Hybrid molecules having translocation region and cell-binding region

• US 6,022,950 A
• Filed: 06/07/1995
• Issued: 02/08/2000
• Est. Priority Date: 06/07/1984
• Status: Expired due to Term

• Current Assignee: Seragen, Inc. (Ligand Pharmaceuticals, Inc.)
• Original Assignee: Seragen, Inc. (Ligand Pharmaceuticals, Inc.)
• Inventors: Murphy, John R.
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): SCHWARTZMAN, ROBERT A
• Application Number:
  US08/479,510
• Time in Patent Office: 1,707 Days
• Field of Search: 530/324, 530/350, 530/402, 530/387.3, 530/388.1, 530/351
• US Class Current: 530/350
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/25 Shigella (G)
• CPC Class Codes: C07K 14/28 from Vibrionaceae (F)
• CPC Class Codes: C07K 14/34 from Corynebacterium (G)
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/55 IL-2
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 2740/16022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12Y 114/16001 Phenylalanine 4-monooxygenase (1.14.16.1)
• CPC Class Codes: C12Y 302/0102 Alpha-glucosidase (3.2.1.20)
• CPC Class Codes: C12Y 302/01052 Beta-N-acetylhexosaminidase (3.2.1.52)

Abstract

Disclosed is a hybrid molecule comprising a first part, a second part, and a third part connected by covalent bonds,

(a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal;

(b) wherein said second part comprises a portion of a translocation domain of naturally occurring protein which translocates said third part across the cytoplasmic membrane into the cytosol of the cell; and

(c) wherein said third part comprises a chemical entity to be introduced into the cell, wherein each of said first part and said third part is non-native with respect to said naturally occurring protein, and further wherein said covalent bond connecting said second part and said third part is a cleavable bond, provided that when said second part comprises a portion of a translocation domain of Pseudomonas exotoxin, said third part is not a polypeptide.

Claims

1. A hybrid molecule comprising a first part, a second part, and a third part connected by covalent bonds,

(a) wherein said first part comprises a portion of the binding domain of a cell-binding ligand effective to cause said hybrid molecule to bind to a cell of an animal;

(b) wherein said second part comprises a portion of a translocation domain of a naturally occurring protein which translocates said third part across the cytoplasmic membrane into the cytosol of the cell; and

(c) wherein said third part comprises a chemical entity to be introduced into the cell, wherein each of said first part and said third part is non-native with respect to said naturally occurring protein, and further wherein said covalent bond connecting said second part and said third part is a cleavable bond, provided that when said second part comprises a portion of a translocation domain of Pseudomonas exotoxin, said third part is not a polypeptide.

2. The hybrid molecule of claim 1, wherein said cell-binding ligand is a polypeptide.

3. The hybrid molecule of claim 1, wherein said third part is a detectable label.

4. The hybrid molecule of claim 1, wherein said third part is a polypeptide.

5. The hybrid molecule of claim 1, wherein the covalent bond connecting said second and third parts is a disulfide bond.

6. The hybrid molecule of claim 1, wherein the covalent bond connecting said second and third parts is a thioether bond.

7. The hybrid molecule of claim 1, wherein said naturally occurring protein of (b) is botulinum neurotoxin.

8. The hybrid molecule of claim 1, wherein said naturally occurring protein of (b) is Shiga-like toxin.

9. The hybrid molecule of claim 2, wherein said first part comprises the binding domain of said cell-binding polypeptide ligand.

10. The hybrid molecule of claim 2, wherein said first part comprises the cell-binding polypeptide ligand.

11. The hybrid molecule of claim 2, wherein said cell-binding polypeptide ligand is a hormone.

12. The hybrid molecule of claim 2, wherein said cell-binding polypeptide ligand is an antigen-binding, single-chain analog of a monoclonal antibody.

13. The hybrid molecule of claim 2, wherein said first part comprises a monoclonal antibody.

14. The hybrid molecule of claim 2, wherein said first part comprises a portion of the binding domain of a polypeptide toxin.

15. The hybrid molecule of claim 3, wherein said detectable label is a fluorescent moiety, a radioactive moiety, or an electron-dense moiety.

16. The hybrid molecule of claim 4, wherein said polypeptide is an antigen-binding, single-chain analog of a monoclonal antibody.

