GLP-1 Fusion Proteins

• US 20040053370A1
• Filed: 05/29/2003
• Published: 03/18/2004
• Est. Priority Date: 12/07/2000
• Status: Active Grant

• Current Assignee: Eli Lilly and Company
• Original Assignee: Eli Lilly and Company
• Inventors: Glaesner, Wolfgang, Micanovic, Radmila, Tschang, Sheng-Hung Rainbow
• Granted Patent: US 7,271,149 B2
• Time in Patent Office: Days
• US Class Current: 435/69.7
• 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• 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
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/735 containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

The present invention relates to glucagon-like-1 compounds fused to proteins that have the effect of extending the in vivo half-life of the peptides. These fusion proteins can be used to treat non-insulin dependent diabetes mellitus as well as a variety of other conditions.

Claims

1. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of

a) human albumin;

b) human albumin analogs; and

c) fragments of human albumin,

and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.

4. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula 1 [SEQ ID NO:

2]

5. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula II (SEQ ID NO:

3);

6. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula III (SEQ ID NO:

4);

7. The heterologous fusion protein of claim 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula IV (SEQ ID NO:

5);

8. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula V (SEQ ID NO:

6)

9. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula VIII (SEQ ID NO:

11).

10. The heterologous fusion protein of claims 1 through 9 wherein the GLP-1 compound has no more than 6 amino acids that are different from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

11. The heterologous fusion protein of claim 10 wherein the GLP-1 compound has no more than 5 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

12. The heterologous fusion protein of claim 11 wherein the GLP-1 compound has no more than 4 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

13. The heterologous fusion protein of claim 12 wherein the GLP-1 compound has no more than 3 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

14. The heterologous fusion protein of claim 13 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

15. The heterologous fusion protein of any one of claims 1 through 14 wherein Xaa at position 8 is glycine or valine.

16. The heterologous fusion protein of claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine or valine and Xaa at position 30 is alanine, glutamic acid, aspartic acid, serine, or histidine.

17. The heterologous fusion protein of claim 16 wherein Xaa at position 30 is Glutamic acid.

18. The heterologous fusion protein of claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine or valine and Xaa at position 37 is histidine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, serine, threonine, arginine, or lysine.

19. The heterologous fusion protein of claim 18 wherein Xaa at position 37 is histidine.

20. The heterologous fusion protein of claim 8 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine, valine, leucine, isoleucine, serine, threonine, or methionine and Xaa at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine or histidine.

21. The heterologous fusion protein of claim 20 wherein Xaa at position 22 is lysine or glutamic acid.

22. The heterologous fusion protein of claim 8 wherein the GLP-1 compound is Val⁸-GLP-1(7-37).

23. The heterologous fusion protein of claim 8 wherein the GLP-1 compound is Gly⁸-GLP-1(7-37).

24. The heterologous fusion protein of any one of claims 1 through 23 wherein the second polypeptide is human albumin.

25. The heterologous fusion protein of claim 24 wherein the second polypeptide has the sequence of SEQ ID NO:

34.

26. The heterologous fusion protein of any one of claims 1 through 23 wherein the second polypeptide is an N-terminal fragment of albumin.

57. A polynucleotide encoding a heterologous fusion protein of any one of claims 1 through 56.

58. A vector comprising the polynucleotide of claim 57.

59. A host cell comprising the vector of claim 58.

60. A host cell expressing at least one heterologous fusion protein of any one of claims 1 through 56.

61. The host cell of claim 60 wherein said host cell is a CHO cell.

62. A process for producing a heterologous fusion protein comprising the steps of transcribing and translating a polynucleotide of claim 57 under conditions wherein the heterologous fusion protein is expressed in detectable amounts.

63. A method for normalizing blood glucose levels in a mammal in need thereof comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of claims 1 through 56.

64. A method of treating a patient with non-insulin dependent diabetes mellitus comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of claims 1 through 56.

65. A method of treating obesity comprising the administration of a therapeutically effective amount of the heterologous fusion protein of any one of claims 1 through 56.

66. The use of a heterologous fusion protein as claimed in any one of claims 1 through 56 for the manufacture of a medicament for the treatment of patients with non-insulin dependent diabetes mellitus.

67. The use of a heterologous fusion protein as claimed in any one of claims 1 through 56 for the manufacture of a medicament for the treatment of patients with obesity.

68. A pharmaceutical formulation adapted for the treatment of patients with non-insulin dependent diabetes comprising a heterologous fusion protein of any one of claims 1 through 56.

69. The heterologous fusion protein of claims 1, 2, or 3 wherein the GLP-1 compound comprises the sequence of formula IX [SEQ ID NO:

12]

70. The heterologous fusion protein of claim 69 wherein the second polypeptide is human albumin.

71. The heterologous fusion protein of claim 70 wherein the second polypeptide has the sequence of SEQ ID NO:

34.

