Vascular endothelial growth factor-B and DNA coding therefor

Abstract

VEGF-B polypeptides from the PDGF family of growth factors having the property of promoting mitosis and proliferation of vascular endothelial cells, DNA sequences encoding these polypeptides, pharmaceutical compositions containing them and antibodies which react with them. The VEGF-B polypeptides are useful in stimulating angiogenesis as well as in diagnostic applications.

What is claimed is:  
     
         1 . An isolated DNA which codes for a protein which comprises a characteristic amino acid sequence 
 Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO:16) and has the property of promoting proliferation of endothelial cells or mesodermal cells, said DNA being selected from the group consisting of the DNA of FIGS. 1 and 2 (SEQ ID NO:1), the DNA of FIG. 3 (SEQ ID NO:4), the DNA of FIG. 5 (SEQ ID NO:6), the DNA of FIG. 7 (SEQ ID NO:8), the DNA of FIG. 10 (SEQ ID NO:10), the DNA of FIG. 12 (SEQ ID NO:12), the DNA of FIG. 14 (SEQ ID NO:14), and DNA which hybridizes under stringent conditions with at least one of the foregoing DNA sequences.    
     
     
         2 . A DNA according to  claim 1 , wherein said DNA is a cDNA.  
     
     
         3 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIGS. 1 and 2 (SEQ ID NO:1).  
     
     
         4 . A DNA according to  claim 1 , wherein said DNA sequence is a mammalian DNA.  
     
     
         5 . A DNA according to  claim 4 , wherein said DNA is a murine DNA.  
     
     
         6 . A DNA according to  claim 4 , wherein said DNA is a human DNA.  
     
     
         7 . A DNA according to  claim 1 , wherein said DNA codes for a protein which promotes proliferation of vascular endothelial cells.  
     
     
         8 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 3 (SEQ ID NO:4).  
     
     
         9 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 5 (SEQ ID NO:6).  
     
     
         10 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 7 (SEQ ID NO:8).  
     
     
         11 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 10 (SEQ ID NO:10).  
     
     
         12 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 12 (SEQ ID NO:12).  
     
     
         13 . A DNA according to  claim 1 , comprising a cDNA corresponding to the DNA of FIG. 14 (SEQ ID NO:14).  
     
     
         14 . A vector comprising a DNA according to  claim 1 .  
     
     
         15 . A vector according to  claim 14 , wherein said vector is a eukaryotic vector.  
     
     
         16 . A vector according to  claim 14 , wherein said vector is a prokaryotic vector.  
     
     
         17 . A vector according to  claim 14 , wherein said vector is a plasmid.  
     
     
         18 . A protein which exhibits a characteristic sequence 
 Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO:16) and has the property of promoting proliferation of endothelial cells or mesodermal cells, said protein comprising a sequence of amino acids substantially corresponding to an amino acid sequence selected from the group consisting of the amino acid sequence of FIG. 1 (SEQ ID NO:2), the amino acid sequence of FIG. 2 (SEQ ID NO:3), the amino acid sequence of FIG. 4 (SEQ ID NO:5), the amino acid sequence of FIG. 6 (SEQ ID NO:7), the amino acid sequence of FIG. 8 (SEQ ID NO:9), the amino acid sequence of FIG. 11 (SEQ ID NO:11), the amino acid sequence of FIG. 13 (SEQ ID NO:13), and the amino acid sequence of FIG. 15 (SEQ ID NO:15).    
     
     
         19 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 1 (SEQ ID NO:2).  
     
     
         20 . A protein according to  claim 18 , wherein said protein comprises an amino acid sequence corresponding to the amino acid sequence of FIG. 2 (SEQ ID NO:3).  
     
     
         21 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 4 (SEQ ID NO:5).  
     
     
         22 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 6 (SEQ ID NO:7).  
     
     
         23 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 8 (SEQ ID NO:9).  
     
     
         24 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 11 (SEQ ID NO:11).  
     
     
         25 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 13 (SEQ ID NO:13).  
     
     
         26 . A protein according to  claim 18 , wherein said protein comprises a sequence of amino acids corresponding to the amino acid sequence of FIG. 15 (SEQ ID NO:15).  
     
     
         27 . A protein according to  claim 18 , wherein said protein is a mammalian protein.  
     
     
         28 . A protein according to  claim 27 , wherein said protein is a murine protein.  
     
     
         29 . A protein according to  claim 27 , wherein said protein is a human protein.  
     
