# US 9,517,273 B2 · Methods of treatment using modified FGF-21 polypeptides comprising non-naturally occurring amino acids

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- Number: US 9,517,273 B2
- Document ID: US9517273B2
- Status: Granted
- Kind: B2
- Application number: 14/680,543
- Filed: 2015-04-07
- Published: 2015-10-01
- Granted: 2016-12-13
- Earliest claimed: 2007-03-30
- Assignees: Ambrx, Inc.
- Applicants: Ambrx, Inc.
- Inventors: Thomas P. Cujec; Roberto Mariani; Anna-Maria A. Hays Putnam; Wiliam M. Keefe; Nick Knudsen; Lillian Skidmore; Jason Pinkstaff; Vadim Kraynov
- Web page: https://patentgrep.com/patent/US9517273B2

## CPC

- C07K 14/50: Fibroblast growth factor [FGF]
- A61K 38/1825: {Fibroblast growth factor [FGF]}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 37/02: Immunomodulators
- A61P 39/00: General protective or antinoxious agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- 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)
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Modified FGF-21 polypeptides and uses thereof are provided.

## Claims

### Claim 1

A method of treating a patient in need thereof, comprising administering an effective amount of a modified FGF-21 polypeptide to said patient, wherein said modified FGF-21 polypeptide maintains the biological activity of human FGF-21 polypeptides and comprises a non-naturally encoded amino acid, wherein:
(a) the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7;
(b) the 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) the polypeptide contains a substitution of an amino acid with the 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;
(d) the non-naturally encoded amino acid is linked to a linker, polymer, or biologically active molecule; and
(e) the polypeptide has an in vivo half-life at least two-fold greater than the human FGF-21 polypeptide in SEQ ID NO: 1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the non-naturally encoded amino acid is a phenylalanine derivative.

### Claim 3 (depends on 1)

The method of claim 1, wherein the non-naturally encoded amino acid is linked to a polymer.

### Claim 4 (depends on 1)

The method of claim 1, wherein the non-naturally encoded amino acid is 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.

### Claim 5 (depends on 1)

The method of claim 1, wherein said patient has a disease selected from type 2 diabetes, obesity, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, a glucose metabolism disorder, or a combination thereof.

### Claim 6

A method of treating a patient in need thereof, comprising administering an effective amount of a modified FGF-21 polypeptide to said patient, wherein said modified FGF-21 polypeptide maintains the biological activity of human FGF-21 polypeptides and comprises the polypeptide of SEQ ID NO:1 except that an amino acid in the polypeptide is substituted by a non-naturally encoded amino acid, wherein:
(a) said non-naturally encoded amino acid is at a position corresponding to residue 108 of SEQ ID NO:1;
(b) said non-naturally encoded amino acid comprises para-acetyl phenylalanine linked to a polymer comprising a poly(ethylene glycol), wherein said poly(ethylene glycol) has an average molecular weight of about 30 kDa; and
(c) said non-naturally encoded amino acid is linked to said polymer through an oxime linkage.

### Claim 7 (depends on 6)

The method of claim 6, wherein said polymer comprises an aminooxy group, and said non-naturally encoded amino acid is linked to said polymer by said oxime linkage formed by a reaction of said para-acetyl phenylalanine with said aminooxy group.

### Claim 8

A method of treating a patient in need thereof, comprising administering an effective amount of a modified FGF-21 polypeptide to said patient, wherein said modified FGF-21 polypeptide maintains the biological activity of human FGF-21 polypeptides and comprises a non-naturally encoded amino acid, wherein:
(a) the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7;
(b) the 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) the polypeptide contains a substitution of an amino acid with the 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;
(d) the non-naturally encoded amino acid is linked to a linker, polymer, or biologically active molecule; and
(e) the polypeptide has an in vivo half-life at least two-fold greater than the human FGF-21 polypeptide in SEQ ID NO: 1.

### Claim 9 (depends on 8)

The method of claim 8, wherein the non-naturally encoded amino acid is a phenylalanine derivative or comprises a first functional group and the linker, polymer, or biologically active molecule comprises a second functional group, wherein the first functional group and second functional group are not identical and each comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

### Claim 10 (depends on 9)

The method of claim 9, wherein the first functional group on the non-naturally encoded amino acid is a carbonyl moiety and the second functional group on the linker, polymer, or biologically active molecule is an aminooxy moiety, and the resultant covalent linkage created by the reaction of the first and second functional groups is an oxime linkage.

