Methods and compositions for diagnosing and treating disorders involving angiogenesis Number:6,803,211 from the United States Patent and Trademark Office (PTO) owispatent

Title: Methods and compositions for diagnosing and treating disorders involving angiogenesis

Abstract: The present invention relates to polynucleotides associated with angiogenesis-related disorders. The present invention also relates to canine endostatin genes, novel genes associated with angiogenesis-related disorders, such as cancer. The invention encompasses endostatin nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof, endostatin gene products and antibodies directed against such gene products, cloning vectors containing mammalian endostatin gene molecules, and hosts that have been genetically engineered to express such molecules. The invention further relates to methods for the identification of compounds that modulate the expression of endostatin genes and gene products and to using such compounds as therapeutic agents in the treatment of angiogenesis-related disorders, e.g., cancer. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of angiogenesis-related disorders, e.g., cancer, and to methods and compositions for the treatment these disorders.

Patent Number: 6,803,211 Issued on 10/12/2004 to Tong,   et al.

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Inventors:	Tong; Xiao (East Brunswick, NJ); Sheppard; Michael G. (Victoria, AU)
Assignee:	Pfizer Inc. (New York, NY) Pfizer Products Inc. (Groton, CT)
Appl. No.:	09/938,391
Filed:	August 24, 2001

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Current U.S. Class:	435/69.1 ; 435/320.1; 435/325; 435/455; 530/350; 530/351; 536/23.1; 536/23.5
Field of Search:	435/69.1,320.1,325,455 530/350,351 536/23.1,23.5

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References Cited [Referenced By]

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Primary Examiner: Fredman; Jeffrey
Assistant Examiner: Kaushal; Sumesh
Attorney, Agent or Firm: Ling; Lorraine B. Wootton; Thomas Kohn & Associates, PLLC

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from United States Provisional Application No. 60/227,924, filed Aug. 25, 2000.

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Claims

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What is claimed:

1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes an endostatin consisting of: a) a polypeptide as shown in SEQ ID NO: 2; or b) a polypeptide as shown in SEQ ID NO: 4.

2. An isolated nucleic acid molecule consisting of: a) a nucleic acid as shown in SEQ ID NO: 1; or b) a nucleic acid as shown in SEQ ID NO: 3.

3. An isolated nucleic acid molecule comprising a complement of the nucleic acid molecule of any one of claims 1-2.

4. A vector comprising the nucleic acid of any one of claims 1-2.

5. An expression vector comprising the nucleic acid of any one of claims 1-2 operatively associated with a regulatory nucleic acid controlling the expression of the polypeptide encoded by said nucleic acid.

6. A host cell genetically engineered to contain the nucleic acid of any on of claims 1-2.

7. A host cell genetically engineered to express the nucleic acid of any one of claims 1-2 operatively associated with a regulatory nucleic acid controlling expression of the polypeptide encoded by said nucleic acid.

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Description

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The present invention relates to polynucleotide sequences which are shown herein to be associated with the regulation of angiogenesis. More specifically, the present invention relates to novel polynucleotide sequences which encode the angiogenesis inhibitor endostatin, and more particularly, the canine angiogenesis inhibitor. The invention encompasses endostatin nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such endostatin nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules. The invention further relates to endostatin gene products and antibodies directed against such gene products. The invention further relates to methods for the identification of compounds that modulate the expression, synthesis and activity of such endostatin nucleic acids, and to methods of using compounds such as those identified herein as therapeutic agents in the treatment of angiogenesis-related disorders, including, but not limited to, cancer. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of an angiogenesis-related disorder, including, but not limited to, cancer.

BACKGROUND OF THE INVENTION

Angiogenesis, defined as the growth or sprouting of new blood vessels from existing vessels, is a complex process that primarily occurs during embryonic development. Under normal physiological conditions in adults, angiogenesis takes place only in very restricted situations such as hair growth and wounding healing (Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther 63(3):265-3 11; Ribatti et al.,1991, Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4). Unregulated angiogenesis has gradually been recognized to be responsible for a wide range of disorders, including, but not limited to, cancer, cardiovascular disease, rheumatoid arthritis, psoriasis and diabetic retinopathy (Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation 99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999, Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2): 158-64). Of particular interest is the observation that angiogenesis is required by solid tumors for their growth and metastases (Folkman, 1986, Cancer Res, 46(2) 467-73. Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6, Folkman, 1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24). A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay `dormant` without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman, 1995, Nat Med 1(1):27-31).

