Therapeutic agents comprising pro-apoptotic proteins

• US 20030086919A1
• Filed: 07/17/2002
• Published: 05/08/2003
• Est. Priority Date: 07/17/2001
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Rosenblum, Michael G., Liu, Yuying
• Granted Patent: US 7,101,977 B2
• Time in Patent Office: Days
• US Class Current: 424/94.63
• 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 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/6467 Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)

Abstract

The present invention relates to targeted killing of a cell utilizing a chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factor. In a preferred embodiment, the signal transduction pathway factor is an apoptosis-inducing factor, such as granzyme B, granzyme A, or Bax.

Claims

1. A chimeric polypeptide comprising a cell-specific targeting moiety and a signal transduction pathway factor.

2. A chimeric polypeptide comprising a cell-specific targeting moiety and an apoptosis-inducing factor, wherein said apoptosis-inducing factor is a granzyme.

3. The polypeptide of claim 2, wherein said granzyme is granzyme B.

4. The polypeptide of claim 3, wherein the amino acid sequence of said granzyme B is SEQ ID NO:

60.

5. The polypeptide of claim 3, wherein the amino acid sequence of said granzyme B is at least 100 contiguous amino acids from SEQ ID NO:

60.

6. The polypeptide of claim 3, wherein the amino acid sequence of said granzyme B is at least 75 contiguous amino acids from SEQ ID NO:

60.

7. The polypeptide of claim 3, wherein the amino acid sequence of said granzyme B is at least 40 contiguous amino acids from SEQ ID NO:

60.

8. The polypeptide of claim 4, wherein SEQ ID NO:

60 further comprises an N-terninal extension comprising SEQ ID NO;

61.

9. The polypeptide of claim 4, wherein the first twenty amino acids are absent from SEQ ID NO:

60.

10. The polypeptide of claim 2, wherein said granzyme is granzyme A.

11. The polypeptide of claim 10, wherein the amino acid sequence of said granzyme A is SEQ ID NO:

25.

12. The polypeptide of claim 10, wherein the amino acid sequence of said granzyme A is at least 100 contiguous amino acids from SEQ ID NO:

25.

13. The polypeptide of claim 10, wherein the amino acid sequence of said granzyme A is at least 75 contiguous amino acids from SEQ ID NO:

25.

14. The polypeptide of claim 10, wherein the amino acid sequence of said granzyme A is at least 40 contiguous amino acids from SEQ ID NO:

25.

15. The polypeptide of claim 2, wherein said cell-specific targeting moiety is a cytokine, an antibody, a ligand, or a hormone.

16. The polypeptide of claim 15, wherein said ligand is VEGF.

17. The polypeptide of claim 16, wherein said VEGF is vegf121.

18. The polypeptide of claim 15, wherein said antibody is a single chain antibody.

19. The polypeptide of claim 18, wherein said single chain antibody is scFvMEL.

20. The polypeptide of claim 2, wherein said granzyme is granzyme B and said cell-specific targeting moiety is vegf121.

21. The polypeptide of claim 2, wherein said granzyme is granzyme B and said cell-specific targeting moiety is scFvMEL.

22. The polypeptide of claim 2, further comprising a linker.

23. The polypeptide of claim 22, wherein the linker comprises SEQ ID NO:

50, SEQ ID NO;

51, or SEQ ID NO;

52.

24. The polypeptide of claim 2, wherein the polypeptide is encoded by a recombinant polynucleotide.

25. An expression cassette comprising a polynucleotide encoding a chimeric polypeptide comprising a cell-specific targeting moiety and an apoptosis-inducing factor, wherein said apoptosis-inducing factor is a granzyme, and wherein said polynucleotide is under control of a regulatory sequence operable in a host cell.

26. The expression cassette of claim 25, wherein said granzyme is granzyme A.

27. The expression cassette of claim 25, wherein said granzyme is granzyme B.

28. The expression cassette of claim 26, wherein said granzyme A is encoded by a polynucleotide of SEQ ID NO:

26, SEQ ID NO;

27, or SEQ ID NO;

28.

29. The expression cassette of claim 27, wherein said granzyme B is encoded by a polynucleotide of SEQ ID NO:

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, or SEQ ID NO;

22.

