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.
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