Protease-resistant forms of VEGF-D, method of making and method of use
The present invention provides modified VEGF-D polypeptide variants that are resistant to serine protease processing and methods of making and using the same, as well pharmaceutical compositions comprising the peptide or a polynucleotide encoding the same. The VEGF-D variants comprise a VEGF homology domain, and at least one of (1) a C-terminal propeptide that is not cleavable by a serine protease, and (2) an N-terminal propeptide that is not cleavable by a serine protease. The VEGF-D variants can be made using site-directed mutagenesis. The VEGF-D variants are useful for the treatment of diseases such as cardiovascular disease and primary and secondary lymphedema, and for the prevention of stenosis and restenosis of blood vessels.
1 . A modified VEGF-D polypeptide that comprises a VEGF homology domain (VHD), and at least one of (1) a C-terminal propeptide that is resistant to cleavage from the VHD by a serine protease and (2) an N-terminal propeptide that is resistant to cleavage from the VHD by a serine protease.
2 . The modified VEGF-D polypeptide according to claim 1 , which comprises a C-terminal propeptide resistant to cleavage from the VHD by a serine protease.
3 . The modified VEGF-D polypeptide according to claim 2 , which further comprises an N-terminal propeptide resistant to cleavage from the VHD by a serine protease.
4 . The modified VEGF-D polypeptide according to claim 1 , which comprises an N-terminal propeptide resistant to cleavage from the VZHD by a serine protease.
5 . The VEGF-D polypeptide according to claim 1 , wherein the serine protease is selected from the group consisting of furin, PC5, PC7 and plasmin.
6 . The VEGF-D polypeptide according to claim 5 , wherein the serine protease is furin.
7 . A VEGF-D polypeptide that comprises an amino acid sequence corresponding to SEQ ID NO: 2 wherein neither amino acid residue at position 85 or 88 is arginine, or neither amino acid residue at position 204 or 205 is arginine.
8 . The VEGF-D polypeptide according to claim 7 , wherein both amino acid residues at positions 85 and 88 are arginine.
9 . The VEGF-D polypeptide according to claim 7 , wherein both amino acid residues at positions 204 and 205 are arginine.
10 . The VEGF-D polypeptide according to claim 7 , wherein none of the amino acid residues at positions 85, 88, 204 and 205 is arginine.
11 . The VEGF-D polypeptide according to claim 7 , wherein at least one of the amino acid residues at positions 85, 88, 204 and 205 is serine.
12 . The VEGF-D polypeptide according to claim 7 , wherein at least two of the amino acid residues at positions 85, 88, 204 and 205 are serine.
13 . The VEGF-D polypeptide according to claim 7 , wherein at least three of the amino acid residues at positions 85, 88, 204 and 20 are serine.
14 . The VEGF-D polypeptide according to claim 7 , wherein each of the amino acid residues at positions 85, 88, 204 and 205 is serine.
15 . A modified VEGF-D polypeptide that comprises an amino acid sequence corresponding to SEQ ID NO:2 wherein each of the amino acid residues at positions 85, 88, 204 and 205 is serine.
16 . A polypeptide comprising the N-terminal propeptide and VHD of VEGF-D wherein neither amino acid residue at positions 85 and 88 is arginine.
17 . The polypeptide of claim 16 wherein each of the amino acid residues at positions 85 and 88 is serine.
18 . A polypeptide comprising the VHD and C-terminal propeptide of VEGF-D wherein neither amino acid residue at positions corresponding to positions 204 and 205 is arginine.
19 . The polypeptide of claim 18 wherein each of the amino acids at positions corresponding to positions 204 and 205 is serine.
20 . A nucleic acid molecule that encodes a modified VEGF-D polypeptide wherein said polypeptide comprises a VEGF homology domain (VHD), and at least one of (1) a C-terminal propeptide that is resistant to cleavage from the VHD by a serine protease and (2) an N-terminal propeptide that is resistant to cleaveage from the VHD by a serine.
21 . A nucleic acid molecule that encodes a VEGF-D polypeptide that comprises an amino acid sequence corresponding to SEQ ID NO: 2 wherein neither amino acid residue at position 85 or 88 is arginine, or neither amino acid residue at position 204 or 205 is arginine.
22 . A vector comprising a nucleic acid molecule that encodes a modified VEGF-D polypeptide wherein said polypeptide comprises a VEGF homology domain (VHD), and at least one of (1) a C-terminal propeptide that is resistant to cleavage from the VHD by a serine protease and (2) an N-terminal propeptide that is resistant to cleaveage from the VHD by a serine, wherein said nucleic acid is operably linked to a regulatory element.
23 . The vector of claim 22 , which is an expression vector.
24 . The vector of claim 23 , which is a prokaryotic expression vector or a eukaryotic expression vector.
25 . The vector of claim 23 , which is viral based, a plasmid, or a mammalian expression vector.
26 . A cell comprising the vector of claim 23 .
27 . The cell according to claim 26 , which is a mammalian cell.
28 . The cell according to claim 27 , which is a human cell.
29 . A method for making the modified VEGF-D polypeptide of claim 1 comprising: preparing a polynucleotide encoding the modified polynucleotide via site-directed mutagenesis; and expressing said polynucleotide.
30 . The method according to claim 29 , wherein the site-directed mutagenesis is PCR-based.
31 . A method for selectively/differentially activating VEGFR-2 or VEGFR-3 in a cell, the method comprising applying to the cell an effective amount of the modified VEGF-D polypeptide of claim 1 .
32 . The method according to claim 31 , wherein VEGFR-2 is selectively activated, and wherein the modified VEGF-D polypeptide comprises a C-terminal propeptide that is not cleavable by a serine protease, and an N-terminal propeptide that is not cleavable by a serine protease.
33 . A method for selectively/differentially activating VEGFR-2 or VEGFR-3 in a cell, the method comprising applying to the cell an effective amount of a nucleic acid of claim 20 .
34 . The method according to claim 33 , wherein the nucleic acid is operatively linked to a regulatory element.
35 . A method of inducing lymphangiogenesis in a mammal comprising administering a composition comprising the polypeptide of claim 1 to said mammal in an amount effective to induce lymphangiogenesis.
36 . The method of claim 35 wherein said mammal is a human.
37 . A method of inducing angiogenesis in a mammal comprising administering a composition comprising the polypeptide of claim 1 to said mammal in an amount effective to induce angiogenesis.
38 . The method of claim 37 wherein said mammal is a human.
39 . A method of inhibiting stenosis or restenosis in a mammal comprising administering a composition comprising the polypeptide of claim 1 to said mammal in an amount effective to inhibit stenosis or restenosis.
40 . The method of claim 39 wherein said mammal is a human.
41 . The method of claim 39 wherein the composition is administered as a stent comprising the polypeptide.
42 . The method of claim 41 wherein the stent is configured to elute the polypeptide.
43 . A composition comprising a modified VEGF-D polypeptide according to claim 1 .
44 . A composition comprising the vector according to claim 22.