WO2025245399A1

WO2025245399A1 - Method of treating cancer with natural killer cells

Info

Publication number: WO2025245399A1
Application number: PCT/US2025/030679
Authority: WO
Inventors: Mark Frohlich; Stefanie MANDL-CASHMAN; Austin BIGLEY
Original assignee: Indapta Therapeutics Inc
Current assignee: Indapta Therapeutics Inc
Priority date: 2024-05-23
Filing date: 2025-05-22
Publication date: 2025-11-27
Anticipated expiration: 2026-11-23

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 CELLS

Cross-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 treatment of 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 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.

[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 that binds 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 (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2C^pos). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg).

[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 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. 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 (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2C^pos). In some of any embodiments, at least 8% of the NK cells are positive for NKG2C (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg).

[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 (NKG2C^pos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2A^neg).

[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 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.

[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 (FcRy^neg) 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 (FcRy^neg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRy^neg) 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 FcRy^pos; 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 FcRy^pos.

[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 NKG2C^pos cells cultured with irradiated HLA-E+ feeder cells, wherein the NKG2C^pos 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 CD3^negNKG2C^pos cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3^negNKG2C^pos cells 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 of any 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 10⁸ cells to at or about 50 x 10⁹ cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 5 x 10⁸ cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 5 x 10⁹ cells of the composition of g-NK cells. In some of any embodiments, each dose of g-NK cells is or is about 10 x 10⁹ cells of the composition of g- NK cells. In some of any embodiments, each dose of g-NK cells is or is about 20 x 10⁹ cells 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 a dose 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/m² body 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/m² body 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/m² body 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/m² body surface area of the subject, daily, and cyclophosphamide at or about 400 mg/m² body 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 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.

[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 NKG2C^pos/NKG2A^neg NK 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 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 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 NKG2C^pos/NKG2A^neg NK 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 cancer cells 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 cell therapies, 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 not result 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 or twice 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 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.

[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 1 the 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 NKG2C^pos, NKG2A^neg and CD16^pos. In some embodiments, cells of the composition that are g-NK cells are characterized as being CD57^pos, CD7^dim/neg, CD161^neg and/or CD38^neg. In some embodiments, cells of the composition of g-NK cells are NKG2A^neg/CD161^neg. In some embodiments, cells of the composition of g-NK cells are CD38^neg. In some embodiments, cells of the composition of g- NK cells have the phenotype CD45^pos/CD3^neg/CD56^pos.

[0145] In some embodiments, the composition contains NKG2C^pos cells. In some embodiments, the compositions contain NKG2A^neg cells. In some embodiments, the composition contains NKG2C^pos/NKG2A^neg cells. In some embodiments, g-NK cells of the composition are NKG2C^pos cells. In some embodiments, g-NK cells of the composition contain NKG2A^neg cells. In some embodiments, g- NK cells of the composition contain NKG2C^pos/NKG2A^neg cells.

[0146] In some embodiments, the composition comprises about 5-99% NKG2C^pos cells. In some embodiments, the composition can include an increased or greater percentages of NKG2C^pos cells relative to total NK cells or total cells compared to the percentage of NKG2C^pos 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. [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% NKG2C^pos cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2C^pos cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C^pos cells 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% NKG2C^pos cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2C^pos cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C^pos cells of the total NK cells in the composition.

[0148] In some embodiments, the provided compositions include those in which the NKG2C^pos 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.

[0149] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2C^pos. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2C^pos.

[0150] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2C^pos. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2C^pos. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2C^pos. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2C^pos. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2C^pos. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2C^pos.

[0151] In some embodiments, the composition comprises about 5-99% NKG2A^neg cells. In some embodiments, the composition can include an increased or greater percentages of NKG2A^neg cells relative to total NK cells or total cells compared to the percentage of NKG2A^neg 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.

[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% NKG2A^neg cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2A^neg cells 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 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% NKG2A^neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2A^neg cells of the total NK cells in the composition.

[0153] In some embodiments, the provided compositions include those in which the NKG2A^neg 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.

[0154] In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2A^neg.

[0155] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2A^neg.

[0156] In some embodiments, the composition comprises about 5-99% NKG2C^pos/NKG2A^neg cells. In some embodiments, the composition can include an increased or greater percentages of NKG2C^pos/NKG2A^neg cells relative to total NK cells or total cells compared to the percentage of NKG2C^pos/NKG2A^neg 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.

[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% NKG2C^pos/NKG2A^neg cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2C^pos/NKG2A^neg cells of the total cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C^pos/NKG2A^neg cells 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% NKG2C^pos/NKG2A^neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2C^pos/NKG2A^neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C^pos/NKG2A^neg cells of the total NK cells in the composition.

[0158] In some embodiments, the provided compositions include those in which the NKG2C^pos/NKG2A^neg 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.

[0159] In some embodiments, of the total cells in the composition greater than at or about 8% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 10% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 15% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 20% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 25% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 30% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 50% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 60% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 70% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 80% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 90% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 95% of the cells are NKG2C^pos/NKG2A^neg.

[0160] In some embodiments, of the total NK cells in the composition greater than at or about 50% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 60% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 70% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 80% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 90% of the cells are NKG2C^pos/NKG2A^neg. In some embodiments, of the total NK cells in the composition greater than at or about 95% of the cells are NKG2C^pos/NKG2A^neg.

[0161] In some embodiments, the composition comprises about 5-99% g-NK cells that are NKG2C^pos/NKG2A^neg cells. In some embodiments, the composition can include an increased or greater percentages of g-NK cells that are NKG2C^pos/NKG2A^neg cells relative to total NK cells or total cells compared to the percentage of g-NK cells that are NKG2C^pos/NKG2A^neg 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.

[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 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 NKG2C^pos/NKG2A^neg cells 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 NKG2C^pos/NKG2A^neg cells 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 NKG2C^pos/NKG2A^neg cells 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 NKG2C^pos/NKG2A^neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 8% NKG2C^pos/NKG2A^neg g-NK cells that are NKG2C^pos/NKG2A^neg cells of the total NK cells in the composition. In some embodiments, the composition can include at least at or about 15% NKG2C^pos/NKG2A^neg g-NK cells that are NKG2C^pos/NKG2A^neg cells 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 NKG2C^pos/NKG2A^neg 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.

[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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. In some embodiments, of the total cells in the composition greater than at or about 40% of the cells are g-NK cells that are NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg.

[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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg. 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 NKG2C^pos/NKG2A^neg.

[0166] In some embodiments, the g-NK cells are CD16^pos. 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 the composition 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 FcRy^pos. 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 FcRy^pos NK 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 FcRy^pos NK 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 FcRy^pos NK 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 FcRy^pos NK 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 FcRy^pos NK 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 FcRy^pos NK 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 are positive 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 FcRy^neg.

[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 FcRy^pos. 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 FcRy^pos.

[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 FcRy^pos.

[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 FcRy^pos.

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

[0179] In some of any of the provided embodiments, the composition comprises at least or about at least 10⁶ cells. In some of any of the provided embodiments, the composition comprises from at or about 10⁶ to at or about 10¹⁰ cells, from at or about 10⁶ to at or about 10⁹ cells, from at or about 10⁶ to at or about 10⁸ cells, from at or about 10⁶ to at or about 10⁷ cells, from at or about 10⁷ to at or about 10¹⁰ cells, from at or about 10⁷ to at or about 10⁹ cells, from at or about 10⁷ to at or about 10⁸ cells, from at or about 10⁸ to at or about IO¹⁰ cells, from at or about 10⁸ to at or about 10⁹ cells, or from at or about 10⁹ to at or about 10¹⁰ cells.

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

[0181] In some of any of the provided embodiments, the composition comprises at least at or about 10⁶ g-NK cells. In some of any of the provided embodiments, the composition comprises from at or about 10⁶ to at or about 10¹⁰ g-NK cells, from at or about 10⁶ to at or about 10⁹ g-NK cells, from at or about 10⁶ to at or about 10⁸ g-NK cells, from at or about 10⁶ to at or about 10⁷ g-NK cells, from at or about 10⁷ to at or about 10¹⁰ g-NK cells, from at or about 10⁷ to at or about 10⁹ g-NK cells, from at or about 10⁷ to at or about 10⁸ g-NK cells, from at or about 10⁸ to at or about 10¹⁰ g-NK cells, from at or about 10⁸ to at or about 10⁹ g-NK cells, or from at or about 10⁹ to at or about 10¹⁰ g-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 provided methods. 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 10⁵ and at or about 10¹² g-NK cells, or from at or about 10⁵ to at or about 10⁸ g-NK cells, or from at or about 10⁶ and at or about 10¹² g-NK cells, or from at or about 10⁸ and at or about 10” g-NK cells, or from at or about 10⁹ and at or about 10¹⁰ g-NK cells. In some embodiments, the composition comprises greater than or greater than at or about 10⁵ g-NK cells, at or about 10⁶ g-NK cells, at or about 10⁷ g-NK cells, at or about 10⁸ g-NK cells, at or about 10⁹ g-NK cells, at or aboutlO¹⁰ g-NK cells, at or about 10” g-NK cells, or at or about 10¹² g-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 NKG2C^pos cells or a subset thereof that is from at or about 10⁵ and at or about 10¹²NKG2C^pos cells or a subset thereof, or from at or about 10⁵ to at or about 10⁸ NKG2C^pos cells or a subset thereof, or from at or about 10⁶ and at or about 10¹² NKG2C^pos cells or a subset thereof, or from at or about 10⁸ and at or about 10” NKG2C^pos cells or a subset thereof, or from at or about 10⁹ and at or about 10¹⁰ NKG2C^pos cells or a subset thereof. In some embodiments, the composition comprises greater than or greater than at or about 10⁵ NKG2C^pos cells or a subset thereof, at or about 10⁶ NKG2C^pos cells or a subset thereof, at or about 10⁷ NKG2C^pos cells or a subset thereof, at or about 10⁸ NKG2C^pos cells or a subset thereof, at or about 10⁹ NKG2C^pos cells or a subset thereof, at or about 10¹⁰ NKG2C^pos cells or a subset thereof, at or about 10” NKG2C^pos cells or a subset thereof, or at or about 10¹² NKG2C^pos cells 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 10⁵ cells/mL, 5 x 10⁵ cells/mL, 1 x 10⁶ cells/mL, 5 x 10⁶ cells/mL, 1 x 10⁷ cells/mL, 5 x 10⁷ cells/mL or 1 x 10⁸ cells/mL. In some embodiments, the cell density of the composition is between or between about 1 x 10⁵ cells/mL to 1 x 10⁸ cells/mL, 1 x 10⁵ cells/mL to 1 x 10⁷ cells/mL, 1 x 10⁵ cells/mL to 1 x 10⁶ cells/mL, 1 x 10⁶ cells/mL to 1 x 10⁷ cells/mL, 1 x 10⁶ cells/mL to 1 x 10⁸ cells/mL, 1 x 10⁶ cells/mL to 1 x 10⁷ cells/mL or 1 x 10⁷ cells/mL to 1 x 10⁸ cells/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 of which 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 NKG2C^pos cells 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 FcRy^neg cells 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 some embodiments, 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 HLA mis-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 gene edited 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-specific endonuclease 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 CD3^negCD57^pos or CD3^negCD56^pos) 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 endonuclease used 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 of RNA-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 also deplete 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 a TGF 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., is complementary 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, these alternative 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 a host 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 an immunomodulator, 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 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 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 in which 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 higher expression 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 to be “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 presence of 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 threshold level. 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 caused by, 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 the presence 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) and according 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 of g-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) a MRD 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-M or 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 IDH2^R140Q is 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/mm³ platelets and >l,000/mm³ neutrophils.

