# Patent

## Title
Methods for inhibiting angiogenesis

## Abstract

The invention provides methods for detecting and inhibiting angiogenesis, endothelial cell adhesion, and endothelial cell migration using agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention further provides methods for screening test compounds for their ability to inhibit angiogenesis, endothelial cell adhesion, or endothelial cell migration by employing agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention additionally relates to methods for isolating endothelial progenitor cells which express integrin α4β1. The methods of the invention are useful in, for example, diagnosing and inhibiting pathological conditions that are associated with angiogenesis, endothelial cell adhesion, and/or endothelial cell migration. The invention's methods are also useful in isolating endothelial progenitor cells, and in determining the mechanisms that underlie angiogenesis, development, wound healing, and the function of the female reproductive system.

## Background
This application is the U.S. National stage filing of PCT Application No. PCT/US02/24573, filed on Aug. 1, 2002, which claims priority to U.S. provisional Patent Application Ser. No. 60/310,645, filed Aug. 6, 2001, now abandoned.


This invention was made, in part, with government support under grant numbers CA71619 and CA 83133 awarded by the National Cancer Institute of the National Institutes of Health. The U.S. government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention provides methods for detecting and inhibiting angiogenesis, endothelial cell adhesion, and endothelial cell migration. In preferred embodiments, the present invention utilizes agents that inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention further relates to methods for screening test compounds for their ability to inhibit angiogenesis, endothelial cell adhesion, or endothelial cell migration by employing agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention additionally relates to methods for isolating endothelial cells which express integrin α4β1. The methods of the invention are useful in, for example, diagnosing and inhibiting pathological conditions that are associated with angiogenesis, endothelial cell adhesion, and/or endothelial cell migration. The invention's methods are also useful in isolating endothelial progenitor cells, and in determining the mechanisms that underlie angiogenesis, development, wound healing, and the function of the female reproductive system.
BACKGROUND OF THE INVENTION
Angiogenesis is essential in the female reproduction system and during development and wound repair. However, inappropriate angiogenesis can have severe consequences. Indeed, the proliferation of new blood vessels from pre-existing capillaries plays a key role in diseases, such as the pathological development of solid tumor cancers, solid tumor metastases, angiofibromas, skin cancer, retrolental fibroplasia, Kaposi's sarcoma, childhood hemangiomas, diabetic retinopathy, neovascular glaucoma, age related macular degeneration, psoriasis, gingivitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and capillary proliferation in atherosclerotic plaques. Because these serious diseases afflict several million people in the United States each year, considerable scientific effort has been directed toward gaining an understanding of the mechanisms regulating angiogenesis and toward developing therapies for such diseases.
With respect to cancer, over six hundred thousand new cases of lung, colon, breast and prostate cancer will be diagnosed in the United States each year, accounting for 75% of new solid tumor cancers and 77% of solid tumor cancer deaths. Although advances in therapy and in our understanding of cancer causes and risk factors have lead to improved outcomes overall, most cancers still have low five year survival rates. Despite these advances in primary tumor management, 50% of patients will ultimately die of their disease largely due to side effects of current therapies or to the metastatic spread of tumors to numerous or inoperable sites through the tumor associated vasculature. It is now known that the growth and spread of solid tumor cancer depends on the development of a tumor-associated vasculature by a process known as angiogenesis.
One of the most significant consequences of tumor angiogenesis is the invasion of tumor cells into the vasculature. Thus, vascularization permits the survival and growth of primary tumors, as well as the metastatic spread of cancer. Metastases arise from tumor cells which enter the tumor's own vasculature to be carried to local and distant sites where they create new tumors. Tumors have typically established a vasculature and metastasized to local and distant sites by the time that primary tumors are detectable.
Current treatments for cancer rely mainly on treatments which are not selective for the disease but which have deleterious effects on other organs of the body. For example, chemotherapeutics reagents or radiation have serious side effects because they kill or impair all proliferating cells in the body, including healthy cells. Side effects are unpleasant and often create health problems that themselves increase patient mortality.
Angiogenesis also plays a major role in the progression and exacerbation of a number of inflammatory diseases. Psoriasis, a disease which afflicts 2 million Americans, is characterized by significant angiogenesis. In rheumatoid arthritis and possibly osteoarthritis, the influx of lymphocytes into joints induces blood vessels of the joint synovial lining to undergo angiogenesis; this angiogenesis appears to permit a greater influx of leukocytes and the destruction of cartilage and bone. Angiogenesis is also likely to play a role in chronic inflammatory diseases such as ulcerative colitis and Crohn's disease. In addition, the growth of capillaries into atherosclerotic plaques is a serious problem; the rupture and hemorrhage of vascularized plaques is thought to cause coronary thrombosis. To date, however, no effective therapies exist for these diseases.
Angiogenesis is also a factor in many ophthalmic disorders which can lead to blindness. In age-related macular degeneration (ARMD), a disorder afflicting 25% of otherwise healthy individuals over the age of 60, and in diabetic retinopathy, a condition prevalent among both juvenile and late onset diabetics, angiogenesis is induced by hypoxic conditions on the choroid or the retina, respectively. Hypoxia induces an increase in the secretion of growth factors including vascular endothelial growth factor (VEGF). It is possible that VEGF expression in the eye induces the migration and proliferation of endothelial cells into regions of the eye where they are not ordinarily found. Vascularization in ocular tissue has adverse effects on vision. New blood vessels on the cornea can induce corneal scarring, whereas new blood vessels on the retina can induce retinal detachment, and angiogenic vessels in the choroid may leak vision-obscuring fluids; these events often lead to blindness.
For other pathological conditions associated with abnormal angiogenesis such as diabetic retinopathy, there are no effective treatments short of retinal transplants. However, even in cases where retinal transplantation is performed, the new retina is subject to the same conditions that resulted in the original retinopathy.
While agents that prevent continued angiogenesis are currently being tested, there remains a need to identify the molecular interactions involved in the undesirable angiogenesis that occurs in certain pathological conditions, and to develop methods for diagnosing and specifically treating such pathologies.
SUMMARY OF THE INVENTION
The present invention satisfies the need in the art for identifying the molecular interactions involved in the undesirable angiogenesis that occurs in certain pathological conditions, and for developing methods for diagnosing and specifically treating such pathologies.
In particularly preferred embodiments, the invention provides a method for inhibiting angiogenesis in a tissue, comprising: a) providing at least one tissue and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the tissue with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and a treated tissue is produced; and c) observing inhibition of angiogenesis in the treated tissue. In one embodiment, the tissue is in a subject. In a preferred embodiment, subject has tissue angiogenesis. In another preferred embodiment, the subject is suspected of being capable of developing angiogenesis in the tissue. In yet another preferred embodiment, the tissue comprises ocular tissue, skin tissue, bone tissue, or synovial tissue. In a more preferred embodiment, the ocular tissue is selected from the retina, macula, cornea, choroids, and vitreous humor. In an alternative embodiment, the tissue comprises a tumor. In a preferred embodiment, the tumor is malignant. In a more preferred embodiment, the malignant tumor is metastatic. In yet another embodiment, the agent comprises a peptide. In an alternative embodiment, the agent comprises an antibody. In a further preferred embodiment, the antibody is an anti-integrin α4β1 antibody. In another preferred embodiment, the antibody is an anti-vascular cell adhesion molecule antibody. In yet another preferred embodiment, the antibody is an anti-fibronectin antibody. In an alternative embodiment, the agent comprises an antisense sequence. In a preferred embodiment, antisense sequence is an integrin α4β1 antisense sequence. In another preferred embodiment, the antisense sequence is a vascular cell adhesion molecule antisense sequence. In yet another preferred embodiment, the antisense sequence is a fibronectin antisense sequence. In yet another alternative embodiment, the agent comprises a ribozyme. In a preferred embodiment, the ribozyme is an integrin α4β1 ribozyme. In another preferred embodiment, the ribozyme is a vascular cell adhesion molecule ribozyme. In yet another preferred embodiment, the ribozyme is a fibronectin ribozyme. In an alternative embodiment, the ligand is vascular cell adhesion molecule. In a preferred embodiment, the ligand is fibronectin.
Also provided herein are methods for inhibiting endothelial cell adhesion, comprising: a) providing endothelial cells and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the endothelial cells with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and treated endothelial cells are produced; and c) observing inhibition of cell adhesion of the treated endothelial cells.
The invention further provides methods for inhibiting endothelial cell migration, comprising: a) providing endothelial cells and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the endothelial cells with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and treated endothelial cells are produced; and c) observing inhibition of cell migration by the treated endothelial cells.
The present invention also provides methods of inhibiting angiogenesis in a subject, comprising: a) providing a subject comprising a tissue and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) administering the agent to the subject under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand in the tissue is inhibited and a treated tissue is produced; c) observing inhibition of angiogenesis in the treated tissue. In one embodiment, the subject has a pathological condition associated with angiogenesis in the tissue. In a preferred embodiment, the tissue comprises ocular tissue, skin tissue, bone tissue, or synovial tissue. In a more preferred embodiment, the ocular tissue is selected from retina, macula, cornea, choroids, and vitreous humor. In another preferred embodiment, the tissue comprises a tumor. In a more preferred embodiment, the tumor is malignant. In a yet more preferred embodiment, the malignant tumor is metastatic. In an alternative preferred embodiment, the malignant tumor is selected form lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer; stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer, muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer, joint cancer, glioblastoma, lymphoma, leukemia, osteosarcoma, and Kaposi's sarcoma. In another embodiment, the agent comprises a peptide. In another embodiment, the agent comprises an antibody. In a preferred embodiment, the antibody is an anti-integrin α4β1 antibody. In another preferred embodiment, the antibody is an anti-vascular cell adhesion molecule antibody. In yet another preferred embodiment, the antibody is an anti-fibronectin antibody. In an alternative embodiment, the agent comprises an antisense sequence. In a preferred embodiment, the antisense sequence is an integrin α4β1 antisense sequence. In another preferred embodiment, the antisense sequence is a vascular cell adhesion molecule antisense sequence. In yet another preferred embodiment, the antisense sequence is a fibronectin antisense sequence. In another embodiment, the agent comprises a ribozyme. In a preferred embodiment, the ribozyme is an integrin α4β1 ribozyme. In an alternative embodiment, the ribozyme is a vascular cell adhesion molecule ribozyme. In a further embodiment, the ribozyme is a fibronectin ribozyme. In another embodiment, the ligand is vascular cell adhesion molecule. In an alternative embodiment, the ligand is fibronectin. In an alternative embodiment, the treatment reduces the severity of the pathological condition. In another alternative embodiment, the subject has angiogenesis in the tissue. In a further alternative embodiment, the subject is suspected of being capable of developing angiogenesis in the tissue. In another embodiment, the subject is human. In a further embodiment, the administering is selected from oral, intranasal, parenteral, and topical. In a further embodiment, the tissue is a tumor, and the administering is into the tumor. In yet another embodiment, the tissue is ocular. In a more preferred embodiment, the pathological condition is selected from diabetic retinopathy and macular degeneration by neovascularization. In an alternative preferred embodiment, the administering is selected from intravenous, oral, and topical. In another embodiment, the tissue is selected from bone tissue and synovial tissue. In a preferred embodiment, the pathological condition is selected from rheumatoid arthritis and osteoarthritis. In an alternative preferred embodiment, the administering is intrasynovial. In yet another embodiment, the tissue is skin tissue. In a j preferred embodiment, the pathological condition is selected from psoriasis and skin cancer. In an alternative preferred embodiment, the administering is selected from intravenous, oral, and topical.
The invention additionally provides methods for detecting angiogenesis in a tissue, comprising: a) providing a tissue and an agent capable of specifically binding to a molecule selected from integrin α4β1 polypeptide and integrin α4β1 mRNA; b) treating the tissue with the agent under conditions such that the agent specifically binds to the molecule to produce a treated tissue; and c) detecting the specific binding of the molecule to the agent in the treated tissue, thereby detecting angiogenesis in the tissue. In one embodiment, the method further comprises d) diagnosing a pathological condition characterized by angiogenesis in the tissue. In an alternative embodiment, the agent comprises a peptide. In another embodiment, the agent comprises an antibody. In a preferred embodiment, the antibody is an anti-integrin α4β1 antibody. In another preferred embodiment, the antibody is an anti-vascular cell adhesion molecule antibody. In yet another preferred embodiment, the antibody is an anti-fibronectin antibody. In yet another embodiment, the agent comprises an antisense sequence. In one preferred embodiment, the antisense sequence is an integrin α4β1 antisense sequence. In another preferred embodiment, the antisense sequence is a vascular cell adhesion molecule antisense sequence. In yet another preferred embodiment, the antisense sequence is a fibronectin antisense sequence. In an alternative embodiment, the agent comprises a ribozyme. In one preferred embodiment, the ribozyme is an integrin α4β1 ribozyme. In another preferred embodiment, the ribozyme is a vascular cell adhesion molecule ribozyme. In yet another preferred embodiment, the ribozyme is a fibronectin ribozyme.
Also provided herein are methods for screening test compounds, comprising: a) providing endothelial cells and a test compound; b) treating the endothelial cells with the compound to produce treated endothelial cells; and c) detecting inhibition of binding of integrin α4β1 to an integrin α4β1 ligand in the treated endothelial cells; and d) identifying the test compound as inhibiting angiogenesis. In one embodiment, the treating is in vivo. In another embodiment, the treating is in vitro. In a further embodiment, the endothelial cells are in a tissue exhibiting angiogenesis, and instead of steps c) and d), the method comprises c) detecting inhibition of angiogenesis in the tissue; and d) identifying the test compound as inhibiting angiogenesis. In yet another embodiment, the endothelial cells are in tumor tissue, and instead of steps c) and d), the method comprises c) detecting a reduction in the tumor tissue; and d) identifying the test compound as anti-tumorigenic. In a further embodiment, instead of steps c) and d), the method comprises c) detecting inhibition of endothelial cell adhesion in the treated endothelial cells; and d) identifying the test compound as inhibiting endothelial cell adhesion to integrin α4β1 ligand. In yet another embodiment, instead of steps c) and d), the method comprises c) detecting inhibition of endothelial cell migration in the treated endothelial cells; and d) identifying the test compound as inhibiting endothelial cell migration to integrin α4β1 ligand.
The invention additionally provides methods for isolating endothelial progenitor cells from a tissue, comprising: a) providing a tissue comprising endothelial progenitor cells and an antibody which specifically binds to integrin α4β1 polypeptide; b) treating the tissue with the agent under conditions such that the agent binds to the endothelial progenitor cells to produce treated endothelial progenitor cells; c) isolating the treated endothelial progenitor cells from the tissue.
Also provided by the invention are methods for inhibiting angiogenesis in a benign tumor, comprising: a) providing: a benign tumor (e.g., hemangioma, glioma, and teratoma); and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the tumor with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and a treated tumor is produced; and c) observing inhibition of angiogenesis in the treated tumor.
The invention further provides methods for inhibiting angiogenesis in a cancer tissue, comprising: a) providing cancer tissue and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the cancer tissue with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and a treated cancer tissue is produced; and c) observing inhibition of angiogenesis in the treated cancer tissue. In one embodiment, the cancer is selected from lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer; stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer, muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer, joint cancer, glioblastoma, lymphoma, leukemia, osteosarcoma, and Kaposi's sarcoma. In another embodiment, the cancer comprises a cell selected from hyperplastic cells, dysplastic cells, and neoplastic cells. In a further embodiment, the cancer is metastatic.
Also provided by the invention are methods for inhibiting angiogenesis in ocular tissue, comprising: a) providing ocular tissue and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the ocular tissue with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and a treated ocular tissue is produced; and c) observing inhibition of angiogenesis in the treated ocular tissue. In one embodiment, the ocular tissue is selected from retina, macula, cornea, choroid, and vitreous humor. In another embodiment, the ocular tissue comprises a cancer.
The invention additionally provides methods for inhibiting angiogenesis in skin tissue, comprising: a) providing skin tissue and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the skin tissue with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited and a treated skin tissue is produced; and c) observing inhibition of angiogenesis in the treated skin tissue. In one embodiment, the skin tissue comprises a cancer. In another embodiment, the skin tissue is injured. In an alternative embodiment, the skin tissue comprises a pathological condition selected from psoriasis, hemangioma, and gingivitis.
The invention further provides methods for reducing symptoms associated with cancer in a subject, comprising: a) providing a subject having cancer tissue; and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the subject with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited in the cancer tissue, and a treated cancer tissue is produced; and c) observing a reduction in one or more symptoms associated with the cancer tissue.
The invention also provides methods for reducing symptoms associated with a pathological condition in ocular tissue, comprising: a) providing: i) a subject having a pathological condition in ocular tissue; ii) an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the subject with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited in the ocular tissue, and a treated ocular tissue is produced; and c) observing a reduction in one or more symptoms associated with the pathological condition in the ocular tissue.
Further provided by the invention are methods for reducing symptoms associated with a pathological condition in skin tissue, comprising: a) providing a subject having a pathological condition in skin tissue, and an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; b) treating the subject with the agent under conditions such that specific binding of integrin α4β1 to the integrin α4β1 ligand is inhibited in the skin tissue, and a treated skin tissue is produced; and c) observing reduction in one or more symptoms associated with the pathological condition in the treated skin tissue.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows inhibition of angiogenesis using (A) antibody against fibronectin C-terminus (Anti-CT) in a chick CAM model and (B) monoclonal antibody directed against the C-terminal CS-1 or heparin binding regions of fibronectin.
FIG. 2 shows an immunohistochemical analysis of integrin α4β1 expression in normal and tumor blood vessels.
FIG. 3 shows inhibition of endothelial cell adhesion (A), and migration (B) by anti-integrin α4 antibody antagonists.
FIG. 4 shows inhibition of angiogenesis by anti-integrin α4 antibody antagonists in chick chorioallantoic membrane (CAM) treated with basic fibroblast growth factor (A), vascular endothelial growth factor (B), interleukin 8 (C), or tumor necrosis alpha (D).
FIG. 5 shows inhibition of murine angiogenesis by antibody (A) and peptide antagonists (B) of integrin α4β1.
FIG. 6 shows the polypeptide sequence (SEQ ID NO:1) of the human α4 subunit, GenBank Accession No. XP—039012.1.
FIG. 7 shows the polypeptide sequence (SEQ ID NO:2) of the human β1 subunit, GenBank Accession No. P05556.
FIG. 8 shows the polypeptide sequence of the human vascular cell adhesion molecule (VCAM), GenBank Accession Nos. P19320 (SEQ ID NO:3) (A) and XP—035774 (SEQ ID NO:96) (B).
FIG. 9 shows the polypeptide sequence (SEQ ID NO:4) of human fibronectin, GenBank Accession No. PO2751.
FIG. 10 shows exemplary agents which inhibit binding of integrin α4β1 to VCAM.
FIG. 11 shows exemplary agents which inhibit binding of integrin α4β1 to its ligands, with IC50 values based on direct binding assays. In this Figure, the abbreviations are as follows: FCA, 9-fluorenecarboxyl; IC, inhibition concentration; PA, phenylacetyl.
FIG. 12 shows exemplary β-turn mimetics which inhibit binding of integrin α4β1 to fibronectin.
FIG. 13 shows the cDNA sequence (SEQ ID NO:5) of the human integrin α4 subunit cDNA, GenBank Accession No. XM—039012.
FIG. 14 shows the cDNA sequence (SEQ ID NO:6) of the human integrin α4 subunit, GenBank Accession No. XM—039012.
FIG. 15 shows the cDNA sequence (SEQ ID NO:7) of the human integrin β1 subunit, GenBank Accession No. X07979.
FIG. 16 shows the human VCAM cDNA sequence (SEQ ID NO:8), GenBank Accession No. X53051.
FIG. 17 shows the sequence of human fibronectin cDNA (SEQ ID NO:9), GenBank Accession No. X02761.
FIG. 18 shows a graph of microvessel density versus antibody treatments (Panel A) and photographs of immunostained cryosections of excised matrigel plugs (Panel B).
FIG. 19 shows a graph of relative area of blood vessel versus antibody treatment (Panel A) and photographs of FITC labeled blood vessels in cryosections of cornea (Panel B).
FIG. 20 shows a graph of percent cells expressing integrin α4β1 versus HUVEC and EPC.
FIG. 21 shows a graph of number of beta-galactosidase positive cells per 100×field versus antibody treatments (Panel A) and photographs of immunostained cryosections of excised matrigel plugs (Panel B).
FIG. 22 shows a graph of average tumor size in cubic millimeters versus days of measurement (Panel A) and a graph of tumor mass versus treatment with anti-α4β1.



