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

Ala TIMP-2 作为癌症治疗剂的临床前开发

• 批准号: 8553037
• 负责人: William Stetler-Stevenson
• 金额: $ 112.81万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553037
• 关键词: A549; Active Sites; Adenocarcinoma; Apoptosis; Basal Cell; Binding; Binding Sites; Biochemical; Biological Assay; Biological Response Modifier Therapy; Cell Proliferation; Cell Surface Receptors; Cells; Chinese Hamster Ovary Cell; Clinical; Development; Dose; Effectiveness; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Enzymes; Exhibits; Extracellular Matrix; Family; Genetically Engineered Mouse; Goals; Growth; Homeostasis; Homologous Gene; Human; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro; Inhibition of Matrix Metalloproteinases Pathway; Integrin alpha3; Laboratories; Lung; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Materials Testing; Matrix Metalloproteinases; Mediating; Metalloproteases; Modeling; Mus; Neoplasm Metastasis; Normal tissue morphology; PECAM1 gene; PTK2 gene; Peptides; Procedures; Process; Production; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Recombinants; Regimen; Relative (related person); Retroviral Vector; Role; System; Testing; Therapeutic; Therapeutic Agents; Time; Tissue Inhibitor of Metalloproteinases; Tissues; Transforming Growth Factor beta; Treatment Effectiveness; Western Blotting; Work; Xenograft procedure; analog; angiogenesis; cell motility; density; high throughput screening; in vivo; inhibitor/antagonist; melanoma; member; mouse model; mutant; neoplastic cell; novel; overexpression; peptide analog; pre-clinical; protein function; research study; small molecule; tumor; tumor growth; tumorigenic

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 modulation of the tumor microenvironment.

致力于我们在体内异种移植生长实验中进行临床前开发TIMP-2的目标。这些实验表明，当使用逆转录病毒载体系统过度充实时，缺乏MMP抑制活性的TIMP-2同源物有效地抑制了人A549肺癌异种移植的生长。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的强迫表达的影响。为了进一步发展ALA+TIMP-2作为治疗性，我们开发了用于生产重组ALA+TIMP-2和TIMP-2的CHO细胞表达系统，并正在继续进行这些努力，以优化在良好的制造程序（GMP）下大量生产的生产。我们已经确定了一个简化的两个步骤Purificaiton Shceme，该Shceme应该允许从CHO和HURN HEK-293细胞中产生大量的GMP级重组TIMP-2。我们的计划是将这些材料作为Xenogfraft和Syngeneic Murine肿瘤模型中的治疗剂进行测试，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的基因工程小鼠模型（GEMM）来测试重组外源性TIMP-2和ALA+TIMP-2的抗肿瘤作用。结果将与TIMP家族的其他成员对效力和功效的影响进行比较。这些模型将集中于肺癌（A549和Lewis Lung）以及黑色素瘤（A2058和B16F10）的治疗。与TIMP-2相比，将利用各种剂量方案比较ALA+TIMP-2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与TIMP-2这样的MMP的活性位点没有结合，因此有效地增加了其对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，我们建议开发高吞吐量筛选测定法，以筛选合成的小分子类似物，可以竞争TIMP-2，ALA+TIMP-2或TIMP肽与细胞表面受体alpha3 beta1结合的TIMP肽，我们已经显示了抗抗激发和抗timpiTITITITITITIT的抗抗激发和抗timp-2- （TIMP-2）属于一个小家族的内源性蛋白质，其功能可抑制一组酶，即基质金属蛋白酶（MMP）。 TIMP-2通过MMP依赖和独立的机制抑制体内体外和血管生成的内皮细胞增殖和迁移。但是，关于这些机制对TIMP-2的抗肿瘤作用的贡献知之甚少。使用逆转录病毒递送系统，我们在人腺癌A549细胞中稳定表达过表达的TIMP-2及其突变体ALA+TIMP-2（没有MMP抑制活性）。使用实时PCR，Western印迹和ELISA，我们通过反向Zymography证实了TIMP-2表达增强及其MMP抑制活性。在体外，生长测定表明TIMP-2和ALA+TIMP-2没有改变基底细胞增殖率，但是，肿瘤细胞迁移和侵袭被抑制。在体内，TIMP-2和ALA+TIMP-2 A549异种移植物均表现出生长速度降低，CD31免疫染色表明肿瘤内微血管密度降低，而TUNEL显示出肿瘤细胞凋亡的增强。免疫印迹和免疫组织化学分析在体外和A549异种移植组织中，具有磷酸化-FAK（Tyr397）或磷酸化AKT（SER473）在TIMP-2和ALA+TIMP-2肿瘤细胞中的激活下降。我们得出的结论是，TIMP-2介导的肿瘤生长的抑制至少部分是独立于MMP抑制作用的，这是TIMP-2对肿瘤细胞的直接影响和肿瘤微环境的调节的结果。