内外联动协同促进sTRAIL抗肿瘤作用的肿瘤微环境触发式融合蛋白的构建及效应机制研究

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily. Its extracellular fragment could be released into body fluid by protease digestion. To date, this soluble protein named soluble TRAIL (sTRAIL) has gained much attention in anti-tumor drug development due to its specific anti-tumor potential without toxic side effects. However, the relatively weak anti-tumor activity in vivo and unfortunate development of sTRAIL resistance in various tumor cells limit both its further development and clinical application. As a result, overcoming the apoptosis resistance and improving in vivo activity of sTRAIL is urgent needed. To solve this problem, a novel fusion protein for cancer therapy was designed and developed based on our innovated MMP2-resistance sTRAIL mutant (msTRAIL) in this work. Briefly, by using literature review and in vitro screening, a target peptide which could sensitize TRAIL-apoptosis in resistant cancer cells will be found out from anti-tumor peptides in preclinical and clinical trial. Following the principle of fusion protein and pro-drug design, the anti-apoptosis-resistance peptide and other functional peptides includes MMP2 sensitive sequence and tumor cell targeted cell penetrating peptide will be fused with msTRAIL by genetic manipulation. Hypothetically, after target delivering into the tumor site, our novel fusion protein would be activated by restricted digestion with MMP2 which is abnormally overexpressed in tumor microenvironment. As a result, the released sTRAIL will induce apoptosis by binding to its cognate death receptor. Meanwhile, under the help of the tumor cell targeted cell penetrating peptide in the rest of fusion protein, the anti-apoptosis-resistance peptide will rapidly diffuse into target cells and synergistically sensitize TRAIL-apoptosis. Moreover, the creative combination of our fusion protein could also provide a new protein skeleton for further design and development new anti-tumor biologicals based on sTRAIL. Its synergistical mechanism of cancer therapy is potentially becoming an innovative theoretical support of multi-targeting cancer biotherapy.

可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）有望成为新一代抗肿瘤生物技术药物，体内抗肿瘤活性不佳以及部分肿瘤对其诱导凋亡的抵抗是阻碍sTRAIL开发和应用的关键。为此，本研究以前期获得的抵抗MMP2的sTRAIL新分子（msTRAIL）为基础，依据肿瘤微环境特性，采用蛋白融合和前药策略，进行创新分子设计，并通过基因重组将msTRAIL与MMP2敏感肽、抗凋亡抵抗肽和肿瘤靶向性细胞穿透肽进行生物融合，制备融合蛋白，并围绕新蛋白的抗肿瘤效应机制展开系统研究。新蛋白可望通过肿瘤靶向性运输、肿瘤微环境触发和细胞内外联动等机制克服凋亡抵抗、促进体内抗肿瘤作用。本研究是对原创性成果的持续性创新，是运用蛋白改造策略解决上述问题的探索性尝试，研究成果可望获得一种新颖的抗肿瘤生物技术候选药物，新蛋白的组合方式及作用机制也将为基于sTRAIL的重组蛋白药物开发和个性化肿瘤生物治疗发展提供新载体、新思路。

重组可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）是一种具有开发潜力的抗肿瘤候选物。部分肿瘤细胞对其诱导凋亡的抵抗限制了sTRAIL的抗肿瘤效应，成为阻碍其应用开发的瓶颈。课题组以自主创新的sTRAIL新分子为骨架，以基于蛋白降解靶向嵌合体（PROTAC）设计思路建立的原创性抗肿瘤多肽为增敏分子，设计并建立了一种新型的sTRAIL融合蛋白，并利用大肠杆菌系统对目的蛋白进行生产制备，最终以人肿瘤细胞系为模型研究了新型融合蛋白的抗肿瘤效应机制。结果表明：本项目获得了一种新颖的抗肿瘤融合蛋白，该蛋白是集合课题组两种原创性分子的二次创新，包含N-端的原创性sTRAIL新分子msTRAIL、C-端的原创性抗肿瘤多肽SHIP以及位于中部包含MMP2敏感序列的连接肽段。其中，msTRAIL是课题组在研究N-端氨基酸对sTRAIL大肠杆菌可溶性表达效率的影响中甄选获得的一种生产效率高且抗肿瘤活性佳的新型突变分子，而SHIP多肽则是课题组在研究中以Hsp90为靶点建立的一种能够促进sTRAIL抗肿瘤活性的PROTAC多肽。该融合蛋白能够在简单、高效、经济和安全的大肠杆菌系统中高效可溶性表达，并可通过简单的纯化工艺得以生产。与msTRAIL相比，融合蛋白在多种人肿瘤细胞系中表现出更好的抗肿瘤效应，不仅能提高msTRAIL在敏感细胞（NCI-H460）中的活性，而且能够克服msTRAIL在耐药细胞中（A549）的凋亡抵抗。机制研究显示，新颖融合蛋白能够被肿瘤微环境中具有代表性的MMP2酶解而释放出msTRAIL和SHIP多肽。其中，游离的msTRAIL通过经典的外途径诱导肿瘤细胞凋亡；同时，游离的SHIP多肽在iRGD引导下渗入肿瘤细胞内通过泛素化蛋白降解途径促进亲和分子Hsp90及其凋亡相关客户蛋白survivin的降解，最终以“内动”的方式促进msTRAIL的抗肿瘤活性。总之，本项目建立了一种能够通过内外联动协同促进sTRAIL抗肿瘤作用的肿瘤微环境触发式融合蛋白，研究成果获得了一种具有后续研究和开发价值的抗肿瘤候选药用蛋白，项目中新蛋白的表达载体、组合方式为基于sTRAIL 的重组蛋白药物开发和个性化肿瘤生物治疗的发展提供新载体和新思路，项目中获得的msTRAIL分子以及靶向Hsp90的PROTAC多肽能为相关的抗肿瘤生物技术药物研究提供新选择。

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