DESCRIPTION OF THE INVENTION
The invention provides methods for detecting and inhibiting angiogenesis, endothelial cell adhesion, and endothelial cell migration using agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention further provides methods for screening test compounds for their ability to inhibit angiogenesis, endothelial cell adhesion, or endothelial cell migration by employing agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention additionally relates to methods for isolating endothelial cells which express integrin α4β1. The methods of the invention are useful in, for example, diagnosing and inhibiting pathological conditions that are associated with angiogenesis, endothelial cell adhesion, and/or endothelial cell migration. The methods of the present invention are also useful in isolating endothelial progenitor cells, and in determining the mechanisms that underlie angiogenesis, development, wound healing, and the function of the female reproductive system.
The methods of the invention are based, in part, on the inventor's fortuitous discovery that integrin α4β1 plays a role in the angiogenic process, including endothelial cell migration and endothelial cell adhesion. The methods provided herein also are based on the inventors' discovery that angiogenesis, endothelial cell migration and endothelial cell adhesion are inhibited by inhibiting the specific binding of integrin α4β1 to one or more of its ligands.
The invention is further discussed below under the headings: (A) Inhibiting Angiogenesis, (B) Inhibiting Endothelial Cell Adhesion And Endothelial Cell Migration, (C) Detecting Angiogenesis, (D) Screening Compounds, and (E) Isolating Endothelial Cell Progenitors.
A. Inhibiting Angiogenesis
Angiogenesis, or neovascularization, is the process by which new blood vessels develop from pre-existing vessels [Varner et al. (1999) Angiogen. 3(1):53-60; Mousa et al. (2000) Angiogen. Stim. & Inhib. 35-42; 44. Kim et al. (2000) Amer. J. Path. 156:1345-1362; Kim et al. (2000) J. Biol. Chem. 275:33920-33928; Kumar et al. (2000) Angiogenesis: From Molecular to Integrative Pharm. 169-180]. Endothelial cells from pre-existing blood vessels or from circulating endothelial stem cells [Takahashi et al. (1995) Nat. Med. 5:434-438; Isner et al. (1999) J. Clin. Invest. 103:1231-1236] become activated to migrate, proliferate, and differentiate into structures with lumens, forming new blood vessels, in response to growth factor or hormonal cues, or hypoxic or ischemic conditions. During ischemia, such as occurs in cancer, the need to increase oxygenation and delivery of nutrients apparently induces the secretion of angiogenic factors by the affected tissue; these factors stimulate new blood vessel formation.
The invention provides methods for inhibiting angiogenesis in a tissue that involve treating the tissue with an agent which inhibits the specific binding of integrin α4β1 to one or more of its ligands. The methods of the invention are useful in, for example, determining the mechanisms which underlie desirable angiogenesis in processes such as development, wound healing, and the function of the female reproductive system. The methods of the present invention are also useful in preventing and inhibiting undesirable angiogenesis in normal angiogenesis processes in a subject, such as scar formation during wound healing or fertility. These methods also find use in inhibiting undesirable angiogenesis which occurs in ocular tissue, skin, synovial tissue, bone, or intestinal tissue, by inhibiting the binding of α4β1 binding to one or more of its ligands (e.g., fibronectin and VCAM) in the tissue. In addition, the methods of the invention are useful for reducing or inhibiting angiogenesis in a neoplasm, which can be benign or malignant and, where malignant, can be a metastatic neoplasm. Preferably, inhibiting angiogenesis results in reducing the severity of the pathological condition that is associated with angiogenesis.
Without limiting the invention to any particular mechanism, and while recognizing that an understanding of the mechanism of the invention is not required, it is contemplated that agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands block the outgrowth of new blood vessels from pre-existing vessels, and/or block the ability of circulating endothelial cells and/or progenitor endothelial cells from leaving the bloodstream and entering and migrating through tissues to sites of hypoxia or growth factor secretion where they may participate in the formation of new blood vessels.
As used herein, the term “tissue exhibiting angiogenesis” refers to a tissue in which new blood vessels are developing from pre-existing blood vessels.
As used herein, the term “inhibiting angiogenesis,” “diminishing angiogenesis,” “reducing angiogenesis,” and grammatical equivalents thereof refer to reducing the level of angiogenesis in a tissue to a quantity which is preferably 10% less than, more preferably 50% less than, yet more preferably 75% than, even more preferably 90% less than, the quantity in a corresponding control tissue, and most preferably is at the same level which is observed in a control tissue. A reduced level of angiogenesis need not, although it may, mean an absolute absence of angiogenesis. The invention does not require, and is not limited to, methods that wholly eliminate angiogenesis.
The level of angiogenesis may be determined using methods well known in the art, including, without limitation, counting the number of blood vessels and/or the number of blood vessel branch points, as discussed herein. An alternative assay involves an in vitro cell adhesion assay that shows whether a compound inhibits the ability of α4β1-expressing cells (e.g. M21 melanoma cells) to adhere to VCAM or fibronectin. Another in vitro assay contemplated includes the tubular cord formation assay that shows growth of new blood vessels at the cellular level [D. S. Grant et al., Cell, 58: 933-943 (1989)]. Art-accepted in vivo assays are also known, and involve the use of various test animals such as chickens, rats, mice, rabbits and the like. These in vivo assays include the chicken chorioallantoic membrane (CAM) assay, which is suitable for showing anti-angiogenic activity in both normal and neoplastic tissues [D. H. Ausprunk, Amer. J. Path., 79, No. 3: 597-610 (1975) and L. Ossonowski and E. Reich, Cancer Res., 30: 2300-2309 (1980)]. Other in vivo assays include the mouse metastasis assay, which shows the ability of a compound to reduce the rate of growth of transplanted tumors in certain mice, or to inhibit the formation of tumors or preneoplastic cells in mice which are predisposed to cancer or which express chemically-induced cancer [M. J. Humphries et al., Science, 233: 467-470 (1986) and M. J. Humphries et al., J. Clin. Invest., 81: 782-790 (1988)].
As discussed herein, data obtained during the development of the present invention demonstrate that angiogenesis is inhibited in vivo when using antibodies to the C-terminus of fibronectin in the chick chorioallantoic membrane (CAM) assay (Example 1), antibodies to integrin α4β1 in the CAM assay and by intravenous injection into chicks (Example 4), and antibodies and peptide antagonists to integrin α4β1 which were applied subcutaneously in a mouse model (Example 5).
While the invention is illustrated using antibodies to the C-terminus of fibronectin and to integrin α4β1, and using exemplary peptide antagonists to integrin α4β1, the invention is not limited to the use of these particular agents. Rather, the invention expressly includes any agent which inhibits the specific binding of integrin α4β1 to one or more integrin α4β1 ligands.
The term “integrin α4β1” is interchangeably used with the terms “CD49d/CD29,” “very late antigen 4,” and “VLA4” to refer to a member of the family of integrins. An “integrin” is an extracellular receptor that is expressed in a wide variety of cells and binds 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 acid-valine (Leu-Asp-Val) tripeptide, and include, for example, fibronectin, vitronectin, osteopontin, tenascin, and von Willebrands's factor. Integrin α4β1 is a heterodimeric cell surface adhesion receptor composed of an α4 and β1 subunits that bind to ligands which are present in the extracellular matrix (ECM) as well as on the cell surface. An exemplary α4 polypeptide sequence is shown in FIG. 6, and an exemplary β1 polypeptide sequence is shown in FIG. 7.
The term “integrin α4β1” is contemplated also to include a portion of α4β1. The term “portion,” when used in reference to a protein (as in a “portion of α4β1”) refers to a fragment of that protein. The fragments may range in size from three (3) contiguous amino acid residues to the entire amino acid sequence minus one amino acid residue. Thus, a polypeptide sequence comprising “at least a portion of an amino acid sequence” comprises from three (3) contiguous amino acid residues of the amino acid sequence to the entire amino acid sequence.
In one preferred embodiment, the portion of integrin α4β1 comprises a portion of the α4 polypeptide sequence. In a more preferred embodiment, the portion of the (4 polypeptide sequence shown in FIG. 6 comprises the sequence IVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFA SCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLG YSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKK (SEQ ID NO:10) (from amino acid 141 to amino acid 301). In a more preferred embodiment, the portion of integrin α4β1 comprises the sequence GHRWKN IFYIKNENKLPTGG (SEQ ID NO:11) (from amino acid 145 to amino acid 164), the sequence YQDYVKKFGENFAS (SEQ ID NO:12) (from amino acid 184 to amino acid 197), the sequence SYWTGS (SEQ ID NO:13) (from amino acid 219 to amino acid 224), the sequence GGAPQHEQIGK (SEQ ID NO:14) (from amino acid 270 to amino acid 280), and the sequence YNVDTES ALLYQGPHNT IFGYSVVLHS HGANRWLLVG APTANWLANA SVINP (SEQ ID NO:54) (from amino acid 34 to amino acid 85). In an alternative embodiment, the invention expressly includes portions of the α4 polypeptide sequence (which is exemplified by the sequence of FIG. 6) that contain the fore-mentioned portions. Such sequences include, for example,








GRPYNVDTESALLYQGPHNTLFGYSVVLHSHGAN
(SEQ ID NO: 55)


 


RWLLVGAPTANWLANASVINPGAIYR,


 


GVPTGRPYNVDTESALLYQGPHNT LFGYSVVLH
(SEQ ID NO: 56)


 


SHGANRWLLVGAPTANWLANASVINPGAIYRCRI


 


GKNPGQT,


 


IVTCGHRWKNIFYIKNENKLPTGGCYG,
(SEQ ID NO: 57)


 


GHRWKNIFYIKNENKLPTGGCYGVPPDLRTELS
(SEQ ID NO: 58)


K,


 


APCYQKYVKKFGENFAS,
(SEQ ID NO: 59)


 


CYQKYVKKFGENFASCQAGISSFYTKDL,
(SEQ ID NO: 60)


 


GSSYWTGSLFVYNI,
(SEQ ID NO: 61)


 


RSQHTTEVVGGAPQHEQIGK,
(SEQ ID NO: 62)


 


GGAPQHEQIGKAYIFSIDEKEL,
(SEQ ID NO: 63)


and/or


 


GGAPQHEQIGKA.
(SEQ ID NO: 64)






