Title:

Anti-angiogenic proteins and fragments and methods of use thereof

Document Type and Number:

United States Patent 7387779

Abstract:

Proteins with anti-angiogenic properties are disclosed, and fragments thereof, and methods of using those proteins and fragments to inhibit or promote angiogenesis.

Inventors:

Kalluri, Raghuram (Boston, MA, US)

Plaque It!

RELATED APPLICATIONS

This application is a continuation-in-part of International Application No. PCT/US01/00565, which designated the United States and was filed on Jan. 8, 2001, to be published in English, which is a continuation-in-part of application Ser. No. 09/625,191, filed Jul. 21, 2000, now U.S. Pat. No. 6,962,974, U.S. Ser. No. 09/543,371, filed Apr. 4, 2000, now abandoned and U.S. Ser. No. 09/479,118, filed Jan. 7, 2000 now abandoned, U.S. Ser. No. 09/625,191 is in turn a Continuation-In-Part of U.S. Ser. No. 09/543,371, which, with U.S. Ser. No. 09/479,118, is a Continuation-In-Part of U.S. Ser. No. 09/335,224, filed Jun. 17, 1999, now U.S. Pat. No. 6,759,047, which in turn claims the benefit of U.S. provisional application No. 60/089,689, filed Jun. 17, 1998 and U.S. provisional application No. 60/126,175, filed Mar. 25, 1999. The entire teachings of all these applications are incorporated herein by reference.

Claims:

What is claimed is:

1. An isolated polypeptide consisting of the amino acid sequence Lys Leu Phe Cys Asn Val Asn Cys Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser (SEQ ID NO:41), wherein said polypeptide has the ability to of inhibit angiogenic activity or the ability to inhibit protein synthesis in endothelial cells.

2. An isolated polypeptide consisting of the amino acid sequence Thr Met Pro Phe Leu Phe Cys Ash Val Asn Asp Val Cys Ash Phe Ala Ser Arg Asn Asp Tyr Ser Tyr Trp Leu (SEQ ID NO:37), wherein said polypeptide has the ability to of inhibit angiogenic activity or the ability to inhibit protein synthesis in endothelial cells.

3. An isolated polypeptide consisting of the amino acid sequence Thr Met Pro Phe Met Phe Cys Asn Ile Ash Ash Val Cys Asn Phe Ala Set Arg Ash Asp Tyr Ser Tyr Trp Leu (SEQ ID NO:38).

4. An isolated polypeptide consisting of the amino acid sequence Lys Gln Arg Phe Thr Thr Met Pro Phe Leu Phe Cys Asn Val Asn Asp Val Cys Asn Phe Ala Ser Arg Asn Asp Tyr Ser (SEQ ID NO:39), wherein said polypeptide has the ability to of inhibit angiogenic activity or the ability to inhibit protein synthesis in endothelial cells.

5. An isolated polypeptide consisting of the amino acid sequence Lys Gln Arg Phe Thr Thr Met Pro Phe Leu Phe Ser Asn Val Asn Asp Val Ser Asn Phe Ala Set Arg Asn Asp Tyr Set (SEQ ID NO:40).

6. An isolated polypeptide consisting of the amino acid sequence Lys Gln Arg Phe Thr Tier Met Pro Phe Leu Phe Asp Asn Vat Asn Asp Val Asp Ash Phe Ala Ser Arg Asn Asp Tyr Ser (SEQ ID NO:42).

7. The isolated polypeptide of claim 2, wherein said polypeptide is capable of inhibiting tumor growth.

8. An isolated, mutated Tumstatin polypeptide of SEQ ID NO: 10 comprising one of the following mutations: (a) wherein the Leu at position 77, Val and position 81 and Asp at position 83 have been substitute for Met, Ile and Asn, respectively; (b) wherein the Leu at position 68 has been substituted for Lys; (c) wherein the Leu at position 68, Cys at positions 79 and 85 have been substituted for Lys, Ser and Ser, respectively; (d) wherein Phe at position 76 and Asp at position 83 have been substituted for Lys and Cys, respectively; or (e) wherein Leu at position 68 and Cys at positions 79 and 85 have been substituted for Lys, Asp and Asp, respectively; and wherein said mutated polypeptide has the ability to inhibit angiogenic activity or the ability to inhibit protein synthesis in endothelial cells.

9. An isolated, mutated Tumstatin polypeptide of SEQ ID NO: 10 having the amino acid sequence of SEQ ID NOs:37-42, wherein said mutated polypeptide has the ability to inhibit angiogenic activity or the ability to inhibit protein synthesis in endothelial cells.

Description:

GOVERNMENT INTERESTS

The invention was supported, in whole or in part, by grants DK-51711, DK-55001, from the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Basement membranes are thin layers of specialized extracellular matrix that provide supporting structure on which epithelial and endothelial cells grow, and that surround muscle or fat (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127). Basement membranes are always associated with cells, and it has been well documented that basement membranes not only provide mechanical support, but also influence cellular behavior such as differentiation and proliferation. Vascular basement membranes are composed of macromolecules such as collagen, laminin, heparan sulfate proteoglycans, fibronectin and entactin (Timpl, R., 1996, Curr. Opin. Cell. Biol. 8:618-24). Functionally, collagen promotes cell adhesion, migration, differentiation and growth (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127), and via these functions is presumed to play a crucial role in endothelial cell proliferation and behavior during angiogenesis, which is the process of formation of new blood vessels from pre-existing ones (Madri, J. A. et al., 1986, J. Histochem. Cytochem. 34:85-91; Folkman, J., 1972, Ann. Surg. 175:409-16). Angiogenesis is a complex process, and requires sprouting and migration of endothelial cells, proliferation of those cells, and their differentiation into tube-like structures and the production of a basement membrane matrix around the developing blood vessel. Additionally angiogenesis is a process critical for normal physiological events such as wound repair and endometrium remodeling (Folkman, J. et al., 1995, J. Biol. Chem. 267:10931-34). It is now well documented that angiogenesis is required for metastasis and growth of solid tumors beyond a few mm ³in size (Folkman, J., 1972, Ann. Surg. 175:409-16; Folkman, J., 1995, Nat. Med. 1:27-31). Expansion of tumor mass occurs not only by perfusion of blood through the tumor, but also by paracrine stimulation of tumor cells by several growth factors and matrix proteins produced by the new capillary endothelium (Folkman, J., 1995, Nat. Med. 1:27-31). Recently, a number of angiogenesis inhibitors have been identified, namely angiostatin (O'Reilly, M. S. et al., 1994, Cell 79:315-28), endostatin (O'Reilly, M. S. et al., 1997, Cell 88:277-85), restin (Ramchandran, R. et al., 1999, Biochem. Biophys. Res. Commun. 255:735-9) and pigment epithelilum-derived factor (PEDF) (Dawson, D. W. et al., 1999, Science 285:245-8).

Type IV collagen is expressed as six distinct α-chains, α1 through α6 (Prockop, D. J. et al., 1995, Annu. Rev. Biochem. 64:403-34), and assembled into triple helices. It further forms a network to provide a scaffold for other macromolecules in basement membranes. These α-chains are composed of three domains, the N-terminal 7S domain, the middle triple helical domain, and the C-terminal globular non-collagenous (NC1) domain (Timpl, R. et al., 1981, Eur. J. Biochem. 120:203-11). Several studies have shown that inhibitors of collagen metabolism have anti-angiogenic properties, supporting the notion that basement membrane collagen synthesis and deposition is crucial for blood vessel formation and survival (Maragoudakis, M. E. et al., 1994, Kidney Int. 43:147-50; Haralabopoulos, G. C. et al., 1994, Lab. Invest. 71:575-82). However, the precise role of collagen in basement membrane organization and angiogenesis is still not well understood.

Integrins are a family of important cell surface adhesion receptors which function as adhesive molecules for many compounds. They are involved in cell-cell or cell-extracellular matrix interactions, and both mediate cells' interactions with the extracellular matrix, and cause cells to bind with it. Integrins are αβ heterodimers, consisting of two non-covalently bound transmembrane glycoprotein subunits, the α subunit and the β subunit. All α subunits exhibit shared homology with each other, as do all of the p subunits. There are currently sixteen α subunits identified (α ₁through α ₉, α _D, α _L, α _M, α _v, α _X, α _IIband α _IELb), and eight β subunits (β ₁through β ₈), which form 22 different known combinations (β ₁and α ₁through α. ₉; β ₁and α _v; β ₂and α _D, α _L, α _Mand .α _X; β ₃and α _vand α _X; β ₄and α ₆; β ₅and α _v; β ₆and α _v; β ₇and α ₄and α _IELb; β ₈and α _v). The pool of the available integrin subunits can be further increased by alternative splicing of the mRNA of some of the integrin subunits.

Integrins generally bind their ligands when the concentration of integrins at a particular spot on the cell surface is above a certain minimum threshold, forming a focal contact, or hemidesmosome. This combination of low binding affinity and formation of focal contacts enables integrins to bind both weakly and strongly, depending on the concentrations of integrin molecules.

SUMMARY OF THE INVENTION

The present invention relates to anti-angiogenic proteins, and their biologically-active fragments. The fragments described herein demonstrate that anti-angiogenic proteins can be subdivided into regions with discrete activities, for example, anti-angiogenic and anti-tumor cell activities, and that these discrete activities may only be apparent upon subdivision of the larger protein molecule. In the case of the α3(IV) NC1 domain of Type IV collagen, these activities are also outside the region of the Goodpasture epitope.

As shown herein, these active fragments have a high specificity for endothelial cells, and in particular, endothelial cells expressing the α _vβ ₃integrin, and these fragments inhibit protein synthesis in such cells. The binding of the active fragments to α _vβ ₃integrin on endothelial cells induces a negative signal which results in the inhibition of protein synthesis in those cells. This is shown to be distinct from the inhibition of protein synthesis that is caused by rapamycin, which affects protein synthesis in all cell types, not just endothelial cells. Such specificity may be useful in inhibiting protein synthesis in situations where protein synthesis is undesirable, e.g., the proteins and peptides described herein can be used as immunosuppressive agents.

