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 polypeptide comprising an amino acid sequence that is at least 95% identical to a chimeric vascular endothelial growth factor (VEGF) 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 is 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 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 129 and 138;
wherein X 3 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 and 139;
wherein X 4 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 131 and 140;
wherein X 5 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 132 and 141;
wherein X 6 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 133 and 142;
wherein X 7 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 143;
wherein X 8 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 135 and 144;
wherein X 9 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 and 145;
wherein the chimeric VEGF binds to at least one receptor selected from the group consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3; 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. A polypeptide according to claim 1 , 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.
3. A polypeptide according to claim 1 , wherein the polypeptide further includes an amino-terminal methionine residue.
4. A polypeptide according to claim 1 , 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.
5. A polypeptide according to claim 1 , 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.
6. A polypeptide comprising an amino acid sequence of the formula X N -V/PHD-X C , wherein X N is selected from the group consisting of amino acids 1-34 of SEQ ID NO: 2, amino acids 1-111 of SEQ ID NO: 22, amino acids 1-34 of SEQ ID NO: 147, or fragments thereof;
wherein V/PHD is a polypeptide according to claim 1 ;
wherein X C is selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
7. A composition comprising a polypeptide according to claim 1 in a pharmaceutically acceptable carrier.
8. A polypeptide according to claim 1 that binds to human VEGFR-1 and VEGFR-3.
9. A polypeptide according to claim 1 that binds to human VEGFR-1, VEGFR-2, and VEGFR-3.
10. The polypeptide of claim 1 , wherein the amino acid sequence of the polypeptide further comprises a sequence selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
11. The polypeptide of claim 1 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
12. A polypeptide according to claim 1 , wherein X 4 is SEQ ID NO: 140, and wherein the polypeptide binds to VEGFR-3.
13. A polypeptide according to claim 12 , wherein X 8 is SEQ ID NO: 144.
14. A polypeptide according to claim 1 , wherein X 2 is SEQ ID NO: 129, and wherein the polypeptide binds to VEGFR-1.
15. A polypeptide according to claim 14 , wherein X 7 is SEQ ID NO: 134.
16. A polypeptide according to claim 14 , wherein X 4 is SEQ ID NO: 140, and wherein the polypeptide binds to VEGFR-3.
17. A polypeptide comprising 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 ID NO: 75; amino acids 1-104 as set forth in SEQ ID NO: 77; amino acids 1-104 as set forth in SEQ ID NO: 153; amino acids 1-105 as set forth in SEQ ID NO: 155; amino acids 1-105 as set forth in SEQ ID NO: 157; amino acids 1-105 as set forth in SEQ ID 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 ID 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.
18. The polypeptide of claim 17 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
19. A polypeptide comprising 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.
20. The polypeptide of claim 19 , herein the amino acid sequence of the polypeptide further comprises a sequence selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
21. A polypeptide comprising an amino acid sequence of the formula X N -V/PHD-X C , wherein X N is selected from the group consisting of amino acids 1-34 of SEQ ID NO: 2, amino acids 1-111 of SEQ ID NO: 22, amino acids 1-34 of SEQ ID NO: 147, and fragments thereof.
wherein V/PHD is a polypeptide according to claim 19 ;
wherein X C is selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 or SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
22. The polypeptide of claim 19 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
23. A polypeptide according to claim 19 that binds to human VEGFR-1, VEGFR-2, and VEGFR-3.
24. The polypeptide of claim 23 , wherein the amino acid sequence of the polypeptide further comprises a sequence selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
25. The polypeptide of claim 23 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
26. A polypeptide that binds human vascular endothelial growth factor receptors VEGFR-1, VEGFR-2, and VEGFR-3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:
amino acids 1-102 of SEQ ID NO: 51; amino acids 1-102 of SEQ ID NO: 63; amino acids 1-104 of SEQ ID NO: 71; amino acids 1-104 of SEQ ID NO: 153; amino acids 1-103 of SEQ ID NO: 161; amino acids 1-102 of SEQ ID NO: 163; amino acids 1-104 of SEQ ID NO: 165; amino acids 1-104 of SEQ ID NO: 167; amino acids 1-104 of SEQ ID NO: 173; and amino acids 1-104 of SEQ ID NO: 175.
