received by the International Bureau on 13 December 2018 ( 13. 12.20 18)

CLAIMS

1. A method of targeting a cancer stem cell, comprising:

providing an antibody having binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state;

providing a genetically modified natural killer (NK) cell that expresses a CD 16

receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions; and

contacting the cancer stem cell with the antibody and the genetically modified NK cell to allow antibody-mediated binding of the genetically modified NK cell to the cancer stem cell.

2. The method of claim 1 wherein the antigen is at least one of calreticulin, PD-L1, and c- MET.

3. The method of any one of the preceding claims wherein the antibody is a human

antibody.

4. The method of any one of the preceding claims wherein the antibody is a bi-spccific antibody having binding specificity against at least two of calreticulin, PD-L1, and c- MET.

5. The method of any one of the preceding claims wherein the genetically modified NK cell is a genetically modified NK92 cell.

6. The method of any one of the preceding claims wherein the genetically modified NK cell is genetically modified to express at least one of a high affinity variant CD 16 and endoplasmic restricted IL-2.

7. The method of any one of the preceding claims wherein the tumor stem cell is from a solid tumor.

8. The method of any one of the preceding claims wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is performed while the cancer stem cell is within a tumor microenvironment.

9. The method of any one of the preceding claims wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is performed while the cancer stem cell is under hypoxic conditions.

10. The method of any one of the preceding claims wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is administration in vivo to a patient in which the cancer stem cell is within a tumor micro environment.

11. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment an immune stimulating cytokine.

12. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment an IL-15, an IL-15 superagonist, and/or an IL-15 superagonist hybrid comprising a chemokine or chemokine portion.

13. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment a chemokine that attracts at least one of a T cell and an NK cell.

14. The method of any one of the preceding claims further comprising a step of administering CXCL14 to the cancer stem cell or a tumor microenvironment.

15. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment additional oxygen, optionally via oxygen hyperbaric treatment.

16. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment a radiosensitizing drug, optionally via coupling of the drug to albumin.

17. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or tumor microenvironment a CD47 antagonist or a SHPS-1 antagonist.

18. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or tumor microenvironment ah agent that up-regulates surface expression of calreticulrn, optionally an anthracycline or thapsigargin.

19. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or tumor microenvironment an antibody or fragment thereof that binds lo the antigen and that further comprises an alpha or beta radioisotope.

20. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or tumor microenvironment an agent that down-regulates suppressor cells, optionally gemcitabine, cis-platinum, or cyclophosphamide.

21. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment a peptide that down-regulates or kills M2 macrophages, optionally a RP-182 or an antibody against B7-H4.

22. The method of any one of the preceding claims further comprising a step of administering to the cancer stem cell or a tumor microenvironment an IL-8 antibody, an IL-8 antagonist, a CXCR1 inhibitor, and/or a CXCR2 inhibitor.

23. A method of treating a cancer, comprising:

co-administering to a patient having the cancer an antibody and a genetically modified natural killer (NK) cell;

wherein the antibody has binding specificity towards an antigen that is specific to a mesenchymal state of a tumor stem cell;

wherein the genetically modified NK cell expresses a CD 16 receptor and has

granulysin and granzyme mediated cytotoxic activity under hypoxic conditions; and

wherein the antibody and the genetically modified NK cell are administered to the patient to allow antibody-mediated binding of the genetically modified NK cell to the cancer stem cell in a tumor microenvironment.

24. The method of claim 23 wherein the antigen is at least one of calreticulin, PD-L1, and c- MET.

25. The method of any one of claims 23-24 wherein the antibody is a human antibody.

26. The method of any one of claims 23-25 wherein the antibody is a bi-specific antibody having binding specificity against at least two of calreticulin, PD-L1, and c-MET.

27. The method of any one of claims 23-26 wherein the genetically modified NK cell is a genetically modified NK92 cell.

28. The method of any one of claims 23-27 wherein the genetically modified NK cell is genetically modified to express at least one of a high affinity variant CD 16 and endoplasmic restricted IL-2.