i. Integrin α4β1 Ligands
The methods of the invention employ agents which inhibit the specific binding of integrin α4β1 with one or more of its ligands. The term “ligand” as used herein in reference to a ligand for the integrin α4β1 receptor, refers to a molecule, or portion thereof, to which α4β1 specifically binds, thereby initiating a specific biological response (e.g., endothelial cell migration, endothelial cell adhesion, angiogenesis, etc.) or the transduction of a signal in a cell. Integrin α4β1 ligands may be present on the cell surface or present in the extracellular matrix (ECM).
In one preferred embodiment, an integrin α4β1 ligand that is present on the cell surface is exemplified by the vascular cell adhesion molecule (VCAM). An example of the polypeptide sequence of VCAM is shown in FIG. 8. In another preferred embodiment, the integrin α4β1 ligand is a portion of VCAM. Preferred portions of VCAM (FIG. 8A, GenBank Accession Nos. P19320) comprise the amino acid sequence RTQIDSPLNG (SEQ ID NO:15) (from amino acid 60 to amino acid 69); the amino acid sequence RTQIDSPLSG (SEQ ID NO:16) (from amino acid 348 to amino acid 357); and the amino acid sequence KLEK (SEQ ID NO:17) (from amino acid 103 to amino acid 106, and from amino acid 391 to amino acid 394). Other portions of VCAM are also contemplated, which preferably contain one of more of the RTQIDSPLNG (SEQ ID NO:15), RTQIDSPLSG (SEQ ID NO:16), or KLEK (SEQ ID NO:17) sequences. These are exemplified by, but not limited to, WRTQIDSPLNGK (SEQ ID NO:65), SWRTQIDSPLNGKV (SEQ ID NO:66), SWRTQIDSPLNGKVT (SEQ ID NO:67), PFFSWRTQIDSPLNGKVTNE (SEQ ID NO:68), SRKLEKGI (SEQ ID NO:69), CESRKLEKGIQV (SEQ ID NO:70), ATCESRKLEKGIQVEI (SEQ ID NO:71), LCTATCESRKLEKGIQVEIYSFPKDPE (SEQ ID NO:72), GHKKLEKGIQVEL (SEQ ID NO:73), VTCGHKKLEKGI (SEQ ID NO:74), TCGHKKLEKGIQVELYSFPRDPE (SEQ ID NO:75), PVSFENEHSYLCTVTCGHKKLEKG (SEQ ID NO:76), RTQIDSPLSGK (SEQ ID NO:77), FSWRTQIDSPLSGKVR (SEQ ID NO:78), and/or ESPSFWWRTQIDSPLSGK (SEQ ID NO:79).
In another preferred embodiment, an integrin α4β1 ligand that is present in the ECM is exemplified by fibronectin. An exemplary polypeptide sequence of fibronectin is shown in FIG. 9. In another preferred embodiment, the integrin α4β1 ligand is a portion of fibronectin. Preferred portions of fibronectin as exemplified in FIG. 9 include the IIICS sequence (SEPLIGRKKTDELPQLVTLPHPNLHGPE ILDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPTTA TPIRHRPRPYPPNVGEEIQIGHIPREDVDYHLYPHGPGLNPNAST) (SEQ ID NO:18) from amino acid 1982 to amino acid 2111, which encodes two α4β1 binding sites. In one more preferred embodiment, the portion comprises the CS-1 sequence which contains the amino acid sequence LDV (SEQ ID NO:19) (from amino acid 2011 to amino acid 2013). In an alternative embodiment, the portion comprises the CS-5 sequence which contains the amino acid sequence REDV (SEQ ID NO:20) (from amino acid 2091 to amino acid 2094). In yet another preferred embodiment, the portion comprises the amino acid sequence IDAPS (SEQ ID NO:21) (from amino acid 1903 to amino acid 1907). The invention further includes portions of fibronectin that contain the fore-mentioned sequences, as exemplified by, but not limited to, the sequences TAIDAPSNLRDAS (SEQ ID NO:80), TAIDAPSNLRFLATTP (SEQ ID NO:81), RSSPVVIDASTAIDAPS (SEQ ID NO:82), IDAPSNLRFLATTPNSLLV (SEQ ID NO:83), IDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYE (SEQ ID NO:84), IDDVPST (SEQ ID NO:85), NLHGPEILDVPSTVQK (SEQ ID NO:86), PHPNLHGPEILDV (SEQ ID NO:87), ILDVPSTVQKTPFVTHPGYD (SEQ ID NO:88), VTLPHPNLHGPEILDVP (SEQ ID NO:89), EILDV (SEQ ID NO:90), IPREDVDY (SEQ ID NO:91), GHIPRDDVD (SEQ ID NO:92), GHIPREDV (SEQ ID NO:93), LDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHG FRRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDV (SEQ ID NO:94), and/or PEILDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPT TTATPIRHRPRPYPPNVGEEIQIGHIPREDVDY (SEQ ID NO:95).
Integrin α4β1 ligands other than VCAM, fibronectin, and portions thereof are also contemplated to be within the scope of the invention. These ligands may be determined using routine methods available to those skilled in the art. For example, the existence of antibodies against VCAM, fibronectin, and integrin α4β1 makes possible methods for isolating other integrin α4β1 and integrin α4β1 ligands. One method takes advantage of an antibody characteristic known as idiotypy. Each antibody contains a unique region that is specific for an antigen. This region is called the idiotype. Antibodies themselves contain antigenic determinants; the idiotype of an antibody is an antigenic determinant unique to that molecule. By immunizing an organism with antibodies, one can raise “anti-antibodies” that recognize antibodies, including antibodies that recognize the idiotype. Antibodies that recognize the idiotype of another antibody are called anti-idiotypic antibodies. Some anti-idiotypic antibodies mimic the shape of the original antigen that the antibody recognizes and are said to bear the “internal image” of the antigen [Kennedy (1986) Sci. Am. 255:48-56]. For example, anti-idiotypic antibodies have been successfully generated against anti-ELAM1 antibodies and were found to recognize the ELAM1 ligand, which (similarly to integrin α4β1) is a molecule expressed on the surface of endothelial cells [U.S. Pat. No. 6,252,043, incorporated in its entirety by reference].
When the antigen is a ligand, certain anti-idiotypes can bind to that ligand's receptor. Several of these have been identified, including anti-idiotypes that bind to receptors for insulin, angiotensin II, adenosine I, adrenalin, and rat brain nicotine and opiate receptors [Carlsson and Glad (1989) Bio/Technology 7:567-73].
ii. Agents Which Inhibit Binding of Integrin α4β1 to its Ligands
Some preferred methods of the present invention include the step of utilizing an agent that inhibits the specific binding of α4β1 to one or more of its ligands. The term “specific binding,” as used herein in reference to the binding of an agent to either integrin α4β1 or an integrin α4β1 ligand, means that the interaction is dependent upon the presence of a particular structure on integrin α4β1 or its ligand, respectively. For example, if an agent is specific for epitope “A,” the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled “A” and the agent will reduce the amount of labeled A bound to the agent.
The terms “inhibit the specific binding” and “reduce the specific binding” when used in reference to the effect of an agent on the specific binding of integrin α4β1 with an integrin α4β1 ligand, mean that the agent reduces the level of specific binding of integrin α4β1 with its ligand to a quantity which is preferably 10% less than, more preferably 50% less than, yet more preferably 75% less than, even more preferably 90% less than, the quantity of specific binding in a corresponding control sample, and most preferably is at the same level which is observed in a control sample, as detected by (for example) an Enzyme Linked Immunosorbant Assay (ELISA). A reduced level of specific binding need not, although it may, mean an absolute absence of specific binding. The invention does not require, and is not limited to, methods that wholly eliminate specific binding of integrin α4β1 with its ligand.
The terms “agent” and “antagonist” are used herein to mean a molecule, (e.g., antibody) which can inhibit the specific binding of a receptor and its ligand. For example, an anti-α4β1 integrin antibody, which inhibits the specific binding of α4β1 with fibronectin, is an example of an α4β1 antagonist. An antagonist can act as a competitive inhibitor or a noncompetitive inhibitor of α4β1 binding to its ligand.
Without intending to limit the invention to any mechanism, and recognizing that an understanding of a mechanism is not required, it is contemplated that an agent can inhibit the specific binding of an integrin α4β1 receptor with its ligand by various mechanisms, including, for example, by binding to the binding site which is located on the ligand (e.g., VCAM) thereby inhibiting the binding of the integrin α4β1 receptor to its binding site on the ligand, or by binding to a site other than the binding site on the ligand and sterically hindering the binding of the integrin α4β1 receptor to the binding site on the ligand. Alternatively, the agent may bind to integrin α4β1 (rather than to the integrin α4β1 ligand) thereby causing a conformational or other change in the receptor that inhibits binding of integrin α4β1 to the ligand.
1. Antibodies
In one embodiment, the agent that inhibits the specific binding of α4β1 to one or more of its ligands is an antibody. The terms “antibody” and “immunoglobulin” are interchangeably used to refer to a glycoprotein or a portion thereof (including single chain antibodies), which is evoked in an animal by an immunogen and which demonstrates specificity to the immunogen, or, more specifically, to one or more epitopes contained in the immunogen. The term “antibody” expressly includes within its scope antigen binding fragments of such antibodies, including, for example, Fab, F(ab′)2, Fd or Fv fragments of an antibody. The antibodies of the invention also include chimeric and humanized antibodies. Antibodies may be polyclonal or monoclonal. The term “polyclonal antibody” refers to an immunoglobulin produced from more than a single clone of plasma cells; in contrast “monoclonal antibody” refers to an immunoglobulin produced from a single clone of plasma cells.
Antibodies contemplated to be within the scope of the invention include 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. Naturally occurring antibodies may be generated in any species including murine, rat, rabbit, hamster, human, and simian species using methods known in the art. 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 previously described [Huse et al., Science 246:1275-1281 (1989)]. 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); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); and Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995).
As used herein, the term “antibody” when used in reference to an anti-integrin antibody, particularly an anti-integrin α4β1 antibody, refers to an antibody which specifically binds to one or more epitopes on an integrin α4β1 polypeptide or peptide portion thereof, and which may or may not include some or all of an RGD binding domain. In one embodiment, an anti-integrin α4β1 antibody, or antigen binding fragment thereof, is characterized by having specific binding activity for integrin α4β1 of at least about 1×105M−1, more preferably at least about 1×106M−1, and yet more preferably at least about 1×107M−1.
Those skilled in the art know how to make polyclonal and monoclonal antibodies that are specific to a desirable polypeptide. For example, monoclonal antibodies may be generated by immunizing an animal (e.g., mouse, rabbit, etc.) with a desired antigen and the spleen cells from the immunized animal are immortalized, commonly by fusion with a myeloma cell.
Immunization with antigen may be accomplished in the presence or absence of an adjuvant (e.g., Freund's adjuvant). Typically, for a mouse, 10 μg antigen in 50-200 μl adjuvant or aqueous solution is administered per mouse by subcutaneous, intraperitoneal or intra-muscular routes. Booster immunization may be given at intervals (e.g., 2-8 weeks). The final boost is given approximately 2-4 days prior to fusion and is generally given in aqueous form rather than in adjuvant.
Spleen cells from the immunized animals may be prepared by teasing the spleen through a sterile sieve into culture medium at room temperature, or by gently releasing the spleen cells into medium by pressure between the frosted ends of two sterile glass microscope slides. The cells are harvested by centrifugation (400×g for 5 min.), washed and counted.
Spleen cells are fused with myeloma cells to generate hybridoma cell lines. Several mouse myeloma cell lines which have been selected for sensitivity to hypoxanthine-aminopterin-thymidine (HAT) are commercially available and may be grown in, for example, Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL) containing 10-15% fetal calf serum. Fusion of myeloma cells and spleen cells may be accomplished using polyethylene glycol (PEG) or by electrofusion using protocols that are routine in the art. Fused cells are distributed into 96-well plates followed by selection of fused cells by culture for 1-2 weeks in 0.1 ml DMEM containing 10-15% fetal calf serum and HAT. The supernatants are screened for antibody production using methods well known in the art. Hybridoma clones from wells containing cells that produce antibody are obtained (e.g., by limiting dilution). Cloned hybridoma cells (4-5×106) are implanted intraperitoneally in recipient mice, preferably of a BALB/c genetic background. Sera and ascites fluids are typically collected from mice after 10-14 days.
The invention also contemplates humanized antibodies that are specific for at least a portion of integrin α4β1 or its ligands. Humanized antibodies may be generated using methods known in the art, including those described in U.S. Pat. Nos. 5,545,806; 5,569,825 and 5,625,126, the entire contents of which are incorporated by reference. Such methods include, for example, generation of transgenic non-human animals which contain human immunoglobulin chain genes and which are capable of expressing these genes to produce a repertoire of antibodies of various isotypes encoded by the human immunoglobulin genes.
In a preferred embodiment, the antibody is specific for integrin α4β1 or a portion thereof. In a more preferred embodiment, the anti-integrin α4β1 antibody binds integrin α4β1 with at least 2 times greater, preferably at least 5 times greater, more preferably at least 10 times greater, and yet more preferably at least 100 times greater, affinity than it binds another integrin, for example, aVβ3 or aVβ5. Anti-integrin α4β1 antibodies include, without limitation, mouse anti-human integrin α4β1 antibodies such as HP2/1, HP1/3, HP 1/1, HP1/7, HP2/4 [Sanchez-Madrid et al. (1986) Eur. J. Immunol. 16, 1342-1349], ALC1/4.1, ALC1/5.1 [Munoz et al. (1997) Biochem J., 327, 27-733], 44H6 [Quackenbush et al. (1985) J. Immunol. 134: 1276-1285], P1H4, P4C2, P4G9 [Wayner et al. (1998) J. Cell Biol. 109:1321], 9C10 [Kinashi et al. (1994) Blood Cells 20: 25-44)], 9F10 [Hemler et al. (1987) J. Biol. Chem. 262: 11478], BSG10 [Hemler et al. (1987) J. Biol. Chem. 262, 3300-3309], 15/7 [Yednock et al. (1995) J. Biol. Chem. 270:28740-28750], SG/73 [Miyake et al. (1992) J. Cell Biol., 119, 653-662]. Also included within the scope of this invention are humanized anti-human integrin α4β1 antibodies, such as “ANTEGRENTM” (also known as natalizumab) [Tubridy et al. (1999) Neurology 53(3):466-72, Sheremata et al. (1999) Neurology 52: No.5, Mar. 23, 1999, and Lin et al. (1998) Current Opinion in Chemical Biology 2:453-457] and the chimeric antibodies disclosed by Newman et al., U.S. Pat. No. 5,750,105, the contents of which are incorporated by reference; rat anti-mouse integrin α4β1 antibodies such as PS/2 [Chisholm et al. (1993) European J. Immunol 23: 682-688]; mouse anti-rat α4β1 antibodies such as TA-2 [Issekutz (1991) J. Immunol 147:4178-4184]; and rat anti-mouse α4β1 antibodies such as R1-2 [Holzmann et al. (1989) Cell 56: 37-46].
In another preferred embodiment, the antibody is specific for VCAM or a portion thereof. In a more preferred embodiment, the anti-VCAM antibody inhibits the binding of VCAM to α4β1 integrin but not to other integrins. Exemplary antibodies include, for example, 4B2 and 1E10, P1B8, and P3C4 [Needham et al. (1994) Cell Adhes. Commun. 2:87-99; Dittel et al. (1993) Blood 81:2272-2282], and the chimeric antibodies disclosed by Newman et al., U.S. Pat. No. 5,750,105, the contents of which are incorporated by reference.
In yet another preferred embodiment, the antibody is specific for fibronectin or a portion thereof. In a more preferred embodiment, the anti-VCAM antibody inhibits the binding of VCAM to α4β1 integrin but not to other integrins. Such antibodies include, without restriction, antibodies against the major and minor integrin α4β1-binding sites in the C-terminal region of fibronectin, and antibodies against neighboring heparin binding sites that interfere with binding of integrin α4β1 to fibronectin. Exemplary antibodies include P1F11 and P3D4 [Garcia-Pardo et al. (1992) Biochemical and Biophysical Research Communications 186(1):135-42]; and the antibodies 20E10, 21E5, 9E9, 16E6, 19B7, 26G10, 30B6, 36C9, and 39B6 [Mostafavi-Pour et al. (2001) Matrix Biology 20(1):63-73].
2. Peptides
In an alternative embodiment, the agent which inhibits the specific binding of integrin α4β1 to one or more of its ligands is a peptide. Data provided herein show that the exemplary peptide EILDVPST (SEQ ID NO:22) inhibits integrin α4β1 binding to its ligand resulting in inhibition of angiogenesis (Example 5). The term “peptide” as used herein is used broadly to refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide bond or an analog of a peptide bond. The term peptide includes oligomers and polymers of amino acids or amino acid analogs. The term peptide also includes molecules which are commonly referred to as peptides, which generally contain from about two to about twenty amino acids. The term peptide also includes molecules which are commonly referred to as polypeptides, which generally contain from about twenty to about fifty amino acids. The term peptide also includes molecules which are commonly referred to as proteins, which generally contain from about fifty to about 3000 amino acids. The amino acids of the peptide antagonists may be L-amino acids or D-amino acids.
The terms “derivative” or “modified” when in reference to a peptide mean that the peptide contains at least one derivative amino acid. A “derivative” of an amino acid and a “modified” amino acid are chemically modified amino acids. Derivative amino acids can be “biological” or “non-biological” amino acids. Chemical derivatives of one or more amino acid members may be achieved by reaction with a functional side group. Illustrative derivatized molecules include for example those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides that contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine for lysine. Other included modifications are amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine), and similar terminal modifications. Terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the peptides in solutions, particularly in biological fluids where proteases may be present. Exemplary modified amino acids include, without limitation, 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine, beta-Aminopropionic acid, 2-Aminobutyric acid, 4-Aminobutyric acid, piperidinic acid, 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2′-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylgilycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-Isoleucine, N-Methylglycine, sarcosine, N-Methylisoleucine, N-Methylavaline, Norvaline, Norleucine, and Ornithine. Derivatives also include peptides containing one or more additions or deletions, as long as the requisite activity is maintained.
The amino acids of the peptides are contemplated to include biological amino acids as well as non-biological amino acids. The term “biological amino acid” refers to any one of the known 20 coded amino acids that a cell is capable of introducing into a polypeptide translated from an mRNA. The term “non-biological amino acid” refers to an amino acid that is not a biological amino acid. Non-biological amino acids are useful, for example, because of their stereochemistry or their chemical properties. The non-biological amino acid norleucine, for example, has a side chain similar in shape to that of methionine. However, because it lacks a side chain sulfur atom, norleucine is less susceptible to oxidation than methionine. Other examples of non-biological amino acids include aminobutyric acids, norvaline and allo-isoleucine, that contain hydrophobic side chains with different steric properties as compared to biological amino acids.
Peptides that are useful in the instant invention may be synthesized by several methods, including chemical synthesis and recombinant DNA techniques. Synthetic chemistry techniques, such as solid phase Merrifield synthesis are preferred for reasons of purity, freedom from undesired side products, ease of production, etc. A summary of the techniques available are found in several references, including Steward et. al., Solid Phase Peptide Synthesis, W. H. Freeman, Co., San Francisco (1969); Bodanszky, et. al., Peptide Synthesis, John Wiley and Sons, Second Edition (1976); J. Meienhofer, Hormonal Proteins and Peptides, 2: 46, Academic Press (1983); Merrifield, Adv. Enzymol. 32: 221-96 (1969); Fields, et. al., Intl. Peptide Protein Res., 35: 161-214 (1990), and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis; and Schroder et al., The Peptides, Vol 1, Academic Press (New York) (1965) for classical solution synthesis. Protecting groups usable in synthesis are described as well in Protective Groups in Organic Chemistry, Plenum Press, New York (1973). Solid phase synthesis methods consist of the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Either the amino or carboxyl group of the first amino acid residue is protected by a suitable selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
