HCC患者肝组织和外周血中TCR组库的组成和特点进行了对比分析，并对特异性TCR进行了研究。本项目将基于前期积累的大量数据，以HCC患者为研究对象，通过全外显子、转录组测序，突变表位对肿瘤浸润CD8+T细胞的刺激和分选，单细胞TCR αβ链配对和病毒包装，新抗原特异性TCR-T制备及系列免疫学实验，利用患者自体肿瘤细胞和自体肿瘤小鼠模型，研究HCC新抗原特异性TCR介导的肿瘤杀伤活性，为HCC患者精准免疫治疗的探索提供支持，具有重要的临床价值和意义。

In our country, the incidence of hepatitis B virus-associated hepatocellular carcinoma (HCC) is high, and the options for effective therapeutic are lack. So new therapeutic approaches are urgently needed. Targeting of cancer neoantigen with specific T cell have shown the high specificity, efficient anti-tumor activity, and promising data in clinical treatment. Whereas data on HCC neoantigen-specific T cell receptor (TCR) and its antitumor activity are lacking. Our previous studies have analyzed TCR repertoires and specific TCR in tissues and peripheral blood mononuclear cells in patients with chronic hepatitis B, liver cirrhosis and HCC. In this project, based on our previous TCR data, HCC patients will be enrolled and studied. Through whole exome sequencing, RNA sequencing by next-generation sequencing, somatic mutations will be selected to stimulate and sort CD8+ T cells from tumor-infiltrating lymphocytes. Then TCR β and α sequences will be paired by single-cell analysis. The paired TCR β and α sequences will be cloned into retroviral vector MP71 and packaged with 293T. Autologous peripheral blood mononuclear cells from the patients will be transduced with retroviral supernatants. Neoantigen specific TCR-T will be cocultured with their corresponding autologous tumor cell lines, or will be intravenously injected into SCID mice with orthotopic HCC patient-derived xenografts. Immune assays including lactate dehydrogenase cytotoxicity and pathological analysis will be used for the final confirmation of antitumor acitivity of HCC neoantigen specific TCR-T. The identification of neoantigen specific TCR will do a great help on immunotherapy of patients with hepatitis B virus-associated HCC.