29. The method of any one of claims 23-28 wherein the tumor stem cell is in a solid tumor

30. The method of any one of claims 23-29 wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is performed while the cancer stem cell is within a tumor microenvironment.

31. The method of any one of claims 23-30 wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK. cell is performed while the cancer stem cell is under hypoxic conditions.

32. The method of any one of claims 23-31 wherein the step of co-administering is performed with the antibody bound to the genetically modified NK cell.

33. The method of any one of claims 23-32 further comprising a step of admmistering to the patient or tumor microenvironment an immune stimulating cytokine.

34. The method of any one of claims 23-33 further comprising a step of administering to the patient or tumor microenvironment an IL-15, an IL-15 superagonist, and/or an IL-15 superagonist hybrid comprising a chemokine or chemokine portion.

35. The method of any one of claims 23-34 further comprising a step of administering to the patient or tumor microenvironment a chemokine that attracts at least one of a T cell and an NK cell.

36. The method of any one of claims 23-35 further comprising a step of administering

CXCL14 to the patient or tumor microenvironment.

37. The method of any one of claims 23-36 further comprising a step of administering to the patient or tumor microenvironment additional oxygen, optionally via oxygen hyperbaric treatment.

38. The method of any one of claims 23-37 further comprising a step of administering to the patient or tumor microenvironment a radiosensitizing drug, optionally via coupling of the drug to albumin.

39. The method of any one of claims 23-38 further comprising a step of administering to the patient or tumor microenvironment a CD47 antagonist or a SHPS-1 antagonist.

40. The method of any one of claims 23-39 further comprising a step of administering to the patient or tumor microenvironment an agent that up-regulates surface expression of calreticulin, optionally an anthracycline or thapsigargin.

41. The method of any one of claims 23-40 further comprising a step of administering to the patient or tumor microenvironment an antibody or fragment thereof that binds to the antigen and that further comprises an alpha or beta radioisotope.

42. The method of any one of claims 23-41 further comprising a step of administering to the patient or tumor microenvironment an agent that down-regulates suppressor cells, optionally gemcitabine, cis-platinum, or cyclophosphamide.

43. The method of any one of claims 23-42 further comprising a step of administering to the patient or tumor microenvironment a peptide that down-regulates or kills M2

macrophages, optionally a RP-182 or an antibody against B7-H4.

44. The method of any one of claims 23-43 further comprising a step of administering to the patient or tumor microenvironment an IL-8 antibody, an IL-8 antagonist, a CXCR1 inhibitor, and/or a CXCR2 inhibitor.

45. A method of targeting a cancer cell in a hypoxic environment in which the cancer cell has reduced cell division and/or activity in an apoptotic pathway, comprising:

identifying the cancer cell as expressing an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor cell;

providing an antibody having binding specificity towards the antigen;

providing a genetically modified natural killer (NK) cell that expresses a CD 16

receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions; and contacting the cancer stem cell with the antibody and the genetically modified NK cell to allow antibody-mediated binding of the genetically modified NK cell to the cancer stem cell.

46. The method of claim 45 wherein the antigen is at least one of calreticulin, PD-L1, and c- MET.

47. The method of any one of claims 45-46 wherein the antibody is a human antibody_^

48. The method of any one of claims 45-47 wherein the antibody is a bi-specific antibody having binding specificity against at least two of calreticulin, PD-L1, and c-MET.

49. The method of any one of claims 45-48 wherein the genetically modified NK cell is a genetically modified NK92 cell.

50. The method of any one of claims 45-49 wherein the genetically modified NK cell is

genetically modified to express at least one of a high affinity variant CD16 and endoplasmic restricted IL-2.

51. The method of any one of claims 45-50 wherein the tumor stem cell is from a solid tumor,

52. The method of any one of claims 45-51 wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is performed while the cancer stem cell is within a tumor micro environment.

53. The method of any one of claims 45-52 wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is performed while the cancer stem cell is under hypoxic conditions.

