WO1999045951A2 - Thy-1 expression in angiogenesis - Google Patents

Thy-1 expression in angiogenesis Download PDF

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Publication number
WO1999045951A2
WO1999045951A2 PCT/US1999/005256 US9905256W WO9945951A2 WO 1999045951 A2 WO1999045951 A2 WO 1999045951A2 US 9905256 W US9905256 W US 9905256W WO 9945951 A2 WO9945951 A2 WO 9945951A2
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thy
ser
seq
mammal
val
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PCT/US1999/005256
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French (fr)
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WO1999045951A3 (en
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Shyh-Yu Shaw
Mukesh K. Jain
Wen-Sen Lee
Edgar Haber
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President And Fellows Of Harvard College
HABER, Carol
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to diseases characterized by undesired angiogenesis .
  • Angiogenesis results from endothelial cell proliferation induced by angiogenic factors .
  • Angiogenic factors bind to receptors on endothelial cells lining blood vessels . This event triggers signals which cause the cells to proliferate; the proliferating endothelial cells secrete proteases which digest the basement membrane surrounding a vessel. The junctions between the endothelial cells are altered, allowing projections from the cells to pass through the space created. These outgrowths then become new blood vessels, e.g., capillaries.
  • Undesired angiogenesis is associated with a variety of diverse disease states, including tumor growth, diabetic retinopathy, rheumatoid arthritis, psoriasis, and coronary atheroma. For example, tumor growth depends on an adequate blood supply which in turn is dependent on the growth of new blood vessels and capillaries into the tumor.
  • the invention is based on the discovery that the endothelial cells which form new blood vessels, e.g., vascular endothelial cells, express the cell surface antigen, Thy-1. Thy-1 is not generally expressed on endothelial cells in non-angiogenic vessels nor on endothelial cells involved in embryonic vasculogenesis .
  • the invention features a method of inhibiting undesired angiogenesis in a mammal (e.g., a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat or human) by administering to the mammal a compound that inhibits Thy-1-associated proliferation of an endothelial cell, e.g., a vascular endothelial cell .
  • a mammal e.g., a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat or human
  • one class of inhibitors of angiogenesis includes single chain Thy-1 binding compounds, e.g., a soluble monovalent Thy- 1 ligand.
  • the compound is characterized as having the property of inhibiting Thy-1 associated proliferation of cultured endothelial cells, e.g., vascular endothelial cells.
  • a single chain polypeptide containing an extracellular fragment of Thy-1 inhibits binding of cell surface Thy-1 to an endogenous ligand which may crosslink cell surface Thy-1 molecules and initiate a signal transduction cascade which results in cell proliferation.
  • the polypeptide contains amino acids 1-113 of rat Thy-1 (SEQ ID NO:l); more preferably, the polypeptide contains amino acids 1-111 of rat Thy-1 (amino acids 1-111 of SEQ ID NO:l) .
  • the polypeptide may contain amino acids 1-113 of human Thy-1 (SEQ ID NO: 2) or amino acids 1-111 of human Thy-1 (amino acids 1-111 of SEQ ID NO:2) .
  • the compound may be a chimeric polypeptide containing a polypeptide fragment of Thy-1 and a polypeptide fragment of an immunoglobulin.
  • an extracellular fragment of Thy- 1 is linked to a second polypeptide containing a constant region of IgG (SEQ ID NO: 11) to make a chimeric polypeptide such as rat Thy-1- Ig (SEQ ID NO:3) or human Thy-l-Ig (SEQ ID N0:4) .
  • the invention also includes a polypeptide with at least 50% (preferably at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100%) sequence identity to SEQ ID NO: 3 or 4 and having the activity (i.e., inhibiting binding of Thy-1 to a Thy-1 ligand) of Thy-l-Ig.
  • a nucleic acid encoding a Thy-l-Ig polypeptide is also encompassed by the invention.
  • the nucleic acid encodes a polypeptide containing the amino acid sequence of rat Thy-l-Ig (SEQ ID NO: 3) or human Thy-l-Ig (SEQ ID NO:4) .
  • the nucleic acid contains a nucleotide sequence of SEQ ID NO: 5 or 6 or a degenerate variant thereof .
  • Gin Arg Val lie Ser Leu Thr Ala Cys Leu Val Asn Gin Asn Leu
  • Gin Arg Val lie Ser Leu Thr Ala Cys Leu Val Asn Gin Asn Leu Arg Leu Asp Cys Arg His Glu Asn Asn Thr Asn Leu Pro lie Gin His Glu Phe Ser Leu Thr Arg Glu Lys Lys Lys His Val Leu Ser Gly Thr Leu Gly Val Pro Gin His Thr Tyr Arg Ser Arg Val Asn Leu Phe Ser Asp Arg Phe lie Lys Val Leu Thr Leu Ala Asn Phe Thr Thr Lys Asp Glu Gly Asp Tyr Met Cys Glu Leu Arg Val Ser Gly Gin Asn Pro Thr Ser Ser Asn Lys Thr He Asn Val He Arg Asp Lys Leu Val Lys Cys Gly Gly Ala Asp Leu Ser Ser Arg Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met He Ser
  • Inhibition of Thy-1 -associated endothelial cell proliferation to reduce angiogenesis can be used to treat cancers, e .g. , by reducing vascularization of a tumor.
  • the compound is locally administered to a mammal at the site of a tumor, e.g., a solid tumor, to reduce the blood supply to the neoplastic tissue, thereby inhibiting tumor growth.
  • therapeutic formulations may be administered by implantation at or near the tumor site or the site from which a tumor was excised.
  • the compound may also be administered to mammals to treat rheumatoid arthritis, the lesions of which are commonly associated with neovascularization.
  • the therapeutic method may be carried out by locally administering the compound at the site of an arthritic lesion to reduce vascular endothelial cell proliferation, and in turn, the severity of the lesion.
  • the method may also be used to treat atherosclerosis by locally administering a compound that reduces Thy-1-associated endothelial cell proliferation (and thus, angiogenesis) to a mammal at the site of an atherosclerotic lesion.
  • a compound that reduces Thy-1-associated endothelial cell proliferation and thus, angiogenesis
  • angiogenesis may be inhibited by administering to a mammal a compound, e.g., a Thy-1 anti-sense nucleic acid, that inhibits expression of Thy-1 in an endothelial cell.
  • the antisense nucleic acid may have a nucleotide sequence that is complementary to DNA encoding amino acids 1-113 of Thy-1 (e.g., SEQ ID NO:1 or 2) .
  • a compound that increases Thy-1 expression in an endothelial cell can be used to promote angiogenesis in a mammal.
  • Angiogenesis can also be inhibited by administering to the mammal a compound, e.g., a Thy-1-specific antibody linked to a cytotoxic agent, that selectively destroys Thy-1 expressing endothelial cells.
  • Thy-1 ligand is any protein or polypeptide that binds to the Thy-1. Such ligands can be soluble or expressed on a cell surface. Thy-1 ligands include any protein, polypeptide, or other molecule that binds to Thy-1, e.g. Thy-1 specific antibodies, Thy-1 specific antibody fragments, and Thy-1 binding peptides. Cell- surface Thy-1 interactions (e.g., those resulting in crosslinking of Thy-1 on the surface of a cell) may by homotypic, i.e., a Thy-1 molecule on the surface of one cell may interact with a Thy-1 molecule on the surface of another cell. Such homotypic interactions may result in crosslinking of endothelial cell surface Thy-1 molecules which potentiates an intracellular signal which culminates in cell proliferation
  • the term “block” means to interfere either partially or completely with a binding interaction between two molecules, e.g., the binding of Thy-1 to a Thy-1 ligand.
  • the term “antibody” encompasses not only an intact antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab) 2 fragment; an engineered single chain Fv molecule.
  • antibodies or antibody fragments can be humanized by methods known in the art .
  • Antibodies or antibody fragments with a desired binding specificity can also be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA) .
  • the term “Fab” or “antibody fragment” refers to the portion of an antibody molecules which includes the variable region of the heavy chain and/or light chain, and which exhibits antigen-binding activity.
  • purified DNA DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • polypeptide any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation) .
  • Thy-1-encoding DNA or Thy-1 antisense DNA may be operably linked to regulatory sequences, e.g., a promoter.
  • the promoter is vascular cell-specific, more preferably, it is vascular endothelial cell-specific.
  • operably linked is meant that a coding sequence and a regulatory sequence (s) are connected in such a way as to permit expression of the DNA when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence (s) .
  • promoter is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible.
  • vector means a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid.
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions .
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions .
  • the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides.
  • Sequence identity is measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .
  • Thy-1-specific antibody means an antibody that binds to a Thy-1 protein or polypeptide and displays no substantial binding to other naturally-occurring proteins other than those sharing the same antigenic determinants as Thy-1.
  • the term includes polyclonal and monoclonal antibodies .
  • substantially pure protein means a protein separated from components that naturally accompany it.
  • the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated.
  • the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight.
  • a substantially pure Thy-l protein can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding an Thy-1 polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • substantially pure proteins include those having sequences derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes .
  • fragment as applied to a protein, means at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length.
  • Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering.
  • Fig. 1 is a bar graph showing induction of Thy-1-associated endothelial cell proliferation (as measured by incorporation of thymidine) by crosslinking of endothelial cell surface Thy-1 molecules using a Thy-1-specific antibody. Incubation of endothelial cells with a control antibody (anti-CD44) resulted in little or no endothelial cell proliferation.
