β2 microglobulin fusion proteins and high affinity variants

• US 6,682,741 B1
• Filed: 03/19/2001
• Issued: 01/27/2004
• Est. Priority Date: 06/10/1998
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Shields, Michael, Ribaudo, Randall K.
• Primary Examiner(s): Gambel, Phillip
• Assistant Examiner(s): ROARK, JESSICA HOPE
• Application Number:
  US09/719,243
• Time in Patent Office: 1,044 Days
• Field of Search: 530/350, 435/325, 435/320.1, 424/184.1, 424/185.1, 424/192.1, 424/93.1, 424/93.2, 424/93.21, 424/93.7, 536/23.1, 536/23.4
• US Class Current: 424/192.1
• CPC Class Codes: A61K 2039/55516 Proteins; Peptides
• CPC Class Codes: A61K 2039/57 characterised by the type of response, e.g. Th1, Th2
• 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 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: C07K 14/70525 ICAM molecules, e.g. CD50, CD54, CD102
• CPC Class Codes: C07K 14/70528 CD58
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence

Abstract

β₂-microglobulin fusion proteins and modified forms of β₂-microglobulin are disclosed. The fusion proteins are shown to incorporate onto the surface of MHC I expressing mammalian cells and to cause an increased cytotoxic T-cell response to antigens presented by such cells. The fusion proteins are useful in methods of tumor therapy. Modified forms of human β₂-microglobulin, particularly a form having a serine to valine transition at amino acid position 55 of the mature protein are shown to have an enhanced affinity for MHC I heavy chain, and are useful both in the disclosed fusion proteins and as a vaccine adjuvant where enhanced cytotoxic T-cell response is desired.

Claims

1. A fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is human β

₂-microglobulin S55V (hβ

₂m S55V) (SEQ ID NO;

     10).

2. The fusion protein of claim 1, wherein the first amino acid sequence comprises B7.1, B7.2, a lymphocyte function-associated (LFA) protein, or an intercellular adhesion molecule (ICAM).

3. The fusion protein of claim 2, wherein the LFA protein comprises LFA-1 or LFA-3.

4. The fusion protein of claim 2, wherein the ICAM comprises ICAM-1 or ICAM-2.

25. A recombinant nucleic acid molecule encoding the fusion protein of claim 1.

29. A vector comprising the recombinant nucleic acid of claim 25.

33. A transgenic cell comprising the recombinant nucleic acid molecule of claim 25.

37. A cell having a cell membrane comprising the fusion protein of claim 1.

38. The cell of claim 37, wherein the cell is a tumor cell.

45. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

contacting the cell with the fusion protein of claim 1 such that the fusion protein is presented on the surface of the cell; and

administering the cell to the mammal.

46. The method of claim 45, wherein the cell is a tumor cell.

53. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

transforming the cell with the recombinant nucleic acid molecule of claim 25, such that expression of the nucleic acid molecule results in expression of a fusion protein encoded by the nucleic acid molecule being presented on the surface of the cell; and

administering the cell to a mammal.

54. The method of claim 53, wherein the cell is a tumor cell.

61. A method of stimulating a tumor-reactive cytotoxic T-cell response, comprising:

isolating T-cells from a patient having a tumor;

isolating tumor cells from the patient;

incubating the tumor cells with the fusion protein of claim 1, such that the fusion protein is presented on the surface of the tumor cells;

incubating the T-cells in the presence of the fusion protein-presenting tumor cells to increase the number of tumor-reactive T-cells; and

administering a therapeutically effective dose of the tumor-reactive T-cells to the patient.

5. A fusion protein comprising first and second domains, wherein the first domain is B7.1, B7.2, a lymphocyte function-associated (LFA) protein, or an intercellular adhesion molecule (ICAM) and the second domain is β

₂m.

6. The fusion protein of claim 5, wherein the LFA protein comprises LFA-1 or LFA-3.

7. The fusion protein of claim 5, wherein the ICAM comprises ICAM-1 or ICAM-2.

8. The fusion protein of claim 5, wherein the first domain is joined to an amino terminus of the second domain.

9. The fusion protein of claim 5, wherein the second domain is hβ

₂m.

10. The fusion protein of claim 9, wherein the hβ

₂m is hβ

₂m S55V (SEQ ID NO;

     10).

11. The fusion protein of claim 9, wherein the protein has an amino acid sequence as shown in SEQ ID NO:

2 or 3.

12. The fusion protein of claim 5, wherein the first and second domains are linked by a peptide linker.

13. The fusion protein of claim 5, wherein the fusion protein further comprises a signal peptide joined to an amino terminus of the first domain.

14. The fusion protein of claim 13, wherein the signal peptide is a β

₂m signal peptide.

26. A recombinant nucleic acid molecule encoding the fusion protein of claim 5.

30. A vector comprising the recombinant nucleic acid of claim 26.

34. A transgenic cell comprising the recombinant nucleic acid molecule of claim 26.

39. A cell having a cell membrane comprising the fusion protein of claim 5.

40. The cell of claim 39, wherein the cell is a tumor cell.

47. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

contacting the cell with the fusion protein of claim 5 such that the fusion protein is presented on the surface of the cell; and

administering the cell to a mammal.