54. The method of any one of claims 45-53 wherein the step of contacting the cancer stem cell with the antibody and the genetically modified NK cell is administration in vivo to a patient in which the cancer stem cell is within a tumor microenvironment.

55. The method of any one of claims 45-54 further comprising a step of adrnmistering to the cancer stem cell or a tumor microenvironment an immune stimulating cytokine.

56. The method of any one of claims 45-55 further comprising a step of administering to the cancer stem cell or a tumor microenvironment an IL-15, an IL-15 superagonist, and/or an IL-15 superagonist hybrid comprising a chemokine or chemokine portion.

57. The method of any one of claims 45-56 further comprising a step of administering to the cancer stem cell or a tumor microenvironment a chemokine that attracts at least one of a T cell and an NK cell.

58. The method of any one of claims 45-57 further comprising a step of administering

CXCL14to the cancer stem cell or a tumor microenvironment.

59. The method of any one of claims 45-58 further comprising a step of administering to the cancer stem cell or a tumor microenvironment additional oxygen, optionally via oxygen hyperbaric treatment.

60. The method of any one of claims 45-59 further comprising a step of administering to the cancer stem cell or a tumor microenvironment a radiosensitizing drug, optionally via coupling of the drug to albumin.

61. The method of any one of claims 45-60 further comprising a step of administering to the cancer stem cell or tumor microenvironment a CD47 antagonist or a SHPS-1 antagonist.

62. The method of any one of claims 45-61 further comprising a step of administering to the cancer stem cell or tumor microenvironment an agent that up-regulates surface expression of calreticulin, optionally an anthracycline or thapsigargin.

63. The method of any one of claims 45-62 further comprising a step of administering to the cancer stem cell or tumor microenvironment an antibody or fragment thereof that binds to the antigen and that further comprises an alpha or beta radioisotope.

64. The method of any one of claims 45-63 further comprising a step of administering to the cancer stem cell or tumor microenvironment an agent that down-regulates suppressor cells, optionally gemcitabine, cis-platinum, or cyclophosphamide.

65. The method of any one of claims 45-64 further comprising a step of administering to the cancer stem cell or a tumor microenvironment a peptide that down-regulates or kills M2 macrophages, optionally a RP-182 or an antibody against B7-H4.

66. The method of any one of claims 45-65 further comprising a step of administering to the cancer stem cell or a tumor microenvironment an IL-8 antibody, an IL-8 antagonist, a CXCR1 inhibitor, and/or a CXCR2 inhibitor.

67. Use of (i) an antibody having binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor stem cell and (ii) a genetically modified natural killer (NK) cell that expresses a CD 16 receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions to target a cancer stem cell.

68. Use of (i) an antibody having binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor stem cell and (ii) a genetically modified natural killer (NK) cell that expresses a CD 16 receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions to treat a cancer.

69. Use of (i) an antibody having binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor stem cell and (ii) a genetically modified natural killer (NK) cell that expresses a CD 16 receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions to target a cancer cell in a hypoxic environment in which the cancer cell has reduced cell division and/or activity in an apoptotic pathway.

70. The use of any one of claims 67-79 wherein the antigen is at least one of calreticulin, PD- Ll, and c-MET.

71. The use of any one of claims 67-70 wherein the antibody is a human antibody.

72. The use of any one of claims 67-71 wherein the antibody is a bi-specific antibody having binding specificity against at least two of calreticulin, PD-L1 , and c-MET.

73. The use of any one of claims 67-72 wherein the genetically modified NK cell is a

genetically modified NK92 cell.

74. The use of any one of claims 67-73 wherein the genetically modified NK cell is genetically modified to express at least one of a high affinity variant CD 16 and endoplasmic restricted IL-2,

75. The use of any one of claims 67-74 wherein the tumor stem cell is from a solid tumor.

76. The use of any one of claims 67-75 wherein the cell is contacted with the antibody and the genetically modified NK cell while the cell is within a tumor microenvironment.

77. The use of any one of claims 67-76 wherein the cell is contacted with the antibody and the genetically modified NK. cell while the cancer stem cell is under hypoxic conditions.