  • Thy-1 is expressed by vascular endothelial cells during angiogenesis in an adult animal.
  • Thy-1 expression was found to be upregulated during physiological angiogenesis, e.g., during pregnancy, as well as during pathological angiogenesis, e.g., ischemia, cancer, and blood vessel disease.
  • Thy-1 expression was detected by an increase in the level of Thy-1 mRNA as well Thy-1 protein.
  • Upregulation was found to be induced by exposure of endothelial cells to inflammatory cytokines, e.g., TNF- ⁇ or IL-1/3. Proliferation of vascular endothelial cells is a critical step in the formation of blood vessels .
  • Thy-1 Engagement of endothelial cell surface Thy-1 with a Thy-1 ligand initiates a cascade of signal transduction events that culminate in endothelial cell proliferation leading to angiogenesis.
  • Crosslinking of Thy-1 on the surface of an vascular endothelial cell e.g., by a bivalent Thy-1-specific antibody was found to induce proliferation of the Thy-1-expressing human microvascular endothelial cells (Fig. 1) .
  • the animal models described below represent a spectrum of conditions that are accompanied by new blood vessel formation.
  • Stimuli such as mechanical injury (carotid injury model) , tumorigenesis (eye model) , and ischemia (renal artery ligation model) are accompanied by the release of inflammatory cytokines such as IL- 1/3 and tumor necrosis factor- ⁇ .
  • Thy-1 was highly expressed in endothelial cells of small blood vessels formed after the pathologic stimuli or during physiological angiogenesis.
  • Thy-1 expression in endothelial cells was studied in four art-recognized rat models of angiogenesis.
  • the model for renal artery stenosis was performed using known methods, e.g., the method of Ilich et al. (Ilich et al., 1979, Circ. Res., 45:579-582).
  • the main renal artery was partially occluded by the application of a silver clip.
  • the silver clip was removed. Ureters were harvested 5 days after ligation.
  • Implantation of a tumor in the cornea was performed according to standard methods (e.g., the method of Gimbrone et al., 1974, J. Nat. Cancer Inst. 52:413-427). Corneas were harvested 10 days after tumor implantation.
  • Balloon angioplasty of the carotid artery was performed using well-known methods of angioplasty (e.g., the method of Clowes et al., 1983, Lab. Invest. 49:327-333 or Clowes et al., 1983, Lab. Invest. 49:208-215) .
  • Carotid arteries were harvested from adult male Sprague-Dawley rats 14 days after they had been subjected to balloon injury.
  • mice anti-rat Thy-1 antibody (clone 0X-7, Pharmingen, San Diego, CA) at 1:300 dilution and biotinylated goat anti-mouse IgGi (Amersham, Arlington Heights, IL) at 1:100 dilution;
  • mouse anti-rat Thy-1 antibody (clone HIS51, Pharmingen) at 1:100 dilution and biotinylated goat anti-mouse IgG 2a (Amersham) at 1:100 dilution;
  • rabbit anti-human von Willebrand factor antibody (DAKO, Carpinteria, CA) at 1:1000 dilution and biotinylated goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA) at 1:2000 dilution;
  • rat anti-mouse CD31 PECAM-1) antibody (Pharmingen) at 1:100 dilution and biot
  • tissue sections were then rinsed twice with high-salt PBS and once with standard PBS for 5 minutes (each wash) and incubated with avidin-biotin complex (ABC reagent, Vector Laboratories) at 1:100 dilution for 1 hour at room temperature. After a wash with PBS, the sections were incubated with 3,3'- diaminobenzidine or 3, 3' -diaminobenzidine plus nickel sulfate in PBS- H 2 0 2 to detect antibody binding. Color development (i.e., brown or blue/black, for 3 , 3 ' -diaminobenzidine or 3, 3' -diaminobenzidine plus nickel sulfate, respectively) indicated positive staining.
  • tissue was incubated with anti-Thy-1 antibody (OX-7) that had been preabsorbed with 25 ⁇ g/ml Thy-l-Ig fusion protein. Preabsorption of the Thy-1- specific antibody with Thy-l-Ig fusion protein blocked all staining. Counter-staining was performed with a solution of 1% methyl green.
  • OX-7 anti-Thy-1 antibody
  • rat Thy-l-Ig expression plasmid (pThy-1-Ig) was constructed by cloning a DNA encoding an extracellular fragment of rat Thy-1 (residues Glnl-Glyll3; SEQ ID N0:1) (Seki et al., 1985, Fed. Proc. 44:2865-2869) in frame to a DNA encoding a human IgGi constant region, e.g., a fragment of IgG containing the hinge, CH2 and CH3 regions (SEQ ID NO: 11) .
  • the rat Thy-1 and human IgGi DNAs were obtained by polymerase chain reaction with templates from rat brain cDNA (Clontech, Palo Alto, CA) and human spleen cDNA (Clontech) , respectively.
  • the Thy-1 forward primer, 5'-GCG CAG AAG CTT ATT GGC ACC ATG AAC CCA GTC ATC-3' (SEQ ID NO:7) corresponded to the N-terminus of the rat Thy-1 signal sequence plus a Hindlll restriction endonuclease site.
  • the Thy-1 reverse primer 5' -CCT CGA GAG ATC TCC ACC ACA CTT GAC CAG CTT GTC-3' (SEQ ID NO: 8) , corresponded to residues Aspl06-Glyll3 of rat Thy-1 plus a Bglll restriction endonuclease site.
  • the IgGj constant region forward primer 5' -A GAT CTC TCG AGT AGA CCC AAA TCT TCT GAC AAA ACT CAC ACA TCC CCA CCG TCC CCA-3' (SEQ ID NO: 9), corresponded to residues
  • Pro227-Pro243 of the human IgGi hinge region plus a Bglll restriction endonuclease site Three mutations were introduced in DNA encoding the IgG hinge region to change cysteines to serines .
  • the PCR products of the Thy-1 and human IgG x constant regions were digested with Hindlll/Bglll and Bglll/Xbal, respectively.
  • the digested fragments were ligated to the Hindlll/Xbal site of pCNDA-lamp (Invitrogen, San Diego, CA) resulting in the final expression plasmid pThy-1-Ig.
  • the same strategy was used to make human Thy-l-Ig (SEQ ID NO:4) .
  • Thy-l-Ig fusion proteins were transiently expressed in COS cells and purified from the culture medium, using standard methods, e.g., by protein G affinity chromatography (Pharmacia, Uppsala, Sweden) . The purity of the protein was evaluated by SDS-PAGE, and its concentration was determined by the DC-protein assay (Biorad,
  • Presence of a Thy-1 epitope on the fusion protein was determined by Western blot with a biotin-conjugated mouse anti-rat Thy-1 primary antibody (clone OX-7) and a streptavidin-conjugated alkaline phosphatase secondary antibody (Pharmingen, San Diego, CA) .
  • RNA Extraction and Northern Blot Analysis were obtained from Clonetics Corporation (San Diego, CA) and grown in microvascular endothelial cell growth medium (Clonetics, No. CC-3125) . Cells were passaged every 4-5 days. Cells from passages 6-7 were used for all experiments. After the cells had grown to 80% confluence, they were placed in quiescence medium (endothelial cell basal medium-2, serum free; Clonetics, No. CC-3156) overnight.
  • quiescence medium endothelial cell basal medium-2, serum free; Clonetics, No. CC-3156
  • Total RNA was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride using methods well known in the art.
  • Total RNA from cells was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose paper.
  • the filters were hybridized with a randomly primed 32 P-labeled human Thy-1 full-length cDNA.
  • the hybridized filters were then washed in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS solution at 55°C and autoradiographed on Kodak XAR film at -80°C.
  • the blots were hybridized with an oligonucleotide probe specific for 18S ribosomal RNA to correct for differences in RNA loading. Filters were scanned on a PhosphorImager running the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) . Thy-l-Ig chimera
  • a recombinant chimeric fusion protein Thy-l-Ig was made by cloning DNA encoding the extracellular portion of rat Thy-1 (residues Glnl-Glyll3; SEQ ID N0:1) to DNA encoding the human IgG constant region (hinge, CH2, and CH3; SEQ ID NO:11) and expressed transiently in COS cells .
  • Three cysteine residues in the IgG hinge region were mutated to serines to prevent nonspecific disulfide linkage with the Cyslll in Thy-1.
  • Chimeric proteins were purified by protein G affinity chromatography and characterized by SDS-polyacrylamide gel electrophoresis .
  • the Coomassie blue-stained gel revealed bands of the expected size: one band at 104 kDa (nonreducing conditions) and one band at 52 kDa (under reducing conditions) .
  • the molecular weight of a Thy-l-Ig monomer approximated the sum of rat Thy-1 at 25 kDa and the human IgFc fragment at 25 kDa.
  • Western blot analysis with OX-7 confirmed the presence of Thy-1 epitopes in both the 104 kDa and the 52 kDa bands.
  • Thy-l-Ig is a disulfide-linked homodimer (104 kDa band under nonreducing conditions) .
  • Thyl-1-Ig was used to verify the specificity of immunostaining of endothelial cells for expression of Thy-1.
  • Preabsorption of a Thy-1-specific antibody (e.g., OX-7) with a Thy-l- Ig fusion protein prior to contacting the tissue sections with the OX-7 antibody solution eliminated all Thy-1 immunoreactivity.
  • Thy-1 immunoreactivity was examined in adult rat tissue during angiogenensis .
  • Expression of Thy-1 was evaluated by immunostaining with an antibody specific for rat Thy-1 (clone OX-7) 14 days after balloon injury to the rat carotid artery.
