Th1 cell adoptive immunotherapy

• US 20030194395A1
• Filed: 09/17/2002
• Published: 10/16/2003
• Est. Priority Date: 09/17/2001
• Status: Abandoned Application

• Current Assignee: Medcell Biologics, LLC
• Original Assignee: Medcell Biologics, LLC
• Inventors: Chavan, Surenda, Gruenberg, Micheal L.
• Application Number:
  US10/246,646
• Publication Number:
  US 20030194395A1
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/57 characterised by the type of response, e.g. Th1, Th2
• 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 39/4644 Cancer antigens
• 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 5/0636 T lymphocytes

Abstract

Methods for consistently producing a pure population of activated, polyclonal, Th1 memory cells for use in adoptive immunotherapy without the use of any exogenous cytokines and without significant subject-to-subject variation are provided. The resulting cells obtain a surface phenotype that enables their trafficking to tumors and other sites of inflammation upon infusion. The cells can be reinfused into the a subject to enhance the cellular immune response and/or switch the predominant immune response from Th2-dominated to a Th1-dominated immune response.

Claims

1. A method for producing a highly pure population of Th1 cells, comprising:

purifying T-cells from the source material; and

activating the T-cells a plurality of times, whereby a highly pure population of Th1 cells is produced.

2. The method of claim 1, wherein:

the source material is collected from a subject;

the T-cells are activated at least minimum of 3 times at 2-4 day intervals to produce a highly pure population of polyclonal Th1 memory cells.

3. The method of claim 2, wherein the T-cells are activated 3 to 5 times at 2-4 day intervals.

4. The method of claim 1, wherein the T-cells are purified CD4+ cells.

5. The method of claim 4, wherein the CD4+ cells are purified by positive selection.

6. The method of claim 5 wherein the CD4+ cells are purged of CD45RO+ cells.

7. The method of claim 1, wherein the source material is purged of platelets.

8. The method of claim 6, wherein the source material is purged of platelets.

9. The method of claim 1, wherein the source material is purged of monocytes.

10. The method of claim 7, wherein the source material is purged of monocytes.

11. The method of claim 1, wherein the activation of T-cells is effected by contacting the cells with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs).

12. The method of claim 10, wherein the anti-CD3 and anti-CD28 mAbs are immobilized.

13. The method of claim 12, wherein the mAbs are immobilized on particulate supports.

14. The method of claim 12, wherein particulate supports are immunomagnetic beads.

15. The method of claim 12, wherein the Mabs are immobilized on colloidal size paramagnetic beads during at least the last activation step.

16. The method of claim 11, wherein the mAbs are immobilized on colloidal size paramagnetic beads during at least two activation steps.

17. The method of claim 11, wherein the beads are initially administered to the purified T-cells at a 3:

1 particulate;

cell ratio and subsequently at a 1;

1 particulate;

cell ratio.

18. The method of claim 1, wherein the source material comprises mononuclear cells.

19. The method of claim 2, wherein the subject is a human cancer patient.

20. The method of claim 19, wherein the cancer is selected from the group consisting of liver, kidney, breast, prostate, melanoma, colon, lymphoma, lung, pancreatic, ovarian, esophageal, head and neck, brain, uterine and stomach cancer.

21. The method of claim 1, wherein the Th1 cells are activated.

22. The method of claim 21, wherein the population comprises at least 10⁹ Th1 cells.

23. The method of claim 22, wherein the 10⁹ cells are in a volume of about 1 liter or less.

66. A composition of polyclonal Th1 memory cells produced by the method of claim 1.

24. A method comprising:

(a) collecting a sample of mononuclear cells from a subject with a disease characterized by either an excess of Th2 cytokine activity or low Th1 cytokine activity; and

(b) processing the mononuclear cells ex vivo without the use of any exogenous cytokines to produce an expanded population of highly pure Th1 memory cells.

25. The method of claim 24, further comprising:

(c) infusing the Th1 memory cells into a subject, thereby altering the Th1/Th2 cell balance of the subject.

