抗明胶酶scFv-LDM双价融合蛋白的分子设计及其抗胰腺癌实验研究

Pancreatic cancer is one of maligancies that is seriously threat to human health, there is no effictive drug other than Gemcitabine in non-surgical treatment of pancreatic cancer, the development of more potent drugs against pancreatic cancer is imperative. It was known that gelatinases was overexpressed on pancreatic cancer and its stromal cells, and was low-expressed on normal tissue which was strictly regulated, therefore, gelatinases was a potential target for cancer theapy of pancreatic cancer.Lidamycin (LDM) was a potent antitumor agent and demonstrated well antitumor efficiency against pancreatic cancer, its conjugates with antibody molecule could signficantly inhibited the tumor growth in vitro and in vivo. This project was designed based on the previous results of a tandem anti-gelatinases scFv-LDM fusion protein, which demonstrated that the fusion protein could not enter the cancer cells efficient. In order to improve the internalization efficacy of bivalent fusion protein scFv-LDM, the activatable cell-penetrating peptide (ACPP) of E9-GPLGMLSQ-R9 was introduced between the tandem scFv and LDM, and thus enhanced the antitumor efficacy of dFv-LDM fusion protein. In this project, a new approach of preinjecting the anti-gelatinases scFv was adopted,with an aim to avoid the non-specific activatble possibility of ACPP. The results of which could provide a theroy and experimental basis for the development of ACPP in future.

胰腺癌是一种严重威胁人类健康的恶性肿瘤，非手术治疗除吉西他滨外还缺乏更有效的治疗药物，开发新型的胰腺癌治疗药物迫在眉睫。明胶酶在胰腺癌及其间质中高表达，在正常组织呈极低表达并受到严格调控，因此是一个潜在的治疗靶点；力达霉素LDM是一个高效的抗肿瘤抗生素，其与抗体分子的偶联物在动物体内展现了良好的抗肿瘤效果。本项目在前期抗明胶酶scFv-LDM双价融合蛋白的基础上，针对其不易被细胞内化的问题，拟采用可剪切的细胞穿透肽ACPPs技术，将明胶酶可切割的E9-GPLGMLSQ-R9多肽序列引入scFv和LDM之间，提高LDM在肿瘤局部释放和进入胞内的效率，以期获得一种新型、高效的抗胰腺癌抗体靶向药物。同时，本项目体内药效评价拟采用提前给予小剂量抗明胶酶scFv的策略，以规避ACPPs非特异剪切释放的问题，为后续基于MMP-2/9 ACPPs的融合蛋白体内给药策略提供新的思路和借鉴作用。

胰腺癌是一种严重威胁人类健康的恶性肿瘤，非手术治疗除吉西他滨外还缺乏更有效的治疗药物，开发新型的胰腺癌治疗药物迫在眉睫。明胶酶在胰腺癌及其间质中高表达，在正常组织呈极低表达并受到严格调控，因此是一个潜在的治疗靶点；力达霉素LDM是一个高效的抗肿瘤抗生素，其与抗体分子的偶联物在动物体内展现了良好的抗肿瘤效果。本研究在前期工作工作基础上，构建和表达了抗明胶酶的双价scFv-LDM（dFv-LDP）、带有细胞穿膜肽（Arg）9的dFv-R-LDP以及有明胶酶可切割的E9-GPLGMLSQ-R9（EMR）多肽系列的dFV-EMR-LDP三种融合蛋白。ELISA和FACS分析表明，三种蛋白dFv-LDP、dFv-R-LDP和dFv-EMR-LDP均能较好的与抗原明胶酶和胰腺癌细胞SW1990结合，R9和EMR的加入不影响scFv和抗原的结合。FACS分析表明，R9和EMR的插入均能提高融合蛋白dFv-LDP进入细胞的效率，在一定时间内以dFv-R-LDP进入细胞效率最高。融合蛋白与发色团AE强化后，dFv-R-LDP-AE表现了最强的体外细胞毒活性。三种强化的融合蛋白均能诱导SW1990和PNCA-1胰腺癌细胞的凋亡，其中以dFv-R-LDP-AE凋亡诱导能力最强。体内动物实验表明，强化融合蛋白dFv-EMR-LDP-AE毒副作用较大，导致实验荷瘤小鼠全部死亡，而dFv-R-LDP-AE能提高dFv-LDP-AE的抗肿瘤活性；免疫组化分析表明，能够进一步下调PCNA和MMP-9的表达。体外免疫印迹分析表明，在胰腺癌细胞SW1990和PNCA-1细胞中，强化融合蛋白dFv-R-LDP-AE能够下调其VEGF、Cyclin D1、Cox-2、Bcl-2的表达，增加Caspase-3和PARP的剪切。小动物活体成像表明，FITC标记的融合蛋白dFv-R-LDP可以具有较好的靶向异位移植的胰腺癌SW1990裸鼠肿瘤以及原位移植胰腺癌Capan-2的裸鼠肿瘤。以上研究表明，以明胶酶为靶的scFv结合能被MMP-2/9切割的ACPP构想，能够带来更大的毒副作用，在将来的药物研发中需要慎重考虑。抗明胶酶的dFv-LDP加入细胞穿梭肽（Arg）9后，能够提高其抗肿瘤的效果。因此，融合蛋白dFv-R-LDP-AE在胰腺癌的治疗方面具有一定的应用前景。