In particular, the invention described herein relates to an isolated fragment of SEQ ID NO:10, having the ability to inhibit tumor growth. The fragment can be T7 (SEQ ID NO:37), T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 (SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

The invention also relates to an isolated mutated fragment of SEQ ID NO:10, where one to five amino acids have been substituted, and where the mutated fragment has the ability to inhibit tumor growth. The fragment can be T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 (SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

The invention further relates to an isolated fragment of SEQ ID NO:10, which has the ability to inhibit angiogenesis. The fragment can be T7 (SEQ ID NO:37), T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 (SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

The invention also relates to an isolated mutated fragment of SEQ ID NO:10, where one to five amino acids have been substituted, and where the mutated fragment has the ability to inhibit angiogenesis. The fragment can be T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

The invention further relates to an isolated fragment of SEQ ID NO:10, which has the ability to inhibit protein synthesis in endothelial cells. The fragment can be T7 (SEQ ID NO:37), T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 (SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid. The protein synthesis can be cap-dependent protein synthesis. The cells can express the α _vβ ₃integrin.

The invention also relates to an isolated mutated fragment of SEQ ID NO:10, where one to five amino acids have been substituted, and where the mutated fragment has the ability to inhibit protein synthesis in endothelial cells. The fragment can be T7-mutant (SEQ ID NO:38), T8 (SEQ ID NO:39), T8-3 (SEQ ID NO:40), TP3 (SEQ ID NO:41) or P2 (SEQ ID NO:42). Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid. The protein synthesis can be cap-dependent protein synthesis. The cells can express the α _vβ ₃integrin.

In an additional aspect, the invention relates to a method for inhibiting angiogenic activity in mammalian tissue, where the method includes contacting the tissue with a composition containing an isolated fragment selected from the group consisting of: (a) SEQ ID NO:10; (b) amino acid 1 through amino acid 244 of SEQ ID NO:10; (c) amino acid 1 through amino acid 124 of SEQ ID NO:10; (d) SEQ ID NO:20; (e) SEQ ID NO:21; (f) SEQ ID NO:22; (g) SEQ ID NO:23; (h) SEQ ID NO:25; (i) SEQ ID NO:26; (j) SEQ ID NO:29; (k) SEQ ID NO:30; (l) SEQ ID NO:33; (m) SEQ ID NO:34; (n) SEQ ID NO:37; (o) SEQ ID NO:38; (p) SEQ ID NO:39; (q) SEQ ID NO:40; (r) SEQ ID NO:41; and/or (s) SEQ ID NO:42. Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

In an additional aspect, the invention relates to a method for inhibiting tumor growth in mammalian tissue, where the method includes contacting the tissue with a composition containing an isolated fragment selected from the group consisting of: (a) SEQ ID NO:10; (b) amino acid 1 through amino acid 244 of SEQ ID NO:10; (c) amino acid 1 through amino acid 124 of SEQ ID NO:10; (d) SEQ ID NO:20; (e) SEQ ID NO:21; (f) SEQ ID NO:22; (g) SEQ ID NO:23; (h) SEQ ID NO:25; (i) SEQ ID NO:26; (j) SEQ ID NO:29; (k) SEQ ID NO:30; (1) SEQ ID NO:33; (m) SEQ ID NO:34; (n) SEQ ID NO:37; (o) SEQ ID NO:38; (p) SEQ ID NO:39; (q) SEQ ID NO:40; (r) SEQ ID NO:41; and/or (s) SEQ ID NO:42. Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid.

In an additional aspect, the invention relates to a method for inhibiting protein synthesis in one or more mammalian cells, where the method includes contacting the cells with a composition containing an isolated fragment selected from the group consisting of: (a) SEQ ID NO:10; (b) amino acid 1 through amino acid 244 of SEQ ID NO:10; (c) amino acid 1 through amino acid 124 of SEQ ID NO:10; (d) SEQ ID NO:20; (e) SEQ ID NO:21; (f) SEQ ID NO:22; (g) SEQ ID NO:23; (h) SEQ ID NO:25; (i) SEQ ID NO:26; (j) SEQ ID NO:29; (k) SEQ ID NO:30; (l) SEQ ID NO:33; (m) SEQ ID NO:34; (n) SEQ ID NO:37; (o) SEQ ID NO:38; (p) SEQ ID NO:39; (q) SEQ ID NO:40; (r) SEQ ID NO:41; and/or (s) SEQ ID NO:42. Such fragments can also be reduced, alkylated, or oxidized. Such fragments can also have one or more of the cysteine residues substituted for another amino acid. The protein synthesis can be cap-dependent protein synthesis. The cells can be endothelial cells. The cells can express the α _vβ ₃integrin.

In another aspect, the invention features a method for inhibiting protein synthesis in one or more mammalian cells, tumor growth, and/or angiogenesis in mammalian tissue, where the method includes contacting the one or more cells with a composition including an isolated fragment such as (a) SEQ ID NO:2, (b) SEQ ID NO:6, or (c) SEQ ID NO:10. The protein synthesis can be cap-dependent protein synthesis. The mammalian cells can express the α _vβ ₃integrin.

The invention also features an isolated peptide of the formula:
R ¹X ¹LFX ²NVNX ³VX ⁴NFR ²(SEQ ID NO:45),

where R ¹is hydrogen or a peptidyl chain of 1 to 17 amino acids, R ²is hydrogen or a peptidyl chain of 1 to 12 amino acids, and X ¹, X ²and X ³are individually an amino acid, where said peptide inhibits tumor growth. X ¹can be an amino acid with a basic side chain or an amino acid with an aromatic side chain. X ¹can be phenylalanaine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine or asparagine. X ¹can also be lysine or phenylalanine. X ², X ³and X ⁴can be independently an amino acid with a hydrophilic side chain or an amino acid with a basic side chain. X ², X ³and X ⁴can be independently cysteine, serine, threonine, aspartic acid or glutamine. X ²and X ⁴can be independently cysteine, serine or aspartic acid and X ³is cysteine or aspartic acid. X ¹can be phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine or asparagine, X ², X ³and X ⁴can be independently cysteine, serine, threonine, aspartic acid or glutamine. R ¹can be one amino acid or a peptidyl chain of 2, 3, 4, 5, 6, 7, or 8 amino acid residues. The amino acid or peptidyl chain represented by R ¹can be selected from the group consisting of: (a) P, (b) MP, (c) TMP, (d) TTMP (SEQ ID NO:46), (e) FTTMP (SEQ ID NO:47), (f) RFTTMP (SEQ ID NO:48), (g) QRFTTMP (SEQ ID NO:49), (h) LQRFTTMP (SEQ ID NO:50), (i) KQRFTTMP (SEQ ID NO:51), and (j) a conservative variant of any of (a)-(i). R ²can be one amino acid or a peptidyl chain of 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues. The amino acid or peptidyl chain represented by R ²can be selected from the group consisting of: (a) A, (b) AS, (c) ASR, (d) ASRN (SEQ ID NO:52), (e) ASRND (SEQ ID NO:53), (f) ASRNDY (SEQ ID NO:54), (g) ASRNDYS (SEQ ID NO:55), (h) ASRNDYSY (SEQ ID NO:56), (i) ASRNDYSYW (SEQ ID NO:57), (j) ASRNDYSYWL (SEQ ID NO:58), and (k) a conservative variant of any of (a)-(j). Such an isolated peptide can be reduced, alkylated, or oxidized. Such an isolated peptide can be used in a method for inhibiting protein synthesis in one or more mammalian cells, tumor growth, and/or angiogenesis in mammalian tissue, where the method includes contacting the tissue with a composition comprising the isolated peptide. The isolated peptide can be combined with a pharmaceutically-acceptable carrier.

The invention also relates to an anti-angiogenic, isolated non-Goodpasture fragment of α3(IV) NC1 domain, which has one or more of the following characteristics: (a) the ability to bind α _vβ ₃integrin; (b) the ability to inhibit proliferation of endothelial cells; and (c) the ability to cause apoptosis of endothelial cells. The isolated non-Goodpasture fragment binds α _vβ ₃integrin by an RGD-independent mechanism, as described herein. Such an isolated fragment of the α3(IV) NC1 domain of Type IV collagen is described herein, and is designated “Tumstatin”. “Tumstatin”, as the term is used herein, comprises the amino acid sequence of SEQ ID NO:10. In addition, another isolated non-Goodpasture fragment, designated herein as “Tum-1”, or “Tumstatin N53” (SEQ ID NO:22), consists of the amino acid sequence of amino acid residue 54 to amino acid 244 of full-length Tumstatin (SEQ ID NO:10). Other isolated fragments disclosed herein include “Tum-2” (SEQ ID NO:23), “Tum-3” (SEQ ID NO:24), “Tum-4” (SEQ ID NO:25), and “Tum-5” (SEQ ID NO:26), which consist of the amino acid sequence of residues 1 to 132 (Tum-2), residues 133 to 244 (Tum-3), residues 181 to 244 (Tum-4), and residues 54 to 132 (Tum-5) of full-length Tumstatin (SEQ ID NO:10), respectively. Peptide fragments are also disclosed herein, including “T1” (SEQ ID NO:27), “T2” (SEQ ID NO:28), “T3” (SEQ ID NO:29), “T4” (SEQ ID NO:30), “T5” (SEQ ID NO:31), “T6” (SEQ ID NO:32) and “T7” (SEQ ID NO:37), which consist of amino acid residues 1 to 20 (T1), 53 to 72 (T2), 68 to 87 (T3), 83 to 102 (T4), 98 to 116 (T5), 113 to 131 (T6) and 73 to 97 (T7), respectively, of full-length Tumstatin (SEQ ID NO:10). Yet another peptide fragment of full-length Tumstatin is designated herein as “Tumstatin-44-131” (SEQ ID NO:33) and consists of amino acid residues 44 to 131 of full-length Tumstatin (SEQ ID NO:10). Another fragment of full-length Tumstatin is designated herein as “Tum-5-125-C-A” (SEQ ID NO:34), and consists of Tumstatin-44-131, where the cysteine at position 125 (of full-length Tumstatin) is mutated via site-directed mutagenesis to alanine. Fragments of Tumstatin which are reduced, e.g., alkaline reduced, are also described herein to possess anti-angiogenic properties. Two other fragments are “Tumstatin 333” (SEQ ID NO:20) and “Tumstatin 334” (SEQ ID NO:21), which consist of residues 1 through 124 (Tumstatin 333) and residues 125 through 244 of full-length Tumstatin (SEQ ID NO:10), respectively. Other fragments of Tumstatin include T7-mutant (SEQ ID NO:38, in which methionine has been substituted for the leucine residue at position 77 of the full-length Tumstatin molecule, and isoleucine has been substituted for valine at position 81, and asparagine has been substituted for aspartic acid at position 83), T8 (SEQ ID NO:39, in which lysine has been substituted for the leucine residue at position 68 of the full-length Tumstatin molecule), T8-3 (SEQ ID NO:40, in which lysine has been substituted for the leucine residue at position 68 of the full-length Tumstatin molecule, and serine has been substituted for the cysteine residues at positions 79 and 85), TP3 (SEQ ID NO:41, in which lysine has been substituted for the phenylalanine residue at position 76 of the full-length Tumstatin molecule, and cysteine has been substituted for the aspartic acid at position 83), and P2 (SEQ ID NO:42, in which lysine has been substituted for the leucine residue at position 68 of the full-length Tumstatin molecule, and aspartic acid has been substituted for the cysteine residues at positions 79 and 85).

