KR20010052566A - Protein oligomer compositions comprising endostatin protein and methods of using the same - Google Patents

Abstract

Protein oligomer compositions comprising an endostatin protein and methods of using the protein oligomer compositions for disruption of tubule formation and inhibition of tumorigenesis are provided. The composition of the present invention constitutes a scattering factor of the kidneys and specifically comprises an endostatin protein dimer and a trimer and optionally a metal ion such as zinc.

Description

Protein oligomer composition comprising the endostatin protein and a method of using the same {PROTEIN OLIGOMER COMPOSITIONS COMPRISING ENDOSTATIN PROTEIN AND METHODS OF USING THE SAME}

Some of the direct evidence suggests that the growth and persistence of solid tumors and their metastases to terminal organs are critically dependent on angiogenesis, or replenishment of neovascularization (Folkman, 1989; Hori et al., 1991; Kim). et al., 1993; Millauer et al., 1994). Angiogenesis not only provides the increased nutrient and waste removal pathways required for tumor expansion, but also promotes tumor metastasis by providing a pathway for tumor cells to leave the primary site and enter the bloodstream (Zetter, 1998). In particular, angiogenesis increases the entry of tumor cells into the bloodstream by providing increased densities of unmatured hyperpermeable vessels with thinner basement membranes and fewer intracellular junction complexes than normal mature vessels (Zetter, 1998).

It is assumed that the angiogenic phenotype is the result of a net balance between both positive and negative regulators of neovascularization (Good et al., 1990; O'Reilly et al., 1994; Parangi et al., 1996; Rastinejad et al. , 1989). The tumor itself, along with other accessory host cells such as macrophages, mast cells and lymphocytes, fibroblast growth factor (FGF) (Kandel et al., 1991) and vascular endothelial growth factor / vascular permeability factor (VEGF / VPF) (Zetter , 1998) to stimulate angiogenesis by upregulating the production of various angiogenesis factors. However, many malignant tumors also produce angiogenesis inhibitors including angiostatin proteins and thrombospondins (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994; US Pat. No. 5,639,725 ).

Some angiogenic and anti-angiogenic proteins are stored as inactive precursors in the blood or basement membrane (Hanahan, 1996). One example of such an inert precursor is endostatin protein. The endostatin protein is a collagen-like protein, approximately 20 kDa C-terminal spherical domain of collagen XVIII (c18) that is concentrated in the basal membrane around blood vessels (Oh et al., 1994). Endostatin is stored in vivo as the C-terminal portion of the c18 protein (Rehn et al., 1994; Muragaki et al., 1995) and it is believed that c18 fragments longer than endostatin do not inhibit endothelial cell proliferation (O ′). Reilly et al., 1997). Endostatin is described in US Pat. It was first isolated from angioendothelial cell line for the proliferative inhibition of capillary endothelial cells as described in 5,854,205.

Although endostatin is known to be stored as an inert precursor, it is not known how endostatin is activated. Existing x-ray crystallographic investigations suggest that endostatin does not contain the characteristic Ca ²⁺ binding site active in the pseudomolecule group, selectin (Hohenester et al., 1998). This same investigation suggests that no metal is bound to the temperature human endostatin.

Metal ions are often involved in the biological activity of proteins. In particular, zinc cations are important components of many proteins and play a role in biological processes in the host (Coleman, 1992; O'Reilly et al., 1996, Vallee et al., 1990). In most cases, zinc is directly involved in catalytic activity, but the structural role for zinc has also been described. For example, zinc participates in the dimer formation of human growth hormone and increases the affinity of human growth hormone for prolactin receptors by approximately 8,000 fold (Cunningham et al., 1990; Cunningham et al., 1991).

Summary of the Invention

The present invention includes a protein oligomer consisting of one or more endostatin protein monomers, wherein the oligomers have scatter factor activity. The endostatin protein is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as measured by reduction gel electrophoresis and 18 kDa as measured by non-reducing gel electrophoresis. In one aspect of the invention, the protein oligomer comprises a dimer of an endostatin monomer. In another embodiment, the protein oligomer comprises one or more NC1 region fragments of collagen XVIII having a molecular weight of approximately 38 kDa under reduced gel electrophoresis such that each NC1 fragment contains an endostatin monomer. In a preferred embodiment, the protein oligomer comprising one or more NC1 region fragments is a trimer. The protein oligomer of the present invention may optionally contain a metal component, preferably zinc.

The novel protein oligomers described herein constitute a renal family of scattering factors. The protein oligomers of the present invention have an anti-tubule producing effect and induce recognition of actin cytoskeleton, destruction of coronal lumens, and elongation of cells. New protein oligomers are also anti-tumorigenic and anti-angiogenic.

Also included in the present invention is a method of using a protein oligomer comprising an endostatin protein. The protein oligomers described herein can be used to inhibit tubule production and tumorigenesis.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the described embodiments and the appended claims.

Related Application

This application is filed on June 3, 1998. 60 / 087,890; Provisional Application No. filed July 10, 1998. 60 / 092,393; And Provisional Application No., filed September 1, 1998. Claim 60 / 098,790 as a priority. Each of the foregoing applications is incorporated herein by reference.

Field of invention

The present invention relates to the field of oncology, angiogenesis and morphogenesis, in particular novel protein oligomers comprising endostatin proteins. Endostatin oligomers are useful for inhibiting endothelial tubule formation, regulating cell morphogenesis, and treating metastatic cancer and angiogenesis-dependent diseases.

Figure 1.Structure of human endostatin

β strands are labeled in A-P order, α helix is indicated, and zinc is spherical.

Figure 2. N-terminal loop and zinc binding site of human endostatin

Place the zinc (black circle) ligand histidines 1, 3, and 11 and aspartic acid 76, and the zinc ligand glutamic acid 175, residue 11 carbonyl oxygen, and arginine 4 from the N-terminal loop of the adjacent molecule into the dimer. The second shell of interaction is represented by a ball-stick model.

3A-B. Zinc-dependent dimers in human endostatin crystals

Figure 3a. The zinc (black sphere) site N-terminal loops of the two monomers contact across the central dyad axis. Glutamine 7, phenylalanine 6, and arginine 5 in the loop protrude from one monomer to the next. Representative are residues 31 and 34, two phenylalanine rings that protrude from the endostatin α helix and form different dimer contacts in the crystal.

