Method of treating cancer with natural killer cells

Abstract

Provided are methods and uses for treating HLA-E expressing cancer with Natural Killer (NK) cells, involving dosing of compositions containing NK cells deficient in FcRγ chain (g-NK cells). Among the provided methods and uses are methods and uses for treating various HLA-E expressing cancers.

Description

METHOD OF TREATING CANCER WITH NATURAL KILLER CELLSCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 651,384, filed May 23, 2024, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 663,669, filed June 24, 2024, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 716,699, filed November 5, 2024, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 729,308, filed December 6, 2024, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” U.S. provisional application No. 63 / 777,605, filed March 25, 2025, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” and U.S. provisional application No. 63 / 795,183, filed April 25, 2025, entitled “METHOD OF TREATING CANCER WITH NATURAL KILLER CELLS,” the contents of each of which are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 776032001840SeqList.xml, created May 21, 2025, which is 108,491 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure provides methods and uses for treating cancer with Natural Killer (NK) cells, involving dosing of compositions containing NK cells deficient in FcRy chain (g-NK cells). Among the provided methods and uses are methods and uses for treating Acute Myeloid Leukemia (AML) and certain other HLA-E expressing cancers.Background

[0004] Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity when the Fc receptor (CD 16; FcyRIII) binds to the Fc portion of antibodies bound to an antigen-bearing cell. NK cells, including specific specialized subsets thereof, can be used in therapeutic methods. Improved methods involving NK cells are needed for therapeutic uses related to the treatmentof cancers, including methods of using the NK cells as a monotherapy. Provided herein are embodiments that meet such needs.Summary

[0005] Provided herein in some embodiments is a method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia.

[0006] In some of any embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody. In some of any embodiments, the method does not comprise administering an antibody to the subject in combination with the composition of g-NK cells. In some of any embodiments, the antibody is a therapeutic antibody. In some of any embodiments, the antibody binds to a target antigen expressed by cells of the AML.

[0007] In some of any embodiments, the g-NK cells are not engineered with an antigen receptor (e.g., a chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the AML. In some of any embodiments, the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the AML. In some of any embodiments, the g-NK cells are not engineered with an antigen receptor (e.g., chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by myeloid stem cell or precursor cells associated with the AML. In some of any embodiments, the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen of expressed by cells of the AML.

[0008] In some of any embodiments, at the time of treatment the subject has measurable residual disease (MRD). In some of any embodiments, the AML is a low burden disease, optionally <25% blasts in peripheral blood and bone marrow and / or white blood cell count < 10,000. In some of any embodiments, the AML is a relapsed or refractory AML. In some of any embodiments, the AML is low burden relapsed or refractory AML. In some of any embodiments, the AML is a relapsed AML, optionally wherein the relapsed AML is characterized by >5% BM blasts, reappearance of blasts in the blood or development of extramedullary disease following achievement of CR, CRi or morphologic leukemia-free state (MLFS). In some of any embodiments, the AML is refractory AML, optionally wherein the subject failed to achieve CR, CRi or MLFS following prior treatment, and blasts >5%. In some of any embodiments, the subject has received one or more prior treatment regimens for treating the AML selected from: (i) at least 1 cycle of purine analogue containing intensive induction chemotherapy regimen, e.g., FLAG-Ida, CLIA or CLAG-M or similar regimens with or without venetoclax; (ii) at least1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX-351 with venetoclax or similar regimens; (iii) at least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax; (iv) 2 cycles of venetoclax with HMA / LDAC + / - other agents; or (v) 4 cycles of HMA alone.

[0009] In some of any embodiments, pathogenesis of the AML is associated with a viral infection. In some of any embodiments, the AML is characterized by B cells or cancer cells with upregulated HLA- E expression. In some of any embodiments, the upregulation of HLA-E expression is caused by a viral infection. In some of any embodiments, the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV). In some of any embodiments, the viral infection is an Epstein-Barr virus (EBV).

[0010] Also provided herein in some embodiments is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an HLA-E expressing cancer. In some of any embodiments, the method further comprises selecting a subject with the HLA-E expressing cancer.

[0011] Also provided herein in some embodiments is a method of treating an HLA-E expressing cancer, the method comprising: (a) selecting a subject with an HLA-E expressing cancer; and (b) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer.

[0012] Also provided herein is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of once a week (QW) in a 7-day cycle; and (b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily at a frequency of once a week (QW) in the 7-day cycle and on the same day as the g-NK cells, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

[0013] Also provided herein is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of once a week (QW) in a 7-day cycle; and (b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily for the first five consecutive days in the 7-day cycle, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

[0014] Also provided herein is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of every other day (Q2D) in a 7- day cycle; and (b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and administered one time daily every other day (Q2D) in the 7-day cycle and on the same day as the g-NK cells, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

[0015] Also provided herein is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of every other day (Q2D) in a 7- day cycle; and (b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and administered twice daily (BID) at a frequency of the first five consecutive days in a first 7-day cycle and one time daily every other day (Q2D) for a second 7-day cycle.

[0016] In some of any embodiments, a method provided herein further comprises selecting a subject with the HLA-E expressing cancer.

[0017] In some of any embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.

[0018] In some of any embodiments, a method provided herein further comprises administering to the subject an antibody directed against a target antigen associated with the HLA-E expressing cancer. In some of any embodiments, the target antigen is a B cell antigen, a plasma cell antigen, or a myeloid cell antigen. In some of any embodiments, the target antigen is a B cell antigen. In some of any embodiments, the target antigen is a plasma cell antigen. In some of any embodiments, the target antigen is a myeloid cell antigen.

[0019] Also provided herein in some embodiments is a method of treating an HLA-E expressing cancer, the method comprising: (a) administering a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer; and (b) administering to the subject an antibody that is directed against a B cell antigen, plasma cell antigen, or myeloid cell antigen.

[0020] In some of any embodiments, the g-NK cells are not engineered with an antigen receptor (e.g., a chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the cancer. In some of any embodiments, the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the cancer. In some of any embodiments, the g-NK cells are not engineered with an antigen receptor (e.g., chimeric antigen receptor) comprising an extracellular binding domain thatbinds to a target antigen expressed by myeloid stem cell or precursor cells associated with the cancer. In some of any embodiments, the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen of expressed by cells of the cancer.

[0021] In some of any embodiments, the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

[0022] In some of any embodiments, the HLA-E expressing cancer is selected from the group consisting of: head and / or neck cancer, gynecological cancer, gastric cancer, colorectal cancer, and laryngeal cancer. In some of any embodiments, the HLA-E expressing cancer is a B-cell marker expressing cancer. In some of any embodiments, the cancer is a lymphoma. In some of any embodiments, the lymphoma is a Non-Hodgkin’ s Lymphoma (NHL).

[0023] In some of any embodiments, the HLA-E expressing cancer is a plasma cell marker expressing cancer. In some of any embodiments, the cancer is a Multiple Myeloma (MM).

[0024] In some of any embodiments, the HLA-E expressing cancer is a myeloid cell marker expressing cancer. In some of any embodiments, the cancer is an acute myeloid leukemia (AML).

[0025] In some of any embodiments, at the time of treatment the subject has measurable residual disease (MRD). In some of any embodiments, the AML is a low burden disease, optionally <25% blasts in peripheral blood and bone marrow and / or white blood cell count < 10,000. In some of any embodiments, the AML is a relapsed or refractory AML. In some of any embodiments, the AML is low burden relapsed or refractory AML. In some of any embodiments, the AML is a relapsed AML, optionally wherein the relapsed AML is characterized by >5% BM blasts, reappearance of blasts in the blood or development of extramedullary disease following achievement of CR, CRi or morphologic leukemia-free state (MLFS). In some of any embodiments, the AML is refractory AML, optionally wherein the subject failed to achieve CR, CRi or MLFS following prior treatment, and blasts >5%.

[0026] In some of any embodiments, the subject has received one or more prior treatment regimens for treating the AML selected from: (i) at least 1 cycle of purine analogue containing intensive induction chemotherapy regimen, e.g., FLAG-Ida, CLIA or CLAG-M or similar regimens with or without venetoclax; (ii) at least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX- 351 with venetoclax or similar regimens; (iii) at least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax; (iv) 2 cycles.

[0027] In some of any embodiments, the antibody is a full-length antibody. In some of any embodiments, the B cell antigen, plasma cell antigen, or myeloid cell antigen is selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.

[0028] In some of any embodiments, the antibody is directed against a lymphoma antigen. In some of any embodiments, the lymphoma antigen comprises an antigen selected from CD 19 or CD20. In some of any embodiments, the antibody is an anti-CD19 antibody. In some of any embodiments, the antibody is inebilizumab, tafasitamab-cxix or obexelimab. In some of any embodiments, the antibody is an anti- CD20 antibody. In some of any embodiments, the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

[0029] In some of any embodiments, the antibody is directed against a multiple myeloma antigen. In some of any embodiments, the multiple myeloma antigen comprises an antigen selected from CD38 or BCMA. In some of any embodiments, the antibody is an anti-CD38 antibody. In some of any embodiments, each dose of the anti-CD38 antibody is about 0.5-10 mg / kg, optionally wherein each dose of the anti-CD38 antibody is about 0.5 mg / kg. In some of any embodiments, the anti-CD38 antibody is daratumumab or is isatuximab. In some of any embodiments, less than 25% of the cells in the composition of g-NK cells are positive for surface CD38. In some of any embodiments, the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression. In some of any embodiments, the antibody is an anti-BCMA antibody.

[0030] In some of any embodiments, the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

[0031] In some of any embodiments, the composition of g-NK cells is dosed at a frequency of once every week (QW).

[0032] Provided herein in some embodiments is a method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia, wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

[0033] Provided herein in some embodiments is a method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia, wherein the composition of g-NK cells is dosed at a frequency of once every week (QW).

[0034] Provided herein in some embodiments is a method of treating lymphoma in a subject, the method comprising: (a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and (b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD20 antibody.

[0035] Provided herein in some embodiments is a method of treating lymphoma in a subject, the method comprising: (a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells isdosed at a frequency of once every week (QW); and (b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD20 antibody. In some of any embodiments, the lymphoma is Non-Hodgkin’s Lymphoma (NHL). In some of any embodiments, the anti-CD20 antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

[0036] Provided herein in some embodiments is a method of treating Multiple Myeloma (MM) in a subject, the method comprising: (a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and (b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti- CD38 antibody.

[0037] Provided herein in some embodiments is a method of treating Multiple Myeloma (MM) in a subject, the method comprising: (a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of once every week (QW); and (b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti- CD38 antibody.

[0038] In some of any embodiments, each dose of the anti-CD38 antibody is about 0.5-10 mg / kg. In some embodiments, each dose of the anti-CD38 antibody is about 0.5 mg / kg. In some of any embodiments, the anti-CD38 antibody is daratumumab or is isatuximab.

[0039] In some of any embodiments, pathogenesis of the HLA-E expressing cancer is associated with a viral infection. In some of any embodiments, the HLA-E expressing cancer is characterized by B cells or cancer cells with upregulated HLA-E expression. In some of any embodiments, the upregulation of HLA-E expression is caused by a viral infection. In some of any embodiments, the subject has been selected as having a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV). In some of any embodiments, the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV). In some of any embodiments, the viral infection is an Epstein-Barr virus (EBV).

[0040] Also provided herein in some embodiments is a method of treating a disease or disorder associated with an Epstein-Barr virus (EBV), the method comprising: (a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an HLA-E expressing cancer. In some of any embodiments, the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos). In some of anyembodiments, at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

[0041] In some of any embodiments, the method further comprises administering to the subject an antibody directed against a target antigen associated with the HLA-E expressing cancer. In some of any embodiments, the target antigen is a B cell antigen, plasma cell antigen, or myeloid cell antigen.

[0042] In some of any embodiments, among cells in the composition of g-NK cells, greater than at or about 20% of the cells are g-NK cells. In some of any embodiments, among cells in the composition of g-NK cells, greater than at or about 30% of the cells are g-NK cells, greater than at or about 40% of the cells are g-NK cells, greater than at or about 50% of the cells are g-NK cells, greater than at or about 60% of the cells are g-NK cells, greater than at or about 70% of the cells are g-NK cells, greater than at or about 80% of the cells are g-NK cells, greater than at or about 90% of the cells are g-NK cells, or greater than at or about 95% of the cells are g-NK cells. In some of any embodiments, at least at or about 15% of the NK cells of the composition are positive for NKG2C (NKG2Cpos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2Aneg).

[0043] In some of any embodiments, the antibody is a full-length antibody. In some of any embodiments, the B cell antigen, plasma cell antigen, or myeloid cell antigen is selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.

[0044] In some of any embodiments, the disease or disorder associated with EBV is a lymphoma. In some of any embodiments, the lymphoma is Non-Hodgkin’s Eymphoma (NHE).

[0045] In some of any embodiments, the antibody is an anti-CD19 antibody. In some of any embodiments, the antibody is inebilizumab, tafasitamab-cxix or obexelimab.

[0046] In some of any embodiments, the antibody is an anti-CD20 antibody. In some of any embodiments, the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

[0047] In some of any embodiments, the antibody is an anti-CD22 antibody. In some of any embodiments, the antibody is epratuzumab.

[0048] In some of any embodiments, the antibody is an anti-BAFF-R antibody. In some of any embodiments, the antibody is belimumab.

[0049] In some of any embodiments, the disease or disorder associated with EBV is Multiple Myeloma (MM).

[0050] In some of any embodiments, the antibody is an anti-CD38 antibody. In some of any embodiments, each dose of the anti-CD38 antibody is about 0.5-10 mg / kg, optionally wherein each dose of the anti-CD38 antibody is about 0.5 mg / kg. In some of any embodiments, the anti-CD38 antibody is daratumumab or is isatuximab. In some of any embodiments, less than 25% of the cells in thecomposition of g-NK cells are positive for surface CD38. In some of any embodiments, the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression.

[0051] In some of any embodiments, the antibody is administered intravenously. In some of any embodiments, the antibody is administered subcutaneously. In some of any embodiments, the antibody is administered once weekly.

[0052] In some of any embodiments, the composition of g-NK cells is administered once weekly for a predetermined number of doses. In some of any embodiments, the composition of g-NK cells is administered twice weekly for a predetermined number of doses. In some of any embodiments, the composition of g-NK cells is administered three times weekly for a predetermined number of doses.

[0053] In some of any embodiments, the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

[0054] In some of any embodiments, the composition of g-NK cells is dosed at a frequency of once every week (QW).

[0055] In some of any embodiments, a second dose of g-NK cells is administered at or about at 24 hours after a first dose of g-NK cells. In some of any embodiments, a third dose of g-NK cells is administered at or about at 24 hours after a second dose of g-NK cells.

[0056] In some of any embodiments, the composition of g-NK cells is administered as two doses in a 7-day cycle. In some of any embodiments, the composition of g-NK cells is administered in a 7-day cycle. In some of any embodiments, the composition of g-NK cells is administered on day 0, day 2, and day 4 in the 7-day cycle. In some of any embodiments, the 7-day cycle is repeated one to three times. In some of any embodiments, the 7-day cycle is repeated one time. In some of any embodiments, the 7-day cycle is repeated two times.

[0057] In some of any embodiments, the composition of g-NK cells is administered from two total doses to six total doses. In some of any embodiments, the composition of g-NK cells is administered as two or four total doses. In some of any embodiments, the composition of g-NK cells is administered as three or six total doses.

[0058] In some of any embodiments, at least at or about 20% of the cells in composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK). In some of any embodiments, at least at or about 40% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

[0059] In some of any embodiments, greater than at or about 70% of the g-NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B. In some of any embodiments, (i) greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B, (ii) greater than at or about 90% of the g-NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B, or (iii) greater than at or about 95% of the g-NK cells are positive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B. In some of any embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos; and / or among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0060] In some of any embodiments, greater than 10% of the cells in the composition of g-NK cells are capable of degranulation against tumor target cells, optionally as measured by CD107a expression, optionally wherein the degranulation is measured in the absence of an antibody against the tumor target cells. In some of any embodiments, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% exhibit degranulation, optionally as measured by CD107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

[0061] In some of any embodiments, greater than 10% of the cells in the composition of g-NK cells are capable of producing interferon-gamma or TNF-alpha against tumor target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of an antibody against the tumor target cells.

[0062] In some of any embodiments, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce an effector cytokine in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

[0063] In some of any embodiments, the effector cytokine is IFN-gamma or TNF-alpha. In some of any embodiments, the effector cytokine is IFN-gamma and TNF-alpha.

[0064] In some of any embodiments, the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD56+ cells are enriched from a biological sample from a donor subject. In some of any embodiments, the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells are enriched from a biological sample from a donor subject. In some of any embodiments, the composition of g-NK cells has been produced by ex vivo expansion of cells that are NKG2Cposcells cultured with irradiated HLA-E+ feeder cells, wherein the NKG2Cposcells are enriched from a biological sample from a donor subject. In some of anyembodiments, the composition of g-NK cells has been produced by ex vivo expansion of cells that are CD3negNKG2Cposcells cultured with irradiated HLA-E+ feeder cells, wherein the CD3negNKG2Cposcells are enriched from a biological sample from a donor subject.

[0065] In some of any embodiments, the donor subject is CMV-seropositive. In some of any embodiments, the donor subject has the CD 16 F / F NK cell genotype. In some of any embodiments, the donor subject has the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype. In some embodiments, the biological sample is from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype.

[0066] In some of any embodiments, at least at or about 15% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2Cpos) and at least 70% of NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2Aneg).

[0067] In some of any embodiments, the irradiated feeder cells are deficient in HLA class I and HLA class II. In some of any embodiments, the irradiated feeder cells are 221. AEH cells.

[0068] In some of any embodiments, the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is interleukin (IL)-2 and at least one recombinant cytokine is IL-21. In some of any embodiments, the recombinant cytokines are IL-21 and IL-2. In some of any embodiments, the recombinant cytokines are IL-21, IL-2, and IL-15.

[0069] In some of any embodiments, the g-NK cells in the composition are from a single donor subject that have been expanded from the same biological sample.

[0070] In some of any embodiments, the composition of g-NK cells is formulated in a serum-free cryopreservation medium comprising a cryoprotectant, optionally wherein the cyroprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v / v).

[0071] In some of any embodiments, the g-NK cells are not engineered with an antigen receptor, optionally wherein the antigen receptor is a chimeric antigen receptor.

[0072] In some of any embodiments, the g-NK cells are not engineered with a secreted cytokine, optionally a cytokine receptor fusion protein, such as IL- 15 receptor fusion (IL-15RF).

[0073] In some of any embodiments, the method does not include exogenous cytokine administration to the subject to support NK cell survival or expansion, wherein the exogenous cytokine is one or more of IL-2, IL-7, IL-15 or IL-21.

[0074] In some of any embodiments, the method further comprises administering exogenous cytokine support to facilitate expansion or persistence of the g-NK cells in vivo in the subject, optionally wherein the exogenous cytokine is or comprises IL- 15 or IL-2.

[0075] In some of any embodiments, the method comprises administering IL-2 to the subject. In some of any embodiments, the IL-2 is administered once a week, two times a week or three times a week. In some of any embodiments, the IL-2 is administered at a frequency of once a week (QW). In some ofany embodiments, the IL-2 is administered at a frequency of every other day (Q2W). In some of any embodiments, for each day of administration the IL-2 is administered once daily. In some of any embodiments, for each day of administration the IL-2 is administered twice daily (BID). In some of any embodiments, the IL-2 is administered in a cycling regimen of one or more 7-day cycles. In some of any embodiments, the IL-2 is administered in three 7-day cycles, optionally wherein the three 7-day cycles are in consecutive weeks. In some of any embodiments, each 7-day cycle is the same. In some of any embodiments, the IL-2 is administered one time daily at a frequency of once per week (QW) on day 0 in one or more 7-day cycles. In some of any embodiments, the IL-2 is administered one time daily for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in one or more 7-day cycles. In some of any embodiments, each 7-day cycle is different. In some of any embodiments, the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in one or more 7-day cycles. In some of any embodiments, the IL-2 is administered twice daily (BID) for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in one or more 7-day cycles. In some of any embodiments, the IL-2 is administered twice daily (BID) for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in a first 7-day cycle; and the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in a second 7-day cycle. In some of any embodiments, the IL-2 is administered to the subject within about 1 hour of the administration of the g- NK cells.

[0076] In some of any embodiments, each dose of the IL-2 is 1 million to 12 million IU. In some of any embodiments, each dose of IL-2 is 4 million IU to 8 million IU. In some of any embodiments, each dose is at or about 6 million IU. In some of any embodiments, the IL-2 is administered subcutaneously. In some of any embodiments, administration of the IL-2 is administered on the same day as the first dose of the g-NK cells.

[0077] In some of any embodiments, each dose of g-NK cells is from at or about from at or about 1 x 108cells to at or about 50 x 109cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 5 x 108cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 5 x 109cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 10 x 109cells of the composition of g- NK cells. In some of any embodiments, each dose of g-NK cells is or is about 20 x 109cells of the composition of g-NK cells.

[0078] In some of any embodiments, prior to the administration of the dose of g-NK cells, the subject has received a lymphodepleting therapy. In some embodiments, the method further comprises administering to the subject a lymphodepleting therapy prior to administering the g-NK cells. In some of any embodiments, administration of a dose of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphodepleting therapy. In some of any embodiments, administration of adose of g-NK cells is initiated within 7 days or at or about 7 days after initiation of the lymphodepleting therapy. In some of any embodiments, before repeating a subsequent cycle, administering to the subject a lymphodepleting therapy. In some of any embodiments, the lymphodepleting therapy comprises fludarabine and / or cyclophosphamide. In some of any embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some of any embodiments, the lymphodepleting comprises the administration of fludarabine at or about 20-40 mg / m2body surface area of the subject, optionally at or about 30 mg / m2, daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days. In some of any embodiments, the lymphodepleting therapy further comprises administration of mesna at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days. In some of any embodiments, the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at or about 400 mg / m2body surface area of the subject and mesna at or about 300 mg / m2, daily, each for 2-4 days, optionally 3 days.

[0079] In some of any embodiments, the method further comprises administration of a bispecific T cell targeting agent to the subject. In some of any embodiments, the bispecific T cell targeting agent is a bispecific T cell engager (BiTE) comprising an anti-CD3 antibody specific to CD3 and a target antigen expressed by cells of the AML, HLA-E expressing cancer, MM, or lymphoma.

[0080] Also provided herein is a method of assessing response following administration of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple myeloma (MM), the method comprising: (1) assessing the level of expression of one or more RNA transcripts or portion thereof in a biological sample from the subject wherein: (a) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of CD28, CLECL1, DEPTOR, DUSP2, DUSP5, FCGR2B, FCRL2, GPR160, HLA-DOB, ITGA6, LY9, MAGEA1, MAGEA12, MAGEC2, PDK1, PTCD2, SLAMF7, SMAD5, TNFRSF17 (BCMA), TNFSF8 (CD30 ligand), and WNT10A, optionally wherein said one or more RNA transcripts negatively correlates to the likelihood of response following administration of the composition; and / or (b) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of EGF, ITGB3, NID2, and PG4, optionally wherein said one or more RNA transcripts positively correlates to the likelihood of response following administration of the composition; and (2) determining the likelihood of response of the subject to administration of the composition, wherein the subject is responsive to administration of the composition if the subject receives a minor response or better based on IMWG.

[0081] Also provided herein is a method of adaptive treatment following administration of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple myeloma (MM), the method comprising: (1) assessing the level of expressionof one or more RNA transcripts or portion thereof in a biological sample from the subject wherein: (a) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of CD28, CLECL1, DEPTOR, DUSP2, DUSP5, FCGR2B, FCRL2, GPR160, HLA-DOB, ITGA6, LY9, MAGEA1, MAGEA12, MAGEC2, PDK1, PTCD2, SLAMF7, SMAD5, TNFRSF17 (BCMA), TNFSF8 (CD30 ligand), and WNT10A, optionally wherein said one or more RNA transcripts negatively correlates to the likelihood of response following administration of the composition; and / or (b) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of EGF, ITGB3, NID2, and PG4, optionally wherein said one or more RNA transcripts positively correlates to the likelihood of response following administration of the composition; (2) determining the likelihood of response of the subject to administration of the composition, wherein the subject is responsive to administration of the composition if the subject receives a minor response or better based on IMWG; and (3) administering to the subject who is determined to not be responsive to the administration of the composition: (a) administration of a dose of IL-2 to the subject, (b) administration of a composition of g- NK cells to the subject, and / or (c) administration of an antibody, optionally wherein the antibody is an anti-CD38 antibody.

[0082] In some of any embodiments, the subject is a human subject.Brief Description of the Drawings

[0083] FIG. 1 depicts the preferential expansion of g-NK cells when starting with >10% preexpansion NKG2C+ / NKG2A- NK-cells. Values are mean + SE (N=8). p < 0.05.

[0084] FIG. 2A and 2B depict representative flow cytometry results, of conventional NK cells (FIG. 2A) and g-NK cells (FIG. 2B), prior to expansion, for intracellular expression of FceRly (abbreviated FcRy) chain and surface expression of NKG2A and NKG2C.

[0085] FIG. 3A shows the correlation between the percentage of g-NK cells and the percentage of NKG2C+ / NKG2A- expanded NK cells.

[0086] FIG. 3B depicts representative flow cytometry histograms of expanded conventional NK cells (Expansion A and Expansion B) and g- NK cells (Expansion C and Expansion D). The percentages of NKG2C+ / NKG2A- expression as well as FceRly expression are shown.

[0087] FIG. 4 depicts images of a tumor biopsy of cecal lesion from a subject with NHL that was administered a combination therapy of g-NK cells, IL-2, and rituximab at baseline (top row, pretreatment) and on day 7 post-administration of the first infusion of g-NK cells (bottom, post-treatment).

[0088] FIG. 5 shows the change in positron emission tomography (PET) standardized uptake units (SUVs) in four different indicated multiple myeloma patients from baseline to either 2 or 5 months after administration of g-NK cell compositions.

[0089] FIG. 6A is a plot of kappa light chain levels (mg / L) over days for patient ID 04. Blue arrows indicate g-NK cell infusions. Patient ID 04 was dosed three times following a weekly dosing schedule and was also administered IL-2.

[0090] FIG. 6B depicts FDG-PET images for Patient ID 03 at baseline (left) and at four months after g-NK cell therapy treatment (right). Both figures contain arrows indicating FDG-avid lesion locations.

[0091] FIG.6C is a plot depicting maximum change in serum tumor biomarker for the multiple myeloma subjects. M-protein was the primary serum tumor biomarker and free light chain (FLC) was used for patients that did not have evaluable M-protein. The percentage max change from baseline is plotted against patient ID.

[0092] FIGS. 7A and 7B depict the enrichment of g-NK cells (cluster 2) in the tumor microenvironment of subjects by tumor grade (FIG. 7A) or by tumor stage (FIG. 7B) in patients with clear cell renal carcinoma. g-NK cells were enriched in patients with low grade and early stage clear cell renal carcinoma. Single-cell protein activity analysis identified recurrence-associated renal tumor macrophages. Conversely, the number of g-NK cells was decreased in high grade and late stage tumor samples.

