JP2008019271A - Anti-angiogenesis protein and method of use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a C-terminal fragment of the NC1 domain of Type IV collagen of α1 chain, α2 chain and α3 chain suspending in vivo growth of tumor, or inhibiting formation of blood capillary in some in vitro models including endothelial tube assay. <P>SOLUTION: The invention relates to the protein isolated from a group comprising the NC1 domain of α1 chain of the Type IV collagen, the NC1 domain of α2 chain of the Type IV collagen, a fragment, an analogue, a derivative or a variant of the same, wherein the protein, fragment, analogue, derivative or variant of the same has the anti-angiogenesis activity. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to anti-angiogenic proteins and methods of use thereof.

RELATED APPLICATIONS This application is based on US Provisional Application No. 60 / 089,689 filed June 17, 1998, and US Provisional Application No. 60 / 126,175, filed March 25, 1999. All the teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION The vascular basement membrane consists of macromolecules such as collagen, laminin, heparan sulfate proteoglycan, fibronectin and entactin (Timpl, R., 1996, Curr Opin Cell Biol 8: 618-24). Functionally, collagen promotes cell adhesion, cell migration, cell differentiation and cell growth (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27: 93-127), and these Via function, it is presumed to play an important role in endothelial cell proliferation and behavior during angiogenesis, the process of forming new blood vessels from pre-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 that requires the sprouting and migration of endothelial cells, their proliferation, and their differentiation into tube-like structures and the generation of a developing perivascular basement membrane matrix. Furthermore, angiogenesis is an important process for normal physiological events such as wound healing and endometrial remodeling (Folkman, J. et al., 1995, J. Biol. Chem. 267: 10931-34) . At present, it is well reported that angiogenesis is necessary for the metastasis and growth of solid tumors larger than a few mm ² (Folkman, 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 anti-angiogenic properties, supporting that basement membrane collagen synthesis and deposition are important for angiogenesis and survival (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 basement membrane tissue and angiogenesis is still not fully understood.

SUMMARY OF THE INVENTION The present invention relates to proteins comprising the NC1 domain of the α chain of type IV collagen having anti-angiogenic properties. In particular, the present invention relates to the novel proteins Arresten, Canstatin, and Tumstatin, and biologically active (eg, anti-angiogenic) fragments, mutants, analogs, homologs And their derivatives, as well as their multimers (eg, dimers) and fusion proteins (also referred to herein as chimeric proteins). All of these proteins contain a C-terminal fragment of NC1 [non-collagenous 1 · (non-collagenous 1)] of type IV collagen. More specifically, Arresten, Canstatin, and Tumstatin are C-terminal fragments of the NC1 domain of type IV collagen α1, α2, and α3 chains, respectively. In particular, Arresten, Canstatin and Tumstatin are monomeric proteins. All three stop tumor growth in vivo and inhibit capillary formation in several in vitro models, including endothelial tube assays.

The gist of the present invention is as follows.
[1] An isolated protein selected from the group consisting of the α1 chain NC1 domain of type IV collagen, the α1 chain NC1 domain of type IV collagen, or a fragment, analog, derivative or variant thereof, wherein the protein, The fragment, analog, derivative or variant has anti-angiogenic activity,
[2] The isolated protein of [1], wherein the protein is a monomer,
[3] The isolated protein of [2], wherein the protein is arrestin,
[4] The isolated protein of [2], wherein the protein is canstatin,
About.

  The present invention includes isolated arresten and recombinantly produced arresten. Arresten, also referred to herein as Arrestin, contains the NC1 domain of the α1 chain of type IV collagen and has anti-angiogenic activity. The invention also encompasses isolated Arresten anti-angiogenic fragments, multimers of isolated Arresten and anti-angiogenic fragments, and polynucleotides encoding their anti-angiogenic proteins. Also encompassed are compositions comprising isolated Arresten, an anti-angiogenic fragment thereof, or both as biologically active ingredients. In another aspect, the invention features a method for treating a proliferative disease, such as cancer, in a mammal wherein the proliferative disease is characterized by angiogenic activity. Such methods include the step of administering to the mammal a composition comprising an anti-angiogenic Arresten or fragment thereof. Anti-angiogenic Arresten and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies against isolated anti-angiogenic Arresten and fragments thereof.

  The invention also encompasses isolated canstatin and recombinantly produced canstatin. Canstatin contains the NC1 domain of α2 chain of type IV collagen and has anti-angiogenic activity. The present invention also includes isolated anti-angiogenic fragments of canstatin, multimers of isolated canstatin and anti-angiogenic fragments, and polynucleotides encoding these anti-angiogenic proteins. Also encompassed are compositions comprising isolated canstatin, an anti-angiogenic fragment thereof, or both as biologically active ingredients. In another aspect, the invention features a method for treating a proliferative disease, such as cancer, in a mammal wherein the proliferative disease is characterized by angiogenic activity. Such methods include the step of administering to the mammal a composition comprising an anti-angiogenic canstatin or a fragment thereof. Anti-angiogenic canstatin and fragments thereof can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies against isolated anti-angiogenic canstatin and fragments thereof.

  The invention also encompasses isolated tumstatins and recombinantly produced tumstatins. Tumstatin contains the α1 chain NC1 domain of type IV collagen and has anti-angiogenic activity. The invention also encompasses isolated tumstatin anti-angiogenic fragments, multimers of isolated tumstatin and anti-angiogenic fragments, and polynucleotides encoding these anti-angiogenic proteins. Also encompassed are compositions comprising isolated tumstatin, an anti-angiogenic fragment thereof, or both as biologically active ingredients. In another aspect, the invention features a method for treating a proliferative disease, such as cancer, in a mammal wherein the proliferative disease is characterized by angiogenic activity. Such a method comprises administering to a mammal a composition comprising an anti-angiogenic tumstatin or a fragment thereof. Anti-angiogenic tumstatin and fragments thereof can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies against isolated anti-angiogenic tumstatin and fragments thereof.

DETAILED DESCRIPTION OF THE INVENTION A variety of diseases occur with undesirable angiogenesis. On the other hand, if it is possible to stop the growth and hypertrophy of capillaries at a certain point in time or in a specific tissue under some condition, many diseases and undesirable conditions can be prevented or ameliorated. Basement membrane structure relies on the assembly of type IV collagen networks presumed to occur through the C-terminal globular non-collagenous (NCl) domain of type IV collagen (Timple, R., 1996, Curr Opin Cell Biol 8: 618-24; Timple, R. et al., 1981, Eur. J. Biochem. 120: 203-211). Type IV collagen is made up of six different gene products, α1 to α6 (Prockop, DJ et al., 1995, Annu. Rev. Biochem. 64: 403-34). α1 and α2 isoforms are universally present in human basement membranes (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27: 93-127), but the other four isoforms are limited (Kalluri, R. etal., 1997, J. Clin. Invest. 99: 2470-8).

In the process of tumor growth and metastasis, the formation of new capillaries from existing blood vessels, ie angiogenesis, is essential (Folkman, 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 are not initially vascularized but may become vascularized because the tumor grows to several mm ³ or more (Folkman, J., 1995, Nat. Med. 1: 27-31; Hanahan, D. etal., 1996, Cell 86: 353-64). Switching to the angiogenic phenotype requires both an upregulation of angiogenic stimulants and a downregulation of angiogenic inhibitors (Folkman, J., 1995, Nat. Med. 1: 27-31). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most commonly expressed angiogenic factors in tumors. Vascularized tumors may overexpress one or more of these angiogenic factors that can synergistically promote tumor growth. Inhibition of a single angiogenic factor such as VEGF with a receptor antagonist does not stop tumor growth. Several angiogenesis inhibitors have recently been identified, including IFN-α, platelet factor 4 (Maione, TEetal., 1990, Science 247: 77-9) and PEX (Brooks, PC et al., 1998, Cell 92 : 391-400) are not intrinsically associated with 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) is a tumor-related angiogenesis inhibitor produced by the tumor tissue itself. Treatment of tumor growth and metastasis with these endogenous angiogenesis inhibitors is a very effective and attractive idea, but must take into account some of the problems that can occur in connection with anti-angiogenic therapy I must. Consideration should also be given to the delayed toxicity caused by long-term anti-angiogenic therapy and the potential for wound healing and impaired reproductive angiogenesis during treatment.

  In the present invention, proteins having anti-angiogenic properties, and fragments, analogs, derivatives, homologues and mutants thereof, as well as proliferative diseases involving angiogenesis using these proteins, analogs, derivatives, homologues and mutants. A method of inhibiting will be described. The protein is composed of the α-chain NCl domain of type IV collagen, or a part of the domain, specifically, the monomers of the α1, α2, and α3 chain NCl domains of type IV collagen. . These proteins, particularly when monomeric, stop tumor growth in in vivo models of cancer and also inhibit capillary formation in several in vitro models, including endothelial tube assays.

  These proteins may contain the junction region of the NCl domain. The α1, α2, or α3 chains are preferred because there is evidence to suggest that the α4, α5, and α6 chains have reduced or no longer detectable anti-angiogenic activity. In general, the monomeric form of the protein is preferred since there is evidence to suggest that the hexameric form also has little or no activity.

  More specifically, the present invention relates to “Arresten”, which is a protein having a length of about 230 amino acids corresponding to the amino acid at the N-terminus of the α1 chain of the NCl domain of human type IV collagen. The called protein is described (Hostikka, SL et al., 1988, J. Biol. Chem. 263: 19488-93).

  As disclosed herein, human Arresten can be produced in E. coli using a bacterial expression plasmid such as pET22b that is capable of periplasmic transport and therefore produces a soluble protein. The protein is expressed as a 29 kDa fusion protein with 6 C-terminal histidine tags. The additional 3 kDa (added to 26 kDa) is derived from the polylinker and histidine tag sequences. Arresten was also produced as a secreted soluble protein in 293 kidney cells using the pcDNA3.1 eukaryotic vector. This 293 produced protein does not have a purification tag or a detection tag.

E. coli-produced Arresten inhibits the growth of bFGF-stimulated endothelial cells in a dose-dependent manner with an ED _{50 of} 0.25 μg / ml. There was no significant effect on the proliferation of renal cancer cells (786-0), prostate cancer cells (PC-3), or human prostate epithelial cells (HPEC). Endostatin inhibited proliferation of C-PAE cells, but not A-498 cancer cells, with an ED ₅₀ of 0.75 μg / ml 3 times higher than Arresten.

  As described herein, the specific inhibition of endothelial cell proliferation and migration indicates that Arresten may function via cell surface proteins or receptors. Inhibition of matrix metalloproteinases or MMPs suggests that Arresten plays a direct role in tumor growth and metastasis, similar to batimastat (BB-94) and mariimastat (BB-2516). Suggests that

  In the present invention, Canstatin, which is the NCl domain of α2 chain of type IV collagen, was used to inhibit angiogenesis. This inhibition inhibited endothelial cell proliferation and migration and endothelial tube formation. Assayed by inhibition of. The specific inhibition of endothelial cell proliferation and migration by canstatin also demonstrates that this substance has anti-angiogenic activity and may function via cell surface proteins / receptors is doing. Integrins can be candidate molecules because of their ability to bind extracellular matrix and regulate cell behavior such as migration and proliferation. In particular, avb3 integrin can be a canstatin receptor because it is induced during angiogenesis and can bind any partner.

  In the present invention, Tumstatin which is the NCl domain of α3 chain of type IV collagen (Timple, R. et al., 1981, Eur. J. Biochem. 120: 203-11; Turner, N. et al. , 1992, J. Clin. Invest. 89: 592-601) were used to regulate vascular endothelial cell proliferation and angiogenesis in in vitro and in vivo angiogenesis and tumor growth models. The distribution of α3 (IV) chain is determined by certain basement membranes such as GBM, several types of cochlear basement membranes, ocular basement membranes such as anterior chamber capsular sac, Descemet's membrane, ovary and testicular basement membrane (Frojdman, K. et al., 1998, Differentiation 83: 125-30), and alveolar capillary basement membrane (Kashtan, CE, 1998, J. Am. Soc. Nephrol. 9: 1736-50). However, this chain is not found in kidney mesangium, cutaneous vascular and epidermal basement membranes, and hepatic vascular basement membranes (Kashtan, C.E., supra). In the wound healing process, α3 and α4 chains of type IV collagen are not “existing”, ie, components of the basement membrane of the skin vasculature, so α chains other than these two chains aggregate to form new capillaries. Form. Since the α3 (IV) chain is not a natural component in normal human skin, the process of collagen assembly and angiogenesis in wound healing lesions is not altered by treatment with tumstatin.

  The α3 (IV) chain is expressed in human kidney vascular basement membrane as well as GBM (Kalluri, R. et al., 1997, J. Clin. Invest. 99: 2470-8). These “existing” blood vessels are presumed to be involved in the progression of primary renal tumors such as renal cell carcinoma. Tumstatin may be effective in the treatment of primary renal tumors by disrupting neovascularization through the assembly of α3 (IV) chains and other α chains. The number of patients diagnosed with renal cell carcinoma is about 30,000 in the US in 1996 (Mulders, P. etal., 1997, Cancer Res. 57: 5189-95), and the prognosis for metastatic cases is very poor. . Despite advances in radiation therapy and chemotherapy, the long-term survival of treated patients has not improved much (Mulders, P., supra). It is very important to establish a new treatment modality because there is no significant treatment option for renal cell carcinoma. In view of this fact, recently the targeting of solid tumor neovascularization has shown promising results in several animal models (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 effectiveness of tumstatin in inhibiting renal cell carcinoma growth in vivo indicates that this molecule can be an effective anti-angiogenic therapy for this tumor type.

  In the present invention, tumstatin specifically inhibited endothelial cell proliferation and did not affect the growth of tumor cell lines PC-3 and 786-O in vitro. Tumstatin did not inhibit endothelial cell migration but significantly suppressed endothelial tube formation in vitro. Taken together, these results indicate that tumstatin inhibits the formation of new blood vessels by inhibiting various stages in the angiogenesis process.

  In in vivo studies, tumstatin inhibited angiogenesis in the matrigelplug assay and suppressed the growth of PC-3 and 786-O tumors in a mouse xenograft model. That tumstatin inhibited the growth of large tumors is particularly significant in view of tumor treatment in clinical settings.

  Tumstatin retains the pathogenic epitope of Goodpascher's syndrome, an autoimmune disease characterized by pulmonary hemorrhage and rapidly progressive glomerulonephritis (Butkowski, RJ et al., 1987, J. Biol. Chem. 262 Saus, J. et al., 1988, J. Biol. Chem. 263: 13374-80; Kalluri, R. et al., 1991, J. Biol. Chem. 266: 24018-24), Short-term or long-term administration of tumstatin can induce this autoimmune disease. Several groups have attempted to map or predict the location of the Goodpascher's syndrome self-epitope on α3 (IV) NC1 and reported that the N-terminal part, middle part, and C-terminal part retain the epitope (Kalluri, R. et al., 1995, J. Am. Soc. Nephrol. 6: 1178-85; Kalluri, R. etal., 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, it was reported that the reactivity of autoantibodies against only the N-terminus of α3 (IV) NCl correlated with kidney viability using recombinant chimeric constructs (Hellmark, T. et al., 1999, Kidney Int. 55: 936-44). Disease-related epitopes for the first 40 amino acids of the N-terminal portion were also identified. To remove the Goodpasture's syndrome epitope, truncated tumstatin lacking the 53 amino acid residues at the N-terminus was synthesized, but this molecule is directed against 786-O tumor growth in a mouse xenograft model. Shows an inhibitory effect. This molecule also did not bind to autoantibodies in critically ill patients with Goodpasture's syndrome. From these results, it can be seen that the antiangiogenic region of tumstatin is preserved even if the 53 amino acids at the N-terminus are removed.

  The specific inhibition of endothelial cell proliferation by tumstatin strongly suggests that tumstatin may function via cell surface proteins / receptors. Angiogenesis is also dependent on specific endothelial cell adhesion events mediated by integrin avb3 (Brooks, P.C. et al., 1994, Cell 79: 1157-64). Tumstatin can lead to apoptosis of endothelial cells because it can disrupt the interaction of proliferating endothelial cells with matrix components (Re, F. et al., 1994, J. Cell. Biol. 127: 537-46). Matrix metalloproteinase (MMP) is presumed to be an important enzyme that regulates 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 suppress tumor growth by inhibiting angiogenesis (Brooks, P.C. et al., Cell 92: 391-400). Tumstatin may function by inhibiting the activity of MMP.

  Tumstatin inhibits angiogenesis in vitro and in vivo resulting in suppression of tumor progression. In order to apply this method to patients, the potential toxicity or side effects of systemic administration must also be considered. The distribution of tumstatin is limited and scarcely present in the skin basement membrane, suggesting that the potential for side effects from tumstatin treatment is low. In addition, the presence of tumstatin in the vascular basement membrane of some organs such as the kidney suggests that it may exhibit unique advantages in targeting tumors that develop in some organs. Ultimately, it is desirable to use gene transfer methods to develop alternative ways of expressing tumstatin genes in tumor vasculature in vivo (Kashihara, N. et al., 1997, Exp. Nephrol. 5: 126- 31; Maeshima, Y. et al., 1996, J. Am. Soc. Nephrol. 7: 2219-29; Maeshima, T. et al., 1998, J. Clin. Invest. 101: 2589-97).

  Since the distribution of α3 (IV) chain is limited to the basement membrane of some organs, considering the mechanism that tumstatin molecules can exhibit in inhibiting α chain assembly, tumstatin may be less harmful high. In addition, α3 (IV) chains are also found in the vascular basement membrane of the kidney (Kalluri, R. et al., 1997, J. Clin. Invest. 99: 2470-8), and these vessels are renal cell carcinoma It is thought to be involved in the progression of primary renal tumors. Therefore, tumstatin may be effective in treating the tumor by destroying the assembly of α3 (IV) chain and other α chains.

  As used herein, the term “angiogenesis” means the creation of new blood vessels in one tissue or organ and includes endothelial cell proliferation. Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific and limited situations. For example, angiogenesis is usually found in wound healing, fetal and embryonic development, and the formation of the corpus luteum, endometrium and placenta. The term “endothelium” means a thin layer of flat epithelial cells that lining the serous cavities, lymphatic vessels, and blood vessels. Thus, “anti-angiogenic activity” refers to the ability of a composition to inhibit blood vessel growth. The growth of blood vessels is a complex sequence of events that involves the local degradation of the basement membrane beneath each endothelial cell, the proliferation of these cells, the migration of the cells to future blood vessels, and the formation of new vascular membranes. Reconstitution of the cells to form, arrest of endothelial cell proliferation, and uptake of cells that support perivascular cells and other new vessel walls. Thus, as used herein, “anti-angiogenic activity” includes inhibition of some or all of these steps, which ultimately inhibits the formation of new blood vessels. .

  Anti-angiogenic activity generally involves the ability of a composition to inhibit angiogenesis, such as cultured bovines in the presence of fibroblast growth factors, angiogenesis-related factors, or other known growth factors. It includes endothelial inhibition activity, which refers to the ability to inhibit the growth or migration of capillary endothelial cells. A “growth factor” is a composition that stimulates cell growth, regeneration, or synthetic activity. “Angiogenesis-related factors” are factors that inhibit or promote angiogenesis. Specific examples of angiogenesis-related factors include angiogenesis growth factors such as basic fibroblast growth factor (bFGF), which is an angiogenesis promoter. Other specific 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, WO96 / 35774, WO95 / 29242, WO96 / 41194, WO97 / 23500) or endostatin (eg An angiogenesis inhibitor such as WO97 / 15666).

