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Methods for detecting and inhibiting angiogenesis Number:7,056,506 from the United States Patent and Trademark Office (PTO) owispatent

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Title: Methods for detecting and inhibiting angiogenesis

Abstract: The present invention provides methods for reducing or inhibiting angiogenesis in a tissue, by contacting .alpha.5.beta.1 integrin in the tissue with an agent that interferes with specific binding of the .alpha.5.beta.1 integrin to a ligand expressed in the tissue; and methods of identifying angiogenesis in a tissue, by contacting the tissue with an agent that specifically binds .alpha.5.beta.1 integrin, and detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue. Also provided are methods of diagnosing a pathological condition characterized by angiogenesis in a tissue in an individual. The invention further provides methods of reducing or inhibiting angiogenesis in a tissue in an individual, by administering to the individual an agent that interferes with the specific binding of .alpha.5.beta.1 integrin to a ligand expressed in the tissue; and methods of reducing the severity of a pathological condition associated with angiogenesis in an individual, by administering to the individual an agent that interferes with specific binding of .alpha.5.beta.1 integrin to a ligand in a tissue associated with the pathological condition. The invention also provides methods of identifying an agent that reduces or inhibits angiogenesis associated with .alpha.5.beta.1 integrin expression in a tissue by contacting a tissue exhibiting angiogenesis associated with .alpha.5.beta.1 integrin expression with an agent, and detecting a reduction or inhibition of angiogenesis in the tissue.

Patent Number: 7,056,506 Issued on 06/06/2006 to Varner


Inventors: Varner; Judith A. (Encinitas, CA)
Assignee: The Regents of the University of California (Oakland, CA)
Appl. No.: 190460
Filed: July 5, 2002


Related U.S. Patent Documents

Application NumberFiling DatePatent NumberIssue Date
09307223May., 19996852318
60084850May., 1998

Current U.S. Class: 424/130.1 ; 424/9.1; 424/9.34
Current International Class: A61K 39/395 (20060101); A61K 49/00 (20060101)
Field of Search: 435/7.1,7.23 424/9.1,9.34,130.1


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5567417 October 1996 Sasisekharan et al.
5627263 May 1997 Ruoslahti et al.
5677181 October 1997 Parish
5753230 May 1998 Brooks et al.
5766591 June 1998 Brooks et al.
5855866 January 1999 Thorpe et al.
5866540 February 1999 Jonczyk et al.
5874081 February 1999 Parish
5922676 July 1999 Pasqualini et al.
6123941 September 2000 Bissell
6177542 January 2001 Ruoslahti et al.
6852318 February 2005 Varner
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0906919 Apr., 1999 EP
WO93/15203 Aug., 1993 WO
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WO96/04304 Feb., 1996 WO
WO 97/10507 Mar., 1997 WO
WO 97/33887 Sep., 1997 WO
WO 98/10795 Mar., 1998 WO
WO 99/13329 Mar., 1999 WO

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Primary Examiner: Siew; Jeffrey
Assistant Examiner: Fetterolf; Brandon
Attorney, Agent or Firm: Townsend and Townsend and Crew, LLP

Government Interests



This invention was made, in part, with government support under grant number R01 CA71619 awarded by the National Cancer Institute. The government has certain rights in the invention.
Parent Case Text



This application is a continuation of application Ser. No. 09/307,223, filed May 7, 1999, now U.S. Pat. No. 6,852,318, which claims the benefit of priority of United States Provisional Application Ser. No. 60/084,850 to Judith A. Varner, filed May 8, 1998, now abandoned, and entitled A NOVEL METHOD FOR DETECTION AND INHIBITION OF ANGIOGENESIS, the entire contents of which are incorporated herein by reference. Appropriate correction is required.
Claims



What is claimed is:

1. A method of diagnosing a pathological condition characterized by angiogenesis in a tissue in an individual, comprising: a) administering an agent that specifically binds .alpha.5.beta.1 integrin to an individual suspected of having the pathological condition, wherein said agent is an antibody; and b) detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue, thereby diagnosing a pathological condition characterized by angiogenesis in the individual.

2. The method of claim 1, wherein the agent is detectably labeled.

3. The method of claim 2, wherein detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue comprises: a) obtaining a sample of the tissue from the individual; and b) detecting specific binding of the agent in the sample.

4. The method of claim 2, wherein detecting specific binding of the agent is performed using an in vivo imaging method.

5. The method of claim 2, wherein the detectably labeled agent comprises the agent linked to a label selected from the group consisting of a radionuclide, a paramagnetic material and an X-ray attenuating material.

6. The method of claim 4, wherein the in vivo imaging method is selected from the group consisting of radionuclide imaging, positron emission tomography, computerized axial tomography, and magnetic resonance imaging.

7. The method of claim 1, wherein detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue comprises: a) obtaining a sample of the tissue from the individual; b) contacting the agent that is specifically bound to .alpha.5.beta.1 integrin with a reagent that specifically interacts with the agent; and c) detecting interaction of the reagent with the agent, thereby diagnosing a pathological condition characterized by angiogenesis in the individual.