One of the most potent angiogenesis inhibitors is endostatin identified by O'Reilly and Folkman (O'Reilly et al., 1997, Cell 88(2):277-85; O'Reilly et al., 1994, Cell 79(2):3 15-28). Its discovery was based on the phenomenon that certain primary tumors can inhibit the growth of distant metastases. O'Reilly and Folkman hypothesized that a primary tumor initiates angiogenesis by generating angiogenic stimulators in excess of inhibitors. However, angiogenic inhibitors, by virtue of their longer half life in the circulation, reach the site of a secondary tumor in excess of the stimulators. The net result is the growth of primary tumor and inhibition of secondary tumor. Endostatin is one of a growing list of such angiogenesis inhibitors produced by primary tumors. It is a proteolytic fragment of a larger protein: endostatin is a 20 kDa fragment of collagen XVIII (amino acid H1132-K1315 in murine collagen XVIII). Endostatin has been shown to specifically inhibit endothelial cell proliferation in vitro and block angiogenesis in vivo. More importantly, administration of endostatin to tumor-bearing mice leads to significant tumor regression, and no toxicity or drug resistance has been observed even after multiple treatment cycles (Boehm et al., 1997, Nature 390(6658):404-407). The fact that endostatin targets genetically stable endothelial cells and inhibits a variety of solid tumors makes it a very attractive candidate for anticancer therapy (Fidler and Ellis, 1994, Cell 79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84; Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3). In addition, angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents (Klement, 2000, J. Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998, Nature 394(6690):287-91).

Cancer is not only devastating to humans, but is also the most common cause of natural death in dogs. (Bronson, 1982, Am J Vet Res, 43(11) 2057-9). Dogs develop tumors twice as frequently as humans and it has been reported that 45-50% of dogs that live to 10 years or older die of cancer; regardless of age, and that 23% of dogs that present for necropsy died of cancer(Bronson, 1982, Am J Vet Res, 43(11) 2057-9). Surgical removal of the tumor is the most common treatment, but the prognosis for invasive/metastatic tumor is very poor, with median survival time ranging from weeks to months. Other treatments, such as radiation therapy and chemotherapy, have only very limited success (Bostock, 1986, Br Vet J 142(6):506-15; Bostock, 1986, Br Vet J 142(1):1-19; MacEwen, 1990, Cancer Metastasis Rev 9(2): 125-36). Thus, more effective treatments for angiogenic diseases, such as, for example, canine cancers, are necessary.

SUMMARY OF THE INVENTION

The present invention encompasses novel nucleotide sequences that are associated with angiogenesis related disorders, e.g., cancer. The invention more specifically relates to nucleotide sequences that encode endostatin. In addition, endostatin nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof are provided herein. The invention also provides vectors, including expression vectors, containing endostatin nucleic acid molecules, and hosts that have been genetically engineered to express and/or contain such endostatin gene products.

The invention further relates to novel endostatin gene products and to antibodies directed against such gene products, or variants or fragments thereof.

The invention further relates to methods for modulation of endostatin-mediated processes and for the treatment of disorders involving angiogenesis, such as cancer, including the amelioration or prevention of at least one symptom of the disorders, wherein such methods comprise administering a compound which modulates the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product. In one embodiment, the invention relates to methods for the use of a novel endostatin gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against an endostatin gene product, to treat or ameliorate a symptom of such disorders.

Such disorders include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; corornary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; diabetic neovascularization; macular degeneration; fractures; vasculogenesis; hematopoiesis; ovulation; menstruation; and placentation.

The invention further relates to methods for modulation of endostatin-mediated processes and for the treatment of disorders involving abnormal stimulation of endothelial cells, including the amelioration or prevention of at least one symptom of the disorders, wherein such methods comprise administering a compound which modulates the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product. In one embodiment, the invention relates to methods for the use of a novel endostatin gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against an endostatin gene product, to treat or ameliorate a symptom of such disorders.

The endothelial cell proliferation inhibiting proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori).

The invention further relates to methods for blocking interactions between endostatin and its respective receptors with analogs that act as receptor antagonists. These antagonists may promote endothelialization and vascularization. Such effects may be desirable in situations including, but not limited to, inadequate vascularization of the uterine endometrium and associated infertilty, wound repair, healing of cuts and incisions, treatment of vascular problems in diabetics, especially retinal and peripheral vessels, promotion of vascularization in transplanted tissue including muscle and skin, promotion of vascularization of cardiac muscle especially following transplantation of a heart or heart tissue and after bypass surgery, promotion of vascularization of solid and relatively avascular tumors for enhanced cytotoxin delivery, and enhancement of blood flow to the nervous system, including but not limited to the cerebral cortex and spinal cord.

The term "endostatin-related disorder" as used herein, refers to disorders involving an endostatin gene or gene product, or an aberrant level of endostatin gene expression, gene product synthesis and/or gene product activity, respectively, relative to levels found in normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose levels represent baseline, average endostatin levels.

The term "endostatin-mediated process" as used herein, includes processes dependent and/or responsive, either directly or indirectly, to the level of expression, gene product synthesis and/or gene product activity of endostatin genes.