  “Substantially the same physiological activity” or “substantially the same or more physiological activity” means that a composition has an anti-angiogenic activity, measured by a conventional assay, It means to behave similarly to statins and tumstatins. “Regular assays” include, but are not limited to, protocols used in the field of molecular biology to assess anti-angiogenic activity, cell cycle arrest, and apoptosis. Such assays include endothelial cell proliferation, endothelial cell migration, cell cycle analysis, and endothelial cell tube formation assays, such as detection of apoptosis by apoptotic cell morphology or annexin V-FITC assay, chorioallantoic membrane (CAM) assay, and nudity Inhibition of renal cancer tumor growth in mice includes, but is not limited to. Such an assay is shown in the examples below.

  “Arresten”, also referred to herein as “arrestin”, includes fragments, variants, homologs, analogs, and allelic variants of Arresten sequences and Arresten amino acid sequences derived from other mammals, and Arresten amino acid sequences Fragments, variants, homologues, analogs and allelic variants.

  As used herein, “canstatin” refers to canstatin sequences as well as fragments, variants, homologues, analogs, and allelic variants of canstatin amino acid sequences from other mammals, and canstatin amino acid sequences. Fragments, variants, homologues, analogs and allelic variants.

  As used herein, “tumstatin” refers to tumstatin sequences as well as fragments, variants, homologs, analogs and allelic variants of tumstatin amino acid sequences from other mammals, and fragments of tumstatin amino acid sequences, Mutants, homologues, analogs and allelic variants are intended to be included.

  The present invention relates to endothelial inhibitory activity (eg, the ability of certain compositions to generally inhibit angiogenesis, such as cultured bovine capillary in the presence of fibroblast growth factor, angiogenesis-related factor, or other known growth factors). It is understood to encompass any derivative of Arresten, Canstatin or Tumstatin that has the ability to inhibit the growth or migration of vascular endothelial cells). The present invention encompasses whole Arresten, Canstatin or Tumstatin proteins, derivatives of these proteins and bioactive fragments of these proteins. These include Arresten, Canstatin or Tumstatin active proteins having amino acid substitutions or having sugars or other molecules attached to amino acid functional groups.

  The present invention also describes Arresten, Canstatin and Tumstatin fragments, variants, homologs and analogs. An Arresten, Canstatin or Tumstatin “fragment” is an amino acid sequence shorter than an Arresten, Canstatin or Tumstatin molecule consisting of at least 25 contiguous amino acids of Arresten, Canstatin or Tumstatin polypeptide. Such molecules may or may not further include additional amino acids obtained from the cloning process, such as amino acid residues or amino acid sequences corresponding to full length or partial linker sequences, for example. In order to be included in the scope of the present invention, such a mutant must have substantially the same physiological activity as the natural or full-length form of the reference polypeptide, regardless of the presence or absence of the additional amino acid residues. Don't be.

  One such fragment, called “tumstatin N-53”, was found to have anti-angiogenic activity equivalent to full-length tumstatin as measured by routine assays. Tumstatin N-53 consists of a tumstatin molecule lacking the N-terminal 53 amino acids. Other mutant fragments described herein have been found to have very high levels of anti-angiogenic activity, as demonstrated by the assays described herein. These fragments, “Tumstatin 333”, “Tumstatin 334”, “12 kDa Arresten Fragment”, “8 kDa Arresten Fragment”, and “10 kDa Canstatin Fragment” are 75 ng / ml, 20 ng / ml, 50 ng / ml, respectively. ED50 values of 50 ng / ml and 80 ng / ml. On the other hand, full length Arresten, Canstatin and Tumstatin were found to have ED50 values of 400 ng / ml, 400 ng / ml and 550 ng / ml, respectively. Tumstatin 333 consists of amino acids 2 to 125 of SEQ ID NO: 10, and tumstatin 334 consists of amino acids 126 to 245 of SEQ ID NO: 10.

  By “variant” of Arresten, Canstatin or Tumstatin is meant a polypeptide having an amino acid sequence change closely related to the amino acid sequence of an equivalent reference Arresten, Canstatin or Tumstatin polypeptide. Such changes may occur naturally or by human manipulation, by chemical energy (eg X-rays), by other forms of chemical mutagenesis, by genetic engineering, mating or other forms of genetic information. As a result of exchange. Mutations include, for example, base changes, deletions, insertions, inversions, transpositions, or duplications. Arresten, Canstatin or Tumstatin variant forms may have increased or decreased anti-angiogenic activity relative to an equivalent reference Arresten, Canstatin or Tumstatin polypeptide, such variants may be, for example, in whole or in part Additional amino acids obtained from the cloning process, such as amino acid residues or amino acid sequences corresponding to the linker sequence, may or may not be included.

  Variants / fragments of the anti-angiogenic proteins of the invention can be generated by PCR cloning. As described in Example 18 below, FIG. As shown in 23 and 24, fragments called “Tumstatin 333” and “Tumstatin 334” were generated in this manner and were found to have superior anti-angiogenic activity over full-length Tumstatin. To create such fragments, PCR primers are designed from known sequences in such a way that each set of primers amplifies known subsequences from the entire protein. These subsequences are then cloned into a suitable expression vector such as the pET22b vector and the anti-angiogenic activity of the expressed protein is tested as described in the assay below.

  Variants / fragments of the anti-angiogenic proteins of the present invention are described by Mariyama, M. et al. (1992, J. Biol. Chem. 267: 1253-8) and as described in Example 24 below. It can also be produced by Pseudomonas elastase digestion. Using this method, 12 kDa and 8 kDa arrestin mutants and 10 kDa canstatin mutants were produced, all three having higher levels of anti-angiogenic activity than the original full-length protein.

  An “analog” of Arresten, Canstatin, or Tumstatin is a non-naturally occurring molecule that is substantially the same as any Arresten, Canstatin, or Tumstatin molecule or a fragment or allelic variant thereof. Or a molecule having a physiological activity higher than that. Such analogs are bioactive Arresten, Canstatin or Tumstatin, and fragments, variants, homologues, and allelic variant derivatives thereof (eg, chemical derivatives as defined above), wherein the unmodified Arresten, Canstatin Or a derivative that exhibits an agonistic or antagonistic effect qualitatively similar to a tumstatin polypeptide, fragment, variant, homolog, or allelic variant.

  An “allele” of Arresten, Canstatin or Tumstatin means a polypeptide sequence containing a native sequence variation relative to the polypeptide sequence of a reference Arresten, Canstatin or Tumstatin polypeptide. An “allele” of a polynucleotide encoding an Arresten, Canstatin or Tumstatin polypeptide is a polynucleotide sequence containing a sequence variation relative to a reference polynucleotide sequence encoding a Reference Arresten, Canstatin and Tumstatin polypeptide. Thus, an allelic variant of a polynucleotide encoding an Arresten, Canstatin or Tumstatin polypeptide means a polynucleotide sequence encoding an allelic form of Arresten, Canstatin or Tumstatin polypeptide.

  The particular polypeptide may be any one of Arresten, Canstatin or Tumstatin fragments, variants, analogs, or allelic variants, or two or more of these, For example, one polypeptide may be an analog and variant of Arresten, Canstatin or Tumstatin polypeptide. For example, shortened versions of Arresten, Canstatin or Tumstatin molecules (eg, Arresten, Canstatin or Tumstatin fragments) can be made in the laboratory. The fragment is then mutagenized by means known in the art to produce molecules that are fragments and variants of Arresten, Canstatin or Tumstatin. In another embodiment, variants can be made that later become apparent in some mammalian individuals as allelic forms Arresten, Canstatin or Tumstatin. Accordingly, such mutant Arresten, Canstatin or Tumstatin molecules are mutant and allelic variants. Such combinations of fragments, variants, allelic variants, and analogs are intended to be included within the scope of the present invention.

  For example, tumstatin produced by the E. coli expression cloning method described in Example 18 below is a monomer. Since E. coli expression cloning methods add polylinker sequences and histidine tags that are not present in the native protein to the expressed protein, it is also a fusion or chimeric protein. The tumstatin fragment “Tumstatin N-53”, also described in Example 18, is a full-length tumstatin protein fragment and deletion mutant, which also adds additional sequences when made by the same E. coli expression cloning method. This is also a fusion or chimeric variant fragment of the full-length tumstatin protein. When this tumstatin N-53 subunit is integrated into, for example, a dimer or trimer, a multimeric fusion of chimeric mutant fragments of tumstatin protein is produced.

  Also included within the scope of the invention are proteins having substantially the same amino acid sequence as Arresten, Canstatin or Tumstatin, or polynucleotides having a nucleic acid sequence substantially identical to a polynucleotide encoding Arresten, Canstatin or Tumstatin. It is. A “substantially identical sequence” means at least about 70% sequence homology with a reference sequence such as another nucleic acid or polypeptide, usually at least about 80% sequence homology with the reference sequence, preferably at least about By nucleic acid or polypeptide is meant 90% sequence homology, more preferably at least about 95% homology, most preferably at least about 97% sequence homology with a reference sequence. The comparative length of the sequence is usually at least 75 nucleotide bases or 25 amino acids, more preferably at least 150 nucleotide bases or 50 amino acids, most preferably 243 to 264 nucleotide bases or 81 to 88 amino acids. is there. As used herein, “polypeptide” refers to a molecular chain of amino acids and does not refer to a product of a particular length. That is, peptides, oligopeptides and proteins are included in the definition of polypeptide. This term also encompasses polypeptides that have already undergone post-expression modifications such as glycation, acetylation, phosphorylation.

  As used herein, “sequence homology” refers to subunit sequence similarity between two polymeric molecules such as, for example, two polynucleotides or two polypeptides. For example, if the position in each of the two peptides is occupied by serine, if the subunit positions in both of the two molecules are occupied by the same monomeric subunit, these are Become homologous. The homology between two sequences is directly proportional to the number of matching or homologous positions, e.g., half of the positions in two peptide or compound sequences (e.g. 5 in a polymer 10 subunits long). If two positions are homologous, the two sequences are 50% homologous, for example 90% of the number of positions, eg 9 out of 10, match the two sequences with 90% sequence homology. There will be. Illustratively, the amino acid sequences R2 R5 R7 R10 R6 R3 and R9 R8 R1 R10 R6 R3 share three of the six positions, so there is 50% sequence homology, and the sequences R2 R5 R7 R10 R6 R3 and R8 R1 R10 R6 R3 have five Since 3 of the positions are shared, there is 60% sequence homology. The homology between two sequences is directly proportional to the number of matching positions, ie the number of homologous positions. Therefore, when a part of the reference sequence deletes a specific peptide, the deleted part is not calculated for the purpose of calculating the sequence homology. For example, R2 R5 R7 R10 R6 R3 and R2 R5 R7 R10 R3 Since 5 of the positions are shared, there is 83.3% sequence homology.

  Homology is often measured using sequence analysis software such as BLASTN or BLASTP (available from http://www.ncbi.nlm.nih.gov/BLAST/). Compare two sequences by BLASTN (for nucleotide sequences) (eg, “Blasting” the two sequences against each other, http://www.ncbi.nlm.nih.gov/gorf/bl2. The default parameters for html) are match reward = 1, mismatch penalty = -2, open gap = 5, extension gap = 2. The default parameters when using BLASTP for protein sequences are match reward = 0, mismatch penalty = 0, open gap = 11, and extension gap = 1.

  When two sequences have “sequence complementarity”, it means that the two sequences differ from each other only by conservative substitutions. In the case of polypeptide sequences, such conservative substitutions can be made by replacing one amino acid at a particular position in the sequence with another amino acid of the same class (eg, valine for leucine, arginine for lysine, hydrophobicity, charge, pK Or other amino acids that share configurational or chemical properties. Or one or more non-conservative amino acid substitutions, deletions or insertions at sequence positions that do not change the configuration or folding of the polypeptide to such an extent that the biological activity of the polypeptide is destroyed. is there. A specific example of “conservative substitution” is that one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine is replaced with another under the condition that the polypeptide exhibits essential bioactivity. Substituted; one polar (hydrophilic) residue substituted with another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; lysine, arginine or histidine One basic residue such as substituted with another; or one acidic residue such as aspartic acid or glutamic acid substituted with another; or in place of a non-derivatized residue And those using chemically derivatized residues. Two sequences having sequence complementarity are sometimes referred to as “sequence homologs”.

  Polypeptide complementarity is typically measured using sequence analysis software (eg, Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Protein analysis software matches similar sequences by assigning degrees of complementarity to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups. That is, glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

  Chemical derivatives of Arresten, Canstatin and Tumstatin are also within the scope of the present invention. “Chemical derivative” refers to a polypeptide of interest having one or more residues chemically derivatized by reaction of a functional side group. As such derivatized residues, for example, a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. Molecule. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-imbenzyl histidine. Chemical derivatives also include peptides containing one or more natural amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be substituted with proline; 5-hydroxylysine may be substituted with lysine; 3-methylhistidine may be substituted with histidine; homoserine is substituted with serine Ornithine may be substituted with lysine.

  The invention also encompasses fusion and chimeric proteins consisting of anti-angiogenic proteins, fragments, variants, homologs, analogs, and allelic variants thereof. Fusion or chimeric proteins can be produced by recombinant expression and cloning processes, for example, proteins consisting of additional amino acids or amino acid sequences corresponding to full-length or partial linker sequences can be produced, eg, Those produced in E. coli (see Example 2 below) have an additional vector sequence containing a histidine tag added to the protein. As used herein, “fusion or chimeric protein” is intended to encompass this type of change to the original protein sequence. Similar changes were made for canstatin and tumstatin proteins (Examples 11 and 18 respectively). A fusion or chimeric protein can consist of a multimer of a single protein, for example, an anti-angiogenic protein or a repeating unit of a fusion and chimeric protein can be several proteins, such as several of the anti-angiogenic proteins It may be made of. The fusion or chimeric protein is a combination of two or more known anti-angiogenic proteins (eg, angiostatin and endostatin, or a bioactive fragment of angiostatin and endostatin), or one anti-angiogenic protein and targeting A combination of agents (eg, endostatin and epidermal growth factor (EGF) or RGD peptide), or a combination of an anti-angiogenic protein and an immunoglobulin molecule (eg, endostatin and IgG, especially IgG with the Fc portion removed) ). Fusion and chimeric proteins can also include anti-angiogenic proteins, their fragments, mutants, homologs, analogs, and allelic variants, as well as other anti-angiogenic proteins such as endostatin or angiostatin. Other anti-angiogenic proteins include restin and apomigren (PCT / US98 / 26058, the teachings of which are incorporated herein by reference) and endostatin fragments (PCT / US98 / 26057, the teachings of this document are also included herein by reference). As used herein, a “fusion protein” or “chimeric protein”, for example, delivers a chemotherapeutic agent in which a polynucleotide encoding a chemotherapeutic agent is linked to a polynucleotide encoding an anti-angiogenic protein. It may also include additional components for. A fusion or chimeric protein may include multimers of anti-angiogenic proteins such as dimers and trimers. Such fusion or chimeric proteins can be linked (eg, chemically linked) via post-translational modifications, but the entire fusion protein may be produced by recombinant methods.

  Multimeric proteins consisting of Arresten, Canstatin, Tumstatin, their fragments, mutants, homologs, analogs and allelic variants are also intended to be within the scope of the present invention. “Multimer” means a protein composed of two or more copies of a subunit protein. The subunit protein may be one of the proteins of the invention, eg a fragment in which Arresten is repeated more than once, or a fragment, variant, homologue, such as a tumstatin variant or fragment such as tumstatin 333, An analog or allelic variant may be repeated more than once. Such multimers may be fusion or chimeric proteins, eg, combining a repeat tumstatin variant with a polylinker sequence that may be present in a single copy, and / or one or more anti-angiogenic peptides. Or may be tandemly repeated, for example, one protein may consist of two or more multimers within the whole protein.

  The present invention relates to compositions consisting of one or more isolated polynucleotides encoding Arresten, Canstatin or Tumstatin, and vectors and host cells containing such polynucleotides, as well as Arresten, Canstatin and Tumstatin, and fragments thereof Also included are methods for producing mutants, homologs, analogs and allelic variants. As used herein, the term “vector” refers to a carrier into which nucleic acid pieces may be inserted or cloned, and which functions to introduce the nucleic acid fragments into a host cell. Means carrier. Such vectors may provide for replication and / or expression of the introduced nucleic acid fragment. Specific examples of vectors include nucleic acid molecules derived from non-viral vectors such as plasmids, bacteriophages, or mammalian, plant or insect viruses, or ligand-nucleic acid conjugates, liposomes, or lipid-nucleic acid complexes. Desirably, the introduced nuclear molecule is operably linked to an expression control sequence to form an expression vector capable of expressing the introduced nucleic acid. Such introduction of nucleic acid is commonly referred to as “transformation” and refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method used for the insertion. For example, direct uptake, transduction or f-mating is included. The foreign polynucleotide can be maintained as a non-integrated vector, such as a plasmid, or alternatively can be integrated into the host genome. “Operably linked” refers to a situation in which the components are in a relationship that allows them to function in an intended manner, eg, a control sequence “operably linked” to a coding sequence Ligation is carried out in such a way that the expression of the sequence is carried out under conditions suitable for the control sequence. A “coding sequence” is a polynucleotide sequence that is transcribed into mRNA and translated into a polypeptide when placed in control of appropriate regulatory sequences (eg, operably linked). The boundaries of the coding sequence are determined by a translation start codon at the 5 'end and a translation stop codon at the 3' end. Such boundaries can occur naturally, but can also be introduced or added to polynucleotide sequences by methods known in the art. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

  The vector from which the cloned polynucleotide is cloned can be selected because it functions in prokaryotes, but may also be selected because it functions in eukaryotes. Two specific examples of vectors that can both clone polynucleotides encoding Arresten, Canstatin, and Tumstatin proteins and express these proteins from the polynucleotides are protein expression in bacteria and yeast, respectively. Possible pET22b and pET28 (a) vectors (Novagen, Madison, Wisconsin, USA) and modified pPICZαA vectors (In Vitrogen, San Diego, California, USA). (See, eg, PCT / US98 / 25892, the teachings of which are incorporated herein by reference).

  Once the polynucleotide is cloned into a suitable vector, it can be transformed into a suitable host cell. By “host cell” is meant a cell that has already been used or can be used as a recipient for introduced nucleic acid via a vector. The host cell can be a prokaryotic or eukaryotic cell, a mammal, a plant, or an insect, and exists as a single cell or as a collection, for example as a culture, or in tissue culture, or in a tissue or organism Can do. Host cells can also be obtained from normal or diseased tissues of multicellular organisms such as mammals. As used herein, a host cell is intended to include not only the original cell transformed with a nucleic acid, but also the progeny of such a cell that still contains the nucleic acid.

  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 can include at least one additional codon encoding at least one additional amino acid. Usually, the linker consists of 1 to 20 or 30 amino acids. The polynucleotide linker is translated in the same manner as the polynucleotide encoding the anti-angiogenic protein, resulting in the expression of an anti-angiogenic protein having at least one additional amino acid residue at the amino or carboxyl terminus of the anti-angiogenic protein. . It is important that the one or more additional amino acids do not reduce the activity of the anti-angiogenic protein.

