Development of Novel VEGF Analogs

描述(申请人提供)：我们之前的结构-功能研究导致了第一个糖蛋白激素类似物的开发，受体结合和生物活性都有显著的提高(“超活性类似物”)。大多数癌症高度依赖血管内皮生长因子A(VEGF-A)与其KDR(激酶域受体)受体的相互作用来支持肿瘤血管生成。完全抑制多种形式的血管内皮生长因子的合成和作用是很难实现的，而更通用的受体拮抗剂的使用似乎在任何抗血管生成治疗中都是至关重要的。以前开发高亲和力的血管内皮生长因子激动剂和拮抗剂的尝试都没有成功，但我们已经开发了几个新的高亲和力的糖蛋白激素类似物，它们在结构上与血管内皮生长因子相关。我们假设我们的新的生长因子修饰方法将完全适用于血管内皮生长因子。新的血管内皮生长因子类似物将由连接的血管内皮生长因子单体组成，在同一分子内有两种类型的修饰。首先，将引入“功能增益”氨基酸替换，以增强与VEGF二聚体一端(第1位)的KDR受体的结合。我们将应用电荷扫描突变血管内皮生长因子(第1位)外周环内的氨基酸残基，以及先前被证明成功用于糖蛋白激素的其他合理设计方法。我们的初步数据表明，可以将血管内皮生长因子与KDR受体的结合亲和力提高10倍。在未来的研究中，通过确定选定的氨基酸替代的最佳组合，这一点可能会大大增强。第二种类型的修饰将包括“功能丧失”突变，有或没有突变，在VEGF二聚体的第二极(第2位)引入额外的糖基化位点或聚乙二醇化。这种一极KDR受体结合增强，第二极结合消失的不对称的血管内皮生长因子分子，不仅可以拮抗血管内皮生长因子的作用，而且可以限制单体KDR受体与其他相关受体的异构化。体外受体结合分析和与人内皮细胞的生物活性研究将用于表征这些新的激动剂和拮抗剂。在随后的第二阶段研究中，我们将使用血管生成和肿瘤形成的动物模型来评估血管内皮生长因子类似物的治疗潜力。

DESCRIPTION (provided by applicant): Our previous structure-function studies resulted in the development of the first analogs of glycoprotein hormones with major increases in receptor binding and bioactivity ("super-active analogs"). The majority of cancers are highly dependent on vascular endothelial growth factor A (VEGF-A) interaction with its KDR (kinase domain receptor) receptor to support tumor angiogenesis. Complete inhibition of synthesis and action of all the multiple forms of VEGF is difficult to achieve, and the use of more general receptor antagonists appears vital as an adjunct in any anti-angiogenic therapy. Previous attempts to develop high affinity VEGF agonists and antagonists were not successful, but we have developed several novel high affinity analogs of glycoprotein hormones that are structurally related to VEGF. We hypothesize that our novel approach of growth factor modifications would be entirely applicable to VEGF. New VEGF analogs will consist of linked monomers of VEGF with two types of modifications within the same molecule. First, "gain-of-function" amino acid substitutions will be introduced to enhance binding to the KDR receptor at one pole of the VEGF dimer (Site 1). We will apply charge scanning mutagenesis of amino acid residues within the peripheral loops of VEGF (Site 1) as well as other rational design methods previously proven successful for glycoprotein hormones. Our preliminary data indicate that it is possible to increase binding affinity of VEGF to the KDR receptor up to ten fold. This could be enhanced considerably in future studies by determining optimal combinations of selected amino acid substitutions. The second type of modifications will include "loss-of-function" mutations with or without mutations introducing additional glycosylation site or pegylation at the second pole of VEGF dimer (Site 2). Such an asymmetrical VEGF molecule with enhanced KDR receptor binding at one pole and elimination of binding at the second pole is expected not only to antagonize the action of VEGF but also to limit the pool of monomeric KDR receptors capable of heterodimerizing with other related receptors. In vitro receptor binding assays and bioactivity studies with human endothelial cells will be used to characterize these novel agonists and antagonists. In the subsequent Phase II studies we will assess the therapeutic potential of VEGF analogs using animal models of angiogenesis and tumorigenesis.