30. The expression cassette of claim 25, wherein the cassette is comprised in a recombinant viral vector.

31. The expression cassette of claim 30, wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

32. A host cell comprising an expression cassette comprising a polynucleotide encoding a chimeric polypeptide comprising a cell-specific targeting moiety and an apoptosis-inducing factor, wherein said apoptosis-inducing factor is a granzyme.

33. The host cell of claim 32, further defined as a prokaryotic host cell.

34. The host cell of claim 32, further defined as an eukaryotic host cell.

35. A method of using a host cell comprising an expression cassette comprising a polynucleotide encoding a chimeric polypeptide comprising a cell-specific targeting moiety and an apoptosis-inducing factor, wherein said apoptosis-inducing factor is a granzyme, the method comprising culturing the host cell under conditions suitable for the expression of the chimeric polypeptide.

36. A method of inducing apoptosis in a cell, comprising administering to said cell an effective amount of a chimeric polypeptide comprising a cell-specific targeting moiety and a granzyme.

37. The method of claim 36, wherein said granzyme is granzyme A or granzyme B.

38. The method of claim 37, wherein said granzyme is granzyme B.

39. The method of claim 36, wherein said cell is in vivo.

40. The method of claim 39, wherein said cell is in a human.

41. A method of inducing apoptosis in a cell, comprising administering to said cell an effective amount of a chimeric polypeptide comprising a cell-specific targeting moiety and a granzyme, wherein said cell-specific targeting moiety is scFvMEL and said granzyme is granzyme B.

42. A method of inducing apoptosis in a cell, comprising administering to said cell an effective amount of a chimeric polypeptide comprising a cell-specific targeting moiety and a granzyme, wherein said cell-specific targeting moiety is vegf121 and said granzyme is granzyme B.

43. A method of inducing apoptosis in a cell, comprising administering to said cell an effective amount of a chimeric polypeptide comprising a cell-specific targeting moiety and a pro-apoptotic member of the Bcl-2 family.

44. The method of claim 43, wherein said pro-apoptotic member of the Bcl-2 family is Bax or a fragment thereof.

45. The method of claim 43, wherein said cell is in vivo.

46. The method of claim 45, wherein said cell is in a human.

47. The method of claim 44, wherein said fragment of Bax lacks at least part of a polypeptide encoded by exon 6 in a Bax polynucleotide sequence.

48. A method of inducing apoptosis in a cell, comprising administering to said cell an effective amount of a chimeric polypeptide comprising a cell-specific targeting moiety and a pro-apoptotic member of the Bcl-2 family, wherein said cell-specific targeting moiety is scFvMEL and said pro-apoptotic member of the Bcl-2 family is Bax or a fragment of Bax.

49. The method of claim 48, wherein said fragment of Bax lacks at least part of exon 6 in a Bax polynucleotide sequence.

50. A method of treating a disease in an individual, comprising the steps of administering to said individual a therapeutically effective amount of a composition comprising:

a) a chimeric polypeptide comprising an apoptosis-inducing moiety and a cell-specific targeting moiety; and

b) a pharmaceutical carrier.

51. The method of claim 50, wherein said pharmaceutical carrier comprises a lipid.

52. The method of claim 50, wherein said disease is cancer, diabetes, arthritis, or inflammatory bowel disease, atherosclerosis, or diabetic retinopathy.

53. The method of claim 52, wherein said disease is cancer.

54. The method of claim 50, wherein said apoptosis-inducing moiety is a granzyme.

55. The method of claim 54, wherein said granzyme is granzyme B or a fragment thereof.

56. The method of claim 50, wherein said apoptosis-inducing moiety is a pro-apoptotic member of the Bcl-2 family.

57. The method of claim 56, wherein said pro-apoptotic member of the Bcl-2 family is Bax or a fragment thereof.

58. The method of claim 57, wherein said fragment of Bax lacks at least part of a polypeptide encoded by exon 6 in a Bax polynucleotide sequence.

59. The method of claim 57, wherein said fragment of Bax lacks at least part of a polypeptide encoded by exons 4, 5, or 6.

60. The method of claim 50, wherein said administration is intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in a creme, or in a lipid composition.

61. The method of claim 50, further comprising administering to said individual an anti-inflammatory composition, chemotherapy, surgery, radiation, hormone therapy, or gene therapy.