Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith

• US 20060233813A1
• Filed: 12/09/2005
• Published: 10/19/2006
• Est. Priority Date: 12/10/2004
• Status: Active Grant

• Current Assignee: Pheromonicin Biotech, Ltd.
• Original Assignee: West China Hospital of Sichuan University (Sichuan University)
• Inventors: Qiu, Xiao-Qing
• Granted Patent: US 8,367,066 B2
• Time in Patent Office: Days
• US Class Current: 424/178.100
• 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: A61K 38/164 from bacteria
• 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: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/212 Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
• CPC Class Codes: C07K 14/24 from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C07K 14/34 from Corynebacterium (G)
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/085 Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2318/10 Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

The present invention relates to molecules that are capable of killing cells. The molecules comprise a targeting agent and a channel-forming moiety. The molecules may be polypeptides. The present invention also relates to polynucleotide sequences encoding the polypeptides of the invention. In a preferred embodiment, the channel-forming moiety comprises a colicin and the targeting agent is an antibody. Methods of treatment by administering the molecules of the present invention are also provided.

Claims

1. A molecule for treating a cell proliferative disorder comprising a targeting agent covalently attached to a channel-forming moiety.

2. The molecule of claim 1, wherein said molecule is a polypeptide.

3. The polypeptide of claim 2, wherein said channel-forming moiety is a channel-forming polypeptide or a channel-forming fragment thereof.

4. The polypeptide of claim 3, wherein said channel-forming polypeptide, or channel-forming fragment thereof, is selected from the group consisting of:

α

-hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and E1 family colicin.

5. The polypeptide of claim 3, wherein said channel-forming peptide, or channel-forming fragment thereof, is E1 family colicin.

6. The polypeptide of claim 5, wherein said E1 family colicin is selected from the group consisting of E1, Ia, Ib, A, K or N.

7. The polypeptide of claim 6, wherein said colicin is colicin Ia.

8. The polypeptide of claim 7, wherein said channel-forming fragment of colicin Ia is 451-626.

9. The molecule of claim 1, wherein said targeting agent is selected from the group consisting of a ligand, an antibody, an antibody fragment, a reconstituted antibody mimetic, and a phage segment.

10. The polypeptide of claim 2, wherein said targeting agent is selected from the group consisting of a ligand, an antibody, an antibody fragment, a reconstituted antibody mimetic, and a phage segment.

11. The polypeptide of claim 12, wherein said antibody is an antibody or engineered antibody variant.

12. The polypeptide of claim 13, wherein said antibody or engineered antibody variant is against Epstein-Barr virus gp350/220 envelope glycoprotein.

13. The polypeptide of claim 10, wherein said targeting agent is a reconstituted antibody mimetic derived from engineered antibody variant.

14. The polypeptide of claim 11, wherein said reconstituted antibody mimetic, is specific for a polypeptide expressed by a virus.

16. The polypeptide of any claims 2-15, comprising non-natural amino acid residues.

17. The polypeptide of claim 16, wherein said non-natural amino acid residues are amino acid analog, or mimetics.

18. The polypeptide of claim 17, wherein said non-natural amino acid residues are D-isomers of natural amino acid residues.

19. A nucleic acid molecule encoding the polypeptide of any claim 2-15.

20. A vector comprising the nucleic acid molecule of claim 19.

21. A host cell comprising the vector of claim 20.

22. The host cell of claim 21, wherein said host cell is an E. coli cell.

23. The host cell of claim 21, wherein said host cell is a mammalian cell.

24. A method of producing the polypeptide of any of claim 2-15, comprising:

Culturing the host cell of claim 21;

such that said polypeptide is produced.

25. The method of claim 24, further comprising purifying said polypeptide.

26. A method of producing the molecule of claim 1, wherein said targeting agent and said channel-forming moiety are produced separately and covalently linked after production.

27. The method of claim 24, wherein said channel-forming moiety is produced recombinantly.

15. A polypeptide for the treatment of a cell proliferative disorder comprising an targeting moiety selected from the group consisting of reconstituted antibody mimetics derived from engineered antibody variants against Epstein-Barr virus gp350/220 envelope glycoprotein and a channel-for mning domain of colicin.

28. A method of treating a subject having a cell proliferative disorder, comprising:

administering to said subject an effective amount of a polypeptide comprising a targeting agent and a channel-forming moiety;

thereby treating said subject.

29. The method of claim 28, wherein said targeting agent is selected from an antibody, an antibody fragment, and a reconstituted antibody mimetic.

30. The method of claim 29, wherein said targeting agent is specific for a polypeptide that is differentially expressed in cancer cells.

31. The method of claim 28, wherein said channel-forming moiety is selected from the group consisting of α

-hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and E1 family colicin, or a channel-forming domain thereof.

32. The method of claim 31, wherein said channel-forming domain of colicin is selected from the group consisting of colicin E1, Ia, Ib, A, K or N.

33. The method of claim 32, wherein said colicin is colicin Ia.

34. The polypeptide of claim 33, wherein said fragment of colicin is selected from the group consisting of amino acid residues 1-626 and 451-626.

35. The method of claim 28, wherein said cell proliferative disorder is cancer.

36. The method of claim 28, wherein said cancer is a viral associated cancer.

37. The method of claim 28, wherein said cancer is selected from the group consisting of a cancer formed in the larynx, the prostate, the stomach, the skin, the oral cavity, the pharynx, the esophagus, the liver, the lung, the head, the neck, the bronchus, the pancreas, the small intestine, the colon, the rectum, the breast, the bladder, the uterus, the brain, the lymph system, the blood, the ovaries, the kidneys, and soft tissue

38. Amolecule comprising a targeting agent covalently attached to a channel-forming moiety, wherein the molecule has the ability to decrease or inhibit cell proliferation in a cell in need thereof.

39. Apolypeptide consisting essentially of the amino acid sequence of SEQ ID NO:

1.

40. A nucleic acid molecule encoding the polypeptide of claim 39.

41. The nucleic acid molecule according to claim 40 having the nucleic acid sequence of SEQ ID NO:

2.

42. The polypeptide according to claim 39, wherein the polypeptide is a channel-forming moiety.

43. A molecule comprising a targeting agent and the channel-forming moiety of claim 39 or 42, wherein the molecule is capable of decreasing or inhibiting cell proliferation in a cell in need thereof.

44. A polypeptide comprising the amino acid sequence of SEQ ID NO:

7.

45. A nucleic acid molecule encoding the polypeptide of claim 44.

46. The nucleic acid molecule according to claim 45 having the nucleic acid sequence of SEQ ID NO:

8.

47. A polypeptide comprising the amino acid sequence of SEQ ID NO:

11.

48. A nucleic acid molecule encoding the polypeptide of claim 47.

49. The nucleic acid molecule of claim 48 having the nucleic acid sequence of SEQ ID NO:

12.