The invention also features an anti-tumor, isolated non-Goodpasture fragment of α3(IV) NC1 domain, which has one or more of the following characteristics: (a) the ability to bind α _vβ ₃integrin, (b) the ability to bind endothelial cells, (c) the ability to inhibit proliferation of tumor cells, and (d) the inability to inhibit proliferation of endothelial cells. The isolated non-Goodpasture fragment can bind α _vβ ₃integrin by an RGD-independent mechanism, as described herein. One isolated non-Goodpasture fragment comprises the amino acid sequence of amino acid residue 185 to amino acid 203 of full-length Tumstatin (SEQ ID NO:10). Another peptide fragment of full-length Tumstatin is designated herein as “T3,” and consists of amino acid residues 68 to 87 of full-length Tumstatin (SEQ ID NO:10). Yet another peptide fragment of full-length Tumstatin is designated herein as “Tumstatin-44-131,” and consists of amino acid residues 44 to 131 of full-length Tumstatin (SEQ ID NO:10). Another fragment of full-length Tumstatin is designated herein as “Tum-5-125-C-A” (SEQ ID NO:34), and consists of Tumstatin-44-131 (SEQ ID NO:33), where the cysteine at position 125 (of full-length Tumstatin) is mutated via site-directed mutagenesis to alanine. Fragments of Tumstatin which are reduced, e.g., alkaline reduced, are also described herein to possess anti-angiogenic properties. Other fragments of Tumstatin include T7-mutant, T8, T8-3, TP3, and P2.

The present invention also relates to receptors, binding proteins, e.g., that interact with (e.g., bind to) anti-angiogenic proteins and peptides, thereby providing targets for assessing anti-angiogenic proteins, peptides and compounds. These receptors and their subunits mediate angiogenesis, tumor growth and metasasis, and endothelial cell proliferation and migration and endothelial cell tube formation. These receptors also mediate cell apoptosis.

In particular, the invention relates to the integrin subunits α ₁, α ₂, α ₃, αv, β ₁and β ₃, which have been found to bind to Arresten, which is the α1 chain of the NC1 domain of Type IV collagen, the integrin subunits α ₁, α ₂and β ₁, which have been found to bind to Canstatin, which is the α2 chain of the NC1 domain of Type IV collagen, and integrin subunits α ₅, α ₆, α _v, β ₁and β ₃, which have been found to bind to Tumstatin, the α3 chain of the NC1 domain of Type IV collagen. Angiogenesis and proliferation of endothelial cells mediated by integrin binding may be inhibited by either administering Arresten, Canstatin or Tumstatin, or administering another protein, peptide or compound that binds to the above-listed integrin subunits, which serve as receptors for Arresten, Canstatin and Tumstatin. Apoptosis of endothelial cells mediated by integrin binding may also be inhibited by either administering Arresten, Canstatin or Tumstatin, or administering another protein, peptide or compound that binds to the above-listed integrin subunits, which serve as receptors for Arresten, Canstatin and Tumstatin. Such compounds can include antibodies, fragments or portions of Arresten, Canstatin or Tumstatin, or proteins or peptides comprising those regions of Arresten, Canstatin or Tumstatin which bind to the above-listed integrin subunits.

The invention also relates to methods of enhancing, promoting or inducing angiogenesis and cell proliferation by administering proteins, peptides or compounds that mimic the integrin subunits that serve as receptors for Arresten, Canstatin or Tumstatin. Such proteins, peptides or compounds include integrin protein composed of the selected subunits, which serves to interact with (e.g., bind to) available Arresten, Canstatin or Tumstatin, and biologically active (e.g., anti-angiogenic) fragments, mutants, analogs, homologs and derivatives thereof, as well as multimers (e.g., dimers) and fusion proteins (also referred to herein as chimeric proteins) thereof. Such proteins, peptides or compounds also include heparan sulfate proteoglycan, which binds Arresten with a Kd ₁value of 8.5×10 ⁻¹M and Bmax ₁of 3×10 ⁶sites per cell. As referred to herein, “available” can mean soluble or circulatory proteins that can contact or interact with (e.g., bind to) the integrins or a subunit or fragment thereof. Angiogenesis and cell proliferation can also be enhanced by administering antibodies to Arresten, Canstatin or Tumstatin, or biologically active (e.g., anti-angiogenic) fragments, mutants, analogs, homologs and derivatives thereof, as well as multimers (e.g., dimers) and fusion proteins (also referred to herein as chimeric proteins) thereof. Such antibodies bind these molecules, thereby preventing them from interacting with their respective integrin receptors and inhibiting angiogenic activity.

The invention also includes kits for identifying anti-angiogenic proteins, peptides and compounds which inhibit angiogenesis in a manner similar to Arresten, Canstatin and Tumstatin, and anti-angiogenic variants and fragments thereof. Such kits comprise appropriate (e.g., α ₁, α ₂, β ₃, etc.) subunits of integrin, and such other components necessary to perform one of the assays described in the Examples below. Exceptional assays to be performed with such a kit would include the Cell Adhesion Assay, described in Examples 12 and 28 below, and the Competition Proliferation Assay, described in Example 26 below.

The invention relates to methods of inhibiting angiogenesis, tumor growth, or tumor metastasis in a tissue (e.g., mammalian or human tissue), wherein the tissue is contacted with one or more a chains (e.g., α1 through α6) of the NC1 domain of Type IV collagen, and wherein the angiogenesis, tumor growth or tumor metastasis is mediated by one or more integrins or integrin subunits.

More specifically, the invention features a method of inhibiting angiogenesis in a tissue, where the angiogenesis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁, α ₃β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, α ₃, α _v, β ₁, β ₃). The method comprises contacting the endothelial cells with Arresten or a fragment, mutant, homolog, analog or allelic variant thereof. The angiogenesis can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention also features a method of inhibiting tumor growth or metastasis in a tissue, where the tumor growth or metastasis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁, α ₃β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, α ₃, α _v, β ₁, β ₃); the method comprises contacting the endothelial cells with Arresten or a fragment, mutant, homolog, analog or allelic variant thereof. The tumor growth can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

In addition, the invention features a method of promoting or inducing endothelial cell apoptosis in a tissue, where the endothelial cell apoptosis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁, α ₂β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, α ₃, α _v, β ₁, β ₃); the method comprises contacting the endothelial cells with Arresten or a fragment, mutant, homolog, analog or allelic variant thereof. The apoptosis can be promoted or induced by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention features a method of inhibiting angiogenesis in a tissue, where the angiogenesis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, β ₁); the method comprises contacting the endothelial cells with Canstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The angiogenesis can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention also features a method of inhibiting tumor growth or metastasis in a tissue, where the tumor growth or metastasis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, β ₁); the method comprises contacting the endothelial cells with Canstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The tumor growth can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

In addition, the invention features a method of promoting or inducing endothelial cell apoptosis in a tissue, where the endothelial cell apoptosis is mediated by one or more endothelial cell integrins (e.g., α ₁β ₁, α ₂β ₁) or one or more endothelial cell integrin subunits (e.g., α ₁, α ₂, β ₁); the method comprises contacting the endothelial cells with Canstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The apoptosis can be promoted or induced by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention features a method of inhibiting angiogenesis in a tissue, where the angiogenesis is mediated by one or more endothelial cell integrins (e.g., α ₅β ₃, α ₆β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₅, α ₆, α _v, β ₁, β ₃); the method comprises contacting the endothelial cells with Tumstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The angiogenesis can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention also features a method of inhibiting tumor growth or metastasis in a tissue, where the tumor growth or metastasis is mediated by one or more endothelial cell integrins (e.g., α ₅β ₃, α ₆β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₅, α ₆, α _v, β ₁, β ₃); the method comprises contacting the endothelial cells with Tumstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The tumor growth can be inhibited by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

In addition, the invention features a method of promoting or inducing endothelial cell apoptosis in a tissue, where the endothelial cell apoptosis is mediated by one or more endothelial cell integrins (e.g., α ₅β ₃, α ₆β ₁, α _vβ ₃) or one or more endothelial cell integrin subunits (e.g., α ₅, α ₆, α _v, β ₁, β ₃); the method comprises contacting the endothelial cells with Tumstatin or a fragment, mutant, homolog, analog or allelic variant thereof. The apoptosis can be promoted or induced by inhibiting one or more of the following: endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation.

The invention further features a method of inhibiting angiogenesis or cell proliferation in a tissue, comprising contacting the tissue with one or more of the following: an antibody or peptide that specifically binds the α ₁subunit of integrin; an antibody or peptide that specifically binds the α ₂subunit of integrin; an antibody or peptide that specifically binds the α ₃subunit of integrin; an antibody or peptide that specifically binds the α ₅subunit of integrin; an antibody or peptide that specifically binds the α ₆subunit of integrin; an antibody or peptide that specifically binds the α _vsubunit of integrin; an antibody or peptide that specifically binds the β ₁subunit of integrin; or an antibody or peptide that specifically binds the β ₃subunit of integrin. This method may be used to treat a condition characterized by angiogenesis or cell proliferation.

Additionally, the invention features a method of promoting or inducing angiogenesis or cell proliferation in a tissue, comprising contacting the tissue with one or more of the following: the α ₁subunit of integrin; the α ₂subunit of integrin; the α ₃subunit of integrin; the α ₅subunit of integrin; the α ₆subunit of integrin; the α _vsubunit of integrin; the β ₁subunit of integrin; or the β ₃subunit of integrin. The one or more of the subunits of integrin can be in soluble form, and they can also be monomers, dimers, trimers, tetramers, or multimers.

