Albumin-insulin fusion proteins

• US 9,505,823 B2
• Filed: 08/07/2007
• Issued: 11/29/2016
• Est. Priority Date: 08/07/2006
• Status: Active Grant

• Current Assignee: Teva Biopharmaceuticals USA, Inc. (Teva Pharmaceutical Industries Limited)
• Original Assignee: Teva Pharmaceutical Industries Limited
• Inventors: Bell, Adam C., Rosen, Craig A., Sanyal, Indrajit, Zhao, Qinghai
• Primary Examiner(s): Xie, Xiaozhen
• Application Number:
  US11/835,114
• Publication Number:
  US 20080057004A1
• Time in Patent Office: 3,402 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

Disclosed are fusion proteins that include albumin fused to a polypeptide that has insulin activity. The fusion proteins may include albumin fused to insulin or an insulin analog. In particular, the fusions proteins may include albumin fused to a single chain insulin analog. The fusion proteins may exhibit extended insulin activity in vivo or in vitro relative to insulin that is not fused to albumin. The fusion proteins may be formulated as aerosol compositions.

Claims

1. A fusion protein having insulin activity and comprising albumin fused to the N-terminus, C-terminus, or both termini of an insulin analog, wherein the insulin analog has a formula of:

B(_1-30)—

X-A(_1-21);

wherein;

X is a polypeptide linking sequence selected from the group consisting of;

NPNLPRLVR (SEQ ID NO;

18), KDDNPNLPRLVR (SEQ ID NO;

19), NDEMPAD (SEQ ID NO;

20), GGGPQT (SEQ ID NO;

21), and GAGSSSRRAPQT (SEQ ID NO;

22);

B(_1-30) is a B-chain of insulin; and

A(_1-21) is an A-chain of insulin.

2. The fusion protein of claim 1, wherein albumin is fused to the N-terminus of the insulin analog.

3. The fusion protein of claim 1, wherein albumin is fused to the C-terminus of the insulin analog.

4. The fusion protein of claim 1, wherein albumin is fused to the N-terminus and C-terminus of the insulin analog.

5. The fusion protein of claim 1, wherein X is NPNLPRLVR (SEQ ID NO:

18).

6. The fusion protein of claim 1, wherein X is KDDNPNLPRLVR (SEQ ID NO:

19).

7. The fusion protein of claim 1, wherein X is NDEMPAD (SEQ ID NO:

20).

8. The fusion protein of claim 1, wherein X is GGGPQT (SEQ ID NO:

21).

9. The fusion protein of claim 1, wherein X is GAGSSSRRAPQT (SEQ ID NO:

22).

10. The fusion protein of claim 1, further comprising an N-terminal leader or signal sequence.

11. The fusion protein of claim 10, wherein the N-terminal leader or signal sequence is selected from the group consisting of albumin leader sequence, insulin leader sequence, MPIF-1 signal sequence, stanniocalcin signal sequence, invertase signal sequence, yeast mating factor alpha signal sequence, K. lactis killer toxin leader sequence, immunoglobulin Ig signal sequence, Fibulin B precursor signal sequence, clusterin precursor signal sequence, insulin-like growth factor-binding protein 4 signal sequence, acid phosphatase (PHO5) leader, pre-sequence of MFoz-1, pre-sequence of 0 glucanase (BGL2), S. diastaticus glucoamylase II secretion leader sequence, S. carlsbergensis alpha-galactosidase (MEL1) secretion leader sequence, Candida glucoamylase leader sequence, gp67 signal sequence for use in a baculovirus expression system, and S. cerevisiae invertase (SUC2) leader.

