KR100682666B1 - Anti-angiogenic proteins and methods of use thereof - Google Patents

Abstract

The present invention relates to proteins having antiangiogenic properties and methods of inhibiting angiogenesis using them.

Description

ANTI-ANGIOGENIC PROTEINS AND METHODS OF USE THEREOF}

The vascular basement membrane consists of polymers such as collagen, laminin, heparan sulfate proteoglycans, fibronectin and entaxin (Timpl, R., 1996, Curr Opin Cell Biol 8: 618-24). Functionally, collagen promotes cell adhesion, migration, differentiation and growth (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27: 93-127), and by these functions angiogenesis It is thought to play an important role in endothelial cell proliferation and behavior. Angiogenesis refers to the process of new blood vessel formation from existing blood vessels (Madri, JA et al ., 1986, J. Histochem. Cytochem. 34:85 -91; Folkman, J., 1972, Ann.Surg. 175: 409-16). Angiogenesis is a complex process and requires the germination and migration of endothelial cells, the proliferation of these cells and their differentiation into tubular structures and the generation of a basement membrane matrix around the developing blood vessels. Angiogenesis is also an important process for normal physiological events such as wound healing and endometrial remodeling (Follkman, J. et al ., 1995, J. Biol. Chem. 267: 10931-34). It is now well known that angiogenesis is required for metastasis and growth of solid tumors larger than several mm ³ in size (Follkman, J., 1972, Ann. Surg. 175: 409-16; Folkman, J., 1995, Nat. Med. 1: 27-31). Several studies have shown that inhibitors of collagen metabolism have antiangiogenic properties, which support the view that basal membrane collagen synthesis and precipitation are important for angiogenesis and survival (see Maragoudakis, ME et al ., 1994, Kidney Int. 43: 147-50; Haralabopoulos, GC et al ., 1994, Lab. Invest. 71: 575-82). However, the exact role of collagen in basal membrane tissue composition and angiogenesis is not well understood.

Summary of the Invention

The present invention relates to a protein comprising the NC1 domain of the alpha chain of type IV collagen with antiangiogenic properties. Specifically, the present invention relates to novel proteins Arresten, Canstatin and Tumstatin, and their biologically active (eg antiangiogenic) fragments, mutants, analogs, homologs and Derivatives as well as their multimers (eg, dimers) and fusion proteins (also referred to herein as chimeric proteins). All of these proteins contain C-terminal fragments of the NC1 (non-collagen 1) domain of type IV collagen. More specifically, Aresten, Canstatin and Tumstatin are each C-terminal fragment of the NC1 domain of the α1 chain, α2 chain and α3 chain of type IV collagen, respectively. In particular, aresten, canstatin and tombstatin are monomeric proteins. All three proteins stop tumor growth in vivo and also inhibit the formation of capillaries in some in vitro models, including endothelial tube assays.

The present invention provides an isolated and recombinantly produced aresten (also referred to herein as "Arrestin), isolated ares, comprising an NC1 domain of the α1 chain of type IV collagen having antiangiogenic activity. Antiangiogenic fragments of ten, multimers of isolated aresten and antiangiogenic fragments, and polynucleotides encoding such antiangiogenic proteins. In another embodiment, the invention features angiogenic activity in a mammal comprising administering to the mammal a composition containing an angiogenic arsten or a fragment thereof. Methods for treating proliferative diseases, such as cancer, and antiangiogenic arestens and fragments thereof It may be used to prevent migration or endothelial cell proliferation The invention also includes antibodies against isolated antiangiogenic aresten and fragments thereof.

The invention also provides an isolated and recombinantly produced canstatin comprising an NC1 domain of the α2 chain of type IV collagen having antiangiogenic activity, an angiogenic fragment of isolated canstatin, an isolated canstatin and antivascularity Multimers of forming fragments and polynucleotides encoding such antiangiogenic proteins. The present invention also includes compositions containing canstatin, antiangiogenic fragments thereof, or both, isolated as biologically active ingredients. In another aspect, the invention includes a method of treating a proliferative disease, such as cancer, characterized by angiogenic activity in a mammal, comprising administering to the mammal a composition containing canstatin or a fragment thereof. Antiangiogenic canstatin and fragments thereof can also be used to prevent cell migration or endothelial cell proliferation. The invention also includes antibodies to isolated antiangiogenic canstatin and fragments thereof.

The present invention also provides an isolated and recombinantly produced tomstatin comprising an NC1 domain of the α3 chain of type IV collagen having antiangiogenic activity, an angiogenic fragment of isolated tomstatin, an isolated tomstatin and antiangiogenic Multimers of forming fragments and polynucleotides encoding such antiangiogenic proteins. The present invention also encompasses compositions containing tumstatin, antiangiogenic fragments thereof, or both, isolated as biologically active ingredients. In another aspect, the present invention includes a method of treating a proliferative disease, such as cancer, characterized by angiogenic activity in a mammal, comprising administering to the mammal a composition containing antiangiogenic tumstatin or a fragment thereof. . Antiangiogenic tumstatin and fragments thereof can also be used to prevent cell migration or endothelial cell proliferation. The present invention also encompasses antibodies against isolated antiangiogenic tumstatin and fragments thereof.

Brief description of the drawings

1A and 1B are schematic diagrams depicting the nucleotide (FIG. 1A, SEQ ID NO: 1) and amino acid (FIG. 1B, SEQ ID NO: 2) sequences of the α1 chain of human type IV collagen. The positions of the pET22b (+) forward (SEQ ID NO: 3) and reverse (SEQ ID NO: 4) primers are underlined, and the pPICZαA forward (SEQ ID NO: 15) and reverse (SEQ ID NO: 16) The position of the primer is indicated by a single underline.

2 is a schematic showing the aresten cloning vector pET22b (+). The sites where the forward (SEQ ID NO: 3) and reverse (SEQ ID NO: 4) primers and aresten are cloned are indicated.

3A and 3B show Aresten (FIG. 3A, 0 μg / ml to 10 μg / ml, x-axis) and endostatin (FIG. 3B, 0 μg / ml to 10 μg /) as an indication of endothelial cell (C-PAE) proliferation. ml, x-axis) is a pair of linear graphs showing the effect of ³ H-thymidine incorporation (y-axis).

4A, 4B, 4C and 4D are four sets of bar charts showing the effect of Aresten and endostatin on ³ H-thymidine incorporation (y-axis) as an indication of endothelial cell proliferation. 4A, 4B and 4C show Aresten (0 μg / ml-50 μg / ml (FIGS. 4A and 4B) and 0 μg / ml-10 μg / ml (FIG. 4) for 786-0, PC-3 and HPEC cells, respectively. 4C)) shows the effect. 4D shows the effect of 0.1-10 μg / ml endostatin on A-498 cells.

Figures 5A, 5B and 5C show endothelial cells induced by FBS-induced chemotaxis in Areman (2 μg / ml, Fig. 5B) and Endostatin (20 μg / ml, Fig. 5C) in human navel endothelial (ECV-304) cells. One set of four micrographs showing the effect on migration. 5A shows untreated control cells.

6 is a bar chart showing the results of FIG. 5 in a graphical form. 6 shows the effect of Aresten (2 μg / ml or 20 μg / ml) and endostatin (2.5 μg / ml and 20 μg / ml) on the migration of ECV-304 endothelial cells.

7 is a linear graph showing the effect of aresten on endothelial tube formation. The percentage of tube formation is plotted on the y-axis and the concentration of inhibitor is plotted on the x-axis. Treatments were as follows: none (control, ◆), BSA (control, Δ), 7S domain (control, X) and aresten (■).

8A and 8B are a pair of micrographs showing the effect of Aresten (0.8 μg / ml, FIG. 8B) on endothelial tube formation compared to the control (FIG. 8A).

9A, 9B, 9C and 9D are four sets of linear graphs showing the effect of Aresten and Entostatin on tumor growth in vivo. 9A shows that tumor volume increases from 700 mm ³ for 10 mg / kg aresten treated (□) mice, BSA treated (+) mice and control (●) mice. 9B shows an increase in tumor volume from 100 mm ³ for 10 mg / kg aresten treated (□) and BSA treated (+) tumors. 9C shows an increase in tumor volume from about 100 mm ³ for 10 mg / kg arestened (□) mice, endostatin treated (▲) mice and control mice (●). 9D shows an increase for 200 mm ³ tumors when treated with Aresten (□) vs. Control (●).

10A and 10B are schematic diagrams depicting the nucleotide (FIG. 10A, SEQ ID NO: 5) and amino acid (FIG. 10B, SEQ ID NO: 6) sequences of the α2 chain of human type IV collagen. The positions of the pET22b (+) forward (SEQ ID NO: 7) and reverse (SEQ ID NO: 8) primers are indicated with double underlining, and the pPICZαA forward (SEQ ID NO: 17) and reverse (SEQ ID NO: 18) primers. The position of is indicated by a single underline.

11 is a schematic showing the canstatin cloning vector pET22b (+). Sites where the forward (SEQ ID NO: 7) and reverse (SEQ ID NO: 8) primers and canstatin are cloned are indicated.

12A, 12B, 12C and 12D show various concentrations of canstatin endothelial (C-PAE) cells (FIGS. 12A and 12C) and non-endothelial (786-0, PC-3 and HEK 293) cells (FIGS. 12B and 12D). It is a bar graph showing the effect (x-axis) on the growth of. Proliferation was measured as a function of ³ H-thymidine incorporation (FIGS. 12A and 12B) and methylene blue staining (FIGS. 12B and 12D).

13 shows treatment of cells without VFGF (with or without VEGF) and VEGF (1% FCS and 10 ng / ml VEGE) cells and 0.01 μg / ml canstatin (1% FCS, 10 ng / ml VEGF and 0.01 μg / bar chart showing the number of migrated endothelial cells per field (y-axis) for treatment of ml canstatin) and 1.0 μg / ml canstatin (1% FCS, 10 ng / ml VEGF and 1 μg / ml canstatin) to be.

FIG. 14 is a linear graph showing the amount of endothelial tube formation as a percentage of control (PBS treated well) tube formation (y-axis) under various treatments of BSA (□), Canstatin (■) and α5NC1 (○). Vertical bars represent standard error of the mean.

Figures 15A, 15B, 15C and 15D show canstatin (■), entostatin (for tumor volume (y-axis, Figures 15C and 15D) expressed in partial tumor volume (y-axis, Figures 15A and 15B) or mm ³ . ○) and control plots plotting the effects of PC-3 cells (FIGS. 15A and 15B) and 786-0 cells (FIGS. 15C and 15D) over treatment day (x-axis).

16A and 16B are schematic diagrams depicting the nucleotide (FIG. 16A, SEQ ID NO: 9) and amino acid (FIG. 16B, SEQ ID NO: 10) sequences of the α3 chain of human type IV collagen. The positions of the pPET22b (+) forward (SEQ ID NO: 11) and reverse (SEQ ID NO: 12) primers are indicated with double underlines. Also shown are the beginning and end of the fragments "Tumstatin 333" and "Tumstatin 334" ("*" = Tumstatin 333; "+" = Tumstatin 334).

FIG. 17 is a schematic showing the tumstatin cloning vector pET22b (+). FIG. Sites where the forward (SEQ ID NO: 11) and reverse (SEQ ID NO: 12) primers and toomstatin are cloned are indicated.

FIG. 18 is a schematic diagram showing the position of amino acids cleaved within the α3 (IV) NC1 monomer of the tomstatin mutant tomstatin N-53. FIG. The filled circle corresponds to the N-terminal 53 amino acid residue deleted from tumstatin to generate this mutant. Disulfide bonds represented by short bars are arranged as they occur in α1 (IV) NC1 and α2 (IV) NC1.

19A, 19B and 19C show C-PAE cells (FIG. 19A), PC-3 cells (FIG. 19B) and 786-0 cells (FIG. 19C) when treated with various concentrations of tumstatin (x-axis). ^Three sets of histograms showing ³ H-thymidine incorporation (y-axis). All groups represent triple samples.

20 is a linear graph showing the effect of varying amounts (x-axis) of tumstatin (●), BSA (control, □) and 7S domains (control, ○) on endothelial tube formation (y-axis).

21A and 21B are a pair of linear graphs showing the effect of tumstatin (•) and endostatin (○) versus control (□) on tumor volume (mm ³ , y-axis) over treatment day (x-axis). to be. Data points marked with an asterisk are important, with a one-sided student test of P <0.05.

FIG. 22 is a graph showing the increase in tumor volume (y-axis) over treatment day (x-axis) for control mice (□) and mice treated with tumstatin mutant N-53 (●). Data points marked with an asterisk are important, with a one-sided student test result of P <0.05.

FIG. 23 shows endothelial tube formation by varying concentrations of Aresten (●), Canstatin (○), 12 kDa Aresten Fragment (■), 8 kDa Aresten Fragment (□) and 10 kDa Canstatin Fragment (▲) Linear graph showing suppression (y-axis).

FIG. 24 shows endothelial tubes with varying concentrations (x-axis) of tomstatin fragment 333 (●), tomstatin fragment 334 (○), BSA (control, ■), α6 (control, □) and toomstatin (▲). Linear graph showing inhibition of formation (y-axis).

Unexpected angiogenesis causes a wide variety of diseases. In other words, under certain conditions, many diseases and undesirable diseases can be prevented or alleviated if one can stop the growth and expansion of capillaries at certain times or in certain tissues. The basal membrane histology depends on the assembly of the type IV collagen network, which is thought to occur through the C-terminal spherical comparative protein (NC1) domain of type IV collagen (Timpl, R., 1996, Curr Opin Cell Biol 8). : 618-24; Timpl, R. et al ., 1981, Eur. J. Biochem. 120: 203-211). Type IV collagen consists of six different gene products (ie α1 to α6) (Prockop, DJ et al ., 1995, Annu. Rev. Biochem. 64: 403-34). α1 and α2 isoforms are localized in the human basement membrane (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27: 93-127), but the remaining four isoforms are limited. (Kalluri, R. et al ., 1997, J. Clin. Invest. 99: 2470-8).

Formation of new capillaries, ie angiogenesis, from existing vessels is essential for the process of tumor growth and metastasis (Follkman, J. et al ., 1992, J. Biol. Chem. 267: 10931-4; Folkman, J. 1995, Nat. Med. 1: 27-31; Hanahan, D. et al ., 1996, Cell 86: 353-64). Human and animal tumors initially do not produce blood vessels, but tumors that grow to a few mm ³ or more can produce blood vessels (see Folkman, J. 1995, Nat. Med. 1: 27-31; Hanahan, D. et al ., 1996, Cell 86: 353-64). Conversion to the angiogenic phenotype requires both upregulation of angiogenic stimulants and downregulation of angiogenesis inhibitors (Follkman, J. 1995, Nat. Med. 1: 27-31). Vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF) are the most commonly expressed angiogenic factors in tumors. Angiogenic tumors may synergistically promote tumor growth by overexpressing one or more of the angiogenic factors. Inhibiting single angiogenic factors such as VEGF with receptor antagonists is not sufficient to stop tumor growth. Numerous angiogenesis inhibitors have recently been identified, including IFN-a, platelet-factor-4 (Maione, TE et al ., 1990, Science 247: 77-9) and PEX (Brooks, PC et al . Certain factors such as 1998, Cell 92: 391-400) are not endogenously related to tumor cells, but angiostatin (O'Reilly, MS et al ., 1994, Cell 79: 315-28) And endostatin (O'Reilly, MS et al ., 1997, Cell 88: 277-85) are tumor-associated angiogenesis inhibitors produced by the tumor tissue itself. Treatment of tumor growth and metastasis with such endogenous angiogenesis inhibitors is a very effective and fascinating idea, but several potential problems associated with antiangiogenic treatments must be considered. Serious consideration should be given to the possibility of delayed toxicity induced by chronic antiangiogenic therapy as well as the treatment of damaged wounds and regenerative angiogenesis that occurs during treatment.

In the present invention, a protein having antiangiogenic properties and fragments, analogs, derivatives, homologs and mutants thereof are used together with the protein, analogs, derivatives, homologs and mutants to inhibit angiogenesis-mediated proliferative disease. Describe how to do this. The protein comprises monomers of the NC1 domain of the α chain of type IV collagen or a portion of the domain, in particular of the NC1 domain of the α1, α2 and α3 chains of type IV collagen. These proteins (particularly when present in monomeric form) inhibit tumor growth in in vivo models of cancer, and also inhibit the formation of capillaries in some in vitro models, including endothelial tube assays.

The proteins also include a linking site of the NC1 domain. A1, α2 and α3 chains are preferred, according to evidence suggesting that α4, α5 and α6 chains have reduced or undetectable antiangiogenic activity. In general, the monomeric form of the protein is preferred, as evidence suggests that the hexameric form has little or no activity.

More specifically, the present invention describes a protein designated "aresten", which is a protein of about 230 amino acids in length and amino acids at the N-terminus of the α1 chain of the NC1 domain of human type IV collagen. Corresponding to Hostikka, SL et al ., 1988, J. Biol. Chem. 263: 19488-93.

As described herein, human arestenine can be produced in Escherichia coli using bacterial expression plasmids such as pET22b, and pET22b can be obtained as a soluble protein because of its periplasmic migration capacity. The protein is expressed as a 29 kDa fusion protein with a C-terminal 6-histitin tag. An additional 3 kDa (over 26 kDa) results from the polylinker and histitin tag sequences. Aresten can also be produced as a soluble protein secreted from 293 kidney cells using pcDNA 3.1 eukaryotic vectors. This 293-generated protein lacks a purification or detection tag.

E. coli-produced arestenes show an ED ₅₀ of 0.25 μg / ml and inhibit the proliferation of bFGF-stimulated endothelial cells in a dose-dependent manner. No significant effect was found on the proliferation of renal carcinoma cells (786-0), prostate cancer cells (PC-3) or human prostate endothelial cells (HPEC). Endostatin inhibited proliferation of C-PAE cells three times higher than Aresten at 0.75 μg / ml of ED ₅₀ and did not inhibit A-498 cancer cells.

As described herein, specific inhibition of endothelial cell proliferation and migration suggests that aresten can function through cell surface proteins or receptors. Inhibition of matrix metalloproteinases or MMPs suggests a direct role of Aresten in tumor growth and metastasis, similar to batimastat (BB-94) and marimastat (BB-2516).

In the present invention, canstatin, the NC1 domain of the α2 chain of type IV collagen, was used to inhibit angiogenesis, as assayed by inhibition of endothelial cell proliferation and migration and inhibition of endothelial tube formation. The specific inhibition of endothelial cell proliferation and migration by canstatin also demonstrates its antiangiogenic activity and demonstrates that canstatin can function through cell surface proteins / receptors. Integrins are potential candidate molecules in terms of their extracellular matrix binding capacity and ability to modulate cellular behavior such as migration and proliferation. In particular, avb3 integrins are possible canstatins due to their induction and their indiscriminate binding capacity during angiogenesis.

In the present invention, tumstatin, NC1 of the α3 chain of type IV collagen (Timpl, R. et al ., 1981, Eur. J. Biochem. 120: 203-11; Turner, N. et al ., 1992, J. Clin. Invest. 89: 592-601) was used to control the proliferation and angiogenesis of vascular endothelial cells using in vitro and in vivo models of angiogenesis and tumor growth. The distribution of α3 (IV) chains can be found in certain basement membranes such as GBM, several basement membranes of the cochlear angle, the basement membrane of the eye such as the anterior lens capsule, Desmet's membrane, ovary and testis basement membrane (Frojdman, K. et al . 1998, Differentiation 83: 125-30) and alveolar capillary basement membranes (Kashtan, CE, 1998, J. Am. Soc. Nephrol. 9: 1736-50). However, the chain is not present in the renal vasculature, the vascular and epithelial basement membranes of the skin and the vascular basement membranes of the liver (Kashtan, CE, supra ). In the course of wound healing, the α chains of type IV collagen, except for the α3 and α4 chains, will assemble to form new capillaries, because these two chains are not components of the 'traditional' basement membrane, the skin vasculature. Because. Since the α3 (IV) chain is not an intrinsic component of normal human skin, the process of collagen assembly and angiogenesis in the treatment of wound damage will not be altered by treatment with tumstatin.

α3 (IV) chain is expressed in GBM as well as in human renal vascular basement membrane (Kalluri, R. et al ., 1997, J. Clin. Invest. 99: 2470-8). These 'traditional' blood vessels are thought to be involved in the development of primary kidney tumors such as renal cell carcinoma. Tumstatin can be effective in the treatment of primary kidney tumors by disrupting angiogenesis mediated by the assembly of the α3 (IV) chain with other α chains. The number of patients diagnosed with renal cell carcinoma was approximately 30,000 in the US in 1996 (Mulders, P. et al ., 1997, Cancer Res. 57: 5189-95), and the prognosis for metastatic cases is very pessimistic. . Despite advances in radiotherapy and chemotherapy, long-term survival of treated patients has not yet improved significantly (Mulders, P., supra ). The lack of important treatment alternatives for renal cell carcinoma stresses the importance of developing new treatment strategies. Considering this fact, the treatment of neovascularization of solid tumors has recently demonstrated promising results in some animal models (see Baillie, CT et al ., 1995, Br. J. Cancer 72: 257-67). Burrows, FJ et al ., 1994, Pharmacol. Ther. 64: 155-74; Thorpe, PE et al ., 1995, Breast Cancer REs. Treat. 36: 237-51). The effect of tumstatin in inhibiting renal cell carcinoma growth in vivo demonstrates the molecule's potential as an effective antiangiogenic treatment for the tumor type.

In the present invention, tumstatin specifically inhibits endothelial cell proliferation and has no effect on the proliferation of tumor cell lines PC-3 and 786-O in vitro. Tumstatin did not inhibit endothelial cell migration but significantly inhibited endothelial tube formation in vitro. Collectively, these results show that tumstatin inhibits the formation of new blood vessels by inhibiting several stages of the angiogenesis process.

In in vivo studies, tumstatin inhibited angiogenesis in Matrigel plug assays and inhibited the growth of PC-3 tumors and 786-O in mouse xenograft models. The fact that tumstatin inhibited the growth of huge tumors is encouraging, especially when considering the treatment of tumors in clinical settings.