[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, 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 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 et al., 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, or PMBCL, (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 a subject 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, Janssen Biotech, 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 (first dose), 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 10⁵ to at about 10¹², or from at or about 10⁵ and at or about 10⁸, or from at or about 10⁶ and at or about 10¹², or from at or about 10⁸ and at or about 10”, or from at or about 10⁹ and at or about IO¹⁰ of 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 10⁶ to IO¹⁰ of 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 10⁵ to at about 10¹², or from at or about 10⁵ and at or about 10⁸, or from at or about 10⁶ and at or about 10¹², or from at or about 10⁸ and at or about 10”, or from at or about 10⁹ and at or about 10¹⁰ of 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 10¹² of NK cells from any of the provided compositions are administered to the individual. In some embodiments, from or from about 10⁶ to 10¹⁰ of 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 x

10⁸ cells to at or about 50 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 10⁸ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 10 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 20 x

10⁹ cells 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 10⁵ to at about 10¹² g-NK cells, or from at or about 10⁵ and at or about 10⁸g-NK cells, or from at or about 10⁶ and at or about 10¹² g-NK cells, or from at or about 10⁸ and at or about 10” g-NK cells, or from at or about 10⁹ and at or about 10¹⁰ g-NK cells. In some embodiments, a dose of cells containing at or greater than at or about 10⁵ g-NK cells, at or greater than at or about 10⁶ g-NK cells, at or greater than at or about 10⁷ g-NK cells, at or greater than at or about 10⁸ g-NK cells, at or greater than at or about 10⁹ g- NK cells, at or greater than at or about 10¹⁰ g-NK cells, at or greater than at or about 10” g-NK cells, or at or greater than at or about 10¹² g-NK cells are administered to the individual. In some embodiments, from or from about 10⁶ to IO¹⁰ g-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 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, such as from at or about 1 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 5 x 10⁵ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 2.5 x 10⁵ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 5 x 10⁵ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 7.5 x 10⁶ cells/kg, or from at or about 7.5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg. In some embodiments, the individual dose is from at or about 1 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, such as from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 7.5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 7.5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 1 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 2.5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, or from at or about 7.5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg. In some embodiments, the individual dose is from at or about 5 x 10⁷ to at or about 10 x 10⁹ cells, such as from at or about 5 x 10⁷ to at or about 5 x 10⁹ cells, from about or about 5 x 10⁷ to at or about 1 x 10⁹ cells, from at or about 5 x 10⁷ to at or about 5 x 10⁸ cells, from about or about 5 x 10⁷ to at or about 1 x 10⁸ cells, 1 x 10⁸ to at or about 10 x 10⁹ cells, from at or about 1 x 10⁸ to at or about 5 x 10⁹ cells, from about or about 1 x 10⁸ to at or about 1 x 10⁹ cells, from at or about 1 x 10⁸ to at or about 5 x

10⁸ cells, from at or about 5 x 10⁸ to at or about 10 x 10⁹ cells, from at or about 5 x 10⁸ to at or about 5 x

10⁹ cells, from about or about 5 x 10⁸ to at or about 1 x 10⁹ cells, from about or about 5 x 10⁸ to at or about 2 x 10¹⁰ cells, from at or about 1 x 10⁹ to at or about 10 x 10⁹ cells, from at or about 1 x 10⁹ to at or about 5 x 10⁹ cells, or from at or about 5 x 10⁹ to at or about 10 x 10⁹ cells. In some embodiments, the individual dose is or is about 5 x 10⁸ cells. In some embodiments, the individual dose is or is about 1 x 10⁹ cells. In some embodiments, the individual dose is or is about 5 x 10⁹ cells. In some embodiments, the individual dose is or is about 1 x 10¹⁰ cells. In some embodiments, the individual dose is or is about 2 x 10¹⁰ cells. 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 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, such as from at or about 1 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 5 x 10⁵ cells/kg, from at or about 1 x 10⁵ cells/kg to at or about 2.5 x 10⁵ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 2.5 x 10⁵ cells/kg to at or about 5 x 10⁵ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁶ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 7.5 x 10⁵ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 2.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 7.5 x 10⁶ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 5 x 10⁶ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 7.5 x

10⁶ cells/kg, or from at or about 7.5 x 10⁶ cells/kg to at or about 1 x 10⁷ cells/kg. In some embodiments, the dose for administration is from at or about 1 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, such as from at or about 2.5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 7.5 x 10⁵ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 1 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 2.5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 7.5 x 10⁶ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 1 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 2.5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, from at or about 5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/kg, or from at or about 7.5 x 10⁷ cells/kg to at or about 1 x 10⁸ cells/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 10⁷ to at or about 10 x 10⁹ cells, such as from at or about 5 x 10⁷ to at or about 5 x 10⁹ cells, from about or about 5 x 10⁷ to at or about 1 x 10⁹ cells, from at or about 5 x

10⁷ to at or about 5 x 10⁸ cells, from about or about 5 x 10⁷ to at or about 1 x 10⁸ cells, 1 x 10⁸ to at or about 10 x 10⁹ cells, from at or about 1 x 10⁸ to at or about 5 x 10⁹ cells, from about or about 1 x 10⁸ to at or about 1 x 10⁹ cells, from at or about 1 x 10⁸ to at or about 5 x 10⁸ cells, from at or about 5 x 10⁸ to at or about 10 x 10⁹ cells, from at or about 5 x 10⁸ to at or about 5 x 10⁹ cells, from about or about 5 x 10⁸ to at or about 1 x 10⁹ cells, from at or about 1 x 10⁹ to at or about 10 x 10⁹ cells, from at or about 1 x 10⁹ to at or about 5 x 10⁹ cells, from at or about 5 x 10⁹ to at or about 10 x 10⁹ cells, or from at or about 5 x 10⁹ to at or about 20 x 10⁹ cells. In some embodiments, the dose for administration is at or about 5 x 10⁸ cells. In some embodiments, the dose for administration is at or about 1 x 10⁹ cells. In some embodiments, the dose for administration is at or about 5 x 10⁹ cells. In some embodiments, the dose for administration is at or about 1 x 10¹⁰ cells. In some embodiments, the dose for administration is at or about 2 x 10¹⁰ cells. In some embodiments, the dose for administration is at or about 5 x 10¹⁰ cells. 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.

[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 10⁶ cells to 10 x 10⁹ cells are administered by intravenous infusion in a volume of 1 mL to 100 mL. In some embodiments, at or about 50 x 10⁶ cells are administered. In some embodiments, at or about 1 x 10⁹ cells are administered. In some embodiments, at or about 5 x 10⁹ cells are administered. In some embodiments, at or about 10 x 10⁹ cells 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 10⁹ cells 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 10⁷ cells/mL (e.g., at or about 5 x 10⁹ cells 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 combinations of 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 about 7 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 an amount 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/m² to about 3000 mg/m² of the antibody (such as about 5- 300 mg/m², about 30-600 mg/m², about 300-1500 mg/m², or about 600-3000 mg/m². In some embodiments, each dose of the antibody is administered in an amount from about 30-600 mg/m². In some embodiments, each dose of the antibody is administered in an amount of about 15 mg/m², about 30 mg/m², about 60 mg/m², about 90 mg/m², about 120 mg/m², about 150 mg/m², about 180 mg/m², about 210 mg/m², about 240 mg/m², about 270 mg/m², about 300 mg/m², about 330 mg/m², about 360 mg/m², about 375 mg/m², about 390 mg/m², about 420 mg/m², about 450 mg/m², about 480 mg/m², about 510 mg/m², about 540 mg/m², about 570 mg/m², about 600 mg/m², about 630 mg/m², about 660 mg/m², about 690 mg/m², about 720 mg/m², about 750 mg/m², about 780 mg/m², about 810 mg/m², about 840 mg/m², about 870 mg/m², about 900 mg/m², about 930 mg/m², about 960 mg/m², or about 990 mg/m², or any value between any of the foregoing.

[0390] In some examples, the subject is administered about 100 to about 2000 mg of the antibody (such as about 100-1000 mg, about 100-800 mg, about 300-700 mg, for example, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg).

[0391] An effective amount of the antibody can be selected by a skilled clinician, taking into consideration the particular antibody, the particular disease or conditions (e.g., HLA-E expressing cancer), 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, such as an effective amount of a composition containing g-NK cells, such as composition of g-NK cells described in Section I. A. Examples of any such effective amounts of g-NK cells are described in Section I.C. Both the antibody and the population of g-NK cells are typically administered parenterally, for example intravenously. One of skill in the art can determine appropriate routes of administration. [0392] Non-limiting antibodies that can be used in the provided methods in combination therapy with a cell composition including g-NK cells include antibodies directed against a B cell antigen. In some embodiments, the B cell antigen is an antigen selected from the group consisting of CD19, CD20, CD38, CD22, BAFF-R, BCMA, and TACI.

[0393] In some embodiments, the antibody is an anti-CD20 antibody. Any anti-CD20 antibody with means for binding CD20 and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, rituximab, ocrelizumab (Ocrevus®), ofatumumab (Kesimpta®), obinutuzumab (Gazyva®; also known as afutuzumab, GA101), ripertamab, tositumomab (Bexxar®), ublituximab (TG-1101). In some embodiments, the antibody is a biosimilar of rituximab. Non-limiting examples of a rituximab biosimilar include MK-8808, CT-P10, GP-2013, PF-05280586, SAIT101, TL011, and B1695500. In some embodiments, the antibody is rituximab or a biosimilar, ocrelizumab, ofatumumab (Kesimpta®), obinutuzumab or ripertamab.

[0394] In some embodiments, the subject is a subject with a lymphoma, such as non-Hodgkin’s lymphoma (NHL). Exemplary features of subjects with lymphoma, such as NHL, for treatment in the provided methods in combination with an antibody include any as described in Section LB.2.

[0395] In some embodiments, the anti-CD20 antibody is used for treating a subject with lymphoma, such as NHL. In some embodiments, the anti-CD20 antibody is used in combination with a cell composition including g-NK cells for treating a subject with NHL. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, rituximab is used for treating a subject with NHL. In some embodiments, rituximab is used in combination with a cell composition including g-NK cells for treating a subject with NHL.

[0396] In some embodiments, the anti-CD20 antibody (e.g., rituximab) used in combination with a cell composition including g-NK cells is administered as a once weekly dose. In some embodiments, the anti-CD20 antibody (e.g., rituximab) is administered once weekly for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, 4 once weekly doses of anti-CD20 antibody are administered. In some embodiments, 6 once weekly doses of anti-CD20 antibody are administered. In some embodiments, 8 once weekly doses of anti-CD20 antibody are administered. In some embodiments, the once weekly doses are administered in consecutive weeks. In some embodiments, each dose of the anti-CD20 antibody (e.g., rituximab) used in combination with a cell composition including g- NK cells is administered at a dose of about 30-600 mg/m². In some embodiments, each dose of the anti-CD20 antibody (e.g., rituximab) is administered at a dose of about 375 mg/m². In some embodiments, the anti-CD20 antibody is administered intravenously (IV).

[0397] In some embodiments, administration of at least one dose of the anti-CD20 antibody (e.g., rituximab) used in combination with a cell composition including g-NK cells is initiated within one month prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the anti-CD20 antibody (e.g., rituximab) is 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 anti-CD20 antibody (e.g., rituximab) is 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 anti-CD20 antibody (e.g., rituximab) of the combination therapy is initiated at or about one week (e.g., 7 days) prior to the first administration of the composition of g-NK cells.

[0398] In some embodiments, each dose of g-NK cells is an effective amount for treating a subject with lymphoma, such as NHL, in combination with an anti-CD20 antibody (e.g., rituximab), such as any effective amount of g-NK cells described in Section I.C. In some examples, each dose of g-NK cells may be from at or about from 5 x 10⁸ cells to at or about 2 x IO¹⁰ or about 5 x 10⁸ cells, about 5 x 10⁹ cells, about 1 x IO¹⁰ cells, or about 2 x IO¹⁰ cells.

