Human monoclonal antibodies to influenza M2 protein and methods of making and using same

1. An isolated antibody that specifically binds to an influenza protein M2 extracellular domain, wherein the antibody binds to at least two peptides selected from M2TGEKS (SEQ ID NO:10), M2TDGEKS (SEQ ID NO:15), M2LTGEKS (SEQ ID NO:9) and M2TGE (SEQ ID NO:19), as determined by a peptide based ELISA assay.

2. An isolated antibody that specifically binds to an influenza protein M2 extracellular domain, wherein the antibody has a binding activity (EC50) of less than 10 −8 M for M2LTGEKS (SEQ ID NO:9) and M2TGEK (SEQ ID NO:13), as determined by an ELISA assay of binding between the antibody and an M2LTGEKS-BSA or M2TGEK-BSA conjugate.

3. An isolated antibody that specifically binds to an influenza protein M2 extracellular domain, wherein the antibody has a binding activity (EC50) of less than 10 −9 M for M2LTGEKS (SEQ ID NO:9) and M2TGEK (SEQ ID NO:13), as determined by an ELISA assay of binding between the antibody and an M2LTGEKS-BSA or M2TGEK-BSA conjugate.

4. The isolated antibody of claim 2 or 3 , wherein the antibody has a binding activity (EC50) of less than 10 −8 M for one or more of M2TGS (SEQ ID NO:12) or M2TGE (SEQ ID NO:19).

5. An isolated antibody that specifically binds to a minimal binding sequence that is SLLTEVETPIRNEWGC (SEQ ID NO:23), wherein the minimal binding sequence for antibody binding is neither LTEVETPIRNEW (SEQ ID NO:71) nor EVETPIRN (SEQ ID NO:72).

6. The antibody of any of claims 1 to 3 , wherein the antibody does not bind to LTEVETPIRNEW (SEQ ID NO:71) and EVETPIRN (SEQ ID NO:72).

7. The antibody of claim 5 , wherein the antibody comprises the heavy and light chain variable region sequence of the antibody produced by the hybridoma L66 (ATCC PTA-5048).

8. The antibody of claim 5 , wherein the antibody comprises the mature portion of heavy chain variable region sequence as shown in SEQ ID NO:30 and the mature portion of light chain variable region sequence as shown in SEQ ID NO:31.

9. An isolated antibody that specifically binds to a minimal binding sequence that is TPIRNE (SEQ ID NO:24), wherein the minimal binding sequence for antibody binding is neither LTEVETPIRNEW (SEQ ID NO:71) nor EVETPIRN (SEQ ID NO:72).

10. The antibody of claim 9 , wherein the antibody comprises the heavy and light chain variable region sequence of the antibody produced by the CHO cell C40G1 (ATCC PTA-5050).

11. The antibody of claim 9 , wherein the antibody comprises the mature portion of heavy chain variable region sequence as shown in SEQ ID NO:26 and the mature portion of light chain variable region sequence as shown in SEQ ID NO:27.

12. The antibody of any of claims 1 to 3 , wherein the antibody is a subclass selected from human IgG1, human IgG2, human IgG3 and human IgG4.

13. The antibody of any of claims 1 to 3 , wherein the subclass of the antibody is human IgG1.

14. The antibody of any of claims 1 to 3 , wherein the antibody is selected from IgG, IgA, IgM, IgE, and IgD isotypes.

15. The antibody of claim 14 , wherein the IgG isotype is selected from IgG 1 , IgG 2 , IgG 3 and IgG 4 .

16. The antibody of any of claims 1 to 3 , wherein the antibody inhibits influenza virus infection of a cell, influenza virus proliferation or influenza virus replication in vitro or in vivo.

17. The antibody of any of claims 1 to 3 , wherein the antibody inhibits influenza binding of a cell in vitro or in vivo.

18. The antibody of any of claims 1 to 3 , wherein the antibody inhibits increases in influenza virus titer, decreases influenza virus titre, decreases influenza virus replication or proliferation, or decreases one or more symptoms or complications associated with influenza virus infection in a subject.

19. The antibody of claim 18 , wherein the symptom or complication is selected from chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache and death.

20. The antibody of any of claims 1 to 3 , wherein the antibody has an EC 50 less than 2.0 to 3.0, 1.0 to 2.0, 0.5 to 1.0, 0.1 to 0.5 or less than 0.1 μg/ml for inhibiting influenza A virus infection of MDCK cells, as determined by a cell based-ELISA assay.

21. The antibody of claim 20 , wherein the influenza virus comprises A/PR/8/34, A/HK8/68, A/HK/1/68, H1N1, H3N2, H5N1, H9N2, H6N2, H7N2, or H7N3.

22. The antibody of any of claims 1 to 3 , wherein the antibody has an EC 50 less than 0.05 to 0.1 μg/ml for inhibiting influenza A virus infection of MDCK cells.

23. The antibody of claim 22 , wherein the influenza virus comprises A/PR/8/34, A/HK8/68, A/HK/1/68, H1N1, H3N2, H5N1, H9N2, H6N2, H7N2, or H7N3.

24. The antibody of any of claims 1 to 3 , wherein the antibody has an EC 50 less than 2.0 to 3.0, 1.0 to 2.0, 0.5 to 1.0, 0.1 to 0.5 or less than 0.1 ug/ml for inhibiting M2 binding to MDCK cells.

25. The antibody of any of claims 1 to 3 , wherein the antibody comprises a human, humanized or chimeric monoclonal antibody.

