MXPA00008209A - Method of producing mouse and human endostatin - Google Patents

Abstract

Methods for producing mouse and human endostation are disclosed. Methods for refolding and purifying endostatin from inclusion bodies expressed in bacteria and nucleic acids encoding full-length and truncated forms of endostatin are also disclosed.

Description

METHOD TO PRODUCE HUMAN ENDOSTATINE AND MOUSE Field of the Invention Methods for producing human and mouse endostatin are described. Methods for renaturing and purifying endostatin in the inclusion bodies, which are expressed in bacteria and nucleic acids encoding the truncated and full-length forms of endostatin, are also described.

Background of the Invention Angiogenesis Angiogenesis, the growth of new blood vessels, plays an important role in the growth of cancer and in metastasis. In humans, the extent of the vasculature in a tumor has been shown to correlate with the patient's prognosis for a variety of cancers (Folkman, J., Semlnars in Medi cine of the Beth Israel Hospi- tal, Boston 3326 (26): 1757- 1763; 1995; Gasparini, G., European Journal of Cancer 32 A (14): REF: 122709 2485-2493, 1996; Pluda, J. M., Seminars in Oncology 24 (2): 203-218, 1997; Norrby, K., APMIS 105: 417-437, 1997). In normal adults, angiogenesis is limited to properly controlled situations, such as wound healing and the female reproductive system (Battegay, EJ, J Mol Med 73: 333-346, 1995; Dvorak, H. F, New Engl J Med, 315: 1650-1659, 1986). Studies in animals suggest that tumors may exist in a latent state, in which tumor growth is limited by a balance between high proliferation rates and high rates of apoptosis (Holmgren, L. et al., Na t. Med. (NY) 1 (2): 149-153, 1995; Hanahan, D. et al., Cell 86 (3): 353-364, 1996). The change to an angiogenic phenotype allows tumor cells to escape their latency and grow rapidly, presumably as a result of a decrease in the apoptotic rate of tumor cells (Bouck, Cancer Cells, 2 (6): 179-185, 1990 Dameron et al, Cold Spring Harb Symp Quant Biol, 59: 483-489, 1994). It is believed that the control of angiogenesis is a balance between the factors that promote the formation of new vessels and the anti-angiogenic factors that suppress the formation of the neovasculature (Bouck, N. et al., Advances in Cancer Research 69: 135 -173, 1996; O'Reilly et al., Cell 79 (2): 315-328, 1994). A variety of pro-angiogenic factors have been characterized because they include basic and acid factors for the growth of fibroblasts (bFGF and aFGF) and the vascular permeability factor / vascular endothelial growth factor (VPF / VEGF) (Potgens, AJG et al. al., Biol. Chem. Hoppe-Seyl er 376: 57-70, 1995; Ferrara, N., European Journal of Cancer 32 A (14): 2413-2442, 1996; Bikfavi, A. Et al., Endocrine Revi Ews 18: 26-45, 1997). Several endogenous anti-angiogenic factors have also been characterized, including angiostatin (O'Reilly et al., CeIJ 79 (2): 315-328, 1994), endostatin (O'Reilly et al, CeJi 88 (2): 277 -285, 1997), interferon-a. (Ezeko itz et al, N. Engl. J. Med., May 28, 326 (22): 1456-1463, 1992), thrombospondin (Good et al, Proc Na ti Acad Sci USA 87 (17): 6624-6628 , 1990, Tolsma et al., J CeIJ Biol 122 (2): 497-511, 1993), and platelet factor 4 (PF4) (Maione et al, Sci ence 247 (4938): 77-79, 1990). Several angiogenic inhibitors are in clinical development (see Shawver et al., Drug Discovery Today 2 (2): 50-63, 1997, and references therein). Polypeptides such as interferon-a and platelet factor 4 are found in clinical trials. Angiostatin, the soluble receptor Flt-1, and the bactericidal protein derivative / permeability enhancer 23 are found in preclinical studies. Monoclonal antibodies, such as humanized anti-avb3 antibody (LM609), anti-VEGF, anti-Flk-1 monoclonal antibody (DC101) are also found in preclinical studies. Tecogalan (DS4152), a sulfated polysaccharide-peptidoglycan complex, is found in clinical studies, and the carbohydrate inhibitor bFGF (GM1474) and the glyceptide mimic inhibitor of bFGF (GL14.2) are in preclinical studies. The antibiotic AGM1470 (TNP470), an analog of. fumagillin, and Suramina, a polyanionic compound, are found in clinical studies. Small molecule inhibitors, such as urokinase receptor antagonists, phosphatidic acid inhibitors, Tlk-1 inhibitors, and VEGF-F11 agglutination inhibitors, are all found in preclinical studies. Thalidomide, and its analogs, and the inhibitors of the matrix. Metalloproteinase, such as Batimastat / Marimastat, are found in clinical trials. Oligonucleotides, such as ribozymes, have a specificity on VEFGF receptors and VEGF antisense oligonucleotides, they are also found in preclinical assays. Anti-angiogenic therapy may offer several advantages over conventional chemotherapy for the treatment of cancer. Anti-angiogenic agents have low toxicity in preclinical assays and no development of drug resistance has been observed (Folkman, J., Seminars in Medicine of the Beth Israel Hospi- tal, Boston 333 (26): 1757-1763, 1995). Angiogenesis is a complex process, made up of several stages that includes the invasion, proliferation and migration of endothelial cells, it can be anticipated that the combination therapies are the most effective. In fact, combinations of chemotherapy with anti-angiogenic therapy already show promising results in preclinical models (Teicher, B. A et al., Breast Cancer Research and Task 36: 227-236, 1995; Teicher, B. A. et al. European Journal of Cancer 32 A (14): 2461-2466, nineteen ninety six) . Endostatin Endostatin is a 20 kDa protein that is derived from the C-terminal fragment of collagen XVIII type alpha 1. It is shown that cell culture media conditioned by a hemangioendothelioma cell line (EOMA) contain a factor that inhibits cell proliferation of the endothelium in vi tro (O'Reilly et al, Cell 88: 277-285, 1997). The factor responsible for this inhibition is called endostatin. A recombinant form of this protein that is expressed in insect cells infected with a baculovirus, inhibits the growth of metastases in the Lewis model of lung tumor and an insoluble form derived from E. coli, of this protein, is shown as efficient in the prevention of primary tumor growth in various tumor models (O'Reilly et al, CeJl 88: 277-285, 1997; Boeh et al., Na ture 390: 404-410, 1997).

