针对肝癌高频突变抗原靶点的肿瘤特异性CTL回输治疗方法研发

Liver cancer ranks second in cancer mortality, resulting in significant social and economic loss every year. The traditional liver cancer treatments, including surgery, radiotherapy and chemotherapy, have limited efficacy, while tumor immunotherapy has recently been considered as a promising new method. Among the different immunotherapy methods, adoptive CTL transfter has been widely used in research and clinic applications due to the following advantages: low side effects, tumor-specific targeting, effective for a longer period of time. Currently the advance of adoptive CTL therapy for hepatocellular carcinoma has 2 major limiting factors: lack of tumor-specific antigens, low immunogenicity of tumor antigen peptides. This project will use HCC gene sequencing databases database to screen high-frequency mutations which can cover 8% HCC population, and construct a stable human liver cancer tissue derived cell line containing these mutation sites by transfecting HepaG2 cells with a lentivirus, and then develop a liver cancer xenograft model in nude mice by injecting the modified HepG2 cell line. Meanwhile, optimal tumor-specific antigen peptide and their affinity to MHC were predicted using bioinformatics model. After that, PBMC were isolated from volunteers PBMC, and CTL which recognizing the high frequency mutation sites were cultured and adoptively transferred into nude mice harboring the xenograft, tumor size and immune response after treatment was monitored. The tumor-specific CTL adoptive transfer therapy method can be translated to clinical use, and provide new way and stragety for the treatment of other type of cancers.

肝癌的死亡率高居我国恶性肿瘤的第二位。肿瘤免疫疗法具有很好的治疗前景，正被深入研究。其中CTL细胞过继疗法因毒副作用小，针对性强，持久性好，适应症广等特性而被广泛研究。肝癌CTL细胞过继疗法的主要制约因素是癌症特异性靶点的缺乏和抗原肽的免疫原性不足。本项目将从肝癌的基因测序数据库出发，筛选可以覆盖8%人群的6个高发高频突变位点，用慢病毒转染HepG2细胞构建一个可表达这些高频突变的细胞系，然后在裸鼠上建立肝癌移植瘤模型。同时利用生物信息模型预测对应的肿瘤抗原肽和MHC分子的亲和力，合成每个突变位点的最优肿瘤抗原肽，利用体外细胞模型对其进行免疫原性筛选。最后从志愿者分离免疫细胞，培养能特异性识别top 6高频突变的抗原肽的CTL，然后回输到裸鼠进行治疗，观察肿瘤的变化和相关免疫反应。本项目开发的针对人肝癌高频突变抗原肽来诱导抗原特异性的CTL方法后期可在临床推广使用，为肝癌的治疗提供新思路。

肝癌死亡率高居我国恶性肿瘤第二位。CTL细胞过继疗法因毒副作用小，针对性强，持久性好，适应症广等特性而被广泛研究。其主要制约因素是癌症特异性靶点的缺乏和抗原肽的免疫原性不足。本项目从肝癌的基因测序数据库出发，筛选可覆盖8%人群的20个高发高频突变位点，利用生物信息模型预测对应的肿瘤抗原肽和MHC分子的亲和力，合成突变位点的最优新生抗原肽，为了增加肝癌患者的覆盖率，进一步合成了肝癌和病毒相关性抗原肽，利用2D体外细胞模型对其进行免疫原性筛选，培养能特异性识别阳性抗原肽的CTL，并用慢病毒载体转染HepG2细胞构建可表达高频突变位点的细胞系，然后利用去细胞肝脏模型再细胞化建立3D体外肝癌模型，最后将成熟的CTL回输到3D体外肝癌模型进行治疗，观察肿瘤模型的变化和相关免疫反应。本项目开发的针对人肝癌高频突变抗原肽来诱导抗原特异性的CTL方法后可在临床推广使用，为肝癌和其他癌症的治疗提供新思路。