Preclinical development of Ala+TIMP-2 as an cancer therapeutic

为了实现我们宣布的TIMP-2作为一种新的癌症治疗药物的临床前开发目标，我们进行了体内异种移植生长实验。这些实验表明，缺乏基质金属蛋白酶抑制活性的TIMP-2同系物，即ALA+TIMP-2，当使用逆转录病毒载体系统过度散布在肿瘤细胞中时，有效地抑制了人A549肺癌移植瘤的生长。正在进行的实验正在利用Tet-on系统来检查TIMP-2和ALA+TIMP-2在已建立的肿瘤系统中强制表达的效果。为了进一步开发Ala+TIMP-2作为治疗药物，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，并正在继续努力在良好的制造工艺(GMP)下优化大批量生产。我们已经确定了一种简化的两步纯化方案，该方案可以从CHO和人HEK-293细胞中大量生产GMP级重组TIMP-2。我们计划在异种和同基因小鼠肿瘤模型中测试这些材料作为治疗剂的作用，以证明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(GEMM)来测试重组外源TIMP-2和ALA+TIMP-2的抗肿瘤作用。结果将与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Lewis肺癌)以及黑色素瘤(A2058和B16F10)的治疗。将采用不同的给药方案来比较ALA+TIMP-2与TIMP-2在体内的相对有效性。初步研究表明，ALA+TIMP-2比TIMP-2更有效，这是因为ALA+TIMP-2不像TIMP-2那样与基质金属蛋白酶的活性部位结合，从而有效地增加了细胞结合部位的浓度。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多肽类似物。此外，我们建议发展一种高通量筛选方法来筛选能够竞争TIMP-2、ALA+TIMP-2或与细胞表面整合素α3β1结合的TIMP-2、ALA+TIMP-2或TIMP肽的合成小分子类似物，我们已经证明ALA+TIMP-2调节ALA+TIMP-2的抗血管生成和抗肿瘤活性。金属蛋白酶组织抑制因子2(TIMP-2)是一小类内源性蛋白，其功能是抑制一组酶，即基质金属蛋白酶(MMPs)。TIMP-2在体外抑制内皮细胞的增殖和迁移，在体内抑制血管生成，其机制可能与基质金属蛋白酶的依赖和独立有关。然而，关于这些机制对TIMP-2抗肿瘤作用的贡献，我们知之甚少。利用逆转录病毒传递系统，我们在人腺癌细胞A549中稳定高表达TIMP-2及其突变体Ala+TIMP-2(缺乏基质金属蛋白酶抑制活性)。通过实时定量聚合酶链式反应、免疫印迹和酶联免疫吸附试验证实TIMP-2的表达增强，并通过反向酶谱分析证实其具有抑制基质金属蛋白酶的活性。体外生长实验表明，TIMP-2和ALA+TIMP-2不改变基础细胞的增殖率，但抑制肿瘤细胞的迁移和侵袭。在体内，TIMP-2和ALA+TIMP-2 A549移植瘤生长速度减慢，CD31免疫染色显示瘤内微血管密度降低，TUNEL显示肿瘤细胞凋亡增加。免疫印迹和免疫组织化学分析显示，TIMP-2和ALA+TIMP-2肿瘤细胞的TIMP-2和ALA+TIMP-2肿瘤细胞活性均降低。我们得出结论，TIMP-2对肿瘤生长的抑制作用至少部分地不依赖于对基质金属蛋白酶的抑制，是TIMP-2直接作用于肿瘤细胞和调节肿瘤微环境共同作用的结果。

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). We have identified a simplified two step purificaiton shceme that should allow production of significant quantities of GMP grade recombinant TIMP-2 from both CHO and human HEK-293 cells. 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 modulationof the tumor microenvironment.