靶向食管癌高频突变抗原的TCR-T治疗方法研发

Esophageal cancer is one of the deadliest malignancies with poor prognosis, and has a high incidence in China. TCR-T therapy has received extensive attention because of its ability to treat solid tumors. The current treatment of esophageal cancer with TCR-T has shown its efficiency. However, TCR-T therapies are mainly based on tumor-associated-antigens which have low tumor-specificity. In addition, TCR-T treatment strategies are mainly designed for patients with HLA-A*02:01 genotype, however, the most frequent HLA genotype is HLA-A*11:01 in china and until now there are few TCR-T treatment strategies designed for patients with HLA-A*11:01 genotype. To overcome these shortcomings, we analyse the NGS sequencing data of 650 Chinese esophageal cancer patients using bioinformatics tools which we developed and optimized, and establish a somatic mutation database of Chinese esophageal cancer patients as well as construct a database of mutation-derived tumor antigens. In addition, an HLA-A*11:01 cell line is constructed to verify the peptide-MHC binding ability and in vitro immunogenicity of the top 20 high-frequency predicted mutation-derived-tumor-antigens. Finally, we will screen, construct the TCR-T clones, and finish functional validation experiments in vitro. Furthermore, tumor-bearing mice will be treated with adoptive TCR-T immunotherapy, and related immune response will be measured to evaluate its efficency. The aim of this project is to provide a new idea for the clinical treatment of esophageal cancer in China based on high frequency mutant antigen targeting TCR-T in Chinese esophageal cancer population.

食管癌是我国高发癌症，中晚期癌症患者预后很差。肿瘤免疫治疗中TCR-T疗法因其能治疗实体瘤而受到广泛关注。当前用于治疗食管癌的TCR-T主要以肿瘤相关抗原为主，疗效不明显。目前肿瘤新抗原受到广泛关注，靶向肿瘤新抗原的TCR-T原理上能提高其治疗效果。TCR-T主要治疗HLA-A*02:01亚型病人，中国以HLA-A*11:01为主，针对此类患者的治疗急需开展。本项目拟利用当前可用的650例中国食管癌病人的测序数据，建立其SNV数据集合，利用生物信息工具建设食管癌患者的抗原数据库，筛出top20高频多肽；通过实验验证多肽-MHC结合能力及体外免疫原性验证，筛选出两个高亲和力TCR序列，用于构建TCR-T，进行体外功能验证；最后功能性的TCR-T回输荷瘤小鼠，观察肿瘤的变化和相关免疫反应。本项目开发的针对中国食管癌人群高频突变抗原靶点TCR-T可为中国本土食管癌的临床治疗提供新思路。

背景：和世界其他国家或地区相比，我国的消化道肿瘤发病率和死亡率偏高，特别是食管癌和结直肠癌。目前，肿瘤细胞免疫治疗得到了广泛的关注。靶向NY-ESO-1抗原的TCR-T细胞制品在滑膜肉瘤上取得了良好疗效，然而用于治疗消化道肿瘤的TCR-T疗效不明显。肿瘤特异性抗原是用于开发TCR-T的优质靶点，即找到一些肿瘤患者人群中高频的肿瘤特异性抗原，开发靶向这些抗原的TCR-T。.主要研究内容：本项目从公共数据库中收集食管癌和结直肠癌患者的测序数据，经生物信息学分析得到高频突变位点，利用亲和力/抗原呈递预测软件预测肿瘤新抗原。将所得候选新抗原经流式荧光染色技术验证其亲和力，并经ELISPOT实验验证其免疫原性。最后基于单细胞TCR测序技术，筛选得到新抗原的TCR功能序列，并构建TCR-T细胞，在体外验证其免疫识别作用。.数据和结果：本项目累计收集到733例食管癌和321例结直肠癌患者的测序数据，并筛选得到205条候选高频突变新抗原序列；合成45个新抗原短肽进行亲和力试验验证，得到11条高亲和力的新抗原；随后挑选10条候选新抗原开展免疫原性筛选试验，得到7条具有免疫原性的新抗原；对2条新抗原阳性的T细胞进行了单细胞TCR测序，对测序得到的TCR序列进行体外免疫原性，获得2个具有免疫识别功能的TCR。.科学意义：该研究获得的肿瘤新抗原及对应的TCR-T，为临床治疗提供新的靶标和TCR序列，为进一步的临床试验验证提供了工作基础，为我国的消化道肿瘤治疗添砖加瓦。