  After the polynucleotide is selected and inserted into the vector, the vector is transformed into an appropriate prokaryotic system and the system is cultured under culture conditions suitable for production of the bioactive anti-angiogenic protein (eg, passage To produce a bioactive anti-angiogenic protein, or a variant, derivative, fragment or fusion protein thereof. In one aspect, the present invention comprises the steps of cloning a polynucleotide encoding an anti-angiogenic protein into the vector pET22b, pET17b or pET28a and then transforming these vectors into bacteria. The bacterial host system then expresses the anti-angiogenic protein. Usually, the anti-angiogenic protein is produced in an amount of about 10-20 milligrams or more per liter of culture solution.

  In another embodiment of the invention, the eukaryotic cell vector consists of a modified yeast vector. One method is to use the pPICzα plasmid containing multiple cloning sites. The multiple cloning site is His. A tag motif is inserted into the multiple cloning site. In addition, the vector can be modified to add an NdeI site or other suitable restriction site. Such sites are known to those skilled in the art. The anti-angiogenic protein produced according to this embodiment comprises a histidine tag motif (His. Tag) consisting of one or more histidines, usually about 5-20 histidines. The tag must not inhibit the anti-angiogenic properties of the protein.

  For example, one method of producing Arresten, Canstatin or Tumstatin amplifies a polynucleotide of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9, respectively, for example, pET22b, pET28 (a), pPICZαA Or cloned into other expression vectors, the vector containing the polynucleotide is transformed into a host cell capable of expressing the polypeptide encoded by the polynucleotide, and the transformed host cell is transformed into the protein Culturing under culture conditions suitable for expression of the protein, and then extracting and purifying the protein from the culture. Representative methods for producing generally anti-angiogenic proteins, specifically Arresten, Canstatin and Tumstatin are shown in the Examples below. Arresten, Canstatin or Tumstatin protein is transgenic, for example as a product of transgenic animals such as as a component of transgenic female cow, goat, sheep or pig milk, or combined or linked with starch molecules in eg corn It may be expressed as a plant product.

  Arresten, canstatin or tumstatin may be produced by a conventionally known chemical synthesis method. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed Arresten, Canstatin or Tumstatin protein sequences share primary, secondary or tertiary structural and / or configurational properties with, for example, recombinantly produced Arresten, Canstatin or Tumstatin Therefore, biological properties including physiological activity may be commonly held with those. That is, synthetically constructed Arresten, Canstatin or Tumstatin protein sequences are recombinantly produced and purified Arresten, for example, in screening therapeutic compounds and in immunological processes for antibody production, It may be used as a biologically active or immunological substitute for canstatin or tumstatin protein.

  Arresten, Canstatin and Tumstatin proteins are useful in inhibiting anti-angiogenesis as measured in routine assays as shown in the Examples below. Arresten, Canstatin or Tumstatin does not inhibit the growth of other cell types, for example cells other than endothelial cells.

  Polynucleotides encoding Arresten, Canstatin or Tumstatin can be cloned from isolated DNA or cDNA libraries. Nucleic acid polypeptides, referred to herein as “isolated,” are substantially free of the biological source material from which they were obtained (eg, present in a mixture of nucleic acids or in a cell). Ie nucleic acids or polypeptides that are completely separated) and may be further processed. “Isolated” nucleic acids or polypeptides include nucleic acids or polypeptides obtained by the methods described herein, similar methods, or other suitable methods, including substantially pure nucleic acids or polypeptides, A nucleic acid or polypeptide produced by chemical synthesis, a nucleic acid or polypeptide produced by a combination of chemical or biological methods, and an isolated nucleic acid or polypeptide produced by a recombinant method included. Thus, an isolated polypeptide refers to one that is relatively free of other proteins, carbohydrates, lipids, and other cellular components with which it is normally associated. Isolated nucleic acid is not directly contiguous (ie, covalently linked) to both nucleic acids that are directly contiguous in the natural genome of the organism from which the nucleic acid was derived. Thus, the term is independent of other nucleic acids, such as nucleic acids incorporated into vectors (eg, self-replicating viruses or plasmids), or nucleic acid fragments produced by chemical means or restriction endonuclease treatment. Including nucleic acids that exist as molecules.

  The polynucleotides and proteins of the invention can also be used to design probes for isolating other anti-angiogenic proteins. A special method is shown in US Pat. No. 5,837,490 of Jacobs et al., The entire teachings of which are hereby incorporated by reference in their entirety. The design of the oligonucleotide probe preferably follows the following items. That is, (a) the probe must be designed for the area of the sequence that has the least number of ambiguous bases ("N") if bases are present. Also, (b) the probe must be designed to have a Tm of about 80 ° C. (assuming 2 ° C. for each A or T, 4 degrees for each G or C).

Oligonucleotides are preferably labeled with g- ³² P-ATP (specific activity 6000 Ci / mmole) and T4 polynucleotide kinase using conventional oligonucleotide labeling techniques. Other labeling techniques can also be used. Unincorporated label is preferably removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe must be quantified by measurement in a scintillation counter. The specific activity of the resulting probe is preferably about 4 × 10 ⁶ dpm / pmole. Preferably, the bacterial culture containing the pool of full-length clones is thawed and inoculated with 100 μl stock solution into a sterile culture flask containing 25 ml of sterile L broth containing 100 μg / ml ampicillin. The culture is preferably cultured at 37 ° C. until saturation, and the saturated culture is preferably diluted with fresh L broth. A solid bacterium containing L broth containing 100 μg / ml ampicillin and 1.5% agar in a 150 mm Petri dish when a certain amount of these dilutions were plated and incubated overnight at 37 ° C. It is preferable to determine the dilution and volume that yield about 5000 distinct and well-separated colonies on the medium. Other known methods for obtaining clear and well-separated colonies can also be used.

  The colonies must then be transferred to nitrocellulose filters and lysed, denatured and heated using routine procedures for colony hybridization. High stringency conditions refer to conditions that are at least as stringent as, for example, treatment with 1 × SSC at 65 ° C., or 1 × SSC and 50% formamide at 42 ° C. Moderate stringent conditions refer to conditions that are at least as stringent as treatment with 4 × SSC at 65 ° C. or 4 × SSC and 50% formamide at 42 ° C. Low stringency conditions refer to conditions that are at least as stringent as treatment with 4 × SSC at 50 ° C., or 6 × SSC and 50% formamide at 40 ° C.

Filters were then added to 6x SSC containing 0.5% SDS, 100 μg / ml yeast RNA, and 10 mM EDTA (20 × stock solution was 175.3 g NaCl per liter, 88.2 g per liter quencher. It is preferable to keep the temperature at 65 ° C. for 1 hour while slowly stirring in acid Na adjusted to pH 7.0 with NaOH (about 10 mL per 150 mm filter). The probe is then preferably added to the hybridization mixture at a concentration greater than or equal to 1 × 10 ⁶ dpm / mL. The filter is then preferably kept overnight at 65 ° C. with slow stirring. The filter is then preferably washed in 500 mL of 2 × SSC / 0.5% SDS at room temperature without agitation followed by 15 in 500 mL of 2 × SSC / 0.1% SDS with shaking at room temperature. It is preferred to wash for a minute. A third wash with 0.1 × SSC / 0.5% SDS at 65 ° C. may be performed for 30 minutes to 1 hour. The filter is then preferably dried and subjected to autoradiography for a time sufficient to visualize positives on X-ray film. Other known hybridization methods can also be used. The positive clones are then picked and cultured, and plasmid DNA is isolated using conventional methods. The clones can then be confirmed by restriction enzyme analysis, hybridization analysis, or DNA sequencing.

  The present invention includes a method of inhibiting angiogenesis in mammalian tissue using Arresten, Canstatin, Tumstatin or a bioactive fragment, analog, homolog, derivative or variant thereof. More specifically, the present invention relates to an angiogenesis-related disease in combination with one or more anti-angiogenic proteins or one or more bioactive fragments thereof, or fragments having anti-angiogenic activity, or agonists. And methods of treating with an effective amount of an antagonist. An effective amount of an anti-angiogenic protein is an amount sufficient to completely or partially ameliorate the disease or condition by inhibiting angiogenesis that causes the disease or condition. An improvement in a disease involving angiogenesis can be determined by observing an improvement in the symptoms of the disease, such as a reduction in tumor size or tumor growth arrest. As used herein, the term “effective amount” refers to a meaningful benefit for the patient, ie the treatment, cure, prevention or alleviation of the associated medical condition, or the rate of treatment, cure, prevention or alleviation of such condition. Or the total amount of each active ingredient in the composition or method sufficient to show an increase in When applied to a combination, the term refers to the total amount of active ingredient that provides the therapeutic effect, whether administered concomitantly, sequentially or simultaneously. Diseases involving angiogenesis include cancer, solid tumors, hematologic tumors (eg leukemia), tumor metastasis, benign tumors (eg hemangiomas, auditory neuromas, neurofibromas, trachoma, and pyogenic granulomas), chronic joints Rheumatism, psoriasis, ocular angiogenic diseases (eg diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, angioplasty glaucoma, post-lens fiber growth, rubeosis), Osler-Weber syndrome, myocardial angiogenesis, new plaque Examples include, but are not limited to, angiogenesis, telangiectasia, hemophilia joints, vascular fiber species, and wound granulation. Anti-angiogenic proteins are useful for the treatment of diseases caused by excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, Crohn's disease, arteriosclerosis, scleroderma, and hyperplastic scars (ie keloids). Anti-angiogenic proteins can be used as birth control agents by preventing angiogenesis necessary for embryo implantation. Anti-angiogenic proteins are useful for the treatment of pathologically angiogenic diseases such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). Anti-angiogenic proteins can also be used to prevent dialysis graft vascular access stenosis and obesity, for example by preventing their hypertrophy by inhibiting capillary formation in adipose tissue . Anti-angiogenic proteins can also be used to treat localized (eg, not metastasized) diseases. “Cancer” means a pathological state of neoplastic growth, hyperplastic or proliferative growth, or abnormal cell growth, including abnormal cell proliferation such as that found in solid tumors, non-solid tumors, and leukemias. Including. As used herein, “cancer” refers to angiogenesis-dependent cancers and tumors, ie, an increase in the number and density of blood vessels that supply blood, for its growth (increase in volume and / or mass). It also means a tumor. “Regression” refers to a reduction in tumor mass and size as measured using methods known to those skilled in the art.

  Alternatively, if increased angiogenesis is desired, for example, in wound healing or in post-infarction heart tissue, antibodies or antisera directed against anti-angiogenic proteins can be used to localize native and anti-angiogenic proteins and Blocking the process can increase the formation of new blood vessels and inhibit tissue atrophy.

  Anti-angiogenic proteins may be used in combination with other compositions and procedures for disease treatment. For example, a tumor may be conventionally treated with surgery, radiation, chemotherapy, or immunotherapy combined with an anti-angiogenic protein, and then the anti-angiogenic protein is administered to the patient to produce microscopically. Metastatic latency may be extended to stabilize and inhibit residual primary tumor growth. Anti-angiogenic proteins, or fragments thereof, antisera, receptor agonists, or receptor antagonists, or combinations thereof may be combined with other anti-angiogenic compounds, or other anti-angiogenic proteins (eg, angiostatin, endo Statins), fragments, antisera, receptor agonists and receptor antagonists. In addition, anti-angiogenic proteins, or fragments thereof, antisera, receptor agonists, receptor antagonists, or combinations thereof, pharmaceutically acceptable excipients, and optionally biodegradable polymers, etc. In combination with a sustained release matrix of to form a therapeutic composition. The compositions of the present invention may further comprise other anti-angiogenic proteins or chemical compounds, such as endostatin or angiostatin, and variants, fragments and analogs thereof. The composition may further contain other agents such as chemotherapeutic agents and radioactive agents that enhance the activity of the protein or enhance its activity or use during treatment. Such additional factors and / or agents can be included in the composition to produce a synergistic effect with the protein of the present invention or to minimize side effects. Also, administration of the compositions of the present invention can be administered in combination with other therapies, such as, for example, in combination with chemotherapy or radiation therapy regimens.

  The present invention includes a method of inhibiting angiogenesis in mammalian tissue by contacting the tissue with a composition comprising the protein of the present invention. By “contacting” is meant not only topical application, but also a mode of delivery that introduces the composition into the tissue or into cells of the tissue.

  The use of slow or sustained release delivery systems is also included in the present invention. Such systems are particularly desirable in situations where surgery is difficult or impossible, such as patients who are debilitating due to age or disease course itself, or where control is required rather than cure in risk / effect analysis.

  As used herein, a sustained release matrix is a matrix made of a material, usually a polymer, that can be degraded by enzymatic or acid / base hydrolysis or by dissolution. The matrix inserted into the body is acted on by enzymes and body fluids. Sustained release matrices include liposomes, polylactide (polylactic acid), polyglycolide (glycolic acid polymer), polylactide coglycolide (copolymer of lactic acid and glycolic acid) polyanhydride, poly (ortho) ester, polyprotein, hyaluron Selected from acids, collagens, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, amino acids such as polyamino acids, phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and biocompatible substances such as silicon It is desirable that A preferred biodegradable matrix includes a matrix of any one of polylactide, polyglycolide, or polylactide coglycolide (copolymer of lactic acid and glycolic acid).

  The angiogenesis regulating composition of the present invention may be a solid, liquid or aerosol, and can be administered by a known administration route. Specific examples of solid compositions include tablets, creams, and implant dosage forms. Tablets can be administered orally and therapeutic creams can be administered topically. The implant dosage form can be administered locally, for example, at the tumor site, but may be placed, for example, subcutaneously for the purpose of systemic release of the angiogenesis modulating composition. Specific examples of liquid compositions include formulations for subcutaneous, intravenous, intraarterial injection, and formulations for topical and intraocular administration. Specific examples of aerosol formulations include inhalation formulations for pulmonary administration.

  The above-mentioned proteins and protein fragments having anti-angiogenic activity are provided as isolated and substantially purified proteins and protein fragments as pharmaceutically acceptable formulations using formulation methods known to those skilled in the art. Can do. These formulations can be administered by conventional routes. Generally, the composition is topical, transdermal, intraperitoneal, intracranial, cerebrospinal fluid, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (eg, intravenous, intraspinal, subcutaneous or It may be administered by the intramuscular) route. Alternatively, the anti-angiogenic protein can be incorporated into a biodegradable polymer that slowly releases the compound and the polymer is implanted near a location where drug delivery is desired, such as a tumor site, or the anti-angiogenic protein. May be implanted so that is gradually released systemically. An osmotic minipump may be used to control delivery of a high concentration of anti-angiogenic protein via a cannula to the site of interest, such as directly to the vasculature to metastases or tumors. Biodegradable polymers and their uses are described in detail, for example, in Brem et al. (1991) (J. Neurosurg. 74: 441-446), which is hereby incorporated by reference in its entirety. .

  A composition containing a polypeptide of the invention can be administered intravenously, such as by unit dose injection. As used with the therapeutic compositions of the present invention, the term “unit dose” refers to a physically distinct unit that is appropriate as a unit dosage for a subject, where each unit is a required diluent, ie, It contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the carrier or vehicle.

  Modes of administration of the compositions of the present invention include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Parenteral injectable pharmaceutical compositions are reconstituted into pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile injectable solutions or dispersions just prior to use. Consists of sterile powder. Specific examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (eg glycerin, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (eg olive oil) And injectable organic esters such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. These compositions may further contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbic acid, may be included to ensure prevention of microbial activity. It is also desirable to include isotonic agents such as sugars and sodium chloride. Long-term absorption of a pharmaceutical dosage form for injection may be caused by including an agent such as aluminum monostearate or gelatin that delays absorption. Accumulated injection forms are made by forming a microcapsule matrix of 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 employed, the rate of drug release can be controlled. Accumulated injectable formulations may be prepared by encapsulating the drug in biohistocompatible liposomes or microemulsions. The injectable formulation is prepared by adding a sterilant, for example by filtration through a bacteria capture filter, or by adding a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. It may be sterilized.

  The therapeutic compositions of the present invention can include pharmaceutically acceptable salts of the components, which may be obtained, for example, from inorganic or organic acids. “Pharmaceutically acceptable salt” is used within the scope of justified medical judgment and is in contact with human and lower animal tissues without undesirable toxicity, irritation, allergic reaction, etc. Means a salt that is suitable for, and corresponds to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are known in the art. For example, S.M. Berge et al. Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1 et seq., Which is incorporated herein by reference. Pharmaceutically acceptable salts include, for example, acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid (formed with the free amino group of the polypeptide) Is mentioned. Salts formed with free carboxyl groups are obtained from, for example, inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine You can also The salts may be prepared in situ when the compound of the invention is finally isolated and purified, or separately by reacting the free base functionality with a suitable organic acid. Typical acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, disulfate, butyrate, camphorate, camphorsulfonate Salt, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxymethanesulfonate (isethion) Acid salt), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, picrin , Pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoic acid They include, but are not limited to. Also, basic nitrogen-containing groups include methyl, ethyl, propyl, and lower alkyl halides such as butyl chloride, bromide and iodide; dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfate; decyl, It can be quaternized with agents such as long chain halides such as lauryl, myristyl and stearyl chlorides, bromides and iodides; allyl alkyl halides such as benzyl and phenethyl bromides. In this way a water or fat-soluble or dispersible product is obtained. Specific examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and succinic acid, maleic acid, succinic acid and Examples include organic acids such as citric acid.

  As used herein, when the terms “pharmaceutically acceptable”, “physiologically tolerated” and their grammatical variations are referred to for compositions, carriers, diluents and reagents, Used interchangeably to indicate that the substance can be administered to mammals with little undesirable physiological effects such as nausea, dizziness, stomach upset. The preparation of a pharmacological composition that contains a dissolved or dispersed active ingredient is obvious in the art and need not be limited depending on the formulation. Such compositions are usually prepared as injections, either as liquid solutions or suspensions, but solid dosage forms suitable for liquid solutions or suspensions prior to use can also be prepared. The preparation can also be emulsified.

  The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerin, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary agents such as wetting or emulsifying agents and pH buffering agents that enhance the effect of the active ingredient.

  The anti-angiogenic protein of the present invention can also be contained in a composition comprising a prodrug. As used herein, the term “prodrug” refers to a compound that is rapidly transformed in vivo to yield the parent compound, for example, by enzymatic hydrolysis in blood. A detailed discussion (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 PermagonPress, 1987 All of these documents are incorporated herein by reference. As used herein, the term “pharmaceutically acceptable prodrug” refers to (1) humans within the scope of sound medical judgment, without undesired toxicity, irritation, allergic reactions, etc. And prodrugs of the compounds of the present invention suitable for use in contact with animal tissues, corresponding to a suitable risk-benefit ratio and effective for their intended use, and (2) In some cases, it refers to the bipolar ion form of the parent compound.

  The dosage of the anti-angiogenic protein of the present invention will depend on other clinical factors, such as the disease state or condition being treated and the weight or condition of the human or animal and the route of administration of the compound. When treating humans or animals, about 10 mg / kg body weight to about 20 mg / kg body weight protein can be administered. For example, in combination therapy, such as using the protein of the invention in combination with radiation therapy, chemotherapy, or immunotherapy, the dosage can be reduced, for example, from about 0.1 mg / kg body weight to about 0.2 mg / kg body weight. Depending on the half-life of the anti-angiogenic protein in a particular animal or human, the anti-angiogenic protein can be administered between several times per day to once a week. It is understood that the present invention has both human and veterinary applications. The method of the present invention allows for single doses as well as multiple doses, whether simultaneous or prolonged. The anti-angiogenic protein can also be administered in combination with other forms of therapy, such as chemotherapy, radiation therapy, or immunotherapy.