27. The polypeptide of claim 26 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
28. A polypeptide that binds human vascular endothelial growth factor receptors VEGFR-1, VEGFR-2, and VEGFR-3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of amino acids 1-102 of SEQ ID NO: 63; amino acids 1-104 of SEQ ID NO: 71; amino acids 1-104 of SEQ ID NO: 167; and amino acids 1-104 of SEQ ID NO: 175.
29. The polypeptide of claim 28 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
30. A polypeptide comprising an amino acid sequence that is at least 95% identical to a chimeric vascular endothelial growth factor (VEGF) amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence of the formula:
5′-N 1 -N 2 -N 3 -N 4 -N 5 -N 6 -N 7 -N 8 -N 9 -3′
where-in N 1 is a nucleotide sequence selected from the group consisting of nucleotides 7-34 of SEQ ID NO: 3 and nucleotides 7-34 of SEQ ID NO: 23;
wherein N 2 is a nucleotide sequence selected from the group consisting of nucleotides 1-32 of SEQ ID NO: 4 and nucleotides 1-35 of SEQ ID NO: 24;
wherein N 3 is a nucleotide sequence selected from the group consisting of nucleotides 1-31 of SEQ ID NO: 5 and nucleotides 1-31 of SEQ ID NO: 25;
wherein N 4 is a nucleotide sequence selected from the group consisting of nucleotides 10-33 of SEQ ID NO: band nucleotides 10-33 of SEQ ID NO: 26;
wherein N 5 is a nucleotide sequence selected from the group consisting of nucleotides 1-37 of SEQ ID NO: 7 and nucleotides 1-37 of SEQ ID NO: 27;
wherein N 6 is a nucleotide sequence selected from the group consisting of nucleotides 3-28 of SEQ ID NO: 8 and nucleotides 3-28 of SEQ ID NO: 28;
wherein N 7 is a nucleotide sequence selected from the group consisting of nucleotides 1-46 of SEQ ID NO: 9 and nucleotides 1-46 of SEQ ID NO: 29;
wherein N 8 is a nucleotide sequence selected from the group consisting of nucleotides 1-43 of SEQ ID NO: 10 and nucleotides 1-49 of SEQ ID NO: 30;
wherein N 9 is a nucleotide sequence selected from the group consisting of nucleotides 1-39 of SEQ ID NO: 11 and nucleotides 1-39 of SEQ ID NO: 31;
wherein 5′-N 1 -N 2 -N 3 -N 4 -N 5 -N 6 -N 7 -N 8 -N 9 -3′ is nut identical to nucleotides 156 to 461 of SEQ ID NO: 1 or nucleotides 685 to 999 of SEQ ID NO: 21;
wherein the chimeric VEGF binds to at least one receptor selected from the group consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3; 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.
31. A polypeptide according to 30 , wherein the polypeptide further includes a signal peptide amino acid sequence.
32. A polypeptide according to claim 30 , wherein the polypeptide further includes an amino-terminal methionine residue.
33. A polypeptide according to claim 30 , wherein the polypeptide further includes a tag amino acid sequence.
34. A polypeptide according to claim 30 , 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.
35. A composition comprising a polypeptide according to claim 30 in a pharmaceutically acceptable carrier.
36. The polypeptide of claim 30 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
37. A polypeptide according to claim 30 , wherein N 4 is nucleotides 10-33 of SEQ ID NO: 26, and wherein the polypeptide binds to VEGFR-3.
38. A polypeptide according to claim 37 , wherein N 8 is nucleotides 1-49 of SEQ ID NO: 30.
39. A polypeptide according to claim 30 , wherein N 2 is nucleotides 1-32 of SEQ ID NO: 4, and wherein the polypeptide binds to VEGFR-1.
40. A polypeptide according to claim 39 , wherein N 7 is nucleotides 1-46 of SEQ ID NO: 9.
41. A polypeptide according to claim 39 , wherein N 4 is nucleotides 10-33 of SEQ ID NO: 26, and wherein the polypeptide binds to VEGFR-3.
