Compositions and Methods for the Preparation of Human Growth Hormone Glycosylation Mutants

• US 20080102083A1
• Filed: 05/07/2004
• Published: 05/01/2008
• Est. Priority Date: 05/09/2003
• Status: Active Grant

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Neose Technologies, Inc.
• Inventors: DeFrees, Shawn, Clausen, Henrik
• Granted Patent: US 7,932,364 B2
• Time in Patent Office: Days
• US Class Current: 424/198.1
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin

Abstract

The present invention relates to mutants of human growth hormone, which contain newly introduced N-linked or O-linked glycosylation site(s), such that these recombinantly produced polypeptides have glycosylation patterns distinctly different from that of the naturally occurring human growth hormone. The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.

Claims

1. An isolated nucleic acid comprising a polynucleotide sequence encoding a mutant human growth hormone, wherein the mutant human growth hormone comprises a newly introduced N-linked or O-linked glycosylation site that does not exist in the corresponding wild-type human growth hormone.

2. The nucleic acid of claim 1, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

3. The nucleic acid of claim 1, wherein the newly introduced glycosylation site is near a proline residue.

4. The nucleic acid of claim 3, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

5. The nucleic acid of claim 1, wherein the mutant human growth hormone comprises the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

6. The nucleic acid of claim 1, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.

7. An expression cassette comprising the nucleic acid of claim 1.

8. A cell comprising the nucleic acid of claim 1.

9. A mutant human growth hormone, comprising a newly introduced N-linked or O-linked glycosylation site that does not exist in the corresponding wild-type human growth hormone.

10. The mutant human growth hormone of claim 9, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

11. The mutant human growth hormone of claim 9, wherein the newly introduced glycosylation site is proximate a proline residue.

12. The mutant human growth hormone of claim 11, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

13. The mutant human growth hormone of claim 9, comprising the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

14. The mutant human growth hormone of claim 9, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.

15. The mutant human growth hormone of claim 9, comprising a water-soluble polymer attached to a glycosylation site through a glycosyl linker.

16. The mutant human growth hormone of claim 15, wherein said glycosyl linker is an intact glycosyl linker.

17. The mutant human growth hormone of claim 15, wherein glycosylation site is a mutant glycosylation site.

18. A method for making a mutant human growth hormone, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type human growth hormone, comprising the steps of:

(a) recombinantly producing the mutant human growth hormone; and

(b) glycosylating the mutant human growth hormone at the newly introduced glycosylation site.

19. The method of claim 18, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

20. The method of claim 18, wherein the newly introduced glycosylation site is near a proline residue.

21. The method of claim 20, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

22. The method of claim 18, wherein the mutant human growth hormone comprises the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

23. The method of claim 18, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.

24. A pharmaceutical composition comprising an effective amount of a mutant human growth hormone, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type human growth hormone.

25. The composition of claim 24, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

26. The composition of claim 24, wherein the newly introduced glycosylation site is near a proline residue.

27. The composition of claim 26, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

28. The composition of claim 24, wherein the mutant human growth hormone comprises the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

29. The composition of claim 24, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.

30. A method for treating human growth hormone deficiency in a patient, comprising the step of administering an effective amount of a mutant human growth hormone to the patient, wherein the mutant human growth hormone comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type human growth hormone.

31. The method of claim 30, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

32. The method of claim 30, wherein the newly introduced glycosylation site is near a proline residue.

33. The method of claim 32, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

34. The method of claim 30, wherein the mutant human growth hormone comprises the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

35. The method of claim 30, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.

36. A method for making a glycoconjugate of a mutant human growth hormone, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type human growth hormone, comprising the steps of:

(a) recombinantly producing the mutant human growth hormone, and

(b) enzymatically glycosylating the mutant human growth hormone with a modified sugar at the newly introduced glycosylation site.

37. The method of claim 36, wherein the modified sugar is modified with a member selected from poly(ethylene glycol) and m-poly(ethylene glycol).

38. The method of claim 36, wherein the corresponding wild-type human growth hormone has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

39. The method of claim 36, wherein the newly introduced glycosylation site is near a proline residue.

40. The method of claim 36, wherein the proline residue is located at position 2, 5, 37, 48, 59, 89, 113, 140, or 190 of SEQ ID NO:

1 or SEQ ID NO;

2.

41. The method of claim 36, wherein the mutant human growth hormone comprises the amino acid sequence of SEQ ID NO:

3, 4, 5, 6, 7, 8, or 9.

42. The method of claim 36, wherein the mutant human growth hormone comprises more than one newly introduced glycosylation site.