CTLA-4纳米抗体增强肿瘤特异性T淋巴细胞抗肝癌作用研究

Adoptive T-cell therapy should be improved on the primary hepatocellular carcinoma. Cytotoxic tlymphocyte-associated antigen 4 (CTLA-4) is an important negative T cell costimulatory molecule and highly expressed in activated T cells. Nanobody is a minimal antigen-binding functional fragment with higher water solubility, stability and targeted ability. A CTLA-4 specific nanobody (CTLA4-Nb16) was screened in our previous study. In our project, the efficient anti-hepatoma effect and mechanism of the specific T lymphocytes enhanced by CTLA-4 nanobody is explored combined with the high efficiency technology of tumor/DC fusion cell. First, the biologic activity of CTLA-4 nanobody is evaluated in vitro and the anti-hepatoma activity is studied in vivo with the nude mice model of bearing human T cell lymphoma Jurkat cells. Then, the proliferation, secretion and killing effect on target cells of specific T lymphocytes induced by DC/HepG2 with CTLA-4 nanobody are evaluated. Last, the therapeutic effect and mechanism of the specific T lymphocytes are investigated. The aim of the project is to provide a potential clinical value of adoptive T-cell therapy on the primary hepatocellular carcinoma.

目前原发性肝癌的过继T细胞疗效亟待提高。纳米抗体是目前可得到的稳定可结合抗原的最小单位，具有高水溶性、稳定性和靶向性。本项目拟用前期工作中筛选出的CTLA-4特异性纳米抗体CTLA-4/Nb16，结合本课题组高效的肿瘤/DC融合细胞诱导特异性T淋巴细胞技术，探索CTLA-4纳米抗体增强DC/HepG2融合细胞诱导的肿瘤特异性T淋巴细胞抗肝癌作用及其机制。首先在体外对CTLA-4纳米抗体生物学活性进行鉴定，其次通过建立荷人T细胞淋巴瘤裸鼠动物模型，对其体内抗肿瘤生物活性加以验证；进一步研究CTLA-4纳米抗体在体外对融合细胞诱导的肿瘤特异性CTL的增殖、分泌及对靶细胞的杀伤效应，最后从体内研究CTLA-4纳米抗体增强肿瘤特异性CTL过继治疗荷人肝癌裸鼠的效果及相关机制。本研究的完成将为肝癌的过继治疗提供一条有潜在临床应用价值的新策略。

细胞毒性T淋巴细胞相关抗原-4(Cytotoxic T Lymphocyte-associated Antigen-4，CTLA-4)是T细胞重要的负性共刺激分子，在活化的T细胞表面高表达，与B7竞争性结合，抑制T细胞的进一步活化，被认为是抑制机体抗肿瘤的免疫因子，也是肿瘤免疫治疗的良好靶标。纳米抗体是目前可以得到的稳定的可结合抗原的最小单位，具有高表达性、水溶性、稳定性、强组织穿透性及较弱的免疫原性等优点，在肿瘤免疫靶向治疗等方面展现了广阔的应用前景。我们成功筛选出了一种CTLA-4纳米抗体(CTLA-4 Nb16)，发现其与CTLA-4抗原具有较好的结合力，进一步使用CTLA-4Nb16来增强高融合效率的DC/HepG2融合细胞诱导的肿瘤特异性的T淋巴细胞有效性，防止CTL活化杀伤过程中CTLA-4对T细胞活化后的抑制，消除其免疫抑制功能，获得更好的抗肝癌效应。研究结果显示：DC/HepG2-FC@CTLA-4 Nb16在体外刺激的自体T淋巴细胞增殖活性增强和分泌IFN-γ的T淋巴细胞数目增多，DC/HepG2-FC@CTLA-4 Nb16诱导产生的特异性CTL能特异杀伤肿瘤细胞。DC/HepG2-FC@CTLA-4 Nb16诱导产生的特异性CTL过继治疗后能明显抑制荷人肝癌NOD SCID小鼠的肿瘤生长，抑制肿瘤组织内细胞增殖，促进肿瘤组织内细胞凋亡，从而延长了小鼠的生存时间。我们的研究显示DC/HepG2-FC@CTLA-4 Nb16能够增强特异性CTL高效抗肝癌作用，为肝癌的过继治疗提供了一条有潜在临床应用价值的新策略。