[0093] FIG. 8A depicts the proportion of samples with either a “g-NK low” or “g-NK high” proportion for trastuzumab (TH) treated pathologic complete response (OCR) of HER2+ breast cancer (CALGB40601) samples. A higher percentage of g-NK cells was associated with a statistically significant increase in the pathologic complete response (pCR) rate of patients treated with trastuzumab (TH). FIG. 8B depicts the distant disease-free survival (DDES) in trastuzumab and chemotherapy treated samples from HER 2+ breast cancer patients, with either a low proportion or a high proportion of g-NK cells. There was a trend toward increased DDES in patients with a high proportion of g-NK cells when treated with trastuzumab and chemotherapy.

[0094] FIGS. 9A-9C depict the migration of g-NK cells by quantifying effector displacement before synapse (pm / min) at 1 effector: 1 target (IE: IT) ratio as shown by FIG. 9A, tSeek (time to synapse from To in minutes) at IE: IT as shown by FIG. 9B, or tSynapse (time to synapse) at IE: IT as shown by FIG. 9C. Alternatively, tSeek is the rate at which g-NK cells find their target and tSynapse is the rate at which g-NK cells formed a synapse. P-values were generated using Fisher’s exact test.

[0095] FIGS. 10A-10C depict the frequency of synapse formation between g-NK cells and target tumor cells in the presence of the monoclonal antibody daratumumab (Dara) or in the absence of any monoclonal antibody (no Ab). FIG. 10A evaluates the frequency using a IE: IT ratio, FIG. 10B evaluates the frequency using a 1E:2T ratio, and FIG. 10C evaluates the frequency using a 1E:3T ratio. P-values were generated using Fisher’s exact test.

[0096] FIGS. 11A-11D demonstrate g-NK cell target killing of conventional and g-NK cells, with or without the addition of daratumumab after synapse formation. FIG. 11A evaluates the killing using a IE: IT ratio. FIG. 11B is a plot of the probability of survival against time of death (minutes) for conventional and g-NK cells, with or without the addition of daratumumab at a IE: IT ratio. FIG. 11C evaluates the killing using a 1E:3T ratio. FIG. 11C is a plot of the probability of survival against time of death (minutes) for conventional and g-NK cells, with or without the addition of daratumumab at a IE: IT ratio. FIG. 11D shows representative images of nano wells for conventional (cNK; top row) versus g-NK cells (bottom row) and alive or dead tumor cells.

[0097] FIGS. 12A-12B demonstrate the mRNA expression profile of different NK cell subsets from subjects with clear cell renal carcinoma, including single cell RNA sequencing data of g-NK cells (cluster 2) from subjects with clear cell renal carcinoma. FIG. 12A depicts positive markers whereas FIG. 12B depicts negative markers. FIG. 12C depicts the CD2 (LFA-1) expression (percentage of CD2+ cells) within the Total NK (CD56+), cNK (FceRly-i-), and g-NK (FceRly-) populations of ex vivo expanded NK cells by flow cytometry. *p<0.05, **p<0.01, One-way ANOVA, Tukey post-hoc test for multiple comparisons.

[0098] FIG. 13 depicts the percentage of g-NK cells bound monoclonal antibody across time (minutes). The data is plotted at % cells with surface-bound mAh normalized to time = 0 (The percentage at t = 0 was about 32%).

[0099] FIGS. 14A-14B depict the post-thaw recovery and expansion of cryopreserved NK cells when cultured with IL-2. FIG. 14A depicts the total number of NK cells post-thaw following cryopreservation over time when cultured with IL-2 at 500 lU / mL or IL-15 at 10 ng / mL. “+cyto” indicates days when new IL-2 was added to the culture. FIG. 14B depicts the viability of NK cells postthaw following cry opreservation over time when cultured with IL-2 at 500 lU / mL or IL- 15 at 10 ng / mL. “+cyto” indicates days when new IL-2 or IL-15 was added to the culture.

[0100] FIGS. 15A-15B depict IL-2 concentrations in serum in subjects receiving different dosing regiments of g-NK cell compositions with or without IL-2 on either a every other day (Q2D) or once a week (QW) schedule. FIG. 15A depicts IL-2 concentration in subjects on a QW schedule. IL-2 levels were not measured in Subject D on day 10 post-g-NK cell administration. FIG. 15B depicts IL-2 concentration in subjects on a Q2D schedule. “D” notes the administration of daratumumab.

[0101] FIGS. 16A-16B depict IL-2 concentrations in subjects receiving dosing regimens of g-NK cell compositions with or without IL-2 and / or daratumumab (“D”) delivered on either a every other day (Q2D) or once a week (QW) schedule at times when IL-2 levels are either at trough (FIG. 16A) or peak (FIG. 16B).

[0102] FIG. 17A-17D for patient ID 03 demonstrate tumor microenvironment remodeling post g- NK cell therapy treatment. FIG. 17A depicts T-cell infiltration (total, CD8+, and CD4+ T cells), pre-and post-treatment. FIG. 17B shows the CD8 to CD4 (CD8:CD4) ratio on the left plot as well as the terminal effector memory / effector memory (Teff) to regulatory T (Treg) cells (Teff:Treg) on the right plot, pre- and post-treatment. MDSCs are myeloid-derived suppressor cells. FIG. 17C depicts the percentage of CD8+ T cells expressing selected inflammatory markers (PD-1, CXCR3, and CD38), pre- and post-treatment. FIG. 17D shows the percentage of polymorphonuclear (PMN)-MDSCs, pre- and post-treatment.

[0103] FIG. 18A-18B for patient ID 13 demonstrate tumor microenvironment remodeling post g- NK cell therapy treatment. FIG. 18A depicts T-cell infiltration (total, CD8+, and CD4+ T cells), pre- and post-treatment (one month). FIG. 18B shows the CD8 to CD4 (CD8:CD4) ratio on the left plot as well as the terminal effector memory / effector memory (Teff) to regulatory T (Treg) cells (Teff:Treg) on the right plot, pre- and post-treatment (one-month).

[0104] FIG. 19 shows a volcano plot showing different gene expression using normalized counts obtained by Nanostring analysis in bone marrow of non-responding and responding patients 28 days following administration of g-NK cell compositions. Response was defined as greater or equal to minor response (MR) by IMWG.

[0105] FIG. 20 shows the difference in gene expression in bone marrow samples using normalized counts obtained by Nanostring analysis between baseline and day 28 post-administration of g-NK cell compositions for non-responders (N) and responders (R). Response was defined as greater or equal to minor response (MR) by IMWG.Detailed Description

[0106] Provided herein are methods of treating cancers, wherein the method includes administering a dose of cells of a composition of Natural Killer (NK) cells deficient in expression of the signaling adaptor Fc.epsilon.RI.gamma (FceRly; also called FcRy or gamma) chain (this subset of NK cells referred to as “g-NK cells”) to a subject having an HLA-E expressing cancer. In some embodiments, the g-NK cells also are high in expression of NKG2C and low or negative in expression of NKG2A. In some embodiments, the g-NK cells are NKG2Cpos / NKG2AnegNK cells. In provided embodiments, the methods of treating the particular subset of cancers that are associated with HLA-E expression with a dose of g- NK cells as described can result in favorable treatment outcomes, including even following administration of g-NK cells either as a monotherapy or with being engineered with an antigen receptor (e.g., CAR) against the cancer. In some cases, additionally administering an antibody directed against the cancer or engineering the cells with an antigen receptor (e.g, CAR) targeting an antigen of the cancer can further improve the methods.

[0107] In some embodiments, the HLA-E expressing cancer can include, but is not limited to, a head and / or neck cancer, a gynecological cancer, a gastric cancer, a colorectal cancer, and a laryngealcancer. In some embodiments, the gynecological cancer can include, but is not limited to, an ovarian cancer, a cervical cancer, or a breast cancer. In some embodiments, the HLA-E expressing cancer can be a B-cell expressing cancer. In particular embodiments, the HLA-E expressing cancer can be a NonHodgkin’s lymphoma (NHL). In some embodiments, the HLA-E expressing cancer can be an acute myeloid leukemia (AML).

[0108] In particular, among provided embodiments, are methods of treating acute myeloid leukemia (AML) by administering a dose of cells of a composition of g-NK cells to a subject having AML. In some embodiments, the administered g-NK cells also exhibit high expression of NKG2C and low or negative expression of NKG2A. In some embodiments, the g-NK cells are NKG2Cpos / NKG2AnegNK cells.

[0109] The provided embodiments are based on the exploitation of unique features of g-NK cells that the inventors have discovered that make g-NK cells particularly suitable for cell therapy methods for treatment of HLA-E expressing cancers, including as a monotherapy. A problem with many existing treatments for HLA-E expressing cancers, including by existing cell therapy approaches, is that many existing treatments are not specific to the cancer cells, such as may act to deplete all B cells. The provided embodiments provide for advantageous methods that are more specific to killing cells associated with the particular cancer to be treated.

[0110] Natural Killer (NK) cells are innate lymphocytes important for mediating immunity responses through cytokine and chemokine secretion, and through the release of cytotoxic granules (Vivier et al. Science 331(6013):44-49 (2011); Caligiuri, Blood 112(3):461-469 (2008); Roda et al., Cancer Res. 66(1):517-526 (2006)). Activation of NK cells can occur through the direct binding of NK cell receptors to ligands on the target cell, or through the crosslinking of the Fc receptor (CD16; also known as CD 16a or FcyRIIIa) by binding to the Fc portion of antibodies bound to an antigen-bearing cell. Upon activation, NK cells produce cytokines and chemokines abundantly and at the same time exhibit potent cytolytic activity. This release of cytokines and chemokines can play a role in the cytolytic activity of NK cells in vivo. NK cells also have small granules in their cytoplasm containing perforin and proteases (granzymes). Upon release from the NK cell, perforin forms pores in the cell membrane of targeted cells through which the granzymes and associated molecules can enter, inducing apoptosis.

[0111] g-NK cells are a specialized subset of NK cells lacking the FcRy adaptor protein, also known as g-NK cells, that are able to mediate robust ADCC responses (see e.g., published Patent Appl. No. US2013 / 0295044). In some embodiments, g-NK cells are cells that do not express substantial FcRy but do express at least one marker for Natural Killer cells. An amino acid sequence for FcRy chain (Homo sapiens, also called the high affinity immunoglobulin gamma Fc receptor I) is available in the NCBI database as accession number NP_ 004097.1 (GL4758344), and is reproduced below as SEQ ID NO:1.MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLT LLYCRLKIQVRKAAITSYEK SDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO:1)

[0112] The mechanism for increased responses of g-NK cells may be due to changes in epigenetic modification that influence the expression of the FcRy chain as well are other factors such as Syk. These epigenetic modifications are promoted at least in part by response to CMV infection where this subset of NK cells arise in about 25% of CMV exposed individuals. This special subset is relatively rare because g-NK cells are detectable at levels of ~3% to 10% of total NK cells in only 25% to 30% of cytomegalovirus (CMV)-seropositive individuals; thus, expansion is generally required for in vivo use (see e.g., Hwang et al. Int Immunol, 24:793-802, 2012; Zhang et al., J Immunol., 190:1402-1406, 2013; Bigley et al., Blood Adv 5:3021-3021, 2021). The g-NK cells express the signaling adaptor CD3 C, (zeta) chain abundantly, but are deficient in the expression of the signaling adaptor FceRly (gamma). This means that, in some cases, all signaling activity upon their activation goes through the CD3^ chain, which contains 3 ITAM motifs (versus 1 ITAM for FcRy). The result is that g-NK cells have been shown to exhibit stronger cell proliferation, more cytokine secretion, more cytolytic enzymes (e.g., perforin and granzyme B) and better antibody-dependent cellular cytotoxicity (ADCC) compared to conventional NK cells (see e.g., International published PCT Application Nos. W02020 / 107002 and WO2021 / 216790). In some embodiments, the unique activity of g-NK cells, compared to conventional NK cells and other cell therapy platforms, is due to their high expression of CD94 / NKG2C and HLA-E targeted activity. In some embodiments, g-NK cells are more effective in eliciting cell-mediated cytotoxicity than are conventional NK cells even in the absence of antibody.

[0113] Human leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I (lb) molecule. Immune cells, such as B cells, T lymphocytes, monocytes, and macrophages, basally express HLA-E. Coupel et al., Blood 109:2806-2814 (2007). In particular, HLA-E is a ligand for receptors CD94 / NKG2A and CD94 / NKG2C receptors, which are receptors expressed on NK cells and bind to HLA-E. Between the two receptors, binding of HLA-E to the inhibitory receptor NKG2A is typically favored. Specifically, interaction and binding of HLA-E with the inhibitory CD94 / NKG2A receptor results in inhibition of NK cell dependent lysis. As such, HLA-E molecules, by binding to CD94 / NKG2A receptors expressed by NK cells, can provide protection to cells seeking to evade NK cell killing. Siemanszko et al., Arch Immunol Ther Exp (Warsz) 71 (1):9 (2023). For example, tumor cells may avoid NK cell lysis through upregulation of HLA-E.

[0114] Provided embodiments are based on recognition that high NKG2C expression and low NKG2A expression on g-NK cells may circumvent the NK cell evasion strategy by certain cancer cells, including those formed in various B cell cancers, while also providing for NK cell lysis of cells expressing HLA-E, including cells associated with many HLA-E expressing cancers. Specifically, among the provided embodiments, g-NK cells can effectuate potent killing of HLA-E expressing cancercells because the g-NK cells have low expression of the CD94 / NKG2A inhibitory receptor. This means that unlike conventional NK cells, g-NK cells are not susceptible to the inhibitory effect of the HLA- E / NKG2A axis that typically has been established in cancer and autoimmune diseases (see e.g., Martmez-Rodnguez et al., Mult Scler. 22(6):741-52 (2016); Vietzen et al., Cell 196(26):5705-5718 (2023); Vietzen et al., Front. Immunol. 14:1183788 (2023)). In embodiments of provided methods, g- NK cells also may exhibit HLA-E-targeted killing activity via the high NKG2C expression on g-NK cells.

[0115] In certain cases, viral targets play important roles in the etiology or development of cancers, and particularly HLA-expressing cancers. For example, cancer subjects who are infected with an Epstein- Barr virus may have cancer cells that are predominantly infected with EBV variants that highly upregulate HLA-E and inhibit NKG2A+ cells, in which EBV isolates carrying GGDPHLPTL (SEQ ID NO:20), GGDPPLPTL (SEQ ID NO:21) and GTDPHLPTL (SEQ ID NO:74) LMP-1 peptide variants are particularly associated with HLA-E upregulation and inhibition of NKG2A+ cells (V Vietzen et al., Front. Immunol. 14:1183788 (2023)). Moreover, g-NK cells are primed by HCMV for potent killing of virally infected cells irrespective of virus. g-NK cells thus can eradicate virally infected cells mediated by anti-viral antibodies as well as by targeting of viral peptides presented on HLA-E via high levels of NKG2C and low NKG2A expression.

[0116] While other existing NK cell therapies may in some cases be able to deplete B cells, existing NK cell therapies indiscriminately kill all B cells. Further, if B cells are infected with a virus, such as with an Epstein-Barr virus (EBV), the cancer cells may exhibit peptide induced HLA-E expression. The enhanced HLA-E expression results in HLA-E / NKG2A inhibitory evasion of NK cell responses mounted by most other NK cell therapies, including in combination with an antibody via ADCC-mediated killing. Evidence indicates that HLA-E expression may be a prognostic factor of certain cancers, indicating its association to certain cancers. For instance, HLA-E has been evaluated as, for example, a prognostic factor for advanced gastric cancer (Morinaga et al., Ann Surg Oncol, 29(8):4951-4960 (2022)). In Morinaga et al., the study described that subjects who were HLA-E positive had significantly worse prognosis of relapse-free survival compared to those subjects who were HLA-E negative. HLA-E expression has also been used as a prognostic factor, in for example, gynecological cancers such as ovarian and breast cancer (Borst et al., Clin Cancer Res, 26(21):5549-5556 (2020); de Kruif et al., J. Immunol, 185(12):7452-7459 (2010); Gooden et al., Proc Natl Acad Sci USA, 108(26): 10656-10661 (2011)); colorectal cancer (Levy et al., Int J Oncol, 32(3):633-41 (2008); Guo et al., Cell Immunol., 293(1): 10-16 (2015)); and laryngeal cancer (Silva et al., Histol Histopathol., 26(12): 1487-1497 (2011)). Moreover, other cell therapies, including T- and NK-cell therapies, require the use of an engineered targeting domain, such as a chimeric antigen receptor (CAR), for targeting the cancer for treatment. However, CAR-engineered cell strategies, including autologous and allogeneic CAR-directed celltherapies, also are not always ideal because the CAR cell therapy also does not exhibit HLA-E targeting. As such CAR cell therapies also exhibit only non-selective or indiscriminate cancer and / or B cell killing based on CAR-directed targeting of B cell antigens. Moreover, compared to T cell therapies, another advantage of NK cell therapy such as g-NK cell therapy is that multiple dosing cycles of NK cells is feasible. In contrast, with CAR T therapy, multiple dosing cycles are not feasible at least in part because there is a risk of immune reactions against the chimeric antigen receptor. In sum, the provided embodiments employing g-NK cells for treating HLA-E expressing cancers are thus highly differentiated from other cell therapy approaches because they provide NKG2C and anti-viral mechanisms due to low expression of NKG2A inhibitory receptor as well as robust killing by ADCC.

[0117] The provided approaches thus allow for multiple mechanisms in which the provided g-NK cells can be used to treat HLA-E expressing cancers including AML, including inhibition and direct lysis of cancer cells that have an upregulation of HLA-E and / or by enhanced control of a latent virus that drives cancer which, in some aspects, is driven by a virus, such as an EBV infection that upregulates HLA-E on infected cells. In some aspects, the above embodiments are based on the unique NKG2C+ / NKG2A- phenotype of g-NK cells. Moreover, in addition to the above mechanisms, the g-NK cells also are able to promote ADCC killing that can further potentiate responses and treatment of HLA-E expressing cancers. In particular, in addition to potent anti-viral properties due to NKG2C+ / NKG2A- phenotype, g-NK cells also exhibit anti-viral properties by enhanced plasma-mediated ADCC against virally infected cells (Lee et al. Immunity, 2015). Also, targeted ADCC killing of cancer cells can be achieved by g-NK cells by administering the g-NK cells in combination with an antibody (e.g., an antibody targeting a B cell antigen, such as CD19, CD20, CD22 and others as described) or by engineering the g-NK cells with a CAR directed against a target antigen, such as a B cell antigen (such as a CAR directed against CD19, CD20, CD22 and others as described).

[0118] NK cells are capable of killing tumor cells via antibody dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when receptors on the NK cell surface (such as CD16) recognize IgGl or IgG3 antibodies bound to the surface of a cell. In addition to activation of NK cells that can occur through the direct binding of NK cell receptors to ligands on the target cell, as seen with direct HLA-E recognition, ADCC can be initiated through the crosslinking of the Fc receptor (CD16; also known as CD 16a or FcyRIIIa) by binding to the Fc portion of antibodies bound to an antigenbearing cell. This triggers release of cytoplasmic granules containing perforin and granzymes, leading to target cell death. Because NK cells express the activating Fc receptor CD16, which recognizes IgG- coated target cells, target recognition is broadened (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001); Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long, Curr Opin Immunol. 20(3):344- 352 (2008)). ADCC and antibody-dependent cytokine / chemokine production are primarily mediated by NK cells.

[0119] In conventional NK cells, the CD16 receptor is able to associate with adaptors, the chain of the TCR-CD3 complex (CD3Q and / or the FcRy chain, to transduce signals through immunoreceptor tyrosine-based activation motifs (IT AMs). In some aspects, CD16 engagement (CD16 crosslinking) initiates NK cell responses via intracellular signals that are generated through one, or both, of the CD 16- associated adaptor chains, FcRy or CD3^. Triggering of CD16 leads to phosphorylation of the y or chain, which in turn recruits tyrosine kinases, SYK and ZAP-70, initiating a cascade of signal transduction leading to rapid and potent effector functions. The most well-known effector function is the release of cytoplasmic granules carrying toxic proteins to kill nearby target cells through the process of antibody-dependent cellular cytotoxicity. CD 16 crosslinking also results in the production of cytokines and chemokines that, in turn, activate and orchestrate a series of immune responses. CD16 also exists in a glycosylphosphatidylinositol-anchored form (also known as FcyRIIIB or CD16B). It is understood that reference to CD 16 herein is with reference to the CD 16a form that is expressed on NK cells and that is involved in antibody-dependent responses (such as NK cell-mediated ADCC), and it is not meant to refer to the glycosylphosphatidylinositol-anchored form.

[0120] The specialized subset of g-NK cells that lack the FcRy adaptor protein are able to mediate robust ADCC responses (see e.g., published Patent Appl. No. US2013 / 0295044). The mechanism for increased responses may be due to changes in epigenetic modification that influence the expression of the FcRy. The g-NK cells express the signaling adaptor CD3 chain abundantly, but are deficient in the expression of the signaling adaptor FceRly chain. In some embodiments, g-NK cells are more effective in eliciting cell-mediated cytotoxicity than are conventional NK cells even in the absence of antibody. When activated by antibodies, y-deficient g-NK cells exhibit dramatically enhanced activity when activated by antibodies, compared to conventional NK cells, e.g., NK cells that are not deficient in the y chain. In particular, when CD 16 is engaged by the Fc region of an antibody, the signaling is mediated by solely the chain of the TCR-CD3 complex (CD3Q, which transduces signals through three immunoreceptor tyrosine-based activation motifs (IT AMs). In some aspects, the g-NK cells produce greater amounts of cytokines (e.g., IFN-y or TNF-a) and chemokines (e.g., MIP-la, MIP-ip, and RANTES) and / or display higher degranulation responses than conventional NK cells expressing the y chain, and thus have a higher capacity to release cytoplasm containing perforin and proteases (granzymes). The g-NK cells provide high expression of Granzyme B, a component of natural killer cell cytotoxic machinery. Moreover, the g-NK cells have a prolonged lifespan, compared to conventional NK cells, and their presence is maintained long-term. In some embodiments, g-NK cells are functionally and phenotypically stable. The provided embodiments thus allow for approaches in which the g-NK cells exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC) as well as antibody-independent cell-mediated cytotoxicity, supporting the utility of such cells for therapeutic applications for treating HLA-E expressing cancers such as AML. Importantly, adoptive transfer of allogeneic NK-cells does notresult in severe graft-versus-host (GVHD), and thus such a cell therapy can be given in an “off-the-shelf’ manner for clinical use.

[0121] The properties of g-NK cells that differentiate them from other cell therapy approaches for cancer are highlighted below in Table 1.Table 1. Modes of Actions of g-NK Cells Compared to Other Cell Therapies

[0122] Moreover, the present embodiments relate to methods of treatment and dosing of the g-NK cells that provide for improved treatments of subjects.

[0123] Among the provided methods are methods that involve a higher frequency dosing of the g- NK cells more than once a week, such as every other day. The cells may be administered in a 7-day cycle, or in some cases further administered in one or two repeat cycles. Results herein demonstrate safety and tolerability of the g-NK cells even at a higher dosing frequency of the g-NK cells. The ability of the g-NK cells to be well tolerated even at a higher frequency of dosing may support improvements in durability of response and overall efficacy, including as a monotherapy or in combination with antibody.

[0124] Also among provided methods are methods that include combination of the g-NK cells with IL-2, particularly low dose IL-2, administered subcutaneously (e.g., about 6 M IU), which is a strategy to improve the pharmacokinetics (PK) of the NK cells in vivo and thereby also increase durability. Results herein demonstrate tolerability and safety of g-NK cells administered with IL-2. In view of the remarkable tolerability with IL-2, the data support higher frequency dosing including daily dosing ortwice a day (BID) dosing in some aspects. The use of IL-2 to improve NK cell PK also is contemplated to support improvements in durability of response and overall efficacy, including as a monotherapy or in combination with antibody.

[0125] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0126] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS OF TREATMENT

[0127] Provided herein are compositions and methods relating to cell compositions comprising g- NK cells for use in treating an HLA-E expressing cancer in a subject. In some embodiments, provided herein is a method of treating an HLA-E expressing cancer in an individual, comprising administering a composition comprising g-NK cells, to an individual in need thereof. In some embodiments, the methods are for treating AML. Provided herein are compositions and methods relating to cell compositions comprising g-NK cells for use in treating a subject that has AML. In some embodiments, provided herein is a method of treating AML in an individual, comprising administering a composition comprising g-NK cells, to an individual in need thereof.

[0128] The composition comprising g-NK cells can include any of the provided compositions. In some embodiments, the composition is produced by the methods provided herein. Such methods and uses include therapeutic methods and uses, for example, involving administration of the therapeutic cells, or compositions containing the same, to a subject having an HLA-E expressing cancer such as AML. In some embodiments, the HLA-E expressing cancer is not caused by or is not exacerbated by an infection. In some embodiments, the HLA-E expressing cancer is caused by or is exacerbated by an infection. In some embodiments, the infection is non-viral. In some embodiments, the infection is viral. In some embodiments, the HLA-E expressing cancer is caused by or is exacerbated by a virus infection. In some embodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to effect treatment of the HLA-E expressing cancer. Uses include uses of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods thereby treat the HLA-E expressing cancer in the subject. The viral infection may be caused by any number of exemplary viruses,including, but not limited to: cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

[0129] In some of any of the provided embodiments, administration of the g-NK cells, cytokines (e.g. IL-2), and / or lymphodepleting therapy can be carried out inpatient (typically requiring a stay in a hospital overnight). In some of any of the provided embodiments, administration of the g-NK cells, cytokines (e.g. IL-2), and / or lymphodepleting therapy can be carried out outpatient (typically taking place at a hospital or clinic setting but not requiring a stay in a hospital overnight so that the patient returns home the same day). In some of any of the provided embodiments, one or more of the doses of the g-NK cells, cytokines (e.g. IL-2), and / or lymphodepleting therapy can occur inpatient and one or more of the doses of the g-NK cells, cytokines (e.g. IL-2), and / or lymphodepleting therapy. For instance, in some aspects the lymphodepleting therapy is outpatient but the administration of the g-NK cells and IL-2 is inpatient. In some embodiments, the lymphodepleting therapy is inpatient and the administration of the g-NK cells and IL-2 is outpatient. In some examples, a dosing regimen involving administration of g-NK cells once a week with IL-2 can be administered outpatient. In other examples, a dosing regimen involving administration of g-NK cells every other day with IL-2 administered BID can be inpatient at least on the days in which IL-2 is administered twice a day. Various embodiments and alternatives are within the level of a skilled artisan and at the discretion of the treating physician.A. G-. A Ceii Compositions

[0130] In some embodiments, the compositions for use in the provided methods contain g-NK cells. In some embodiments, the compositions of g-NK cells for use in the provided methods contain a plurality of g-NK cells. In some embodiments, the compositions are pharmaceutical compositions for use in treating an HLA-E expressing cancers. Also provided herein are uses of any of the provided pharmaceutical compositions for manufacture of a medicament for use in treating an HLA-E expressing cancer in a subject.