3b. Contact at the interface of dimers seen in the crystals of human endostatin. Zinc (black sphere) ligands have open bonds, and interfacial residues have tight bonds. The pathways of the polypeptide chains of both monomers are shown in tubes. The solvent accessible surface embedded at this dimer interface is 403 x ² (probe size = 1.4 ms) per monomer.

4. Oligomeric endostatin showing scattering factor activity

HUVEC tubules on Matrigel were preformed for 16 hours and human endostatin monomer, human Fc-endostatin dimer, human Fc dimer, human Fc-endostatin (C7) dimer, human endostatin (C7) dimer, human endostatin / NC1 Treatment with trimers, human Fc collagen 15 dimers, or Fc angiostatin dimers. Only endostatin dimers and trimers showed scattering activity and inhibited tubule formation.

The present invention provides a renal family of scattering factors comprising an endostatin oligomer. Such intra-oleus scattering factor oligomers can act as "anti-matrix" scattering factors and inhibit endothelial tubule assembly caused by extracellular matrix proteins. Endostatin oligomers are also anti-angiogenic and anti-tumorogenic. Although endostatin oligomers are functionally defined, it should be understood that this functional definition never limits the bioactivity of an endostatin oligomer.

A protein oligomer of one aspect of the invention comprises a plurality of endostatin monomers, wherein the endostatin monomer is approximately 20 kDa protein as measured under reduced gel electrophoresis or approximately 18 kDa protein as measured under non-reduced gel electrophoresis. It is characterized by the ability to inhibit cultured endothelial cell proliferation. Endostatin oligomers are carboxy-region fragments of collagen-like molecules, such as collagen XVIII, and can be derived from any mammal. In a preferred embodiment, the endostatin monomer is disclosed at approximately amino acid position 1132 of mouse collagen XVIII and relates to the human endostatin fragment of SEQ ID NO: 1 below.

HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVG

LAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSW

EALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSD

PNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASC

HHAYIVLCIENSFMTAS

As described below, amino acid substitutions may occur in the sequence of endostatin that still produces the functional endostatin protein. For example, when the gene sequence is recombinantly expressed, the observable doublet of the protein produces both versions that are functional endostatin proteins. In addition to the endostatin protein, the following endostatin variants occur, which are missing the first four amino acids from the preceding protein. This demonstrates the variability of the functional endostatin protein molecule. Thus, in another embodiment, the endostatin monomer relates to the human endostatin fragment of SEQ ID NO: 2 below.

DFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAG

TFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEAL

FSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNG

RRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHA

YIVLCIENSFMTAS

The terms "endostatin" and "endostatin monomer" are synonymous and include natural, recombinant, or synthetic endostatin proteins that contain integrity, or "silent" amino acid substitutions, deletions, and additions and still retain scattering factor activity upon oligomerization. . The term "scatter factor activity" refers to the disruption of endothelial tubule formation as measured by Matrigel tube-forming assay. In a preferred embodiment, the endostatin monomer is modified at the seventh amino acid such that glutamine is substituted with cysteine. Substitution of glutamine by cysteine promotes dimerization or oligomerization between endostatin monomers. The term "endostatin monomer" includes a shortened protein wherein one or more amino acids are removed at one or both ends of the endostatin monomer, or in the internal region of the protein, and still retain scattering factor activity upon oligomerization. The term “endostatin monomer” also includes extended proteins or peptides that add one or more amino acids to one or both ends of the endostatin monomer, or to the internal region, and still retain scattering factor activity upon oligomerization. . One example of such a modification is the addition of tyrosine at the first position. Tyrosine labeled molecules can be further labeled with ¹²⁵ I for use in the assay. Labeling by other radioactive isotopes or chemicals such as lysine may also be useful in providing molecular tools for the destruction of target cells containing endothin oligomer receptors. Endostatin monomers may also be recombinantly fused to other proteins or peptides, such as the Fc portion of an antibody, as described in the Examples below.

Silent substitutions of amino acids are also well known in the art and are intended to be included in the claims of the present invention. Silent substitutions occur when substituting amino acids for structurally or chemically similar amino acids does not seriously change the structure, form, or activity of the protein. The term "endostatin monomer" also includes modifications of proteins, subunits and peptide fragments thereof. Such modifications include substituting other molecules for natural amino acids at certain sites, including, but not limited to, natural and non-natural amino acids. Such substitutions can modify the bioactivity of the endostatin oligomer, such as increasing or decreasing scattering factor activity, and can produce biological or pharmacological agonists or antagonists.

In one aspect, the protein oligomer is an endostatin dimer. The present invention encompasses a renal family of scattering factors comprising an endostatin dimer. The endostatin dimers of the present invention gradually disperse established tubes into constituent cells, with no apparent toxicity, after 2-3 hours. While endostatin dimers induce spawning, significant recognition of cytoskeleton can be observed with the destruction of tubul lumens and cellular elongation. Endostatin oligomers differ from the previously described hepatocyte growth factor / scattering factor (HGF / SF) class of scattering factors. In contrast to the HGF / SF class of scattering factors that promote tube formation, the endostatin oligomers of the invention have an anti-tubule producing effect. Endostatin oligomers also affect different types of cells compared to the HGF / SF class of scattering factors. In addition to the scattering activity, the endostatin dimer of the present invention can exert anti-tumorigenic and anti-angiogenic activity when bound to a metal such as zinc.

In another aspect, the present invention provides a protein oligomer comprising a plurality of endostatin / NC1 proteins. The endostatin / NC1 protein of the invention is an approximately 38 kDa C-terminal region fragment of collagen XVIII, each containing approximately 20 kDa endostatin protein described above. The present invention describes for the first time that endostatin / NC1 trimers are included in the renal flow of the aforementioned scattering factors. Endostatin / NC1 trimers induce scattering of endothelial tube structure and morphogenic changes in other cells in a manner similar to endostatin dimers. The anti-tubule producing activity of endostatin / NC1 trimers can also be inhibited by endostatin monomers.

In another embodiment, the aforementioned protein oligomers comprise an endostatin monomer that is a fusion protein. The endostatin fusion protein may include endostatin and anti-angiogenic molecules, angiogenic molecules, and / or molecules that promote dimerization of endostatin monomers. In a preferred embodiment, the endostatin fusion protein comprises the endostatin and the Fc portion of the antibody, wherein the Fc portion of the antibody promotes dimerization. The Fc moiety can be derived from an IgG, IgE, IgA or IgM isotype, but the preferred isotype is IgG.