Tumstatin has a pathogenic epitope for Goodpasture syndrome, an autoimmune disease characterized by pulmonary bleeding and rapidly progressive glomerulonephritis (Butkowski, RJ et al . 1987, J. Biol. Chem. 262: 7874-77). Saus, J. et al ., 1988, J. Biol. Chem. 263: 13374-80; Kalluri, R. et al ., 1991, J. Biol. Chem. 266: 24018-24), acute of tumstatin Or chronic administration can cause this autoimmune disease. Several groups were tested to predict the mapping of the Goodpasture self-epitope on α3 (IV) NC1 or the location of the epitope, with the N-terminal part, the middle part and the C-terminal part having the epitope. Have been reported (Kalluri, R. et al ., 1995, J. Am. Soc. Nephrol. 6: 1178-85; Kalluri, R, et al ., 1996, J. Biol. Chem. 271: 9062-8 Levy, JB et al ., 1997, J. Am. Soc. Nephrol. 8: 1698-1705; Quinones, S. et al ., 1992, J. Biol. Chem. 267: 19780-4; Kefalides, NA et. al ., 1993, Kidney Int. 43: 94-100; Netzer, KO et al ., 1999, J. Biol. Chem. 274: 11267-74). Recently, the use of recombinant chimeric constructs has been reported to correlate with the renal survival rate of autoantibodies specific for the N-terminus of α3 (IV) NC1 (Hellmark, T. et al ., 1999, Kidney Int. 55: 936-44). Disease-related epitopes have also been identified for the first 40 amino acids of the N-terminal portion. Truncated tumstatin was synthesized by lacking the N-terminal 53 amino acid residues to eliminate epitopes for Goodpasture syndrome, which showed an inhibitory effect on 786-O tumor growth in a mouse xenograft model. The molecule also did not bind autoantibodies from patients with severe Goodpasture syndrome. These results suggest that the anti-angiogenic site of toomstatin is preserved even when the N-terminal 53 amino acids are removed.

Specific inhibition of endothelial cell proliferation by tumstatin strongly suggests that tumstatin can function through cell surface proteins / receptors. Angiogenesis also depends on specific endothelial cell adhesion events mediated by integrin avb3 (Brooks, PC et al ., 1994, Cell 79: 1157-64). Tumstatin can lead to endothelial cells initiating apoptosis by disrupting the interaction of matrix components with proliferating endothelial cells (Re, F. et al ., 1994, J. Cell.Biol 127: 537-46). Matrix metalloproteinases (MMPs) are involved as key enzymes that regulate the formation of new blood vessels in tumors (Ray, JM et al ., 1994, Eur. Respir. J. 7: 2062-72). Recently, it has been demonstrated that inhibitors of MMP-2 (PEX) can inhibit tumor growth by inhibiting angiogenesis (Brooks, PC et al ., Cell 92: 391-400). Tumstatin can function by inhibiting the activity of MMPs.

Tumstatin inhibits tumor progression by inhibiting angiogenesis in vitro and in vivo. In addition, to apply this method to patients, the potential toxicity or side effects of tumstatin by systemic administration must be considered. The limited distribution of tumstatin and the absence of most of the basal membranes of the skin suggest a low likelihood of side effects from treatment with tumstatin. In addition, the presence of tumstatin in the vascular basement membrane of restricted organs, such as the kidney, suggests its potential unique advantage in targeting increasing tumors in restricted organs. Ultimately, it is desirable to develop another method for the expression of the tumstatin gene in vivo in tumor vasculature using gene transfer approaches (see Kashihara, N. et al ., 1997, Exp. Nephrol. 5). : 126-31; Maeshima, Y. et al ., 1996, J. Am. Soc.Neprol. 7: 2219-29; Maeshima, T. et al ., 1998, J. Clin.Invest. 101: 2589-97 ).

The distribution of the α3 (IV) chain is limited to the basal membrane of selected organs, and therefore, it is thought that tumstatin is of low hazard considering the possible mechanism of this molecule by inhibiting the assembly of the α chains. In addition, α3 (IV) chains are found in the basal membranes of the kidneys (Kalluri, R. et al ., 1997, J. Clin. Invest. 99: 2470-8), and these vessels are associated with primary cells such as renal cell carcinoma. It is thought to be related to the progression of renal tumors. Therefore, tumstatin can be effective in the treatment of such tumors by disrupting the assembly of other α chains and α3 (IV) chains.

As used herein, the term “angiogenesis” refers to the creation of new blood vessels in tissues or organs and is associated with endothelial cell proliferation. Under normal physiological conditions, humans or animals initiate angiogenesis only in very specific limited circumstances. For example, angiogenesis is normally observed in wound healing, fetal and abdominal development, and in the formation of the corpus luteum, endometrium and placenta. The term "endothelium" means a thin layer of flat epithelial cells that are aligned in the mesenteric cavity, lymphatic vessels and blood vessels. Thus "antiangiogenic activity" refers to the ability of a composition to inhibit the growth of blood vessels. Vascular growth is a series of complex events, including the local destruction of the basement membrane underneath individual endothelial cells, the proliferation of these cells, the subsequent movement of cells to the location of blood vessels, the reconstitution of cells to form new vascular membranes, and endothelial cell proliferation. And the binding of perivascular and other cells to support new vessel walls. Thus "antiangiogenic activity" as used herein includes the blocking of any or all of the above steps with the final result that the formation of new blood vessels is inhibited.

Antiangiogenic activity may be associated with endothelial inhibitory activity, which is generally small capillary endothelial cells in culture in the presence of fibroblast growth factors, angiogenesis related factors or other known growth factors that inhibit angiogenesis. Refers to the ability of a composition to inhibit growth or migration. A "saint factor" is a composition that stimulates the growth, reproduction or synthetic activity of a cell. "Angiogenesis related factors" are factors that inhibit or promote angiogenesis. Examples of angiogenesis related factors include angiogenic growth factors such as basic fibroblast growth factor (bFGF), which factors are angiogenesis promoters. Other examples of angiogenesis related factors include, for example, angiostatin (eg, US Pat. No. 5,801,012, US Pat. No. 5,837,682, US Pat. No. 5,733,876, US Pat. No. 5,776,704, US Pat. No. 5,639,725, US Pat. No. 5,792,845). Anti-angiogenic factors such as WO 96/35774, WO 95/29242, WO 96/41194, WO 97/23500) or endostatin (see, eg, WO 97/15666).

"Substantially the same biological activity" or "substantially the same or superior biological activity" means that the composition has antiangiogenic activity and acts similarly to aresten, canstatin and tomstatin, as determined in standard assays. . “Standard assays” include, but are not limited to, protocols used in the field of molecular biology to assess antiangiogenic activity, cell cycle arrest, and apoptosis. Such assays include endothelial cell proliferation, endothelial cell migration, cell cycle analysis, and endothelial cell tube formation, such as apoptotic cell morphology or annexin V-FITC assay, chorionic membrane (CAM) assay, and renal cancer tumors in athymic nude mice. Assays for detection of apoptosis by inhibition of growth are included, but are not limited to these. The assays are provided in the examples below.

"Arsten" (also referred to herein as "arrestin") includes allelic variants of fragments, mutants, homologs, analogs and amino acid sequences as well as aresten from other mammals, and aresten amino acid sequences. Fragments, mutants, homologs, analogs and allelic variants of are included.

As used herein, “canstatin” includes fragments of tanstatin sequences, allelic variants of mutants, homologs, analogs and amino acid sequences, as well as canstatins from other mammals, and fragments of canstatin amino acid sequences, Mutants, homologs, analogs and allelic variants are included.

As used herein, “tumstatin” includes fragments of tumstatin sequences, allelic variants of mutants, homologs, analogs and amino acid sequences, as well as tombstatins from other mammals, and fragments of tumstatin amino acid sequences, Mutants, homologs, analogs and allelic variants are included.

The present invention inhibits the growth or migration of bovine capillary endothelial cells in culture in the presence of endothelial inhibitory activity (e.g., generally inhibiting angiogenesis such as fibroblast growth factor, angiogenesis related factors or other known saint factors). It is to be understood that it is intended to include any derivative of aresten, canstatin or tombstatin). The present invention includes all aresten, canstatin or tombstatin proteins, derivatives of these proteins and biologically active fragments of these proteins. These proteins are proteins with Aresten, Canstatin or Tumstatin activity with sugar or other molecules attached to amino acid substituents or amino acid functional groups.

The present invention also describes fragments, mutants, homologs and analogs of aresten, canstatin and tombstatin. A “fragment” of aresten, canstatin or tombstatin is an amino acid sequence that is shorter than the aresten, canstatin or tombstatin molecule and comprises at least 25 consecutive amino acids of an aresten, canstatin or tombstatin polypeptide. In addition, these molecules may or may not include additional amino acids derived from the process of cloning, such as amino acid residues or amino acid sequences corresponding to complete or partial linker sequences. In order to be encompassed by the present invention, the mutants with or without the additional amino acid residues must have a biological activity that is substantially the same as the native or full length protein of the reference polypeptide.

One such fragment, designated "Tumstatin N-53", was found to have antiangiogenic activity equivalent to that of full length tumstatin as determined by standard assays. Tumstatin N-53 includes a tumstatin molecule deleted with an N-terminal 53 amino acid. Other mutant fragments described herein have been found to have very high levels of antiangiogenic activity, as indicated by the assays described herein. These fragments "Tumstatin 333", "Tumstatin 334", "12 kDa Aresten Fragment", "8 kDa Aresten Fragment" and "10 kDa Canstatin Fragment" are 75 ng / ml, 20 ng / ml, respectively, ED ₅₀ values of 50 ng / ml, 50 ng / ml and 80 ng / ml. In contrast, the full length aresten, canstatin and tombstatin were found to have ED ₅₀ values of 400 ng / ml, 400 ng / ml and 550 ng / ml, respectively. Tumstatin 333 comprises 2 to 125 amino acids of SEQ ID NO: 10, and tumstatin 334 comprises 126 to 245 amino acids of SEQ ID NO: 10.

A “mutant” of aresten, canstatin or tombstatin means a polypeptide that contains any change in amino acid sequence compared to the amino acid sequence of an equivalent reference aresten, canstatin or tombstatin polypeptide. Such changes may be spontaneously or by human manipulation, by chemical energy (eg, X-rays), or by other forms of chemical mutagenesis, or by genetic manipulation, or by mating genetic information or Can occur as a result of other forms of exchange. Mutations include, for example, base changes, deletions, insertions, inversions, translocations, or duplications. Mutant forms of Aresten, Canstatin or Tombstatin may exhibit increased or decreased antiangiogenic activity compared to equivalent reference Aresten, Canstatin or Tombstatin polynucleotides, and these mutations are also derived from the process of cloning Additional amino acids, such as amino acid residues or amino acid sequences corresponding to a complete or partial linker sequence, may or may not be included.

Mutants / fragments of antiangiogenic proteins of the invention can be produced by PCR cloning. Fragments designated “tumstatin 333” and “tumstatin 334” were generated in this manner, which were superior to the activity of full length tomstatin, as described in Example 18 below and shown in FIGS. 23 and 24. Has angiogenic activity. To prepare the fragments, PCR primers were designed from known sequences in such a way as to amplify known subsequences from the entire protein with each set of primers. These subsequences were then cloned into appropriate expression vectors, such as the pET22b vector, and the expressed protein was tested for its antiangiogenic activity as described in the assay below.

Mutants / fragments of the anti-angiogenic proteins of the invention are also described in Mariyama, M. et al. (1992, J. Biol. Chem. 267: 1253-8) and Example 24 below. As described in, it may be produced by Pseudomonas elastase digestion. This method was used to generate 12 kDa and 8 kDa Arestin mutants and 10 kDa canstatin mutants, all three having higher levels of antiangiogenic activity than the original full length proteins.

“Analogue” of aresten, canstatin or tomstatin means a non-natural molecule that is substantially similar to an aresten, canstatin or tombstatin molecule or fragment or allelic variant thereof, which are substantially the same or have excellent biological activity Have. These analogs are intended to include biologically active derivatives of aresten, canstatin or tombstatin (eg, chemical derivatives as defined above) as well as fragments, mutants, homologues and allelic variants thereof. A promoter or antagonist effect that is qualitatively identical to that of an unmodified aresten, canstatin or tombstatin polypeptide, fragment, mutant, homologue or allelic variant.

"Allele" of aresten, canstatin or tombstatin means a polypeptide sequence comprising a naturally occurring sequence change compared to the polypeptide sequence of a reference aresten, canstatin or tombstatin polypeptide. “Allele” of a polynucleotide encoding an aresten, canstatin or tombstatin polypeptide means a polypeptide comprising a sequence change compared to a reference polynucleotide sequence encoding a reference aresten, canstatin and tombstatin polypeptide, Wherein the allele of a polynucleotide encoding an aresten, canstatin or tombstatin polypeptide encodes an allelic form of an aresten, canstatin or tombstatin polypeptide.

Provided polypeptides are fragments, mutants, analogs or allelic variants of aresten, canstatin or tomstatin, or two or more of these, such as polypeptides are both analogs and mutants of aresten, canstatin or tomstatin polypeptides There is a possibility. For example, shortened proteins (eg, fragments of aresten, canstatin or tombstatin) of aresten, canstatin or tombstatin molecules can be produced in the laboratory. If this fragment is then mutated through means known in the art, the molecule may be produced as both fragments and mutants of aresten, canstatin or tombstatin. In another example, a mutant can be produced, which was later found to exist as an allelic form of aresten, canstatin or tombstatin in some mammalian individuals. Therefore, such mutant aresten, canstatin or tombstatin molecules will be both mutant and allelic variants. Such combinations of fragments, mutants, allelic variants and analogs are intended to be included in the present invention.

For example, the tumstatin produced by the E. coli expression cloning method described in Example 18 below is a monomer. In addition, because the E. coli expression cloning method adds a polylinker sequence and histitin tag to the expressed protein that does not exist in the original protein, the tumstatin is a fusion or chimeric protein. Also the tomstatin fragment "tumstatin N-53" described in Example 18 is a fragment and a deletion mutant of the full length tomstatin protein, which has an additional sequence added thereto when prepared by the same E. coli expression cloning method. Thus, they are also fusions or chimeric mutant fragments of full-length tumstatin proteins. For example, when bound to each other as dimers, trimers, etc., the subunits of this tomstatin N-53 will produce a multimeric fusion of chimeric mutant fragments of the tumstatin protein.

Included by the present invention is a protein having an amino acid sequence substantially identical to Aresten, Canstatin or Toomstatin or a polynucleotide having a nucleic acid sequence substantially identical to a polynucleotide encoding Aresten, Canstatin or Tombstatin . "Substantially identical sequence" means at least about 70% sequence identity, typically at least about 80% sequence identity, preferably at least about 90% sequence identity, more preferably, with a reference sequence, such as another nucleic acid or polypeptide Preferably nucleic acid or polypeptide that exhibits at least 95% identity, most preferably at least about 97% sequence identity with a reference sequence. The comparative length for the sequences is generally at least 75 nucleotide bases or 25 amino acids, more preferably at least 150 nucleotide bases or 50 amino acids, most preferably 243-264 nucleotide bases or 81-88 amino acids would. As used herein, "polypeptide" refers to the molecular chain of amino acids and does not refer to products of a particular length. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. The term is also intended to include polypeptides that have undergone post-expression modification such as, for example, glycosylation, acetylation, phosphorylation, and the like.

As used herein, “sequence identity” refers to subunit sequence similarity between two polymer molecules, such as two polynucleotides or two polypeptides. When the subunit positions of both molecules are filled with the same monomer subunits, for example, if the positions of each of the two peptides are occupied with serine, they are the same at this position. Identity between two sequences is a direct function of the number of identical or identical positions (eg, if half of these positions in the two peptide or compound sequences (eg, five positions in a polymer 10 subunits long) are identical, 2 Dog sequences are 50% identical; If 90% of the positions (eg, 9 out of 10 match) are identical, the two sequences share 90% sequence identity]. By way of example, the amino acid sequences R ₂ R ₅ R ₇ R ₁₀ R ₆ R ₃ and R ₉ R ₈ R ₁ R ₁₀ R ₆ R ₃ have ₃ of 6 positions in common and thus share 50% sequence identity. While the sequences R ₂ R ₅ R ₇ R ₁₀ R ₆ R ₃ and R ₈ R ₁ R ₁₀ R ₆ R ₃ have _three of the five positions in common and thus share 60% sequence identity. Identity between two sequences is a direct function of the number of identical or identical positions. Thus, if a portion of the reference sequence is deleted in a particular peptide, that deleted portion is not included in the sequence identity calculation (eg, R ₂ R ₅ R ₇ R ₁₀ R ₆ R ₃ and R ₂ R ₅ R ₇ R ₁₀ R ₃ has 5 of the 6 positions in common, resulting in 83.3% sequence identity).

Identity is often measured using sequencing software, such as BLASTN or BLASTP (available at http://www.ncbi.nlm.nih.gov/BLAST/). BLASTN (for nucleotide sequences) Comparing two sequences (eg, “Blast” -ing two sequences together (“Blast” -ing), http://www.ncbi.nlm.nih.gov/gorf/bl2). The default parameters for the html are 1 for the match, 1 for the mismatch -2, 5 for the open gap, and 2 for the extension gap. When using BLASTP for protein sequences, the default parameters are zero for match, zero for mismatch, one for open gap and one for extension gap.

When two sequences share "sequence homology", sequence homology means that the two sequences differ from each other only by conservative substitutions. In a polypeptide sequence, such conservative substitutions are those that substitute for another amino acid of the same class (e.g., amino acids that share hydrophobicity, charge, pK or other morphological or chemical properties) with one amino acid at a given position. (Eg, replacing leucine with lysine, lysine with arginine), or one or more non-conservative amino acid substitutions, deletions, or at positions of sequences that do not change the form or folding of the polypeptide to the extent that destroys the biological activity of the polypeptide. It consists of an insert. Examples of “conservative substitutions” include replacing one nonpolar (hydrophobic) residue, such as isoleucine, valine, leucine or methionine, with another nonpolar (hydrophobic) residue; Replacing one polar (hydrophilic) residue with another polar (hydrophilic) residue, such as substitution between arginine and lysine, substitution between glutamine and asparagine, and substitution between threonine and serine; Replacing one basic residue, such as lysine, arginine or histidine, with another basic residue; Replacing one acidic residue, such as aspartamic acid or glutamic acid, with another residue; Or the use of chemically derivatized residues in place of non-derivatized residues, provided that the polypeptide exhibits the necessary biological activity. Two sequences that share sequence homology are sometimes referred to as “sequence homologs”.

Homology for polypeptides is typically sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Is measured using. Protein analysis software matches similar sequences by specifying degrees of homology to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; Valine, isoleucine, leucine; Aspartamic acid, glutamic acid, asparagine, glutamine; Serine, threonine; Lysine, arginine; And phenylalanine, tyrosine.

The present invention also includes chemical derivatives of aresten, canstatin and tombstatin. A “chemical derivative” is a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. For example, these derivatized moieties include molecules in which free amino groups are derivatized to form amine hydrochloride, p-toluene sulfonyl group, carbenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. Included. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histitin can be derivatized to form N-imbenzyl histidine. The invention also includes proteins comprising one or more naturally occurring amino acid derivatives of twenty standard amino acids as chemical derivatives. For example, 4-hydroxyproline can substitute proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine can substitute histidine; Homoserine may substitute serine; Ornithine may substitute for lysine.

The invention also includes fusion proteins and chimeric proteins, including anti-angiogenic proteins, fragments thereof, mutants, homologs, analogs and allelic variants. Fusion proteins or chimeric proteins may be produced as a result of recombinant expression and cloning processes, eg, such proteins may comprise additional amino acids or amino acid sequences corresponding to complete or partial linker sequences (eg, aresten) of the present invention. And, when produced in E. coli (see Example 2 below), includes additional vector sequences added to the protein, including histidine tags. As used herein, the term "fusion protein or chimeric protein" is intended to include such types of changes in the original protein sequence. Similar changes can occur for canstatin and toomstatin proteins (Examples 11 and 18, respectively). The fusion protein or chimeric protein may consist of a multimer of a single protein, such as a repeat of an angiogenic protein, or the fusion protein and chimeric protein may consist of several proteins, such as several antiangiogenic proteins. Fusion proteins or chimeric proteins are combinations of two or more known antiangiogenic proteins (e.g., angiostatin and endostatin, or biologically active fragments of angiostatin and endostatin), or antiangiogenic proteins in combination with targeting agents ( For example, epidermal growth factor (EGF) or RGD peptides with endostatin), or anti-angiogenic proteins that bind immunoglobulin molecules (eg, endostatin and IgG, specifically the removed Fc moiety). Fusion proteins and chimeric proteins may also include antiangiogenic proteins, fragments, mutants, homologues, analogs and allelic variants thereof, and other antiangiogenic proteins such as endostatin, or angiostatin. Other anti-angiogenic proteins include restin and apomigren (see WO 99/29856, teaching about which is incorporated herein by reference); And fragments of endostatin (see WO 99/29855, teaching about which is incorporated herein by reference). The term "fusion protein" or "chimeric protein" as used herein may also include additional components, for example, for delivering a chemotherapeutic agent, wherein the polynucleotide encoding the chemotherapeutic agent refers to an antiangiogenic protein. It is linked to the polynucleotide encoding. Fusion proteins or chimeric proteins may also include multimers, such as dimers or trimers, of anti-angiogenic proteins. Such fusion proteins or chimeric proteins may be linked to one another (eg, chemically linked) through post-translational modifications, or the entire fusion protein may be recombinantly produced.

It is also intended to include multimeric proteins including aresten, canstatin, tombstatin, fragments, mutants, homologs, analogs and allelic variants thereof. "Multimer" means a protein comprising two or more copies of a subunit protein. Subunit proteins are proteins of the invention, such as aresten, which repeats two or more times, or fragments, mutants, homologs, analogs or allelic variants that repeat two or more times, such as toomstatin mutants or fragments, such as tombs. It may be one of statins 333. Such multimers may also be fusion proteins or chimeric proteins. For example, repeated thumstatin mutants may be combined with one or more polylinker sequences and / or anti-angiogenic peptides that may be present in a single copy, or may be repeated in line (eg, a protein may have two or more multimers within the entire protein). It may include).