[0131] In some embodiments, the composition comprises about 5-99% g-NK cells, or any percentage of g-NK cells between 5 and 99%, inclusive. In some embodiments, the composition can comprise about 5-99% g-NK cells, inclusive, prior to expansion. In specific embodiments, most of the NK cells in a composition, prior to expansion, can be g-NK cells. In specific embodiments, the composition, prior to expansion, can comprise about 30% g-NK cells, 40% g-NK cells, 50% g-NK cells, 60% g-NK cells, 70% g-NK cells, 80% g-NK cells, 90% g-NK cells, or up to 99% g-NK cells. In some embodiments, the composition can comprise about 5-99% g-NK cells, inclusive, after expansion. In specific embodiments, the composition, after expansion, can comprise about 30% g-NK cells, 40% g-NK cells, 50% g-NK cells, 60% g-NK cells, 70% g-NK cells, 80% g-NK cells, 90% g-NK cells, or up to 99% g-NK cells.

[0132] In some embodiments, the composition can include an increased or greater percentages of g- NK cells relative to total NK cells or total cells compared to the percentage of g-NK relative to total NK cells or total cells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0133] In some embodiments, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total NK cells in the composition.

[0134] In some embodiments, prior to expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition.

[0135] In some embodiments, prior to expansion, the composition can include at least at or about20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at leastat or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g- NK cells of the total NK cells in the composition.

[0136] In some embodiments, after expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total cells in the composition. In some embodiments, after expansion, the composition can include at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells of the total NK cells in the composition.

[0137] In some embodiments, the provided compositions include those in which the g-NK cells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, prior to expansion, the provided compositions include those in which the g-NK cells make up at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, after expansion, the provided compositions include those in which the g-NK cells make up at least at or about 20%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition. In some embodiments, after expansion, the provided compositions include those in which 1the g-NK cells make up at least at or about 20% or more of the cells in the composition or of the NK cells in the composition.

[0138] In some embodiments, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0139] In some embodiments, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0140] In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition. Greater than at or about 90% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0141] In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, prior to expansion, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0142] In some embodiments, after expansion, of the total cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, after expansion, of the total cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0143] In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells. In some embodiments, after expansion, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells.

[0144] In some of any embodiments, cells of the composition that are g-NK cells also are characterized as NKG2Cpos, NKG2Anegand CD16pos. In some embodiments, cells of the composition that are g-NK cells are characterized as being CD57pos, CD7dim / neg, CD161negand / or CD38neg. In some embodiments, cells of the composition of g-NK cells are NKG2Aneg / CD161neg. In some embodiments, cells of the composition of g-NK cells are CD38neg. In some embodiments, cells of the composition of g- NK cells have the phenotype CD45pos / CD3neg / CD56pos.

[0145] In some embodiments, the composition contains NKG2Cposcells. In some embodiments, the compositions contain NKG2Anegcells. In some embodiments, the composition contains NKG2Cpos / NKG2Anegcells. In some embodiments, g-NK cells of the composition are NKG2Cposcells. In some embodiments, g-NK cells of the composition contain NKG2Anegcells. In some embodiments, g- NK cells of the composition contain NKG2Cpos / NKG2Anegcells.

[0146] In some embodiments, the composition comprises about 5-99% NKG2Cposcells. In some embodiments, the composition can include an increased or greater percentages of NKG2Cposcells relative to total NK cells or total cells compared to the percentage of NKG2Cposcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0147] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cposcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cposcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cposcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cposcells of the total NK cells in the composition.

[0148] In some embodiments, the provided compositions include those in which the NKG2Cposcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0149] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Cpos. In someembodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Cpos. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Cpos.

[0150] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Cpos. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Cpos.

[0151] In some embodiments, the composition comprises about 5-99% NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages of NKG2Anegcells relative to total NK cells or total cells compared to the percentage of NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0152] In some embodiments, the composition can include at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Anegcells of the total NK cells in the composition.

[0153] In some embodiments, the provided compositions include those in which the NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0154] In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Aneg.

[0155] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Aneg.

[0156] In some embodiments, the composition comprises about 5-99% NKG2Cpos / NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages ofNKG2Cpos / NKG2Anegcells relative to total NK cells or total cells compared to the percentage of NKG2Cpos / NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0157] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegcells of the total NK cells in the composition.

[0158] In some embodiments, the provided compositions include those in which the NKG2Cpos / NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0159] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater thanat or about 10% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2Cpos / NKG2Aneg.

[0160] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2Cpos / NKG2Aneg.

[0161] In some embodiments, the composition comprises about 5-99% g-NK cells that are NKG2Cpos / NKG2Anegcells. In some embodiments, the composition can include an increased or greater percentages of g-NK cells that are NKG2Cpos / NKG2Anegcells relative to total NK cells or total cells compared to the percentage of g-NK cells that are NKG2Cpos / NKG2Anegcells naturally present in the subject from which the cells were isolated. In some embodiments, the percentage is increased at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more.

[0162] In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at leastat or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% g- NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8%, at least at or about 10%, at least at or about 15%, at least at or about 20%, at least at or about 25%, at least at or about 30%, at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 65%, at least at or about 70%, at least at or about 75%, at least at or about 80%, at least at or about 81%, at least at or about 82%, at least at or about 83%, at least at or about 84%, at least at or about 85%, at least at or about 86%, at least at or about 87%, at least at or about 88%, at least at or about 89%, at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, at least at or about 99%, or substantially 100% g-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2Cpos / NKG2Anegg-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2Cpos / NKG2Anegg-NK cells that are NKG2Cpos / NKG2Anegcells of the total NK cells in the composition.

[0163] In some embodiments, the provided compositions include those in which the g-NK cells that are NKG2Cpos / NKG2Anegcells make up at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, at least at or about 85%, at least at or about 90%, at least at or about 95% or more of the cells in the composition or of the NK cells in the composition.

[0164] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are g- NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are g-NK cells that areNKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg.

[0165] In some embodiments, of the total NK cells in the composition, greater than at or about 50% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 60% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 70% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 80% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 90% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg. In some embodiments, of the total NK cells in the composition, greater than at or about 95% of the cells are g-NK cells that are NKG2Cpos / NKG2Aneg.

[0166] In some embodiments, the g-NK cells are CD16pos. In some embodiments, the genotype of the CD 16 protein is one in which there is a substitution of valine (V) for phenylalanine (F) at position 158 in the mature (processed) form of the protein (F158V). In some embodiments, the NK cells bear the CD16 158V polymorphism in both alleles (called 158V / V herein). In some embodiments, the g-NK cells comprise CD16 158V / V (V158). In some embodiments, the g-NK cells are CD16 158V / F. In some embodiments the g-NK cells comprise CD16 158 F / F (F158).

[0167] In some embodiments, the g-NK cells of the composition, or a certain percentage thereof, e.g., greater than about 70%, are positive for perforin and / or granzyme B. Methods for measuring the number of cells positive for perforin or granzyme B are known to a skilled artisan. Methods include, for example, intracellular flow cytometry. In an example, the percentage or number of cells positive for perforin or granyzme B may be determined by the permeabilization of cells, for instance using the Inside Stain Kit from Miltenyi Biotec, prior to staining with antibodies against perforin and granzyme B. Cell staining can then be resolved for instance using flow cytometry.

[0168] In some embodiments, greater than at or about 70% of the g-NK cells of the composition are positive for perforin, and greater than at or about 70% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 75% of the g-NK cells of the composition are positive for perforin, and greater than at or about 75% of the g-NK cells of thecomposition are positive for granzyme B. In some embodiments, greater than at or about 80% of the g- NK cells of the composition are positive for perforin, and greater than at or about 80% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 85% of the g-NK cells of the composition are positive for perforin, and greater than at or about 85% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 90% of the g-NK cells of the composition are positive for perforin, and greater than at or about 90% of the g-NK cells of the composition are positive for granzyme B. In some embodiments, greater than at or about 95% of the g-NK cells of the composition are positive for perforin, and greater than at or about 95% of the g-NK cells of the composition are positive for granzyme B.

[0169] In some embodiments, perforin and granzyme B expression levels by NK cells, for instance g-NK cells, can be measured by intracellular flow cytometry and levels measured based on levels of mean fluorescence intensity (MFI). In some embodiments, perforin and granzyme B expression levels based on MFI will differ between g-NK cells and cells that are FcRypos. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about two times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about three times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for perforin express a mean level of perforin, based on MFI levels, at least at or about four times the mean level of perforin expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about two times the mean level of granzyme B expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about three times the mean level of granzyme B expressed by FcRyposNK cells. In some embodiments, the g-NK cells of the composition that are positive for granzyme B express a mean level of granzyme B, based on MFI levels, at least at or about four times the mean level of granzyme B expressed by FcRyposNK cells.

[0170] In some embodiments, at least at or about 50% of the cells in the composition are FcRy- deficient NK cells (g-NK), wherein greater than at or about 70% of the g-NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 90% of the g-NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B. In some embodiments, greater than at or about 95% of the g-NK cells arepositive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B. In some embodiments, the g-NK cells are FcRyneg.

[0171] In some of any embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos. In some of any embodiments, among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0172] In some of any of the preceding embodiments, greater than at or at about 80% of the cells are positive for perforin. In some of any of the preceding embodiments, greater than at or at about 90% of the cells are positive for perforin. In some of any of the preceding embodiments, among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos.

[0173] In some of any of the preceding embodiments, greater than at or at about 80% of the cells are positive for granzyme B. In some of any of the preceding embodiments, greater than at or at about 90% of the cells are positive for granzyme B. In some of any of the preceding embodiments, among the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

[0174] In some of any of the provided embodiments, it is understood that the terms positive, pos or + with reference to a marker or protein expressed on or in a cell are used interchangeably herein. Likewise, it is understood that the terms negative, neg or - with reference to a marker or protein expressed on or in a cell are used interchangeably herein. Further, it is understood that reference to cells that are markernegherein may refer to cells that are negative for the marker as well as cells expressing relatively low levels of the marker, such as a low level that would not be readily detectable compared to control or background levels. In some aspects, expression of any of the provided markers can be determined by their expression on the surface of the cells (surface expression) or in the cells (intracellular expression). In some embodiments, the expression can be determined by flow cytometry, for example, by staining with an antibody that specifically bind to the marker and detecting the binding of the antibody to the marker. Similar methods can be carried out to assess expression of intracellular markers, except that such methods typically include methods for fixation and permeabilization before staining to detect intracellular proteins by flow cytometry.

[0175] In some embodiments, a cell (e.g., NK cell subset) is positive (pos) for a particular marker if there is detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In embodiments, surface expression is positive if staining is detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to, or in some cases higher than, a cell known to be positive for the marker and / or at a level higher than that for a cell known to be negative for the marker.

[0176] In some embodiments, a cell (e.g., NK cell subset) is negative (neg) for a particular marker if there is an absence of detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In embodiments, surface expression is negative if staining is not detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially lower than a cell known to be positive for the marker and / or at a level substantially similar to a cell known to be negative for the marker.

[0177] In some embodiments, a cell (e.g., NK cell subset) is low (lo or min) for a particular marker if there is a lower level of detectable presence on or in the cell of a particular marker compared to a cell known to be positive for the marker. In embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically bind to the marker and detecting the binding of the antibody to the marker, wherein expression, either surface or intracellular depending on the method used, is low if staining is at a level lower than a cell known to be positive for the marker.

[0178] In some of any of the provided embodiments, the composition comprises from at or about 106cells to at or about 1012cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about 10” cells, from at or about 106to at or about 1010cells, from at or about 106to at or about 109cells, from at or about 106to at or about 108cells, from at or about 106to at or about 107cells, from at or about 107to at or about 1012cells, from at or about 107to at or about 10” cells, from at or about 107to at or about 1010cells, from at or about 107to at or about 109cells, or from at or about 107to at or about 108cells, from at or about 108to at or about 1012cells, from at or about 108to at or about 10” cells, from at or about 108to at or about 1010cells, from at or about 108to at or about 109cells, from at or about 109to at or about 1012cells, from at or about 109to at or about 10” cells, from at or about 109to at or about 1010cells, from at or about 1010to at or about 1012cells, from at or about 1010to at or about 10” cells, or from at or about 10” to at or about 1012cells.

[0179] In some of any of the provided embodiments, the composition comprises at least or about at least 106cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about 1010cells, from at or about 106to at or about 109cells, from at or about 106to at or about 108cells, from at or about 106to at or about 107cells, from at or about 107to at or about 1010cells,from at or about 107to at or about 109cells, from at or about 107to at or about 108cells, from at or about 108to at or about IO10cells, from at or about 108to at or about 109cells, or from at or about 109to at or about 1010cells.

[0180] In some of any of the provided embodiments, the composition comprises at least or about at least 108cells. In some of any of the provided embodiments, the composition comprises at least at or about 109cells. In some of any of the provided embodiments, the composition comprises at least at or about 1010cells. In some of any of the provided embodiments, the composition comprises at least at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 1010cells. In some of any of the provided embodiments, the composition comprises from at or about 108to at or about 109cells. In some of any of the provided embodiments, the composition comprises from at or about 109to at or about 10” cells. In some of any of the provided embodiments, the composition comprises from at or about 109to at or about 1010cells. In some of any of the provided embodiments, the composition comprises from at or about 1010to at or about 10” cells.

[0181] In some of any of the provided embodiments, the composition comprises at least at or about 106g-NK cells. In some of any of the provided embodiments, the composition comprises from at or about 106to at or about 1010g-NK cells, from at or about 106to at or about 109g-NK cells, from at or about 106to at or about 108g-NK cells, from at or about 106to at or about 107g-NK cells, from at or about 107to at or about 1010g-NK cells, from at or about 107to at or about 109g-NK cells, from at or about 107to at or about 108g-NK cells, from at or about 108to at or about 1010g-NK cells, from at or about 108to at or about 109g-NK cells, or from at or about 109to at or about 1010g-NK cells.

[0182] In some embodiments, the cells in the described composition of g-NK cells are for allogenic cell therapy. In some embodiments, the cells in the described composition of g-NK cells are from a donor or donors that are different from the subject to be treated. In some embodiments, the donor or donors are not known to have the HLA-E expressing cancer. In particular embodiments of any of the provided compositions, the cells in the composition are from the same donor. As such, the compositions do not include a mixed population of cells from one or more different donors.

[0183] In some embodiments, the g-NK cells are primary g-NK cells from a subject. According to some embodiments, the primary g-NK cells can be obtained from a sample from a mammalian subject, such as a human subject. The sample or source can be, for example, but not limited to, cord blood, bone marrow or peripheral blood. In particular, among the provided compositions are compositions of cells that are enriched for g-NK cells. In some embodiments, the compositions for use in the provided methods contain g-NK cells that are expanded NK cells such as produced by any of the providedmethods. In some embodiments, the g-NK cells are selected and expanded such as by methods described in Section II.

[0184] In some embodiments, the composition of g-NK cells are produced by an ex vivo expansion method that enriches and expands for g-NK cells from a donor subject. In some embodiments, the method of expansion include those as described in Section II. As provided here, the methods of expansion result in high yield expansion of at or greater than 500-fold, at or greater than 600-fold, at or greater than 700-fold, at or greater than 800-fold, at or greater than 900-fold, at or greater than 1000-fold or more of g-NK cells. In some of any embodiments, the increase is at or about 1000-fold greater. In some of any embodiments, the increase is at or about 2000-fold greater. In some of any embodiments, the increase is at or about 2500-fold greater. In some of any embodiments, the increase is at or about 3000-fold greater. In some of any embodiments, the increase is at or about 5000-fold greater. In some of any embodiments, the increase is at or about 10000-fold greater. In some of any embodiments, the increase is at or about 15000-fold greater. In some of any embodiments, the increase is at or about 20000-fold greater. In some of any embodiments, the increase is at or about 25000-fold greater. In some of any embodiments, the increase is at or about 30000-fold greater. In some of any embodiments, the increase is at or about 35000-fold greater.

[0185] In certain embodiments, the number of such cells in the composition is a therapeutically effective amount. In some embodiments, the amount is an amount that reduces the severity, the duration and / or the symptoms associated with an HLA-E expressing cancer.

[0186] In some embodiments, the composition comprises an amount of g- NK cells that is from at or about 105and at or about 1012g-NK cells, or from at or about 105to at or about 108g-NK cells, or from at or about 106and at or about 1012g-NK cells, or from at or about 108and at or about 10” g-NK cells, or from at or about 109and at or about 1010g-NK cells. In some embodiments, the composition comprises greater than or greater than at or about 105g-NK cells, at or about 106g-NK cells, at or about 107g-NK cells, at or about 108g-NK cells, at or about 109g-NK cells, at or aboutlO10g-NK cells, at or about 10” g-NK cells, or at or about 1012g-NK cells. In some embodiments, such an amount can be administered to a subject having a disease or condition, such as to a subject with an HLA-E expressing cancer.

[0187] In some embodiments, the composition comprises an amount of NKG2Cposcells or a subset thereof that is from at or about 105and at or about 1012NKG2Cposcells or a subset thereof, or from at or about 105to at or about 108NKG2Cposcells or a subset thereof, or from at or about 106and at or about 1012NKG2Cposcells or a subset thereof, or from at or about 108and at or about 10” NKG2Cposcells or a subset thereof, or from at or about 109and at or about 1010NKG2Cposcells or a subset thereof. In some embodiments, the composition comprises greater than or greater than at or about 105NKG2Cposcells or a subset thereof, at or about 106NKG2Cposcells or a subset thereof, at or about 107NKG2Cposcells or a subset thereof, at or about 108NKG2Cposcells or a subset thereof, at or about 109NKG2Cposcells or asubset thereof, at or about 1010NKG2Cposcells or a subset thereof, at or about 10” NKG2Cposcells or a subset thereof, or at or about 1012NKG2Cposcells or a subset thereof. In some embodiments, such an amount can be administered to a subject having a disease or condition, such as to a subject with an HLA- E expressing cancer.

[0188] In some embodiments, the volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL or 500 mL, such as is from or from about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL, each inclusive. In some embodiments, the composition has a cell density of at least or at least about 1 x 105cells / mL, 5 x 105cells / mL, 1 x 106cells / mL, 5 x 106cells / mL, 1 x 107cells / mL, 5 x 107cells / mL or 1 x 108cells / mL. In some embodiments, the cell density of the composition is between or between about 1 x 105cells / mL to 1 x 108cells / mL, 1 x 105cells / mL to 1 x 107cells / mL, 1 x 105cells / mL to 1 x 106cells / mL, 1 x 106cells / mL to 1 x 107cells / mL, 1 x 106cells / mL to 1 x 108cells / mL, 1 x 106cells / mL to 1 x 107cells / mL or 1 x 107cells / mL to 1 x 108cells / mL, each inclusive.

[0189] Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. In some embodiments, the engineered cells are formulated with a pharmaceutically acceptable carrier.

[0190] A pharmaceutically acceptable carrier can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical carrier should be one that is suitable for NK cells, such as a saline solution, a dextrose solution or a solution comprising human serum albumin.

[0191] In some embodiments, the pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which the NK cells can be maintained, or remain viable, for a time sufficient to allow administration of live NK cells. For example, the pharmaceutically acceptable carrier or vehicle can be a saline solution or buffered saline solution. The pharmaceutically acceptable carrier or vehicle can also include various biomaterials that may increase the efficiency of NK cells. Cell vehicles and carriers can, for example, include polysaccharides such as methylcellulose (M. C. Tate, D. A. Shear, S. W. Hoffman, D. G. Stein, M. C. LaPlaca, Biomaterials 22, 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (Suh J K F, Matthew H W T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford D S, et al., J Biomed Mater Res, 51, 586, 2000, each ofwhich is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P(NIPAM- co-AA) (Y. H. Bae, B. Vernon, C. K. Han, S. W. Kim, J. Control. Release 53, 249, 1998; H. Gappa, M. Baudys, J. J. Koh, S. W. Kim, Y. H. Bae, Tissue Eng. 7, 35, 2001, each of which is incorporated herein by reference in its entirety), as well as Poly(oxyethylene) / poly(D,L-lactic acid-co-gly colic acid) (B. Jeong, K. M. Lee, A. Gutowska, Y. H. An, Biomacromolecules 3, 865, 2002, which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs L J, Mikos A G. Cell Trans, 8, 345, 1999, which is incorporated herein by reference in its entirety), PEO / PEG (Mann B K, Gobin A S, Tsai A T, Schmedlen R H, West J L., Biomaterials, 22, 3045, 2001; Bryant S J, Anseth K S. Biomaterials, 22, 619, 2001, each of which is incorporated herein by reference in its entirety), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005, which is incorporated herein by reference in its entirety), collagen (Lee C R, Grodzinsky A J, Spector M., Biomaterials 22, 3145, 2001, which is incorporated herein by reference in its entirety), and / or alginate (Bouhadir K H, Lee K Y, Alsberg E, Damm K L, Anderson K W, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990, each of which is incorporated herein by reference in its entirety).

[0192] In some embodiments, the NK cells such as NKG2Cposcells or a subset thereof can be present in the composition in an effective amount. In some embodiments, the composition contains an effective amount of g-NK cells, such as FcRynegcells or cells having a g-NK surrogate marker profile thereof. An effective amount of cells can vary depending on the patient, as well as the type, severity and extent of disease. Thus, a physician can determine what an effective amount is after considering the health of the subject, the extent and severity of disease, and other variables.

[0193] In some embodiments, the composition, including pharmaceutical composition, is sterile. In some embodiments, isolation, enrichment, or culturing of the cells is carried out in a closed or sterile environment, for example and for instance in a sterile culture bag, to minimize error, user handling and / or contamination. In some embodiments, sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. In some embodiments, culturing is carried out using a gas permeable culture vessel. In some embodiments, culturing is carried out using a bioreactor.

[0194] Also provided herein are compositions that are suitable for cryopreserving the provided NK cells. In some embodiments, the NK cells are cryopreserved in a serum-free cry opreservation medium. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, the cryopreservation medium is between at or about 5% and at or about 10% DMSO (v / v). In some embodiments, the cry opreservation medium is at or about 5% DMSO (v / v). In some embodiments, the cry opreservation medium is at or about 6% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 7% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 8% DMSO (v / v). In some embodiments, the cryopreservation medium is at or about 9% DMSO (v / v). In someembodiments, the cryopreservation medium is at or about 10% DMSO (v / v). In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor™ CS10). CryoStor™ CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). In some embodiments, compositions formulated for cry opreservation can be stored at low temperatures, such as ultra-low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0195] In some embodiments, the compositions can be preserved at ultra-low temperature before the administration to a patient. In some aspects, NK cell subsets, such as g-NK cells, can be isolated, processed and expanded, such as in accord with the provided methods, and then stored at ultra-low temperature prior to administration to a subject.

[0196] A typical method for the preservation at ultra-low temperature in small scale is described, for example, in U.S. Pat. No. 6,0168,991. For small-scale, cells can be preserved at ultra-low temperature by low density suspension (e.g., at a concentration of about 200xl06 / ml) in 5% human albumin serum (HAS) which is previously cooled. An equivalent amount of 20% DMSO can be added into the HAS solution. Aliquots of the mixture can be placed into vials and frozen overnight inside an ultra-low temperature chamber at about -80° C.

[0197] In some embodiments, the cryopreserved NK cells are prepared for administration by thawing. In some cases, the NK cells can be administered to a subject immediately after thawing. In such an embodiment, the composition is ready-to-use without any further processing. In other cases, the NK cells are further processed after thawing, such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activating or stimulating agent, or are activated washed and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

[0198] In one embodiment, cytokines can be administered to a subject prior to isolating primary NK cells. For example, IL-12, IL-15, IL-18, IL-2, and / or CCL5 can be administered to a subject prior to isolating the primary NK cells.1. Gene Editing

[0199] Among the provided composition of g-NK cells are compositions in which the g-NK cells are engineered g-NK cells.

[0200] In some embodiments, the g-NK cells described herein may be genetically engineered by gene editing to alter (e.g., reduce) expression of one or more genes by the g-NK cells, thereby altering one or more properties or activities of the NK cells. For instance, strategies for gene editing can include one or more strategy that reduced fratricide (self-killing) due to expression of target antigen on g-NK cells; reduces undesired immunoreactivity that may result in graft vs. host disease (GvHD) particularly when infused into immune-compromised HLA-matched or, in some cases, also when infused into HLAmis-matched recipients; or reduces immunosuppression by host factors, particularly in the tumor microenvironment. In some embodiments, the engineered g-NK cells, including those engineered by one or more gene editing strategy, exhibit enhanced NK cell response characteristics as compared to similar NK cells without the gene editing, e.g., enhanced target recognition, enhanced NK cell response level and / or duration, improved NK cell survival, delayed NK cell exhaustion, and / or enhanced target recognition.

[0201] In some embodiments, the g-NK cells described herein can be gene edited to reduce FcRy chain expression, activity and / or signaling in the cell. For example, methods of gene editing may comprise introducing a genetic disruption of a gene encoding FcRy chain, a gene encoding a protein that regulates expression or activity of FcRy signaling adaptor (e.g., a transcription factor, such as PLZF or HELIOS) and / or a gene encoding a protein that is involved in FcRy-mediated signaling (e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2) as described. In some embodiments, method of engineering may comprise introducing an inhibitory nucleic acid molecule that targets a gene encoding FcRy chain, a gene encoding a protein that regulates expression or activity of FcRy signaling adaptor (e.g., a transcription factor, such as PLZF or HELIOS) and / or a gene encoding a protein that is involved in FcRy-mediated signaling (e.g., a downstream signaling molecule, such as SYK, DAP2 or EAT2) as described. In some embodiments, the g-NK cells described herein are gene edited to be deficient in or reduced in FcRy chain expression, activity and / or signaling in the cell. Methods for reduction of FcRy chain expression, such as knockout or disruption of FcRy chain in NK cells, are described in PCT. Pub. No. WO2018 / 148462 and Liu et al. iScience, 2020; 23:101709, the disclosures of each of which are incorporated by reference in their entireties. For example, in some embodiments, cells are gene edited to knockout the FcRy chain using a CRISPR-Cas9 system. In some embodiments, cells are gene edited to knockout the FcRy chain by introducing a caspase effector nuclease, such as a Cas9, and guide RNA, such as a guide RNA comprising the sequence set forth in SEQ ID NO: 75 and / or a guide RNA comprising the sequence set forth in SEQ ID NO: 76. In some embodiments, the caspase effector nuclease and guide RNA are introduced by delivering a ribonucleoprotein (RNP) complex comprising the caspase effector nuclease and guide RNA to the cell by electroporation of the RNP.

[0202] In some embodiments, the method provided herein comprises obtaining a primary NK cell or anNK cell line, and gene editing the cell to reduce expression of FcRy expression, activity and / or signaling in the cell in accord with the provided methods. In some embodiments, the methods provided herein comprises isolating an NK cell from a subject, such as by the methods as described above or known to a skilled artisan, and reducing the expression of FcRy chain expression, activity and / or signaling in the cell in accord with the provided methods. In some embodiments, primary cells derived from a subject may be expanded and / or cultured before gene editing. In some embodiments the geneedited primary cells are cultured and / or expanded following gene editing and prior to administration to a patient.

