Preclinical development of AlaTIMP-2 as an cancer therapeutic

AlaTIMP-2 作为癌症治疗剂的临床前开发

• 批准号: 8349393
• 负责人: William Stetler-Stevenson
• 金额: $ 96.44万
• 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/8349393
• 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; 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; 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). 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.

为了实现我们既定的 TIMP-2 作为一种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生长实验。这些实验表明，缺乏 MMP 抑制活性的 TIMP-2 同源物（称为 Ala+TIMP-2）当使用逆转录病毒载体系统在肿瘤细胞中过度表达时，可有效抑制人 A549 肺癌异种移植物的生长。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 作为治疗剂，我们开发了 CHO 细胞表达系统，用于生产重组 Ala+TIMP-2 和 TIMP-2，并继续努力在良好的制造程序下优化大量生产（GMP）。我们计划在异种移植和同基因小鼠肿瘤模型中测试这些材料作为治疗剂，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功效效果进行比较。这些模型将专注于肺癌（A549 和 Lewis Lung）以及黑色素瘤（A2058 和 B16F10）的治疗。将利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，这是因为Ala+TIMP-2不像TIMP-2那样与MMP的活性位点结合，从而有效增加其对细胞的浓度结合位点。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，我们建议开发一种高通量筛选方法来筛选合成的小分子类似物，这些类似物可以竞争 TIMP-2、Ala+TIMP-2 或 TIMP 肽与细胞表面受体整合素 α3 β1 的结合，我们已经证明它可以调节抗-Ala+TIMP-2的血管生成和抗肿瘤生成活性。金属蛋白酶组织抑制剂 2 (TIMP-2) 属于一小类内源性蛋白质，其功能是抑制一组酶，即基质金属蛋白酶 (MMP)。 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，人们对这些机制对 TIMP-2 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 细胞中稳定过表达 TIMP-2 及其突变体 Ala+TIMP-2（缺乏 MMP 抑制活性）。使用实时 PCR、蛋白质印迹和 ELISA，我们通过反向酶谱证实了 TIMP-2 表达的增强及其 MMP 抑制活性。体外生长测定表明TIMP-2和Ala+TIMP-2不会改变基底细胞增殖率，但肿瘤细胞迁移和侵袭受到抑制。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，CD31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。体外免疫印迹和免疫组织化学分析以及使用磷酸-FAK (Tyr397) 或磷酸-AKT (Ser473) 的 A549 异种移植组织显示 TIMP-2 和 Ala+TIMP-2 肿瘤细胞的激活减少。我们得出的结论是，TIMP-2介导的肿瘤生长抑制至少部分独立于MMP抑制，并且是TIMP-2对肿瘤细胞的直接作用和肿瘤微环境调节的结果。