Anti-angiogenic proteins and methods of use thereof

Abstract

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

What is claimed is:  
     
         1 . An isolated protein, selected from the group consisting of: the NC1 domain of the α1 chain of Type IV collagen, the NC1 domain of the α2 chain of Type IV collagen, or the NC1 domain of the α3 chain of Type IV collagen, or a fragment, analog, derivative or mutant thereof, wherein the protein, fragment, analog, derivative or mutant thereof has anti-angiogenic properties.  
     
     
         2 . The isolated protein of  claim 1 , wherein the protein is a monomer.  
     
     
         3 . The isolated protein of  claim 2 , wherein the protein is Arresten.  
     
     
         4 . The isolated protein of  claim 2 , wherein the protein is Canstatin.  
     
     
         5 . The isolated protein of  claim 2 , wherein the protein is Tumstatin.  
     
     
         6 . A multimer of the protein of  claim 1 , or of a fragment, analog, derivative or mutant thereof, wherein the multimer has anti-angiogenic activity.  
     
     
         7 . A chimeric protein, comprising one or more of the proteins of  claim 1 , or of a fragment, analog, derivative or mutant thereof, wherein the chimeric protein has anti-angiogenic activity.  
     
     
         8 . The chimeric protein of  claim 7 , further comprising at least one protein molecule selected from the group consisting of: endostatin or fragments thereof, angiostatin or fragments thereof, restin or fragments thereof, apomigren or fragments thereof, or other anti-angiogenic proteins, or fragments thereof.  
     
     
         9 . A composition comprising, as a biologically active ingredient, one or more of the proteins of  claim 1 .  
     
     
         10 . The composition of  claim 9 , and a pharmaceutically-compatible carrier.  
     
     
         11 . A composition comprising, as a biologically active ingredient, one or more of the proteins of  claim 1 , and further comprising at least one protein molecule selected from the group consisting of: endostatin or fragments thereof, angiostatin or fragments thereof, restin or fragments thereof, apomigren or fragments thereof, or other anti-angiogenic proteins, or fragments thereof.  
     
     
         12 . A composition comprising, as a biologically active ingredient, the multimer of  claim 6 .  
     
     
         13 . A composition comprising, as a biologically active ingredient, the chimeric protein of  claim 7 .  
     
     
         14 . An isolated polynucleotide encoding the protein of  claim 1 , or a fragment, analog, derivative or mutant thereof, wherein the protein or fragment, analog, derivative or mutant thereof has anti-angiogenic activity.  
     
     
         15 . An isolated polynucleotide of  claim 14 , wherein the polynucleotide is operably linked to an expression control sequence.  
     
     
         16 . A host cell transformed with the polynucleotide of  claim 15 .  
     
     
         17 . The host cell of  claim 16 , where the cell is selected from the group comprising bacterial, yeast, mammalian, insect or plant cells.  
     
     
         18 . An isolated polynucleotide encoding the protein of  claim 3 .  
     
     
         19 . An isolated polynucleotide encoding the protein of  claim 4 .  
     
     
         20 . An isolated polynucleotide encoding the protein of  claim 5 .  
     
     
         21 . A process for producing a protein encoded by the polynucleotide of  claim 14 , wherein the process comprises: 
 (a) growing a culture of a host cell transformed with the polynucleotide of  claim 14 , where the host cell is selected from the group comprising bacterial, yeast, mammalian, insect or plant cells; and    (b) purifying the protein from the culture;    thereby producing the protein encoded by the polynucleotide of  claim 14 .    
     
     
         22 . An isolated polynucleotide produced according to the process of: 
 (a) preparing one or more polynucleotide probes that hybridize under conditions under moderate stringency to the polynucleotide of  claim 14;     (b) hybridizing said probe(s) to mammalian DNA; and    (c) isolating the DNA polynucleotide detected with the probe(s);    wherein the nucleotide sequence of the isolated polynucleotide corresponds to the nucleotide sequence of the polynucleotide of  claim 14 .    
     