[0203] In some of any of the preceding embodiments, the g-NK cell can further comprise nucleic acid encoding a heterologous CD16. In some of any of the preceding embodiments, the heterologous CD 16 can comprise a CD16-activating mutation, wherein the mutation can result in higher affinity to IgGl. In some of any of the preceding embodiments, the heterologous CD16 can comprise a 158V mutation. In some of any of the preceding embodiments, the engineered g-NK cells can be derived from a primary cell obtained from a human subject.

[0204] One of ordinary skill in the art will appreciate that there are many ways of decreasing the expression or activity of FcRy. For example, the level of transcription can be decreased. One method of decreasing gene expression, such as FcRy chain expression, involves modifying an endogenous gene to decrease transcription. For example, the FcRy chain gene may be deleted, disrupted, or mutated. In addition to targeting the FcRy RNA, mutating, or modifying the FcRy gene, FcRy protein level can be decreased by effecting a molecule that increases FcRy gene expression or activity, such as a transcription factor that regulates transcription of FcRy. In some embodiments a gene that regulates transcription or translation of the FcRy chain gene may be deleted, disrupted, or mutated. In some of these embodiments, the gene is a transcription factor that regulates expression of the FcRy chain gene. Specifically, inhibition of a transcription factor that positively regulates FcRy expression will result in decreased FcRy expression. Transcription factors that regulate FcRy transcription include HELIOS and PLZF.

[0205] One of ordinary skill in the art will understand that there are many suitable methods for disrupting FcRy chain gene or other gene, such as those described herein. For example, the entire gene locus, such as FcRy locus, may be deleted. In some cases, it is also suitable to delete a portion of the gene, for example an exon, or a domain. Specifically, the IT AM signaling domain of FcRy may be deleted. Alternatively, the provided methods also include introducing one or more amino acid substitutions into the gene locus, such as FcRy locus, such as an inactivating mutation. In some embodiments, a stop codon can be introduced into the mRNA, such as FcRy mRNA, to produce a truncated and / or inactivated form of the expressed gene, such as FcRy signaling adaptor. In some embodiments, regulatory elements of the gene, such as FcRy gene, can also be mutated or deleted in order to reduce expression, activity and / or signaling of FcRy signaling adaptor.

[0206] In some embodiments, gene disruption can be carried out in mammalian cells using sitespecific endonucleases. Endonucleases that allow for site-specific deletion of a gene are well known in the art and may include TAL nucleases, meganucleases, zinc-finger nucleases, Cas9, and Argonaute. Methods for producing engineered, site-specific endonucleases are known in the art. The site-specificendonuclease can be engineered to recognize and delete or modify a specific gene, such as the FcRy chain gene.

[0207] In some embodiments, provided g-NK cells are engineered by editing the genome of the g- NK cells. In some embodiments, the editing of the genome may be carried out in a method that enriches for g-NK cell subset from a starting sample of NK cells. Thus, it is understood that the provided methods do not require selecting editing the genome only of g-NK cells that have been selected for NK cells that are deficient in the FcRy chain (or only that have been selected or identified by a g-NK surrogate marker profile), but may involve gene editing of a composition of NK cells that are to be, or that have been, preferentially expanded or enriched in g-NK cells. As such, the final composition of cells that are enriched in g-NK cells include g-NK cells that have been gene edited. Exemplary methods for preparing and expanding a composition enriched in g-NK cells is provided in Section II.

[0208] In some embodiments, the editing of the genome may take place at any suitable time during the methods of expanding the g-NK cells, such as described in Section II. In some embodiments, the gene editing is carried out after the selection of cells from a subject (e.g. selecting or enriching cells that are CD3negCD57posor CD3negCD56pos) and prior to incubating or culturing the selected or enriched cells with feeder cells (e.g. HLA-E-expressing feeder cells) for proliferation or expansion of the NK cells. In some embodiments, the gene editing is carried out after the incubation or culture with the feeder cells (e.g. HLA-E-expressing feeder cells) and thus after selected or enriched cells have proliferated or expanded.

[0209] Methods for knocking out (e.g., deleting) a target gene expression include, but not limited to, a zinc finger nuclease (ZFN), a Tale-effector domain nuclease (TALEN), and CRIPSR / Cas system. Such methods typically comprise administering to the cell one or more polynucleotides encoding one or more nucleases such that the nuclease mediates modification of the endogenous gene, for example in the presence of one or more donor sequence, such that the donor is integrated into the endogenous gene targeted by the nuclease. Integration of one or more donor molecule(s) occurs via homology-directed repair (HDR) or by non-homologous end joining (NHEJ) associated repair. In certain embodiments, one or more pairs of nucleases are employed, which nucleases may be encoded by the same or different nucleic acids.

[0210] In one embodiment, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA- binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence, about or approximately 18 base pairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)).

[0211] In other embodiments, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA- binding domain fused to the Fokl nuclease domain (reviewed in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair. Because ZFNs and TALENs are heterodimeric so that the production of a single functional nuclease in a cell requires co-expression of two protein monomers, compact TALENs provide an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4: 1762). A compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.

[0212] In some embodiments, engineered endonucleases based on the CRISPR / Cas9 system are also known in the art and can be employed in the provided methods to gene edit the cells (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al. (2013) Nat Methods. 10:957- 63). A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" that directs the nuclease to a location of interest in the genome. In some embodiments, the guide RNA comprises an approximately 20 nucleotide targeting sequence. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in in the genome. Methods of using CRISPR-Cas9 are well known in the art.

[0213] In some embodiments, gene editing is carried out using an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is an RNA-guided DNA endonuclease. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-guided nucleases include any as described in PCT publication No. W02020 / 168300 (e.g., Table 2 therein). In some embodiments, the RNA-guided nuclease is a Cas9 or Casl2 nuclease. In some embodiments, the RNA- guided nuclease is Cpfl (Casl2a). In some embodiments, Cpfl is Acidaminococcus sp. Cpfl (AsCpfl).

[0214] In some embodiments, gene editing is carried out with an RNA-guided nuclease and a guide RNA (gRNA). These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation. In some embodiments, the gRNA includes a crRNA and, optionally, a tracrRNA. In some embodiments, the RNA-guided nuclease (e.g., Cas9 or a Casl2) and one or more gRNAs form ribonucleoprotein (RNP) complexes that associate with (i.e., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the gRNA (e.g., crRNA). In some embodiments, the Cas is a Cas9 nuclease, such as from Streptococcus pyogenes. It is understood that the endonucleaseused herein is not limited to the Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9. In one aspect, the Cas9 can come from a different bacterial source. Substitution of the Cas9 can also be used to increase the targeting specificity so less gRNA needs to be used. Thus, for example, the Cas can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus sp. (AsCpfl), Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpfl) derived from Lachnospiracase bacterium (LbCpfl), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacter jejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HFl, Fokl-Fused dCas9, or an expanded Cas9 (xCas9). Additionally other Cas endonucleases can be used in place of a Cas9 system such as, for example, CasX, CasY, Casl4, Cas4, Csn2, Cas 13a, Cas 13b, Cas 13c, Cas 13d, C2cl, or C2c3 or using any other type of engineered Cas protein including prime editing.

[0215] In some embodiments, a genome editing system containing an RNA-guided nucleases (e.g., a Cas) and a gRNA is implemented, in certain embodiments, as a protein / RNA complex (a ribonucleoprotein, or RNP) that is introduced into the cell to be edited. In some embodiments, the RNP complex is introduced into the cells in an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, or liposome. In certain embodiments, a genome editing system containing an RNA- guided nucleases (e.g., a Cas) and a gRNA is implemented as one or more nucleic acids encoding the RNA-guided nuclease and guide RNA components. For instance, in certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus.

[0216] In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM.” The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA-guided nuclease / gRNA combinations. Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. For example, Cas9 nucleases recognize PAM sequences that are 3’ of the protospacer, while Cpfl, on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer. In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA- guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. F. novicida Cpfl recognizes a TTN PAM sequence. PAM sequences have been identified for a variety ofRNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov el al, 2015, Molecular Cell 60, 385-397, November 5, 2015.

[0217] It is understood and herein contemplated that the use of a particular Cas can change the PAM sequence which the Cas endonuclease (or alternative) uses to screen for targets. As used herein, suitable PAM sequences comprises NGG (SpCas9 PAM) NNGRRT (SaCas9 PAM) NNNNGATT (NmCAs9 PAM), NNNNRYAC (CjCas9 PAM), NNAGAAW (St), TTTV (LbCpfl PAM and AsCpfl PAM); TYCV (LbCpfl PAM variant and AsCpfl PAM variant); where N can be any nucleotide; V = A, C, or G; Y = C or T; W = A or T; and R = A or G.

[0218] In some embodiments, the gRNA promotes the specific association (or “targeting”) of an RNA-guided nuclease (e.g., a Cas, such as a Cas9 or a Cpfl) to a target sequence such as a genomic sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA) and a tracrRNA, which are usually associated with one another, for instance by duplexing). Guide RNAs, whether unimolecular or modular, include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. For instance, in connection with a Cas9 the crRNA is the guide RNA that provides the targeting domain that is a nucleotide sequence complementary to the target DNA, and also can include a tracrRNA that serves as a binding scaffold for the Cas nuclease. In connection with Cpfl, which induces double stranded DNA breaks under the guidance of a single crRNA, a tracrRNA is not required and instead the crRNA includes a 5 '-handle engaging Cpfl recognition and a guide segment interacting with targeted DNA sequences through complementary binding. Targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length).

[0219] In some embodiments, the gRNA, in some cases the crRNA, is any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity, when optionally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal 1W, Clustal X, BLAT, and others known to a skilled artisan. The ability of a guide sequence (within a nucleic-acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay.For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence. Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.

[0220] Methods for designing gRNAs are known to a skilled artisan (see e.g., Cui et al. (2018) Interdisciplinary Sciences: Computational Life Sciences, 10:455-465); PCT publication No. W02019 / 010384). Methods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al, 2014 Nat Biotechnol 32(3): 279- 84, Heigwer et al, 2014 Nat methods 11(2): 122-3 ; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. As a non-limiting example, gRNA design may involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s target sequence, e.g., to minimize total off-target activity across the genome. While off-target activity is not limited to cleavage, the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.

[0221] For example, a guide RNA comprising a targeting sequence of RNA nucleotides would include the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and this contains uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence of RNA nucleotides, and described by a DNA sequence that includes thymidine molecules would have a targeting domain of the corresponding RNA sequence that is the same but including uracil instead of thymidine. As will be apparent to the skilled artisan, such a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA / tracerRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For Cpfl, for example, a suitable scaffold sequence comprises the sequence U A AUUU CU ACUCUU GU AG AU (SEQ ID NO:77), added to the 5’- terminus of the targeting domain.

[0222] In some embodiments, efforts to enhance the clinical ADCC response to antibodies, including MM antibodies, have been challenging because NK-cells also express certain antigens that are the same as the tumor targets. These antigens include, for example, CD38 and SLAMF7. Thus, when an NK cell therapy is combined with an antibody against the target antigen (e.g., daratumumab and elotuzumab for targeting CD38 and SLAMF7, respectively), or when the NK cells express a CAR as provided herein against the target antigen, the therapy may not only target the cancer, but can alsodeplete the patient’s NK cell population. For instance, high CD38 expression particularly results in rapid depletion of NK cells early in the daratumumab treatment course, largely eliminating this source of innate immune cells which could potentially drive even more complete tumor eradication.

[0223] In some embodiments, the NK cells are edited to reduce expression of a target antigen that is known or suspected of also being expressed at some level by the NK cells. In some embodiments, gene editing is carried out with a gRNA that targets the target antigen known or suspected of being expressed at some level by the NK cells. In some embodiments, the NK cells express a CAR directed against CD38 and CD38 expression is reduced or eliminated in the NK cells. In some embodiments, the gRNA for use in the disclosure is a gRNA targeting CD38 (see e.g., WO2019 / 222503, WO2021 / 087466 and WO2021 / 113853 for exemplary gRNA targeting CD38).

[0224] In some embodiments, the gRNA targets a molecule involved in immunoreactivity of the NK cell. In some embodiments, HLA class I expression on the surface of the engineered g-NK cell is reduced. The human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. The HLA class I proteins all have a long alpha chain and a short beta chain, B2M. Little HLA class I can be expressed in the absence of B2M and the expression of B2M is required for HLA class I proteins to present peptides from inside the cell. The present disclosure provides g-NK cells engineered to reduce expression of B3M. Thus, these cells avoid the immune surveillance and attach by cytotoxic T cells. In some embodiments, the gRNA for use in the disclosure is a gRNA targeting beta 2 microglobulin (B2M) (see e.g., W02020 / 168300, WO2018 / 064694, WO2015 / 161276, or W02017 / 152015) for exemplary gRNA targeting B2M).

[0225] In some embodiments, the gRNA targets a molecule involved in immunosuppression of the NK cell activity. Suitably, engineered NK cells comprise reduced or absent checkpoint inhibitory receptor function. Suitably, the checkpoint inhibitory receptors with reduced or absent function comprise one or more or all of CD96 (TACTILE), CD 152 (CTLA4), CD223 (LAG-3), CD279 (PD-1), CD328 (SIGLEC7), SIGLEC9, TIGIT, and / or TIM-3. Suitably, the NK cell cells comprise reduced or absent checkpoint inhibitory receptor function for two or more checkpoint inhibitory receptors. Suitably, the two or more checkpoint inhibitory receptors comprise CD96 (TACTILE), CD 152 (CTLA4), or CD328 (SIGLEC7) or CD279 (PD-1).

[0226] In some embodiments the gRNA for use in the disclosure is a gRNA targeting TIGIT (see e.g., W02020 / 168300 for exemplary gRNA targeting TIGIT). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting PD-1 (see e.g., WO2015 / 161276, or W02017 / 152015 for exemplary gRNA targeting PD-1).

[0227] In some embodiments the gRNA for use in the disclosure is a gRNA targeting an adenosine receptor, such as adenosine A2a receptor (ADORA2a) (see e.g., W02020 / 168300 for exemplary gRNA targeting ADORA2a). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting aTGF beta receptor, such as TGFbetaR2 (see e.g., W02020 / 168300 for exemplary gRNA targeting TGFbetaR2). In some embodiments, the gRNA for use in the disclosure is a gRNA targeting the gene encoding cytokine-inducible SH2-containing protein (CISH) (see e.g., W02020 / 168300 for exemplary gRNA targeting CISH).

[0228] In some embodiments, RNA-guided nuclease-encoding and / or gRNA encoding DNA, can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof. In some embodiments the nucleic acid encoding the RNA-guided nuclease (e.g., a Cas) and / or gRNA is delivered by AAV. Nucleic acids for gene editing can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells.

[0229] In some embodiments the RNA-guided nuclease and gRNA are delivered into cells as a ribonucleoprotein (RNP) complex. In some embodiments, the Cas and gRNA are separately purified and then assembled to form the RNP. In some embodiments, one or more RNP complexes are delivered to the cell sequentially in any order, or simultaneously. In some embodiments the RNP complex is delivered into cells by electroporation. In some embodiments the RNP complex is delivered into cells using lipid nanoparticles.

[0230] In one non-limiting example, to make the RNP complex, crRNA and tracrRNA can be mixed at a 1:1, 2:1, or 1:2 ratio of concentrations between about 50 pM and about 500pM (for example, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 35, 375, 400, 425, 450, 475, or 500pM), preferably between 100 pM and about 300 pM, most preferably about 200 pM at 95C for about 5 min to form a crRNA:tracrRNA complex (i.e., the guide RNA). The crRNA:tracrRNA complex can then be mixed with between about 20pM and about 50pM (for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 48,49, or 50pM) final dilution of a Cas endonuclease (such as, for example, Cas9).

[0231] In particular embodiments, introduction of an RNP complex into NK cells, such as expanded NK cells enriched for g-NK cells as described in Section II, is by electroporation. Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane. The application of the electric filed cause a charge gradient across the membrane which draws the charged molecules such as, nucleic acid, across the cell membrane. Thus, in one aspect, disclosed herein are methods of genetically modifying an NK cell comprising obtaining guide RNA (gRNA) specific for a target DNA sequence in the NK cell; and b) introducing via electroporation into a target NK cell, a ribonucleoprotein (RNP) complex comprising a Cas endonuclease (e.g., Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the target sequence within the genomic DNA of the NK cell.

[0232] In some aspects, the guide sequence is any polynucleotide sequence comprising at least a sequence portion that has sufficient complementarity with a target polynucleotide sequence, such as a gene encoding FcRy, PLZF, HELIOS, SYK, DAB2 or EAT2, to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. Typically, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0233] In some embodiments, a CRISPR enzyme (e.g., Cas9 nuclease) in combination with (and optionally complexed with) a guide sequence is delivered to the cell. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus.

[0234] In one embodiment of the invention, the DNA break-inducing agent is an engineered homing endonuclease (also called a "meganuclease"). Homing endonucleases are a group of naturally-occurring nucleases which recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA- repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757- 3774). The LAGLID ADG homing endonucleases with a single copy of the LAGLID ADG motif form homodimers, whereas members with two copies of the LAGLID ADG motif are found as monomers.

[0235] Another method of decreasing FcRy chain expression, activity and / or signaling involves introducing an inhibitory nucleic acid, such as an inhibitory RNA, into the cell that targets, e.g., iscomplementary to, a target gene transcript, such as an FcRy, PLZF, HELIOS, SYK, DAB2 or EAT2 gene transcript, thereby reducing expression of the gene product. For example, the nucleic acid may target FcRy chain mRNA. In other embodiments, the inhibitory nucleic acid may target the mRNA of a gene that regulates transcription or translation of the FcRy chain gene, such as a transcription factor, for example PLZF or HELIOS mRNA. In some embodiments the nucleic acid targets the mRNA of gene encoding a protein involved in FcRy-mediated signaling, such as SYK, DAB2 or EAT-2 mRNA.

[0236] The presently disclosed subject matter takes advantage of RNAi technology (for example shRNA, siRNA and miRNA molecules and ribozymes) to cause the down regulation of cellular genes, a process referred to as RNA interference (RNAi). As used herein, “RNA interference” (RNAi) refers to a process of sequence-specific post-transcriptional gene silencing mediated by a small interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules, miRNA molecules or synthetic hammerhead ribozymes. See generally Fire et al., Nature 391:806-811, 1998, and U.S. Pat. No. 6,506,559. The process of RNA interference (RNAi) mediated post-transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism that has evolved to prevent the expression of foreign genes (Fire, Trends Genet 15:358-363, 1999).

[0237] In some embodiments, a recombinant virus comprising nucleic acid encoding the RNA can be produced. Engineering retroviral vectors is known to those having ordinary skill in the art. Such a skilled artisan would readily appreciate the multiple factors involved in selecting the appropriate virus and vector components needed to optimize recombinant virus production for use with the presently disclosed subject matter without the necessity of further detailed discussion herein. As one non-limiting example, a retrovirus can be engineered comprising DNA encoding an shRNA comprising an siRNA.

[0238] The gene expression may be reduced permanently, transiently, or inducibly. Suitable inducible systems are well known and include eukaryotic promoters responsive to heavy metals, Lac / VP16, and the tetracycline repressor system.

[0239] On the other hand, it may be beneficial to permanently reduce expression of the gene, for example by producing a cell line with a deletion, substitution, or insertion that causes inactivation of the gene.

[0240] Retroviral systems can be used to introduce cDNAs into NK cells. Methods of eukaryotic cell transfection and prokaryotic cell transformation are well known in the art. The choice of host cell dictates the preferred technique for introducing the polynucleotide of interest. Introduction of polynucleotides into an organism may also be done with ex vivo techniques that use an in vitro method of transfection, as well as established genetic techniques, if any, for that particular organism.

[0241] Other vectors and packaging cell lines have been used in the preparation of genetically modified variants of NK cells and can be used equivalently herein. Retroviral transduction systems have also been successfully used to transduce a variety of genes into NK cells. By way of example, thesealternative methods include, but are not limited to, the p-JET vector in conjunction with FLYA13 packaging cells (Gerstmayer et al., 1999), the plasmid-based kat retroviral transduction system, and DFG-hIL-2-neo / CRIP (Nagashima et al., 1998). Electroporation and “gene gun” introduction of the vector into the packaging cells is also practiced. Use of the pBMN-IRES-EGFP vector in combination with the Phoenix- Amphotropic packaging cell line is convenient in that it provides high efficiencies of Phoenix- Amphotropic cell transfection. The use of Moloney ETR promoters results in a high level of CD16 expression; the virus is produced at high titers. The efficiency of NK transduction is improved over other vectors that have been used to transduce NK cells; and the vector provides adequate space to accommodate the CD16 cDNA or alternative inserts. The pBMN-IRES-EGFP vector further incorporates genes for enhanced green fluorescent protein (EGFP), which can be used as an endogenous surrogate marker for gene expression. The Phoenix cell line stably expresses this vector in episomal form along with producing other viral components, thus allowing the cells to stably produce virus for an extended period of time.

[0242] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0243] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Eaboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0244] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0245] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0246] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into ahost cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0247] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 deg. C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0248] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise gene edit the NK cell in accord with the provided methods, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR or "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots.

[0249] In some embodiments, the gene edited g-NK cells can be further selected and expanded such as by methods described in Section II.

[0250] In some embodiments, the engineered g-NK cells of the composition express a CAR. In some embodiments, the g-NK cell is engineered with a bispecific CAR or multiple different CARs. In some embodiments, the CAR or CARs are directed to target antigens expressed by cells of the HLA-E expressing cancers. In particular embodiments, the CAR or CARs are directed to a B cell antigen. Exemplary CARs and methods for engineering cells are described in Section IV.

[0251] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid encoding a CAR.

[0252] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR. In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid encoding a CAR.

[0253] In some embodiments, the engineered g-NK cells of the composition express one or more other additional heterologous protein agent. In some embodiments, the engineered g-NK cells express animmunomodulator, such as a cytokine. In some embodiments, the engineered g-NK cells also express a secreted antibody. In some embodiments, the immunomodulator is an agent that is capable of regulating immune function of the NK cell. In some embodiments, an immunomodulator may be an immunoactivator. In other embodiments, an immunomodulator may be an immunosuppressant. In some embodiments, the immunomodulator is an exogenous cytokine, such as an interleukin or a functional portion thereof. Exemplary immunomodulators and methods for engineering cells are described in Section IV.

[0254] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described).

[0255] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid encoding animmunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid encoding an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described).

[0256] In some embodiments, greater than at or about 20% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 50% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 80% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of total cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described).

[0257] In some embodiments, greater than at or about 20% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 30% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 40% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 50% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 60% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 70% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described). In some embodiments, greater than at or about 80% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 90% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membrane-bound as described). In some embodiments, greater than at or about 95% of g-NK cells in the composition comprise a heterologous nucleic acid(s) encoding a CAR and an immunomodulator (e.g., cytokine, either secreted or membranebound as described).B. Cancers

[0258] In some embodiments, the provided methods relate to treating an HLA-E expressing cancer. In some embodiments, the HLA-E expressing cancer can be a solid tumor. In some embodiments, the HLA-E expressing cancer can include, but is no limited due, a head and / or neck cancer, a gynecological cancer, a gastric cancer, a colorectal cancer, and a laryngeal cancer. In some embodiments, the gynecological cancer can include, but is not limited to, an ovarian cancer, a cervical cancer, or a breast cancer. In some embodiments, the HLA-E expressing cancer can be pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the HLA-E expressing cancer is a hematologic malignancy, such as a leukemia, lymphoma or a myeloma. In some embodiments, the HLA-E expressing cancer can be a B-cell expressing cancer. In particular embodiments, the HLA-E expressing cancer can be a Non-Hodgkin’s lymphoma (NHL). In some embodiments, the HLA-E expressing cancer can be an acute myeloid leukemia (AML). In some embodiments, the HLA-E expressing cancer can be a multiple myeloma (MM). In some of any of the provided embodiments, the subject has a relapsed / refractory cancer inwhich the subject has failed (relapsed or is refractory to) one or more prior lines of treatment (e.g. one or more prior therapy regimens) for treating the cancer. In some embodiments, a prior treatment has not worked (refractory to treatment) or the cancer has returned after the prior treatment or treatments (relapsed). In some embodiments, the subject has received 2 to 12 prior treatment regimens, such as 3 to 12 prior treatment regimens. In some embodiments, the subject has failed due to relapse or being refractory to the prior regimens. In particular embodiments, the subject is in relapse or is refractory to the immediate prior therapy prior to be treated in accord with the prior methods. In some embodiments, at the time of treatment, the subject has either progressive disease or best response to most recent chemotherapy containing regimen is stable disease (SD) for less than or equal to 12 months, and has failed at least 2 lines of systemic chemotherapy.

[0259] In some embodiments, HLA-expressing cancer is associated with expression of human leukocyte antigen-E (HLA-E). In some embodiments, HLA-E is also referred to as MHC-E. HLA-E (or MHC-E) is a major histocompatibility complex lb (MHC lb) cell surface protein which performs an essential role in the adaptive immune system. There are several non-classical MHC molecules (including HLA-E, HLA-F, and HLA-G), which have immune regulatory functions. HLA-E, which is encoded by an HLA-E gene (i.e., NCBI Gene ID: 3133), is a heterodimer class lb molecule that primarily functions as a ligand for the NK cell receptors CD94 / NKG2A (NKG2A) and CD94 / NKG2C (NKG2C). Specifically, HLA-E enables NK cells to monitor other MHC class I molecule expression and to tolerate self-expression. A peptide that binds to HLA-E is one which associates with HLA-E on a cell surface, forming a complex that is capable of interacting with a specific cell receptor on an immune cell. In some aspects, HLA-E can bind to peptides also recognized by MHC class I, albeit with lower affinity (Pietra et al. (2010) Journal of Biomedicine and Biotechnology, 1-8). The expression of other class I MHC molecules can regulate the expression of HLA-E, thereby allowing NK cells to monitor the state of the MHC class I dependent antigen presentation pathway in potential target cells. The level of cell surface HLA -E can regulate the NK cell cytotoxicity towards autoreactive immune cells and virally infected cells.

[0260] Natural killer cells modulate their activity through cell-surface receptors such as CD94 / NKG2A (NKG2A) and CD94 / NKG2C (NKG2C). NKG2A and NKG2C bind to the non-classical MHC-Ib HLA-E protein: peptide complexes. In their classical role, NK cells bind HLA-E in complex with a constrained set of peptides (largely resembling VMX1PRTX2X3L (SEQ ID NO: 19), wherein Xi is A or P, X2 is L or V and X3 is I, L, F, or V), derived signal peptides from signal peptides of MHC class la molecules. NKG2A binding of HLA-E inhibits NK cells whereas NKG2C binding activates NK cells. NKG2A typically possess higher peptide binding affinity that the NKG2C.

[0261] In some embodiments the HLA-E-peptide complex interacts with a NKG2A receptor, a NKG2C receptor, or both. The g-NK cells described herein are superior because they have higherexpression of NKG2C and lower expression of NKG2A, effectively skewing NKG2A / NKG2C regulation, and enabling the described g-NK cells to be activated as opposed to be inhibited upon NKG2C and HLA-E binding.

