SERUM ALBUMIN BINDING MOLECULES

• US 20150152147A1
• Filed: 11/25/2014
• Published: 06/04/2015
• Est. Priority Date: 05/03/2010
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: FABRIZIO, David, CAMPHAUSEN, Ray, FURFINE, Eric, GOSSELIN, Michael L., SWAIN, Joanna F., MITCHELL, Tracy, CLOAD, Sharon T., MORIN, Paul E., MUKHERJEE, Ranjan, TAYLOR, Simeon I.
• Granted Patent: US 9,540,424 B2
• Time in Patent Office: Days
• 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 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• 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 3/06 Antihyperlipidemics
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present invention relates to an antibody-like protein based on the tenth fibronectin type III domain (¹⁰Fn3) that binds to serum albumin. The invention further relates to fusion molecules comprising a serum albumin-binding ¹⁰Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.

Claims

1. A polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain binds to domain 1 or 2 of human serum albumin with a K_D of 1 uM or less, and wherein the serum half-life of the polypeptide in the presence of albumin is at least 5-fold greater than the serum half-life of the polypeptide in the absence of serum albumin.

2. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a modified amino acid sequence in one or more of the BC, DE and FG loops relative to the wild-type ¹⁰Fn3 domain.

3. The polypeptide of claim 1 or 2, wherein the ¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

4. The polypeptide any one of claims 1-3, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 200 nM or less at pH 5.5.

55. A pharmaceutical composition comprising the polypeptide of any one of claims 1-54.

56. The pharmaceutical composition of claim 55, further comprising a physiologically acceptable carrier.

59. A nucleic acid encoding a polypeptide of any one of claim 1-26, 45-51 or 53, or a fusion polypeptide of any one of claim 27-45 or 52.

61. A vector comprising a nucleic acid of claim 59 or 60.

62. The vector of claim 61, wherein the vector is an expression vector.

63. A host cell comprising the vector of claim 61 or 62.

64. The host cell of claim 63, wherein the cell is a bacterial cell.

65. The host cell of claim 63, wherein the cell is a mammalian cell.

5. A polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain binds to human serum albumin and comprises an amino acid sequence at least 70% identical to SEQ ID NO:

2.

6. The polypeptide of claim 5, wherein the ¹⁰Fn3 domain comprises one or more of a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

5, a DE loop comprising the amino acid sequence set forth in SEQ ID NO;

6, and an FG loop comprising the amino acid sequence set forth in SEQ ID NO;

7.

7. The polypeptide of claim 5, wherein the ¹⁰Fn3 domain comprises a sequence selected from SEQ ID NO:

8, 12, 16, 20, and 24-44.

8. The polypeptide of any one of claims 5-7, wherein the ¹⁰Fn3 domain also binds to one or more of rhesus serum albumin (RhSA), cynomolgous monkey serum albumin (CySA), or murine serum albumin (MuSA).

9. The polypeptide of any one of claims 5-7, wherein the ¹⁰Fn3 domain does not cross-react with one or more of RhSA, CySA or MuSA.

10. The polypeptide of any one of claims 5-9, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 1 uM or less.

11. The polypeptide of any one of claims 5-10, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 500 nM or less.

12. The polypeptide of any one of claims 5-11, wherein the ¹⁰Fn3 domain binds to domain I or II of HSA.

13. The polypeptide of any one of claims 5-12, wherein the ¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

14. The polypeptide of any one of claims 5-13, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 200 nM or less at pH 5.5.

15. The polypeptide of any one of claims 5-14, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 5-fold greater than the serum half-life of the polypeptide in the absence of serum albumin.

16. The polypeptide of any one of claims 5-15, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 2 hours.

17. A polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain binds to human serum albumin and comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

5, a DE loop comprising the amino acid sequence set forth in SEQ ID NO;

6, and an FG loop comprising the amino acid sequence set forth in SEQ ID NO;

7.

18. The polypeptide of claim 17, wherein the ¹⁰Fn3 domain also binds to one or more of rhesus serum albumin (RhSA), cynomolgous monkey serum albumin (CySA), or murine serum albumin (MuSA).

19. The polypeptide of claim 17, wherein the ¹⁰Fn3 domain does not cross-react with one or more of RhSA, CySA or MuSA.

20. The polypeptide of any one of claims 17-19, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 1 uM or less.

21. The polypeptide of any one of claims 17-20, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 500 nM or less.

22. The polypeptide of any one of claims 17-21, wherein the ¹⁰Fn3 domain binds to domain I or II of HSA.

23. The polypeptide of any one of claims 17-22, wherein the ¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

24. The polypeptide of any one of claims 17-23, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 200 nM or less at pH 5.5.

25. The polypeptide of any one of claims 17-24, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 5-fold greater than the serum half-life of the polypeptide in the absence of serum albumin.

26. The polypeptide of any one of claims 17-25, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 2 hours.

27. A fusion polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain and a heterologous protein, wherein the ¹⁰Fn3 domain binds to human serum albumin with a K_D of 1 uM or less.

28. The fusion polypeptide of claim 27, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:

4.