In a further embodiment, the protein oligomers, dimers and monomers are bound to metal ions, preferably zinc ions. The present invention first provides the fact that endostatin contains a zinc binding site and requires metal bonding for its activity. Prior attempts to investigate crystallized endostatin have resulted in membranes with irregular residues and inaccurate results, but we have expressed endostatin monomers in such a way that the residues are not irregular and identified zinc binding domains on endostatin. In the present invention, human endostatin is expressed as a secreted protein in a rat myeloma cell line in chimeric form with the Fc domain of IgG-1 and released from the Fc portion by enterokinase digestion (described in more detail below). In addition, human endostatin crystals were grown at high pH where the N-terminal histidine was not charged. This modification of the prior art method allows the inventor to accurately identify endostatin as a zinc binding protein.

Using this method, the inventors found that the zinc binding site of human endostatin is the three ligands from the N-terminal loop, namely the fourth ligand from the loop between histidine 1, 3 and 11, and the E and F β strands, namely Aspartic acid 76 and tetrahedron were found (FIGS. 1 and 2). We also describe for the first time zinc-dependent dimer contacts between endostatin monomers in human endostatin crystals. Based on these findings, the present invention provides novel endostatin compositions in combination with zinc which are essential for the anti-tumorigenic activity of the endostatin compositions, and the use of such compositions. In a preferred embodiment, the Fc-endostatin dimer is bound to zinc. In another preferred embodiment, the NC1 / endostatin trimer is bound to zinc.

The invention also includes methods for in vitro or in vivo activation of endostatin, comprising binding one or more metal ions to an endostatin oligomer. In a preferred embodiment, the metal ion is zinc, and in a more preferred embodiment, one zinc molecule is bound per endostatin monomer. Activation of endostatin comprises increased anti-angiogenic activity, increased oncogenic activity and / or increased scattering factor activity. In one embodiment, the zinc bond results in dimerization of the endostatin monomer and thus activating the endostatin. The present invention also includes a method for forming an endostatin oligomer by addition of metal ions, preferably zinc ions.

Typically, the isolated endostatin oligomers of the invention have a purity of at least about 80%, typically at least about 90%, preferably at least about 95%, as measured by band intensity on silver stained gels. The phrase “isolated”, “biologically pure” or “substantially pure” refers to a substance that is essentially free of at least some of the components normally involved when found in its natural state. Other important terms used herein are defined as follows. The articles “a”, “an” and “the” are defined to mean one or more and include the plural unless the context is inappropriate. "Anti-tumor activity" and "anti-tumor development" mean the tumor degeneration of the endostatin oligomer. "Degeneration" means a reduction in tumor mass or size. The term "anti-angiogenesis" refers to the inhibition of angiogenesis. Additionally, "substantial sequence homology" means at least about 70% homology, preferably at least about 80% homology, more preferably at least about 90% homology between known amino acid residue sequences in the endostatin oligomer. . Homology can be determined by sequence identity or by using well-known computer programs such as DNA Star or GeneJockey for lack setting parameters.

The endostatin and NC1 monomers that make up the aforementioned protein oligomer moiety can be separated from body fluids and tissues, including but not limited to serum, urine and ascites fluids. Endostatin monomers and NC1 can also be produced from recombinants, genetically modified cells transplanted into animals, tumors, cell cultures and other sources. Recombinant techniques include gene amplification from DNA using amplification techniques such as polymerase chain reaction (PCR), and gene amplification from RNA sources using amplification techniques such as reverse transcriptase / PCR. The endostatin monomer may be produced by polypeptide synthesis or may be induced by in vitro enzymatic catalysis of a larger included polypeptide such as collagen XVIII to yield an active endostatin monomer. In a preferred embodiment, the endostatin monomer of the invention is produced from a recombinant expression system and the cells are Pchia pastoris.

The endostatin oligomers of the present invention have multiple uses. Endostatin oligomers are particularly useful for endothelial tubule formation inhibition and morphogenesis studies. Endostatin oligomers, and active fragments thereof, are also useful for the treatment of metastatic cancer and tumors and angiogenesis-related cancers and diseases.

For example, endostatin oligomers can be used for the treatment of metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases. One example of a metastatic disease is metastatic cancer. Angiogenesis-related diseases include, for example, angiogenesis-dependent cancer, including solid tumors, hematologic tumors (eg leukemia), and tumor metastasis; Benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and purulent granulomas; Rheumatoid arthritis; psoriasis; Angiovascular diseases of the eye, such as diabetic retinopathy, prematurity retinopathy, spot degeneration, corneal graft rejection, renal vascular glaucoma, fibroproliferation behind the lens, dermal scarring; Osler-Weber syndrome; Myocardial angiogenesis; Plaque neovascularization; Capillary dilator; Hemophilia patients joints; Fibrotic hemangioma; And wound granulation. The endostatin oligomers of the invention are useful for treating diseases of excessive or abnormal stimulation of endothelial cells. Such diseases include but are not limited to intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, ie keloids. They are also useful in the treatment of diseases with angiogenesis as pathological consequences such as Rochele minalia quintosa and ulcers (Helobacter pylori).

Endostatin oligomers can also be used in the development of affinity columns for the separation of antibodies directed to endostatin. Such antibodies can be isolated and purified and then analyzed for amino acids. In addition, polypeptides that bind to highly specific and active endostatin oligomers can be labeled with a label or reporter group and then used for visualization and quantification in the assays described herein using detection techniques such as radiographic and membrane binding techniques. Reporter groups or labels are typically fluorescent or radioactive groups or enzymes. This application provides important diagnostic and research tools.

Endostatin oligomers can also be used in the development of affinity columns for the separation of endostatin oligomer receptors. After separation and purification of these receptors, amino acid sequencing can be performed. Using this information, the gene encoding the receptor can be identified and isolated. Then, a cloned nucleic acid sequence for insertion into a vector capable of expressing the receptor can be developed.

Applicants' invention also encompasses recombinant expression systems in which a nucleic acid encoding a protein component of an endostatin oligomer described herein is contained in a single cell. Nucleic acids encoding oligomeric components can be in a single vector or multiple vectors. In a preferred embodiment, the recombinant expression system is Pchia pastoris. Nucleic acid sequences corresponding to biologically active endostatin oligomers and proteins are useful for in vivo regulation of endothelial cell progression and for the diagnosis and treatment of endothelial cell-related diseases, eg, by gene therapy.