Purification and Renaturing of Endostatin Although various types of expression systems have been developed over the last twenty years, bacterial systems, particularly those based on E. coli, are widely used for the production of proteins on an industrial scale. Vectors that allow high level expression and the ability to carry out fermentations at high cell densities and at low cost have contributed to the extensive development and use of expression systems based on E. coli. However, a significant problem is the tendency of E. coli to form inclusion bodies that contain the desired recombinant protein. The formation of inclusion bodies requires further processing in the 3 'direction, such as in vitro renaturation, before the biologically active proteins can be recovered. The tendency to form insoluble agglomerates seems to correlate with factors such as size, hygrophobicity, subunit structure, or the use of fusion domains (Kane J. F. and Harley, D.L., Tibtech 6: 95, 1988). The formation of inclusion bodies seems to be determined by the rates of protein synthesis, renaturation, agglomeration, and proteolytic degradation, solubility and thermodynamics to naturalize intermediates and natural proteins, and the interactions of these classes, with the chaperone proteins (Rainer Rudolph, in Protein Engineering: Principles and Practice, Edited by Jeffrey L. Cleland and Charles S. Craik, p 283-298, Wiley-Liss, Inc., New York, New York, 1996). In general, inclusion bodies are formed in the cytoplasm of cells that express, at high levels, a recombinant protein. These refract light when observed in a phase contrast microscope, therefore, sometimes they are called refractile bodies. The inclusion bodies are characterized by a relatively high specific density and can be formed in sediments, from Used cells, by centrifugation. The formation of inclusion bodies can protect the recombinant proteins from proteolysis, since they do not readily disintegrate under physiological conditions in a solvent. High concentrations of renaturing agents, such as 6 M guanidine hydrochloride or 6-8 M urea, have been commonly used to solubilize the proteins that are present in the inclusion bodies. We have compared a diversity of protocols for the solubilization of inclusion bodies (Fisher, B., Summer, L. and Goodenough, P. Biotechnol. Bioeng. 1: 3-13, 1992). Although the desired product of the foreign gene is the main component of the inclusion bodies, other proteins of the host cells, such as the small heat shock proteins, the outer membrane proteins, the elongation factor EF-Tu, and the RNA polymerases can also be enriched in these preparations (Alien, SP, Polazzi, JO Gierse, JK, and Easton, AM, J. Bacteriol., 174: 6938-6947, 1992); Hart, R.A., Riñas, U., and Bailey, J.E., J. Bi ol. Chem. 265: 12728-12733, 1990; Hartley, D. L., and Kane, J. F., Biochem. Soc. Trans. 16: 101, 1988). Worldwide Patent Application WO 97/15666 describes the expression, purification, and characterization of endostatin from insect cells infected with a baculovirus and E. coli. Bacterially derived endostatin is not renatured to its natural state, but is used as an insoluble suspension for most of these studies. This description also describes the purification of the natural endostatin in a conditioned medium of a murine cell line of EOMA hemangioendothelioma. Endostatin is purified from the conditioned medium through a basic purification methodology. No successful attempts to renature endostatin have been described (O'Reilly et al, Cell 88: 277-285, 1997). The recombinant endostatin derived from E. coli characterized by these authors is precipitated after dialysis- in PBS. The precipitated material (not renatured) can not be analyzed in vi tro due to its insolubility in the culture medium. A small percentage (not specified) of the material is spontaneously solubilized in the PBS during dialysis. This material has an inhibitory activity comparable to the activities of the endothelial cell, both the natural endostatin, and the soluble endostatin derived from the baculovirus. When recombinant endostatin derived from E. coli is renatured in the presence of 0.1 M sodium phosphate, at pH 7.4, 150 M NaCl, 0.6 M urea, 2 mM reduced glutathione, 0.02 mM oxidized glutathione, and 0.5 M of arginine to a final concentration of 0.1 mg / ml, more than 99% of the protein is lost. This great loss precludes the use of this material for in vivo assessments. Instead, the authors use the insoluble, uncharacterized (non-renatured) form of endostatin for most of their in vi ve studies. It is observed that the precipitated product of endostatin, derived from E. coli, dissolves gradually during 5 days and produces a sustained anti-angiogenic effect in the chorioallantoic membrane evaluations of chickens (CAM). A suspension of the same material forms a pellet at the injection site of the mice, which is slowly reabsorbed for a period of 24-48 hours. Subsequent studies in the same group demonstrate that drug resistance does not develop when mice have lung carcinoma, T241 fibrosarcoma, or B16F10 melanoma if they are treated with mouse endostatin (Boehm, T., et al., Na ture 390: 404-407, 1997). The recombinant murine endostatin derived from E. coli is prepared as described above (O'Reilly et al, CeIJ 88: 277-285, 1997) except that the bacteria are pelleted and resuspended in 8 M urea, 10 mM of beta mercaptoethanol, and 10 mM at pH 8.0, and incubated for 1 to 2 hours. Beta mercaptoethanol is eliminated in subsequent stages. The recombinant mouse endostatin is supplied in the mice as a suspension in PBS. Mice suffering from one of the three tumor types are injected with the suspension of purified endostatin, but poorly soluble in the subcutaneous torso at a site away from the inoculated tumors. The treatment is discontinued when the tumors regress, then they are allowed to grow again. Tumor growth does not recur after 6, 4, or 2 treatment cycles with endostatin, respectively, when therapy is terminated. Recently, the circulating form of human endostatin is isolated and characterized (Standker et al., FEBS Letters 420: 129-133, 1997). High molecular weight peptides (1-20 kDa) are isolated from 2,500 liters of an ultrafiltered amount of human blood (hemofiltration, HF) obtained from patients with chronic renal failure. The concentrate is agglutinated in a preparative cation exchange column and the mixture is eluted with pH fractionation (7 buffer solutions with a pH increasing from 3.6 to 9.0). High molecular weight peptides are detected in well 8, which is eluted with water, and subsequently purified by reverse phase HPLC. The aliquots are subjected to matrix-assisted mass desorption mass ionization (MALDI-MS) spectrometry and the exact molecular mass is determined by electro-spray mass spectroscopy (ES-MS) and is observed to be 18,494 Da . It is found that the cysteine residues 1-3 and 2-4 in the molecule are bound by disulfide bridges. It is estimated that the final recovery during the purification is in the order of 20%, which results in a concentration of >10"nM in the hemofiltered product The resulting concentration of endostatin in the patient's plasma is estimated to be in the range of 10" 10M or more. It is not known if there is a tissue volume bound to endostatin, as proposed for angiostatin (Kost et al., Eur J. Biochem, 236: 682-688, 1996). The biological characterization in vi tro of the natural human endostatin (which is 12 amino acids shorter than the mouse endostatin) does not show any anti-proliferative activity in the different cell types of the endothelium. The characterization of the recombinant forms of human endostatin is not reported. The authors speculate that the differences in activity reported for the human and mouse form of endostatin may be due to several factors: (i) the two forms may have been isolated from different sources and may have a different selectivity, specificity , or efficacy in in vi tro and in vi ve valuations, (ii) differences in post-translational purifications found in peptides may be the reason for the discrepancy in reported activities, (iii) human endostatin it can not necessarily inhibit the proliferation of endothelial cells, but it can indirectly influence other cellular components that can only be observed in a complex system in vivo.

Brief Description of the Invention One objective of the invention is to describe a method for expressing high levels of endostatin in bacteria. Another aspect of the invention is to describe an efficient method by which the endostatin inclusion bodies can be solubilized, renatured, and subsequently purified to generate a biologically active material. Preferably, the steps of solubilizing the inclusion bodies with human or mouse endostatin are carried out at a high pH. Preferably, the elevated pH in the solubilization step is carried out at a pH ranging from about pH 9 to about pH 11.5. Even more preferably, the range of the high pH ranges from about pH 10 to about pH 11. More preferably still, the high pH is about 10.5. Preferably, steps to renature the inclusion bodies of human or mouse endostatin are carried out at an almost neutral pH. Preferably the almost neutral pH in the solubilization step is carried out at a pH ranging from about pH 6 to about pH 8.5. Even more preferably still, the quasi-neutral pH for renaturation of mouse endostatin ranges from about pH 7.0 to about pH 8.0. More preferably, the range of neutral pH to renature the mouse endostatin is about pH 7.5. Even more preferably still, the almost neutral pH range for renaturation of human endostatin is from about pH 7.0 to about pH 8.0. More preferably, the almost neutral pH range for renaturing human endostatin is about pH 7.5. Preferably, the concentration of the endostatin gene product is present at a concentration ranging from about 0.2 to about 20 mg / ml during the solubilization step. Even more preferably, the concentration is about 2.5 mg / ml. Preferably, the concentration of the endostatin gene product is present at a concentration ranging from about 0.02 to about 2 mg / ml during the renaturation step. Even more preferably, the concentration is about 0.25 mg / ml. Preferably, a renaturant selected from urea and guanidine hydrochloride is used during the stages of solubilization and renaturation. Even more preferably, the renaturant is urea.

Preferably, the urea concentration is from about 4 M to about 10 M during the solubilization step. Even more preferably, the concentration is about 6 M. Preferably, the concentration of urea is from about 2 M to about 4 M during the renaturation step. Even more preferably, the concentration is about 3.5 M. Preferably, the concentration of the guanidine hydrochloride is from about 2 M to about 8 M during the solubilization step. Even more preferably, the concentration is about 4 M. Preferably the concentration of the guanidine hydrochloride is from about 0.2 M to about 2 M during the renaturation step. Even more preferably, the concentration is about 1.5 M. Preferably, the solubilization and reduction steps are carried out in the presence of a reducing agent capable of reducing disulfide bonds to sulfhydryl groups. Preferably, the reducing agent is selected from the group consisting of DTT, BME, cysteine, and reduced glutathione. Even more preferably, the reducing agent is DTT or cysteine. Preferably, DTT is present at a concentration ranging from about 2 mM to about 10 mM during the solubilization step. Even more preferably, the concentration is about 5 mM. Preferably, DTT is present at a concentration ranging from about 0.5 mM to about 2 mM during the renaturation step. Even more preferably, the concentration is about 0.5 mM. Preferably, the reduced glutathione is present in a concentration ranging from about 5 mM to about 20 mM during the solubilization step. Even more preferably, the concentration is about 10 mM. Preferably, the reduced glutathione is present at a concentration ranging from about 0.5 mM to about 4 mM during the renaturation step. Even more preferably, the concentration is about 1 mM. Preferably, the cysteine is present at a concentration ranging from about 5 mM to about 20 mM during the solubilization step.

