Materials and methods involving hybrid vascular endothelial growth factor DNAs and proteins
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 selected from the group consisting of SEQ ID NO: 241 (construct 11-3) or SEQ ID NO: 209 (construct 13-1),
wherein the polypeptide binds to human VEGFR-1 and human VEGFR-2, and
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.
2. The polypeptide according to claim 1 , wherein the polypeptide includes a prepro-VEGF-C amino-terminal propeptide and a prepro-VEGF-C carboxy-terminal propeptide.
3. The polypeptide according to claim 1 , wherein the polypeptide comprises a prepro-VEGF-C signal peptide and VEGF-C amino-terminal pro-peptide.
4. The polypeptide according to claim 1 , wherein the polypeptide comprises a prepro-VEGF-C carboxy terminal propeptide.
5. The polypeptide of claim 1 that is purified.
6. A composition comprising the polypeptide according to claim 1 in a pharmaceutically acceptable carrier.
7. A polypeptide comprising an amino acid sequence of the formula:
X N —V/PHD-X C ,
wherein X N comprises amino acids 1-111 of SEQ ID NO: 22 or a fragment thereof;
wherein V/PHD comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 241 (construct 11-3) or SEQ ID NO: 209 (construct 13-1);
wherein X C comprises amino acids 217-419 of SEQ ID NO: 22 or a fragment thereof;
wherein X N and X C are each identical to an amino acid sequence in a naturally occurring human VEGF-C precursor protein; and
wherein the polypeptide binds human VEGFR-1 and human VEGFR-2.
8. The polypeptide of claim 7 that is purified.
9. A composition comprising the polypeptide according to claim 7 in a pharmaceutically acceptable carrier.
10. A polynucleotide that comprises a nucleotide sequence that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to a chimeric vascular endothelial growth factor (VEGF) amino acid sequence selected from the group consisting of SEQ ID NO: 241 (construct 11-3) or SEQ ID NO: 209 (construct 13-1),
wherein the polypeptide binds to human VEGFR-1 and human VEGFR-2, and
wherein the polypeptide further includes a prepro-VEGF-C amino-terminal propeptide and a prepro-VEGF-C carboxy-terminal pro-peptide.
11. A polynucleotide that comprises a nucleotide sequence that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence of the formula:
X N —V/PHD-X C ,
wherein X N comprises amino acids 1-111 of SEQ ID NO: 22 or a fragment thereof;
wherein V/PHD comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 241 (construct 11-3) or SEQ ID NO: 209 (construct 13-1);
wherein X C comprises amino acids 217-419 of SEQ NO: 22 or a fragment thereof;
wherein X N and X C are each identical to an amino acid sequence in a naturally occurring human VEGF-C precursor protein; and
wherein the polypeptide binds human VEGFR-1 and human VEGFR-2.
12. A polypeptide comprising an amino acid sequence of the formula:
X N —V/PHD-X C ,
wherein X N comprises amino acids 1-111 of SEQ ID NO: 22 or a fragment thereof;
wherein V/PHD comprises an amino acid sequence at least 95% identical to an amino acid sequence of the formula:
NH2-X 1 —X 2 —X 3 —X 4 —X 5 —X 6 —X 7 —X 8 —X 9 —COOH
wherein X 1 is amino acids 3-11 of SEQ ID NO: 128; wherein X 2 is SEQ ID NO: 129; wherein X 3 is SEQ ID NO: 130; wherein X 4 is SEQ ID NO: 131; wherein X 5 is SEQ ID NO: 132; wherein X 6 is SEQ ID NO: 142; wherein X 7 is SEQ ID NO: 134; wherein X 8 is SEQ ID NO 135; and wherein X 9 is SEQ ID NO 136; or
wherein X 1 is amino acids 3-11 of SEQ ID NO: 128; wherein X 2 is SEQ ID NO: 129; wherein X 3 is SEQ ID NO: 130; wherein X 4 is SEQ ID NO: 131; wherein X 5 is SEQ ID NO: 141; wherein X 6 is SEQ ID NO: 133; wherein X 7 is SEQ ID NO: 134; wherein X 8 is SEQ ED NO 135; and wherein X 9 is SEQ ID NO 136;
wherein X C comprises amino acids 217-419 of SEQ ID NO: 22 or a fragment thereof;
wherein X N and X C are each identical to an amino acid sequence in a naturally occurring human VEGF-C precursor protein; and
wherein the polypeptide binds to human VEGFR-1 and human VEGFR-2.
13. A polynucleotide comprising a nucleotide sequence at least 95% identical to a nucleotide sequence encoding a chimeric VEGF 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 the VEGF nucleotide sequence of A1-F (SEQ ID NO: 3); wherein N 2 is the VEGF nucleotide sequence of A2-F (SEQ ID NO: 4); wherein N 3 is the VEGF nucleotide sequence of A3-F (SEQ ID NO: 5); wherein N 4 is the VEGF nucleotide sequence of A4-F (SEQ ID NO: 6); wherein N 5 is the VEGF-C nucleotide sequence of C5-F (SEQ ID NO: 27); wherein N 6 is the VEGF nucleotide sequence of A6-F (SEQ ID NO: 8); wherein N 7 is the VEGF nucleotide sequence of A7-F (SEQ ID NO: 9); wherein N 8 is the VEGF nucleotide sequence of A8-F (SEQ ID NO: 10); and wherein N 9 is the VEGF nucleotide sequence of A9-F (SEQ ID NO: 11); or
wherein N 1 is the VEGF nucleotide sequence of A1-F (SEQ ID NO: 3); wherein N 2 is the VEGF nucleotide sequence of A2-F (SEQ ID NO: 4); wherein N 3 , is the VEGF nucleotide sequence of A3-F (SEQ ID NO: 5); wherein N 4 is the VEGF nucleotide sequence of A4-F (SEQ ID NO: 6); wherein N 5 is the VEGF nucleotide sequence of A5-F (SEQ ID NO: 7); wherein N 6 is the VEGF-C nucleotide sequence of C6-F (SEQ ID NO: 28); wherein N 7 is the VEGF nucleotide sequence of A7-F (SEQ ID NO: 9); wherein N 8 is the VEGF nucleotide sequence of A8-F (SEQ ID NO: 10); and wherein N 9 is the VEGF nucleotide sequence of A9-F (SEQ ID NO: 11);
wherein the encoded polypeptide binds to human VEGFR-1 and human VEGFR-2, and
wherein the polypeptide encoded by the polynucleotide further includes a prepro-VEGF-C amino-terminal propeptide, a prepro-VEGF-C carboxy-terminal pro-peptide, or a prepro-VEGF-C amino-terminal propeptide and a prepro-VEGF-C carboxy-terminal pro-peptide.