Fusion proteins for blood-brain barrier delivery

• US 8,142,781 B2
• Filed: 10/07/2005
• Issued: 03/27/2012
• Est. Priority Date: 10/07/2005
• Status: Active Grant

• Current Assignee: JCR Pharmaceuticals Co., Ltd.
• Original Assignee: ArmaGen Technologies, Inc.
• Inventors: Boado, Ruben J., Pardridge, William M.
• Primary Examiner(s): Kolker, Daniel E
• Application Number:
  US11/245,546
• Publication Number:
  US 20070081992A1
• Time in Patent Office: 2,363 Days
• Field of Search: None
• US Class Current: 424/134.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 16/2869 against hormone receptors for antibodies against neuromediator receptors C07K16/286
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention provides compositions, methods, and kits for increasing transport of agents across the blood brain barrier while allowing their activity once across the barrier to remain substantially intact. The agents are transported across the blood brain barrier via one or more endogenous receptor-mediated transport systems. In some embodiments the agents are therapeutic, diagnostic, or research agents.

Claims

1. A composition comprising an agent covalently linked at its amino terminus to an antibody that crosses the blood brain barrier (BBB) through an endogenous BBB receptor, wherein the agent is selected from the group consisting of a brain derived neurotrophic factor (BDNF) comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24, nerve growth factor (NGF), neurotrophin-4/5, neurotrophin (NT)-3, epidermal growth factor (EGF), transforming growth factor (TGF)-α

, glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), artemin, bone morphogenetic proteins (BMPs), hepatocyte growth factor (HGF), erythropoietin (EPO), heregulin, neuregulin, granulocyte-colony stimulating factor (CSF), and saposins, the composition produces an average elevation of concentration of the agent in the brain of at least 5 ng/gram brain following peripheral administration, wherein the agent binds to a receptor and induces neuroprotection, and wherein the antibody and the agent each retain at least 30% of their activities, compared to their activities as separate entities.

2. The composition of claim 1 wherein the agent has a molecular weight greater than about 400 Daltons.

3. The composition of claim 1 wherein the agent alone does not cross the BBB in a therapeutically effective amount following peripheral administration.

4. The composition of claim 1 wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

5. The composition of claim 4 wherein the endogenous BBB receptor is the insulin BBB receptor.

6. The composition of claim 1 wherein the antibody is a monoclonal antibody (MAb).

7. The composition of claim 6 wherein the MAb is a chimeric MAb.

8. The composition of claim 7 wherein the insulin receptor is a human insulin receptor and wherein the BDNF is a human BDNF.

9. The composition of claim 6 wherein the BDNF is covalently linked at its amino terminus to the carboxy terminus of the light chain of the MAb.

10. The composition of claim 6 wherein the BDNF is covalently linked at its amino terminus to the carboxy terminus of the heavy chain of the MAb.

11. The composition of claim 10 wherein the heavy chain of the MAb comprises a sequence that is at least 80% identical to amino acids 20-462 of SEQ ID NO:

24.

12. The composition of claim 11 further comprising a linker between the heavy chain of the MAb and the BDNF.

13. The composition of claim 12 wherein the linker is S-S-M.

14. The composition of claim 13 wherein the light chain comprises a sequence that is at least 80% identical to amino acids 21-234 of SEQ ID NO:

36.

15. The composition of claim 14 wherein the MAb is glycosylated.

16. The composition of claim 1 wherein the agent comprises amino acids 466-582 of SEQ ID NO:

24.

17. The composition of claim 1 wherein the BDNF is a two amino acid carboxyl-truncated variant.

18. The composition of claim 1 wherein the BDNF is a human BDNF.

19. The composition of claim 1 wherein the BDNF comprises a sequence that is at least 95% identical to the sequence of amino acids 466-582 of SEQ ID NO:

24.

20. The composition of claim 19 wherein the agent is BDNF.

21. The composition of claim 20 wherein the MAb is an antibody to the insulin BBB receptor.

22. A composition comprising the composition of claim 1 and a second composition comprising a second agent covalently linked to a second antibody that crosses the blood brain barrier (BBB).

23. The composition of claim 22 wherein the first and second agents are different, and the first and second antibodies that cross the blood brain barrier (BBB) are the same antibody.

24. The composition of claim 23 wherein the first agent is covalently linked to the heavy chain of the first antibody and the second agent is covalently linked to the heavy chain of the second antibody.

25. The composition of claim 1, wherein the composition comprises BDNF, GDNF, or EGF.

26. The composition of claim 1, wherein the composition comprises EGF.

27. The composition of claim 1, wherein the composition comprises EPO.

28. The composition of claim 1, wherein the composition comprises an agent selected from the group consisting of:

BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO;

24, NGF, neurotrophin-4/5, NT-3, EGF, TGF-α

, GDNF, neurturin, PDGF, artemin, BMPs, and saposins.

29. A pharmaceutical composition comprising the composition of claim 1, 19, 10, or 9 and a pharmaceutically acceptable excipient.