8. The method of claim 1, wherein the individual is a human.
Description



BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates generally to methods for detecting and treating conditions involving undesirable angiogenesis and more specifically to methods of detecting or inhibiting angiogenesis by interfering with specific binding of .alpha.5.beta.1 integrin to a ligand.

BACKGROUND INFORMATION

Angiogenesis is the process whereby new blood vessels are formed. Angiogenesis, also called neovascularization, occurs normally during embryogenesis and development, and occurs in fully developed organisms during wound healing and placental development. In addition, angiogenesis occurs in various pathological conditions, including in ocular diseases such as diabetic retinopathy and macular degeneration due to neovascularization, in conditions associated with tissue inflammation such as rheumatoid arthritis and inflammatory bowel disease, and in cancer, where blood vessel formation in the growing tumor provides oxygen and nutrients to the tumor cells, as well as providing a route via which tumor cells metastasize throughout the body. Since millions of people around the world are afflicted by these diseases, a considerable effort has been made to understand the mechanisms involved in angiogenesis in the hope that such an understanding will allow the development of methods for detecting and inhibiting such undesirable angiogenesis.

Angiogenesis occurs in response to stimulation by one or more known growth factors, and also may involve other as yet unidentified factors. Endothelial cells, which are the cells that line mature blood vessels, normally do not proliferate. However, in response to an appropriate stimulus, the endothelial cells become activated and begin to proliferate and migrate into unvascularized tissue, to form new blood vessels. In some cases, precursor cells can be activated to differentiate into endothelial cells, which form new blood vessels.

Blood vessels are surrounded by an extracellular matrix. In addition to stimulation by growth factors, angiogenesis depends on interaction of the endothelial cells with the extracellular matrix, as well as with each other. The activation of endothelial cells by growth factors and the migration into and interaction with the extracellular matrix and with each other is dependent on cell surface receptors expressed by the endothelial cells. These cell surface receptors, which include growth factor receptors and integrins, interact specifically with particular molecules.

In pathological conditions such as age-related macular degeneration and diabetic retinopathy, decreasing availability of oxygen to the retina results in a hypoxic condition that stimulates the secretion of angiogenic growth factors such as vascular endothelial growth factors (VEGF), which induce abnormal migration and proliferation of endothelial cells into tissues of the eye. Such vascularization in ocular tissues can induce corneal scarring, retinal detachment and fluid accumulation in the choroid, each of which can adversely affect vision and lead to blindness.

Angiogenesis also is associated with the progression and exacerbation of inflammatory diseases, including psoriasis, rheumatoid arthritis, osteoarthritis, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In inflammatory arthritic disease, for example, influx of lymphocytes into the region surrounding the joints stimulates angiogenesis in the synovial lining. The increased vasculature provides a means for greater influx of leukocytes, which facilitate the destruction of cartilage and bone in the joint. Angiogenic vascularization that occurs in inflammatory bowel disease results in similar effects in the bowel.

The growth of capillaries into atherosclerotic plaques in the coronary arteries represents another pathological condition associated with growth factor induced angiogenesis. Excessive blood flow into neovascularized plaques can result in rupture and hemorrhage of the blood-filled plaques, releasing blood clots that can result in coronary thrombosis.

The involvement of angiogenesis in such diverse diseases as cancer, ocular disease and inflammatory diseases has led to an effort to identify methods for specifically inhibiting angiogenesis as a means to treat these diseases. For cancer patients, such methods of treatment can provide a substantial advantage over currently used methods such as chemotherapy, which kill or impair not only the target tumor cells, but also normal cells in the patient, particularly proliferating normal cells such as blood cells, epithelial cells, and cells lining the intestinal lumen. Such non-specific killing by chemotherapeutic agents results in side effects that are, at best, unpleasant, and can often result in unacceptable patient morbidity, or mortality. In fact, the undesirable side effects associated with cancer therapies often limit the treatment a patient can receive.

For other pathological conditions associated with abnormal angiogenesis such as diabetic retinopathy, there are no effective treatments short of retinal transplants. However, even if retinal transplantation is performed, the new retina would be subject to the same conditions that resulted in the original retinopathy. Thus, there exists a need to identify the molecular interactions involved in the undesirable angiogenesis that occurs in certain pathological conditions such that methods for diagnosing and specifically treating such pathologies can be developed. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods for reducing or inhibiting angiogenesis in a tissue, by contacting .alpha.5.beta.1 integrin associated with blood vessels in the tissue with an agent that interferes with specific binding of the .alpha.5.beta.1 integrin to a ligand expressed in the tissue, thereby reducing or inhibiting angiogenesis in the tissue. In one embodiment, the agent is an .alpha.5.beta.1 antagonist that does not substantially interfere with the specific binding of an integrin other than .alpha.5.beta.1 integrin to its ligand, for example, .alpha.V.beta.3 integrin binding to vitronectin. In another embodiment, the .alpha.5.beta.1 integrin ligand is fibronectin.