Even more preferably, the concentration is about 10 mM. Preferably, the cysteine is present at a concentration ranging from about 0.5 mM to about 4 mM during the renaturation step. Even more preferably, the concentration is about 1 mM. Preferably, an agent capable of enhancing the exchange of disulfide bonds is present during the renaturation step. Preferably, the agent is selected from cystine and oxidized glutathione. Even more preferably, the agent is cystine. Preferably, the cystine is present at a concentration ranging from about 0.2 mM to about 5 M during the renaturation step. Even more preferably, the concentration is about 1 mM. Preferably, the disulfide bonds are formed through atmospheric oxidation during the renaturation step. Preferably, the atmospheric oxidation step is carried out from about 12 to about 96 hours. Even more preferably, the atmospheric oxidation step is carried out from about 24 to about 72 hours. More preferably, the atmospheric oxidation step is carried out for about 60 hours. Preferably, the renatured endostatin is further purified by a process selected from but not limited to the group consisting of ion exchange chromatography, hydrophobic interaction chromatography and RP-HPLC.

Preferably, the method of expression, solubilization, renaturation and purification uses endostatin genes that are either mouse or human. Even more preferably, these genes are selected from the group consisting of SEQ ID Nos: 5-9. The novel proteins of this invention are modified amino acid sequences of human or mouse endostatin, and this protein can, optionally, be immediately preceded by (methionine "1), (alanine" 1), (methionine "2, alanine" 1), (serine "1), (methionine" 2, serine "), (cysteine" 1), or (methionine "2, cysteine" 1). In addition, the present invention relates to recombinant expression vectors comprising nucleotide sequences coding for endostatin, to variants and mutins of endostatin, related to microbial and eukaryotic expression systems, and to processes to do (comprising the steps of expression, solubilization, renaturation, and purification) these proteins. The cloning of the DNA sequences coding for these proteins can be achieved by the use of intermediate vectors. Alternatively, a gene can be cloned directly into a vector that contains the other gene. The linkers and adapters can be used to join the DNA sequences, as well as to replace the sequences that are lost, where a restriction sequence is within the region of interest. Therefore, the genetic material (DNA) encoding a polypeptide, a peptide linker and the other polypeptide, are inserted into a suitable expression vector and used to form the bacterium, yeast, insect cell or mammalian cell. The transformed organism, by the cell line, is cultured and the protein is isolated by conventional techniques. The resulting product, therefore, is a new protein that has all, or a portion of a protein that is bound by a binding region to all, or a portion of a second protein. Another aspect of the present invention includes plasmid vectors of DNA for use in the expression of these proteins. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Suitable vectors that can transform the microorganisms or cell lines capable of expressing for the proteins, include the expression vectors that comprise the nucleotide sequences that code for the proteins that bind to the transcriptional or translational regulatory sequences., and which are selected according to the host cells that are used. Vectors that incorporate modified sequences, as described above, are included in the present invention and are useful for the production of the proteins. The vectors that are used in the method also contain the selected regulatory sequences in a functional association with the sequences coding for the DNA of the invention, and which are capable of controlling the replication and expression of these in selected host cells. Methods for producing these proteins is another aspect of the present invention. The method of the present invention involves culturing suitable cells or cell lines, which have been transformed as a vector containing a DNA sequence and encode for the expression of a novel multifunctional protein. Suitable cells or cell lines can be bacterial cells. For example, the various strains of E. coli are well known as host cells in the field of biotechnology. Examples of these strains include strains of E. coli M101 (Yanisch-Perron et al Gene 33: 103-119, 1985) and MON105 (Obukowicz et al., Appli ed Environmental Microbiology 58: 1511-1523, 1992). Also included in the present invention,. the expression of multifunctional proteins that use a chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126: 25-33, 1993). The various strains of B. subtilis can also be used in this methodology. Also available as host cells are various strains of yeast cells that are known to those skilled in the art for the expression of the polypeptides of the present invention. When expressed in the cytoplasm of E. coli, the gene encoding the proteins of the present invention can also be structured such that the 5 'end of the gene codons are added to encode Met "2-Ala_1, Met-2-Ser_1, Met "2-Cys_1, or Met" 1 in the N-terminus of the protein The terminus of proteins that form in the cytoplasm of E. coli, is affected by post-translational processing of methionine aminopeptidase (Ben Bassat et al., J Bacteriol., 169: 751-757, 1987) and possibly by other peptidases, such that, in expression, methionine is cleaved from the? -terminus. present invention may include polypeptides having Met-2, Ala-1, Ser "1, Cys" 1, Met "2-Ala-1, Met-Ser" 1, Met "2-Cys-1, or Met" 1 In the? -terminus, these mutant proteins can also be expressed in E. coli by fusing a secretion signal peptide to the? -terminus.This signal peptide is cleaved from the polypeptide, as a e of the secretion process.

Definitions What follows is a list of abbreviations and the corresponding meanings as used interchangeably herein: g = grams HPLC = high performance liquid chromatography mg = milligrams ml = milliliter DTT = dithiothreitol RT = room temperature PBS = phosphate buffered saline solution What follows is a list of definitions of various terms used in the present: The term "anti-tumor" means that it has an activity that reduces or inhibits the growth of, or that annihilates, or otherwise damaging to tumors in vi The term "natural sequence" refers to an amino acid or nucleic acid sequence which is identical to the wild type or to the natural form of a gene or protein. The terms 'mutant amino acid sequence', 'mutant protein', 'variant protein', 'mutin', or "mutant polypeptide" refers to a polypeptide having an amino acid sequence that varies from the natural sequence due to amino acid additions, deletions and substitutions, or any combination thereof, or which is encoded by a nucleotide sequence from an intentionally made variant that is derived from a natural sequence or from a chemically synthesized sequence The term 'endostatin' means a protein fragment of collagen XVIII which has anti-angiogenic activity. The activity of these fragments can be determined by micropocket titration for angiogenesis of the mouse cornea or by inhibition of endothelial cell growth or migration in vi tro. Preferably, mouse endostatin is the sequence shown in SEQ ID 10, and human endostatin is the sequence shown in SEQ ID NOll.

Brief Description of the Figures Figure 1 shows a schematic view of the cloned fragment of endostatin. The C-terminal fragment of collagen XVIII is shown. Plasmid pMON24345 (SEQ ID NO: 8) codes for the C-terminal fragment of mouse collagen XVIII, plasmid pMON20440 (SEQ ID NO: 9) codes for the C-terminal fragment of human collagen XVIII.

Figure 2 shows a schematic view of the renaturing process of endostatin. The optimal conditions for solubilization in the presence of urea and DTT or cysteine and renaturation in the presence of cystine are highlighted.

Figures 3-3b show the renaturation products of mouse endostatin under various pH conditions. The RP-HPLC traces of the renaturalization products of mouse endostatin at pH 7.5, pH 8.0, and at pH 8.5 in 3.5 M urea.

Figures 4-4b show the produtos of renaturation of mouse endostatin under various concentrations of urea. The RP-HPLC traces of the renaturalization products of mouse endostatin at 3.0, 3.5, and 4.0 M urea a, a pH 7.5.

"Figures 5-5b show the products of renaturation of human endostatin under various pH conditions.

The traces of RP-HPLC of the renaturation products of human endostatin at pH 7.5, pH 8.0, and pH 7.5 in 3.5 M urea.

Figures 6-6b show the produtos of the renaturation of human endostatin under various concentrations of urea. The RP-HPLC traces of the renaturation products of human endostatin at 3.0, 3.5, and 4.0 M urea at pH 7.5.

Figure 7 shows the inhibition of HMEC migration by mouse endostatin. Purified mouse endostatin that is assessed in the HMEC cell migration assay at 15 and 30 ug / ml. The inhibition of migration is observed in both concentrations.

Figure 8 shows the inhibition of CPAE by mouse endostatin.

