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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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.