METHODS AND COMPOSITIONS RELATED TO PEPTIDES AND PROTEINS WITH C-TERMINAL ELEMENTS

• US 20090226372A1
• Filed: 02/20/2009
• Published: 09/10/2009
• Est. Priority Date: 02/21/2008
• Status: Active Grant

• Current Assignee: Sanford-Burnham Medical Research Institute (Sanford Burnham Prebys Medical Discovery Institute)
• Original Assignee: Burnham Institute For Medical Research
• Inventors: Ruoslahti, Erkki, Sugahara, Kazuki, Teesalu, Tambet
• Granted Patent: US 11,260,133 B2
• Time in Patent Office: Days
• US Class Current: 424/9.100
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/6923 the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
• CPC Class Codes: A61P 35/00 Antineoplastic agents

Abstract

Disclosed are compositions and methods useful for targeting and internalizing molecules into cells of interest and for penetration by molecules of tissues of interest. The compositions and methods are based on peptide sequences that are selectively internalized by a cell, penetrate tissue, or both. The disclosed internalization and tissue penetration is useful for delivering therapeutic and detectable agents to cells and tissues of interest.

Claims

1. A method of forming a CendR conjugate, the method comprising

(a) selecting an amino acid sequence for internalization into a cell, tissue penetration, or both, wherein the amino acid sequence comprises a CendR element,

(b) causing a cargo composition to be covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence, wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element,

wherein the CendR conjugate comprises the protein or peptide and the coupled or associated cargo composition.

2. The method of claim 1, wherein the protein or peptide of step (b) can be internalized into a cell, penetrate tissue, or both when the selected amino acid sequence of step (a) is present in the protein or peptide but not when the selected amino acid is not present in the protein or peptide.

3. The method of claim 1, wherein the protein or peptide of step (b) can penetrate tissue when the selected amino acid sequence of step (a) is present in the protein or peptide but not when the selected amino acid is not present in the protein or peptide.

4. The method of claim 1, wherein the protein or peptide of step (b) can be internalized into a cell and penetrate tissue when the selected amino acid sequence of step (a) is present in the protein or peptide but not when the selected amino acid is not present in the protein or peptide.

5. The method of claim 1, wherein the selected amino acid sequence of step (a) can be internalized into a cell, penetrate tissue, or both without being associated with the cargo composition of step (b).

6. The method of claim 1, wherein the selected amino acid sequence of step (a) can penetrate tissue without being associated with the cargo composition of step (b).

7. The method of claim 1, wherein the selected amino acid sequence of step (a) can be internalized into a cell and penetrate tissue without being associated with the cargo composition of step (b).

8. The method of claim 1, wherein the selected amino acid sequence of step (a) is the only functional internalization element in the protein or peptide of step (b).

9. The method of claim 1, wherein the selected amino acid sequence of step (a) is the only functional internalization element in the CendR conjugate.

10. The method of claim 1, wherein the CendR element is an activatable CendR element.

11. The method of claim 10, wherein the activatable CendR element is a protease-activatable CendR element.

12. The method of claim 1, wherein the protein or peptide is circular.

13. The method of claim 1, wherein the CendR element is at the C-terminal end of the protein or peptide.

14. The method of claim 1, wherein the cargo composition of step (b) is an anti-angiogenic agent, a pro-angiogenic agent, a nanoparticle, a cancer chemotherapeutic agent, a cytotoxic agent, an anti-inflammatory agent, or an anti-arthritic agent.

15. The method of claim 1, wherein the cargo composition of step (b) comprises a homing sequence.

16. The method of claim 15, wherein the cargo composition selectively homes to a tumor.

17. The method of claim 16, wherein the cargo composition selectively homes to tumor vasculature.

18. The method of claim 1, wherein the amino acid sequence is selected for internalization into a cell.

19. The method of claim 1, wherein the amino acid sequence is selected for tissue penetration.

20. The method of claim 1, wherein the amino acid sequence is selected for internalization into a cell and tissue penetration.