  • renal arteries were clipped for 5 days, to induce new blood vessel formation in the periureteric area using known methods, (e.g., the method of Ilich et al., 1979, Circ. Res. 45:579-582 or Hollenberg et al., 1985,
  • Thy-1 immunostaining was observed in small blood vessels beneath the ureteral epithelium in a tissue sample taken from a rat with a clipped renal artery but not in a sample taken from a control rat (without a clipped renal artery) .
  • Thv-1 expression during rat embrvogenesis The fourth model of adult angiogenesis is pregnancy. Using a rat model, new blood vessels which formed in the rat uterus during pregnancy were evaluated for Thy-1 expression. At days 8 and 11 of pregnancy, strong Thy-1 staining was observed in small blood vessels of the uterus . Absence of Thy-1 Immunoreactivity During Blood Vessel Development in
  • Thy-1 expression during physiologic vasculogenesis and angiogenesis in developing rat embryos was also examined.
  • Vasculogenesis refers to origination of the vascular system, i.e., development of new blood vessels from angioblasts, whereas angiogenesis refers to the growth of new blood vessels from existing blood vessels .
  • Adjacent rat embryonic tissue sections (at embryonic day 11) were stained for von Willebrand factor (as a positive control) and Thy-1. Strong staining was observed in the large blood vessels
  • Thy-1 immunostaining was detectable in the aorta or the small vessels of the yolk sac.
  • Thy-1 immunoreactivity was also not detected in the endothelium of large or small blood vessels at embryonic days 10 and 12-18. Thy-1 immunoreactivity was detected in the developing brain and, as described above, in small blood vessels of the pregnant uterus.
  • Northern analysis was performed with 7.5 ⁇ g of total RNA per lane. After electrophoresis, RNA was transferred to nitrocellulose filters and hybridized with 32 P-labelled human Thy-1 cDNA. A single Thy-1 transcript was visible at 1.8 kB (the filters were also hybridized with a probe specific for 18S ribosomal RNA to verify equivalent sample loading) .
  • Thy-1 mRNA expression was detected in control microvascular endothelial cells, whereas incubation of endothelial cells with IL-1 / 3 increased Thy-1 mRNA expression by 8-fold at 24 hours after stimulation. Similarly, stimulation of endothelial cells with tumor necrosis factor- ⁇ resulted in 5- and 14-fold in Thy-1 mRNA expression at 8 and 24 hours (respectively) after stimulation. In contrast, VEGF or bFGF did not induce Thy-1 mRNA expression in these cells, nor did platelet-derived growth factor (PDGF) and transforming growth factor-/? (TGF-/3) .
  • PDGF platelet-derived growth factor
  • TGF-/3 transforming growth factor
  • angiogenesis in an animal can be inhibited by administering to the animal a therapeutically effective amount of a composition which blocks engagement of endothelial cell surface Thy-1 with a Thy-1 ligand, selectively reduces Thy-1 expression in endothelial cells, or destroys Thy-1 expressing endothelial cells.
  • Monomeric peptides that bind to endothelial cell surface Thy-1 e.g., soluble Thy-1 or fragments thereof (e.g., amino acids 1- 113 of Thy-1) , or a chimeric polypeptide containing a portion of Thy- 1 or another Thy-1 ligand linked to a non-Thy-1 peptide
  • Thy-1 ligand may be present on a second cell or may be soluble.
  • Synthetic ligands, antibodies or antigen-binding fragments thereof, which are specific for either the extracellular domain of Thy-1 or a ligand to which Thy-1 binds, are also useful to block Thy- 1 crosslinking.
  • Such peptides will ordinarily be at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 contiguous amino acids in length.
  • Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering, e.g., cloning and expression of a fragment of Thy-1 cDNA.
  • a composition containing a cytotoxic Thy-binding compound e.g., a Thy- 1 specific antibody coupled to a cytotoxic agents such as ricin A chain, abrin A chain, modeccin A chain, gelonin, melphalan, bleomycin, adriamycin, daumomycin, or pokeweed anti-viral proteins (PAP, PAPII, PAP-S) , may be administered to the animal to kill Thy-1- expressing cells.
  • a cytotoxic Thy-binding compound e.g., a Thy- 1 specific antibody coupled to a cytotoxic agents such as ricin A chain, abrin A chain, modeccin A chain, gelonin, melphalan, bleomycin, adriamycin, daumomycin, or pokeweed anti-viral proteins (PAP, PAPII, PAP-S)
  • PAP pokeweed anti-viral proteins
  • cytotoxic agents including radioisotopes or chemotherapeutic agents
  • target-specific antibodies can be coupled to target- specific antibodies by well-known techniques, and delivered to the animal (either systemically or locally) to specifically destroy targeted tissue (e.g., Thy-1 expressing endothelial cells).
  • targeted tissue e.g., Thy-1 expressing endothelial cells.
  • Therapeutic compositions are typically administered in a pharmaceutically acceptable carrier (e.g., physiological saline). Carriers are selected on the basis of mode and route of administration and standard pharmaceutical practice.
  • a therapeutically effective amount of a therapeutic composition e.g., Thy-l-Ig
  • a therapeutically effective amount of a therapeutic composition is an amount which is capable of producing a medically desirable result in a treated animal .
  • compositions of the invention can be administered locally i.e., at the site of angiogenesis or systemically. Administration will generally be parenterally, e.g., intravenously.
  • the compositions may also be administered directly to a tissue site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in a blood vessel.
  • an intravenous dosage of approximately 1 to 100 ⁇ moles of the peptide of the invention would be administered per kg of body weight per day.
  • the formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal .
  • Other methods of delivery e.g., liposomal delivery or diffusion from a device impregnated with therapeutic compound, are known in the art.
  • the anti-Thy-1 antibodies useful in the present invention can be obtained by techniques well known in the art. Such antibodies can be polyclonal or preferably monoclonal . Polyclonal antibodies can be obtained, for example, by the methods described in Ghose et al., Methods in Enzymology, Vol.
  • Thy-1-reactive polyclonal antibodies can be used as the immunogen to stimulate the production of Thy-1-reactive polyclonal antibodies in the antisera of animals such as rabbits, goats, sheep, rodents and the like.
  • the monoclonal antibodies can be obtained by the process described by Milstein and Kohler, 1975, Nature 256:495- 97.
  • Anti-Thy-1 antibodies are well known in the art and can be purchased commercially, e.g., OX-7 (Pharmingen, San Diego, CA) and HIS51 (Pharmingen, San Diego, CA) .
  • Antibody fragments e.g., a fragment containing only the constant region, for use in construction of chimeric polypeptides are generated using methods well known in the art.
  • Analogs of the above peptides may also be used to block Thy- 1 binding to a ligand.
  • Analogs can differ from the native peptides of Thy-1 by amino acid sequence, or by modifications which do not affect the sequence, or both. Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivitization of polypeptides, e.g., acetylation or carboxylation.
  • glycosylation e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
  • the peptide bonds of a peptide may be replaced with an alternative type of covalent bond (a "peptide mimetic") .
  • a peptide mimetic an alternative type of covalent bond
  • replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable, and thus more useful as a therapeutic.
  • mimetics, and methods of incorporating them into polypeptides are well known in the art.
  • the replacement of an L-amino acid residue with a D-amino acid is a standard way of rendering the polypeptide less sensitive to proteolysis.
  • amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4, -dinitrophenyl.
  • Peptides may be administered to a subject intravenously in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. Modulation of Thy-1 expression
  • Thy-1 expression was detected in endothelial cells participating in the process of new blood vessel formation.
  • manipulation of Thy-1 expression is a therapeutic strategy to prevent or inhibit angiogenesis.
  • administration of compounds which inhibit Thy-1 expression are useful to treat conditions characterized by pathologic angiogenesis.
  • administration of compounds which increase expression of Thy-1 would be useful to promote angiogenenis, e.g., to treat ischemia and arteriosclerosis .
  • Thy-1 DNA or Thy-1 antisense DNA may be introduced into target cells e.g., vascular endothelial cells, of the subject animal by standard vectors and/or gene delivery systems. Expression of exogenous Thy-1 DNA in an endothelial cell would increase the amount of Thy-1 protein on the cell's surface. Expression of Thy-1 antisense DNA in an endothelial cell decreases the amount of Thy-1 protein on the cell's surface by inhibiting translation of endogenous Thy-1 mRNA.
  • Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenovirus, and adeno-associated virus, among others .
  • a therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal, e.g., increased or decreased expression of Thy-1.
  • dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, sex, and general health, the particular compound to be administered, the time and route of administration, and other drugs being administered concurrently. Dosages for therapeutic nucleic acids will vary, but a preferred dosage for intravenous administration is from approximately 10 6 to 10 22 copies of the nucleic acid molecule .
  • a phospholipase can be administered to release Thy-1 from a plasma membrane by hydrolyzing its glycosylphosphatidyl-inositol anchor.
  • a phospholipase can be administered to release Thy-1 from a plasma membrane by hydrolyzing its glycosylphosphatidyl-inositol anchor.
  • engagement of two or more Thy-1 molecules on the surface of an endothelial cell triggers proliferation. Proliferation of endothelial cells contributes to angiogenesis.
  • a monomeric fragment of Thy-1 or a monomeric Thy-1 fusion protein, e.g., Thy-l-Ig can be used to inhibit Thy-1 binding to a Thy-1 ligand (e.g., a cell surface or soluble Thy-1 ligand) .