Nucleic acid sequences corresponding to and encoding the protein components of the endostatin oligomer can be prepared based on the knowledge of the amino acid sequence and the corresponding recognition in the art between codons (sequences of three nucleic acid bases) and amino acids. Although the third base of the codon can vary, due to the degeneracy of the genetic code that still encodes the same amino acid, many different possible coding nucleic acid sequences are derivable for a particular protein or peptide fragment. Alternatively, nucleic acid sequences can be derived from gene banks using oligonucleotide probes designed based on techniques well known for N-terminal amino acid sequences and genetic cloning. Nucleic acid sequences are synthesized using automated systems well known in the art. The entire sequence may be synthesized or a smaller series of oligonucleotides may be made and subsequently linked together to obtain a full length sequence. Genes encoding components of the endostatin oligomers can also be used to (1) isolate messenger RNA from tissue, (2) generate corresponding DNA sequences using reverse transcriptase, and (3) use PCR to sequence the active sequences with appropriate primers. By amplifying a DNA sequence encoding a, it can be isolated from cells or tissues expressing high levels of endostatin monomer or NC1.

The isolated recombinant or synthetic endostatin oligomers described herein are useful for producing antibodies specific for endostatin oligomers. Such antibodies that specifically bind endostatin oligomers can be used in diagnostic methods and kits well known to those skilled in the art for detecting or quantifying endostatin oligomers in body fluids or tissues. The results of these tests can be used to diagnose or predict the onset or recurrence of cancer and other angiogenic mediated diseases. In addition, passive antibody therapy using antibodies that specifically bind to endostatin oligomers can lead to morphogenesis processes such as metastatic cancer and angiogenesis-dependent progression such as proliferation, development, wound healing, tissue repair and angiogenesis-dependent diseases. It can be used to control the process. In addition, antisera directed to the Fab region of an endostatin oligomer antibody can be administered to block the ability of endogenous endostatin oligomer antisera to bind endostatin oligomers.

Antibodies specific for an endostatin oligomer may be polyclonal or monoclonal and are prepared according to techniques and protocols well known in the art. A preferred method of making monoclonal antibodies is a modified version of the method of Kearney et al. (1979), which is incorporated herein by reference. Monoclonal antibodies can be used in well known immunoassay formats such as competitive and noncompetitive immunoassays, including ELISA, sandwich immunoassay and radioimmunoassay (RIA), to detect the presence or absence of the present endostatin oligomers in body fluids and tissues. Decide Examples of body fluids include, but are not limited to, blood, serum, synovial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterine fluid, saliva, mucus and free fluid.

Polyclonal antisera are also produced using established techniques known to those skilled in the art. For example, polyclonal antiserum can be produced by administering antigens to animals in mice, rabbits, rats, goats, sheep, guinea pigs, chickens and other animals, most preferably mice and rabbits. Isolated recombinant or synthetic endostatin oligomers in combination with carrier molecules such as bovine serum albumin can be combined and emulsified with the adjuvant mixture to subcutaneously inject into various parts of the animal's back, neck, flanks and sometimes footpads. The second shot is injected at regular intervals, such as every two to four weeks. Blood samples are obtained by venipuncture using, for example, marginal ear veins after inflation approximately 7 to 10 days after each injection. Blood samples are coagulated and centrifuged, serum removed, divided and stored under refrigeration for immediate use or frozen for subsequent analysis.

Techniques for producing single chain antibodies are known to those skilled in the art and are described in U.S. Pat. It can be used in single chain antibody production against the endostatin oligomers described in patent 4,946,778 and described herein. Bispecific antibodies have two antigen binding domains in which each domain is directed against a different epitope. Phage display technology can be used to select antibody genes having binding activity for endostatin oligomers from either a pure library screened for the presence or absence of antibodies against endostatin oligomers or from PCR-amplified v genes of lymphocytes in humans.

Once labeled with a detectable biological molecule or chemical, the aforementioned endostatin oligomers and antibodies are useful for purposes such as in vivo and in vitro diagnostic and laboratory studies using the methods and assays described below. Various types of labels and methods of binding labels to endostatin oligomers and antibodies are well known to those skilled in the art. Several specific markers are described below.

For example, endostatin oligomers and antibodies are combined with radiolabels such as, but not limited to, ³² P, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I or ¹³¹ I. Labels can be detected by methods such as scintillation coefficient, gamma-ray spectroscopy or radiographs.

Bioluminescent labels, such as derivatives of firefly luciferin, are also useful. The bioluminescent material is covalently attached to the endostatin oligomer or antibody in a conventional manner, and the labeled endostatin oligomer or antibody is detected when an enzyme such as luciferase catalyzes the reaction with ATP to cause the bioluminescent material to emit photons.

Fluorescent sources can also be used as labels. Examples of fluorescent sources include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine and Texas Red. Fluorescent sources are generally detected by fluorescence combiners.

Endostatin oligomers and antibodies may alternatively be labeled with a dye source to provide an enzyme or affinity label. For example, endostatin oligomers or antibodies can be biotinylated and used in a biotin-avidin reaction that can also be coupled with a label such as an enzyme or fluorescent source. Alternatively, endostatin oligomers or antibodies may be labeled with peroxidase, alkaline phosphatase or other enzymes that cause a chromogen or fluorescence reaction upon the addition of a substrate. Additives such as 5-amino-2,3-dihydro-1,4-phthalazinedione (also known as Luminol ^™ ) (Sigma Chemical Company, St. Louis, MO) and p-hydroxybiphenyl (p- Rate increasing agents such as phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through fluorescence reactions; Luminescent or fluorescent dioxetane derivatives of enzyme substrates may also be used. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting color changes with the aid of a spectrophotometer. In addition, the endostatin oligomers or antibodies can be labeled with colloidal gold for use in immunoelectron microscopy according to methods well known to those skilled in the art.

The proteins and antibodies of the invention are useful for the diagnosis and treatment of metastatic and angiogenic-related diseases. One example of a metastatic disease is metastatic cancer. Angiogenesis-related diseases include, for example, angiogenesis-dependent cancers including solid tumors, hematologic tumors such as leukemia and tumor metastasis; Benign tumors such as hemangiomas, acoustically neuromas, neurofibromas, trachomas and purulent granulomas; Rheumatoid arthritis; psoriasis; Angiogenic diseases of the eye such as diabetic retinopathy, prematurity retinopathy, spot degeneration, corneal graft rejection, neovascular glaucoma, fibroplasia behind the lens, dermal scarring; Osler-Weber syndrome; Myocardial angiogenesis; Plaque neovascularization; Capillary dilatation; Hemophilia patients joints; Fibrotic hemangioma; And wound granulation. The endostatin oligomers of the invention are useful for the treatment of diseases due to excessive or abnormal stimulation of endothelial cells. Such diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma and hypertrophic scars, ie keloids. They are also useful for the treatment of diseases in which angioplasty occurs with pathological consequences such as Rochele minalia quintosa and ulcers (Helobacter pylori).