21. A CendR conjugate made by the method comprising

(a) selecting an amino acid sequence for internalization into a cell, tissue penetration, or both, wherein the amino acid sequence comprises a CendR element,

(b) causing a cargo composition to be covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence, wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element,

wherein the CendR conjugate comprises the protein or peptide and the coupled or associated cargo composition.

22. The CendR conjugate of claim 21, wherein the CendR element is an activatable CendR element.

23. The CendR conjugate of claim 22, wherein the activatable CendR element is a protease-activatable CendR element.

24. The CendR conjugate of claim 21, wherein the protein or peptide is circular.

25. The CendR conjugate of claim 21, wherein the CendR element is at the C-terminal end of the protein or peptide.

26. The CendR conjugate of claim 21, wherein the amino acid sequence is selected for internalization into a cell.

27. The CendR conjugate of claim 21, wherein the amino acid sequence is selected for tissue penetration.

28. The CendR conjugate of claim 21, wherein the amino acid sequence is selected for internalization into a cell and tissue penetration.

29. A method of delivering a cargo composition into a cell, the method comprising:

(a) coupling a CendR element to the cargo composition thus forming a CendR conjugate; and

(b) exposing the cell to the CendR conjugate, wherein the CendR conjugate can then enter the cell, thereby delivering the cargo composition into the cell.

30. The method of claim 29, wherein the CendR element is an activatable CendR element.

31. The method of claim 30, wherein the activatable CendR element is a protease-activatable CendR element.

32. The method of claim 29, wherein the protein or peptide is circular.

33. The method of claim 30, wherein a cleaving agent activates the activatable CendR element of the CendR conjugate when the cell is exposed to the CendR element.

34. The method of claim 29, wherein the CendR element is at the C-terminal end of the protein or peptide.

35. A method of identifying a cell that can internalize a CendR element, the method comprising:

(a) exposing a cell to a CendR element;

(b) determining if the CendR element was internalized.

36. The method of claim 35, wherein the cell is in an assay.

37. The method of claim 35, wherein the CendR element is coupled to a protein or peptide.

38. The method of claim 35, wherein the CendR element is an activatable CendR element.

39. The method of claim 38, wherein the activatable CendR element is a protease-activatable CendR element.

40. The method of claim 35, wherein the protein or peptide is circular.

41. The method of claim 35, wherein the activatable CendR element is activated before exposure to the cell.

42. The method of claim 35, wherein the CendR element is at the C-terminal end of the protein or peptide.

43. The method of claim 38, wherein the activatable CendR element is a protease-activatable CendR element.

44. A method of identifying a cancer cell as a candidate for CendR-based therapy, the method comprising:

(a) exposing the cancer cell to a CendR element;

(b) determining if the CendR element was internalized by the cancer cell, wherein an internalized CendR element identifies the cancer cell as being a candidate for CendR-based therapy.

45. The method of claim 44, wherein the cell is in an assay.

46. The method of claim 44, wherein the cell is in a subject.

47. The method of claim 44, wherein the CendR element is coupled to a protein or peptide.

48. The method of claim 44, wherein the CendR element is an activatable CendR element.

49. The method of claim 48, wherein the activatable CendR element is a protease-activatable CendR element.

50. The method of claim 44, wherein the protein or peptide is circular.

51. The method of claim 44, wherein the CendR element is at the C-terminal end of the protein or peptide.

52. A method of producing an activatable CendR element that can be activated in proximity to a cell of interest, the method comprising forming an activatable CendR element wherein a blocking group is coupled to a CendR element via a cleavable bond, wherein the cleavable bond is cleavable by an enzyme present in proximity to the cell of interest.

53. The method of claim 52, wherein the cell is in a subject.

54. The method of claim 52, further comprising, prior to forming the activatable CendR element, identifying the enzyme that is present in proximity to the cell of interest.

55. The method of claim 52 further comprising, prior to forming the activatable CendR element, selecting the cleavable bond based on the enzyme that is present in proximity to the cell of interest.