The resultant linear peptides may then be reacted to form their corresponding cyclic peptides. A method for cyclizing peptides is described in Zimmer et. al., Peptides, 393-394 (1992), ESCOM Science Publishers, B. V., 1993. To cyclize peptides containing two or more cysteines through the formation of disulfide bonds, the methods described by Tam et al., J. Am. Chem. Soc., 113: 6657-6662 (1991); Plaue, Int. J. Peptide Protein Res., 35: 510-517 (1990); Atherton, J. Chem. Soc. Trans. 1: 2065 (1985); and B. Kamber et. al., Helv. Chim. Acta 63: 899 (1980) are useful. Polypeptide cyclization is a useful modification to generate modified peptides (e.g., peptidomimetics) because of the stable structures formed by cyclization and in view of the biological activities observed for cyclic peptides.
Alternatively, selected compounds of the present invention are produced by expression of recombinant DNA constructs prepared in accordance with well-known methods once the peptides are known. Such production can be desirable to provide large quantities or alternative embodiments of such compounds. Production by recombinant means may be more desirable than standard solid phase peptide synthesis for peptides of at least 8 amino acid residues. The DNA encoding the desired peptide sequence is preferably prepared using commercially available nucleic acid synthesis methods. Following these nucleic acid synthesis methods, DNA is isolated in a purified form that encodes the peptides. Methods to construct expression systems for production of peptides in recombinant hosts are also generally known in the art. Preferred recombinant expression systems, when transformed into compatible hosts, are capable of expressing the DNA encoding the peptides. Other preferred methods used to produce peptides comprise culturing the recombinant host under conditions that are effective to bring about expression of the encoding DNA to produce the peptide of the invention and ultimately to recover the peptide from the culture.
Expression can be effected in either prokaryotic or eukaryotic hosts. The prokaryotes are most frequently represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacterial strains. In such prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host are used. For example, a workhorse vector for E. coli is pBR322 and its derivatives. Commonly used prokaryotic control sequences, which contain promoters for transcription initiation, optionally with an operator, along with ribosome binding-site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system, and the lambda-derived P.sub.L promoter and N-gene ribosome binding site. However, any available promoter system compatible with prokaryote expression is suitable for use.
Expression systems useful in eukaryotic hosts comprise promoters derived from appropriate eukaryotic genes. A class of promoters useful in yeast, for example, includes promoters for synthesis of glycolytic enzymes (e.g., those for 3-phosphoglycerate kinase). Other yeast promoters include those from the enolase gene or the Leu2 gene obtained from YEp13. Suitable mammalian promoters include the early and late promoters from SV40 or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers may also be used. In the event plant cells are used as an expression system, the nopaline synthesis promoter, for example, is appropriate.
Once the expression systems are constructed using well-known restriction and ligation techniques, transformation of appropriate host cells is done using standard techniques appropriate to such cells. The cells containing the expression systems are cultured under conditions appropriate for production of the peptides, and the peptides are then recovered and purified.
In a preferred embodiment, the agent that specifically binds integrin α4β1 finds use in methods of the invention where the peptide binds to integrin α4β1 with at least about a two-fold greater, more preferably at least about five-fold greater, even more preferably at least about ten-fold greater, and most preferably at least about one hundred-fold greater, specificity for integrin α4β1 than for another integrin such as αVβ3. As such, the various RGD and RLD containing peptides that have been identified based on their relatively high binding affinity for integrin αVβ63 or for integrin αVβ5 (PCT/US94/13542) are not considered peptide antagonists of integrin α4β1 binding to its ligand, as defined herein.
Exemplary peptides which inhibit the specific binding of integrin α4β1 to one or more of its ligands include, without limitation, CS-1 fibronectin and fragments of CS-1 fibronectin, such as DELPQLVTLPHPNLHGPEILDVPST (SEQ ID NO:23), HGPEILDVPST (SEQ ID NO:24), and EILDV (SEQ ID NO:25) [Wayner et al., J. Cell Biol. (1989) 109(3):1321-30]; LDVP (SEQ ID NO:26) [Clements et al., J. Cell Sci. (1994) 107 (Pt 8):2127-35], LDV (SEQ ID NO:27) [Wayner et al., J. Cell Biol. (1992) 116(2):489-97]; IDAP (SEQ ID NO:28) and RDV (SEQ ID NO:29) [Clements et al., J. Cell Sci. (1994) 107 (Pt 8):2127-35]; GPEYLDVP (SEQ ID NO:30) [Bochner et al., J. Exp. Med. (1991) 173(6):1553-7]; (X)C*DPC* (SEQ ID NO:40) where X is any amino acid or modified amino acid, (X) C*(X)PC* (SEQ ID NO:31) where X is any amino acid, RC*DPC* (SEQ ID NO:32), C*WLDVC* (SEQ ID NO:33), YC*APC* (SEQ ID NO:34) and YC*DPC* (SEQ ID NO:35), and phenyacyl-C*DfC* (SEQ ID NO:36) (where “f” is D-Phe) [Jackson et al., J. Med. Chem. (1997) 40(21):3359-68]; RC*D[ThioP]C* (SEQ ID NO:37) (Nowlin et al., J. Biol. Chem. (1993) Sep 25, 268(27):20352-9]; 9-fluorenecarboxylRC*D[ThioP]C* (SEQ ID NO:38) [Cardarelli et al., J. Biol. Chem. (1994) 269(28):18668-73]; EGYYGNYGVYA (SEQ ID NO:39) and C*YYGNC* (SEQ ID NO:97) where * indicates cyclization points; and modifications thereof [Thorsett et al., Inhibitors of leukocyte adhesion (1996) WO9602644]; 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (SEQ ID NO:41) [Cardarelli et al., J. Biol. Chem. (1994) 269(28):18668-73]. Other exemplary peptides include snake disintegrins, which are exemplified by, but not limited to, EC3 from Echis carinatus, EC3B which is a subunit of EC3 and which has the sequence NSVHPCCDPVTCEPREGEHClSGPCCRNCKFLNAGTICKRAMLDGLNDYCTGKSSD CPRNRYKGKED (SEQ ID NO:42), MLDG (SEQ ID NO:43), a peptide fragment of EC3; and modifications thereof [Brando et al., Biochem. Biophys. Res. Commun. (2000) 267(1):413-417, and Marcinkiewicz et al., J. Biol. Chem. (1999) 274(18):1 2468-73]; soluble VCAM [Rose et al. (2000) Blood 95:602-609]; soluble VCAM fragments [Dudgeon et al., Eur. J. Biochem. (1994) 226(2):517-23]; VCAM peptide sequences RTQIDSPLN (SEQ ID NO:44), TQIDSP (SEQ ID NO:45), QIDS (SEQ ID NO:46), IDSP (SEQ ID NO:47) and KLEK (SEQ ID NO:48) [Clements et al., J. Cell Sci. (1994) 107 (Pt 8): 2127-35].
Further exemplary modified peptides which inhibit the specific binding of integrin α4β1 to one or more of its ligands include a peptidomimetic (i.e., an organic molecules that mimics the structure of a peptide); or a peptoid such as a vinylogous peptoid. Examples of cyclic peptides and peptidomimetics which are within the scope of the invention include, without limitation, those which are based on the peptide structure GPEYLDVP (SEQ ID NO:49), such as the compound named TBC722 [Kogan et al., WO9600581], based on the peptide structure LDVP (SEQ ID NO:50) including phenylacetyl LDFp [Arrhenius et al., WO9515973; Arrhenius et al., WO9606108], based on the peptide structure ILDV (SEQ ID NO:51) [Dutta, WO9702289), BIO1211 [4-(2-methylphenylluriedo) phenylacetyl LDVP] BIO1272 [Lin et al., WO9200995; Lin et al., WO9622966], CY9652 a CS-1 peptidomimetic, TBC3342, ZD-7349 [Curley et al. (1999) Cell. Mol. Life Sci., 56:427-441]; and others [EP-842943-A2, WO9842656-A1, WO9620216-A1, WO9600581-A1, Souers et al. (1998) Bioorg. Med. Chem. Lett., 8:2297-2302]. Exemplary peptides and modified peptides are illustrated in FIG. 10 [see, Lin et al. (1999) J. Med. Chem., 42:920-934], FIG. 11 [See, Lin et al. (1998) Curr. Opin. Chem. Biol., 2:453-457], and FIG. 12 [See, Souers et al. (1998) Bioorg. Med. Chem. Lett., 8:2297-2302]. Methods for generating libraries of mimetics and for evaluating the library of mimetics for inhibiting the binding of receptors to their ligands are known in the art [Souers et al. (1998) supra].
Other peptides useful as α4β1 antagonists that reduce or inhibit angiogenesis can be purchased from commercial sources, and can be identified by screening libraries of peptides, which can be prepared using well known methods of chemical synthesis [Koivunen et al. J. Cell Biol., 124: 373-380 (1994)]. For example, peptide agonists of integrin α4β1 other than those specifically disclosed herein may be identified using methods known in the art, such as by panning phage-display peptide libraries as described in U.S. Pat. No. 5,780,426 to Palladino et al., the entire contents of which are herein incorporated by reference. For example, phage-display peptide libraries are panned with the integrin α4β1 receptor attached to a solid support, such as small diameter (1 μm) polystyrene latex beads. Phage selected by this method can then be tested for specific binding to integrin α4β1 via ELISA or other immunologically-based assays. Individual peptide sequences are then determined via sequencing of phage DNA. Further analysis of the minimal peptide sequence required for binding can be assessed via deletion and site-directed mutagenesis, followed by testing of the phage for binding to integrin α4β1 via ELISA. Since the identified peptide candidates are fused to the major phage coat protein, soluble peptides are then chemically synthesized and the activity of these free peptides are tested in various in vitro and in vivo assays for the ability to act as antagonists of the integrin α4β1 receptor.
3. Nucleic Acid Sequences
In an alternative embodiment, the agent that inhibits the specific binding of α4β1 to one or more of its ligands is a nucleic acid sequence. The terms “nucleic acid sequence” and “nucleotide sequence” as used herein refer to two or more nucleotides that are covalently linked to each other. Included within this definition are oligonucleotides, polynucleotide, and fragments or portions thereof, DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Nucleic acid sequences that are particularly useful in the instant invention include, without limitation, antisense sequences and ribozymes. The nucleic acid sequences are contemplated to bind to genomic DNA sequences or RNA sequences that encode integrin α4β1 or one or more of its ligands, thereby inhibiting the binding of integrin α4β1 with one or more of its ligands. Antisense and ribozyme sequences may be delivered to cells by transfecting the cell with a vector that expresses the antisense nucleic acid or the ribozyme as an mRNA molecule. Alternatively, delivery may be accomplished by entrapping ribozymes and antisense sequences in liposomes.
a. Antisense Sequences
Antisense sequences have been successfully used to inhibit the expression of several genes [Markus-Sekura (1988) Anal. Biochem. 172:289-295; Hambor et al. (1988) J. Exp. Med. 168:1237-1245; and patent EP 140 308], including the gene encoding VCAM1, one of the integrin α4β1 ligands [U.S. Pat. No. 6,252,043, incorporated in its entirety by reference]. The terms “antisense DNA sequence” and “antisense sequence” as used herein interchangeably refer to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5′ to 3′ orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex. A “sense strand” of a DNA duplex refers to a strand in a DNA duplex that is transcribed by a cell in its natural state into a “sense mRNA.” Sense mRNA generally is ultimately translated into a polypeptide. Thus, an “antisense DNA sequence” is a sequence which has the same sequence as the non-coding strand in a DNA duplex, and which encodes an “antisense RNA” (i.e., a ribonucleotide sequence whose sequence is complementary to a “sense mRNA” sequence). The designation (−) (i.e., “negative”) is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., “positive”) strand. Antisense RNA may be produced by any method, including synthesis by splicing an antisense DNA sequence to a promoter that permits the synthesis of antisense RNA. The transcribed antisense RNA strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation, or promote its degradation.
Any antisense sequence is contemplated to be within the scope of this invention if it is capable of reducing the level of expression of integrin α4β1 and/or one or more of its ligands (e.g., VCAM and fibronectin) to a quantity which is less than the quantity of integrin α4β1 or integrin α4β1 ligand expression in a corresponding control tissue which is (a) not treated with the antisense integrin α4β1 or integrin α4β1 ligand sequence, (b) treated with a corresponding sense integrin α4β1 or integrin α4β1 ligand sequence, or (c) treated with a nonsense sequence.
The terms “reducing the level of expression of integrin α4β1 or integrin α4β1 ligand,” “diminishing integrin α4β1 or integrin α4β1 ligand expression” and grammatical equivalents thereof, refer to reducing the level of integrin α4β1 or integrin α4β1 ligand expression to a quantity which is preferably 20% less than the quantity in a corresponding control tissue, more preferably is 50% less than the quantity in a corresponding control tissue, yet more preferably is 90% less than the quantity in a corresponding control tissue, and most preferably is at the background level of, or is undetectable by, a Western blot analysis of integrin α4β1 or integrin α4β1 ligand, by immunofluorescence for detection of integrin α4β1 or integrin α4β1 ligand, by reverse transcription polymerase chain (RT-PCR) reaction for detection of integrin α4β1 or integrin α4β1 ligand mRNA, or by in situ hybridization for detection of integrin α4β1 or integrin α4β1 ligand mRNA. When a background level or undetectable level of integrin α4β1 or integrin α4β1 ligand peptide or mRNA is measured, this may indicate that integrin α4β1 or integrin α4β1 ligand is not expressed. A reduced level of integrin α4β1 or integrin α4β1 ligand need not, although it may, mean an absolute absence of expression of integrin α4β1 or integrin α4β1 ligand. The invention does not require, and is not limited to, antisense integrin α4β1 or integrin α4β1 ligand sequences that eliminate expression of integrin α4β1 or integrin α4β1 ligand.
Antisense integrin α4β1 or integrin α4β1 ligand sequences capable of reducing the level of integrin α4β1 expression include, for example, sequences which are capable of hybridizing with at least a portion of integrin α4β1 cDNA or integrin α4β1 ligand cDNA under high stringency or low stringency conditions. Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4—H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5× Denhardt's reagent [50× Denhardt's contains the following per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.2×SSPE, and 0.1% SDS at room temperature when a DNA probe of about 100 to about 1000 nucleotides in length is employed. High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE, and 0.1% SDS at 68° C. when a probe of about 100 to about 1000 nucleotides in length is employed.
The term “equivalent” when made in reference to a hybridization condition as it relates to a hybridization condition of interest means that the hybridization condition and the hybridization condition of interest result in hybridization of nucleic acid sequences which have the same range of percent (%) homology. For example, if a hybridization condition of interest results in hybridization of a first nucleic acid sequence with other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization condition of interest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence.
Antisense integrin α4β1 sequences and antisense integrin α4β1 ligand sequences within the scope of this invention may be designed using approaches known in the art. In a preferred embodiment, the antisense integrin α4β1 sequences and antisense integrin α4β1 ligand sequences are designed to be hybridizable to integrin α4β1 mRNA or to integrin α4β1 ligand mRNA which is encoded by the coding region of the integrin α4β1 gene and the integrin α4β1 ligand gene, respectively. Alternatively, antisense integrin α4β1 or integrin α4β1 ligand sequences may be designed to reduce transcription by hybridizing to upstream nontranslated sequences, thereby preventing promoter binding to transcription factors.
In a preferred embodiment, the antisense oligonucleotide sequences of the invention range in size from about 8 to about 100 nucleotide residues. In yet a more preferred embodiment, the oligonucleotide sequences range in size from about 8 to about 30 nucleotide residues. In a most preferred embodiment, the antisense sequences have 20 nucleotide residues.
However, the invention is not intended to be limited to the number of nucleotide residues in the oligonucleotide sequence disclosed herein. Any oligonucleotide sequence that is capable of reducing expression of integrin α4β1 or of integrin α4β1 ligand is contemplated to be within the scope of this invention. For example, oligonucleotide sequences may range in size from about 3 nucleotide residues to the entire integrin α4β1 or integrin α4β1 ligand cDNA sequence. The art skilled know that the degree of sequence uniqueness decreases with decreasing length, thereby reducing the specificity of the oligonucleotide for the integrin α4β1 mRNA, or integrin α4β1 ligand mRNA.
The antisense oligonucleotide sequences that are useful in the methods of the instant invention may comprise naturally occurring nucleotide residues as well as nucleotide analogs. Nucleotide analogs may include, for example, nucleotide residues that contain altered sugar moieties, altered inter-sugar linkages (e.g., substitution of the phosphodiester bonds of the oligonucleotide with sulfur-containing bonds, phosphorothioate bonds, alkyl phosphorothioate bonds, N-alkyl phosphoramidates, phosphorodithioates, alkyl phosphonates and short chain alkyl or cycloalkyl structures), or altered base units. Oligonucleotide analogs are desirable, for example, to increase the stability of the antisense oligonucleotide compositions under biologic conditions since natural phosphodiester bonds are not resistant to nuclease hydrolysis. Oligonucleotide analogs may also be desirable to improve incorporation efficiency of the oligonucleotides into liposomes, to enhance the ability of the compositions to penetrate into the cells where the nucleic acid sequence whose activity is to be modulated is located, in order to reduce the amount of antisense oligonucleotide needed for a therapeutic effect thereby also reducing the cost and possible side effects of treatment.
Antisense oligonucleotide sequences may be synthesized using any of a number of methods known in the art, as well as using commercially available services (e.g., Genta, Inc.). Synthesis of antisense oligonucleotides may be performed, for example, using a solid support and commercially available DNA synthesizers. Alternatively, antisense oligonucleotides may also be synthesized using standard phosphoramidate chemistry techniques. For example, it is known in the art that for the generation of phosphodiester linkages, the oxidation is mediated via iodine, while for the synthesis of phosphorothioates, the oxidation is mediated with 3H-1,2-benzodithiole-3-one,1,-dioxide in acetonitrile for the step-wise thioation of the phosphite linkages. The thioation step is followed by a capping step, cleavage from the solid support, and purification on HPLC, e.g., on a PRP-1 column and gradient of acetonitrile in triethylammonium acetate, pH 7.0.
In one embodiment, the antisense DNA sequence is an “integrin α4α1 antisense DNA sequence” (i.e., an antisense DNA sequence which is designed to bind with at least a portion of the integrin α4β1 genomic sequence or with integrin α4β1 mRNA). The design of integrin α4β1 antisense DNA sequences is facilitated by the availability of the sequences for the integrin α4 subunit cDNA (FIG. 13 and FIG. 14), and integrin G31 cDNA (FIG. 15). Particularly preferred antisense sequences are those which hybridize with genomic DNA or with RNA encoding a portion of integrin α4β1 which is involved in the specific binding with one or more of its ligands. Such integrin α4β1 portions are exemplified by, but not limited to, the sequences (see FIG. 6) which comprises the sequence IVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFA ASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYL GYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKK (SEQ ID NO:10) (from amino acid 141 to amino acid 301), GHRWKN IFYIKNENKLPTGG (SEQ ID NO:11) (from amino acid 145 to amino acid 164), YQDYVKKFGENFAS (SEQ ID NO:12) (from amino acid 184 to amino acid 197), SYWTGS (SEQ ID NO:13) (from amino acid 186 to amino acid 224), GGAPQHEQIGK (SEQ ID NO:14) (from amino acid 270 to amino acid 280), YNVDTES ALLYQGPHNT IFGYSVVLHS HGANRWLLVG APTANWLANA SVINP (SEQ ID NO:54) (from amino acid 34 to amino acid 85), GRPYNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVG APTANWLANASVINPGAIYR (SEQ ID NO:55), GVPTGRPYNVDTESAL LYQGPHNT LFGYSVVLHSHGANRWLLVGAPTANWLANASVI NPGAIYRCRIGKNPGQT (SEQ ID NO:56), IVTCGHRWKNIFYIKNENKLPTGGCYG (SEQ ID NO:57), GHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSK (SEQ ID NO:58), APCYQDYVKKFGENFAS (SEQ ID NO:59), CYQDYVKKFGENFASCQA GISSFYTKDL (SEQ ID NO:60), GSSYWTGSLFVYNI (SEQ ID NO:61), RSQHTTEVVGGAPQHEQIGK (SEQ ID NO:62), GGAPQHEQIGKAYIFSIDEKEL (SEQ ID NO:63), and/or GGAPQHEQIGKA (SEQ ID NO:64).