The invention also features a method of inhibiting a proliferative disease in a vertebrate, where the disease is characterized by angiogenesis that is mediated by receptors to Arresten (e.g., α ₁β ₁integrins, α ₂β ₁integrins, α ₃β ₃, integrins, α _vβ ₃integrins); the method comprises inhibiting Arresten receptor-mediated angiogenesis, thereby inhibiting the proliferative disease. The inhibition of Arresten receptor-mediated angiogenesis can result in the inhibition of tumor growth, metastasis, or the regression of an established tumor. The inhibition of the Arresten receptor-mediated angiogenesis can be accomplished by contacting the proliferating cells with a molecule that inhibits Arresten receptor-mediated angiogenesis, e.g., an antibody (e.g., polyclonal or monoclonal antibody), antibody fragment or a peptide that specifically binds to the Arresten receptor.

The invention additionally features a method of promoting angiogenesis in a tissue, comprising contacting the tissue with a composition comprising one or more soluble receptors that bind Arresten.

In another aspect, the invention features a method of inhibiting a proliferative disease in a vertebrate, where the disease is characterized by angiogenesis that is mediated by receptors to Canstatin (e.g., α ₁β ₁integrins, α ₂β ₁integrins); the method comprises inhibiting Canstatin receptor-mediated angiogenesis, thereby inhibiting the proliferative disease. The inhibition of Canstatin receptor-mediated angiogenesis can result in the inhibition of tumor growth, metastasis, or the regression of an established tumor. The inhibition of the Canstatin receptor-mediated angiogenesis can be accomplished by contacting the proliferating cells with a molecule that inhibits Canstatin receptor-mediated angiogenesis, e.g., an antibody (e.g., polyclonal or monoclonal antibody), antibody fragment or a peptide that specifically binds to the Canstatin receptor.

The invention additionally features a method of promoting angiogenesis in a tissue, comprising contacting the tissue with a composition comprising one or more soluble receptors that bind Canstatin.

In another aspect, the invention features a method of inhibiting a proliferative disease in a vertebrate, where the disease is characterized by angiogenesis that is mediated by receptors to Tumstatin (e.g., α ₅β ₁integrins, α ₆β ₁integrins, α _vβ ₃integrins); the method comprises inhibiting Tumstatin receptor-mediated angiogenesis, thereby inhibiting the proliferative disease. The inhibition of Tumstatin receptor-mediated angiogenesis can result in the inhibition of tumor growth, metastasis, or the regression of an established tumor. The inhibition of the Tumstatin receptor-mediated angiogenesis can be accomplished by contacting the proliferating cells with a molecule that inhibits Tumstatin receptor-mediated angiogenesis, e.g., an antibody (e.g., polyclonal or monoclonal antibody), antibody fragment or a peptide that specifically binds to the Tumstatin receptor.

The invention additionally features a method of promoting angiogenesis in a tissue, comprising contacting the tissue with a composition comprising one or more soluble receptors that bind Tumstatin.

In another aspect, the invention features a method of inhibiting angiogenesis in a tissue, comprising contacting the tissue with a molecule that decreases FLIP levels in the tissue.

The invention also features a composition comprising, as a biologically active ingredient, one or more molecules (e.g., antibodies, antibody fragments, peptides) that specifically bind to one or more Arresten receptors or Arresten receptor subunits (e.g., α ₁β ₁integrin, α ₂β ₁integrin, α ₃β ₁integrin, α _vβ ₃integrin, α ₁integrin subunit, α ₂integrin subunit, α ₃integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to inhibit a disease characterized by angiogenic activity, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

In another aspect, the invention features a composition comprising, as a biologically active ingredient, one or more Arresten receptors or Arresten receptor subunits (e.g., α ₁β ₁integrin, α ₂β ₁integrin, α ₃β ₁integrin, α _vβ ₃integrin, α ₁, integrin subunit, α ₂integrin subunit, α ₃integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to promote or induce angiogenesis, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

The invention also features a composition comprising, as a biologically active ingredient, one or more molecules (e.g., antibodies, antibody fragments, peptides) that specifically bind to one or more Canstatin receptors or Canstatin receptor subunits (e.g., α ₁β ₁integrin, α ₂β ₁integrin, α ₃β ₁integrin, α _vβ ₃integrin, α ₁, integrin subunit, α ₂integrin subunit, α ₃integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to inhibit a disease characterized by angiogenic activity, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

In another aspect, the invention features a composition comprising, as a biologically active ingredient, one or more Canstatin receptors or Canstatin receptor subunits (e.g., α ₁β ₁integrin, α ₂β ₁integrin, α ₃β ₁integrin, α _vβ ₃integrin, α ₁integrin subunit, α ₂integrin subunit, α ₃integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to promote or induce angiogenesis, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

The invention also features a composition comprising, as a biologically active ingredient, one or more molecules (e.g., antibodies, antibody fragments, peptides) that specifically bind to one or more Tumstatin receptors or Tumstatin receptor subunits (e.g., α ₅β ₁integrin, α ₆β ₁integrin, α _vβ ₃integrin, α ₅integrin subunit, α ₆integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to inhibit a disease characterized by angiogenic activity, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

In another aspect, the invention features a composition comprising, as a biologically active ingredient, one or more Tumstatin receptors or Tumstatin receptor subunits (e.g., α ₅β ₁integrin, α ₆β ₁integrin, α _vβ ₃integrin, α ₅integrin subunit, α ₆integrin subunit, α _vintegrin subunit, β ₁integrin subunit, β ₃integrin subunit). The composition may optionally include a pharmaceutically-acceptable carrier. The composition can be used in a method to promote or induce angiogenesis, where the method comprises administering the composition to a patient with the disease. The disease may be characterized by angiogenic activity, and the composition can be administered to a patient in conjunction with radiation therapy, chemotherapy or immunotherapy.

In further aspects, the invention features a method of determining if a cell (e.g., a cancer cell) will be susceptible to the action of Arresten, comprising the steps of: (a) providing a sample (e.g., from a mammal) containing the cell, (b) reacting the sample with one or more antibodies (e.g., antibodies to α ₁β ₁integrin, α ₂β ₁integrin, α ₃β ₁integrin, α _vβ ₃integrin, the α ₁integrin subunit, the α ₂integrin subunit, the α ₃integrin subunit, the α _vintegrin subunit, the β ₁integrin subunit, the β ₃integrin subunit) for sufficient time and under conditions suitable for binding of the one or more antibodies to the cell; and where if the cell is susceptible to the action of Arresten a cell-antibody complex is formed; and then (c) detecting the presence of the cell-antibody complex; so that the presence of the cell-antibody complex in the sample is indicative of the cell's susceptibility to the action of Arresten. The mammal may have a condition characterized at least in part by undesired angiogenesis.

In further aspects, the invention features a method of determining if a cell (e.g., a cancer cell) will be susceptible to the action of Canstatin, comprising the steps of: (a) providing a sample (e.g., from a mammal) containing the cell, (b) reacting the sample with one or more antibodies (e.g., antibodies to α ₁β ₁integrin, α ₂β ₁integrin, the α ₁integrin subunit, the α ₂integrin subunit, the β ₁integrin subunit) for sufficient time and under conditions suitable for binding of the one or more antibodies to the cell; and where if the cell is susceptible to the action of Canstatin a cell-antibody complex is formed; and then (c) detecting the presence of the cell-antibody complex; so that the presence of the cell-antibody complex in the sample is indicative of the cell's susceptibility to the action of Canstatin. The mammal may have a condition characterized at least in part by undesired angiogenesis.

In further aspects, the invention features a method of determining if a cell (e.g., a cancer cell) will be susceptible to the action of Tumstatin, comprising the steps of: (a) providing a sample (e.g., from a mammal) containing the cell, (b) reacting the sample with one or more antibodies (e.g., antibodies to α β ₁integrin, α ₆β ₁integrin, α _vβ ₃integrin, α ₁, integrin subunit, the α ₅integrin subunit, the α ₆integrin subunit, the α _vintegrin subunit, the β ₁integrin subunit, the β ₃integrin subunit) for sufficient time and under conditions suitable for binding of the one or more antibodies to the cell; and where if the cell is susceptible to the action of Tumstatin a cell-antibody complex is formed; and then (c) detecting the presence of the cell-antibody complex; so that the presence of the cell-antibody complex in the sample is indicative of the cell's susceptibility to the action of Tumstatin. The mammal may have a condition characterized at least in part by undesired angiogenesis.

The present invention also relates to proteins comprising the NC1 domain of an a chain of Type IV collagen having anti-angiogenic properties. In particular, the present invention relates to the novel proteins Arresten, Canstatin and Tumstatin, and to biologically active (e.g., anti-angiogenic) fragments, mutants, analogs, homologs and derivatives thereof, as well as multimers (e.g., dimers) and fusion proteins (also referred to herein as chimeric proteins) thereof. These proteins all comprise the C-terminal fragment of the NC1 (non-collagenous 1) domain of Type IV collagen. More specifically, Arresten, Canstatin and Tumstatin are each a C-terminal fragment of the NC1 domain of the α1 chain, α2 chain and α3 chain, respectively, of Type IV collagen. In particular, Arresten, Canstatin and Tumstatin are monomeric proteins. All three arrest tumor growth in vivo, and also inhibit the formation of capillaries in several in vitro models, including the endothelial tube assay.

The present invention encompasses the integrin or integrin subunits (e.g., the α ₁β ₁, α ₁β ₂and α ₂β ₁integrins) as the receptor for Arresten in endothelial cells, mediating anti-angiogenic activity, including endothelial cell apoptosis, in these cells. Arresten also specifically binds and inhibits the basement membrane-degrading activities of matrix metalloproteinases 2, 3 and 9; such degradative activity is an integral part of angiogensis.

The present invention also encompasses isolated and recombinantly-produced Arresten, which comprises the NC1 domain of the α1 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Arresten, multimers of the isolated Arresten and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Arresten, its anti-angiogenic fragments, or both, as biologically active components. In another embodiment, the invention features a method of treating a proliferative disease such as cancer, in a mammal where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Arresten or its fragments. The anti-angiogenic Arresten and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Arresten and its fragments.

The present invention also encompasses the integrins or integrin subunits (e.g., α ₁β ₁and α ₁β ₂integrins) as the cell adhesion receptors for Canstatin in endothelial cells, mediating anti-angiogenic activity, including endothelial cell apoptosis, in these cells.

