Development of TIMP-2 derivatives or strategies as biologic therapies for cancer

开发 TIMP-2 衍生物或作为癌症生物疗法的策略

• 批准号: 10702503
• 负责人: William Stetler-Stevenson
• 金额: $ 80.78万
• 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/10702503
• 关键词: A549; Address; Affinity Chromatography; Animal Model; Area; Bacteria; Biochemical; Biological; Biological Assay; Biological Models; Biological Products; Biological Response Modifier Therapy; Bioreactors; Blinded; Breast; Breast Carcinoma; C-terminal; Carbon Dioxide; Cell Line; Cells; Chinese Hamster; Chinese Hamster Ovary Cell; Cloning; Code; Codon Nucleotides; Collaborations; Color; Complementary DNA; Culture Techniques; Development; Diagnosis; Disease Progression; Dose; Drug Delivery Systems; Economics; Elements; Embryo; Endotoxins; Enzyme Inhibition; Enzyme-Linked Immunosorbent Assay; Equilibrium; Excision; Extracellular Matrix; Formulation; Glioblastoma; Goals; Growth; Guidelines; High Pressure Liquid Chromatography; Homeostasis; Human; Immobilization; Immunologics; In Vitro; Injections; Insecta; Kidney; Laboratories; Luciferases; Luminescent Measurements; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Mammalian Cell; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Metalloproteases; Metals; Metastatic Neoplasm to the Lung; Methods; Mission; Modeling; Monitor; Mus; NOD/SCID mouse; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Nonmetastatic; Normal tissue morphology; Outcome; Ovary; Palpation; Pancreas; Pancreatic carcinoma; Patients; Phase; Plasmids; Post-Translational Protein Processing; Preparation; Primary Neoplasm; Process; Production; Proteins; Protocols documentation; Publishing; Radiation therapy; Recombinant Proteins; Recombinants; Reporting; Research; Research Personnel; Serum-Free Culture Media; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Study models; Survival Rate; Suspension Culture; System; Techniques; Temperature; Testing; Time; Tissue Inhibitor of Metalloproteinases; Toxic effect; Transfection; Vial device; Viral; Work; Xenograft procedure; Yeasts; angiogenesis; anticancer research; base; bioprocess; cancer therapy; cell growth; drug development; efficacy evaluation; enteropeptidase; experimental study; expression vector; feasibility trial; flasks; good laboratory practice; human tissue; improved; in vitro testing; in vivo; in vivo Model; in vivo evaluation; inhibitor; large scale production; lung Carcinoma; malignant breast neoplasm; method development; milligram; mouse model; neoplastic cell; novel; novel therapeutic intervention; novel therapeutics; phase I trial; preclinical study; quality assurance; seal; therapeutic development; therapeutic evaluation; tumor; tumor growth; tumor progression; tumor xenograft; tumorigenic; vector

Major Activities/Specific Objectives The principal goals of our current research effort are to evaluate the efficacy of exogenous, recombinant TIMP-2 based therapies to inhibit tumor growth, angiogenesis and metastasis using murine models. To accomplish these goals we have identified three specific objectives. These are: 1) Optimize in vitro expression of recombinant TIMP-2 using mammalian expression systems; 2) Develop efficient production and purification methods for recombinant, human TIMP-2 utilizing GMP principals, as well as developing methods for quality assurance analysis (in vitro biochemical and cellular testing, endotoxin testing, etc.) of recombinant TIMP-2; 3) Develop and test therapeutic drug delivery method and dosing for recombinant TIMP-2 or derivatives thereof; 4) implement in vivo testing of the angio-inhibitory, tumor growth inhibitory and anti-metastatic activity of recombinant, human TIMP-2 or derivatives in murine tumor models. Significant Results. Optimization of in vitro expression of recombinant TIMP-2 using mammalian expression systems. The principal obstacle to the use of endogenous MMP inhibitors (TIMPs) as biologic therapeutics has been the inability to produce sufficient quantities of recombinant protein for testing and development. Our ongoing work is the development of an expression system for recombinant human TIMP-2 that will allow rapid and simple purification of milligram quantities using the Organization for Economic Co-operational and Development (OECD) guidelines for good laboratory practice grade proteins suitable for animal model studies. This process development can then be transferred to a GMP lab for production of recombinant TIMP-2 sufficient for feasibility trials and early Phase I trials. We envision this as the preliminary steps necessary for successful development of TIMP-2 as a biopharmaceutical. The first issue that we need to address is how to produce sufficient quantities of TIMP-2 and Ala+TIMP-2 suitable for our preclinical studies that can be readily transitioned to bioscale manufacturing. In terms of expression systems for recombinant