Purified mouse endostatin is assessed by its inhibition of CPAE migration at 5 and 30 ug / ml. The inhibition of migration is observed in both concentrations.

Figure 9 shows the inhibition of endothelial cell proliferation by mouse endostatin. It is the inhibition of endothelial cell proliferation by mouse endostatin. Significant inhibition is observed at 20 ug / ml.

Figure 10 shows the inhibition of endothelial cell proliferation by human endostatin. It is the inhibition of endothelial cell proliferation by human endostatin. Significant inhibition is observed at 10 ug / ml endostatin.

Detailed Description of the Invention The following examples illustrate in greater detail the invention, although it is understood that the invention is not limited by these specific examples. Several more examples are apparent to persons skilled in the art upon reading the present description without departing from the spirit and scope of the invention. It is intended that all other examples be included within the scope of the appended claims.

General Methods The general methods for cloning, expressing, and characterizing the proteins are found in T. Maniatis, et al., Molecular Cloning. A Labora tory Manual, Cold Spring Harbor Laboratory, 1982, and references cited there, are incorporated there for your reference; and in J. Sambrook, et al., Mol ecular Cloning. A Labora tory Manual 2nd edition, Cold Spring Harbor Laboratory, 1989, and references are cited there, and are incorporated herein for reference. Unless otherwise indicated, all specialty chemicals are obtained from Sigma, Co. (St. Louis, MO). Restriction endonucleases and T4 DNA ligase are obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN) or Promega. { Madison, Wl).

Strains and Plasmids The bacterial strains that are used in these studies are listed in Table 1. The plasmids that are used in or that are constructed for this study are listed in Table 2.

Transformation of Strains of E. csll Strains of E. coli (Table 1), such as DH5 alpha and DH10B (Life Technologies, Rockville, MD), and TG1 (Amersham Corp., Arlington Heights, IL) are used for the transformation of the ligation reactions and are the hosts that are used to prepare the plasmid DNA to transport the mammalian cells. E. coli strains such as JM101 (Yanisch-Perron et al., Gene, 33: 103-119, 1985) and MON105 (Obukowicz, et al., Appl. And Envir. Mi cr., 58: 1511- 1523, 1992) can be used to express the proteins of the present invention in the cytoplasm or in the periplasmic space. The cells for subcloning efficiency DH5 alpha and DH10B are purchased as competent cells and are ready for transformation using the manufacturer's protocol. Strains of E. coli JM101 and MONI05 become competent to appropriate the DNA using the CaCl2 method. Typically, 20 to 50 ml of cells are cultured in an LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 150 mM NaCl) at a density of approximately 1.0 absorbance units at 600 nanometers ( OD600) as quantified by a spectrophotometer Baush & Lomb (Rochester, NY). The cells are harvested by centrifugation and resuspended in 1/5 of the culture volume of a CaCl 2 solution [50 mM CaCl 2, 10 mM Tris-Cl ((10 mM of 2-amino-2- (hydroxymethyl) 1,3-propanediol hydrochloride, pH 7.4] and kept at 4 ° C for 30 minutes The cells are harvested again by centrifugation and resuspended in 1 ml. / 10 of the culture volume in a CaCl2 solution.The ligated DNA is added to 0.2 mL of these cells, and the samples are kept at 4 ° C for 30-60 minutes.The samples are changed at 42 ° C for 2 minutes and add 1.0 mL of LB before shaking the samples at 37 ° C for one hour.The cells of these samples are spread on plates (medium LB plus 1.5% Bacto-agar) and contain either ampicillin (100 micrograms / mL, ug / mL) when selected for ampicillin-resistant transformants, or spectinomycin (75 ug / mL) when selected for spectinomycin-resistant transformants.The plates are incubated overnight at 37 [deg.] C. The colonies are selected and inoculate in LB plus an appropriate antibiotic (100 ug / mL of ampicillin or 75 ug / mL of espe ctinomycin) and cultured at 37 ° C while shaking.

Isolation and Characterization of DNA Plasmid DNA can be isolated by a variety of different methods and commercially available sets of implements known to those skilled in the art can be used. The plasmid DNA is isolated using the Promega Wizard ™ Miniprep kit (Madison, Wl), the Qiagen QIAwell plasmid isolation kit (Chatsworth, CA) or the Qiagen Plasmid Midi or Mini kit. These sets of implements follow the same general procedure for the isolation of plasmid DNA. Briefly, the cells are pelleted by centrifugation (5000 x g), the plasmid DNA is released by consecutive NaOH / acid treatment, and the cell debris is removed by centrifugation (10000 x g). The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA binding resin, the column is washed, and the plasmid DNA is cleaved. After detecting the colonies with the plasmid of interest, the E. coli cells are inoculated in 50-100 ml of LB plus an appropriate antibiotic for an overnight culture at 37 ° C in an air incubator while stirring. The purified plasmid DNA is used for DNA sequencing, for further digestion with restriction enzymes, and for further subcloning of DNA fragments from a transfection within E. coli, mammalian cells, or other cell types.

Sequence Confirmation The purified plasmid DNA is resuspended in deionized H20 and its concentration is determined by quantifying the absorbance at 260/280 nm on a Baush and Lomb Spectronic 601 UV ultraviolet spectrometer. DNA samples are sequenced using AB * I PRISM ™ and DyeDeoxy ™ sequencing chemicals (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) (Part Number 401388 or 402078) according to the protocol suggested by the manufacturer, is probably modified by the addition of 5% DMSO to the sequencing mixture. The sequencing reactions are carried out in a DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess stained terminators with Centri-Sep ™ centrifuge columns (Princeton Separations, Adelphia, NJ) and lyophilized. Sequencing reactions labeled with a fluorescent dye are resuspended in deionized formamide, and sequenced in renaturing urea gels with 4.75% polyacrylamide-8M using the ABL Model 373 A and Model 377 automated DNA sequencers. The spliced fragments of DNA sequences are analyzed and assembled in contiguous DNA masters using a Sequencher computer program for DNA analysis (Gene Codes Corporation, Ann Atbor, MI).

Small Scale Expression of Endostatin in E. coli The strain of E. coli MON105 or JM101 that harbors the plasmid of interest and is grown at 37 ° C in an M9 medium plus casamino acids with shaking in a model G25 air incubator. Brunswick Scientific (Edison, NJ). Growth is monitored at OD600 until it reaches a value of 1.0, at which time nalidixic acid (10 mg / mL) is added in 0.1N NaOH to a final concentration of 50 μg / mL. The cultures are then stirred at 37 ° C for an additional 3 to 4 hours. A high degree of aeration is maintained throughout the growing period in order to achieve a maximum production of the desired gene product. The cells are examined under a light microscope to see the presence of inclusion bodies (IB). The 1 mL aliquots of the culture are removed for analysis of the protein content by boiling the pelleted cells, subjecting them to treatment with a reductive buffer and electrophoresis by means of SDS-PAGE (see Maniatis et al., 'Molecular Cloning: A Laboratory Manual ", 1982) The culture is centrifuged (5000 xg) to pellet the cells.

Large Scale Expression (10 L) of Endostatin in E. coli The endostatin molecules progressively increase in a 10 L Biostat E ™ fermentor (B. Braun Biotech Inc., Allentown PA). The fermentation medium used is M9 salts that are supplemented with 2% casamino acids (Difco Laboratories, Detroit MI) and glucose. Approximately 1 ml of the thaw culture is transferred to a 3.8 L Fernbach stirring flask containing 1.0 L of the medium and then incubated at 37 ° C at a stirring speed of 250 rpm for 12 hours. This culture of the shake flask is then used to inoculate a 10L fermenter containing 9.0 L of the medium. The fermentation conditions are as follows: stirring = 1000 rpm, air flow velocity in the sprayer = 15 liters / min, control of the pH = 7.0 by the addition of NH40H, back pressure = 10 psi, dissolved oxygen control > 30%, and temperature = 37 ° C. The glucose is initially grouped at 15 g / 1 and controlled at 2-5 g / 1 by the addition of a forage with 50% glucose. The fermentation culture is grown to an OD (550 nm) = 12-15 and is induced with 50 mg / l nalidixic acid. Fermentation is harvested 4 hours after induction by continuous flow centrifugation. 10L Scale Isolation of Inclusion Bodies The cell pellet of a 10L fermentation is resuspended in approximately 6.0 L of a 50 mM Tris / 150 mM EDTA buffer. The resuspension is passed once through a Microfluidizer ™ (Newton, MA) at 10,000 psi and the temperature is kept below 8 ° C. The recovered homogeneous substance is then spun at 15, ooo x G for 25 minutes and mixed until made consistent with an Ultr-Turrax mixer. Resuspension of the cell paste is then homogenized by passing the cell suspension twice through a Microfluidizer ™ (Newton, MA) operating at 10,000 psi pressure. The temperature of the homogenized substance is maintained at less than 6 ° C when collected in a stainless steel container that is placed in an ice bath. The inclusion bodies are isolated by centrifugation of the homogenate of cells at 15,000 x g for 30 minutes and the removal of the supernatant. The inclusion bodies are then washed a total of two times by resuspending the inclusion bodies in an ice-cold D.I. and subjecting them to centrifugation at 15,000 x g for 30 minutes. The inclusion bodies of endostatin are then frozen at -70 ° C.