[0399] In some embodiments, the g-NK cells are administered once weekly (QW). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. In some embodiments, the g-NK cell composition is administered in consecutive weeks. As one specific example, the g-NK cell composition is administered on Days 0, 7, and 14, and the anti-CD20 antibody (e.g., rituximab) is administered on Days -7, 0, 7, 14, 21 and 28.

[0400] In some embodiments, the g-NK cells are administered every other day (Q2D). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. As one specific example, the g-NK cell composition is administered on Days 0, 2, and 4, and the anti-CD20 antibody (e.g., rituximab) is administered on Days -7, 0, 7, 14, 21 and 28.

[0401] In some embodiments, the antibody is an anti-CD19 antibody. Any anti-CD19 antibody with means for binding CD 19 and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, inebilizumab (Uplizna®; also called MEDI-551), tafasitamab (tafasitamab-cxix; Monjuvi®; also called MOR208 or XmAb 5574), obexelimab (also known as XmAb 5871), MDX-1342, DI-B4 or LY3541860. In some embodiments, the antibody is inebilizumab, tafasitamab, or obexelimab. [0402] In some embodiments, the anti-CD19 antibody is used for treating a subject with lymphoma, such as NHL. In some embodiments, the anti-CD19 antibody is used in combination with a cell composition including g-NK cells for treating a subject with NHL.

[0403] In some embodiments, the anti-CD19 antibody is used in combination with a cell composition including g-NK cells is administered as a once weekly dose. In some embodiments, the anti- CD19 antibody is administered once weekly for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, 4 once weekly doses of anti-CD19 antibody are administered. In some embodiments, 6 once weekly doses of anti-CD19 antibody are administered. In some embodiments, 8 once weekly doses of anti-CD19 antibody are administered. In some embodiments, the once weekly doses are administered in consecutive weeks. In some embodiments, the anti-CD19 antibody is administered intravenously (IV).

[0404] In some embodiments, administration of at least one dose of the anti-CD20 antibody used in combination with a cell composition including g-NK cells is initiated within one month prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the anti-CD19 antibody is 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 anti-CD19 antibody is 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 anti-CD19 antibody of the combination therapy is initiated at or about one week (e.g., 7 days) prior to the first administration of the composition of g-NK cells.

[0405] In some embodiments, each dose of g-NK cells is an effective amount for treating a subject with lymphoma, such as NHL, in combination with an anti-CD19 antibody, such as any effective amount of g-NK cells described in Section I.C. In some examples, each dose of g-NK cells may be from at or about from 5 x 10⁸ cells to at or about 2 x 10¹⁰ or about 5 x 10⁸ cells, about 5 x 10⁹ cells, about 1 x 10¹⁰ cells, or about 2 x 10¹⁰ cells.

[0406] In some embodiments, the g-NK cells are administered once weekly (QW). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. In some embodiments, the g-NK cell composition is administered in consecutive weeks. As one specific example, the g-NK cell composition is administered on Days 0, 7, and 14, and the anti-CD19 antibody is administered on Days -7, 0, 7, 14, 21 and 28. [0407] In some embodiments, the g-NK cells are administered every other day (Q2D). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. As one specific example, the g-NK cell composition is administered on Days 0, 2, and 4, and the anti-CD19 antibody is administered on Days -7, 0, 7, 14, 21 and 28.

[0408] In some embodiments, the antibody is an anti-CD38 antibody. Any anti-CD38 antibody with means for binding CD38 and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, daratumumab and isatuximab.

[0409] In some embodiments, the dose of the antibody administered is the same or similar to its approved dose, for the duration when it is administered in accord with the provided methods and uses. For instance, each of daratumumab and isatuximab are approved antibody therapies that include a once weekly administration for the initial cycles of administration. An approved dosing regimen of daratumumab includes administration at or about 16 mg/week when it is administered once a week (QW). In some embodiments, in provided methods in combination with g-NK cells, daratumumab may be administered once a week at a dose of about 16 mg/kg, such as for multiple consecutive weeks, for example 8 weeks. As another example, an approved dosing regimen of isatuximab includes administration at or about 10 mg/kg when it is administered once a week. In some embodiments, in provided methods in combination with g-NK cells, isatuximab may be administered once a week at a dose of about 10 mg/kg, such as for multiple consecutive weeks, for example 4 to 6 weeks.

[0410] In other embodiments, the dose of the antibody that is administered is reduced compared to an approved dose. In some embodiments, the dose administered is reduced to as low as about 2.5% of an approved dose of the antibody. For instance, in embodiments in which the dose is 16 mg/kg (e.g., administered once a week), the dose may be reduced to as low as 0.5 mg/kg (e.g., administered once a week). In some embodiments, the dose is reduced to 2.5% to 50% of the approved dose of an antibody, such as reduced to about 2.5% to about 25%, about 2.5% to about 10%, about 2.5% to about 5%, about 5% to about 50%, about 5% to about 25%, about 5% to about 10%, about 10% to about 50%, about 10% to about 25% or about 25% to about 50 % of the approved dose of an antibody.

[0411] In particular, it is believed that the potent ADCC activity of g-NK cells supports activity even at lower administered doses of antibody. Moreover, in the case of CD38, a lower dose of administered antibody is believed to reduce any fratricide risk against CD38 that could be expressed on the g-NK cells. CD38 is a marker that is expressed on conventional NK cells and other NK cell sources that can result in “fratricide,” whereby ADCC activity leads to elimination of NK cells in addition to tumor. In fact, other reported NK cell compositions are reported to express a high percentage (e.g. >90%) of CD38high NK cells. In contrast, enriched g-NK cells are known to have a substantially reduced percentage of CD38pos cells, which thus leads to markedly reduced anti-CD38 (e.g. daratumumab)- mediated fratricide by the g-NK cells related to the conventional NK cell. Despite the lower fratricide, it is believed that administering a lower dose of an anti-CD38 antibody in combination with g-NK cells also can further reduce the fratricide and be therapeutically effect to lead to potent ADCC activity and killing of the CD38-expressing tumor cells.

[0412] In some embodiments, the subject is a subject with multiple myeloma (MM). In some embodiments, the anti-CD38 antibody is used for treating a subject with multiple myeloma (MM). In some embodiments, the anti-CD38 antibody is used in combination with a cell composition including g- NK cells for treating a subject with multiple myeloma (MM). In some embodiments, the anti-CD38 antibody is daratumumab.

[0413] In some embodiments, daratumumab is used for treating a subject with multiple myeloma (MM). In some embodiments, daratumumab is used in combination with a cell composition including g- NK cells for treating a subject with multiple myeloma (MM).

[0414] In some embodiments, daratumumab used in combination with a cell composition including g-NK cells is administered as a once weekly dose. In some embodiments, the daratumumab is administered once weekly for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, 4 once weekly doses of daratumumab are administered. In some embodiments, 6 once weekly doses of daratumumab are administered. In some embodiments, 8 once weekly doses of daratumumab are administered. In some embodiments, the once weekly doses are administered in consecutive weeks. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-50 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 10-50 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 8-16 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-10 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-8 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-5 mg/kg. In some embodiments, each dose of daratumumab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-1 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 0.5 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 1 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 1.5 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 2 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 3 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 4 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 5 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 8 mg/kg. In some embodiments, each dose of daratumumab is administered at a dose of about 16 mg/kg. In some embodiments, daratumumab is administered intravenously (IV).

[0415] In some embodiments, administration of at least one dose of daratumumab used in combination with a cell composition including g-NK cells is initiated within one month prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of daratumumab is 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 daratumumab is 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 daratumumab of the combination therapy is initiated at or about one week (e.g., 7 days) prior to the first administration of the composition of g-NK cells.

[0416] In some embodiments, each dose of g-NK cells is an effective amount for treating a subject with MM, in combination with daratumumab, such as any effective amount of g-NK cells described in Section I.C. In some examples, each dose of g-NK cells may be from at or about from 5 x 10⁸ cells to at or about 2 x IO¹⁰ or about 5 x 10⁸ cells, about 5 x 10⁹ cells, about 1 x IO¹⁰ cells, or about 2 x IO¹⁰ cells.

[0417] In some embodiments, the g-NK cells are administered once weekly (QW). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. In some embodiments, the g-NK cell composition is administered in consecutive weeks. As one specific example, the g-NK cell composition is administered on Days 0, 7, and 14, and daratumumab is administered on Days -7, 0, 7, 14, 21 and 28.

[0418] In some embodiments, the g-NK cells are administered every other day (Q2D). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. As one specific example, the g-NK cell composition is administered on Days 0, 2, and 4, and daratumumab is administered on Days -7, 0, 7, 14, 21 and 28.

[0419] In some embodiments, the anti-CD38 antibody is isatuximab. In some embodiments, isatuximab is used for treating a subject with multiple myeloma (MM). In some embodiments, isatuximab is used in combination with a cell composition including g-NK cells for beating a subject with multiple myeloma (MM).

[0420] In some embodiments, isatuximab used in combination with a cell composition including g- NK cells is administered as a once weekly dose. In some embodiments, the isatuximab is administered once weekly for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In some embodiments, 4 once weekly doses of isatuximab are administered. In some embodiments, 6 once weekly doses of isatuximab are administered. In some embodiments, 8 once weekly doses of isatuximab are administered. In some embodiments, the once weekly doses are administered in consecutive weeks. In some embodiments, each dose of isatuximab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-20 mg/kg. In some embodiments, each dose of isatuximab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-10 mg/kg. In some embodiments, each dose of isatuximab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-8 mg/kg. In some embodiments, each dose of isatuximab used in combination with a cell composition including g- NK cells is administered at a dose of about 0.5-5 mg/kg. In some embodiments, each dose of isatuximab used in combination with a cell composition including g-NK cells is administered at a dose of about 0.5-1 mg/kg. In some embodiments, each dose of isatuximab used in combination with a cell composition including g-NK cells is administered at a dose of about 1-20 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 0.5 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 1 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 1.5 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 2 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 3 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 4 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 5 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 6 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 7 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 8 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 9 mg/kg. In some embodiments, each dose of isatuximab is administered at a dose of about 10 mg/kg. In some embodiments, isatuximab is administered intravenously (IV). [0421] In some embodiments, administration of at least one dose of isatuximab used in combination with a cell composition including g-NK cells is initiated within one month prior to the first administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of isatuximab is 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 isatuximab is 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 isatuximab of the combination therapy is initiated at or about one week (e.g., 7 days) prior to the first administration of the composition of g-NK cells.

[0422] In some embodiments, each dose of g-NK cells is an effective amount for treating a subject with MM, in combination with isatuximab, such as any effective amount of g-NK cells described in Section I.C. In some examples, each dose of g-NK cells may be from at or about from 5 x 10⁸ cells to at or about 2 x IO¹⁰ or about 5 x 10⁸ cells, about 5 x 10⁹ cells, about 1 x IO¹⁰ cells, or about 2 x IO¹⁰ cells.

[0423] In some embodiments, the g-NK cells are administered once weekly (QW). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. In some embodiments, the g-NK cell composition is administered in consecutive weeks. As one specific example, the g-NK cell composition is administered on Days 0, 7, and 14, and isatuximab is administered on Days -7, 0, 7, 14, 21 and 28.

[0424] In some embodiments, the g- NK cells are administered every other day (Q2D). In some embodiments, the g-NK cell composition is administered from two to six total doses. In some embodiments, the g-NK cell composition is administered in two total doses. In some embodiments, the g- NK cell composition is administered in three total doses. In some embodiments, the g-NK cell composition is administered in four total doses. In some embodiments, the g-NK cell composition is administered in five total doses. In some embodiments, the g-NK cell composition is administered in six total doses. As one specific example, the g-NK cell composition is administered on Days 0, 2, and 4, and isatuximab is administered on Days -7, 0, 7, 14, 21 and 28.

