Long Acting VEGF Binding Proteins for Treating Rheumatoid Arthritis

描述（由申请人提供）：类风湿关节炎（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的特征之一是滑膜内新血管网络的发展，该血管网络允许将细胞和营养物质输送到入侵的输卵管。这种新血管的形成（也称为血管生成）是一个受宿主微环境和许多分泌介质影响的高度调控的过程。许多促血管生成介质在RA中表达。其中一种介质，血管内皮生长因子（VEGF）似乎在关节滑膜内皮细胞的分化和血管系统的发育中起着关键作用，因此是RA患者治疗干预的合理目标。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。在第一阶段，我们利用已公布的可溶性VEGF受体I（也称为sFlt-1）的结构信息，利用半胱氨酸反应性PEG合理设计聚乙二醇(PEG)-sFlt-1偶联物。我们在sFlt-1被认为对生物活性不是必需的区域使用定点诱变引入了一个新的“自由”半胱氨酸。“游离”半胱氨酸残基作为使用巯基反应性PEG对蛋白质进行共价修饰的位点。该技术允许创建具有明确结构的新颖、全活性PEG-Cys-sFlt-1类似物，并克服了使用标准胺反应性peg修饰蛋白质时降低生物活性和异质性的问题。在第一阶段，我们确定了sFlt-1中可以在不影响蛋白质体外生物活性的情况下进行修饰的位点。我们还进行了一项药代动力学研究，以验证PEGylation延长了sFlt-1的循环半衰期。在第二阶段，我们将开发一种具有成本效益的制造工艺，并生产足够数量的聚乙二醇化重组sFlt-1，用于RA动物疾病模型的测试。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。血管内皮生长因子（VEGF）似乎在这一炎症过程中起着关键作用，因此是RA患者治疗干预的合理目标。VEGF抑制剂已经在人体中进行了测试，并被证明可以延缓疾病的进展。我们的总体目标是创造一种长效VEGF抑制剂，可以每周一次或每两周服用一次。

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. During Phase I, we used the published structural information for soluble VEGF Receptor I (also known as sFlt-1) to rationally design polyethylene glycol (PEG)-sFlt-1 conjugates using cysteine-reactive PEGs. We introduced a new "free" cysteine using site-directed mutagenesis in regions of sFlt-1 that were believed to be non-essential for biological activity. The "free" cysteine residue served 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 identified sites in sFlt-1 that can be modified without affecting the protein's in vitro bioactivity. We also performed a pharmacokinetic study to verify that PEGylation extends the circulating half-life of sFlt-1. During Phase II, we will develop a cost effective manufacturing process and produce sufficient quantities of PEGylated recombinant sFlt-1 for testing in animal disease models of RA. Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes progressive joint destruction, deformities and disability. Vascular endothelial growth factor (VEGF) appears to play a critical role in this inflammatory process and therefore, is a reasonable target for therapeutic intervention in RA patients. VEGF inhibitors have been tested in humans and have been shown to delay the progression of the disease. Our overall goal is to create a long acting VEGF inhibitor that can be dosed once a week or once every two weeks.