In another embodiment, such methods can comprise modulating the level of expression or the activity of an endostatin gene product in a cell such that the endostatin-mediated process or the disorder is treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a nucleic acid molecule encoding an endostatin gene product to increase the level, expression or activity of the endostatin gene product within the cell such that the endostatin-mediated process or the disorder is treated, e.g., a symptom is ameliorated. The nucleic acid molecule encoding the endostatin gene product can encode a mutant endostatin gene product with increased activity or expression levels.

The invention still further relates to methods for modulation of endostatin-mediated processes or the treatment of endostatin-related disorders, such as cancer, including, but not limited to, disorders resulting from endostatin gene mutations, and/or an abnormal levels of endostatin expression or activity and disorders involving one or more endostatin genes or gene products, wherein treatment includes the amelioration or prevention of at least one symptom of such disorders. In one embodiment, such methods can comprise supplying a mammal in need of treatment with a nucleic acid molecule encoding an unimpaired endostatin gene product such that the unimpaired endostatin gene product is expressed and the disorder is treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a mammal in need of treatment with a cell comprising a nucleic acid molecule that encodes an unimpaired endostatin gene product such that the cell expresses the unimpaired endostatin gene product and the disorder is treated, e.g., a symptom is ameliorated. In yet another embodiment, such methods comprise supplying a mammal in need of treatment with a modulatory compound, such as, for example, a small molecule, peptide or antibody that is capable of modulating the activity of an endostatin gene or gene product.

In addition, the present invention is directed to methods that utilize endostatin gene sequences and/or endostatin gene product sequences for the diagnostic evaluation, genetic testing and/or prognosis of angiogenesis-related disorders, such as cancer. For example, the invention relates to methods for diagnosing angiogenesis-related disorders, e.g., cancer, wherein such methods can comprise measuring endostatin gene expression in a patient sample, or detecting an endostatin mutation that correlates with the presence or development of such a disorder, in the genome of a mammal suspected of exhibiting such a disorder.

The present invention also is directed to utilizing the endostatin gene sequences and/or gene products as markers for mapping of the human chromosome.

The invention still further relates to methods for identifying compounds capable of modulating the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product, wherein such methods comprise contacting a compound with a cell that expresses such an endostatin gene, measuring the levels of endostatin gene expression, gene product expression or gene product activity, and comparing such levels to the levels of endostatin gene expression, gene product, or gene product activity produced by the cell in the absence of such compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the endostatin gene and/or the synthesis or activity of the endostatin gene product has been identified.

DEFINITIONS

As used herein, the following terms shall have the abbreviations indicated.

BAC: bacterial artificial chromosome bp: base pair(s) dbEST: expressed sequence tag data base (National Center for Biotechnology Information) EST: expressed sequence tag RT-PCR: reverse transcriptase PCR SSCP: single-stranded conformational polymorphism SNP: single nucleotide polymorphism YAC: yeast artificial chromosome

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: RT-PCR analysis of dog liver RNA. FIG. 1 shows results of an amplification reaction of dog liver RNA. A region of canine collagen XVIII which contains endostatin (proendo) were specifically amplified. The positions of PCR products of expected size are indicated by an arrow.

FIG. 2: The nucleotide sequence of canine pro-endostatin (SEQ ID NO: 1).

FIG. 3: The amino acid sequence of canine pro-endostatin translated from the sequence of FIG. 2 (SEQ ID NO:2). The region corresponding to endostatin is in bold (amino acid residues 47-230). The stop codon is indicated by *.

FIG. 4: The nucleotide sequence of canine endostatin (SEQ ID NO:3).

FIG. 5: The amino acid sequence of canine endostatin translated from the sequence of FIG. 4 (SEQ ID NO:4). The stop codon is indicated by *.

FIG. 6: An amino acid alignment of endostatin from canine, chicken, human and mouse. The program used is Lasergene MegAlign, aligned by Clustal method (DNA Star Inc., Madision, Wis.).

FIG. 7: Immunofluorescence analysis of canine and murine endostatin. 293 cells were transfected with HA-tagged canine endostatin (ca-endo) and murine endostatin (mu-endo). The cells were stained with antibody against the HA epitope and TRITC conjugated secondary antibody.

FIG. 8: Immunoblot analysis of 293 cells transfected with HA-tagged canine endostatin (ca-endo) and murine endostatin (mu-endo). Intracellular proteins from cell lysates and secreted proteins from culture supernatants (sup) were run on SDS-PAGE and analyzed by immunoblot using HA antibody and alkaline phosphatase conjugated secondary antibody.