[0262] In certain embodiments, surface expression of HLA-E is sufficient to protect target cells from lysis by CD94 / NKG2A+ NK cells. In particular embodiments, the described g-NK cells in the provided methods herein are CD94 / NKG2A- NK cells. In certain embodiments, surface expression of HLA-E does not protect target cells from lysis by CD94 / NKG2C+ NK cells. In particular embodiments, the described g-NK cells in the provided methods herein are CD94 / NKG2C+ NK cells. In particular embodiments, the target cells are not protected by lysis from the described g-NK cells.

[0263] In some embodiments, the target cells are cancer cells. In certain embodiments, the target cells are B cells. In some embodiments, the g-NK cells described herein can effectuate potent killing of HLA-E expressing cells because the g-NK cells have low expression of the CD94 / NKG2A inhibitory receptor.

[0264] In some embodiments, the methods provided herein involve a determination, detection, quantification, or other assessment of the HLA-E expression. A subject to be treated in accord with the provided methods can be treated with or without a prior detection step to assess expression of HLA-E on the surface of cells, such as B cells or cancer cells. In some embodiments, the provided methods include a step of detecting an HLA-E nucleic acid or polypeptide in a biological sample from the subject (e.g., on a target cell) from an individual. A determination that a biological sample expresses HLA-E (e.g., prominently expresses; expresses HLA-E at a high level, high intensity of staining with an anti-HLA-E antibody, compared to a reference) indicates that the patient has an HLA-E expressing cancer that may have a strong benefit from treatment in accord with provided methods. In one embodiment, the method comprises determining the level of expression of an HLA-E nucleic acid or polypeptide in a biological sample and comparing the level to a reference level (e.g., a value, weak cell surface staining, etc.) corresponding to a healthy individual or to an individual that does not have a virus infection (e.g., EBV infection). A determination that a biological sample expresses HLA-E nucleic acid or polypeptide at a level that is increased compared to the reference level indicates that the subject has an HLA-E expressing cancer that can be treated in accord with provided methods. In some embodiments, a subject has an HLA-E expressing cancer that can be treated in accord with the provided methods if the determination of HLA-E expression shows that cells from a biological sample from the subject prominently expresses HLA-E nucleic acid or polypeptide. “Prominently expressed”, when referring to an HLA-E polypeptide, means that the HLA-E polypeptide is expressed in a substantial number of cells (e.g., B cells or cancer cells) taken from a biological sample (e.g., PBMCs) from a subject. While the definition of the term “prominently expressed” is not bound by a precise percentage value, in some examples a receptor said tobe “prominently expressed” will be present on at least 30%, 40%, 50° %, 60%, 70%, 80%, or more of the cells from a biological sample from the subject.

[0265] In some embodiments, cells of the subject with the HLA-E expressing cancer described in the methods provided herein has intermediate or high HLA-E expression. In some embodiments, the cells are B cells. In some embodiments, the cells are cancer cells. In certain embodiments, an increased HLA-E expression is an expression of HLA-E that is greater than a threshold, e.g., a predetermined threshold or a threshold value based on a reference. HLA-E expression can be assessed with techniques such as, but not limited to, flow cytometry, PCR-based methods include RT-PCR, immunohistochemistry, and confocal microscopy.

[0266] In some embodiments, HLA-E expression is assessed on cells from a peripheral blood biological sample from the subject. In some embodiments, the cells are PBMCs that include T cells and B cells. In some embodiments, HLA-E expression is assessed on T cells enriched or isolated from peripheral blood from the subject, e.g., by selection or isolation of cells that are CD3+, CD4+ or CD8+ cells. In some embodiments, HLA-E expression is assessed on B cells enriched or isolated from peripheral blood of the subject, e.g., by selection or isolation of cells that are CD19+. In some embodiments, HLA-E expression is assessed on total lymphocytes enriched or isolated from peripheral blood from the subject, e.g., by selection or isolation of cells that are CD45+.

[0267] In some embodiments, the HLA-E allele is HLA-E*01:01. In some embodiments, the HLA- E allele is HLA-E*01:03. The HLA-E*01:01 and HLA-E*01:03 differ in one amino acid substitution at position 107, in which an arginine for HLA-E*01:01 is substituted by a glycine for HLA-E*01:03 (Kraemer et al. J Immunol Res, 2014:Article ID 352160, 2014). In some embodiments, HLA-E genotyping is carried out by RT-PCR using HLA-E*01:01 and HLA-E*01:03 specific primers and probes (see e.g., Vietzen et al. J Infect Dis, 217:802-806, 2018; Paquay et al., Tissue Antigens, 74:514- 519, 2009).

[0268] In some embodiments, HLA-expression is assessed by flow cytometry using an antibody directed against HLA-E. Any of a variety of anti-HLA-E antibodies are known. Exemplary anti-HLA-E antibodies include, but are not limited to, clone 3D12 (IgGl), MEM-E / 07, MEM-E / 06, MEM-E / 08, or 1A4G3. In some embodiments, the antibody also may be an anti-HLA-E polyclonal antibody. In some embodiments, a cell, such as an immune cells (e.g., B cell) or cancer cells, is increased for expression of HLA-E if there is detectable presence of HLA-E on or in the cell that is at a level that is higher than the level of HLA-E expression on such cells from a healthy subject detected carrying out the same procedures under otherwise identical conditions. In some embodiments, the level of increased expression is increased at least or at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2.0-fold, 2.5-fold, 3.0-fold, 4.0-fold, 5.0-fold or more. In some embodiments, a cell, such as an immune cells (e.g., T cell or B cell), is increased for expression of HLA-E if there is detectable presenceof HLA-E on or in the cell that is at a level substantially similar to a reference level detected carrying out the same procedures under otherwise identical conditions, in which the reference level of expression is known to be higher than a level of HLA-E expression on normal or healthy cells or higher than a median or mean level among a plurality of normal or healthy cells.

[0269] In certain embodiments, HLA-E expression is assessed in a tissue sample obtained from the subject with the HLA-E expressing cancer. In particular embodiments, the tissue sample is a biopsy sample. In some embodiments, the HLA-E expression is assessed by immunofluorescence or immunohistochemistry analysis of cells of a biological sample. In some embodiments, the analysis is quantitative. In some embodiments, the HLA-E expression is measured, detected, and / or quantified by surface and / or intracellular staining. In some embodiments, staining of HLA-E of tumor cells was scored for by analyzing expression intensity and percentage surface area expression. In some embodiments, subjects considered to have high and low HLA-E expression can be distinguished based on 75th percentile of HLA-E expression scores of all analyzed tumor tissues.

[0270] In some embodiments, the biological sample is a PBMC sample and an antibody directed against HLA-E is incubated with smears of peripheral blood cells. In some embodiments, the biological sample is a tissue sample and the tissue to be assessed is fixed with a cell fixative agent. The fixative agent can include, but are not limited to, a fixative solution or a solution containing a chemical such as formaldehyde, glutaraldehyde or the like.

[0271] In some embodiments, the cells to be analyzed for HLA-E expression can be homogenized and prepared for Western blot analysis. Protein lysates can be probed with an anti-HLA-E antibody.

[0272] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein has an increased HLA-E polypeptide expression level compared to a reference level corresponding to a healthy individual. In some examples, the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and / or is within one or two standard deviation(s) below the median or mean value of HLA-E in samples obtained from a group of healthy individuals.

[0273] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein exhibits a fold increase in HLA-E polypeptide expression level compared to a reference expression level corresponding to a healthy individual. In particular embodiments, the fold increase is at least or at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or more than the reference expression level corresponding to a healthy individual. In alternative embodiments, the fold increase is 1.2-fold, 1.3-fold, 1.4-fold, 1.5- fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold more than the median or mean expression level corresponding to a group of healthy individuals.

[0274] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein has an increased HLA-E polypeptide expression level relative to a thresholdlevel. In some embodiments, the threshold level is the presence of a high number of cells in the stained tissue exhibiting the HLA-E staining. In some embodiments, the threshold level is a percentage of cells exhibiting HLA-E staining that is greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% or higher.

[0275] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein has similar HLA-E polypeptide expression level compared to that of a reference level in which the reference level of expression is known to be higher than a level of HLA-E expression on normal or healthy cells or higher than a median or mean level among a plurality of normal or healthy cells. In some embodiments, the reference level corresponds to the level of HLA-E expression as determined carrying out the same procedures under otherwise identical conditions on or in cell of a subject with an HLA-E expressing cancer known to have high expression of HLA-E on such cells. In some embodiments, the reference level corresponds to the level of HLA-E expression as determined carrying out the same procedures under otherwise identical conditions on or in cell of another subject with the same HLA-E expressing cancer that benefited from administered g-NK cells as described herein. In some examples, the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and / or is within one or two standard deviation(s) above or below the median or mean value of HLA-E in cells obtained from a group of subjects with the same HLA-E expressing cancer known to have high expression of HLA-E on such cells or from a group of subjects with the same HLA-E expressing cancer benefitting from administered g-NK cells as described herein. In particular embodiments, the fold increase is at least or at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or more than the reference expression level.

[0276] In some embodiments, the HLA-E expression is assessed by quantitative RT-PCR. In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein has increased HLA-E polynucleotide expression level compared to a reference level corresponding to a healthy individual. In some examples, the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and / or is within one or two standard deviation(s) below the median or mean value of HLA-E in samples obtained from a group of healthy individuals.

[0277] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein exhibits a fold increase in HLA-E polynucleotide expression level compared to a reference expression level corresponding to a healthy individual. In particular embodiments, the fold increase is at least or at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more than the reference expression level corresponding to a healthy individual. In alternative embodiments, the fold increase is at least or at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 2-fold,3-fold, 4-fold, or 5-fold more than the median or mean expression level corresponding to a group of healthy individuals.

[0278] In certain embodiments, the subject with the HLA-E expressing cancer described in the methods provided herein has a similar HLA-E polynucleotide expression level compared to that of a reference level corresponding to that of another subject with the same HLA-E expressing cancer benefitting from administered g-NK cells as described herein. In some examples, the reference level is within 25%, within 20%, within 15%, within 10% or within 5% and / or is within one or two standard deviation(s) below the median or mean value of HLA-E in samples obtained from a group of subjects with the same HLA-E expressing cancer benefitting from administered g-NK cells as described herein.

[0279] In some embodiments, the HLA-E expressing cancer is not associated with a viral infection. For example, cancer cells may upregulate HLA-E as a response to selective pressure by immune surveillance and in response to IFNg. In some embodiments, the HLA-E expressing cancer is associated with an infection. In some embodiments, the infection is non-viral. In some embodiments, the infection is viral.

[0280] In some embodiments, the HLA-E expressing cancer is associated with a viral infection, and in particular a viral infection that produce HLA-E stabilizing peptides to increase HLA-E infection on infected cancer and B cells that may otherwise exacerbate the disease or condition. Without wishing to be bound by theory, certain viral infections result in a restricted set of virus-specific peptides that can be presented on the surface of HLA-E present on the surface of virally-infected cells. The result is that the peptides can act to stabilize HLA-E on the surface of the cells, which is a viral infection strategy that commonly protects target cells from lysis by engagement of the HLA-E with the inhibitory receptor CD94 / NKG2A on NK cells. This then can drive infection and disease. However, in provided method the described g-NK cells compositions are enriched for cells that have reduced expression of the inhibitory receptor NKG2A (e.g., CD94 / NKG2A- NK cells) and increased expression of the activating receptor NKG2C (e.g., CD94 / NKG2C+ NK cells). In some embodiments, the g-NK cells described herein can effectuate potent killing of HLA-E expressing virally infected cells, such as cancer cells or B cells, because of the low expression of the CD94 / NKG2A inhibitory receptor and the high expression of the CD94 / NKG2C activating receptor. In particular embodiments, the infected target cells are not protected by lysis from the described g-NK cells.

[0281] In some embodiments, the viral infection is one that is implicated in the pathogenesis and / or known to increase the likelihood of a subject developing an HLA-E expressing cancer. In some embodiments, the HLA-E expressing cancer is one in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the HLA-E expressing cancer. In some embodiments, the viral infection can be causedby, but is not limited to, a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

[0282] In some embodiments, the subject is identified to have a viral infection associated with the HLA-E expressing cancer. In certain embodiments, the viral infection associated with the HLA-E expressing cancer can be, but is not limited to a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

[0283] In some embodiments, the viral infection is a cytomegalovirus (CMV) infection. In some embodiments, the HLA-E expressing cancer is one that is associated with a cytomegalovirus (CMV) infection, in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the HLA-E expressing cancer. In some of any such embodiments, the subject for treatment is or has been selected as having CMV infected cells.

[0284] In some embodiments, the viral infection is a Human papillomavirus (HPV). In some embodiments, the HLA-E expressing cancer is one that is associated with a Human papillomavirus (HPV), in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the HLA-E expressing cancer. In some of any such embodiments, the subject for treatment is or has been selected as having HPV infected cells. In some embodiments, the HLA-E expressing cancer is a head and / or neck cancer, a gynecological cancer, a gastric cancer, a colorectal cancer, and / or a laryngeal cancer.

[0285] In some embodiments, the viral infection is an influenza virus. In some embodiments, the HLA-E expressing cancer is one that is associated with an influenza virus, in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the HLA-E expressing cancer. In some of any such embodiments, the subject for treatment is or has been selected as having influenza virus infected cells.

[0286] In some embodiments, the viral infection is an Epstein-Barr virus (EBV) infection. In some embodiments, the HLA-E expressing cancer is one that is associated with EBV infection, in which it is probable or likely that a majority of the subjects with the disease or condition have a viral infection that is implicated in the pathogenesis or susceptibility to the HLA-E expressing cancer. In some of any such embodiments, the subject for treatment is or has been selected as having EBV infected cells.

[0287] Epstein-Barr virus (EBV) is a y-herpes virus that primarily infects B cells and human epithelial cells. The prominent hallmark of herpesviruses is the capacity to readily establish lifelong infection (latency) in their host, with EBV establishing latency mainly in B lymphocytes. In a latent state, herpesviruses usually do not produce disease. Once EBV’s initial lytic infection is brought under control, EBV latency persists in the individual's B cells for the rest of their life. In certain subjects,increased frequencies of EBV specific, HLA-E restricted CD8+ T-cells are found. Based on seroprevalence, 95% of adults carry EBV world- wide.

[0288] The virus has a well-established oncogenic potential and is associated with ~ 1 % of all human cancers and can cause a broad range of diseases ranging from lymphoproliferative diseases, inflammatory immune dysregulations, epithelial cancers to autoimmune diseases (Farrell, P. J. (2019) Annu. Rev. Pathol. Meeh. Dis. 14, 29-53; Wald A. & Corey L. (2007) Herpesviruses; Biology, Therapy and Immunoprohylacis, Cambridge University Press; Zhang, T. et al. (2014) Pathology - Research and Practice 210, 69-73).

[0289] In some embodiments, the provided methods are for treating an EBV associated disease or condition. In some embodiments, an EBV associated disease or condition is characterized by an EBV infection in the subject. In some embodiments, an EBV infection can be a primary EBV infection, a latent EBV infection or a latent EBV infection with a lytic EBV component. In some embodiments, the provided methods relate to prevention or reduction of latent EBV infection of B cells, and thus the treatment of diseases associated with EBV infection. In some embodiments, an EBV associated disease or condition is a disease associated with any one or more of the following: a) ill-controlled or uncontrolled EBV infection in a subject; b) latent EBV infection with a lytic EBV component in a subject; and c) uncontrolled proliferation of B cell lymphocytes latently infected with EBV in a subject.

[0290] In some embodiments, an EBV associated disease or condition is an HLA-E expressing cancer. In some embodiments, the HLA-E expressing cancer is a head and / or neck cancer, a gynecological cancer, a gastric cancer, a colorectal cancer, and / or a laryngeal cancer. In some embodiments, the gynecological cancer can include, but is not limited to, an ovarian cancer, a cervical cancer, or a breast cancer. In some embodiments, the HLA-E expressing cancer can be a B-cell expressing cancer. In particular embodiments, the HLA-E expressing cancer can be a Non-Hodgkin’s lymphoma (NHL). In some embodiments, the HLA-E expressing cancer can be an acute myeloid leukemia (AML).

[0291] In some embodiments, a subject to be treated has an EBV infection. In some embodiments, a subject is selected for treatment by identifying a subject that has an EBV infection. An EBV infection in a subject can be determined using methods known in the art. In some embodiments, a subject has a longterm EBV infection. In some embodiments, a subject can have an EBV infection for about 6 months or longer, about 9 months or longer, about 1 year or longer, about 2 years or longer, about 3 years or longer. In some embodiments, the EBV infection is asymptomatic.

[0292] In some embodiments, active EBV infection is detected in peripheral B cell populations. In some embodiments, active EBV infection is detected in CSF B cell populations. Methods for detection of active EBV infection can include, without limitation, detection of EBV proteins on the surface of B cells, where such markers include, without limitation: BILF-1, LMP1 and LMP2. In some embodiments,methods for detection of active EBV infection can include determining the presence of transcripts associated with active infection. In some embodiments, latent infection is characterized by limited expression of viral proteins, apart from, for example EBNA1, LMP1 and LMP2. In some embodiments, active infection can result in expression of a broader range of viral proteins, including for example BILF- 1, LMP1, LMP2, etc. Detection of such proteins or transcripts can be indicative of an EBV-driven HLA- E expressing cancer.

[0293] In some embodiments, a subject to be treated is EBV seropositive. In some embodiments, a subject is diagnosed for the presence of EBV-associated disease or condition, such as an EBV-associated HLA-expressing cancer, by detecting the presence of antibodies in serum. In some embodiments, the antibodies detected are IgG antibodies. In some embodiments, the antibodies detected are IgM antibodies. In some embodiments, the antibodies are anti-VCA IgM, anti-VCA IgG or anti-EBNA-1 IgG. The determination is optionally combined with detection of active EBV infection. A variety of methods may be utilized for the detection of antibodies. In some embodiments, any of a variety of immunoassays can be used to detect antibodies, such as by using ELISA.

[0294] In some embodiments, the subject has detectable EBV- viremia. In some embodiments, EBV load can be determined by assessing viral DNA from plasma samples and detected and quantified by PCR-based methods (see e.g., Aberle et al. J Clin Cirol., 25: S79-85, 2002). In any of the embodiments, a subject determined to have an EBV infection has an EBV DNA load of greater than or equal to about 5,000 copies / pg DNA in blood, such greater than or equal to about 10000 copies / pg, 25000 copies / pg, 50000 copies / pg, 75000 copies / pg, 100000 copies / pg, 125000 copies / pg or 150000 copies / pg, or any value between any of the foregoing. In some embodiments, a subject determined to have an EBV infection has greater than or equal to about 1,000 copies / 100 pl plasma, such as greater than or equal to about 1500 copies / 100 pl plasma, 2000 copies / 100 pl plasma, 2500 copies / 100 pl plasma, 3000 copies / 100 pl plasma, 3500 copies / 100 pl plasma, 4000 copies / 100 pl plasma, or 4500 copies / 100 pl plasma, or any value between any of the foregoing. In any of the methods described herein, the EBV DNA load in a subject in need of a treatment as described herein can be increasing over time. EBV DNA load can be measured using techniques known in the art.

[0295] In some embodiments, the subject is infected with an EBV strain encoding for a peptide variant that results in stable upregulation of HLA-E on the surface of immune cells, such as B cells, or of cancer cells. Cell surface stabilization of HLA-E requires loading with peptides, which can be derived from the signal sequences of MHC class I molecules or other proteins such as HSP60 at steady state. In some embodiments, the subject described in the provided methods herein may be infected by a virus (e.g., EBV), wherein the virus may give rise to HLA-E stabilizing peptides. In some embodiments, the peptide is an LMP-1 peptide. In some embodiments, the LMP-1 peptide is GGDPHLPTL (SEQ ID NO:20) or GGDPPLPTL (SEQ ID NO:21). In some embodiments, the subject is selected for thepresence of an EBV strain encoding for one or both peptides GGDPHLPTL (SEQ ID NO:20) or GGDPPLPTL (SEQ ID NO:21). In some embodiments, the peptide is an BZLFl-derived peptide. In some embodiments, the BZLFl-derived peptide is SQAPLPCVL (SEQ ID NO:22). In some embodiments, the subject is selected for the presence of an EBV strain encoding for the peptide SQAPLPCVL (SEQ ID NO:22). In some embodiments, peptides can be detected by PCR-based methods, such as by gene amplification using nested PCR, followed by sequencing (see e.g., Mbiribindi et al. Scientific Reports, 10:19973, 2020 for detection of LMP-1 variants and Lorenzetti et al., Clin Microb Infec, 20:0861-0869, 2014 for detection of BZLF1 peptides).1. Acute Myeloid Lymphoma

[0296] In some embodiments, the provided methods relate to treating an HLA-E expressing cancer, wherein the HLA-E expressing cancer is an acute myeloid leukemia (AML). In some embodiments, the methods relate to treating an acute myeloid leukemia (AML).

[0297] In one aspect, disclosed herein is a method of treating AML, wherein the method includes administering a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having AML. In some embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody. In other embodiments, the method further includes administering to the subject an antibody directed against a target antigen expressed by cells of the AML cancer, for example to promote ADCC by the co-administered g-NK cells. Examples of antibodies in such a provided combination therapy include any described in Section I.D.

[0298] Acute myeloid leukemia (AML) is a heterogeneous hematologic disorder characterized by clonal expansion of myeloid blasts in bone marrow, peripheral blood and other tissues, leading to transformed leukemia-initiating cells (LICs). However, the genetic makeup of AML cells and properties of the LIC population is heterogenous among patients. The presenting symptoms are usually nonspecific (e.g., fatigue, fever, malaise, weight loss) and reflect the failure of normal hematopoiesis. Anemia and thrombocytopenia are very common (75 to 90%). The whole blood cell (WBC) count may be decreased, normal, or increased.

[0299] Current treatment of AML remains unsatisfactory with a 5-year relapse-free survival rate lower than 30%. The basic induction regimen for AML includes cytarabine; along with daunorubicin or idarubicin. Some regimens include 6-thioguanine, etoposide, vincristine, and prednisone. FDA approved targeted therapies for AML also include the targeted BCL2 inhibitor venetoclax in combination with azacitidine or cytarabine (Wang et al., Nat. Comm., 2024).

[0300] In provided embodiments, AML diagnosis can be performed by a physician according to guidelines available, for example according to the World Health Organization (WHO) classification of AML (Brunning et al., World Health Organization Classificaiton of Tumors, 3, pp77-80; eds. Jaffe et al., Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues) andaccording to guidelines available for example at National Comprehensive Cancer Network (http: / / _www_nccn.Org / _professionals / _physician_gls / _f_guidelines_asp#site). The WHO classification incorporates clinical features, cytogenetics, immunophenotype, morphology and genetics in order to define biologically homogenous subgroups having therapeutic and prognostic relevance, and divides AML to four main subtypes: AML with recurrent genetic abnormalities, AML with multilineage dysplasia, therapy-related AML, and not otherwise categorized AML.

[0301] In some embodiments, the AML is associated with expression of human leukocyte antigen-E (HLA-E). Different types or subtypes of AML have different HLA-E expression or different expression of IFNy signaling, which is directly correlated to HLA-E expression (Wang et al., Nat. Comm., 2024). For example, it is known that acute monolytic AML (AML-M5), such as diploid monocytic AML, AML with a del 7 / 7q mutation, AML with a del 5 / 5q mutation, and AML that is relapsed or refractory (e.g., relapsed or refractory AML that has been treated with venetoclax), has higher expression of HLA-E and / or IFNy signaling as compared to non-monocytic AML (Wang et al., Nat. Comm., 2024; Wang et al., Blood, 2023). In some embodiments, a subject is selected for treatment according to the provided embodiments that has acute monolytic AML (AML-M5), such as diploid monocytic AML, AML with a del 7 / 7q mutation, AML with a del 5 / 5q mutation, or AML that is relapsed or refractory (e.g., relapsed or refractory AML that has been treated with venetoclax). In some embodiments, a subject is selected that has an AML that is relapsed or refractory (e.g., relapsed or refractory AML that has been treated with venetoclax).

[0302] In some embodiments, AML for treatment in accord with the provided methods is acute monocytic leukemia (M5). In some embodiments, AML for treatment in accord with the provided methods is diploid monocytic AML. In some embodiments, AML for treatment in accord with the provided methods is an AML that has a del7 / 7q mutation. In some embodiments, AML for treatment in accord with the provided methods is AML that has a del 5 / 5q mutation.

[0303] In provided embodiments, the subject for treatment is a subject that had an initial morphologic diagnosis of AML (“MDS / AML”) and then the time of treatment with g-NK cells either has (1) measurable residual disease (MRD), including MRD with various features as described further below; or (2) low burden relapsed or refractory (R / R) AML. In some embodiments, MRD means a complete response <5% BM blasts and molecularly measurable residual disease.

[0304] In some embodiments, AML for treatment in accord with the provided methods is relapsed or refractory AML. In some embodiments, the AML is relapsed AML. In some embodiments, the AML is refractory AML. While some relapsed or refractory AML (e.g., relapsed or refractory AML that has been treated with venetoclax) are known to be less susceptible to NK-cell mediated killing or NK cell therapy due to upregulation of HLA-E on the relapsed or refractory AML (Chandra et al., Transplantation and Cell Therapy, 2024), the present embodiments are based on the superior activity ofg-NK cells in this patient population. The g-NK cells described herein are superior for treatment of AML because the described g-NK cells have features, such as higher expression of NKG2C and lower expression of NKG2A, that enables the described g-NK cells to be activated as opposed to be inhibited upon increased HLA-E binding due to higher expression or upregulation of HLA-E.

[0305] In some embodiments, a subject selected for treatment in accord with the provided methods has relapsed or refractory AML. In some embodiments, the subject received 1 prior treatment regimen to treat the AML and replaced or was refractory to the prior treatment regimen. In some embodiments, the subject received 2 prior treatment regimens to treat the AML and replaced or was refractory to the prior treatment regimens. In some embodiments, the subject received 3 prior treatment regimens to treat the AML and replaced or was refractory to the prior treatment regimens. In some embodiments, the prior treatment regimen is any treatment regimen described herein. In some embodiments, the prior treatment regimen comprises idarubicin, cytrabine or hydroxyurea. In some embodiments, the relapsed or refractory AML has been treated with idarubicin, cytrabine or hydroxyurea. In some embodiments, the prior treatment regimen comprises BCL2 inhibitor. In some embodiments, the relapsed or refractory AML has been treated with a BCL2 inhibitor. In some embodiments, the prior treatment regimen comprises venetoclax. In some embodiments, the relapsed or refractory AML has been treated with venetoclax. Subjects with relapsed or refractory AML may have higher NKG2A expression on peripheral NK cells (Sandoval-Borrego et al., Arch. Med Res., 2016; Stringaris et al., Haematologica, 2014).