48. The method of claim 47, wherein the cell is a tumor cell.

55. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

transforming the cell with the recombinant nucleic acid molecule of claim 26, such that expression of the nucleic acid molecule results in expression of a fusion protein encoded by the nucleic acid molecule being presented on the surface of the cell; and

administering the cell to a mammal.

56. The method of claim 55, wherein the cell is a tumor cell.

62. A method of stimulating a tumor-reactive cytotoxic T-cell response, comprising:

isolating T-cells from a patient having a tumor;

isolating tumor cells from the patient;

incubating the tumor cells with the fusion protein of claim 5, such that the fusion protein is presented on the surface of the tumor cells;

incubating the T-cells in the presence of the fusion protein-presenting tumor cells to increase the number of tumor-reactive T-cells; and

administering a therapeutically effective dose of the tumor-reactive T-cells to the patient.

15. A fusion protein comprising first and second domains, wherein the first domain comprises B7.1, B7.2, LFA-3, or ICAM-1, and the second domain comprises β

₂m.

16. A fusion protein comprising a structure X-Y, wherein X is B7.1, B7.2, a lymphocyte function-associated (LFA) protein, or an intercellular adhesion molecule (ICAM), and Y is a β

₂m.

17. The fusion protein of claim 16, wherein the LFA protein comprises LFA-1 or LFA-3.

18. The fusion protein of claim 16, wherein the ICAM comprises ICAM-1 or ICAM-2.

19. The fusion protein of claim 16, wherein the protein comprises a structure X-L-Y wherein L is a linker peptide.

20. The fusion protein of claim 19, wherein the protein comprises a structure S-X-L-Y wherein S is a signal peptide.

21. The fusion protein of claim 20, wherein the signal peptide is a β

₂m signal peptide.

22. The fusion protein of claim 16 wherein the protein has an amino acid sequence as shown in SEQ ID NO:

2 or 3.

27. A recombinant nucleic acid molecule encoding the fusion protein of claim 16.

31. A vector comprising the recombinant nucleic acid of claim 27.

35. A transgenic cell comprising the recombinant nucleic acid molecule of claim 27.

41. A cell having a cell membrane comprising the fusion protein of claim 16.

42. The cell of claim 41, wherein the cell is a tumor cell.

49. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

contacting the cell with the fusion protein of claim 16 such that the fusion protein is presented on the surface of the cell; and

administering the cell to a mammal.

50. The method of claim 49, wherein the cell is a tumor cell.

57. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

transforming the cell with the recombinant nucleic acid molecule of claim 27, such that expression of the nucleic acid molecule results in expression of a fusion protein encoded by the nucleic acid molecule being presented on the surface of the cell; and

administering the cell to a mammal.

58. The method of claim 57, wherein the cell is a tumor cell.

63. A method of stimulating a tumor-reactive cytotoxic T-cell response, comprising:

isolating T-cells from a patient having a tumor;

isolating tumor cells from the patient;

incubating the tumor cells with the fusion protein of claim 16, such that the fusion protein is presented on the surface of the tumor cells;

incubating the T-cells in the presence of the protein-presenting tumor cells to increase the number of tumor-reactive T-cells; and

administering a therapeutically effective dose of the tumor-reactive T-cells to the patient.

23. An isolated human β

₂-microglobulin molecule having a valine residue present at position 55 in mature wild-type hβ

₂m instead of a serine residue.

24. The isolated human β

₂-microglobulin molecule of claim 23, wherein the molecule comprises the amino acid sequence shown in SEQ ID NO;

10.

28. A recombinant nucleic acid molecule encoding the hβ

₂m molecule of claim 23.

32. A vector comprising the recombinant nucleic acid of claim 28.

36. A transgenic cell comprising the recombinant nucleic acid molecule of claim 28.

43. A cell having a cell membrane comprising the hβ

₂m molecule of claim 23.

44. The cell of claim 43, wherein the cell is a tumor cell.

51. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

contacting the cell with the hβ

₂m molecule of claim 23 such that the hβ

₂m molecule is presented on the surface of the cell; and

administering the cell to a mammal.

52. The method of claim 51, wherein the cell is a tumor cell.

59. A method of enhancing an immune response of a mammal to an antigen presented on a surface of a cell, comprising:

transforming the cell with the recombinant nucleic acid molecule of claim 28, such that expression of the nucleic acid molecule results in expression of a protein encoded by the nucleic acid molecule being presented on the surface of the cell; and

administering the cell to a mammal.

60. The method of claim 59, wherein the cell is a tumor cell.

64. A method of stimulating a tumor-reactive cytotoxic T-cell response, comprising:

isolating T-cells from a patient having a tumor;

isolating tumor cells from the patient;

incubating the tumor cells with the hβ

₂m molecule of claim 23, such that the hβ

₂m molecule is presented on the surface of the tumor cells;

incubating the T-cells in the presence of the protein-presenting tumor cells to increase the number of tumor-reactive T-cells; and

administering a therapeutically effective dose of the tumor-reactive T-cells to the patient.