The invention also relates to compositions comprising at least one isolated polynucleotide (s) encoding aresten, canstatin or toomstatin as well as a vector and host cell comprising said polynucleotide, and aresten, canstatin and tombstatin And methods for making fragments, mutants, homologs, analogs and allelic variants thereof. As used herein, the term "vector" refers to a carrier into which a fragment of nucleic acid can be inserted or cloned, which carrier functions to transport the fragment of nucleic acid into a host cell. Such vectors may also replicate and / or express migrated nucleic acid fragments. Examples of vectors include, for example, nucleic acid molecules derived from plasmids, bacteriophages or mammalian, plant or insect viruses, or nonviral vectors such as ligand-nucleic acid conjugates, liposomes or lipid-nucleic acid complexes. The migrated nucleic acid molecules are preferably operably linked to expression control sequences to form an expression vector capable of expressing the migrated nucleic acid. This transfer of nucleic acid is generally referred to as “transformation,” and transformation refers to the insertion of exogenous polynucleotides into a host cell, regardless of the method used for insertion. For example, direct uptake, transduction or f-mating are included. Exogenous polynucleotides may be maintained as unintegrated vectors, such as plasmids, or integrated into the host genome. “Operably linked” refers to a situation in which a described component is in a relationship that allows them to function in an intended manner, eg, a control sequence “operably linked” to a coding sequence indicates that the expression of the coding sequence is a control sequence. It is connected in a way that can be achieved under conditions that are compatible with. A “coating sequence” is a polynucleotide sequence that is translated into a polypeptide when transcribed into mRNA and placed under control (eg, operably linked) under appropriate regulatory sequences. The boundaries of the coding sequence are determined by the translation start codon at the 5'-end and the translation stop codon at the 3'-end. Such boundaries can be naturally occurring or polynucleotide sequences can be introduced or added by methods known in the art. Coding sequences may include, but are not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

The vector to which the cloned polypeptide is cloned may be selected because of its function in prokaryotes or because of its function in eukaryotes. Two examples of vectors that allow for both cloning of polynucleotides encoding Aresten, canstatin and tomstatin proteins and expression of these proteins from the polynucleotides are pET22b and pET28 (a) vectors [Novagen, Madison, Wisconsin, USA) and modified pPICZαA vectors (InVitrogen, San Diego, California, USA), which allow expression of these proteins in bacteria and yeast, respectively (eg, WO 99 / 29878, the teachings of which are incorporated herein by reference).

Once the polynucleotide has been cloned into a suitable vector, it can be transformed into a suitable host cell. "Host cell" means a cell that can be used or can be used as a recipient cell of a nucleic acid carried by a vector. The host cell may be a prokaryote or eukaryote, mammalian, plant or insect, and may exist as a single cell or as an aggregate, such as a culture or tissue culture, or as a tissue or organism. Host cells can also be derived from multicellular organisms, such as normal tissue or diseased tissue of a mammal. As used herein, host cells include not only the original cell transformed with the nucleic acid, but also cells that still have the nucleic acid as a progeny of such cells.

In one embodiment, the isolated polynucleotide encoding the anti-angiogenic protein further comprises a polynucleotide linker encoding the peptide. Such linkers are known to those skilled in the art, for example the linker may comprise one or more additional codons encoding one or more additional amino acids. Typically the linker contains 1 to 20 or 30 amino acids. Like polynucleotides encoding anti-angiogenic proteins, polynucleotide linkers are also translated, expressed as anti-angiogenic proteins with one or more additional amino acid residues at the amino or carboxyl terminus of the anti-angiogenic protein. Importantly, these additional amino acids or amino acids do not impair the activity of antiangiogenic proteins.

After inserting the selected polynucleotide into the vector, the vector is transformed into an appropriate prokaryotic strain and the strain is cultured (eg maintained) under suitable culture conditions to produce a biologically active antiangiogenic protein, thereby biologically Active antiangiogenic proteins, or mutants, derivatives, fragments or fusion proteins thereof, were generated. In one embodiment, the invention comprises cloning a polynucleotide encoding an antiangiogenic protein into vector pET22b, pET17 / b or pET28a and then transforming it into bacteria. The bacterial host strain then expresses the angiogenic protein. Typically anti-angiogenic proteins are produced in an amount of at least about 10-20 milligrams per liter of culture.

In another aspect of the invention, the eukaryotic cell vector comprises a modified yeast vector. One method is to use a pPICzα plasmid containing multiple cloning sites. The multiple cloning site has a His.Tag motif inserted within the multiple cloning site. In addition, the vector can be modified to add an Nde I site or other suitable restriction sites. These sites are well known to those skilled in the art. Antiangiogenic proteins produced by this embodiment include one or more histidines, typically a Histidine tag motif (His.tag) comprising about 5-20 histidines. The tag should not interfere with the antiangiogenic properties of the protein.

For example, one method of producing arestenen, canstatin or tombstatin amplifies the polynucleotides of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 9, respectively, and expresses them as expression vectors, such as Cloning into pET22b, pET28 (a), pPICZαA or some other expression vector, transforming the vector containing the polynucleotide into a host cell capable of expressing the polypeptide encoded by the polynucleotide, and expressing the protein The transformed host cell is cultured under appropriate culture conditions, and then the protein is extracted and purified from the culture. General antiangiogenic proteins and exemplary methods for producing aresten, canstatin and tombstatin are provided in the Examples below. Aresten, canstatin or tomstatin proteins may also be linked to or associated with products of transgenic animals, such as components of the milk of transgenic cattle, goats, sheep or pigs, or products of transgenic plants (e.g., starch molecules in corn). Can be expressed as

Aresten, canstatin or tombstatin can also be produced by commonly known chemical synthesis methods. Methods for preparing the proteins of the invention by synthetic methods are known to those skilled in the art. Synthesically produced aresten, canstatin or tombstatin protein sequences may, for example, share primary, secondary or tertiary structural and / or morphological features with recombinantly produced aresten, canstatin or tombstatin. Including biological activity, they may have common biological properties with them. Thus, synthetically produced aresten, canstatin or tombstatin protein sequences can be recombinantly generated and purified, for example, in an immunological method for the screening of therapeutic compounds and for the development of antibodies. It can instead be used as a biologically active or immunological substitute.

Aresten, canstatin and tombstatin proteins are useful for inhibiting angiogenesis as determined in standard assays and provided in the Examples below. Aresten, canstatin or tombstatin do not inhibit the growth of other cell types, such as non-endothelial cells.

Polynucleotides encoding Aresten, canstatin or tombstatin can be cloned from an isolated DNA or cDNA library. As used herein, an “isolated” nucleic acid polypeptide is essentially free of (eg, isolated from, such a substance of a biological source that makes it possible to obtain them, such as a mixture of nucleic acids or a substance present in a cell). ) Nucleic acids or polypeptides, which can be further processed. An “isolated” nucleic acid or polypeptide is described herein, including essentially a nucleic acid or polypeptide, nucleic acid or polypeptide produced by a combination of chemical synthesis, chemical or biological methods, and isolated recombinantly generated nucleic acid or polypeptide. Nucleic acids or polypeptides obtained by methods of, by analogous methods, or other suitable methods. Thus, isolated polypeptide is meant to be relatively free of other proteins, carbohydrates, lipids and other cellular components that are normally associated. In the naturally occurring genome of the organism from which the nucleic acid is derived, the nucleic acid is in direct contact (ie covalently) with both nucleic acids, but the isolated nucleic acid is not in direct contact with both nucleic acids. Thus, the term refers to a molecule isolated independently from other nucleic acids, such as, for example, nucleic acids (eg, self-replicating viruses or plasmids) integrated in a vector or nucleic acid fragments produced by chemical means or restriction enzyme treatment. Nucleic acids present are included.

The polynucleotides and proteins of the invention can also be used to design probes for isolating other antiangiogenic proteins. Excellent methods are provided in US Pat. No. 5,837,490, described by Jacobs et al., The entire teachings of which are hereby incorporated by reference in their entirety. The design of the oligonucleotide probe should preferably follow the following parameters: (a) design with a region of the sequence having the least amount, even if it has an unknown base ("N"), and (b) 2 for each of A or T Assuming 4 ° C for each of ° C, G or C), the design should be such that T _m is about 80 ° C.

It is preferred to label the oligonucleotides using g- ³² P-ATP (specific activity 6000 Ci / mmole) and T4 polynucleotide kinase using techniques commonly used to label oligonucleotides. Other labeling techniques can also be used. It is desirable to remove unincorporated labels by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantified by measuring with a scintillation counter. Preferably, the specific activity of the probes obtained should be about 4 × 10 ⁶ dpm / pmole. It is preferred to dissolve bacterial cultures containing a mixture of full length clones and inoculate a sterile culture flask containing 25 ml of sterile L-culture solution containing ampicillin at 100 µg per ml using 100 µl of this preservation culture. . It is desirable to grow this culture to saturate at 37 ° C. and to dilute the saturated culture in fresh L-culture. Aliquots of these dilutions were plated and grown overnight at 37 ° C. on solid bacterial medium containing 100 μg Ampicillin per ml in 150 mm petri dish and 1.5% agar. The dilution and volume were determined to yield about 5000 clearly separated colonies. Also other known methods of producing clearly separated colonies can be used.

The colonies should then be transferred to nitrocellulose filters using standard colony hybridization methods and dissolved, denatured and baked. For example, highly stringent conditions are those using 1 × SSC at least 65 ° C. or 1 × SSC at 50 ° C. and 50% formamide. Moderate stringent conditions are those using 4 × SSC at least 65 ° C. or 4 × SSC and 50% formamide at 42 ° C. Mild stringent conditions are those using 4x SSC at least 50 ° C or 6x SSC and 50% formamide at 40 ° C.

The filter was then placed into a 6-fold SSC containing 0.5% SDS, 100 μg / ml yeast RNA and 10 mM EDTA (about 10 mL per 150 mm filter) (20 × stock reagents were 173.3 g NaCl / liter, 88.2 g Na citrate). / Liter, adjusted to pH 7.0 with NaOH), incubating at 65 ° C. for 1 hour with gentle stirring. The probe is then added to the hybridization mixture, preferably at a concentration of at least 1 × 10 ⁶ dpm / mL. It is then preferred to incubate overnight at 65 ° C. with gentle stirring. It is then preferred to wash in 500 ml of 2 × SSC / 0.5% SDS at room temperature without stirring, followed by washing in 500 mL of 2 × SSC / 0.1% SDS for 15 minutes at room temperature with gentle stirring. Washing with 0.1 × SSC / 0.5% SDS at 65 ° C. for 30 minutes to 1 hour is optional. The filter is then dried and autoradiography performed for a sufficient time to visualize the positive on the X-ray film. Other known hybridization methods can also be used. Positive colonies are then taken, grown in culture, and plasmid DNA is isolated using standard methods. The clones can then be identified by restriction analysis, hybridization analysis or DNA sequencing.

The present invention includes methods for inhibiting angiogenesis in mammalian tissue using aresten, canstatin, tombstatin or biologically active fragments, analogs, homologs, derivatives or mutants thereof. In particular, the present invention utilizes at least one antiangiogenic protein, at least one biologically active fragment thereof, a combination of fragments having antiangiogenic activity or an effective amount of an accelerator and an antagonist to treat an angiogenic-mediated disease. It includes a method. An effective amount of the anti-angiogenic protein is an amount sufficient to inhibit angiogenesis resulting in the disease or condition and thus alleviate the disease or condition completely or partially. Mitigation of angiogenesis-mediated disease may be determined by mitigating the manifestation of the disease, eg, by reducing tumor size or inhibiting tumor growth. Also as used herein, the term “effective amount” refers to an advantage that is meaningful to a patient, that is, an increase in the rate of treatment, cure, prevention or improvement of a related medical disease, or treatment, cure, prevention or improvement of such a disease. A total amount of each active ingredient of the composition or method sufficient to represent. When applied to a combination, the term refers to the combined amount of the active ingredient that produces a therapeutic effect, whether administered continuously or in concert together. Angiogenesis-mediated diseases include cancer, solid tumors, blood-generating tumors (eg, leukemia), tumor metastases, benign tumors (eg, hemangiomas, neurofibromas, trachoma and purulent granulomas), rheumatoid arthritis, psoriasis, Ocular angiogenic diseases (eg diabetic retinopathy, prematurity retinopathy, macular degeneration, corneal graft rejection, neovascular glaucoma, posterior capsular fibrosis, melanoma), Osler-Weber syndrome, myocardial angiogenesis, plaque angiogenesis, capillary dilator , Hemophilia arthrosis, vascular fibrosis and trauma granulation. Antiangiogenic proteins are useful for treating diseases that excessively or abnormally stimulate endothelial cells. Such diseases include, but are not limited to, intestinal adhesions, Crohn's disease, arteriosclerosis, scleroderma, and hypertrophic reactions (ie, keloids). Antiangiogenic proteins can be used as birth control agents by preventing the angiogenesis necessary for embryo implantation. Antiangiogenic proteins are useful for treating diseases with angiogenesis as pathological consequences such as Cat scratch disease ( Rochele minalia quintosa ) and ulcers ( Heliobacter pylori ). . Antiangiogenic proteins may also be used to prevent dialysis graft vascular pathway stenosis and obesity, for example by inhibiting capillary formation in adipose tissue and preventing the expansion of capillaries. Antiangiogenic proteins can also be used to treat local (eg, non-metastatic) disease. "Cancer" refers to a pathological condition of neoplastic growth, hyperplastic or proliferative growth or abnormal cell development and includes solid tumors, non-solid tumors and any abnormal cell proliferation as seen in leukemia. As also used herein, "cancer" refers to angiogenesis-dependent cancers and tumors, ie, the number and density of blood vessels that supply them with blood for their growth (expansion in volume and / or mass). It means a tumor requiring an increase of. "Regression" refers to a reduction in tumor mass and size as determined using methods well known to those skilled in the art.

In addition, where an increase in angiogenesis is desired, such as wound healing or post-infarct heart tissue, antibodies to antiangiogenic proteins or antiserum, interfere with local, native antiangiogenic proteins and processes, thereby forming new blood vessels. Can be used to inhibit tissue degeneration.

Antiangiogenic proteins can be used with other compositions and methods for the treatment of diseases. For example, tumors are typically treated using surgery, radiation, chemotherapy or immunotherapy with antiangiogenic proteins, followed by administration of antiangiogenic proteins to the patient to prolong the dormant phase of micrometastasis and the remaining primary tumors. Can be stabilized and stabilized. Antiangiogenic proteins, or fragments thereof, antisera, receptor promoters or receptor antagonists, or combinations thereof, may be other antiangiogenic compounds, or proteins, fragments, antisera, other antiangiogenic proteins (eg, angiostatin, endostatin). ) Receptor promoter, receptor antagonist. Additionally, antiangiogenic proteins or fragments thereof, antisera, receptor promoters, receptor antagonists or combinations thereof combine with optionally sustained release matrices such as pharmaceutically acceptable excipients and biodegradable polymers to form a therapeutic composition. The compositions of the present invention may also contain other antiangiogenic proteins or chemical compounds such as endostatin or angiostatin and mutants, fragments and analogs thereof. The compositions may further contain other agents for use in therapy, such as drugs that enhance or highlight the activity of the protein or are chemotherapy or radioactive agents. Such additional factors and / or agents may be included in the composition to produce synergistic effects or minimize side effects with the proteins of the invention. In addition, administration of the compositions of the invention may be combined with other therapies, eg, in combination with chemotherapy or radiotherapy.

The present invention includes a method for inhibiting angiogenesis in mammalian tissue by contacting tissue with a composition containing a protein of the invention. "Contact" means a mode of transport that introduces the composition into a tissue or tissue of a tissue as well as topical application.

The invention also encompasses the use of timed or sustained release shear systems. Such a system is highly desirable in situations where surgery is difficult or impossible, such as when a patient is weakened by age or course of illness, or when a risk-benefit analysis determines that adjustment is needed over treatment.

As used herein, a sustained-release matrix is a matrix composed of materials that are commonly polymers, which can be degraded by enzymatic or acid / base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted on by enzymes and body fluids. Sustained release matrices include liposomes, polylactide (polylactic acid), polyglycolide (polymers of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides ), Poly (ortho) esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinyl propylene It is preferably selected from biocompatible materials such as vinylpyrrolidone and silicone. Preferred biodegradable matrices are those of either polylactide, polyglycolide or polylactide coglycolide (copolymers of lactic acid and glycolic acid).

Angiogenesis control compositions of the invention may be solids, liquids or aerosols and may be administered via any known route of administration. Examples of solid compositions include pills, creams, and injectable dosage units. Pills can be administered orally and therapeutic creams can be administered topically. Injectable dosage units may be administered locally, such as at a tumor site, or injected, eg subcutaneously, for systemic release of the angiogenesis-modulating composition. Examples of liquid compositions include formulations suitable for subcutaneous, intravenous, intraarterial injection and formulations for topical and intraocular administration. Aerosol formulations include inhalation formulations for administration to the lungs.

The proteins and protein fragments having the antiangiogenic activity described above can be provided as substantially purified proteins and protein fragments isolated in pharmaceutically acceptable formulations using formulation methods known to those skilled in the art. . Generally, the compositions are topical, percutaneous, intraperitoneal, intracranial, intraventricular, cerebral, vaginal, intrauterine, oral, rectal or parenteral (eg, intravenous, spinal, subcutaneous or muscular). Intra) route. In addition, anti-angiogenic proteins may be incorporated into biodegradable polymers to enable sustained release of the compound, which may be injected or injected near a location where drug delivery is desired, such as the location of a tumor The forming protein is slowly released systemically. Osmotic minipumps can also be used to control the transport of high concentrations of anti-angiogenic proteins via cannula within a site of direct metastatic growth or at a location of interest such as within a blood vessel supplied to the tumor. Biodegradable polymers and their use are described in detail in, eg, Brem et al. (1991, J. Neurosurg. 74: 441-446), the entirety of which is incorporated herein.

Compositions containing a polypeptide of the invention can be administered intravenously, such as by unit dose injection. The term “unit dose” when used as a reference to a therapeutic composition herein refers to the physical separation of appropriate units as unit doses for the subject, each unit being associated with the required diluent, ie, carrier or vehicle. It contains a predetermined amount of active substance calculated to produce the desired therapeutic effect.

Modes of administration of the compositions of the present invention include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarterial injections and infusions. Pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or sterile non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile water or dispersion injectable immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyisols (eg glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (eg olive oil) And injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of spraying and by the use of surfactants. The compositions may also include adjuvant such as preservatives, wetting agents, emulsifiers and sprays. Prevention of the action of microorganisms can be ensured by the inclusion of various antibiotics and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and the like. It is also preferable to contain isotonic agents, such as sugars, sodium chloride and the like. Prolonged absorption of injectable pharmaceutical forms is possible by containing agents such as aluminum monostearate and gelatin that delay absorption. Injectable depot forms are prepared by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides). Depending on the ratio of drug to polymer and the nature of the particular polymer used, the ratio of drug release can be controlled. Depot depot formulations may also be prepared by placing the drug in liposomes or microemulsions that are compatible with body tissue. Injectable formulations may be sterilized, for example, by incorporation of a sterilizing agent in the form of a sterile solid composition that can be dissolved or sprayed in sterile water or other sterile injectable media immediately by use or by filtration through a bacterial-residual filter. .

Therapeutic compositions of the invention may include pharmaceutically acceptable salts of the components in the composition, such as salts that can be derived from inorganic or organic acids. “Pharmaceutically acceptable salts” are within the scope of normal medical judgment and are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc. It means a salt forming. Pharmaceutically acceptable salts are well known in the art. For example, S. M. SM Berge et al., J. Pharmaceutical Sciences (1997) 66: 1 et seq ., Describe in detail pharmaceutically acceptable salts, which are incorporated herein by reference. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, tartaric acid, mandelic acid and the like. Salts formed with free carboxyl groups also derive from, for example, inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Can be. The salts can be prepared by reacting the free base with a suitable organic acid in situ or separately during the final isolation and purification of the compounds of the invention. Representative acid addition salts include acetates, adipates, alginates, citrate, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, camphorsulfonates, digluconates, glycerophosphates, hemisulfates, Heptonoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxymethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2 Naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, phosphate, glutamate, bicarbonate , but not limited to p-toluenesulfonate and undecanoate . Basic nitrogen-containing groups also include methyl, ethyl, propyl and lower alkyl halides such as butyl chloride, bromide, iodide; Dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; Long chain halides such as decyl, lauryl, myristyl and stearyl chloride, bromide and iodide; It may be quaternized with agents such as arylalkyl halides such as benzyl and phenethyl bromide. This gives a water soluble, oil soluble or dispersible product. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid.

As used herein, when referring to compositions, carriers, diluents and reagents, the terms "pharmaceutically acceptable", "physiologically acceptable" and their grammatical variations are used interchangeably and include nausea, dizziness, vomiting, and the like. Such substances can be administered to a mammal while minimizing such undesirable physiological effects. The preparation of pharmacological compositions containing the active ingredient dissolved or dispersed is well understood in the art and need not be limited based on the formulation. Typically the compositions are prepared as injections, such as solutions or suspensions, but may also be prepared in solid form in a liquid suitable for solution or suspension before use. The preparation may also be emulsified.

The active ingredient may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient in an amount suitable for use in the methods of treatment described herein. For example, suitable excipients contain water, saline, dextrose, glycerol or ethanol and the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, and the like, which enhance the effectiveness of the active ingredient.

In addition, the anti-angiogenic proteins of the present invention can be included in compositions containing prodrugs. As used herein, the term “prodrug” refers to a compound that is rapidly transformed in vivo to produce the parent compound, eg, by enzymatic hydrolysis in blood. A full commentary is available in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems , Vol. 14 of the ACS Symposium Series and Edward B. Roche, ed., Bioreversible Carriers in Drug Design , American Pharmaceutical Association and Permagon Press , 1987), both of which are incorporated herein by reference. As used herein, the term "pharmaceutically acceptable prodrug" (1) is used for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the scope of normal medical judgment Prodrugs of the compounds of the invention and (2) amphoteric ionic forms of the parent compounds, if possible, which are suitable for their intended use and which are balanced against the ratio of the advantages to suitable risks.