72. The heterologous fusion protein of claim 69 wherein the second polypeptide is an N-terminal fragment of albumin.

80. The heterologous fusion protein of any one of claims 69 through 79 wherein the GLP-1 compound is selected from the group consisting of Gly⁸-GLP-1(7-37), Val⁸-Tyr¹²-GLP-1(7-37), Val⁸-Tyr¹²-GLP-1(7-36), Val⁸-Trp¹²-GLP-1(7-37), Val⁸-Leu¹⁶-GLP-1(7-37), Val⁸-Val¹⁶-GLP-1(7-37), Val⁸-Tyr¹⁶-GLP-1(7-37), Gly⁸-Glu²²-GLP-1(7-37), Val⁸-Glu²²-GLP-1(7-37), Val⁸-Leu²⁵-GLP-1(7-37), Val⁸-Tyr¹²-Tyr¹⁶-GLP-1(7-37), Val⁸-Trp¹²-Glu²²-GLP-1(7-37), Val⁸-Tyr¹²-Glu²²-GLP-1(7-37), Val⁸-Tyr¹⁶-Phe¹⁹-GLP-1(7-37), Val⁸-Tyr¹⁶-Glu²²-GLP-1(7-37), Val8-Trp¹⁶-Glu²²-GLP-1(7-37), Val⁸-Leu¹⁶-Glu²²-GLP-1(7-37), Val¹⁸-Ile¹⁶-Glu²²-GLP-1(7-37), Val⁸-Phe¹⁶-Glu²²-GLP-1(7-37), Val⁸-Trp¹⁸-Glu²²-GLP-1(7-37), Val⁸-Tyr¹⁸-Glu²²-GLP-1(7-37), Val⁸-Phe¹⁸-Glu²²-GLP-1(7-37), Val⁸-Ile¹⁸-Glu²²-GLP-1(7-37), Val⁸-Lys¹⁸-Glu²²-GLP-1(7-37), Val⁸-Trp¹⁹-Glu²²-GLP-1(7-37), Val⁸-Phe¹⁹-Glu²²-GLP-1(7-37), Val⁸-Phe²⁰-Glu²²-GLP-1(7-37), Val⁸-Glu²²-Leu²⁵-GLP-1(7-37), Val⁸-Glu²²-Ile²⁵-GLP-1(7-37), Val⁸-Glu²²-Val²⁵-GLP-1(7-37), Val⁸-Glu²²-Ile²⁷-GLP-1(7-37), Val⁸-Glu²²-Ala²⁷-GLP-1(7-37), Val⁸-Glu²²-Ile³³-GLP-1(7-37), Val⁸-Asp⁹-Ile¹¹-Tyr¹⁶-Glu²²-GLP-1(7-37), Val⁸-Tyr¹⁶-Trp¹⁹-Glu²²-GLP-1(7-37), Val⁸-Trp¹⁶-Glu²²-Val²⁵-Ile³³-GLP-1(7-37), Val⁸-Trp¹⁶-Glu²²-Ile³³-GLP-1(7-37), Val⁸-Glu²²-Val²⁵-Ile³³-GLP-1(7-37), Val⁸-Trp¹⁶-Glu²²-Val²⁵-GLP-1(7-37), and Val⁸-Cys¹⁶-Lys²⁶-GLP-1(7-37).

2. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of

a) human albumin;

b) human albumin analogs; and

c) fragments of human albumin, and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker.

3. The heterologous fusion protein of the claim 2 wherein the peptide linker is selected from the group consisting of:

a) a glycine rich peptide;

b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]_n where n is 1, 2, 3, 4, 5 or 6; and

c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]₃.

27. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of

a) the Fc portion of an immunoglobulin;

b) an analog of the Fc portion of an immunoglobulin; and

c) fragments of the Fc portion of an immunoglobulin,

and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide.

30. The heterologous fusion protein of any one of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula 1 [SEQ ID NO:

2].

31. The heterologous fusion protein of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula II (SEQ ID NO:

3);

32. The heterologous fusion protein of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula III (SEQ ID NO:

4);

33. The heterologous fusion protein of claim 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula IV (SEQ ID NO:

5);

34. The heterologous fusion protein of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula V (SEQ ID NO:

6)

35. The heterologous fusion protein of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula VIII (SEQ ID NO:

11).

36. The heterologous fusion protein of claims 27 through 35 wherein the GLP-1 compound has no more than 6 amino acids that are different from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

37. The heterologous fusion protein of claim 36 wherein the GLP-1 compound has no more than 5 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

38. The heterologous fusion protein of claim 37 wherein the GLP-1 compound has no more than 4 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

39. The heterologous fusion protein of claim 38 wherein the GLP-1 compound has no more than 3 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

40. The heterologous fusion protein of claim 39 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.

41. The heterologous fusion protein of any one of claims 27 through 40 wherein Xaa at position 8 is glycine or valine.

42. The heterologous fusion protein of claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine or valine and Xaa at position 30 is alanine, glutamic acid, aspartic acid, serine, or histidine.