In another embodiment, the antisense DNA sequence is a “vascular cell adhesion molecule antisense DNA sequence,” i.e., and antisense DNA sequence which is designed to bind with at least a portion of the VCAM genomic sequence or with VCAM α4β1 mRNA. The selection and design of these antisense sequences is made possible by the availability of VCAM cDNA sequences (FIG. 16). Exemplary preferred antisense sequences are those which hybridize with genomic DNA or with RNA encoding a portion of VCAM which is involved in the specific binding of VCAM with integrin α4β1, such as the VCAM (FIG. 8A, GenBank Accession Nos. P19320) comprise the amino acid sequence RTQIDSPLNG (SEQ ID NO:15) (from amino acid 60 to amino acid 69); RTQIDSPLSG (SEQ ID NO:16) (from amino acid 348 to amino acid 357), KLEK (SEQ ID NO:17) (from amino acid 103 to amino acid 106, and from amino acid 391 to amino acid 394), RTQIDSPLNG (SEQ ID NO:15), RTQIDSPLSG (SEQ ID NO:16), KLEK (SEQ ID NO:17), WRTQIDSPLNGK (SEQ ID NO:65), SWRTQIDSPLNGKV (SEQ ID NO:66), SWRTQIDSPLNGKVT (SEQ ID NO:67), PFFSWRTQIDSPLNGKVTNE (SEQ ID NO:68), SRKLEKGI (SEQ ID NO:69), CESRKLEKGIQV (SEQ ID NO:70), ATCESRKLEKGIQVEI (SEQ ID NO:71), LCTATCESRKLEKGIQVEIYSFPKDPE (SEQ ID NO:72), GHKKLEKGIQVEL (SEQ ID NO:73), VTCGHKKLEKGI (SEQ ID NO:74), TCGHKKLEKGIQVELYSFPRDPE (SEQ ID NO:75), PVSFENEHSYLCTVTCGHKKLEKG (SEQ ID NO:76), RTQIDSPLSGK (SEQ ID NO:77), FSWRTQIDSPLSGKVR (SEQ ID NO:78), and/or ESPSFWWRTQIDSPLSGK (SEQ ID NO:79).
In yet another embodiment, the antisense DNA sequence is a “fibronectin α4β1 antisense DNA sequence” (i.e., an antisense DNA sequence which is designed to bind with at least a portion of the fibronectin genomic sequence or with fibronectin α4β1 mRNA). The selection and design of these antisense sequences is made possible by the availability of the sequence for fibronectin cDNA (FIG. 17). Exemplary nucleic acid sequences which may be targeted are those which encode the following sequences shown in FIG. 9, the IIICS sequence (SEPLIGRKKTDELPQLVTLPHPNLHGPE ILDVPSTVQKTPFVTHPGYDTGNGIQLPGGTSGQQPSVGQQMIFEEHGFRRTTPPTT ATPIRHRPRPYPPNVGEEIQIGHIPREDVVDYHLYPHGPGLNPNAST) (SEQ ID NO:18) from amino acid 1982 to amino acid 2111, the CS-1 sequence which contains the amino acid sequence LDV (SEQ ID NO:19) (from amino acid 2011 to amino acid 2013), the CS-5 sequence which contains the amino acid sequence REDV (SEQ ID NO:20) (from amino acid 2091 to amino acid 2093), IDAPS (SEQ ID NO:21) (from amino acid 1903 to amino acid 1907), TAIDAPSNLRDAS (SEQ ID NO:80), TAIDAPSNLRFLATTP (SEQ ID NO:81), RSSPVVIDASTAIDAPS (SEQ ID NO:82), IDAPSNLRFLATTPNSLLV (SEQ ID NO:83), IDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYE (SEQ ID NO:84), IDDVPST (SEQ ID NO:85), NLHGPEILDVPSTVQK (SEQ ID NO:86), PHPNLHGPEILDV (SEQ ID NO:87), ILDVPSTVQKTPFVTHPGYD (SEQ ID NO:88), VTLPHPNLHGPEILDVP (SEQ ID NO:89), EILDV (SEQ ID NO:90), IPREDVDY (SEQ ID NO:91), GHIPRDDVD (SEQ ID NO:92), GHIPREDV (SEQ ID NO:93), LDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHG FRRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDV (SEQ ID NO:94), and/or PEILDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPT TTATPIRHRPRPYPPNVGEEIQIGHIPREDVDY (SEQ ID NO:95).
b. Ribozyme
In some alternative embodiments, the agent that inhibits the specific binding of integrin α4β1 to its ligand is a ribozyme. Ribozyme sequences have been successfully used to inhibit the expression of several genes including the gene encoding VCAM1, which is one of the integrin α4β1 ligands [U.S. Pat. No. 6,252,043, incorporated in its entirety by reference].
The term “ribozyme” refers to an RNA sequence that hybridizes to a complementary sequence in a substrate RNA and cleaves the substrate RNA in a sequence specific manner at a substrate cleavage site. Typically, a ribozyme contains a “catalytic region” flanked by two “binding regions.” The ribozyme binding regions hybridize to the substrate RNA, while the catalytic region cleaves the substrate RNA at a “substrate cleavage site” to yield a “cleaved RNA product.” The nucleotide sequence of the ribozyme binding regions may be completely complementary or partially complementary to the substrate RNA sequence with which the ribozyme binding regions hybridize. Complete complementarity is preferred, in order to increase the specificity, as well as the turnover rate (i.e., the rate of release of the ribozyme from the cleaved RNA product), of the ribozyme. Partial complementarity, while less preferred, may be used to design a ribozyme binding region containing more than about 10 nucleotides. While contemplated to be within the scope of the claimed invention, partial complementarity is generally less preferred than complete complementarity since a binding region having partial complementarity to a substrate RNA exhibits reduced specificity and turnover rate of the ribozyme when compared to the specificity and turnover rate of a ribozyme which contains a binding region having complete complementarity to the substrate RNA. A ribozyme may hybridize to a partially or completely complemetary DNA sequence but cannot cleave the hybridized DNA sequence since ribozyme cleavage requires a 2′-OH on the target molecule, which is not available on DNA sequences.
The ability of a ribozyme to cleave at a substrate cleavage site may readily be determined using methods known in the art. These methods include, but are not limited to, the detection (e.g., by Northern blot analysis as described herein, reverse-transcription polymerase chain reaction (RT-PCR), in situ hybridization and the like) of reduced in vitro or in vivo levels of RNA which contains a ribozyme substrate cleavage site for which the ribozyme is specific, compared to the level of RNA in controls (e.g., in the absence of ribozyme, or in the presence of a ribozyme sequence which contains a mutation in one or both unpaired nucleotide sequences which renders the ribozyme incapable of cleaving a substrate RNA).
Ribozymes contemplated to be within the scope of this invention include, but are not restricted to, hammerhead ribozymes [See e.g., Reddy et al., U.S. Pat. No. 5,246,921; Taira et al., U.S. Pat. No. 5,500,357, Goldberg et al., U.S. Pat. No. 5,225,347, the contents of each of which are herein incorporated by reference], Group I intron ribozyme [Kruger et al. (1982) Cell 31: 147-157], ribonuclease P [Guerrier-Takada et al. (1983) Cell 35: 849-857], hairpin ribozyme [Hampel et al., U.S. Pat. No. 5,527,895 incorporated by reference], and hepatitis delta virus ribozyme [Wu et al. (1989) Science 243:652-655].
A ribozyme may be designed to cleave at a substrate cleavage site in any substrate RNA so long as the substrate RNA contains one or more substrate cleavage sequences, and the sequences flanking the substrate cleavage site are known. In effect, expression in vivo of such ribozymes and the resulting cleavage of RNA transcripts of a gene of interest reduces or ablates expression of the corresponding gene.
For example, where the ribozyme is a hammerhead ribozyme, the basic principle of a hammerhead ribozyme design involves selection of a region in the substrate RNA which contains a substrate cleavage sequence, creation of two stretches of antisense oligonucleotides (i.e., the binding regions) which hybridize to sequences flanking the substrate cleavage sequence, and placing a sequence which forms a hammerhead catalytic region between the two binding regions.
In order to select a region in the substrate RNA which contains candidate substrate cleavage sites, the sequence of the substrate RNA needs to be determined. The sequence of RNA encoded by a genomic sequence of interest is readily determined using methods known in the art. For example, the sequence of an RNA transcript may be arrived at either manually, or using available computer programs (e.g., GENEWORKS,.from IntelliGenetic Inc., or RNADRAW available from the internet), by changing the T in the DNA sequence encoding the RNA transcript to a U.
Substrate cleavage sequences in the target RNA may be located by searching the RNA sequence using available computer programs. For example, where the ribozyme is a hammerhead ribozyme, it is known in the art that the catalytic region of the hammerhead ribozyme cleaves only at a substrate cleavage site which contains a NUH, where N is any nucleotide, U is a uridine, and H is a cytosine (C), uridine (U), or adenine (A) but not a guanine (G). The U-H doublet in the NUH cleavage site does not include a U-G doublet since a G would pair with the adjacent C in the ribozyme and prevent ribozymne cleavage. Typically, N is a G and H is a C. Consequently, GUC has been found to be the most efficient substrate cleavage site for hammerhead ribozymes, although ribozyme cleavage at CUC is also efficient.
In a preferred embodiment, the substrate cleavage sequence is located in a loop structure or in an unpaired region of the substrate RNA. Computer programs for the prediction of RNA secondary structure formation are known in the art and include, for example, “RNADRAW”, “RNAFOLD”, “DNASIS” (Hitachi), and The Vienna RNA Package.
In addition to the desirability of selecting substrate cleavage sequences which are located in a loop structure or an unpaired region of the substrate RNA, it is also desirable, though not required, that the substrate cleavage sequence be located downstream (i.e., at the 3′-end) of the translation start codon (AUG or GUG) such that the translated truncated polypeptide is not biologically functional.
In a preferred embodiment, the ribozyme is an “integrin α4β1 ribozyme” (i.e., a ribozyme whose substrate cleavage sequence is designed to hybridize with a portion of integrin α4β1 that is involved in the specific binding of integrin α4β1 with one or more of its ligands). Such integrin α4β1 portions are exemplified by, but not limited to, the sequences (see FIG. 6) which comprises the sequence IVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFA ASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYL GYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKK (SEQ ID NO:10) (from amino acid 141 to amino acid 301), GHRWKN IFYIKNENKLPTGG (SEQ ID NO:11) (from amino acid 145 to amino acid 164), YQDYVKKFGENFAS (SEQ ID NO:12) (from amino acid 184 to amino acid 197), SYWTGS (SEQ ID NO:13) (from amino acid 186 to amino acid 224), GGAPQHEQIGK (SEQ ID NO:14) (from amino acid 270 to amino acid 280), YNVDTES ALLYQGPHNT IFGYSVVLHS HGANRWLLVG APTANWLANA SVINP (SEQ ID NO:54) (from amino acid 34 to amino acid 85), GRPYNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVG APTANWLANASVINPGAIYR (SEQ ID NO:55), GVPTGRPYNVDTESAL LYQGPHNT LFGYSVVLHSHGANRWLLVGAPTANWLANASVI NPGAIYRCRIGKNPGQT (SEQ ID NO:56), IVTCGHRWKNIFYIKNENKLPTGGCYG (SEQ ID NO:57), GHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSK (SEQ ID NO:58), APCYQDYVKKFGENFAS (SEQ ID NO:59), CYQDYVKKFGENFASCQA GISSFYTKDL (SEQ ID NO:60), GSSYWTGSLFVYNI (SEQ ID NO:61), RSQHTTEVVGGAPQHEQIGK (SEQ ID NO:62), GGAPQHEQIGKAYIFSIDEKEL (SEQ ID NO:63), and/or GGAPQHEQIGKA (SEQ ID NO:64).
In an alternative embodiment, the substrate cleavage sequence is designed to hybridize with a portion of an integrin α4β1 ligand, wherein the portion is involved in the specific binding of the ligand with integrin α4β1.
In a more preferred embodiment, the ribozyme is a “vascular cell adhesion molecule ribozyme” (i.e., a ribozyme whose substrate cleavage sequence is designed to hybridize with a portion of VCAM that is involved in the specific binding of VCAM with integrin α4β1). Exemplary portions of the ligand VCAM (FIG. 8A, GenBank Accession Nos. P19320) comprise the amino acid sequence RTQIDSPLNG (SEQ ID NO:15) (from amino acid 60 to amino acid 69); RTQIDSPLSG (SEQ ID NO:16) (from amino acid 348 to amino acid 357), KLEK (SEQ ID NO:17) (from amino acid 103 to amino acid 106, and from amino acid 391 to amino acid 394), RTQIDSPLNG (SEQ ID NO:15), RTQIDSPLSG (SEQ ID NO:16), KLEK (SEQ ID NO:17), WRTQIDSPLNGK (SEQ ID NO:65), SWRTQIDSPLNGKV (SEQ ID NO:66), SWRTQIDSPLNGKVT (SEQ ID NO:67), PFFSWRTQIDSPLNGKVTNE (SEQ ID NO:68), SRKLEKGI (SEQ ID NO:69), CESRKLEKGIQV (SEQ ID NO:70), ATCESRKLEKGIQVEI (SEQ ID NO:71), LCTATCESRKLEKGIQVEIYSFPKDPE (SEQ ID NO:72), GHKKLEKGIQVEL (SEQ ID NO:73), VTCGHKKLEKGI (SEQ ID NO:74), TCGHKKLEKGIQVELYSFPRDPE (SEQ ID NO:75), PVSFENEHSYLCTVTCGHKKLEKG (SEQ ID NO:76), RTQIDSPLSGK (SEQ ID NO:77), FSWRTQIDSPLSGKVR (SEQ ID NO:78), and/or ESPSFWWRTQIDSPLSGK (SEQ ID NO:79).
In an alternative preferred embodiment, the ribozyme is a “fibronectin ribozyme” (i.e., a ribozyme whose substrate cleavage sequence is designed to hybridize with a portion of fibronectin that is involved in the specific binding of fibronectin with integrin α4β1). Exemplary portions of the ligand fibronectin comprise the following sequences shown in FIG. 9, the IIICS sequence (SEPLIGRKKTDELPQLVTLP HPNLHGPEILDVPSTVQKTPFVTHPGYDTGNGIQLPGGTSGQQPSVGQQMIFEEHGF RRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDVVDYHLYPHGPGLNPN AST) (SEQ ID NO:18) from amino acid 1982 to amino acid 2111, the CS-1 sequence which contains the amino acid sequence LDV (SEQ ID NO:19) (from amino acid 2011 to amino acid 2013), the CS-5 sequence which contains the amino acid sequence REDV (SEQ ID NO:20) (from amino acid 2091 to amino acid 2093), IDAPS (SEQ ID NO:21) (from amino acid 1903 to amino acid 1907), TAIDAPSNLRDAS (SEQ ID NO:80), TAIDAPSNLRFLATTP (SEQ ID NO:81), RSSPVVIDASTAIDAPS (SEQ ID NO:82), IDAPSNLRFLATTPNSLLV (SEQ ID NO:83), IDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYE (SEQ ID NO:84), IDDVPST (SEQ ID NO:85), NLHGPEILDVPSTVQK (SEQ ID NO:86), PHPNLHGPEILDV (SEQ ID NO:87), ILDVPSTVQKTPFVTHPGYD (SEQ ID NO:88), VTLPHPNLHGPEILDVP (SEQ ID NO:89), EILDV (SEQ ID NO:90), IPREDVDY (SEQ ID NO:91), GHIPRDDVD (SEQ ID NO:92), GHIPREDV (SEQ ID NO:93), LDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHG FRRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDV (SEQ ID NO:94), and/or PEILDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPT TTATPIRHRPRPYPPNVGEEIQIGHIPREDVDY (SEQ ID NO:95).
It is known in the art that the specificity of ribozyme cleavage for a substrate RNA molecule is determined by the sequence of nucleotides which flank the substrate cleavage site and which hybridize with the ribozyme binding regions. Thus, ribozymes can be designed to cleave at different locations within a substrate RNA molecule by altering the sequence of the binding regions that surround the ribozyme catalytic region of the ribozyme such that the binding regions hybridize with any known sequence on the substrate RNA.
In addition to varying the sequence of the binding regions to effect binding to different locations on the RNA substrate, the number of nucleotides in each of the ribozyme binding regions may also be altered in order to change the specificity of the ribozyme for a given location on the RNA substrate. The number of nucleotides in a binding region is preferably between about 5 and about 25 nucleotides, more preferably between about 11 and about 15 nucleotides, yet more preferably between about 7 nucleotides and about 10 nucleotides.
One of skill in the art appreciates that it is not necessary that the two binding regions that flank the ribozyme catalytic region be of equal length. Binding regions that contain any number of nucleotides are contemplated to be within the scope of this invention so long as the desirable specificity of the ribozyme for the RNA substrate and the desirable cleavage rate of the RNA substrate are achieved. One of skill in the art knows that binding regions of longer nucleotide sequence, while increasing the specificity for a particular substrate RNA sequence, may reduce the ability of the ribozyme to dissociate from the substrate RNA following cleavage to bind with another substrate RNA molecule, thus reducing the rate of cleavage. On the other hand, though binding regions with shorter nucleotide sequences may have a higher rate of dissociation and cleavage, specificity for a substrate cleavage site may be compromised.
It is well within the skill of the art to determine an optimal length for the binding regions of a ribozyme such that a desirable specificity and rate of cleavage are achieved. Both the specificity of a ribozyme for a substrate RNA and the rate of cleavage of a substrate RNA by a ribozyme may be determined by, for example, kinetic studies in combination with Northern blot analysis or nuclease protection assays.
In a preferred embodiment, the complementarity between the ribozyme binding regions and the substrate RNA is complete. However, the invention is not limited to ribozyme sequences in which the binding regions show complete complementarity with the substrate RNA. Complementarity may be partial, so long as the desired specificity of the ribozyme for a substrate cleavage site and the rate of cleavage of the substrate RNA are achieved. Thus, base changes may be made in one or both of the ribozyme binding regions as long as substantial base pairing with the substrate RNA in the regions flanking the substrate cleavage sequence is maintained and base pairing with the substrate cleavage sequence is minimized. The term “substantial base pairing” means that greater than about 65%, more preferably greater than about 75%, and yet more preferably greater than about 90% of the bases of the hybridized sequences are base-paired.
It may be desirable to increase the intracellular stability of ribozymes expressed by an expression vector. This is achieved by designing the expressed ribozyme such that it contains a secondary structure (e.g., stem-loop structures) within the ribozyme molecule. Secondary structures which are suitable for stabilizing ribozymes include, but are not limited to, stem-loop structures formed by intra-strand base pairs. An alternative to the use of a stem-loop structure to protect ribozymes against ribonuclease degradation is by the insertion of a stem loop at each end of the ribozyme sequence [Sioud and Drlica (1991) Proc. Natl. Acad. Sci. USA 88:7303-7307]. Other secondary structures which are useful in reducing the susceptibility of a ribozyme to ribonuclease degradation include hairpin, bulge loop, interior loop, multibranched loop, and pseudoknot structure as described in “Molecular and Cellular Biology,” Stephen L. Wolfe (Ed.), Wadsworth Publishing Company (1993) p. 575. Additionally, circularization of the ribozyme molecule protects against ribonuclease degradation since exonuclease degradation is initiated at either the 5′-end or 3′-end of the RNA. Methods of expressing a circularized RNA are known in the art [see, e.g., Puttaraju et al. (1993) Nucl. Acids Res. 21:4253-4258].
Once a ribozyme with desirable binding regions, a catalytic region and nuclease stability has been designed, the ribozyme may be produced by any known means including chemical synthesis. Chemically synthesized ribozymes may be introduced into a cell by, for example, microinjection electroporation, lipofection, etc. In a preferred embodiment, ribozymes are produced by expression from an expression vector that contains a gene encoding the designed ribozyme sequence.
4. Other Agents
While the present invention is illustrated herein using antibody, peptide, and nucleic acid sequences which inhibit the specific binding of integrin α4β1 to one or more of its ligands, the invention expressly contemplates within its scope other agents (e.g., organic molecules, inorganic molecules, etc.) so long as the agent is capable of inhibiting the specific binding of integrin α4β1 to one or more of its ligands. Such agents may be identified by screening libraries of test compounds using a competitive binding assay or a cell adhesion assay. In a competitive binding assay, for example, integrin α4β1 is coated on plastic microtiter plates and contacted with a labeled known integrin α4β1 ligand (e.g., CS-1 fibronectin or VCAM). The test compounds are tested for their ability to inhibit binding of the labeled ligand to integrin α4β1. Compounds that inhibit such binding are identified as agents that are capable of inhibiting the specific binding of integrin α4β1 to the ligand.
Alternatively, in a cell adhesion assay, a labeled known integrin α4β1 ligand (e.g., CS-1 fibronectin or VCAM) is coated on culture plates, and cells which express integrin α4β1 are allowed to adhere to the ligand for 20-30 minutes in the presence of libraries of test compounds. Compounds that inhibit the binding of the integrin α4β1-expressing cells to the coating of integrin α4β1 ligand are identified as agents that inhibit the specific binding of integrin α4β31 to the ligand.
iii. Inhibiting Angiogenesis in a Subject
The invention provides methods for inhibiting undesirable angiogenesis in a subject by inhibiting the binding of α4β1 to one or more of its ligands (e.g., fibronectin and VCAM) in the tissue in the subject.