The dosage of the anti-angiogenic protein of the invention will depend on the route of administration of the compound and other clinical factors such as the condition or situation of the disease being treated and the weight and condition of the human or animal. To treat a human or animal, about 10 mg to about 20 mg of protein per kg of body weight may be administered. In combination with the therapies, for example, in combination with the proteins of the invention and radiotherapy, chemotherapy or immunotherapy, the dosage can be reduced, for example, from 0.1 mg to about 0.2 mg per kg body weight. Depending on the half-life of anti-angiogenic proteins in certain animals or humans, anti-angiogenic proteins may be administered several times per day once a week. It should be understood that the present invention is applied for both human and veterinary use. The method of the present invention contemplates multiple administrations alone as well as over extended periods of time. Additionally, antiangiogenic proteins can be administered in conjunction with other forms of therapy, such as chemotherapy, radiotherapy or immunotherapy.

Antiangiogenic protein formulations include oral, rectal, eye (including intravitreal or anterior), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (subcutaneous, abdominal) Formulations suitable for intramuscular, intramuscular, intravenous, intracranial, intratracheal and epidural) administration. Antiangiogenic protein formulations may conveniently be presented in unit dose form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of combining the active ingredient with the pharmaceutical carrier (s) or excipient (s). Generally, formulations are prepared by uniformly and directly combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then molding the product, if necessary.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents and solvents that render the formulation isotonic with the blood of the intended subject; And aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickeners. The formulations may be presented in unit doses or in multi-dose containers, such as sealed ampoules and vials, and may be stored in lyophilized condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Instant injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type described above.

When an effective amount of a protein of the invention is administered orally, the anti-angiogenic proteins of the invention will be in the form of tablets, capsules, powders, solutions or elixirs. When administered in tablet form, the pharmaceutical compositions of the present invention may further contain a solid carrier such as gelatin or adjuvant. Tablets, capsules and powders contain about 5 to 95%, preferably 25 to 90%, of the protein of the present invention. When administered in liquid form, liquid carriers such as synthetic oils and oils derived from animals or plants such as water, petroleum, peanut oil, mineral oil, soybean oil or sesame oil may be added. The liquid form of the pharmaceutical composition may further contain physiological saline, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight, preferably about 1 to 50% by weight of the protein of the present invention.

When an effective amount of a protein of the invention is administered by intravenous, dermal or subcutaneous injection, the protein of the invention will be in the form of a parenterally acceptable aqueous solution, excluding pyrogen. The preparation of such parenterally acceptable protein solutions suitable for pH, isotonicity, stability and the like is known in the art. Preferred pharmaceutical compositions for intravenous, dermal or subcutaneous injections are in addition to isotonic vehicles such as injections of sodium chloride, Ringer's injection, dextrose injection, dextrose and sodium chloride, injection of lactate linkers in addition to the proteins of the present invention. It should contain other vehicles known in the art. The pharmaceutical compositions of the present invention may also contain stabilizers, preservatives, buffers, antioxidants or additives known to those skilled in the art. The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend on the nature and severity of the disease being treated and the nature of the previous treatment received by the patient. Ultimately, the attending physician will determine the amount of protein of the invention for treating each individual patient. Initially, the attending physician will administer a small amount of the protein of the invention and observe the patient's response. Patients may be administered higher amounts of the protein of the invention until the optimal therapeutic effect is achieved, at which point no further increase in dosage.

The duration of intravenous treatment using the pharmaceutical compositions of the invention will depend on the severity of the disease being treated and the condition and potential specificity of each individual patient. The duration of each application of the protein of the invention will range from 12 to 24 hours of continuous intravenous administration. Ultimately, the attending physician will determine the appropriate duration of intravenous treatment using the pharmaceutical composition of the present invention.

Preferred unit dose formulations contain a daily dose or unit, daily subdosage, or an appropriate subdivision thereof of the administered components. In addition to the components mentioned above in detail, the formulations of the present invention may contain other agents conventional in the art having a type of the formulation. Optionally, cytotoxic agents may be incorporated or combined with antiangiogenic proteins or biologically functional protein fragments thereof to provide dual treatment to the patient.

Therapeutic compositions are also useful for veterinary applications at present. In particular human as well as livestock and hematopoietic horses are preferred patients for this treatment using the proteins of the invention.

Cytotoxic agents, such as lysine, can provide a tool for destroying cells that bind anti-angiogenic proteins by binding to anti-angiogenic proteins and fragments thereof. These cells can be found in many locations including, but not limited to, micrometastases and primary tumors. Proteins bound to cytotoxic agents are injected in a manner designed to maximize delivery to the desired location. For example, lysine-bound high affinity fragments are delivered via cannula into the target or directly into the blood vessel providing the target site. The medicament is also delivered in a controlled manner via an osmotic pump connected to the infusion cannula. Binding of the antagonist to the anti-angiogenic protein may increase the angiogenesis of the tissue by co-application with an angiogenic stimulant. This treatment regime provides an effective means of destroying metastatic cancer.

Additional methods of treatment include antiangiogenic proteins, fragments, analogs, antiserum or receptor promoters and antagonists bound to cytotoxic agents. It should be understood that the source of antiangiogenic proteins may be human or animal. Antiangiogenic proteins can also be produced synthetically by chemical reactions or by recombinant techniques associated with expression systems. Antiangiogenic proteins can also be produced by enzymatic cleavage of isolated Type IV collagen to produce proteins with antiangiogenic activity. Antiangiogenic proteins may also be produced by compounds that mimic the action of endogenous enzymes that cleave type IV collagen against antiangiogenic proteins. The production of antiangiogenic proteins may also be modified by compounds that affect the activity of the cleavage enzyme.

The present invention also encompasses gene therapy wherein polynucleotides encoding antiangiogenic proteins or mutants, fragments or fusion proteins thereof are introduced into and controlled by a patient. Various methods of transferring or transporting DNA to cells for expressing gene product proteins, or methods referred to as gene therapy, are described in Gene Transfer into Mammalian Somatic Cells in vivo , N. Yang (1992) Crit. Rev. Biotechn . 12 (4): 335-356, which is incorporated herein by reference. Gene therapy involves the incorporation of DNA sequences into somatic or germ cells for use in vitro or in vivo therapy. Gene therapy serves to replace genes, increase normal or abnormal gene function, and fight infectious diseases and other pathologies.

Strategies for treating these medical problems using gene therapy include therapeutic strategies such as identifying defective genes and then adding functional genes to replace defective genes or to augment weakly functioning genes; Or preventive strategies that include the addition of genes for the production protein to treat the disease or to allow tissues or organs to better accept the treatment regime. By way of example of a prevention strategy, the occurrence of angiogenesis may be prevented by placing in the patient a gene encoding one or more antiangiogenic proteins; A gene is inserted that makes tumor cells more sensitive to radiation and then the tumor is examined to increase the death of tumor cells.

Many protocols for the implantation of DNA or regulatory sequences of antiangiogenic proteins are envisioned in the present invention. In addition, transfection of promoter sequences or other sequences other than normally found promoter sequences specifically related to anti-angiogenic proteins that increase production of anti-angiogenic proteins is planned as a method of gene therapy. Examples of such techniques are found in techniques using homologous recombination techniques (see Transkaryotic Therapies, Inc., of Cambridge, Mass.) To insert "gene switches" that drive erythropoietin in cells (Genetic Engineering News, Apr. 15, 1994). The "gene switch" can be used to activate anti-angiogenic proteins (or their receptors) in cells that normally do not express the protein (or receptors).

Gene transfer methods for gene therapy fall into three broad categories: physical (eg, electroporation, direct gene transfer and particle bombardment), chemical (eg, lipid based carriers or other non-viral vectors) and biological ( Virus-derived vectors and receptor uptake). For example, nonviral vectors comprising liposomes coated with DNA can be used. The liposome / DNA complex can be injected directly into the patient intravenously. The liposome / DNA complex is concentrated in the liver where it transports DNA to macrophages and Kupffer cells. Additionally, the vector or "naked" DNA of the gene can be injected directly into organs, tissues or tumors that are desirable for targeted delivery of therapeutic DNA.

Gene therapy methodologies can also be described by delivery sites. Basic methods for delivering genes include ex vivo gene transplantation, in vivo gene transplantation, and in vitro gene transplantation. In ex vivo gene transplantation, cells are taken from a patient and grown in cell culture. The DNA is transfected into cells and the transfected cells are inflated numerically and then reinjected into the patient. In in vitro gene transplantation, transformed cells are not specific cells from a particular patient, but are cells growing in culture, such as tissue culture cells. Such “lab cells” are transfected and the transfected cells are selected and expanded to be injected into patients or used for other purposes.

In vivo gene transplantation involves introducing DNA into a patient's cells when the cells are present in the patient. The method includes using a virus mediated gene transplant using an uninfected virus to deliver the gene to the patient or inject naked DNA into the patient's site, wherein the DNA is dependent on the percentage of cells in which the gene producing protein is expressed. Taken by. Additionally, other methods described herein, such as the use of a "gene gun," can be used to insert DNA or regulatory sequences that control the production of antiangiogenic proteins in vitro.

Chemical methods of gene therapy involve lipid-based compounds (not necessarily liposomes), which allow DNA to move through cell membranes. Lipofectin or cytofectin, cations based on lipids that bind to negatively charged DNA, form complexes that can pass DNA into the cell through the cell membrane. Another chemical method utilizes receptor-based endocytosis, which involves binding specific ligands to cell surface receptors and surrounding them and moving across the cell membrane. The ligand binds to the DNA and transfers the entire complex into the cell. The ligand gene complex will be injected into the bloodstream and then the target cell with the receptor will specifically bind to the ligand and move the ligand-DNA complex into the cell.

Many gene therapy methodologies use viral vectors to insert genes into cells. For example, modified retroviral vectors have been used in ex vivo methods for introducing genes into and around lymphocytes, hepatocytes, epithelial cells, myocytes or other somatic cells in which the tumor penetrates into and around the tumor. The modified cells are then introduced into the patient to provide a genetic product from the inserted DNA.

Viral vectors are also used to insert genes into cells using in vivo protocols. For tissue specific expression of foreign genes, cis-acting regulatory elements known as tissue specific can be used. This can also be accomplished by using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo. For example, gene transfer into blood vessels in vivo has been achieved by implanting transduced endothelial cells in selected sites in the arterial wall. The virus infects surrounding cells that express the gene product. Viral vectors can be delivered directly to a site in vivo, eg, via a catheter, allowing only certain sites to be infected by the virus to provide long term site specific gene expression. In vivo gene transfer using retroviral vectors has also been demonstrated in mammalian and liver tissues by injecting modified viruses into blood vessels to reach organs.

Viral vectors used for gene therapy protocols include retroviruses, polioviruses or other RNA viruses such as Sindbisvirus, adenoviruses, adeno-associated viruses, herpes virus, SV 40, vaccinia and other DNA viruses. It is not limited to this. Replication Defective murine retroviral vectors are the most widely used gene transfer vectors. The murine leukemia retrovirus consists of single-stranded RNA complexed with nuclear core proteins and polymerases (pol), surrounded by glycoprotein envelopes that are enclosed by protein cores and determine host range. have. The genome structure of retroviruses includes gag , pol and env surrounded by 5 'and 3' long terminal repeats (LTRs). Retroviral vector systems allow for minimal packaging, including 5 'and 3' LTRs and packaging signals, to allow integration into vector packaging, infection and target cells when viral structural proteins are supplied to the packaging cell line in a trans manner. Use the fact that it is enough. Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, integration of sophisticated single copy vectors into target cell chromosomal DNA, and ease of manipulation of retroviral genomes.

Adenoviruses consist of a linear double stranded DNA complexed with a core protein and a capsid protein surrounding it. The development of molecular virology has enabled the biology of these organisms to be used to produce vectors capable of transducing new gene sequences into target cells in vivo. Vectors based on adenoviruses will express high levels of genes produced. Adenovirus vectors have low virus titer but high infectivity. In addition, since the virus is sufficiently infectious as acellular virions, no injection of producer cell lines is necessary. Another potential advantage of adenovirus vectors is the long-term expression of heterologous genes in vivo.

Mechanical methods of transporting DNA include fusogenic lipid vehicles such as liposomes or other vehicles for membrane fusion, lipid particles of DNA incorporating cationic lipids such as lipofectin, polylysine-mediated transplantation of DNA, germ cells or Direct injection of DNA, such as microinjection of DNA into somatic cells, delivery using compressed air of DNA coded particles, such as gold particles used in "gene guns", and inorganic chemical approaches such as calcium phosphate transfection. Particle-mediated gene transplantation methods were first used to transform plant tissue. In particle bombardment devices or "gene guns", power was generated to accelerate DNA-coated dense particles (eg, gold or tungsten) at high speeds that could penetrate target organs, tissues, or cells. Particle bombardment can be used in in vitro systems or with in vitro or in vivo techniques to introduce DNA into cells, tissues or organs. Another method, ligand-mediated gene therapy, involves the complexation of specific ligands and DNA, whereby specific cells or tissues and DNA directly form ligand-DNA conjugates.

Injection of plasmid DNA into muscle cells has been found to increase the percentage of cells that are transfected to sustain expression of the marker gene. The DNA of the plasmid may or may not be integrated in the genome of the cell. Expression and transfection of gene-producing proteins in non-proliferative tissue differentiated at the end of prolonged periods without fear of mutant insertion, deletion or change in unintegrated cell genome or mitochondrial genome of transfected DNA Will make it possible. Although not necessarily permanent, long-term transplantation of therapeutic genes into specific cells can provide treatment for genetic disease or prophylactic use. The DNA may be periodically reinoculated to maintain the level of the gene product without mutations occurring in the genome of the recipient cell. Several other exogenous DNA constructs in a single, unintegrated cell of exogenous DNA and constructs that express several gene products can coexist.

Electroporation for gene transplantation uses electrical current to make cells or tissues suitable for electroporation-mediated gene transplantation. Short electrical shocks using a given field strength are used to increase the permeability of the membrane in a way that can penetrate DNA molecules into cells. This technique can be used in vitro systems or in conjunction with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.

In vivo, carrier mediated gene transfer can be used to transfect foreign DNA into cells. The carrier-DNA complex can typically be introduced into the liquor or blood and then site-specifically treated with the target organ or tissue in the body. Both liposomes and polycations such as polylysine, lipofectin or cytofectin can be used. Liposomes can be developed to be cell specific or organ specific so that foreign DNA carried by liposomes will be taken by the target cell. Injection of immunoliposomes targeted to specific receptors on a particular cell can be used as a convenient way to insert DNA into cells bearing the receptor. Another carrier system in use is an asialo glycoprotein / polylysine conjugate system for delivering DNA to hepatocytes for in vivo gene transplantation.

Transfected DNA can also be present in the cytoplasm or in the nucleus by complexing with other types of carriers and then transporting the DNA to recipient cells. DNA can be transported directly into the nucleus by coupling with a carrier nuclear protein in a specially engineered vehicle complex.

Gene regulation of antiangiogenic proteins is performed by administering a gene that encodes one of the antiangiogenic proteins, a control region associated with the gene, or a compound that binds to its corresponding RNA transcript to alter the rate of transcription or translation. Can be. In addition, cells transfected with a DNA sequence encoding an angiogenic protein can be administered to a patient to provide an in vivo source of said proteins. For example, the cells can be transfected with a vector comprising a nucleic acid sequence encoding an anti-angiogenic protein. Transfected cells can be normal tissue of the patient, cells derived from the diseased tissue of the patient, or non-patient cells.

For example, tumor cells removed from a patient can be transfected using a vector capable of expressing a protein of the invention and reintroduced into the patient. Transfected tumor cells produce proteins at levels that inhibit tumor growth in the patient's body. The patients can be human or non-human animals. Cells can also be transfected by physical or chemical methods known in the art, such as using no vector or by means of electroporation, ionophoresis, or “gene guns”. The DNA may also be injected directly into the patient, without the aid of a carrier. Specifically, the DNA can be injected into the skin, muscle or blood.

Gene therapy protocols for transfecting anti-angiogenic proteins into a patient's body may be performed by integrating the anti-angiogenic protein DNA into the genome of cells, into minichormosomes, or by isolated replication in the cytoplasm or nucleus of cells It can be set as a non-replicating DNA construct. Expression of antiangiogenic proteins may continue for a long time or may be periodically reinoculated to maintain the desired level of protein (s) at the cell, tissue or organ or at a determined blood level.

In addition, the present invention includes antibodies and antisera, which may be used to test new antiangiogenic proteins and are used for the diagnosis, prognosis of diseases or disorders associated with or characterized by antiangiogenic activity or lack thereof. (Iii) or may be used for treatment. The antibodies and antisera may also be used to upregulate angiogenesis in preferred sites, such as post-infarct heart tissue, and antibodies or antisera to proteins of the invention may be directed to localized native antiangiogenic proteins and processes. It can be used to interfere with and increase the formation of new blood vessels to inhibit degeneration of heart tissue.

The antibody and antiserum may be combined with pharmaceutically acceptable compositions to form a diagnostic, prognostic or therapeutic composition. The term “antibody” or “antibody molecule” refers to an immunoglobulin molecule and / or an immunologically active portion of an immunoglobulin molecule, ie, a cluster of molecules containing an antibody binding site or paratope.

Passive antibody therapy using antibodies that specifically bind antiangiogenic proteins can be used to modulate angiogenesis dependent processes such as reproductive, developmental and wound healing and tissue treatment. In addition, direct administration of antisera to the Fab region of an agent of an antiangiogenic protein may interfere with the ability of endogenous antisera to proteins that bind to the protein.

The antiangiogenic proteins of the invention can also be used to generate antibodies specific for inhibitor (s) and receptor (s). The antibodies can be polyclonal antibodies or monoclonal antibodies. Antibodies that specifically bind antiangiogenic proteins or their receptors may be used in diagnostic methods and kits well known to those of skill in the art to detect and quantify antiangiogenic proteins or their receptors in body fluids or tissues. have. The results from the test can be used to diagnose or predict the occurrence or recurrence of cancer and other angiogenesis mediated diseases.

The present invention also encompasses the use of compositions containing the proteins and / or antibodies thereof in the diagnosis or prognosis of anti-angiogenic proteins, antibodies to the proteins and diseases characterized by angiogenic activity. As used herein, the term "prognostic method" means a method that allows for the prediction of the progression of the disease, in particular human or animal diagnosed as having angiogenic dependent disease. As used herein, the term "diagnostic method" means a method that enables the determination of the presence and type of angiogenic dependent disease in humans or animals.

The antiangiogenic proteins can be used in diagnostic methods and kits for detecting and quantifying antibodies capable of binding to the protein. The kit will allow detection of circulating antibodies against antiangiogenic proteins that exhibit a distribution of micrometastasis in the presence of antiangiogenic proteins secreted by the primary tumor in situ. Patients with the circulating antiprotein antibodies are likely to develop multiple tumors and cancers, and are likely to recur after treatment or palliation. Fab fragments of the antiprotein antibodies can be used as antigens to generate antiprotein Fab fragment antisera that can be used to neutralize antiprotein antibodies. This method will effectively raise the level of circulation of anti-angiogenic proteins by reducing the elimination of circulating proteins by antiprotein antibodies.

The invention also encompasses the isolation of receptors specific for antiangiogenic proteins. Protein fragments with high affinity binding to tissue can be used to isolate receptors of anti-angiogenic proteins on an affinity column. Isolation and purification of the receptor (s) is a fundamental step to elucidate the mechanism of action of antiangiogenic proteins. Isolation of receptors and identification of promoters and antagonists will facilitate the development of drugs to modulate the activity of these receptors, which is the final pathway for biological activity. Isolation of the receptor allows for the preparation of nucleotide probes so that in situ and solution hybridization techniques can be used to monitor the location and synthesis of the receptor. In addition, the genes for these receptors are isolated, integrated into expression vectors and transfected into cells, such as patient tumor cells, to increase the ability and locality of cell types, tissues or tumors to bind antiangiogenic proteins. Can inhibit angiogenesis.

Antiangiogenic proteins are used to develop an affinity column for the isolation of receptor (s) for antiangiogenic proteins from cultured tumor cells. Isolation and purification of the receptor is followed by amino acid sequencing. This information can be used to identify and isolate the gene or genes encoding the receptor. Next, the cloned nucleic acid sequence is developed for insertion into a vector capable of expressing the receptor. Such methods are well known to those skilled in the art. Transfection of the nucleic acid sequence (s) encoding said receptor into tumor cells and expression of said receptor by transfected tumor cells enhance metastatic growth by enhancing the reactivity of said cells to endogenous or exogenous antiangiogenic proteins. Decreases the speed.

Antiangiogenic proteins of the present invention can be synthesized in standard microchemical equipment and purity can be tested using HPLC and mass spectrophotometry. Methods of protein synthesis, HPLC purification and mass spectrophotometry are universally known to those skilled in the art. Antiangiogenic proteins and their receptor proteins are also produced in recombinant E. coli or yeast expression systems and purified using column chromatography.

Other protein fragments of intact antiangiogenic proteins include antigens for the development of specific antisera, promoters and antagonists active at the binding sites of antiangiogenic proteins, and cytotoxic agents for targeted killing of cells that bind antiangiogenic proteins. It may be synthesized for use in some applications, including but not limited to proteins linked to or used with the cytotoxic agent.

Synthetic protein fragments of anti-angiogenic proteins have a variety of uses. Proteins that bind to the receptor (s) of high specificity and binding anti-angiogenic proteins are radiolabeled and used for visualization and quantification of binding sites using autoradiophotography and membrane binding techniques. This application provides an important diagnostic and research tool. Knowledge of the binding properties of the receptor (s) has facilitated the study of transduction mechanisms linked to the receptor (s).

Antiangiogenic proteins and proteins derived from these receptors can be coupled with other molecules using standard methods. Both the amino and carboxyl termini of antiangiogenic proteins include tyrosine and lysine residues and many techniques, such as conventional techniques (tyrosine residues-chloramine T, iodogen, lactoperoxidase; lysine residues-bolton- Labeled with isotopes and non-isotopes using radiolabeling using Hunter reagent). Such coupling techniques are well known to those skilled in the art. In addition, tyrosine or lysine is added to fragments without these residues to promote labeling of radioactive amino and hydroxyl groups on the protein. Coupling techniques are selected based on the functional groups available on amino acids, including but not limited to amino, sulfhydral, carboxyl, amide, phenol and imidazole. Several reagents used to achieve this coupling include, among others, glutaraldehyde, diazotized benzidine, carbodiimide and p-benzoquinone.

