Long Acting VEGF Binding Proteins for Treating Rheumatoid Arthritis

描述（由申请人提供）：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。在I期，我们使用可溶性VEGF受体I（也称为sFlt-1）的已发表结构信息，使用半胱氨酸反应性PEG合理设计聚乙二醇（PEG）-sFlt-1缀合物。我们使用定点诱变在sFlt-1的被认为是生物活性非必需的区域中引入了新的“游离”半胱氨酸。“游离”半胱氨酸残基用作使用硫醇反应性PEG对蛋白质进行共价修饰的位点。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，我们确定了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。我们还进行了药代动力学研究以验证PEG化延长了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生产工艺，并生产足够数量的聚乙二醇化重组sFlt-1，用于在RA动物疾病模型中进行测试。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发挥关键作用，因此，是RA患者治疗干预的合理靶点。VEGF抑制剂已在人体中进行了测试，并已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，可以每周或每两周给药一次。

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.