Novel therapeutic molecules

• US 20060183687A1
• Filed: 04/24/2006
• Published: 08/17/2006
• Est. Priority Date: 09/17/1997
• Status: Active Grant

• Current Assignee: Andreas Strasser, David C.S. Huang, Hamsa Puthalakath, Jerry Adams, Liam O'Connor, Lorraine O'Reilly, Suzanne Cory
• Original Assignee: Andreas Strasser, David C.S. Huang, Hamsa Puthalakath, Jerry Adams, Liam O'Connor, Lorraine O'Reilly, Suzanne Cory
• Inventors: Huang, David C.S., Strasser, Andreas, O'Connor, Liam, Cory, Suzanne, Adams, Jerry, O'Reilly, Lorraine, Puthalakath, Hamsa
• Granted Patent: US 7,582,439 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins

Abstract

The present invention relates generally to novel molecules capable of, inter alia, modulating apoptosis in mammalian cells and to genetic sequences encoding same. More particularly, the present invention relates to a novel member of the Bcl-2 family of proteins, referred to herein as “Bim”, and to genetic sequences encoding same. The molecules of the present invention are useful, for example, in therapy, diagnosis, antibody generation and as a screening tool for therapeutic agents capable of modulating physiological cell death or survival and/or modulating cell cycle entry.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having one or more of the identifying characteristics of Bim or a derivative or homologue thereof.

2. A nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises a nucleotide sequence encoding or complementary, to a sequence encoding an amino acid sequence substantially as set forth in one of SEQ ID NO:

2, 4 or 6 or a derivative or homologue thereof or having at least about 45% or greater similarity to one or more of SEQ ID NO;

2, 4 or 6 or derivative or homologue thereof.

3. A nucleic acid molecule according to claim 1 comprising a nucleotide sequence substantially as set forth in one of SEQ ID NO:

1, 3 or 5 or a derivative or homologue thereof capable of hybridising to one of SEQ ID NO;

1, 3 or 5 under low stringency conditions at 42°

C.

4. A nucleic acid molecule according to claim 3 which further encodes an amino acid sequence corresponding to an amino acid sequence set forth in one or SEQ ID NO:

2, 4 or 6 or a derivative or homologue thereof or having at least about 45% or greater similarity, to one or more of SEQ ID NO;

2, 4 or 6 or derivative or homologue thereof.

5. A nucleic acid molecule according to claim 3 substantially as set forth in one of SEQ ID NO:

1, 3 or 5.

6. A nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises a nucleotide sequence encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth is one of SEQ ID NO:

8 or 10 or a derivative or homologue thereof or having at least about 45% or greater similarity to one or more of SEQ ID NO0;

8 or 10 or derivative or homologue thereof.

7. A nucleic acid molecule according to claim 1 comprising a nucleotide sequence substantially as set forth in SEQ ID NO:

7 or 9 or a derivative or homologue thereof capable of hybridising to one of SEQ ID NO;

7 or 9 under low stringency conditions at 42°

C.

8. A nucleic acid molecule according to claim 7 which further encodes an amino acid sequence corresponding to an amino acid sequence substantially as set forth in one of SEQ ID NO:

8 or 10 or a derivative or homologue thereof or having at least about 45% or greater similarity to one or more of SEQ ID NO;

8 or 10 or a derivative or homologue thereof.

9. A nucleic acid molecule according to claim 7 substantially as set forth in one of SEQ ID NO:

7 or 9.

21. A variant of an isolated Bim nucleic acid molecule as claimed in claim 1 comprising one or more nucleotide mutations in said nucleic acid molecule resulting in at least one amino acid addition, substitution and/or deletion to the polypeptide encoded by said variant wherein said polypeptide cannot bind, couple or otherwise associate with a dynein light chain.

22. A variant according to claim 21 wherein said mutation results in an amino acid addition, substitution and/or deletion in the region of the polypeptide chain which binds the dynein light chain.

23. A variant according to claim 22 wherein said Bim is murine or human Bim_L, and said region is defined by amino acid residue numbers 42 to 71.

24. A variant according to claim 23 wherein said mutation is a substitution of one or more of D51, S53, T54 and/or N65.

25. A variant according to claim 24 wherein said substitution is one or more of D51G, S53P, T54A, T52I and/or N65S.

26. A variant according to claim 25 wherein said substitution is D51G or S53P or T54A or T54I and N65S.

27. A variant according to claim 22 wherein said Bim is murine Bim_EL and said region is defined by amino acid residue numbers 42 to 127.

28. A variant according to claim 22 wherein said Bim is human Bim_EL and said region is defined by amino acid residue numbers 42 to 131.

10. A polypeptide comprising the amino acid sequence of Bim or having one or more of the identifying characteristics thereof or derivative or homologue thereof.

11. A polypeptide according to claim 10 comprising an amino acid sequence substantially as set forth in SEQ ID NO:

2, 4 or 6 or derivative or homologue thereof or a sequence having at least about 45% similarity to one or more of SEQ ID NO;

2, 4 or 6.

12. A polypeptide according to claim 10 encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:

1, 3 or 5 or a derivative or homologue thereof under low stringency conditions at 42°

C.

13. A polypeptide according to claim 12 further comprising an amino acid sequence substantially as set forth in SEQ ID NO:

2, 4 or 6 or derivative or homologue thereof or a sequence having at least about 45% similarity to one or more of SEQ ID NO;

2, 4 or 6.