### Claim 11 (depends on 8)

The method of claim 8, wherein the non-naturally encoded amino acid is linked to a polymer comprising poly(ethylene glycol) or is linked to a polymer comprising poly(ethylene glycol) having an average molecular weight of between about 0.1 kDa and about 100 kDa.

### Claim 12 (depends on 11)

The method of claim 11, wherein the poly(ethylene glycol) has an average molecular weight of about 30 kDa.

### Claim 13 (depends on 8)

The method of claim 8, wherein the polypeptide has an in vivo half-life at least five-fold greater than the human FGF-21 polypeptide in SEQ ID NO: 1.

### Claim 14 (depends on 8)

The method of claim 8, wherein the non-naturally encoded amino acid is at a position corresponding to residue 108 of SEQ ID NO:1.

### Claim 15 (depends on 8)

The method of claim 8, wherein the non-naturally encoded amino acid is 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.

### Claim 16 (depends on 15)

The method of claim 15, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

### Claim 17 (depends on 8)

The method of claim 8, wherein the polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, containing a substitution of an amino acid with a non-naturally encoded amino acid.

### Claim 18 (depends on 8)

The method of claim 8, wherein said patient has a disease selected from type 2 diabetes, obesity, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, a glucose metabolism disorder, or a combination thereof.

### Claim 19 (depends on 8)

The method of claim 8, further comprising administering another active agent comprising anti-diabetic agent to the patient.

### Claim 20 (depends on 8)

The method of claim 8, further comprising administering to the patient another active agent selected from insulin, biguanides, thiazolidinediones, sulfonylureas, benzoic acid derivatives, glucosidase inhibitors, DPP-4 inhibitors, human insulin, zinc insulin, protamine zinc insulin, a fragment or derivative of insulin, INS-1, oral insulin, insulin sensitizers, pioglitazone or a salt thereof, pioglitazone hydrochloride, rosiglitazone or a salt thereof, rosiglitazone maleate, netoglitazone, Rivoglitazone (CS-011), FK-614, Tesaglitazar (AZ-242), Ragaglitazar (N,N-622), Muraglitazar (BMS-298585), Edaglitazone (BM-13-1258), Metaglidasen (MBX-102), Naveglitazar (LY-519818), MX-6054, LY-510929, AMG-131(T-131), THR-0921, alpha-glucosidase inhibitors, voglibose, acarbose, miglitol, emiglitate, phenformin, metformin, metformin HCl, buformin or a salt thereof, buformin hydrochloride, buformin fumarate, buformin succinate, insulin secretagogues, sulfonylurea, tolbutamide, glyburide, micronized glyburide, glimepiride, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride, AG-EE 623 Z, glipizide, glipizide GITS, glybuzole, meglitinides, repaglinide, nateglinide, mitiglinide, mitiglinide calcium salt hydrate, dipeptidyl peptidase IV inhibitors, Vidagliptin (LAF237), P32/98, Sitagliptin (MK-431), P93/01, PT-100, Saxagliptin (BMS-477118), T-6666, TS-021, beta3 agonists, AJ-9677, GPR40 agonists, glucagon-like polypeptides (I) (glp I), glp2, diabetogenic peptide hormones, GLP-1 receptor agonists, GLP-1, GLP-1MR agent, N,N-2211, AC-2993 (exendin-4), BIM-51077, Aib(8,35)hGLP-1 (7,37)NH2, GLP-1(7-36)amide, CJC-[131], amylin agonists, pramlintide, phosphotyrosine phosphatase inhibitors, sodium vanadate, gluconeogenesis inhibitors, glycogen phosphorylase inhibitors, glucose-6-phosphatase inhibitors, glucagon antagonists, SGLUT (sodium-glucose cotransporter) inhibitors, T-1095, 11beta-hydroxysteroid dehydrogenase inhibitors, BVT-3498, adiponectin, adiponectin agonists, IKK inhibitors, AS-2868, leptin resistance improving drugs, somatostatin receptor agonists, glucokinase activators, Ro-28-1675, Glucose-dependent insulinotropic peptide (GIP), Vildagliptin (Galvus) (LAF237), Sitagliptin (Januvia), Saxagliptin, Alogliptin, 1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(- S)-pyrrolidine (NVP DPP728), troglitazone, carnitine palmitoyl-transferase I inhibitors, and etomoxir.