CHIMERIC FIBROBLAST GROWTH FACTORS WITH ALTERED RECEPTOR SPECIFICITY

• US 20110104152A1
• Filed: 10/15/2010
• Published: 05/05/2011
• Est. Priority Date: 10/15/2009
• Status: Active Grant

• Current Assignee: F Hoffmann-La Roche AG (Roche Holding AG)
• Original Assignee: F Hoffmann-La Roche AG (Roche Holding AG)
• Inventors: SONODA, Junichiro
• Granted Patent: US 8,535,912 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• 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 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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: C07K 16/22 : against growth factors ; against growth regulators
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention is directed to novel chimeric fibroblast growth factor (FGF) polypeptides, novel DNA encoding chimeric FGF polypeptides, and to the recombinant production of chimeric FGF polypeptides, and to methods, compositions and assays utilizing chimeric FGF polypeptides for the therapeutic treatment of metabolic-related disorders and other conditions, and for producing pharmaceutically active compositions including chimeric FGF polypeptides, the compositions having therapeutic and pharmacologic properties including those associated with the treatment of metabolic-related disorders and other conditions.

Claims

1. A chimeric fibroblast growth factor 19 (FGF19) polypeptide, the sequence of the polypeptide comprising:

a C-terminal portion that comprises a C-terminal portion of the hFGF19 polypeptide sequence; and

an N-terminal portion that comprises an N-terminal portion of the hFGF21 polypeptide sequence, wherein

the C-terminal portion of the hFGF19 polypeptide sequence is from about 45 to about 185 residues in length, the C-terminal portion of the hFGF19 polypeptide sequence having a first position and a final position,

the N-terminal portion of the hFGF21 polypeptide sequence is from about 7 to about 140 residues in length, the C-terminal portion of the hFGF21 polypeptide sequence having a first position and a final position.

2. The chimeric FGF19 polypeptide of claim 1, wherein:

the first position of the C-terminal portion of the hFGF19 polypeptide sequence corresponds to a position of SEQ ID NO;

1 selected from the group consisting of;

10, 11, 25, 26, 27, 28, 30, 33, 35, 37, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56, 57, 58, 59, 72, 73, 74, 79, 80, 81, 143, 144, 145 and 146; and

the final position of the C-terminal portion of the hFGF19 polypeptide sequence corresponds to about position 194 of SEQ ID NO;

1.

3. The chimeric FGF19 polypeptide of claim 2, wherein:

the first position of the C-terminal portion of the hFGF19 polypeptide sequence corresponds to position 25 of SEQ ID NO;

1, and

the final position of the C-terminal portion of the hFGF19 polypeptide sequence corresponds to about position 194 of SEQ ID NO;

1.

4. The chimeric FGF19 polypeptide of any one of claims 1-3, wherein:

the first position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to about position 1 of SEQ ID NO;

2; and

the final position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to a position of SEQ ID NO;

2 selected from the group consisting of;

7, 8, 20, 21, 22, 23, 25, 27, 29, 31, 34, 35, 36, 37, 38, 39, 46, 47, 48, 50, 51, 52, 53, 66, 67, 68, 73, 74, 75, 135, 136, 137 and 138.

5. The chimeric FGF19 polypeptide of claim 4, wherein:

the first position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to about position 1 of SEQ ID NO;

2; and

the final position of the N-terminal portion of the hFGF21 polypeptide sequence corresponds to position 20 of SEQ ID NO;

2.

6. The chimeric FGF19 polypeptide of claim 1, wherein:

the C-terminal portion of the hFGF19 polypeptide sequence corresponds to about position 25 to about position 194 of SEQ ID NO;

1; and

the N-terminal portion of the hFGF21 polypeptide sequence corresponds to about position 1 to about 20 of SEQ ID NO;

2.

15. The chimeric FGF19 polypeptide of any one of claims 1-14, wherein the chimeric hFGF19 polypeptide further comprises a substitution of the β

1-β

2 loop of the first polypeptide, a substitution of the β

10-β

12 segment of the first polypeptide, and/or a substitution of amino acid residues 38-42 of the first polypeptide with the β

1-β

2 loop of the second polypeptide, the β

10-β

12 segment of the second polypeptide, and/or the corresponding sequence amino acid residues of the second polypeptide.