[0425] In some embodiments, the antibody is an anti-CD22 antibody. Any anti-CD22 antibody with means for binding CD22 and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, epratuzumab, SM03 or inotuzumab. In some embodiments, the antibody is epratuzumab. [0426] In some embodiments, the antibody is an anti-BAFF-R antibody. Any anti-BAFF-R antibody with means for binding BAFF-R and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, belimumab and ianalumab (VAY-736).

[0427] In some embodiments, the antibody is an anti-TACI antibody. Any anti-TACI antibody with means for binding TACI and engaging CD16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, GenSci-X002.

[0428] In some embodiments, the antibody is an anti-BCMA antibody. Any anti-BCMA antibody with means for binding BCM A and engaging CD 16 via the Fc domain can be used. Exemplary antibodies include, but are not limited to, belantamab.

[0429] In some embodiments, the additional agent is a bispecific antibody. In some embodiments, the subject is administered a population of g-NK cells described herein and an effective dose of a bispecific antibody. In some embodiments, the bispecific antibody comprises a first binding domain and a second binding domain, the first binding domain specifically binding to a surface antigen on an NK cell. In some embodiments, the first binding domain specifically binds to an activating receptor, for instance CD16 (CD16a), on an NK cell. In some embodiments, the second binding domain specifically binds to a B cell antigen.

[0430] In some embodiments, the additional agent is a bispecific NK cell engager (BiKE). In some embodiments, the subject is administered a population of g-NK cells described herein and an effective dose of a bispecific NK cell engager (BiKE). In some embodiments, the BiKE comprises a first binding domain and a second binding domain, the first binding domain specifically binding to a surface antigen on an NK cell. In some embodiments, the first domain specifically binds to a NK cell surface antigen CD16, NKG2D, NKG2C, NKp30, or NKp46. In some embodiments, the first domain specifically binds to CD16. In some embodiments, the first binding domain specifically binds to an activating receptor, for instance CD16 (CD16a), on an NK cell. In some embodiments, the first domain specifically binds to NKG2D.

[0431] In some embodiments, the second binding domain specifically binds to a target antigen expressed by cells of a cancer that expresses HLA-E. Such exemplary target antigens expressed by cells of a cancer that expresses HLA-E can be found herein in Section IV. A. Exemplary cancers include, but are not limited to, lymphomas (e.g., Non-Hodgkin’s Lymphoma), myeloma (e.g., multiple myeloma), and acute myeloid leukemia (AML). In some embodiments, the target antigen is a lymphoma antigen. In some embodiments, the target antigen is a non-Hodgkin’s lymphoma (NHL) antigen. In some embodiments, the second binding domain specifically binds to a lymphoma antigen, such as an NHL antigen. Exemplary lymphoma antigens, such as NHL antigens, include CD19, CD20, and CD22. In some embodiments, the second binding domain specifically binds to CD19, CD20, or CD22. In some embodiments, the target antigen is a multiple myeloma (MM) antigen. In some embodiments, the second binding domain specifically binds to a multiple myeloma (MM) antigen. Exemplary multiple myeloma (MM) antigens include BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DL, and Kappa light chain. In some embodiments, the second binding domain specifically binds to BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DE, or Kappa light chain. In some embodiments, the target antigen is acute myeloid leukemia (AME). In some embodiments, the second binding domain specifically binds to an AME antigen. Exemplary AME antigens include CD123, CD33, and CLL-E In some embodiments, the second binding domain specifically binds to CD123, CD33, and CLL-E

[0432] In some embodiments, the second binding domain specifically binds to a T cell antigen. In some embodiments, the second binding domain specifically binds to a B cell antigen. In certain embodiments, the B cell antigen is BCMA. In some embodiments, the second binding domain specifically targets a B cell, for instance, by targeting BCMA. In any of the preceding embodiments, any bispecific NK cell engager (BiKE) with means for targeting a B cell and engaging CD 16 via the Fc domain can be used.

[0433] In some embodiments, the subject is administered a population of g-NK cells described herein and an effective dose of an additional T cell-targeting therapy, such as a bispecific T cell-targeting therapy, such as a T cell engager (e.g., BiTE). The provided embodiments are based on the discovery of the inventors that g-NK cell compositions alter tumor microenvironment to allow the infiltration of nonexhausted T cells to assist in the treatment or control of cancer that expresses HLA-E, such as multiple myeloma (see, e.g., Example 9). Accordingly, a combination therapy that both exploits the ability of g- NK cells to improve infiltration of T cells to cancer cells and further enhances antigen-directed cytotoxic activity of the T cells using T cell-targeting therapy to cancer cells may further enhance the treatment of the target cancer cells.

[0434] In some embodiments, a bispecific T cell targeting agent is a bispecific antibody. Bispecific antibodies may be designed to bind with a first “arm” (a first binding domain) to a component of the T cell receptor (TCR) complex, such as CD3, and a second “arm” (a second binding domain) to a surface target antigen on a target cell. The simultaneous binding of such an antibody to both its targets by both arms will bring in proximity the cell expressing the component of the TCR complex, e.g., a T cell, and the target cell. In some embodiments, the simultaneous binding of such an antibody will bring in proximity a T cell and the target cell.

[0435] In some embodiments, the bispecific T cell targeting agent includes a first binding portion that binds to the CD3 ectodomain expressed by a T cell and a second binding portion that binds to a target antigen on a target cell. In some embodiments, the bispecific T cell targeting agent comprises two antibody variable domains on a single polypeptide chain. [0436] In some embodiments, a first portion of the bispecific T cell targeting agent binds to the CD3 ectodomain expressed by a T cell. In some embodiments, the first portion of the bispecific T cell targeting agent comprises a first binding domain. In some embodiments, the first binding domain of the bispecific T cell targeting agent can be derived from an antibody. In particular embodiments, the first binding domain of the bispecific T cell targeting agent may include all or at least a portion of a variable heavy chain (VH) and/or a variable light chain (VL). In some embodiments, the first binding domain of the bispecific T cell targeting agent comprises a portion of an antibody, such as an antibody fragment. In some embodiments, the first binding domain of the bispecific T cell targeting agent comprises a singlechain variable fragment (scFv).

[0437] In some embodiments, the first binding domain of the bispecific T cell targeting agent engages or binds to a CD3 ectodomain of T cells. In particular embodiment, the CD3 ectodomain comprises an epsilon (CD3e) ectodomain.

[0438] In some embodiments, the first binding domain of a bispecific T cell targeting agent comprises an anti-CD3 antibody or antigen-binding fragment thereof, including, e.g., a VH, VL, SCFV, light chain, or heavy chain (such as IgGl, IgG2, or IgG4).

[0439] Any of the known anti-CD3 antibodies may be used in the present invention, including but not limited to, the Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766), Otelixizumab and Foralumab.

[0440] In some embodiments, the anti-CD3 antibody is OKT3 or the antigen-binding fragment is derived from OKT3. OKT3 (Brown, WM, 2006, Curr Opin Investig Drugs 7:381-388; Ferran, C et al., 1993 Exp Nephrol 1:83-89; Kung, P. et al., 1991, Science 206:347-349; Salmeron, A. et al., 1991, J Immunol 147:3047-3052) is the first monoclonal antibody approved for human therapeutic use, and is clinically used as an immunomodulator for the treatment of allogenic transplant rejection. U.S. Pat. No. 4,658,019, describes a hybridoma (designated OKT3, ATCC Accession No. CRL-8001) which is capable of producing a murine monoclonal antibody against an antigen found on most normal human peripheral T cells.

[0441] In some embodiments, the anti-CD3 antibody is SP34 or the antigen-binding fragment is derived from SP34. A SP34 mouse monoclonal antibody may bind specifically to human CD3 in denatured form (western blot or dot blot) and in native form (on T cells) (Pressano, S. et al., 1985 The EMBO J. 4:337-344, ; Alarcon, B. et al., 1991 The EMBO J. 10:903-912). SP34 mouse monoclonal antibody also binds to CD3e singly transfected COS cells as well as CD3e/yor CD3e/8 double transfectants (Salmeron A. et al., 1991, J. Immunol. 147:3047-52). SP34 antibody also cross reacts nonhuman primates (Yoshino N. et al., 2000, Exp. Anim 49:97-110; Conrad ML. et al., 2007 , Cytometry 71A:925-33,). In addition, SP34 activates T cell when cross-linked (Yang et al., 1986, J. Immunol. 137:1097-1100).

[0442] Further CD3 binding molecules contemplated herein include UCHT-1 (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334, SP34 (Silvana et. al. (1985) The EMBO Journal.' 4:337 -344) and CD3 binding molecules described in W02004/106380; W02010/037838; W02008/119567; W02007/042261; W02010/0150918; WO2018/052503; WO2016/204966.

[0443] A second portion of the bispecific T cell targeting agent is capable of binding to a target antigen on a target cell. In some embodiments, the second portion of the bispecific T cell targeting agent comprises a second binding domain. In some embodiments, the second binding domain of the bispecific T cell targeting agent can be derived from an antibody. In particular embodiments, the second binding domain of the bispecific T cell targeting agent may include all or at least a portion of a variable heavy chain (VH) and/or a variable light chain (VL). In some embodiments, the second binding domain of the bispecific T cell targeting agent comprises a portion of an antibody, such as an antibody fragment. In some embodiments, the second binding domain of the bispecific T cell targeting agent comprises a single-chain variable fragment (scFv).

[0444] In some embodiments, the second binding domain of the bispecific T cell targeting agent engages or binds to a target antigen expressed by cells of a cancer. Such exemplary target antigens expressed by cells of a cancer can be found herein in Section IV. A. In some embodiments, the target antigen is a lymphoma antigen. In some embodiments, the target antigen is a non-Hodgkin’ s lymphoma (NHL) antigen. In some embodiments, the second binding domain specifically binds to a lymphoma antigen, such as an NHL antigen. Exemplary lymphoma antigens, such as NHL antigens, include CD19, CD20, and CD22. In some embodiments, the second binding domain specifically binds to CD19, CD20, or CD22. In some embodiments, the target antigen is a multiple myeloma (MM) antigen. In some embodiments, the second binding domain specifically binds to a multiple myeloma (MM) antigen. Exemplary multiple myeloma (MM) antigens include BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DL, and Kappa light chain. In some embodiments, the second binding domain specifically binds to BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DL, or Kappa light chain. In some embodiments, the target antigen is acute myeloid leukemia (AML). In some embodiments, the second binding domain specifically binds to an AML antigen. Exemplary AML antigens include CD 123, CD33, and CLL-1. In some embodiments, the second binding domain specifically binds to CD123, CD33, and CLL-1.

[0445] In some embodiments, the bispecific T cell targeting agent is a bispecific T cell engager (BiTE). Bispecific T-cell engager antibodies (BiTE) have been explored as a means to recruit cytolytic T-cells to kill tumor cells. This is based on the simultaneous recognition of an antigen on tumor cells and binding to the CD3 epsilon chain, or CD3, within the T-cell receptor complex on T-cells that bridges malignant tumor cells directly to CD3+ T-cells. Blinatumomab, or BLINCYTO®, the first bispecific T- cell engager reactive with the B-cell antigen CD19, was approved by the FDA in 2014 for the treatment of neoplasms.

[0446] A bispecific T cell engager (BiTE) can comprise an antibody or antigen-binding fragment. In some embodiments, the bispecific T cell engager (BiTE) is a bispecific antibody containing at least one antigen-binding domain binding to an activating component of the T cell (e.g. a T cell surface molecule) and at least one antigen-binding domain binding to a surface antigen on a target cell, such as a surface antigen on a tumor or cancer cell, for example any of the listed antigens as described herein.