The present invention also encompasses isolated and recombinantly produced Canstatin, which comprises the NC1 domain of the α2 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Canstatin, multimers of the isolated Canstatin and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Canstatin, its anti-angiogenic fragments, or both, as biologically active ingredients. In another embodiment, the invention features a method of treating a proliferative disease such as cancer, in a mammal, where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Canstatin or its fragments. The anti-angiogenic Canstatin and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Canstatin and its fragments.

The present invention also encompasses the integrins and integrin subunits (e.g., α ₅β ₁, α ₆β ₁and α _vβ ₃integrins) as receptors of Tumstatin in endothelial cells, mediating anti-angiogenic activity, including endothelial cell apoptosis, in these cells.

The invention likewise also encompasses isolated and recombinantly-produced Tumstatin, comprising the NC1 domain of the α3 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Tumstatin, multimers of the isolated Tumstatin and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Tumstatin, its anti-angiogenic fragments, or both, as biologically active ingredients. In another embodiment, the invention features a method of treating a proliferative disease such as cancer in a mammal, where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Tumstatin or its fragments. The anti-angiogenic Tumstatin and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Tumstatin and its fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are diagrams depicting the nucleotide (FIG. 1A, SEQ ID NO:1) and amino acid (FIG. 1B, SEQ ID NO:2) sequences of the α1 chain of human Type IV collagen. The locations of the pET22b(+) forward (SEQ ID NO:3) and reverse (SEQ ID NO:4) primers are indicated by double underlining, and the locations of the pPICZαA forward (SEQ ID NO:15) and reverse (SEQ ID NO:16) primers are indicated by single underlining.

FIG. 2 is a schematic diagram representing the Arresten cloning vector pET22b(+). Forward (SEQ ID NO:3) and reverse (SEQ ID NO:4) primers and site into which Arresten was cloned are indicated.

FIGS. 3A and 3B are a pair of line graphs showing the effects of Arresten (FIG. 3A, 0 μg/ml to 10 μg/ml, x-axis) and endostatin (FIG. 3B, 0 μg/ml to 10 μg/ml, x-axis) on ³H-thymidine incorporation (y-axis) as an indicator of endothelial cell (C-PAE) proliferation.

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

FIGS. 5A, 5 B and 5 C are a set of four photomicrographs showing the effects of Arresten (2 μg/ml, FIG. 5B) and endostatin (20 μg/ml, FIG. 5C) on endothelial cell migration via FBS-induced chemotaxis in human umbilical endothelial (ECV-304) cells. FIG. 5A shows untreated control cells.

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

FIG. 7 is a line graph showing the effect of Arresten on the endothelial tube formation. Percent tube formation is shown on the y-axis, and concentration of inhibitor on the x-axis. The treatments were: none (control, ♦), BSA (control, Δ), 7S domain (control, X) and Arresten (▪).

FIGS. 8A and 8B are a pair of photomicrographs showing the effect of Arresten (0.8 μg/ml, FIG. 8B) on endothelial tube formation relative to control (FIG. 8A).

FIGS. 9A, 9 B, 9 C and 9 D are a set of four line graphs showing the effect of Arresten and endostatin on tumor growth in vivo. FIG. 9A is a plot showing the increase in tumor volume from 700 mm ³for 10 mg/kg Arresten-treated (□), BSA-treated (+), and control mice (●). FIG. 9B shows the increase in tumor volume from 100 mm ³for 10 mg/kg Arresten-treated (□) and BSA-treated (+) tumors. FIG. 9C shows the increase in tumor volume from about 100 mm ³for 10 mg/kg Arresten-treated (□), Endostatin-treated (▴), and control mice (●). FIG. 9D shows the increase for 200 mm ³tumors when treated with Arresten (□) versus controls (●).

FIGS. 10A and 10B are a pair of histograms showing the amount of Caspase-3 activity as a function of absorbance at OD ₄₀₅(y-axis) for C-PAE cells (FIG. 10A) and PC-3 cells (FIG. 10B) under various treatments (x-axis). Each column represents the mean the standard error of the mean of triplicate well.

FIGS. 11A and 11B are diagrams depicting the nucleotide (FIG. 11A, SEQ ID NO:5) and amino acid (FIG. 11B, SEQ ID NO:6) sequences of the α2 chain of human Type IV collagen. The locations of the pET22b(+) forward (SEQ ID NO:7) and reverse (SEQ ID NO:8) primers are indicated by double underlining, and the location of the pPICZαA forward (SEQ ID NO:17) and reverse (SEQ ID NO:18) primers are indicated by single underlining.

FIG. 12 is a schematic diagram representing the Canstatin cloning vector pET22b(+). Forward (SEQ ID NO:7) and reverse (SEQ ID NO:8) primers and site into which Canstatin was cloned are indicated.

FIGS. 13A, 13 B, 13 C and 13 D are histograms showing the effect of varying concentrations of Canstatin (x-axis) on proliferation of endothelial (C-PAE) cells (FIGS. 13A and 13C) and non-endothelial (786-O, PC-3 and HEK 293) cells (FIGS. 13B and 13D). Proliferation was measured as a function of ³H-thymidine incorporation (FIGS. 13A and 13B) and methylene blue staining (FIGS. 13C and 13D).

FIG. 14 is a bar chart showing the number of migrated endothelial cells per field (y-axis) for treatments of no VEGF (no VEGF or serum), and VEGF (1% FCS and 10 ng/ml VEGF) cells, and for treatments of 0.01 Canstatin (1% FCS and 10 ng/ml VEGF and 0.01 μg/ml Canstatin) and 1.0 μg/ml Canstatin (1% FCS and 10 ng/ml VEGF and 1 μg/ml Canstatin).

FIG. 15 is a line graph showing the amount of endothelial tube formation as a percent of control (PBS-treated wells) tube formation (y-axis) under varying treatments of BSA (□), Canstatin (▪), and α5NC1 (◯). Vertical bars represent the standard error of the mean.

FIG. 16 is a graph of the FLIP (FLICE-Inhibitory Protein, or FADD-Like Interleukin-1β-Converting Enzyme-Inhibitory Protein) levels as a function of the level of vinculin as a percentage of the protein present at t=0 (y-axis), over time (x-axis).

FIGS. 17A, 17 B, 17 C and 17 D are line graphs depicting the effect on PC-3 cells (FIGS. 17A and 17B) and 786-O cells (FIGS. 17C and 17D) of Canstatin (▪), endostatin (∘) and controls (□) on fractional tumor volume (y-axis, FIGS. 17A and 17B) or tumor volume in mm ³(y-axis, FIGS. 17C and 17D), plotted over the days of treatment x-axis).

FIGS. 18A and 18B are diagrams depicting the nucleotide (FIG. 18A, SEQ ID NO:9) and amino acid (FIG. 18B, SEQ ID NO:10) sequence of the α3 chain of human Type IV collagen. The locations of the pET22b(+) forward (SEQ ID NO:11) and reverse (SEQ ID NO:12) primers are indicated by double underlining. The beginning and end of the “Tumstatin 333” (SEQ ID NO:20) and “Tumstatin 334” (SEQ ID NO:21) fragments are also indicated (“*”=Tumstatin 333; “+”=Tumstatin 334).

FIG. 19 is a schematic diagram representing the Tumstatin cloning vector pET22b(+). Forward (SEQ ID NO:11) and reverse (SEQ ID NO:12) primers and site into which Tumstatin was cloned are indicated.

FIG. 20 is a schematic diagram showing the location of truncated amino acids within the α3(IV) NC1 monomer in the Tumstatin mutant Tumsatin N-53 (Tum-1). The filled circles correspond to the N-terminal 53 amino acid residues deleted from Tumstatin to generate this mutant. The disulfide bonds, marked by short bars, are arranged as they occur in α1(IV) NC1 and α2(IV) NC1.

FIGS. 21A, 21 B and 21 C are a set of three histograms showing ³H-thymidine incorporation (y-axis) for C-PAE cells (FIG. 21A), PC-3 cells (FIG. 21B) and 786-O cells (FIG. 21C) when treated with varying concentrations of Tumstatin (x-axis). All groups represent triplicate samples.

FIG. 22 is a histogram showing on the x-axis the effect of 0.1 μg/ml Tumstatin combined with increasing amounts of α _vβ ₃on the uptake of dye by C-PAE cells. Absorbance at OD ₆₅₅is shown on the y-axis. “0.1% FCS” represents the 0.1% FCS-treated (unstimulated) control, and “20% FCS” is the 20% FCS-treated (stimulated) control. The remaining bars represent a control of α _vβ ₃alone, and treatments with Tumstatin plus increasing concentrations of α _vβ ₃. Each bar represents the mean ±the standard error of the mean for triplicate well. The experiments were repeated three times. An asterisk indicates that P<0.05 by the one-tailed Student's t-test.

FIGS. 23A and 23B are a pair of histograms showing the amount of Caspase-3 activity as a function of absorbance at OD ₄₀₅(y-axis) for C-PAE cells (FIG. 23A) and PC-3 cells (FIG. 23B) under various treatments (x-axis). Each column represents the mean the standard error of the mean of triplicate well.

FIGS. 24A, 24 B, 24 C and 24 D are a set of four histograms showing binding of HUVEC cells to plates coated with Tumstatin (FIG. 24A), or controls of type IV collagen (FIG. 24B), vitronectin (FIG. 24C) or laminin-1 (FIG. 24A) in the presence of integrin subunits α1 through α ₆, β ₁, or α _vβ ₃integrin blocking antibody. The plate coating is listed at the top of each graph, and the antibodies used for incubation are on the x-axis of each graph. BSA-coated plates were used as negative controls.

FIG. 25 is a histogram showing binding of C-PAE cells to Tumstatin-coated plates. BSA-coated plates were used as negative controls.

FIG. 26 is a line graph showing the effect on endothelial tube formation (y-axis) of varying amounts (x-axis) of Tumstatin (●), BSA (control, □) and 7S domain (control, ∘).

FIGS. 27A and 27B are a pair of line graphs showing the effects on tumor volume (mm ³, y-axis) against days of treatment (x-axis) of Tumstatin (●) and endostatin (∘) versus controls (□). Data points marked with an asterisk are significant, with P<0.05 by one-tailed Student's test.

FIG. 28 is a graph showing increase in tumor volume (y-axis) against day of treatment (x-axis) for control mice (□) and mice treated with the Tumstatin mutant N-53 (●). Data points marked with an asterisk are significant, with P<0.05 by one-tailed Student's test.

