Soluble inhibitors of vascular endothelial growth factor and use thereof

Abstract

The present invention relates to cDNA encoding a soluble neuropilin protein (sNP) which is isolated from neuropilin (NP) producing cells or is recombinantly engineered from NP-encoding DNA. NP-1 and NP-2 are preferred NPs but any neuropilin or VEGF receptor (VEGFR), where the constituents share at least about 85% homology with either of the above VEGF 165 R/NP-1 and NP-2. More preferably, such constituent shares at least 90% homology. Still more preferably, each constituent shares at least 95% homology.

1 .- 26 . (canceled) 
     
     
         27 . An isolated cDNA encoding a human soluble neuropilin polypeptide which specifically binds VEGF 165  and reduces VEGF-mediated HUVEC proliferation. 
     
     
         28 . The isolated cDNA of  claim 27  wherein the encoded soluble neuropilin polypeptide is a soluble neuropilin-2 polypeptide. 
     
     
         29 . The isolated cDNA of  claim 28 , wherein the encoded soluble neuropilin-2 polypeptide comprises amino acids 277-594 of SEQ ID NO: 4, or a fragment thereof that reduces VEGF 165  mediated HUVEC proliferation and binds to a VEGF protein encoded by SEQ ID NO: 15. 
     
     
         30 . The isolated cDNA of  claim 28 , wherein the encoded soluble neuropilin-2 polypeptide comprises a neuropilin-2 fragment that binds to VEGF 165  and inhibits VEGF 165 -mediated HUVEC proliferation, the fragment lacking the c domain (amino acids 642-800 of SEQ ID NO: 4), transmembrane domain (amino acids 865-893 of SEQ ID NO: 4) and cytoplasmic domain (amino acids 885-923 of SEQ ID NO: 4) and comprising the b1 domain (amino acids 277-433 of SEQ ID NO: 4) and b2 domain (amino acids 434-594 of SEQ ID NO: 4) of human neuropilin-2. 
     
     
         31 . The isolated cDNA of  claim 30 , wherein the encoded neuropilin-2 polypeptide consists of amino acids 277-594 of SEQ ID NO: 4. 
     
     
         32 . The isolated cDNA of  claim 28 , wherein the encoded soluble neuropilin-2 consists of the amino acid sequence of SEQ ID NO:8. 
     
     
         33 . The isolated cDNA of  claim 28 , having the nucleotide sequence of SEQ ID NO:7. 
     
     
         34 . A vector comprising the cDNA of  claim 28 . 
     
     
         35 . The vector of  claim 34  that is an expression vector. 
     
     
         36 . A host cell comprising the vector of  claim 34 . 
     
     
         37 . The host cell of  claim 36  that expresses the cDNA. 
     
     
         38 . The host cell of  claim 36 , wherein the host cell is selected from the group consisting of a bacteria cell, a yeast cell, a mammalian cell and an insect cell. 
     
     
         39 . The isolated cDNA of  claim 27  wherein the encoded soluble neuropilin polypeptide is a soluble neuropilin-1 polypeptide. 
     
     
         40 . The isolated cDNA of  claim 39 , wherein the encoded soluble neuropilin-1 polypeptide comprises amino acids 275-587 of SEQ ID NO: 2, or a fragment thereof that reduces VEGF 165  mediated HUVEC proliferation and binds to a VEGF protein encoded by SEQ ID NO: 15. 
     
     
         41 . The isolated cDNA of  claim 39 , wherein the encoded soluble neuropilin-1 polypeptide comprises a neuropilin-1 fragment that binds to VEGF 165  and inhibits VEGF 165 -mediated HUVEC proliferation, the fragment lacking the c domain (amino acids 646-809 of SEQ ID NO: 2), transmembrane domain (amino acids 857-884 of SEQ ID NO: 2) and cytoplasmic domain (amino acids 885-923 of SEQ ID NO: 2) and comprising the b1 domain (amino acids 275-430 of SEQ ID NO: 2) and b2 domain (amino acids 431-587 of SEQ ID NO: 2) of human neuropilin-1. 
     
     
         42 . The isolated cDNA of  claim 41 , wherein the encoded neuropilin-1 polypeptide consists of amino acids 275-587 of SEQ ID NO: 4. 
     
     
         43 . The isolated cDNA of  claim 39 , wherein the encoded soluble neuropilin-1 consists of the amino acid sequence of SEQ ID NO:6. 
     
     
         44 . The isolated cDNA of  claim 39 , having the nucleotide sequence of SEQ ID NO:5.