[0447] A BiTE antibody construct can be a recombinant protein construct, comprising two flexibly linked antibody derived binding domains. In some embodiments, the binding domain of a BiTE comprises two scFv molecules fused together by a flexible linker. In some embodiments, a binding domain of the BiTE is specific for a subunit of the T cell receptor complex on the T cells. In some embodiments, another binding domain of a BiTE is specific for a selected surface antigen, expressed by target cells.

[0448] In some embodiments, the binding domain specific for a subunit of the T cell receptor complex on the T cells includes an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0449] In some embodiments the binding domain specific for a subunit of the T cell receptor complex on the T cells for the bispecific T cell engager is an anti-CD3. In some aspects, the binding domain on the bispecific T cell engager is an anti-CD3s. In some aspects, the anti-CD3 domain is an scFv. In some aspects, the anti- CD3s domain is an scFv. In some embodiments, the anti-CD3s domain of the bispecific T cell engager binds to the CD3s on a T cell.

[0450] In some embodiments, the bispecific T cell engager (BiTE) is a bispecific antibody containing at least one antigen-binding domain binding to the engineered CD3 expressed by a T cell and at least one antigen-binding domain binding to a antigen on a target cell, such as a target antigen expressed by cells of a cancer, for example any of the target antigens as described herein. In some aspects, the engagement of the engineered CD3 protein on the T cells redirects the T cells to the tumor. In some aspects, the binding of the bispecific T cell engager with the T cells expressing the CD3 protein stimulates and/or activates the T cells. In some embodiments, the bispecific T cell engager (BiTE) engages or binds to the T cells, recruiting T cells in proximity to target cells. In some embodiments, the simultaneous or near simultaneous binding of such an antibody to both of its targets can result in a temporary interaction between the T cell and target cell, thereby resulting in activation of the T cell. [0451] Numerous methods of producing bispecific T cell engagers are known, including fusion of two different hybridomas (Milstein and Cuello, Nature 1983;305:537-540), and chemical tethering though heterobifunctional cross linkers (Staerz et al. Nature 1985; 314:628-631). Exemplary bispecific T cell engager (BiTE) molecules may include tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further comprise an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C- terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010).

[0452] In some embodiments, the bispecific T cell engager is blinatumomab or AMG 330 (CD33xCD3; Ravandi et al. J Clin Oncol. 38, 7508 (2020)). In some embodiments, the bispecific T cell engager is Epcoritamab, also known as GEN3013 or DuoBody® (CD20xCD3; Hutchings M. et al., Blood 136, 45-6 (2020)). In some embodiments, the bispecific T cell engager is Odronextamab or REGN1979 (CD20xCD3; Bannerji et al., Blood 136, 42-3 (2020)). In some embodiments, the bispecific T cell engager is Mosunetuzumab or RG7828 (CD20xCD3; Assouline et al., Blood 136, 42^1 (2020)). In some embodiments, the bispecific T cell engager is Plamotamab or XmAbl3676 (CD20xCD3; Patel et al., Blood 134, 4079 (2019)). In some embodiments, the bispecific T cell engager is Glofitamab or RG6026 (CD20xCD3; Hutchings et al. Blood 136, 46-8 (2020)). In some embodiments, the bispecific T cell engager is Flotetuzumab or MGD006 (CD123xCD3; Aldoss et al., Blood 136, 16-8 (2020)). In some embodiments, the bispecific T cell engager is IGM-2323 (CD20/CD3). In some embodiments, the bispecific T cell engager is AMG 673 (CD33/CD3; Subklewe et al. Blood 134, 833 (2019)). In some embodiments, the bispecific T cell engager is AMG 420 (BCMA/CD3; Topp et al., J Clin Oncol. 38, 775-83 (2020)). In some embodiments, the bispecific T cell engager is AMG 701 (BCMA/CD3; Harrison et al., Blood 136, 28-9 (2020)). In some embodiments, the bispecific T cell engager is Teclistamab (BCMA/CD3; Garfall et al., Blood. 136, 27 (2020)). In some embodiments, the bispecific T cell engager is REGN5458 (BCMA/CD3; Madduri et al., Blood. 136, 41-2 (2020)). In some embodiments, the bispecific T cell engager is Elranatamab or PF-06863136 (BCMA/CD3). In some embodiments, the bispecific T cell engager is TNF-383B (BCMA/CD3; Rodriguez C et al. Blood 136, 43-4 (2020)) In some embodiments, the bispecific T cell engager is Cevostamab (FcRH5/CD3; Cohen AD, Harrison et al., Blood 136, 42-3 (2020)) In some embodiments, the bispecific T cell engager is Talquetamab (GPRC5D/CD3; Chari et al., Blood 136, 40-1 (2020)). Any of such bispecific T cell engagers can be used in used in the provided methods, compositions, combinations, kits, or articles of manufacturers. [0453] The bispecific T cell targeting agent can be also be administered separately by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, the T cell engaging therapy is administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intrathoracic, intracranial, or subcutaneous administration.

[0454] In certain embodiments, one or more doses of a bispecific T cell targeting agent are administered. In particular embodiments, between or between about 0.001 pg and about 5,000 pg, inclusive, of the bispecific T cell targeting agent is administered. In particular embodiments, between or between about 0.001 pg and 1,000 pg, 0.001 pg tol pg, 0.01 pg tol pg, 0.1 pg tolO pg, 0.01 pg tol pg, 0.1 pg and 5 pg, 0.1 pg and 50 pg, 1 pg and 100 pg, 10 pg and 100 pg, 50 pg and 500 pg, 100 pg and 1,000 pg, 1,000 pg and 2,000 pg, or 2,000 pg and 5,000 pg of the bispecific T cell targeting agent is administered. In some embodiments, the dose of the bispecific T cell targeting agent is or includes between or between about 0.01 pg/kg and 100 mg/kg, 0.1 pg/kg and 10 pg/kg, 10 pg/kg and 50 pg/kg, 50 pg/kg and 100 pg/kg, 0.1 mg/kg and 1 mg/kg, 1 mg/kg and 10 mg/kg, 10 mg/kg and 100 mg/kg, 100 mg/kg and 500 mg/kg, 200 mg/kg and 300 mg/kg, 100 mg/kg and 250 mg/kg, 200 mg/kg and 400 mg/kg, 250 mg/kg and 500 mg/kg, 250 mg/kg and 750 mg/kg, 50 mg/kg and 750 mg/kg, 1 mg/kg and 10 mg/kg, or 100 mg/kg and 1,000 mg/kg, each inclusive. In some embodiments, the dose of the bispecific targeting agent is at least or at least about or is or is about 0.1 pg/kg, 0.5 pg/kg, 1 pg/kg, 5 pg/kg, 10 pg/kg, 20 pg/kg, 30 pg/kg, 40 pg/kg, 50 pg/kg, 60 pg/kg, 70 pg/kg, 80 pg/kg, 90 pg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1,000 mg/kg.

[0455] In particular embodiments, the bispecific T cell targeting agent is administered orally, intravenously, intraperitoneally, transdermally, intrathecally, intramuscularly, intranasally, transmucosally, subcutaneously, or rectally.

E. Cytokines or Growth Factors

[0456] In some embodiments provided herein, the g- NK cells can be administered to an individual in combination with cytokines and/or growth factors. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL- 15, IL-12, IL-21, IL-18, and IL-27. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, IL- 21, and IL-27. In some embodiments, the cytokines and/or growth factors are the cytokine IL-2. In particular embodiments recombinant IL-2 is administered to the subject. In other particular embodiments, recombinant IL-15 is administered to the subject. In other particular embodiments, recombinant IL-21 is administered to the subject. In some embodiments, the g-NK cells and the cytokines or growth factors are administered sequentially. For example, the g-NK cells may be administered first, followed by administration of the cytokines and/or growth factors. In some embodiments, the g-NK cells are administered simultaneously with the cytokines or growth factors. In some embodiments, the cytokines and/or growth factors are administered first, followed by administration of the g-NK cells.

[0457] In some embodiments, the subject is administered one or more cytokines (such as IL-2, IL- 15, IL-21, IL-18, IL-27, and/or IL-12) to support survival and/or growth of NK cells. The cytokine(s) can be administered before, after, or substantially simultaneously with the NK cells. In some examples, the cytokine(s) can be administered after the NK cells. In one specific example, the cytokine(s) is administered to the subject within about 1-8 hours (such as within about 1-4 hours, about 2-6 hours, about 4-6 hours, or about 5-8 hours) of the administration of the NK cells.

[0458] In some examples, the cytokine(s) can be administered before the NK cells. In one specific example, the cytokine(s) is administered to the subject within about 1 hour of the administration of the g- NK cells.

[0459] In some embodiments, the subject is administered one or more cytokines once a week, two times a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In some embodiments, the subject is administered one or more cytokines once a day, every two days, every three days, every four days, every five days, every six days, or every seven days. In some embodiments, the cytokine (e.g., IL-2) is administered once every two days (every other day; Q2D). In some embodiments, the cytokine (e.g., IL-2) is administered once every three days. In some embodiments, the cytokine (e.g., IL-2) is administered once a week. In some embodiments, the cytokine (e.g., IL-2) is administered at the same frequency as the dose of g-NK cells. In some embodiments, the cytokine (e.g., IL-2) is administered on the same day as the g-NK cells, such as typically within 12 hours of administering the g-NK cells. In some embodiments, the cytokine (e.g., IL-2) is administered 0-6 hours prior to administering each dose of the g-NK cells, such as 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour, 1-4 hours, 1-3 hours or 1-2 hours prior to administering the g-NK cells. In some embodiments, the cytokine (e.g., IL-2) is administered about or within 1 hour prior to administering each dose of the g-NK cells. [0460] In some embodiments, the cytokine is administered in a cycling regimen involving administering a dose of the cytokine (e.g., IL-2) one or more times in a cycle, in which the cycle may be optionally repeated. In some embodiments, the cycle is a 7 day cycle. In some embodiments, the cycle is a 14 days cycle. In some embodiments, the cycle is a 21 day cycle. In some embodiments, the cycle is a 28 day cycle. In some embodiments, a dose of the cytokine (e.g., IL-2) is administered one or more times in a first cycle, which is then repeated one or two times (for a total of 2 or 3 cycling regimens). In some embodiments, the cytokine (e.g., IL-2) is administered once per day for consecutive days in a cycle, such as for 2, 3, 4 or 5 consecutive days of a 7-day cycle. In some embodiments, the cytokine (e.g., IL-2) is administered twice per day (BID) for consecutive days in a cycle, such as for 2, 3, 4, 5, 6 or 7 days of consecutive days of a 7-day cycle. In some embodiments, the cytokine (e.g., IL-2) is administered once every two days (Q2D) in a cycle, such as Q2D in a 7-day cycle. In some embodiments, the cytokine (e.g., IL-2) is administered once a week (QW) in a cycle, such as one time in a 7-day cycle. In some embodiments, there is more than one cycle in a cycling regimen, wherein each cycle can be the same or different.