A method of the invention is useful, for example, for reducing or inhibiting angiogenesis in ocular tissue such as retina, macula or cornea; in skin; in synovial tissue; in intestinal tissue; or in bone. In addition, a method of the invention is useful for reducing or inhibiting angiogenesis in a neoplasm, which can be benign or malignant and, where malignant, can be a metastatic neoplasm. As such, the invention provides medicaments, which contain .alpha.5.beta.1 antagonists and are useful for reducing or inhibiting angiogenesis in an individual. An agent useful in practicing a method of the invention can be a peptide, for example, a peptide containing the amino acid sequence CRRETAWAC (SEQ ID NO: 1); an antibody, for example, an anti-.alpha.5.beta.1 integrin antibody or an .alpha.5.beta.1 integrin binding fragment thereof; or a nonpeptide, small organic molecule, for example, (S)-2-{(2,4,6-trimethyl phenyl)sulfonyl}amino-3-{7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1- -oxy-2,7-diazaspiro-{4,4}-non-2-en-3-yl}carbonylamino}propionic acid. An agent useful as an .alpha.5.beta.1 antagonist can be linked to a cytotoxin, for example, a cancer chemotherapeutic drug.

The invention also provides methods of identifying the presence of angiogenesis in a tissue by contacting the tissue with an agent that specifically binds .alpha.5.beta.1 integrin, and detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue. The agent can be a peptide, an antibody, or a nonpeptide, small organic molecule, and can be linked to a detectable label, which can be detected directly, or the presence of which can be detected due to its interaction with a particular reagent. Such a method is useful for identifying the presence of angiogenesis in various tissues, including in normal tissues such as embryonic tissue or placental tissue, in granulation tissue, or in a tissue involved in a pathological condition such as a neoplasm, a retinopathy, or an arthritic condition or other inflammatory condition.

The invention further provides methods of diagnosing a pathological condition characterized by angiogenesis in a tissue in an individual. A method of diagnosis can be performed, for example, by obtaining a sample of the tissue from the individual, wherein, in an individual having the pathological condition, the tissue exhibits angiogenesis; contacting the sample with an agent that specifically binds .alpha.5.beta.1 integrin; and detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue, thereby diagnosing a pathological condition characterized by angiogenesis in the individual. The pathological condition can involve the eye, for example, diabetic retinopathy or macular degeneration; the skin, for example, a hemangioma or psoriasis; a joint, for example, rheumatoid arthritis or osteoarthritis; or the intestine, for example Crohn's isease or ulcerative colitis; or can be a neoplasm, which can be benign or malignant. A malignant neoplasm, which can be metastatic, can be, for example, a breast carcinoma, colon carcinoma, ovarian carcinoma, or pancreatic carcinoma.

A method of diagnosing a pathological condition characterized by angiogenesis in a tissue in an individual also can be performed by administering an agent that specifically binds .alpha.5.beta.1 integrin to an individual suspected of having the pathological condition; and detecting specific binding of the agent to .alpha.5.beta.1 integrin associated with a blood vessel in the tissue. The agent can be detectably labeled, for example, by linking it to a moiety such as a radionuclide, a paramagnetic material or an X-ray attenuating material. The method of detecting can be an in vivo imaging method such as a radionuclide imaging, positron emission tomography, computerized axial tomography, or magnetic resonance imaging method, or can be an ex vivo method, wherein, following administration of the agent, a sample of the tissue is obtained from the individual, and specific binding of the agent in the sample is detected. Agent that is specifically bound to .alpha.5.beta.1 integrin in such a sample can be detected directly, for example, by detecting radioactivity due to the moiety linked to the agent, or can be detected indirectly by contacting the specifically bound agent with a reagent that specifically interacts with the agent, or with the moiety, and detecting an interaction of the reagent with the agent or the moiety.

The present invention further provides methods of reducing or inhibiting angiogenesis in a tissue in an individual, by administering to the individual an agent that interferes with the specific binding of .alpha.5.beta.1 integrin to a ligand expressed in the tissue, thereby reducing or inhibiting angiogenesis in the tissue in the individual. Also provided is a method of reducing the severity of a pathological condition associated with angiogenesis in an individual, by administering to the individual an agent that interferes with specific binding of .alpha.5.beta.1 integrin to a ligand in a tissue associated with the pathological condition, thereby reducing or inhibiting angiogenesis in the tissue and, consequently, reducing the severity of the pathological condition. The condition can be any pathological condition associated with angiogenesis, including a neoplasm, which can be a malignant neoplasm, for example, a carcinoma such as breast carcinoma, colon carcinoma, ovarian carcinoma or pancreatic carcinoma, or a sarcoma, mesothelioma, teratocarcinoma, an astrocytoma, glioblastoma, or other neoplasm, including a metastatic malignant neoplasm. The agent can be administered by various routes, for example, intravenously, orally, or directly into the region to be treated, for example, directly into a neoplastic tumor; via eye drops, where the pathological condition involves the eye; or intrasynovially, where the condition involves a joint.