Antiangiogenic proteins are chemically coupled with isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent agents, bioluminescent agents and other compounds for various applications. The efficiency of the coupling reaction is determined using other techniques suitable for the particular reaction. For example, radiolabeling of proteins of the invention using ¹²⁵ I is performed using chloramine T and Na ¹²⁵ I of high specific activity. The reaction is terminated using sodium metabisulfite and the mixture is desalted on a disposable column. Labeled proteins are eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity is measured in a gamma counter. In this method, unreacted Na ¹²⁵ I is separated from the labeled protein. Protein nuclei with the highest specific radioactivity are stored for future use, such as analysis of the ability of the antiangiogenic proteins to bind antiserum.

In addition, the labeling of anti-angiogenic proteins using short isotopes is characterized by the location of the tumor using the binding site of the protein by visualizing receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques. Can be determined.

Systemic substitution of amino acids within the scope of the synthesized protein results in high affinity protein promoters and antagonists of the receptor (s) of the anti-angiogenic proteins that enhance or decrease binding to the receptor (s). The promoters are used to limit the spread of cancer by inhibiting the growth of micrometastases. Antagonists against anti-angiogenic proteins are applied in situations of insufficient angiogenesis, thereby inhibiting the inhibitory effects of anti-angiogenic proteins to promote angiogenesis. For example, such treatment may have a therapeutic effect of promoting wound healing in diabetes.

The invention is further illustrated by the following examples, which do not imply that they are construed in a limiting manner. In contrast, the spirit of the present invention may have many other aspects, modifications, and equivalents thereof, and after reading the description herein, the present invention may be made without departing from the spirit and / or scope of the appended claims. It should be clearly understood that it can be suggested to those skilled in the art.

Example 1 Isolation of Natural Aresten

Aresten can be produced from the placenta and amnion tissue in amounts in milligrams. Protocols for isolating such and similar proteins are already described in other literature, for example in Langveld, JP et al ., 1988, J. Biol. Chem . 263: 10481-10488; Saus, J. et al ., 1988, J. Biol. Chem . 263: 13374-13380; Gunwar, S. et al ., 1990, J. Biol. Chem . 265: 5466-5469; Gunwar S. et al ., 1991, J. Biol. Chem . 266: 15318-15324; Kahsai, TZ et al ., 1997, J. Biol. Chem. 272: 17023-17032. The generation of recombinant arestens is described by Neilson et al . 1993, J. Biol. Chem . 268: 8402-8406. Proteins are also described, eg, in Hohenester, E. et al ., 1998, EMBO J. 17: 1656-1664 can also be expressed in 293 kidney cells. Aresten is also described in Phylajaniemi, T. et al . 1985, J. Biol. Chem . 260: 7681-7687 can also be isolated according to the method described.

The nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) of the α1 chain of the NC1 domain of type IV collagen is shown in FIG. 1. Aresten generally comprises the NC1 domain of the α1 chain of type IV collagen and possibly also the conjugation region, which is the 12 amino acids immediately preceding the NC1 domain.

Native arestens were subjected to bacterial collagenase, anion exchange chromatography, gel filtration chromatography, HPLC and affinity chromatography [Reference: Gunwar, S. et al. , 1991, J. Biol. Chem. 266: 15318-24; Weber, S. et al ., 1984, Eur. J. Biochem. 139: 401-10], isolated from the human placenta. Type IV collagen monomers isolated from the human placenta were HPLC purified using a C-18 hydrophobic column (Pharmacia, Piscataway, New Jersey, USA). The constituent proteins were distributed in an acetonitrile gradient (32% -39%). One main peak and a small double peak were observed. SDS-PAGE analysis confirmed that there were two bands in the first peak and no observable protein in the second peak. No immunodetectable protein was observed in the second peak through immunoblotting and the main peak was confirmed to be aresten.

Example 2 Recombinant Production of Aresten in Escherichia Coli

The sequences encoding aresten are expressed as forward primer 5'-CGG GAT CCT TCT GTT GAT CAC GGC TTC-3 '(SEQ ID NO: 3) and reverse primer 5'-CCC AAG CTT TGT TCT TCT CAT ACA GAC-3' ( Amplified by PCR from α1 NC1 (IV) / pDS vector using SEQ ID NO: 4) (Neilson, EG et al ., 1993, J. Bio. Chem. 268: 8402-5. The resulting cDNA fragment was digested with Bam HI and Hin dIII and linked to pre-digested pET22b (+) (Novagen, Madison, Wisconsin, USA). This configuration is shown in FIG. This localizes and expresses soluble proteins in periplasm by positioning aresten in frame downstream of the pelB leader sequence. Another vector sequence was added to the amino acid MDIGINSD (SEQ ID NO: 13) encoding the protein. The 3 'end of the sequence was linked in frame with the polyhistidine tag sequence. An additional vector sequence between the 3 'end of the cDNA and his-tag encodes amino acid KLAAALE (SEQ ID NO: 14). Positive clones were sequenced on both strands.

The plasmid construct encoding Aressten was first transformed into E. coli HMS174 (Novagen, Madison, Wisconsin, USA) and then transformed into BL21 (Novagen, Madison, Wisconsin, USA) for expression. Bacteria cultured overnight were used to inoculate 500 ml of culture in LB medium. The culture was grown for about 4 hours until the OD ₆₀₀ of the cells reached 0.6. The protein was then expressed by adding IPTG to a final concentration of 1-2 mM. After expressing the protein for 2 hours, cells were obtained by centrifugation at 5000 × g and the cells were lysed by resuspension in 6M guanidine, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl, pH 8.0. . Resuspended cells were sonicated briefly and centrifuged at 12,000 × g for 30 minutes. The supernatant fractions were passed four to six times through a 5 ml Ni-NTA agarose column (Qiagen, Hilden, Germany) at a rate of 2 ml per minute. Nonspecifically bound proteins were removed by washing with 10 mM and 25 mM imidazole, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl (pH 8.0) in 8 M urea. Aresten proteins were eluted from the column with increasing concentrations of imidazole (50 mM, 125 mM and 250 mM), 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl (pH 8.0) in 8 M urea. Eluted protein was dialyzed twice against PBS at 4 ° C. Small amounts of total protein precipitated during dialysis. The dialyzed protein was collected and centrifuged at about 3500 × g to separate the pellet and supernatant fractions. Protein concentration in each fraction was determined by BCA assay (Pierce Chemical Co., Rockford, Illinois, USA) and quantitative SDS-PAGE analysis. The fraction of total protein in the pellet was about 22% and the remaining 78% was recovered as soluble protein. The total yield of protein was about 10 mg / l.

E. coli expressed proteins were isolated primarily as soluble proteins, and SDS-PAGE showed monomer bands at 29 kDa. Additional 3kDa was derived from the polylinker and histidine tag sequences and was immunodetected by both aresten and 6-histidine tag antibodies.

Example 3 Expression of Aresten in 293 Embryonic Kidney Cells

Aresten was amplified using a pDS plasmid containing α1 (IV) NC1 by adding the leader signal sequence in-frame into the pcDNA 3.1 eukaryotic expression vector (InVitrogen, San Diego, California, USA). The leader sequence from the 5 'end of the full length α1 (IV) chain was cloned into 5 ′ of the NC1 domain to allow protein secretion into the culture medium. Aresten containing recombinant vectors were sequenced using flanking primers. Error-free cDNA clones were further purified and used for in vitro translation studies to determine protein expression. Aresten containing plasmids and control plasmids were used to transfect 293 cells using the calcium chloride method. Transfected clones were selected by geneticin antibiotic treatment (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA). The cells were passaged for 3 weeks in the presence of antibiotics until no cell death occurred. The clones were then spread into T-225 flasks and grown until confluence. The supernatant was then collected and concentrated using Amicon concentrator (Amicon). The concentrated supernatants were analyzed by ELDS for SDS-PAGE, immunoblotting and Aresten expression. Strong binding in the supernatant was detected by ELISA. SDS-PAGE analysis showed that one outstanding band appeared at about 30 kDa. Aresten containing supernatants were treated by affinity chromatography using aresten specific antibodies [Gunwar, S. et al ., 1991, J. Biol. Chem. 266: 15318-24. A major peak containing about 30 kDa monomer immunized with Aresten antibody was identified. About 1-2 mg of recombinant aresten was produced per liter of culture.

Example 4 Aresten Inhibits Endothelial Cell Proliferation

C-PAE cells were grown in DMEM containing 10% fetal calf serum (FCS) to confluence and remain in contact for 48 hours. As control cells, 786-0 (renal carcinoma) cells, PC-3 cells, HPEC cells and A-498 (renal carcinoma) cells were used. These cells were collected by treatment with trypsin (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA) at 37 ° C. for 5 minutes. A suspension of 12,500 cells in DMEM containing 1% FCS was added to each well of a 24-well plate coated with 10 μg / ml fibronectin. These cells were incubated for 24 hours at 37 ° C. with 5% CO ₂ and 95% humidity. The medium was removed and replaced with DMEM containing 0.5% FCS and 3 ng / ml bFGF (R & D Systems, Minneapolis, Minnesota, USA). Non-irritating controls did not receive any bFGF. Cells were treated with aresten or endostatin at concentrations ranging from 0.01-50 μg / ml. All wells received 1 μ Curie of ³ H-thymidine upon treatment. After 24 hours, the medium was removed and these wells were washed with PBS. Cells were extracted with 1N NaOH and added to scintillation vials containing 4 ml of ScintiVerse II (Fisher Scientific, Pittsburgh, Pennsylvania, USA). The thymidine incorporation amount was measured using a scintillation counter. The results are shown in FIGS. 3A and 3B, a pair of graphs showing the incorporation of ³ H-thymidine into C-PAE cells treated with variable amounts of aresten (FIG. 3A) and endostatin (FIG. 3B). Aresten, like endostatin, has been shown to inhibit thymidine incorporation into C-PAE. The behavior of control cells treated with aresten and endostatin is shown in Figures 4a, 4b, 4c and 4d, where aresten is 786-0 cells (Figure 4a), PC-3 cells (Figure 4b) or HPEC cells (Figure Had little effect on 4c). Endostatin had little effect on A-498 cells (FIG. 4D). All groups in FIGS. 3 and 4 represent triplicate samples.

Example 5 Aresten Inhibits Endothelial Cell Migration

Human umbilical endothelial cells (ECV) using Boyden chamber assay (Neuro-Probe, Inc., Cabin John, Maryland, USA) to investigate the inhibitory effects of Aresten and endostatin on FBS induced chemotaxis -304 cells, ATCC 1998-CRL, American Type Culture Collection, 10801 University Boulevard, Manassas, VA, 20110-2209, USA.Tested ECV-304 cells with 10% FBS and 5ng / ml of DiIC18 (3 ) Grown overnight in M199 medium containing natural fluorescent dye (Molecular Probes, Inc., Eugene, Oregon, USA) Cells were treated with trypsin, washed, diluted in M199 with 0.5% FBS and then 60,000 Cells were seeded into the upper chamber wells with or without arestenene or endostatin (2-40 μg / ml) M199 medium containing 2% FBS as the lower chamber as a chemotactic agent Cell containing compartments were 8 micron pore sized polycarbonate The chemotaxis was isolated by a filter (Poretics Corp., Livemore, California, USA) The chamber was incubated for 4.5 hours at 37 ° C. with 5% CO ₂ and 95% humidity The discarded cells were discarded and the top wells were PBS. After washing with the filter, the filter was scraped off with a plastic blade, fixed in 4% formaldehyde in PBS, and placed on a glass slide.When using a fluorescent high performance field, several independent homogeneous images were generated using image processing software (Image Processing Software) was recorded by a Digital Sens. ™ SenSys camera operated with PMIS (Roper Scientific / Photometrics, Tucson, Arizona, USA.) Representative photographs are shown in FIGS. 5A, 5B and 5C. It is shown that Aresten at 20 μg / ml is as effective as endostatin at 20 μg / ml. Cells were counted using OPTIMIZE 6.0 software (Media Cybernetics, Rochester, NY) and the results are shown in FIG. 6, which shows the results shown in micrographs in graphical form.

Example 6 Aresten to Inhibit Endothelial Tube Formation

To measure the inhibitory effect of endothelial tube formation, 320 μl of Matrigel (Collaborative Biomedical Products, Bedford, Massachusetts, USA) was added to each well of a 24-well plate to allow polymerization to occur [Ref. Grant, DS. et al ., 1994, Pathol. Res. Pract. 190: 854-63. A suspension of 25,000 mouse aortic endothelial cells (MAEs) suspended in antibiotic-free EGM-2 medium (Clonetics Corporation, San Diego, California, USA) was passed into each well coated with Matrigel. These cells were treated with increasing concentrations of any of Aresten, BSA, sterile PBS or 7S domains. All assays were performed in triplicate. Cells were incubated at 37 ° C. for 24-48 hours and observed using a CK2 Olympus microscope (3.3 eyepiece, 10 × objective). The cells were then photographed using a 400DK coated TMAX film (Kodak). Cells were stained with a diff-quik fixative (Sigma Chemical Co., St. Louis, Missouri, USA) and photographed again. Ten fields were observed, the tubes were counted and averaged. These results are shown in FIG. 7, which shows that Aresten (■) inhibits endothelial tube formation compared to controls (sterile PBS, ◆; BSA, Δ; 7S domain, X). A representative well formed endothelial tube can be seen in FIG. 8A, which shows cells (100 × magnification) treated with the 7S domain. On the other hand, FIG. 8B shows that poor endothelial tubes were formed or no endothelial tubes were formed (100 × magnification) in MAE cells treated with 0.8 μg / ml arestenene.

Matrigel assays were also performed in C57 / BL6 mice in vivo. Matrigel was dissolved overnight at 4 ° C. This was followed by 20 U / ml of heparin (Pierce Chemical Co., Rockford, Illinois, USA), 150 ng / ml of bFGF (R & D Systems, Minneapolis, Minnesota, USA), and 1 μg / ml of Aresten or 10 μg / It was mixed with either ml of endostatin. Matrigel mixture was injected subcutaneously using a 21 g needle. The control group received the same mixture but did not accept any inhibitor that could be measured by angiogenesis. After 14 days, mice were sacrificed and Matrigel plugs removed. Matrigel plugs were fixed in 4% paraformaldehyde in PBS for 4 hours at room temperature and then converted to PBS for 24 hours. The plug was embedded in paraffin, sliced and colored with H & E. The flakes were examined under a light microscope to count and average the number of blood vessels from 10 high performance fields.

When Matrigel was in the presence of bFGF with or without increasing the concentration of Aresten, a 50% reduction in the number of blood vessels was observed at 1 μg / ml Aresten and 10 μg / ml. These results show that Aresten influences the formation of new blood vessels by inhibiting various stages during angiogenesis. These results also show that at 1 μg / ml Aresten is as effective as 10 μg / ml endostatin, which inhibits new blood vessel formation in vivo.

Example 7: Aresten inhibits tumor metastasis in vivo

C57 / BL6 mice were injected intravenously with 1 million MC38 / MUC1 [Ref. Gong, J. et al ., 1997, Nat. Med. 3: 558-61. Every other day for 26 days, control mice received 10 mM sterile PBS and 6 experimental mice received 4 mg / ml aresten. After 26 days of treatment, lung tumor nodules were counted for each mouse and averaged for both groups. Two mice were sacrificed in each group. Aresten significantly reduced the average primary nodule number from 300 to 200 in control mice.

Example 8 Aresten Inhibits Tumor Growth in Vivo

Two million 786-0 cells were injected subcutaneously into male athymic nude mice aged 7-9 weeks. In a first group of six mice, the tumors were about 700 mm ³ . In the second group of six mice, the tumors were 100 mm ³ . Aresten in sterile PBS was injected intraperitoneally daily for 10 days at a concentration of 20 mg / kg for mice with a tumor volume of 700 mm ³ and a concentration of 10 mg / kg for mice with a tumor volume of 100 mm ³ . Control mice were injected with either BSA or PBS vehicle. The results are shown in FIGS. 9A and 9B. 9A shows a graph of increasing tumor volume from 700 mm ³ for 10 mg / kg of Aresten treated mice (□), BSA treated mice (+) and control mice (●). Tumor volume in the mice treated with Aresten decreased from 700 mm ³ to 500 mm ³ , while the tumor volume in BSA treated and control mice increased to about 1200 mm ³ after 10 days. 9b shows that in mice with 100 mm ³ tumors, Aresten (□) reduced tumors to about 80 mm ³ while the size of tumors (+) in mice treated with BSA was nearly 500 mm ³ after 10 days. It shows an increase.

Approximately 500 million PC-3 cells (human prostate carcinoma cells) were collected and injected subcutaneously into male athymic nude mice aged 7-9 weeks. Tumors were grown for 10 days and then sized by vernier calipers. Standard formula (width ² × length × 0.52 [Reference: O'Reilly, MS et al ., 1997, Cell 88: 277-85; O'Reilly, M / S. Et al ., 1994, Cell 79: 315- 28]) was used to calculate the volume of the tumor. Animals were divided into groups of 5-6 animals. The experimental groups were intraperitoneally injected with Aresten (10 mg / kg / day) or Endostatin (10 mg / kg / day) daily. The control group was injected with PBS daily. The results are shown in FIG. 9C, which shows that Aresten (□) inhibited tumor growth as well as or slightly better than endostatin (▲) or control (●). This experiment was repeated in the same manner except that the Aresten dose was 4 mg / kg / day. Results are shown in FIG. 9D (aresten, □; control, ●). After 8 days had elapsed (marked with arrows in the figure), this treatment was discontinued, but 12 days more significant inhibition persisted without further Aresten treatment. After 12 days without any treatment, the tumor began to deviate from the inhibitory effect of Aresten.

Example 9 Cyclic Half-Life of Aresten

Natural Aresten isolated from the human placenta was injected intravenously into rats weighing 200 g. Each rat was injected with 5 mg of aresten from humans. Serum was analyzed by ELISA directly at various time points using anti- Aresten antibodies to determine the presence of circulating Aresten. As a control, serum albumin was also evaluated at each time point to ensure that the same amount of serum was used in the assay. This experiment revealed that aresten circulates in the serum with a half-life of about 36 hours.

Another group of rats were injected intraperitoneally and / or subcutaneously with 200 μg of human aresten and evaluated for signs of disease pathogenesis in lung, kidney, liver, pancreas, spleen, brain, testes, ovaries, and the like. Direct ELISA was performed and Aresten antibody was detected in the serum of these rats and some endogenous IgG deposits were detected on the renal glomerular basement membrane as previously observed [Kalluri, R, et al ., 1994, Proc. Natl. Acad. Sci. USA 91: 6201-5. Antibody deposits in the kidney did not involve any signs associated with inflammation of the kidneys or deterioration of renal function. This experiment suggests that aresten is nonpathogenic.

Example 10 Binding and Inhibition of MMP-2 Enzyme by Aresten

MMP-2, MMP-9, antibodies against these enzymes were purchased from Oncogene, Inc. Direct ELISA was performed using natural aresten isolated from human placenta as previously described (Kalluri, R. et al ., 1994, Proc. Natl. Acad. Sci. USA 91: 6201-5. Both MMP-2 and MMP-9 specifically bound to aresten. They did not bind to the 7S domain. Such binding depends on TIMP-2 and TIMP-1 binding, respectively.

To evaluate Aresten's ability to degrade the basement membrane, the Matrigel was incubated with MMP-2 and MMP-9 at 37 ° C. for 6 hours with gentle shaking and stirring. Supernatants were analyzed by SDS-PAGE and immunoblotted with antibodies to the α2 chain of type IV collagen. At the beginning of the degradation assay, aresten was added at increasing concentrations and it was observed that MMP-2 activity was inhibited. The NC1 domain was dissolved in SDS-PAGE gel as a 26kDa monomer and a 56kDa dimer, which could be visualized by western blotting using type IV collagen antibodies. Increasing concentrations of aresten inhibited the breakdown of the basal membrane by MMP-2, indicating that aresten could bind to MMP-2 and prevent the breakdown of the basal membrane collagen. Similar results were obtained for MMP-9.

Example 11 Recombinant Production of Canstatin in Escherichia Coli

The nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences for the α2 NC1 domain of type IV collagen are shown in FIG. 10. Sequences encoding canstatin include forward primer 5'-CGG GAT CCT GTC AGC ATC GGC TAC CTC-3 '(SEQ ID NO: 7) and reverse primer 5'-CCC AAG CTT CAG GTT CTT CAT GCA CAC-3' ( Amplified by PCR from α2 NC1 (IV) / pDS vector using SEQ ID NO: 8) (Neilson, EG et al ., 1993, J. Biol. Chem. 268: 8402-5. The resulting cDNA fragment was digested with Bam HI and Hin dIII and linked to pre-digested pET22b (+) (Novagen, Madison, Wisconsin, USA). This configuration is shown in FIG. It localizes and expresses soluble proteins in periplasm by positioning canstatin in-frame downstream of the pelB leader sequence. Another vector sequence was added to the amino acid MDIGINSD (SEQ ID NO: 13) encoding the protein. The 3 'end of the sequence was linked in frame with the polyhistidine tag sequence. Another vector sequence between the 3 'end of the cDNA and his-tag encodes amino acid KLAAALE (SEQ ID NO: 14). Positive clones were sequenced on both strands.