[0306] Various qualitative and / or quantitative methods may be used to determine if a subject has relapsed, is resistant, has developed or is susceptible to developing a resistance to treatment with a drug or a therapeutic. Symptoms that may be associated with relapse and / or resistance include, for example, a decline or plateau of the well-being of the patient, an increase in the size of a tumor or tumor burden, increase in the number of cancer cells, arrested or slowed decline in growth of a tumor or tumor cells, and / or the spread of cancerous cells in the body from one location to other organs, tissues or cells. Reestablishment or worsening of various symptoms associated with tumor may also be an indication that a subject has relapsed or has developed or is susceptible to developing resistance to a drug or a therapeutic. The symptoms associated with cancer may vary according to the type of cancer. For example, symptoms associated with AML may include weakness, tiredness, feeling dizzy or cold, headaches, frequent nosebleeds, excess bruising or bleeding gums.

[0307] In some embodiments, subjects with relapsed or refractory AML have measurable residual disease (MRD). In some embodiments, the subject with MRD is further characterized by (a) composite complete remission (cCR) including complete remission, complete remission with partial hematologic recovery (CRh), and complete remission with incomplete hematologic recovery (CRi); (b) bone marrow with MRD >0.1%, such as shown by multi-parameter flow cytometry difference from a normal assay; (c) a subject with second or higher cCR for AML have received at least one cycle of salvage therapy; (d) aMRD relapse after allogeneic stem cell transplantation (allo-SCT) or during consolidation or maintenance therapy; or (e) a subject with first remission cCR have had adverse risk AML per Dohner, 2022 criteria and must have received at least 1 cycle of intensive induction and 1 cycle of consolidation chemotherapy with intermediate or high-dose cytarabine based regimen; or 4 cycles of venetoclax-based lower intensity regimen containing hypomethylating agent (HMA) or low dose cytarabine (LDAC); or 4 cycles of HMA-based regimen.

[0308] In some embodiments, the AML is low disease burden AML. In some embodiments, the low disease burden AML is a relapsed or refractory (R / R) AML. Low burden AML, such as low burden relapsed or refractory AML, is indicative of a progressive AML disease, such as with <5% blast counts but not extremely high white counts in the peripheral blood. In some embodiments, a subject with low burden AML may be characterized by (a) having <25% blasts in peripheral blood and bone marrow; (b) relapsed or refractory disease in which (i) relapse is bone marrow (BM) blasts >5%, reappearance of blasts in the blood or development of extramedullary disease following achievement of CR / Cri / morphologic leukemia-free state (MLFS) or (ii) refractory is failure to achieve CR / Cri / MLFS following initial treatment with evidence of persistent leukemia by blood and / or BM evaluation with blasts >5%; (c) white blood cell (WBC) count that is 10,000 cells or under; (d) received an appropriate prior therapy for treating the AML, such as any prior therapy described herein; (e) for subjects that are younger (e.g., less than 40 years) or fit have had a first relapse following intensive chemotherapy eligible if the first remission (CR1) duration was < 12 months; (f) a subject that has relapsed with a persistent or new TP53 mutation irrespective of CR1 duration, primarily due to poor outcomes with TP53 mutation; (g) an older subject (e.g., greater than 40 years) or not fit that have relapsed on HMA+ venetoclax based regimen; (h) a subject with relapsed or refractory AML has not received more than 3 prior lines of therapies for active disease and 1 prior allo-SCT; (i) a subject with AML that has relapsed after allo-SCT if it has been >100 days since prior allo-SCT at the time of lymphodepletion and the subject has recovered from all transplant-related toxicities and is no longer on immunosuppression, with no more than grade 1 chronic GVHD, although a physiologic dose of steroids (e.g., < 5 mg prednisone or equivalent daily) is acceptable); and / or (j) subject has received a cytoreductive therapy until the day of lymphdepleting conditioning. In some embodiments, a subject with low burden relapsed AML is characterized by: >5% bone marrow blasts, reappearance of blasts in the blood, and / or development of extramedullary disease following a CR, CRi, or morphologic leukemia-free state (MLFS). In some embodiments, a subject with low burden refractory AML is characterized by a failure to achieve CR, CRi, of MLFS following initial treatment, with evidence of persistent leukemia by blood and / or a bone marrow evaluation with >5% blasts.

[0309] In some embodiments, the prior treatment regimen comprises: at least 1 cycle of purine analogue comprising an intensive induction chemotherapy regimen, e.g., FLAG-Ida, CLIA or CLAG-Mor similar regimens with or without venetoclax; at least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX-351 with venetoclax or similar regimens; at least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax; 2 cycles of venetoclax with HMA / LDAC + / - other agents; 4 cycles of HMA alone. In some embodiments, the prior treatment regimen comprises venetoclax. In some embodiments, the prior treatment regimen comprises venetoclax and / or a hypomethylating agent.

[0310] In some embodiments, a subject with AML, such as with low burden relapsed or refractory AML, is a young subject. In some embodiments, the subject is younger than 40 years old. In some embodiments, the subject is 15-39 years old. In some embodiments, the young subject had a remission duration following chemotherapy of <12 months. In some embodiments, a subject with AML, such as low burden relapsed or refractory AML, has persistent or new TP53 mutations. In some embodiments, a subject with AML, such as with low burden relapsed or refractory AML, relapsed following a prior treatment regimen comprising HMA and ventoclax.

[0311] In some embodiments, a subject with AML has AML with actionable mutations with available therapies, e.g., FLT3 or IDH1 / 2 inhibitors that have been exhausted or failed. In some embodiments, the subject with AML was using venetoclax and / or a hypomethylating agent until the day of conditioning. In some embodiments, the subject with AML has antecedent hematological disorder (AHD), e.g., aplastic anemia, myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) or myeloproliferative disorder or neoplasm.

[0312] In some embodiments, AML is AML with at least one genetic abnormality.

[0313] AML may be associated with a translocation between chromosomes 8 and 21, translocation or inversion in chromosome 16, translocation between chromosomes 15 and 17, or changes in chromosome 11. Common chromosomal rearrangements associated with AML are translocations t(8; 21)(q22; q22) ( AML1 / ETO), inv(16)(pl3; q22) or t(16; 16)(pl3; q22); (CBFp / MYHl l) or t(15; 17)(q22; ql2); (PML / RARA). Patients with these favorable chromosomal translocations may be more susceptible to treatment and achieve higher complete remission (CR) rates. In some embodiments, AML is associated with a translocation between chromosomes 8 and 21, translocation or inversion in chromosome 16, translocation between chromosomes 15 and 17, or changes in chromosome 11. In some embodiments, AML is associated with a chromosomal abnormality t(8; 21)(q22; q22) ( AML1 / ETO), inv(16)(pl3; q22) or t(16; 16)(pl3; q22); (CBFp / MYHl l) or t(15; 17)(q22; ql2); (PML / RARA).

[0314] Somatic mutations in various genes have been identified as being relevant to AML pathogenesis. These include mutations in fms-related tyrosine kinase 3 (FLT3), nucleophosmin (NPM1), isocitrate dehydrogenase 1(IDH1), isocitrate dehydrogenase 2 (IDH2), DNA (cytosine-5)- methyltransferase 3 (DNMT3A), CCAAT / enhancer binding protein alpha (CEBPA), U2 small nuclear RNA auxiliary factor 1(U2AF1), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2),structural maintenance of chromosomes 1A (SMC1A) and structural maintenance of chromosomes 3 (SMC3) (The Cancer Genome Atlas Research Network; N Engl J Med 368:2059-74, 2013).

[0315] Activating mutations in the FLT3 gene have been described in approximately 20- 30% of newly diagnosed AML patients. These include FLT3-ITD, internal tandem duplication mutations as a result of duplication and tandem insertion of parts of the juxtamembrane domain of the FET3 gene (Schnittger et al., Blood 100:59-66, 2002) and D835 mutations in the FET3 kinase domain. Patients with FET3-ITD mutations appear to have reduced overall survival (OS) with increased relapse rate (Kottaridis et al., Blood 98: 1752-9, 2001; Yanada et al., Eeukemia 19: 1345-9, 2005).

[0316] Mutations in IDH1 and IDH2 are present in about 15% of newly diagnosed patients. IDH1 mutations include substitutions R132H, R132X (X being any amino acid) and R100Q / R104V / F108E / R119Q / I130V and IDH2 mutations include substitutions R140Q and R172. IDH1 / 2 mutations are associated with poorer prognosis, except that IDH2R140Qis associated with somewhat prolonged survival (Molenaar et al., Biochim Biophys Acta 1846: 326-41, 2014). IDH1 / 2 mutation frequency increases with disease progression (Molenaar et al., Biochim Biophys Acta 1846: 326-41, 2014). In some embodiments, AME is associated with one or more mutations in a fms-related tyrosine kinase 3 (FET3), nucleophosmin (NPM1), isocitrate dehydrogenase 1(IDH1), isocitrate dehydrogenase 2 (IDH2), DNA (cytosine-5)- methyltransferase 3 (DNMT3A), CCAAT / enhancer binding protein alpha (CEBPA), U2 small nuclear RNA auxiliary factor 1(U2AF1), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), structural maintenance of chromosomes 1A (SMC1A) and structural maintenance of chromosomes 3 (SMC3).

[0317] In some embodiments, AME is associated with one or more mutations in fms-related tyrosine kinase 3 (FLT3) or FLT3-ITD. In some embodiments, AML is associated with one or more mutations in isocitrate dehydrogenase 1(IDH1) or isocitrate dehydrogenase 2 (IDH2). In some embodiments, AML is associated with mutations R132H, R132X or R100Q / R104V / F108L / R119Q / I130V in isocitrate dehydrogenase 1 (IDH1). In some embodiments, AML is associated with mutations R140Q and R172 in isocitrate dehydrogenase 2 (IDH2).

[0318] In some embodiments, AML is AML with multilineage dysplasia. AML associated with multilineage dysplasia is characterized by dysplasia in two or more myeloid cell lineage, and by at least 20% increased blasts in either the blood or bone marrow.

[0319] In some embodiments, AML is therapy-related AML. Therapy-related AML is a result of prior chemotherapy and / or radiation therapy, and may occur several years after exposure to the mutagenic agent. More than 90% of patients with therapy-related AML exhibit chromosomal abnormalities, including those of chromosomes 5 and / or 7. Chromosomal rearrangements may be identified using well-known methods, for example fluorescent in situ hybridization, karyotyping, Southern blot, or sequencing.

[0320] In some embodiments, AML is undifferentiated AML (MO), AML with minimal maturation (Ml), AML with maturation (M2), acute myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute erythroid leukemia (M6), acute megakaryoblastic leukemia (M7), acute basophilic leukemia, acute panmyelosis with fibrosis or myeloid sarcoma. In some embodiments, AML is acute monocytic leukemia (M5). In some embodiments, AML is diploid monocytic AML.

[0321] In some embodiments, AML is adult AML. In some embodiments, AML is pediatric AML.

[0322] In some embodiments, AML is in remission. AML in remission is typically defined as normocellular marrow with less than 5% blasts, normal peripheral blood count with >100,000 / mm3platelets and >l,000 / mm3neutrophils.

[0323] In some embodiments, the subject is undergoing hematopoietic stem cell transplantation (HSCT). In some embodiments, the HSCT is allogeneic, autologous or synegeneic, i.e. the donor is a twin. Autologous HSCT comprises the extraction of HSC from the subject and freezing of the harvested HSC. After myeloablation, the subject's stored HSC are transplanted into the subject. Allogeneic HSCT involves HSC obtained from an allogeneic HSC donor who has an HLA type that matches the subject.

[0324] "Hematopoietic stem cell transplantation" is the transplantation of blood stem cells derived from the bone marrow (in this case known as bone marrow transplantation), blood (such as peripheral blood and umbilical cord blood), or amniotic fluid.

[0325] “Undergoing hematopoietic stem cell transplantation” means that the patient did already receive, is receiving or will receive HSCT.

[0326] In some embodiments, the patient has completed chemotherapy and / or radiation therapy prior to HSCT. Patients may be treated with chemotherapy and / or radiation therapy prior to HSCT (so- called pre-transplant preparation) to eradicate some or all of the patient’s hematopoietic cells prior to transplant. The patient may also be treated with immunosuppressants in case of allogeneic HSCT. An exemplary pre-transplant preparation therapy is high-dose melphalan (see for example Skinner et al., Ann Intern Med 140:85-93, 2004; Gertz et al., Bone Marrow Transplant 34: 1025-31, 2004; Perfetti et al., Haematologica 91:1635-43, 2006). The radiation therapy that may be employed in pretransplant treatment may be carried out according to commonly known protocols in this field.2. Lymphoma

[0327] In some embodiments, the provided methods relate to treating an HLA-E expressing cancer, wherein the HLA-E expressing cancer is a lymphoma. In some embodiments, the lymphoma is nonHodgkin’s lymphoma (NHL). In some embodiments, the methods relate to treating an NHL.

[0328] In one aspect, disclosed herein is a method of treating a lymphoma, such as NHL, wherein the method includes administering a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having a lymphoma, such as NHL. In some embodiments, thecomposition of g-NK cells is administered as a monotherapy without co-administration of an antibody. In other embodiments, the method further includes administering to the subject an antibody directed against a target antigen expressed by cells of the lymphoma cancer, for example to promote ADCC by the coadministered g-NK cells. Examples of antibodies in such a provided combination therapy include any described in Section I.D. In some embodiments, the lymphoma is NHL.

[0329] In some embodiments, the NHL includes any of the known NHL subtypes, including those based on the WHO classification which in some cases categorizes subtypes based on cell type. In particular embodiments, the NHL is a B-cell lymphoma, including aggressive or indolent lymphomas. Examples of aggressive B-cell lymphomas include, but are not limited to diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), lymphoblastic lymphoma, Burkitt lymphoma, primary mediastinal largest B cell lymphoma (PMBCL), transformed follicular and transformed mucosa- associated lymphoid tissue (MALT) lymphoma, high-grade B cell lymphoma with double or triple hits (HBL), primary cutaneous DLBCL, primary DLBCL of the central nervous system, primary central nervous system (CNS) lymphoma, and acquired immunodeficiency syndrome (AIDS)-associated lymphoma. Examples of indolent B-cell lymphomas include, but are not limited to, follicular lymphoma (FL), marginal zone lymphoma (MZL), chronic lymphocytic leukemia / small-cell lymphocytic lymphoma (CLL / SLL), gastric musosa-associated lymphoid tissue (MALT) lymphoma, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia (WM), nodal martinal zone lymphoma (NMZL) and splenic marginal zone lymphoma (SMZL).

[0330] In some embodiments, the lymphoma is an advanced B-cell lymphoma, such as stage III or IV. Advanced B-cell malignancies include diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and marginal zone lymphomas (MZL). In 2016, the incidence of each in the US was: DLBLC (26.0%), FL (13%), MZL (7%), and MCL (3%) (Swerdlow, 2016). From initial diagnosis and treatment, recurrence of the B-cell lymphomas typically happens within a 3-year period. The first-line standard of treatment for DEBCE is rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (Cancer.gov, 2023). Treatment with rituximab may improve a patient’s overall survival (OS), but 30% to 40% of those treated will relapse and lose sensitivity to chemotherapy. While patients may be treated with autologous stem cell therapy, it is estimated that 50% of these patients’ relapse, with an OS rate of 5.7 months (Cancer.gov, 2023). Recently, drugs that inhibit Bruton tyrosine kinase have been approved for use in B-cell lymphomas including MCL (for patients who have received at least 1 prior therapy), and MZL (de Weerdt, 2017). While this inhibitor may be tolerable to patients, it does not cure the disease, and the addition of salvage drugs brings new adverse events (AEs) (de Weerdt, 2017).

[0331] The Eugano Classification may be used for evaluation, staging, and response assessment of subjects with lymphoma, such as non-Hodgkin’s lymphoma (NHE), as previously described (Cheson etal., J. Clin Oncol., 2014). For example, CT-based response is preferred for histologies with low or variable fluorodeoxy glucose (FDG) avidity and in regions of the world where PET-CT is unavailable. However, in the absence of a PET-CT scan, a mass that has decreased in size but persists is considered at best a PR in the absence of biopsy documenting absence of lymphoma, and the former term “CRu” is not to be considered. In studies exploring new agents in multiply relapsed disease where data are lacking regarding PET-CT and where assessment of disease control is more important than likelihood of cure, CT-based response may also be more relevant.

[0332] In some embodiments, NHL for treatment in accord with the provided methods is relapsed or refractory NHL. In some embodiments, the NHL is relapsed NHL. In some embodiments, the NHL is refractory NHL. In particular embodiments, the subject has relapsed or is refractory to one or more prior therapy. In some embodiments, a prior treatment or treatments have not worked (refractory to treatment) or the cancer has returned after the prior treatment or treatments (relapsed). In some embodiments, the subject is refractory to first-line chemotherapy or relapsed within 12 months of first-line chemotherapy. In some embodiments, the subject is relapsed or refractory to two or more prior lines of systemic therapy. In some embodiments, the subject has R / R disease and failed > 3 prior lines of therapy, such as 3 to 12 prior therapies. In some embodiments, the subject has either progressive disease or best response to most recent chemotherapy containing regimen is stable disease (SD) for less than or equal to 12 months, and has failed at least 2 lines of systemic chemotherapy.

[0333] In some embodiments, NHL for treatment in accord with the provided methods is advanced NHL.

[0334] In some embodiments, the NHL is associated with expression of human leukocyte antigen-E (HLA-E). Although high HLA-E expression is thought to inhibit the function of NK and T cells, leading to tumor immune escape, the present embodiments are based on the superior activity of g-NK cells in this patient population. The g-NK cells described herein are superior for treatment of NHL with high HLA-E expression because the described g-NK cells have features, such as higher expression of NKG2C and lower expression of NKG2A, that enables the described g-NK cells to be activated as opposed to be inhibited upon increased HLA-E binding due to higher expression or upregulation of HLA-E.

[0335] In some embodiments, a subject selected for treatment in accord with the provided methods has NHL, wherein the subject meets at least one or all of the following criteria: (a) a relapsed or refractory (R / R) NHL that is of any of the following types: DLBCL, high grade B-cell lymphoma (HGBL), transformed follicular lymphoma (tFL), primary mediastinal large B-cell lymphoma (PMBCL), FL, MZL, or MCL; (b) progressive disease or best response to most recent chemotherapy containing regimen was stable disease <12 months; (c) must have failed at least 2 lines of systemic chemotherapy and have the following additional criteria depending on type: (1) must have failed a line of chemoimmunotherapy that includes an anti-CD20 mAh plus anthracycline for DLBCL, HGBL, tFL, orPMBCL, (2) must have failed a line of chemoimmunotherapy that includes an anti-CD20 mAh plus an alkylating agent (i.e., anti-CD20 alone is not sufficient) for FL or MZL, (3) must have failed a line of chemoimmunotherapy that includes an anti-CD20 mAh plus an alkylating agent, as well as a Bruton’ s tyrosine kinase inhibitor for MCL; and (d) has at least one measurable lesion according to the revised International Working Group Response Criteria for Malignant Lymphoma (Cheson, 2014), wherein lesions that have been previously irradiated will be considered measurable only if progression has been documented following completion of radiation therapy. In some embodiments, the subject has previously received a prior CAR T-cell and / or CD3 bispecific therapy.

[0336] In one aspect, disclosed herein is a method of treating non-Hodgkin’ s lymphoma (NHL), wherein the method includes administering a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having NHL. In some embodiments, the composition of g- NK cells are administered as a monotherapy without co-administration of an antibody.

[0337] In other embodiments, the method further includes administering to the subject an antibody directed against a target antigen expressed by cells of the NHL cancer, for example to promote ADCC by the co-administered g-NK cells.

[0338] In one particular example, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the population of g-NK cells. An effective amount of the antibody can be selected by a skilled clinician, taking into consideration the particular antibody, the particular disease or condition (e.g. tumor or other disorder), the general condition of the subject, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is also administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, for example intravenously; however, injection or infusion to a tumor or close to a tumor (local administration) or administration to the peritoneal cavity can also be used. One of skill in the art can determine appropriate routes of administration.

[0339] The g-NK cells and the antibody can be administered sequentially or simultaneously. In some embodiments, the initiation of administration of the antibody can be before administration of the g- NK cells. In some embodiments, the initiation of administration of the antibody can be after administration of the g-NK cells. In some embodiments, the initiation of administration of the antibody can be simultaneously with the g-NK cells. In some cases, the g-NK cells can be administered at selected times that are distinct from the times when antibodies specific for the selected cancer type are administered.

[0340] In some embodiments, the NHL is associated with CD20. In some embodiments, a cell composition including g-NK cells can be targeted to tumors by combination therapy involving administration with an antibody that recognizes a tumor associated antigen that is CD20 for treating asubject with MM. In some embodiments, the NHL is associated with CD19. In some embodiments, a cell composition including g-NK cells can be targeted to tumors by combination therapy involving administration with an antibody that recognizes a tumor associated antigen that is CD 19 for treating a subject with MM. In some embodiments, the NHL is associated with CD30. In some embodiments, a cell composition including g-NK cells can be targeted to tumors by combination therapy involving administration with an antibody that recognizes a tumor associated antigen that is CD30 for treating a subject with MM. Examples of any such exemplary antibodies are described in Section I.D.3. Multiple Myeloma (MM)

[0341] In some embodiments, the provided methods relate to treating an HLA-E expressing cancer, wherein the HLA-E expressing cancer is a multiple myeloma (MM). In some embodiments, the methods relate to treating a multiple myeloma (MM).

[0342] In one aspect, disclosed herein is a method of treating MM, wherein the method includes administering a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having MM. In some embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody. In other embodiments, the method further includes administering to the subject an antibody directed against a target antigen expressed by cells of the MM cancer, for example to promote ADCC by the co-administered g-NK cells. Examples of antibodies in such a provided combination therapy include any described in Section I.D.

[0343] Advanced multiple myeloma (MM) is an incurable cancer of monoclonal plasma cells that reside primarily in the bone marrow. These monoclonal cells accumulate and eventually destroy the normal architecture of the BM, thereby disrupting the proper physiological function of bone (Durie, 2003). In terms of blood cancers, MM is the third-most frequent blood cancer in the United States (US) (Maiese, 2018).

[0344] The cytogenetically defined MM risk factor, patient transplant eligibility, serves as the primary method of allocating treatment. Treatments for MM include: a triple induction therapy, which includes a proteasome inhibitor, steroids, and an immunomodulatory drug, followed by an autologous transplant and maintenance therapy (e.g, lenalidomide). Multiple agents are often combined, including combinations of new or older therapies with steroids and / or conventional chemotherapy. Other treatments include chimeric antigen receptor (CAR) T-cell therapies and bispecific T-cell engaging molecules have been approved for use in patients with advanced MM. Two CAR T-cell products have been approved by FDA for treatment of relapsed and / or refractory (R / R) MM: Idecaptagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel). Both drugs are approved for use in R / R MM after 4 or more lines of therapy, including a proteasome inhibitor, immunomodulatory agent, and an anti-CD38 directed therapy. The FDA also granted accelerated approval to teclistamab-cqyv (Tecvayli, JanssenBiotech, Inc.) as the first bispecific B-cell maturation antigen-directed CD3 T-cell engager for adult patients with R / R MM who received at least 4 prior lines of therapy, including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 monoclonal antibody (mAh). Despite these treatments, MM remains incurable in most patients and represents a major unmet medical need (Willenbacher, 2018).

[0345] In some embodiments, MM for treatment in accord with the provided methods is relapsed or refractory MM. In some embodiments, the MM is relapsed MM. In some embodiments, the MM is refractory MM. In particular embodiments, the subject has relapsed or is refractory to one or more prior therapy. In some embodiments, a prior treatment or treatments have not worked (refractory to treatment) or the cancer has returned after the prior treatment or treatments (relapsed). In some embodiments, the subject is refractory to first-line chemotherapy or relapsed within 12 months of first-line chemotherapy. In some embodiments, the subject is relapsed or refractory to two or more prior lines of systemic therapy. In some embodiments, the subject has R / R disease and failed > 3 prior lines of therapy, such as 3 to 12 prior therapies. In some embodiments, the prior treatments include proteasome inhibitors, immunomodulatory agents and / or anti-CD38 monoclonal antibody (mAh). In some embodiments, the subject is triple refractory to prior treatment with > 1 proteasome inhibitors, > 1 immunomodulatory agents, and > 1 anti-CD38 mAh.

[0346] In some embodiments, the subject with MM, such as the subject with relapsed or refractory (R / R) MM, has at least one genetic abnormality.

[0347] In some embodiments, the subject with MM has a complex karyotype. In some embodiments, the complex karyotype includes the presence of three or more chromosomal abnormalities. In some embodiments, the complex karyotype includes the presence of five or more chromosomal abnormalities.

[0348] In some embodiments, the subject with MM has a TP53 mutation. In some embodiments, the subject with MM has a p53 deletion.

[0349] In some embodiments, the MM is associated with expression of human leukocyte antigen-E (HLA-E). It is known that greater HLA-E expression during MM, such as relapsed or refractory MM, correlates with worse progression-free survival in newly diagnosed patients with MM (Lagana et al., Blood, 2018). Although MM with high HLA-E expression is thought to inhibit the function of NK and T cells, leading to tumor immune escape, the present embodiments are based on the superior activity of g- NK cells in this patient population. The g-NK cells described herein are superior for treatment of MM (e.g., MM with high HLA-E expression) because the described g-NK cells have features, such as higher expression of NKG2C and lower expression of NKG2A, that enables the described g-NK cells to be activated as opposed to be inhibited upon increased HLA-E binding due to higher expression or upregulation of HLA-E.

[0350] In some embodiments, a subject selected for treatment in accord with the provided methods has MM, wherein the subject meets at least one or all of the following criteria: (a) documented diagnosis of MM requiring systemic therapy; (b) R / R disease after >3 prior lines of therapy, wherein induction with or without high-dose chemotherapy followed by autologous stem cell rescue and with or without maintenance therapy is a single regimen; (c) exposure to >1 proteasome inhibitors, >1 immunomodulatory agents, and >1 anti-CD38 mAb; (d) the subject achieved a response (minimal response or better) to at least 1 prior treatment regimen; (e) diagnosis of MM must be evidenced in end organ damage or tissue impairment following the established International Myeloma Working Group (IMWG) criteria; and (f) presence of a measurable M-protein in serum and / or urine and clonal plasma cells in the bone marrow or > 1 clonal plasmacytoma.

[0351] In one aspect, disclosed herein is a method of treating multiple myeloma (MM), wherein the method includes administering a composition of Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple myeloma (MM). In some embodiments, the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.

[0352] In some embodiments, the method further includes administering to the subject an antibody directed against a target antigen expressed by cells of the MM cancer, for example to promote ADCC by the co-administered g-NK cells.

[0353] In one particular example, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the population of g-NK cells. An effective amount of the antibody can be selected by a skilled clinician, taking into consideration the particular antibody, the particular disease or condition (e.g. tumor or other disorder), the general condition of the subject, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is also administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, for example intravenously; however, injection or infusion to a tumor or close to a tumor (local administration) or administration to the peritoneal cavity can also be used. One of skill in the art can determine appropriate routes of administration.

[0354] The g-NK cells and the antibody can be administered sequentially or simultaneously. In some embodiments, the initiation of administration of the antibody can be before administration of the g- NK cells. In some embodiments, the initiation of administration of the antibody can be after administration of the g-NK cells. In some embodiments, the initiation of administration of the antibody can be simultaneously with the g-NK cells. In some cases, the g-NK cells can be administered at selected times that are distinct from the times when antibodies specific for the selected cancer type are administered.

