Vascular endothelial growth factor-like protein from ORF viruses binds and activates mammalian VEGF receptor-2, and uses thereof

Abstract

The invention is based on the discovery that a viral VEGF-like protein from the orf virus strain NZ2 and from the orf virus strain NZ10 is capable of binding to the extracellular domain of the VEGF receptor-2 to form bioactive complexes which mediate useful cellular responses and/or antagonize undesired biological activities. Disclosed are methods which stimulate or inhibit these biological activities, methods for therapeutic applications and antagonists of ORFV2-VEGF and/or NZ10.

What is claimed is:  
     
         1 . A method for stimulating proliferation of endothelial or mesodermal cells, comprising the step of exposing said endothelial cells to an effective endothelial or mesodermal cell proliferation stimulating amount of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         2 . A method for activation of VEGF receptor 2, comprising the step of exposing cells bearing said receptor to an effective receptor activating dose of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         3 . A method according to  claim 2 ,wherein said method is carried out in vivo.  
     
     
         4 . A method according to  claim 2 ,wherein said method is carried out in vitro.  
     
     
         5 . A method for specific activation of VEGF receptor  2 , comprising the step of exposing cells bearing said receptor to an effective receptor activating dose of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         6 . A method according to  claim 5 , wherein the VEGF receptor 1 is not activated.  
     
     
         7 . A method for modulating vascular permeability, comprising the step of administering an effective vascular permeability modulating amount of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         8 . A method for treatment of pustular dermatitis, comprising the step of administering a therapeutically effective amount of an antagonist to a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         9 . A method for treatment of pustular dermatitis, comprising the step of administering a therapeutically effective amount of an antagonist to the VEGF receptor 2.  
     
     
         10 . A method for treatment of fluid accumulation caused by viral infection, comprising the step of administering a therapeutically effective amount of an antagonist to a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         11 . A method of  claim 10 , wherein the viral infection is caused by an orf virus.  
     
     
         12 . A method of  claim 11 , wherein said orf virus is a NZ2 strain.  
     
     
         13 . A method of  claim 11 , wherein said orf virus is a NZ10 strain.  
     
     
         14 . A host cell transformed or transfected with a vector comprising a nucleic acid sequence which encodes a polypeptide which has the property of promoting proliferation of endothelial cells, said nucleic acid consisting of the sequence of FIG. 8 (SEQ ID NO:1) or of the sequence of FIG. 10 (SEQ ID NO:10) and nucleic acids which hybridize under stringent conditions with said sequence, which said sequence is operably linked to a promoter sequence.  
     
     
         15 . A host cell according to  claim 14 , wherein said host cell expresses a polypeptide having the property of promoting proliferation of endothelial cells by binding specifically to a VEGF receptor-2.  
     
     
         16 . A host cell according to  claim 14 , wherein said host cell is a eukaroytic cell.  
     
     
         17 . A host cell according to  claim 14 , wherein said host cell is a COS cell.  
     
     
         18 . A host cell according to  claim 14 , wherein said host cell is a 293EBNA cell.  
     
     
         19 . A host cell according to  claim 14 , wherein said host cell is a prokaryotic cell.  
     
     
         20 . A host cell according to  claim 14 , wherein said host cell is an insect cell.  
     
     
         21 . A diagnostic test kit for orf viral infection in sheep, goats and humans comprising a specific binding reagent for a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10 and means for detecting binding to said reagent.  
     
     
         22 . An isolated polypeptide dimer comprising a polypeptide according to FIG. 9 (SEQ ID NO:2) or to FIG. 11 (SEQ ID NO:2).  
     
     
         23 . An isolated polypeptide dimer according to  claim 22 , wherein said polypeptide dimer is a homodimer of said polypeptide.  
     
     
         24 . An isolated polypeptide dimer according to  claim 22 , wherein said polypeptide dimer is a heterodimer of said polypeptide and at least one further growth factor selected from the group consisting of VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, NZ10 and ORFV2-VEGF.  
     
     
         25 . An isolated polypeptide dimer according to  claim 22 , wherein said polypeptide dimer is a disulfide-linked dimer.  
     
     
         26 . A pharmaceutical composition comprising an effective endothelial cell proliferation promoting amount of a polypeptide according to FIG. 9 (SEQ ID NO:2) or according to FIG. 11 (SEQ ID NO:11), and at least one further growth factor selected from the group consisting of VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, NZ10 and ORFV2-VEGF.  
     
     
         27 . A pharmaceutical composition according to  claim 26 , further comprising heparin.  
     
     
         28 . A pharmaceutical composition comprising an effective endothelial or mesodermal cell proliferation promoting amount of polypeptide according to FIG. 9 (SEQ ID NO:2) or according to FIG. 11 (SEQ ID NO:11), and at least one pharmaceutical carrier or diluent.  
     