The plasmid construct encoding the canstatin was first transformed with E. coli HMS174 (Novagen, Madison, Wisconsin, USA) and then transformed with BL21 (Novagen, Madison, Wisconsin, USA) for expression. Bacteria cultured overnight were used to inoculate with 500 ml of culture in LB medium. The culture was grown for about 4 hours until the OD ₆₀₀ of the cells reached 0.6. The protein was then expressed by adding IPTG to a final concentration of 0.5 mM. After expressing the protein for 2 hours, cells were obtained by centrifugation at 5000 × g and the cells were lysed by resuspension in 6M guanidine, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl, pH 8.0. . Resuspended cells were sonicated briefly and centrifuged at 12,000 × g for 30 minutes. The supernatant fractions were passed four to six times through a 5 ml Ni-NTA agarose column (Qiagen, Hilden, Germany) at a rate of 2 ml per minute. Nonspecifically bound proteins were removed by washing with 10 mM, 25 mM and 50 mM imidazole, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl (pH 8.0) in 8M urea. Canstatin protein was eluted from the column increasing to two concentrations of imidazole (125 mM and 250 mM) in 8 M urea, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris-HCl, pH 8.0. Eluted protein was dialyzed twice against PBS at 4 ° C. Small amounts of total protein precipitated during dialysis. The dialyzed protein was collected and centrifuged at about 3,500 × g to separate the pellet and supernatant fractions. Protein concentration in each fraction was determined by BCA assay (Pierce Chemical Co. Rockford, Illinois, USA) and quantitative SDS-PAGE analysis. SDS-PAGE analysis showed monomer bands at 29 kDa, with additional 3 kDa derived from polylinker and histidine tag sequences. Eluents containing canstatin were dialyzed against PBS for recombination for subsequent assays. Canstatin proteins analyzed by SDS-PAGE and western blotting were detected by poly-histidine tagged antibodies. In addition, recombinant canstatin proteins expressed bacterially with a collagen type IV NC1 antibody were detected.

E. coli expressed proteins were preferentially isolated as soluble proteins. The fraction of total protein in the pellet was about 40% and the remaining 60% was recovered as soluble protein. The total yield of protein was about 15 mg / l.

Example 12 Expression of Canstatin in 293 Embryonic Kidney Cells

PDS plasmid containing α2 (IV) NC1 by adding the leader signal sequence in-frame into the pcDNA 3.1 eukaryotic expression vector (InVitrogen, San Diego, California, USA) [Neilson, EG et al ., 1993. , J. Biol. Chem. 268: 8402-5] was used to PCR amplify canstatin. The leader sequence from the 5 'end of the full length α2 (IV) chain was cloned into 5' of the NC1 domain to allow protein secretion into the culture medium. Canstatin-containing recombinant vectors were sequenced using flanking primers. Error-free cDNA clones were further purified and used for in vitro translation studies to determine protein expression. Canstatin containing plasmids and control plasmids were used to transfect 293 cells using the calcium chloride method [Kingston, RE, 1996, "Calcium Phosphate Transfection", pp. 9.1.4-9.1.7, in: Current Protocols in Molecular Biology , Ausubel, FM, et al ., Eds., Wiley and sons, Inc. New York, New York, USA]. Transfected clones were selected by geneticin antibiotic treatment (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA). The cells were passaged for 3 weeks in the presence of antibiotics until no cell death occurred. The clones were then spread into T-225 flasks and grown until confluence. The supernatant was then collected and concentrated using Amicon Concentrator (Amicon, Inc.). The concentrated supernatants were analyzed by SDS-PAGE, immunoblotting and ELISA for canstatin expression. Strong binding in the supernatant was detected by ELISA. Supernatants containing canstatin were treated by affinity chromatography using canstatin specific antibodies [Gunwar, S. et al ., 1991, J. Biol. Chem. 266: 15318-24. A major peak containing about 24 kDa of pure monomers immunized with canstatin antibody (anti-α2 NC1 antibody, diluted 1: 200) was identified.

Example 13 Canstatin Inhibits Endothelial Cell Proliferation

Calf aortic endothelial (CPAE) cells were grown to confluence in DMEM containing 10% Fetal Bovine Serum (FCS) and left out for 48 hours. These cells were collected by treatment with trypsin (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA) at 37 ° C. for 5 minutes. A suspension of 12,500 cells suspended in DMEM containing 0.5% FCS was added to each well of a 24-well plate coated with 10 μg / ml fibronectin. Cells were incubated for 24 hours at 37 ° C. with 5% CO ₂ and 95% humidity. The medium was removed and replaced with DMEM containing FCS (non-stimulated) or 10% FCS (stimulated and treated cells). 786-0, PC-3 and HEK 293 cells were used as controls, which were also grown in the same manner, confluenced, trypsinized and plated. Cells were treated in triplicate with canstatin or endostatin at concentrations ranging from 0.025 to 40 mg / ml. In the thymidine incorporation experiment, all wells received 1 μ Curie of ³ H-thymidine upon treatment. After 24 hours, the medium was removed and the wells washed three times with PBS. Radioactive cells were extracted with 1N NaOH and added to scintillation vials containing 4 ml of ScintiVerse II (Fisher Scientific, Pittsburgh, Pennsylvania, USA). The thymidine incorporation amount was measured using a scintillation counter.

The results are shown in FIGS. 12A and 12B. 12A is a bar graph showing the effect of variable amounts of canstatin on proliferation of CPAE cells. The thymidine incorporation counted per minute is shown on the y-axis. "0.5%" on the x-axis represents a 0.5% FCS (non-stimulated) control and "10%" represents a 10% FCS (stimulated) control. Treatment performed with increasing concentrations of canstatin continued to reduce thymidine incorporation. 12B shows a bar graph showing the effect of increasing amounts of canstatin on thymidine incorporation into non-endothelial cells 786-0 (stained bars), PC-3 (hatched bars) and HEK 293 (white bars). . The thymidine incorporation, expressed as counts per minute, is shown on the y-axis, the x-axis shows 0.5% FCS (non-stimulated) and 10% FCS (stimulated) controls for each of the three cell lines, followed by canstatin To increase the concentration. All groups are triple samples and bars represent mean coefficients per minute ± standard error of the mean.

Methylene blue coloring test was also performed. After adding 3,100 cells to each well and treating as described above, the method of Oliver et al ., Oliver, MH et al ., 1989, J. Cell. Science 92: 513-8] was used to count the cells. Wash all wells once with 100 ml of 1 × PBS and fix cells by adding 100 ml of 10% formalin in neutral buffered saline (Sigma Chemical Co., St. Louis, Missouri, USA) for 30 minutes at room temperature. I was. After removing formalin, cells were stained with 1% methylene blue solution (Sigma Chemical Co., St. Louis. Missouri, USA) in 0.01 M borate buffer, pH 8.5 for 30 minutes at room temperature. After removing the staining solution, the cells were washed 5 times with 100 ml of 0.01 M borate buffer (pH 8.5). Methylene blue was extracted from the cells using 100 ml of 0.1N HCl / ethanol (1: 1 mixture) for 1 hour at room temperature. The amount colored by methylene blue was measured in a microplate reader using absorbance at 655 nm wavelength.

The results are shown in FIGS. 12C and 12D. 12C is a bar graph showing the effect of increasing the amount of canstatin with respect to the amount of colorant uptake by CPAE cells. The absorbance at OD ₆₅₅ is shown on the y-axis. "0.1%" refers to a 0.1% FCS treated (non-stimulated) control and "10%" refers to a 10% FCS treated (stimulated) control. The remaining bars represent treatments performed with increasing concentrations of canstatin. In CPAE cells, the colorant uptake gradually dropped to the level seen in unstimulated cells at the canstatin treatment level of about 0.625-1.25 μg / ml. 12D is a bar graph showing the effect of varying the concentration of canstatin on non-endothelial cells HEK 293 (white bars) and PC-3 (hatched bars). The absorbance at OD ₆₅₅ is shown on the y-axis. "0.1%" refers to a 0.1% FCS treated (non-stimulated) control and "10%" refers to a 10% FCS treated (stimulated) control. Bars represent standard error for 8 wells for relative absorbance units ± treatment concentration at 655 nm.

Dose dependent inhibition of 10% serum stimulated endothelial cells was detected with an ED ₅₀ value of about 0.5 μg / ml (FIGS. 12A and 12C). No significant effect was observed on the proliferation of renal carcinoma cells (786-0), prostate carcinoma cells (PC-3) or human-born cells (HEK 293) at doses below 40 mg / ml (FIG. 12B). And 12d). This endothelial cell specificity indicates that canstatin may be a particularly effective antiangiogenic agent.

Example 14 Canstatin Inhibits Endothelial Cell Migration

During angiogenesis, endothelial cells migrate as well as proliferate. Therefore, the effect of canstatin on endothelial cell migration was evaluated. The inhibitory effect of canstatin and endostatin on FBS induced chemotaxis was tested on human navel endothelial cells (HUVEC) using the Boyden chamber assay (Neuro-Probe, Inc., Cabin John, Maryland, USA) It was. HUVEC cells were overnight in M199 (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA) containing 10% FBS and 5 ng / ml of DiIC18 (3) natural fluorescent stain (Molecular Probes, Inc., Eugene, Oregon, USA). Grown. After trypsinizing, washing and diluting in M199 with 0.5% FBS, 60,000 cells were seeded in the upper chamber wells with or without canstatin (0.01 or 1.00 mg / ml). M199 medium containing 2% FBS was placed in the lower chamber as a chemotactic material. The cell containing compartments were isolated from the chemotactic material by a polycarbonate filter of 8 μm pore size (Poretics Corp., Livemore, California, USA). The chamber was incubated for 4.5 hours at 37 ° C. with 5% CO ₂ and 95% humidity. After unmoved cells were discarded and the top wells were washed with PBS, the filters were scrapped with plastic blades, fixed in 4% formaldehyde in PBS and placed on glass slides. When using fluorescent high-performance fields, several independent uniform images can be placed on a digital sensing camera powered by Image Processing Software PMIS (Roper Scientific / Photometrics, Tucson, Arizona, USA). Was recorded. Cells were counted using the OPTIMIZE 6.0 software-program (Media Cybernetics, Rochester, NY) (Klemke, RL et al ., 1994, J. Cell. Biol. 127: 859-66).

The results are shown in FIG. 13, which shows the case when treated without VEGF (without VEGF or serum) and with VEGF (1% FCS and 10ng / ml VEGF) cells, and 0.01 canstatin (1% FCS and Number of migrated endothelial cells per field for treatment with 10 ng / ml VEGF and 0.01 μg / ml canstatin) and 1.0 μg / ml canstatin (1% FCS and 10 ng / ml VEGF and 1 μg / ml canstatin) Bar graph showing (y-axis).

Canstatin inhibited the migration of HUVECs and a significant effect was observed at 10 ng / ml. The ability of canstatin to inhibit both proliferation and migration of endothelial cells suggests that it acts at one or more stages during angiogenesis. Alternatively, canstatin may function as an apoptotic signal for stimulated endothelial cells that may act on both proliferation and migration. Apoptosis induction has been reported for another angiogenesis inhibiting molecule, angiostatin. O'Reilly, M.S. et al., 1994, Cell 79: 315-28; Lucas, R. et al., 1998, Blood 92: 4730-41.

Example 15 Canstatin Inhibits Endothelial Tube Formation

As a first test of the ability of canstatin to inhibit angiogenesis, it was evaluated for its ability to disrupt tube formation by endothelial cells in matrigel, a solid gel of mouse basement membrane proteins. When mouse aortic endothelial cells were cultured on Matrigel, they rapidly aligned to form a hollow tube-like structure.

Matrigel (Collaborative Biomedical Products, Bedford, Massachusetts, USA) was added to each well of a 24-well plate (320 mL) and polymerized. Grant, D.S. et al., 1994, Pathol. Res. Pract. 190: 854-63. A suspension of 25,000 mouse aortic endothelial cells (MAE) in EGM-2 (Clonetics Corporation, San Diego, California, USA) medium without antibiotics was transferred into each well coated with Matrigel. Cells were treated with either canstatin, BSA, sterile PBS or α5-NC1 domains at increasing concentrations. All assays were performed in triplicate. Cells were incubated at 37 ° C. for 24-48 hours and observed using a CK2 Olympus microscope (3.3 × eyepiece, 10 × objective). The cells were then photographed using a 400 DK coated TMAX film (Kodak). Cells were stained with diff-quik fixative (Sigma Chemical Co., St. Louis, Missouri, USA) and photographed once again. Grant, D.S. et al., 1994, Pathol. Res. Pract. 190: 854-63. Ten fields were observed, the tubes were counted and averaged.

The results are shown in FIG. 14, which plots the amount of tube formation under different treatments of BSA (□), canstatin (■) and α5NC1 (○) (PBS treated wells) Percentage of tube formation (y-axis It is a graph shown as). Vertical bars represent standard error of the mean. The results indicate that canstatin significantly reduces endothelial tube formation compared to the control.

Canstatin inhibited endothelial tube formation in a dose dependent manner and almost complete inhibition was observed when 1 g of canstatin protein was added (FIG. 14). The control protein, bovine serum albumin (BSA), as well as the NC1 domain of the type IV collagen α5 chain, did not act on endothelial tube formation, which is not due to the added protein content of the inhibitory effect of canstatin in this assay. Demonstrates specificity for statins.                 

Example 16: Canstatin Inhibits Tumor Growth In Vivo

Human prostate cancer cells (PC-3 cells) were collected from the culture and 2 million cells in sterile PBS were injected subcutaneously into male SCID mice aged 7-9 weeks. After tumor growth for about 4 weeks, four animals were grouped by group. The experimental groups were intraperitoneally injected daily with a 10 mg / kg dose of canstatin in 0.1 mL total volume of PBS. The control group was injected daily with the same volume of PBS. At the start of treatment (day 0), tumors had a volume between 88 mm 3 and 135 mm 3 for control mice and 108 mm 3 and 149 mm 3 for canstatin treated mice. Each group included 5 mice. Tumor volume fraction (V / V _O ) was calculated by dividing the calculated tumor volume on the specific day by the volume at day 0 treatment. The results are shown in FIG. 15A, which is a graph showing tumor volume fraction (y-axis) ± standard error plotted against date of treatment (x-axis). Canstatin treated (■) tumors only slightly increased in size compared to the control (□).

In the second PC-3 experiment, PC-3 cells were collected from the culture, 3 million cells were injected into 6 to 7 week old athymic nude mice, and tumors were grown subcutaneously for about 2 weeks, Four animals were grouped by group. The experimental group (4 mice) was intraperitoneally injected daily with a 3 mg / kg dose of canstatin in a total volume of 0.2 ml PBS or an endostatin dose of 8 mg / kg in an equal volume of PBS. Controls (4) were injected daily with the same volume of PBS. Tumor length and width were measured using vernier calipers and calculated using the following standard formula: length x width ² x 0.52. Tumor volume ranged from 26 mm to 73 mm 3 and the tumor volume fraction (V / V ₀ ) as described above was calculated by dividing the calculated tumor volume on the specific day by the volume on day 0 treatment. The results are shown in FIG. 15B, which is a graph showing the tumor volume fraction (y-axis) ± standard error plotted against the day of treatment (x-axis). Compared to the control (□), canstatin treated (■) tumors only increased slightly in size, and the results are comparable to the results obtained using endostatin (○).

In the renal cell carcinoma cell model, 2 million 786-0 cells were injected subcutaneously into male athymic nude mice aged 7-9 weeks. Tumors were grown from about 100 mm 3 to about 700 mm 3. Each group included 6 mice. Canstatin in sterile PBS was injected daily intraperitoneally at a concentration of 10 mg / kg for 10 days. The control group was injected with the same volume of PBS. Results are shown in FIG. 15C (100 mmW tumor) and 15D (700 mm tumor). In both groups, canstatin treated (■) tumors were actually smaller than control (□).

Canstatin produced in Escherichia coli inhibited the growth of small (100 mm 3, FIG. 15c) renal cell carcinoma (786-0) tumor and large (700 mm 3, FIG. 15d) renal cell carcinoma (786-0) tumor. For human prostate (PC-3) tumors in mice with severe combined immunodeficiency disease (SCID), 10 mg / kg of canstatin maintained a tumor volume fraction of up to 55% of vehicle-injected mice. For athymic (nu / nu) mice, low doses of both canstatin and endostatin were used, and 3 mg / kg canstatin showed the same inhibitory effect as 8 mg / kg endostatin. For all in vivo experiments, mice were considered healthy without showing signs of wasting, and no one died during treatment.

Example 17 CD31 Immunohistochemistry on Canstatin Treated Mice

Reduction of tumor size in vivo suggested an inhibitory effect on angiogenesis in these tumors. To detect tumor vessels, immunocytochemistry with an alkaline phosphatase conjugated anti-CD31 antibody was performed on paraffin embedded tumor sections. The isolated tumors were dissected into multiple pieces of about 3-4 mm thick with scafels and then fixed for 24 hours in 4% paraformaldehyde. The tissues were then switched to PBS for 24 hours, then dehydrated and paraffin embedded. After embedding in paraffin, 3 mm tissue sections were cut out and mounted. Sections were paraffin-depleted, rehydrated and pretreated with 300 mg / kg protease XXIV (Sigma Chemical Co., St. Louis, Missouri, USA) for 5 minutes at 37 ° C. The degradation was stopped in 100% ethanol. Sections were air dried, rehydrated and blocked with 10% rabbit serum. The slides were then incubated overnight at 4 ° C. with a 1:50 dilution of rabbit anti-mouse CD31 monoclonal antibody (PharMingen, San Diego, California, USA), followed by rabbit anti-rat immunoglobulin (DAKO) and Incubation was performed successively for 30 min at 37 ° C. with a 1:50 dilution of rat APAAP (DAKO). The color reaction was carried out using a new fuchsin. Sections were counterstained with hematoxylin.

A decrease in the number of blood vessels was seen in canstatin treated tumors compared to control tumors.                 

Example 18 Recombinant Production of Tumstatin and Tumstatin Variants in Escherichia Coli

The nucleotide (SEQ ID NO: 9) and amino acid (SEQ ID NO: 10) sequences for the α3 chain of the NC1 domain of type IV collagen are shown in FIG. 16. The sequence encoding the tumstatin was identified by forward primer 5'-CGG GAT CCA GGT TTG AAA GGA AAA CGT-3 '(SEQ ID NO: 11) and reverse primer 5'-CCC AAG CTT TCA GTG TCT TTT CTT CAT-3' ( Amplified by PCR from α3 NCI (IV) / pDS vector (Neilson, EG et al., 1993, J. Biol. Chem. 268: 8402-5) using SEQ ID NO; The resulting cDNA fragments were digested with Bam HI and Hin dIII and linked to pre-digested pET22b (+) (Novagen, Madison, Wisconsin, USA). The construct is shown in FIG. 17. The linkage localizes and expresses the soluble protein in periplasm by positioning the tumstatin in-frame downstream of the pelB leader sequence. Additional vector sequences were added to the protein coding amino acid MDIGINSD (SEQ ID NO: 13). The 3 'end of the sequence was linked in-frame with the poly-histidine-tag sequence. An additional vector sequence between the 3 'end of the cDNA and his-tag encodes amino acid KLAAALE (SEQ ID NO: 14). Positive clones were sequenced on both strands. Plasmid constructs encoding tumstatin were first transformed into E. coli HMS174 (Novagen, Madison, Wisconsin, USA) and then transformed into BL21 (Novagen, Madison, Wisconsin, USA). Overnight cultured bacterial cultures were used to inoculate 500 ml cultures in LB medium (Fisher Scientific, Pittsburgh, Pennsylvania, USA). This culture was grown for about 4 hours until the OD ₆₀₀ of the cells reached 0.6. Protein expression was then induced by adding IPTG to a final concentration of 1 mM. After 2 hours induction, cells were collected by centrifugation at 5,000 × g and lysed by resuspending in 6M guanidine, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl, pH 8.0. Resuspended cells were briefly sonicated and centrifuged at 12,000 × g for 30 minutes. The supernatant fractions were transferred to 5 ml Ni-NTA agarose columns (Qiagen, Hilden, Germany) four to six times at a rate of 2 ml per minute. Unspecifically bound proteins were removed by washing with 8M urea, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl, both 10 mM and 25 mM imidazole in pH 8.0. Tumstatin protein was eluted from the column with increasing concentrations of imidazole in 8M urea, 0.1M NaH ₂ PO ₄ , 0.01M Tris-HCl, pH 8.0 (50mM, 125mM and 250mM). Eluted protein was dialyzed twice against PBS at 4 ° C. Some of the total protein precipitated during dialysis. The dialyzed proteins were collected, centrifuged at about 3,500 × g and separated into insoluble (pellet) and soluble (supernatant) fractions.

E. coli expressed tumstatin was isolated primarily as a soluble protein, and SDS-PAGE analysis showed a monomer band at 31 kDa. Additional 3kDa appeared from the polylinker and histidine tag sequences. Eluted fractions containing this band were used in subsequent experiments. Protein concentration in each fraction was determined by quantitative SDS-PAGE analysis using BCA assay (Pierce Chemical Co., Rockford, Illinois, USA) and scanning density measurement. Under reducing conditions, a band is observed at about 60 kDa, which represents a dimer of tumstatin in non-reducing conditions analyzed as a single band of 31 kDa. The overall yield of protein was about 5 mg per liter.

Truncated recombinant tomstatin (Tumstatin-N53) lacking 53 N terminal amino acids is generated in E. coli and, as is already known for another variant (Kalluri, R. et al ., 1996, J. Biol. Chem. 271: 9062-8). This variant is shown in FIG. 18, which is a complex diagram showing the position of the truncated amino acid in the α3 (IV) NC1 monomer. The black circle corresponds to the N-terminal 53 amino acid residues deleted from tumstatin to produce tumstatin-N53 '(Kalluri, R. et al ., 1996, J. Biol. Chem. 271: 9062- 8). The disulfide bonds indicated by the short bars are arranged as they appear in α1 (IV) NC1 and α2 (IV) NC1 (see Siebold, B. et al ., 1988, Eur. J. Biochem. 176: 617- 24). For accurate understanding, only one of the two possible disulfide arrangements is shown.

Rabbit antibodies directed against human α3 (IV) NC1 were prepared as previously known (Kalluri, R. et al ., 1997, J. Clin. Invest. 99: 2470-8). Monoclonal rat anti-mouse CD31 (platelet endothelial cell adhesion molecule, PECAM-1) antibody was purchased from Pharmingen (PharMingen, San Diego, California, USA). FITC conjugated goat anti-rat IgG antibodies, FITC conjugated goat anti-rabbit IgG antibodies, and goat anti rabbit IgG antibodies conjugated to horseradish peroxidase were prepared by Sigma Chemical Co., St. Louis, Missouri, USA).

