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

• US 9,975,936 B2
• Filed: 10/13/2016
• Issued: 05/22/2018
• Est. Priority Date: 03/30/2007
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Cujec, Thomas P., Mariani, Roberto, Hays Putnam, Anna-Maria A., Keefe, William M., Knudsen, Nick, Skidmore, Lillian, Pinkstaff, Jason, Kraynov, Vadim
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US15/292,700
• Publication Number:
  US 20170096463A1
• Time in Patent Office: 586 Days
• Field of Search: None
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Modified FGF-21 polypeptides and uses thereof are provided.

Claims

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;

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;

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.