A METHOD FOR OBTAINING NATURAL KILLER CELLS FROM PLURIPOTENT STEM CELLS

Claims

1. A method for obtaining a cell population enriched in NK cells, wherein the method comprises the following steps: (A) culturing the pluripotent stem cell population under a first set of conditions resulting in a cell population comprising at least 20% CD34+ HPC (total HPC mass); and (B) changing the first set of conditions to a second set of conditions, thereby obtaining a cell population comprising at least 5% CD4 cells; and (C) changing the second set of conditions to a third set of conditions, thereby obtaining a cell population enriched in NK cells.

2. The method according to paragraph 1, wherein the method is carried out without the isolation step.

3. The method of claim 1, comprising isolating CD4 cells after step (B) before contacting the isolated cells with a third set of conditions in step (C).

4. The method of claim 3, wherein isolating CD4 cells comprises removing CD4+ cells from the cell population.

5. A method for obtaining a cell population enriched in NK cells, wherein the method comprises the following steps: (A) culturing the pluripotent stem cell population under a first set of conditions resulting in a cell population comprising at least 20% CD34+ HPC (total HPC mass); and (B) changing the first set of conditions to a second set of conditions, thereby obtaining a cell population comprising at least 5% CD4 cells; and (C) changing the second set of conditions to a third set of conditions, thereby obtaining a cell population enriched in NK cells, wherein the method is carried out without an isolation step.

6. The method according to any one of the preceding claims, wherein the population of cells comprising at least 5% CD4- cells further comprises at least 5% CD4+ cells.

7. The method according to any one of the preceding claims, wherein the NK cell-enriched cell population comprises at least 30% NK cells.

8. The method according to any one of the preceding claims, wherein the cell population obtained in step (B) comprises at least 10%, 15% or 20% CD4 cells.

9. The method according to any one of the preceding claims, wherein the CD4- cells in step (B) are CD8+ cells.

10. The method according to any one of claims 1 to 8, wherein the CD4 cells in step (B) are CD8 cells.

11. The method according to any one of claims 1-8, wherein the cell population obtained in step (B) contains from 20 to 55% CD4- / CD8+ cells.

12. The method according to claim 11, wherein the resulting cell population contains from 25 to 55% CD4- / CD8- cells.

13. The method according to claim 1, wherein the NK cells are CD56+ / CD3- cells.

14. The method according to any one of the preceding claims, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

15. The method according to any one of the preceding claims, wherein the first set of conditions comprises a culture medium containing at least one compound selected from bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), ascorbic acid, Flt3 ligand (Flt3L), thrombopoietin (TPO), and a TGFβ inhibitor.

16. The method of claim 15, wherein the first set of conditions comprises a culture medium containing BMP4 at a concentration of 5 ng / ml - 500 ng / ml.

17. The method according to claim 16, wherein BMP4 is at a concentration of 50 ng / ml.

18. The method according to any one of claims 15-17, wherein the first set of conditions comprises a culture medium containing VEGF at a concentration of 5 ng / ml - 500 ng / ml.

19. The method according to claim 18, wherein the VEGF is at a concentration of about 50 ng / ml.

20. The method according to any one of claims 15-19, wherein the first set of conditions comprises a culture medium containing bFGF at a concentration of 5 ng / ml - 500 ng / ml.

21. The method according to claim 20, wherein the bFGF is at a concentration of 50 ng / ml.

22. The method according to any one of paragraphs 15-21, wherein the first set of conditions includes a culture medium containing ascorbic acid at a concentration of 5 μg / ml - 500 μg / ml.

23. The method according to claim 22, wherein the ascorbic acid is at a concentration of 50 μg / ml.

24. The method according to any one of claims 15-23, wherein the first set of conditions comprises a culture medium containing Flt3L at a concentration of 1 ng / ml - 100 ng / ml.

25. The method according to claim 24, wherein Flt3L is at a concentration of 50 ng / ml.

26. The method according to any one of paragraphs 15-25, wherein the first set of conditions includes a culture medium containing TPO at a concentration of 1 ng / ml - 200 ng / ml.