  • Thy-1 ligand e.g., a cell surface or soluble Thy-1 ligand
  • Decreasing or preventing endothelial cell surface Thy-1 from associating with or binding to a Thy-1 ligand inhibits endothelial cell proliferation which, in turn, inhibits angiogenesis.
  • the Thy-1 fusion protein can also be used to detect Thy-1 specific binding by antibodies.
  • the fusion protein can be added to a Thy-1-specific immunostaining assay, to compete with native Thy-1 for binding to the antibody. A reduction in Thy-1 detection would indicate that the antibody binds Thy-1 specifically.
  • a Thy-1 fusion protein is also useful in purifying substances which bind Thy-1. For example, a mixture containing a Thy-1-binding substance is contacted with a sample of immobilized Thy-1 fusion protein, the unbound components of the mixture are then separated from the bound substance, and the Thy-1 binding substance is recovered from the immobilized Thy-1 fusion protein. Screening assays
  • a screening assay to identify compounds which are capable of modulating angiogenesis is carried out as follows .
  • a sample of endothelial cells e.g., cultured microvascular endothelial cells
  • a sample of control cells is incubated in the absence of the compound.
  • Each sample of cells is evaluated for the expression of Thy-1.
  • each sample of cells can be incubated with a Thy-1-specific antibody and the cells evaluated for binding of the antibody by methods well known in the art, e.g., immunofluorescent staining.
  • the amount of antibody binding correlates with the level of expression of Thy-1.
  • Thy-1 expression can also be measured at the level of gene transcription.
  • Thy-1 transcripts can be measured by Northern blotting techniques using Thy-1-specific DNA probes or by PCR using Thy-1-specific DNA primers.
  • a decrease in the amount of Thy-1 expression in cells contacted with a candidate compound compared to the amount in untreated cells indicates that the candidate compound is capable of inhibiting the expression of Thy-1 in endothelial cells (and of inhibiting angiogenesis) .
  • an increase in the amount of Thy-1 expression in treated cells, compared to the amount in untreated cells indicates that the candidate compound is capable of upregulating the expression of Thy-1 in endothelial cells (and therefore promoting angiogenesis) .
  • Thy-1-associated endothelial cell proliferation can be induced by crosslinking of endothelial cell surface Thy-1 molecules using a Thy-1-specific antibody
  • monovalent Thy-1 ligands e.g., small non-peptide compounds which bind to Thy-1 which are capable of inhibiting undesired angiogenesis can be identified by screening for those that prevent or inhibit crosslinking of Thy-1 on the surface of an endothelial cell.
  • a sample of endothelial cells e.g., cultured microvascular endothelial cells
  • a conventional bivalent Thy-1 specific antibody i.e., Thy-1 specific monoclonal antibody of the IgG isotype
  • a sample of control cells is incubated in the presence of anti-Thy-1 but in the absence of the compound.
  • Each sample of cells is evaluated for proliferation.
  • a decrease in the amount of proliferation the sample of cells cultured in the presence of anti-Thy-1 and a candidate compound compared to the amount of proliferation of the sample of cells cultured in the presence of anti-Thy-1 in the absence of the candidate compound indicates that the compound inhibits Thy-1 associated endothelial cell proliferation.

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Abstract

A method of inhibiting angiogenesis in a mammal by administering to the mammal a compound that inhibits Thy-1-associated proliferation of endothelial cells.

Description

THY-1 EXPRESSION IN ANGIOGENESIS Statement as to Federally Sponsored Research This invention was made with Government support under grant number HL57664 awarded by the National Institutes of Health. The Government has certain rights in the invention.
Background of the Invention The invention relates to diseases characterized by undesired angiogenesis .
Angiogenesis results from endothelial cell proliferation induced by angiogenic factors . Angiogenic factors bind to receptors on endothelial cells lining blood vessels . This event triggers signals which cause the cells to proliferate; the proliferating endothelial cells secrete proteases which digest the basement membrane surrounding a vessel. The junctions between the endothelial cells are altered, allowing projections from the cells to pass through the space created. These outgrowths then become new blood vessels, e.g., capillaries. Undesired angiogenesis is associated with a variety of diverse disease states, including tumor growth, diabetic retinopathy, rheumatoid arthritis, psoriasis, and coronary atheroma. For example, tumor growth depends on an adequate blood supply which in turn is dependent on the growth of new blood vessels and capillaries into the tumor.
Summary of the Invention The invention is based on the discovery that the endothelial cells which form new blood vessels, e.g., vascular endothelial cells, express the cell surface antigen, Thy-1. Thy-1 is not generally expressed on endothelial cells in non-angiogenic vessels nor on endothelial cells involved in embryonic vasculogenesis .
The invention features a method of inhibiting undesired angiogenesis in a mammal (e.g., a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat or human) by administering to the mammal a compound that inhibits Thy-1-associated proliferation of an endothelial cell, e.g., a vascular endothelial cell . Without wishing to limit ourselves to a single mechanism, the data described herein indicate that engagement of endothelial cell surface Thy-1 with an endogenous Thy-1 ligand may result in crosslinking of cell surface Thy-1 molecules which leads to endothelial cell proliferation. According to this mechanism, blocking cell surface crosslinking of Thy-1 reduces or inhibits Thy- 1-associated endothelial cell proliferation. Regardless of the mechanism involved, one class of inhibitors of angiogenesis includes single chain Thy-1 binding compounds, e.g., a soluble monovalent Thy- 1 ligand. The compound is characterized as having the property of inhibiting Thy-1 associated proliferation of cultured endothelial cells, e.g., vascular endothelial cells. For example, a single chain polypeptide containing an extracellular fragment of Thy-1 inhibits binding of cell surface Thy-1 to an endogenous ligand which may crosslink cell surface Thy-1 molecules and initiate a signal transduction cascade which results in cell proliferation.
Preferably, the polypeptide contains amino acids 1-113 of rat Thy-1 (SEQ ID NO:l); more preferably, the polypeptide contains amino acids 1-111 of rat Thy-1 (amino acids 1-111 of SEQ ID NO:l) . Alternatively, the polypeptide may contain amino acids 1-113 of human Thy-1 (SEQ ID NO: 2) or amino acids 1-111 of human Thy-1 (amino acids 1-111 of SEQ ID NO:2) . The compound may be a chimeric polypeptide containing a polypeptide fragment of Thy-1 and a polypeptide fragment of an immunoglobulin. For example, an extracellular fragment of Thy- 1 is linked to a second polypeptide containing a constant region of IgG (SEQ ID NO: 11) to make a chimeric polypeptide such as rat Thy-1- Ig (SEQ ID NO:3) or human Thy-l-Ig (SEQ ID N0:4) . The invention also includes a polypeptide with at least 50% (preferably at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100%) sequence identity to SEQ ID NO: 3 or 4 and having the activity (i.e., inhibiting binding of Thy-1 to a Thy-1 ligand) of Thy-l-Ig.
