基于肿瘤靶向及特异性激活的“前药”性大分子药物给药系统治疗结肠癌的研究

Chemotherapy has thus far attained little success in treating cancers, primarily due to lack clinical efficacy and presence of drug-induced toxicity. In this project, we have integrated innovative strategies into a drug delivery system to overcome such obstacles in the treatment of colorectal cancers. This drug delivery system comprises of two components. One is a targeting component composed of T84.66, a mouse mAb against human carcinoembryonic antigen (CEA). Literature reports demonstrated that CEA, a surface antigen known to associate with tumor growth/metastasis, was overly expressed in colonic cancers but limited in normal tissues. Our previous findings also showed that >85% of the injected T84.66, a mouse mAb against human CEA, accumulated at the colorectal tumor target via an antigen-mediated elimination pathway. The second component of this DDS comprises of a potent toxin (gelonin) or a siRNA drug covalently linked with LMWP, a proven but non-toxic CPP, via S-S bond. This LMWP-Drug conjugate will be linked to the targeting T84.66 through a peptide linker containing a legumain-degradable specific AANL sequence. The protease legumain was recently reported to express profoundly on the surface of colorectal tumors but is almost undetectable in normal tissues. With this design, the LMWP-Drug-T84.66 complex would exhibit a prodrug behavior during tumor targeting, since LMWP is embedded between the antibody and drug molecule, thus being unable to transverse the drug through tumor cells in exerting cytotoxic effects. Following tumor accumulation via T84.66 targeting, release of LMWP-Drug from T84.66 will take place on tumor surface automatically via cleavage of the AANL linker by surface-expressed legumain. LMWP-Drug would then internalize into tumor cells via LMWP-mediated cell entry action, initiating tumor apoptosis. The specific aims are to validate the pre-clinical capability of this system in managing specifically colorectal cancers, through extensive in vitro and in vivo studies using a well-established LS174T xenograft mouse model; thus paving the road for future clinical translation.

化疗因其效力欠佳且呈现全身毒性而难以取得良好的癌症治疗效果。其原因主要有：药物缺乏靶向性；药物自身的效力低；大分子药物难以透过细胞膜以发挥药效；药物对正常组织具有毒性。本项目旨在建立可有效克服上述限制的药物传递系统，以达到肿瘤治疗的最佳效果和最低毒性。本项目拟以结肠癌为研究模型，选择具有潜力的抗癌大分子药物siRNA，设计了一种以肿瘤表面特异性表达的抗原和内肽酶为双重靶标，且具有“前药”特性的药物传递系统——siRNA-LMWP-Linker-T84.66。该系统以特异性抗体T84.66高效靶向结肠癌细胞并作为前药保护基团，暂时封闭穿膜肽的作用，用结肠癌细胞表面特异性表达的legumain 酶作为前药激活因子，使药物在肿瘤部位被特异性激活而发挥作用，从而杜绝肿瘤治疗药物的全身毒性，提高药物治疗的效果和安全性。本项目中的研究技术具有普适性，对其它肿瘤给药系统亦具有一定参考价值。

本课题研究目标是构建双重酶敏感的前药型抗体靶向递药体系(siRNA -LMWP- Linker-Antibody)，实现药物的高效靶向递送及定点释放。体系以还原酶敏感的siRNA-LMWP前体药物为基础，利用癌细胞表面过表达的Legumain或MMP2酶的底物肽(Linker)连接该前体药物与抗体，借助穿膜肽促进药物入胞及其胞浆释放，提高对靶细胞的基因沉默效率。.首先开发了新型的前体药物siRNA-S-S-PEG-LMWP，有效解决了体系聚沉问题，实现了细胞穿膜肽与siRNA的一对一共价键连，实验结果证明该前体药物具有基因沉默效率高和细胞毒性低的良好特性，鉴于该技术兼具创新性与实用性，目前已完成成果技术转移。进一步将前体药物通过酶特异性底物肽与抗体共价偶联，成功构建了siRNA-S-S-PEG-LMWP-Linker-Antibody递药体系。实验证实了所构建递药体系的抗体特异性及肿瘤酶响应性，即在抗体与细胞表面抗原特异性结合后，含有底物肽的前药体系被对应酶切，激活前体药物，细胞穿膜肽介导siRNA入胞，随后二硫键在胞浆被还原并释放siRNA，继而产生良好的基因沉默效果。本研究中多项技术具有普适性，可推广到其他药物传递系统，也为近一步的临床转化提供了理论支持和技术参考。.目前，该课题相关研究成果已发表在《Theranostics》、 《Journal of Controlled Release》、《ACS Applied Materials & Interfaces》等高水平杂志上，共发表SCI学术论文11篇（均标注基金号），累计影响因子为59.845，平均每篇影响因子为5.44，单篇最高影响因子为9.222。申请专利2项，成果技术转移1项，大会报告3次，发表会议论文3篇。培养毕业博士生1名，培养毕业硕士生2名，在读硕士生2名。