  Anti-angiogenic protein formulations include oral, rectal, intraocular (including intravitreal or intraocular), intranasal, topical (including buccal and sublingual), intrauterine, intravaginal or parenteral (subcutaneous) And those suitable for administration (including intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural). Anti-angiogenic protein formulations can be conveniently administered as a unit dosage form and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing the active ingredient into association with one or more pharmaceutical carriers or one or more excipients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

  Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulation can be administered in unit dose or multi-dose containers such as enclosed ampoules and vials, for example, in a lyophilized state by simply adding a sterile liquid carrier such as water for injection just prior to use. Can be saved. Formulation injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

  When an effective amount of the protein of the invention is administered orally, the anti-angiogenic protein of the invention will take the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further contain a solid carrier such as gelatin or an adjuvant. The tablets, capsules and powders contain about 5 to 95% of the protein of the present invention, preferably about 25 to 95% of the protein of the present invention. When administered in liquid form, liquid carriers such as water, petroleum, peanut oil, mineral oil, soybean oil, oil of animal or vegetable origin such as sesame oil, or synthetic oil can be added. The pharmaceutical composition in liquid form may further contain physiological saline, dextrose or other sugar solution, 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 of the protein of the invention, but preferably contains about 1 to 50% of the protein of the invention.

  When an effective amount of the protein of the invention is administered by intravenous, intradermal or subcutaneous injection, the protein of the invention will take the form of a pyrogen-free parenterally acceptable aqueous solution. Such parenterally acceptable protein solutions are considered with respect to pH, isotonicity, stability, etc., and their preparation is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal or subcutaneous injection are “sodium chloride injection”, “Ringer injection”, “dextrose injection” as known in the art, in addition to the protein of the present invention. Must contain an isotonic vehicle such as “dextrose / sodium chloride injection”, “lactated Ringer's injection”, or other vehicle. The pharmaceutical composition of the present invention may further contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art.

  The amount of the protein of the invention in the pharmaceutical composition of the invention will depend on the nature and extent of the condition being treated and the nature of the prior treatment that the patient has received. Ultimately, the attending physician will decide the amount of protein of the invention to use in treating each patient. First, the attending physician administers the protein of the present invention at a low dose and observes the patient's reaction. The protein of the invention is administered at higher doses until the optimal therapeutic effect for the patient is obtained, at which point the dosage is not increased further.

  The duration of intravenous therapy using the pharmaceutical composition of the present invention depends on the extent of the disease being treated, the condition of each patient and the potential idiosyncratic response. The duration of each administration of the protein of the present invention ranges from 12 to 24 hours with continuous intravenous administration. Ultimately, the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

  Preferred unit dosage formulations are those containing a daily dose or unit of administration component, a sub-dose per day, or an appropriate fraction thereof. In particular, in addition to the above-described components, the formulation of the present invention can contain other conventionally known agents in the field appropriate for the target formulation type. Optionally, combination therapy may be provided to the patient by adding cytotoxic agents or otherwise in combination with anti-angiogenic proteins or their physiologically functional protein fragments.

  The therapeutic composition is currently useful for veterinary applications. In addition to humans, particularly livestock animals and thoroughbred horses are desirable for such treatment with the protein of the present invention.

  By linking a cytotoxic agent such as ricin to the anti-angiogenic proteins and fragments thereof, a means of destroying cells that bind to the anti-angiogenic proteins can be provided. These cells are found in many places including, but not limited to, micrometastases and primary tumors. Proteins linked to cytotoxic agents are administered by infusion in a manner designed to maximize delivery to the desired location. For example, a lysine-linked high affinity fragment is delivered via a cannula to a blood vessel supplying blood to the target site or directly to the target. Such agents may be delivered in a controlled manner via an osmotic pump connected to an infusion cannula. Combinations of antagonists to anti-angiogenic proteins may be co-administered with an angiogenesis simulator to increase tissue angiogenesis. This treatment regimen provides an effective means of destroying metastatic cancer.

  Other therapeutic methods include administration of anti-angiogenic proteins, their fragments, analogs, antisera, or receptor agonists and antagonists linked to cytotoxic agents. It is understood that the anti-angiogenic protein may be of human or animal origin. Anti-angiogenic proteins can also be produced synthetically by chemical reactions or by combining recombinant techniques with expression systems. Anti-angiogenic proteins can also be produced by enzymatically degrading isolated type IV collagen to produce proteins with anti-angiogenic activity. Anti-angiogenic proteins may be produced by compounds that mimic the action of endogenous enzymes that degrade type IV collagen to produce the anti-angiogenic proteins. The production of anti-angiogenic proteins may be regulated by compounds that affect the activity of degrading enzymes.

  The present invention also encompasses gene therapy in which a polynucleotide encoding an anti-angiogenic protein or variant, fragment or fusion protein thereof is introduced and regulated in a patient. Various methods, also called gene therapy, that transduce or deliver DNA to cells to express gene product proteins 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 includes the incorporation of DNA sequences into somatic or germ cells for use in either ex vivo or in vivo therapy. Gene therapy replaces genes, enhances normal or abnormal gene function, and functions to combat infections and other pathologies.

  As a method of treating these medical problems by gene therapy, treatments such as adding a functional gene after identifying a defective gene to replace it with the function of the defective gene or strengthening a slightly functioning gene Prophylaxis; or prophylaxis such as treating the disease state or adding a gene for a product protein that makes the tissue or organ more sensitive to the treatment regimen. A specific example of a prophylactic regime may be to prevent the occurrence of angiogenesis by introducing a gene, such as one encoding one or more anti-angiogenic proteins, into the patient's body; After inserting a gene to make it more sensitive, the tumor can be irradiated with radiation to increase the mortality of the tumor cells.

  Many protocols for the introduction of DNA or regulatory sequences of anti-angiogenic proteins are also included in the present invention. Transduction of promoter sequences other than those normally associated with anti-angiogenic proteins or other sequences that increase the production of anti-angiogenic proteins are also considered methods of gene therapy. As a specific example of this technology, Transkaryotic Therapies, Inc., of Cambridge, Mass., Which uses a homologous recombination method in which a “gene switch” that changes to an erythropoietin gene in a cell is inserted is shown. See Genetic Engineering News, Apr. 15, 1994. Such “gene switches” can be used to activate a protein (or receptor) in cells that normally do not express the anti-angiogenic protein (or receptor).

  Gene transfer methods for gene therapy are roughly divided into three categories. That is, physical methods (eg, electroporation, direct gene transfer and particle bombardment), chemical methods (eg, lipid-based carriers, or other non-viral vectors) and biological methods (eg, virus-derived vectors and receptor uptake) ). For example, non-viral vectors comprising liposomes coated with DNA can be used. Such liposome / DNA complexes can be directly injected intravenously into a patient. Liposome / DNA complexes are thought to be concentrated in the liver and deliver DNA to macrophages and Kupffer cells. These cells have a long life, resulting in long-term expression of the delivery DNA. Alternatively, targeted delivery of therapeutic DNA may be achieved by directly injecting the vector, or “naked” DNA of the gene, into the desired organ, tissue or tumor.

  Gene therapy methods can also be described by delivery site. Basic gene delivery means include ex vivo gene transfer, in vivo gene transfer, and in vitro gene transfer. In ex vivo gene transfer, cells are collected from a patient and grown in cell culture. The DNA is transfected into the cells and the transfected cells are allowed to grow several times before returning to the patient. In in vitro gene transfer, cells that grow in a cultured state, such as tissue culture cells, become transformed cells, and specific cells of a specific patient do not fall under this. These “laboratory cells” are transfected and the transfected cells are selected, expanded and used for patient implantation or other applications.

  In vivo gene transfer is the introduction of DNA into cells when the patient's cells are in the patient's body. Methods include using virus-mediated gene transfer with non-infectious viruses to deliver the gene to the patient or injecting bare DNA into a site in the patient's body so that the gene product protein is expressed. Incorporated into a certain ratio of cells. Also, other methods can be used for in vitro insertion of DNA or regulatory sequences that control the production of anti-angiogenic proteins, such as the use of a “gene gun” as described herein.

  As a chemical method of gene therapy, there is a method of allowing DNA to pass through a cell membrane using a lipid-based compound that is not necessarily a liposome. Lipofectin or cytofectin, a lipid-based cation that binds to negatively charged DNA, forms a complex that can cross the cell membrane and deliver DNA into the cell. Another chemical method utilizes receptor-based endocytosis in which specific ligands are bound to cell surface receptors, which are encapsulated and transported through the cell membrane. The ligand binds to DNA and the entire complex is transported into the cell. After the ligand gene complex is injected into the bloodstream, the target cell with the receptor specifically binds to the ligand and transports the ligand-DNA complex into the cell.

  Many gene therapy methods use viral vectors to insert genes into cells. For example, altered retroviral vectors are used in an ex vivo manner to introduce genes into peripheral and tumor infiltrating lymphocytes, hepatocytes, epithelial cells, muscle cells, or other somatic cells. These altered cells are then introduced into the patient to provide a gene product derived from the inserted DNA.

  Viral vectors have also been used to insert genes into cells by in vivo protocols. To cause tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters known to be tissue specific can be used. Alternatively, this expression can be performed in vivo by delivering a DNA or viral vector in situ to a specific anatomical site. For example, in vivo gene transfer into blood vessels has been performed by transplanting transduced endothelial cells in vitro at selected sites on the arterial wall. The virus infected surrounding cells and these cells also expressed the gene product. Viral vectors can be delivered directly to an in vivo site, for example by a catheter, so that only a specific area is infected with the virus, resulting in long-term site-specific gene expression. In vivo gene transfer using retroviral vectors has also been shown to occur in mammalian and liver tissues by injecting modified viruses into blood vessels leading to organs.

  Viral vectors that have been used in gene therapy protocols include other RNA viruses such as retrovirus, poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes virus, SV40, vaccinia and other DNA viruses. . Replication deficient murine retroviral vectors are the most widely used gene transfer vectors. The murine leukemia retrovirus is a single-stranded RNA complexed with a nuclear core protein and a polymerase (pol) enzyme, enclosed in a protein core (gag), and surrounded by a glycoprotein envelope (env) that determines the host range. It consists of what is being. The retroviral genomic structure includes the gag, pol, and env genes closed at the 5 'and 3' long terminal repeat units (LTR). The retroviral vector system is based on the packaging of a vector, as long as there is a minimal vector containing 5 'and 3' LTR and a packaging signal, provided that viral structural proteins are provided in trans positions in the packaging cell line. Take advantage of the fact that it is sufficient to cause migration, infection and uptake into target cells. The basic advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, accurate incorporation of single-copy vectors into target cell chromosomal DNA, and retroviruses. It is easy to manipulate the genome.

  Adenoviruses consist of linear double-stranded DNA complexed with a core protein and surrounded by a capsid protein. Advances in molecular virology have made it possible to take advantage of the biological properties of these organisms to create vectors that can transduce new gene sequences into target cells in vivo. Adenovirus-based vectors express high levels of gene product proteins. Adenoviral vectors have high potency infectivity even with low titer viruses. In addition, since the virus is completely infectious as a cell-free virion, it is not necessary to inject a producer cell line. Another advantage of adenoviral vectors is that long-term expression of heterologous genes can be performed in vivo.

  Mechanical methods of DNA delivery include fusion-inducing lipid vesicles such as liposomes or other membrane fusion vesicles, lipid particles of DNA containing cationic lipids such as lipofectin, polylysine-mediated introduction of DNA, to reproductive or somatic cells Direct injection of DNA such as microinjection of DNA, pneumatic delivery DNA coated particles such as gold particles used in “gene guns”, and inorganic chemical approaches such as calcium phosphate transfection. The particle-mediated gene transfer method was initially used for transformation of plant tissues. A particle bombardment device or “gene gun” generates power and accelerates the DNA-coated high density particles (such as gold or tungsten) to a high speed that allows entry into the target organ, tissue or cell. Particle bombardment can be used in in vitro systems or in conjunction with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs. Another method, ligand-mediated gene therapy, delivers DNA to specific cells or tissues by complexing the DNA with a specific ligand to form a ligand-DNA conjugate.

  It has been found that when plasmid DNA is injected into muscle cells, a high proportion of cells are transfected and the marker gene is expressed persistently. The DNA of the plasmid may or may not be incorporated into the cell genome. If the transfected DNA is not taken up, transfection and expression of the gene product protein can be achieved over a long period of time in a terminally differentiated non-proliferating tissue without fear of mutated insertions, deletions or substitutions of the cell or mitochondrial genome. It becomes possible. Introduction of a therapeutic gene into a specific cell that is long-term but not necessarily permanent provides a therapeutic or prophylactic use for genetic diseases. Periodic re-injection of DNA can be used to maintain gene product levels without mutating the genome of the recipient cell. If no foreign DNA is taken up, several different foreign DNA constructs can be present in one cell, with all constructs expressing various gene products.

  Electroporation for gene transfer utilizes current to sensitize cells or tissues to electroporation-mediated intergene transfer. A short electrical impulse with a constant electric field strength is used to increase the permeability of the membrane in such a way that DNA molecules can enter the cell. The technology can be used in in vitro systems or in conjunction with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.

  Exogenous DNA can be transfected into cells using carrier-mediated gene transfer in vivo. After the carrier-DNA complex is simply introduced into a body fluid or blood stream, it can be site-specifically delivered to a target organ or tissue in the body. Any of liposomes and polycations such as polylysine, lipofectin or cytofectin can be used. Since liposomes that are cell-specific or organ-specific can be prepared, the foreign DNA retained by the liposomes is taken up by the target cells. Injection of immunoliposomes targeted to specific receptors on certain cells can be used as a convenient method for inserting DNA into receptor-bearing cells. Another carrier system that has been used is the asialoglycoprotein / polylysine conjugate system for delivering DNA to hepatocytes for in vivo gene transfer.

  The transfected DNA may be complexed with other types of carriers so that the DNA remains in the cytoplasm or nucleoplasm after it is delivered to the recipient cell. DNA can be bound to the carrier nucleoprotein in a specifically made vesicle complex and delivered directly to the nucleus.

  Gene regulation of an anti-angiogenic protein involves transcription or translation by administering a compound that encodes one of the anti-angiogenic proteins, or a regulatory region associated with that gene, or its corresponding RNA transcript. This can be done by changing the speed. Also, cells transfected with a DNA sequence encoding anti-angiogenic proteins can be administered to a patient to provide an in vivo source of those proteins. For example, the cells can be transfected with a vector containing a nucleic acid sequence encoding an anti-angiogenic protein. The transfected cell may be a cell derived from a normal tissue of a patient or a diseased tissue of a patient, or may be a cell other than a patient.

  For example, tumor cells taken from a patient can be transfected with a vector capable of expressing the protein of the invention and returned to the patient. Transfected tumor cells produce levels of protein in the patient that inhibit tumor growth. The patient may be a human or non-human animal. Cells may be transfected by non-vector or by physical or chemical methods known in the art such as electroporation, ionoporation, or via a “gene gun”. DNA may also be injected directly into the patient without the assistance of a carrier. In particular, the DNA can be injected into skin, muscle or blood.

  Gene therapy protocols for transfecting anti-angiogenic proteins into patients are by incorporating anti-angiogenic protein DNA into the cell's genome or into the minichromosome, or separate replication in the cell's cytoplasm or nucleoplasm Alternatively, it can be performed as a non-replicating DNA construct. Expression of the anti-angiogenic protein may be sustained over time or may be periodically re-injected to maintain the desired cell, tissue or organ protein level or a predetermined blood level.

  The present invention also includes antibodies and antisera. Such antibodies and antisera can be used to test new anti-angiogenic proteins and are used in the diagnosis, prognosis, or treatment of diseases and conditions characterized by or associated with angiogenic activity or lack thereof can do. Such antibodies and antisera also block the localization and process of natural anti-angiogenic proteins using antibodies or antisera against the proteins of the invention in the desired, eg post-infarction heart tissue, It can be used for the upstream regulation of angiogenesis by increasing angiogenesis as well as inhibiting atrophy of heart tissue.

  Such antibodies and antisera can be combined with pharmaceutically acceptable compositions and carriers to make diagnostic, prognostic or therapeutic compositions. The term “antibody” or “antibody molecule” refers to an immunoglobulin molecule and / or an immunologically active portion of an immunoglobulin molecule, ie, a population of molecules comprising an antibody binding site or paratope.

  Passive antibody therapy using antibodies that specifically bind anti-angiogenic proteins can be performed to regulate reproduction, development, and angiogenesis-dependent processes such as wound healing and tissue repair. In addition, an antiserum against the Fab region of an antibody of an anti-angiogenic protein can be administered to block the ability of endogenous antisera against the protein to bind to the protein.

  The anti-angiogenic proteins of the present invention can also be used to generate antibodies specific for one or more inhibitors and one or more receptors. Such antibodies can be polyclonal antibodies or monoclonal antibodies. These antibodies that specifically bind to anti-angiogenic proteins or their receptors are diagnostic methods and diagnostics known to those skilled in the art for detecting or quantifying anti-angiogenic proteins or their receptors in body fluids or tissues. Can be used in kits. Results from these tests can be used to diagnose or predict the occurrence or regeneration of cancer and other angiogenesis-mediated diseases.

  The present invention also includes the use of anti-angiogenic proteins, antibodies to those proteins, and compositions comprising these proteins and / or their antibodies in the diagnosis or prognosis of diseases characterized by angiogenic activity. As used herein, the term “prognostic method” means a method that allows a prediction regarding the progression of a disease in a human or animal diagnosed with a disease, particularly an angiogenesis-dependent disease. As used herein, the term “diagnostic method” means a method that allows the determination of the presence or type of angiogenesis-dependent disease in or on a human or animal.

  Anti-angiogenic proteins can be used in diagnostic methods and diagnostic kits to detect and quantify antibodies capable of binding the protein. These kits will allow the detection of circulating antibodies against anti-angiogenic proteins that show the spread of micrometastasis in the presence of anti-angiogenic proteins secreted by primary tumors in situ. Patients with such circulating anti-protein antibodies may be more likely to have a variety of tumors and cancers, and may have cancer regeneration after a period of treatment or remission May be higher. These anti-protein antibody Fab fragments may be used as antigens to produce anti-protein Fab fragment antisera that can be used to neutralize anti-protein antibodies. Such a method would reduce the removal of circulating proteins by anti-protein antibodies, thereby effectively improving the circulating levels of anti-angiogenic proteins.

  The invention also includes the isolation of receptors specific for anti-angiogenic proteins. Protein fragments with highly neoplastic binding to tissue can be used to isolate receptors for anti-angiogenic proteins on affinity columns. Isolation and purification of one or more receptors is a fundamental step in elucidating the mechanism of action of anti-angiogenic proteins. Isolation of receptors and identification of agonists and antagonists will facilitate the development of drugs to modulate receptor activity, which is the final pathway to biological activity. Receptor isolation allows the construction of nucleotide probes to follow receptor location and synthesis using in situ and solution hybridization techniques. In addition, the gene of the receptor can be isolated and incorporated into an expression vector, and to increase the ability to bind cell type, tissue or tumor anti-angiogenic proteins and to inhibit local angiogenesis. Can be transfected into cells such as tumor cells.

