Preclinical development of TIMP-2 as a biologic therapy for cancer

TIMP-2 作为癌症生物疗法的临床前开发

• 批准号: 9153818
• 负责人: William Stetler-Stevenson
• 金额: $ 95.16万
• 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/9153818
• 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; Brain; Buffers; C-terminal; Carbon Dioxide; Cell Line; Cells; Chinese Hamster; Cloning; Code; Codon Nucleotides; Collaborations; Color; Complementary DNA; Culture Techniques; Development; Diagnosis; Disease Progression; Dose; Drug Delivery Systems; Drug Formulations; Economics; Elements; Embryo; Endotoxins; Enzyme Inhibition; Enzyme-Linked Immunosorbent Assay; Equilibrium; Excision; Extracellular Matrix; Glioblastoma; Goals; Growth; Guidelines; High Pressure Liquid Chromatography; Homeostasis; Human; Immunologics; Implant; In Vitro; Injection of therapeutic agent; Insecta; Kidney; Laboratories; Left; Lewis Lung Carcinoma; Luciferases; Luminescent Measurements; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of lung; Mammalian Cell; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Metalloproteases; Metals; Methods; Mission; Modeling; Monitor; Mus; NOD/SCID mouse; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Outcome; Ovary; Palpation; Patients; Phase; Plasmids; Post-Translational Protein Processing; Preparation; Process; Production; Proteins; Protocols documentation; Publishing; Pump; Radiation therapy; Recombinant Proteins; Recombinants; Reporting; Research; Research Personnel; Serum-Free Culture Media; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Staging; Survival Rate; Suspension Culture; System; Techniques; Temperature; Testing; Therapeutic; Time; Tissue Inhibitor of Metalloproteinases; Toxic effect; Transfection; Vial device; Viral; Work; Xenograft Model; Xenograft procedure; Yeasts; adaptive immunity; angiogenesis; anticancer research; base; bioprocess; cancer therapy; cell growth; drug development; enteropeptidase; expression vector; flasks; good laboratory practice; human tissue; improved; in vitro testing; in vivo; in vivo Model; inhibitor/antagonist; large scale production; milligram; monoclonal antibody LL2; neoplastic cell; novel; novel therapeutics; phase I trial; pre-clinical; preclinical study; quality assurance; research study; seal; subcutaneous; therapeutic development; treatment strategy; 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 therapy 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; 4) implement in vivo testing of the angio-inhibitory, tumor growth inhibitory and anti-metastatic activity of recombinant, human TIMP-2 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. TIMP-2 or Ala+TIMP-2 were tested in experiments utilizing Alzet pumps to maintain continuous systemic delivery in a syngeneic murine Lewis lung carcinoma model. We determined if Alzet pump administration of TIMP-2 and Ala+TIMP-2 would inhibit growth in the presence of intact innate and adaptive immune responses. C57bl/6 mice were injected with 0.25 million LL2-LUC cells and on the following day subcutaneous Alzet pumps containing buffer control, or purified human recombinant TIMP-2 or Ala+TIMP-2 were implanted. Results obtain 24 days after tumor inoculation show that the administration of recombinant human TIMP-2 or Ala+TIMP-2 can suppress the growth of this highly aggressive murine tumor model in a statistically significant fashion. These findings confirm our observations in the A549 xenograft model using the systemic administration of TIMP-2 and Ala+TIMP-2. Furthermore, they suggest that additional methods for systemic delivery of recombinant TIMP-2 or Ala+TIMP-2 should be successful in reducing tumor growth and growth of metastasis.

