GPC3嵌合抗原受体基因修饰的T细胞靶向治疗肝细胞癌的研究

项目名称： GPC3嵌合抗原受体基因修饰的T细胞靶向治疗肝细胞癌的研究

项目编号： No.81472830

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 张文敏

作者单位： 福建医科大学

项目金额： 72万元

中文摘要： 嵌合抗原受体（CAR）将肿瘤相关抗原的单链抗体和T细胞的活化基序相结合，通过基因转导将特异性抗体锚定在T细胞上，CAR-T细胞过继疗法是肿瘤免疫治疗的热点。课题组在前期对嵌合抗原受体基因以及肝癌特异性磷脂酰肌醇蛋白聚糖（GPC3）研究的基础上，首次提出将GPC3-CAR修饰的T细胞应用于肝癌治疗的新思路。本项目设计人源化GPC3 scFv第二代和第三代嵌合抗原受体，构建GPC3-CAR慢病毒表达载体并导入人脐带血/外周血T细胞，采用流式细胞术、WB、ELISA、51Cr释放检测GPC3-CAR修饰的T细胞增殖情况以及对肝癌细胞的杀伤作用，进一步通过动物实验和人体标本检测经GPC3-CAR修饰的T细胞对肝癌治疗的疗效以及对肝癌转移的抑制作用，筛选并优化培养靶向性好、杀伤性强、安全性能佳的CAR-T细胞，为GPC3-CAR T细胞过继疗法进一步应用于临床肝癌治疗提供有效性、安全性的实验依据。

中文关键词： 嵌合抗原受体；磷脂酰肌醇蛋白聚糖；肝细胞癌；T细胞；过继免疫疗法

英文摘要： Hepatocellular carcinoma (HCC), one of the most common malignancies worldwide, is the third leading cause of cancer-related deaths globally. There is an urgent need for developing a new therapeutic strategy to treat patient with HCC. Chimeric antigen receptors (CAR) modified T cell therapy has been emerged as a promising regiment for cancers. CD19-specific CAR-T cells have demonstrated their therapeutic effect in treating B-cell leukemia in the clinical trails. CAR is comprised of an antigen combined domain (single chain variable fragment,scFv), an extracellular spacer/hinge region, a trans-membrane domain, an intracellular costimulator domain, and a T cell activation domain. There are several advantages for CAR-modified T cell immunotherapy, such as HLA-independent to tumor antigens, more available tumor targets including proteins, carbohydrates and glycolipids, applicable to a broad range of patients. CAR-modified T cell therapy requires a tumor-specific antigen or molecular target. Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate (HS) proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. In the previous study, we found that GPC3 was highly expressed on HCC but not on normal tissues, suggesting that GPC3 is a specific tumor marker and a potential therapeutic target for HCC. In this proposal, we will first engineer the second and third generation of GPC3 specific CAR T cells by transfecting human umbilical cord blood T cells with a GPC3-CAR lentiviral vector. The expression of GPC3-CAR on the T cells will be confirmed by both Western blot analysis and flow cytometry.Meanwhile,GPC3 expressing HCC cell lines will be identified and selected for in vitro experiments and in vivo mouse models. The specific cytolytic effect of GPC3-CAR T cell targeting HCC cells will be determined by 51Cr releasing assay, and by ELISA measuring cytokines (IFN-γ、IL-2、GM-CSF). To evaluate the efficacy of GPC3-CAR T cells against HCC tumors in vivo, we will establish a primary HCC,and a HCC lung metastases mouse model, respectively. The GPC3-CAR T cells will be labeled with CM-Dil and intravenously injected into the mice. After CAR-T cell therapy, the primary liver tumors and metastatic lung tumors as well as heart, spleen, kidney tissues will be removed and examined by using HE staining and florescence microscopy. Antitumor effects of GPC3-CAR T cells will be evaluated by tumor sizes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The potential side effects of GPC3-CAR-T cells in normal tissues and organs will also be evaluated. In summary, this proposal will investigate the antitumor effects of GPC3 CAR-modified T cells targeting HCC, aiming to develop a potential CAR-based T cell therapy to treat HCC patients.

英文关键词： chimeric antigen receptor(CAR);glypican 3(GPC3);hepatocellular carcinoma;T cell;adoptive immunotherapy