[0355] In some embodiments, the MM is associated with CD38. In some embodiments, a cell composition including g-NK cells can be targeted to tumors by combination therapy involving administration with an antibody that recognizes a tumor associated antigen that is CD38 for treating a subject with MM. Examples of any such exemplary antibodies are described in Section I.D.C Methods of Dosing G-NK Cell Compositions

[0356] In some embodiments, a single dose of g-NK cells is administered to the subject. In some embodiments, multiple doses of g-NK cells are administered to the subject in a predetermined number of doses. In some embodiments, the composition of g-NK cells is administered as a plurality of doses. In some embodiments, the doses of the plurality are for a predetermined number of doses. In some embodiments, the g-NK cells are administered once a week, twice a week, three times a week, once every two weeks, once every three weeks or once a month. In some embodiments, the g-NK cells are administered once a week. In some embodiments, the g-NK cells are administered once every 1 day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days. In some embodiments, the g-NK cells are administered once every 2 days. In some embodiments, the g-NK cells are administered once every 7 days. In some embodiments, the number of doses of the g-NK cells is two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, 9 doses, 10 doses, 11 doses or 12 doses. In some embodiments, the number of doses is 2 doses of g-NK cells. In some embodiments, the number of doses is 3 doses of g-NK cells. In some embodiments, the number of doses is 4 doses of g-NK cells. In some embodiments, the number of doses is 5 doses. In some embodiments, the number of doses is 6 doses. In some embodiments, the number of doses is 7 doses. In some embodiments, the number of doses is 8 doses. In some embodiments, the number of doses is 9 doses. In some embodiments, all doses of the g-NK cells are administered within a month of the first dose.

[0357] In some embodiments, a dose of g-NK is administered once weekly (Q1W or QW). In some embodiments, a dose of g-NK cells is administered twice weekly. In some embodiments, a dose of g-NK cells is administered three times weekly (or thrice weekly), which can be administered every other day (Q2D).

[0358] In some embodiments, the number of doses is 3 doses in a cycle, which may be repeated. For example, a dose of g-NK cells is administered on Day 0 (first dose), Day 7 and Day 14. In some embodiments, the cycle is a 28-day cycle. In some embodiments, the cycle is repeated at least one time. In some embodiments, a dose of g-NK cells is dosed at a frequency of every two days (i.e. every other day, Q2D) for a predetermined number of doses. In some embodiments, the number of doses is 3 doses in a cycle, which may be repeated. For example, a dose of g-NK cells is administered on Day 0 (firstdose), Day 2 and Day 4. In some embodiments, the cycle is a 7-day cycle. In some embodiments, the cycle is repeated at least one time.

[0359] In some embodiments, a second dose of g-NK cells is administered at or about at 24 hours after a first dose of g-NK cells. In some embodiments, a third dose of g-NK cells is administered at or about at 24 hours after a second dose of g-NK cells.

[0360] In some embodiments, the doses of g-NK cells are administered as part of a 7-day cycle. In some embodiments, the 7-day cycle is repeated one to three times. In some embodiments, the 7-day cycle is repeated one time (for two 7-day cycles total).

[0361] In some embodiments, doses of the composition of g-NK cells are administered as two doses in a 7-day cycle. In some embodiments, doses of the composition of g-NK cells are administered as three doses in a 7-day cycle.

[0362] In some embodiments, the composition of g-NK cells is administered from two total doses to six total doses. In some embodiments, the composition of g-NK cells is administered as two or four total doses. In some embodiments, the composition of g-NK cells is administered as three or six total doses.

[0363] In some embodiments, the g-NK cells are administered once weekly. In some embodiments, the number of once weekly doses is two doses. In some embodiments, the number of once weekly doses is three doses. In some embodiments, the number of once weekly doses is four doses. In some embodiments, the once weekly doses are administered in consecutive weeks. For example, the g-NK cells may be administered in a cycling regimen involving more than one 7-day cycle carried out consecutively, each with once weekly administration of the g-NK cells. In some embodiments, the number of consecutive weeks (or consecutive 7-days cycles) is 2, 3, 4 or 5. In some embodiments, the g- NK cells are administered on Day 0 (first dose), Day 7 and Day 14. In some embodiments, the g-NK cells are administered on Day 0 (first dose), Day 7, Day 14 and Day 21. In some embodiments, the g-NK cells are administered on Day 0 (first dose), Day 7, Day 14, Day 21 and Day 28. In some embodiments, the g-NK cells are administered on Day 0 (first dose), Day 7, Day 14, Day 21, Day 28 and Day 35.

[0364] In some embodiments, the g-NK cells are administered twice a week (twice weekly). In some embodiments, the predetermined number of twice weekly doses is two doses. In some embodiments, the predetermined number of twice weekly doses is four doses. In some embodiments, the twice weekly doses are administered for 1 week, 2 weeks, 3 weeks, 4 weeks or more. In some embodiments, one (1) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, two (2) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, three (3) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, four (4) twice weekly dose of the composition of g-NK cells is administered. In some embodiments, the twice weekly doses are administered in consecutive weeks.

[0365] In some embodiments, the g-NK cells are administered three times a week (thrice weekly). In some embodiments, the g-NK cells are administered every other day (Q2D), such as on Day 0 (first dose), Day 2 and Day 4 of a week (such as a 7-day cycle). In some embodiments, the predetermined number of thrice weekly doses is three doses. In some embodiments, the predetermined number of thrice weekly doses is six doses. In some embodiments, the thrice weekly doses, such as administered Q2D, are administered in consecutive weeks. For example, the g-NK cells may be administered in a cycling regimen involving more than one 7-day cycle carried out consecutively, each with thrice weekly, such as Q2D, administration of the g-NK cells. In some embodiments, the thrice weekly doses are administered for 1 week, 2 weeks, 3 weeks, 4 weeks or more. In some embodiments, one (1) thrice weekly dose of the composition of g-NK cells is administered (e.g, Day 0, Day 2 and Day 4 of the first week). In some embodiments, two (2) thrice weekly dose of the composition of g-NK cells is administered (e.g, Day 0, Day 2 and Day 4 of the first week and Day 0, Day 2 and Day 4 of the second week). In some embodiments, three (3) thrice weekly dose of the composition of g-NK cells is administered (e.g, Day 0, Day 2 and Day 4 of the first week; Day 0, Day 2 and Day 4 of the second week; Day 0, Day 2 and Day 4 of the third week). In some embodiments, four (4) thrice weekly dose of the composition of g-NK cells is administered (e.g, Day 0, Day 2 and Day 4 of the first week; Day 0, Day 2 and Day 4 of the second week; Day 0, Day 2 and Day 4 of the third week; and Day 0, Day 2 and Day 4 of the fourth week). In some embodiments, the thrice weekly doses are administered in consecutive weeks.

[0366] In some embodiments the twice weekly dose is administered in a cycling regimen. In some embodiments, the cycling regimen is a 7 day cycle. In some embodiments, the twice weekly dose is administered two times in the 7 day cycle. In some embodiments, the 7 day cycle is repeated twice. In some embodiments, the 7 day cycle is repeated three times. In some embodiments, the cycling regimen is a 14 day cycle. In some embodiments, the twice weekly dose is administered four times in the 14 day cycle. In some embodiments, the 14 day cycle is repeated twice. In some embodiments, the 14 day cycle is repeated three times.

[0367] In some embodiments the thrice weekly dose is administered in a cycling regimen. In some embodiments, the cycling regimen is a 7 day cycle. In some embodiments, the thrice weekly dose is administered three times in the 7 day cycle. In some embodiments, the 7 day cycle is repeated twice. In some embodiments, the 7 day cycle is repeated three times. In some embodiments, the cycling regimen is a 14 day cycle. In some embodiments, the thrice weekly dose is administered six times in the 14 day cycle. In some embodiments, the 14 day cycle is repeated twice. In some embodiments, the 14 day cycle is repeated three times.

[0368] In some embodiments, the methods of treatment or uses involve administration of an effective amount of a composition containing a composition of expanded NK cells produced by the provided method to an individual. In some embodiments, from at or about 105to at about 1012, or from ator about 105and at or about 108, or from at or about 106and at or about 1012, or from at or about 108and at or about 10”, or from at or about 109and at or about IO10of such expanded NK cells is administered to an individual subject. In some embodiments, a dose of cells containing at or greater than at or about 105, at or greater than at or about 106, at or greater than at or about 107, at or greater than at or about 108, at or greater than at or about 109, at or greater than at or about IO10, at or greater than at or about 10”, or at or greater than at or about 1012of such expanded NK cells are administered to the individual. In some embodiments, from or from about 106to IO10of such expanded NK cells per kg are administered to the subject.

[0369] In some embodiments, the methods of treatment or uses involve administration of an effective amount of any of the provided NK cell compositions, including any as described herein, to an individual. In some embodiments, from at or about 105to at about 1012, or from at or about 105and at or about 108, or from at or about 106and at or about 1012, or from at or about 108and at or about 10”, or from at or about 109and at or about 1010of NK cells from any of the provided compositions is administered to an individual subject. In some embodiments, a dose of cells containing at or greater than at or about 105, at or greater than at or about 106, at or greater than at or about 107, at or greater than at or about 108, at or greater than at or about 109, at or greater than at or about 1010, at or greater than at or about 10”, or at or greater than at or about 1012of NK cells from any of the provided compositions are administered to the individual. In some embodiments, from or from about 106to 1010of NK cells of any of the provided compositions per kg are administered to the subject.

[0370] In some embodiments, each dose of g-NK cells may be from at or about from at or about 1 x108cells to at or about 50 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 108cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 10 x 109cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 20 x109cells of the composition of g-NK cells.

[0371] In some embodiments, the methods of treatment comprises administering an effective amount of a composition containing g- NK cells to an individual. In some embodiments, from at or about 105to at about 1012g-NK cells, or from at or about 105and at or about 108g-NK cells, or from at or about 106and at or about 1012g-NK cells, or from at or about 108and at or about 10” g-NK cells, or from at or about 109and at or about 1010g-NK cells. In some embodiments, a dose of cells containing at or greater than at or about 105g-NK cells, at or greater than at or about 106g-NK cells, at or greater than at or about 107g-NK cells, at or greater than at or about 108g-NK cells, at or greater than at or about 109g- NK cells, at or greater than at or about 1010g-NK cells, at or greater than at or about 10” g-NK cells, orat or greater than at or about 1012g-NK cells are administered to the individual. In some embodiments, from or from about 106to IO10g-NK cells / kg are administered to the subject.

[0372] In some cases, expansion achieved by the provided methods from an initial source of NK cells obtained from a single donor can produce a composition of g-NK cells to provide a plurality of individual doses for administration to a subject in need. As such, the provided methods are particularly suitable for allogeneic methods. In some cases, a single expansion from a starting population of NK cells isolated from one donor in accord with the provided methods can result in greater than or greater than about 20 individual doses for administration to a subject in need, such as at or about 30 individual doses, 40 individual doses, 50 individual doses, 60 individual doses, 70 individual doses, 80 individual doses, 90 individual doses, 100 individual doses, or an individual dose that is a value between any of the foregoing. In some embodiments, the individual dose is from at or about 1 x 105cells / kg to at or about 1 x 107cells / kg, such as from at or about 1 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 1 x 105cells / kg to at or about 5 x 105cells / kg, from at or about 1 x 105cells / kg to at or about 2.5 x 105cells / kg, from at or about 2.5 x 105cells / kg to at or about 1 x 107cells / kg, from at or about 2.5 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 2.5 x 105cells / kg to at or about 5 x 105cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 107cells / kg, from at or about 5 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 1 x 106cells / kg to at or about 1 x 107cells / kg, from at or about 1 x 106cells / kg to at or about 7.5 x 106cells / kg, from at or about 1 x 106cells / kg to at or about 5 x 106cells / kg, from at or about 1 x 106cells / kg to at or about 2.5 x 106cells / kg, from at or about 2.5 x 106cells / kg to at or about 1 x 107cells / kg, from at or about 2.5 x 106cells / kg to at or about 7.5 x 106cells / kg, from at or about 2.5 x 106cells / kg to at or about 5 x 106cells / kg, from at or about 5 x 106cells / kg to at or about 1 x 107cells / kg, from at or about 5 x 106cells / kg to at or about 7.5 x 106cells / kg, or from at or about 7.5 x 106cells / kg to at or about 1 x 107cells / kg. In some embodiments, the individual dose is from at or about 1 x 105cells / kg to at or about 1 x 108cells / kg, such as from at or about 2.5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 7.5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 1 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 2.5 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 106cells / kg to at or about 1 x 108cells / kg,from at or about 7.5 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 1 x 107cells / kg to at or about 1 x 108cells / kg, from at or about 2.5 x 107cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 107cells / kg to at or about 1 x 108cells / kg, or from at or about 7.5 x 107cells / kg to at or about 1 x 108cells / kg. In some embodiments, the individual dose is from at or about 5 x 107to at or about 10 x 109cells, such as from at or about 5 x 107to at or about 5 x 109cells, from about or about 5 x 107to at or about 1 x 109cells, from at or about 5 x 107to at or about 5 x 108cells, from about or about 5 x 107to at or about 1 x 108cells, 1 x 108to at or about 10 x 109cells, from at or about 1 x 108to at or about 5 x 109cells, from about or about 1 x 108to at or about 1 x 109cells, from at or about 1 x 108to at or about 5 x108cells, from at or about 5 x 108to at or about 10 x 109cells, from at or about 5 x 108to at or about 5 x109cells, from about or about 5 x 108to at or about 1 x 109cells, from about or about 5 x 108to at or about 2 x 1010cells, from at or about 1 x 109to at or about 10 x 109cells, from at or about 1 x 109to at or about 5 x 109cells, or from at or about 5 x 109to at or about 10 x 109cells. In some embodiments, the individual dose is or is about 5 x 108cells. In some embodiments, the individual dose is or is about 1 x 109cells. In some embodiments, the individual dose is or is about 5 x 109cells. In some embodiments, the individual dose is or is about 1 x 1010cells. In some embodiments, the individual dose is or is about 2 x 1010cells. In any of the above embodiments, the dose is given as the number of cells, g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described above, or a number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of expanded cells produced by the method, or a number of viable cells of any of the foregoing.

[0373] In some embodiments, the dose for administration in accord with any of the provided methods of treatment or uses is from at or about 1 x 105cells / kg to at or about 1 x 107cells / kg, such as from at or about 1 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 1 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 1 x 105cells / kg to at or about 5 x 105cells / kg, from at or about 1 x 105cells / kg to at or about 2.5 x 105cells / kg, from at or about 2.5 x 105cells / kg to at or about 1 x 107cells / kg, from at or about 2.5 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 2.5 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 2.5 x 105cells / kg to at or about 5 x 105cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 107cells / kg, from at or about 5 x 105cells / kg to at or about 7.5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 2.5 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 106cells / kg, from at or about 5 x 105cells / kg to at or about 7.5 x 105cells / kg, from at or about 1 x 106cells / kg to at or about 1x 107cells / kg, from at or about 1 x 106cells / kg to at or about 7.5 x 106cells / kg, from at or about 1 x 106cells / kg to at or about 5 x 106cells / kg, from at or about 1 x 106cells / kg to at or about 2.5 x 106cells / kg, from at or about 2.5 x 106cells / kg to at or about 1 x 107cells / kg, from at or about 2.5 x 106cells / kg to at or about 7.5 x 106cells / kg, from at or about 2.5 x 106cells / kg to at or about 5 x 106cells / kg, from at or about 5 x 106cells / kg to at or about 1 x 107cells / kg, from at or about 5 x 106cells / kg to at or about 7.5 x106cells / kg, or from at or about 7.5 x 106cells / kg to at or about 1 x 107cells / kg. In some embodiments, the dose for administration is from at or about 1 x 105cells / kg to at or about 1 x 108cells / kg, such as from at or about 2.5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 7.5 x 105cells / kg to at or about 1 x 108cells / kg, from at or about 1 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 2.5 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 7.5 x 106cells / kg to at or about 1 x 108cells / kg, from at or about 1 x 107cells / kg to at or about 1 x 108cells / kg, from at or about 2.5 x 107cells / kg to at or about 1 x 108cells / kg, from at or about 5 x 107cells / kg to at or about 1 x 108cells / kg, or from at or about 7.5 x 107cells / kg to at or about 1 x 108cells / kg.

[0374] In some embodiments, the dose is given as the number of g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or a number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of expanded cells produced by the provided method, or a number of viable cells of any of the foregoing.

[0375] In some embodiments, the dose for administration in accord with any of the methods of treatment or uses is from at or about 5 x 107to at or about 10 x 109cells, such as from at or about 5 x 107to at or about 5 x 109cells, from about or about 5 x 107to at or about 1 x 109cells, from at or about 5 x107to at or about 5 x 108cells, from about or about 5 x 107to at or about 1 x 108cells, 1 x 108to at or about 10 x 109cells, from at or about 1 x 108to at or about 5 x 109cells, from about or about 1 x 108to at or about 1 x 109cells, from at or about 1 x 108to at or about 5 x 108cells, from at or about 5 x 108to at or about 10 x 109cells, from at or about 5 x 108to at or about 5 x 109cells, from about or about 5 x 108to at or about 1 x 109cells, from at or about 1 x 109to at or about 10 x 109cells, from at or about 1 x 109to at or about 5 x 109cells, from at or about 5 x 109to at or about 10 x 109cells, or from at or about 5 x 109to at or about 20 x 109cells. In some embodiments, the dose for administration is at or about 5 x 108cells. In some embodiments, the dose for administration is at or about 1 x 109cells. In some embodiments, the dose for administration is at or about 5 x 109cells. In some embodiments, the dose for administration is at or about 1 x 1010cells. In some embodiments, the dose for administration is at or about 2 x 1010cells. In some embodiments, the dose for administration is at or about 5 x 1010cells. In some embodiments, the dose is given as the number of g-NK cells or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, such as any of the NK cell subsets described herein, or a number ofviable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of expanded cells produced by the provided method, or a number of viable cells of any of the foregoing.

[0376] In some embodiments, the composition containing expanded NK cells are administered to an individual soon after expansion according to the provided methods. In other embodiments, the expanded NK cells are stored or expanded by growth in culture prior to administration, such as by methods described above. For example, the NK cells can be stored for greater than 6, 12, 18, or 24 months prior to administration to the individual.

[0377] In some embodiments, the provided compositions containing NK cells and subsets thereof, such as g-NK cells, can be administered to a subject by any convenient route including parenteral routes such as subcutaneous, intramuscular, intravenous, and / or epidural routes of administration.

[0378] In particular embodiments, the provided compositions are administered by intravenous infusion. In some embodiments, at or about 10 x 106cells to 10 x 109cells are administered by intravenous infusion in a volume of 1 mL to 100 mL. In some embodiments, at or about 50 x 106cells are administered. In some embodiments, at or about 1 x 109cells are administered. In some embodiments, at or about 5 x 109cells are administered. In some embodiments, at or about 10 x 109cells are administered. It is within the level of a skilled artisan to determine the volume of cells for infusion to administer the number of cells. In one example, 0.5 x 109cells is administered by intravenous infusion of a volume of about 20 mL from a composition, such as a thawed cryopreserved composition, formulated at a concentration of at or about 2.5 x 107cells / mL (e.g., at or about 5 x 109cells in 200 mL).D. Combination Therapy

[0379] In some embodiments, the subject is administered a population of g-NK cells described herein and an effective dose of an additional agent. In provided embodiments, the additional agent is an antibody, such as a monoclonal antibody. In some of the provided methods as described, cells of a composition of g-NK cells as described are administered in combination with an antibody that targets an antigen expressed on cells associated with the HLA-E expressing cancer. In some embodiments, the combination of g-NK cells with an antibody results in antibody-dependent cell-mediated cytotoxicity (ADCC). Cytotoxic killing occurs with the cells which the antibody is targeted against or binds to. In some embodiments, the targeted cells are B cells. In some embodiments, the targeted cells are cancer cells. Thus, also provided herein are pharmaceutical compositions of g-NK cells for combination therapy with an antibody for use in treating an HLA-E expressing cancer in a subject in accord with any of the provided methods. Also provided herein are uses of any of the provided pharmaceutical compositions of g-NK cells for manufacture of a medicament for use in combination therapy with an antibody for treating an HLA-E expressing cancer in a subject. In some embodiments, also provided herein are combinationsof a pharmaceutical composition of g-NK cells as provided herein and an antibody each manufactured as a medicament for use in combination therapy for treating an HLA-E expressing cancer in a subject.

[0380] In particular embodiments, the antibody contains an Fc domain for binding to CD16. In some embodiments, compositions containing g- NK cells as provided herein exhibit enhanced activity when activated by or contacted with antibodies or Fc-containing proteins, such as compared to conventional NK cells. For example, the g-NK cells can be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells. Suitable antibodies may include polyclonal antibodies or monoclonal antibodies. In particular embodiments, the antibody is a full-length antibody.

[0381] In such embodiments, the composition containing g-NK cells as provided herein can be administered prior to, concurrently with or subsequent (after) the administration of one or more antibodies.

[0382] The g-NK cells and the additional agent, such as an antibody, can be administered sequentially or simultaneously. In particular examples, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the population of g-NK cells. In some embodiments, the additional agent, such as an antibody, can be administered before administration of the g-NK cells. In some embodiments, the additional agent, such as an antibody, can be administered after administration of the g-NK cells. In some embodiments, the g-NK cells can be administered simultaneously with antibodies specific for a selected HEA-E expressing cancer. Alternatively, the g-NK cells can be administered at selected times that are distinct from the times when antibodies specific for a selected HEA-E expressing cancer are administered.

[0383] In some embodiments, administration of at least one dose of the antibody (e.g. first dose of the antibody of the combination therapy) may be initiated within one month prior to the first administration of the composition of g-NK cells. In some embodiments, at least one dose of the antibody (e.g. first dose of the antibody of the combination therapy) may be initiated within three weeks prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the antibody (e.g. first dose of the antibody of the combination therapy) may be initiated within two weeks prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the antibody (e.g. first dose of the antibody of the combination therapy) may be initiated within two weeks prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the antibody (e.g. first dose of the antibody of the combination therapy) may be initiated within one week prior to the first administration of the composition of g-NK cells. In some embodiments, administration of the first dose of the antibody of the combination therapy is initiated at or about 14 days, at or about 13 days, at or about 12 days, at or about 11 days, at or about 10 days, at or about 9 days, at or about 8 days, at or about7 days, at or about 6 days, at or about 5 days, at or about 4 days, at or about 3 days, at or about 2 days, or at or about 1 day prior to the first administration of the composition of g-NK cells.

[0384] In some embodiments, the antibody may be administered as a once weekly dose. In some embodiments, the antibody is administered once weekly for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11, weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks or more. In some embodiments, four (4) once weekly doses of the antibody is administered. In some embodiments, five (5) once weekly doses of the antibody is administered. In some embodiments, six (6) once weekly doses of the antibody is administered. In some embodiments, seven (7) once weekly doses of the antibody is administered. In some embodiments, eight (8) once weekly doses of the antibody is administered. In some embodiments, the once weekly doses are administered in consecutive weeks.

[0385] In some embodiments, administration of the first dose of the antibody of the combination therapy is initiated at or about 14 days prior to the first administration of the composition of g-NK cells. In some embodiments, 2 doses of the antibody is administered prior to administering the g-NK cells in a once weekly cycle (e.g., on day -14 and day -7, in which the first administration of g-NK cells is day 0). In some embodiments, administration of the first dose of the antibody of the combination therapy is initiated at or about 7 days prior to the first administration of the composition of g-NK cells. In some embodiments, 1 dose of the antibody is administered prior to administering the g-NK cells in a once weekly cycle (e.g., on day -7, in which the first administration of g-NK cells is day 0).

[0386] In some embodiments, the antibody may be administered in a cycling regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the 28-days cycle begins on the day of administration of the g-NK cells (not including any prior administration of the antibody prior to administration of the g-NK cells). In some embodiments, the antibody is administered for one or two 28- day cycles. In some embodiments, the antibody is administered once weekly in each cycle, such as for one or two 28-day cycles.

[0387] In some embodiments, the antibody is administered once weekly on day -7, on day 0 (same day as the first administration of the g-NK cells), day 7, day 14, day 21 and day 28. In some embodiments, at least one further 28-day cycle of the antibody may be carried out. In some embodiments, the antibody is administered once weekly on day -7, day 0 (same day as the first administration of the g-NK cells), day 7, day 14, day 21, day 28, day 35, day 42, day 49 and day 56.

[0388] In some examples, each dose of the antibody that is administered is about 0.1 mg / kg to about 100 mg / kg of the antibody (such as about 0.5-10 mg / kg, about 1-20 mg / kg, about 10-50 mg / kg, or about 20-100 mg / kg. In some embodiments, each dose of the antibody that is administered is in an amount from about 0.5-10 mg / kg. In some embodiments, each dose of the antibody that is administered is in anamount from about 0.5-8 mg / kg. In some embodiments, each dose of the antibody that is administered is in an amount from about 0.5-1 mg / kg. In some embodiments, each dose of the antibody is administered in an amount from about 10-50 mg / kg. In some embodiments, each dose of the antibody is administered in an amount of about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 22 mg / kg, about 24 mg / kg, about 32 mg / kg, about 36 mg / kg, about 40 mg / kg, about 44 mg / kg, about 48 mg / kg, about 52 mg / kg, about 56 mg / kg, about 60 mg / kg, about 64 mg / kg, about 68 mg / kg, about 72 mg / kg, about 80 mg / kg, about 88 mg / kg, about 96 mg / kg, or about 100 mg / kg, or any value between any of the foregoing.

[0389] In some examples, each dose of the antibody that is administered is about 3 mg / m2to about 3000 mg / m2o...

Claims

1. CLAIMS1. A method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia.

2. The method of claim 1 , wherein the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.

3. The method of claim 1, wherein the method does not comprise administering an antibody to the subject in combination with the composition of g-NK cells.

4. The method of claim 2 or claim 3, wherein the antibody is a therapeutic antibody.

5. The method of any one of claims 2-4, wherein the antibody binds to a target antigen expressed by cells of the AML.

6. The method of any one of claims 1-5, wherein the g-NK cells are not engineered with an antigen receptor (e.g., a chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the AML.

7. The method of any one of claims 1-6, wherein the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the AML.

8. The method of any one of claims 1-7, wherein the g-NK cells are not engineered with an antigen receptor (e.g., chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by myeloid stem cell or precursor cells associated with the AML.

9. The method of any one of claims 1-8, wherein the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the AML.

10. The method of any one of claims 1-9, wherein at the time of treatment the subject has measurable residual disease (MRD).

11. The method of any one of claims 1-10, wherein the AML is a low burden disease, optionally <25% blasts in peripheral blood and bone marrow and / or white blood cell count < 10,000.

12. The method of any one of claims 1-11, wherein the AML is a relapsed or refractory AML.

13. The method of any one of claims 1-11, wherein the AML is low burden relapsed or refractory AML.

14. The method of claim 12 or claim 13, wherein the AML is a relapsed AML, optionally wherein the relapsed AML is characterized by >5% BM blasts, reappearance of blasts in the blood or development of extramedullary disease following achievement of CR, CRi or morphologic leukemia-free state (MLFS).