The invention also provides methods of identifying an agent that reduces or inhibits angiogenesis associated with .alpha.5.beta.1 integrin expression in a tissue. Such a method, which is useful as a screening assay, can be performed by contacting a tissue exhibiting angiogenesis associated with .alpha.5.beta.1 integrin expression with an agent, and detecting a reduction or inhibition of angiogenesis in the tissue. Contacting of the tissue with the agent can occur in vivo or ex vivo. Where the method is performed using an in vitro format, it readily can be adapted for automated, high throughput screening assays. The tissue can be any tissue that undergoes angiogenesis associated with .alpha.5.beta.1 integrin expression, for example, malignant neoplastic tissue, and can be from any individual, including, for example, from a mammal, bird, reptile or amphibian.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the inhibitory effect of the nonpeptide small organic molecule, SJ749, on .alpha.5.sup.+ HT29 tumor cell adhesion to fibronectin. .alpha.5.sup.+ HT29 tumor cells were produced by transfecting HT29 cells with .alpha.5.sup.+ cDNA.

FIG. 2 demonstrates the dose dependent inhibitory effect of SJ749 on blood vessel branch point formation in chorioallantoic membranes (CAM's). Angiogenesis was stimulated by treatment of the CAM's with basic fibroblast growth factor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for detecting angiogenesis in a tissue by identifying .alpha.5.beta.1 binding to a ligand in a blood vessel in the tissue. Methods of diagnosing the presence of angiogenesis in an individual also are provided. The invention further provides methods for reducing or inhibiting angiogenesis in a tissue by interfering with the specific binding of .alpha.5.beta.1 integrin to a ligand expressed in the tissue. Methods of reducing or inhibiting angiogenesis, which can be associated with a pathological condition, in an individual, also are provided.

Angiogenesis depends on the cooperation of various growth factors and cell adhesion events. The .alpha.V integrins have been shown to play critical roles in angiogenesis, although studies using .alpha.V integrin null mice have suggested that other adhesion receptors and their ligands also may be involved in angiogenesis. As disclosed herein, the integrin .alpha.5.beta.1 and its ligand fibronectin are coordinately upregulated during growth factor stimulated angiogenesis and on blood vessels present in human tumor biopsies, and the interaction of these molecules is required for the angiogenesis that occurs during and supports tumor growth in vivo, as well as angiogenesis associated with various pathological conditions.

The development of vascular networks during embryogenesis or normal and pathological angiogenesis depends on stimulation induced by growth factors (Breier and Risau, Trends in Cell Biology 6:454 456 (1996); Breier et al., Thromb. Haemost. 78:678 683 (1997); Folkman, Nature Med. 1:27 31 (1995); Risau, Nature 386:671 674 (1997)) and on cellular interactions with the extracellular matrix (Stromblad and Cheresh, Chemistry and Biology 3:881 885 (1996); Varner, Exs. 79:361 390 (1997); each of the publications cited in this disclosure is incorporated herein by reference). Genetic and functional analyses indicate that extracellular components and cell surface receptors regulate endothelial cell growth, survival and differentiation in vasculogenesis and in angiogenesis (George et al., Development 119:1079 1091 (1993); Yang et al., Development 119:1093 1105 (1993); Stromblad and Cheresh, supra, 1996; Bloch et al., J. Cell Biol. 139: 265 278 (1997); Varner, supra, 1997; Risau, supra, 1997; Bader et al., Cell 95:507 519 (1998)).

Blood vessels arise during embryogenesis by two processes, vasculogenesis and angiogenesis (Risau, supra, 1997), and the role of growth factors in both processes is well established. For example, vascular endothelial growth factor (VEGF; Ferrara et al., Nature 380:439 442 (1996)) and its receptors (de Vries et al., Science 255:989 991 (1992); Fong et al., Nature 376:66 70 (1995); Millauer et al., Cell 72:835 846 (1993); Shalaby et al., Cell 89:981 990 (1997)), and basic fibroblast growth factor (bFGF; Basilico and Moscatelli, Adv. Cancer Res. 59:115 165 (1992)) promote the initial development of the embryonic vascular network, and are involved in the formation of new blood vessels from pre-existing vessels during development, wound healing and the female reproductive cycle. VEGF (Warren et al., J. Clin. Invest. 95:1789 1797 (1995); Yoshida et al., Mol. Cell. Biol. 17:14015 4023 (1997); Kong et al., Human Gene Ther. 9:823 833 (1998)), bFGF (Stan et al., J. Neurosurg. 82:1044 1052 (1995); Chopra et al., J. Canc. Res. Clin. Oncol. 123:167 172 (1997); Czubayko et al., Nature Med. 0.3:1137 1140 (1997); Yoshida et. al., supra, 1997), Interleukin-8 (IL-8; Arenberg et al., J. Clin. Invest. 97: 2792 2802 (1996); Luca et al., Am. J. Path. 151:1105 1113 (1997); Keane et al.,. J. Immunol. 159:1437 43(1997);Yatsunami et al., Cancer Lett. 120:101 108 (1997); Yoshida et al., Invest. Ophthamol. Vis. Sci. 39:1097 1106 (1998)), and tumor necrosis factor-.alpha. (TNF.alpha.; Yoshida et. al., supra, 1997) are some of the growth factors that have a role in the angiogenesis that is associated with various pathological conditions, including, for example, solid tumor growth, diabetic retinopathy, and rheumatoid arthritis.