FIG. 29 is a graph showing cell viability (as a function of OD ₅₉₀, y-axis) at increasing concentrations of Tumstatin and Numstatin N-53 (x-axis). Each point represents the mean ±the standard error of the mean for triplicate well. An asterisk indicates P<0.05 by the one-tailed Student's t test.

FIG. 30 is a line graph showing the inhibition of endothelial tube formation (y-axis) by varying concentrations (x-axis) of Arresten (●), Canstatin (∘), the 12 kDa Arresten fragment (▪), the 8 kDa Arresten fragment (□), and the 10 kDa Canstatin fragment (▴).

FIG. 31 is a line graph showing the inhibition of endothelial tube formation (y-axis) by varying concentrations (x-axis) of Tumstatin fragment 333 (●), Tumstatin fragment 334 (∘), BSA (control, ▪), α6 (control, □), and Tumstatin (▴).

FIGS. 32A, 32 B and 32 C are the set of three histograms showing the effect of increasing concentrations of Tumstatin (x-axis) on proliferation (y-axis) of HPE (FIG. 32A), C-PAE (FIG. 32B) and WM-164 (FIG. 32C) cells.

FIGS. 33A and 33B are a pair of graphs showing the effect of increasing concentration (x-axis) of Tumstatin (♦), Tum-1 (□), Tum-2 (●), Tum-3 (⋄) and Tum-4 (▴) on the relative number (y-axis) of C-PAE cells (FIG. 33A) and WM-164 cells (FIG. 33B).

FIGS. 34A and 34B are a pair of graphs showing the effect of increasing concentration (x-axis) of Tumstatin (♦), Tum-1 (□), Tum-2 (●), Tum-3 (⋄) and Tum-4 (▴) on the cell viability (y-axis) of C-PAE cells (FIG. 34A) and WM-164 cells (FIG. 34B). Each point represents the mean ±the standard error of the mean for triplicate wells.

FIG. 35 is a histogram showing Caspase-3 activity as a measure of absorbance at OD ₄₀₅(y-axis) of C-PAE cells treated (x-axis) with 5 μg/ml Tum-1, Tum-2, Tum-3 or Tum-4, or 80 ng/ml TNF-α or PBS buffer (control).

FIGS. 36A, 36 B and 36 C are a set of three histograms. FIGS. 36A, 36 B and 36 C show the percent binding of C-PAE cells (y-axis) to plates coated with Tum-1 (FIG. 36A), Tum-2 (FIG. 36B) and Tum-4 (FIG. 36C) in the presence of control IgG, αvβ ₃, α _vβ ₅and BSA.

FIG. 37 is a histogram showing the level of methylene blue staining by absorbance at OD ₆₅₅(y-axis) for WM-164 cells that attached to plates coated with PBS, Tumstatin, Tum-1, Tum-2, Tum-4 or BSA (x-axis).

FIGS. 38A, 38 B, 38 C, 38 D and 38 E are a set of five histograms showing proliferation of C-PAE cells (y-axis) treated with 1.5 μg/ml Tum-1 (FIG. 38A) or Tum-2 (FIG. 38B) that had been preincubated with anti-Tum-4 antibody (1:100, 1:200, 1:500 dilution) (x-axis), or α _vβ ₃protein (FIG. 38C), or WM-164 cells treated with Tumstatin (FIG. 38D) or Tum-4 (FIG. 38E).

FIG. 39 is a graph showing concentration of Tumstatin (●), endostatin (A), anti-α _vβ ₃(□) antibody and IgG (♦) (control) on the x-axis, versus relative cell number on the y-axis. Each point represents the mean ±the standard error of the mean for triplicate wells. The experiments were repeated three times. Asterisks indicate P<0.05 by one-tailed Student's t-test.

FIG. 40 is a graph showing the effect of increasing concentrations of Canstatin (♦), Can-1 (▪) and Can-2 (▴) (x-axis) on the relative cell number (y-axis) of C-PAE cells. Each concentration of each protein was tested in quadruplicate.

FIG. 41 is a histogram showing the mean number of vessels per plug (y-axis) for treatments with PBS (control), Canstatin, Can-1 and Can-2.

FIG. 42 is a diagram of the Tumstatin protein sequence, with the locations of the T1, T2, T3, T4, T5 and T6 peptides indicated. GP-A=first Goodpasture epitope. GP-B=second Goodpasture epitope.

FIGS. 43A, 43 B, 43 C and 43 D are four histograms showing inhibition of endothelial cell proliferation (FIGS. 43A, 43 B and 43 C) and induction of endothelial cell apoptosis (FIG. 43D) by the T3 peptide. FIG. 43A shows proliferation of C-PAE cells (y-axis) treated with 10 μg/ml of peptide T2, T3, T4, T5 or T6 (x-axis). FIG. 43B shows proliferation of C-PAE cells (y-axis) treated with 0.1, 1.0 or 10 μg/ml T3 peptide. FIG. 43C shows cell growth of C-PAE cells (y-axis) when treated with T3 peptide that has been pre-incubated with varying concentrations (x-axis) of α _vβ ₃integrin. FIG. 43D shows cell viability (y-axis) of C-PAE cells as determined by MTT assay, after treatment of the cells with 10 μg/ml of peptides T2, T3, T4, T5 or T6. All columns represent the mean ±SEM of triplicate wells.

FIGS. 44A, 44 B, 44 C, 44 D, 44 E, 44 F and 44 G are a set of seven histograms showing attachment of C-PAE cells when treated with anti-human integrin antibodies, mouse IgG (control), or peptides T2, T3, T4, T5 or T6. FIG. 44A shows binding (y-axis) of HUVEC cells to plates coated with Tum-5 peptide (10 μg/ml), in the presence of BSA (control), no antibody (control), mouse IgG (control) and α _vβ ₃integrin antibody (x-axis). FIG. 44B is a histogram showing attachment of C-PAE cells (y-axis) to 96-well plates that were coated with 10 μg/ml recombinant Tum-5 peptide (x-axis). FIG. 44C is a histogram showing binding of C-PAE cells (y-axis) to 96-well plates coated (x-axis) with Tum-5 and treated with 2.5 μg/ml peptides T2, T3, T4, T5 or T6, or Tum-4-coated plates treated with T3. PBS treatment served as control. FIG. 44D shows the effect on binding of C-PAE cells (y-axis) to Tum-5-coated plates of varying concentrations of T3 peptide (x-axis). PBS treatment served as a control. FIG. 44E shows the binding of C-PAE cells (y-axis) to T2, T3, T4, T5 or T6-coated plates (x-axis) in the presence of PBS (control), IgG (control), or α _vβ ₃integrin antibody. FIG. 44F shows binding of C-PAE cells (y-axis) to T3-coated plates when incubated with PBS (control), IgG (control), or α _vintegrin antibody, β ₁integrin antibody, β ₃integrin antibody, α _vβ ₅integrin antibody, or BSA (control) (x-axis). FIG. 44G shows binding of C-PAE cells (y-axis) to plates coated with vitronectin (2.5 μg/ml) when incubated with PBS (control), BSA (control) or varying (0.1, 1.0, 10.0 μg/ml) concentrations of T3 peptide or varying (0.1, 1.0, 10.0 μg/ml) concentrations of T6 peptide (x-axis). Each column represents the mean ±the SEM of triplcate wells. The experiments were repeated three times. *P<0.05 by one-tailed Student's t-test.

FIG. 45 is a histogram showing adhesion of HUVEC cells to Tumstatin-N53-coated (20 μg/ml) plates, in the presence of PBS (control), α _vβ ₃integrin antibodies, β ₁integrin antibodies, α ₆integrin antibodies, or BSA (control).

FIG. 46 is a graph showing mean tumor volume in mm ³(y-axis) for PC3 prostate tumors (PC3 prostate xenograft model) over 15 days (x-axis) for tumors treated with vehicle (control, °), Tumstatin-N53 at 5 mg per kilogram per day (□), or Tumstatin-N53 at 20 mg per kilogram per day (⋄).

FIG. 47 is a graph showing the mean tumor volume in mm ³(y-axis) for MDA-MB435 breast cancer carcinoma tumors over 22 days (x-axis) for tumors treated with vehicle (control, °), Tumstatin-N53 at 20 mg per kilogram per day (□), or Tumstatin-N53 at 5 mg per kilogram per day (⋄).

FIG. 48 is a histogram showing the percent of C-PAE cells in S-phase (y-axis) when treated with PBS (control), buffer (control), 20 μg/ml Tumstatin-N53, 10 μg/ml Tumstatin-44-131, and 5 μg/ml Tumstatin-44-131 (x-axis). The cell cycle assay was performed in the presence of 10% FBS.

FIG. 49 is a histogram showing adhesion (in OD ₅₉₅, y-axis) of HUVEC cells (y-axis) to Tumstatin-44-131-coated (20 μg/ml) plates, in the presence of PBS (control), α _vβ ₃integrin antibodies, β ₁integrin antibodies, α ₆integrin antibodies, or BSA (control).

FIGS. 50A and 50B are a set of two histograms showing the effect of Tumstatin-44-131 on cell proliferation. FIG. 50A shows cell proliferation measured by BrdU assay (at OD ₄₅₀, y-axis), with C-PAE cells treated with E. Coli -expressed Tumstatin-44-131 (black bars), or 293 cell-expressed full-length Tumstatin (white bars), at concentrations of 0, 0.125, 0.250, 0.500, 1.0 or 2.0 μM (x-axis). FIG. 50B shows cell proliferation as measured by methylene blue staining (at OD ₆₅₅), with C-PAE cells treated with Pichia -expressed Tumstatin-44-131 at concentrations of 0, 0.1, 1.0, 5.0 and 10.0 μg/ml (x-axis). Unstimulated C-PAE cells served as the control.

FIG. 51 is a histogram showing the effect of E. Coli -expressed Tumstatin-44-131 and Tum-5-125-C-A on progression of the cell cycle. The percentage of C-PAE cells in S phase (y-axis) is shown at hour 0 (control), and after treatment by 0, 1, 10 and 20 μg/ml (x-axis) Tumstatin-44-131 (black bars) or Tum-5-125-C-A (white bars). The experiments were repeated three times.

