基于单体双重异质修饰的TRAIL偶联药物及其靶向抗肿瘤活性机制

After binding with death receptors (DR4 and DR5), homotrimeric tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand (TRAIL) could induce apoptosis in various cancer cells instead of most normal human cell types, followed with the internalization of ligand-receptor complex by tumor cells. However, poor pharmacokinetics and resistance within some tumor cell lines have been the major obstacles during the preclinical or clinical application of TRAIL. The half-life of TRAIL114-281 (114 to 281 amino acids) was revealed to be no more than 30 minutes across species. To overcome TRAIL resistance resulted from the defects of intrinsic apoptotic pathway in some tumor cells, the internalization process has been utilized to deliver highly toxic chemical (Monomethyl Auristatin E, MMAE) into cytoplasm via TRAIL-MMAE conjugates in the previous study, and the results were published in top journal Advanced Materials (IF 15.4). The released MMAE could inhibit tumor cell division by blocking the polymerization of tubulin, after the enzymatic hydrolysis of conjugates in lysosome. Therefore, maleimido activated PEG (polyethylene glycol) and MMAE (Monomethyl Auristatin E) were applied to site-specifically conjugate with the mutated cysteines from different monomers of TRAIL successively, taking advantage of steric effects involved within TRAIL mutant conjugations. What’s more, the affinity and activity of PEG-TRAIL-MMAE conjugates could be altered along with different PEG/MMAE ratios, followed by further changed pharmacokinetics (PK) and pharmacodynamics (PD). Thus this project will attribute the fundamental mechanisms of antitumor activities of TRAIL conjugates to their cellular metabolic behaviors, after the synthesis of PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios as model molecules.

TRAIL能够诱导大部分肿瘤细胞凋亡，而对正常细胞无损伤，是极具潜力的新型大分子靶向药物。然而，半衰期短（5~30 min）及肿瘤细胞先天和获得性耐药的存在制约了TRAIL的进一步应用。在前期研究中，我们利用TRAIL偶联药物（海兔毒素，MMAE）的靶向抗肿瘤策略使其耐药性问题得以改善，相关成果发表于Advanced Materials（IF 15.4）。但TRAIL偶联药物的深层次活性和代谢机制仍待挖掘，且TRAIL的药代动力学缺陷也亟需克服。因此，本研究将针对TRAIL三聚体的结构特征，利用偶联反应中的空间位阻效应在各个单体的突变位点上先后修饰PEG和MMAE，以达到双重异质修饰TRAIL三聚体的目的，并借此同时解决半衰期短和耐药性的难点。此外，本研究将以PEG/MMAE比例为唯一初始变量，研究TRAIL偶联药物细胞水平代谢机制，结合组织分布和药效学阐释其靶向抗肿瘤活性的内在机制。

针对TRAIL三聚体的结构特征，利用偶联反应中的空间位阻效应在各个单体的突变位点上先后修饰PEG和MMAE，以达到双重异质修饰TRAIL三聚体的目的，并借此同时解决半衰期短和耐药性的难点。以PEG/MMAE比例为唯一初始变量，研究TRAIL偶联药物细胞水平代谢机制，结合组织分布和药效学阐释其靶向抗肿瘤活性的内在机制。.我们通过偶联条件摸索和研究，得到了不同PEG/MMAE修饰比例的TRAIL偶联药物，并利用圆二色谱分析了不同PEG/MMAE修饰比例的TRAIL偶联药物的二级结构变化；完成了不同PEG/MMAE修饰比例的TRAIL偶联药物与肿瘤细胞的亲和力比较；完成了不同PEG/MMAE修饰比例的TRAIL偶联药物在体外活性评价、以及DR4-Fc和DR5-Fc重组蛋白的竞争性活性抑制实验；完成了不同PEG/MMAE修饰比例的TRAIL偶联药物在体内的药代动力学研究；完成了不同PEG/MMAE修饰比例的TRAIL偶联药物的荷瘤小鼠体内药效研究以及毒性评价。.通过对各种TRAIL偶联药物的体内外活性、毒性、药代动力学性质评价等，深入研究了PEG和MMAE两种修饰对TRAIL偶联药物的影响，最终阐明了它们的抗肿瘤活性机制：MMAE主要贡献了TRAIL偶联药物的活性，因此体外活性与MMAE含量成正相关；PEG则极大提高TRAIL偶联药物的半衰期，因此增强了TRAIL偶联药物体内生物利用度，但会影响偶联物的体外活性；当PEG/MMAE比例为1：2时，TRAIL偶联药物的抗肿瘤活性达到最佳；各种TRAIL偶联药物均未发现明显的毒性；通过靶标蛋白的体外竞争性抑制，我们阐明了TRAIL偶联药物通过DR4和DR5的内吞实现抗肿瘤活性，而通过caspase的全局抑制实验，我们揭示TRAIL偶联药物最终通过caspase通路发挥抗肿瘤活性。