While growth factors stimulate new blood vessel growth, adhesion to the extracellular matrix (ECM) regulates endothelial cell survival, proliferation and motility during new blood vessel growth (Stromblad and Cheresh, supra, 1996; Varner, supra, 1997). Specific integrins or their ligands also influence vascular development and angiogenesis. For example, the .alpha.V integrins participate in angiogenesis by providing survival signals to activated endothelial cells (Arap et al., Science 279:377 380 (1997); Brooks et al., Science 264: 569 571 (1994a); Carron et al., Cancer Res. 58:1930 1955 (1998); Clark et al., Amer. J. Pathol. 148:1407 1421 (1997); Drake et al., Devel. Dyn. 193:83 91 (1992); Clark et al., J. Cell Science 108:2655 2661 (1995); Friedlander et al., Science 270:1500 1502 (1995)). However, some aspects of angiogenesis also can proceed in the absence of .alpha.V integrins (Bader et. al., supra, 1998), suggesting that other molecules, including the .beta.1 integrin family, may compensate for the absence .alpha.V integrins during development (Drake et al., supra, 1992; Bloch et al., supra, 1997; Senger et al., Proc. Natl. Acad. Sci., USA 94:13612 13617 (1997)).

While active roles for integrins in the promotion of angiogenesis have been identified, the cognate ECM ligands for integrins that are involved in angiogenesis in vivo are less well described. One ECM protein, fibronectin, is expressed in provisional vascular matrices and provides proliferative signals to vascular cells during wound healing, atherosclerosis, and hypertension (Magnusson and Mosher, Arterioscler. Thromb. Vasc. Biol. 18:1363 1370 (1998)). Fibronectin expression is upregulated on blood vessels in granulation tissues during wound healing (Clark et al., J. Invest. Dermatol. 79:269 276 (1982)), and an isoform of fibronectin, the ED-B splice variant, is preferentially expressed on blood vessels in fetal and tumor tissues, but not on normal quiescent adult blood vessels (Castellani et al., Int. J. Cancer 59:612 618 (1994); Kaczmarek et al., Int. J. Cancer 58:11 16 (1994); Neri et al., Nature Biotech. 15:1271 1275 (1997)). These observations suggest that fibronectin may have a role in angiogenesis. In addition, animals that lack fibronectin die early in development from a collection of defects, including missing notochord and somites as well as an improperly formed vasculature (George et al., supra, 1993). Prior to the present disclosure, however, a direct functional role for fibronectin in vasculogenesis or in angiogenesis was not established.

Several integrins bind to fibronectin (Hynes, Cell 69:11 25 (1992)), and integrin .alpha.5.beta.1 generally is selective for fibronectin (Pytela et al., Cell 40:191 98 (1985)). Studies have demonstrated that loss of the gene encoding the integrin .alpha.5 subunit is embryonic lethal in mice and is associated with a complete absence of the posterior somites and with some vascular and cardiac defects (Yang et al., supra, 1993; Goh et al., Development 124: 4309 4319 (1997)). It was unclear, however, whether integrin .alpha.5.beta.1 has a direct role in the regulation of vascular development or of angiogenesis in particular.

As disclosed herein, both fibronectin and its receptor, .alpha.5.beta.1 integrin, directly regulate angiogenesis. Moreover, the specific interaction of fibronectin and .alpha.5.beta.1 is central to the contribution of these two molecules to angiogenesis. Integrin .alpha.5.beta.1 participates in pathways of angiogenesis that are the same as those of integrin .alpha.V.beta.3, but distinct from the pathways involving .alpha.V.beta.5. It is further disclosed herein that agents that interfere with the specific binding of .alpha.5.beta.1 and fibronectin can reduce or inhibit growth factor stimulated angiogenesis and the angiogenesis that occurs in tumors and, therefore, can be useful for treating various pathological conditions, including malignant neoplasms.