17. The chimeric FGF19 polypeptide of any one of claims 1-16, wherein the chimeric hFGF19 polypeptide is fused to a second polypeptide, the second polypeptide is selected from the group consisting of:

the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin and one or more fragments of the Fc portion of an immunoglobulin.

18. The chimeric FGF19 polypeptide of claim 17, wherein the immunoglobulin is selected from the group consisting of:

IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2, IgE, IgD and IgM.

19. The chimeric FGF19 polypeptide of claim 17 or 18, wherein the Fc portion is human or humanized.

20. The chimeric FGF19 polypeptide of any one of claims 17-19, wherein the C-terminus of the chimeric hFGF19 polypeptide is fused to the N-terminus of the second polypeptide.

21. The chimeric FGF19 polypeptide of claim 20, wherein the C-terminus of the chimeric hFGF19 polypeptide is fused to the N-terminus of the second polypeptide via a linker, the linker is selected from the group consisting of:

a [Gly]n linker, a [Gly3Ser]m linker and a [Gly4Ser]m linker, wherein n is an integer from 1-30 and m is an integer from 1-6.

22. The chimeric FGF19 polypeptide of any one of claims 1-21, wherein the chimeric hFGF19 polypeptide does not substantially activate FGFR4 in either a Klotho-beta independent or Klotho-beta dependent manner.

23. The chimeric FGF19 polypeptide of any one of claims 1-21, wherein the chimeric FGF19 polypeptide activates FGFR1c in a Klotho-beta dependent manner.

24. The chimeric FGF19 polypeptide of any one of claims 1-23, wherein the chimeric FGF19 polypeptide when administered to an individual does not reduce the level of phosphorylated STAT5 polypeptide in the individual.

25. The chimeric FGF19 polypeptide of any one of claims 1-23, wherein the chimeric FGF19 polypeptide when administered to an individual reduces the amount of phosphorylated STAT5 polypeptide in the individual but this amount of phosphorylated STAT5 polypeptide is greater than the amount of phosphorylated STAT5 polypeptide upon administration of native hFGF21 to the individual.

26. The chimeric FGF19 polypeptide of any one of claims 1-23 and 25, wherein the chimeric FGF19 polypeptide when administered to an individual reduces the amount of phosphorylated STAT5 polypeptide to an amount that is any of:

from 100% to 5%, from 100% to 10%, from 100% to 20%, from 100% to 30%, from 100% to 40%, from 100% to 50%, from 100% to 60%, from 100% to 70%, from 100% to 80%, from 100% to 90% or from 100% to 95%, of the amount of phosphorylated STAT5 polypeptide in the individual without such administration.

27. The chimeric FGF19 polypeptide of any one of claims 1-23, 25 and 26, wherein the chimeric FGF19 polypeptide when administered to an individual, the reduction in the amount of phosphorylated STAT5 polypeptide is less than reduction in the amount of phosphorylated STAT5 polypeptide upon administration of native hFGF21.

28. The chimeric FGF19 polypeptide of claim 27, wherein the reduction of the phosphorylated STAT5 polypeptide when the chimeric hFGF19 polypeptide is administered to the individual is by any of:

from 0% to 5%, from 0% to 10%, from 0% to 20%, from 0% to 30%, from 0% to 40%, from 0% to 50%, from 0% to 60%, from 0% to 70%, from 0% to 80%, from 0% to 90% or from 0% to 95%, of the reduction in the amount of phosphorylated STAT5 polypeptide upon administration of native hFGF21.

29. The chimeric FGF19 polypeptide of any one of claims 1-28, wherein the chimeric FGF19 polypeptide when administered to an individual does not induce growth hormone resistance.

30. The chimeric FGF19 polypeptide of any one of claims 1-29, wherein the in vivo physiological half-life of the chimeric FGF19 polypeptide is at least or about the same as FGF19.

31. The chimeric FGF19 polypeptide of any one of claims 1-30, wherein the in vivo physiological half-life of the chimeric FGF19 polypeptide is at least or about the same as FGF21.

32. A pharmaceutical composition comprising:

(a) a therapeutically effective amount of the chimeric FGF19 polypeptide of any one of claims 1-31; and

(b) an acceptable pharmaceutical carrier.