The concentrated supernatant obtained as above was analyzed by SDS-PAGE and immunoblotting for tumstatin expression as already known (Kalluri, R. et al., 1996, J. Biol. Chem. 271: 9062-8). One-dimensional SDS-PAGE was performed using a 12% assay gel and a discrete buffer system. The separated protein was transferred to nitrocellulose membrane and blocked with 2% BSA for 30 minutes at room temperature. After blocking the remaining binding site, the membrane was washed thoroughly with wash buffer and incubated with the primary antibody at a dilution of 1: 1000 in PBS containing 1% BSA. Incubation was performed overnight at room temperature with a shaker. The blot was then washed thoroughly with wash buffer and incubated with a secondary antibody conjugated to horseradish peroxidase for 3 hours at room temperature with a shaker. The blot was washed once again thoroughly and the substrate (diaminobenzidine in 0.05M phosphate buffer containing 0.01% cobalt chloride and nickel ammonium) was added and incubated for 10 minutes at room temperature. The substrate solution was then decanted and the substrate buffer containing hydrogen peroxide was added. After developing the band, the reaction was stopped with distilled water and the blot was dried. A single band of 31 kDa appeared.

Example 19 Expression of Tumstatin in 293 Embryonic Kidney Cells

Human tumstatin was also generated as a soluble protein secreted from 293 embryonic kidney cells using the pcDNA 3.1 eukaryotic vector. These recombinant proteins (without any purification or detection tag) were isolated using affinity chromatography and pure monomeric forms were detected as major bands by SDS-PAGE and immunoblot analysis.

PDS plasmid containing α3 (IV) NCI (Neilson, EG et al., 1993, J) in such a way that the leader signal sequence is added in-frame into the pcDNA 3.1 eukaryotic expression vector (InVitrogen, San Diego, California, USA). Biol.Chem. 268: 8402-5) was used to PCR amplify the tumstatin. The leader sequence from the 5 'end of the full length α3 (IV) chain was cloned into 5' of the NC1 domain to allow the protein to be secreted into the medium. Tumstatin-containing recombinant vectors were sequenced on both strands using flanking primers. Error-free cDNA clones were further purified and used for in vitro translation experiments to confirm protein expression. 293 cells were transfected using the calcium chloride method using the tumstatin containing plasmid and control plasmid. See Sambrook, J. et al ., 1989, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold. Spring Harbor, New York, USA, pps. 16.32-16.40]. Transfected clones were selected by antibiotic treatment with Geneticin (Life Technologies / Gibco BRL, Gaithersburg, Maryland, USA). Cells were passaged for 3 weeks in the presence of antibiotics until cell death was not evident. Clones were spread into T-225 flasks and grown until confluence. The supernatant was then collected and concentrated using an Amicon concentrator (Amicon, Inc., Beverly, Massachusetts, USA). Concentrated supernatants were analyzed for tumstatin expression by SDS-PAGE, immunoblotting and ELISA. Strong binding in the supernatant was detected by ELISA.

The tumstatin-containing supernatant was subjected to affinity chromatography and both anti-tumstatin and anti-6-histitin tagged antibodies (Gunwar, S. et al., 1991, J. Biol. Chem. 266: 15318-24) Immunodetection was performed. A major peak was identified, which contained about 31 kDa monomers that were immunoreactive with the tumstatin antibody.

Example 20 Tumstatin Inhibits Endothelial Cell Proliferation

The antiproliferative effect of tumstatin on C-PAE cells was examined by ³ H-thymidine incorporation assay using E. coli generated soluble protein.

Cell line and culture medium . 786-0 (renal clearance cell carcinoma system), PC-3 (human prostate cancer cell line), C-PAE (bovine pulmonary endothelial cell line), MAE (mouse aortic endothelial cell line) were all obtained from American Type Culture Collection (ATCC). . 786-0 and C-PAE cell lines were maintained in DMEM (Life Technologies / Gibco BRL, Gaithersburg, Maryland, USA), ECV-304 cell lines were maintained in M199, MAE cells were 10% fetal bovine serum (FCS), 100 It was maintained in EGM-2 (Clonetics Corporation, San Diego, California, USA) containing units / ml penicillin and 100 mg / ml streptomycin.

Proliferation assay . C-PAE cells were grown in DMEM containing 10% FCS until confluence and contact inhibited for 48 hours. C-PAE cells were used between the second and fourth passages. 786-0 and PC-3 cells were used as controls that were not endothelial in this experiment. Cells were collected by trypsinization (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA) at 37 ° C. for 5 minutes. A suspension of 12,500 cells in DMEM containing 0.1% FCS was added to each well of a 24-well plate coated with 10 μg / ml fibronectin. Cells were incubated for 24 hours at 37 ° C. with 5% CO ₂ and 95% humidity. The medium was removed and replaced with DMEM containing 20% FCS. Unstimulated control cells were incubated with 0.1% FCS. Cells were treated with tumstatin at various concentrations ranging from 0.01 to 10 mg / ml. Twelve hours after the start of treatment, all wells were fed with ¹ mCi of ³ H-thymidine. After 24 hours, the medium was removed and the wells washed three times with PBS. Cells were extracted with 1N NaOH and added to scintillation vials containing 4 ml of ScintiVerse II (Fisher Scientific, Pittsburgh, Pennsylvania, USA) solution. Thymidine incorporation was measured using a scintillation counter.

Results are shown in FIGS. 19A, 19B and 19C, which show C-PAE cells (FIG. 19A), PC-3 cells (FIG. 19B) and 786-when treated with various concentrations of tumstatin (x-axis). Histogram showing ³ H-histidine incorporation (y-axis) for 0 cells (FIG. 19 ^c ). All groups represent triple samples. Tumstatin markedly inhibited 20% FCS stimulated ³ H-thymidine incorporation in a dose dependent manner, with an ED ₅₀ of about 0.01 mg / ml (FIG. 19A). In addition, prostate cancer cells (PC-3) or renal cancer cells (786-0) did not show a significant proliferation inhibitory effect even at a dose of tumstatin of 20 mg / ml or less (FIGS. 19B and 19C). The difference between the mean value of ³ H-thymidine incorporation in the tumstatin treated (0.1-10 mg / ml) group and the control group was significant (P <0.05). When PC-3 cells or 786-0 cells were treated with toomstatin, no inhibitory effect was observed (FIGS. 19B, 19C). Each column represents the mean ± SE of the triple wells. This experiment was repeated three times. Bars marked with an asterisk are important, with P <0.05 when using the one-sided student's test.

Example 21: Tumstatin Inhibits Endothelial Tube Formation

Matrigel (Collaborative Biomedical Products, Bedford, Massachusetts, USA) was added to each well of a 24-well plate (320 mL) and polymerized. Grant, DS et al ., 1994, Pathiol. Res. Pract. 190: 854-63. Suspensions of 25,000 MAE cells in antibiotic-free EGM-2 medium (Clonetics Corporation, San Diego, California, USA) were delivered into each of the Matrigel coated wells. Grant, DS et al ., 1994, Pathiol. Res. Pract. 190: 854-63. Cells were treated with increasing concentrations with tumstatin, BSA or 7S domains. Control cells were incubated with sterile PBS. All assays were performed in triplicate. Cells were incubated at 37 ° C. for 24-48 hours and observed using a CK2 Olympus microscope (3.3 × eyepiece, 10 × objective). The cells were then photographed using a 400 DK coated TMAX film (Kodak). Cells were stained with Deep-Quick Fixer (Sigma Chemical Co., St. Louis, Missouri, USA) and photographed once again. Grant, DS et al., 1994, Pathol. Res. Pract. 190: 854-63. Ten fields were observed and the number of tubes was counted by two investigators who did not know the experimental protocol and averaged.

The results are shown in FIG. When mouse aortic endothelial cells are cultured on Matrigel (solid gel of mouse basement membrane protein), they quickly align to form a hollow tube-like structure. See Haralabopoulos, G.C. et al., 1994, Lab. Invest. 71: 575-82. Tumstatin produced in 293 cells significantly inhibited endothelial tube formation in MAE cells in a dose dependent manner compared to the BSA control (FIG. 20). The percentage of tube formation after treatment with 1 mg / ml protein was 98.0 ± 4.0 for BSA and 14.0 ± 4.0 for tumstatin. Similar results were also obtained when using tumstatin produced in E. coli. The 7S domain (N-terminal non-collagenic domain) of type IV collagen showed no effect on endothelial tube formation. Maximum inhibition with tumstatin was obtained at 800-1000 ng / ml. The difference between the mean percent values of the tumstatin treated (●, 0.1-10 mg / mL) group and the control group (BSA (□), 7S domain (○)) was significant (P <0.05). Each point represents the mean ± SE of the triple wells. This experiment was repeated three times. Data points marked with an asterisk are important, with P <0.05 by the one-sided student test. Well-formed tubes were observed for 7S domain treatment. MAE cells treated with 0.8 mg / ml tumstatin showed reduced tube formation.

To evaluate the in vivo effects of tumstatin on the formation of new capillaries, Matrigel plug assays were performed. Passaniti, A. et al., 1992, Lab Invest. 67: 519-29. Male C57 / BL6 mice from 5 to 6 weeks of age (Jackson Laboratories, Bar Harbor, Maine, USA) were purchased. Matrigel (Collaborative Biomedical Products, Bedford, Massachusetts, USA) was thawed overnight at 4 ° C. Prior to injection into C57 / BL6 mice, this was 20 U / ml of heparin (Pierce Chemical Co., Rockford, Illinois, USA), 150 ng / ml of bFGF (R & D Systems, Minneapolis, Minnesota, USA) and 1 mg / ml Mixed with thumstatin. The control group was not injected with an angiogenesis inhibitor. Matrigel mixture was injected subcutaneously using a 21 gauge needle. After 14 days, mice were sacrificed and Matrigel plugs removed. Matrigel plugs were fixed in 4% para-formaldehyde (in PBS) at room temperature for 4 hours and then switched to PBS for 24 hours. Plugs were embedded in paraffin, dissected and H & E stained. Sections were examined under an optical microscope, the number of vessels from ten high performance fields was counted and averaged. All sections were coded and allowed to be observed by a pathologist who did not know the experimental protocol.

When Matrigel was positioned in the presence of bFGF and heparin and with or without E. coli-produced tumstatin, a 67% reduction in blood vessel count was observed by treatment with 1 mg / ml of tumstatin. The number of blood vessels per high performance field was 2.25 ± 1.32 for tumstatin and 7.50 ± 2.17 for control. Each column represents the mean ± SE of 5-6 mice per group. Tumstatin (1 mg / ml) significantly inhibited angiogenesis in vivo compared to the control treated with PBS. The difference between the mean percentage values of the tumstatin treated and control animals was significant (P <0.05). Tumstatin treatment was significant and was P <0.05 by the one-sided student's test.

Example 22: Tumstatin and Tumstatin Variants Inhibit Tumor Growth In Vivo

Five million PC-3 cells were collected and injected subcutaneously on the back of male athymic nude mice aged 7-9 weeks. Tumors were measured using vernier calipers and volume was calculated using standard width ² × length × 0.52. After growing the tumor to about 100 mm 3, the animals were divided into groups of 5 or 6 mice. Tumstatin or mouse endostatin was intraperitoneally injected (20 mg / kg) for 10 days daily in the form contained in sterile PBS. The control group was injected with vehicle (either BSA or PBS). Tumor volume was calculated every 2 or 3 days over 10 days. The results are shown in FIG. 21A, which shows tumor volume (X-axis) versus treatment day (x-axis) for PBS control (□), 20 mg / kg toomstatin (●) and 20 mg / kg endostatin (○). ) Is a graph. Tumstatin produced in E. coli significantly inhibited the growth of PC-3 human prostate tumors (FIG. 21A). Tumstatin at 20 mg / kg inhibited tumor growth to a similar extent as 20 mg / kg endostatin (FIG. 21A). Significant inhibitory effects on tumor growth were observed at day 10 (control 202.8 ± 50.0 mm 3, tumstatin 82.9 ± -25.2 mm 3, endostatin 68.9 ± 16.7 mm 3). Daily intraperitoneal injection of tumstatin or endostatin inhibited human prostate cancer cell (PC-3) tumors compared to controls. This experiment was initiated when the tumor volume was less than 100 mm 3.

The effect of tumstatin on another primary tumor induced in mice was examined. Two million 786-0 renal cell carcinoma cells were injected subcutaneously on the back of male athymic nude mice aged 7-9 weeks. After growing the tumor to about 600-700 mm 3, the animals were divided into six groups. Tumstatin was injected daily intraperitoneally in sterile PBS (6 mg / kg). The control group was injected with BSA. The results are shown in FIG. 21B, which is a graph showing tumor volume (y-axis) versus treatment day (x-axis) for PBS control (□) and 6 mg / kg tumstatin (●). . E. coli produced tumstatin at 6 mg / kg inhibited tumor growth of 786-0 human renal cell carcinoma compared to the BSA control (FIG. 21B). Significant inhibitory effects on tumor growth were observed at day 10 (control 1096 ± 179.7 kPa, tumstatin 619 ± 120.7 kPa). Intraperitoneal injection of tumstatin daily inhibited tumor growth of human kidney cell carcinoma (786-0) compared to the control. This experiment was initiated when the tumor volume was between 600 and 700 mm 3. Each point represents the mean ± SE of 5-6 mice per group. Data points marked with an asterisk are important, with P <0.05 by the one-sided student test.

Part of the NC1 domain (α3 (IV) NC1) of the α3 chain of type IV collagen is a pathogenic epitope of Goodpassture syndrome. Butkowski, RJ et al ., 1987, J. Biol. Chem. 262: 7874-7; Saus, J. et al ., 1988, J. Biol. Chem. 263: 13374-80; Kalluri, R. et al ., 1991, J. Biol. Chem. 266: 24018-24. Goodpasture syndrome is an autoimmune disease characterized by pulmonary bleeding and / or rapid progressive glomerulonephritis. Wilson, C. & F. Dixon, 1986, The Kidney , WB Sanders Co., Philadelphia, Pennsylvania, USA, pps . 800-89; Hudson, BG et al ., 1993, J. Biol. Chem. 268: 16033-6. These syndromes are caused by the destruction of glomeruli and alveolar basement membranes through the binding of autoantibodies to α3 (IV) NC1. Wilson, 1986, supra ; Hudson, 1993, supra ). Several groups have done research to map or predict the location of Good Pathure autoantigens for α3 (IV), see Kalluri, R. et al ., 1995, J. Am. Soc. Nephrol. 6: 1178-85; Kalluri, R. et al ., 1996, J. Biol. Chem. 271: 9062-8; Levy, JB et al ., 1997, J. Am. Soc. Nephrol. 8: 1698-1705; Kefalides, NA et al ., 1993, Kidney Int. 43: 94-100; Quinones, S. et al ., 1992, J. Biol. Chem. 267: 19780-4 (erratum in J. Biol. Chem 269: 17358); Netzer, KO et al ., 1999, J. Biol. Chem. 274: 11267-74, residues at the N-terminus, C-terminus and middle portions have been reported to be epitope positions. Recently, the most probable disease related pathogenic epitopes have been identified in the N-terminal part, see Hellmark, T. et al ., 1999, Kidney Int. 55: 936-44, further defined as being the N-terminal 40 amino acids. Truncated tombstatin lacking the N-terminal 53 amino acids (tumstatin-N53) corresponding to the pathogenic Goodpasture autoepitopes was designed. This variant protein was used in the following experiment.

Two million 786-0 renal cell carcinoma cells were injected subcutaneously on the back of male athymic nude mice aged 7-9 weeks. Tumors were grown to about 100-150 mm 3. Animals were then divided into five groups and E. coli-expressed truncated tumstatin lacking 53 N-terminal amino acids (Kalluri, R. et al., 1996, J. Biol. Chem. 271: 9062-8 20 mg / kg was intraperitoneally injected daily for 10 days. The control group was injected with PBS. The results are shown in FIG. 22, which shows a graph showing the rate of increase of tumor volume (y-axis) over the day of treatment (x-axis) for control mice (□) and mice treated with toomstatin variant N26 (x). to be. E. coli-produced Tumstatin-N53 at 20 mg / kg significantly inhibited 786-0 human kidney tumors from day 4 to day 10 compared to the control group (Day 10: Toomstatin-N53 110.0 ± 29.0 kPa, control group) 345.0 ± 24.0 Hz) (FIG. 22). Each point represents the mean ± SE of 5-6 mice per group. Data points marked with an asterisk are important, with P <0.05 by the one-sided student test.

Example 23 Immunohistochemical Staining for α3 (IV) NC1 and CD31

Kidney and skin tissues from 7 week old male C57 / BL6 mice were treated for evaluation by immunofluorescence microscopy. Tissue samples were frozen with liquid nitrogen and 4 mm thick sections were used. Tissues were treated by known indirect immunofluorescence techniques. Kalluri, R. et al ., 1996, J. Biol. Chem. 271: 9062-8. Frozen sections were stained with primary antibody, polyclonal anti-CD31 antibody (1: 100 dilution) or polyclonal anti-α3 (IV) NC1 antibody (1:50 dilution), followed by secondary antibody, FITC conjugate Stained with gated anti-rat IgG antibody or FITC conjugated anti-human IgG antibody. Immunofluorescence was examined by Olympus fluorescence microscopy (Tokyo, Japan). Negative controls were performed by replacing the primary antibody with irrelevant preimmune serum.

For mouse kidneys, expression of α3 (IV) NC1 was observed in GBM and vascular basement membrane. Expression of CD31, PECAM-1 was observed in glomerular and vascular endothelium. For mouse skin, α3 (IV) NC1 was not present in the epidermal basement membrane and the vascular basement membrane. Expression of CD31 was observed in the vascular endothelium of the skin. CD31 expression was observed in the glomeruli of the mouse kidney and in the endothelium of small blood vessels, and α3 (IV) NC1 expression was observed in the glomerular basement membrane and extravascular glomerular basement membrane. Expression of CD31 was observed in the endothelium of dermal small blood vessels in mouse skin. α3 (IV) NC1 expression was not present in the epidermal basement membrane and hardly observed in the basement membrane of the dermal small blood vessels. These results show an example of a limited distribution of toomstatin.

Example 24 Mutants and Fragments of Angiogenesis Inhibitory Proteins

Fragments and mutants of aresten and canstatin are described in Mariyama et al., 1992, J. Biol. Chem. 267: 1253-8] were also prepared according to Pseudomonas (Pseudomonas) elastase decomposition. The digest was analyzed by gel filtration HPLC and the resulting fragments were analyzed by SDS-PAGE and evaluated by endothelial tube assay as described above. These fragments included aresten of 12kDa fragment, aresten of 8kDa fragment, and canstatin of 10kDa fragment. In addition, two fragments of tumstatin ('333' and '334') were generated by PCR cloning.

As shown in FIG. 23, according to the endothelial tube assay performed as described above, two arestenic fragments (12kDa (■) and 8kDa (□)) and canstatin fragments (19kDa (▲)) were treated with aresten ( Or) inhibiting the formation of endothelial tubes to a higher degree than canstatin (○). FIG. 24 shows that the tumstatin fragments "333" (●) and "334" (○) likewise outperformed toomstatin (▲), and BSA (■) and α6 chains (□) serve as controls. do.

All references, patents and patent applications are incorporated herein by reference in their entirety. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will understand that various changes in formal and substantial forms may be made without departing from the spirit and scope of the invention as defined by the appended claims.