Small Scale Isolation of Inclusion Bodies The cell pellet of a 330 mL E. coli culture is resuspended in 15 mL of a buffer by sound treatment (50 M Tris-HCl, pH 8.0, 1 mM EDTA). These resuspended cells are subjected to sound treatment using the microtip probe of a Disruptor Sonicador Celular (Model W-375, Heat Systems-Ultrasonics, Inc., Farmingdale, New York). Three rounds of sound treatment are used in a shock absorber with sound treatment followed by a centrifugation to destabilize the cells and wash the inclusion bodies (IB). The first round of sound treatment is a rumble of three minutes followed by a rumble of one minute, and the final two rounds of sound treatment that are one minute each.

Extraction and Renaturing of Human and Mouse Proteins from Sediments of Inclusion Bodies All steps are carried out at 4 ° C. The inclusion bodies of mouse endostatin are dissolved in 6 M urea, 5 mM DTT, 50 mM Bis-Tris Propane, at pH 10.8 in 2.5 mg / ml endostatin. This solution is stirred for 2 hours followed by an addition of cystine (from strain 0.2 M, at a pH of 10.5) to 10 mM. This is mixed for 5 minutes and then diluted to 0.25 mg / ml endostatin in 3.5 M urea, 100 mM Bis-Tris propane, at pH 7.0. It is then stirred for 0 hours to complete the renaturation of the protein as assessed by a reversible phase HPLC. The inclusion bodies of human endostatin are dissolved in 10 mM cysteine, 6 M urea, 50 mM Bis-Tris propane, at a pH of 10.8 in 2.5 mg / ml of endostatin. This is stirred for 2 hours followed by an addition of 10 mM cystine (0.2 M strain, pH 10.5) and mixed for 5 minutes. This solution is then diluted in 0.25 mg / ml endostatin in 3.0 M urea, 100 mM Bis-Tris propane, pH 7.5 and stirred for 60 hours to complete the renaturation step.

Purification The purification of human or mouse endostatin is achieved by using the same process used in acid precipitation followed by chromatography, from a column on a sulfo-propyl column. The renatured sample is concentrated approximately 10 times by ultrafiltration and the pH is decreased to pH 5.0 with acetic acid. This is then subjected to extensive dialysis in 5 mM acetic acid, at pH 5.0. The precipitated product is removed by filtration and the filtrate is applied to an HP column of Pharmacia S-Sepharose. The column is washed with a column volume of a balancing buffer and the protein eluted using a gradient of 20 column volumes of 50 mM phosphate, at pH 6.5 to the same buffer containing 0.4 M NaCl. The fractions are analyzed by SDS and RP-HPLC gel electrophoresis and combined and dialyzed in PBS and frozen. In some cases the renatured proteins can be purified by affinity when using affinity reagents such as monoclonal antibodies or receptor subunits adhered to the appropriate matrix. Purification can also be achieved by using any variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic interaction chromatography or reversible phase HPLC. These and other methods of protein purification are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification', edited by Murray Deutscher, Academic Press, San Diego, California, 1990.

Characterization of Proteins The purified protein is analyzed by RP-HPLC, mass spectrometry by electroaspersion, amino acid sequencing and SDS-PAGE. The protein quantification is carried out by an amino acid composition, RP-HPLC, and the Bradford protein determination. In some cases, the peptide correlation of peptides is carried out in conjunction with mass spectrometry by electroaspersion to confirm the identity of the protein.

Proliferation Evaluation of Endothelial Cells The assessment of endothelial cell proliferation is carried out as described by Cao et al. (J. Biol. Chem. 271: 29461-29467, 1996). Briefly, human microvascular dermal endothelial cells (HdMVEC, Clonetics) or bovine microvascular endothelial cells of the adrenal cortex (bacEnd, Incell, San Antonio, TX) are maintained in MCDB131 containing 5% thermally inactivated fetal bovine serum (FBS, Hyclone), antibiotics, 100 ug / ml heparin (Sigma) and 100 ug / ml endothelial mitogen (Biomedical Technologies). The confluent monolayers in passages 2-5 are ersed in 0.05% trypsin and resuspended in a complete medium. Five hundred ul of the complete medium containing 1.25 x 104 cells are seeded in the wells of a 4-well tissue culture plate coated with 0.1% gelatin (Sigma). The cells are incubated overnight at 37 ° C / 5% C02 at which time the medium is replaced with 250 ul of a medium containing 5% FDS and various concentrations of inhibitors. After 30 minutes of incubation, 250 ul of a medium containing 1 ng / ml bFGF (R & D Systems) are added and the cells are incubated for an additional 72 hours, at which time they are trypsinized and counted with a Coulter counter.

Validation of Endothelial Cell Migration Endothelial cell migration assessment is carried out essentially as previously described by (Gately et al., Cancer Res. 56: 4887-4890, 1996). To determine the ability of endostatin to inhibit the migration of endothelial cells, the titration for migrations is carried out in a chamber with permeable well supports ('Trans-wells' from Costar) containing polycarbonate membranes with a size of 8 mm pore The cells that are used in the titration whether they are human microvascular endothelial cells (Emory University, Atlanta, CA) or bovine pulmonary artery endothelial cells (Monsanto, St. Louis, MO). The cells are starved overnight before being used in MCDB131 + 0.1% BSA (human cells) or DMEM + 0.1% BSA (bovine cells), which are cultured, and resuspended in the same medium to 106 cells / ml. The lower side with the permeable well supports is coated with 0.1% gelatin for 30 minutes at 37 ° C before the addition of 2 x 105 cells in the upper chamber. The permeable supports for wells are moved to a well containing the chemoattractant (bFGF or VEGF) in the lower chamber. Migration is allowed to occur overnight at 37 ° C. Then the membranes are fixed and stained, and the number of cells migrating to the underside of the membrane are counted in three highly amplified lenses.