33. A method of treating an individual exhibiting one or more of obesity, type 1 diabetes, type 2 diabetes, high blood glucose, metabolic syndrome, atherosclerosis, hypercholesterolemia, stroke, osteoporosis, osteoarthritis, degenerative joint disease, muscle atrophy, sarcopenia, decreased lean body mass, baldness, wrinkles, increased fatigue, decreased stamina, decreased cardiac function, immune system dysfunction, cancer, Parkinson'"'"'s disease, senile dementia, Alzheimer'"'"'s disease and decreased cognitive function, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of claim 32.

34. A method of lowering the blood glucose of an individual in need of such treatment, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of claim 32.

35. The method of any one of claims 24-29, 33 and 34, wherein the individual is a human.

48. An antibody that binds specifically to any one of the chimeric FGF19 polypeptides of claims 1-31.

49. The antibody of claim 48, wherein the antibody is monoclonal.

50. The antibody of claim 48 or claim 49, wherein the antibody does not bind native FGF19 polypeptide or native FGF21 polypeptide.

7. A chimeric FGF19 polypeptide, the sequence of the polypeptide comprising:

a C-terminal portion that comprises a C-terminal portion of the hFGF21 polypeptide sequence; and

an N-terminal portion that comprises an N-terminal portion of the hFGF19 polypeptide sequence, wherein the C-terminal portion of the hFGF21 polypeptide sequence is from about 8 to about 145 residues in length, the C-terminal portion of the hFGF21 polypeptide sequence having a first position and a final position, and

wherein the N-terminal portion of the hFGF19 polypeptide sequence is from about 45 to about 175 residues in length, the N-terminal portion of the hFGF19 polypeptide sequence having a first position and a final position.

8. The chimeric FGF19 polypeptide of claim 7, wherein:

the first position of the C-terminal portion of the hFGF21 polypeptide sequence corresponds to a position of SEQ ID NO;

2 selected from the group consisting of;

8, 9, 21, 22, 23, 24, 35, 36, 37, 38, 39, 40, 47, 48, 49, 51, 52, 53, 54, 67, 68, 69, 74, 75, 76, 146, 147, 148 and 149; and

the final position of the C-terminal portion of the hFGF21 polypeptide sequence corresponds to about position 181 of SEQ ID NO;

2.

9. The chimeric FGF19 polypeptide of claim 7 or 8, wherein:

the first position of the N-terminal portion of the hFGF19 polypeptide sequence corresponds to about position 1 of SEQ ID NO;

1; and

the final position of the N-terminal portion of the hFGF19 polypeptide sequence corresponds to a position of SEQ ID NO;

1 selected from the group consisting of;

9, 10, 24, 25, 26, 27, 39, 40, 41, 42, 43, 44, 51, 52, 53, 55, 56, 57, 58, 71, 72, 73, 78, 79, 80, 142, 143, 144 and 145.

10. A chimeric FGF19 polypeptide, wherein the sequence of the polypeptide comprises:

a first polypeptide sequence having at least 85% sequence identity to SEQ ID NO;

1, wherein a portion of the first polypeptide sequence is substituted with a portion of a second polypeptide sequence, the second polypeptide sequence having at least 85% sequence identity to SEQ ID NO;

2, the substituted portion of the first polypeptide sequence has a first position and last position,

the first position of the substituted portion corresponds to a position in SEQ ID NO;

1 selected from the group consisting of;

1, 10, 11, 17, 18, 21, 22, 25, 26, 27, 28, 40, 41, 42, 43, 44, 45, 52, 53, 54, 56, 57, 58, 59, 63, 72, 73, 74, 79, 80, 81, 143, 144, 145 and 146,

the last position of the substituted portion corresponds to a position in SEQ ID NO;

1 selected from the group consisting of;

9, 10, 24, 25, 26, 27, 29, 31, 32, 34, 36, 39, 40, 41, 42, 43, 44, 51, 52, 53, 55, 56, 57, 58, 66, 71, 72, 73, 78, 79, 80, 142, 143, 144, 145 and 194, and

the first position of the substituted portion and the last position of the substituted portion are selected such that the substituted portion of the first polypeptide sequence is from about 3 to about 185 residues in length.