Undesirable angiogenesis includes normal angiogenesis processes (e.g., scar formation during wound healing or during fertility), and angiogenesis, which is associated with a pathological condition, such as that which occurs in ocular tissue (e.g., retina, macular or cornea), in skin such as occurs with psoriasis, in synovial tissue, in bone, in intestinal tissue, or in a tumor. Preferably, inhibiting angiogenesis results in reducing the severity of the undesirable angiogenesis and/or pathological condition that is associated with angiogenesis.
An advantage of the methods of the present invention is that inhibition of angiogenesis in a subject is inherently less toxic using the methods of the present invention, as compared with currently available treatments. This is because the methods of the present invention specifically target those cells that are involved in angiogenesis in the disease site, thus sparing healthy tissues from deleterious side effects. In addition, inhibition of angiogenesis offers a therapeutic approach to the treatment of diseases such as rheumatoid arthritis, neovascular eye disease, and psoriasis, for which no effective therapy exits.
The terms “pathological condition” and “angiogenic disease” are used broadly herein to mean any abnormal physical or physiological condition characterized, at least in part, by angiogenesis associated with α4β1 integrin expression on newly forming blood vessels in a tissue. Such pathological conditions are exemplified by, but not limited to, neoplasms, ocular diseases such as diabetic retinopathy and macular degeneration associated with neovascularization, skin diseases such as psoriasis and hemangiomas, gingivitis, arthritic conditions such as rheumatoid arthritis and osteoarthritis, and inflammatory bowel diseases.
The terms “neoplasm” and “tumor” refer to a tissue growth that is characterized, in part, by angiogenesis. Neoplasms may be benign and are exemplified, but not limited to, a hemangioma, glioma, teratoma, and the like. Neoplasms may alternatively be malignant, for example, a carcinoma, sarcoma, glioblastoma, astrocytoma, neuroblastoma, retinoblastoma, and the like.
The terms “malignant neoplasm” and “malignant tumor” refer to a neoplasm that contains at least one cancer cell. A “cancer cell” refers to a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression as previously described [H. C. Pitot (1978) in “Fundamentals of Oncology,” Marcel Dekker (Ed.), New York pp 15-28]. The features of early, intermediate and advanced stages of neoplastic progression have been described using microscopy. Cancer cells at each of the three stages of neoplastic progression generally have abnormal karyotypes, including translocations, inversion, deletions, isochromosomes, monosomies, and extra chromosomes. A cell in the early stages of malignant progression is referred to as “hyperplastic cell” and is characterized by dividing without control and/or at a greater rate than a normal cell of the same cell type in the same tissue. Proliferation may be slow or rapid, but continues unabated. A cell in the intermediate stages of neoplastic progression is referred to as a “dysplastic cell.” A dysplastic cell resembles an immature epithelial cell, is generally spatially disorganized within the tissue and loses its specialized structures and functions. During the intermediate stages of neoplastic progression, an increasing percentage of the epithelium becomes composed of dysplastic cells. “Hyperplastic” and “dysplastic” cells are referred to as “pre-neoplastic” cells. In the advanced stages of neoplastic progression a dysplastic cell become a “neoplastic” cell. Neoplastic cells are typically invasive (i.e., they either invade adjacent tissues, or are shed from the primary site and circulate through the blood and lymph) to other locations in the body where they initiate one or more secondary cancers (i.e., “metastases”). Thus, the term “cancer” is used herein to refer to a malignant neoplasm, which may or may not be metastatic. Malignant neoplasms that can be diagnosed using a method of the invention include, for example, carcinomas such as lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer; stomach cancer, esophageal cancer, mouth cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of the choroids, cancer of the macula, vitreous humor cancer, etc.), joint cancer (e.g., synovium cancer), glioblastoma, lymphoma, and leukemia. Malignant neoplasms are further exemplified by sarcomas (such as osteosarcoma and Kaposi's sarcoma). The invention expressly contemplates within its scope any malignant neoplasm, so long as the neoplasm is characterized, at least in part, by angiogenesis associated with α4β1 expression by the newly forming blood vessels.
The terms “reducing the severity of a pathological condition,” “diminishing the severity of a pathological condition, and “reducing symptoms associated with a pathological condition” mean that adverse clinical signs or symptoms associated with the pathological condition are reduced, delayed, or eliminated, as compared to the level of the pathological condition in the absence of treatment with the particular composition or method. The effects of diminishing the severity of a pathological condition may be determined by methods routine to those skilled in the art including, but not limited to, angiography, ultrasonic evaluation, fluoroscopic imaging, fiber optic endoscopic examination, biopsy and histology, blood tests, which can be used to determine relevant enzyme levels or circulating antigen or antibody, imaging tests which can be used to detect a decrease in the growth rate or size of a neoplasm, or an ophthalmic procedure which can be used to identify a reduction in the number of blood vessels in the retina of a diabetic patient. Such clinical tests are selected based on the particular pathological condition being treated. For example, it is contemplated that the methods of the invention result in a “reduction in tumor tissue” (e.g., a decrease in the size, weight, and/or volume of the tumor tissue) as compared to a control tumor tissue (e.g., the same tumor prior to treatment with the invention's methods, or a different tumor in a control subject). A reduction in the severity of a pathological condition also can be detected based on comments made by the patient being treated, for example, that a patient suffering from arthritis feels less pain or has greater joint mobility, or that a patient with diabetic retinopathy or with macular degeneration due to neovascularization can see more clearly, or the like.
Pathological conditions that are amenable to prevention and/or treatment with the invention's methods include any pathological condition whose development or progression in a tissue involves angiogenesis. The invention's methods offer a therapeutic approach to the management of these exemplary diseases and other diseases which are associated with angiogenesis that is controlled by integrin α4β1.
Exemplary pathological conditions include, for example, solid tumor cancers, solid tumor metastases, angiofibromas, skin cancer, retrolental fibroplasia, Kaposi's sarcoma, childhood hemangiomas, diabetic retinopathy, neovascular glaucoma, age related macular degeneration, psoriasis, gingivitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and atheroscrelotic plaques.
Other pathological conditions include those that entail injury to tissue. The term “injured” in reference to a tissue refers to tissue in which the cellular organization of the tissue has been altered as compared to the cellular organization in normal tissue. Such injury may result, for example, from a breaking of the skin tissue (e.g., a cut, slash, laceration) such as accidental cuts or cuts associated with planned surgery, burns, etc. Injured tissues include those any tissue which undergoes angiogenesis as a result of injury, such as lung, breast, prostate, cervical, pancreatic, colon, ovarian, stomach, esophagus cancer, mouth cancer, tongue cancer, gum, muscle, etc. In particular, skin injury that is associated with undesirable angiogenesis and the formation of scar tissue is particularly amenable to the invention's therapeutic approaches.
Disclosed herein are methods for inhibiting angiogenesis, tumors and metastases in a mouse model (Example 11), a nude mouse model (Examples 6 and 10), a SCID mouse model (Example 7), and a chick CAM model (Example 9). Also disclosed herein are methods for inhibiting angiogenesis in arthritis disease using mouse and rabbit models (Example 13), and in ocular disease using mouse and rabbit models (Example 14).
An agent useful for detecting or inhibiting angiogenesis associated with α4β1 integrin expression, or a pharmaceutical composition thereof containing the agent, can be used for treating any pathological condition that is characterized, at least in part, by such angiogenesis. One skilled in the art would know that the agent can be administered by various routes including, for example, orally, intranasally, or parenterally, including intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intrasynovially, intraperitoneally, intracistemally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis. Furthermore, the agent can be administered by injection, intubation, via a suppository, orally or topically, the latter of which can be passive, for example, by direct application of an ointment or powder containing the agent, or active, for example, using a nasal spray or inhalant. The agent can also be administered as a topical spray, if desired, in which case one component of the composition is an appropriate propellant. The pharmaceutical composition also can be incorporated, if desired, into liposomes, microspheres or other polymer matrices [Gregoriadis, “Liposome Technology,” Vol. 1, CRC Press, Boca Raton, Fla. 1984]. Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Liposomes are lipid-containing vesicles having a lipid bilayer as well as other lipid carrier particles that can entrap chemical agents. Liposomes may be made of one or more phospholipids, optionally including other materials such as sterols. Suitable phospholipids include phosphatidyl cholines, phosphatidyl serines, and many others that are well known in the art. Liposomes can be unilamellar, multilamellar or have an undefined lamellar structure.
For example, where angiogenesis associated with integrin α4β1 expression is localized to the retina, the agent can be formulated in a pharmaceutical composition convenient for use as eye drops, which can be administered directly to the eye. In comparison, in an individual suffering from a metastatic carcinoma, the agent in a pharmaceutical composition can be administered intravenously, orally or by another method that distributes the agent systemically.
Agents that inhibit the specific binding of integrin α4β1 to one or more of its ligands may be administered in conjunction with other therapies. For example, in the case of cancer therapy, the agent may be administered in conjunction with conventional drug therapy and/or chemotherapy that is directed against solid tumors and for control of establishment of metastases. In one embodiment, the agent is administered during or after chemotherapy. In a more preferred embodiment, the agent is administered after chemotherapy, at a time when the tumor tissue will be responding to the toxic assault. The tumor will attempt to induce angiogenesis to recover by the provision of a blood supply and nutrients to the tumor tissue. Such recovery will be thwarted by the administration of agents which inhibit angiogenesis by inhibiting the specific binding of integrin α4β1 to one or more of its ligands. In an alternative embodiment, the agent may be administered after surgery in which solid tumors have been removed as a prophylaxis against future metastases.
An agent is administered in a “therapeutic amount” (i.e., in an amount which is sufficient to achieve a desired result). In particular, a therapeutic amount is that amount which inhibits the specific binding of α4β1 integrin to its specific ligand in a tissue of a subject, and which results in the reduction, delay, or elimination of undesirable pathologic effects in the subject. One of ordinary skill recognizes that a “therapeutically effective” amount varies depending on the therapeutic agent used, the subject's age, condition, and sex, and on the extent of the disease in the subject. Generally, the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. The dosage can also be adjusted by the individual physician or veterinarian to achieve the desired therapeutic goal.
A therapeutic amount may be determined using in vitro and in vivo assays known in the art and disclosed herein. These are exemplified, without limitation, to administering different amounts of the agent to determine inhibition of angiogenesis using the CAM assay (Examples 4 and 8), inhibition of angiogenesis by intravenous administration of the agent into chicks (Example 4), inhibition of angiogenesis in mice or rabbits (Examples 5, 13 and 14), inhibition of tumor growth in nude mice (Example 6) or in SCID mice (Example 7), inhibition of tumor and metastasis growth using the CAM assay (Examples 8 and 9), in nude mouse (Example 10), or in mice which spontaneously develop tumors (Example 11). Generally, an agent antagonist is administered in a dose of about 0.0001 to 100 mg/kg body weight.
The “subject” to whom the agents are administered includes any animal which is capable of developing angiogenesis in a tissue, including, without limitation, human and non-human animals such simians, rodents, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. Preferred non-human animals are members of the Order Rodentia (e.g., mouse and rat). Thus, the compounds of the invention may be administered by human health professionals as well as veterinarians.
The agents which inhibit angiogenesis may be applied to any tissue which is capable of developing angiogenesis including, for example, skin, ocular tissue (e.g., retina, macular or cornea), skin, synovial tissue, bone, intestinal tissue, muscle tissue, gut tissue, connective tissue, blood vessels, tumor tissue (including benign and cancer tissue), etc.
B. Inhibiting Endothelial Cell Adhesion and Endothelial Cell Migration
The invention further provides methods for inhibiting endothelial cell adhesion and/or for inhibiting endothelial cell migration by employing an agent that inhibits the specific binding of integrin α4β1 to its ligand. Data provided herein demonstrates that endothelial cell adhesion to, and migration across, CS-1 fibronectin was inhibited using anti-integrin α4β1 antibody (Example 3).
Endothelial cell adhesion and migration are known to regulate endothelial cell survival, proliferation, and motility during new blood vessel growth in normal and pathologic conditions that involve angiogenesis. Thus, the invention's methods for inhibiting endothelial cell adhesion and/or for inhibiting endothelial cell migration are useful in determining the mechanisms that underlie desirable and undesirable angiogenesis, and in inhibiting undesirable angiogenesis in normal angiogenesis processes or in pathologic conditions that are associated with angiogenesis.
The term “endothelial cell adhesion” as used herein refers to the adhesion of an endothelial cell to one or more components of the extracellular matrix (e.g., fibronectin, collagens I-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrands's factor, etc.), to a ligand which is expressed on the cell surface (e.g., VCAM, ICAM, LI-CAM, VE-cadherin, integrin a2, integrin a3, etc.) and/or to another cell (e.g., another endothelial cell, to a fibroblast cell, stromal cell, tumor cell, etc.) The terms “inhibiting endothelial cell adhesion” and “reducing endothelial cell adhesion” refer to reducing the level of adhesion of an endothelial cell to one or more components of the extracellular matrix (e.g., fibronectin, collagens I-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrands's factor, etc.), and/or to another cell (e.g., another endothelial cell, fibroblast cell, stromal cell, tumor cell, etc.) to a quantity which is preferably 10% less than, more preferably 50% less than, yet more preferably 75% than, even more preferably 90% less than, the quantity in a corresponding control endothelial cell, and most preferably is at the same level which is observed in a control endothelial cell. A reduced level of endothelial cell adhesion need not, although it may, mean an absolute absence of cell adhesion. The invention does not require, and is not limited to, methods that wholly eliminate cell adhesion. The level of endothelial cells adhesion may be determined using methods well known in the art, including those disclosed herein (Example 3).
The term “endothelial cell migration” as used herein refers to the translocation of an endothelial cell across one or more components of the extracellular matrix (e.g., fibronectin, collagens I-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrands's factor, etc.), or along the surface of another cell (e.g., another endothelial cell, fibroblast cell, stromal cell, tumor cell, etc.).
The terms “inhibiting endothelial cell migration” and “reducing endothelial cell migration” refer to reducing the level of migration of an endothelial cell to a quantity which is preferably 10% less than, more preferably 50% less than, yet more preferably 75% less than, and even more preferably 90% less than, the quantity in a corresponding control endothelial cells, and most preferably is at the same level which is observed in a control endothelial cell. A reduced level of endothelial cell migration need not, although it may, mean an absolute absence of cell migration. The invention does not require, and is not limited to, methods that wholly eliminate cell migration. The level of endothelial cells migration may be determined using methods well known in the art, such as time lapse video microscopy, scratch type wound assay, and methods disclosed herein (Example 3).
C. Detecting Angiogenesis
The invention additionally provides methods for detecting angiogenesis which involve the use of an agent which specifically binds to integrin α4β1 polypeptides or to integrin α4β1 mRNA. Such a method is useful for identifying the presence of angiogenesis in various tissues, including, for example, normal tissues such as embryonic tissue or placental tissue, granulation tissue, and a tissue involved in a pathological condition. As such, the invention further provides methods of diagnosing a pathological condition characterized by angiogenesis associated with integrin α4β1 expression in a tissue in an individual.
Data provided herein demonstrate using immunohistochemical analysis that human melanoma cells are characterized by expression of integrin α4β1 (Example 2). However, the invention is not restricted to the detection of melanoma cells. Rather, any pathological condition, which is characterized by angiogenesis associated with integrin α4β1 expression, can be identified by the invention's methods. For example, angiognesis in a tissue may be detected by detecting the presence of integrin α4β1 polypeptide using Western blot analysis or immunofluorescence. Alternatively, angiognesis in a tissue may be detected by detecting the presence of integrin α4β1 mRNA using reverse transcription polymerase chain (RT-PCR), or in situ hybridization. These methods are well known and well within the ordinary skill of those in the art.
In one embodiment, the agent which is used in detecting the presence of integrin α4β1 polypeptide and/or mRNA can be detectably labeled, for example, by linking the agent to a moiety, which is selected based, for example, on whether specific binding of the agent is to be detected in vivo or whether a tissue to which the agent is suspected of binding is to be removed (e.g., by biopsy) and examined ex vivo.
A moiety useful for labeling an agent antagonist can be a radionuclide, a paramagnetic material, an X-ray attenuating material, a fluorescent, chemiluminescent or luminescent molecule, a molecule such as biotin, or a molecule that can be visualized upon reaction with a particular reagent, for example, a substrate for an enzyme or an epitope for an antibody. The moiety can be linked to an agent using well known methods, which are selected, in part, based on the chemical nature of the agent and the moiety. For example, where the moiety is an amino acid sequence such as a hexahistidine (His6) sequence, and the agent is a peptide, the His6 sequence can be synthesized as part of the peptide, and the His6-labeled agent can be identified by the binding of a nickel ion reagent to the His6 moiety.