主要活动/具体目标 我们当前研究工作的主要目标是使用小鼠模型评估外源重组 TIMP-2 疗法抑制肿瘤生长、血管生成和转移的功效。为了实现这些目标，我们确定了三个具体目标。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) 利用GMP原理开发重组人TIMP-2的高效生产和纯化方法，以及开发重组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）开发并测试重组TIMP-2的治疗药物递送方法和剂量； 4) 在小鼠肿瘤模型中实施重组人TIMP-2的血管抑制、肿瘤生长抑制和抗转移活性的体内测试。显着的成果。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) 作为生物疗法的主要障碍是无法生产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 的表达系统，该系统将允许使用经济合作与发展组织 (OECD) 适合动物模型研究的良好实验室实践级蛋白质指南快速、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。我们认为这是成功开发 TIMP-2 作为生物制药所需的初步步骤。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，并可以轻松过渡到生物规模生产。就重组蛋白的表达系统而言，有多种选择，包括酵母、细菌、昆虫和哺乳动物细胞。然而，其中一些由于最终需要适合治疗开发的 GMP 级材料以及包含生物活性所需的适当翻译后修饰的能力而被消除。表达系统的选择还应由最终的生物制药工艺开发决定，因为早期选择正确的表达系统可以加快生产时间并消除稍后时间点的监管问题。在可用于重组蛋白生产的许多哺乳动物细胞系中，中国仓鼠卵巢（CHO）和人胚胎肾-293（HEK-293）是应用最广泛的。已经描述了用于快速生产重组蛋白的哺乳动物细胞的大规模瞬时转染。然而，需要解决的其他参数包括生产优化细胞的克隆选择、适合悬浮培养的细胞系的使用、优化的表达载体构建（即密码子优化）、无血清培养基的使用、温度、pH和CO2水平的控制以及生物反应器的选择/优化。主要成果：密码子优化的合成 TIMP-2 cDNA 构建体增强了重组 TIMP-2 蛋白的产量。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，用于框内克隆 TIMP-2-肠激酶切割位点 (EK)-6XHis 标签 (TIMP-2-EK-6XHis) cDNA，其中包含我们最初于 1990 年报道的人类野生型、人类 TIMP-2 cDNA 序列 (Stetler-Stevenson，W.G.等人，JBC 1990；265：13933-13938)。通过消除肠激酶切割位点并合成密码子优化的 TIMP-2 cDNA 构建体（再次包含 6X-His 标签以便于纯化）（coTIMP-2-6X-His），对表达构建体进行了精炼。用于从 C 末端去除 His 标签的肠激酶位点将在重组蛋白制剂的 C 末端留下肠激酶切割序列。此外，如先前报道的，6XHis标签的存在不会干扰TIMP-2的C末端环6区域的抗血管生成活性（Fernandez, CA等人，JBC 2010；285:41886-95）。将表达质粒构建于pcDNA3.3Topo载体中。通过直接测序验证载体构建体，并通过免疫学方法和MALDI-质谱法确认TIMP-2蛋白的序列。 HEK-293-F 摇床培养的温度和摇床培养条件均经过优化。通过 ELISA 测定的这些实验的产量表明，通过有限稀释并使用悬浮培养技术选择稳定表达克隆，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。生物加工方法的进一步改进是将摇瓶培养转变为使用旋转瓶。关键成果。这些实验的结果表明，仅用简单的C端6XHis标签，我们就可以获得高产量的TIMP-2重组蛋白（40 mg/L），并且可以通过固定化金属亲和层析系统（IMAC）和反相制备型HPLC轻松纯化，从而实现该项目的前两个目标。为了实现我们测试重组 TIMP-2 治疗对肿瘤生长和转移的影响的目标，我们检查了外源 TIMP-2 对肺肿瘤异种移植物生长的影响。在本实验中，我们使用 HEK293 细胞中产生的纯化 C 端 His 标记的 TIMP-2 或 Ala+TIMP-2。小瓶用颜色编码的盖子密封，研究人员对治疗不知情。 NOD-SCID 小鼠（总共 30 只小鼠）在右侧胁腹区域接种 100 万个 A549-LUC（表达荧光素酶）细胞。使用发光测量来跟踪肿瘤生长。生长两周后，通过触诊很容易检测到肿瘤，并开始每天腹膜内注射 100 微升颜色编码治疗，持续两周，并连续监测肿瘤生长。结果表明，1 微克/小鼠/天（4 毫克/千克/天）的 TIMP-2 和 Ala+TIMP-2 均以时间和治疗依赖性方式显着抑制 A549 异种移植物生长。我们的发现很重要，因为我们证明外源重组 TIMP-2 或 Ala+TIMP-2 可以抑制体内肿瘤生长。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）来证明对肿瘤生长和肺转移的抑制。在实验中使用 Alzet 泵对 TIMP-2 或 Ala+TIMP-2 进行了测试，以在同基因小鼠 Lewis 肺癌模型中维持连续的全身递送。我们确定 Alzet 泵给予 TIMP-2 和 Ala+TIMP-2 是否会在存在完整先天性和适应性免疫反应的情况下抑制生长。 C57bl/6小鼠被注射25万个LL2-LUC细胞，并在第二天皮下植入含有缓冲液对照的Alzet泵，或纯化的人重组TIMP-2或Ala+TIMP-2。肿瘤接种24天后获得的结果表明，施用重组人TIMP-2或Ala+TIMP-2可以以统计学上显着的方式抑制这种高度侵袭性的小鼠肿瘤模型的生长。这些发现证实了我们在使用 TIMP-2 和 Ala+TIMP-2 全身给药的 A549 异种移植模型中的观察结果。此外，他们建议全身递送重组 TIMP-2 或 Ala+TIMP-2 的其他方法应能成功减少肿瘤生长和转移生长。