Long-Acting Endostatin for Treating Cancer

描述（由申请人提供）： 内皮抑素是一种20 kDa的非糖基化蛋白，是内皮细胞增殖的特异性抑制剂和有效的抗血管生成剂。内皮抑制素的治疗效果由于几个原因而难以评估。内皮抑制素的可溶性制剂迅速失去活性，并且受到与储存、处理和纯化方法相关的技术困难的困扰。此外，由于其相对较短的血清半衰期，需要大量的蛋白质才能在体内观察到显著的生物活性。我们的研究目标是创造新的内皮抑制素蛋白质，既改善物理特性和增强在体外和体内的活动。我们实现这一任务的方法涉及通过聚乙二醇（PEG）部分的位点特异性共价加成来修饰内皮抑素。通过使用内皮抑制素的已知结构，我们已经能够将特定的半胱氨酸突变工程化到蛋白质中，以作为巯基反应PEG的附着位点。该策略允许合理设计具有确定结构的完全活性蛋白质类似物，并克服了使用标准赖氨酸反应性PEG试剂修饰蛋白质时生物活性损失和产物异质性的问题。在第二阶段的部分，我们将评估这些聚乙二醇化内皮抑制素突变蛋白在体外和体内。 根据我们的经验和文献报道，聚乙二醇化人内皮抑制素应具有增强的稳定性、更大的效力和更低的剂量要求。这将使更多的患者获得药物，因为据估计，超过900万癌症患者可能受益于抗血管生成治疗。

DESCRIPTION (provided by applicant): Endostatin is a 20 kDa non-glycosylated protein that is a specific inhibitor of endothelial proliferation and a potent anti-angiogenesis agent. The therapeutic effects of endostatin have been difficult to evaluate for several reasons. Soluble preparations of endostatin quickly lose activity and are plagued by technical difficulties related to storage, handling, and purification methods. In addition, because of its relatively short serum half-life, large quantities of protein are needed to see significant bioactivity in vivo. The goal of our research is to create novel endostatin proteins with both improved physical characteristics and enhanced in vitro and in vivo activities. Our approach towards accomplishing this task involves modifying endostatin by the site-specific covalent addition of a polyethylene glycol (PEG) moiety. By using the known structure of endostatin, we have been able to engineer into the protein, specific cysteine mutations to serve as attachment sites for thiol-reacfive PEGs. This strategy allows for the rational design of fully active protein analogues of defined structure and overcomes the problem of loss of bioactivity and product heterogeneity when proteins are modified using standard lysine-reactive PEG reagents. During the Phase II portion of this grant we will evaluate a number of these PEGylated endostatin muteins both in vitro and in vivo. Based upon our experience and literature reports, PEGylated human endostatin should have enhanced stability, greater potency and lower dose requirements. This will allow a larger number of patients access to the drug since it is estimated that more than 9 million cancer patients may benefit from anti-angiogenesis therapy.