proteins there are several options ranging from yeast, bacteria, insect and mammalian cells. However, some of these are eliminated by the eventual need for GMP grade material suitable for therapeutic development, and ability to include the appropriate post-translational modifications needed for biological activity. The choice of the expression system should also be dictated by the eventual biopharmaceutical process development, in that the early choice of the correct expression system can speed time to production and obviate regulatory problems at a later time point. Among the many mammalian cell lines that can be employed for recombinant protein production, Chinese Hamster Ovary (CHO) and Human Embryonic Kidney-293 (HEK-293) are the most widely utilized. Large-scale transient transfection of mammalian cells for the fast production of recombinant proteins have been described. However, other parameters that need to be addressed are clonal selection of production optimized cells, the use of cell lines adapted to suspension culture, optimized expression vector constructs (i.e. codon optimization), use of serum-free culture media, control of temperature, pH and CO2 levels and selection/ optimization of bioreactors. Key outcome: Codon-optimized, synthetic TIMP-2 cDNA construct enhances recombinant TIMP-2 protein production. To develop bioprocessing methods for large-scale production of TIMP-2 (using GMP adaptable methods) we started by constructing an expression plasmid using the pcDNA expression plasmid for in frame cloning of a TIMP-2-Enterokinase cleavage site (EK)-6XHis tag (TIMP-2-EK-6XHis) cDNA containing the human wild type, human TIMP-2 cDNA sequence we originally reported in 1990 (Stetler-Stevenson, W. G., et al., JBC 1990; 265: l3933-l3938). Refinement of the expression construct was obtained by eliminating the enterokinase cleavage site and synthesis of a codon-optimized TIMP-2 cDNA construct again containing the 6X-His-tag for ease of purification (coTIMP-2-6X-His). The enterokinase site, for removal of the His-tag from the C-terminus, would leave behind the enterokinase cleavage sequence at the C-terminus of the recombinant protein preparation. Furthermore, the presence of a 6XHis-tag did not interfere with the anti-angiogenic activity of the C-terminal loop 6 region of TIMP-2, as previously reported (Fernandez, CA, et al., JBC 2010; 285: 41886-95). The expression plasmid was constructed in pcDNA3.3Topo vector. The vector construct was verified by direct sequencing, and the sequence of the TIMP-2 protein was confirmed by immunologic methods and MALDI-mass spectrometry. Both temperature and shaker culture conditions were optimized for the HEK-293-F shaker cultures. Yields from these experiments, as determined by ELISA, demonstrated that selection of a stable expressing clone via limiting dilution, and using suspension culture techniques, resulted in an approximate doubling of the yield of TIMP-2 over that obtained using CHO-S cell suspension culture. Further refinement of the bioprocessing methods was to convert from shaker flask culture to the use of spinner flasks. Key Outcome. The results of these experiments suggest that we can obtain substantial yields of TIMP-2 recombinant protein (40 mg/L) with a simple C-terminal 6XHis-tag alone that can be readily purified by immobilized metal affinity chromatography system (IMAC) and reverse phase preparative HPLC, thus accomplishing the first two goals of this project. To address our goal of testing recombinant TIMP-2 treatment on tumor growth and metastasis we examined the effect of exogenous TIMP-2 on lung tumor xenograft growth. In this experiment we used purified, C-terminal His-tagged TIMP-2 or Ala+TIMP-2 produced in HEK293 cells. Vials were sealed with color-coded caps and investigators were blinded to the treatment. NOD-SCID mice (30 mice total) were inoculated with 1 million A549-LUC (luciferase expressing) cells in the right flank area. Tumor growth was followed using luminescence measurements. After 2 weeks of growth, the tumors were readily detectable by palpation and daily 100 microliter i.p injections of the color-coded treatments for two weeks were begun with continuous to monitoring of tumor growth. The results shown that both TIMP-2 and Ala+TIMP-2 at 1 microgram/mouse/day ( 4 mg/kg/day) significantly inhibited A549 xenograft growth in a time and treatment dependent fashion. Our findings are important in that we demonstrate exogenous, recombinant TIMP-2 or Ala+TIMP-2 can inhibit tumor growth in vivo. Most published experiments have used forced expression of TIMP-2 (by tumor cell transfection or viral transduction), to demonstrate inhibition of tumor growth and lung metastasis. Our goal is to test our recombinant TIMP-2 in murine models of lung, breast and pancreatic carcinoma for effects on primary tumor growth and metastasis formation.

