Preclinical development of AlaTIMP-2 as an cancer therapeutic

朝着我们所述的TIMP-2作为一种新型癌症的临床前开发目标努力 治疗我们已经进行了体内异种移植物生长实验。这些实验 显示缺乏MMP抑制活性的TIMP-2同系物（称为Ala+TIMP-2）有效地 当在肿瘤细胞中过表达时，抑制人A549肺癌异种移植物的生长 使用逆转录病毒载体系统。为了进一步开发Ala+TIMP-2作为治疗药物，我们 已经开发了杆状病毒和哺乳动物细胞表达系统用于生产 重组Ala+TIMP-2和TIMP-2。我们的计划是测试这些材料的治疗效果 在异种移植物和同系小鼠肿瘤模型中证明药物的有效性 外源性重组蛋白治疗。这些模型将侧重于治疗 肺癌（A549和刘易斯肺）以及黑色素瘤（A2058和B16 F10）。各种给药 方案将用于比较Ala+TIMP-2的相对体内有效性， TIMP-2初步研究表明Ala+TIMP-2比TIMP-2更有效， 这归因于Ala+TIMP-2不像TIMP-2那样与MMP的活性位点结合， 因此有效地增加了其细胞结合位点的浓度。另一个重要 这个项目的一个方面是确定我们是否可以开发肽类似物， 用于体内治疗。此外，我们建议开发一种高通量筛选方法， 筛选可竞争TIMP-2、Ala+TIMP-2或 TIMP肽与细胞表面受体整合素α 3 β 1结合，我们已经证明 调节Ala+TIMP-2的抗血管生成和抗肿瘤生成活性。

Working towards our stated goal of preclinical development of TIMP-2 as a novel cancer therapeutic we have performed in vivo xenograft growth experiments. These experiments have shown that the TIMP-2 homolog lacking MMP inhibitory activity known as Ala+TIMP-2 effectively inhibits growth of the human A549 lung cancer xenograft, when overexperssed in the tumor cells using a retroviral vector system. To further the development of Ala+TIMP-2 as a therapeutic we have developed both baculovirus and mammalian cell expression systems for the production of recombinant Ala+TIMP-2 and TIMP-2. It is our plan to test these materials as therapeutic agents in both xenogfraft and syngeneic murine tumor models to demonstrate the effectiveness of treatment with exogenous recombinant proteins. These models will focus on the treatment of lung cancer (A549 and Lewis Lung) as well as melanoma (A2058 and B16F10). Various dosing regimens will be utilized to compare the relative in vivo effectiveness of Ala+TIMP-2 compared to TIMP-2. Preliminary studies indicate that Ala+TIMP-2 is more effective than TIMP-2, which is attributed to the fact that Ala+TIMP-2 does not bind to the active site of MMP like TIMP-2, therefore effectively increasing its concentration for cell binding sites. Another important aspect of this project is to determine if we can develop peptide analogs that could be utilized for in vivo therapy. Furthermore we propose to develop a high throughput screening assay to screen synthetic small molecule analogs that can compete for TIMP-2, Ala+TIMP-2, or TIMP peptide binding to the cell surface receptor integrin alpha3 beta1, that we have shown modulates the anti-angiogenic and anti-tumorigenic activity of Ala+TIMP-2.