A nucleic acid encoding a Thy-l-Ig polypeptide is also encompassed by the invention. For example, the nucleic acid encodes a polypeptide containing the amino acid sequence of rat Thy-l-Ig (SEQ ID NO: 3) or human Thy-l-Ig (SEQ ID NO:4) . Preferably, the nucleic acid contains a nucleotide sequence of SEQ ID NO: 5 or 6 or a degenerate variant thereof . TABLE 1: Components of rat Thy-l-Ig
Met Asn Pro Val lie Ser lie Thr Leu Leu Leu Ser Val Leu Gin Met Ser Arg Gly (signal sequence; SEQ ID NO:l2)
Gin Arg Val lie Ser Leu Thr Ala Cys Leu Val Asn Gin Asn Leu
Arg Leu Asp Cys Arg His Glu Asn Asn Thr Asn Leu Pro lie Gin His Glu
Phe Ser Leu Thr Arg Glu Lys Lys Lys His Val Leu Ser Gly Thr Leu Gly
Val Pro Gin His Thr Tyr Arg Ser Arg Val Asn Leu Phe Ser Asp Arg Phe lie Lys Val Leu Thr Leu Ala Asn Phe Thr Thr Lys Asp Glu Gly Asp Tyr
Met Cys Glu Leu Arg Val Ser Gly Gin Asn Pro Thr Ser Ser Asn Lys Thr lie Asn Val lie Arg Asp Lys Leu Val Lys Cys Gly Gly Ala Asp Leu Ser Ser Arg (rat Thy-1 amino acids 1-113; SEQ ID NO:l)
Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys
Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly
Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys (human IgG constant region; SEQ ID N0:11)
TABLE 2 : Mature Rat Thy-l-Ig
Gin Arg Val lie Ser Leu Thr Ala Cys Leu Val Asn Gin Asn Leu Arg Leu Asp Cys Arg His Glu Asn Asn Thr Asn Leu Pro lie Gin His Glu Phe Ser Leu Thr Arg Glu Lys Lys Lys His Val Leu Ser Gly Thr Leu Gly Val Pro Gin His Thr Tyr Arg Ser Arg Val Asn Leu Phe Ser Asp Arg Phe lie Lys Val Leu Thr Leu Ala Asn Phe Thr Thr Lys Asp Glu Gly Asp Tyr Met Cys Glu Leu Arg Val Ser Gly Gin Asn Pro Thr Ser Ser Asn Lys Thr He Asn Val He Arg Asp Lys Leu Val Lys Cys Gly Gly Ala Asp Leu Ser Ser Arg Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys (SEQ ID N0:3)
TABLE 3: Components of human Thv-l-Ig
Met Asn Leu Ala He Ser He Ala Leu Leu Leu Thr Val Leu Gin Val Ser Arg Gly (signal sequence; SEQ ID NO: 13)
Gin Lys Val Thr Ser Leu Thr Ala Cys Leu Val Asp Gin Ser Leu Arg Leu Asp Cys Arg His Glu Asn Thr Ser Ser Ser Pro He Gin Tyr Glu Phe Ser Leu Thr Arg Glu Thr Lys Lys His Val Leu Phe Gly Thr Val Gly Val Pro Glu His Thr Tyr Arg Ser Arg Thr Asn Phe Thr Ser Lys Tyr His Met Lys Val Leu Tyr Leu Ser Ala Phe Thr Ser Lys Asp Glu Gly Thr Tyr
Thr Cys Ala Leu His His Ser Gly His Ser Pro Pro He Ser Ser Gin Asn Val Thr Val Leu Arg Asp Lys Leu Val Lys Cys Glu Gly Ala Asp Leu Ser Ser Arg (human Thy-1 amino acids 1-113; SEQ ID NO: 2)
Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys (human IgG constant region; SEQ ID NO: 11)
TABLE A : Mature human Thy-l-Ig
Gin Lys Val Thr Ser Leu Thr Ala Cys Leu Val Asp Gin Ser Leu Arg Leu Asp Cys Arg His Glu Asn Thr Ser Ser Ser Pro He Gin Tyr Glu
Phe Ser Leu Thr Arg Glu Thr Lys Lys His Val Leu Phe Gly Thr Val Gly
Val Pro Glu His Thr Tyr Arg Ser Arg Thr Asn Phe Thr Ser Lys Tyr His
Met Lys Val Leu Tyr Leu Ser Ala Phe Thr Ser Lys Asp Glu Gly Thr Tyr
Thr Cys Ala Leu His His Ser Gly His Ser Pro Pro He Ser Ser Gin Asn Val Thr Val Leu Arg Asp Lys Leu Val Lys Cys Glu Gly Ala Asp Leu Ser
Ser Arg Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys
Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly
Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys (SEQ ID NO: )
TABLE 5 : DNA encoding rat Thy-l-Ig
ATG AAC CCA GTC ATC AGC ATC ACT CTC CTG CTT TCA GTC TTG CAG ATG TCC CGA GGA CAG AGG GTG ATC AGC CTG ACA GCC TGC CTG GTG AAC CAG AAC CTT CGA CTG GAC TGC CGT CAT GAG AAT AAC ACC AAC TTG CCC ATC CAG CAT GAG TTC AGC CTG ACC CGA GAG AAG AAG AAG CAC GTG CTG TCA GGC ACC CTG GGG GTT CCC CAG CAC ACT TAC CGC TCC CGC GTC AAC CTT TTC AGT GAC CGC T T ATC AAG GTC CTT ACT CTA GCG AAC TTC ACC ACC AAG GAT GAG GGC GAC TAC ATG TGT GAA CTT CGA GTC TCG GGC CAG AAT CCC ACA AGC TCC AAT AAA ACT ATC AAT GTG ATC AGA GAC AAG CTG GTC AAG TGT GGT GGC GCA GAT CTC TCG AGT AGA CCC AAA TCT TCT GAC AAA ACT CAC ACA TCC CCA CCG TCC GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGG GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA (SEQ ID NO: 5) TABLE 6 : DNA encoding human Thy-l-Ig Sequence
ATG AAC CTG GCC ATC AGC ATC GCT CTC CTG CTA ACA GTC TTG CAG GTC TCC CGA GGA CAG AAG GTG ACC AGC CTA ACG GCC TGC CTA GTG GAC CAG AGC CTT CGT CTG GAC TGC CGC CAT GAG AAT ACC AGC AGT TCA CCC ATC CAG TAC GAG TTC AGC CTG ACC CGT GAG ACA AAG AAG CAC GTG CTC ITT GGC ACT GTG GGG GTG CCT GAG CAC ACA TAC CGC TCC CGA ACC AAC TTC ACC AGC AAA TAC CAC ATG AAG GTC CTC TAC TTA TCC GCC TTC ACT AGC AAG GAC GAG GGC ACC TAC ACG TGT GCA CTC CAC CAC TCT GGC CAT TCC CCA CCC ATC TCC TCC CAG AAC GTC ACA GTG CTC AGA GAC AAA CTG GTC AAG TGT GAG GGC GCA GAT CTC TCG AGT AGA CCC AAA TCT TCT GAC AAA ACT CAC ACA TCC CCA CCG TCC GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGG GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA (SEQ ID NO: 6)
Inhibition of Thy-1 -associated endothelial cell proliferation to reduce angiogenesis can be used to treat cancers, e .g. , by reducing vascularization of a tumor. For example, the compound is locally administered to a mammal at the site of a tumor, e.g., a solid tumor, to reduce the blood supply to the neoplastic tissue, thereby inhibiting tumor growth. For example, therapeutic formulations may be administered by implantation at or near the tumor site or the site from which a tumor was excised. The compound may also be administered to mammals to treat rheumatoid arthritis, the lesions of which are commonly associated with neovascularization. For example, the therapeutic method may be carried out by locally administering the compound at the site of an arthritic lesion to reduce vascular endothelial cell proliferation, and in turn, the severity of the lesion.
The method may also be used to treat atherosclerosis by locally administering a compound that reduces Thy-1-associated endothelial cell proliferation (and thus, angiogenesis) to a mammal at the site of an atherosclerotic lesion. In addition to reducing Thy-1-associated endothelial cell proliferation by inhibiting or blocking crosslinking of cell surface Thy-1, angiogenesis may be inhibited by administering to a mammal a compound, e.g., a Thy-1 anti-sense nucleic acid, that inhibits expression of Thy-1 in an endothelial cell. For example, the antisense nucleic acid may have a nucleotide sequence that is complementary to DNA encoding amino acids 1-113 of Thy-1 (e.g., SEQ ID NO:1 or 2) . A compound that increases Thy-1 expression in an endothelial cell can be used to promote angiogenesis in a mammal. Angiogenesis can also be inhibited by administering to the mammal a compound, e.g., a Thy-1-specific antibody linked to a cytotoxic agent, that selectively destroys Thy-1 expressing endothelial cells.
A "Thy-1 ligand" is any protein or polypeptide that binds to the Thy-1. Such ligands can be soluble or expressed on a cell surface. Thy-1 ligands include any protein, polypeptide, or other molecule that binds to Thy-1, e.g. Thy-1 specific antibodies, Thy-1 specific antibody fragments, and Thy-1 binding peptides. Cell- surface Thy-1 interactions (e.g., those resulting in crosslinking of Thy-1 on the surface of a cell) may by homotypic, i.e., a Thy-1 molecule on the surface of one cell may interact with a Thy-1 molecule on the surface of another cell. Such homotypic interactions may result in crosslinking of endothelial cell surface Thy-1 molecules which potentiates an intracellular signal which culminates in cell proliferation
As used herein, the term "block" means to interfere either partially or completely with a binding interaction between two molecules, e.g., the binding of Thy-1 to a Thy-1 ligand. As used herein, the term "antibody" encompasses not only an intact antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule. For administration to human patients, antibodies or antibody fragments can be humanized by methods known in the art . Antibodies or antibody fragments with a desired binding specificity can also be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA) . As used herein, the term "Fab" or "antibody fragment" refers to the portion of an antibody molecules which includes the variable region of the heavy chain and/or light chain, and which exhibits antigen-binding activity.
By "purified DNA" is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation) .
Thy-1-encoding DNA or Thy-1 antisense DNA may be operably linked to regulatory sequences, e.g., a promoter. Preferably, the promoter is vascular cell-specific, more preferably, it is vascular endothelial cell-specific. By "operably linked" is meant that a coding sequence and a regulatory sequence (s) are connected in such a way as to permit expression of the DNA when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence (s) . By "promoter" is meant a minimal DNA sequence sufficient to direct transcription. Promoters may be constitutive or inducible. As used herein, "vector" means a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid.
As used herein, "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions . When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10- nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .
As used herein, "Thy-1-specific antibody" means an antibody that binds to a Thy-1 protein or polypeptide and displays no substantial binding to other naturally-occurring proteins other than those sharing the same antigenic determinants as Thy-1. The term includes polyclonal and monoclonal antibodies .
As used herein, "substantially pure protein" means a protein separated from components that naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure Thy-l protein can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding an Thy-1 polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically- synthesized protein or a recombinant protein produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure proteins include those having sequences derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes .
As used herein, "fragment", as applied to a protein, means at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent form the following detailed description and from the claims. Brief Description of the Drawing
Fig. 1 is a bar graph showing induction of Thy-1-associated endothelial cell proliferation (as measured by incorporation of thymidine) by crosslinking of endothelial cell surface Thy-1 molecules using a Thy-1-specific antibody. Incubation of endothelial cells with a control antibody (anti-CD44) resulted in little or no endothelial cell proliferation.