47. The composition of claim 1 or 44, wherein said agent binds to a receptor and induces neuroprotection in a human.

79. A method of transport of an agent active in the CNS from the peripheral circulation across the BBB in an effective amount, comprising peripherally administering to an individual the composition of claim 1.

80. The method of claim 79 wherein the agent is a neurotherapeutic agent.

81. A method for treating a CNS disorder in an individual comprising peripherally administering to the individual an effective amount of the composition of claim 1.

82. The method of claim 81 wherein the antibody is an antibody to an insulin receptor and the therapeutic agent comprises a BDNF.

83. The method of claim 82 wherein the administering is selected from the group consisting of oral, intravenous, intramuscular, subcutaneous, intraperitoneal, rectal, transbuccal, intranasal, transdermal, and inhalation administering.

84. The method of claim 83 wherein the administering is intravenous, intramuscular, or subcutaneous.

85. The method of claim 81 wherein the CNS disorder is an acute CNS disorder.

86. The method of claim 85 wherein the acute CNS disorder is selected from the group consisting of spinal cord injury, brain injury, focal brain ischemia and global brain ischemia.

87. The method of claim 85 wherein the composition is administered only once.

88. The method of claim 85 wherein the composition is administered at a frequency of no greater than about once per week.

89. The method of claim 81 wherein the CNS disorder is a chronic disorder.

90. The method of claim 89 wherein the chronic disorder is selected from the group consisting of chronic neurodegenerative disease, retinal ischemia, and depression.

91. The method of claim 90 wherein the chronic neurodegenerative disease is selected from the group consisting of prion diseases, Alzheimer'"'"'s disease, Parkinson'"'"'s disease, amyotrophic lateral sclerosis, Huntington'"'"'s disease, multiple sclerosis, transverse myelitis, motor neuron disease, Pick'"'"'s disease, tuberous sclerosis, lysosomal storage disorders, Canavan'"'"'s disease, Rett'"'"'s syndrome, spinocerebellar ataxias, Friedreich'"'"'s ataxia, optic atrophy, and retinal degeneration.

92. The method of claim 81 wherein the individual is a human.

93. The method of claim 92 wherein the individual is administered a dose of the composition that is about 1 to about 100 mg.

30. A composition comprising an agent covalently linked at its amino terminus to a MAb to the human BBB insulin receptor, wherein the agent is selected from the group consisting of a BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24, neurotrophin-4/5, neurotrophin (NT)-3, epidermal growth factor (EGF), transforming growth factor (TGF)-α

, artemin, glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMPs), and saposins, wherein the MAb comprises a heavy chain and a light chain, wherein the agent binds to a receptor and induces neuroprotection.

31. The composition of claim 30, wherein the agent is the BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24.

32. The composition of claim 31, wherein the BDNF comprises an amino acid sequence at least 95% identical to amino acids 466-582 of SEQ ID NO:

24.

33. The composition of claim 32 wherein the BDNF comprises amino acids 466-582 of SEQ ID NO:

24.

34. The composition of claim 32 wherein the BDNF is a two amino acid carboxyl-terminal truncated BDNF.

35. The composition of claim 31 wherein the heavy chain of the MAb is covalently linked to the BDNF to form a fusion protein, and wherein the sequence of said fusion protein comprises a first sequence that is at least 80% identical to amino acids 20-462 of SEQ ID NO:

24 and a second sequence that is at least 80% identical to amino acids 466-582 of SEQ ID NO;

24.

36. The composition of claim 35 wherein the MAb is glycosylated.

37. The composition of claim 35 wherein the light chain of the MAb comprises a sequence that is at least 80% identical to amino acids 21-234 of SEQ ID NO:

36.

38. The composition of claim 37 further comprising a peptide linker between the carboxyl terminus of the first sequence and the amino terminus of the second sequence.

39. The composition of claim 38 wherein the linker comprises S-S-M.

40. The composition of claim 39 wherein the MAb is glycosylated.

41. The composition of claim 30, wherein the antibody and the agent each retain an average of at least 40% of their activities.

42. A composition for treating a neurological disorder comprising a BDNF covalently linked at its amino terminus to an immunoglobulin that crosses the blood brain barrier, wherein the BDNF binds to a TrkB receptor and is effective to induce neuroprotection, and wherein the covalently linked BDNF crosses the BBB in an amount that is effective in treating the neurological disorder.

43. The composition of claim 42, wherein the antibody and the agent each retain an average of at least 40% of their activities.

44. A composition comprising an agent covalently linked at its amino terminus to an immunoglobulin, wherein the agent in the composition has a serum half-life that is an average of at least 5-fold greater than the serum half-life of the agent alone, the agent is selected from the group consisting of a BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24, artemin, neurotrophin-4/5, neurotrophin (NT)-3, epidermal growth factor (EGF), transforming growth factor (TGF)-α

, glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMPs), and saposins, wherein the agent binds to a receptor and induces neuroprotection and wherein the immunoglobulin is an antibody to an endogenous BBB receptor.

45. The composition of claim 44 wherein the agent is the BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24.

46. The composition of claim 44, wherein the antibody and the agent each retain an average of at least 40% of their activities.