Example 1: Construction of pMON24345 (SEQ IS NO: 8) and the selection of strains that produce high levels of mouse endostatin. 5 μg of mouse total RNA (Clontech Laboratories Ine, Palo Alto, CA) is mixed with 500 ng of a random hexamer primer (Promega Corporation, Madison, Wl), are heated for 10 minutes at 65 ° C, then cooled for 2 minutes on ice. 20 units of Rnasina are added to the RNA / primer mixture (Promega), SuperScript II buffer, DTT at a final concentration of 0.01M, a mixture of dNTP (Boehringer) at a final concentration of 0.005 M and 200 units of SuperScript II transcriptase (Life Technologies Inc.). The reaction is incubated at 42 ° C for 1. 5 hours and the enzyme is inactivated by incubating the reaction at 70 ° C for 5 minutes. The RNA is removed by adding 2 units of E. coli Rnasa H (Life Technologies Inc.) and the reaction is incubated at 37 ° C for 20 minutes. The double-stranded DNA is generated by the polymerase chain reaction with the vision of dNTP at a final concentration of 1.6 mM, 50 pmol of a 5 'primer of mouse endostatin (SEQ ID NO: 1), 50 pmol of a 3 'primer of mouse endostatin (SEQ ID NO: 2), a High Fidelity PCR buffer, and 2.5 units of a High Fidelity enzyme (Boehringer). The reaction mixture is incubated at 95 ° C for 3 minutes, then cycled 10 times through incubation at 94 ° C for 1) 5 seconds, incubation at 50 ° C for 30 seconds, incubation at 72 ° C. ° C for 4 minutes and then cycled 15 times through an incubation at 94 ° C for 15 seconds, an incubation at 50 ° C for 30 seconds, an incubation of 72 ° C for 4 minutes plus a 20-second extension per cycle. Finally, the reaction is incubated at 72 ° C for 7 minutes. The double-stranded DNA is subcloned into the pCRII vector (Invitrogen) by adding 1 ul of the PCR reaction to 25 ng of the vector, one unit of T4 ligase from the ligation buffer. The ligase reaction is incubated at 12 ° C overnight. The ligated DNA is transformed into competent DH5 alpha cells (Life Technologies Inc., Rockville, MD) and grown on LB amplified plates. Two isolates with inserts as determined by digestion by coRI are further characterized. Of the cDNA inserts of the two isolates, pMON24342 (SEQ ID NO: 5) and pMON24343 (SEQ ID NO: 6), are analyzed by DNA sequencing using conventional dideoxy technology. To construct the correct coding sequence for the DNA, both pMON24342 and pMON24343 are digested with Apal. The Apal mouse endostatin DNA fragment and the pMON24342 vector plus the 5 'mouse endostatin encoding the DNA sequence are isolated using a set of QiaexII gel extraction implements (Qiagen, Germany). The fragments are ligated together in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 ug / ml BSA and one unit of T4 ligase. The reconstructed plasmid is transformed into competent DH5 alpha cells to generate pMON24344 (SEQ ID NO: 7). Plasmid pMON24344 is digested with Ncol and HindIII and the fragment is isolated with the set of Qiaex II gel extraction implements. the fragment pMO? 24344 Ncoi / iindIII is ligated into a dephosphorylated pMO? 5723 expression vector of E. coli digested by Ncoi / Hindl ll in 50 mM tris pH 7.5, 10 mM MgCl2, 50 ug / ml and one unit of T4 ligase. The AD? bound is transformed into MO? 105 and the isolates are selected on Spec LB plates to generate pMO? 24345 (SEQ ID? O: 8). The strain E. coli MO? 105 harboring pMO? 24345 is induced with 10 mg / ml nalidixic acid and mouse endostatin which is expressed in these cells is monitored by SDS-PAAGE.

Example 2: Construction of pMON 20440 (SEQ ID NO: 9) encoding human endostatin. 5 ug of fully human RNA (Clontech Laboratories Ine, Palo Alto, CA) is mixed with 500 ng of a random primer of hexamers, heated for 10 minutes at 65 ° C and then cooled for 2 minutes on ice. 20 units of Rnasin (Promega) are added to the RNA / primer mixture., SuperScript II buffer, and DTT at a total concentration of 0.01 M, dNTP (Boehringer) is mixed at a final concentration of 0.005 M and 20 units of SuperScript II transcriptase. The reaction is incubated at 42 ° C for 1.5 hours and the enzyme is inactivated by incubating the reaction at 70 ° C for 5 minutes. The RNA is removed by adding 2 units of E. coli Rnasa H and the reaction is incubated at 37 ° C for 20 minutes. The double-stranded DNA is generated by the polymerase chain reaction with the final addition of 1.6 mM dNTR, 50 pmoles of a 5 'primer of human endostatin (SEQ ID NO: 3), 50 pmoles of a human primer 3 '(SEQ ID NO: 4), a High Fidelity PCR buffer, and 2.5 units of a High Fidelity enzyme (Boehringer). The reaction mixture is incubated at 95 ° C for 3 minutes, then cycled 10 times in incubation at 9 ° C for 15 seconds, incubation at 50 ° C for 30 seconds, incubation at 72 ° C for 4 minutes , and then cycled 15 times during an incubation at 94 ° C for 15 seconds, an incubation at 50 ° C for 30 seconds, an incubation at 72 ° C for 4 minutes plus an extension of 20 seconds per cycle. Finally, the reaction is incubated at 72 ° C for 7 minutes. The double-stranded DNA is digested with Ncoi and ífindlll and ligated into a dephosphorylated pMON2341 expression vector of E. coli digested by Ncoi / ífindl II in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50ug / ml BSA and a T4 ligase unit. The ligated DNA is transformed into the E. coli strain MON105 and the isolates are selected on Amp LB plates to generate pMON20440 (SEQ ID NO: 9). The E. coli strain MON105 harboring pMON20440 is induced with 10 mg / ml nalidixic acid and the human endostatin that is expressed in these cells is monitored by SDS-PAGE.

Example 3: Method for Renaturing Mouse Endostatin All steps are carried out at 4 ° C. The inclusion bodies of mouse endostatin are dissolved in 6M urea, 5 mM DTT, 50 mM Bis-Tris Propane, pH 10.8 in 2.5 mg / ml. This solution is stirred for 2 hours and followed by an addition of cystine (strain 0.2M, pH 10.5) for 10 mM. This is mixed for 5 minutes and then diluted to 0.25 mg / ml endostatin in 3.5 M urea, 100 mM Bis-Tris propane, pH 7.0. It is then stirred for 60 hours to complete the renaturation of the protein as evaluated by the reversible phase HPLC. Other conditions of pH and urea concentrations can be used, but at a lower efficiency. Figures 3 and 4 show the HPLC traces of the renatured products of mouse endostatin under various pH conditions and urea concentrations, respectively. Figures 7, 8 and 9 show the inhibitory activity of purified mouse endostatin on endothelial cell proliferation and on cell migration valuations.

Example 4: Method for Renaturing Human Endostatin All steps are carried out at 4 ° C. The inclusion bodies of human endostatin are dissolved in 10 mM cysteine, 6 M urea, 50 mM Bis-Tris propane, pH 10.8 in 2.5 mg / ml endostatin. This is stirred for 2 hours after an addition of 10 mM of cystine (strain 0.2M, pH 10.5) and mixed for 5 minutes. This solution is then diluted to 0.25 mg / ml endostatin in 3.0 M urea, 100 mM Bis-Tris propane, pH 7.5 and stirred for 60 hours to complete the renaturation step. Other conditions of pH and urea concentrations can be used, but at a lower efficiency. Figures 5 and 6 show the HPLC traces of the renaturation products of human endostatin under various pH conditions and urea concentrations, respectively. Figure 10 shows the inhibitory activity of purified human endostatin in a titration for endothelial cell proliferation.

Those skilled in the art recognize that the conditions of solubilization, renaturation, and purification conditions that are outlined above represent dramatic improvements over published methods for purifying endostatin from bacteria. The production of endostatin on a commercial scale for use in preclinical studies and clinical trials requires vast quantities of properly renatured soluble material possessing the desired biological characteristics. The commercial development of endostatin as a therapeutic product requires a thoroughly characterized material that behaves in the same way in all applications. Soluble endostatin, properly renatured, is therefore more desirable than suspensions of insoluble material for in vitro valuations and for in vivo studies of the effectiveness, potency, pharmacokinetics, and pharmacodynamics of this protein. The dramatically improved processes for expressing, solubilizing, renaturing, purifying and characterizing this material that is outlined in this description greatly facilitates subsequent studies that focus on developing endostatin, endostatin fragments, utins, insertins, permutins, or the chimeras of these, or the conjugates of these with other anti-angiogenic proteins or small molecules that are useful as compounds for the treatment of angiogenesis disorders, including cancer. All references, patents or applications cited herein are incorporated in their entirety here for your reference, as if they were written here.

