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

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.

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。

嵌合抗原受体（chimeric antigen receptor, CAR）修饰的T细胞过继疗法是近年来肿瘤免疫治疗的热点，其在治疗实体性恶性肿瘤方面面临巨大挑战。肝细胞癌（hepatocellular carcinoma, HCC）是我国常见的高度恶性肿瘤，为了研究CAR修饰的T细胞过继疗法在HCC中的可行性，本课题选择HCC细胞表面高表达的肿瘤抗原磷脂酰肌醇聚糖（glypican-3, GPC3），通过体内外实验探讨GPC3-CAR修饰的T细胞对HCC治疗的靶向性和有效性。主要研究内容和结果有：①以人源化GPC3 单链抗体GC33和HN3为胞外抗原结合区，分别设计4种第二代和第三代GPC3-CAR，第二代CAR的共刺激分子选用4-1BB分子，第三代CAR增加CD28，构建GPC3-CAR慢病毒表达载体；②分选人外周血T细胞，将GPC3-CAR慢病毒表达载体导入T细胞，优化GPC3-CAR修饰的T细胞培养条件，使CAR-T细胞的感染效率明显提高，存活时间延长；③为了确认GPC3-CAR修饰的T细胞对肝癌细胞的特异性杀伤作用，课题组筛选不同程度表达GPC3基因的肝癌细胞：HepG2、Huh-7、原代培养两株HCC细胞1222和1214，不表达GPC3基因的Sk-Hep1和敲除GPC3基因的Huh-7-KO，从体外分析GPC3-CAR-T细胞对这些肝癌细胞的杀瘤作用，并检测细胞因子IFN-γ、IL-2的分泌情况，结果提示GPC3-CAR修饰的T细胞对表达GPC3基因的各种HCC细胞杀伤性强，分泌更多的细胞因子，统计学差异显著（P<0.05）。④ HepG2和Sk-Hep1皮下成瘤，构建SCID小鼠肝癌荷瘤模型，尾静脉注射GPC3-CAR-T细胞，结果显示第二代和第三代GPC3-CAR T细胞均能明显抑制HepG2细胞的生长，肿瘤组织内有T细胞浸润，而对Sk-Hep1组及对照组无明显抑制作用，第三代CAR-T效果更好，对各重要器官无明显损伤。.本课题证实构建的第二代和第三代GPC3-CAR-T细胞对HCC杀伤性强、靶向性好，第三代CAR强于第二代，以GC33为胞外抗原结合区的第三代CAR效果最好，基本完成了预定目标。课题组还对负性共刺激分子B7-H3及肿瘤坏死因子超家族成员（LIGHT）进行初步研究，以期为提升CAR-T细胞活性、提高CAR-T细胞对HCC疗效奠定基础。

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