The participation of the central cell binding domain of fibronectin and its receptor .alpha.5.beta.1 in angiogenesis is disclosed herein. Expression of both integrin .alpha.5.beta.1 and fibronectin were significantly enhanced on blood vessels of human tumors and in growth factor stimulated tissues, while these molecules were minimally expressed on normal human vessels and on unstimulated tissues (Example I). In addition, antibody antagonists, which bind the central cell binding domain of fibronectin and anti-.alpha.5.beta.1 antibodies, as well as two other classes of .alpha.5.beta.1 antagonists (peptides and nonpeptide, small organic molecule antagonists) blocked growth factor stimulated angiogenesis in chick chorioallantoic membrane (CAM; Example II) and in human skin grown on SCID mice (Example III). Antagonists of integrin .alpha.5.beta.1 blocked bFGF, TNF.alpha. and IL-8 stimulated angiogenesis, but had a minimal effect on VEGF-induced angiogenesis. Each of these .alpha.5.beta.1 antagonists inhibited tumor angiogenesis and resulted in tumor regression in animal model systems (Example IV). Antagonists of fibronectin function also blocked both bFGF and VEGF angiogenesis, suggesting that other fibronectin receptors are involved in VEGF-mediated angiogenesis The results disclosed herein demonstrate that the expression of integrin .alpha.5.beta.1 and fibronectin in angiogenesis is coordinated. When the expression of each molecule is minimal, as on unstimulated, quiescent blood vessels, antagonists of each molecule and addition of fibronectin to chick chorioallantoic membranes (CAM's) had little effect on angiogenesis. In contrast, after stimulation with growth factors, .alpha.5.beta.1 and fibronectin expression are enhanced and blood vessels become sensitive to agents that act as antagonists of either molecule, as well as to the effects of exogenously added fibronectin. VEGF stimulation does not increase .alpha.5.beta.1 expression, supporting the observation that VEGF angiogenesis is refractory to antagonists of .alpha.5.beta.1. This result is substantiated by a report that in vitro expression of integrin .alpha.5.beta.1 on endothelial cells was upregulated in response to bFGF (Collo and Pepper, J. Cell Sci. 112:569 578 (1999)), and that VEGF failed to upregulate .alpha.5.beta.1 expression (Senger et al., Am. J. Pathol. 149:1 7 (1996); Senger et al., Proc. Natl. Acad. Sci., USA 94:13612 13617 (1997)). Thus, the functional roles of integrin .alpha.5.beta.1 and fibronectin in angiogenesis likely are a direct consequence of their growth factor induced expression.

Antibodies directed against the central cell binding fragment of fibronectin, which contains the RGD integrin binding site, inhibited angiogenesis (Examples II and III). These antibodies likely interfere with the specific binding of .alpha.5.beta.1 integrin to fibronectin, and, consequently, with possible downstream signal transduction events in vivo. Stimulation of bFGF angiogenesis by fibronectin and its cell binding domain in an .alpha.5.beta.1-dependent manner indicate that .alpha.5.beta.1 is the integrin receptor for fibronectin during angiogenesis. The absence of integrin .alpha.5.beta.1 expression in VEGF stimulated angiogenesis likely accounts for the failure of fibronectin to enhance VEGF angiogenesis, even though antibodies directed against the cell binding peptide of fibronectin blocked VEGF angiogenesis. The results disclosed herein are the first demonstration of a direct in vivo role for fibronectin in angiogenesis.

The results disclosed herein also are the first to clearly identify a role for an extracellular matrix protein in the promotion of angiogenesis. Although collagens have been suggested to have roles in vascular development, intact collagens do not support endothelial cell outgrowth, survival or proliferation (Ilan et al., J. Cell Sci.111:3621 3631 (1998); Isik et al., J. Cell. Phys.175:149 155 (1999)). In fact, inhibition of the collagen receptors integrins .alpha.2.beta.1 and .alpha.1.beta.1 prevented the formation of large blood vessels and promoted the formation of small vessels (Senger et al., supra, 1997). Those results suggest that .alpha.2.beta.1, .alpha.1.beta.1, and their ligand, collagen, are involved in blood vessel maturation, rather than in the promotion of new blood vessel sprouts.

A functional role for integrin .alpha.5.beta.1 in angiogenesis was established by demonstrating that agents that antagonize .alpha.5.beta.1 binding to its ligand blocked angiogenesis induced by growth factors and angiogenesis in tumor fragments (Examples II, III and IV). Like .alpha.5.beta.1, .alpha.V.beta.3 can serve as a fibronectin receptor (Charo et al., J. Cell Biol. 111:2795 800 (1990)), although, as disclosed herein, endothelial cells use .alpha.5.beta.1 as the major fibronectin receptor when both integrins are expressed.

The expression of .alpha.5.beta.1 and .alpha.V.beta.3 is regulated by similar growth factors, and both integrins have a significant role in bFGF, TNF.alpha., IL-8 and tumor-induced angiogenesis, but not in VEGF-induced angiogenesis (see Examples; see, also, Brooks et al., supra, 1994a; Brooks et al., Cell 79:1157 1164 (1994b); Friedlander et al., supra, 1995). These two integrins likely influence the same angiogenesis pathways, since combinations of their antagonists in angiogenesis animal models were neither additive nor synergistic (see Example II).

Binding of integrins to extracelluar matrix proteins promotes cell attachment, migration, invasion, survival and proliferation (Varner, supra, 1997), and antagonists of .alpha.V.beta.3 induce apoptosis of proliferating endothelial cells in vitro and in vivo (Brooks et al., supra, 1994b; Stromblad et al., supra, 1996). As disclosed herein, .alpha.5.beta.1 antagonists also induce apoptosis of growth factor stimulated endothelial cells in vitro and in vivo.