48. A composition for treating a neurological disorder comprising a agent covalently linked at its amino terminus to an immunoglobulin that crosses the blood-brain barrier (BBB), wherein the agent is selected from the group consisting of a brain derived neurotrophic factor (BDNF) comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24, nerve growth factor (NGF), neurotrophin-4/5, neurotrophin (NT)-3, and glial-derived neurotrophic factor (GDNF), wherein the agent binds to a receptor and induces neuroprotection, and wherein the covalently-linked agent crosses the BBB in an amount that is effective in treating the neurological disorder and wherein the agent and the immunoglobulin each retain at least 30% of their activities, compared to their activities as separate entities.

49. The composition of claim 48, wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

50. The composition of claim 48, wherein the immunoglobulin is a MAb to the human BBB insulin receptor and wherein the MAb comprises a heavy chain and a light chain.

51. The composition of claim 48, wherein the neurotrophin in the composition has a serum half-life that is an average of at least 5-fold greater than the serum half-life of the neurotrophin alone.

52. A fusion protein comprising:

(i) an antibody that crosses the blood-brain barrier (BBB), covalently linked to

(ii) the amino terminus of an agent selected from the group consisting of a brain derived neurotrophic factor (BDNF) comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO;

24, nerve growth factor (NGF), neurotrophin-4/5, neurotrophin (NT)-3, epidermal growth factor (EGF), transforming growth factor (TGF)-β

, glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), artemin, bone morphogenetic proteins (BMPs), hepatocyte growth factor (HGF), erythropoietin (EPO), heregulin, neuregulin, granulocyte-colony stimulating factor (CSF), and saposins,

wherein the antibody and the agent each retain at least 30% of their activities, compared to their activities as separate entities, and wherein the agent binds to a receptor and induces neuroprotection.

53. The fusion protein of claim 52 wherein the immunogloblulin crosses the BBB on an endogenous BBB receptor.

54. The fusion protein of claim 53 wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

55. The fusion protein of claim 54 wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor and the transferrin receptor.

56. The fusion protein of claim 55 wherein the endogenous BBB receptor is the insulin receptor.

57. The fusion protein of claim 56 wherein the insulin receptor is the human insulin receptor.

58. The fusion protein of claim 52 wherein the antibody is a MAb.

59. The fusion protein of claim 58 wherein the MAb is a chimeric MAb.

60. The fusion protein of claim 52 wherein the antibody is an antibody to an endogenous BBB receptor-mediated receptor.

61. The fusion protein of claim 60 wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

62. The fusion protein of claim 61 wherein the endogenous BBB receptor is selected from the group consisting of the insulin receptor and the transferrin receptor.

63. The fusion protein of claim 62 wherein the endogenous BBB receptor is the insulin receptor.

64. The fusion protein of claim 63 wherein the insulin receptor is the human insulin receptor.

65. The fusion protein of claim 52 wherein the agent is the BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO:

24.

66. The fusion protein of claim 65 wherein the BDNF is a truncated BDNF.

67. The fusion protein of claim 66 wherein the truncated BDNF is a carboxyl-truncated BDNF.

68. The fusion protein of claim 67 wherein the carboxyl-truncated BDNF is lacking the two carboxyl terminal amino acids.

69. The fusion protein of claim 52 wherein the antibody and the agent are covalently linked by a peptide linker.

70. The fusion protein of claim 52, wherein the fusion protein comprises an agent selected from the group consisting of:

BDNF comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO;

24, NGF, neurotrophin-4/5, NT-3, EGF, TGF-α

, GDNF, neurturin, PDGF, artemin, BMPs, and saposins.

71. The fusion protein of claim 52, wherein the fusion protein comprises BDNF, GDNF, or EGF.

72. The fusion protein of claim 52, wherein the fusion protein comprises EGF.

73. The fusion protein of claim 52, wherein the fusion protein comprises EPO.

78. The fusion protein of claim 52 or 74, wherein said agent binds to a receptor and induces neuroprotection in a human.

74. A fusion protein comprising:

(i) an antibody that crosses the blood brain barrier (BBB), covalently linked to

(ii) the amino terminus of an agent selected from the group consisting of a brain derived neurotrophic factor (BDNF) comprising an amino acid sequence at least 80% identical to amino acids 466-582 of SEQ ID NO;

24, nerve growth factor (NGF), neurotrophin-4/5, neurotrophin (NT)-3, and glial-derived neurotrophic factor (GDNF),

wherein the antibody and the neurotrophin each retain at least 30% of their activities, compared to their activities as separate entities, and wherein the agent binds to a receptor and induces neuroprotection.

75. The fusion protein of claim 74, wherein the antibody crosses the BBB by binding an endogenous BBB receptor selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

76. The fusion protein of claim 75, wherein the antibody is a MAb to the human BBB insulin receptor and wherein the MAb comprises a heavy chain and a light chain.

77. The fusion protein of claim 74, wherein the neurotrophin in the fusion protein has a serum half-life that is an average of at least 5-fold greater than the serum half-life of the neurotrophin alone.