  Anti-angiogenic proteins are used to develop affinity columns for the isolation of one or more receptors for anti-angiogenic proteins from cultured tumor cells. Amino acid sequence analysis is performed after receptor isolation and purification. Using this information, one or more genes encoding the receptor can be identified and isolated. The cloned nucleic acid sequence is then developed for insertion into a vector capable of expressing the receptor. These techniques are well known to those skilled in the art. Transfection of a nucleic acid sequence encoding a receptor into a tumor cell and expression of the receptor by the transfected tumor cell enhances the responsiveness of the cell to endogenous or exogenous anti-angiogenic proteins, thereby metastasizing The rate of growth is reduced.

  The angiogenesis-inhibiting proteins of the present invention can be synthesized in a standard microchemical apparatus and can be examined for purity by HPLC and mass spectroscopic optics. Methods of protein synthesis, HPLC purification and mass spectroscopic techniques are generally known to those skilled in the art. High angiogenic proteins and their receptor proteins are produced in recombinant E. coli or yeast expression systems and purified by column chromatography.

  Different protein fragments of intact anti-angiogenic proteins can be synthesized for use in several applications, including but not limited to: as antigens for the development of specific antisera As an agonist and antagonist active at the binding site of an anti-angiogenic protein, as a protein for linking to or in combination with a cytotoxic agent for targeted killing of cells that bind the anti-angiogenic protein.

  Synthetic protein fragments of anti-angiogenic proteins have a variety of uses. Proteins that bind to receptors for anti-angiogenic proteins with high specificity and affinity are radiolabeled and used for visualization and quantification of binding sites using autoradiography and membrane binding techniques. This application provides important diagnostic and research tools. Recognition of the binding properties of one or more receptors facilitates the investigation of transduction mechanisms associated with one or more receptors.

  Anti-angiogenic proteins and proteins derived therefrom can bind to other molecules using standard methods. Both the amino terminus and the carboxyl terminus of the anti-angiogenic protein contain tyrosine and lysine residues, and many techniques such as conventional techniques (tyrosine residue-chloramine T, iodogen, lactoperoxidase; lysine residue-bolton -Radiolabeled with a Hunter reagent) and labeled with isotopes and non-isotopes. These coupling techniques are well known to those skilled in the art. On the other hand, tyrosine or lysine is added to fragments that do not have these residues to facilitate labeling of reactive amino and hydroxyl groups on the protein. The coupling technique is not particularly limited, but is selected based on the functional groups available for amino acids including amino, sulfhydral, carboxyl, amide, phenol, and imidazole. Various reagents used to act on these bonds include, among others, glutaraldehyde, diazotized benzidine, carbodiimide, and p-benzoquinone.

Anti-angiogenic proteins are chemically conjugated to isotopes, enzymes, carrier proteins, cytotoxic reagents, fluorescent molecules, chemiluminescent materials, bioluminescent materials and other compounds for a variety of applications. The efficiency of the binding reaction is determined using different techniques suitable for the specific reaction. For example, ¹²⁵ radiolabeled proteins of the present invention with I is accomplished using chloramine T and Na ¹²⁵ I of high specific activity. The reaction is terminated with sodium metabisulfite and desalted with a disposable column. The labeled protein is eluted from the column and the fraction is collected. Aliquots are removed from each fraction and the radioactivity is measured with a gamma counter. In this manner, unreacted Na ¹²⁵ I is separated from the labeled protein. The protein fraction with the highest specific radioactivity is saved for subsequent use, such as analysis of the ability to bind anti-angiogenic proteins.

  In addition, anti-angiogenic protein labeling with short-lived isotopes allows the localization of tumors with protein binding sites by in vivo visualization of receptor binding sites using positron emission tomography or other modern radiographic techniques. To.

  The systematic substitution of amino acids within these synthetic proteins allows high affinity protein agonists and antagonists to one or more receptors of anti-angiogenic proteins that enhance or eliminate binding to one or more receptors. Arise. Such agonists are used to inhibit the growth of micrometastasis, thereby limiting the spread of cancer. Antagonists to anti-angiogenic proteins are applied in the case of insufficient angiogenesis to block the inhibitory action of anti-angiogenic proteins and promote angiogenesis. For example, this treatment may have a therapeutic effect that promotes wound healing in diabetes.

  The invention is further illustrated by the following examples. Such examples are not intended to be construed as any limitation within the scope of the invention. On the other hand, various other embodiments, modifications, and equivalents thereof will be apparent to those of ordinary skill in the art without departing from the spirit of the invention and / or the scope of the appended claims after reading the description herein. It should be clearly understood that they may have room to suggest themselves.

Example
Example 1: Isolation of natural Arresten.
Arresten can be obtained in milligram quantities from human placenta and amniotic tissue. Protocols for isolating Arresten and similar proteins have been described by other investigators (eg, Langeveld, 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. 15318-15324; Kahsai, TZ et al., 1997, J. Biol. Chem. 272: 17023-17032). The production of recombinant Arresten is described in Neilson et al. (1993, J. Biol. Chem. 268: 8402-8406). This protein can also be expressed in 293 kidney cells (eg, according to the method described in Hohenester, E. et al., 1998, EMBO J. 17: 1656-1664). Arresten has been published by Pihlajaniemi, T .; (1985, J. Biol. Chem. 260: 7681-7687).

  The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the α1 chain of the NC1 domain of type IV collagen are shown in FIG. It is shown in 1. The approximate beginning and end of Arresten are marked. Arresten generally contains the NC1 domain of the α1 chain of type IV collagen and probably also contains a junction region that is 12 amino acids immediately preceding the NC1 domain.

  Natural Arresten was isolated from human placenta using bacterial collagenase, anion exchange chromatography, gel filtration chromatography, HPLC and affinity chromatography (Gunwar, S. et al., 1991, J. Biol. Chem. 266). 15318-24; Weber, S. et al., 1984, Eur. J. Biohem. 139: 401-10). Type IV collagen monomer isolated from human placenta was HPLC purified using a C-18 hydrophobic column (Pharmacia, Piscataway, NJ, USA). The constituent proteins were separated with an acetonitrile gradient (32% -39%). A major peak was observed, which was a small double peak. In SDS-PAGE analysis, two bands were observed in the first peak, and no protein was detected in the second peak. Immunoblotting also found no immunodetectable protein in the second peak, and the main peak was identified as Arresten.

Example 2: Arresten recombinant production in E. coli.
Arsenten-encoding sequence was obtained by PCR from the α1NC1 (IV) / pDS vector (Neilson, EG, et al., 1993, J. Bio. Chem. 268: 8402-5) by forward primer 5′-CGG GAT CCT TCT. Amplification was performed using GTT GAT CAC GGC TTC-3 ′ (SEQ ID NO: 3) and reverse primer 5′-CCC AAG CTT TGT TCT TCT CAT ACA GAC-3 ′ (SEQ ID NO: 4). The resulting cDNA fragment was digested with Bam HI and Hin dIII, and ligated into pET22b which had been previously digested (+) (Novagen, Madison, Wis., USA). This structure is shown in FIG. It is shown in 2. As a result, Arresten was placed in-frame with the pelB leader sequence downstream of the pelB leader sequence, enabling periplasmic localization and soluble protein expression. An additional vector sequence encoding the amino acid MDIGINSD (SEQ ID NO: 13) was added to the protein. The 3 'end of the sequence was ligated in-frame with the polyhistidine tag sequence. The additional vector sequence between the 3 ′ end of the cDNA and the his-tag encoded the amino acid KLAAALE (SEQ ID NO: 14). Both strands of positive clones were sequenced.

  The plasmid construct encoding Arresten was first transformed into E. coli HMS174 (Novagen, Madison, Wis., USA), and then transformed into BL21 for expression (Novagen, Madison, Wis., USA). An overnight bacterial culture was used to inoculate a 500 ml culture of LB medium. This culture was grown for about 4 hours until the cells reached an OD600 of 0.6. Next, protein expression was induced by adding IPTG to a final concentration of 1-2 mM. After 2 hours of induction, cells were harvested by centrifugation at 5000 × g and lysed by resuspension in 6M guanidine, 0.1M NaH 2 PO 4, 0.01M Tris-HCl (pH 8.0). did. The resuspended cells were sonicated briefly and centrifuged at 12,000 × g for 30 minutes. The supernatant fraction was passed 4-6 times through a 5 ml Ni-NTA agarose column (Qiagen, Hilden, Germany) at a flow rate of 2 ml per minute. Non-specifically bound proteins were removed by washing with both 10 mM and 25 mM imidazole in 8 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl (pH 8.0). Arresten protein was eluted from the above column by sequentially increasing the imidazole concentration in 50 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl (pH 8.0) (50 mM, 125 mM and 250 mM). The eluted protein was dialyzed twice against PBS at 4 ° C. A small portion of the total protein precipitated during dialysis. The dialyzed protein was collected and centrifuged at approximately 3500 × g to separate into a pellet fraction and a supernatant fraction. The protein concentration in each fraction was determined by BCA assay (Pierce Chemical Co., Rockford, Ill., USA) and quantitative SDS-PAGE analysis. Approximately 22% of the total protein was in the pellet and the remaining 78% was recovered as soluble protein. The total protein yield was about 10 mg / liter.

  E. coli expressed protein was isolated mainly as a soluble protein, and a monomer band was observed at 29 kDa by SDS-PAGE. The additional 3 kDa was attributed to the polylinker sequence and the histidine tag sequence and was immunodetected by both Arresten antibody and 6-histidine tag antibody.

Example 3: Arresten expression in 293 fetal kidney cells.
Using a pDS plasmid containing α1 (IV) NC1, Arresten is amplified in such a way that the leader signal sequence is added in-frame into a pcDNA3.1 eukaryotic expression vector (InVitrogen, San Diego, Calif.). did. A leader sequence derived from the 5 ′ end of the full-length α1 (IV) chain was cloned into the 5 ′ side of the NC1 domain to allow protein secretion into the culture medium. The Arresten-containing recombinant vector was sequenced using flanking primers. An error free cDNA clone was further purified and used for in vitro translation experiments to confirm protein expression. Using the Arresten-containing plasmid and a control plasmid, 293 cells were transfected by the calcium chloride method. Transfected clones were selected by geneticin antibiotic treatment (Life Technologies / Gibco BRL, Gaithersburg, MD, USA). The cells were left in the presence of the antibiotic for 3 weeks until no cell death was evident. The clones were then expanded into T-225 flasks and grown to confluence. The supernatant was then collected and concentrated using an Amicon concentrator (Amicon). The concentrated supernatant was analyzed for Arresten expression by SDS-PAGE, immunoblotting and ELISA. Strong binding was detected in the supernatant by ELISA. In SDS-PAGE analysis, a single major band was observed at about 30 kDa. Arresten-containing supernatants were subjected to affinity chromatography using Arresten-specific antibodies (Gunwar, S. et al., 1991, J. Biol. Chem. 266: 15318-24). A major peak containing an approximately 30 kDa monomer showing immunoreactivity with Arresten antibody was identified. Approximately 1-2 mg of recombinant Arresten was produced per liter of culture.

Example 4: Arresten inhibits endothelial cell proliferation.
C-PAE cells were grown to confluence in DMEM containing 10% fetal calf serum (FCS) and contact inhibition was maintained for 48 hours. Control cells were 786-0 (renal cancer) cells, PC-3 cells, HPEC cells, and A-498 (renal cancer) cells. Cells were harvested by trypsinization (Life Technologies / Gibco BRL, Gaithersburg, MD, USA) for 5 minutes at 37 ° C. 12,500 cells suspended in DMEM containing 1% FCS were added to each well of a 24-well plate coated with 10 μg / ml fibronectin. The 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, Minn., USA). Unstimulated controls did not receive bFGF. Cells were treated with Arresten or endostatin at a concentration of 0.01-50 μg / ml. At the time of processing, 1 microcurie of ³ H-thymidine was added to all wells. After 24 hours, the medium was removed and the 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, PA) solution. Thymidine incorporation was measured using a scintillation counter. The results are shown in FIG. 3A and FIG. Shown in 3B. FIG. 3A and FIG. 3B is a set of graphs showing ³ H-thymidine incorporation into C-PAE cells treated with various amounts of Arresten (FIG. 3A) or Endostatin (FIG. 3B). Arresten appeared to inhibit thymidine incorporation in C-PAE as did endostatin. The behavior of control cells treated with Arresten and Endostatin is also shown in FIG. As shown in 4A, 4B, 4C and 4D, Arresten had little effect on 786-0 cells (Fig. 4A), PC-3 cells (Fig. 4B) or HPEC cells (Fig. 4C). . Endostatin had little effect on A-498 cells (FIG. 4D). FIG. 3 and FIG. All groups shown in 4 represent triplicate samples.

Example 5: Arresten inhibits endothelial cell migration.
The inhibitory effect of Arresten and endostatin on chemotaxis induced by FBS was determined by comparing human umbilical vein endothelial cells (ECV-304 cells, ATCC 1998-CRL, ATCC (American Type Culture Collection, USA 20110-2209, Manassas, VA). City Boulevard 10801)) using the Boyen chamber measurement method (Neuro-Probe, Inc., Cabin John, Maryland, USA). ECV-304 cells were grown overnight in M199 medium containing 10% FBS and 5 ng / ml DilC18 (3) live cell fluorescent dye (Molecular Probes, Inc., Eugene, Oreg., USA). After trypsinization and washing and dilution of the cells with M199 containing 0.5% FBS, 60,000 cells were added with Arresten or Endostatin (2-40 μg / ml) or with Arresten or Endostatin Without seeding into the upper chamber well. M199 medium containing 2% FBS was placed in the lower chamber as a chemoattractant. The cell-containing compartment was isolated from the chemoattractant with an 8 μm pore size polycarbonate filter (Portetics Corp., Livermore, Calif., USA). The chamber was incubated for 4.5 hours at 37 ° C. with 5% CO 2 and 95% humidity. After non-migrated cells were discarded and the upper wells were washed with PBS, the filter was scraped with a plastic blade, fixed in 4% formaldehyde / PBS, and placed on a glass slide. Several independent homogeneous images were recorded with a digital SenSys ^™ camera operated with the image processing software PMIS (Roper Scientific / Photometrics, Tucson, Arizona, USA) using a fluorescent intense field of view. Representative images are shown in FIG. Shown in 5A, 5B and 5C. These figures show that 2 μg / ml Arresten is as effective as 20 μg / ml endostatin. Cell counts were counted using OPTIMAS 6.0 software (Media Cybernetics, Rochester, NY). The results are shown in FIG. It is shown in FIG. This figure is a graphical representation of the results found in micrographs.

Example 6: Arresten inhibits endothelial tube formation.
To measure inhibition of endothelial tube formation, 320 μl of Matrigel (Collaborative Biomedical Products, Bedford, Mass., USA) was added to each well of a 24-well plate and allowed to polymerize (Grant, DS et al., 1994, Pathol.Res.Pract. 190: 854-63). 25,000 mouse aortic endothelial cells (MAE) suspended in EGM-2 medium without antibiotics (Clonetics Corporation, San Diego, Calif., USA) were transferred to each well coated with Matrigel. The cells were treated with Arresten, BSA, sterile PBS or 7S domain in increasing concentrations. All assays were done in triplicate. The cells were incubated at 37 ° C. for 24-48 hours and observed using a CK2 Olympus microscope (eyepiece 3.3 ×, objective lens 10 ×). The cells were then photographed using 400DK coated TMAX film (Kodak). Cells were stained with diff-quik fixative (Sigma Chemical Company, St. Louis, MO) and photographed again. Ten fields of view were observed and the number of tubes was counted and averaged. The results are shown in FIG. 7 shows. This figure shows that Arresten inhibits tube formation compared to the control. A representative example of a well-formed tube is shown in FIG. It can be seen at 8A (magnification 100 ×). In contrast, FIG. 8B shows that MAE cells treated with 0.8 μg / ml Arresten did not form well or did not form at all (magnification 100 ×).

  Matrigel assay was also performed in vivo in C57 / BL6 mice. Matrigel was melted overnight at 4 ° C. It was then treated with 20 U / ml heparin (Pierce Chemical Co., Rockford, Ill., USA), 150 ng / ml bFGF (R & D Systems, Minneapolis, Minn., USA) and 1 μg / ml Arresten or 10 μg / ml endostatin. Mixed with. The matrigel mixture was injected subcutaneously using a 21 gauge needle. The control group received the same mixture except that it did not contain an angiogenesis inhibitor. After 14 days, the mice were sacrificed and the matrigel plugs were removed. The matrigel plug was fixed in a 4% paraformaldehyde / PBS solution at room temperature for 4 hours and then switched to PBS for 24 hours. The plug was embedded in paraffin, sectioned and H & E stained. The sections were examined with an optical microscope, and the number of blood vessels was counted from 10 fields of high magnification and averaged.

  When Matrigel was placed in the presence of bFGF with or without increasing concentrations of Arresten, a 50% reduction in blood vessel count was observed with 1 μg / ml Arresten and 10 μg / ml Endostatin. It was. These results indicate that Arresten affects the formation of new blood vessels by inhibiting various stages of the angiogenesis process. These results also show that 1 μg / ml Arresten is as effective as 10 μg / ml endostatin in inhibiting neovascularization in vivo.

Example 7: Arresten inhibits tumor metastasis in vivo.
C57 / BL6 mice were injected intravenously with 1 million MC38 / MUCl (Gong, J. et al., 1997, Nat. Med. 3: 558-61). Every other day for 26 days, 5 control mice were injected with 10 mM sterile PBS, and 6 experimental mice were administered 4 mg / ml Arresten. 26 days after treatment, the number of lung tumor nodules was counted for each mouse and averaged for the two groups described above. Two deaths were recorded in each group. Arresten significantly reduced the average number of primary bars from 300 to 200 in control mice.

Example 8: Arresten inhibits tumor growth in vivo.
Two million 786-0 cells were injected subcutaneously into 7-9 week old male athymic nude mice. In the first group of 6 mice, tumors were grown to about 700 mm ³ . In the second group of 6 mice, tumors were grown to 100 mm ³ . Arresten in sterile PBS was administered daily for 10 days at a concentration of 20 mg / kg for mice with 700 mm ³ tumors and 10 mg / kg for mice with 100 mm ³ tumors. P. Injected. Control mice received BSA or PBS vehicle. The results are shown in FIG. 9A and FIG. Shown in 9B. FIG. 9A is a plot showing the increase in tumor volume from 700 mm ^{3 for} 10 mg / kg Arresten treated mice (open squares), BSA treated mice (+), and control mice (filled circles). Arresten-treated mouse tumors shrunk from 700 mm ³ to 500 mm ³ , while BSA-treated and control mouse tumors grew to approximately 1200 mm ³ in 10 days. FIG. 9B shows that even in mice with 100 mm ³ tumors, Arresten (white squares) contracted the tumors to about 80 mm ³ , while BSA-treated tumors (+) increased in size to almost 500 mm ^{3 in} 10 days. ing.