27. The method according to claim 26, wherein the TPO is at a concentration of 100 ng / ml.

28. The method according to any one of the preceding claims, wherein the second set of conditions comprises a culture medium containing at least one compound selected from the group consisting of ascorbic acid, stem cell factor (SCF), IL-7, Flt3L, thrombopoietin (TPO), p38 inhibitor and SDF-1.

29. The method according to claim 28, wherein the second set of conditions includes a culture medium containing ascorbic acid at a concentration of 5 μg / ml 500 μg / ml.

30. The method according to claim 29, wherein the ascorbic acid is at a concentration of 50 μg / ml.

31. The method according to any one of claims 28-30, wherein the second set of conditions comprises a culture medium containing SCF at a concentration of 5 ng / ml - 100 ng / ml.

32. The method according to claim 31, wherein the SCF is at a concentration of 50 ng / ml.

33. The method according to any one of claims 28-32, wherein the second set of conditions comprises a culture medium containing IL-7 at a concentration of from 1 ng / ml to 100 ng / ml.

34. The method according to claim 33, wherein IL-7 is at a concentration of 50 ng / ml.

35. The method according to any one of claims 28-34, wherein the second set of conditions comprises a culture medium containing Flt3L at a concentration of 1 ng / ml - 100 ng / ml.

36. The method according to claim 35, wherein Flt3L is at a concentration of 50 ng / ml.

37. The method according to any one of paragraphs 28-36, wherein the second set of conditions includes a culture medium containing TPO at a concentration of 1 ng / ml - 200 ng / ml.

38. The method according to claim 37, wherein the TPO is at a concentration of 100 ng / ml.

39. The method according to any one of claims 28-38, wherein the second set of conditions comprises a culture medium containing a p38 inhibitor at a concentration of from 0.5 μM to 100 μM.

40. The method of claim 39, wherein the p38 inhibitor is SB203580.

41. The method according to claim 40, wherein SB203580 is at a concentration of 15 μM.

42. The method according to any one of paragraphs 28-41, wherein the second set of conditions comprises a culture medium containing SDF-1 at a concentration of 10 ng / ml - 100 ng / ml.

43. The method according to claim 42, wherein SDF-1 is at a concentration of 30 nM.

44. The method according to any one of the preceding claims, wherein the third set of conditions comprises a culture medium containing at least one compound selected from the group consisting of a CD3 activator, IL-2 and IL-7.

45. The method of claim 44, wherein the third set of conditions includes IL-2 at a concentration of 1 ng / ml - 100 ng / ml.

46. ​​The method according to claim 45, wherein IL-2 is at a concentration of 10 ng / ml.

47. The method according to any one of paragraphs 44-46, wherein the third set of conditions comprises a culture medium containing IL-7 at a concentration of 1 ng / ml - 100 ng / ml.

48. The method according to claim 47, wherein IL-7 is at a concentration of 10 ng / ml.

49. The method of any one of the preceding claims, wherein each of the culturing steps is carried out at an oxygen level of about 5%.

50. The method according to any one of paragraphs 1-48, wherein each of the cultivation stages is carried out at an oxygen level of more than 14%.

51. The method according to any one of paragraphs 1-48, wherein each of the cultivation stages is carried out at atmospheric oxygen levels.

52. The method according to any one of paragraphs 1-48, wherein each of the cultivation steps is carried out at an oxygen level of less than 5%.

53. The method according to any one of the preceding claims, wherein the culturing of pluripotent stem cells under the first set of conditions to obtain a total mass of pluripotent stem cells lasts for more than 10 days.

54. The method according to claim 53, wherein the cultivation of pluripotent stem cells in the first set of conditions for obtaining the total mass of pluripotent stem cells lasts from 11 to 15 days.

55. The method according to claim 54, wherein the cultivation of pluripotent stem cells in the first set of conditions to obtain a total mass of NPCs lasts for 14 days.

56. The method according to any one of the preceding claims, wherein the iPSCs are obtained from peripheral blood mononuclear cells.

57. The method according to any one of the preceding claims, wherein at least about 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 97% or more of the cells obtained are CD56+ / CD3- NK cells without an enrichment step.