43. The heterologous fusion protein of claim 42 wherein Xaa at position 30 is Glutamic acid.

44. The heterologous fusion protein of claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine or valine and Xaa at position 37 is histidine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, serine, threonine, arginine, or lysine.

45. The heterologous fusion protein of claim 44 wherein Xaa at position 37 is histidine.

46. The heterologous fusion protein of claim 34 wherein the GLP-1 compound has no more than 2 amino acids that differ from the corresponding amino acid in GLP-1(7-37)OH or GLP-1(7-36)OH and Xaa at position 8 is glycine, valine, leucine, isoleucine, serine, threonine, or methionine and Xaa at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine or histidine.

47. The heterologous fusion protein of claim 46 wherein Xaa at position 22 is lysine or glutamic acid.

48. The heterologous fusion protein of claim 34 wherein the GLP-1 compound is Val-8-GLP-1(7-37).

49. The heterologous fusion protein of claim 34 wherein the GLP-1 compound is Gly-8-GLP-1(7-37).

50. The heterologous fusion protein of any one of claims 27 through 49 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of:

IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM.

51. The heterologous fusion protein of any one of claims 27 through 50 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of:

IgG1, IgG2, IgG3, and IgG4.

52. The heterologous fusion protein of claim 51 wherein the second polypeptide is the Fc portion of an IgG1 immunoglobulin.

53. The heterologous fusion protein of claim 51 wherein the second polypeptide is the Fc portion of an IgG4 immunoglobulin.

54. The heterologous fusion protein of claims 50 through 53 wherein the IgG is human.

55. The heterologous fusion protein of any one of claims 27 through 54 wherein the Fc portion comprises the hinge, CH2, and CH3 domains.

56. The heterologous fusion protein of claim 52 wherein the second polypeptide has the sequence of SEQ ID NO:

32.

73. The heterologous fusion protein of any one of claims 27, 28, or 29 wherein the GLP-1 compound comprises the sequence of formula IX [SEQ ID NO:

12]

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Gln-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Lys-Gly-Arg-Xaa₃₇  

 

Formula IX (SEQ ID NO;

     12)

wherein;

Xaa₇ is;

L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β

-hydroxy-histidine, homohistidine, α

-fluoromethyl-histidine, or α

-methyl-histidine;

Xaa₈ is;

Ala, Gly, Val, Leu, Ile, Ser, or Thr;

Xaa₁₂ is;

Phe, Trp, or Tyr;

Xaa₁₆ is;

Val, Trp, Ile, Leu, Phe, or Tyr;

Xaa₁₈ is;

Ser, Trp, Tyr, Phe, Lys, Ile, Leu, Val;

Xaa₁₉ is;

Tyr, Trp, or Phe;

Xaa₂₀ is;

Leu, Phe, Tyr, or Trp;

Xaa₂₂ is;

Gly, Glu, Asp, or Lys;

Xaa₂₅ is;

Ala, Val, Ile, or Leu;

Xaa₂₇ is;

Glu, Ile, or Ala;

Xaa₃₀ is;

Ala or Glu

Xaa₃₃ is;

Val, or Ile; and

Xaa₃₇ is;

Gly, His, NH₂, or is absent.

74. The heterologous fusion protein of claim 73 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of:

IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM.

75. The heterologous fusion protein of claim 73 or 74 wherein the second polypeptide is the Fc portion of an Ig selected from the group consisting of:

IgG1, IgG2, IgG3, and IgG4.

76. The heterologous fusion protein of claim 75 wherein the second polypeptide is the Fc portion of an IgG1 immunoglobulin.

77. The heterologous fusion protein of claim 75 wherein the second polypeptide is the Fc portion of an IgG4 immunoglobulin.

78. The heterologous fusion protein of claims 73 through 78 wherein the IgG is human.

79. The heterologous fusion protein of claim 75 wherein the second polypeptide has the sequence of SEQ ID NO:

32.

28. A heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a GLP-1 compound and the second polypeptide is selected from the group consisting of

a) the Fc portion of an immunoglobulin;

b) an analog of the Fc portion of an immunoglobulin; and

c) fragments of the Fc portion of an immunoglobulin,

and wherein the C-terminus of the first polypeptide is fused to the N-terminus of the second polypeptide via a peptide linker.

29. The heterologous fusion protein of the claim 28 wherein the peptide linker is selected from the group consisting of:

a) a glycine rich peptide;

b) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]_n where n is 1, 2, 3, 4, 5, or 6; and

c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]₃.