In accordance with the present invention, endostatin oligomers can be used in combination with other compositions and procedures for the treatment of diseases and conditions described above. For example, tumors may be conventionally treated with surgery, radiation or chemotherapy in parallel with endostatin oligomers and then endostatin oligomers may be administered to prolong the resting state of micrometastasis and stabilize the remaining primary tumors.

The endostatin oligomers described above can be provided as proteins and protein fragments that are separated and substantially purified in pharmaceutically acceptable formulations using formulation methods known to those skilled in the art. Such formulations may be administered by standard routes. In general, the formulations are administered by topical, vascular, intraperitoneal, intracranial, intraventricular, intracranial, intravaginal, intrauterine, oral, rectal or parenteral (eg intravenous, intravertebral, subcutaneous or intramuscular) routes. Can be. In addition, endostatin oligomers can be introduced into biodegradable polymers that allow sustained release of the compound, such that the polymer is injected around the site where the drug is delivered, for example, to the site of the tumor or the endostatin oligomer is slowly released systemically. It is preferable. Osmotic minipumps are also used to regulate and deliver high concentrations of endostatin oligomers via cannula to the vessels that supply the site, such as directly to metastatic growth sites or to the tumor. Biodegradable polymers and their use are described in detail in, for example, Brem et al. (1991), which is incorporated herein by reference in its entirety.

The dosage of the endostatin oligomer of the present invention will depend on the condition or condition to be treated and other clinical factors such as the weight and condition of the human or animal and the route of administration of the compound. To treat a human or animal, approximately 0.5 mg / kg to 500 mg / kg of endostatin oligomer and peptide may be administered. A more preferred range is 1 mg / kg to 100 mg / kg and the most preferred range is 2 mg / kg to 50 mg / kg. Depending on the half-life of the endostatin oligomers and peptides in certain animals or humans, it may be administered several times a day to once a week. It is understood that the present invention applies to both human and veterinary uses. The methods of the present invention include single administration and multiple administrations simultaneously or over long periods of time.

Endostatin oligomer formulations are oral, rectal, eye (including intravitreal or in-camera), nasal, topical (including oral and sublingual), intrauterine, vaginal or parenteral (subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, Intracranial, intratracheal and epidural). The endostatin oligomer formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient with the pharmaceutical carrier (s) or excipient (s). Generally, formulations are prepared by uniformly and intimately combining the active ingredient with a liquid carrier or finely divided solid carrier or both, and then molding the product, if necessary.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes that make the formulation isotonic with the intended recipient blood: and suspensions and thickening agents. Aqueous and non-aqueous sterile suspensions which may include. The formulations may be provided in unit or multidose containers, such as sealed ampoules and vials, and may be freeze-dried (frozen) only requiring the addition of a sterile liquid carrier such as water for injection immediately before use. Dry) conditions. Instant injections and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit dose, up to a daily dose, or as appropriate fractions thereof, as described herein above. In particular, in addition to the components described above, it should be understood that the formulations of the present invention may include other agents conventional in the art in view of the formulation type in question.

In addition to the methods of treatment, the present invention also relates to methods of diagnosing endothelial cell-related diseases and disorders using antibodies that specifically bind endostatin oligomers and active fragments thereof and endostatin oligomers and peptides thereof. Diagnosis is accomplished by detection of an endostatin oligomer or antibody against it in body fluids or tissues. Detection can be accomplished by comparing the detection level of an endostatin oligomer or antibody thereto with the normal level of an endostatin oligomer or antibody thereto.

Kits for the determination of endostatin oligomers are also included as part of the present invention. Antisera that can detect endostatin oligomers in plasma, urine, tissue extracts and cell cultures with the highest titer and specificity can be prepared for rapid, reliable, sensitive and specific measurements and localization of endostatin oligomers. Additional tests are made for ease of use. These assay kits include the following techniques: competitive and noncompetitive assays, radioimmunoassays, bioluminescence and chemiluminescence assays, fluorescence assays, sandwich assays, immunoradiometric assays, dot blots, enzyme binding assays including ELISA, microtiter plates, urine Or antibody coated strips or dipsticks for rapid monitoring of blood and immunocytochemistry. Intra- and intra-assay deviations are set at 20%, 50% and 80% points on the standard curve of substitution or activity.

The assay kit provides instructions for the use of the instructions, antiserum, endostatin oligomers and possibly radiolabeled endostatin oligomers or peptides and / or binding antibody complexes. The kits are useful for the determination of endostatin oligomers in biological fluids and tissue extracts of animals and humans with or without tumors or angiogenesis-dependent diseases.

Kits for locating endostatin oligomers or peptides in tissues and cells are also included in the present invention. This endostatin oligomeric immunohistochemistry kit provides a secondary antiserum in combination with an instruction manual, an endostatin oligomeric antiserum and possibly fluorescent molecules such as blocking serum and fluorescent isothiocyanate or some other reagent used to visualize primary antiserum. do. Immunohistochemical techniques are well known to those skilled in the art. This endostatin oligomer and peptide immunohistochemistry kit allows for localization of endostatin oligomers in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, the tumor is biopsied or collected and the tissue sections are cut into microtomes to examine the site of hyaluronic acid binding link module generation. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.

The invention further includes methods for identifying and quantifying endostatin oligomer-specific receptors. Endostatin oligomer labels with short-lived isotopes allow visualization of receptor binding sites in vivo using positron emission tomography or other current radiographic techniques. This method is important for the study of angiogenesis and metastasis of cancer and other angiogenesis-related diseases.

The invention is specifically illustrated by the following examples and should not be construed as limiting its scope in any way. On the contrary, there may be various other aspects, modifications, and equivalents thereof, and after reading the description herein, one skilled in the art will be able to explain it without departing from the spirit of the invention and / or the scope of the appended claims. It is clearly understood.