Detailed Description Thy-1 is expressed by vascular endothelial cells during angiogenesis in an adult animal. For example, Thy-1 expression was found to be upregulated during physiological angiogenesis, e.g., during pregnancy, as well as during pathological angiogenesis, e.g., ischemia, cancer, and blood vessel disease. Thy-1 expression was detected by an increase in the level of Thy-1 mRNA as well Thy-1 protein. Upregulation was found to be induced by exposure of endothelial cells to inflammatory cytokines, e.g., TNF-α or IL-1/3. Proliferation of vascular endothelial cells is a critical step in the formation of blood vessels . Engagement of endothelial cell surface Thy-1 with a Thy-1 ligand initiates a cascade of signal transduction events that culminate in endothelial cell proliferation leading to angiogenesis. Crosslinking of Thy-1 on the surface of an vascular endothelial cell (e.g., by a bivalent Thy-1-specific antibody) was found to induce proliferation of the Thy-1-expressing human microvascular endothelial cells (Fig. 1) .
The animal models described below represent a spectrum of conditions that are accompanied by new blood vessel formation. Stimuli such as mechanical injury (carotid injury model) , tumorigenesis (eye model) , and ischemia (renal artery ligation model) are accompanied by the release of inflammatory cytokines such as IL- 1/3 and tumor necrosis factor-α. In each model tested, Thy-1 was highly expressed in endothelial cells of small blood vessels formed after the pathologic stimuli or during physiological angiogenesis. Animal Models
Thy-1 expression in endothelial cells was studied in four art-recognized rat models of angiogenesis. The model for renal artery stenosis was performed using known methods, e.g., the method of Ilich et al. (Ilich et al., 1979, Circ. Res., 45:579-582). The main renal artery was partially occluded by the application of a silver clip. In "sham" -operated rats, the silver clip was removed. Ureters were harvested 5 days after ligation.
Implantation of a tumor in the cornea was performed according to standard methods (e.g., the method of Gimbrone et al., 1974, J. Nat. Cancer Inst. 52:413-427). Corneas were harvested 10 days after tumor implantation.
Balloon angioplasty of the carotid artery was performed using well-known methods of angioplasty (e.g., the method of Clowes et al., 1983, Lab. Invest. 49:327-333 or Clowes et al., 1983, Lab. Invest. 49:208-215) . Carotid arteries were harvested from adult male Sprague-Dawley rats 14 days after they had been subjected to balloon injury.
In the model for neovascularization in the early pregnant uterus, uteruses (including placentas and embryos) were harvested after 6-18 days gestation according to known methods (e.g., Klauber et al., 1997, Nat. Med. 3:443-446). Immunocvtochemistry
All tissues were fixed with 4% paraformaldehyde and processed for paraffin embedding in an automated system (Hypercenter XP, Shandon Scientific, Pittsburgh, PA) . Immunocytochemical staining was performed using standard techniques . Paraffin was removed from the tissue sections, and the tissue sections were incubated with 10% normal goat serum for 30 minutes at room temperature to reduce nonspecific binding. The sections were incubated with one of four primary antibodies for 1 hour at room temperature and then overnight at 4°C, after which they were rinsed twice with high-salt PBS (0.5M NaCl) and once with regular PBS for 5 minutes (each wash) . The sections were then incubated with one of four secondary antibodies for 1 hour at room temperature. Primary/secondary antibody combinations were as follows: (a) mouse anti-rat Thy-1 antibody (clone 0X-7, Pharmingen, San Diego, CA) at 1:300 dilution and biotinylated goat anti-mouse IgGi (Amersham, Arlington Heights, IL) at 1:100 dilution; (b) mouse anti-rat Thy-1 antibody (clone HIS51, Pharmingen) at 1:100 dilution and biotinylated goat anti-mouse IgG2a (Amersham) at 1:100 dilution; (c) rabbit anti-human von Willebrand factor antibody (DAKO, Carpinteria, CA) at 1:1000 dilution and biotinylated goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA) at 1:2000 dilution; and (d) rat anti-mouse CD31 (PECAM-1) antibody (Pharmingen) at 1:100 dilution and biotinylated rabbit anti-rat IgG (H+L) (Vector Laboratories) at 1:100 dilution. The tissue sections were then rinsed twice with high-salt PBS and once with standard PBS for 5 minutes (each wash) and incubated with avidin-biotin complex (ABC reagent, Vector Laboratories) at 1:100 dilution for 1 hour at room temperature. After a wash with PBS, the sections were incubated with 3,3'- diaminobenzidine or 3, 3' -diaminobenzidine plus nickel sulfate in PBS- H202 to detect antibody binding. Color development (i.e., brown or blue/black, for 3 , 3 ' -diaminobenzidine or 3, 3' -diaminobenzidine plus nickel sulfate, respectively) indicated positive staining. To control for anti-Thy-1 antibody specificity, tissue was incubated with anti-Thy-1 antibody (OX-7) that had been preabsorbed with 25 μg/ml Thy-l-Ig fusion protein. Preabsorption of the Thy-1- specific antibody with Thy-l-Ig fusion protein blocked all staining. Counter-staining was performed with a solution of 1% methyl green. Construction and Expression of Rat Thv-l-Iq Fusion Protein A rat Thy-l-Ig expression plasmid (pThy-1-Ig) was constructed by cloning a DNA encoding an extracellular fragment of rat Thy-1 (residues Glnl-Glyll3; SEQ ID N0:1) (Seki et al., 1985, Fed. Proc. 44:2865-2869) in frame to a DNA encoding a human IgGi constant region, e.g., a fragment of IgG containing the hinge, CH2 and CH3 regions (SEQ ID NO: 11) . The rat Thy-1 and human IgGi DNAs were obtained by polymerase chain reaction with templates from rat brain cDNA (Clontech, Palo Alto, CA) and human spleen cDNA (Clontech) , respectively. The Thy-1 forward primer, 5'-GCG CAG AAG CTT ATT GGC ACC ATG AAC CCA GTC ATC-3' (SEQ ID NO:7) , corresponded to the N-terminus of the rat Thy-1 signal sequence plus a Hindlll restriction endonuclease site. The Thy-1 reverse primer, 5' -CCT CGA GAG ATC TCC ACC ACA CTT GAC CAG CTT GTC-3' (SEQ ID NO: 8) , corresponded to residues Aspl06-Glyll3 of rat Thy-1 plus a Bglll restriction endonuclease site. The IgGj constant region forward primer, 5' -A GAT CTC TCG AGT AGA CCC AAA TCT TCT GAC AAA ACT CAC ACA TCC CCA CCG TCC CCA-3' (SEQ ID NO: 9), corresponded to residues
Pro227-Pro243 of the human IgGi hinge region plus a Bglll restriction endonuclease site. Three mutations were introduced in DNA encoding the IgG hinge region to change cysteines to serines . The IgGi constant region reverse primer, 5' -TCT AGA CGG CGG TCG CAC TCA TTT AAC-3' (SEQ ID NO:10), corresponded to the C-terminus of the human IgGi CH3 regions plus an Xbal restriction endonuclease site . The PCR products of the Thy-1 and human IgGx constant regions were digested with Hindlll/Bglll and Bglll/Xbal, respectively. The digested fragments were ligated to the Hindlll/Xbal site of pCNDA-lamp (Invitrogen, San Diego, CA) resulting in the final expression plasmid pThy-1-Ig. The same strategy was used to make human Thy-l-Ig (SEQ ID NO:4) .
Thy-l-Ig fusion proteins were transiently expressed in COS cells and purified from the culture medium, using standard methods, e.g., by protein G affinity chromatography (Pharmacia, Uppsala, Sweden) . The purity of the protein was evaluated by SDS-PAGE, and its concentration was determined by the DC-protein assay (Biorad,
Hercules, CA) . Presence of a Thy-1 epitope on the fusion protein was determined by Western blot with a biotin-conjugated mouse anti-rat Thy-1 primary antibody (clone OX-7) and a streptavidin-conjugated alkaline phosphatase secondary antibody (Pharmingen, San Diego, CA) . Cell Culture
Human microvascular endothelial cells were obtained from Clonetics Corporation (San Diego, CA) and grown in microvascular endothelial cell growth medium (Clonetics, No. CC-3125) . Cells were passaged every 4-5 days. Cells from passages 6-7 were used for all experiments. After the cells had grown to 80% confluence, they were placed in quiescence medium (endothelial cell basal medium-2, serum free; Clonetics, No. CC-3156) overnight. RNA Extraction and Northern Blot Analysis
Total RNA was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride using methods well known in the art. Total RNA from cells was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose paper. The filters were hybridized with a randomly primed 32P-labeled human Thy-1 full-length cDNA. The hybridized filters were then washed in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS solution at 55°C and autoradiographed on Kodak XAR film at -80°C. The blots were hybridized with an oligonucleotide probe specific for 18S ribosomal RNA to correct for differences in RNA loading. Filters were scanned on a PhosphorImager running the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) . Thy-l-Ig chimera
As described above, a recombinant chimeric fusion protein Thy-l-Ig was made by cloning DNA encoding the extracellular portion of rat Thy-1 (residues Glnl-Glyll3; SEQ ID N0:1) to DNA encoding the human IgG constant region (hinge, CH2, and CH3; SEQ ID NO:11) and expressed transiently in COS cells . Three cysteine residues in the IgG hinge region were mutated to serines to prevent nonspecific disulfide linkage with the Cyslll in Thy-1.