78. The use of any one of claims 67-77 further comprising use of an immune stimulating cytokine.

79. The use of any one of claims 67-78 further comprising use of IL-15, an IL-15

superagonist, and/or an IL-15 superagonist hybrid comprising a chemokine or chemokine portion.

80. The use of any one of claims 67-79 further comprising use of a chemokine that attracts at least one of a T cell and an NK cell.

81. The use of any one of claims 67-80 further comprising use of a CXCL14.

82. The use of any one of claims 67-81 further comprising use of additional oxygen,

optionally via oxygen hyperbaric treatment.

83. The use of any one of claims 67-82 further comprising use of a radiosensitizing drug, optionally via coupling of the drug to albumin.

84. The use of any one of claims 67-83 further comprising use of a CD47 antagonist or a SHPS-1 antagonist.

85. The use of any one of claims 67-84 further comprising use of an agent that up-regulates surface expression of calreticulin, optionally an anthracycline or thapsigargin.

86. The use of any one of claims 67-85 further comprising use of an antibody or fragment thereof that binds to the antigen and that further comprises an alpha or beta radioisotope.

87. The use of any one of claims 67-86 further comprising use of an agent that down- regulates suppressor cells, optionally gemcitabine, cis-platinum, or cyclophosphamide.

88. The use of any one of claims 67-87 further comprising use of a peptide that down- regulates or kills M2 macrophages, optionally a RP- 182 or an antibody against B7-H4.

89. The use of any one of claims 67-88 further comprising use of an IL-8 antibody, an IL-8 antagonist, a CXCR1 inhibitor, and/or a CXCR2 inhibitor.

90. A genetically modified natural killer (NK) cell that expresses a CD 16 receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions, wherein the cell further comprises an antibody bound to the CD 16 receptor, and wherein the antibody has binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor cell.

91. The genetically modified NK cell of claim 90 wherein the antigen is at least one of

calreticulin, PD-L1, and c-MET.

92. The genetically modified NK cell of any one of claims 90-91 wherein the antibody is a human antibody.

93. The genetically modified NK cell of any one of claims 90-92 wherein the antibody is a bi- specific antibody having binding specificity against at least two of calreticulin, PD-L1, and c-MET.

94. The genetically modified NK cell of any one of claims 90-93 wherein the genetically modified NK cell is a genetically modified NK92 cell.

95. The genetically modified NK cell of any one of claims 90-94 wherein the genetically modified NK cell is genetically modified to express at least one of a high affinity variant CD 16 and endoplasmic restricted 1L-2.

96. A pharmaceutical composition or kit, comprising:

an antibody having binding specificity towards an antigen that is specific to a protein that is specifically or preferentially expressed on a tumor stem cell in a mesenchymal state of the tumor stem cell; and a genetically modified natural killer (NK) cell that expresses a CD 16 receptor and that has granulysin and granzyme mediated cytotoxic activity under hypoxic conditions.

97. The composition or kit of claim 96 wherein the antigen is at least one of calreticulin, PD- Ll, and c-MET.

98. The composition or kit of any one of claims 96-97 wherein the genetically modified NK cell is genetically modified to express at least one of a high affinity variant CD 16 and endoplasmic restricted IL-2.

99. The composition or kit of any one of claims 96-98 further comprising at least one of an immune stimulating cytokine, an IL-15, an IL-15 superagonist, an IL-15 superagonist hybrid comprising a chemokine or chemokine portion, a chemokine that attracts at least one of a T cell and an NK cell, a CXCL14, a radiosensitizing drug, a CD47 antagonist, a SHPS-1 antagonist, an agent that up-regulates surface expression of calreticulin, and an antibody or fragment thereof that binds to the antigen and that further comprises an alpha or beta radioisotope.

100. The composition or kit of any one of claims 96-99 further comprising at least one of an agent that down-regulates suppressor cells, a peptide that down-regulates or kills M2 macrophages, an IL-8 antibody, an IL-8 antagonist, a CXCR1 inhibitor, and a CXCR2 inhibitor.