靶向肿瘤干细胞和肿瘤血管的多功能融合蛋白的制备及抗肿瘤作用

Clinical study shows that multi-target anticancer drugs are better than single-target drugs. Multi-target drugs for cancer therapy is becoming an attractive trend of anticancer drugs.We plan to prepare a fusion protein targeted both cancer stem cells and tumor angiogenesis.Cancer stem cells are the potenial origin of tumor development, relapse and metastasis.As known, tumor blood vessels supply nutrition and oxygen for tumor growth and routes for tumor metastasis.Recent studies show that CD13 is the marker of human liver cancer stem cells,and previous reports also show that CD13 is expressed in vascular endothelial cells and can induce angiogenesis.In this study,we will use NGR peptide, a CD13 ligand, as targeting molecule and use the highly potent antitumor antibiotic lidamycin as "warhead" molecule to prepare a fusion protein. As the lidamycin molecule comoposes of apoprotein and chromophore and they can be dissociated and reconstituted in vitro, we will adopt a 2-step procedure to prepare energized fusion protein: firstly, to construct the fusion protein composed of CNGRC peptide and LDP by DNA recombination; secondly, to reload AE extracted from lidamycin to the fusion protein. Then we will study the targeting activity of NGR-LDP-AE to CD13 which expresses both on liver cancer stem cells and tumor blood vessels, and investigate the therapeutic effect of NGR-LDP-AE on human liver cancer xenograft in nude mice and its mechanism.The combination efficacy of NGR-LDP-AE and other anticancer drugs will also be evaluated. The final goal of this study is set to develop a new anticancer drug which could target both cancer stem cells and tumor angiogenesis; in addition, to explore a new approach to the development of multiple targeted drugs.

临床研究显示，多靶向药物的疗效优于单靶向药物，多靶向药物是抗肿瘤药物的发展趋势。本项目拟制备同时靶向肿瘤干细胞和肿瘤血管的多靶向药物。肿瘤干细胞是肿瘤发生、发展、复发与转移的根源，肿瘤血管为肿瘤的生长提供物质基础及转移的通道。研究显示CD13是人肝癌干细胞的表面标记物，同时与肿瘤新生血管的形成密切相关。本项目以CD13亲和肽NGR为靶向分子，以强效抗肿瘤抗生素力达霉素为"弹头"药物，制备NGR肽与力达霉素的强化融合蛋白NGR-LDP-AE。根据力达霉素由辅基蛋白和发色团构成，且二者可以拆分和重建的特点，运用DNA重组和分子组装相结合的创新路线制备NGR-LDP-AE；研究NGR-LDP-AE对肝癌干细胞及肿瘤血管的抑制活性,建立人肝癌裸鼠移植模型，研究NGR-LDP-AE对肝癌的疗效及其作用机制，同时研究其与常规化疗药物联合用药对肝癌的疗效，以获得一种新型的多靶向抗肿瘤药物。

研究表明CD13与肿瘤新生血管的形成密切相关，同时又是人肝癌干细胞的标记物，以CD13为靶标可同时将药物靶向肝癌干细胞和血管内皮细胞。本项目以CD13 亲和肽NGR为靶向分子，以强效抗肿瘤抗生素力达霉素为"弹头"药物，制备强化融合蛋白NGR-LDP-AE。根据力达霉素由辅基蛋白（LDP）和发色团（AE）构成，且二者可以拆分和重建的特点，运用DNA 重组技术制备融合蛋白NGR-LDP，再组装AE制备NGR-LDP-AE。流式及ELISA实验表明NGR-LDP能与高表达CD13的细胞结合，而LDP结合较弱；免疫组化研究表明NGR-LDP能与肿瘤血管内皮细胞特异结合。CD13分子上的WM15表位是NGR肽的结合位点，免疫印迹和免疫荧光研究表明在人肝癌细胞和微血管内皮细胞中有两种含WM-15表位的CD13分子表达，而人正常肝细胞中则表达缺失。NGR-LDP-AE对上述培养的细胞均表现出较强的细胞毒活性，IC50值介于0.34-3.08 nM。NGR-LDP-AE对人肝癌Huh7细胞的细胞球形成能力有显著的抑制活性，半数抑制剂量为0.225 nM，显著低于细胞毒活性的IC50值（3.08 nM），且细胞球体积变小；与等细胞毒性的LDM相比，抑制活性也显著增强。NGR-LDP-AE在并未显示出明显细胞毒活性的剂量下，能够显著抑制内皮细胞管腔样结构的形成。NGR-LDP-AE可诱导肝癌细胞中LC3-Ⅰ向LC3-Ⅱ的转化及P62、PARP的降解，表明NGR-LDP-AE可诱导肝癌细胞自噬和凋亡。体内实验表明NGR-LDP-AE能够抑制人肝癌裸鼠移植瘤的生长，免疫组化实验表明NGR-LDP-AE处理组CD13表达下调，CD13+细胞数量减少；体外实验也表明NGR-LDP-AE能够诱导肝癌细胞中CD13的下调。NGR-LDP-AE与阿霉素联合，对体外培养的肝癌细胞的生长表现出协同抑制活性，在IC50及IC90抑制浓度时，两药协同指数分别0.930和0.334。以上实验结果表明NGR-LDP-AE可同时靶向肝癌干细胞和血管内皮细胞，具有单一分子多靶向的特点。在我国肝癌发病率位居恶性肿瘤的第二位，每年导致约38万人死亡。NGR-LDP-AE及其与化疗药物的联合用药有望为原发性肝癌尤其是复发及转移肝癌的治疗甚至根治提供新的治疗策略。