Chimeric proteins were purified by protein G affinity chromatography and characterized by SDS-polyacrylamide gel electrophoresis . The Coomassie blue-stained gel revealed bands of the expected size: one band at 104 kDa (nonreducing conditions) and one band at 52 kDa (under reducing conditions) . The molecular weight of a Thy-l-Ig monomer approximated the sum of rat Thy-1 at 25 kDa and the human IgFc fragment at 25 kDa. Western blot analysis with OX-7 confirmed the presence of Thy-1 epitopes in both the 104 kDa and the 52 kDa bands. These experiments indicate that Thy-l-Ig is a disulfide-linked homodimer (104 kDa band under nonreducing conditions) . Thyl-1-Ig was used to verify the specificity of immunostaining of endothelial cells for expression of Thy-1. Preabsorption of a Thy-1-specific antibody (e.g., OX-7) with a Thy-l- Ig fusion protein prior to contacting the tissue sections with the OX-7 antibody solution eliminated all Thy-1 immunoreactivity. These data confirm the specificity of the Thy-1 immunostaining.
Immunohistochemical analysis of Thy-1 Expression after blood vessel injury
Thy-1 immunoreactivity was examined in adult rat tissue during angiogenensis . Expression of Thy-1 was evaluated by immunostaining with an antibody specific for rat Thy-1 (clone OX-7) 14 days after balloon injury to the rat carotid artery.
Following balloon injury to the carotid artery, new blood vessels form in the tunica adventitia. No Thy-1 immunoreactivity was visible in the endothelium of an uninjured carotid artery or in the few small blood vessels of the tunica adventitia. After injury, strong staining with OX-7 (as well as with another Thy-1-specific antibody, HIS51) was visible in many of the small blood vessels of the tunica adventitia. Immunohistochemical analysis of Thv-1 expression after renal artery ligation
In the second rat model of angiogenesis , renal arteries were clipped for 5 days, to induce new blood vessel formation in the periureteric area using known methods, (e.g., the method of Ilich et al., 1979, Circ. Res. 45:579-582 or Hollenberg et al., 1985,
Investigative Radiology, 20:58-61). Strong Thy-1 immunostaining was observed in small blood vessels beneath the ureteral epithelium in a tissue sample taken from a rat with a clipped renal artery but not in a sample taken from a control rat (without a clipped renal artery) .
Immunostaining for von Willebrand factor in an adjacent tissue section from the experimental rat (with clipped artery) was also detected in some small blood vessels. The small vessels expressing Thy-1 were distinct from those expressing von Willebrand factor.
This differential pattern of Thy-1 expression also was also observed in the other angiogenesis models described herein.
Immunohistochemical analysis of Thv-1 expression after glioblastoma placement in a rat cornea Implantation of a tumor in a rat cornea is a well established model of pathologic angiogenesis . Placement of a glioblastoma in the anterior chamber of the normally avascular rat cornea resulted in new blood vessel formation at the site. Corneas harvested 10 days after implantation showed strong Thy-1 immunoreactivity in the small blood vessels of the invading tumor in the experimental sample. In contrast, Thy-1 staining was absent in the control sample.
Immunohistochemical analysis of Thv-1 expression during rat embrvogenesis The fourth model of adult angiogenesis is pregnancy. Using a rat model, new blood vessels which formed in the rat uterus during pregnancy were evaluated for Thy-1 expression. At days 8 and 11 of pregnancy, strong Thy-1 staining was observed in small blood vessels of the uterus . Absence of Thy-1 Immunoreactivity During Blood Vessel Development in
Rat Embryos
Thy-1 expression during physiologic vasculogenesis and angiogenesis in developing rat embryos was also examined.
Vasculogenesis refers to origination of the vascular system, i.e., development of new blood vessels from angioblasts, whereas angiogenesis refers to the growth of new blood vessels from existing blood vessels .
Adjacent rat embryonic tissue sections (at embryonic day 11) were stained for von Willebrand factor (as a positive control) and Thy-1. Strong staining was observed in the large blood vessels
(e.g., the dorsal aorta) and in small vessels of the yolk sac with antibody specific for von Willebrand factor. In contrast, no Thy-1 immunostaining was detectable in the aorta or the small vessels of the yolk sac. Thy-1 immunoreactivity was also not detected in the endothelium of large or small blood vessels at embryonic days 10 and 12-18. Thy-1 immunoreactivity was detected in the developing brain and, as described above, in small blood vessels of the pregnant uterus. These data indicate that Thy-1 is not expressed in the vasculature of a developing embryo. Upregulation of Thv-1 mRNA by Cytokines but Not Growth Factors
The effect of inflammatory cytokines and growth factors on Thy-1 expression in cultured microvascular endothelial cells was examined in cultured human microvascular endothelial cells . Cells were made quiescent for 24 hours before stimulation with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and the cytokines IL-10 and tumor necrosis factor-α (TNF-α) . RNA was harvested 48 hours after incubation with a cytokine or growth factor. Northern analysis was performed with 7.5 μg of total RNA per lane. After electrophoresis, RNA was transferred to nitrocellulose filters and hybridized with 32P-labelled human Thy-1 cDNA. A single Thy-1 transcript was visible at 1.8 kB (the filters were also hybridized with a probe specific for 18S ribosomal RNA to verify equivalent sample loading) .
Negligible Thy-1 mRNA expression was detected in control microvascular endothelial cells, whereas incubation of endothelial cells with IL-1/3 increased Thy-1 mRNA expression by 8-fold at 24 hours after stimulation. Similarly, stimulation of endothelial cells with tumor necrosis factor-α resulted in 5- and 14-fold in Thy-1 mRNA expression at 8 and 24 hours (respectively) after stimulation. In contrast, VEGF or bFGF did not induce Thy-1 mRNA expression in these cells, nor did platelet-derived growth factor (PDGF) and transforming growth factor-/? (TGF-/3) .
Since macrophages are an important source of inflammatory cytokines, the presence of macrophages in tissues undergoing angiogenesis was evaluated. To show that Thy-1 expression during angiogenesis occurred in the context of inflammation, rat carotid arteries were subjected to balloon injury and tissue sections stained with rat macrophage-specific antibody ED-1. Strong staining for macrophages was detected adjacent to newly formed blood vessels in the tunica adventitia. Proliferating cell nuclear antigen (PCNA) staining indicated that the cells were proliferating. Small blood vessels of the tunica adventitia also stained positively for Thy-1 after injury to the blood vessel wall. Inhibition of Thy-1-associated angiogenesis
The data described herein indicate that Thy-1 is directly involved promoting angiogenesis in adult mammals. Thus, angiogenesis in an animal can be inhibited by administering to the animal a therapeutically effective amount of a composition which blocks engagement of endothelial cell surface Thy-1 with a Thy-1 ligand, selectively reduces Thy-1 expression in endothelial cells, or destroys Thy-1 expressing endothelial cells.
Monomeric peptides that bind to endothelial cell surface Thy-1 (e.g., soluble Thy-1 or fragments thereof (e.g., amino acids 1- 113 of Thy-1) , or a chimeric polypeptide containing a portion of Thy- 1 or another Thy-1 ligand linked to a non-Thy-1 peptide) can be used to inhibit angiogenesis by blocking the binding of Thy-1 to a Thy-1 ligand. The Thy-1 ligand may be present on a second cell or may be soluble. Synthetic ligands, antibodies or antigen-binding fragments thereof, which are specific for either the extracellular domain of Thy-1 or a ligand to which Thy-1 binds, are also useful to block Thy- 1 crosslinking. Such peptides will ordinarily be at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 contiguous amino acids in length. Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering, e.g., cloning and expression of a fragment of Thy-1 cDNA.
Methods of killing target cells (e.g. Thy-1-expressing endothelial cells) are well known in the art. For example, a composition containing a cytotoxic Thy-binding compound, e.g., a Thy- 1 specific antibody coupled to a cytotoxic agents such as ricin A chain, abrin A chain, modeccin A chain, gelonin, melphalan, bleomycin, adriamycin, daumomycin, or pokeweed anti-viral proteins (PAP, PAPII, PAP-S) , may be administered to the animal to kill Thy-1- expressing cells. Numerous other cytotoxic agents, including radioisotopes or chemotherapeutic agents, can be coupled to target- specific antibodies by well-known techniques, and delivered to the animal (either systemically or locally) to specifically destroy targeted tissue (e.g., Thy-1 expressing endothelial cells). See, e.g., Blattler et al., U.S. Patent No. 4,542,225. Therapeutic compositions are typically administered in a pharmaceutically acceptable carrier (e.g., physiological saline). Carriers are selected on the basis of mode and route of administration and standard pharmaceutical practice. A therapeutically effective amount of a therapeutic composition (e.g., Thy-l-Ig) is an amount which is capable of producing a medically desirable result in a treated animal . As is well known in the medical arts, dosage for any one animal depends on many factors, including the animal's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently (e.g., a Thy-l-Ig fusion protein) is 0.1 to 100 mg/kg body weight. The compositions of the invention can be administered locally i.e., at the site of angiogenesis or systemically. Administration will generally be parenterally, e.g., intravenously. The compositions may also be administered directly to a tissue site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in a blood vessel. It is expected that an intravenous dosage of approximately 1 to 100 μmoles of the peptide of the invention would be administered per kg of body weight per day. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal . Other methods of delivery, e.g., liposomal delivery or diffusion from a device impregnated with therapeutic compound, are known in the art. The anti-Thy-1 antibodies useful in the present invention, can be obtained by techniques well known in the art. Such antibodies can be polyclonal or preferably monoclonal . Polyclonal antibodies can be obtained, for example, by the methods described in Ghose et al., Methods in Enzymology, Vol. 93, 326-327, 1983. For example, soluble Thy-1 or an antigenic fragment thereof, can be used as the immunogen to stimulate the production of Thy-1-reactive polyclonal antibodies in the antisera of animals such as rabbits, goats, sheep, rodents and the like. The monoclonal antibodies can be obtained by the process described by Milstein and Kohler, 1975, Nature 256:495- 97. Anti-Thy-1 antibodies are well known in the art and can be purchased commercially, e.g., OX-7 (Pharmingen, San Diego, CA) and HIS51 (Pharmingen, San Diego, CA) . Antibody fragments, e.g., a fragment containing only the constant region, for use in construction of chimeric polypeptides are generated using methods well known in the art.
