Materials and methods involving hybrid vascular endothelial growth factor DNAs and proteins
View Patent ↗The present invention provides polypeptides that bind cellular receptors for vascular endothelial growth factor polypeptides; polynucleotides encoding such polypeptides; compositions comprising the polypeptides and polynucleotides; and methods and uses involving the foregoing. Some polypeptides of the invention exhibit unique receptor binding profiles compared to known, naturally occurring vascular endothelial growth factors.
1. A method of modulating the growth of mammalian endothelial cells or mammalian endothelial precursor cells, comprising the step of contacting the cells with a polypeptide in an amount effective to modulate the growth of mammalian endothelial cells, wherein the polypeptide comprises an amino acid sequence at least 95% identical to a chimeric vascular endothelial growth factor amino acid sequence of the formula:
NH 2 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH
wherein X 1 comprises an amino acid sequence selected from the group consisting of amino acids 3-11 of SEQ ID NO: 128 and amino acids 3-11 of SEQ ID NO: 137;
wherein X 2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 129 and 138;
wherein X 3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 and 139;
wherein X 4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 131 and 140;
wherein X 5 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 132 and 141;
wherein X 6 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 133 and 142;
wherein X 7 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 143;
wherein X 8 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135 and 144;
wherein X 9 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 and 145;
wherein NH 2 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH is not identical to amino acids 34 to 135 of SEQ ID NO: 2 or amino acids 112 to 216 of SEQ ID NO: 22; and
wherein the polypeptide binds to at least one receptor selected from the group consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3.
2. The method according to claim 1 wherein the contacting comprises administering a composition comprising said polypeptide and a pharmaceutically acceptable carrier to a mammalian subject.
3. The method of claim 2 wherein the polypeptide binds to a vascular endothelial receptor selected from the group consisting of human VEGFR- 1 and human VEGFR-3
wherein the polypeptide does not bind human VEGFR-2.
4. A method of modulating the growth of mammalian hematopoietic progenitor cells, comprising the step of contacting the cells with a polypeptide in an amount effective to modulate the growth of mammalian endothelial cells, wherein the polypeptide comprises an amino acid sequence at least 95% identical to a chimeric vascular endothelial growth factor amino acid sequence of the formula:
NH 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH
wherein X 1 comprises an amino acid sequence selected from the group consisting of amino acids 3-11 of SEQ ID NO: 128 and amino acids 3-11 of SEQ ID NO: 137;
wherein X 2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 129 and 138;
wherein X 3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 and 139;
wherein X 4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 131 and 140;
wherein X 5 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 132 and 141;
wherein X 6 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 133 and 142;
wherein X 7 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 143;
wherein X 8 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135 and 144;
wherein X9 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 and 145;
wherein NH 2 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH is not identical to amino acids 34 to 135 of SEQ ID NO: 2 or amino acids 112 to 216 of SEQ ID NO: 22; and
wherein the polypeptide binds to at least one receptor selected from the group consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3.
5. The method according to claim 4 , wherein the contacting administering a composition comprising said polypeptide and a pharmaceutically acceptable carrier to a mammalian subject.
6. The method of claim 5 , wherein the polypeptide binds to a vascular endothelial receptor selected from the group consisting of human VEGFR- 1 and human VEGFR-3
wherein the polypeptide does not bind human VEGFR-2.
7. The method according to claim 1 or 4 , wherein the polypeptide further includes a signal peptide amino acid sequence connected to the amino acid sequence of the formula NH 2 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH.
8. The method according to claim 1 or 4 , wherein the polypeptide further includes an amino-terminal methionine residue.
9. The method according to claim 1 or 4 , wherein the polypeptide further includes a tag amino acid sequence connected to the amino acid sequence of the formula NH 2 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -COOH.
10. The method according to claim 1 or 4 , wherein the polypeptide further comprises additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ lID NO: 22).
11. The method according to claim 1 or 4 , wherein the polypeptide further includes one or more amino acid sequences selected from the group consisting of a prepro-VEGF-C signal peptide, a prepro-VEGF-C amino-terminal propeptide, and a prepro-VEGF-C carboxy-terminal pro-peptide.
12. The method according to claim 1 or 4 , wherein the polypeptide binds to human VEGFR-1 and human VEGFR-3.
13. The method of claim 12 , wherein the polypeptide stimulates phosphorylation of human VEGFR-1 and human VEGFR-3.
14. The method according to claim 1 or 4 , wherein the polypeptide binds to human VEGFR-1, human VEGFR-2, and human VEGFR-3.
15. The method of claim 14 , wherein the polypeptide stimulates phosphorylation of human VEGFR- 1, human VEGFR-2, and human VEGFR-3.
16. The method according to claim 1 or 4 , wherein the polypeptide binds to exactly one of human VEGFR- 1, human VEGFR-2 and human VEGFR-3.
17. The method of claim 16 , wherein the polypeptide stimulates phosphorylation of exactly one of human VEGFR- 1, human VEGFR-2 and human VEGFR-3.
18. The method according to claim 1 or 4 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of amino acids 1-102 as set forth in SEQ ID NO: 51; amino acids 1-102 as set forth in SEQ ID NO: 59; amino acids 1-102 as set forth in SEQ ID NO: 63; amino acids 1-104 as set forth in SEQ ID NO: 67; amino acids 1-104 as set forth in SEQ ID NO: 71; amino acids 1-104 as set forth in SEQ TD NO: 75; amino acids 1-104 as set forth in SEQ TD NO: 77; amino acids 1-104 as set forth in SEQ ID NO: 153; amino acids 1-105 as set forth in SEQ TD NO: 155; amino acids 1-105 as set forth in SEQ ID NO: 157; amino acids 1-105 as set forth in SEQ TD NO: 159; amino acids 1-103 as set forth in SEQ ID NO: 161; amino acids 1-102 as set forth in SEQ ID NO: 163; amino acids 1-104 as set forth in SEQ ID NO: 165; amino acids 1-104 as set forth in SEQ TD NO: 167; amino acids 1-104 as set forth in SEQ ID NO: 169; amino acids 1-104 as set forth in SEQ ID NO: 171; amino acids 1-104 as set forth in SEQ ID NO: 173; and amino acids 1-104 as set forth in SEQ ID NO: 175.
19. The method according to claim 1 or 4 , wherein the polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 175, wherein said polypeptide binds at least one human receptor selected from the group consisting of VEGFR-1, VEGFR-2, and VEGFR-3.