14. A polypeptide according to claim 12 substantially as set forth in SEQ ID NO:

2, 4 or 6.

15. A polypeptide according to claim 10 comprising an amino acid sequence substantially as set forth in SEQ ID NO:

8 or 10 or derivative or homologue thereof or a sequence having at least about 45% similarity to one or more of SEQ ID NO;

8 or 10.

16. A polypeptide according to claim 10 encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:

7 or 9 or a derivative or homologue thereof under low stringency conditions at 42°

C.

17. A polypeptide according to claim 16 further comprising an amino acid sequence substantially as set forth in SEQ ID NO:

8 or 10 or derivative or homologue thereof or a sequence having at least about 45% similarity to one or more of SEQ ID NO;

8 or 10.

18. A polypeptide according to claim 16 substantially as set forth in SEQ ID NO:

8 or 10.

19. A polypeptide according to claim 10 in homodimeric form.

20. A polypeptide according to claim 10 in heterodimeric form.

29. A variant of an isolated Bim polypeptide as claimed in claim 10 comprising at least one amino acid addition, substitution and/or deletion wherein said variant cannot bind, couple or otherwise associate with the dynein light chain.

30. A variant according to claim 29 wherein said amino acid addition, substitution and/or deletion occurs in the region of the polypeptide chain which binds the dynein light chain.

31. A variant according to claim 30 wherein said Bim is murine or human Bimk and said region is defined by amino and residue numbers 42 to 71.

32. A variant according to claim 31 wherein said mutation is a substitution of one or more of D51, S53, T54 or N65.

33. A variant according to claim 32 wherein said substitution is one or more of D51G, S53P, T54A, T52I and/or N65S.

34. A variant according to claim 33 wherein said substitution is D51G or S53P or T54A or T54I and N65S.

35. A variant according to claim 30 wherein said Bim is murine Bim_EL and said region is defined by amino acid residue numbers 42 to 127.

36. A variant according to claim 30 wherein said Bim is human Bim_EL and said region is defined by amino acid residue numbers 42 to 131.

37. A method of modulating activity of Bim in a mammal said method comprising administering to said mammal a modulating effective amount of an agent for a time and under conditions sufficient to increase, decrease or otherwise modulate Bim activity.

38. A method of modulating expression of Bim in a mammal, said method comprising administering to said mammal a modulating effective amount of an agent for a time and under conditions sufficient to up-regulate, down-regulate, or otherwise modulate expression of Bim.

39. A method of modulating apoptosis in mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a nucleotide sequence encoding Bim.

40. A method of modulating apoptosis in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of Bim.

41. A method of modulating apoptosis in a mammal said method comprising administering to said mammal an effective amount of Bim or Bim or derivative thereof.

42. A method of treating a mammal said method comprising to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of Bim wherein said modulation results in modulation of apoptosis.

43. A method of treating a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of Bim wherein said modulation results in modulation of apoptosis.

44. A method of treating a mammal said method comprising administering to said mammal an effective amount of Bim or Bim or derivative thereof for a time and under conditions sufficient to modulate apoptosis.

45. An agent for use in modulating Bim expression wherein modulating expression of said Bim modulates apoptosis.

46. An agent for use in modulating Bim expression wherein modulating expression of said Bim modulates apoptosis.

47. A composition comprising Bim or Bim or derivative thereof for use in modulating apoptosis.

48. A pharmaceutical composition comprising Bim, Bim or derivative thereof or an agent capable of modulating Bim expression or Bim activity together with one or more pharmaceutically acceptable carriers and/or diluents.

49. An immunointeractive molecule comprising an antigen binding portion having specificity for Bim or Bim or derivative thereof.

50. The immunointeractive molecule according to claim 49 wherein said immunointeractive molecule is a monoclonal antibody.

51. A monoclonal antibody according to claim 50 wherein said specificity is specificity for Bim_L.

52. A method of detecting an immunointeractive molecule, in a sample, specific for a protein of interest produced by a cell said method comprising contacting the sample to be tested with a population of cells comprising a defined ratio of cells producing the protein of interest and cells not producing the protein of interest for a time and under conditions sufficient for the immunointeractive molecule if present in said sample to interact with said protein of interest and then subjecting said immunointeractive molecule-protein complex to detecting means.

53. A method according to claim 52 wherein said immunoreactive molecule is an antibody.

54. A method according to claim 53 wherein the detecting means comprises an anti-immunoglobin antibody labelled with a reporter molecule capable of giving a detactable signal.

55. A method according to claim 53 wherein the population of cells is subjected to flow cytometric analysis to produce a fluorescent signal wherein a differential fluorescent signal is indicative of antibody binding to said target protein.

56. The method for detecting Bim or Bim in a biological sample from a subject said method comprising contacting said biological sample with an immunointeractive molecule as hereinbefore defined specific for Bim, Bim or its derivatives thereof for a time and under conditions sufficient for an immunoreactive molecute-Bim or immunoreactive molecule-Bim complex to form, and then detecting said complex.

57. A peptide comprising at least 4 contiguous amino acids corresponding to at least 4 contiguous amino acids in SEQ ID NOS:

2, 4, 5, 8 or 10 or derivative or homologue thereof.

58. A nucleic acid molecule encoding a peptide according to claim 60.