FIGS. 52A, 52 B, 52 C and 52 D are a set of four histograms showing the effects of Tumstatin-44-131 and Tum-5-125-C-A on cell viability. FIG. 52A shows cell viability as measured at OD ₅₆₂(y-axis) in an MTT assay, for C-PAE cells treated with 0, 3, 6, 12, 25 and 50 μg/ml (x-axis) Tumstatin-44-131 (black bars) and Tumstatin-44-131 that was alkylated and reduced (white bars). FIG. 52B shows cell viability as measured at OD ₅₆₂(y-axis) in an MTT assay, for C-PAE cells treated with 0, 3, 6, 12, 25 and 50 μg/ml (x-axis) Tum-5-125-C-A. FIG. 52C shows cell viability as measured at OD ₅₆₂(y-axis) in an MTT assay, for PC-3 cells treated with 0, 3, 6, 12, 25 and 50 μg/ml (x-axis) Tumstatin-44-131. FIG. 52D shows cell viability as measured at OD ₅₆₂(y-axis) in an MTT assay, for DU-145 cells treated with 0, 3, 6, 12, 25 and 50 μg/ml (x-axis) Tumstatin-44-131.

FIG. 53 is a histogram showing caspase-3 activity (as measured at OD ₄₀₅, y-axis) of (x-axis) the control, control+DEVD-fmk, TNF-α, TNF-α+DEVD-fmk, Tumstatin-44-131 (1 μg/ml and 10 μg/ml), and Tumstatin-44-131 (10 μg/ml)+DEVD-fmk.

FIG. 54 is a line graph showing the fractional tumor volume (y-axis) in terms of V/V ₀(mean tumor volume/initial tumor volume) at 0, 5, 10, 15 and 20 days (x-axis) of treatment with vehicle (control, □), 1 mg/kg Tumstatin-44-131 (♦), 1 mg/kg Tum-5-125-C-A (●), 20 mg/kg endostatin (°) and mini-pump administered Tumstatin-44-131 (1 mg/kg, Δ).

FIGS. 55A and 55B are a pair of histograms showing C-PAE cell binding to tissue culture plates coated with 293-produced Tumstatin, in the presence of various peptide subunits of Tumstatin. PBS and BSA served as positive and negative controls, respectively. FIG. 55A shows cell binding in the presence of 10 μg/ml peptides T1, T2, T3, T4, T5, T6, Tum-4, and FIG. 55B shows cell binding in the presence of 0.1, 2.0 or 10.0 μg/ml T3 peptide.

FIG. 56 is a histogram showing the proliferation of C-PAE cells (as a percentage of unstimulated control cells treated with 0.1% FCS) when treated with 0, 1, 10 and 20 μg/ml T3 peptide (black bars), and T3 folded peptide (white bars).

FIG. 57 is a histogram showing the proliferation of C-PAE cells (as a percentage of unstimulated control cells treated with 0.1% FCS) when treated with full-length Tumstatin (black bars), Tumstatin-44-131(white bars), T7 peptide (cross-hatched bars) and T3 peptide (stippled bars). Each column represents the mean ±the SEM of triplicate wells. Tumstatin was not tested at the 5 μM concentration.

FIGS. 58A-58H are a series of eight histograms showing ³⁵S-methionine incorporation (y-axes) in cells under various treatments (x-axes). The experiments were repeated three times and representative data are shown. Each column consists of the mean ±SEM of triplicates. In FIG. 58A, C-PAE cells were treated for either 12 hours (black bars) or 24 hours (cross-hatched bars) with T3 peptide (4.5 μM), Tumstatin-44-131 (4.5 μM), endostatin (4.5 μM) or rapamycin (100 ng/ml). In FIG. 58B, HUVECs were treated for 24 hours with T3 peptide (4.5 μM), Tumstatin-44-131 (4.5 μM), endostatin (4.5 μM) or rapamycin (100 ng/ml). In FIG. 58C, C-PAEs were serum-starved for 12 or 24 hours, and then incubated with medium containing 10% FCS for 24 hours in the presence of T3 peptide at 0 μM (control, black bars), 4.5 μM (bars with horizontal cross-hatching), or 22.7 μM (bars with slanted cross-hatching). In FIGS. 58D-H, PC-3 cells, (FIG. 58D), 786-O cells (FIG. 58E), NIH3T3 cells (FIG. 58F), HRE cells (FIG. 58G) and WM-164 cells (FIG. 58H) were treated for 24 hours with T3 peptide (4.5 μM), Tumstatin-44-131 (4.5 μM), T7 peptide (FIG. 58D, FIG. 58E, FIG. 58H), endostatin (4.5 μM) or rapamycin (100 ng/ml).

FIGS. 59A and 59B are a pair of histograms showing the reporter activity (y-axis) for translation of luciferase (LUC; cap-dependent translation; black bars) or chloramphenicol acetyltransferase (CAT; cap-independent translation; cross-hatched bars), under treatment by T3 peptide (4.5 μM), Tumstatin-44-131 (4.5 μM), T7 peptide (4.5 μM), endostatin (4.5 μM) or rapamycin (100 ng/ml). Luciferase and CAT activity relative to the control group is shown. These experiments were repeated three times and representative data are shown. Each column consists of mean ±SEM of triplicates.

FIGS. 60A-60H are a set of eight histograms. FIGS. 60A-60D show total protein synthesis in terms of ³⁵S-methionine incorporation (y-axis) in endothelial cells (MLEC) (FIGS. 60A and 60B) and embryonic fibroblasts (MEF) (FIGS. 60C and 60D) from wild-type (FIGS. 60A and 60C) and β ₃-integrin knockout (FIGS. 60B and 60D) littermate mice, where the cells were treated (x-axis) with Tumstatin-44-131 (4.5 μM), T3 (4.5 μM), T7 (4.5 μM), T7-mutant peptide (4.5 μM), endostatin (4.5 μM) or rapamycin (100 ng/ml). FIGS. 60E-60G show reporter activity of either luciferase (Luc; black bars) or chloramphenicol acetyltransferase (CAT; cross-hatched bars) as a percentage of control (y-axis) in endothelial cells (MLEC) (FIGS. 60E and 60F) and embryonic fibroblasts (MEF) (FIGS. 60G and 60H) from wild-type (FIGS. 60E and 60G) and P ₃-integrin knockout (FIGS. 60F and 60H) littermate mice, where the cells were treated (x-axis) with Tumstatin-44-131 (4.5 μM), T3 (4.5 μM), T7 (4.5 μM), T7-mutant peptide (4.5 μM), endostatin (4.5 μM) or rapamycin (100 ng/ml). These experiments were repeated three times and the representative data are shown. Each column consists of mean ±SEM of triplicates.

FIGS. 61A-61F are a series of eight histograms. FIG. 61A shows the relative density of pFAK/FAK (y-axis) under treatments of (x-axis) no attachment time to vitronectin-coated plates and in the absence of T3 peptide (“0−” bar), 30 minutes attachment time and no T3 peptide (“30−” bar), 30 minutes attachment time and 50 μg/ml T3 peptide (“30+” bar), 60 minutes attachment time and no T3 peptide (“60−” bar) and 60 minutes attachment time and 50 μg/ml T3 peptide (“60+” bar). FIG. 61B shows PI3-kinase activity (y-axis) under treatments of (x-axis) no attachment time to vitronectin-coated plates and in the absence of T3 peptide (“0−” bar), 30 minutes attachment time and no T3 peptide (“30−” bar), 30 minutes attachment time and 50 μg/ml T3 peptide (“30+” bar), 60 minutes attachment time and no T3 peptide (“60−” bar) and 60 minutes attachment time and 50 μg/ml T3 peptide (“60+” bar). FIG. 61C shows the relative density of pFAK/FAK (y-axis) under the same treatments as in FIG. 61A (x-axis). FIG. 61D shows mTOR-kinase activity (y-axis) under treatments of (x-axis) no mTOR transfection and no peptide treatment (“−−” bar), mTOR transfection and no peptide treatment (“±” bar), mTOR transfection and treatment with Tumstatin-44-131 (“+Tum-5” bar) and mTOR transfection and treatment with peptide T3 (“+T3” bar). FIG. 61E shows the density of eIF4E-bound 4E-BP1 (y-axis) in C-PAE cells after treatment with no FBS, T3, Tumstatin-44-131, Rapamycin, Endostatin or FBS (x-axis). FIG. 61F shows the percent luciferase activity relative to CAT activity (y-axis) for C-PAEs that were either infected with adenoviral vectors containing cDNAs of control lacZ (shaded bars), constitutive active Akt (cross-hatched bars), or not infected at all (black bars). The cells were then serum starved, transfected with pcDNA-LUC-pol-CAT, and treated with T3 peptide in the presence of medium containing 10% FCS.

FIG. 62 is a graph showing mean tumor volume (mm ³; y-axis) for various days after treatment onset (x-axis) for treatment with control vehicle (°), and treatment with 1 mg/kg (□) or 2.5 mg/kg (⋄) T8.

FIG. 63 is a graph showing tumor volume ratio (V/Vo; y-axis) for various days after treatment onset (x-axis) for control treatment (°), and treatment with 1 mg/kg TP3 daily (⋄), 5 mg/kg TP3 daily (X), 5 mg/kg T8 daily (□), or 5 mg/kg T8 administered twice weekly (+).

FIGS. 64A and 64B are a pair of graphs showing mean tumor volume ratio (mm ³; y-axis) for various days after treatment onset (x-axis) for various treatments. In FIG. 64A, the treatments were: the stock vehicle used for T7 (°), T7 daily (□), stock vehicle used for T8 (⋄), T8 daily (X), TP3 daily (+), SP1 daily (Δ) and SP2 daily (●). In FIG. 64B, the treatments were: the stock vehicle for T8 (°), T8 daily (□); T8 twice weekly (⋄) and T8 weekly (X).