     
         29 . An orf VEGF-like polypeptide antagonist having the capacity to inhibit the action of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         30 . An orf VEGF-like polypeptide antagonist according to  claim 29 , wherein said antagonist is an antibody specifically reactive with a polypeptide according to amino acid sequence of FIG. 9 (SEQ ID NO:2).  
     
     
         31 . An orf VEGF-like polypeptide antagonist according to  claim 29 , wherein said antagonist is an antibody specifically reactive with a polypeptide according to amino acid sequence of FIG. 11 (SEQ ID NO:11).  
     
     
         32 . An antibody according to  claim 30 , wherein said antibody is a polyclonal antibody.  
     
     
         33 . An antibody according to  claim 30 , wherein said antibody is a monoclonal antibody.  
     
     
         32 . An antibody according to  claim 31 , wherein said antibody is a polyclonal antibody.  
     
     
         34 . An antibody according to  claim 31 , wherein said antibody is a monoclonal antibody.  
     
     
         35 . An antibody according to  claim 30 , wherein said antibody is labeled with a detectable label.  
     
     
         36 . An antibody according to  claim 31 , wherein said antibody is labeled with a detectable label.  
     
     
         37 . An antibody according to  claim 35 , wherein said detectable label is radioactive isotope.  
     
     
         38 . A orf VEGF-like polypeptide antagonist according to  claim 29 , wherein said antagonist is an anti-sense nucleotide sequence complementary to at least a part of the nucleotide sequence encoding a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10 or to the promoter region of ORFV2-VEGF or of ORFV10-VEGF.  
     
     
         39 . An orf VEGF-like polypeptide antagonist according to  claim 29 , wherein said antagonist is an isolated polypeptide which comprises a sequence of amino acids substantially corresponding to the amino acid sequence of FIG. 9 (SEQ ID NO:2) or FIG. 11 (SEQ ID NO:11), wherein said polypeptide has the ability to bind to a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10 and to prevent biological activity of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.  
     
     
         40 . An isolated polypeptide which comprises a sequence of amino acids substantially corresponding to the amino acid sequence of FIG. 9 (SEQ ID NO:2) or FIG. 11 (SEQ ID NO:11), wherein said polypeptide has the ability to bind to endothelial cells but is unable to induce vascular permeability.  
     
     
         41 . An isolated ORFV2-VEGF and VEGF receptor 2 complex.  
     
     
         42 . An isolated NZ10 and VEGF receptor 2 complex.  
     
     
         43 . A method of making a polypeptide of FIG. 9 (SEQ ID NO:2) or of FIG. 11 (SEQ ID NO:11), said method comprising the steps of: 
 culturing a host cell transformed or transfected with a vector comprising a nucleic acid sequence encoding said polypeptide operably associated with a promoter sequence such that the nucleic acid sequence encoding said polypeptide is expressed; and    isolating said polypeptide from said host cell or from a growth medium in which said host cell is cultured.    
     
     
         44 . An isolated nucleic acid molecule comprising a polynucleotide sequence having at least 85% identity with the sequence of FIG. 10 (SEQ ID NO:10).  
     
     
         45 . An isolated nucleic acid molecule which encodes a polypeptide molecule comprising an amino acid sequence having at least 85% identity with the amino acid sequence of FIG. 11 (SEQ ID NO11), or a fragment or analog thereof having the biological activity of NZ10.  
     
     
         46 . A vector comprising a nucleic acid according to  claim 44 , which nucleic acid is operably linked with a promoter sequence.  
     
     
         47 . A vector comprising a nucleic acid according to  claim 45 , which nucleic acid is operably linked with a promoter sequence.  
     
     
         48 . A method of making a vector which expresses a polypeptide comprising an amino acid sequence having at least 85% identity with the amino acid sequence of FIG. 11 (SEQ ID NO:11), or fragment or analog thereof having the biological activity of NZ10, said method comprising incorporating an isolated nucleic acid according to  claim 44  into said vector in operatively linked relation with a promoter.  
     
     
         49 . An isolated polypeptide comprising at least 85% identity with the amino acid sequence of FIG. 11 (SEQ ID NO:11), or a fragment or analog thereof having the biological activity of NZ10.  
     
     
         50 . An isolated polypeptide produced by expression of a polynucleotide comprising the polynucleotide sequence having at least 85% identity with the FIG. 10 (SEQ ID NO:10), or a polynucleotide which hybridizes under stringent conditions with said DNA sequence.  
     
     
         51 . A means for amplifying a polynucleotide according to  claim 44  in a test sample, said means comprising at least one pair of primers complementary to a nucleic acid according to  claim 44 .  
     
     
         52 . A method for identifying an orf VEGF-like polypeptide antagonist comprising: 
 admixing a substantially purified preparation of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10 with a test agent; and    monitoring, by any suitable means, an inhibition in the biological activity of a polypeptide selected from the group consisting of ORFV2-VEGF and NZ10.