                                SEQUENCE LISTING        <110> Beth Israel Deaconess Medical Center              Raghuram Kalluri        <120> ANTI-ANGIOGENIC PROTEINS AND METHODS OF          USE THEREOF        <130> BIDMC98-11pA PCT        <140> PCT / US99 / 13737        <141> 1999-06-17        <150> US 60 / 089,689        <151> 1998-06-17        <150> US 60 / 126,175        <151> 1999-03-25        <160> 18        <170> FastSEQ for Windows Version 3.0        <210> 1        <211> 690        <212> DNA        <213> Homo sapiens        <220>        <221> CDS        (222) (1) ... (687)        <400> 1  tct gtt gat cac ggc ttc ctt gtg acc agg cat agt caa aca ata gat 48  Ser Val Asp His Gly Phe Leu Val Thr Arg His Ser Gln Thr Ile Asp   1 5 10 15  gac cca cag tgt cct tct ggg acc aaa att ctt tac cac ggg tac tct 96  Asp Pro Gln Cys Pro Ser Gly Thr Lys Ile Leu Tyr His Gly Tyr Ser               20 25 30  ttg ctc tac gtg caa ggc aat gaa cgg gcc cat gga cag gac ttg ggc 144  Leu Leu Tyr Val Gln Gly Asn Glu Arg Ala His Gly Gln Asp Leu Gly           35 40 45  acg gcc ggc agc tgc ctg cgc aag ttc agc aca atg ccc ttc ctg ttc 192  Thr Ala Gly Ser Cys Leu Arg Lys Phe Ser Thr Met Pro Phe Leu Phe       50 55 60  tgc aat att aac aac gtg tgc aac ttt gca tca cga aat gac tac tcg 240  Cys Asn Ile Asn Asn Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser   65 70 75 80  tac tgg ctg tcc acc cct gag ccc atg ccc atg tca atg gca ccc atc 288  Tyr Trp Leu Ser Thr Pro Glu Pro Met Pro Met Ser Met Ala Pro Ile                   85 90 95  acg ggg gaa aac ata aga cca ttt att agt agg tgt gct gtg tgt gag 336  Thr Gly Glu Asn Ile Arg Pro Phe Ile Ser Arg Cys Ala Val Cys Glu              100 105 110  gcg cct gcc atg gtg atg gcc gtg cac agc cag acc att cag atc cca 384  Ala Pro Ala Met Val Met Ala Val His Ser Gln Thr Ile Gln Ile Pro          115 120 125  ccg tgc ccc agc ggg tgg tcc tcg ctg tgg atc ggc tac tct ttt gtg 432  Pro Cys Pro Ser Gly Trp Ser Ser Leu Trp Ile Gly Tyr Ser Phe Val      130 135 140  atg cac acc agc gct ggt gca gaa ggc tct ggc caa gcc ctg gcg tcc 480  Met His Thr Ser Ala Gly Ala Glu Gly Ser Gly Gln Ala Leu Ala Ser  145 150 155 160  ccc ggc tcc tgc ctg gag gag ttt aga agt gcg cca ttc atc gag tgt 528  Pro Gly Ser Cys Leu Glu Glu Phe Arg Ser Ala Pro Phe Ile Glu Cys                  165 170 175  cac ggc cgt ggg acc tgc aat tac tac gca aac gct tac agc ttt tgg 576  His Gly Arg Gly Thr Cys Asn Tyr Tyr Ala Asn Ala Tyr Ser Phe Trp              180 185 190  ctc gcc acc ata gag agg agc gag atg ttc aag aag cct acg ccg tcc 624  Leu Ala Thr Ile Glu Arg Ser Glu Met Phe Lys Lys Pro Thr Pro Ser          195 200 205  acc ttg aag gca ggg gag ctg cgc acg cac gtc agc cgc tgc caa gtc 672  Thr Leu Lys Ala Gly Glu Leu Arg Thr His Val Ser Arg Cys Gln Val      210 215 220  tgt atg aga aga aca taa 690  Cys Met Arg Arg Thr  225        <210> 2        <211> 229        <212> PRT        <213> Homo sapiens        <400> 2  Ser Val Asp His Gly Phe Leu Val Thr Arg His Ser Gln Thr Ile Asp   1 5 10 15  Asp Pro Gln Cys Pro Ser Gly Thr Lys Ile Leu Tyr His Gly Tyr Ser              20 25 30  Leu Leu Tyr Val Gln Gly Asn Glu Arg Ala His Gly Gln Asp Leu Gly          35 40 45  Thr Ala Gly Ser Cys Leu Arg Lys Phe Ser Thr Met Pro Phe Leu Phe      50 55 60  Cys Asn Ile Asn Asn Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser  65 70 75 80  Tyr Trp Leu Ser Thr Pro Glu Pro Met Pro Met Ser Met Ala Pro Ile                  85 90 95  Thr Gly Glu Asn Ile Arg Pro Phe Ile Ser Arg Cys Ala Val Cys Glu              100 105 110  Ala Pro Ala Met Val Met Ala Val His Ser Gln Thr Ile Gln Ile Pro          115 120 125  Pro Cys Pro Ser Gly Trp Ser Ser Leu Trp Ile Gly Tyr Ser Phe Val      130 135 140  Met His Thr Ser Ala Gly Ala Glu Gly Ser Gly Gln Ala Leu Ala Ser  145 150 155 160  Pro Gly Ser Cys Leu Glu Glu Phe Arg Ser Ala Pro Phe Ile Glu Cys                  165 170 175  His Gly Arg Gly Thr Cys Asn Tyr Tyr Ala Asn Ala Tyr Ser Phe Trp              180 185 190  Leu Ala Thr Ile Glu Arg Ser Glu Met Phe Lys Lys Pro Thr Pro Ser          195 200 205  Thr Leu Lys Ala Gly Glu Leu Arg Thr His Val Ser Arg Cys Gln Val      210 215 220  Cys Met Arg Arg Thr  225        <210> 3        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) forward oligonucleotide primer for              Arresten        <400> 3  cgggatcctt ctgttgatca cggcttc 27        <210> 4        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) reverse oligonuceotide primer for              Arresten        <400> 4  cccaagcttt gttcttctca tacagac 27        <210> 5        <211> 684        <212> DNA        <213> Homo sapiens        <220>        <221> CDS        <222> (1) ... (681)        <400> 5  gtc agc atc ggc tac ctc ctg gtg aag cac agc cag acg gac cag gag 48  Val Ser Ile Gly Tyr Leu Leu Val Lys His Ser Gln Thr Asp Gln Glu   1 5 10 15  ccc atg tgc ccg gtg ggc atg aac aaa ctc tgg agt gga tac agc ctg 96  Pro Met Cys Pro Val Gly Met Asn Lys Leu Trp Ser Gly Tyr Ser Leu               20 25 30  ctg tac ttc gag ggc cag gag aag gcg cac aac cag gac ctg ggg ctg 144  Leu Tyr Phe Glu Gly Gln Glu Lys Ala His Asn Gln Asp Leu Gly Leu           35 40 45  gcg ggc tcc tgc ctg gcg cgg ttc agc acc atg ccc ttc ctg tac tgc 192  Ala Gly Ser Cys Leu Ala Arg Phe Ser Thr Met Pro Phe Leu Tyr Cys       50 55 60  aac cct ggt gat gtc tgc tac tat gcc agc cgg aac gac aag tcc tac 240  Asn Pro Gly Asp Val Cys Tyr Tyr Ala Ser Arg Asn Asp Lys Ser Tyr   65 70 75 80  tgg ctc tct acc act gcg ccg ctg ccc atg atg ccc gtg gcc gag gac 288  Trp Leu Ser Thr Thr Ala Pro Leu Pro Met Met Pro Val Ala Glu Asp                   85 90 95  gag atc aag ccc tac atc agc cgc tgt tct gtg tgt gag gcc ccg gcc 336  Glu Ile Lys Pro Tyr Ile Ser Arg Cys Ser Val Cys Glu Ala Pro Ala              100 105 110  atc gcc atc gcg gtc cac agt cag gat gtc tcc atc cca cac tgc cca 384  Ile Ala Ile Ala Val His Ser Gln Asp Val Ser Ile Pro His Cys Pro          115 120 125  gct ggg tgg cgg agt ttg tgg atc gga tat tcc ttc ctc atg cac acg 432  Ala Gly Trp Arg Ser Leu Trp Ile Gly Tyr Ser Phe Leu Met His Thr      130 135 140  gcg gcg gga gac gaa ggc ggt ggc caa tca ctg gtg tca ccg ggc agc 480  Ala Ala Gly Asp Glu Gly Gly Gly Gln Ser Leu Val Ser Pro Gly Ser  145 150 155 160  tgt cta gag gac ttc cgc gcc aca cca ttc atc gaa tgc aat gga ggc 528  Cys Leu Glu Asp Phe Arg Ala Thr Pro Phe Ile Glu Cys Asn Gly Gly                  165 170 175  cgc ggc acc tgc cac tac tac gcc aac aag tac agc ttc tgg ctg acc 576  Arg Gly Thr Cys His Tyr Tyr Ala Asn Lys Tyr Ser Phe Trp Leu Thr              180 185 190  acc att ccc gag cag agc ttc cag ggc tcg ccc tcc gcc gac acg ctc 624  Thr Ile Pro Glu Gln Ser Phe Gln Gly Ser Pro Ser Ala Asp Thr Leu          195 200 205  aag gcc ggc ctc atc cgc aca cac atc agc cgc tgc cag gtg tgc atg 672  Lys Ala Gly Leu Ile Arg Thr His Ile Ser Arg Cys Gln Val Cys Met      210 215 220  aag aac ctg tga 684  Lys Asn Leu  225        <210> 6        <211> 227        <212> PRT        <213> Homo sapiens        <400> 6  Val Ser Ile Gly Tyr Leu Leu Val Lys His Ser Gln Thr Asp Gln Glu   1 5 10 15  Pro Met Cys Pro Val Gly Met Asn Lys Leu Trp Ser Gly Tyr Ser Leu              20 25 30  Leu Tyr Phe Glu Gly Gln Glu Lys Ala His Asn Gln Asp Leu Gly Leu          35 40 45  Ala Gly Ser Cys Leu Ala Arg Phe Ser Thr Met Pro Phe Leu Tyr Cys      50 55 60  Asn Pro Gly Asp Val Cys Tyr Tyr Ala Ser Arg Asn Asp Lys Ser Tyr  65 70 75 80  Trp Leu Ser Thr Thr Ala Pro Leu Pro Met Met Pro Val Ala Glu Asp                  85 90 95  Glu Ile Lys Pro Tyr Ile Ser Arg Cys Ser Val Cys Glu Ala Pro Ala              100 105 110  Ile Ala Ile Ala Val His Ser Gln Asp Val Ser Ile Pro His Cys Pro          115 120 125  Ala Gly Trp Arg Ser Leu Trp Ile Gly Tyr Ser Phe Leu Met His Thr      130 135 140  Ala Ala Gly Asp Glu Gly Gly Gly Gln Ser Leu Val Ser Pro Gly Ser  145 150 155 160  Cys Leu Glu Asp Phe Arg Ala Thr Pro Phe Ile Glu Cys Asn Gly Gly                  165 170 175  Arg Gly Thr Cys His Tyr Tyr Ala Asn Lys Tyr Ser Phe Trp Leu Thr              180 185 190  Thr Ile Pro Glu Gln Ser Phe Gln Gly Ser Pro Ser Ala Asp Thr Leu          195 200 205  Lys Ala Gly Leu Ile Arg Thr His Ile Ser Arg Cys Gln Val Cys Met      210 215 220  Lys Asn Leu  225        <210> 7        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) forward oligonucleotide primer for              Canstatin        <400> 7  cgggatcctg tcagcatcgg ctacctc 27        <210> 8        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) reverse oligonucleotide primer for              Canstatin        <400> 8  cccaagcttc aggttcttca tgcacac 27        <210> 9        <211> 738        <212> DNA        <213> Homo sapiens        <220>        <221> CDS        <222> (4) ... (735)        <221> misc_feature        <222> (160) ... (735)        <223> Tumstatin N53        <221> misc_feature        (222) (4) ... (375)        <223> Tumstatin 333        <221> misc_feature        <222> (376) ... (735)        <223> Tumstatin 334        <400> 9  cca ggt ttg aaa gga aaa cgt gga gac agt gga tca cct gca acc tgg 48      Gly Leu Lys Gly Lys Arg Gly Asp Ser Gly Ser Pro Ala Thr Trp       1 5 10 15  aca acg aga ggc ttt gtc ttc acc cga cac agt caa acc aca gca att 96  Thr Thr Arg Gly Phe Val Phe Thr Arg His Ser Gln Thr Thr Ala Ile                   20 25 30  cct tca tgt cca gag ggg aca gtg cca ctc tac agt ggg ttt tct ttt 144  Pro Ser Cys Pro Glu Gly Thr Val Pro Leu Tyr Ser Gly Phe Ser Phe               35 40 45  ctt ttt gta caa gga aat caa cga gcc cac gga caa gac ctt gga act 192  Leu Phe Val Gln Gly Asn Gln Arg Ala His Gly Gln Asp Leu Gly Thr           50 55 60  ctt ggc agc tgc ctg cag cga ttt acc aca atg cca ttc tta ttc tgc 240  Leu Gly Ser Cys Leu Gln Arg Phe Thr Thr Met Pro Phe Leu Phe Cys       65 70 75  aat gtc aat gat gta tgt aat ttt gca tct cga aat gat tat tca tac 288  Asn Val Asn Asp Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser Tyr   80 85 90 95  tgg ctg tca aca cca gct ctg atg cca atg aac atg gct ccc att act 336  Trp Leu Ser Thr Pro Ala Leu Met Pro Met Asn Met Ala Pro Ile Thr                  100 105 110  ggc aga gcc ctt gag cct tat ata agc aga tgc act gtt tgt gaa ggt 384  Gly Arg Ala Leu Glu Pro Tyr Ile Ser Arg Cys Thr Val Cys Glu Gly              115 120 125  cct gcg atc gcc ata gcc gtt cac agc caa acc act gac att cct cca 432  Pro Ala Ile Ala Ile Ala Val His Ser Gln Thr Thr Asp Ile Pro Pro          130 135 140  tgt cct cac ggc tgg att tct ctc tgg aaa gga ttt tca ttc atc atg 480  Cys Pro His Gly Trp Ile Ser Leu Trp Lys Gly Phe Ser Phe Ile Met      145 150 155  ttc aca agt gca ggt tct gag ggc acc ggg caa gca ctg gcc tcc cct 528  Phe Thr Ser Ala Gly Ser Glu Gly Thr Gly Gln Ala Leu Ala Ser Pro  160 165 170 175  ggc tcc tgc ctg gaa gaa ttc cga gcc agc cca ttt cta gaa tgt cat 576  Gly Ser Cys Leu Glu Glu Phe Arg Ala Ser Pro Phe Leu Glu Cys His                  180 185 190  gga aga gga acg tgc aac tac tat tca aat tcc tac agt ttc tgg ctg 624  Gly Arg Gly Thr Cys Asn Tyr Tyr Ser Asn Ser Tyr Ser Phe Trp Leu              195 200 205  gct tca tta aac cca gaa aga atg ttc aga aag cct att cca tca act 672  Ala Ser Leu Asn Pro Glu Arg Met Phe Arg Lys Pro Ile Pro Ser Thr          210 215 220  gtg aaa gct ggg gaa tta gaa aaa ata ata agt cgc tgt cag gtg tgc 720  Val Lys Ala Gly Glu Leu Glu Lys Ile Ile Ser Arg Cys Gln Val Cys      225 230 235  atg aag aaa aga cac tga 738  Met Lys Lys Arg His  240        <210> 10        <211> 244        <212> PRT        <213> Homo sapiens        <220>        <221> PEPTIDE        <222> (54) ... (245)        <223> Tumstatin N53        <221> PEPTIDE        <222> (2) ... (125)        <223> Tumstatin 333        <221> PEPTIDE        <222> (126) ... (245)        <223> Tumstatin 334        <400> 10  Gly Leu Lys Gly Lys Arg Gly Asp Ser Gly Ser Pro Ala Thr Trp Thr   1 5 10 15  Thr Arg Gly Phe Val Phe Thr Arg His Ser Gln Thr Thr Ala Ile Pro              20 25 30  Ser Cys Pro Glu Gly Thr Val Pro Leu Tyr Ser Gly Phe Ser Phe Leu          35 40 45  Phe Val Gln Gly Asn Gln Arg Ala His Gly Gln Asp Leu Gly Thr Leu      50 55 60  Gly Ser Cys Leu Gln Arg Phe Thr Thr Met Pro Phe Leu Phe Cys Asn  65 70 75 80  Val Asn Asp Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser Tyr Trp                  85 90 95  Leu Ser Thr Pro Ala Leu Met Pro Met Asn Met Ala Pro Ile Thr Gly              100 105 110  Arg Ala Leu Glu Pro Tyr Ile Ser Arg Cys Thr Val Cys Glu Gly Pro          115 120 125  Ala Ile Ala Ile Ala Val His Ser Gln Thr Thr Asp Ile Pro Pro Cys      130 135 140  Pro His Gly Trp Ile Ser Leu Trp Lys Gly Phe Ser Phe Ile Met Phe  145 150 155 160  Thr Ser Ala Gly Ser Glu Gly Thr Gly Gln Ala Leu Ala Ser Pro Gly                  165 170 175  Ser Cys Leu Glu Glu Phe Arg Ala Ser Pro Phe Leu Glu Cys His Gly              180 185 190  Arg Gly Thr Cys Asn Tyr Tyr Ser Asn Ser Tyr Ser Phe Trp Leu Ala          195 200 205  Ser Leu Asn Pro Glu Arg Met Phe Arg Lys Pro Ile Pro Ser Thr Val      210 215 220  Lys Ala Gly Glu Leu Glu Lys Ile Ile Ser Arg Cys Gln Val Cys Met  225 230 235 240  Lys Lys Arg His        <210> 11        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) forward oligonucleotide primer for              Tumstatin        <400> 11  cgggatccag gtttgaaagg aaaacgt 27        <210> 12        <211> 27        <212> DNA        <213> Artificial Sequence        <220>        <223> pET22b (+) reverse oligonucleotide primer for              Tumstatin        <400> 12  cccaagcttt cagtgtcttt tcttcat 27        <210> 13        <211> 8        <212> PRT        <213> Artificial Sequence        <220>        <223> Additional vector sequence added to protein        <400> 13  Met Asp Ile Gly Ile Asn Ser Asp   1 5        <210> 14        <211> 7        <212> PRT        <213> Artificial Sequence        <220>        <223> Additional vector sequence added to protein        <400> 14  Lys Leu Ala Ala Ala Leu Glu   1 5        <210> 15        <211> 28        <212> DNA        <213> Artificial Sequence        <220>        <223> pPICZaA forward oligonucleotide primer for              Arresten        <400> 15  ttcggaattc tctgttgatc acggcttc 28        <210> 16        <211> 35        <212> DNA        <213> Artificial Sequence        <220>        <223> pPICZaA reverse oligonucleotide primer for              Arresten        <400> 16  tgctctagag gtgttcttct catacagact tggca 35        <210> 17        <211> 31        <212> DNA        <213> Artificial Sequence        <220>        <223> pPICZaA forward oligonucleotide primer for              Canstatin        <400> 17  ttcggaattc gtcagcatcg gctacctcct g 31        <210> 18        <211> 32        <212> DNA        <213> Artificial Sequence        <220>        <223> pPICZaA reverse oligonucleotide primer for              Canstatin        <400> 18  ggggtacccc caggttcttc atgcacacct gg 32

Claims (47)

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide having the amino acid sequence of SEQ ID NO: 6; (c) a polypeptide having the amino acid sequence of SEQ ID NO: 10; And (d) an isolated antiangiogenic polypeptide selected from the group consisting of antiangiogenic fragments of the polypeptides of (a), (b) or (c). The isolated polypeptide of claim 1, wherein the polypeptide is a monomer. The isolated polypeptide of claim 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 2. 4. The isolated polypeptide of claim 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 6. The isolated polypeptide of claim 2, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 10. 4. A multimer of a polypeptide according to claim 1, wherein the multimer has antiangiogenic activity. A fusion protein, wherein the polypeptide according to claim 1 is fused to a polypeptide selected from the group consisting of endostatin, angiostatin, and restin, wherein the polypeptide has antiangiogenic activity. delete A pharmaceutical composition for treating a disease or condition characterized by angiogenesis comprising the antiangiogenic polypeptide according to claim 1 as a biologically active ingredient. 10. The pharmaceutical composition of claim 9, further comprising a pharmaceutically acceptable carrier. For treating a disease or condition characterized by angiogenesis comprising as an biologically active component an antiangiogenic polypeptide according to claim 1 and a polypeptide molecule selected from the group consisting of endostatin, angiostatin, and apomigren. Pharmaceutical composition. A pharmaceutical composition for treating a disease or condition characterized by angiogenesis comprising the multimer according to claim 6 as a biologically active ingredient. A pharmaceutical composition for treating a disease or condition characterized by angiogenesis comprising the fusion protein according to claim 7 as a biologically active ingredient. An isolated polynucleotide encoding an antiangiogenic polypeptide according to claim 1. The isolated polynucleotide of claim 14, wherein the polynucleotide is operably linked to an expression control sequence. A host cell transformed with the polynucleotide according to claim 15. The host cell of claim 16, wherein the cell is selected from the group consisting of bacterial cells, yeast cells, mammalian cells, insect cells or plant cells. An isolated polynucleotide encoding a polypeptide according to claim 3. An isolated polynucleotide encoding a polypeptide according to claim 4. An isolated polynucleotide encoding a polypeptide according to claim 5. (a) growing a culture of a host cell transformed with the polynucleotide according to claim 14, wherein the host cell is selected from the group comprising bacterial cells, yeast cells, mammalian cells, insect cells or plant cells do); (b) producing a polypeptide encoded by the polynucleotide according to claim 14, comprising producing a polypeptide encoded by the polynucleotide according to claim 14 by purifying the polypeptide from the culture. delete A pharmaceutical composition for treating a disease or condition characterized by angiogenesis comprising a polynucleotide according to claim 14. delete The pharmaceutical composition of claim 23, wherein the polynucleotide is included in a mammalian cell. The pharmaceutical composition of claim 25, wherein the polynucleotide is inserted into the mammalian cell by a viral vector. An antibody that specifically binds to the isolated polypeptide of claim 1. The NC1 domain of the α1 chain of human type IV collagen having the sequence of SEQ ID NO: 2; The NC1 domain of the α2 chain of human type IV collagen having the sequence of SEQ ID NO: 6; The NC1 domain of the α3 chain of human type IV collagen having the sequence of SEQ ID NO: 10; And an antiangiogenic polypeptide selected from the group consisting of one of these antiangiogenic fragments, wherein the pharmaceutical composition for treating a disease or condition characterized by angiogenesis, wherein the pharmaceutical composition has antiangiogenic activity. 29. The method of claim 28, wherein the disease or condition characterized by angiogenesis is angiogenesis-dependent cancer, benign tumor, rheumatoid arthritis, diabetic retinopathy, psoriasis, ocular angiogenic disease, Osler-Weber syndrome, myocardial angiogenesis, Plaque neovascularization, capillary dilatation, hemophilia arthrosis, hemangiofibroma, trauma granulation, intestinal adhesions, arteriosclerosis, scleroderma, thickening scars, cat scratch disease, Heliobacter pylori ulcers, dialysis graft vascular stenosis, Composition selected from the group consisting of contraception and obesity. 30. The composition of claim 29, wherein the disease or condition is cancer. delete delete A polynucleotide encoding an anti-angiogenic polypeptide comprising amino acids 2 to 125 of SEQ ID NO: 10. delete A polynucleotide encoding an anti-angiogenic polypeptide comprising amino acids 126-245 of SEQ ID NO: 10. delete delete An antiangiogenic fragment of the NC1 domain of the α3 chain of type IV human collagen, prepared by PCR cloning method using polynucleotides comprising nucleotides 4 to 375 or nucleotides 376 to 735 of SEQ ID NO: 9 as a template. Fragment produced by Pseudomonas elastase digestion method as an antiangiogenic fragment of NC1 domain of α1 chain of human type IV collagen of SEQ ID NO: 2. 40. The antiangiogenic fragment of claim 39, wherein the size of the fragment is 12 kDa as measured by SDS-PAGE. 40. The anti-angiogenic fragment of claim 39, wherein the size of the fragment is 8 kDa as measured by SDS-PAGE. Fragment produced by Pseudomonas elastase digestion method as an antiangiogenic fragment of NC1 domain of α2 chain of human type IV collagen of SEQ ID NO: 6. 43. The fragment of claim 42, wherein the fragment is 10 kDa as measured by SDS-PAGE. delete The method of claim 1, wherein the antiangiogenic fragment is a) the amino acid sequence of amino acids 54 to amino acid 245 of SEQ ID NO: 10; b) amino acid sequence of amino acids 2 to 245 of SEQ ID NO: 10; And c) an isolated polypeptide selected from the group consisting of amino acid 126 to amino acid 245 of SEQ ID NO: 10. The composition of claim 25, wherein said mammalian cell is selected from the group consisting of blood cells, TIL cells, bone marrow cells, vascular cells, tumor cells, liver cells, muscle cells, and fibroblasts. The antiangiogenic fragment of claim 38, having a sequence of amino acids 2 to 125 of SEQ ID NO: 10 or a sequence of amino acids 126 to 245 of SEQ ID NO: 10. 39.

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