Long Acting VEGF Inhibitor for Treating RA

描述（由申请人提供）： 类风湿性关节炎（RA）是一种慢性炎症性疾病，可导致进行性关节破坏、畸形和残疾。最早观察到的RA特征之一是滑膜内新血管网络的发展，其允许将细胞和营养物质递送到侵入的血管翳。这种新血管的形成（也称为血管生成）是在宿主微环境和许多分泌的介质的影响下高度调节的过程。许多促血管生成介质在RA中表达。这些介质之一，特别是血管内皮生长因子（VEGF）似乎在内皮细胞的分化和关节滑膜衬里血管系统的发育中发挥关键作用，因此，是RA患者治疗干预的合理靶点。我们的总体目标是创造一种长效VEGF抑制剂，并在RA动物模型中测试其疗效。利用已发表的可溶性VEGF受体I（也称为sFlt-1）的结构信息，我们将使用半胱氨酸反应性PEG合理地设计聚乙二醇（PEG）-sFlt-1缀合物。我们将引入一个新的“自由”半胱氨酸使用定点诱变的区域sFlt-1被认为是非必需的生物活性。“游离”半胱氨酸残基将用作使用硫醇反应性PEG共价修饰蛋白质的位点。该技术允许产生具有确定结构的新型、完全活性的PEG-Cys-sFlt-1类似物，并克服了使用标准胺反应性PEG修饰蛋白质时生物活性降低和异质性降低的问题。在第一阶段，我们将确定sFlt-1中可以被修饰而不影响蛋白质的体外生物活性的位点。我们还将进行药代动力学研究以验证PEG化延长sFlt-1的循环半衰期。在第二阶段，我们开发了一种具有成本效益的生产工艺，并生产了足够数量的聚乙二醇化重组sFlt-1，用于动物疾病模型和生化表征的测试。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes progressive joint destruction, deformities and disability. One of the earliest observed features of RA is the development of a new vascular network within the synovium that allows for the delivery of cells and nutrients to the invading pannus. This formation of new blood vessels (also known as angiogenesis) is a highly regulated process under the influence of the host microenvironment and a number of secreted mediators. Many proangiogeneic mediators are expressed in RA. One of these mediators in particular, vascular endothelial growth factor (VEGF) appears to play a critical role in the differentiation of endothelial cells and the development of the vascular system in the synovial lining of the joints and therefore, is a reasonable target for therapeutic intervention in RA patients. Our overall goals are to create a long acting VEGF inhibitor and test its efficacy in animal models of RA. Using the published structural information for soluble VEGF Receptor I (also known as sFlt-1), we will rationally design polyethylene glycol (PEG)-sFlt-1 conjugates using cysteine-reactive PEGs. We will introduce a new "free" cysteine using site-directed mutagenesis in regions of sFlt-1 that are believed to be non-essential for biological activity. The "free" cysteine residue will serve as the site for the covalent modification of the protein using a thiol-reactive PEG. This technology allows for the creation of novel, fully active PEG-Cys-sFlt-1 analogues of defined structure and overcomes the problems of reduced bioactivity and heterogeneity when proteins are modified using standard amine-reactive PEGs. During Phase I we will identify sites in sFlt-1 that can be modified without affecting the protein's in vitro bioactivity. We will also perform a pharmacokinetic study to verify that PEGylation extends the circulating half-life of sFlt-1. During Phase II, we develop a cost effective manufacturing process and produce sufficient quantities of PEGylated recombinant sFlt-1 for testing in animal disease models and biochemical characterization.