TABLES Table 1: Strains Designation Description or Genotype Reference / Fuen te DH5a TM F, phi80 dJacZdeltaM15, Life delta (JacZYA-argF) U169, deoR, Technologies, recAl, endAl, hsdR17 (rk ', mk +), Rockville, phoA, st.pE44, lambda-, thi-1, Maryland gyr96, relAl DH10B F mcrA? (mrr-? sdRMS-incrBC) Life FdlacZ? M15? JacX74 deoR recAl Technologies, endAl araD139? (ara, leu) 7697 Rockville, Maryland gaJU gaJK? ". RpsL nupG JMl01 (ATCC Delta (pro lac), supE, thi, Yanisch-Perron, # 33876) F '(traD36, proA + B +, laclq, et al., Gene, JacZdeltaM15) 33: 103-119, 1985 MONI05 F ", lambda-, IN (rrnD, rrnE) l, Obukowicz, et (ATCC # rpoD +, rpoH358 al., AppJ. and 55204) Envir. Micr. , 58: 1511-1523, 1992 TG1 Delta (lac pro), supE thi-1, Amersham Corp., hsdD5 / F '(traD36, proA + B +, Arlington Jaclq, JacZdeltaK15) Heights, Illinois Table 2: Plasmid Plasmid No. Marker Description Source ID SEC pMON2341 Amp Vector Collection A generic expression of E. coli Amp-resistant laboratory containing the recAl promoter of E. coli and the ribosome binding site G10L, the chloramphenicol acetyl transferase gene (cat3) and the ori M13 gene pMON5723 SpecR Vector from (Olins and expression of E. Rangwalia, Methods-resistant coli Spectinomycin Enzymol., containing 185 (Gene promoter recAl from Expression E. coli and site Technol.): Binding of 115-119, ribosomes G10L 1990) pCRII AmpR Commercial vector Invitrogen for cloning the PCR (TA) fragment pMON24342 # 5 AmpR Vector cloning This for pCR II (TA) + working PCR fragment encoding mouse endostatin, which includes the C-terminal region of the Collagen XVIII natural mouse. Fragment £ .coRI in pCRII (amino acids 1104-1288) pMON24343 # 6 AmpF Vector This cloning for pCR work II (TA) + PCR fragment encoding mouse endostatin, which includes the C-terminal region of collagen XVIII natural mouse. Fragment EcóRI in pCRII (amino acids 1104-1288) pMON24344 # 7 AmpF Vector of This cloning for pCR work II (TA) + PCR fragment encoding mouse endostatin, which includes the C-terminal region of the mouse collagen XVIII natural. Fragment EcoRl in pCRII (amino acids 1104-1288) pMON24345 # 8 Spec "Fragment This pMON5723NcoI / ífindI work II + NcoI / ífindlII of mouse endostatin, which includes the C-terminal region of mouse natural collagen XVIII.EcoRI fragment in pCRII (amino acids 1104-1288) PMON20440 # 9 Spec Fragment This pMON234INco 1 / Hindl work II + NcoI HindIII that codes for human endostatin, which includes the C-terminal region of collagen XVIII natural of human Table 3: SEC ID Correlation Table NO: Sequence ID Description SEC gcgcgcccatggctcatactcatcaggac; Primer 5 'PCR for mouse endostatin gcgcgcaagcttattatttggagaaagaggtc Primer 3' PCR atgaag; for mouse endostatin GCGCGCCCAT GGCTCACAGC CACCGCGAT 5 'PCR Primer TCCAGCCGGT GCTCCACCTG GTTGCGCTCA ACAGCCCCCT for endostatin G; human GCGCGCAAGC TTATTACTTG GAGGCAGTCA 3 'PCR primer TGAAGCTGTT CTCAATGCAG AGCACGATGT AGGCGTGATG GCAGCTCGC; for human endostatin Insert pMON42342 EcoRI encoding mouse endostatin PMON42343 EcoRI insert encoding mouse endostatin PMON42344 EcoRI insert encoding mouse endostatin pMON42345 EcoRI insert encoding mouse endostatin pMON20440 insert encoding human endostatin 0 AHTHQDFQPVI_HLVAL TPLSGGMRGIRGADFQC QOARA Sequence of VGLSGTFRAFLSS RLQSLYSIVRRADRGSVPIVNLKDEVLSPSWDSLFSGSQG amino acids of QLQPGARIFSFDGRDV endostatin LRHPAWPQKSVWHGSDPSGRRLMESYCET RTETTGATGQ mouse ASS LSGRLEEQK AASCHNSYIVLCIENSFMTSFSK; 11 AHSHRDFQPVLHLVA SPLSGGMRGIRGASFQCFQQARA Sequence of VGLAGTFRAFLSS R QDLYSIVRRADRAAV IVN KDEL FPSWEA FSGSEG amino acids of PLKPGARIFDFSGKDV endostatin LRHPT PQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQ human ASSLLGGR LGQS AASCHHAYIVLCIENSFMTA-SK; It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (97)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for producing endostatin, characterized in that it comprises the steps of: (a) culturing host cells expressing for an endostatin gene; (b) recovering the product from this gene expression; (c) solubilizing this gene product at a high pH; (d) renaturing this solubilized gene product to an almost neutral pH; and (e) isolating properly renatured forms of this gene product.

2. A method for producing endostatin, characterized in that it comprises the steps of: (a) culturing the host cells that are expressing for an endostatin gene; (b) recover the product of this gene expression; (c) renaturing this gene product in approximately an almost neutral pH; and (d) isolating properly renatured forms of this gene product.

3. The method according to claim 1, characterized in that the high pH in the solubilization step is from about pH 9 to about pH 11.5.

4. The method according to claim 3, characterized in that this high pH is from about pH IO to about pH 11.

5. The method according to claim 4, characterized in that this pH is about pH 10.5.

6. The method according to claim 1 or claim 2, characterized in that this almost neutral pH in the renaturation step is from about pH 6 to about pH 8.5.

7. The method according to claim 6, characterized in that this almost neutral pH in the renaturation step is from about pH 7.0 to about pH 8.0.

8. The method according to claim 7, characterized in that this almost neutral pH in the renaturation step is about pH 7.5.

9. The method according to claim 6, characterized in that the almost neutral pH in the renaturation step is from about pH 7.0 to about pH 8.0.

10. The method according to claim 9, characterized in that the almost neutral pH in the renaturation step is about pH 7.5.

11. The method according to claim 1 or claim 2, characterized in that the urea is present at a concentration ranging from about 4M to about 10M during the solubilization step.

12. The method according to claim 11, characterized in that the urea is present at a concentration of approximately 6M during the solubilization step.

13. The method according to claim 1 or claim 2, characterized in that the urea is present at a concentration ranging from about 0.5M to about 5M during the renaturation step.

14. The method according to claim 13, characterized in that the urea is present at a concentration of about 3.5M during the renaturation step.

15. The method according to claim 1 or claim 2, characterized in that the guanidine hydrochloride is present at a concentration of about 2M to about 11M during the solubilization step.

16. The method according to claim 15, characterized in that the guanidine hydrochloride is present at a concentration of approximately 4M during the solubilization step.

17. The method according to claim 1 or claim 2, characterized in that the guanidine hydrochloride is present at a concentration of about 0.2M to about 2M during the renaturation step.

18. The method according to claim 17, characterized in that the guanidine hydrochloride is present at a concentration of about 1.5M during the renaturation step.

19. The method according to claim 1 or claim 2, which is carried out in the presence of a reducing agent, characterized in that it is capable of reducing disulfide bonds to sulfhydryl groups.

20. The method according to claim 19, characterized in that the reducing agent is selected from the group consisting of DTT, BME, cysteine and reduced glutathione.

* 21. The method according to claim 20, characterized in that the DTT is present at a concentration ranging from about 2mM to about 10mM during the step of solubilization.

22. The method according to claim 21, characterized in that the DTT is present at a concentration of 5 mM during the step of solubilization.

23. The method according to claim 20, characterized in that the DTT is present at a concentration ranging from about 0.2 mM to about 2 mM during the renaturation step.

24. The method according to claim 23, characterized in that the DTT is present at a concentration ranging from about 0.5 mM during the renaturation step.

25. The method according to claim 20, characterized in that the reduced glutathione is present at a concentration ranging from about 5 mM to about 20 mM during the solubilization step.

26. The method according to claim 25, characterized in that the reduced glutathione is present at a concentration ranging from about 10 mM during the solubilization step.

27. The method according to claim 20, characterized in that the reduced glutathione is present at a concentration ranging from about 1 mM to about 4 mM during the renaturation step.

28. The method according to claim 27, characterized in that the reduced glutathione is present at a concentration of approximately 2 M during the renaturation step.

29. The method according to claim 20, characterized in that the cysteine is present at a concentration ranging from approximately 5 mM to approximately a concentration of 20 mM during the solubilization step.

30. The method according to claim 19, characterized in that the cysteine is present at a concentration ranging from about 10 mM during the solubilization step.

31. The method according to claim 20, characterized in that the cysteine is present at a concentration ranging from about 0.5 mM to about a concentration of 4 mM during the renaturation step.