58. The method of claim 57, wherein less than about 25% of the cells obtained are CD3+ cells.

59. The method according to claim 57 or 58, wherein the percentage of cells obtained is determined using flow cytometry.

60. The method according to claim 57 or 58, wherein the percentage of cells obtained is determined using single cell RNA sequencing (scRNAseq).

61. The method according to any one of the preceding claims, further comprising isolating CD56+ / CD3- cells.

62. The method according to any one of claims 3, 4 and 61, wherein the isolation comprises fluorescence-activated cell sorting (FACS) or magnetic sorting.

63. The method according to any one of the preceding claims, wherein the NK cells are genetically modified to express one or more chimeric antigen receptors (CARs).

64. The method of claim 63, wherein the antigen is CD19.

65. The method according to claim 63 or 64, wherein the NK cells are further genetically modified to express the IL-15Rα / IL-15 complex.

66. A method for producing NK cells derived from induced pluripotent stem cells (iPSCs), comprising the following steps: (1) culturing iPSCs in a first set of conditions comprising at least one compound selected from vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and ascorbic acid to obtain a population comprising at least 20% CD34+ HPCs (total HPC mass); (2) culturing the bulk of HPC obtained in (1) under a second set of conditions comprising a culture medium containing one or more of ascorbic acid, a p38 inhibitor, and SDF-1 to obtain a cell population comprising at least 5% CD4 cells; and (3) culturing the population from (2) under a third set of conditions comprising a culture medium containing at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL-7.

67. A method for producing NK cells derived from induced pluripotent stem cells (iPSCs), comprising the following steps: (1) culturing a total population of cells containing hematopoietic progenitor cells (total HPCs) under a second set of conditions comprising a culture medium containing one or more of ascorbic acid, a p38 inhibitor, and SDF-1 to obtain a population of cells containing at least 5% CD4 cells; (2) isolation of CD4 cells from (1); and (3) culturing the isolated CD4 cells in an NK induction medium containing at least one compound selected from the group consisting of a CD3 activator, IL-2, and IL-7.

68. The method of claim 66 or 67, wherein about 15-30% of the cells in the cell population obtained in step (1) are CD4 cells.

69. The method of claim 68, wherein about 20% of the cells in the cell population obtained in step (1) are CD4 cells.

70. The method according to any one of the preceding claims, wherein the population of cells comprising CD4- cells comprises CD4- / CD8- cells and CD4- / CD8+ cells.

71. A population of NK cells obtained using the method of any of the preceding claims.

72. The NK cell population according to claim 71, wherein the NK cell population is cryopreserved in a cryopreservation medium.

73. A population of unsorted cells containing pluripotent stem cell-derived CD56+ / CD3- cells in a ratio of at least 60% of the total number of pluripotent stem cell-derived CD56+ cells.

74. The unsorted cell population of claim 73, wherein less than 25% of the cells in the unsorted cell population are CD3+ cells.

75. The unsorted cell population of claim 73 or 74, wherein less than 5% of the cells in the unsorted cell population are monocytes.

76. The unsorted cell population of claim 73 or 74, wherein less than 5% of the cells in the unsorted cell population are B cells.

77. The unsorted cell population of any one of claims 73-76, wherein the percentage of cells in the unsorted cell population is determined using flow cytometry.

78. The unsorted cell population according to paragraphs 73-76, wherein the percentage of cells is determined using single-cell RNA sequencing (scRNAseq).

79. A method of treating a subject in need of cell therapy, comprising administering to the subject NK cells according to any one of the preceding claims.

80. The method of claim 79, wherein the subject has cancer.

81. The method according to claim 80, wherein the cancer is leukemia or lymphoma.

82. A method for obtaining a population of cells containing CD34+ hematopoietic progenitor cells (HPCs), comprising culturing a population of pluripotent stem cells in a first set of conditions comprising a medium containing at least one compound selected from vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and ascorbic acid, to obtain a population containing CD34+ HPCs (total HPC mass).

83. A population of cells containing NPCs obtained by the method according to paragraph 82.

84. The cell population of claim 83, wherein the cell population comprises at least 20% CD34+ cells.