26. An amino acid subsequence of the antibody of claim 1 , wherein the subsequence is selected from heavy and light chain variable regions (V H and V L ), Fab, Fab', F(ab') 2 , Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (sdFv), trispecific (Fab 3 ), bispecific (Fab 2 ), diabody ((V L -V H ) 2 or (V H -V L ) 2 ), triabody (trivalent), tetrabody (tetravalent), minibody ((scF v -C H 3) 2 ), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc or (scFv) 2 -Fc.

27. The antibody of any of claims 1 to 3 , wherein the antibody comprises an antibody multimer.

28. The antibody of any of claims 1 to 3 or a subsequence thereof, further comprising one or more heterologous domains.

29. The antibody or the subsequence of claim 28 , wherein the heterologous domain comprises an amino acid sequence.

30. The antibody or the subsequence of claim 28 , wherein the heterologous domain comprises a binding protein, an enzyme activity, a drug, an antiviral, a toxin, an immune-modulator, a detectable moiety or a tag.

31. The antibody of any of claims 1 to 3 , wherein the antibody is a bispecific or bifunctional antibody.

32. An isolated host cell that expresses an antibody of any of claims 1 to 3 .

33. The host cell of claim 32 , wherein the cell is bacteria, yeast, plant or animal.

34. An isolated nucleic acid encoding an antibody produced by a hybridoma or a CHO cell line denoted as N547 (ATCC Deposit No. PTA-5049; American Type Culture Collection, Manassas, Va., USA), L66 (ATCC Deposit No. PTA-5048; American Type Culture Collection, Manassas, Va., USA), or C40G1 (ATCC Deposit No. PTA-5050; American Type Culture Collection, Manassas, Va., USA).

35. The nucleic acid of claim 34 , further comprising a vector.

36. A composition comprising the antibody of any of claims 1 to 3 , and an antiviral agent.

37. A composition comprising the antibody of any of claims 1 to 3 , and an agent that inhibits or reduces one or more symptoms or complications associated with influenza virus infection.

38. The composition of claim 37 , wherein a symptom or complication is selected from chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache and death.

39. A pharmaceutical composition comprising the antibody of any of claims 1 to 3 , and a pharmaceutically acceptable carrier or excipient.

40. A kit comprising the antibody of any of claims 1 to 3 , and instructions for treating, inhibiting or decreasing susceptibility of infection of a subject by one or more influenza virus strains or isolates.

41. The kit of claim 40 , further comprising an article of manufacture for delivery of the antibody into a mucosal tissue.

42. The kit of claim 41 , wherein the article of manufacture comprises an inhaler, aerosol, spray or squeeze bottle suitable for inhalation or nasal administration to a subject.

43. The kit of claim 41 , wherein the mucosal tissue comprises nasal passages, sinuses, mouth, throat, larynx or lungs.

44. The kit of claim 40 , further comprising an antiviral agent.

45. A method for treating influenza virus infection of a subject, comprising administering to the subject an amount of an antibody of any of claims 1 to 3 effective to treat influenza virus infection of the subject.

46. The method of claim 45 , wherein the antibody is administered prior to, substantially contemporaneously with or following infection of the subject.

47. The method of claim 45 , wherein the antibody is administered substantially contemporaneously with or following infection of the subject.

48. The method of claim 45 , wherein the administration provides a therapeutic benefit.

49. The method of claim 48 , wherein the therapeutic benefit comprises inhibiting increases in virus titer, decreasing virus titer, inhibiting increases in virus replication, decreasing virus replication, inhibiting increases in virus proliferation or decreasing virus proliferation, or decreasing one or more symptoms or complications associated with virus infection in a subject.

50. The method of claim 49 , wherein a symptom or complication is selected from chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache and death.

51. The method of claim 45 , wherein the antibody is produced by a hybridoma or a CHO cell line denoted as N547 (ATCC Deposit No. PTA-5049; American Type Culture Collection, Manassas, Va., USA), L66 (ATCC Deposit No. PTA-5048; American Type Culture Collection, Manassas, Va., USA), or C40G1 (ATCC Deposit No. PTA-5050; American Type Culture Collection, Manassas, Va., USA).

52. The method of claim 45 , wherein the influenza strain comprises A/PR/8/34, A/HK8/68, A/HK/1/68, H1N1, H3N2, H5N1, H9N2, H6N2, H7N2, or H7N3.

53. An antibody comprising a heavy-chain variable sequence or a light-chain variable sequence encoded by the nucleic acid sequences of SEQ ID NO:36 and SEQ ID NO:37.

54. An antibody comprising a heavy-chain variable sequence and a light-chain variable sequence as set forth in SEQ ID NO:30 and SEQ ID NO:31.

55. An isolated antibody that specifically binds to a minimal binding sequence that is LLTEVETPIRNEWGC (SEQ ID NO:25), wherein the minimal binding sequence for antibody binding is neither LTEVETPIRNEW (SEQ ID NO:71) nor EVETPIRN (SEQ ID NO:72).

56. The antibody of claim 55 , wherein the antibody comprises the heavy and light chain variable region sequence of the antibody produced by the hybridoma N547 (ATCC PTA-5049).

57. The antibody of claim 55 , wherein the antibody comprises the mature portion of heavy chain variable region sequence as shown in SEQ ID NO:28 and the mature portion of light chain variable region sequence as shown in SEQ ID NO:29.

58. A method of producing an influenza protein M2 antibody, comprising:

a) providing a hybridoma or a CHO cell line denoted as no. N547 (ATCC Deposit No. PTA-5049; American Type Culture Collection, Manassas, Va., USA), L66 (ATCC Deposit No. PTA-5048; American Type Culture Collection, Manassas, Va., USA), or C40G1 (ATCC Deposit No. PTA-5050; American Type Culture Collection, Manassas, Va., USA) that produces a human M2 antibody; and

b) isolating an antibody produced by one of the hybridoma or the CHO cell lines.