Example 1

Production of endostatin monomers and dimers and oligomers

To produce an endostatin monomer, polymerase chain reaction (PCR) is used to modify the endostatin cDNA for expression in a pdCs-Fc (D4K) vector containing the enterokinase recognition site Asp4-Lys at the junction of the fusion protein. . The forward primer is 5′-C AAG CTT CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 3) comprising the Hind III site, followed by the sequence encoding the N-terminus of endostatin (HSHRDFQPVLHL, SEQ ID NO: 4). The reverse primer is a 5'-C CTC GAG CTA CTT GGA GGC AGT CAT G (SEQ ID NO: 5) position immediately following the C-terminus of the endostatin, followed by the Xho I site (CTCGAG). )to be.

The PCR product is cloned and sequenced and the Hind III-Xho I fragment encoding endostatin is linked with the pdCsFc (D4K) vector. Stable clones expressing Fc (D4K) -endostatin are obtained by electroporation of NS / O cells and then selected in growth medium containing 100 nM methotrexate. Protein expression levels are analyzed by anti-human Fc ELISA as described in Lo, K. M. et al. (1998) Protein Engineering 11: 495-500 and confirmed by SDS-PAGE. Top clones are cloned at limiting dilution.

Fc-human endostatin fusion protein is purified on Protein A Sepharose using 100 mM citric acid, pH = 3.0 as elution buffer. Enterokinase and trypsin digestion are performed to form two forms of cleaved endostatin. For trypsin, the ratio of enzyme to protein (w / w) is 1: 200 and 18 hours room temperature results in 4 amino acids, HSHR, cleaved from the N-terminus in the endostatin molecule. Enterokinase degradation leads to products having an additional one amino acid leucine at the N-terminus of endostatin. Both cleaved products are further purified using heparin sepharose and SP sepharose (Pharmacia, Bridgewater, New Jersey).

Disulfide-linked human endostatin dimers are produced in pdCs-Fc (D4K) -hu Endo by replacing a restriction fragment encoding the N-terminal sequence of human endostatin with a double-stranded oligonucleotide encoding the same sequence, but the Q7 codon is To generate pdCs-Fc (D4K) -hu Endo (C7). This plasmid is transfected with myeloma cells and the Fc-endostatin dimer is purified from conditioned media using Protein A affinity chromatography as described in Lo, K. M. et al. (1998) Protein Engineering 11: 495-500. After cleaving the Fc-endostatin dimer with enterokinase, the endostatin dimer is dissolved from the Fc portion by gel permeation in S-200.

An endostatin-NC1 trimer is generated as follows. NC1 protein consists of two primers 5 'GAT CGG CCC AGC CGG CCC ATC ATC ACC ATC ACC ATG ACG ATG ACG ATA AGT CAG GGC AGG TGA GGC TCG CTA CAC GCA GG 3' (SEQ ID NO: 6) and 5 'GAT CGG ATC CCT ACT TGG AGG CAG TCA TGA AGA 3 '(SEQ ID NO: 7). Such primers are used to amplify using PCR from the SGQVRLWATRQ amino acid sequence (SEQ ID NO: 8) through the C terminus of human collagen 18. PCR products are cloned at the Sfil-BamH1 site of pSecTag2a (Invitrogen, San Diego, California). Stable 293T cell transfectants are selected from Zeosin and the conditioned medium is eluted from Ni-agarose (QIAGEN, Valencia, California) with 250 mM imidazole / PBS and then dialyzed with PBS.

Example 2

Zinc-dependent dimers found in crystals of human endostatin

10 mg / ml protein in 20 mM Tris-HCl pH 8.5, 150 mM NaCl and 50 mM Tris-HCl pH 8.5, 6% PEG 8K, 150 mM NaCl and 2 mM MgCl, mixed by dropping the X-ray structure at room temperature Dynamic diffusion of the mixture was obtained at 4 ° C. and determined from crystals prepared in parallel with 0.5 ml of the latter buffer. Crystals (C2 a = 92.76 Å, b = 74.27 Å, c = 137.80 Å, β = 102.56) have 4 monomers in the asymmetric unit. The crystals are transferred to 15% PEG 6K, 20% glycerol, 50 mM Tris-HCl pH 8.5 and 150 mM NaCl for about 20 seconds and then flash cooled with a stream of cold nitrogen. Data is collected at a CHESS Al station (λ = 0.92 Hz) with an 80 mm 2K CCD (complex mode) (0.5 degree vibration). Integrate the data and compare with DENZO and SCALEPACK (HKL Research, Charlottesville, Virginia). Most subsequent processes use the CCP4 program (T. A. Jones (1992) "Molecular Replacement" CCP4, 92-105; G. J. Kleywegt and T. A. Jones (1994) "From First Map to Final Model" CCP4, 59-66).

After this method, the zinc binding site of human endostatin is tetrahedral with 3 zinc ligand histitin 1, 3 and 11 from the N-terminal loop and the fourth ligand aspartic acid 76 from the loop between the E and F-β chains. Is determined (FIGS. 1 and 2). The zinc binding site most closely resembles that of the zinc protease. Atomic absorption spectroscopy confirms that zinc is a constituent of the Fc-endostatin chimeras described above of both human and rat endostatin in the solutions shown in Table 1 below.

protein N-terminal sequence Endostatin concentration (mg / ml) Zinc concentration (micromolar) Zinc per endostatin monomer hE-Ek LHSHRD 4 176 0.9 hE-Tryp D 3.8 14 0.1 hFc-hE 0.4 18 0.9 mFc-mE 2 79 0.9

The first line of Table 1 also explains that Fc-human endostatin treated with enterokinase, which produces full length endostatin, also shows approximately 1 zinc atom per endostatin monomer. Trypsin truncated Fc-human endostatin resulting in the loss of the first 4 residues of endostatin does not contain detectable zinc (second line in Table 1). This result is consistent with the human endostatin crystal structure showing histidine at two of the zinc ligands, residues 1 and 3.

Many 2-fold symmetric dimers are evident in the packaging of 4 endostatin monomers in asymmetric units of human endostatin crystals. One dimeric interface is formed by the contact of phenylalanine exposed to two solvents on the α1α helix from each monomer, and the prominent nonpolar patch is described as a potential interdomain interaction site (residues F31 and F34 in FIG. 3) in murine endodotin. Another dimer contact, which is zinc dependent, is formed between the protruding N-terminal loops of the two monomers; Each loop is ordered around the zinc ions (center in FIG. 3A). This contact is mainly formed by three residues: arginine 4, phenylalanine 6 and glutamine 7 which protrude from the edge of the N-terminal loop (FIG. 3B). At residue 7 of each monomer, the glutamine contacts each other to form a hydrogen bond at the center of the interface. Each ring of phenylalanine at residue 7 is contacted with a nonpolar patch containing Lewisson 78 and valine 72 of adjacent monomers. Arginine 4 from each monomer forms a hydrogen bond with two main chain carbonyl oxygen (carbonyls of residues 75 and 76) on a loop containing zinc aspartate ligand at residue 76 (FIG. 3B). This dimer interaction can only occur when the N-terminal loop is ordered as a result of the zinc bond, and then the zinc bond is preferably stabilized by the dimer interface.