Methods for chemically linking a moiety to an agent also can be utilized. For example, methods for conjugating polysaccharides to peptides are exemplified by, but not limited to coupling via alpha- or epsilon-amino groups to NaIO4-activated oligosaccharide, using squaric acid diester (1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent, coupling via a peptide linker wherein the polysaccharide has a reducing terminal and is free of carboxyl groups (U.S. Pat. No. 5,342,770), coupling with a synthetic peptide carrier derived from human heat shock protein hsp65 (U.S. Pat. No. 5,736,146), and using the methods of U.S. Pat. No. 4,639,512. Methods for conjugating proteins to proteins include coupling with a synthetic peptide carrier derived from human heat shock protein hsp65 (U.S. Pat. No. 5,736,146), the methods used to conjugate peptides to antibodies (U.S. Pat. Nos. 5,194,254; 4,950,480), the methods used to conjugate peptides to insulin fragments (U.S. Pat. No. 5,442,043), the methods of U.S. Pat. No. 4,639,512, and the method of conjugating the cyclic decapeptide polymyxin B antibiotic to and IgG carrier using EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]-mediated amide formation [Drabick et al. (1998) Antimicrob. Agents Chemother. 42:583-588]. Approaches to conjugate nucleic acids to proteins are also known in the art, such as those described in U.S. Pat. Nos. 5,574,142; 6,117,631; 6,110,687; each of is incorporated in its entirety by reference. Methods for conjugating lipids to peptides have been described in the art including, but not limited to, the use of reductive amination and an ether linkage which contains a secondary or tertiary amine (U.S. Pat. No. 6,071,532), the methods of U.S. Pat. No. 4,639,512, the methods used for covalently coupling peptides to unilamellar liposomes [Friede et al. (1994) Vaccine 12:791-797], of coupling human serum albumin to liposomes using the hetero-bifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA) [Kamps et al. (1996) Biochim. Biophys. Acta 1278:183-190], of coupling antibody Fab′ fragments to liposomes using a phospholipid-poly(ethylene glycol)-maleimide anchor [Shahinian et al. (1995) Biochim. Biophys. Acta 1239:157-167], and of coupling Plasmodium CTL epitope to palmitic acid via cysteine-serine spacer amino acids [Verheul et al. (1995) J. Immunol. Methods 182:219-226].
A specifically bound agent can be detected in an individual using an in vivo imaging method, such as a radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, or can be detected using an ex vivo method, wherein, following administration, a sample of the tissue is obtained from the individual, and specific binding of the agent in the sample is detected (e.g., by immunohistochemical analysis; Example 3).
An agent that is specifically bound to α4β1 integrin in a sample can be detected directly by detecting the agent, or indirectly by detecting the presence of a moiety such as by detecting radioactivity emitted by a radionuclide moiety. Specifically bound agent also can be detected indirectly by further contacting it with a reagent that specifically interacts with the agent, or with a moiety linked to the agent, and detecting interaction of the reagent with the agent or label. For example, the moiety can be detected by contacting it with an antibody that specifically binds the moiety, particularly when the moiety is linked to the agent. The moiety also can be, for example, a substrate, which is contacted by an enzyme that interacts with and changes the moiety such that its presence can be detected. Such indirect detection systems, which include the use of enzymes such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase and the like, are well known in the art and commercially available, as are the methods for incorporating or, linking the particular moiety to a particular type of agent.
D. Screening Compounds
The invention further provides methods for identifying compounds which are capable of inhibiting angiogenesis, inhibiting endothelial cells adhesion, and/or inhibiting endothelial cell migration.
A screening assay of the invention can be performed by contacting a tissue exhibiting angiogenesis associated with integrin α4β1 expression with a test compound, and detecting inhibition of angiogenesis in the tissue, thereby identifying the compound as inhibiting angiogenesis associated with α4β1 integrin expression. A tissue can be contacted with the agent in vivo or ex vivo (see, for example, U.S. Pat. No. 5,622,699, incorporated by reference). Where a screening method of the invention is performed using an in vitro format, it can be adapted to automated procedure, thus allowing high throughput screening assays for examining libraries of molecules to identify potential α4β1 antagonists, which can reduce or inhibit angiogenesis associated with α4β1 expression. The tissue can be any tissue that undergoes angiogenesis associated with α4β1 integrin expression, for example, malignant tumor tissue.
Alternatively, a screening assays is carried out by contacting endothelial cells with a test compound, and detecting inhibition of endothelial cell adhesion and/or of endothelial cell migration, thereby identifying the compound a inhibiting endothelial cell adhesion and/or endothelial cell migration.
Methods for preparing libraries of molecules, which can be screened using a method of the invention to identify α4β1 antagonists that inhibit angiogenesis, endothelial cells adhesion and/or endothelial cell migration processes which are associated with α4β1 expression, are known in the art. These are exemplified by methods for preparing oligonucleotide libraries (Gold et al., U.S. Pat. No. 5,270,163, incorporated by reference); peptide libraries (Koivunen et al., supra, 1993, 1994); peptidomimetic libraries (Blondelle et al., Trends Anal. Chem. 14:83-92 (1995)) oligosaccharide libraries (York et al., Carb. Res. 285:99-128 (1996); Liang et al., Science 274:1520-1522 (1996); and Ding et al., Adv. Expt. Med. Biol. 376:261-269 (1995)); lipoprotein libraries (de Kruif et al., FEBS Lett., 399:232-236 (1996)); glycoprotein or glycolipid libraries (Karaoglu et al., J. Cell Biol. 130:567-577 (1995)); or chemical libraries containing, for example, drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem. 37:1385-1401 (1994); Ecker and Crook, Bio/Technology 13:351-360 (1995), U.S. Pat. No. 5,760,029, incorporated by reference). Libraries of diverse molecules also can be obtained from commercial sources.
E. Isolating Endothelial Cell Progenitors
The invention further provides a method for isolating endothelial cell progenitors from a tissue by treating a tissue which contains endothelial cell progenitors with an agent capable of binding to integrin α4β1 and isolating endothelial cell progenitors to which the agent binds. These methods are based, in part, on the inventor's discovery that endothelial cells which are involved in angiogenesis express integrin α4β1.
The term “endothelial cell progenitor” as used herein refers to an endothelial cell which expresses cell surface markers that are characteristic of these cells as understood in the art. For example, human endothelial cell progenitors express the surface molecules CD34, flk-1, and/or tie-2 [Isner et al., U.S. Pat. No. 5,980,887, the entire contents of which are herein incorporated by reference]. Mouse endothelial cell progenitors express the TM gene, tie-2 gene, and/or fgf3 gene, and/or stain with the GSL I B4 lectin [Hatzopoulos et al. (1998) Development 125:1457-1468].
Endothelial cell progenitors that are isolated in accordance with the invention's methods are useful in regulating angiogenesis [Isner et al., U.S. Pat. No. 5,980,887, incorporated by reference]. Heterologous, homologous, and autologous endothelial cell progenitor grafts incorporate in vivo into sites of active angiogenesis or blood vessel injury (i.e., they selectively migrate to such locations [Isner et al., U.S. Pat. No. 5,980,887]).
Thus, in one embodiment, the endothelial cell progenitors can be used to enhance angiogenesis or to deliver an angiogenesis modulator (e.g., anti- or pro-angiogenic agents, respectively), to sites of pathologic or utilitarian angiogenesis. Additionally, in another embodiment, endothelial cell progenitors can be used to induce re-endothelialization of an injured blood vessel, and thus reduce restenosis by indirectly inhibiting smooth muscle cell proliferation [Isner et al., U.S. Pat. No. 5,980,887].
In one preferred embodiment, the endothelial cells can be used alone to potentiate a patient for angiogenesis. Some patient populations, typically elderly patients, may have either a limited number of endothelial cells or a limited number of functional endothelial cells. Thus, if one desires to promote angiogenesis, for example, to stimulate vascularization by using a potent angiogenesis such as VEGF, such vascularization can be limited by the lack of endothelial cells. However, by administering the endothelial cell progenitors one can potentiate the vascularization in those patients.
Because endothelial cell progenitors home to foci of angiogenesis, these cells are also useful as autologous vectors for gene therapy and diagnosis of ischemia or vascular injury. For example, these cells can be utilized to inhibit as well as augment angiogenesis. For anti-neoplastic therapies, for example, endothelial cell progenitors can be transfected with or coupled to cytotoxic agents, cytokines or co-stimulatory molecules to stimulate an immune reaction, other anti-tumor drugs, or angiogenesis inhibitors. For treatment of regional ischemia, angiogenesis could be amplified by prior transfection of endothelial cell progenitors to achieve constitutive expression of angiogenic cytokines and/or selected matrix proteins. In addition, the endothelial cell progenitors may be labeled (e.g., radiolabeled), administered to a patient and used in the detection of ischemic tissue or vascular injury.
Autologous endothelial cell progenitor transplants have been successfully used, and endothelial cell progenitors have been shown to be easily manipulated and expanded ex vivo [Isner et al., U.S. Pat. No. 5,980,887; U.S. Pat. No. 5,199,942, the disclosure of which is incorporated by reference].
Endothelial cell progenitors are present in a number of tissues including, for example, peripheral blood, bone marrow, and umbilical cord blood. Endothelial cell progenitors may be isolated in accordance with the invention's methods by treating a tissue (e.g., peripheral blood, bone marrow, umbilical cord blood, etc.) which contains endothelial cell progenitors with an antibody which is capable of specific binding to at least a portion of integrin α4β1 polypeptide, followed by isolating cells which bind to the antibody. The endothelial cell progenitor nature of the isolated cells may be confirmed by determining the presence of endothelial cell progenitor-specific antigens (e.g., CD34, flk-1, and/or tie-2) on the surface of the isolated cells using commercially available antibodies to these antigens. It may be desirable, but not necessary, to expand endothelial cell progenitors in vivo prior to treating the tissue that contains endothelial cell progenitors by administration of recruitment growth factors (e.g., GM-CSF and IL-3) to the patient.
Once isolated in accordance with the invention's methods, these compositions may be administered to a patient to treat a number of conditions including, for example, unregulated angiogenesis or blood vessel injury. The cells may also be stored in cryogenic conditions. Optionally, the cells may be expanded ex vivo using, for example, the method disclosed by U.S. Pat. No. 5,541,103, the disclosure of which is incorporated by reference.
The endothelial cell progenitors are administered to the patient by any suitable means, including, for example, intravenous infusion, bolus injection, and site directed delivery via a catheter. Preferably, the progenitor cells obtained from the patient are readministered. Generally, from about 106 to about 1018 progenitor cells are administered to the patient for transplantation.
In one embodiment, various genetic material may be delivered to the endothelial cell progenitors. The genetic material that is delivered to the endothelial cell progenitors may be genes, for example, those that encode a variety of proteins including anticancer agents. Such genes include those encoding various hormones, growth factors, enzymes, cytokines, receptors, MHC molecules and the like. The term “genes” includes nucleic acid sequences both exogenous and endogenous to cells into which a virus vector, for example, a pox virus such as swine pox containing the human TNF gene may be introduced. Additionally, it is of interest to use genes encoding polypeptides for secretion from the endothelial cell progenitors so as to provide for a systemic effect by the protein encoded by the gene. Specific genes of interest include those encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12 etc., GM-CSF, G-CSF, M-CSF, human growth factor, co-stimulatory factor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, EPO, β-globin, endothelial cell mitogens and the like, as well as biologically active modifications of these proteins. The gene may further encode a product that regulates expression of another gene product or blocks one or more steps in a biological pathway. In addition, the gene may encode a toxin fused to a polypeptide (e.g., a receptor ligand), or an antibody that directs the toxin to a target, such as a tumor cell. Similarly, the gene may encode a therapeutic protein fused to a targeting polypeptide, to deliver a therapeutic effect to a diseased tissue or organ.
In another embodiment, the endothelial cell progenitors can also be used to deliver genes to enhance the ability of the immune system to fight a particular disease or tumor. For example, the cells can be used to deliver one or more cytokines (e.g., IL-2) to boost the immune system and/or one or more antigens.
In yet another embodiment, the endothelial cell progenitors may also be used to selectively administer drugs, such as an antiangiogenesis compound such as O-chloroacetyl carbamoyl fumagillol (TNP-470). Preferably, the drug would be incorporated into the cell in a vehicle such as a liposome, a timed released capsule, etc. The endothelial cell progenitor would then selectively target a site of active angiogenesis such as a rapidly growing tumor where the compound would be released. By this method, one can reduce undesired side effects at other locations.
In a further embodiment, the endothelial cell progenitors may be used to enhance blood vessel formation in ischemic tissue (i.e., a tissue having a deficiency in blood as the result of an ischemic disease). Such tissues can include, for example, muscle, brain, kidney and lung. Ischemic diseases include, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia. Methods for inducing the formation of new blood vessels in ischemic tissue are disclosed in Isner et al., U.S. Pat. No. 5,980,887, herein incorporated by reference.
EXPERIMENTAL
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml (milliliters); A1 (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec. (seconds); % (percent); FITC (fluorescein isothiocyanate); H & E (haematoxylin and eosin); and (Ig) immunoglobulin.
Example 1
Inhibition of Angiogenesis by Anti-Fibronectin Antibody
Angiogenesis was stimulated on the surface of the chick chorioallantoic membrane (“CAM”) by applying a 5 mm filter disk on the CAM saturated with basic fibroblast growth factor (“bFGF”), vascular endothelial growth factor (“VEGF”), interleukin 8 (“IL-8”), tumor necrosis factor alpha (“TNF-α”) or saline. Twenty four hours after angiogenesis had been stimulated saline, 25 μg control IgG, 25 μg of an antibody directed against the cell binding peptide of fibronectin (Anti-CPB), 25 μg of an antibody directed against the fibronectin C-terminus (Anti-CT), 25 lag of an antibody directed against the cell binding peptide of fibronectin (Anti-CBP) or 25 μg of an antibody directed against vitronectin (Anti-VN) were applied to the CAM of eight to ten eggs each in a volume of 25 μl. Forty eight hours after this, CAMs were excised from the egg and the number of blood vessels in each filter disk were counted. The results are shown in FIG. 1A. These results demonstrate that antibody directed against the fibronectin C-terminus (Anti-CT) inhibited angiogenesis.
In another experiment, twenty four hours after angiogenesis had been stimulated, saline 25 μg control IgG, of 25 μg of monoclonal antibodies directed against the C-terminal CS-1 or heparin binding regions of fibronectin were applied. Forty eight hours after this, CAMs were excised from the egg and the number of blood vessels in each filter disk were counted. Each experiment was performed at least three times. The results are shown in FIG. 1B. These results demonstrate that antibodies directed against the C-terminal CS-1 or heparin binding regions of fibronectin inhibited angiogenesis.
These initial studies of the role of fibronectin in angiogenesis revealed that the C-terminus of fibronectin was critical for angiogenesis. Antibody inhibitors of the C-terminus of fibronectin showed that this region of fibronectin plays a role in angiogenesis. As this region of fibronectin interacts with integrin α4β1, the inventor hypothesized that this integrin regulated angiogenesis (FIG. 1A). In addition, antibodies that bind to secondary integrin α4β1 binding sites in the C-terminus of fibronectin and antibodies that bind to nearby heparin binding sites in the C-terminus of fibronectin also blocked angiogenesis (FIG. 1B), confirming the inventor's hypothesis.
Example 2
Immunohistochemical Analysis of Integrin α4β1 Expression in Normal and Tumor Tissue
Five micron frozen sections of human normal thymus (FIG. 2A) and melanoma (FIG. 2B) were fixed for 2 minutes in acetone, air dried and rehydrated for 5 minutes in phosphate buffered saline. Sections were then blocked for 2 hours in a 2% bovine serum albumin in phosphate buffered saline and incubated with 5 μg/ml anti-α4β1 monoclonal antibody and anti-von Willebrand Factor (a marker of endothelial cells used by pathologists to identify blood vessels) polyclonal antibody for one hour room temperature. Sections were washed in PBS and incubated in 1:400 dilution of goat anti-rabbit-FITC and in goat anti-mouse-rhodamine for 1 hour at room temperature. Slides were well washed, and coverslips were mounted in one drop of Fluoromount, prior to photography under fluorescent illumination. Blood vessels positive vWF (von Willebrand Factor) only are green, cells positive for integrin α4β1 only are red and blood vessels positive for both are yellow.
The results in FIG. 2 demonstrate that integrin α4β1 expression is minimal on blood vessels present in normal human tissues (FIG. 2A) or in unstimulated CAM tissues (results not shown). Integrin α4β1 is expressed on the lymphocytes in lymph nodes, as is well accepted. Integrin α4β1 expression is dramatically upregulated on blood vessels in tumor tissues such as in malignant melanoma (FIG. 2B) and in CAM tissue in response to angiogenic growth factors such as basic fibroblast growth factor (results not shown).
Example 3
Inhibition of Human Neonatal Cell Adhesion to, and Migration of Human Vascular Endothelial Cells on, CS-1 Fibronectin by Anti-Integrin α4β1 Antibody
1. Endothelial Cell Adhesion:
The wells of 48 well culture dishes were coated with 10 μg/ml CS-1 fibronectin for one hour at 37° C. and blocked with 2% bovine serum albumin in phosphate buffered saline for one hour. Fifty thousand cells in 250 μl of adhesion buffer were added in triplicate to wells containing 250 μl of a solution of 50 μg/ml of an anti-α4β1 blocking antibody in adhesion buffer, 50 μg/ml of control antibody in adhesion buffer or adhesion buffer (Hepes buffered Hanks balanced salt solution, HBSS). Human neonatal umbilical vein endothelial cells were allowed to adhere to dishes for ten to twenty minutes at 37° C. Nonadherent cells were removed by washing each well four times with 500 μl of adhesion buffer. Adherent cells were then fixed for 15 minutes with 3.7% paraformaldehyde in phosphate buffered saline and stained with a 2% crystal violet solution. After extensive water washing to remove excess crystal violet, plates were dried overnight. Crystal violet was extracted by incubation for 15 minutes in 10% acetic acid and absorbance at 650 nm determined as an indicator of number of cells bound. The results are shown in FIG. 3A.
These results demonstrate that inhibitors of integrin α4β1 blocked the adhesion of proliferating human neonatal umbilical vein endothelial cells to CS-1 fibronectin (FIG. 3).
2. Endothelial Cell Migration:
The lower side of 8 μm pore transwell inserts were coated with 5 μg/ml of CS-1 fibronectin (B) for one hour and were blocked with 2% bovine serum albumin in phosphate buffered saline for one hour. The inserts were then placed into 24 culture dishes containing 500 μl migration buffer in the lower chamber. Twenty-five thousand human vascular endothelial cells (HUVECS) in 50 μl of migration buffer were added to the upper chamber of duplicate inserts containing 50 μl of a solution of 50 μg/ml of an anti-α4β1 function blocking antibody in migration buffer, 50 μg/ml of control antibodies or migration buffer alone. Cells were allowed to migrate from the upper to the lower chamber for four hours at 37° C. Nonmigratory cells were removed from the upper surface by wiping the upper side with an absorbent tip. Cells which had migrated to the lower side of the transwell insert were then fixed for 15 minutes with 3.7% paraformaldehyde in phosphate buffered saline and stained with a 2% crystal violet solution. After extensive water washing to remove excess crystal violet, the number of cells that had migrated was counted in three representative high power fields per insert. The results are shown in FIG. 3B.
These results demonstrate that inhibitors of integrin α4β1 blocked the migration of human vascular endothelial cells to CS-1 fibronectin (FIG. 3B).
Example 4
