新生血管周细胞靶向、长效TRAIL的构建及其抗肿瘤作用和机制研究

As an anticancer drug candidate, the clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was limited by its poor tumor-targeting and short serum half-life. In previous works conducted by the applicant, the antitumor effect of TRAIL was improved by tumor-associated pericytes-targeted delivery mediated by the artificial affibody ZPDGFRβ. In another work performed by the co-workers of the applicant, the antitumor effect of TRAIL was also improved through extending the serum half-life of TRAIL by using albumin-binding domain (ABD)-mediated endogenous albumin-binding. If the tumor-targeting and serum half-life of TRAIL could be improved simultaneously, it is hopeful to produce a novel protein with superior antitumor effect. Consequently, a fusion protein Z-ABD-TRAIL had been produced by fusing ZPDGFRβ and ABD to the TRAIL. And it was found that the fusion protein could bind pericytes and albumin. It was also cytotoxic in tumor cells. In this project, the tumor-targeting and serum half-life of this fusion protein would be examined at first. Alternatively, the molecular structure of the fusion protein would be optimized. The antitumor effect and mechanism, and biosafety of the fusion protein would be evaluated in detail. It is hopeful to produce a tumor-targeting and long-acting protein, which will be alternative for clinical tumor-targeted therapy.

靶向性差、半衰期短是TRAIL临床抗肿瘤效果不佳的主要原因。申请人前期利用人工抗体ZPDGFRβ将TRAIL靶向递送至新生血管周细胞，使其抗肿瘤效果得以提高。所在课题组通过白蛋白结合域ABD介导TRAIL与内源性白蛋白结合，延长半衰期，也提高了TRAIL抗肿瘤活性。若能同时改造TRAIL靶向性和半衰期，可望推出具有超强抗肿瘤活性的新型蛋白药物。本项目提出将ZPDGFRβ和ABD同时与TRAIL连接，组建Z-ABD-TRAIL，对TRAIL靶向性和半衰期实行双改造。申请人前期已构建一种融合蛋白，拟进一步分析其组织分布和药代动力学，确定其是否具有更好的肿瘤靶向性和显著延长的半衰期。若不理想，进一步优化分子设计，组建新的融合蛋白，最终通过体内靶向性，半衰期、抗肿瘤效果及安全性测试，评价其作为新型抗肿瘤药物的可能性。若获实施，可望推出靶向、长效TRAIL蛋白，为肿瘤靶向治疗提供新选择。

肿瘤坏死因子凋亡相关诱导配体TRAIL因具有高效选择性杀伤肿瘤细胞的能力而被认为是极具潜力的抗肿瘤候选药物。临床试验表明TRAIL在人体内的耐受性很好，但其在体内的肿瘤靶向性不足和半衰期过短，大大限制了其体内抗肿瘤活性。本项目前期工作中，将识别PDGFRβ的亲合体ZPDGFRβ与TRAIL融合，成功将TRAIL靶向递送至肿瘤微血管周细胞，大大提高了抗肿瘤效果。同时将结合白蛋白的ABD结构域与TRAIL融合，使TRAIL进入血液后与白蛋白结合，显著延长了TRAIL半衰期，提高了抗肿瘤效果。在此基础上，本项目拟利用蛋白质工程技术，将ZPDGFRβ和ABD结合域同时融合至TRAIL，通过靶向肿瘤新生血管周细胞和结合体内白蛋白，在增强TRAIL靶向性的同时，延长其半衰期。研究结果显示，新型融合蛋白Z-ABD-TRAIL能够经大肠杆菌表达系统大量可溶性表达，亲和层析后可获得高纯度的蛋白，在天然状态下能够正确形成三聚体状态。体外杀伤实验发现，Z-ABD-TRAIL与TRAIL相似，仍然保持高效选择性诱导肿瘤细胞凋亡的能力。在半衰期方面，Z-ABD-TRAIL具有结合白蛋白的能力，在与内源性白蛋白结合后，显著延长了TRAIL的体内半衰期。在靶向性方面，Z-ABD-TRAIL能够与PDGFRβ阳性的肿瘤相关周细胞结合，明显增强了TRAIL的肿瘤靶向能力。最后，Z-ABD-TRAIL比TRAIL表现出显著增强的体内抗肿瘤作用，且没有引起明显的急性毒性反应。这些结果表明，这种新型的TRAIL变异体蛋白Z-ABD-TRAIL同时具备良好的肿瘤靶向能力和超长的体内半衰期，在体内能够发挥强大的抗肿瘤作用，有望作为一种抗肿瘤候选药物继续研究评估，为肿瘤治疗提供新选择。

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