Antagonists of .alpha.5.beta.1 blocked tumor angiogenesis and growth (Example IV), similar to antagonists of integrin .alpha.V.beta.3 (Brooks et al., supra, 1994b, 1995). The tumor cell lines used for in vivo tumorigenicity and angiogenesis studies (Example IV) were integrin .alpha.5.beta.1 negative, to discount any direct effect of the antagonists on the tumor cells, and remained .alpha.5.beta.1 negative through the course of their culture on CAM's. HT29 tumors express a variety of growth factors, including VEGF, TNF.alpha., TGF-.alpha., TGF.beta., PDGF and IL-8; it is not known whether HT29 cells also express bFGF. VEGF is most commonly associated with the hypoxic core of the tumor, and is transcriptionally regulated by hypoxia, whereas bFGF and other factors are associated with the growing edge of the tumor (Shweiki, et. al., supra, 1992; Kumar et al., Oncol. Res. 10:301 311 (1998)). As observed for growth factor stimulated CAM's, .alpha.5.beta.1 antagonists did not impact large pre-existing vessels on the CAM that underlie the transplanted tumors. These results demonstrate that agents that interfere with specific binding of .alpha.5.beta.1 to its ligands, particularly fibronectin, can reduce or inhibit angiogenesis. The use of such agents, therefore, can provide a clinical benefit to individuals suffering from various pathological conditions, including to cancer patients.

As used herein, the term "integrin" refers to the extracellular receptors that are expressed in a wide variety of cells and bind to specific ligands in the extracellular matrix. The specific ligands bound by integrins can contain an arginine-glycine-aspartic acid tripeptide (Arg-Gly-Asp; RGD) or a leucine-aspartic acidvaline tripeptide, and include, for example, fibronectin, vitronectin, osteopontin, tenascin, and von Willebrand's factor. The integrins comprise a superfamily of heterodimers composed of an .alpha. subunit and a .beta. subunit. Numerous a subunits, designated, for example, .alpha.V, .alpha.5 and the like, and numerous .beta. subunits, designated, for example, .beta.1, .beta.2, .beta.3, .beta.5 and the like, have been identified, and various combinations of these subunits are represented in the integrin superfamily, including .alpha.5.beta.1, .alpha.V.beta.3 and .alpha.V.beta.5. The superfamily of integrins can be subdivided into families, for example, as .alpha.V-containing integrins, including .alpha.V.beta.3 and .alpha.V.beta.5, or the Pi-containing integrins, including .alpha.5.beta.1 and .alpha.V.beta.1. Integrins are expressed in a wide range of organisms, including C. elegans, Drosophila sp., amphibians, reptiles, birds, and mammals, including humans.

As disclosed herein, antibody, peptide and nonpeptide small organic molecule antagonists of .alpha.5.beta.1 can interfere with the specific binding of .alpha.5.beta.1 integrin with its ligands, particularly fibronectin, in vascular tissue, and can reduce or inhibit angiogenesis (see Examples II, III and IV). Such molecules that interfere with the specific binding of .alpha.5.beta.1 with its ligands are referred to herein generally as "agents," "agent antagonists" or ".alpha.5.beta.1 antagonists." As used herein, the term "specific binding" or "binds specifically," when used in reference to the interaction of two or more molecules, means that the molecules can associate with each other under in vivo conditions and in vitro when incubated under appropriate conditions, which can mimic in vivo conditions. The terms "specifically interact" and "specific association" also are used to refer to molecules that specifically bind.

For purposes of the present invention, the molecules that specifically interact with each other generally are a receptor-type molecule and its ligand, including, for example, an integrin and its particular ligand or ligands, or an antibody and its particular antigen or antigens. It is recognized, however, that other molecules, for example, an .alpha. integrin subunit and a .beta. integrin subunit also interact specifically to form an integrin heterodimer, as can an .alpha.5.beta.1 antagonist and an .alpha.5.beta.1 integrin. Methods for determining whether two molecules specifically interact are disclosed herein, and methods of determining binding affinity and specificity are well known in the art (see, for example, Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (W.H. Freeman and Co. 1976)).

Antibodies, peptides and nonpeptide small organic molecule antagonists that interfere with the specific binding of .alpha.5.beta.1 with fibronectin are exemplified (see Example II). As used herein, the term "interfere," when used in reference to the action of an agent antagonist on the specific interaction of a receptor and its ligand, means that the affinity of the interaction is decreased below the level of binding that occurs in the absence of the agent. The skilled artisan will recognize that the association of a receptor and its ligand is a dynamic relationship that occurs among a population of such molecules such that, at any particular time, a certain proportion of receptors and ligands will be in association. An agent that interferes with the specific interaction of a receptor and its ligand, therefore, reduces the relative number of such interactions occurring at a given time and, in some cases, can completely inhibit all such associations.