     
         23 . A process for providing a mammal with an anti-angiogenic protein, the process comprising introducing mammalian cells into a mammal, said mammalian cells having been treated in vitro to insert therein the polynucleotide of  claim 14  and expressing in vivo in said mammal a therapeutically effective amount of the anti-angiogenic protein in an amount sufficient to inhibit angiogenic activity in the mammal.  
     
     
         24 . The method of  claim 23  wherein the expression of the anti-angiogenic protein is transient expression.  
     
     
         25 . The process of  claim 23 , wherein the cells are chosen from the group consisting of: blood cells, TIL cells, bone marrow cells, vascular cells, tumor cells, liver cells, muscle cells, fibroblast cells.  
     
     
         26 . The process of  claim 25 , wherein the polynucleotide is inserted into the cells by a viral vector.  
     
     
         27 . Antibodies that specifically bent to an isolated protein of  claim 1 , analog, derivative homolog or mutant thereof.  
     
     
         28 . A method for inhibiting angiogenic activity in mammalian tissue, the method comprising contacting the tissue with a composition comprising one or more of the following: one or more of the isolated proteins of  claim 1 , a fragment, analog, derivative or mutant thereof, of the isolated protein of  claim 1 , a multimer of the protein of  claim 1 , a multimer of the fragment of  claim 1 , a chimeric protein comprising one or more of the proteins of  claim 1 , or a chimeric protein comprising a fragment of the protein of  claim 1 .  
     
     
         29 . The method of  claim 28 , wherein the disease is selected from the group comprising angiogenesis-dependent cancers, benign tumors, rheumatoid arthritis, diabetic retinopathy, psoriasis, ocular angiogenesis diseases, Osler-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemopheliac joints, angiofibroma, wound granulation, intestinal adhesions, atherosclerosis, scleroderma, hypertrophic scars, cat scratch disease,  Heliobacter pylori  ulcers, dialysis graft vascular access stenosis, contraception and obesity.  
     
     
         30 . The method of  claim 29 , wherein the disease is cancer.  
     
     
         31 . A method of using the composition of  claim 28  to inhibit a disease characterized by angiogenic activity, the method comprising administering to a patient with the disease, the composition in conjunction with radiation therapy, chemotherapy, or immunotherapy.  
     
     
         32 . A polypeptide comprising amino acid 2 to amino acid 125 of SEQ ID NO:10, the polypeptide having anti-angiogenic activity.  
     
     
         33 . A polynucleotide encoding the polypeptide of  claim 32 .  
     
     
         34 . A polypeptide comprising amino acid 125 to amino acid 245 of SEQ ID NO:11, the polypeptide having anti-angiogenic activity.  
     
     
         35 . A polynucleotide encoding the polypeptide of  claim 34 .  
     
     
         36 . An anti-angiogenic fragment of the NC1 domain of the α1 chain of Type IV collagen, the fragment made by the process of PCR cloning.  
     
     
         37 . An anti-angiogenic fragment of the NC1 domain of the α2 chain of Type IV collagen, the fragment made by the process of PCR cloning.  
     
     
         38 . An anti-angiogenic fragment of the NC1 domain of the α3 chain of Type IV collagen, the fragment made by the process of PCR cloning.  
     
     
         39 . An anti-angiogenic fragment of the NC1 domain of the α1 chain of Type IV collagen, the fragment made by the process of Pseudomonas elastase digestion.  
     
     
         40 . The anti-angiogenic fragment of  claim 39 , wherein the fragment is 12 kDa in size.  
     
     
         41 . The anti-angiogenic fragment of  claim 39 , wherein the fragment is 8 kDa in size.  
     
     
         42 . An anti-angiogenic fragment of the NC1 domain of the α2 chain of Type IV collagen, the fragment made by the process of Pseudomonas elastase digestion.  
     
     
         43 . The anti-angiogenic fragment of  claim 42 , wherein the fragment is 10 kDa in size.  
     
     
         44 . An anti-angiogenic fragment of the NC1 domain of the α3 chain of Type IV collagen, the fragment made by the process of Pseudomonas elastase digestion.