Approximately 5 million PC-3 cells (human prostate adenocarcinoma cells) were collected and injected subcutaneously into 7-9 week old male athymic nude mice. Tumors were grown for 10 days and then measured with calipers. Tumor volume was calculated using the standard formula (width ² x length x 0.52) (O 'Reilly, MS et al., 1997, Cell 88: 277-85; O' Reilly, M / S). Et al., 1994, Cell 79: 315-28). The mice were divided into groups of 5-6 animals. Experimental groups received Arresten (10 mg / kg / day) or endostatin (10 mg / kg / day) daily. P. Injected. PBS was administered daily to the control group. The results are shown in FIG. Shown in 9C. This indicates that Arresten (white squares) was comparable to or slightly stronger than endostatin (black triangles) and inhibited tumor growth. The experiment was repeated with the Arresten dose changed to 4 mg / kg / day. The results are shown in FIG. Shown in 9D. Treatment was stopped after 8 days (arrow), but significant inhibition continued for an additional 12 days without additional Arresten treatment. After 12 days of no treatment, the tumor began to escape the inhibitory effects of Arresten.

Example 9: Arresten circulation half-life.
Natural Arresten isolated from human placenta was injected intravenously to a 200 g size rate. Each rat was administered 5 mg human Arresten. Serum was analyzed by direct ELISA for the presence of circulating Arresten at various time points using anti-Arresten antibodies. Serum albumin was also evaluated at each time point as a control to ensure that the same amount of serum was used for analysis. Arresten was found to circulate in serum with a half-life of about 36 hours.

  Another group of rats received 200 μg human Arresten. P. And / or subcutaneous injections and assessed for signs of pathogenesis in lung, kidney, liver, pancreas, spleen, brain, testis, ovary, etc. Upon direct ELISA, Arresten antibody was detected in the sera of these rats and as previously observed (Kalluri, R. et al., 1994, Proc. Natl. Acad. Sci. USA 91: 6201). -5) Some endogenous IgG deposition was found in the glomerular basement membrane. This antibody deposition in the kidney was not accompanied by any signs of inflammation or decreased kidney function. These experiments suggest that Arresten is non-pathogenic.

Example 10: Binding and inhibition of MMP-2 enzyme by Arresten.
Antibodies against MMP-2, MMP-9, and their enzymes were obtained from Oncogene, Inc. Purchased from. As described previously (Kalluri, R., et al., 1994, Proc. Natl. Acad. Sci. USA 91: 6201-5), a direct ELISA was performed using natural Arresten isolated from human placenta. . Both MMP-2 and MMP-9 specifically bound Arresten. These enzymes did not bind the 7S domain. This binding was independent of TIMP-2 and TIMP-1 binding, respectively.

  To assess Arresten's basement membrane resolution, Matrigel was incubated with MMP-2 and MMP-9 for 6 hours at 37 ° C. with gentle shaking. Supernatants were analyzed by immunoblotting with antibodies against SDS-PAGE and α2 chain of type IV collagen. At the start of the degradation assay, Arresten was added in increasing concentrations, and inhibition of MMP-2 activity was observed. The NC1 domain was separated in a SDS-PAGE gel as a 26 kDa monomer and a 56 kDa dimer and could be visualized by Western blot using a type IV collagen antibody. Arresten, which was gradually increased in concentration, inhibited the degradation of the basement membrane by MMP-2, indicating that Arresten can bind MMP-2 and prevent it from degrading the basement membrane collagen. . Similar results were obtained for MMP-9.

Example 11: Recombinant production of canstatin in E. coli.
The nucleotide sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of the α2NC1 domain of type IV collagen are shown in FIG. 10 shows. The sequence encoding canstatin was obtained from the α2NCI (IV) / pDS vector (Neilson, EG et al., 1993, J. Biol. Chem. 268: 8402-5) by PCR from the forward primer 5′-CGG. Amplification was performed using 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 ′ (SEQ ID NO: 8). The resulting cDNA fragment was digested with Bam HI and Hin dIII, and ligated into pET22b which had been previously digested (+) (Novagen, Madison, Wis., USA). The structure is shown in FIG. 11 shows. Such ligation allowed canstatin to be located in-frame with the pelB leader sequence downstream of the pelB leader sequence, allowing for periplasmic localization and soluble protein expression. An additional vector sequence encoding the amino acid MDIGINSD (SEQ ID NO: 13) was added to the protein. The 3 'end of the sequence was ligated in-frame with the polyhistidine tag sequence. The additional vector sequence between the 3 ′ end of the cDNA and the his-tag encoded the amino acid KLAAALE (SEQ ID NO: 14). Both strands of positive clones were sequenced.

  The plasmid construct encoding canstatin was first transformed into E. coli HMS174 (Novagen, Madison, Wis., USA) and then transformed into BL21 for expression (Novagen, Madison, Wis., USA). An overnight bacterial culture was used to inoculate a 500 ml culture of LB medium. This culture was grown for about 4 hours until the cells reached an OD600 of 0.6. Next, protein expression was induced by adding IPTG to a final concentration of 0.5 mM. After induction for 2 hours, cells were harvested by centrifugation at 5,000 × g and resuspended in 6M guanidine, 0.1M NaH 2 PO 4, 0.01M Tris-HCl (pH 8.0). Was dissolved. The resuspended cells were sonicated briefly and centrifuged at 12,000 × g for 30 minutes. The supernatant fraction was passed 4-6 times at a flow rate of 2 ml / min through a 5 ml Ni-NTA agarose column (Qiagen, Hilden, Germany). Nonspecifically bound proteins were removed by washing with 15 ml each of 10 M, 25 mM, and 50 mM imidazole in 8 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl, pH 8.0. Canstatin protein was eluted from the column using two concentrations of imidazole (125 mM and 250 mM) in 8 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl, pH 8.0. The eluted protein was dialyzed twice against PBS at 4 ° C. Some of the total protein precipitated during dialysis. The dialyzed protein was collected and centrifuged at approximately 3,500 × g to separate the pellet fraction and the supernatant fraction. The protein concentration in each fraction was determined by BCA assay (Pierce Chemical Co., Rockford, Ill., USA) and quantitative SDS-PAGE analysis. In SDS-PAGE analysis, a monomer band was observed at 29 kDa, and the extra 3 kDa was attributed to the polylinker sequence and the histidine tag sequence. The eluates containing canstatin were combined and dialyzed against PBS for use in subsequent assays. Canstatin protein analyzed by SDS-PAGE and Western blotting was detected by polyhistidine tag antibody. The type IV collagen NC1 antibody also detected recombinant constatin protein expressed by bacteria.

  E. coli expressed protein was isolated primarily as a soluble protein. About 40% of the total protein was in the pellet and the remaining 60% was recovered as soluble protein. The total protein yield was about 15 mg / liter.

Example 12: Expression of canstatin in 293 fetal kidney cells.
Using a pDS plasmid (Neilson, EG, et al., 1993, J. Iol. Chem. 268: 8402-5) containing α2 (IV) NC1, the leader signal sequence is a pcDNA3.1 eukaryotic expression vector ( Canstatin was PCR amplified in such a way that it was added in-frame in InVitrogen, San Diego, Calif.). A leader sequence derived from the 5 ′ end of the full-length α2 (IV) chain was cloned into the 5 ′ side of the NC1 domain to allow protein secretion into the culture medium. The canstatin-containing recombinant vector was sequenced using flanking primers. An error free cDNA clone was further purified and used for in vitro translation experiments to confirm protein expression. Using the canstatin-containing plasmid and a control plasmid, 293 cells were transformed into the calcium chloride method (Kingston, RE, 1996, “Calcium Phosphate Transfection”, Current Protocols in Molecular Biology (Ausubel, F.). M. et al., Wiley and Sons, Inc., New York, NY, USA, 9.1.4-9.17). Transfected clones were selected by geneticin (Life Technologies / Gibco BRL, Gaithersburg, MD, USA) antibiotic treatment. The cells were left in the presence of the antibiotic for 3 weeks until no cell death was evident. Clones were expanded into T-225 flasks and grown to confluence. The supernatant was then collected and concentrated using an Amicon concentrator (Amicon, Inc.). Concentrated supernatants were analyzed for canstatin expression by SDS-PAGE, immunoblotting and ELISA. Strong binding was detected in the supernatant by ELISA. Canstatin-containing supernatants were subjected to affinity chromatography using canstatin specific antibodies (Gunwar, S. et al., 1991, J. Biol. Chem. 266: 15318-24). A major peak containing about 24 kDa pure monomer showing immunoreactivity with canstatin antibody (anti-α2 NC1 antibody, 1: 200 dilution) was identified.

Example 13: Canstatin inhibits endothelial cell proliferation.
Calf aortic endothelial (CPAE) cells were grown to confluence in DMEM containing 10% fetal calf serum (FCS) and contact inhibition was maintained for 48 hours. Cells were harvested by trypsinization (Life Technologies / Gibco BRL, Gaithersburg, MD, USA) for 5 minutes at 37 ° C. 12,500 cells suspended in DMEM containing 0.5% FCS were added to each well of a 24-well plate coated with 10 μg / ml fibronectin. The 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 (unstimulated cells) or 10% FCS (stimulated and treated cells). 786-0, PC-3 and HEK293 cells served as controls and were grown to confluence in the same way, trypsinized and seeded on plates. Cells were treated 3 times with canstatin or endostatin at a concentration of 0.025-40 mg / ml. For thymidine incorporation experiments, 1 millicurie of ³ H-thymidine was added to all wells during processing. After 24 hours, the medium was removed and the wells were washed 3 times with PBS. Radioactivity was extracted with 1N NaOH and added to a scintillation vial containing 4 ml of ScintiVerse II (Fisher Scientific, Pittsburgh, PA) solution. Thymidine incorporation was measured using a scintillation counter.

  The results are shown in FIG. 12A and FIG. Shown in 12B. FIG. 12A is a histogram showing the effect of various amounts of canstatin on the proliferation of CPAE cells. Thymidine incorporation is shown on the y-axis in units of counts per minute. “0.5%” on the x-axis is a 0.5% FCS (unstimulated) control and “10%” is a 10% FCS (stimulated) control. Thymidine incorporation was steadily reduced by treatment with increasing concentrations of canstatin. FIG. 12B is a histogram showing the effect of canstatin with increasing amounts on thymidine incorporation in non-endothelial cells 786-0, PC-3 and HEK293. Thymidine incorporation is shown on the y-axis in units of counts per minute, and the x-axis shows 0.5% FCS (unstimulated) and 10% FCS (stimulated) controls for each of the three cell lines, followed by Sequentially increased concentrations of canstatin are shown. All groups represent triplicate samples and bars represent mean counts per minute ± standard error of the mean.

  A methylene blue staining test was also performed. After 3,100 cells were added to each well and treated as described above, the cells were used using the method of Oliver et al. (Oliver, MH et al., 1989, J. Cell. Science 92: 513-8). I counted the number. All wells were washed once with 100 ml 1 × PBS and cells were fixed by adding 100 ml of 10% formalin / neutral buffered saline (Sigma Chemical Co., St. Louis, Mo., USA) for 30 minutes at room temperature. did. After removing the formalin, the cells were stained with a 1% methylene blue (Sigma Chemical Co., St. Louis, MO) solution in 0.01 M borate buffer (pH 8.5) for 30 minutes at room temperature. After removing the staining solution, the wells 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 0.1 N HCl / ethanol (1: 1 mixture) for 1 hour at room temperature. The amount of methylene blue staining was measured with a microplate reader (BioRad, Hercules, Calif., USA) using absorbance at a wavelength of 655 nm.

  The results are shown in FIG. 12C and FIG. Shown in 12D. FIG. 12C is a histogram showing the effect of canstatin in increasing amounts on pigment uptake by CPAE cells. The absorbance at OD655 is shown on the y-axis. “0.1%” represents a 0.1% FCS treated (unstimulated) control and “10%” is a 10% FCS treated (stimulated) control. The remaining bars represent treatment with canstatin in increasing concentrations. In CPAE cells, dye uptake decreased to levels seen in unstimulated cells at canstatin treatment levels of about 0.625 to 1.25 μg / ml. FIG. 12D is a histogram showing the effect of various canstatin concentrations on non-endothelial cells HEK293 (open bars) and PC-3 (hatched bars). The absorbance at OD655 is shown on the y-axis. “0.1%” represents a 0.1% FCS treated (unstimulated) control and “10%” represents a 10% FCS treated (stimulated) control. Bars represent the mean ± standard error of relative absorbance units at 655 nm for 8 wells per treatment concentration.

  A dose-dependent inhibition of 10% serum-stimulated endothelial cells was detected with an ED50 value of approximately 0.5 μg / ml (FIG. 12A and FIG. 12C). At canstatin doses up to 40 mg / ml, no significant effect on the growth of renal cancer cells (786-0), prostate cancer cells (PC-3) or human fetal kidney cells (HEK293) was observed (FIG. 12B). And Fig. 12D). This endothelial cell specificity indicates that canstatin is probably a particularly effective anti-angiogenic agent.

Example 14: Canstatin inhibits endothelial cell migration.
In the process of angiogenesis, endothelial cells not only proliferate but also migrate. Therefore, the influence of canstatin on endothelial cell migration was evaluated. Inhibitory effects of canstatin and endostatin on chemotaxis induced by FBS using human umbilical vein endothelial cells (HUVEC) using the Boyen chamber assay (Neuro-Probe, Inc., Cabin John, MD, USA) I investigated. HUVEC cells in M199 medium (Life Technologies / Gibco BRL, Gaithersburg, MD) with 10% FBS and 5 ng / ml DiIC18 (3) live cell fluorescent dye (Molecular Probes, Inc., Eugene, OR, USA) Was grown overnight. After trypsinization and washing and dilution of cells with M199 containing 0.5% FBS, 60,000 cells can be combined with canstatin (0.01 or 1.00 mg / ml) or with canstatin Without seeding into the upper chamber well. M199 medium containing 2% FBS was placed in the lower chamber as a chemoattractant. The cell-containing compartment was isolated from the chemoattractant with an 8 μm pore size polycarbonate filter (Portetics Corp., Livermore, Calif., USA). The chamber was incubated for 4.5 hours at 37 ° C. with 5% CO 2 and 95% humidity. After non-migrated cells were discarded and the upper wells were washed with PBS, the filter was scraped with a plastic blade, fixed in 4% formaldehyde / PBS, and placed on a glass slide. Using a fluorescent intense field of view, several independent homogeneous images were recorded with a digital SenSys ^™ camera operated with the image processing software PMIS (Roper Scientific / Photometrics, Tucson, Arizona, USA). Cell numbers 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. It is shown in FIG. This figure shows treatment of cells with no VEGF (no VEGF or serum) and VEGF (1% FCS and 10 ng / ml VEGF) and 0.01 μg / ml canstatin (1% FCS and 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) in a bar graph showing the number of migrated endothelial cells per field (y-axis) is there.

  Canstatin inhibited HUVEC migration, and a significant effect was observed at 10 ng / ml. Canstatin's ability to inhibit both endothelial cell proliferation and migration suggests that canstatin acts at more than one stage of the angiogenesis process. Alternatively, canstatin may act as an apoptosis signal that can affect both proliferation and migration for stimulated endothelial cells. Apoptosis induction is related to angiostatin (O 'Reilly, MS et al., 1994, Cell 79: 315-28; Lucas, R. et al., 1998, Blood 92: 4730-41), another anti-angiogenic molecule. It has been reported.

Example 15: Canstatin inhibits endothelial tube formation.
As an initial test for the anti-angiogenic ability of canstatin, we evaluated its ability to block tube formation by endothelial cells in Matrigel, a solid gel of mouse basement membrane protein. When mouse aortic endothelial cells are cultured on Matrigel, they quickly align and form a hollow tube-like structure.

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

  The results are shown in FIG. 14 shows. This figure shows the amount of tube formation with various treatments with BSA (white squares), canstatin (black squares) and α5NC1 (white circles) as a percentage of control (PBS-treated wells) tube formation (y-axis). It is a graph to represent. The vertical line segment represents the standard error of the mean. This result indicates that canstatin significantly reduces endothelial tube formation compared to the control.

  Canstatin selectively inhibited endothelial tube formation in a dose-dependent manner, and almost complete inhibition of tube formation was observed with the addition of 1 mg canstatin protein (FIG. 14). Since the control protein, neither bovine serum albumin (BSA) nor the NC1 domain of type IV collagen α5 chain affected endothelial tube formation, the inhibitory effect of canstatin in this assay was specific to canstatin, It was proved not to be due to the content of added protein. These results indicate that canstatin is an anti-angiogenic agent.

Example 16: Canstatin inhibits tumor growth in vivo.
Human prostate adenocarcinoma cells (PC-3 cells) were collected from the culture and 2 million cells in sterile PBS were injected subcutaneously into 7-9 week old male SCID mice. After the tumors had grown for about 4 weeks, the animals were divided into groups of 4 animals. The experimental group received a daily dose of canstatin at a dose of 10 mg / kg in a total volume of 0.1 ml PBS. P. Injected. The control group received an equal volume of PBS daily. At the start of treatment (day 0), the volume of the tumors in the control mice 88mm ³ ~135mm ^3, in the case of Canstatin treated mice was in the range of 108mm ³ ~149mm ^3. Each group contained 5 mice. Tumor volume ratio (V / V0) was determined by dividing the calculated tumor volume on a given day by the volume on day 0 of treatment. The results are shown in FIG. Shown in 15A. This figure is a graph showing the tumor volume ratio (y-axis) ± standard error plotted against the treatment day (x-axis). Canstatin-treated (black squares) tumors increased only slightly in size compared to controls (white squares).

In the second PC-3 experiment, PC-3 cells were collected from culture, 3 million cells were injected into 6-7 week old male athymic nude mice, and tumors were allowed to grow subcutaneously for approximately 2 weeks. After that, the mice were divided into groups of 4 mice. The experimental group (4 animals) received daily doses of 3 mg / kg canstatin in a total volume of 0.2 ml PBS or 8 mg / kg endostatin in the same volume of PBS. P. Injected. A control group (4 animals) received an equal volume of PBS daily. Tumor length and width were measured using calipers and tumor volume was calculated using the standard formula: length x width ² x 0.52. Tumor volume is in the range of 26mm ³ ~73mm ^3, was determined tumor volume ratio (V / V0) divided by the volume of the processing day 0 the tumor volume calculated value of a given day, as described above. The results are shown in FIG. Shown in 15B. This figure is a graph showing the tumor volume ratio (y-axis) ± standard error plotted against the treatment day (x-axis). Compared to the control (open squares), canstatin-treated (black squares) tumors increased only slightly in size, and the results were comparable to those achieved with endostatin (open circles).

As a renal cell carcinoma cell model, 2 million 786-0 cells were injected subcutaneously into 7-9 week old male athymic nude mice. Tumors were grown to about 100 mm ³ or about 700 mm ³ . Each group contained 6 mice. Canstatin in sterile PBS at a concentration of 10 mg / kg daily for 10 days P. Injected. The control group received the same volume of PBS. The results are shown in FIG. 15C (100 mm ³ tumor) and FIG. Shown in 31D (700 mm ³ tumor). In both groups, canstatin-treated (black squares) tumors actually shrunk compared to controls (white squares).

Canstatin produced in E. coli inhibits the growth of small renal cell carcinoma (786-0) tumor (100 mm ³ , Fig. 15C) and large renal cell carcinoma (786-0) tumor (700 mm ³ , Fig. 15D) did. For human prostate (PC-3) tumors in severe combined immunodeficiency (SCID) mice, 10 mg / kg canstatin kept the tumor volume ratio at 55% of mice injected with vehicle alone. When a lower dose of canstatin and endostatin was used in athymic (nu / nu) mice, 3 mg / kg canstatin showed the same inhibitory effect as 8 mg / kg endostatin. In any in vivo test, the mice looked healthy with no signs of wasting and none of the mice died during the treatment.