Example 3

Zinc binding of endostatin is essential for anti-angiogenic activity.

As described in Example 2, it was found that histitin 1, 3, 11 and aspartic acid 76 coordinate with zinc ions. Following this finding, point mutations in the endostatin protein convert the Zn ²⁺ ligand to alanine. One double (H1 / 3A) and two single (H11 A and D76A) mutants are generated in E. coli expression vectors using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California). Mutations are confirmed by sequencing.

The wild type endostatin and three mutants were purified in parallel and were modeled on Lewis lung carcinoma as already described in O'Reilly, MS et al., Cell 88: 277 (1997) and T. Boehm et al., Nature 390: 404 (1997). Test at Different endostatin preparations were compared on sodium dodecyl sulfate (SDS) gels under reducing and non-reducing conditions and no significant differences in purity and crosslinking pattern were observed. SDS-PAGE of endostatin purified from Escherichia coli shows protein ladders including monomers, dimers, trimers and high molecular weight complexes caused by crosslinking of endostatin and other endostatin molecules through disulfide bond formation.

Unlike wild type endostatin, three mutants of endostatin did not regress Lewis lung carcinoma (data not shown). Although the H 1/3 A endostatin mutant is weakly active, the weak activity may be due to the twisted structure resulting from the use of 2 histidines from His-tags that coordinate with Zn ²⁺ ions or loss of ability to bind zinc.

Example 4

Scattering of Preformed Endothelial Structures by Endostatin Dimers

To measure the ability of endostatin monomers and dimers to influence morphogenesis, Matrigel tube-forming assays are used. This assay uses endothelial cells plated on extracellular matrix proteins that spontaneously aggregate and assemble into dense multicellular capillary-like tubular structures. The analysis is performed as follows.

Isolated from single donors (Clonetics, San Diego, California) and <10 subculture was HUVEC (human umbilical vein endothelial cells), a 37 ℃, 5% FCS, hVEGF , hFGF, (10 ng / ㎖) of 5% CO _2, Hydro Cultured in EBM medium (Clonetics, San Diego, California) supplemented with EGM-2-MV growth factors, including cortisone (1 μg / ml) and GA-1000 (0.1%). Cells are plated 1 ml at 40,000 cells / well in 24-well plates containing complete medium as described above. These wells are precoated with Matrigel at 4 ° C. and the dissolved base membrane preparation is extracted at 300 μl / well from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Becton Dickinson / Collaborative, Franklin Lakes, New Jersey). Human endostatin derivatives in PBS (10-2000 nM) are added simultaneously with the cells to Matrigel-coated plates or 16 hours after tube formation. Recombinant hHGF (R & D Systems, Minneapolis, Minnesota) is added at 50 ng / ml.

For inhibition of endostatin oligomers by endostatin monomer, the preformed tube is preincubated for 30 minutes with endostatin monomer (2000 nM) prior to oligomer treatment. The wells are then incubated at 37 ° C., 5% CO ₂ and photographed with a 40-2000 × phase contrast microscope. Human microvascular, pulmonary and umbilical artery primary endothelial cultures yield the same results and processes.

Antibody Fc-endostatin fusion proteins containing endostatin at the C-terminus potentially inhibit tube formation when cultured, including when plating cells that are actually more dispersed over hours rather than adhering to the tube structure (data Not shown). The same result is observed with the primary culture of human microvascular, umbilical vein, umbilical artery and pulmonary artery endothelial cells (data not shown). The rate of this effect has been debated about the main effect on cell growth, and cell counts, BrdU staining and ³ H introduction do not account for significant proliferation triggers compared to untreated cells during this period (data not shown).

Interestingly, after enterokinase cleavage of Fc-endostatin fusion to Fc and endostatin, either Fc or endostatin alone or together with Fc and endostatin showed no inhibitory activity (FIG. 4). Recombinant endostatin from baculovirus and yeast that is not fused with Fc is also ineffective (data not shown). However, it has been found that artificially formed dimer endostatin exhibits scattering activity by using close contact between adjacent glutamine-7 residues predicted by the dimer crystal structure of endostatin. Mutation of endostatin glutamine-7 to cysteine and fusion with antibody Fc form a new intermolecular cysteine bond in cysteine-7, and enterokinase cleavage is a free endostatin dimer that migrates at 40 kDa and 20 kDa under non-reducing and reducing conditions, respectively (Data not shown). Both endostatin dimers in mice and humans significantly regenerate the inhibitory activity of Fc-endostatin fusion proteins upon tube formation with an IC ₅₀ of approximately 10 nM and are inactive for bovine capillary endothelial cells or HUVEC proliferation (data not shown).

During endostatin dimer-derived acid, rapidly demarginating cells appear 1-2 hours faster than prominent lamellipod and limb formation (data not shown). Confocal micrographs of paloydine-FITC show dramatic reconstitution of actin cell structure within 2 hours of pronounced stress fiber formation consistent with signaling pathways associated with rapid actin polymerization (data not shown). As visualized by electron micrographs, endostatin oligomer-induced scattering is achieved by destruction of tubule lumens and cell extension (data not shown).

Scattering factor activity of the endostatin oligomers described herein is specific. Collagen XV (c15) and collagen XVII from which the endostatin monomer is derived show 60% amino acid identity in the endostatin domain and share the NC1 trimerization domain, but the collagen XV oligomer does not act as a scattering factor (FIG. 4). Fc fusion proteins containing c-15 endostatin-like domains are not able to scatter tubules as opposed to Fc endostatin dimers, nor can they counteract the effects of Fc endostatin dimers (data not shown). Fc- angiostatin or monomeric angiostatin also show no tube formation, consistent with no structural homology between endostatin and angiostatin (data not shown). In total, only the oligomeric endostatin composition exhibits scattering activity.