11. The chimeric FGF19 polypeptide of claim 10, wherein the substituted portion of the first polypeptide sequence corresponds to a portion in SEQ ID NO:

1 selected from the group consisting of;

from position 11 to position 24, from position 11 to position 29, from position 11 to position 31, from position 11 to position 32, from position 11 to position 34, from position 11 to position 36, from position 11 to position 39, from position 11 to position 42, from position 11 to position 51, from position 11 to position 55, from position 11 to position 71, from position 18 to position 24, from position 18 to position 29, from position 18 to position 31, from position 18 to position 32, from position 18 to position 34, from position 18 to position 36, from position 22 to position 24, from position 22 to position 29, from position 22 to position 31, from position 22 to position 32, from position 22 to position 34, from position 22 to position 36, from position 28 to position 39, from position 28 to position 42, from position 28 to position 51, from position 28 to position 55, from position 28 to position 71 and from position 63 to position 66.

12. The chimeric FGF19 polypeptide of claim 10 or 11, wherein the substituting portion of the second polypeptide sequence has a first position and last position,

the first position of the substituted portion corresponds to a position in SEQ ID NO:

2 selected from the group consisting of;

1, 8, 9, 13, 14, 17, 18, 21, 22, 23, 24, 35, 36, 37, 38, 39, 40, 47, 48, 49, 51, 52, 53, 54, 58, 67, 68, 69, 74, 75, 76, 136, 137, 138 and 139,

the last position of the substituted portion corresponds to a position in SEQ ID NO;

2 selected from the group consisting of;

7, 8, 20, 21, 22, 23, 24, 25, 27, 29, 31, 34, 35, 36, 37, 38, 39, 46, 47, 48, 50, 51, 52, 53, 61, 66, 67, 68, 73, 74, 75, 135, 136, 137, 138 and 181, and

the first position of the substituting portion and the last position of the substituting portion are selected such that the substituting portion of the second polypeptide sequence is from about 3 to about 174 residues in length.

13. The chimeric FGF19 polypeptide of any one of claims 10-12, wherein the substituting portion of the second polypeptide sequence corresponds to a portion in SEQ ID NO:

2 selected from the group consisting of;

from position 9 to position 20, from position 9 to position 25, from position 9 to position 27, from position 9 to position 29, from position 9 to position 31, from position 9 to position 34, from position 9 to position 37, from position 9 to position 46, from position 9 to position 50, from position 9 to position 66, from position 14 to position 20, from position 14 to position 25, from position 14 to position 27, from position 14 to position 29, from position 14 to position 31, from position 18 to position 20, from position 18 to position 25, from position 18 to position 27, from position 18 to position 29, from position 18 to position 31, from position 24 to position 34, from position 24 to position 43, from position 24 to position 46, from position 24 to position 50, from position 24 to position 66 and from position 57 to position 61.

14. The chimeric hFGF19 polypeptide of claim 13, wherein the substituted portion of the first polypeptide sequence and the portion of the second polypeptide that is substituting therefor are selected from the group consisting of:

the first polypeptide sequence portion from position 11 to position 24 is substituted with the second polypeptide sequence portion from position 9 to position 20,

the first polypeptide sequence portion from position 11 to position 29 is substituted with the second polypeptide sequence portion from position 9 to position 25,

the first polypeptide sequence portion from position 11 to position 31 is substituted with the second polypeptide sequence portion from position 9 to position 27,

the first polypeptide sequence portion from position 11 to position 32 is substituted with the second polypeptide sequence portion from position 9 to position 27,

the first polypeptide sequence portion from position 11 to position 34 is substituted with the second polypeptide sequence portion from position 9 to position 29,

the first polypeptide sequence portion from position 11 to position 36 is substituted with the second polypeptide sequence portion from position 9 to position 31,

the first polypeptide sequence portion from position 11 to position 39 is substituted with the second polypeptide sequence portion from position 9 to position 34,

the first polypeptide sequence portion from position 11 to position 42 is substituted with the second polypeptide sequence portion from position 9 to position 37,

the first polypeptide sequence portion from position 11 to position 51 is substituted with the second polypeptide sequence portion from position 9 to position 46,

the first polypeptide sequence portion from position 11 to position 55 is substituted with the second polypeptide sequence portion from position 9 to position 50,

the first polypeptide sequence portion from position 11 to position 71 is substituted with the second polypeptide sequence portion from position 9 to position 66,

