Nucleic acids encoding and methods of producing fusion proteins

• US 20070082380A1
• Filed: 10/07/2005
• Published: 04/12/2007
• Est. Priority Date: 10/07/2005
• Status: Active Grant

• Current Assignee: JCR Pharmaceuticals Co., Ltd.
• Original Assignee: ArmaGen Technologies, Inc.
• Inventors: Pardridge, William, Boado, Ruben
• Granted Patent: US 8,053,569 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 single nucleic acid sequence comprising a first sequence coding for a light chain of an immunoglobulin and second sequence coding for a heavy chain of the immunoglobulin, wherein either the first sequence further codes for a peptide that is expressed as a fusion protein of the peptide covalently linked to the light chain or the second sequence further codes for a peptide that is expressed as a fusion protein of the peptide covalently linked to the heavy chain.

2. The nucleic acid of claim 1 wherein the first sequence codes for a peptide that is expressed as a fusion protein of the peptide covalently linked to the light chain.

3. The nucleic acid of claim 1 wherein the second sequence codes for a peptide that is expressed as a fusion protein of the peptide covalently linked to the heavy chain.

4. The nucleic acid of claim 1 wherein the peptide is a therapeutic peptide.

5. The nucleic acid of claim 4 wherein the therapeutic peptide is a neurotherapeutic peptide.

6. The nucleic acid of claim 5 wherein the neurotherapeutic peptide is a neurotrophin.

7. The nucleic acid of claim 6 wherein the neurotrophin is BDNF.

8. The nucleic acid of claim 7 wherein the second sequence codes for the nucleic acid coding for the BDNF.

9. The nucleic acid of claim 8 wherein the BDNF is a two amino acid carboxy-truncated BDNF.

10. The nucleic acid of claim 1 wherein the immunoglobulin is an IgG.

11. The nucleic acid of claim 1 wherein the immunoglobulin is a MAb.

12. The nucleic acid of claim 11 wherein the MAb is chimeric.

13. The nucleic acid of claim 1 wherein the immunoglobulin is an antibody to a transport system.

14. The nucleic acid of claim 13 wherein the transport system is an endogenous BBB receptor-mediated transport system.

15. The nucleic acid of claim 14 wherein the endogenous BBB receptor mediated transport system is selected from the group consisting of the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and the IGF receptor.

16. The nucleic acid of claim 14 wherein the endogenous BBB receptor-mediated transport system is the endogenous BBB receptor-mediated insulin transport system.

17. The composition of claim 16 wherein the endogenous BBB receptor-mediated insulin transport system is a human endogenous BBB receptor-mediated insulin transport system and wherein the peptide to which the immunoglobulin heavy chain is covalently linked is human BDNF.

18. The composition of claim 17 wherein the BDNF comprises a sequence that is at least about 80% identical to the sequence of amino acids 466-582 of SEQ ID NO:

24.

19. The composition of claim 18 wherein the BDNF is linked at its amino terminus to carboxy terminus of the heavy chain of the MAb.

20. The composition of claim 19 wherein the heavy chain of the MAb comprises a sequence that is at least about 80% identical to amino acids 20-462 of SEQ ID NO:

24.

21. The composition of claim 20 wherein the light chain comprises a sequence that is at least about 80% identical to amino acids 21-234 of SEQ ID NO:

36.

22. The composition of claim 21 further comprising a nucleic acid sequence that codes for a peptide linker between the heavy chain of the MAb and the BDNF.

23. The composition of claim 22 wherein the linker is S-S-M.

24. The composition of claim 23 further comprising a nucleic acid sequence coding for a signal peptide, wherein the signal peptide is linked to the heavy chain.

25. The composition of claim 24 wherein the signal peptide comprises a sequence that is at least about 80% identical to amino acids 1-19 of SEQ ID NO:

24.

26. The composition of claim 25 further comprising a nucleic acid sequence coding for another signal peptide, wherein the other signal peptide is linked to the light chain.

27. The composition of claim 26 wherein the signal peptide that is linked to the light chain comprises a sequence that is at least about 80% identical to amino acids 1-20 of SEQ ID NO:

36.

28. The composition of claim 27 further comprising a nucleic acid sequence coding for a selectable marker.

29. The composition of claim 28 wherein the selectable marker is DHFR and the sequence of the DHFR is at least about 80% identical to amino acids 1-187 of SEQ ID NO:

38.

37. A vector comprising a nucleic acid of claim 1, 17, 30, or 36.

38. A cell comprising the vector of claim 37.

39. The cell of claim 38 wherein the cell is a eukaryotic cell.

40. The cell of claim 39 wherein the cell is a Chinese hamster ovary cell.

30. A nucleic acid comprising a first sequence that is at least about 80% identical to nucleotides 58-1386-of SEQ ID NO:

33 and a second sequence that is at least 80% identical to nucleotides 1396-1746 of SEQ ID NO;

33.

31. The nucleic acid of claim 30 further comprising a third sequence that is at least about 80% identical to nucleotides 61-702 of SEQ ID NO:

35.

32. The nucleic acid of claim 30 further comprising a fourth sequence that codes for a first signal peptide and a fifth sequence that codes for a second signal peptide.

33. The nucleic acid of claim 32 wherein the fourth sequence is at least about 80% identical to nucleotides 1-57 of SEQ ID NO:

33 and the fifth sequence is at least about 80% identical to nucleotides 1-60 of SEQ ID NO;

35.

34. The nucleic acid of claim 30 or 33 further comprising a sequence that codes for a selectable marker.

35. The nucleic acid of claim 34 wherein the selectable marker is dihydrofolate reductase (DHFR).

36. The nucleic acid of claim 35 wherein the sequence that codes for the DHFR is at least about 80% identical to nucleotides 1-561 of SEQ ID NO:

37.

41. A method of manufacturing an immunoglobulin fusion protein, wherein the fusion protein comprises an immunoglobulin heavy chain fused to a therapeutic agent or an immunoglobulin light chain fused to a therapeutic agent, comprising permanently integrating into a eukaryotic cell a single tandem expression vector, wherein both the gene for the fusion protein and another gene comprising the gene for the immunoglobulin light chain or the gene for the immunoglobulin heavy chain, are incorporated into a single piece of DNA.

42. The method of claim 41 wherein the fusion protein comprises an immunoglobulin heavy chain fused to a therapeutic agent and wherein both the gene for the fusion protein and the gene for the immunoglobulin light chain are incorporated into a single piece of DNA.

43. The method of claim 41 wherein the fusion protein comprises an immunoglobulin light chain fused to a therapeutic agent and wherein both the gene for the fusion protein and the gene for the immunoglobulin heavy chain are incorporated into a single piece of DNA.

44. The method of claim 41 wherein said permanently introducing is acheived by permanently integrating the tandem vector into the eukaryotic cell.

45. The method of claim 41 wherein said permanently introducing is achieved by introducing a replicating episomal genetic element containing the tandem vector into the eukaryotic cell.

46. The method of claim 41 wherein the therapeutic agent is a neurotherapeutic agent.

47. The method of claim 41 further comprising incorporating one or more genes for selectable markers in said single piece of DNA.

48. The method of claim 41 further comprising incorporating one or more amplification genes in said single piece of DNA.

49. The method of claim 41 wherein the immunoglobulin is an IgG.

50. The method of claim 41 wherein the immunoglobulin is a MAb.

51. The method of claim 48 wherein the MAb is a chimeric MAb.

52. The method of claim 41 further comprising expressing the immunoglobulin fusion protein.

53. The method of claim 50 further comprising purifying the immunoglobulin fusion protein.