26. The method of claim 25, wherein the subject is the donor.

27. The method of claim 24, wherein the expanded population comprises at least 10⁹ Th1 cells.

28. The method of claim 27, wherein the 10⁹ cells are in a volume of about 1 liter or less.

29. The method of claim 24, wherein the subject is a human cancer patient.

30. The method of claim 24, wherein the disease is selected from the group consisting of diseases characterized by suppression of the cellular immune response or by over-expression of the humoral immune response.

31. The method of claim 24, wherein the disease is selected from the group consisting of cancer, infectious diseases, autoimmune and allergic diseases.

32. The method of claim 24, wherein processing is effected by a method, comprising:

purifying CD3+ cells from the mononuclear cells.

33. The method of claim 24, wherein processing is effected by a method, comprising purifying CD3+ CD4+ cells from the mononuclear cells.

34. The method of claim 24, wherein processing is effected by a method, comprising purifying CD3+, CD4+, CD45RA+ cells from the mononuclear cells.

35. The method of claim 24, wherein processing is effected by a method, comprising:

(i) reducing the platelet concentration in the sample;

(ii) purging the CD45RO+ cells from the population of mononuclear cells;

(iii) purifying by positive selection a population of CD4+, CD45RA+ cells;

(iv) activating the resulting CD4+ cells in the absence of exogenous cytokines with immobilized anti-CD3/anti-CD28 mAbs;

(v) periodically restimulating with immobilized anti-CD3/anti-CD28 mAbs.

36. The method of claim 35, wherein the cells are restimulated every 2-3 days with immobilized anti-CD3/anti-CD28 mAb for a total of 10-14 days.

37. The method of claim 36, further comprising:

(c) infusing the Th1 memory cells into a subject, thereby altering the Th1/Th2 cell balance of the subject.

38. The method of claim 37, wherein the subject is the donor.

39. The method of claim 37, wherein the expanded population comprises at least 10⁹ Th1 cells.

40. The method of claim 39, wherein the 10⁹ cells are in a volume of about 1 liter or less.

41. The method of claim 24, wherein processing is effected by a method, comprising:

(i) reducing the number of platelets in the sample;

(ii) purging macrophages from the sample;

(iii) purging the CD45RO+ cells from the sample

(iv) purifying by positive selection a population of CD4+, CD45RA+ cells;

(v) activating the CD4+ cells in the absence of exogenous cytokines with immobilized anti-CD3/anti-CD28 mAb; and

(vi) periodically restimulating with immobilized anti-CD3/anti-CD28 mAb.

42. The method of claim 41, wherein the cells are restimulated every 2-3 days with immobilized anti-CD3/anti-CD28 mAb for a total of 10-14 days.

43. The method of claim 41, further comprising:

(c) infusing the Th1 memory cells into a subject, thereby altering the Th1/Th2 cell balance of the subject.

44. The method of claim 41, wherein the subject is the donor.

45. The method of any of claims 41-44, wherein the expanded population comprises at least 10⁹ Th1 cells.

46. The method of claim 45, wherein the, 10⁹ cells are in a volume of about 1 liter or less.

74. The method of claim 35, wherein the mAbs are immobilized on particulate supports.

75. The method of claim 74, wherein particulate supports are immunomagnetic beads.

76. The method of claim 74, wherein particulate supports are colloidal sized paramagnetic beads.

77. The method of claim 36, wherein the mAbs are immobilized on colloidal size paramagnetic beads.

81. The method of claim 24, wherein the Th1 cells are activated.

82. The method of claim 25, wherein the Th1 cells are activated.

47. A composition, comprising at least about 90% activated Th1 cells.

48. The composition of claim 47, comprising at least about 95% Th1 cells.

49. The composition of claim 47, wherein the cells are polyclonal memory Th1 cells.

50. A method of treating a disease, comprising:

infusing a composition of claim 47 into a subject with symptoms of a disease, wherein;

the disease is characterized by suppression of the cellular immune response, by over-expression of the humoral immune response, excess Th2 activity or a lack or decreased Th1 activity.

51. The method of claim 50, wherein the disease is selected from the group consisting of cancer, infectious diseases and allergic diseases.

