TARGETING ANGIOGENESIS WITH TOXIN-VEGF FUSION PROTEINS

该项目的总体目标是开发一种商业上可行的细菌毒素- vegf（血管内皮生长因子）融合蛋白，用于选择性靶向各种病理血管生成部位的内皮细胞。VEGF通过内皮细胞特异性KDR/flk-1受体与内皮细胞相互作用，与静止内皮细胞相比，KDR/flk-1受体在血管生成部位在内皮细胞中过表达。我们的数据表明，VEGF融合蛋白优先与过表达KDR/Flk-1受体的细胞相互作用。为了利用这一靶向性，我们提出制备携带志贺样毒素I （SLT-I）催化片段的VEGF融合蛋白。SLT-I通过破坏内皮细胞导致出血性结肠炎和溶血性尿毒症综合征，这表明内皮细胞对SLT I特别敏感。在I期研究中，我们将构建和测试两种携带SLT-I不同催化片段的SLT- vegf融合蛋白。我们的具体目标是为一种新的血管生成抑制剂提供“原理证明”。此外，关于过表达KDR/flk-1受体的细胞结合和加工SLT-VEGF融合蛋白的信息将为优化这些蛋白建立途径。在II期研究中，我们将优化SLT-VEGF融合蛋白，并将在血管生成的动物模型中进行测试。拟议的商业应用：该项目开发的毒素- vegf融合蛋白可用于治疗几种主要的血管生成依赖性疾病。本项目开发的方法可扩展到其他细胞因子-毒素融合蛋白。

The overall goal of this project is to develop a commercially viable bacterial toxin-VEGF (vascular endothelial growth factor) fusion protein for selective targeting of endothelial cells at the sites of angiogenesis in various pathologies. VEGF interacts with endothelial cells via endothelial cell specific KDR/flk-1 receptor that is overexpressed in endothelial cells at the sites of angiogenesis as compared with quiescent endothelial cells. Our data indicate the VEGF fusion proteins preferentially interact with cells overexpressing KDR/Flk-1 receptors. To exploit this targeting specificity we propose to make VEGF fusion protein carrying catalytic fragment of Shiga-like toxin I (SLT-I). SLT-I causes hemorrhagic colitis and hemolytic uremic syndrome by damaging endothelial cells suggesting that endothelial cells are particularly sensitive to SLT I. In Phase I we will construct and test two SLT-VEGF fusion proteins carrying different catalytic fragments of SLT-I. Our specific aims are designed to provide the "proof-of-principle" for a novel inhibitor of angiogenesis. Furthermore, information about binding and processing of SLT-VEGF fusion proteins by cells overexpressing KDR/flk-1 receptors will establish the routes for optimization of these proteins. In Phase II studies we will optimize SLT-VEGF fusion proteins and will test them in animal models of angiogenesis. PROPOSED COMMERCIAL APPLICATION: Toxin-VEGF fusion protein developed as a result of this project may be used for treatment of several major angiogenesis-dependent pathologies. The methodology developed in this project may be expanded to other cytokine-toxin fusion proteins.