主要活动/特定目标我们当前的研究工作的主要目标是评估外源性重组TIMP-2疗法的疗效，以抑制使用鼠模型抑制肿瘤生长，血管生成和转移的疗效。为了实现这些目标，我们已经确定了三个特定目标。这些是：1）使用哺乳动物表达系统优化重组TIMP-2的体外表达； 2）开发有效的生产和纯化方法，用于利用GMP主体的重组，人类TIMP-2，以及开发质量保证分析的方法（体外生化和细胞测试，内毒素测试等）的重组TIMP-2； 3）开发和测试重组TIMP-2或其衍生物的治疗药物输送方法和给药； 4）在鼠肿瘤模型中，重组，人类TIMP-2或衍生物的血管抑制，肿瘤生长抑制和抗转移活性的体内测试。重大结果。使用哺乳动物表达系统优化重组TIMP-2的体外表达。使用内源性MMP抑制剂（TIMP）作为生物治疗剂的主要障碍是无法生产足够数量的重组蛋白进行测试和发育。 我们正在进行的工作是开发重组人TIMP-2的表达系统，该系统将使用适用于适合动物模型研究的良好实验室实践等级蛋白质的经济合作与发展组织（OECD）准则快速而简单地纯化毫克量。然后可以将该过程开发转移到GMP实验室，以生产重组TIMP-2足以进行可行性试验和早期I期试验。我们将其视为成功开发TIMP-2作为生物制药所必需的初步步骤。我们需要解决的第一个问题是如何生产足够数量的TIMP-2和ALA+TIMP-2，适合我们的临床前研究，这些研究很容易过渡到Bioscale制造。在重组蛋白的表达系统方面，酵母，细菌，昆虫和哺乳动物细胞的几种选择。但是，其中一些是由于最终需要适合治疗性开发的GMP级材料而消除的，并且能够包括生物活性所需的适当翻译后修饰。表达系统的选择还应由最终的生物制药过程开发决定，因为正确的表达系统可以加快生产时间，并在以后的时间点消除调节问题。在可用于重组蛋白质生产的许多哺乳动物细胞系中，中国仓鼠卵巢（CHO）和人类胚胎肾脏293（HEK-293）是最广泛使用的。已经描述了哺乳动物细胞快速产生重组蛋白的大规模瞬时转染。但是，需要解决的其他参数是生产优化细胞的克隆选择，适用于悬浮培养的细胞系，优化的表达载体构建体（即密码子优化），无血清培养基的使用，温度和CO2的控制，pH和CO2水平的控制以及生物反应器的选择/优化。主要结果：密码子优化的合成TIMP-2 cDNA构建体增强了重组TIMP-2蛋白的产生。为了开发用于大规模生产TIMP-2（使用GMP适应能力的方法）的生物处理方法，我们首先使用使用pcDNA表达质粒构建表达质粒，以在Timp-2-触发酶裂解位点（EK）的框架克隆中进行以下器（Stetler-Stevenson，W。G.等，JBC 1990; 265：L3933-L3938）。通过消除肠球菌裂解位点并再次含有6倍HIS-TAG的密码子优化的TIMP-2 cDNA构建体来获得表达构建体的细化，以易于纯化（Cotimp-2-6x-His）。用于从C末端移除HIS标签的肠球菌位点将留下重组蛋白制备的C末端的肠动酶裂解序列。 此外，正如先前报道的（Fernandez，CA等，JBC 2010; 285：41886-95），6xhis-tag的存在不会干扰TIMP-2的C端环6区域的抗血管生成活性（285：41886-95）。表达质粒是在pCDNA3.3TOPO载体中构建的。通过直接测序验证了矢量构建体，并通过免疫学方法和MALDI-MASS光谱法证实了TIMP-2蛋白的序列。温度和振荡器培养条件均针对HEK-293-F振动培养物进行了优化。通过ELISA确定的这些实验的产量表明，通过限制稀释和使用悬浮培养技术的稳定表达克隆的选择导致TIMP-2的产率远比使用CHO-S-S细胞悬浮培养物获得的timp-2的产量大致增加了一倍。 生物处理方法的进一步完善是从振动烧瓶培养物转换为使用旋转瓶。关键结果。这些实验的结果表明，我们可以使用简单的C端6XHIS-TAG获得TIMP-2重组蛋白（40 mg/L）的大量收益率，可以通过固定的金属亲和力色谱系统（IMAC）和反向相位准备的HPLC轻松纯化，从而实现了该项目的前两个目标。为了解决我们测试重组TIMP-2治疗对肿瘤生长和转移的目标，我们检查了外源性TIMP-2对肺肿瘤异种移植生长的影响。在此实验中，我们使用了HEK293细胞中产生的纯化的，具有纯化的C末端His标签的TIMP-2或ALA+TIMP-2。用颜色编码的帽子密封小瓶，研究人员对治疗视而不见。在右侧面积的100万A549-LUC（荧光素酶表达）细胞中接种NOD-SCID小鼠（总计30只小鼠）。使用发光测量进行肿瘤生长。生长2周后，可以通过触诊容易检测到肿瘤，每天100微氧化i.p注射颜色编码处理两周的时间，开始不断监测肿瘤生长。结果表明，在1微克/鼠标/天（4 mg/kg/day）下的TIMP-2和ALA+TIMP-2都显着抑制了A549异种移植的生长，并以依赖于治疗的方式抑制。我们的发现很重要，因为我们证明了外源，重组TIMP-2或ALA+TIMP-2可以抑制体内肿瘤的生长。 大多数已发表的实验都使用了TIMP-2的强制表达（通过肿瘤细胞转染或病毒转导），以证明抑制肿瘤生长和肺转移。我们的目标是在肺，乳腺癌和胰腺癌的鼠模型中测试我们的重组TIMP-2，以对原发性肿瘤生长和转移形成的影响。