Inhibition of Angiogenesis In Ovo in Chick Chorioallantoic Membrane, and In Vivo in Chicks, by Anti-Integrin α4β1 Antibody
1. Chick Chorioallantoic Membrane Model
Angiogenesis was stimulated on the surface of the chick chorioallantoic membrane (CAM) by applying a 5 mm filter disc saturated with basic fibroblast growth factor (FIG. 4A), vascular endothelial growth factor (FIG. 4B), interleukin 8 (FIG. 4C), tumor necrosis alpha (FIG. 4D) or saline saturated filter disk on the CAM. Twenty-four hours after angiogenesis had been stimulated saline, 10 μg control IgG, and 10 μg anti-α4β1 monoclonal antibody with reactivity against chick cells were applied to the CAM of eight to ten eggs each in a volume of 25 μl. Forty-eight hours after this, CAMs were excised from the egg and the number of blood vessel branch points within the 5 mm area were counted.
The data in FIG. 4 demonstrates the inhibition of angiogenesis in chick chorioallantoic membrane (CAM) by anti-integrin α4 antibody. Furthermore, inhibition was dose-dependent (data not shown).
2. In Vivo Inhibition in Chicks
The inventor subsequently tested anti-integrin α4β1 monoclonal antibody, saline, and control IgG for their abilities to inhibit angiogenesis by intravenous injection into the chick vasculature using this same cytokine stimulated filter disk model. Indeed, when injected into the bloodstream, these reagents inhibited angiogenesis, demonstrating that the integrin α4β1 is directly present on the vasculature.
Example 5
Inhibition of Murine Angiogenesis by Antibody and Peptide Agonists to Integrin α4β1
Angiogenesis was induced by subcutaneous injection of 400 μl of growth factor depleted matrigel supplemented 0.4 μg/ml bFGF and saline, 25 μg/ml function blocking anti-α4β1 antibodies or control antibodies. Alternatively, mice were injected with matrigel containing bFGF and 1 mM of the sequence EILDVPST (SEQ ID NO:52) or the control sequence EILEVPST (SEQ ID NO:53). After 72 hours, mice were injected with Bandiera Simplicifolia lectin-FITC conjugate. After 15 minutes, matrigel plugs were excised and homogenized. Fluorescence intensity within the lysed plug was determined and correlated with degree of angiogenesis as determined by standard immunohistochemical analyses. The results are shown in FIG. 5.
FIG. 5 demonstrates that growth factor-stimulated angiogenesis in the mouse model is selectively and specifically inhibited both by anti-integrin α4β1 antibody and by the peptide sequence EILDVPST, and not by control antibodies and peptides. This demonstrates that inhibition of integrin α4β1 function using antagonists of integrin α4β1 ligand binding results in the suppression of angiogenesis.
Example 6
Inhibition of Angiogenesis and Tumor Growth in vivo using a Nude Mouse Tumor Model
Integrin α4β1 inhibitors can be used to inhibit tumor growth, as in immunocompromised mouse tumor growth models. In this model, tumor cells (such as carcinomas of any tissue of origin, melanomas, sarcomas or lymphomas) are injected subcutaneously in nude mice. Mice are then treated (e.g., daily, every other day, weekly, monthly, etc.) by intravenous, intraperitoneal or subcutaneous injection of integrin α4β1 inhibitors such as 200 μg rat anti-mouse α4β1, peptide or small molecule antagonists, control immunoglobulin or saline. Tumor growth is monitored by measurement with calipers every weekday. The experiment is continued for four to eight weeks. Tumors are excised and weighed. Representative tumors are photographed using a stereo microscope. Tumors are fixed in neutral buffered formalin, sectioned and H&E stained. Other tumors are flash frozen and sectioned for immunohistochemical detection and quantification of blood vessels. Integrin α4β1 inhibitors are expected to inhibit tumor growth in this model while control substances are not expected to inhibit tumor growth. All data are analyzed for statistical significance using Student's T-test and Wilcox rank mean sum analyses.
Example 7
Inhibition of Angiogenesis and Tumor Growth in vivo Using a SCID Mouse Tumor Model
Six-week old Balb/c SCID mice are anesthetized, shaved and swabbed with alcohol. An 8 mm by 13 mm section of dorsal skin is removed down to the fascia. A similar-sized piece of full thickness neonatal foreskin is placed on the wound bed and sutured into place. The transplantation site is covered with an adhesive bandage and allowed to heal for four weeks. After four weeks, the human skin is inoculated intradermally with human tumor cells or with growth factor-depleted Matrigel containing angiogenic growth factors. Animals are then injected (e.g., daily, every other day, weekly, monthly, etc.) intravenously or intraperitoneally with α4β1 antagonists, such as 200 μg mouse anti-human integrin α4β1 antibody, peptide or small molecule antagonists, control substances or saline. Animals bearing tumors are treated for up to four weeks. Tumor size is monitored throughout the period. Tumor mass data is analyzed for statistical significance using Student's T-test and Wilcox Rank mean sum analyses. Representative tumors are analyzed for induction of angiogenesis by immunohistochemical methods after preparation of frozen sections. Vascular density is assessed on all sections by staining for expression of the endothelial marker CD31 in a blinded fashion. Tumors are also be evaluated by hematoxylin and eosin staining and by Ki67 and apoptag staining to determine the impact of treatments on tumor cell fate. All data are analyzed for statistical significance using Student's T-test and Wilcox rank mean sum analyses.
Example 8
Inhibition of Angiogenesis and Tumor Growth Using an In Ovo Chick Chorioallantoic Membrane Tumor Model
Integrin α4β1 inhibitors can also be used to inhibit tumor growth on the chick chorioallantoic membrane. 50 mg fragments of human tumors are placed on chick chorioallantoic membranes. Eggs are then injected with integrin-o4β1 inhibitors, with saline or control substances. Tumors grow for one week, then tumors are excised and weighed. Representative tumors are photographed using a stereo microscope. Tumors are fixed in neutral buffered formalin, sectioned and H&E stained. Other tumors are flash frozen and sectioned for immunohistochemical detection and quantification of blood vessels. Integrin α4β1 inhibitors are expected to inhibit tumor growth in this model while control substances are not expected to inhibit tumor growth. All data are analyzed for statistical significance using Student's T-test and Wilcox rank mean sum analyses.
Example 9
Inhibition of Metastases Using an In Ovo Chick Chorioallantoic Membrane Tumor Model
Fifty mg fragments of tumors are placed on fresh CAMS. Eggs are then injected with integrin-α4β1 inhibitors, with saline or control substances. Original tumor weights are determined after 7 days. Tumor metastases to the embryonic lung and other tissues are quantified after 7 days by preparing a single cell suspension of the lung and other tissues. Flow cytometry analysis with antibodies specific for human cell surface markers, such as W6/32, are performed to quantify tumor metastasis.
Example 10
Inhibition of Metastases Using an In Vivo Nude Mouse Tumor Model
Tumor cells are inoculated intravenously, subcutaneously, or orthotopically (i.e., in the appropriate tissue of origin) in nude mice. Mice are treated with integrin-α4β1 inhibitors, with saline or control substances. Primary tumors are removed after three weeks of growth to allow metastases to grow for an additional five. The numbers and sizes of metastases to lung and liver are assessed at this end point. All data are analyzed for statistical significance using student's t-test, Wilcox rank mean sum and power analyses.
Example 11
Inhibition of Spontaneous Tumors and Metastases in an In Vivo Mouse Tumor Model
Tumors arise spontaneously in several strains of mice. Integrin α4β1 inhibitors are used to inhibit tumor growth in many of these. Mtag and APC mice are excellent models of spontaneous tumor growth and metastasis. MTAG female mice carrying the polyoma middle T-oncogene (PyV+/−) transgene bear detectable mammary carcinoma tumors measuring 2-4 mm in all mammary glands. APC mice develop multiple colon polyps. Up to ten mice per group will be enrolled in this study. Mice receive intravenous or intraperitoneal injections of putative antibody, peptide and small molecule antagonists of integrin α4β1 or fibronectin. In each experiment the compounds consist of at least one putative angiogenesis inhibitor and one matched control compound and are administered in a blinded fashion. Tumor dimensions and tumor number are measured every other day where palpable. Mouse weight is measure daily. After up to 30 days of treatment, mice are euthanized, photographed if necessary and tumors surgically resected. In addition, lungs and liver are surgically resected. Tumors are weighed. Metastatic nodules on lungs and liver are counted. Portions of representative tumors or lungs are frozen. Frozen sections are stained with antibodies directed CD31 and Factor VIII related antigen to obtain an indication of angiogenic index.
Example 12
Inhibition of Endothelial Progenitor Cell Migration in In Vivo Mouse and Rat Animal Models
Integrin α4β1 inhibitors can be used to prevent endothelial cell precursors from exiting the blood stream and entering sites of neovascularization. Angiogenesis assay are performed in mouse or nude rats transplanted with murine Tie2-LacZ bone marrow by injecting matrigel, a viscous extracellular matrix that solidifies at body temperature, containing angiogenic growth factors. Mice are treated by intravenous injection with anti-murine α4β1 and control antibodies or other inhibitors of α4β1. α4β1 inhibitors are anticipated to block LacZ staining cells from incorporating into blood vessels, indicating that α4β1 regulates endothelial precursor cell egress from the circulation. Frozen sections of the matrigel are stained with antibodies directed CD31 and Factor VIII related antigen to obtain an indication of angiogenic index.
Example 13
Inhibition of Angiogenesis In Vivo in a Rabbit and Mouse Animal Models of Arthritis
Antigen induced arthritis is induced in rabbits or mice by multiple subcutaneous injections of ovalbumin in Freund's complete adjuvant, followed by booster injections in Freund's incomplete adjuvant two weeks later. Arthritis is induced one week later by intra-articular injection of ovalbumin with basic fibroblast growth factor. After arthritis induction, animals are treated intra-articularly or systemically with anti-murine α4β1 and control antibodies or other inhibitors of α4β1. Frozen sections of joints are stained with antibodies directed CD31 and Factor VIII related antigen to obtain an indication of angiogenic index.
Example 14
Inhibition of Angiogenesis In Vivo in Rabbit and Mouse Animal Models of Ocular Angiogenesis
Hydron pellets containing Carafate-stabilized growth factors such as bFGF or VEGF and integrin α4β1 inhibitors or control substances are surgically implanted into rabbit or mouse corneas and observed daily for 10-15 days. Photographs are taken periodically after implantation. Angiogenesis is quantified by determining the mean area of neovascularization.
Example 15
Antagonists of α4β1 Block Murine bFGF and VEGF Induced Angiogenesis
Murine angiogenesis was induced by subcutaneous injection 400 μl of growth factor depleted matrigel containing 400 ng/ml bFGF or VEGF into the rear dorsal flanks of inbred mice of the strain FVB/N. Animals were treated on day 0 and day 3 by intravenous injection of 200 μg in 100 μg of endotoxin-free rat anti-murine α4β1 antibody (PS-2) or control isotype matched rat anti-murine integrin b2 antibody on days 1 and 4 (n=10). After 5 days, matrigel plugs were excised, embedded in OCT, frozen and sectioned. Thin sections (5 μm) were immunostained with rat anti-murine CD31 followed by Alexa 565-conjugated goat anti-rat immunoglobulin and then counterstained with DAPI. CD31 positive vessel density per 200×microscopic field was determined in 5 fields per matrigel plug. Mean vessel density per field+/−SEM was determined in five randomly selected microscopic fields for each plug in each treatment group and graphed versus treatment condition (n=8) (FIG. 18A). Photographs were taken of representative fields, with red indicating CD31 positive blood vessels and blue representing nuclei of all cells (FIG. 18B).
Antibody antagonists of integrin α4β1 (anti-α4β1) inhibited the ability of either bFGF or VEGF, two pro-angiogenic factors, to induce the growth of new blood vessels. Numerous new blood vessels stimulated by VEGF or bFGF were identified in tissue sections by their reactivity with anti-CD31, a specific marker of endothelial cells that line blood vessels. Few CD31 positive vessels could be identified in tissue sections from anti-α4β1 treated mice, while many blood vessels were observed in tissue sections from control antibody treated mice (cIgG). Quantification of the number of vessels induced in the presence of anti-α4β1 or control antibodies indicated that anti-α4β1 inhibited angiogenesis.
Example 16
Antagonists of α4β1 Block Murine Corneal Angiogenesis
Polymerized pellets containing 400 ng/ml VEGF were implanted into the corneas of inbred mice of the strain FVB/N. Animals were treated by intravenous injection of 200 μg in 100 μl of endotoxin free rat anti-murine α4β1 antibody (PS-2) or control isotype matched antibody (n=3) on days 1 and 4. Mice were perfused on day 5 with FITC-Bandeira simplicifolia, a lectin that selectively binds to all endothelium. Mice were sacrificed, corneas excised and cryopreseved. Thin cryosections sections were the photographed and mean area of fluorescence in the cornea was determined.
Antibody antagonists of integrin α4β1 (anti-α4β1) inhibited the ability of VEGF to induce the growth of new blood vessels in the cornea. Microvessel density was determined as a function of involved area for each treatment group (FIG. 19A). Numerous FITC-labeled new blood vessels stimulated by VEGF could be seen in tissue sections in control treated animals (FIG. 19B). Fewer fluorescent vessels were observed in anti-α4β1 treated mice (FIG. 19B). Quantification of the area invaded by blood vessels (green fluorescent area) indicated that anti-α4β1 inhibited corneal angiogenesis.
Example 17
Endothelial Progenitor Cells (EPC) Express Integrin α4β1
Purified human umbilical vein endothelial cells (“HUVECS”) (Clonetics, San Diego, Calif.) and endothelial progenitor cells (“EPCs”) cultured on fibronectin from circulating CD34+ stem cells [see Asahara et al., Science, 275:964-967, (1997)], were incubated with mouse anti-human integrin α4β1 antibodies for 60 minutes on ice, washed twice with PBS and then incubated for 30 minutes on ice in rhodamine-labeled goat anti-mouse IgG. Cells were washed twice with cold PBS then analyzed on a FACSCAN analyzer for expression of integrin α4β1. The percent cells expressing this integrin was determined and plotted according to cell type (FIG. 20).
Thirty-three percent of endothelial progenitor cells were positive for integrin α4β1 expression while only 12% of HUVECS were positive. These results showed that the inhibitory effect of α4β1 antagonists in angiogenesis result from an inhibition of the participation of endothelial progenitor cells in angiogenesis.
Example 18
α4β1 Antagonists Block Endothelial Stem Cell Contribution to Angiogenesis Murine angiogenesis was induced by subcutaneous injection 400 μl of growth factor depleted matrigel containing 400 ng/ml bFGF or VEGF into the rear dorsal flanks of inbred mice of the strain FVB/N or into FVB/N mice that had been irradiated and transplanted with bone marrow from Tie2LacZ mice. Animals were treated by intravenous injection on day 0 and day 3 with 200 μg of endotoxin free rat anti-murine α4β1 antibody (PS-2) in 100 μl or control isotype matched rat anti-murine integrin beta 2 antibody on days 1 and 4 (n=10). After 5 days, matrigel plugs were excised, embedded in OCT, frozen and sectioned. Thin sections (5 μm) were immunostained with rat anti-murine CD31 followed by Alexa 565-conjugated goat anti-rat immunoglobulin. CD31 positive vessel density per 200×microscopic field was determined in 5 fields per matrigel plug. Mean vessel density per field+/−SEM was graphed versus treatment condition. Photographs were taken of representative fields of control IgG and anti-α4β1 treated bFGF or VEGF containing plugs stained for beta galactosidase expression, with red indicating CD31 positive blood vessels and blue representing nuclei of all cells (FIG. 21B). Sections from Tie2/LacZ transplanted mice were analyzed for presence of bone marrow derived endothelial cells by staining sections for expression of beta galactosidase using a kit from Life Technologies. Blue cells in the plugs that arose from the transplanted bone marrow were counted (FIG. 21A) with bFGF stimulating angiogenesis.
Antagonists of integrin α4β1 prevent the participation of endothelial progenitor cells in angiogenesis. Beta galactosidase expressing endothelial cells derive from bone marrow because these mice were irradiated to kill their own bone marrow prior to transplantation with bone marrow from mice that express LacZ under an endothelial specific promoter, the Tie2 promoter. Thus, endothelial cells that arise from bone marrow will turn blue in tissues incubated in a substrate for beta galactosidase. These data showed that fewer blue endothelial cells were induced by growth factors in mice treated with anti-α4β1 than in mice treated with control antibodies. Therefore, anti-α4β1 inhibited the participation in angiogenesis of endothelial progenitors derived from bone marrow.
Example 19
Antagonists of α4β1 Inhibit Tumor Growth
Seven-week-old Balb/c nu/nu mice were inoculated subcutaneously with 2 million integrin-a4β1 negative HT29 colon carcinoma cells in DMEM culture medium. When tumors were approximately 50 cubic mm in size (2 weeks later), treatment began with intravenous injection twice weekly with saline or three concentrations of rat anti-mouse α4β1 antibody (PS-2). The doses were 200, 400 or 600 μg of antibody per injection. Ten mice were included in each treatment group. Tumor dimensions were measured every other day and tumor volumes were calculated. See FIG. 22A. After four weeks of treatment, tumors were excised and mass were determined. Mean tumor volumes per group were plotted versus time for the entire growth period. Mean tumor mass was plotted per group. See FIG. 22B.
Integrin α4β1 antagonists inhibited growth of integrin α4β1 negative tumors. As these antagonists inhibit angiogenesis, these studies showed that integrin α4β1 antagonists block tumor angiogenesis.
From the above, it is clear that the invention provides methods and compositions for detecting and inhibiting angiogenesis, endothelial cell adhesion, and endothelial cell migration. In some preferred embodiments, the present invention utilizes agents that inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention further provides methods and compositions for screening test compounds for their ability to inhibit angiogenesis, endothelial cell adhesion, and/or endothelial cell migration, by employing agents which inhibit the specific binding of integrin α4β1 to one or more of its ligands. The invention additionally provides methods and compositions for isolating endothelial cells which express integrin α4β1. The methods of the invention are useful in, for example, diagnosing and inhibiting pathological conditions that are associated with angiogenesis, endothelial cell adhesion, and/or endothelial cell migration. The methods and compositions of the present invention are also useful in isolating endothelial progenitor cells, and in determining the mechanisms that underlie angiogenesis, development, wound healing, and the function of the female reproductive system.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiment, it should be understood that the invention as claimed should not be unduly limited to such specific embodiment. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.

## Claims

1. A method for inhibiting angiogenesis in colon tissue, comprising:
a) providing: i) angiogenic colon tissue comprising endothelial cells that express integrin α4β1; and ii) an agent which inhibits specific binding of integrin α4β1 to an integrin α4β1 ligand; and
b) inhibiting angiogenesis in said colon tissue by treating said tissue with said agent under conditions that inhibit specific binding of said integrin α4β1 that is expressed by said endothelial cells to said integrin α4β1ligand.

2. The method of claim 1, wherein said colon tissue is in a subject.

3. The method of claim 2, wherein said colon tissue comprises a tumor.

4. The method of claim 3, wherein said tumor is malignant.

5. The method of claim 4, wherein said malignant tumor is metastatic.

6. The method of claim 1, wherein said agent comprises a peptide.

7. The method of claim 1, wherein said agent comprises an antibody.

8. The method of claim 7, wherein said antibody is an anti-integrin α4β1 antibody.

9. The method of claim 7, wherein said antibody is an anti-vascular cell adhesion molecule antibody.

10. The method of claim 7, wherein said antibody is an anti-fibronectin antibody.

11. The method of claim 1, wherein said ligand is vascular cell adhesion molecule.

12. The method of claim 1, wherein said ligand is fibronectin.