the first polypeptide sequence portion from position 18 to position 24 is substituted with the second polypeptide sequence portion from position 14 to position 20,

the first polypeptide sequence portion from position 18 to position 29 is substituted with the second polypeptide sequence portion from position 14 to position 25,

the first polypeptide sequence portion from position 18 to position 31 is substituted with the second polypeptide sequence portion from position 14 to position 27,

the first polypeptide sequence portion from position 18 to position 32 is substituted with the second polypeptide sequence portion from position 14 to position 27,

the first polypeptide sequence portion from position 18 to position 34 is substituted with the second polypeptide sequence portion from position 14 to position 29,

the first polypeptide sequence portion from position 18 to position 36 is substituted with the second polypeptide sequence portion from position 14 to position 31,

the first polypeptide sequence portion from position 22 to position 24 is substituted with the second polypeptide sequence portion from position 18 to position 20,

the first polypeptide sequence portion from position 22 to position 29 is substituted with the second polypeptide sequence portion from position 18 to position 25,

the first polypeptide sequence portion from position 22 to position 31 is substituted with the second polypeptide sequence portion from position 18 to position 27,

the first polypeptide sequence portion from position 22 to position 32 is substituted with the second polypeptide sequence portion from position 18 to position 27,

the first polypeptide sequence portion from position 22 to position 34 is substituted with the second polypeptide sequence portion from position 18 to position 29,

the first polypeptide sequence portion from position 22 to position 36 is substituted with the second polypeptide sequence portion from position 18 to position 31,

the first polypeptide sequence portion from position 28 to position 39 is substituted with the second polypeptide sequence portion from position 24 to position 34,

the first polypeptide sequence portion from position 28 to position 42 is substituted with the second polypeptide sequence portion from position 24 to position 43,

the first polypeptide sequence portion from position 28 to position 51 is substituted with the second polypeptide sequence portion from position 24 to position 46,

the first polypeptide sequence portion from position 28 to position 55 is substituted with the second polypeptide sequence portion from position 24 to position 50,

the first polypeptide sequence portion from position 28 to position 71 is substituted with the second polypeptide sequence portion from position 24 to position 66, and

the first polypeptide sequence portion from position 63 to position 66 is substituted with the second polypeptide sequence portion from position 57 to position 61.

16. A chimeric FGF19 polypeptide comprising the sequence with SEQ ID NO:

5.

36. An isolated nucleic acid molecule comprising DNA having any of:

at least an 80%, at least a 90%, at least a 95% or at least a 99% sequence identity to (a) a DNA molecule encoding a polypeptide having the amino acid sequence with SEQ ID NO;

5, or (b) the complement of the DNA molecule of (a).

37. The isolated nucleic acid molecule of claim 36 comprising the nucleic acid sequence with SEQ ID NO:

7.

38. The isolated nucleic acid molecule of claim 36 comprising a DNA molecule encoding a polypeptide comprising the sequence of amino acid residues from 1 to 190 of SEQ ID NO:

5, or (b) the complement of the DNA molecule of (a).

39. The isolated nucleic acid molecule of claim 36 comprising a DNA molecule encoding a polypeptide having at least 85% amino acid sequence identity to residues from 1 to 190 of SEQ ID NO:

5, or (b) the complement of the DNA molecule of (a).

40. The isolated nucleic acid molecule of claim 36 comprising DNA having any of:

at least an 80%, at least a 90%, at least a 95% or at least a 99% sequence identity to the sequence with SEQ ID NO;

7.

41. The isolated nucleic acid of any one of claims 36-40, wherein the encoded polypeptide further comprises the amino acid residues corresponding to the Fc portion of an immunoglobulin.

42. An expression system comprising the nucleic acid molecule of any one of claims 36-41.

43. A host cell comprising the expression system of claim 42.

44. A host cell comprising the nucleic acid molecule of any one of claims 36-41.

45. An isolated polypeptide encoded by the nucleic acid molecule of any one of claims 36-41.

46. A process for producing an isolated polypeptide comprising:

culturing the host cell of claim 43 or 44 under conditions suitable for expression of the encoded polypeptide; and

recovering the encoded polypeptide from the cell culture.

47. An isolated polypeptide produced by the process of claim 46.