42. A polypeptide comprising an amino acid sequence of the formula X N -V/PHD-X C , wherein X N is selected from the group consisting of amino acids 1-34 of SEQ ID NO: 2, amino acids 1-111 of SEQ ID NO: 22, amino acids 1-34 of SEQ ID NO: 147, and fragments thereof;
wherein V/PHD is a polypeptide according to claim 30 ;
wherein X C is selected from the group consisting of amino acids 136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, amino acids 136-232 of SEQ ID NO: 147, and fragments thereof.
43. A polypeptide comprising an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence that is at least 95% identical to nucleotides 8-322 of SEQ ID NO: 174, 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.
44. The polypeptide of claim 43 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
45. A polypeptide comprising an amino acid sequence that is encoded by a polynucleotide comprising a nucleotide sequence at least 95% identical to a nucleotide sequence of the formula:
5′-N 1 -N 2 -N 3 -N 4 -N 5 -N 6 -N 7 -N 8 -N 9 -3′
wherein N 1 is a nucleotide sequence selected from the group consisting of nucleotides 7-34 of SEQ ID NO: 3 and nucleotides 7-34 of SEQ ID NO: 23;
wherein N 2 is a nucleotide sequence selected from the group consisting of nucleotides 1-32 of SEQ ID NO: 4 and nucleotides 1-35 of SEQ ID NO: 24;
wherein N 3 is a nucleotide sequence selected from the group consisting of nucleotides 1-31 of SEQ ID NO: 5 and nucleotides 1-31 of SEQ ID NO: 25;
wherein N 4 is a nucleotide sequence selected from the group consisting of nucleotides 10-33 of SEQ ID NO: 6 and nucleotides 10-33 of SEQ ID NO: 26;
wherein N 5 is a nucleotide sequence selected from the group consisting of nucleotides 1-37 of SEQ ID NO: 7 and nucleotides 1-37 of SEQ ID NO: 27;
wherein N 6 is a nucleotide sequence selected from the group consisting of nucleotides 3-28 of SEQ ID NO: 8 and nucleotides 3-28 of SEQ ID NO: 28;
wherein N 7 is a nucleotide sequence selected from the group consisting of nucleotides 1-46 of SEQ ID NO: 9 and nucleotides 1-46 of SEQ ID NO: 29;
wherein N 8 is a nucleotide sequence selected from the group consisting of nucleotides 1-43 of SEQ ID NO: 10 and nucleotides 1-49 of SEQ ID NO: 30;
wherein N 3 is a nucleotide sequence selected from the group consisting of nucleotides 1-39 of SEQ ID NO: 11 and nucleotides 1-39 of SEQ ID NO: 31;
wherein 5′-N 1 -N 2 -N 3 -N 4 -N 5 -N 6 -N 7 -N 8 -N 9 -3′ is not identical to nucleotides 156 to 461 of SEQ ID NO: 1 or nucleotides 685 to 999 of SEQ ID NO: 21; 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.
46. The polypeptide of claim 45 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 22).
47. A polypeptide comprising an amino acid sequence at least 95% identical to 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 ID NO: 75; amino acids 1-104 as set forth in SEQ ID NO: 77; amino acids 1-104 as set forth in SEQ ID NO: 153; amino acids 1-105 as set forth in SEQ ID NO: 155; amino acids 1-105 as set forth in SEQ ID NO: 157; amino acids 1-105 as set forth in SEQ ID 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 ID 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,
wherein the polypeptide binds to at least one human vascular endothelial growth factor receptor selected from VEGFR-1, VEGFR-2, and VEGFR-3.
48. The polypeptide of claim 47 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
49. A polypeptide comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from tho group consisting of:
amino acids 1-102 of SEQ ID NO: 51; amino acids 1-102 of SEQ ID NO: 63; amino acids 1-104 of SEQ ID NO: 71; amino acids 1-104 of SEQ ID NO: 153; amino acids 1-103 of SEQ ID NO: 161; amino acids 1-102 of SEQ ID NO: 163; amino acids 1-104 of SEQ ID NO: 165; amino acids 1-104 of SEQ ID NO: 167; amino acids 1-104 of SEQ ID NO: 173; and amino acids 1-104 of SEQ ID NO: 175,
wherein the polypeptide binds human vascular endothelial growth factor receptors VEGFR-1, VEGFR-2, and VEGFR-3.
50. The polypeptide of claim 49 , further including additional flanking sequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).