56. A method of forming an activatable CendR element, the method comprising:

(a) selecting an amino acid sequence for internalization into a cell, tissue penetration, or both, wherein the amino acid sequence comprises a CendR element,

(b) causing a blocking group to be covalently coupled to the CendR element, wherein a bond coupling the blocking group and the CendR element is cleavable, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell, tissue penetration, or both,

wherein the activatable CendR element comprises the selected amino acid sequence and the blocking group.

57. The method of claim 56, wherein the CendR element comprises a terminal carboxyl group, wherein the blocking group is coupled to the terminal carboxyl group.

58. The method of claim 57, further comprising, prior to step (b), selecting the bond coupling the blocking group and the terminal carboxyl group to be cleavable by a protease present in proximity to the cell of interest.

59. The method of claim 56, wherein the blocking group is coupled to the C-terminal amino acid of the CendR element.

60. The method of claim 56, wherein the blocking group is coupled to an amino acid of the CendR element other than the C-terminal amino acid of the CendR element.

61. The method of claim 56, wherein a cargo composition is covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence, wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element.

62. The method of claim 56, wherein the amino acid sequence is selected for internalization into a cell.

63. The method of claim 56, wherein the amino acid sequence is selected for tissue penetration.

64. The method of claim 56, wherein the amino acid sequence is selected for internalization into a cell and tissue penetration.

65. The method of claim 56, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell.

66. The method of claim 56, wherein the blocking group covalently coupled to the CendR element reduces or prevents tissue penetration.

67. The method of claim 56, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell and tissue penetration.

70. The CendR element of claim 67, wherein the method further comprises, prior to step (b), selecting the bond coupling the blocking group and the terminal carboxyl group to be cleavable by a protease present in proximity to the cell of interest.

68. An activatable CendR element made by the method comprising

(a) selecting an amino acid sequence for internalization into a cell, tissue penetration, or both, wherein the amino acid sequence comprises a CendR element,

(b) causing a blocking group to be covalently coupled to the CendR element, wherein a bond coupling the blocking group and the CendR element is cleavable, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell, tissue penetration, or both,

wherein the activatable CendR element comprises the selected amino acid sequence and the blocking group.

69. The CendR element of claim 68, wherein the CendR element comprises a terminal carboxyl group, wherein the blocking group is coupled to the terminal carboxyl group.

71. The CendR element of claim 68, wherein the blocking group is coupled to the C-terminal amino acid of the CendR element.

72. The CendR element of claim 68, wherein the blocking group is coupled to an amino acid of the CendR element other than the C-terminal amino acid of the CendR element.

73. The CendR element of claim 68, wherein a cargo composition is covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence, wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element.

74. The CendR element of claim 68, wherein the amino acid sequence is selected for internalization into a cell.

75. The CendR element of claim 68, wherein the amino acid sequence is selected for tissue penetration.

76. The CendR element of claim 68, wherein the amino acid sequence is selected for internalization into a cell and tissue penetration.

77. The CendR element of claim 68, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell.

78. The CendR element of claim 68, wherein the blocking group covalently coupled to the CendR element reduces or prevents tissue penetration.

79. The CendR element of claim 68, wherein the blocking group covalently coupled to the CendR element reduces or prevents internalization into a cell and tissue penetration.

80. A method of identifying a tissue that can be penetrated by a CendR element, the method comprising

(a) exposing a tissue to a CendR element, and

(b) determining if the CendR element penetrated the tissue.

81. A method of identifying a tumor as a candidate for CendR-based therapy, the method comprising

(a) exposing the tumor to a CendR element, and

(b) determining if the CendR element penetrated the tumor, wherein a CendR element that penetrated identifies the tumor as being a candidate for CendR-based therapy.

82. A method of identifying a tumor as a candidate for CendR-based therapy, the method comprising

(a) exposing a cell from the tumor to a CendR element, and

(b) determining if the CendR element was internalized by the cell, wherein an internalized CendR element identifies the tumor as being a candidate for CendR-based therapy.