15. The method of claim 12 or claim 13, wherein the AML is refractory AML, optionally wherein the subject failed to achieve CR, Cri or MLFS following prior treatment, and blasts >5%.

16. The method of any one of claims 1-15, wherein the subject has received one or more prior treatment regimens for treating the AML selected from:(i) At least 1 cycle of purine analogue containing intensive induction chemotherapy regimen, e.g.,FLAG-Ida, CLIA or CLAG-M or similar regimens with or without venetoclax;(ii) at least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX-351 with venetoclax or similar regimens;(iii) At least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax;(iv) 2 cycles of venetoclax with HMA / LDAC + / - other agents; or(v) 4 cycles of HMA alone.

17. The method of any one of claims 1-16, wherein pathogenesis of the AML is associated with a viral infection.

18. The method of claim 17, wherein the AML is characterized by B cells or cancer cells with upregulated HLA-E expression.

19. The method of claim 18, wherein the upregulation of HLA-E expression is caused by a viral infection.

20. The method of any one of claims 17-19, wherein the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

21. The method of any one of claims 17-20, wherein the viral infection is an Epstein-Barr virus (EBV).

22. A method of treating an HLA-E expressing cancer, the method comprising administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an HLA-E expressing cancer.

23. The method of claim 22, further comprising selecting a subject with the HLA-E expressing cancer.

24. A method of treating an HLA-E expressing cancer, the method comprising:(a) selecting a subject with an HLA-E expressing cancer; and(b) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer.

25. A method of treating an HLA-E expressing cancer, the method comprising:(a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of once a week (QW) in a 7-day cycle; and(b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily at a frequency of once a week (QW) in the 7-day cycle and on the same day as the g-NK cells, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

26. A method of treating an HLA-E expressing cancer, the method comprising:(a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of once a week (QW) in a 7-day cycle; and(b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and each dose is administered one time daily for the first five consecutive days in the 7-day cycle, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

27. A method of treating an HLA-E expressing cancer, the method comprising:(a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of every other day (Q2D) in a 7- day cycle; and(b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and administered one time daily every other day (Q2D) in the 7-day cycle and on the same day as the g- NK cells, wherein the 7-day cycle is repeated twice, and each 7-day cycle is the same.

28. A method of treating an HLA-E expressing cancer, the method comprising:(a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer, wherein the g-NK cells are administered one time daily at a frequency of every other day (Q2D) in a 7- day cycle; and(b) administering a dose of IL-2 to the subject, wherein the dose is 3 million IU to 9 million IU and administered twice daily (BID) at a frequency of the first five consecutive days in a first 7-day cycle and one time daily every other day (Q2D) for a second 7-day cycle.

29. The method of any one of claims 22-28, wherein the composition of g-NK cells is administered as a monotherapy without co-administration of an antibody.

30. The method of any one of claims 22-28, further comprising administering to the subject an antibody directed against a target antigen associated with the HLA-E expressing cancer.

31. The method of claim 30, wherein the target antigen is a B cell antigen, plasma cell antigen, or myeloid cell antigen.

32. A method of treating an HLA-E expressing cancer, the method comprising:(a) administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having the HLA-E expressing cancer; and(b) administering to the subject an antibody that is directed against a B cell antigen, plasma cell antigen, or myeloid cell antigen.

33. The method of any one of claims 22-32, wherein the g-NK cells are not engineered with an antigen receptor (e.g., a chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the cancer.

34. The method of any one of claims 22-33, wherein the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the cancer.

35. The method of any one of claims 22-34, wherein the g-NK cells are not engineered with an antigen receptor (e.g., chimeric antigen receptor) comprising an extracellular binding domain that binds to a target antigen expressed by myeloid stem cell or precursor cells associated with the cancer.

36. The method of any one of claims 22-35, wherein the g-NK cells are not engineered with a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a target antigen expressed by cells of the cancer.

37. The method of any one of claims 1-36, wherein the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

38. The method of any one of claims 1-37, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos).

39. The method of any one of claims 1-38, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

40. The method of any one of claims 22-39, wherein the HLA-E expressing cancer is selected from the group consisting of: head and / or neck cancer, gynecological cancer, gastric cancer, colorectal cancer, and laryngeal cancer.

41. The method of any one of claims 22-39, wherein the HLA-E expressing cancer is a B- cell marker expressing cancer.

42. The method of any one of claims 22-39 and 41, wherein the cancer is a lymphoma.

43. The method of claim 42, wherein the lymphoma is a Non-Hodgkin’s Lymphoma (NHL).

44. The method of any one of claims 22-39, wherein the HLA-E expressing cancer is a plasma cell marker expressing cancer.

45. The method of claim any one of claims 22-39 and 44, wherein the cancer is a Multiple Myeloma (MM).

46. The method of any one of claims 22-39, wherein the HLA-E expressing cancer is a myeloid cell marker expressing cancer.

47. The method of any one of claims 22-39 and claim 46, wherein the cancer is an acute myeloid leukemia (AML).

48. The method of claim 47, wherein at the time of treatment the subject has measurable residual disease (MRD).

49. The method of claim 47 or claim 48, wherein the AML is a low burden disease, optionally <25% blasts in peripheral blood and bone marrow and / or white blood cell count < 10,000.

50. The method of any one of claims 47-49, wherein the AML is a relapsed or refractory AML.

51. The method of any one of claims 47-50, wherein the AML is low burden relapsed or refractory AML.

52. The method of claim 50 or claim 51 , wherein the AML is a relapsed AML, optionally wherein the relapsed AML is characterized by >5% BM blasts, reappearance of blasts in the blood or development of extramedullary disease following achievement of CR, CRi or morphologic leukemia-free state (MLFS).

53. The method of claim 50 or claim 51, wherein the AML is refractory AML, optionally wherein the subject failed to achieve CR, CRi or MLFS following prior treatment, and blasts >5%.

54. The method of any one of claims 47-53, wherein the subject has received one or more prior treatment regimens for treating the AML selected from:(i) At least 1 cycle of purine analogue containing intensive induction chemotherapy regimen, e.g.,FLAG-Ida, CLIA or CLAG-M or similar regimens with or without venetoclax;(ii) at least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX-351 with venetoclax or similar regimens;(iii) At least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax;(iv) 2 cycles of venetoclax with HMA / LDAC + / - other agents; or(v) 4 cycles of HMA alone.

55. The method of any one of claims 30-54, wherein the antibody is a full-length antibody.

56. The method of any one of claims 31-55, wherein the B cell antigen, plasma cell antigen, or myeloid cell antigen is selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.

57. The method of claim 55 or claim 56, wherein the antibody is directed against a lymphoma antigen.

58. The method of claim 57, wherein the lymphoma antigen comprises an antigen selected from CD 19 or CD20.

59. The method of any one of claims 30-58, wherein the antibody is an anti-CD19 antibody.

60. The method of claim 59, wherein the antibody is inebilizumab, tafasitamab-cxix or obexelimab.

61. The method of any one of claims 30-58, wherein the antibody is an anti-CD20 antibody.

62. The method of claim 61, wherein the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

63. The method of claim 55 or claim 56, wherein the antibody is directed against a multiple myeloma antigen.

64. The method of claim 63, wherein the multiple myeloma antigen comprises an antigen selected from CD38 or BCMA.

65. The method of any one of claims 30-56, 63, and 64, wherein the antibody is an anti- CD38 antibody.

66. The method of claim 65, wherein each dose of the anti-CD38 antibody is about 0.5-10 mg / kg, optionally wherein each dose of the anti-CD38 antibody is about 0.5 mg / kg.

67. The method of claim 65 or claim 66, wherein the anti-CD38 antibody is daratumumab or is isatuximab.

68. The method of any one of claims 65-67, wherein less than 25% of the cells in the g-NK cell composition are positive for surface CD38.

69. The method of any one of claims 64-68, wherein the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression.

70. The method of any one of claims 30-56, 63, and 64, wherein the antibody is an anti- BCMA antibody.

71. The method of any one of claims 1-70, wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

72. The method of any one of claims 1-70, wherein the composition of g-NK cells is dosed at a frequency of once every week (QW).

73. The method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia, wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

74. The method of treating Acute Myeloid Leukemia in a subject, the method comprising administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having Acute Myeloid Leukemia, wherein the composition of g-NK cells is dosed at a frequency of once every week (QW).

75. A method of treating lymphoma in a subject, the method comprising:(a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and(b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD20 antibody.

76. A method of treating lymphoma in a subject, the method comprising:(a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of once every week (QW); and(b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD20 antibody.

77. The method of claim 75 or claim 76, wherein the lymphoma is Non-Hodgkin’ s Lymphoma (NHL).

78. The method of any one of claims 61, 62, and 71-73, wherein the anti-CD20 antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

79. A method of treating Multiple Myeloma (MM) in a subject, the method comprising:(a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D); and(b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD38 antibody.

80. A method of treating Multiple Myeloma (MM) in a subject, the method comprising:(a) administering at least one dose of a composition of allogeneic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells), wherein the composition of g-NK cells is dosed at a frequency of once every week (QW); and(b) administering to the subject an antibody directed against a target antigen associated with the lymphoma, wherein the antibody is an anti-CD38 antibody.

81. The method of claim 79 or claim 80, wherein each dose of the anti-CD38 antibody is about 0.5-10 mg / kg, optionally wherein each dose of the anti-CD38 antibody is about 0.5 mg / kg.

82. The method of any one of claims 65, 66, 68, 69, 79, and 80, wherein the anti-CD38 antibody is daratumumab or is isatuximab.

83. The method of any one of claims 22-82, wherein pathogenesis of the HLA-E expressing cancer is associated with a viral infection.

84. The method of claim 83, wherein the HLA-E expressing cancer is characterized by B cells or cancer cells with upregulated HLA-E expression.

85. The method of claim 84, wherein the upregulation of HLA-E expression is caused by a viral infection.

86. The method of any one of claims 1-85, wherein the subject has been selected as having a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

87. The method of claim 85, wherein the viral infection is a cytomegalovirus (CMV), a Human papillomavirus (HPV), an influenza virus, or an Epstein-Barr virus (EBV).

88. The method of any one of claims 85-87, wherein the viral infection is an Epstein-Barr virus (EBV).

89. A method of treating a disease or disorder associated with an Epstein-Barr virus (EBV), the method comprising: administering at least one dose of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having an HLA-E expressing cancer.

90. The method of claim 89, wherein the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

91. The method of claim 89 or claim 90, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos).

92. The method of any one of claims 89-91, wherein at least 8% of the NK cells are positive for NKG2C (NKG2Cpos) and / or negative or low for NKG2A (NKG2Aneg).

93. The method of any one of claims 22-28 and 89-92, further comprising administering to the subject an antibody directed against a target antigen associated with the HLA-E expressing cancer.

94. The method of claim 93, wherein the target antigen is a B cell antigen, plasma cell antigen, or myeloid cell antigen.

95. The method of any one of claims 1-94, wherein, among cells in the composition of g-NK cells, greater than at or about 20% of the cells are g-NK cells.

96. The method of any one of claims 1-95, wherein, among cells in the composition of g-NK cells, greater than at or about 30% of the cells are g-NK cells, greater than at or about 40% of the cells are g-NK cells, greater than at or about 50% of the cells are g-NK cells, greater than at or about 60% of the cells are g-NK cells, greater than at or about 70% of the cells are g-NK cells, greater than at or about 80% of the cells are g-NK cells, greater than at or about 90% of the cells are g-NK cells, or greater than at or about 95% of the cells are g-NK cells.

97. The method of any one of claims 1-96, wherein at least at or about 15% of the NK cells of the composition are positive for NKG2C (NKG2Cpos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2Aneg).

98. The method of any one of claims 30-54, 56-72, 75-88, and 93-97, wherein the antibody is a full-length antibody.

99. The method of any one of claims 94-98, wherein the B cell antigen, plasma cell antigen, or myeloid cell antigen is selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD38, BCMA, and TACI.

100. The method of any one of claims 89-99, wherein the disease or disorder associated with EBV is a lymphoma.

101. The method of claim 100, wherein the lymphoma is Non-Hodgkin’s Lymphoma (NHL).

102. The method of any one of claims 93-101, wherein the antibody is an anti-CD19 antibody.

103. The method of claim 102, wherein the antibody is inebilizumab, tafasitamab-cxix or obexelimab.

104. The method of any one of claims 30-58, 71, 72, and 93-101, wherein the antibody is an anti-CD20 antibody.

105. The method of claim 104, wherein the antibody is rituximab or a biosimilar thereof, ocrelizumab, ofatumumab, or obinutuzumab.

106. The method of any one of claims 30-58, 71, 72, and 93-101, wherein the antibody is an anti-CD22 antibody.

107. The method of claim 106, wherein the antibody is epratuzumab.

108. The method of any one of claims 30-58, 71, 72, and 93-101, wherein the antibody is an anti-BAFF-R antibody.

109. The method of claim 108, wherein the antibody is belimumab.

110. The method of any one of claims 89-99, wherein the disease or disorder associated with EBV is Multiple Myeloma (MM).

111. The method of any one of claims 30-56, 63, 64, 69, 71, 72, 93-101, and 110, wherein the antibody is an anti-CD38 antibody.

112. The method of claim 111, wherein each dose of the anti-CD38 antibody is about 0.5-10 mg / kg, optionally wherein each dose of the anti-CD38 antibody is about 0.5 mg / kg.

113. The method of claim 111 or claim 112, wherein the anti-CD38 antibody is daratumumab or is isatuximab.

114. The method of claim 112 or claim 113, wherein less than 25% of the cells in the composition of g-NK cells are positive for surface CD38.

115. The method of any one of claims 112-114, wherein the cells in the composition of g-NK cells are not engineered to reduce or eliminate CD38 expression.

116. The method of any one of claims 30-72, 75-88, and 93-115, wherein the antibody is administered intravenously.

117. The method of any one of claims 30-72, 75-88, and 93-115, wherein the antibody is administered subcutaneously.

118. The method of any one of claims 30-72, 75-88, and 93-117, where in the antibody is administered once weekly.

119. The method of any one of claims 89-118, wherein the composition of g-NK cells is dosed at a frequency of every other day (Q2D).

120. The method of any one of claims 89-118, wherein the composition of g-NK cells is dosed at a frequency of once every week (QW).

121. The method of any one of claims 1-120, wherein the composition of g-NK cells is administered in a 7-day cycle.

122. The method of claim 121, wherein the composition of g-NK cells is administered on day 0, day 2, and day 4 in the 7-day cycle.

123. The method of claim 121 or claim 122, wherein the 7-day cycle is repeated one to three times.

124. The method of claim 123, wherein the 7-day cycle is repeated one time.

125. The method of claim 123, wherein the 7-day cycle is repeated two times.

126. The method of any one of claims 1-125, wherein the composition of g-NK cells is administered from two total doses to six total doses.

127. The method of claim 1-24 and 29-126, wherein the composition of g-NK cells is administered as two or four total doses.

128. The method of claim 1-26 and 29-126, wherein the composition of g-NK cells is administered as three or six total doses.

129. The method of any one of claim 1-128, wherein at least at or about 20% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

130. The method of any one of claims 1-123, wherein at least at or about 40% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRyneg) NK cells (g-NK).

131. The method of any one of claims 1-130, wherein greater than at or about 70% of the g- NK cells are positive for perforin and greater than at or about 70% of the g-NK cells are positive for granzyme B.

132. The method of claim 131, wherein (i) greater than at or about 80% of the g-NK cells are positive for perforin and greater than at or about 80% of the g-NK cells are positive for granzyme B, (ii) greater than at or about 90% of the g-NK cells are positive for perforin and greater than at or about 90% of the g-NK cells are positive for granzyme B, or (iii) greater than at or about 95% of the g-NK cells are positive for perforin and greater than at or about 95% of the g-NK cells are positive for granzyme B.

133. The method of claim 131 or claim 132, wherein: among the cells positive for perforin, the cells express a mean level of perforin as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of perforin expressed by cells that are FcRypos; and / oramong the cells positive for granzyme B, the cells express a mean level of granzyme B as measured by intracellular flow cytometry that is, based on mean fluorescence intensity (MFI), at least at or about two times the mean level of granzyme B expressed by cells that are FcRypos.

134. The method of any one of claims 1-133, wherein greater than 10% of the cells in the composition of g-NK cells are capable of degranulation against tumor target cells, optionally as measured by CD 107a expression, optionally wherein the degranulation is measured in the absence of an antibody against the tumor target cells.

135. The method of any one of claims 1-134, wherein, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% exhibit degranulation, optionally as measured by CD 107a expression, in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

136. The method of any one of claims 1-135, wherein greater than 10% of the cells in the composition of g-NK cells are capable of producing interferon-gamma or TNF-alpha against tumor target cells, optionally wherein the interferon-gamma or TNF-alpha is measured in the absence of an antibody against the tumor target cells.

137. The method of any one of claims 1-136, wherein, among the cells in the composition of g-NK cells, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce an effector cytokine in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (antitarget antibody).

138. The method of claim 137, wherein the effector cytokine is IFN-gamma or TNF-alpha.

139. The method of claim 137, wherein the effector cytokine is IFN-gamma and TNF-alpha.

140. The method of any one of claims 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD56+ cells are enriched from a biological sample from a donor subject.

141. The method of any one of claims 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of CD3- / CD57+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells are enriched from a biological sample from a donor subject.

142. The method of any one of claims 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of cells that are NKG2Cposcells cultured with irradiated HLA-E+ feeder cells, wherein the NKG2Cposcells are enriched from a biological sample from a donor subject.

143. The method of any one of claims 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of cells that are CD3negNKG2Cposcells cultured with irradiated HLA-E+ feeder cells, wherein the CD3negNKG2Cposcells are enriched from a biological sample from a donor subject.

144. The method of any one of claims 140-143, wherein the donor subject is CMV- seropositive.

145. The method of any one of claims 140-143, wherein the donor subject has the CD 16 F / F NK cell genotype.

146. The method of any one of claims 140-143, wherein the donor subject has the CD 16 158 V / V NK cell genotype or the CD 16 158 V / F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype.

147. The method of any one of claims 140-146, wherein at least at or about 15% of natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2Cpos) and at least 70% of NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2Aneg).

148. The method of any one of claims 140-147, wherein the irradiated feeder cells are deficient in HEA class I and HEA class IE149. The method of any one of claims 140-148, wherein the irradiated feeder cells are221. AEH cells.

150. The method of any one of claims 140-149, wherein the culturing is performed in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is interleukin (IL)-2 and at least one recombinant cytokine is IL-21.

151. The method of claim 150, wherein the recombinant cytokines are IL-21 and IL-2.

152. The method of claim 150, wherein the recombinant cytokines are IL-21, IL-2, and IL- 15.

153. The method of any one of claims 1-152, wherein the g-NK cells in the composition are from a single donor subject that have been expanded from the same biological sample.

154. The method of any one of claims 1-153, wherein the composition of g-NK cells is formulated in a serum-free cryopreservation medium comprising a cryoprotectant, optionally wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v / v).

155. The method of any one of claims 1-154, wherein the g-NK cells are not engineered with an antigen receptor, optionally wherein the antigen receptor is a chimeric antigen receptor.

156. The method of any one of claims 1-155, wherein the g-NK cells are not engineered with a secreted cytokine, optionally a cytokine receptor fusion protein, such as IL- 15 receptor fusion (IL- 15RF).

157. The method of any one of claims 1-156, wherein the method does not include exogenous cytokine administration to the subject to support NK cell survival or expansion, wherein the exogenous cytokine is one or more of IL-2, IL-7, IL-15 or IL-21.

158. The method of any one of claims 1-156, further comprising administering exogenous cytokine support to facilitate expansion or persistence of the g-NK cells in vivo in the subject, optionally wherein the exogenous cytokine is or comprises IL- 15 or IL-2.

159. The method of any one of claims 1-156 and 158, wherein the method comprises administering IL-2 to the subject.

160. The method of claim 159, wherein the IL-2 is administered once a week, two times a week or three times a week.

161. The method of claim 159 or claim 160, wherein the IL-2 is administered at a frequency of once a week (QW).

162. The method of claim 159 or claim 160, wherein the IL-2 is administered at a frequency of every other day (Q2W).

163. The method of any one of claims 159-162, wherein for each day of administration the IL-2 is administered once daily.

164. The method of any one of claims 159-162, wherein for each day of administration the IL-2 is administered twice daily (BID).

165. The method of any one of claims 159-164, wherein the IL-2 is administered in a cycling regimen of one or more 7-day cycles.

166. The method of any one of claims 159-165, wherein the IL-2 is administered in three 7- day cycles, optionally wherein the three 7-day cycles are in consecutive weeks.

167. The method of claim 165 or claim 166, wherein each 7-day cycle is the same.

168. The method of any one of claims 159-167, wherein the IL-2 is administered one time daily at a frequency of once per week (QW) on day 0 in one or more 7-day cycles.

169. The method of any one of claims 159-167, wherein the IL-2 is administered one time daily for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in one or more 7-day cycles.

170. The method of claim 165 or claim 166, wherein each 7-day cycle is different.

171. The method of any one of claims 159-167 and 170, wherein the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in one or more 7-day cycles.

172. The method of any one of claims 159-167 and 170, wherein the IL-2 is administered twice daily (BID) for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in one or more 7-day cycles.

173. The method of any one of claims 159-166 and 170, wherein the IL-2 is administered twice daily (BID) for the first five consecutive days of day 0, day 1, day 2, day 3, and day 4 in a first 7- day cycle; and the IL-2 is administered one time daily at a frequency of every other day (Q2D) on day 0, day 2, and day 4 in a second 7-day cycle.

174. The method of any one of claims 25-28 and 159-173, wherein the IL-2 is administered to the subject within about 1 hour of the administration of the g-NK cells.

175. The method of any one of claims 159-174, wherein each dose of the IL-2 is 1 million to 12 million IU.

176. The method of any one of claims 25-28 and 159-175, wherein each dose of IL-2 is 4 million IU to 8 million IU.

177. The method of any one of claims 25-28 and 159-176, wherein each dose of IL-2 is at or about 6 million IU.

178. The method of any one of claims 25-28 and 159-177, wherein the IL-2 is administered subcutaneously.

179. The method of any one of claims 159-178, wherein administration of the IL-2 is administered on the same day as the first dose of the g-NK cells.

180. The method of any one of claims 1-179, each dose of the composition of g-NK cells is from at or about from at or about 1 x 108cells to at or about 50 x 109cells.

181. The method of any one of claims 1-180, wherein each dose of the composition of g-NK cells is or is about 5 x 108cells.

182. The method of any one of claims 1-180, wherein each dose of the composition of g-NK cells is or is about 5 x 109cells.

183. The method of any one of claims 1-180, wherein each dose of the composition of g-NK cells is or is about 10 x 109cells.

184. The method of any one of claims 1-180, wherein each dose of the composition of g-NK cells is or is about 20 x 109cells.

185. The method of any one of claims 1-184, wherein: prior to the administration of the dose of the composition of g-NK cells, the subject has received a lymphodepleting therapy; or the method further comprises administering to the subject a lymphodepleting therapy prior to administering the g-NK cells.

186. The method of claim 185, wherein administration of the at least one dose of the composition of g-NK cells is initiated within two weeks or at or about two weeks after initiation of the lymphodepleting therapy.

187. The method of claim 185 or claim 186, wherein administration of the at least one dose of the composition of g-NK cells is initiated within 7 days or at or about 7 days after initiation of the lymphodepleting therapy.

188. The method of any one of claims 110-187, wherein the lymphodepleting therapy comprises fludarabine and / or cyclophosphamide.

189. The method of any of claims 185-188, wherein the lymphodepleting therapy comprises fludarabine and cyclophosphamide.

190. The method of any of claims 185-189, wherein the lymphodepleting comprises the administration of fludarabine at or about 20-40 mg / m2body surface area of the subject, optionally at or about 30 mg / m2, daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days.

191. The method of claim 190, wherein the lymphodepleting therapy further comprises administration of mesna at or about 200-400 mg / m2body surface area of the subject, optionally at or about 300 mg / m2, daily, for 2-4 days.

192. The method of any of claims 185-191, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at or about 400 mg / m2body surface area of the subject and mesna at or about 300 mg / m2, daily, each for 2-4 days, optionally 3 days.

193. The method of any of claims 1-192, wherein the method further comprises administration of a bispecific T cell targeting agent to the subject.

194. The method of claim 193, wherein the bispecific T cell targeting agent is a bispecific T cell engager (BiTE) comprising an anti-CD3 antibody specific to CD3 and a target antigen expressed by cells of the AML, HLA-E expressing cancer, MM, or lymphoma.

195. A method of assessing response following administration of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple myeloma (MM), the method comprising:(1) assessing the level of expression of one or more RNA transcripts or portion thereof in a biological sample from the subject wherein:(a) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of CD28, CLECL1, DEPTOR, DUSP2, DUSP5, FCGR2B, FCRL2, GPR160, HLA-DOB, ITGA6, LY9, MAGEA1, MAGEA12, MAGEC2, PDK1, PTCD2, SLAMF7, SMAD5, TNFRSF17 (BCMA), TNFSF8 (CD30 ligand), and WNT10A, optionally wherein said one or more RNA transcripts negatively correlates to the likelihood of response following administration of the composition; and / or(b) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of EGF, ITGB3, NID2, and PG4, optionally wherein said one or more RNA transcripts positively correlates to the likelihood of response following administration of the composition; and(2) determining the likelihood of response of the subject to administration of the composition, wherein the subject is responsive to administration of the composition if the subject receives a minor response or better based on IMWG.

196. A method of adaptive treatment following administration of a composition of allogenic Natural Killer (NK) cells deficient in expression of FcRy chain (g-NK cells) to a subject having multiple myeloma (MM), the method comprising:(1) assessing the level of expression of one or more RNA transcripts or portion thereof in a biological sample from the subject wherein:(a) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of CD28, CLECL1, DEPTOR, DUSP2, DUSP5, FCGR2B, FCRL2, GPR160, HLA-DOB, ITGA6, LY9, MAGEA1, MAGEA12, MAGEC2, PDK1, PTCD2, SLAMF7, SMAD5, TNFRSF17 (BCMA), TNFSF8 (CD30 ligand), and WNT10A, optionally wherein said one or more RNA transcripts negatively correlates to the likelihood of response following administration of the composition; and / or(b) at least one of the one or more RNA transcripts is transcribed from a gene selected from a group consisting of EGF, ITGB3, NID2, and PG4, optionally wherein said one or more RNA transcripts positively correlates to the likelihood of response following administration of the composition;(2) determining the likelihood of response of the subject to administration of the composition, wherein the subject is responsive to administration of the composition if the subject receives a minor response or better based on IMWG; and(3) administering to the subject who is determined to not be responsive to the administration of the composition:(a) administration of a dose of IL-2 to the subject,(b) administration of a composition of g-NK cells to the subject, and / or(c) administration of an antibody, optionally wherein the antibody is an anti-CD38 antibody.

197. The method of any of claims 1-196, wherein the subject is a human subject.

Images