Analogs of the above peptides may also be used to block Thy- 1 binding to a ligand. Analogs can differ from the native peptides of Thy-1 by amino acid sequence, or by modifications which do not affect the sequence, or both. Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivitization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.
To render the therapeutic peptides less susceptible to cleavage by peptidases, the peptide bonds of a peptide may be replaced with an alternative type of covalent bond (a "peptide mimetic") . Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable, and thus more useful as a therapeutic. Such mimetics, and methods of incorporating them into polypeptides, are well known in the art. Similarly, the replacement of an L-amino acid residue with a D-amino acid is a standard way of rendering the polypeptide less sensitive to proteolysis. Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4, -dinitrophenyl. Peptides may be administered to a subject intravenously in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. Modulation of Thy-1 expression
An increase in Thy-1 expression was detected in endothelial cells participating in the process of new blood vessel formation. Thus, manipulation of Thy-1 expression is a therapeutic strategy to prevent or inhibit angiogenesis. For example, administration of compounds which inhibit Thy-1 expression are useful to treat conditions characterized by pathologic angiogenesis. Likewise, administration of compounds which increase expression of Thy-1 would be useful to promote angiogenenis, e.g., to treat ischemia and arteriosclerosis .
To modulate expression of Thy-1 in an endothelial cell, a Thy-1 DNA or Thy-1 antisense DNA may be introduced into target cells e.g., vascular endothelial cells, of the subject animal by standard vectors and/or gene delivery systems. Expression of exogenous Thy-1 DNA in an endothelial cell would increase the amount of Thy-1 protein on the cell's surface. Expression of Thy-1 antisense DNA in an endothelial cell decreases the amount of Thy-1 protein on the cell's surface by inhibiting translation of endogenous Thy-1 mRNA. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenovirus, and adeno-associated virus, among others . A therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal, e.g., increased or decreased expression of Thy-1. As is well known in the medical arts, dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, sex, and general health, the particular compound to be administered, the time and route of administration, and other drugs being administered concurrently. Dosages for therapeutic nucleic acids will vary, but a preferred dosage for intravenous administration is from approximately 106 to 1022 copies of the nucleic acid molecule .
In addition to the foregoing, compounds which modulate Thy-1 expression by targeting protein synthesis, proper post-translational modification, or translocation and anchoring of the Thy-1 protein product with respect to the cellular membrane can be used. For example, a phospholipase can be administered to release Thy-1 from a plasma membrane by hydrolyzing its glycosylphosphatidyl-inositol anchor. As shown in Fig. 1, engagement of two or more Thy-1 molecules on the surface of an endothelial cell triggers proliferation. Proliferation of endothelial cells contributes to angiogenesis. A monomeric fragment of Thy-1 or a monomeric Thy-1 fusion protein, e.g., Thy-l-Ig, can be used to inhibit Thy-1 binding to a Thy-1 ligand (e.g., a cell surface or soluble Thy-1 ligand) . Decreasing or preventing endothelial cell surface Thy-1 from associating with or binding to a Thy-1 ligand inhibits endothelial cell proliferation which, in turn, inhibits angiogenesis.
The Thy-1 fusion protein can also be used to detect Thy-1 specific binding by antibodies. For example, the fusion protein can be added to a Thy-1-specific immunostaining assay, to compete with native Thy-1 for binding to the antibody. A reduction in Thy-1 detection would indicate that the antibody binds Thy-1 specifically. A Thy-1 fusion protein is also useful in purifying substances which bind Thy-1. For example, a mixture containing a Thy-1-binding substance is contacted with a sample of immobilized Thy-1 fusion protein, the unbound components of the mixture are then separated from the bound substance, and the Thy-1 binding substance is recovered from the immobilized Thy-1 fusion protein. Screening assays
A screening assay to identify compounds which are capable of modulating angiogenesis is carried out as follows . A sample of endothelial cells, e.g., cultured microvascular endothelial cells, can be incubated in the presence of a candidate compound. A sample of control cells is incubated in the absence of the compound. Each sample of cells is evaluated for the expression of Thy-1. For example, each sample of cells can be incubated with a Thy-1-specific antibody and the cells evaluated for binding of the antibody by methods well known in the art, e.g., immunofluorescent staining. The amount of antibody binding correlates with the level of expression of Thy-1. Thy-1 expression can also be measured at the level of gene transcription. For example, Thy-1 transcripts can be measured by Northern blotting techniques using Thy-1-specific DNA probes or by PCR using Thy-1-specific DNA primers. A decrease in the amount of Thy-1 expression in cells contacted with a candidate compound compared to the amount in untreated cells indicates that the candidate compound is capable of inhibiting the expression of Thy-1 in endothelial cells (and of inhibiting angiogenesis) . Likewise, an increase in the amount of Thy-1 expression in treated cells, compared to the amount in untreated cells, indicates that the candidate compound is capable of upregulating the expression of Thy-1 in endothelial cells (and therefore promoting angiogenesis) .
Based on the observation that Thy-1-associated endothelial cell proliferation can be induced by crosslinking of endothelial cell surface Thy-1 molecules using a Thy-1-specific antibody, monovalent Thy-1 ligands (e.g., small non-peptide compounds which bind to Thy-1) which are capable of inhibiting undesired angiogenesis can be identified by screening for those that prevent or inhibit crosslinking of Thy-1 on the surface of an endothelial cell. A sample of endothelial cells, e.g., cultured microvascular endothelial cells, can be incubated in the presence of a conventional bivalent Thy-1 specific antibody, i.e., Thy-1 specific monoclonal antibody of the IgG isotype, and in the presence a candidate compound. A sample of control cells is incubated in the presence of anti-Thy-1 but in the absence of the compound. Each sample of cells is evaluated for proliferation. A decrease in the amount of proliferation the sample of cells cultured in the presence of anti-Thy-1 and a candidate compound compared to the amount of proliferation of the sample of cells cultured in the presence of anti-Thy-1 in the absence of the candidate compound indicates that the compound inhibits Thy-1 associated endothelial cell proliferation. Other Embodiments
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:
I. A method for inhibiting angiogenesis in a mammal, comprising administering to said mammal a compound that inhibits Thy- 1-associated proliferation of an endothelial cell.
2. The method of claim 1, wherein said endothelial cell is a vascular endothelial cell .
3. The method of claim 1, wherein said compound is a soluble monovalent Thy-1 ligand.
4. The method of claim 3, wherein said compound is a polypeptide comprising an extracellular fragment of Thy-1.
5. The method of claim 4 , wherein said fragment comprises SEQ ID N0:1.
6. The method of claim 4 , wherein said fragment comprises SEQ ID NO:2.
7. The method of claim 4, wherein said polypeptide is monovalent Thy-l-Ig.
8. The method of claim 7, wherein said Thy-l-Ig comprises of the amino acid sequence of SEQ ID NO: 3 or SEQ ID N0:4.
9. The method of claim 1, wherein said mammal is characterized as having a tumor.
10. The method of claim 9, wherein said compound is locally administered to said mammal at the site of said tumor.
II. The method of claim 1, wherein said mammal is characterized as suffering from rheumatoid arthritis.
12. The method of claim 11, wherein said compound is locally administered to said mammal at the site of an arthritic lesion.
13. The method of claim 1, wherein said mammal is characterized as having atherosclerosis .
14. The method of claim 13 , wherein said compound is locally administered to said mammal at the site of an atherosclerotic lesion.
15. A method for inhibiting angiogenesis in a mammal, comprising administering to said mammal a compound that inhibits expression of Thy-1 in an endothelial cell.
16. The method of claim 15, wherein said compound comprises a Thy-1 anti-sense nucleic acid.
17. A method of inhibiting angiogenesis in a mammal, comprising administering to said mammal a compound that selectively destroys a Thy-1 expressing endothelial cell.
18. The method of claim 17, wherein said compound comprises a Thy-1-specific antibody linked to a cytotoxic agent.
19. A method for promoting angiogenesis in a mammal, comprising administering to said mammal a compound that increases
Thy-1 expression in an endothelial cell.
20. A chimeric protein comprising a polypeptide fragment of Thy-1 and a polypeptide fragment of an immunoglobulin.
21. The method of claim 20, wherein said compound is a polypeptide comprising an extracellular fragment of Thy-1.
22. The method of claim 21, wherein said fragment comprises SEQ ID N0:1.
23. The method of claim 21, wherein said fragment comprises SEQ ID NO:2.
24. The method of claim 21, wherein said polypeptide is
Thy-l-Ig.
25. The method of claim 24, wherein said Thy-l-Ig comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
26. A substantially pure DNA comprising a sequence encoding a Thy-l-Ig polypeptide.
27. The DNA of claim 26, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
28. The DNA of claim 27, wherein said DNA comprises the sequence of SEQ ID NO: 5 or 6.
29. A substantially pure DNA comprising a nucleotide sequence having at least 50% sequence identity to SEQ ID NO: 5 or 6, said nucleotide sequence encoding a polypeptide having the biological activity of a Thy-l-Ig polypeptide.
30. A substantially pure DNA comprising (a) the sequence of SEQ ID NO: 5 or 6 or (b) a degenerate variant thereof.
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