[0461] In some embodiments, each dose of the one or more cytokines is between at or about 1 million IU and at or about 50 million IU, such as between at or about 1 million IU and at or about 25 million IU, between at or about 1 million IU and at or about 20 million IU, between at or about 1 million IU and at or about 15 million IU, between at or about 1 million IU and at or about 14 million IU, between at or about 1 million IU and at or about 13 million IU, between at or about 1 million IU and at or about 12 million IU, between at or about 1 million IU and at or about 11 million IU, or between at or about 1 million IU and at or about 10 million IU. In some embodiments, each dose of one or more cytokines is between at or about 2 million IU and at or about 20 million IU, between at or about 3 million IU and at or about 20 million IU, between at or about 4 million IU and at or about 20 million IU, between at or about 5 million IU and at or about 20 million IU, or between at or about 6 million IU and at or about 20 million IU. In some embodiments, each dose of one or more cytokines is between at or about 5 million IU and at or about 12 million IU, between at or about 5 million IU and at or about 11 million IU, between at or about 5 million IU and at or about 10 million IU, between at or about 5 million IU and at or about 9 million IU, between at or about 5 million IU and at or about 8 million IU, between at or about 5 million IU and at or about 7 million IU, between at or about 5 million IU and at or about 6 million IU, or between at or about 6 million IU and at or about 7 million IU. In some embodiments, each dose of one or more cytokines is between at or about 1 million IU and at or about 12 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 5 million IU and at or about 10 million IU. In some embodiments, each dose of the one or more cytokines is between at or about 4 million IU and at or about 8 million IU.

Ill [0462] In some embodiments, each dose of the one or more cytokines is at or about 0.25 million IU, is at or about 0.5 million IU, is at or about 1 million IU, is at or about 1.5 million IU, is at or about 2 million IU, is at or about 2.5 million IU, is at or about 3 million IU, is at or about 3.5 million IU, is at or about 4 million IU, is at or about 4.5 million IU, is at or about 5 million IU, is at or about 5.5 million IU, is at or about 6 million IU, is at or about 6.5 million IU, is at or about 7 million IU, is at or about 7.5 million IU, is at or about 8 million IU, is at or about 8.5 million IU, is at or about 9 million IU, is at or about 9.5 million IU, is at or about 10 million IU, is at or about 10.5 million IU, is at or about 11 million IU, is at or about 11.5 million IU, is at or about 12 million IU, is at or about 12.5 million IU, is at or about 13 million IU, is at or about 13.5 million IU, is at or about 14 million IU, is at or about 14.5 million IU, is at or about 15 million IU, is at or about 15.5 million IU, is at or about 16 million IU, is at or about 16.5 million IU, is at or about 17 million IU, is at or about 17.5 million IU, is at or about 18 million IU, is at or about 18.5 million IU, is at or about 19 million IU, is at or about 19.5 million IU, is at or about 20 million IU, or any value between any of the foregoing. In some embodiments, each dose of the one or more cytokines is at or about 5 million IU. In some embodiments, each dose of one or more cytokines is at or about 6 million IU. In some embodiments, each dose of one or more cytokines is at or about 10 million IU.

[0463] In some embodiments, the one or more cytokines is IL-2, IL- 15, IL-21, IL-27, and/or IL-12. In some embodiments, the one or more cytokines is IL-2.

[0464] In particular embodiments, IL-2 is administered to subjects in accord with provided methods. In some embodiments, the dosing of IL-2 in conjunction with adoptive NK cell therapy supports in vivo expansion and augments NK cell persistence. In some embodiments, the administered IL-2 is the recombinant IL-2 aldesleukin (also known as Proleukin), which is a recombinant form of human IL-2. Compared to native human IL-2, the N-terminal alanine is deleted and the sequence contains a cysteine substituted with serine at amino acid position 125 (Cl 25 S mutation) to prevent cysteine mispairing in E. coli, but this does not affect biological activity.

[0465] In some embodiments, the subject is administered IL-2 once a week, two times a week, three times a week, four times a week, five times a week, six times a week, or seven times a week. In some embodiments, the subject is administered IL-2 once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.

[0466] In some embodiments, each dose of IL-2 is between at or about 1 million IU and at or about 50 million IU, such as between at or about 1 million IU and at or about 25 million IU, between at or about 1 million IU and at or about 20 million IU, between at or about 1 million IU and at or about 15 million IU, between at or about 1 million IU and at or about 14 million IU, between at or about 1 million IU and at or about 13 million IU, between at or about 1 million IU and at or about 12 million IU, between at or about 1 million IU and at or about 11 million IU, between at or about 1 million IU and at or about 10 million IU. In some embodiments, each dose of IL-2 is between at or about 2 million IU and at or about 20 million IU, between at or about 3 million IU and at or about 20 million IU, between at or about 4 million IU and at or about 20 million IU, between at or about 5 million IU and at or about 20 million IU, or between at or about 6 million IU and at or about 20 million IU. In some embodiments, each dose of IL-2 is between at or about 5 million IU and at or about 12 million IU, between at or about 5 million IU and at or about 11 million IU, between at or about 5 million IU and at or about 10 million IU, between at or about 5 million IU and at or about 9 million IU, between at or about 5 million IU and at or about 8 million IU, between at or about 5 million IU and at or about 7 million IU, between at or about 5 million IU and at or about 6 million IU, or between at or about 6 million IU and at or about 7 million IU. In some embodiments, each dose of IL-2 is between at or about 1 million IU and at or about 12 million IU. In some embodiments, each dose of IL-2 is between at or about 5 million IU and at or about 10 million IU. In some embodiments, each dose of IL-2 is between at or about 4 million IU and at or about 8 million IU.

[0467] In some embodiments, each dose of IL-2 is at or about 0.25 million IU, is at or about 0.5 million IU, is at or about 1 million IU, is at or about 1.5 million IU, is at or about 2 million IU, is at or about 2.5 million IU, is at or about 3 million IU, is at or about 3.5 million IU, is at or about 4 million IU, is at or about 4.5 million IU, is at or about 5 million IU, is at or about 5.5 million IU, is at or about 6 million IU, is at or about 6.5 million IU, is at or about 7 million IU, is at or about 7.5 million IU, is at or about 8 million IU, is at or about 8.5 million IU, is at or about 9 million IU, is at or about 9.5 million IU, is at or about 10 million IU, is at or about 10.5 million IU, is at or about 11 million IU, is at or about 11.5 million IU, is at or about 12 million IU, is at or about 12.5 million IU, is at or about 13 million IU, is at or about 13.5 million IU, is at or about 14 million IU, is at or about 14.5 million IU, is at or about 15 million IU, is at or about 15.5 million IU, is at or about 16 million IU, is at or about 16.5 million IU, is at or about 17 million IU, is at or about 17.5 million IU, is at or about 18 million IU, is at or about 18.5 million IU, is at or about 19 million IU, is at or about 19.5 million IU, is at or about 20 million IU, or any value between any of the foregoing. In some embodiments, each dose of IL-2 is at or about 5 million IU. In some embodiments, each dose of IL-2 is at or about 6 million IU. In some embodiments, each dose of IL- 2 is at or about 10 million IU.

[0468] In some embodiments, the IL-2 is administered before the administration of the g-NK cells. In some embodiments, the IL-2 is administered within about one hour of the administration of the g-NK cells. In some embodiments, each dose of IL-2 is about 6 million IU.

[0469] In some embodiments provided herein, the IL-2 may be administered in a cycling regimen.

[0470] In some embodiments, the cycling regimen is a 7 day cycle. In some embodiments, the 7 day cycle begins on the day of administration of the g-NK cells (not including any prior administration of the IL-2 prior to administration of the g-NK cells). In some embodiments, the IL-2 is administered once in in a 7-day cycle, i.e. one time in 7 days or once a week (Q1W). In some embodiments, the IL-2 is administered once a week (Q1W). In some embodiments, the IL-2 is administered as one dose in a 7-day cycle.

[0471] In some embodiments, the IL-2 is administered as two doses in a 7-day cycle. In some embodiments, the IL-2 is administered as three doses in a 7-day cycle. In some embodiments, the IL-2 is administered as four doses in a 7-day cycle. In some embodiments, the IL-2 is administered as five doses in a 7-day cycle. In some embodiments, the IL-2 is administered as six doses in a 7-day cycle. In some embodiments, the IL-2 is administered as seven doses in a 7-day cycle. In some embodiments, the IL-2 is administered as eight doses in a 7-day cycle. In some embodiments, the IL-2 is administered as nine doses in a 7-day cycle. In some embodiments, the IL-2 is administered as ten doses in a 7-day cycle. In some embodiments, the IL-2 is administered as eleven doses in a 7-day cycle. In some embodiments, the IL-2 is administered as twelve doses in a 7-day cycle. In any of such embodiments, the IL-2 can be administered on consecutive days of the 7-day cycle. In some embodiments, the IL-2 can be administered only one time each day it is administered in the cycle. In some embodiments, the IL-2 is administered twice daily (i.e. BID) on each day it is administered in the 7-day cycle.

[0472] In some embodiments, the IL-2 is administered twice daily (i.e., BID) for a number of consecutive days in a 7 day cycle, e.g., twice on day 0 (same day as the first administration of the g-NK cells), twice on day 1, twice on day 2, twice on day 3, twice on day 4, twice on day 5, and twice on day 6. In some embodiments, the IL-2 is administered twice daily (i.e., BID) for 2, 3, 4, 5, 6, or 7 days of consecutive days in a 7-day cycle. In some embodiments, the IL-2 is administered twice daily (i.e., BID) for the first 5 consecutive days in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 1, day 2, day 3, and day 4. In some embodiments, the IL-2 is administered one time daily for a number of consecutive days in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 1, day 2, day 3, day 4, day 5, and day 6. In some embodiments, the IL-2 is administered daily for 2, 3, 4, 5, 6, or 7 days of consecutive days in a 7-day cycle. In some embodiments, the IL-2 is administered one time daily for the first 5 consecutive days in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 1, day 2, day 3, and day 4. In some embodiments, the IL-2 is administered one time daily every other day (i.e., Q2D) in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells), day 2, and day 4. In some embodiments, the IL-2 is administered one time daily once a week (e.g., QW), in a 7-day cycle, e.g., on day 0 (same day as the first administration of the g-NK cells). In some embodiments, the 7-day cycle is repeated twice. In some embodiments, the 7-day cycle is repeated three times.

[0473] In some embodiments, the cycling regimen includes one or more 7-day cycles. In some embodiments, the cycling regimen includes one, two or three 7-day cycles. In some embodiments involving two or three cycles, each 7-day cycle is the same or different. In some embodiments, the 7-day cycle is repeated, wherein each 7-day cycle is the same. For example, in some embodiments, the 7-day cycle is repeated twice for a total of three 7-day cycles, wherein IL-2 is administered on the same schedule, e.g., one time every other day (e.g., Q2D), in the first, second, and third 7-day cycles. In some embodiment, at least one 7-day cycle is different from another 7-day cycle in the regimen. For example, in some embodiments, there are three 7-day cycles, wherein IL-2 is administered on a different schedule in the first, second, and third 7-day cycles, e.g., IL-2 is administered twice daily (i.e., BID) for the first 5 consecutive days in a first 7-day cycle, then IL-2 is administered one time daily Q2D for a second 7-day cycle, and then is not administered in the third 7-day cycle (where g-NK cells may be administered during a third cycle).

[0474] In some embodiments, the g- NK cells and IL-2 are administered to an individual in a 7 day cycle.

[0475] In some embodiments, both the g-NK cells and IL-2 are administered one time daily once a week (i.e., QW) in a 7-day cycle, e.g., g-NK cells and IL-2 are administered on day 0 (same day as the first administration of the g- NK cells). In some embodiments, the cycling regimen includes more than one 7-day cycle, which may be the same or different. In some embodiments, the 7-day cycle is repeated two times (e.g., for a total of three 7-day cycles). In some embodiments, each 7-day cycle is the same, e.g., g-NK cells and IL-2 are administered one time daily once a week (i.e., QW) in the first and second 7-day cycles, and optionally also a third 7-day cycle. In some embodiments, the IL-2 is administered before the administration of the g-NK cells. In some embodiments, the IL-2 is administered within about one hour of the administration of the g-NK cells. In some embodiments, each dose of IL-2 is about 6 million IU.

