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: 10486788
• 负责人: William Stetler-Stevenson
• 金额: $ 101.15万
• 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/10486788
• 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; Structure; 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/antagonist; 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.

我们目前的主要研究目标是利用小鼠模型评估外源性重组TIMP-2疗法抑制肿瘤生长、血管生成和转移的效果。为了实现这些目标，我们确定了三个具体目标。这包括：1)利用哺乳动物表达系统优化重组TIMP-2的体外表达；2)利用GMP原理开发重组人TIMP-2的高效生产和纯化方法，开发重组人TIMP-2的质量保证分析方法（体外生化和细胞检测、内毒素检测等）；3)开发和测试重组TIMP-2或其衍生物的治疗药物递送方法和剂量；4)在小鼠肿瘤模型中对重组、人TIMP-2或其衍生物的血管抑制、肿瘤生长抑制和抗转移活性进行体内测试。重要的结果。重组TIMP-2在哺乳动物表达系统中的体外表达优化。使用内源性MMP抑制剂（TIMPs）作为生物疗法的主要障碍是无法产生足够数量的重组蛋白用于测试和开发。我们正在进行的工作是开发重组人TIMP-2表达系统，该系统将允许使用经济合作与发展组织（OECD）适用于动物模型研究的良好实验室规范级蛋白质指南快速、简单地纯化毫克量。然后，该工艺开发可以转移到GMP实验室，用于生产足以进行可行性试验和早期I期试验的重组TIMP-2。我们设想这是成功开发TIMP-2作为生物药物所必需的初步步骤。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，可以很容易地过渡到生物规模生产。就重组蛋白的表达系统而言，有几种选择，包括酵母、细菌、昆虫和哺乳动物细胞。然而，由于最终需要适合治疗开发的GMP级材料，以及包括生物活性所需的适当翻译后修饰的能力，其中一些被消除了。表达系统的选择也应该由最终的生物制药工艺发展决定，因为早期选择正确的表达系统可以加快生产时间，并在以后的时间点避免监管问题。在许多可以用于重组蛋白生产的哺乳动物细胞系中，中国仓鼠卵巢（CHO）和人胚胎肾-293 （HEK-293）是应用最广泛的。大规模瞬时转染的哺乳动物细胞快速生产重组蛋白已被描述。然而，其他需要解决的参数是生产优化细胞的克隆选择，适合悬浮培养的细胞系的使用，优化表达载体构建（即密码子优化），使用无血清培养基，控制温度，pH和CO2水平以及生物反应器的选择/优化。关键结果：密码子优化，合成的TIMP-2 cDNA构建增强了重组TIMP-2蛋白的生产。为了开发大规模生产TIMP-2的生物加工方法（使用GMP适应性方法），我们首先使用pcDNA表达质粒构建表达质粒，用于在框架内克隆TIMP-2肠激酶裂解位点(EK)-6XHis标签（TIMP-2-EK-6XHis） cDNA，该表达质粒含有我们最初于1990年报道的人类野生型TIMP-2 cDNA序列（steler - stevenson， W. G.等，JBC 1990; 265: l3933-l3938）。通过去除肠激酶切割位点和合成密码子优化的TIMP-2 cDNA构建体（coTIMP-2-6X-His标记便于纯化），获得了表达构建体的细化。肠激酶位点，用于从c端去除his标签，将在重组蛋白制备的c端留下肠激酶裂解序列。此外，正如先前报道的那样，6xhis标签的存在不会干扰TIMP-2 c端环6区域的抗血管生成活性（Fernandez， CA, et al., JBC 2010; 285: 41886-95）。在pcDNA3.3Topo载体上构建表达质粒。通过直接测序验证载体构建，通过免疫学方法和maldi质谱法确定TIMP-2蛋白的序列。对HEK-293-F摇床培养的温度和摇床培养条件进行了优化。这些实验的产量，如ELISA测定的那样，表明通过限制稀释选择稳定表达的克隆，并使用悬浮培养技术，导致TIMP-2的产量比使用CHO-S细胞悬浮培养获得的产量大约翻倍。生物处理方法的进一步改进是从摇瓶培养转化为使用旋转瓶。关键的结果。这些实验结果表明，我们可以用一个简单的c端6XHis-tag单独获得大量的TIMP-2重组蛋白（40 mg/L），该蛋白易于用固定化金属亲和色谱系统（IMAC）和反相制备HPLC纯化，从而实现了本项目的前两个目标。为了验证重组TIMP-2对肿瘤生长和转移的影响，我们检测了外源性TIMP-2对肺癌异种移植物生长的影响。在本实验中，我们使用HEK293细胞中纯化的c端his标记的TIMP-2或Ala+TIMP-2。小瓶用彩色编码的瓶盖密封，调查人员对治疗不知情。将100万个表达荧光素酶的A549-LUC细胞接种于NOD-SCID小鼠右侧区域，共30只小鼠。用发光测量法跟踪肿瘤生长情况。肿瘤生长2周后，通过触诊可以很容易地检测到肿瘤，开始每天100微升滴注彩色编码治疗，持续两周，持续监测肿瘤生长。结果显示，1微克/只/天（4 mg/kg/天）的TIMP-2和Ala+TIMP-2均能显著抑制A549异种移植物生长，且呈时间依赖性。我们的发现很重要，因为我们证明了外源性重组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TIMP-2的强制表达（通过肿瘤细胞转染或病毒转导）来证明抑制肿瘤生长和肺转移。我们的目标是在小鼠肺癌、乳腺癌和胰腺癌模型中测试重组TIMP-2对原发肿瘤生长和转移形成的影响。