FIGS. 65A and 65B are a pair of graphs showing mean tumor volume (mm ³; y-axis) in an MDAMB-435 orthotopic human breast tumor xenograft model in nude mice (FIG. 65A) and PC3 human prostate tumor xenograft model in nude mice (FIG. 65B) for various days after treatment onset (x-axis) for control treatment (°), and daily treatment with T8 peptide at 5 mg/kg (□), SP2 at 5 mg/kg (⋄), T8-3 at 1 mg/kg (X) or 5 mg/kg (+), or P2 at 1 mg/kg (Δ) or 5 mg/kg (●). FIGS. 65A and 65B show the results for the MDAMB-435 and PC3 xenograft models, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A wide variety of diseases are the result of undesirable angiogenesis. Put another way, many diseases and undesirable conditions could be prevented or alleviated if it were possible to stop the growth and extension of capillary blood vessels under some conditions, at certain times, or in particular tissues. Basement membrane organization is dependent on the assembly of a type IV collagen network which is speculated to occur via the C-terminal globular non-collagenous (NC1) domain of type IV collagen (Timpl, R., 1996, Curr. Opin. Cell. Biol. 8:618-24; Timpl, R. et al., 1981, Eur. J. Biochem. 120:203-11). Type IV collagen is composed of six distinct gene products, namely, α1 through α6 (Prockop, D. J. et al., 1995, Annu. Rev. Biochem. 64:403-34). The α1 and α2 isoforms are ubiquitously present in human basement membranes (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127), while the other four isoforms exhibit restricted distributions (Kalluri, R. et al., 1997, J. Clin. Invest. 99:2470-78).

The formation of new capillaries from pre-existing vessels, angiogenesis, is essential for the process of tumor growth and metastasis (Folkman, J. et al., 1992, J. Biol. Chem. 267:10931-34; Folkman, J. 1995, Nat. Med. 1:27-31; Hanahan, D. et al., 1996, Cell 86:353-64). Human and animal tumors are not vascularized at the beginning, however, and for a tumor to grow beyond few mm it must vascularize (Folkman, J. 1995, Nat. Med. 1:27-31; Hanahan, D. et al., 1996, Cell 86:353-64). The switch to an angiogenic phenotype requires both upregulation of angiogenic stimulators and downregulation of angiogenesis inhibitors (Folkman, J. 1995, Nat. Med. 1:27-31). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most commonly expressed angiogenic factors in tumors. Vascularized tumors may overexpress one or more of these angiogenic factors which can synergistically promote tumor growth. Inhibition of a single angiogenic factor such as VEGF with a receptor antagonist is not enough to arrest tumor growth. A number of angiogenesis inhibitors have been recently identified, and certain factors such as IFN-α, platelet-factor-4 (Maione, T. E. et al., 1990, Science 247:77-9) and PEX (Brooks, P. C. et al., 1998, Cell 92:391-400) are not endogenously associated with tumor cells, whereas angiostatin (O'Reilly, M. S. et al., 1994, Cell 79:315-28) and endostatin (O'Reilly, M. S. et al., 1997, Cell 88:277-85) are tumor associated angiogenesis inhibitors generated by tumor tissue itself. Although treatment of tumor growth and metastasis with these endogenous angiogenesis inhibitors is very effective and an attractive idea, some potential problems associated with anti-angiogenic therapies must be considered. Delayed toxicity induced by chronic anti-angiogenic therapy as well as the possibility of impaired wound healing and reproductive angiogenesis occurring during treatment are to be considered seriously.

Integrins generally have a short C-terminal cytoplasmic domain linking the receptor to the cytoskeleton of the cell, and a long N-terminal extracellular domain for binding the ligand. Both the α and the β subunits are involved in ligand binding, and a wide array of potential ligands exists. Some common ligands include fibronectin, vitronectin, laminin, and various types of collagen. Some of these (e.g., fibronectin and laminin) are bound by multiple integrins. Collagen I is known to be bound by integrins α ₁β ₁, α ₂β ₁, and α ₃β ₁and collagen IV is bound by integrins α ₂β ₁, and α ₂β ₁. Epithelial cells are bound by integrins α ₂β ₁, α ₆β ₁, α _vβ ₃and α ₆β ₄. Cytokine-activated endothelial cells are bound by α ₄β ₁and α _Lβ ₂, and vascular endothelium is bound by the α _Mβ ₂integrin.

In the present invention, cell surface receptors that interact, e.g., specifically bind, anti-angiogenic proteins and peptides are disclosed, particularly the integrins and integrin subunits that bind the anti-angiogenic proteins Arresten, Canstatin and Tumstatin. These integrins provide targets for assessing new anti-angiogenic proteins, peptides and compounds, or more potent variants and fragments of currently-known anti-angiogenic proteins, peptides and compounds, especially more potent variants and fragments of Arresten, Canstatin and Tumstatin. Specifically, the invention relates to the integrin subunits α ₁, α ₂, α ₃, α _v, β ₁and β ₃, which have been found to bind to Arresten, which is the α1 chain of the NC1 domain of Type IV collagen. The invention also relates to the integrin subunits α ₁, α ₂and β ₁, which have been found to bind to Canstatin, which is the α2 chain of the NC1 domain of Type IV collagen. In addition, the invention relates to integrin subunits α ₅, α ₆, α _v, β ₁and β ₃, which have been found to bind to Tumstatin, the α3 chain of the NC1 domain of Type IV collagen. Other integrins or integrin subunits may also bind to Arresten, Canstatin or Tumstatin, and these may be identified by using the methods described herein (see, e.g., Examples 12, 26 and 28, below).

Angiogenesis and proliferation of endothelial cells may be inhibited, or endothelial cell apoptosis may be promoted or induced, by either administering Arresten, Canstatin or Tumstatin, or administering another protein, peptide or compound that binds to the above-listed integrin subunits, which serve as receptors for Arresten, Canstatin and Tumstatin. Such proteins, peptides and compounds include antibodies, fragments or portions of Arresten, Canstatin or Tumstatin, or proteins or peptides comprising those regions of Arresten, Canstatin or Tumstatin which specifically bind to the above-listed integrin subunits. “Specifically binds” means having high avidity and/or high affinity binding of a ligand (e.g., antigen) to a specific binding protein (e.g., antibody or receptor). For example, antibody binding to its epitope on this specific antigen is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific antigen of interest. Antibodies which bind specifically to a molecule of interest may be capable of binding other molecules at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the molecule of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the molecule of interest, e.g. by use of appropriate controls.

Antibodies to particular peptides are commonly made, and the methods of producing antibodies to a given protein are well-known to those of ordinary skill in the art. See, e.g., Chapter 11 of Ausubel, F. M. et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (John Wiley & Sons, Inc., 1987, with Supplements through 1999), especially pages 11.4.2-11.11.5 (“Preparation of Monoclonal Antibodies”), 11.12.1-11.13.4 (“Preparation of Polyclonal Antisera”) and most especially pages 11.14.1-11.15.4 (“Preparation of Antipeptide Antibodies”). Custom antibodies can also be purchased commercially from a number of suppliers, e.g., from Berkeley Antibody Co., Richmond, Calif., USA. Methods of making antibodies to integrins and integrin subunits are also well known, and methods of making such antibodies are described in Gallatin, W. M. et al. (U.S. Pat. No. 5,817,515), and Kim, K. J. et al. (U.S. Pat. No. 5,652,110; No. 5,652,109; No. 5,578,704), the entire contents of all of which are incorporated herein by reference.

The integrins and integrin subunits described herein can be made recombinantly, and in soluble form. Methods of making soluble receptors and proteins are well-known in the art, and methods of making integrins and integrin receptors in soluble form are described in Briesewitz, R. et al. (1993, J. Biol. Chem. 268:2989-96), Kern, A. et al. (1994, J. Biol. Chem. 269:22811-6); and also in Gallatin, W. M. et al. (U.S. Pat. No. 5,728,533 and No. 5,831,029) and Duong, L. T. et al. (U.S. Pat. No. 5,895,754), the entire contents of all of which are incorporated herein by reference.

The invention also relates to methods of enhancing angiogenesis and cell proliferation, or inhibiting cell apoptosis, by administering proteins, peptides or compounds that mimic the integrin subunits that serve as receptors for Arresten, Canstatin or Tumstatin. Such proteins, peptides or compounds include integrin proteins composed of the selected subunits, which serves to bind available Arresten, Canstatin or Tumstatin, and biologically active (e.g., anti-angiogenic) fragments, mutants, analogs, homologs and derivatives thereof, as well as multimers (e.g., dimers) and fusion proteins (also referred to herein as chimeric proteins) thereof, thereby preventing them from interacting with their respective integrin receptors and inhibiting angiogenic activity. The proteins, peptides or compounds binding to Arresten, Canstatin or Tumstatin, or variants and fragments thereof can also include antibodies to Arresten, Canstatin and Tumstatin, or to the variants or fragments thereof. Such antibodies bind these molecules, thereby preventing them from interacting with their respective integrin receptors and inhibiting angiogenic activity.

In the present invention, Arresten, Canstatin and/or Tumstatin, or their fragments or mutants, may be used alone or in combination to inhibit angiogenesis, endothelial cell proliferation, endothelial cell migration, or endothelial cell tube formation in a tissue, or to induce or promote apoptosis in a tissue, e.g., Arresten and Canstatin can be combined in a pharmaceutical composition, Tum-4 and T7 can be combined in a composition, etc. The combination of Arresten, Canstatin and/or Tumstatin can be further combined with other collagen domains or NC1 chains, or other forms of therapy, e.g., radiotherapy, chemotherapy, immunotherapy, or other active molecules, e.g., endostatin, angiostatin, restin. These molecules decrease levels of the anti-apoptotic protein, FLIP (FLICE-Inhibitory Protein, or FADD-Like Interleukin-1β-Converting Enzyme-Inhibitory Protein). Angiogenesis is therefore inhibited by molecules that decrease levels of FLIP, thereby triggering caspase activation and delivering a terminal apoptotic signal.

The receptors to Arresten, Canstatin and Tumstatin described herein (e.g., the α ₁β ₁, α ₂β ₁, α ₃β ₁, α ₅β ₁, α ₆β ₁, and α _vβ ₃integrins) and/or their subunits (e.g., α ₁, α ₂, α ₃, α ₅, α ₆, α _v, β ₁, β ₃) can be used in combination to promote or induce angiogenesis. The antibodies to Arresten, Canstatin, and/or Tumstatin can also be combined into a single therapeutic regimen, as can the antibodies to the receptors to Arresten, Canstatin and Tumstatin, and their receptor subunits.

The invention also includes kits for identifying anti-angiogenic proteins, peptides and compounds which inhibit angiogenesis in a manner similar to Arresten, Canstatin and Tumstatin, and anti-angiogenic variants and fragments thereof. Such kits comprise appropriate (e.g., α ₁, α ₂, β ₃, etc.) subunits of integrin, and such other ingredients necessary to perform one of the assays described in the Examples below. Exceptional assays to be performed with such a kit would include the Cell Adhesion Assay, described in Examples 12 and 28 below, and the Competition Proliferation Assay, described in Ex

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