Serum albumin binding molecules

• US 8,969,289 B2
• Filed: 05/02/2011
• Issued: 03/03/2015
• Est. Priority Date: 05/03/2010
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Gosselin, Michael L., Fabrizio, David, Swain, Joanna F., Mitchell, Tracy, Camphausen, Ray, Cload, Sharon T., Furfine, Eric, Morin, Paul E., Mukherjee, Ranjan, Taylor, Simeon I.
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US13/098,851
• Publication Number:
  US 20110305663A1
• Time in Patent Office: 1,401 Days
• Field of Search: None
• US Class Current: 514/2
• 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 human serum albumin with a K_D of 1 μ

M or less, 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 human ¹⁰Fn3 domain (SEQ ID NO;

     1), and wherein the polypeptide comprises an amino acid sequence at least 75% identical to an amino acid sequence selected from SEQ ID NOs;

4, 8, 12, 16, 20, and 24-44.

2. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain binds to domain 1 or 2 of human serum albumin with a KD of 1 μ

M or less.

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

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

5. The polypeptide of claim 1, 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).

6. The polypeptide of claim 1, wherein the serum half-life of the polypeptide in the presence of serum albumin is at least 2 hours.

13. A pharmaceutical composition comprising the polypeptide of claim 1.

14. The polypeptide of claim 1, wherein the BC, DE and FG loops of the ¹⁰Fn3 domain are selected from the group consisting of:

(a) 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,

(b) a BC loop comprising the amino acid sequence set forth in SEQ ID NO;

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

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

11,

(c) a BC loop comprising the amino acid sequence set forth in SEQ ID NO;

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

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

15,

(d) a BC loop comprising the amino acid sequence set forth in SEQ ID NO;

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

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

19, and

(e) a BC loop comprising the amino acid sequence set forth in SEQ ID NO;

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

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

23.

15. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs:

4, 8, 12, 16, 20, and 24-44.

16. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs:

4, 8, 12, 16, 20, and 24-44.

17. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a amino sequence selected from SEQ ID NOs:

4, 8, 12, 16, 20, and 24-44.

18. The polypeptide of claim 1, 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.

19. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain further comprises an N-terminal extension sequence selected from any one of the amino acid sequences set forth in SEQ ID NOs:

45-53.

20. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain further comprises a C-terminal extension sequence selected from any one of the amino acid sequences set forth in SEQ ID NOs:

54-64 and 215.

21. The polypeptide of claim 18, wherein the ¹⁰Fn3 domain comprises an amino acid sequence selected from SEQ ID NOs:

89-116 and 222-225.

22. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain 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.

23. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

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

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

11.

24. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

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

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

15.

25. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

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

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

19.

26. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO:

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

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

23.

27. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:

4.

28. The polypeptide of claim 15, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:

4.

29. The polypeptide of claim 16, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:

4.

30. The polypeptide of claim 17, wherein the ¹⁰Fn3 domain comprises the amino sequence of SEQ ID NO:

4.

33. The polypeptide of claim 21, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

90.

7. 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 μ

M or less and comprises an amino acid sequence at least 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs;

4, 8, 12, 16, 20, and 24-44.

8. The fusion polypeptide of claim 7, wherein the ¹⁰Fn3 domain comprises an amino acid sequence selected from SEQ ID NOs:

4, 8, 12, 16, 20, and 24-44.

9. The fusion polypeptide of claim 7, 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.

10. The fusion polypeptide of claim 7, 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 (PYY3-36), 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.

11. The fusion polypeptide of claim 10, wherein the heterologous protein is FGF21.

31. The polypeptide of claim 7, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:

4.

32. The polypeptide of claim 8, wherein the ¹⁰Fn3 domain comprises the amino acid sequence of SEQ ID NO:

4.

12. 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.