Modified vegf-a with improved angiogenic properties

Abstract

The present invention is directed to methods and compositions for making and using chimeric polypeptides that comprise a VEGFR-2 ligand. The chimeric molecules of the present invention retain VEGFR-2 binding activity and an enhanced angiogenic activity as compared to native VEGF-A.

1 . A construct comprising:
 an RTK binding domain, at least one flanking domain, and at least one linkage that connects the RTK binding domain to the at least one flanking domain;   wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: mammalian VEGF-A RTK binding domain amino acid sequences; mammalian VEGF-B RTK binding domain amino acid sequences; mammalian VEGF-C RTK binding domain amino acid sequences; mammalian VEGF-D RTK binding domain amino acid sequences; mammalian VEGF-E RTK binding domain amino acid sequences; mammalian P1GF RTK binding domain amino acid sequences; mammalian PDGF-A RTK binding domain amino acid sequences; mammalian PDGF-B RTK binding domain amino acid sequences; mammalian PDGF-C RTK binding domain amino acid sequences; and mammalian PDGF-D RTK binding domain amino acid sequences;   wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta; and   wherein the at least one flanking domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a mammalian VEGF-C amino-terminal propeptide; a mammalian VEGF-C carboxy-terminal propeptide; a mammalian VEGF-D amino-terminal propeptide; a mammalian VEGF-D carboxy-terminal propeptide; a mammalian TGF-β1 LAP peptide; and fragments thereof that are effective to bind extracellular matrix proteins or neuropilin proteins;   with the proviso that when the RTK binding domain is at least 90% identical to a VEGF-C RTK binding domain amino acid sequence, the at least one flanking domain is not at least 90% identical to a VEGF-C pro-peptide; and when the RTK binding domain is at least 90% identical to a VEGF-D RTK binding domain amino acid sequence, the at least one flanking domain is not at least 90% identical to a VEGF-D pro-peptide.   
     
     
         2 - 18 . (canceled) 
     
     
         19 . A construct comprising:
 an RTK binding domain, a CUB domain, and at least one linkage that connects the RTK binding domain to the CUB domain;   wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: mammalian VEGF-A RTK binding domain amino acid sequences; mammalian VEGF-B RTK binding domain amino acid sequences; mammalian VEGF-C RTK binding domain amino acid sequences; mammalian VEGF-D RTK binding domain amino acid sequences; mammalian VEGF-E RTK binding domain amino acid sequences; mammalian P1GF RTK binding domain amino acid sequences; mammalian PDGF-A RTK binding domain amino acid sequences; and mammalian PDGF-B RTK binding domain amino acid sequences;   wherein the CUB domain comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of PDGF-C CUB domain amino acid sequences and PDGF-D CUB domain amino acid sequences; and   wherein the construct and the RTK binding domain bind to the extracellular domain of at least one receptor tyrosine kinase selected from the group consisting of: VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta.   
     
     
         20 . The construct according to  claim 19 , wherein the CUB domain is connected to the N-terminus of the RTK binding domain. 
     
     
         21 . The construct according to  claim 19 , wherein the CUB domain is connected to the C-terminus of the RTK binding domain. 
     
     
         22 . The construct according to  claim 19 , wherein the CUB domain comprises the amino acid sequence set forth in SEQ ID NO: 53. 
     
     
         23 . The construct according to  claim 19 , wherein the CUB domain comprises the amino acid sequence set forth in SEQ ID NO: 55. 
     
     
         24 . The construct according to  claim 19 , wherein the RTK binding domain comprises an amino acid sequence that is at least 90% identical to amino acids 27 to 127 of the VEGF109 amino acid sequence of SEQ ID NO: 52. 
     
     
         25 . The construct according to  claim 19 , further comprising a heparin binding domain connected to the construct by a linkage. 
     
     
         26 . The construct according to  claim 19 , wherein each of the linkages comprises a peptide bond, whereby the construct comprises a chimeric polypeptide. 
     
     
         27 . The construct according to  claim 26 , wherein the chimeric polypeptide further comprises a signal peptide. 
     
     
         28 . The construct of  claim 27 , wherein the chimeric polypeptide further comprises a peptide tag. 
     
     
         29 . The construct according to  claim 24 , wherein the construct comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 59 and 61. 
     
     
         30 . The construct according to  claim 19 , wherein the CUB domain is attached to the growth factor via a recognition sequence specifically recognized by a proteolytic enzyme such that the proteolytic enzyme if present cleaves at the recognition sequence to remove the CUB domain and produce an activated growth factor. 
     
     
         31 - 33 . (canceled) 
     
     
         34 . A composition comprising the construct of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         35 . A polynucleotide comprising a nucleotide sequence that encodes the construct of  claim 1 , wherein the construct comprises a chimeric polypeptide. 
     
     
         36 - 44 . (canceled) 
     
     
         45 . A composition comprising the polynucleotide of  claim 35  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         46 - 50 . (canceled) 
     
     
         51 . A method of modulating the growth of mammalian endothelial cells or mammalian endothelial precursor cells, comprising contacting the cells with a composition comprising a member selected from the group consisting of:
 (a) the construct of  claim 1 ;   (b) a polynucleotide that encodes (a);   (c) an expression vector containing (b) operatively linked to an expression control sequence; and   (d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a).   
     
     
         52 - 53 . (canceled) 
     
     
         54 . A method of modulating angiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of angiogenesis a composition comprising a member selected from the group consisting of:
 (a) a construct of  claim 1 ;   (b) a polynucleotide that encodes (a);   (c) an expression vector containing (b) operatively linked to an expression control sequence; and   (d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a);   wherein the composition is administered in an amount effective to modulate angiogenesis.   
     
     
         55 . A method of modulating lymphangiogenesis in a mammalian subject comprising administering to a mammalian subject in need of modulation of lymphangiogenesis a composition comprising a member selected from the group consisting of:
 (a) a construct of  claim 1 ;   (b) a polynucleotide that encodes (a);   (c) an expression vector containing (b) operatively linked to an expression control sequence; and   (d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a);   wherein the composition is administered in an amount effective to modulate lymphangiogenesis.   
     
     
         56 - 59 . (canceled) 
     
     
         60 . A method for antagonizing in a cell at least one receptor selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and PDGFR-β, wherein the method comprises administering to the cell an agent selected from the group consisting of:
 (a) the construct of  claim 19 ; 
 (b) a polynucleotide that encodes (a); 
 (c) an expression vector containing (b) operatively linked to an expression control sequence; and 
 (d) a cell transformed or transfected with (b) or (c) and that expresses the polypeptide of (a). 
 
     
     
         61 - 73 . (canceled)