融合高亲和性肿瘤导向肽增强sTRAIL抗肿瘤作用及机制研究

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) is cytotoxic in a varitey of tumor cells, but non-toxic in normal cells. Due to the high selectivity, sTRAIL is attractive in being developed as novel anti-tumor drugs. However, sTRAIL is only promising in clinical therapy for partial types of tumors. In fact, sTRAIL induce apoptosis in tumor cells mainly through the pathway mediated by the death receptor TRAIL R1. In addition, sTRAIL had low affinity for another death receptor TRAIL R2. Therefor, neumorious tumor cells evolved resistance to sTRAIL by prompted expression of TRAIL R2. This resistance might be overcomed by fusion sTRAIL to a ligand recongizing specific molecule on tumor cells, which resulted in improvement of the affinity of sTRAIL for TRAIL R2. In previous works performed by the applicant, many chimeric proteins had been constructed by fusion sTRAIL to bombesin, a natural tumor-homing peptide with high affinity for bombesin receptor that widely expressed on tumor cells but not normal cells. The chimeric protein showed much higher cytotoxicity in tumor cells, expecially in sTRAIL-resistant tumor cells, than that of sTRAIL. It indicates that the cytotoxicity of sTRAIL was significantly enhanced by fusion to the tumor-homing peptide bombesin. In this project, chimeric proteins consists of sTRAIL and bombesin will be further characterized in cytotoxicity in a wide variety of tumor cells, the binding ability to TRAIL receptors, the relationship between cell binding and cytotoxicity, the mechianism of chimiric protein-induced apoptosis, in vivo anti-tumor effect when it was used as a single agent as well as combined with other agents. The possibility of developing these chimeric proteins as novel anti-tumor drugs will be evaluated in this project. Taking the advantages of small molecule, high affinity for receptor and lacking Cystine amino acid in molecule, the natural tumor-homing peptide bombesin is probably more promising than other molecules described in previous works in improvement of cytotoxicity of sTRAIL in tumor cells. And the chimeric proteins containing bombesin and sTRAIL might be developed as novel drugs that is alternative for cancer tagerted biotherapy.

sTRAIL对多种肿瘤细胞显示超强选择性杀伤活性,但临床应用仅对部分肿瘤有确切治疗效果。sTRAIL主要激活TRAIL R1诱导细胞凋亡，多数肿瘤高表达TRAIL R2而对其产生抗性。融合肿瘤细胞特异性识别分子，可提高sTRAIL激活TRAIL R2的能力而增强其抗肿瘤作用。申请人前期选择高亲和力、高选择性、不含半胱氨酸的天然肿瘤导向肽蛙皮素与sTRAIL融合，显著增强了sTRAIL对肿瘤细胞，尤其是抗性肿瘤细胞的结合和杀伤能力。本项目拟进一步研究利用蛙皮素等天然肿瘤导向肽与sTRAIL构建的融合蛋白，确定其对抗性肿瘤细胞杀伤活性的增强作用及机制，单独或与药物联用体内抗肿瘤效果，评价将其开发为新型抗肿瘤药物的可能性。项目选择的天然肿瘤导向肽具有分子小、亲和力强、不含半胱氨酸等优点，将其与sTRAIL融合，可望推出系列自主知识产权，高选择性、无或低毒性的抗肿瘤蛋白，为肿瘤靶向治疗提供新选择。

人源可溶性TRAIL（hTRAIL）因能选择性杀伤肿瘤细胞而被认为是一种极具潜力的抗肿瘤候选药物。体外条件下，hTRAIL在极低浓度（nM）就对肿瘤细胞显示杀伤而对正常细胞无害。但体内实验，尤其是临床试验发现hTRAIL的抗肿瘤效果并理想。原因之一是血液中的hTRAIL大量被表达诱骗受体的正常细胞消耗，使得肿瘤部位的hTRAIL量不足。融合肿瘤导向肽，改善hTRAIL的肿瘤靶向性可能提高其在肿瘤部位累积量而增强其抗肿瘤效果。血小板衍生生长因子受体β（PDGFRβ）通常在肿瘤微血管周细胞上高表达，可能作为靶标分子实现抗肿瘤药物的靶向运输。项目筛选出一种能够特异性识别PDGFRβ并具有肿瘤靶向性的肽ZPDGFRβ。将其与hTRAIL连接后制备了融合蛋白Z-hTRAIL。与hTRAIL不同，Z-hTRAIL能够识别PDGFRβ并与周细胞结合。但其对死亡受体DR4和DR5的结合能力与hTRAIL相当。Z-hTRAIL与周细胞结合后，可通过旁分泌方式或从周细胞上解离下来再杀伤临近的肿瘤细胞。尾静脉注射的Z-hTRAIL首先富集在肿瘤微血管周细胞上，然后再扩散至邻近的肿瘤细胞。由于能识别周细胞，Z-hTRAIL具有明显的肿瘤靶向性。相应地，Z-hTRAIL的肿瘤摄取量和抗肿瘤效果也显著高于hTRAIL。此外，项目还根据白蛋白的肿瘤富集特性和长效性，利用一种白蛋白结合肽ABD改造hTRAIL，制备了融合蛋白ABD-hTRAIL。研究发现，ABD-hTRAIL能够与白蛋白结合进而更多地富集到肿瘤部位，显示了更强的抗肿瘤作用。这些结果说明，融合肿瘤导向肽可以改善hTRAIL的肿瘤靶向性，进而增强其抗肿瘤效果。