29. The fusion polypeptide of claim 27 or 28, wherein the ¹⁰Fn3 domain comprises an amino acid sequence selected from SEQ ID NO:

8, 12, 16, 20, and 24-44.

30. The fusion polypeptide of claim 27 or 28, wherein the ¹⁰Fn3 domain comprises a BC loop having the amino acid sequence set forth in SEQ ID NO:

5, a DE loop having the amino acid sequence set forth in SEQ ID NO;

6, and an FG loop having the amino acid sequence set forth in SEQ ID NO;

7.

31. The fusion polypeptide of any one of claims 27-30, wherein the heterologous protein is selected from fibroblast growth factor 21 (FGF21), insulin, insulin receptor peptide, (bone morphogenetic protein 9 (BMP-9), amylin, peptide YY (PYY_3-36), pancreatic polypeptide (PP), interleukin 21 (IL-21), glucagon-like peptide 1 (GLP-1), Plectasin, Progranulin, Atstrrin, Osteocalcin (OCN), Apelin, or a polypeptide comprising a ¹⁰Fn3 domain.

32. The fusion polpeptide of claim 31, wherein the heterologous protein is FGF21.

33. The fusion polypeptide of claim 32, wherein the heterologous protein comprises the sequence set forth in SEQ ID NO:

118.

34. The fusion polypeptide of any one of claims 27-30, wherein the heterologous protein comprises a second ¹⁰Fn3 domain that binds to a target protein other than HSA.

35. The fusion polypeptide of claim 34, further comprising a third ¹⁰Fn3 domain that binds to a target protein.

36. The fusion polypeptide of claim 35, wherein the third ¹⁰Fn3 domain binds to the same target as the second ¹⁰Fn3 domain.

37. The fusion polypeptide of claim 35, wherein the third ¹⁰Fn3 domain binds to a different target than the first and second ¹⁰Fn3 domains.

38. The fusion polypeptide of any one of claims 27-37, wherein the ¹⁰Fn3 domain also binds to one or more of rhesus serum albumin (RhSA), cynomolgous monkey serum albumin (CySA), or murine serum albumin (MuSA).

39. The polypeptide of any one of claims 27-37, wherein the ¹⁰Fn3 domain does not cross-react with one or more of RhSA, CySA or MuSA.

40. The fusion polypeptide of any one of claims 27-39, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 1 uM or less.

41. The fusion polypeptide of any one of claims 27-40, wherein the ¹⁰Fn3 domain binds to domain I or II of HSA.

42. The fusion polypeptide of any one of claims 27-41, wherein the ¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

43. The fusion polypeptide of any one of claims 27-42, wherein the ¹⁰Fn3 domain binds to HSA with a K_D of 200 nM or less at pH 5.5.

44. The fusion polypeptide of any one of claims 27-43, wherein half-life of the fusion in the presence of serum albumin is at least 5-fold greater than half-life of the heterologous protein alone.

45. The fusion polypeptide of any one of claims 27-44, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 2 hours.

52. The fusion polypeptide of claim 32, wherein the polypeptide comprises a sequence selected from SEQ ID NOs:

132-174.

53. The fusion polypeptide of claim 31, wherein the heterologous protein is PYY_3-36, PP or amylin.

57. A method for treating diabetes, treating or lowering insulin resistance, treating obesity, reducing weight or preventing weight gain, treating dyslipidemia, increasing glucose uptake in adipose tissue, or slowing the progression of diabetes, comprising administering to a subject a therapeutically effective amount of a fusion polypeptide of any one of claim 32-33 or 52.

58. A method for treating hypoglycemia, obesity, diabetes, eating disorders, insulin-resistance syndrome, or cardiovascular disease, comprising administering to a subject a therapeutically effective amount of a fusion polypeptide of claim 53.

60. The nucleic acid of claim 58, wherein the nucleic acid comprises a sequence set forth in Table 3.

46. A polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain (i) comprises a modified amino acid sequence in one or more of the AB, BC, CD, DE, EF and FG loops relative to the wild-type ¹⁰Fn3 domain, (ii) binds to a target molecule not bound by the wild-type ¹⁰Fn3 domain, and (iii) comprises a C-terminal tail having a sequence (ED)_n, wherein n is an integer from 3 to 7.

47. The polypeptide of claim 46, wherein the ¹⁰Fn3 domain comprises an amino acid sequence having at least 60% identity with the amino acid sequence set forth in residues 9-94 of SEQ ID NO:

1.

48. The polypeptide of claim 46 or 47, wherein the C-terminal tail further comprises an E, I or EI at the N-terminus.

49. The polypeptide of any one of claims 46-48, wherein the C-terminal tail enhances solubility or reduces aggregation, or both.

50. The polypeptide of any one of claims 46-49, wherein the ¹⁰Fn3 domain comprises a modified amino acid sequence in each of the BC, DE and FG loops relative to the wild-type ¹⁰Fn3 domain.

51. The polypeptide of any one of claims 46-50, wherein the polypeptide binds to the target with a K_D of 1 uM or less.

54. A polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain binds to human serum albumin and comprises an amino acid sequence at least 70% identical to SEQ ID NO:

8, 12, 16, 20, and 24-44.