Antiangiogenic Gene Therapy of Ovarian and Breast Cancers

描述（申请人提供）：血管生成对肿瘤的生长和转移很重要。因此，抑制血管生成可以补充卵巢癌和乳腺癌的辅助化疗。目前研究了两大类内源性血管生成抑制剂；胶原蛋白和凝血相关蛋白的蛋白水解裂解产物。内皮抑素是XVIII型胶原非胶原结构域（NC1）的20 kda -c端片段。在许多临床前肿瘤模型中，内皮抑素治疗导致不同程度的肿瘤生长抑制。反应不一致的原因之一是蛋白质折叠受制备抗血管生成分子的表达系统类型的影响。我们已经确定并表征了一个突变的内皮抑素，在125位含有脯氨酸取代丙氨酸残基。与天然分子相比，p125a -内皮抑素与内皮细胞的结合增加，抑制血管生成的效果更好。NCI和我们实验室的独立研究证实，在三种不同的肿瘤模型系统中，突变体内皮抑素比天然蛋白更好地抑制肿瘤生长。因此，了解在这个位置上替换的功能重要性是很重要的。XVIII胶原NC1结构域的多态性主要涉及截断并导致与Knobloch综合征相关的血管问题。唯一已知的另一个突变是D104N，但它不影响内皮抑素的生物活性。P125位点尚未发现多态性。在具体目标1中，我们建议系统地生成其他P125替代并研究突变体的相对抗血管生成效力。将进行机制研究以确定内皮抑素生物活性的突变特异性变化。在具体目标2中，我们建议评估在哺乳动物细胞中表达的突变内皮抑素的治疗和药物毒理学特性。最初的研究将集中在比较使用Alzet泵和aav介导的基因治疗的蛋白质治疗的疗效。基于这些结果，我们将在临床相关的卵巢癌和乳腺癌模型中严格评估选定的突变内皮抑素。最近的研究表明，当化疗与突变型内皮抑素治疗相结合时，肿瘤生长的抑制作用得到改善。我们将研究这两种方法的协同作用机制，以便加快该方法的临床开发。

DESCRIPTION (provided by applicant): Angiogenesis is important for the growth and metastasis of cancer. Inhibition of angiogenesis can therefore complement adjuvant chemotherapy of ovarian and breast cancer. Two major groups of endogenous angiogenesis inhibitors are currently investigated; proteolytic cleavage products of collagens and coagulation related proteins. Endostatin is a 20 kDa-C-terminal fragment of the non-collagenous domain (NC1) of collagen type XVIII. Endostatin treatment had resulted in varying degree of tumor growth inhibition in a number of preclinical tumor models. One of the reasons for the inconsistencies in response is due to protein folding which is affected by the type of expression system used to prepare the antiangiogenic molecule. We have identified and characterized a mutant endostatin containing a substitution of proline to alanine residue at position 125. P125A-endostatin showed increased binding to endothelial cells and inhibited angiogenesis better than the native molecule. Independent studies at NCI and in our laboratory confirmed that the mutant endostatin is better in inhibiting tumor growth than the native protein in three different tumor model systems. Therefore, it is important to understand the functional importance of substitutions at this position. Polymorphism in the NC1 domain of collagen XVIII mostly involves truncations and leads to vascular problems associated with Knobloch syndrome. The only other known mutation is D104N which however does not affect the biological activity of endostatin. No polymorphism has been yet identified at P125. In specific aim 1 we propose to systematically generate other P125 substitutions and investigate the relative antiangiogenic potency of the mutants. Mechanistic studies will be carried out to identify mutation specific changes in the biological activity of endostatin. In specific aim 2 we propose to evaluate the therapeutic and pharmacotoxicologic properties of mutant endostatins expressed in mammalian cells. Initial studies will focus on comparing the efficacy of protein therapy using Alzet pumps and AAV-mediated gene therapy. Based on these results we will critically evaluate a selected mutant endostatin in clinically relevant ovarian and breast cancer models. Recent studies have shown improved inhibition of tumor growth when chemotherapy was combined with mutant endostatin treatment. The mechanism of synergy between these two methods will be investigated so as to facilitate expedited clinical development of this approach.