[0476] In some embodiments, the g-NK cells are administered one time daily once a week (i.e., QW) in a 7-day cycle (e.g., g-NK cells are administered on day 0), and IL-2 is administered one time daily for the first consecutive 5 days in a 7 day cycle (e.g., IL-2 is administered on day 0, day 1, day 2, day 3, and day 4). In some embodiments, the cycling regimen includes more than one 7-day cycle, which may be the same or different. In some embodiments, the 7-day cycle is repeated two times (e.g., for a total of three 7-day cycles). In some embodiments, each 7-day cycle is the same, e.g., g-NK cells are administered one time daily once a week (i.e., QW) in the first and second 7-day cycles, and IL-2 is administered once time daily for the first consecutive 5 days in the first and second 7-day cycles. In some embodiments, the IL-2 is administered before the administration of the g-NK cells. In some embodiments, the IL-2 is administered within about one hour of the administration of the g-NK cells. In some embodiments, each dose of IL-2 is about 6 million IU.

[0477] In some embodiments, both the g-NK cells and IL-2 are administered one time daily every other day (i.e., Q2D) in a 7 day cycle, e.g., g-NK cells and IL-2 are administered on day 0 (same day as the first administration of the g- NK cells), day 2, and day 4. In some embodiments, the Q2D administration is a thrice weekly dose. In some embodiments, the cycling regimen includes more than one 7-day cycle, which may be the same or different. In some embodiments, the 7-day cycle is repeated two times (e.g., for a total of three 7-day cycles). In some embodiments, each 7-day cycle is the same, e.g., g-NK cells and IL-2 are administered one time daily every other day (i.e., Q2D) in the first and second 7-day cycles, and optionally also a third 7-day cycle. In some embodiments, the IL-2 is administered before the administration of the g- NK cells. In some embodiments, the IL-2 is administered within about one hour of the administration of the g-NK cells. In some embodiments, each dose of IL-2 is about 6 million IU.

[0478] In some embodiments, there is more than one 7-day cycle, which can be the same or different (e.g., for a total of two 7-day cycles or a total of three 7-day cycles). In some embodiments, each 7-day cycle is different, e.g., the first and second 7-day cycles are different. In some embodiments, the g-NK cells are administered one time daily every other day (i.e., Q2D) in the first 7-day cycle, e.g., g- NK cells are administered on day 0, day 2, and day 4. In some embodiments, the Q2D administration is a thrice weekly dose. In some embodiments, the g-NK cells are not administered in the second 7-day cycle and optionally also not administered in a third 7-day cycle. In some embodiments, the IL-2 is administered twice daily (i.e., BID) for the first consecutive 5 days in the first 7-day cycle, e.g. IL-2 is administered twice on day 0 (e.g., the same day as first administration of the g-NK cells), twice on day 1, twice on day 2, twice on day 3, and twice on day 4. In some embodiments, the IL-2 is administered one time daily every other day (i.e., Q2D) in the second 7-day cycle, e.g., IL-2 is administered one time on day 0, day 2, and day 4). In some embodiments, the IL-2 is not administered in the third 7-day cycle. In some embodiments, when IL-2 is administered twice daily (i.e., BID), the first dose of IL-2 for the day is administered before the administration of the g-NK cells. In some embodiments, the first dose of IL-2 for the day is administered within about one hour of the administration of the g-NK cells. In some embodiments, the second dose of IL-2 for the day (i.e., the second of two doses for IL-2 administered BID) is administered around 12 hours after the first dose of IL-2 for the day.

[0479] Table 2 shows exemplary schedules for the administration of g-NK cells and IL-2, wherein an “X” indicates the administration of one dose and “XX” indicates the administration of two doses on the indicated day of a schedule for either the composition of g-NK cells (g-NK) or IL-2. In some embodiments, each dose of g-NK cells may be from at or about from at or about 1 x 10⁸ cells to at or about 50 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 10⁸ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 5 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of g-NK cells may be or may be about 10 x 10⁹ cells of the composition of g- NK cells. In some embodiments, each dose of g-NK cells may be or may be about 20 x 10⁹ cells of the composition of g-NK cells. In some embodiments, each dose of IL-2 is between at or about 1 million IU and at or about 50 million IU, such as between at or about 1 million IU and at or about 25 million IU, between at or about 1 million IU and at or about 20 million IU, between at or about 1 million IU and at or about 15 million IU, between at or about 1 million IU and at or about 14 million IU, between at or about 1 million IU and at or about 13 million IU, between at or about 1 million IU and at or about 12 million IU, between at or about 1 million IU and at or about 11 million IU, between at or about 1 million IU and at or about 10 million IU. In some embodiments, each dose of IL-2 is at or about 4 million IU, is at or about 4.5 million IU, is at or about 5 million IU, is at or about 5.5 million IU, is at or about 6 million IU, is at or about 6.5 million IU, is at or about 7 million IU, is at or about 7.5 million IU, is at or about 8 million IU, is at or about 8.5 million IU, is at or about 9 million IU, is at or about 9.5 million IU, is at or about 10 million IU, is at or about 10.5 million IU, is at or about 11 million IU, is at or about 11.5 million IU, is at or about 12 million IU, or is at or about 12.5 million IU.

Table 2. Exemplary schedules for administration of g-NK cells and IL-2

[0480] In any of the provided embodiments, it is understood that one or more doses of the IL-2 may be omitted, such as at the discretion of the physician or clinician. For instance, in some aspects in a strategy involving BID dosing, one of the doses of BID can be omitted at the discretion of the physician or clinician such as depending on patient availability for treatment.

F. L ymphodep/eting Therapy

[0481] In some embodiments, the provided methods also can include administering g-NK cells with another treatment, such as with a chemotherapeutic agent or cytotoxic agent or other treatment. [0482] In some aspects, the provided methods can further include administering one or more lymphodepleting therapies, such as prior to or simultaneous with initiation of administration of the composition of g-NK cells. In some embodiments, the lymphodepleting therapy comprises administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymphodepleting therapy can include administration of fludarabine. In some embodiments, the lymphodepleting therapy can include administration of mesna (sodium 2-mercapto ethane sulfonate).

[0483] In some aspects, preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies can improve the effects of adoptive cell therapy (ACT). In some embodiments, the lymphodepleting therapy includes combinations of cyclosporine and fludarabine.

[0484] Such preconditioning can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy. These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL-2, IL-7, and/or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).

[0485] Thus in some embodiments, the provided method further involves administering a lymphodepleting therapy to the subject. In some embodiments, the method involves administering the lymphodepleting therapy to the subject prior to the administration of the dose of cells. In some embodiments, the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and/or cyclophosphamide. In some embodiments, the administration of the cells and/or the lymphodepleting therapy is carried out via outpatient delivery.

[0486] In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the administration of the dose of cells. For example, the subject may be administered a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the administration of the dose of cells. In some embodiments, the subject is administered a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, no more than 14 days prior, such as no more than 13, 12, 11, 10, 9 or 8 days prior, to the administration of the dose of cells.

[0487] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide. In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 200 mg/m² and 600 mg/m², such as between or between about 200 mg/m² and 400 mg/m². In some aspects, the subject is preconditioned with or with about 300 mg/m² of cyclophosphamide. In some aspects, the subject is preconditioned with or with about 400 mg/m² of cyclophosphamide. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, the cyclophosphamide is administered once daily for one or two days.

[0488] In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 mg/m² and 100 mg/m², such as between or between about 10 mg/m² and 75 mg/m², 15 mg/m² and 50 mg/m², 20 mg/m² and 30 mg/m², or 24 mg/m² and 26 mg/m². In some instances, the subject is administered 25 mg/m² of fludarabine. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days.

[0489] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 60 mg/kg (~2 g/m²) of cyclophosphamide and 3 to 5 doses of 25 mg/m² fludarabine prior to the dose of cells. In other aspects, the subject is administered 30 mg/m² fludarabine and 300 mg/m² or 400 mg/m² cyclophosphamide.

[0490] In some embodiments, prior to the administration of the dose of g-NK cells, the subject has received a lymphodepleting therapy. In some embodiments, the lymphodepleting therapy includes fludarabine and/or cyclophosphamide. In some embodiments, the lymphodepleting includes the administration of fludarabine at or about 20-40 mg/m² body 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/m² body surface area of the subject, optionally at or about 300 mg/m², daily, for 2-4 days.

[0491] In some embodiments, the lymphodepleting therapy further includes mesna. In some embodiments, mesna is administered at a dose between or between about 200 mg/m² and 600 mg/m², such as between or between about 200 mg/m² and 400 mg/m². In some aspects, the subject is preconditioned with or with about 300 mg/m² of mesna. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, the cyclophosphamide is administered once daily for one or two days. [0492] In some embodiments, the lymphodepleting therapy includes fludarabine and cyclophosphamide. In some embodiments, the lymphodepleting therapy includes the administration of fludarabine at or about 30 mg/m² body surface area of the subject, daily, and cyclophosphamide at or about 300 mg/m² body surface area of the subject, daily, each for 2-4 days, optionally 3 days. In some embodiments, the lymphodepleting therapy includes the administration of fludarabine at or about 30 mg/m² body surface area of the subject, daily, and cyclophosphamide at or about 400 mg/m² body surface area of the subject, daily, each for 2-4 days, optionally 3 days, and mesna at or about 300 mg/m² body surface area of the subject, daily, each for 2-4 days, optionally 3 days. In some embodiments, the subject is administered a conditioning chemotherapy as a lymphodepleting therapy that includes fludarabine 30 mg/m2 per day, cyclophosphamide 400 mg/m2 per day and mesna 300 mg/m2 per day on Days -5, -4, and -3 prior to the first dose of the g-NK cells.

[0493] In some embodiments, the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment. For example, in some aspects, preconditioning, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, improves the efficacy of treatment with the dose or increases the persistence of the NK cells in the subject. In some embodiments, preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the dose of cells. In some embodiments, the time to median disease-free survival is increased.

II. METHODS OF SELECTING AND EXPANDING NATURAL KILLER CELL SUBSETS

[0494] In some embodiments, the composition of g-NK cells for use in the provided methods are expanded ex vivo from a subset of NK cells from a biological sample from a human subject. In some embodiments, the methods for expanding and producing a composition of g-NK cells can include expanding a subset of cells that are FcRy-deficient NK cells (g N K) from a biological sample from a human subject. In some embodiments, the methods can include expanding a subset of NK cells that are NKG2C^pos from a biological sample from a human subject. In some embodiments, the methods can include expanding a subset of NK cells that are NKG2A^neg from a biological sample from a human subject. In some embodiments, the method includes isolating a population of cells enriched for natural killer (NK) cells from a biological sample from a human subject and culturing the cells under conditions in which preferential growth and/or expansion of the g-NK cell subject and/or an NK cell subset that overlaps or shares extracellular surface markers with the g-NK cell subset. For example, the NK cells may be cultured using feeder cells, or in the presence of cytokines to enhance the growth and/or expansion of g-NK cell subject and/or an NK cell subset that overlaps or shares extracellular surface markers with the g-NK cell subset. In some aspects, the provided methods also can expand other subsets of NK cells, such as any NK cell that is NKG2C^pos and/or NKG2A^neg.

[0495] In some embodiments, the sample, e.g., biological sample, is one containing a plurality of cell populations that includes an NK cell population. In some embodiments, the biological sample is or comprises blood cells, e.g., peripheral blood mononuclear cells. In some aspects, the biological sample is a whole blood sample, an apheresis product or a leukapheresis product. In some embodiments, the sample is a sample of peripheral blood mononuclear cells (PBMCs). Thus, in some embodiments of the provided methods, a population of peripheral blood mononuclear cells (PBMCs) can be obtained. The sample containing a plurality of cell populations that includes an NK cell population can be used as the cells for enriching or selecting an NK cell subset for expansion in accord with the provided methods.