     
         30 . A protein according to  claim 18 , wherein said protein promotes proliferation of vascular endothelial cells.  
     
     
         31 . A protein produced by expression of a DNA selected from the group consisting of the DNA of FIGS. 1 and 2 (SEQ ID NO:1), the DNA of FIG. 3 (SEQ ID NO:4), the DNA of FIG. 5 (SEQ ID NO:6), the DNA of FIG. 7 (SEQ ID NO:8), the DNA of FIG. 10 (SEQ ID NO:10), the DNA of FIG. 12 (SEQ ID NO:12), the DNA of FIG. 14 (SEQ ID NO:14), and DNA which hybridizes under stringent conditions with at least one of the foregoing DNA sequences.  
     
     
         32 . A pharmaceutical composition comprising an effective endothelial or mesodermal cell proliferation promoting amount of a protein according to  claim 18 , and at least one conventional pharmaceutical carrier or diluent.  
     
     
         33 . An antibody which reacts with a protein according to  claim 18 .  
     
     
         34 . An antibody according to  claim 33 , wherein said antibody is a monoclonal antibody.  
     
     
         35 . A host cell transformed or transfected with a vector according to  claim 14 , such that said host cell expresses a protein having the property of promoting proliferation of endothelial or mesodermal cells.  
     
     
         36 . A transfected host cell according to  claim 35 , wherein said host cell is a eukaryotic cell.  
     
     
         37 . A transfected host cell according to  claim 35 , wherein said host cell is a COS cell.  
     
     
         38 . A transformed host cell according to  claim 35 , wherein said host cell is a prokaryotic cell.  
     
     
         39 . A transformed host cell according to  claim 35 , wherein said host cell is a 293EBNA cell.  
     
     
         40 . A transformed host cell according to  claim 35 , wherein said host cell is an insect cell.  
     
     
         41 . A diagnostic means for quantitatively detecting VEGF-B in a test sample, said means comprising an antibody according to  claim 33 , which reacts with VEGF-B in order to detect the amount of VEGF-B in the sample.  
     
     
         42 . A diagnostic means according to  claim 41 , wherein said antibody is a labelled antibody.  
     
     
         43 . A diagnostic means for detecting VEGF-B in a test sample, said means comprising at least one pair of primers complementary to a DNA sequence according to  claim 1 , for amplifying a test VEGF-B gene by polymerase chain reaction in order to facilitate a sequence comparison of the test VEGF-B gene with the DNA sequence according to  claim 1 .  
     
     
         44 . A pharmaceutical composition comprising an effective VEGF-B-binding amount of antibodies according to  claim 33 .  
     
     
         45 . A pharmaceutical composition comprising an effective endothelial or mesodermal cell proliferation promoting amount of a protein according to  claim 18 , and VEGF.  
     
     
         46 . A pharmaceutical composition according to  claim 45 , further comprising an amount of heparin effective to assure that a cell proliferation promoting amount of said protein is present free from association with a cell.  
     
     
         47 . A pharmaceutical composition according to  claim 32 , further comprising an amount of heparin effective to assure that a cell proliferation promoting amount of said protein is present free from association with a cell.  
     
     
         48 . A pharmaceutical composition comprising an effective angiogenesis stimulating amount of a protein according to  claim 18 , and heparin.  
     
     
         49 . A protein dimer comprising a protein according to  claim 18 .  
     
     
         50 . A protein dimer according to  claim 49 , wherein said protein dimer is a homodimer of said protein.  
     
     
         51 . A protein dimer according to  claim 49 , wherein said protein dimer is a heterodimer of said protein and VEGF.  
     
     
         52 . A protein dimer according to  claim 49 , wherein said protein dimer is a disulfide-linked dimer.  
     
     
         53 . A method of promoting release of at least one protein selected from the group consisting of proteins according to  claim 18 , and VEGF from a cell which expresses said at least one protein, said method comprising exposing the cell to heparin.  
     
     
         54 . A vector comprising an anti-sense nucleotide sequence complementary to at least part of a DNA sequence according to  claim 1 .  
     
     
         55 . A method of retarding expression of VEGF-B from a cell which expresses VEGF-B, said method comprising transfecting said cell with a vector according to  claim 54 .  
     
     
         56 . A method according to  claim 55 , wherein said cell is a tumor cell.  
     
     
         57 . An isolated DNA comprising a nucleotide sequence corresponding to the sequence of FIG. 25 (SEQ ID NO:17) or a DNA which hybridizes under stringent conditions to said nucleotide sequence.