Remodeling and glycopegylation of fibroblast growth factor (FGF)

• US 9,200,049 B2
• Filed: 12/21/2011
• Issued: 12/01/2015
• Est. Priority Date: 10/29/2004
• Status: Active Grant

• Current Assignee: 89BIO LTD
• Original Assignee: Novo Nordisk A/S
• Inventors: DeFrees, Shawn
• Primary Examiner(s): GOON, SCARLETT Y
• Application Number:
  US13/332,708
• Publication Number:
  US 20120172300A1
• Time in Patent Office: 1,441 Days
• Field of Search: 514/9.1, 514/20.9, 514/54
• US Class Current: 1/1
• 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 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• 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 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic 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: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present invention relates to mutants of Fibroblast Growth Factor (FGF), particularly FGF-20 and FGF-21, which contain newly introduced N-linked or O-linked glycosylation site(s). The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.

Claims

1. A Fibroblast Growth Factor-21 (FGF-21) conjugate comprising a mutant FGF-21 peptide and a modifying group, wherein the mutant FGF-21 peptide has the sequence of SEQ ID NO:

146 except for the presence of at least one potential O-linked or N-linked glycosylation site not present in SEQ ID NO;

146, wherein the mutant FGF-21 peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs;

323-325, wherein the O-linked glycosylation site is a serine or threonine residue and the N-linked glycosylation site is an asparagine, wherein the O-linked or N-linked glycosylation site is present at a site within SEQ ID NO;

323, 324, or 325, wherein said modifying group is selected from the group consisting of water-soluble polymers, therapeutic moieties, diagnostic moieties, targeting moieties, and biomolecules, wherein said modifying group is covalently attached to said peptide at a preselected glycosyl or amino acid residue of said peptide via an intact glycosyl linking group, wherein said modifying group is not a naturally occurring saccharide moiety, and wherein the FGF-21 conjugate retains a biological activity of SEQ ID NO;

146.

2. The FGF-21 conjugate of claim 1, wherein said FGF-21 peptide is at least 95% homologous to the amino acid sequence of SEQ ID NO:

146.

3. The FGF-21 conjugate of claim 1, wherein said modifying group is a water-soluble polymer.

4. The FGF-21 conjugate of claim 1, wherein said modifying group is covalently attached at said preselected glycosyl residue.

5. The FGF-21 conjugate of claim 4, wherein said modifying group is a non-glycosidic modifying group.

6. The FGF-21 conjugate of claim 5, wherein said non-glycosidic modifying group is a linear PEG or a branched PEG.

7. The FGF-21 conjugate of claim 6, wherein said non-glycosidic modifying group is a linear PEG and said linear PEG has a structure according to the following formula:

8. The FGF-21 conjugate of claim 1, wherein said modifying group covalently attached to said intact glycosyl linking group together is a modifying group-intact glycosyl linking group cassette having a structure according to the following formula:

9. The FGF-21 conjugate of claim 8, wherein said modifying group covalently attached to said intact glycosyl linking group together is a modifying group-intact glycosyl linking group cassette having a structure according to the following formula:

10. The FGF-21 conjugate of claim 1, wherein the mutant FGF-21 peptide contains SEQ ID NO:

323.

11. The FGF-21 conjugate of claim 1, wherein the mutant FGF-21 peptide contains SEQ ID NO:

324.

12. The FGF-21 conjugate of claim 11, wherein said modifying group is a water-soluble polymer.

13. The FGF-21 conjugate of claim 12, wherein said water-soluble polymer is a linear PEG or a branched PEG.

14. The FGF-21 conjugate of claim 13, wherein said water-soluble polymer is a linear PEG and said linear PEG has a structure according to the following formula:

15. The FGF-21 conjugate of claim 13, wherein said modifying group covalently attached to said intact glycosyl linking group together is a modifying group-intact glycosyl linking group cassette having a structure according to the following formula:

16. The FGF-21 conjugate of claim 1, wherein the mutant FGF-21 peptide contains SEQ ID NO:

325.

17. A pharmaceutical composition comprising the FGF-21 conjugate of claim 1 and a carrier therefor.

18. A method of making the FGF-21 conjugate of claim 1, comprising the steps of:

(a) providing a nucleic acid encoding the mutant FGF-21 peptide in a host cell,

(b) culturing the host cell under conditions suitable for the expression of the nucleic acid to produce the mutant FGF-21 peptide,

(c) isolating the mutant FGF-21 peptide from the host cell, and

(d) enzymatically glycosylating the isolated mutant FGF-21 peptide with a sugar that has been covalently functionalized with the modifying group, wherein said glycosylating is a cell free, in vitro process,

thereby making the FGF-21 conjugate.

19. A method of treating FGF-21 deficiency in a patient in need thereof, comprising administering an effective amount of the FGF-21 conjugate of claim 1 to the patient, thereby treating the FGF-21 deficiency in the patient.