Activation and expansion of T-cells using an engineered multivalent signaling platform

• US 20030224520A1
• Filed: 01/03/2003
• Published: 12/04/2003
• Est. Priority Date: 01/03/2002
• Status: Active Grant

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: June, Carl, Thomas, Anna, Riley, James, Maus, Marcela
• Granted Patent: US 7,638,325 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4621 immunosuppressive or immunotolerising
• CPC Class Codes: A61K 39/46433 Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• CPC Class Codes: A61K 39/464499 Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• CPC Class Codes: C12N 2501/51 B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

The present invention relates generally to methods for activating and expanding cells, and more particularly, to a novel method to activate and/or stimulate cells using an engineered multivalent signaling platform. Compositions of cells activated and expanded by the methods herein are further provided.

Claims

1. An engineered multivalent signaling platform (EMSP) for use in stimulating and/or activating T cells comprising an EMSP that expresses or displays on its surface one or more agents that ligate a cell surface moiety of at least a portion of T-cells and stimulates said T-cells.

2. The EMSP of claim 1 wherein said platform comprises a human cell line.

3. The EMSP of claim 2 wherein said human cell line is selected from the group consisting of K562, U937, 721.221, T2, and C1R cells.

4. The EMSP of claim 3 wherein said human cell line is K562.

5. The EMSP of claim 1 wherein said EMSP is a cell that is genetically modified to express a human Fcγ

receptor.

6. The EMSP of claim 5 wherein said human Fcγ

receptor comprises CD32.

7. The EMSP of claim 1 wherein said EMSP is a cell that is genetically modified to express CD32 and said one or more agent is an antibody that binds to a cell surface molecule on the surface of T cells.

8. The EMSP of claim 7 wherein said EMSP is further genetically modified to express a co-stimulatory molecule.

9. The EMSP of claim 8 wherein said co-stimulatory molecule is selected from the group consisting of CD80, CD86, 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1 and PD-L2.

10. The EMSP of claim 7 wherein said one or more agent is an antibody that is displayed on the surface of said EMSP via interaction with the Fcγ

receptor.

11. The EMSP of claim 7 wherein said one or more agent is a natural ligand for a cell surface molecule on the surface of a T cell.

12. The EMSP of claim 11 wherein said natural ligand comprises the natural ligand for CD28.

13. The EMSP of claim 11 wherein said natural ligand comprises 4-1BBL.

14. The EMSP of claim 1 wherein said EMSP is a cell that has been modified to express a cytokine.

15. The EMSP of claim 14 wherein said cytokine is selected from the group consisting of IL-2, GM-CSF, IL-4, TNF-α

, and IFN-γ

.

16. The EMSP of claim 1 wherein said T cells comprise CD4+ T cells.

17. The EMSP of claim 1 wherein said T cells comprise CD8+ T cells.

18. The EMSP of claim 1 wherein said T cells comprise regulatory T cells.

19. The EMSP of claim 10 wherein said antibody is an anti-CD28 antibody.

20. The EMSP of claim 10 wherein said antibody is an anti-CD3 antibody.

21. The EMSP of claim 10 wherein said antibody comprises anti-CD3 and anti-CD28.

22. The EMSP of claim 1 wherein said one or more agent comprises at least an antigen.

23. The EMSP of claim 22 wherein said antigen is selected from the group consisting of a tumor antigen, a bacterial antigen, a fungal antigen, a viral antigen, and an autoantigen.

24. A method for activating or stimulating a population of T-cells by cell surface moiety ligation, comprising:

a. providing a population of cells wherein at least a portion thereof comprises T-cells;

b. contacting said population of cells with an EMSP, said EMSP having on its surface one or more agents that ligate a cell surface moiety of at least a portion of said T-cells and activates or stimulates said T-cells.

25. The method of claim 24, wherein said EMSP comprises a cell.

26. The method of claim 25, wherein said cell is a human cell.

27. The method of claim 26, wherein said cell is a human cell line.

28. The method of claim 24, further comprising expanding said T-cells by incubating in the presence of EMSP.

29. The method of claim 24, further comprising separating said T-cells from said EMSP and subsequently incubating said T-cells with an agent that facilitates T-cell expansion.

30. The method of any one of claims 24-28, wherein the T-cell is a T-cell line.

31. The method of any one of claims 24-28, wherein said T-cell is a T-cell clone.

78. The method of any one of claims 24-70 wherein the natural functionality of said T-cells is preserved following stimulation and expansion

84. The method of claim 29, wherein said agent that facilitates T-cell expansion comprises IL-2.

32. A method for maintaining T-cell repertoire comprising providing a population of cells having T-cells present therein, exposing said T-cells to an EMSP for a time sufficient to induce activation and subsequently expanding said T-cells.

33. The method of claim 32, wherein said expansion occurs in the absence of said EMSP.

34. The method of claim 32, wherein said expansion occurs in the presence of said EMSP.

35. A method for maintaining the viability of T-cells during culture, comprising contacting said T-cells with EMSP.

36. The method of claim 35, wherein said T-cells are CD4+ T-cells.

37. The method of claim 35, wherein said T-cells are CD8+ T-cells.

38. The method of claim 37, wherein said contacting occurs by sequential stimulation of T-cells by an EMSP.

39. The method of claim 38, wherein the viability of said T-cells is greater than 50% after at least one, two, three, four, five, six, seven, or eight rounds of stimulation.

40. The method of claim 38, wherein the viability of said T-cells is greater than 60% after at least one, two, three, four, five, six, seven, or eight rounds of stimulation.

41. The method of claim 38, wherein the viability of said T-cells is greater than 70% after at least one, two, three, four, five, six, seven, or eight rounds of stimulation.