Example 17: CD31 immunohistochemistry in canstatin treated mice.
Reduction of tumor size in vivo suggested an inhibitory effect on blood vessel formation in these tumors. To detect tumor vessels, immunohistochemistry with anti-CD31 antibody alkaline phosphatase conjugate was performed on paraffin-embedded tumor sections. The excised tumor was cut into several pieces having a thickness of about 3 to 4 mm with a scapel and fixed in 4% paraformaldehyde for 24 hours. The tissue was then switched to PBS for 24 hours, then dehydrated and embedded in paraffin. After embedding in paraffin, 3 mm tissue sections were cut and mounted. Sections were deparaffinized, rehydrated and pretreated with 300 mg / ml protease XXIV (Sigma Chemical Co., St. Louis, MO, USA) for 5 minutes at 37 ° C. Digestion was stopped in 100% ethanol. Sections were air-dried, rehydrated and blocked with 10% rabbit serum. Slides were then incubated overnight at 4 ° C. with a 1:50 dilution of rat anti-mouse CD31 monoclonal antibody (PharMingen, San Diego, Calif., USA), followed by 1 of rabbit anti-rat immunoglobulin (DAKO) and rat APAAP (DAKO). : Incubated sequentially for 30 minutes at 37 ° C. with 50 dilutions. A color reaction was performed with new fuchsin. Sections were counterstained with hematoxylin.

  Canstatin-treated tumors had a reduced number of blood vessels compared to control tumors.

Example 18: Recombinant production of tumstatin and tumstatin variants in E. coli.
The nucleotide sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 10) of the α1 chain of the NC1 domain of type IV collagen are shown in FIG. 16 shows. The sequence encoding tumstatin was obtained from the α3NCI (IV) / pDS vector (Neilson, EG et al., 1993, J. Biol. Chem. 268: 8402-5) by PCR from the forward primer 5′-CGG GAT CCG. Amplification was performed using 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 ′ (SEQ ID NO: 12). The resulting cDNA fragment was digested with Bam HI and Hin dIII, and ligated into pET22b which had been previously digested (+) (Novagen, Madison, Wis., USA). The structure is shown in FIG. 17 shows. Such ligation allowed tumstatin to be located in-frame with the pelB leader sequence downstream of the pelB leader sequence, allowing periplasmic localization and soluble protein expression. An additional vector sequence encoding the amino acid MDIGINSD (SEQ ID NO: 13) was added to the protein. The 3 'end of the sequence was ligated in-frame with the polyhistidine tag sequence. The additional vector sequence between the 3 ′ end of the cDNA and the his-tag encoded the amino acid KLAAALE (SEQ ID NO: 14). Both strands of positive clones were sequenced. The plasmid construct encoding tumstatin was first transformed into E. coli HMS174 (Novagen, Madison, Wis., USA) and then transformed into BL21 for expression (Novagen, Madison, Wis., USA). The overnight bacterial culture was used to inoculate a 500 ml culture of LB medium (Fisher Scientific, Pittsburgh, PA, USA). This culture was grown for about 4 hours until the cells reached an OD600 of 0.6. Next, protein expression was induced by adding IPTG to a final concentration of 1 mM. After induction for 2 hours, cells were harvested by centrifugation at 5,000 × g and resuspended in 6M guanidine, 0.1M NaH 2 PO 4, 0.01M Tris-HCl (pH 8.0). Was dissolved. The resuspended cells were sonicated briefly and centrifuged at 12,000 × g for 30 minutes. The supernatant fraction was passed 4-6 times through a 5 ml Ni-NTA agarose column (Qiagen, Hilden, Germany) at a flow rate of 2 ml per minute. Non-specifically bound proteins were removed by washing with both 10 mM and 25 mM imidazole in 8 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl (pH 8.0). Tumstatin protein was eluted from the above column by sequentially increasing the imidazole concentration (50 mM, 125 mM and 250 mM) in 8 M urea, 0.1 M NaH2 PO4, 0.01 M Tris-HCl (pH 8.0). The eluted protein was dialyzed twice against PBS at 4 ° C. Some of the total protein precipitated during dialysis. The dialyzed protein was collected and centrifuged at approximately 3,500 × g to separate the insoluble (pellet) and soluble (supernatant) fractions.

  E. coli-expressed tumstatin was isolated mainly as a soluble protein, and a monomer band was observed at 31 kDa in SDS-PAGE analysis. The extra 3 kDa is due to the polylinker sequence and the histidine tag sequence. The elution fraction containing this band was used in subsequent experiments. The protein concentration in each fraction was determined by quantitative SDS-PAGE analysis using BCA assay (Pierce Chemical Co., Rockford, IL, USA) and scanning densitometry. Under reducing conditions, a band observed around 60 kD corresponding to a dimer of tumstatin under non-reducing conditions was separated as a single band of 31 kDa. The total protein yield was about 5 mg per liter.

  Recombinant truncated tumstatin lacking 53 N-terminal amino acids (Tumstatin-N53) was produced in E. coli and purified as previously described for other mutants (Kalluri, R. et al., 1996, J. Biol. Chem. 271: 9062-8). This mutant is a structural diagram showing the position of the truncated amino acid in the α3 (IV) NC1 monomer. 18. The filled circles correspond 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). Disulfide bonds marked with short bars are arranged to be present in α1 (IV) NC1 and α2 (IV) NC1 (Siebold, B. et al., 1988, Eur. J. Biochem. 176: 617 -twenty four). For clarity, only one of the two possible disulfide configurations is shown.

  Rabbit antibodies against human α (IV) NC1 were prepared as previously described (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, San Diego, California, USA). FITC-conjugated goat anti-rat IgG antibody, FITC-conjugated goat anti-rabbit IgG antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA).

  The resulting concentrated supernatant was analyzed by SDS-PAGE and immunoblotting for expression of tumstatin as previously described (Kalluri, R. et al., 1996, J. Biol. Chem. 271: 9062). -8). One-dimensional SDS-PAGE was performed using a 12% separation gel and a discontinuous buffer system. The separated protein was transferred to a nitrocellulose membrane and blocked with 2% BSA for 30 minutes at room temperature. After blocking the remaining binding sites, the membrane was washed thoroughly with wash buffer and incubated with primary antibody diluted 1: 1000 in PBS containing 1% BSA. Incubations were performed overnight at room temperature on a shaker. The blot was then washed thoroughly with wash buffer and incubated with secondary antibody conjugated to horseradish peroxidase on a shaker at room temperature for 3 hours. The blot was thoroughly washed again and 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 poured out and a hydrogen peroxide-containing substrate buffer was added. After developing the band, the reaction was stopped with distilled water and the blot was dried. A single band of 31 kDa was observed.

Example 19: Expression of tumstatin in 293 fetal liver cells.
Human tumstatin was also produced as a soluble protein secreted in 293 fetal hepatocytes using the pcDNA3.1 eukaryotic vector. This recombinant protein (without any purification or detection tag) was isolated using affinity chromatography and the pure monomeric form was detected in the main peak by SDS-PAGE and immunoblot analysis.

  Using a α3 (IV) NC1-containing pDS plasmid (Neilson, EG et al., 1993, J. Biol. Chem. 268: 8402-5), a pcDNA3.1 eukaryotic cell expression vector (InVitrogen, San Diego, California, USA) Tumstatin was amplified by PCR so that a leader signal sequence was added in-frame to (.). A leader sequence from the 5 'end of the full length α3 (IV) chain was cloned 5' of the NC1 domain so that protein secretion occurred in the culture 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 studies to confirm protein expression. Tumstatin-containing plasmids and control plasmids were used to transfect 293 cells using the calcium chloride method (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 treatment with Geneticin (Life Technologies / Gibco BRL, Gaithersburg, Maryland, USA). Cells were left in the presence of antibiotics for 3 weeks until there was no apparent cell death. Clones were expanded into T-225 flasks and grown to confluence. The supernatant was then collected and concentrated using an Amicon concentrator (Amicon, Inc., Beverly, Massachusetts, USA). The concentrated supernatant was analyzed by SDS-PAGE, immunoblotting and ELISA for expression of tumstatin. Strong binding in the supernatant was detected by ELISA.

  The tumstatin-containing supernatant is subjected to affinity chromatography and immunized with both anti-tumstatin and anti-6-histidine tag antibodies (Gunwar, S. et al., 1991, J. Biol. Chem. 266: 15318-24) Detected. A major peak containing an approximately 31 kDa monomer that immunoreacts with tumstatin antibody was identified.

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 produced soluble protein.
Cell lines and cultures. 786-0 (clear cell carcinoma cell line), PC-3 (human prostate adenocarcinoma cell line), C-PAE (bovine pulmonary artery endothelial cell line), MAE (mouse aortic endothelial cell line) were all obtained from American Type Culture Collection It was. 786-0 and C-PAE cell lines are maintained in DMEM (Life Technologies / Gibco BRL, Gaithersburg, Maryland, USA), ECV-304 cells are maintained in M199, and MAE cells are 10% Maintained in EGM-2 (Clonetics Corporation, San Diego, California, USA) supplemented with fetal calf serum (FCS), 100 units / ml penicillin, and 100 mg / ml streptomycin.
Proliferation assay. C-PAE cells were grown to confluence in DMEM containing 10% FCS and contact inhibition was continued for 48 hours. C-PAE cells were used between the second and fourth passages. 786-0 cells and PC-3 cells were used as non-endothelial controls in this experiment. Cells were harvested by trypsinization (Life Technologies / Gibco BRL, Gaithersberg, Maryland, USA) for 5 minutes at 37 ° C. 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 degrees with 5% CO2 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 various concentrations of tumstatin ranging from 0.01 to 10 mg / ml. 1 m Curie ³ H-thymidine was added to all wells 12 hours after the start of treatment. After 24 hours, the medium was removed and the wells were washed 3 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.

The results were compared with ³ H for 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). -Figure 5 is a histogram showing thymidine incorporation (y-axis). Shown in 19A, 19B and 19C. All groups show triplicate samples. Tumstatin significantly inhibited 20% FCS stimulated ³ H-thymidine incorporation in a dose dependent manner. The ED50 was about 0.01 mg / ml (Fig. 19A). Also, no significant antiproliferative effects were observed in prostate cancer cells (PC-3) or renal cancer cells (786-0), even at concentrations up to 20 mg / ml tumstatin (Fig. 19B and 19C). The difference between the mean ³ H-thymidine incorporation and the control mean in tumstatin treatment (0.1-10 mg / ml) was significant (P <0.05). When PC-3 cells or 786-0 cells were treated with tumstatin, no inhibitory effect was observed (FIG. 19B, 19C). Each column represents the mean ± SE of triplicate wells. This experiment was repeated three times. Bars marked with an asterisk are significant at P <0.05 by one-sided Student's t-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 allowed to polymerize (Grant, DS et al., 1994, Pathol. Res. Pract. 190: 854-63). A suspension of 25,000 MAE cells in EGM-2 medium without antibiotics (Clonetics Corporation, San Diego, California, USA) was transferred to each matrigel-coated well (Grant, DS et al., 1994). , Pathol.Res.Pract. 190: 854-63 ・). Cells were treated with tumstatin, BSA or 7S domain in increasing concentrations. Control cells were incubated with sterile BSA. 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 × magnification of objective lens). The cells were then photographed using TMAX film (Kodak •) coated with 400DK. Cells were stained with diff-quik fixative (Sigma Chemical Co., St. Louis, Missouri, USA) and photographed again (Grant, DS et al., 1994, Pathol. Res. Pract. 190: 854-63 ・). Ten fields were observed and the number of tubes was allowed to be counted by two investigators who did not know the experimental protocol and averaged.

  The results are shown in FIG. 20 shows. When mouse aortic endothelial cells are cultured on Matrigel, a solid gel of mouse basement membrane proteins, they rapidly align and form a hollow tube-like structure (Haralabopoulos, GC 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 rate of tube formation after treatment with 1 mg / ml protein was BSA 98.0 ± 4.0 and tumstatin 14.0 ± 4.0. Similar results were also obtained using tumstatin produced in E. coli. The 7S domain of type IV collagen (N-terminal non-collagenous domain) did not affect endothelial tube formation. Maximum inhibition by tumstatin was reached between 800-1000 ng / ml. The difference between the average ratio value of tumstatin treatment (black circle, 0.1-10 mg / ml) and the average ratio value of the control [BSA (white square), 7S domain (white circle)] is significant (p <0. 05). Each point represents the mean ± SE of triplicate wells. This experiment was repeated three times. Data points marked with an asterisk were significant at p <0.05 by one-sided Student's t-test. Well formed tubes were observed with 7S domain treatment. MAE cells treated with 0.8 mg / ml tumstatin showed reduced tube formation.

  To evaluate the in vivo effect of tumstatin on the formation of new capillaries, a matrigel plug assay was performed (Passaniti, A. et al., 1992, Lab Invest. 67: 519-29 ). 5-6 week old male C57 / BL6 mice (Jackson Laboratories, Bar Harbor, Maine, USA) were obtained. Matrigel (Collaborative Biochemical Products, Bedford, Massachusetts, USA) was dissolved overnight at 4 ° C. Prior to injection into C57 / BL6 mice, 20 U / ml heparin (Pierce Chemical Co., Rockford, Illinois, USA), 150 ng / ml bFGF (R & D Systems, Minneapolis, Minnesota, USA), and 1 mg / ml Of tumstatin. The control group received no angiogenesis inhibitors. The Matrigel mixture was injected subcutaneously using a 21 gauge needle. After 14 days, the mice were sacrificed and the Matrigel plugs were removed. Matrigel plugs were fixed with 4% paraformaldehyde (in PBS) for 4 hours at room temperature and then switched to PBS for 24 hours. Plugs were embedded in paraffin, sectioned and H & E stained. Sections were examined with a light microscope and the number of blood vessels was counted from 10 high power fields and averaged. All sections were encoded and observed by a pathologist who did not know the study protocol.

  When Matrigel was placed in the presence of bFGF and heparin with or without tumstatin produced in E. coli, a 67% reduction in blood vessel number was observed with 1 mg / ml tumstatin treatment. The number of blood vessels per strong magnification field was tumstatin, 2.25 ± 1.32, and control, 7.50 ± 2.17. Each column represents the mean ± SE of 5-6 mice per group. Tumstatin (1 mg / ml) significantly inhibited in vivo angiogenesis compared to PBS treated controls. The difference between the mean percentage value of tumstatin treated animals and the mean percentage of control animals was significant (p <0.05). Tumstatin treatment was significant at p <0.05 with a one-sided student's t-test.

Example 22: Tumstatin and variants of Tumstatin inhibit tumor growth in vivo.
Five million PC-3 cells were collected and injected subcutaneously into the back of 7-9 week old male athymic nude mice. The volume was calculated using a standard caliper and the volume was calculated using the standard formula width ² x length x 0.52. Tumors were grown to approximately 100 mm ³ and the animals were then divided into groups of 5 or 6 mice. Tumstatin or mouse endostatin in sterile PBS was injected intraperitoneally (20 mg / kg) daily for 10 days for each experimental group. Control groups received vehicle injections (BSA or PBS). Tumor volume was calculated every 2 or 3 days over 10 days. Results are graphs showing tumor volume in mm ³ (y-axis) versus days of treatment (x-axis) for PBS control (open squares), 20 mg / kg tumstatin (filled circles) and 20 mg / kg endostatin (open circles). FIG. Shown in 21A. 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 as in endostatin 20 mg / kg (FIG. 21A). Significant inhibitory effect on tumor growth was observed on day 10 (control 202.8 ± 50.0 mm ^3, tumstatin 82.9 ± 25.2 mm ^3, endostatin 68.9 ± 16.7mm ^3). Daily intraperitoneal injections of tumstatin or endostatin inhibited the growth of human prostate adenocarcinoma cell (PC-3) tumors compared to controls. This experiment was started when the tumor volume was less than 100 mm ³ .

The effect of tumstatin on other primary tumors established in mice was also studied. Two million 786-0 renal cell carcinoma cells were injected subcutaneously into the back of 7-9 week old male athymic nude mice. Tumors were grown to about 600 to about 700 mm ³ and the animals were then divided into groups of 6 animals. Tumstatin in sterile PBS was injected intraperitoneally (6 mg / kg) daily for 10 days. The control group was injected with BSA. Results are graphs showing tumor volume in mm ³ (y-axis) versus days of treatment (x-axis) for PBS control (open squares) and 6 mg / kg tumstatin (black circles) FIG. It is shown in 21B. Tumstatin produced in E. coli was 6 mg / kg and inhibited tumor growth of 786-0 human renal cell carcinoma compared to the BSA control (FIG. 21B). A significant inhibitory effect on tumor growth was observed on day 10 (control 1096 ± 179.7 mm ³ , tumstatin 619 ± 120.7 mm ³ ). Daily intraperitoneal injection of tumstatin inhibited tumor growth of human renal cell carcinoma (786-0) compared to controls. This experiment started when the tumor volume was 600-700 mm ³ . Each point represents the mean ± SE of 5-6 mice per group. The star data point markers were significant at one-sided Student's t test with p <0.05.

  The portion of the α3 chain NC1 domain [α3 (IV) NC1] of type IV collagen is a pathogenic epitope of Goodpasture 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.). Goodpascher syndrome is an autoimmune disease characterized by pulmonary hemorrhage and / or rapidly progressing glomerulonephritis (Wilson, C. & F. Dixon, 1986, The Kidney, WB Sanders Co., Philadelphia, Pennsylvania, USA pps. 80-89; Hudson, BG et al., 1993, J. Biol. Chem. 268: 16033-6.). These symptoms are caused by disruption of the glomeruli and alveolar basement membrane via autoantibody binding to α3 (IV) NC1 (Wilson, 1986, supra; Hudson, 1993, supra). Several groups have attempted to locate or predict the location of the Goodpasture autoantigen on α3 (IV) (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 (J. Biol. Chem. 269: 17358 ); Netzer, KO et al., 1999, J. Biol. Chem. 274: 11267-74), N-terminal, C-terminal, and central residues have been reported as epitope positions. The most likely disease-related pathogenic epitope has been identified at the N-terminal position (Hellmark, T. et al., 1999, Kidney Int. 55: 936-44), and further limited to 40 amino acids at the N-terminus Truncated tumstatin is an N-terminal 53 amino acid corresponding to the pathogenic Goodpasture self-epitope Designed to lack (Tumstatin-N53). The mutant protein was used in the following experiments.

Two million 786-0 renal cell carcinoma cells were injected subcutaneously into the back of 7-9 week old male athymic nude mice. Tumors were grown to a size of about 100-150 mm ³ . The mice were then divided into groups of 5 and daily intraperitoneal injections of truncated tumstatin expressed in E. coli lacking 20 mg / kg of 53 N-terminal amino acids for 10 days (Kalluri, R. et al. 1996, J. Biol. Chem. 271: 9062-8). Control mice received PBS injections. Results are graphs showing the increase in tumor volume (y-axis) relative to treatment days (x-axis) for control mice (white squares) and mice treated with tumstatin mutant N26 (black circles) FIG. 22 shows. Tumstatin-N53 produced in E. coli was 20 mg / kg, and significantly inhibited the growth of 786-0 human renal tumors from day 4 to day 10 compared to control (day 10: tumstatin-N53 110.0 ± 29 0.0 mm ³ , control 345.0 ± 24.0 mm ³ ) (FIG. 22). Each point represents the mean ± SE of 5-6 mice / group. The asterisk data point marker was significant at p <0.05 with a one-sided student t-test.