12. The fusion protein of claim 10, wherein the N-terminal leader or signal sequence is selected from the group consisting of a hybrid signal sequence (MKWVSFISLLFLFSSAYSRSLEKR;

SEQ ID NO;

3);

an HSA/MF alpha-1 hybrid signal sequence (MKWVSFISLLFLFSSAYSRSLDKR;

SEQ ID NO;

4);

a K. lactis killer/MFalpha-1 fusion leader sequence (MNIFYIFLFLLSFVQGSLDKR;

SEQ ID NO;

5);

SEQ ID NO;

6;

SEQ ID NO;

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

a modified HSA leader (MKWVTFISLLFLFAGVSG;

SEQ ID NO;

10);

a modified HSA leader (MKWVTFISLLFLFGGVSG;

SEQ ID NO;

11);

a modified HSA (S14) leader (MKWVTFISLLFLFSGVSG;

SEQ ID NO;

12);

a modified HSA (G14) leader (MKWVTFLSLLFLFGGVSG;

SEQ ID NO;

13);

a modified HSA (G14) leader (MKWVTFISLLFLFGGVLGDLHKS;

SEQ ID NO;

14);

a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG;

SEQ ID NO;

15);

K. lactis killer toxin prepro sequence (MNIFYIFLFLLSFVQGLEHTHRRGSLDKR;

SEQ ID NO;

16); and

inulinase sequence (MKLAYSLLLPLAGVSASVINYKR;

SEQ ID NO;

17).

13. The fusion protein of claim 1, wherein the fusion protein:

(a) does not bind to IGF-I receptor or binds with no more than 80% affinity, relative to the affinity of IGF-I for the IGF-I receptor;

or

(b) does not exhibit mitogenic activity or exhibits mitogenic activity of no more than 50% relative to IGF-I.

14. The fusion protein of claim 13, wherein the fusion protein exhibits mitogenic activity of no more than 30% relative to IGF-I.

15. The fusion protein of claim 13, wherein the fusion protein exhibits mitogenic activity of no more than 10% relative to IGF-I.

16. The fusion protein of claim 13, wherein the fusion protein binds with no more than 60% affinity, relative to the affinity of IGF-I for the IGF-I receptor.

17. The fusion protein of claim 13, wherein the fusion protein binds with no more than 40% affinity, relative to the affinity of IGF-I for the IGF-I receptor.

18. The fusion protein of claim 13, wherein the fusion protein binds with no more than 20% affinity, relative to the affinity of IGF-I for the IGF-I receptor.

19. The fusion protein of claim 13, wherein the fusion protein binds with no more than 10% affinity, relative to the affinity of IGF-I for the IGF-I receptor.

20. The fusion protein of claim 13, wherein the fusion protein binds with no more than 5% affinity, relative to the affinity of IGF-I for the IGF-I receptor.

21. A polynucleotide encoding a fusion protein of claim 1.

22. An expression vector comprising the polynucleotide of claim 21.

23. An isolated host cell comprising the expression vector of claim 22.

24. A method of preparing an albumin-insulin fusion protein comprising fermenting the host cell of claim 23.

25. A pharmaceutical composition comprising a fusion protein of claim 1 and a pharmaceutical excipient.

26. A pharmaceutical aerosol composition comprising a fusion protein of claim 1 and a pharmaceutical excipient.

27. The pharmaceutical aerosol composition of claim 26, formulated for nasal delivery, pulmonary delivery, or both.

28. The pharmaceutical aerosol composition of claim 27, wherein the composition is a liquid aerosol or a dry powder aerosol.

29. The pharmaceutical aerosol composition of claim 28, wherein the liquid aerosol comprises droplets having an average diameter selected from the group consisting of:

(a) about 2 to about 10 microns;

(b) about 2 to about 6 microns;

(c) less than about 2 microns;

(d) about 5 to about 100 microns; and

(e) 30 to about 60 microns.

30. The pharmaceutical aerosol composition of claim 28, wherein the dry powder aerosol is a spray-dried powder aerosol or a freeze-dried powder aerosol.

31. A pharmaceutical aerosol composition for use in a propellant-based pMDI comprising a fusion protein of claim 1.

32. The pharmaceutical aerosol composition of claim 31, wherein the propellant is a non-CFC propellant.

33. A method of treating diabetes comprising administering the pharmaceutical composition of claim 25 to a patient in need thereof.