42. The method of claim 38, wherein the viability of said T-cells is greater than 80% after at least one, two, three, four, five, six, seven, or eight rounds of stimulation.

43. A method for decreasing apoptosis of a population of CD8+ T-cells, comprising stimulating said cells with EMSP, said EMSP having on its surface at least one primary stimulatory agent and at least one co-stimulatory agent.

44. The method of claim 43, wherein said CD8+ T-cells are expanded in the presence of said EMSP.

45. The method of claim 43, wherein said CD8+ T-cells are expanded in the absence of said EMSP.

46. The method of claim 43, wherein said CD8+ T-cells demonstrate an increase in Bcl-XL levels compared to CD8+ T-cells expanded in the absence of initial stimulation with said EMSP.

47. The method of any one of claims 43-46, wherein at least one co-stimulatory agent comprises 4-1BB ligand.

48. The method of claim 47, wherein the primary stimulatory agent comprises an agent that binds CD3.

49. The method of claim 48, wherein said EMSP further comprises a second co-stimulatory agent that binds CD28.

50. The method of any one of claims 43-49, further comprising at least one sequential round of restimulation.

51. A method for expanding a population of T-cells by cell surface moiety ligation, comprising:

a. providing a population of cells wherein at least a portion thereof comprises T-cells;

b. contacting said population of cells with an EMSP, said EMSP having on its surface one or more agents that ligate a cell surface moiety of at least a portion of said T-cells and activates or stimulates said T-cells; and

c. culturing said T-cells under conditions and time sufficient to induce cell division, wherein said contacting and/or said culturing occurs in the absence of exogenously added cytokines.

52. The method of claim 51, wherein said EMSP comprises a cell.

53. The method of claim 51, wherein said cell is a human cell.

54. The method of claim 53, wherein said cell is a human cell line.

55. The method of claim 51, further comprising sequentially stimulating said T-cells with EMSP either by previously purifying T-cells from originally added EMSP and subsequently adding additional EMSP or by adding additional EMSP to previously stimulated cells without separation of originally added EMSP.

56. The method of claim 51, further comprising separating said T-cells from said EMSP and subsequently incubating said T-cells with an agent that facilitates T-cell expansion, followed by restimulation with EMSP.

57. The method of any one of claims 51-56, wherein the T-cell is a T-cell line.

58. The method of any one of claims 51-56, wherein said T-cell is a T-cell clone.

59. The method of any one of claims 51-58, wherein said EMSP comprises a cell displaying a ligand for any one of CD3, CD28, or 4-1BB.

71. A population of T-cells expanded by the method of claim 51 or 60, wherein said T-cells are substantially free of EMSP.

60. A method for expanding a population of T-cells by cell surface moiety ligation, comprising:

a. providing a population of cells wherein at least a portion thereof comprises T-cells;

b. contacting said population of cells with an EMSP, said EMSP having on its surface one or more agents that ligate a cell surface moiety of at least a portion of said T-cells and activates or stimulates said T-cells; and

c. culturing said T-cells under conditions and time sufficient to induce cell division.

61. The method of claim 60, wherein said EMSP comprises a cell.

62. The method of claim 60, wherein said cell is a human cell.

63. The method of claim 62, wherein said cell is a human cell line.

64. The method of claim 60, further comprising sequentially stimulating said T-cells with EMSP either by previously purifying T-cells from originally added EMSP and subsequently adding additional EMSP or by adding additional EMSP to previously stimulated cells without separation of originally added EMSP.

65. The method of claim 60, further comprising separating said T-cells from said EMSP and subsequently incubating said T-cells with an agent that facilitates T-cell expansion, followed by restimulation with EMSP.

66. The method of any one of claims 60-65, wherein the T-cell is a T-cell line.

67. The method of any one of claims 60-65, wherein said T-cell is a T-cell clone.

68. The method of any one of claims 60-67, wherein said EMSP comprises a cell displaying a ligand for any one of CD3, CD28, or 4-1BB.

69. The method of claim 60, wherein following initial stimulation or activation said T-cells are separated from EMSP and then expanded in the presence of exogenously added cytokines.

70. The method of claim 69, wherein said T-cells are substantially free of EMSP prior to expansion.

72. A method of immunotherapy comprising administering an EMSP to a subject in need thereof, thereby stimulating a population of T-cells within the subject to expand.

73. A method of enhancing reactivity to an antigen comprising administering an EMSP to a subject in need thereof concurrently, prior to or following inoculation of the subject with an antigen of interest, thereby stimulating a population of antigen specific T-cells within the subject to expand.

74. A method for breaking immune tolerance comprising administering an EMSP to a subject either systemically or locally at a site of interest.

75. A method for increasing uptake of an exogenously added nucleic acid molecule in T-cells, comprising contacting said T-cells with an EMSP and contacting said T-cells with said nucleic acid molecule thereby, said contacting of EMSP with said T-cells rendering cells more amenable to uptake of nucleic acid.

76. The method of claim 75, wherein said exogenously added nucleic acid is operably linked to a promoter

77. The method of claim 76, wherein said nucleic acid molecule provides gene replacement for abnormal gene product.

79. A method of activating antigen specific T-cells, comprising contacting a population of T-cells an antigen and an EMSP under conditions and for time sufficient to induce activation of T-cells specific to said antigen.

80. The method of claim 79, wherein said activation occurs ex vivo after which the T-cells are administered to a patient substantially free or in combination with said EMSP.

81. The method of claim 79, wherein said antigen is displayed by said EMSP.

82. The method of claim 81, wherein said display is by way of a fusion protein.

83. The method of claim 79, wherein said antigen is in the form of a complex with MHC derived peptides.