32. The method according to claim 31, characterized in that the cysteine is present at a concentration ranging from about 1 mM during the renaturation step.

33. The method according to claim 1 or claim 2, characterized in that an agent capable of enhancing the exchange of disulfide bonds is present during the renaturation step.

34. The method according to claim 33, characterized in that the agent is selected from cystine and oxidized glutathione.

35. The method according to claim 34, characterized in that the cystine is present at a concentration ranging from about 0.2 mM to about 5 mM during the renaturation step.

36. The method according to claim 35, characterized in that the cystine is present at a concentration ranging from about 1 mM during the renaturation step.

37. The method according to claim 1 or claim 2, characterized in that the disulfide bonds are formed through atmospheric oxidation during the renaturation step.

38. The method according to claim 37, characterized in that the step of atmospheric oxidation is carried out from about 12 hours to about 96 hours.

39. The method according to claim 38, characterized in that the step of atmospheric oxidation is carried out from about 24 hours to about 72 hours.

40. The method according to claim 39, characterized in that the step of atmospheric oxidation is carried out for 60 hours.

41. The method according to claim 1 or claim 2, characterized in that the gene product is present from a concentration of about 1 to about 20 mg / ml during the solubilization step.

42. The method according to claim 41, characterized in that the gene product is present at a concentration ranging from about 2.5 mg / ml during the solubilization step.

43. The method according to claim 1 or claim 2, characterized in that the gene product is present from a concentration of about 0.1 to about 5 mg / ml during the renaturation step.

44. The method according to claim 43, characterized in that the gene product is present from a concentration of about 0.25 mg / ml during the renaturation step.

45. The method according to claim 1 or claim 2, characterized in that it further includes the step of purifying endostatin by a process selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography and RP-HPLC.

46. The method according to claim 1 or claim 2, characterized in that the endostatin gene is comprised of DNA encoding the non-human animal endostatin.

47. The method according to claim 1 or claim 2, characterized in that the endostatin gene is selected from the group consisting of SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; and SEQ ID NO:

48. The method according to claim 1 or claim 2, characterized in that the endostatin gene is comprised of DNA encoding human endostatin.

49. The method according to claim 1 or claim 2, characterized in that the endostatin gene is comprised of SEQ ID NO: 9.

50. A method for producing endostatin of inclusion bodies that are prepared from bacteria, characterized in that it comprises the steps of: (a) culturing host cells that are expressing an endostatin gene; (b) recover the product of this gene expression; (c) solubilizing the gene product at a high pH; (d) renaturing the solubilized gene product at approximately an almost neutral pH; and (e) isolating properly renatured forms of the gene product.

51. The method according to claim 50, characterized in that the high pH in the solubilization step is from about pH 9 to about pH 11.5.

52. The method according to claim 51, characterized in that the high pH is from about pH 10 to about pH 11.

53. The method according to claim 52, characterized in that the high pH is about pH 10.5.

54. The method according to claim 50, characterized in that the almost neutral pH in the renaturation step is from about pH 6 to about pH 10.5.

55. The method according to claim 54, characterized in that the almost neutral pH in the renaturation step is from about pH 7.0 to about pH 8.0.

56. The method according to claim 55, characterized in that the almost neutral pH, in the renaturation step is about pH 7.5.

57. The method according to claim 54, characterized in that the almost neutral pH in the renaturation step is from about pH 7.0 to about pH 8.0.

58. The method according to claim 57, characterized in that the almost neutral pH in the renaturation step is about pH 7.5.

59. The method according to claim 50, characterized in that the urea is present at a concentration ranging from about 4M to about 10M during the solubilization step.

60. The method according to claim 59, characterized in that the urea is present at a concentration of approximately 6M during the renaturation step.

61. The method according to claim 50, characterized in that the urea is present at a concentration ranging from about 0.5M to about 5M during the renaturation step.

62. The method according to claim 61, characterized in that the urea is present at a concentration of about 3.5M during the renaturation step.

63. The method according to claim 50, characterized in that the guanidine hydrochloride is present at a concentration ranging from approximately 2M to approximately a concentration of 8M during the solubilization step.

64. The method according to claim 63 characterized in that the guanidine hydrochloride is present at a concentration ranging from about 4M during the step of solubilization.

65. The method according to claim 50, characterized in that the guanidine hydrochloride is present at a concentration ranging from approximately 0. 2M to approximately a concentration of 2M during the renaturation step.

66. The method, according to claim 65 characterized in that the guanidine hydrochloride is present at a concentration of about 1.5M during the renaturation step.

67. The method according to claim 50, which is carried out in the presence of a reducing agent characterized in that it is capable of reducing the disulfide bonds to sulfhydryl groups.

68. A method according to claim 67, characterized in that the reducing agent is selected from the group consisting of DTT, BME, cysteine, and reduced glutathione.

69. The method according to claim 68, characterized in that the DTT is present at a concentration ranging from about 2mM to about 10mM during the solubilization step.

70. The method according to claim 69, characterized in that the DTT is present at a concentration of 5 mM during the step of solubilization.

71. The method according to claim 68, characterized in that the DTT is present at a concentration ranging from about 0.2 mM to about 2 mM during the renaturation step.

72. The method according to claim 23, characterized in that the DTT is present at a concentration ranging from about 0.5 mM during the renaturation step.

73. The method according to claim 68, characterized in that the reduced glutathione is present at a concentration ranging from about 5 mM to about 20 mM during the solubilization step.

74. The method according to claim 73, characterized in that the reduced glutathione is present at a concentration ranging from approximately 10 mM during the solubilization step.

75. The method according to claim 68, characterized in that the reduced glutathione is present at a concentration ranging from about 1 mM to about 4 mM during the renaturation step.

76. The method according to claim 75, characterized in that the reduced glutathione is present at a concentration ranging from about 2 mM during the renaturation step.

77. The method according to claim 68, characterized in that the cysteine is present at a concentration ranging from about 5 mM to about a concentration of 20 mM during the solubilization step.

78. The method according to claim 77, characterized in that the cysteine is present at a concentration ranging from about 10 mM during the solubilization step.

79. The method according to claim 68, characterized in that the cysteine is present at a concentration ranging from about 0.5 mM to about a concentration of 4 mM during the renaturation step.

80. The method according to claim 79, characterized in that the cysteine is present at a concentration ranging from about 1 mM during the renaturation step.

81. The method according to claim 50, characterized in that an agent capable of enhancing the exchange of disulfide bonds is present during the renaturation step.

82. The method according to claim 81, characterized in that the agent is selected from cystine and oxidized glutathione.

83. The method according to claim 82, characterized in that the cystine is present at a concentration ranging from about 0.2 mM to about 5 mM during the renaturation step.

84. The method according to claim 83, characterized in that the cystine is present at a concentration ranging from about 1 mM during the renaturation step.

85. The method according to claim 50, characterized in that the disulfide bonds are formed through atmospheric oxidation during the renaturation step.

86. The method according to claim 85, characterized in that the step of atmospheric oxidation is carried out from about 12 hours to about 96 hours.

87. The method according to claim 86, characterized in that the step of atmospheric oxidation is carried out from about 24 hours to about 72 hours.

88. The method according to claim.87, characterized in that the step of atmospheric oxidation is carried out for 60 hours.

89. The method according to claim 50, characterized in that the gene product is present from a concentration of about 1 to about 20 mg / ml during the step of solubilization.

90. The method according to claim 89, characterized in that the gene product is present at a concentration of about 2.5 mg / ml during the step of solubilization.

91. The method according to claim 50, characterized in that the gene product is present from a concentration of about 0.1 to about 5 mg / ml during the renaturation step.

92. The method according to claim 91, characterized in that the gene product is present at a concentration of about 0.25 mg / ml during the renaturation step.

93. The method according to claim 50, characterized in that it further includes the step of purifying the endostatin by a process selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography and RPHPLC.

94. The method according to claim 50, characterized in that the endostatin gene is comprised of DNA encoding the non-human animal endostatin.

95. The method according to claim 50, characterized in that the endostatin gene is selected from the group consisting of SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; and SEQ ID NO: 8.

96. The method according to claim 50, characterized in that the endostatin gene is comprised of DNA encoding human endostatin.

97. The method according to claim 50, characterized in that the endostatin gene is comprised of SEQ ID NO: 9.