Nucleic acids encoding modified FGF-21 polypeptides comprising non-naturally occurring amino acids

Patent metadata

Field	Value
Publication number	US-9975936-B2
Application number	US-201615292700-A
Country	US
Kind code	B2
Filing date	Oct 13, 2016
Priority date	Mar 30, 2007
Publication date	May 22, 2018
Grant date	May 22, 2018

Abstract

Modified FGF-21 polypeptides and uses thereof are provided.

First claim

What is claimed is:

1. An isolated nucleic acid that encodes a biologically active modified FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein:
(a) the modified FGF-21 polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 1 fused to an N-terminal methionine, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 fused to an N-terminal methionine, SEQ ID NO: 6, or SEQ ID NO: 7;
(b) said non-naturally encoded amino acid has the structure:

wherein the R group is any substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine, or selenocysteine;
(c) said modified FGF-21 polypeptide contains a substitution of an amino acid with said non-naturally encoded amino acid at a position in said modified FGF-21 polypeptide corresponding to residue 72, 77, 86, 108, 110, 131, or 146 of SEQ ID NO: 1; and
(d) said non-naturally encoded amino acid is encoded by a selector codon.

2. The isolated nucleic acid of claim 1 , wherein said selector codon is selected from the group consisting of: an amber codon, an ochre codon, an opal codon, a five-base codon, and a four-base codon.

3. An isolated cell comprising the isolated nucleic acid of claim 1 .

4. The isolated cell of claim 3 , wherein said cell is a prokaryotic cell.

5. The isolated cell of claim 3 , wherein said cell further comprises an orthogonal tRNA comprising an anti-codon complementary to said selector codon.

6. The isolated cell of claim 5 , wherein said orthogonal tRNA is linked to a non-naturally encoded amino acid selected from a para-substituted, ortho-substituted, or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

7. The isolated cell of claim 5 , wherein said orthogonal tRNA is linked to a non-naturally encoded amino acid comprising para-acetyl-L-phenylalanine.

8. An isolated vector comprising the nucleic acid of claim 1 .

9. An isolated nucleic acid that encodes a biologically active modified FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein:
(a) the modified FGF-21 polypeptide comprises the sequence of SEQ ID NO: 1 fused to an N-terminal methionine having said non-naturally encoded amino acid substituted at position 108 of SEQ ID NO: 1;
(b) said non-naturally encoded amino acid has the structure:

wherein the R group is any substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine, or selenocysteine; and
(c) said non-naturally encoded amino acid is encoded by a selector codon.

10. An isolated cell comprising the isolated nucleic acid of claim 9 .

11. The isolated cell of claim 10 , wherein said cell is a prokaryotic cell.

12. The isolated cell of claim 10 , wherein said cell further comprises an orthogonal tRNA comprising an anti-codon complementary to said selector codon and linked to a non-naturally encoded amino acid comprising para-acetyl-L-phenylalanine.

13. An isolated nucleic acid that encodes a modified FGF-21 polypeptide, wherein:
(a) the modified FGF-21 polypeptide comprises SEQ ID NO: 1 fused to an N-terminal methionine, except that residue 108 of SEQ ID NO: 1 is substituted by a non-naturally encoded amino acid comprising para-acetyl-L-phenylalanine; and
(b) said non-naturally encoded amino acid is encoded by a selector codon.

14. The isolated nucleic acid of claim 13 , wherein said selector codon is selected from the group consisting of: an amber codon, an ochre codon, an opal codon, a five-base codon, and a four-base codon.

15. An isolated cell comprising the isolated nucleic acid of claim 13 .

16. The isolated cell of claim 15 , wherein said cell is a prokaryotic cell.

17. The isolated cell of claim 16 , wherein said cell is E. coli.

18. The isolated cell of claim 15 , wherein said cell further comprises an orthogonal tRNA comprising an anti-codon complementary to said selector codon.

19. The isolated cell of claim 18 , wherein said orthogonal tRNA is linked to a non-naturally encoded amino acid comprising para-acetyl-L-phenylalanine.

20. An isolated vector comprising the nucleic acid of claim 13 .

Assignees

• Ambrx Inc

Inventors

• Cujec Thomas P
• Mariani Roberto
• Hays Putnam Anna-Maria A
• Keefe William M
• Knudsen Nick
• Skidmore Lillian
• Pinkstaff Jason
• Kraynov Vadim

Classifications

• A61P37/02

  Immunomodulators · CPC title

• A61P3/08

  for glucose homeostasis (pancreatic hormones A61P5/48) · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P39/00

  General protective or antinoxious agents · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title