52. A method of treating a disease, comprising:

infusing a composition of claim 47 into a subject with symptoms of disease, wherein;

the disease is characterized by suppression of the cellular immune response, by over-expression of the humoral immune response, excess Th2 activity or a lack or decreased Th1 activity.

53. The method of claim 52, wherein the disease is selected from the group consisting of cancer, infectious diseases and allergic diseases.

68. A combination, comprising a composition of claim 47 and an immunizing antigen.

54. A process for producing compositions comprising at least 70% Th1 cells, comprising:

(a) collecting a sample of mononuclear cells from a subject with a disease characterized by either an excess of Th2 cytokine activity or lack of Th1 cytokine activity;

(b) removing platelets from the sample;

(c) removing macrophages from the sample;

(c) depleting CD45RO+ cells from the sample by negative selection;

(d) selecting the CD4+ cells by positive selection; and

(e) expanding and differentiating the selected CD4+ cells by repeatedly stimulating the selected CD4+ cells with immobilized anti-CD3/anti-CD28 antibodies.

55. The method of claim 54, wherein the polyclonal Th1 memory cells are activated.

56. The method of claim 54, wherein the disease is selected from the group consisting of diseases characterized by excess Th2 activity or a lack or decreased Th1 activity.

69. The method of claim 54, wherein:

at least 10¹⁰ cells are produced; and

at least 70% are positive for internal interferon-γ

.

70. The method of claim 69, wherein the cells are in a volume of about 1 liter or less.

71. The method of claim 69, wherein the density of cells is at least 10¹¹ cells/liter.

57. A process for producing compositions that have an enhanced population of activated polyclonal Th1 memory cells, comprising:

(a) collecting a sample of mononuclear cells from a subject;

(b) expanding and differentiating the mononuclear cells by repeatedly activating T-cells in the mononuclear cell sample in the absence of exogenous growth or differentiation factors, thereby producing a highly pure population of activated polyclonal Th1 memory cells.

58. The method of claim 57, wherein prior to expanding an differentiating the T-cells are purified from the mononuclear cells.

59. The method of claim 58, wherein the T-cells purified from the mononuclear cells are selected from the group consisting of CD3+ cells, CD4+ cells, CD4+, CD45RA+ cells and CD4+, CD45RO+ cells.

67. A composition of activated polyclonal Th1 memory cells produced by the method of claim 57.

60. A method for expanding T-cells from cancer patients without the use of exogenous cytokines, comprising:

(a) collecting a mononuclear cell sample from a cancer patient;

(b) purging platelets from the mononuclear cells; and

(c) activating the cells with immobilized anti-CD3/anti-CD28 mAbs, wherein all steps are performed in the absence of exogenous cytokines.

61. The method of claim 60, wherein the disease is cancer.

62. The method of claim 61, wherein the disease is selected from the group consisting of liver, kidney, breast, prostate, melanoma, colon, lymphoma, lung, pancreatic, ovarian, esophageal, head and neck, brain, uterine and stomach cancer.

78. The method of claim 60, wherein the mAbs are immobilized on particulate supports.

79. The method of claim 78, wherein particulate supports are immunomagnetic beads.

80. The method of claim 78, wherein the particulate supports are colloidal size paramagnetic beads.

63. A composition of cells, comprising at least about 10⁹ cells, wherein at least about 70% of the cells are polyclonal Th1 memory cells.

64. The composition of claim 63, wherein the Th1 cells are activated.

65. The composition of claim 63 that is in a volume of about a liter or less.

72. A highly pure population of Th1 cells, wherein at least 70% are positive for internal interferon-γ

and the cells are at a density of at least 10¹⁰ cells/liter.

73. The cells of claim 72, wherein the density of cells is at least 10¹¹ cells/liter.

83. A composition, comprising at least 70% polyclonal memory Th1 cells.

84. The composition of claim 83, comprising at least 10⁹ Th1 memory cells.

85. The composition of claim 84, wherein the Th1 cells are CD3+, CD4+, CD45RO+, CD62L−

, CD44+ and CD25+.

86. The composition of claim 84 that has density of cells greater than about 10⁶ cells per ml or 10⁷ cells per mol or 10⁸ cells per ml.