TARGETING TOXIN POLYPEPTIDES TO TUMOR VASCULATURE

对于直径超过2-3毫米的持续生长，固体 肿瘤依赖于新的血液供应的建立。肿瘤 细胞分泌多种血管生成生长因子诱导局部 从预先存在的宿主维管组织中萌发的维管组织 (新生血管)。最近的研究表明，血管内皮细胞 生长因子(VEGF)/血管渗透因子(VPF)是一种重要的 肿瘤新生血管和间质发展的介体。血管内皮生长因子结合 受体酪氨酸激酶(Flt-1和KDR/Flk-1)，它们是 肿瘤血管内皮细胞的差异过表达 与周围正常组织的血管相比。由于每个 微血管是大量肿瘤细胞的生命线，靶向 肿瘤血管对肿瘤的生长将产生深远的影响。论 在这些研究的基础上，我们通过化学方法制备了一种细胞毒性结合物。 将血管内皮生长因子的剪接变体和截短形式的白喉毒素联系起来。 血管内皮生长因子-毒素结合物选择性抑制血管内皮细胞增殖 体外培养内皮细胞株和体内实验血管生成。 受体阴性的细胞系，如肿瘤细胞，不受 共轭疗法。在这些研究之后，我们调查了 血管内皮生长因子-毒素结合物对两种实体瘤生长的影响 动物模型。腹膜腔内注射血管内皮生长因子-毒素结合物7 肿瘤细胞移植后几天，肿瘤的出现和 随后的增长。初步组织病理学检查显示血管内皮生长因子- 毒素选择性地影响肿瘤血管，导致肿瘤坏死。 取自相同动物的正常组织显示没有血管 肝、肾或肺损伤/出血或坏死。这些数据 支持血管内皮细胞生长因子毒素可以安全使用。差异化 血管内皮生长因子-毒素结合物的作用与细胞增殖状态的关系 内皮细胞。静止的正常内皮对血管内皮生长因子毒素具有抵抗力 共轭。在目前的提案中，我们将严格评估和 通过优化合成工艺提高血管内皮生长因子-毒素结合物的抗肿瘤活性 剂量，给药途径，并通过改善组成 用基因工程毒素分子构建。此外，我们 将通过广泛的毒理学研究来确定这种方法的局限性 研究并制定克服这些问题的策略。最后，血管内皮生长因子- 将准备融合毒素以促进进一步的临床前研究 发展。

For a sustained growth beyond the size of 2 - 3 mm in diameter, solid tumors are dependent on the establishment of new blood supply. Tumor cells secrete a number of angiogenic growth factors to induce localized vascular sprouting from pre-existing host vasculature (neovascularization). Recent studies have shown that Vascular endothelial growth factor (VEGF)/Vascular permeability factor (VPF) is an important mediator of tumor neovascularization and stroma development. VEGF binds to receptor tyrosine kinases (flt-1 and KDR/flk-1) which are differentially overexpressed on the endothelial cells of tumor vasculature when compared to blood vessels of surrounding normal tissues. Since each microvessel is a life line for a large number of tumor cells, targeting the tumor vasculature will have a profound effect on tumor growth. On the basis of these studies, we prepared a cytotoxic conjugate by chemically linking a splice variant of VEGF and a truncated form of diphtheria toxin. VEGF-toxin conjugate selectively inhibited the proliferation of endothelial cell lines in vitro and experimental angiogenesis in vivo. Receptor negative cell lines such as tumor cells were not affected by the conjugate treatment. Following these studies, we investigated the effect of VEGF-toxin conjugate on the growth of two solid tumors in experimental animal models. Intraperitoneal administration of VEGF-toxin conjugate 7 days after tumor cell transplantation delayed tumor appearance and subsequent growth. Preliminary histopathological examination showed VEGF- toxin selectively affected the tumor vasculature and induced necrosis. Normal tissues obtained from the same animals showed no vascular damage/hemorrhage or necrosis in liver, kidney or lung. These data support that VEGF-toxin can be safely administered. The differential effect of VEGF-toxin conjugate correlated with the proliferative status of endothelium. Quiescent normal endothelium is resistant to VEGF-toxin conjugate. In the current proposal we will critically evaluate and improve the anti-tumor activity of VEGF-toxin conjugate by optimizing the dose, route of administration and by improving the composition of the construct with genetically engineered toxin molecules. In addition, we will identify the limitations of this approach by extensive toxicological studies and develop strategies to overcome the problems. Finally, VEGF- fusion toxins will be prepared to facilitate further preclinical development.