Preclinical development of AlaTIMP-2 as an cancer therapeutic

为了实现我们所述的TIMP-2作为新型癌症治疗剂的临床前开发目标，我们进行了体内异种移植物生长实验。这些实验已经表明，当使用逆转录病毒载体系统在肿瘤细胞中过表达时，称为Ala+TIMP-2的缺乏MMP抑制活性的TIMP-2同系物有效地抑制人A549肺癌异种移植物的生长。正在进行的实验利用TET-On系统来检查TIMP-2和Ala+TIMP-2在已建立的肿瘤系统中的强制表达的效果。为了进一步开发Ala+TIMP-2作为治疗剂，我们已经开发了用于生产重组Ala+TIMP-2和TIMP-2的CHO细胞表达系统，并且正在继续这些努力以优化在良好生产程序（GMP）下的大量生产。我们计划在异种移植和同系小鼠肿瘤模型中测试这些材料作为治疗剂，以证明用外源重组蛋白治疗的有效性。我们还计划利用新开发的肺癌基因工程小鼠模型（GEMMs）来测试重组外源性TIMP-2和Ala+TIMP-2的抗肿瘤作用。将结果与TIMP家族其他成员的效力和疗效进行比较。这些模型将专注于肺癌（A549和刘易斯肺）以及黑色素瘤（A2058和B16 F10）的治疗。将使用各种给药方案来比较Ala+TIMP-2与TIMP-2相比的相对体内有效性。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那样结合MMP的活性位点，因此有效地增加了其用于细胞结合位点的浓度。该项目的另一个重要方面是确定我们是否可以开发可用于体内治疗的肽类似物。此外，我们建议开发高通量筛选测定以筛选合成的小分子类似物，其可以竞争TIMP-2、Ala+TIMP-2或TIMP肽与细胞表面受体整合素α 3 β 1的结合，我们已经显示整合素α 3 β 1调节Ala+TIMP-2的抗血管生成和抗肿瘤发生活性。金属蛋白酶组织抑制剂2（TIMP-2）属于内源性蛋白质的一个小家族，其功能是抑制一组酶，即基质金属蛋白酶（MMPs）。TIMP-2通过MMP依赖性和非依赖性机制抑制体外内皮细胞增殖和迁移以及体内血管生成。然而，关于这些机制对TIMP-2的抗肿瘤作用的贡献知之甚少。使用逆转录病毒传递系统，我们在人腺癌A549细胞中稳定过表达TIMP-2及其突变体Ala+TIMP-2（缺乏MMP抑制活性）。通过真实的时间PCR、Western blot和ELISA，我们证实了TIMP-2的表达增强，并且通过反向酶谱法证实了其MMP抑制活性。在体外，生长实验表明，TIMP-2和Ala+TIMP-2并没有改变基底细胞增殖率，但抑制肿瘤细胞的迁移和侵袭。在体内，TIMP-2和Ala+TIMP-2 A549异种移植物均表现出生长速率降低，CD 31免疫染色表明肿瘤内微血管密度降低，TUNEL显示肿瘤细胞凋亡增强。在体外和具有磷酸化FAK（Tyr 397）或磷酸化AKT（Ser 473）的A549异种移植物组织的免疫印迹和免疫组织化学分析显示TIMP-2和Ala+TIMP-2肿瘤细胞中的活化降低。我们的结论是，TIMP-2介导的抑制肿瘤生长的发生，至少部分地，独立于MMP抑制，并且是TIMP-2对肿瘤细胞的直接作用和肿瘤微环境的调制的结果。

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. Ongoing experiments are utilizing the TET-On system to examine the effect of forced expression of TIMP-2 and Ala+TIMP-2 in established tumor systems. To further the development of Ala+TIMP-2 as a therapeutic we have developed CHO cell expression systems for the production of recombinant Ala+TIMP-2 and TIMP-2, and are continuing these efforts to optimize production of large quantities under good manufacturing procedures (GMP). 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. We also plan to utilize newly developed genetically engineered mouse models (GEMMs) of lung cancer for testing the anti-tumor effects of recombinant exogenous TIMP-2 and Ala+TIMP-2. The results will be compared with the effects of other members of the TIMP family for potency and efficacy. 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. Tissue inhibitor of metalloproteinase 2 (TIMP-2) belongs to a small family of endogenous proteins that function to inhibit a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP dependent and independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, western blot and ELISA we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intra-tumoral microvascular density and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and Immunohistochemistry analyses in vitro and of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.