FIG. 9: Endothelial Cell Proliferation Assay. The figure shows a summary of inhibition of endothelial cell proliferation by HA-tagged canine endostatin (HA-ca-endo) and murine endostatin (HA-mu-endo). 293 cells were transfected with angiogenesis inhibitors or green fluorescent protein (GFP) as control. The supernatants were harvested 48 hours post transfection and incubated with bFGF stimulated CPAE bovine endothelial cells or 293 cells for 72 hours. The total numbers of cells were counted and plotted. Four independent experiments were carried out and each experiment was done in duplicate.

FIG. 10: Endothelial Cell Proliferation Assay. The figure shows a summary of inhibition of endothelial cell proliferation by untagged canine endostatin (ca-endo). 293 cells were transfected with angiogenesis inhibitors or green fluorescent protein (GFP) as control. The supernatants were harvested 48 hours post transfection and incubated with bFGF stimulated CPAE bovine endothelial cells or 293 cells for 72 hours. The total numbers of cells were counted and plotted. Three independent experiments were carried out and each experiment was done in duplicate.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods relating to nucleic acid sequences associated with disorders involving angiogenesis are described herein. Novel genes which are associated with angiogenesis-related disorders have been identified. Such genes encode endostatin. In particular, described below are endostatin nucleic acid molecules, as well as vectors comprising these molecules, host cells engineered to contain and/or express such molecules, endostatin gene products, and antibodies that specifically recognize such gene products. Also described are various uses of these nucleic acids, polypeptides, and antibodies, as well as methods for their detection. For example, methods for the use of these molecules for modulation of angiogenesis-related processes and for treatment of angiogenesis-related disorders, such as cancer, are described. Screening assays for compounds that interact with an endostatin gene or gene product, or modulate endostatin gene or gene product activity also are described below. Methods of treatment of an angiogenesis-related disorder using the compositions of the invention and compositions identified by the methods of the invention are further described. Finally, pharmaceutical compositions for use with the compositions of the invention are described.

Endostatin nucleic acid molecules are described in this section. Unless otherwise stated, the term "endostatin nucleic acid" refers collectively to the sequences described herein.

The endostatin nucleic acid molecules of the invention include:

(a) a nucleic acid molecule containing the DNA sequence of endostatin (FIG. 4 (SEQ ID NO:3)) and fragments thereof;

(b) a nucleic acid molecule comprising an endostatin nucleic acid sequence (e.g., the nucleic acid sequences depicted in FIG. 2 (SEQ ID NO:1)) or a fragment thereof;

(c) a nucleic acid molecule that encodes an endostatin gene product;

(d) a nucleic acid molecule that comprises at least one exon of an endostatin gene;

(e) a nucleic acid molecule that comprises endostatin gene sequences of upstream untranslated regions, intronic regions, and/or downstream untranslated regions, or fragments thereof, of the endostatin nucleotide sequences in (b) above;

(f) a nucleic acid molecule comprising the novel endostatin sequences disclosed herein that encodes mutants of the endostatin gene products in which all or a part of one or more of the domains is deleted or altered, as well as fragments thereof;

(g) nucleic acid molecules that encode fusion proteins comprising an endostatin gene product, or a fragment thereof, fused to a heterologous polypeptide;

(h) nucleic acid molecules within the endostatin genes described in b), above (e.g., primers), or within chromosomal nucleotide sequences flanking the endostatin gene, which can be utilized as part of the methods of the invention for identifying and diagnosing individuals at risk for, or exhibiting an angiogenesis-related disorder, such as cancer, or can be used for mapping human chromosomes; and;

(i) nucleic acid molecules within the endostatin genes described in b), above, or within chromosomal nucleotide sequences flanking the endostatin genes, which correlate with an angiogenesis-related disorder, such as cancer.

The endostatin nucleotide sequences of the invention further include nucleotide sequences corresponding to the nucleotide sequences of (a)-(i) above wherein one or more of the exons, or fragments thereof, have been deleted.

The endostatin nucleotide sequences of the invention also include nucleotide sequences greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, or more base pairs long that have at least 85%, 90%, 95%, 98%, or more nucleotide sequence identity to the endostatin nucleotide sequences of (a)-(i) above, with the proviso that the endostatin is not chicken, human, or mouse endostatin.

The endostatin nucleotide sequences of the invention further include nucleotide sequences that encode polypeptides having at least 85%, 90%, 95%, 98%, or higher amino acid sequence identity to the polypeptides encoded by the endostatin nucleotide sequences of (a)-(i) above.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical overlapping positions/total # of overlapping positions.times.100%). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997, supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997, supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

The endostatin nucleotide sequences of the invention further include: (a) any nucleotide sequence that hybridizes to an endostatin nucleic acid molecule of the invention under stringent conditions, e.g., hybridization to filter-bound DNA in 6.times.sodium chloride/sodium citrate (SSC) at about 45.degree. C. followed by one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree. C., or (b) under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6.times.SSC at about 45.degree. C. followed by one or more washes in 0.1.times.SSC/0