靶向FGFR1重组溶瘤病毒基因疫苗抗肿瘤血管生成研究

Herpes simplex virus type 1 (HSV1) is one of the most effective oncolytic virus, which can especially kill tumor cells without damaging normal cells, however, oncolytic virus alone is not enough to induce the anti-tumor immune responses. MIP-3α is the most specific chemokines of dendritic cells (DC), and can effectively improve the antigen presenting capacity of DC. FGFR1 is the important target for anti-angiogenesis of tumor. Previous studies showed that the immunotherapy targeting FGFR1 had obvious anti-tumor effect, and FGFR1 single chain antibody had stronger effect in tumor targeting and penetration. Based on the previous work, this study aims to construct the HSV1/MIP-3α recombinant virus by reverse genetic (virus rescue) technology, after the nano liposome coated and PEG modified, conjugated with FGFR1 single chain antibody to form a kind of new composite gene vaccine. Thus, we will investigate the multiple anti-tumor efficacy of the targeting vaccine in oncolytic, anti-tumor angiogenesis as well as strong chemotaxis of DC to induce immune response both in vivo and in vitro, and then further explore its molecular mechanism. This study will provide a new biotheraputic strategy for tumor.

单纯疱疹病毒1型(HSV1)是最为有效的溶瘤病毒之一,可以特异性杀灭肿瘤细胞而对正常细胞没有损害，但是单纯性溶瘤所激发的抗肿瘤免疫反应通常不理想。MIP-3α 是树突状细胞（DC）最重要的特异性趋化因子, 能够有效提高DC递呈抗原的能力。FGFR1是抗肿瘤血管生成的重要靶点。申请者前期研究表明，靶向FGFR1免疫治疗具有显著抗肿瘤作用，并且发现FGFR1单链抗体具有更强的肿瘤靶向性和穿透力。本项目拟在前期基础上，以肿瘤新生血管为靶点，结合HSV1溶瘤特点，利用反向遗传（病毒拯救）技术构建高效表达MIP-3α 的重组病毒HSV1/MIP-3α ，经纳米脂质体包被、PEG修饰后与FGFR1单链抗体偶联成复合型基因疫苗，在体内外观察该疫苗是否兼具靶向溶瘤、抗肿瘤血管生成以及体内趋化募集DC强力诱导特异性免疫反应等多重抗肿瘤功效，并进一步探讨其分子作用机理，寻找一种肿瘤生物治疗的新策略。

本项目利用基因工程技术获得兼具靶向溶瘤、抗肿瘤血管生成以及体内趋化募集 DC强力诱导特异性免疫反应等多种生物活性的复合型基因疫苗（sFR1-HSV1-MIP3α）进行体内外实验。完成的主要工作包括：（1）构建MIP-3α的克隆载体并鉴定；（2）制备抗FGFR1单链抗体并进行活性鉴定；（3）体外HSV1/MIP3α重组病毒活性鉴定并检测MIP3α的表达和分泌情况；（4）制备sFR1-HSV1-MIP3α疫苗并在几个动物模型中进行抗肿瘤作用观察。研究发现，sFR1-HSV1-MIP3α作为疫苗具有肿瘤新生血管靶向性和抑制肿瘤血管生成作用；具有较好溶瘤作用且安全性好；还发现其具有树突状细胞趋化募集作用。此外，课题组完成了“MIP3α-Fc 融合蛋白及其用途”研究并申请技术专利，发现其能够趋化未成熟的树突状细胞并具有长效血浆半衰期；构建了FGFR1/MIP3α-Fc融合蛋白真核表达载体并进行初步抗肿瘤作用研究。阶段性研究成果将为深入研究靶向FGFR1抗肿瘤生物学功能奠定基础，并为肿瘤生物靶向治疗提供了新的策略。