In addition, pretreatment of established tubes with excess endostatin monomer (1000 nM) strongly blocks the ability of endostatin dimers (20-50 nM) to scatter cells (data not shown). It is potentially consistent with endostatin monomers by unproductively occupying receptors that act to block the endostatin oligomers in a mainly negative manner, eg, requiring cross-linked oligomers for activity.

Example 5

Scattering by Endostatin-NC1 Trimers of Preformed Endothelial Structure

Human and murine NC1 proteins are isolated from the transfected 293T cell supernatant as described above in Example 1. Trimerization is confirmed by crosslinking the trimerized complexes to partition the product by non-reducing modified polyacrylamide gel electrophoresis (PAGE). Crosslinking is carried out for 20 minutes at room temperature in the presence of 5 mM ethylene glycol-bis (succinidiisuccinate) (EGS). EGS is dissolved in DMSO and diluted 10-fold in PBS. The reaction is stopped by adding Tris at a final concentration of 100 mM. The resulting gel produces approximately 120 kDa species upon EGS crosslinking (data not shown).

The murine and human NC1-endostatin trimers equally show endothelial tube formation with an IC ₅₀ of approximately 10 nM (FIG. 4), which in turn is consistent with the dependence of activity upon endostatin domain oligomerization. NC1-endostatin trimers are slightly less effective than endostatin dimers at high concentrations and possibly show a potential reduction from noncovalent compared to covalent oligomerization.

Scattering results obtained with endostatin / NC1 trimers are similar to endostatin dimers. Endostatin / NC1 trimers induce spawning, and rapidly demagnetizing cells appear 1-2 hours faster than prominent lamelipod and limb formation (data not shown). Confocal micrographs of paloydine-FITC show dramatic reconstitution of actin cell structure within 2 hours of pronounced stress fiber formation consistent with signaling pathways associated with rapid actin polymerization (data not shown). As visualized by electron micrographs, endostatin oligomer-induced scattering is achieved by destruction of tubule lumens and cell extension (data not shown).

Pretreatment of established tubules with excess endostatin monomer (1000 nM) strongly blocks the ability of NCI-endostatin trimers (20-50 nM) to scatter cells (data not shown). As mentioned above, this is potentially consistent with endostatin monomers by non-productively occupying receptors that act to block the endostatin oligomers in a predominantly negative way, eg, requiring cross-linked oligomers for activity.

Example 6

Endostatin oligomers include a new class of scattering factors

Oligomeric endostatin-induced cell scattering and cell structure reconstitution reminds of morphogenic changes caused by hepatocyte growth factor / scattering factor (HGF / SF), and associated macrophage stimulating proteins (MSPs) regulate cell motility, dispersion, proliferation and apoptosis Include scattering factor classes. Human umbilical vein endothelial cells (HUVECs) express large amounts of the HGF receptor c-met 16 as opposed to endostatin oligomers, but HGF cannot spawn HUVEC tubules at all (data not shown). In contrast, while HGF potentially disperses and scatters MDCK and HepG2 cell aggregates cultured in plastics, oligomeric endostatin derivatives are inactive (data not shown). MSP also cannot derive HUVEC scattering (data not shown).

Clearly, it is well established that HGF actually promotes tube formation in the collagen matrix, as opposed to the anti-tubule producing effects of endostatin described herein. Thus, endostatin oligomers define novel scattering factors that are structurally and functionally distinct from previous unique HGF / MSP strains.

Modifications and variations of the method will be apparent to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to be included within the scope of the appended claims.

Reference

Claims (29)

An isolated protein oligomer comprising at least one endostatin monomer, wherein the oligomer retains scattering factor activity. The isolated protein oligomer of claim 1, wherein the endostatin monomer is a carboxy-terminal region fragment of collagen XVIII having a molecular weight of approximately 20 kDa as measured by reduction gel electrophoresis and 18 kDa as measured by non-reducing gel electrophoresis. The isolated protein oligomer of claim 1, wherein the oligomer is a dimer of an endostatin monomer. 4. The isolated protein oligomer of claim 3, further comprising a metal component. The isolated protein oligomer of claim 4, wherein the metal component is zinc. 6. The isolated protein oligomer of claim 5, wherein the oligomer has anti-tumorogenic activity. The protein oligomer of claim 1, wherein the protein oligomer comprises at least one NC1 region fragment of collagen XVIII having a molecular weight of approximately 38 kDa under reduced gel electrophoresis such that each NC1 fragment contains an endostatin monomer. 8. The isolated protein oligomer of claim 7, wherein the oligomer is a trimer. The isolated protein oligomer of claim 8, further comprising a metal component. 10. The isolated protein oligomer of claim 9, wherein the metal component is zinc. The isolated protein oligomer of claim 10, wherein the oligomer retains anti-tumor activity. The protein oligomer of claim 1, wherein the endostatin monomer is a fusion protein. 13. The protein oligomer of claim 12, wherein the endostatin monomer contains endostatin and the Fc portion of the antibody. A method for inhibiting tubule production, comprising administering to the endothelial cells an amount of a tubule production inhibitor comprising a one or more endostatin monomers, the oligomer having scattering factor activity. The method of claim 14, wherein the protein oligomer is a dimer of an endostatin monomer. The method of claim 15, wherein the protein oligomer further comprises a metal component. The method of claim 16, wherein the metal component is zinc. The method of claim 14, wherein the protein oligomer comprises at least one NC1 region fragment of collagen XVIII having a molecular weight of approximately 38 kDa under reduced gel electrophoresis such that each NC1 fragment contains an endostatin monomer. The method of claim 18, wherein the oligomer is a trimer. The method of claim 19, wherein the protein oligomer further comprises a metal dimerization component. The method of claim 20, wherein the metal dimerization component is zinc. A method of inhibiting tumorigenesis, comprising administering to the endothelial cells an oncogenic inhibitory amount of a protein oligomer comprising at least one endostatin monomer, wherein the oligomer has scattering factor activity. The method of claim 22, wherein the protein oligomer is a dimer of an endostatin monomer. The method of claim 23, wherein the protein oligomer further comprises a metal component. The method of claim 24, wherein the metal component is zinc. The method of claim 22, wherein the protein oligomer comprises one or more NC1 region fragments of collagen XVIII having a molecular weight of approximately 38 kDa under reduced gel electrophoresis such that each NC1 fragment contains an endostatin monomer. 27. The method of claim 26, wherein the oligomer is a trimer. The method of claim 27, wherein the protein oligomer further comprises a metal dimerization component. The method of claim 28, wherein the metal dimerization component is zinc.