17. The hybrid molecule of claim 4, wherein said polypeptide comprises the enzymatically active portion of an enzyme.

18. The hybrid molecule of claim 4, wherein said polypeptide comprises the enzymatically active portion of a protease.

19. The hybrid molecule of claim 4, wherein said polypeptide comprises the enzymatically active portion of a nuclease.

20. The hybrid molecule of claim 4, wherein said polypeptide comprises the enzymatically active portion of a toxin.

21. The hybrid molecule of claim 4, wherein the covalent bond connecting said second and third parts is a disulfide bond.

22. The hybrid molecule of claim 4, wherein the covalent bond connecting said second and third parts is a peptide bond or a proteolytically-sensitive disulfide loop.

23. The hybrid molecule of claim 8, wherein said naturally occurring protein of (b) is ricin.

24. The hybrid molecule of claim 11, wherein said hormone is selected from the group consisting of insulin, interleukin II, interleukin IV, interleukin VI and, epidermal growth factor (EGF).

25. The hybrid molecule of claim 14, wherein said toxin is, diphtheria toxin.

26. The hybrid molecule of claim 20, wherein said toxin is selected from the group consisting of cholera toxin, LT toxin, C3 toxin, Shiga toxin, Shiga-like toxin, ricin toxin, pertussis toxin, tetanus toxin, diphtheria toxin, and Pseudomonas exotoxin A.

27. The hybrid molecule of claim 22, wherein said first part is a polypeptide and the covalent bond connecting said first and second parts is a peptide bond.

28. The hybrid molecule of claim 24, wherein said hormone is interleukin II.

29. The hybrid molecule of claim 24, wherein said hormone is EGF.

30. The hybrid molecule of claim 26, wherein said toxin is cholera toxin.

31. The hybrid molecule of claim 26, wherein said toxin is Shiga-like toxin.

32. The hybrid molecule of claim 26, wherein said toxin is ricin toxin.

33. The hybrid molecule of claim 27, which is a recombinant protein.

42. The hybrid molecule of claim 32, wherein the covalent bond connecting said second and third parts is a disulfide bond.

34. A hybrid molecule comprising a first part, a second part and a third part connected by covalent bonds,

(a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal;

(b) wherein said second part comprises a portion of the translocation domain of diphtheria toxin which translocates said third part across the cytoplasmic membrane and into the cytosol of the cell; and

(c) wherein said third part comprises a chemical entity to be introduced into the cell, wherein said chemical entity and said first part are non-native with respect to said diphtheria toxin, and further wherein said covalent bond connecting said second part and said third part is a cleavable bond.

35. The hybrid molecule of claim 34, wherein said first part comprises a portion of the binding domain of interleukin II effective to cause said hybrid molecule to bind to an interleukin II receptor-bearing cell.

36. The hybrid molecule of claim 34, wherein said first part comprises a portion of the binding domain of EGF.

37. The hybrid molecule of claim 34, wherein said second part comprises Fragment B'"'"' of diphtheria toxin illustrated in FIG. 3.

38. The hybrid molecule of claim 34, wherein said third part comprises the enzymatically active portion of cholera toxin.

39. The hybrid molecule of claim 34, wherein said third part comprises the enzymatically active portion of ricin toxin.

40. The hybrid molecule of claim 34, wherein said third part comprises the enzymatically active portion of Shiga-like toxin.

41. The hybrid molecule of claim 34, wherein the covalent bond connecting said second and third parts is a thioether bond.

43. The hybrid molecule of claim 34, wherein said third part is a polypeptide and the covalent bond connecting said second and third parts is a peptide bond or a proteolyticaily-sensitive disulfide loop.

44. The hybrid molecule of claim 43, wherein said first part is a polypeptide and the covalent bond connecting said first and second parts is a peptide bond.

45. The hybrid molecule of claim 44, which is a recombinant hybrid protein.

46. The hybrid molecule of claim 45, which is CTA/DTB'"'"'/IL2.

47. The hybrid molecule of claim 45, which is SLA/DTB'"'"'/IL2.

48. The hybrid molecule of claim 45, which is ricin A/DTB'"'"'/IL2.

49. A hybrid molecule comprising a first part, a second part, and a third part connected by covalent bonds,

(a) wherein said first part comprises a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal;

(b) wherein said second part comprises a portion of a translocation domain of Shiga-like toxin which translocates said third part across the cytoplasmic membrane into the cytosol of the cell; and

(c) wherein said third part comprises an enzymatically active domain of Shiga-like toxin.

50. The hybrid molecule of claim 49, wherein said cell-binding polypeptide ligand is interleukin-2.

51. The hybrid molecule of claim 49, wherein said covalent bond comprises a proteolytically sensitive disulfide loop.