以DLL4及VEGF为双靶点的双特异性抗体的抗肿瘤活性及机制研究

Currently，blocking DLL4/Notch signal pathway to inhibit tumor develoment, tumor angiogenesis and cancer stem cell self-renewing is a focus on tumor-targetted therapy. Besides, VEGF blockade is also an important way of tumor-targetted therapy by inhibiting abnormal angiogenesis. Therefore, in this project, anti-VEGF single chain antibody will be screened out from the human scFv phage display library, and on the basis of our humanized anti-DLL4 mAb, with the help of our skilled gene engineering antibody preparation technology, the anti-DLL4/anti-VEGF bispecific antibody will be constructed and expressed by eukaryotic system to achieve bispecific antibody with high affinity and stable structure. Then, anti-tumor activity of the bispecific antibody in vitro and in vivo will be studied. Further more, how the bispecific antibody regulates structure and function of tumor angiogenesis will also be studied by tumor-bearing mice models and whole genome expression microarray with the help of up-to-date molecular approaches. The anti-tumor mechanism will be studied by both anti-angiogenesis effect and anti-cancer stem cell effect. We expect the new bispecific antibody can inhibit tumor growth by double targetting both DLL4 and VEGF on surface of tumor angiogenesis, block the two signaling pathways so as to strengthen the blocking effect, has better anti-tumor activity than single monoclonal antibody and can effectively avoid or reduce the tolerance of the tumor cells. It will achieve the purpose of better treatment of tumors. This project aims to provide technological platform for anti-tumor antibody drugs of independent intellectual property rights in China.

目前，阻断DLL4/Notch通路抑制肿瘤发生、肿瘤血管生成及肿瘤干细胞增殖成为靶向治疗肿瘤的热点。同时，阻断 VEGF通路抑制血管异常增生亦是肿瘤靶向治疗的重要策略。因此，本项目拟在课题组前期获得的人源化抗DLL4单克隆抗体基础上，首先从全人源噬菌体展示库筛选抗VEGF单链抗体，然后运用本实验室成熟的抗体制备技术获得亲和力高、结构稳定的抗DLL4/抗VEGF双特异性抗体，研究其抗肿瘤活性并用荷瘤小鼠模型及全基因组表达谱芯片结合分子生物学手段探索其对肿瘤血管结构和功能的调控，从抗血管生成和抗肿瘤干细胞两方面研究其抗肿瘤机制。我们预期该双特异性抗体能通过双靶向肿瘤新生血管表面的DLL4与VEGF同时阻断这两条通路从而加强阻断效果，抗肿瘤活性将优于单一的单克隆抗体，并能有效避免或减少肿瘤细胞对单克隆抗体的耐受性，达到更好的治疗效果，同时也为发展我国具有自主知识产权的抗肿瘤抗体药物搭建技术平台。

Notch/DLL4和血管内皮生长因子（VEGF）信号通路在血管生成和肿瘤生长中扮演着重要的角色。由于VEGF和DLL4在脉管系统中的不同监管效应，对DLL4或VEGF信号的阻断可以通过不同的机制抑制肿瘤的生长。抗DLL4治疗会导致不良血管的异常增加，从而导致非生产性血管生成不能支持肿瘤生长，而抗VEGF疗法则显著减少脉管系统，减少肿瘤的供血。此外，DLL4-Notch信号显示在维持癌症干细胞方面的关键作用。因此，本课题在前期获得的人源化抗DLL4单克隆抗体的基础上，利用基因工程技术，设计靶向VEGF和DLL4分子的CrossMab，得到了双靶向VEGF和DLL4的双特异性抗体HB-32。通过基因工程技术将目的基因整合至表达载体pCApuro和pMH3，将其电穿孔共转染CHO-S细胞，挑选单克隆细胞株，进行阳性筛选，连续培养十代以上，进而获得表达量稳定的细胞株；采用Protein A柱对蛋白进行纯化。进行 SDS-PAGE 电泳及 Western blot 检测，验证融合抗体的纯度及装配，ELISA检测双特异性抗体HB-32与两种抗原VEGF和DLL4的结合能力。在体外抗血管生成活性研究过程中，MTT 实验结果表明HB-32对HUVEC 细胞的增殖有明显的抑制作用，且抑制作用呈剂量依赖性，且抑制效果优于Bevacizumab。 划痕损伤修复实验结果显示，加药作用后，与不加药的阴性对照组相比，加药组细胞迁移速度随加药浓度增大而逐渐减慢，表明HB-32对 HUVEC 细胞迁移能力的影响是时间和剂量依赖的，HB-32能显著抑制HUVEC 细胞的迁移。小管形成实验中，加药组的小管形成数与对照组相比显著减少。此外，通过构建MDA-MB-231 乳腺癌移植瘤裸鼠模型研究HB-32的体内抗肿瘤活性。肿瘤生长曲线及抑瘤率显示HB-32可有效抑制肿瘤生长。免疫组化检测HB-32对肿瘤细胞增殖标志 Ki67 及凋亡标志 cleaved caspase-3 表达的影响时，发现HB-32能抑制肿瘤细胞增殖并促进肿瘤细胞的凋亡。应用免疫荧光双染CD31/α-SMA 检测HB-32对肿瘤血管生成的影响时发现HB-32显著减少了功能性血管的生成。因此，本项目获得的以DLL4及VEGF为双靶点的双特异性抗体HB-32具有良好的应用前景。