The term "antagonist" is used herein to mean an agent, which can be an antibody, a peptide or a nonpeptide small organic molecule, that can interfere with the specific interaction of a receptor and its ligand. An anti-.alpha.5.beta.1 integrin antibody, which can interfere with the binding of .alpha.5.beta.1 with fibronectin, thereby reducing or inhibiting the association of .alpha.5.beta.1 integrin with fibronectin, is an example of an .alpha.5.beta.1 antagonist. An antagonist can act as a competitive inhibitor or a noncompetitive inhibitor of .alpha.5.beta.1 binding to its ligand.

It can be difficult to distinguish whether an antagonist completely inhibits the association of a receptor with its ligand or reduces the association below the limit of detection of a particular assay. Thus, the term "interfere" is used broadly herein to encompasses reducing or inhibiting the specific binding of a receptor and its ligand. Furthermore, an agent can interfere with the specific binding of a receptor and its ligand by various mechanism, including, for example, by binding to the ligand binding site, thereby interfering with ligand binding; by binding to a site other than the ligand binding site of the receptor, but sterically interfering with ligand binding to the receptor; by binding the receptor and causing a conformational or other change in the receptor, which interferes with binding of the ligand; or by other mechanisms. Similarly, the agent can bind to or otherwise interact with the ligand to interfere with its specifically interacting with the receptor. For purposes of the methods disclosed herein for interfering with the specific interaction of an .alpha.5.beta.1 integrin and its ligand, an understanding of the mechanism by which the interfering occurs is not required and no mechanism of action is proposed.

An agent that acts as an antagonist for .alpha.5.beta.1 integrin binding to its ligand can be an antibody, particularly an anti-.alpha.5.beta.1 antibody or an anti-fibronectin antibody. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. With regard to an anti-integrin antibody, particularly an anti-.alpha.5.beta.1 antibody, the term "antigen" means an integrin, particularly an .alpha.5.beta.1 integrin protein, polypeptide, or peptide portion thereof, which may or may not include some or all of an RGD binding domain. An anti-.alpha.5.beta.1 antibody, or antigen binding fragment thereof, is characterized by having specific binding activity for an .alpha.5.beta.1 integrin of at least about 1.times.10.sup.5 M.sup.-1, generally at least about 1.times.10.sup.6 M.sup.-1, and particularly at least about 1.times.10.sup.7 M.sup.-1. Fab, F(ab').sub.2, Fd or Fv fragments of an anti-.alpha.5.beta.1 antibody, which retain specific binding activity for the .alpha.5.beta.1 integrin are included within the definition of an antibody.

The term "antibody" as used herein encompasses naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275 1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243 246 (1993); Ward et al., Nature 341:544 546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

Anti-integrin antibodies, including anti-.alpha.5.beta.1 antibodies, can be purchased from a commercial source, for example, Chemicon, Inc. (Temecula Calif.), or can be raised using as an immunogen a substantially purified full length integrin, which can be a human integrin, mouse integrin or other mammalian or nonmammalian integrin that is prepared from natural sources or produced recombinantly, or a peptide portion of an integrin, which can include a portion of the RGD binding domain, for example, a synthetic peptide. A non-immunogenic peptide portion of an integrin such as a human .alpha.5.beta.1 can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described, for example, by Harlow and Lane (supra, 1988).

Particularly useful antibodies for performing a method of the invention are those that specifically bind to an .alpha.5.beta.1 integrin. Such antibodies are particularly useful where they bind .alpha.5.beta.1 with at least an order of magnitude greater affinity than they bind another integrin, for example, .alpha.V.beta.3 or .alpha.V.beta.5. An anti-fibronectin antibody also can be useful in a method of the invention, particularly an anti-fibronectin antibody that interferes with binding of fibronectin to .alpha.5.beta.1 integrin, but not to .alpha.V.beta.3 or other integrins.

As disclosed herein, an anti-.alpha.5.beta.1 antibody was used to detect regions of growth factor stimulated angiogenesis, as occurs in a pathological condition (see Example I). The presence or amount of .alpha.5.beta.1 integrin expression can be identified, for example, in a tissue sample, which can be a histological section obtained from a tissue or organ of an individual suspected of having a pathology characterized, at least in part, by undesirable angiogenesis. The identification of the presence or level of an .alpha.5.beta.1 integrin expression in the sample can be made using well known immunoassay or immunohistochemical methods (Harlow and Lane, supra, 1988). An anti-.alpha.5.beta.1 antibody, particularly an antibody that prevents ligand binding to the .alpha.5.beta.1 integrin, also can be used in a screening assay to identify agents that compete for ligand binding to the integrin. As disclosed herein, such agents can be useful for inhibiting .alpha.5.beta.1 mediated angiogenesis.

Peptides that specifically bind to .alpha.5.beta.1 also are useful as antagonists of .alpha.5.beta.1 binding to its ligands, including fibronectin. As discussed for anti-.alpha.5.beta.1 antibodies, a peptide that specif


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