Example 23: Immunohistochemical staining of α3 (IV) NC1 and CD31.
Kidney and skin tissue from 7 week old male C57 / BL6 mice were processed for evaluation by immunofluorescence microscopy. Tissue samples were frozen in liquid nitrogen and 4 mm thick sections were used. Tissues were processed by indirect immunofluorescence techniques as previously described (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-conjugated anti-rat IgG antibody or FITC-conjugated Stained with anti-human IgG antibody. Immunofluorescence was examined under an Olympus fluorescence microscope (Tokyo, Japan). Negative controls were performed by replacing the primary antibody with an irrelevant preimmune serum.

  In mouse kidney, expression of a3 (IV) NC1 was observed in GBM and vascular basement membrane. CD31, PECAM-1 expression was observed in glomerular and vascular endothelium. In mouse skin, α3 (IV) NC1 was absent from the epidermal basement membrane and vascular basement membrane. CD31 expression was observed in the vascular endothelium of the skin. CD31 expression was observed in glomerular and tubule endothelium in mouse kidney, and α3 (IV) NC1 expression was observed in glomerular and extraglomerular vascular basement membranes. CD31 expression was observed in the endothelium of dermal tubules in mouse skin. α3 (IV) NC1 expression was not present in the endothelial basement membrane and was hardly observed in the basement membrane of the dermal tubule. These results show examples where the distribution of tumstatin is limited.

Example 24: Anti-angiogenic protein variants and fragments Arresten and Canstatin fragments and variants were also obtained by digestion with Pseudomonas elastase from Mariyama et al. (1992, J. Biol. Chem. 267: 1253-8). Produced. The digests were separated by gel filtration HPLC, and the resulting fragments were analyzed by SDS-PAGE and evaluated by the endothelial tube assay. These fragments include Arresten's 12 kDa fragment, Arresten's 8 kDa fragment, and Canstatin's 10 kDa fragment. Two fragments of tumstatin (“333” and “334”) were also generated by PCR cloning.

  FIG. 23, in the endothelial tube assay performed as described above, two arresten fragments [12 kDa (black square) and 8 kDa (white square)] and canstatin fragment [19 kDa (black triangle)] (Black circles) or canstatin (white circles) inhibited endothelial tube formation to an even greater extent. FIG. 24 similarly shows that the tumstatin fragments, “333” (black circle) and “334” (white circle) outperform tumstatin (black triangle). BSA (black squares) and α6 chain (white squares) were used as controls.

  All references, patents, and patent applications are hereby incorporated by reference in their entirety. While the invention has been shown and described in detail with reference to preferred embodiments thereof, various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will understand that the invention may be made.

The following are mentioned as an aspect of this invention.
[1] An isolated protein selected from the group consisting of the α1 chain NC1 domain of type IV collagen, the α1 chain NC1 domain of type IV collagen, or the NC1 domain of α3 chain of type IV collagen, or a fragment or analog thereof An isolated protein, which is a derivative or variant, wherein the protein, fragment, analog, derivative or variant thereof has anti-angiogenic properties.
[2] The isolated protein according to [1], wherein the protein is a monomer.
[3] The isolated protein according to [2], wherein the protein is Arresten.
[4] The isolated protein according to [2], wherein the protein is canstatin.
[5] The isolated protein of [2], wherein the protein is tumstatin.
[6] A multimer of the protein according to [1], having an anti-angiogenic activity, or a fragment, analog, derivative or mutant thereof.
[7] A chimeric protein comprising one or more proteins according to [1] having anti-angiogenic activity, or fragments, analogs, derivatives or variants thereof.
[8] Further comprising at least one protein molecule selected from the group consisting of endostatin or a fragment thereof, angiostatin or a fragment thereof, restin or a fragment thereof, apomiglen or a fragment thereof, or other anti-angiogenic protein or a fragment thereof [7] The chimeric protein according to
[9] A composition comprising one or more proteins according to [1] as a biologically active ingredient.
[10] A carrier that is pharmaceutically compatible with the composition according to [9].
[11] A biologically active ingredient comprising one or more proteins according to [1], and endostatin or a fragment thereof, angiostatin or a fragment thereof, restin or a fragment thereof, apomiglen or a fragment thereof Or at least one protein molecule selected from the group consisting of other anti-angiogenic proteins, or fragments thereof.
[12] A composition comprising the multimer according to [6] as a biologically active ingredient.
[13] A composition comprising the chimeric protein according to [7] as a biologically active ingredient.
[14] The protein according to [1] or a fragment, analog, derivative or variant thereof, wherein the protein or fragment, analog, derivative or variant thereof has anti-angiogenic activity. An isolated polynucleotide.
[15] The isolated polynucleotide of [14], which is operably linked to an expression control sequence.
[16] A host cell transformed with the polynucleotide according to [15].
[17] The host cell according to [16], which is selected from the group consisting of bacterial cells, yeast cells, mammalian cells, insect cells or plant cells.
[18] An isolated polynucleotide encoding the protein according to [3].
[19] An isolated polynucleotide encoding the protein according to [4].
[20] An isolated polynucleotide encoding the protein according to [5].
[21] A step of growing a culture of host cells transformed with the polynucleotide according to [a] [14], wherein the host cells are bacterial cells, yeast cells, mammalian cells, insect cells or plant cells And (b) purifying the protein from the culture;
A protein encoded by the polynucleotide according to [14], wherein the protein encoded by the polynucleotide according to [14] is produced by such a step.
[22] (a) producing one or more polynucleotide probes that hybridize to the polynucleotide of [14] under moderately stringent conditions;
(B) hybridizing the one or more probes to mammalian DNA; and (c) isolating a DNA polynucleotide detected with the one or more probes;
An isolated polynucleotide produced by the method, wherein the nucleotide sequence corresponds to the nucleotide sequence of the polynucleotide described in [14].
[23] A step of introducing a mammalian cell treated in vitro and inserted with the polynucleotide according to [14] into the mammal, and an anti-angiogenic protein in an amount sufficient to inhibit angiogenic activity in the mammal A method for providing an anti-angiogenic protein to a mammal, comprising the step of expressing a therapeutically effective amount of said in vivo in said mammal.
[24] The method according to [23], wherein the expression of the anti-angiogenic protein is transient expression.
[25] The method according to [23], wherein the cells are selected from the group consisting of blood cells, TIL cells, bone marrow cells, vascular cells, tumor cells, hepatocytes, muscle cells, and fibroblasts.
[26] The method according to [25], wherein the polynucleotide is inserted into the cell by a viral vector.
[27] An antibody that specifically binds to the isolated protein, analog, derivative homologue or variant thereof according to [1].
[28] One or more of the following: one or more isolated proteins according to [1], fragments, analogs, derivatives or variants of the isolated protein according to [1], A multimer of the protein of [1], a multimer of the fragment of [1], a chimeric protein comprising one or more proteins of [1], or a fragment of the protein of [1] A method for inhibiting anti-angiogenic activity in mammalian tissue, comprising the step of contacting the tissue with a composition comprising the chimeric protein comprising:
[29] The disease is angiogenesis-dependent cancer, benign tumor, rheumatoid arthritis, diabetic retinopathy, psoriasis, ocular angiogenesis disease, Osler-Weber syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, blood Group consisting of hemophilia joint, hemangiofibroma, wound granulation, intestinal adhesion, arteriosclerosis, scleroderma, hyperplastic scar, cat scratch disease, Helicobacter pylori ulcer, dialysis graft vascular access stenosis, infertility, obesity The method according to [28], which is selected from the above.
[30] The method according to [29], wherein the disease is cancer.
[31] The composition according to [28] for inhibiting a disease characterized by angiogenic activity, which comprises the step of administering a combination of radiation therapy, chemotherapy, or immunotherapy to a patient having a disease. How to use.
[32] A polypeptide comprising amino acid position 2 to amino acid position 125 of SEQ ID NO: 10 having anti-angiogenic activity.
[33] A polynucleotide encoding the polypeptide according to [32].
[34] A polypeptide comprising amino acid position 125 to amino acid position 245 of SEQ ID NO: 10 having anti-angiogenic activity.
[35] A polynucleotide encoding the polypeptide according to [34].
[36] An anti-angiogenic fragment of the NC1 domain of the α1 chain of type IV collagen produced by the method of PCR cloning.
[37] An anti-angiogenic fragment of the NC1 domain of α2 chain of type IV collagen produced by the method of PCR cloning.
[38] An anti-angiogenic fragment of the NC1 domain of the α3 chain of type IV collagen produced by the method of PCR cloning.
[39] An anti-angiogenic fragment of the NC1 domain of the α1 chain of type IV collagen, produced by Pseudomonas elastase digestion.
[40] The anti-angiogenic fragment according to [39], which has a size of 12 kDa.
[41] The anti-angiogenic fragment according to [39], which has a size of 8 kDa.
[42] An anti-angiogenic fragment of the NC1 domain of α2 chain of type IV collagen produced by digestion with Pseudomonas elastase.
[43] The anti-angiogenic fragment according to [42], which has a size of 10 kDa.
[44] An anti-angiogenic fragment of the NC1 domain of α3 chain of type IV collagen produced by digestion with Pseudomonas elastase.

FIG. 1A and 1B show the nucleotide sequence (FIG. 1A, SEQ ID NO: 1) and amino acid sequence (FIG. 1B, SEQ ID NO: 2) of the α1 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) are shown. FIG. 1A and 1B show the nucleotide sequence (FIG. 1A, SEQ ID NO: 1) and amino acid sequence (FIG. 1B, SEQ ID NO: 2) of the α1 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) are shown. FIG. 2 is a schematic diagram showing the Arresten cloning vector pET22b (+). The forward primer (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) and the site where Arresten is cloned are shown. FIG. 3A and 3B are Arresten (Fig. 3A, 0 μg / ml to 10 μg / ml, x-axis) and endostatin (FIG. ³ ) for ³ H-thymidine incorporation (y-axis) as an indicator of endothelial cell (C-PAE) proliferation. 3B, a set of line graphs showing the effect of 0 μg / ml to 10 μg / ml, x-axis). FIG. 4A, 4B, 4C and 4D are a set of four bar graphs showing the effect of Arresten and Endostatin on ³ H-thymidine incorporation (y-axis) as an indicator of endothelial cell proliferation. FIG. 4A, 4B and 4C are Arresten [0 μg / ml to 50 μg / ml (FIG. 4A and 4B) and 0 μg / ml to 10 μg / ml (FIG. 4C) for 786-0 cells, PC-3 cells and HPEC cells, respectively. ] Shows the influence. FIG. 4D shows the effect of 0.1-10 μg / ml endostatin on A-498 cells. FIG. 4A, 4B, 4C and 4D are a set of four bar graphs showing the effect of Arresten and Endostatin on ³ H-thymidine incorporation (y-axis) as an indicator of endothelial cell proliferation. FIG. 4A, 4B and 4C are Arresten [0 μg / ml to 50 μg / ml (FIG. 4A and 4B) and 0 μg / ml to 10 μg / ml (FIG. 4C) for 786-0 cells, PC-3 cells and HPEC cells, respectively. ] Shows the influence. FIG. 4D shows the effect of 0.1-10 μg / ml endostatin on A-498 cells. FIG. 4A, 4B, 4C and 4D are a set of four bar graphs showing the effect of Arresten and Endostatin on ³ H-thymidine incorporation (y-axis) as an indicator of endothelial cell proliferation. FIG. 4A, 4B and 4C are Arresten [0 μg / ml to 50 μg / ml (FIG. 4A and 4B) and 0 μg / ml to 10 μg / ml (FIG. 4C) for 786-0 cells, PC-3 cells and HPEC cells, respectively. ] Shows the influence. FIG. 4D shows the effect of 0.1-10 μg / ml endostatin on A-498 cells. FIG. 4A, 4B, 4C and 4D are a set of four bar graphs showing the effect of Arresten and Endostatin on ³ H-thymidine incorporation (y-axis) as an indicator of endothelial cell proliferation. FIG. 4A, 4B and 4C are Arresten [0 μg / ml to 50 μg / ml (FIG. 4A and 4B) and 0 μg / ml to 10 μg / ml (FIG. 4C) for 786-0 cells, PC-3 cells and HPEC cells, respectively. ] Shows the influence. FIG. 4D shows the effect of 0.1-10 μg / ml endostatin on A-498 cells. FIG. 5A, 5B and 5C are Arresten (2 μg / ml, FIG. 5B) and Endostatin (20 μg / ml, FIG. 5) for endothelial cell migration via FBS-induced chemotaxis in human umbilical vein endothelial (ECV-304) cells. 5C) is a set of four photomicrographs showing the effect of 5C). FIG. 5A shows untreated control cells. FIG. 6 is shown in FIG. 5 is a bar graph showing the results of 5 in a graph format. FIG. 6 shows the effect of Arresten (2 μg / ml or 20 μg / ml) and endostatin (2.5 μg / ml or 20 μg / ml) on ECV-304 endothelial cell migration. FIG. 7 is a line graph showing the effect of Arresten on endothelial tube formation. The rate of tube formation is shown on the y-axis and the concentration of inhibitor on the x-axis. The treatments were: none (control, black diamond), BSA (control, white triangle), 7S domain (control, X) and Arresten (black square). FIG. 8A and 8B are a set of photomicrographs showing the effect of Arresten (0.8 μg / ml, FIG. 8B) on endothelial tube formation compared to the control (FIG. 8A). FIG. 9A, 9B, 9C and 9D are a set of four line graphs showing the effects of Arresten and Endostatin on tumor growth in vivo. FIG. 9A is a plot showing the increase in tumor volume from 700 mm ³ for 10 mg / kg Arresten treatment (open squares), BSA treatment (+), and control mice (filled circles). FIG. 9B shows an increase in tumor volume from 100 mm ³ for 10 mg / kg Arresten-treated (open squares) and BSA-treated (+) tumors. FIG. 9C shows an increase in tumor volume from approximately 100 mm ³ for 10 mg / kg Arresten treatment (open squares), endostatin treatment (filled triangles), and control mice (filled circles). FIG. 9D shows the increase for 200 mm ³ tumors when treated with Arresten (open squares) vs. control (filled circles). FIG. 10A and 10B are diagrams showing the nucleotide sequence (FIG. 10A, SEQ ID NO: 5) and amino acid sequence (FIG. 10B, SEQ ID NO: 6) of the α2 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8) are shown. FIG. 10A and 10B are diagrams showing the nucleotide sequence (FIG. 10A, SEQ ID NO: 5) and amino acid sequence (FIG. 10B, SEQ ID NO: 6) of the α2 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8) are shown. FIG. 11 is a schematic diagram showing canstatin cloning vector pET22b (+). The forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8) and the site where canstatin is cloned are shown. FIG. 12A, 12B, 12C and 12D are various for the proliferation of endothelial (C-PAE) cells (Fig. 12A and 12C) and non-endothelial (786-0, PC-3 and HEK293) cells (Fig. 12B and 12D). It is a histogram which shows the influence of concentration of canstatin (x-axis). Proliferation was measured as a function of ³ H-thymidine incorporation (FIG. 12A and 12B) and methylene blue staining (FIG. 12B and 12D). FIG. 13 for treatment of VEGF-free (no VEGF or serum), and VEGF (1% FCS and 10 ng / ml VEGF) cells, and 0.01 μg / ml canstatin (1% FCS and 10 ng / ml VEGF and 0. Shows the number of migrating endothelial cells per field (y-axis) for treatment of 01 μg / ml canstatin) and 1.0 μg / ml canstatin (1% FCS and 10 ng / ml VEGF and 1 μg / ml canstatin) It is a bar graph. FIG. 14 is the amount of endothelial tube formation as a percentage of control (PBS treated well) tube formation (y axis) under various treatments of BSA (white squares), canstatin (black squares), and α5NC1 (white circles). It is a line graph which shows. The vertical line segment indicates the standard error of the average. FIG. 15A, 15B, 15C and 15D are tumor volume fractions (y-axis, FIGS. 15A and 15B) or tumor volumes in mm ³ (y-axis, FIGS. 15C and 15D) plotted against the number of treatment days (x-axis). ) For canstatin (black squares), endostatin (white circles) and control (white squares) on PC-3 cells (FIG. 15A and 15B) and 786-0 cells (FIG. 15C and 15D). It is. FIG. 16A and 16B are diagrams showing the nucleotide sequence (Fig. 16A, SEQ ID NO: 9) and amino acid sequence (Fig. 16B, SEQ ID NO: 10) of the α3 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 12) are shown. The beginning and end of the “Tumstatin 333” and “Tumstatin 334” fragments are also shown. FIG. 16A and 16B are diagrams showing the nucleotide sequence (Fig. 16A, SEQ ID NO: 9) and amino acid sequence (Fig. 16B, SEQ ID NO: 10) of the α3 chain of human type IV collagen. The positions of the forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 12) are shown. The beginning and end of the “Tumstatin 333” and “Tumstatin 334” fragments are also shown. FIG. 17 is a schematic diagram showing a tumstatin cloning vector pET22b (+). The sites where the forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 12) and tumstatin are cloned are shown. FIG. 18 is a schematic diagram showing the position of the truncated amino acid in the α3 (IV) NC1 monomer in the tumstatin mutant tumstatin N-53. The filled circle corresponds to the N-terminal 53 amino acid residues deleted from tumstatin to create this mutant. The disulfide bonds marked with short bars are positioned such that they occur at α1 (IV) NC1 and α2 (IV) NC1. FIG. 19A, 19B and 19C are 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). Is a set of three histograms showing ³ H-thymidine incorporation for (y-axis). All groups show triplicate samples. FIG. 20 is a line graph showing the effect of varying amounts of tumstatin (filled circles), BSA (control, open squares) and 7S domain (control, open circles) (x axis) on endothelial tube formation (y axis). FIG. 21A and 21B are a set of line graphs showing the effect on tumor volume (mm ³ , y axis) versus days of treatment (x axis) with tumstatin (filled circles) and endostatin (open circles) versus control (open squares) is there. Data points marked with an asterisk are significant at P <0.05 by one-sided Student's test. FIG. 22 is a line graph showing the increase relative to control mice (white squares), tumor treated with tumstatin mutant N-53 (black circles) with respect to tumor volume (y axis) plotted against days of treatment (x axis). is there. Data points marked with an asterisk are significant at P <0.05 by one-sided Student's test. FIG. 23 is due to concentration changes (x axis) of Arresten (black circle), Canstatin (white circle), Arresten 12 kDa fragment (black square), Arresten 8 kDa fragment (white square), and Canstatin 10 kDa fragment (black triangle). It is a line graph which shows inhibition of endothelial tube formation (y-axis). FIG. 24, endothelium due to changes in the concentration of tumstatin fragment 333 (black circle), tumstatin fragment 334 (white circle), BSA (control, black square), α6 (control, white square), and tumstatin (black triangle) (x axis) It is a line graph which shows inhibition of tube formation (y-axis).

Claims (4)

  An isolated protein selected from the group consisting of the α1 chain NC1 domain of type IV collagen, the α1 chain NC1 domain of type IV collagen, or a fragment, analog, derivative or variant thereof, wherein the protein, fragment, analog The derivative or variant has anti-angiogenic activity.   2. The isolated protein of claim 1, wherein the protein is a monomer.   The isolated protein of claim 2, wherein the protein is arrestin. The isolated protein of claim 2, wherein the protein is canstatin.