[0496] In some embodiments, the biological sample is from a donor subject that is a healthy subject. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the donor subject. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom. In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.

[0497] In some examples, cells from the circulating blood of a donor subject are obtained. The samples, in some aspects, contain lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the donor subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient, such as by using a Histopaque® density centrifugation.

[0498] In some embodiments, the biological sample is from an enriched leukapheresis product collected from normal peripheral blood. In some embodiments, the enriched leukapheresis product can contain fresh cells. In some embodiments, the enriched leukapheresis product is a cryopreserved sample that is thawed for use in the provided methods. [0499] In some embodiments, the source of biological cells contains from at or about 5 x 10⁵ to at or about 5 x 10⁸ NK cells or a g-NK cell subset or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells. In some embodiments, the number of NK cells, or a g-NK cell subset or an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, in the biological sample is from at or about 5 x 10⁵ to at or about 1 x 10⁸, from at or about 5 x 10⁵ to at or about 5 x 10⁷, from at or about 5 x 10⁵ to at or about 1 x 10⁷, from at or about 5 x 10⁵ to at or about 5 x 10⁶, from at or about 5 x 10⁵ to at or about 1 x 10⁶, from at or about 1 x 10⁶ to at or about 1 x 10⁸, from at or about 1 x 10⁶ to at or about 5 x 10⁷, from at or about 1 x 10⁶ to at or about 1 x 10⁷, from at or about 1 x 10⁶ to at or about 5 x 10⁶, from at or about 5 x 10⁶ to at or about 1 x 10⁸, from at or about 5 x 10⁶ to at or about 5 x 10⁷, from at or about 5 x 10⁶ to at or about 1 x 10⁷, from at or about 1 x 10⁷ to at or about 1 x 10⁸, from at or about 1 x 10⁷ to at or about 5 x 10⁷, or from at or about 5 x 10⁷ to at or about 1 x 10⁸.

[0500] In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 3%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 5%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 10%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 12%. In some embodiments, the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 14%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 16%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 18%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 20%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 22%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 24%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 26%. In some embodiments, the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 28%. In some embodiments, the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 30%.

[0501] In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 3%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 5%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 10%. In some embodiments, a donor subject is selected if the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 12%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 14%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 16%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 18%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 20%. In some embodiments, a donor subject is selected if the percentage of g- NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 22%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 24%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 26%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 28%. In some embodiments, a donor subject is selected if the percentage of g-NK cells, or of an NK cell subset that is associated with or includes a surrogate marker for g-NK cells, among NK cells in the biological sample is greater than at or about 30%.

[0502] In some embodiments, the biological sample is from a donor subject that is CMV seropositive. CMV infection can result in phenotypic and functional differentiation of NK cells, including development of high fractions of NK cells expressing NKG2C that exhibit enhanced antiviral activity. CMV-associated NK cells expressing NKG2C display altered DNA methylation patterns and reduced expression of signaling molecules, such as FcRy (Schlums et al., Immunity (2015) 42:443-56). These NK cells are linked to more potent antibody-dependent activation, expansion, and function relative to conventional NK-cell subsets. In some cases, the biological sample can be from a donor subject that is CMV seronegative as NK cells with reduced expression of FcRy can also be detected in CMV seronegative individuals, albeit generally at lower levels. In some cases, the biological sample can be from CMV seropositive individuals.

[0503] In some embodiments, a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 20% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 25% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 30% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 35% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 40% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 45% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 50% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 55% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 65% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 70% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 75% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 80% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 85% of NK cells in the peripheral blood sample are positive for NKG2C. In some embodiments, the donor subject is selected if at least at or about 90% of NK cells in the peripheral blood sample are positive for NKG2C.

[0504] In some embodiments, a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected based on the percentage of NK cells in a peripheral blood sample that are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 70% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 75% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 80% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 85% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, a donor subject is selected if at least at or about 90% of NK cells in the peripheral blood sample are negative or low for NKG2A.

[0505] In some embodiments, a donor subject is selected based on both the percentage of NK cells in a peripheral blood sample that are positive for NKG2C and the percentage of NK cells in the peripheral blood sample that are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 60% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 15% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 70% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 20% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 70% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 30% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 75% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 40% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 80% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 50% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 85% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 90% of NK cells in the peripheral blood sample are negative or low for NKG2A. In some embodiments, the donor subject is selected if at least at or about 60% of NK cells in the peripheral blood sample are positive for NKG2C and at least at or about 95% of NK cells in the peripheral blood sample are negative or low for NKG2A.

[0506] In some embodiments, a donor subject is selected for expansion of cells in accord with the provided methods if the donor subject is CMV seropositive, and if among NK cells in a peripheral blood sample from the donor subject, the percentage of g-NK cells is greater than at or about 30%, the percentage of NKG2C^pos cells is greater than at or about 20%, and the percentage of NKG2A^neg cells is greater than at or about 70%.

[0507] In some embodiments, NK cells from the donor subject bear a single nucleotide polymorphism (SNP rs396991) in the CD 16 gene, nucleotide 526 [thymidine (T) guanine (G)] resulting in an amino acid (aa) substitution of valine (V) for phenylalanine (F) at position 158 in the mature (processed) form of the protein (F158V). In some embodiments, NK cells bear the CD16 158V polymorphism in both alleles (called 158V/V herein). In some embodiments, NK cells bear the CD16 158V polymorphism in a single allele (called 158V/F herein). It is understood that reference to a 158V+ genotype herein refers to both the 158V/V genotype and the 158V/F genotype. It has been found that the CD16 F158V polymorphism is associated with substantially higher affinity for IgGl antibodies and have the ability to mount more robust NK cell-mediated ADCC responses (Mellor et al. (2013) Journal of Hematology & Oncology, 6:1; Musolino et al. (2008) Journal of Clinical Oncology, 26:1789-1796 and Hatjiharissi et al. (2007) Blood, 110:2561-2564). In some embodiments, antibody-directed targeting of CD16 158V+/g-NK cells leads to improved outcomes for patients due to the improved affinity, cytotoxic and/or cytokine-mediated effect functions of the CD16 158V+/g-NK cell subset.

[0508] In some embodiments, the provided methods include enriching or isolating NK cells or a subset thereof from a biological sample of a donor subject identified as having the CD16 158V+ NK cell genotype. In some embodiments, the method includes screening donor subjects for the presence of the CD16 158V+ NK cell genotype. In some embodiments, genomic DNA is extracted from a sample from a donor subject that is or includes NK cells, such as blood sample or bone marrow sample. In some embodiments, the sample is or comprises blood cells, e.g., peripheral blood mononuclear cells. In some embodiments, the sample is or comprises isolated NK cells. In some embodiments, the sample is a sample from a healthy donor subject. Any method for extracting DNA from the sample can be employed. For instance, nucleic acids can be readily isolated from a sample, e.g., cells, using standard techniques such as guanidium thiocyanate -phenol-chloroform extraction (Chomocyznski et al. (1987) Anal. Biochem. 162: 156). Commercially available kits also are readily available for extracting genomic DNA, such as the Wizard genomic DNA purification kit (Promega, Madison, WI). [0509] Genotyping can be performed on any suitable sample. In any of the embodiments described herein, the genotyping reaction can be, for example, a pyrosequencing reaction, DNA sequencing reaction, MassARRAY MALDI- TOF, RFLP, allele-specific PCR, real-time allelic discrimination, or microarray. In some embodiments, a PCR-based technique, such as RT-PCR, of genomic DNA is carried out using allele-specific primers for the polymorphism. The PCR method for amplifying target nucleic acid sequences in a sample is well known in the art and has been described in, e.g., Innis et al. (eds.) PCR Protocols (Academic Press, NY 1990); Taylor (1991) Polymerase chain reaction: basic principles and automation, in PCR: A Practical Approach, McPherson et al. (eds.) IRL Press, Oxford; Saiki et al. (1986) Nature 324: 163; as well as in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,889,818, all incorporated herein by reference in their entireties.

[0510] Primers for detecting the 158V+ polymorphism are known or can be easily designed by a skilled artisan, See. e.g., International published PCT Appl. No. W02012/061814; Kim et al. (2006) Blood, 108:2720-2725; Cartron et al. (2002) Blood, 99:754-758; Koene et al. (1997) Blood, 90:1109- 1114; Hatijiharissi et al. (2007) Blood, 110:2561-2564; Somboonyosdech et al. (2012) Asian Biomedicine, 6:883-889). In some embodiments, PCR can be carried out using nested primers followed by allele-specific restriction enzyme digestion. In some embodiments, the first PCR primers comprise nucleic acid sequences 5’ -ATA TTT ACA GAA TGG CAC AGG -3’ (SEQ ID NO:2) and 5’-GAC TTG GTA CCC AGG TTG AA-3’ (SEQ ID NOG), while the second PCR primers are 5’-ATC AGA TTC GAT CCT ACT TCT GCA GGG GGC AT-3’ (SEQ ID NO:4) and 5’-ACG TGC TGA GCT TGA GTG ATG GTG ATG TTC AC-3’ (SEQ ID NOG), which, in some cases, generates a 94-bp fragment depending on the nature of allele. In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NOG (GAAATCTACC TTTTCCTCTA ATAGGGCAAT). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NOG (GAAATCTACC TTTTCCTCTA ATAGGGCAA). In some embodiments, the primer pair comprises the nucleic acid sequences set forth in SEQ ID NOG (CCCAACTCAA CTTCCCAGTG TGAT) and SEQ ID NO:9 (GAAATCTACC TTTTCCTCTA ATAGGGCA). In some embodiments, genotyping can be carried out by quantitative real-time RT-PCR following extraction of RNA using primer sequences as follows: CD16 sense set forth in SEQ ID NO: 10 (5'- CCAAAAGCCACACTCAAAGAC-3') and antisense set forth in SEQ ID NO: 11 (5'- ACCCAGGTGGAAAGAATGATG-3') and TaqMan probe set forth in SEQ ID NO: 12 (5'- AACATCACCATCACTCAAGGTTTGG-3').

[0511] To confirm the genotyping, allele specific amplification can be used with a set of V allele specific primers (e.g., forward primer set forth in SEQ ID NO:13, 5’-CTG AAG ACA CAT TTT TAC TCC CAAA-3’; and reverse primer set forth in SEQ ID NO: 14, 5’-TCC AAA AGC CAC ACT CAA AGA C-3’) or a set of F allele specific primers (e.g., forward primer set forth in SEQ ID NO: 15, 5’-CTG AAG ACA CAT TTT TAC TCC CAAC-3’ ; and reverse primer set forth in SEQ ID NO: 14, 5’-TCC AAA AGC CAC ACT CAA AGA C-3’).

[0512] The genomic sequence for CD 16a is available in the NCBI database at NG_009066.1. The gene ID for CD16A is 2214. Sequence information for CD16, including gene polymorphisms, is available at UniProt Acc. No. P08637. The sequence of CD16 (F158) is set forth in SEQ ID NO: 16 (residue F158 is bold and underlined). In some embodiments, CD16 (F158) further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO: 17).

GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDA ATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKV TYLQNGKGRKYFHHNSDFYIPKAI'LKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFF PPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 16)

[0513] The sequence of CD16 158V+ (polymorphism resulting in F158V) is known as VAR_003960 and has the sequence set forth in SEQ ID NO: 18 (158V+ polymorphism is in bold and underline). In some embodiments, CD16 (158V+) further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO: 17).

GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFI DAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTA LHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLA VSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 18)

[0514] In some embodiments, single nucleotide polymorphism (SNP) analysis is employed on genomic deoxyribonucleic acid (DNA) samples using allele-specific probes containing a fluorescent dye label (e.g., FAM or VIC) on the 5’ end and a minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3’ end and unlabeled PCR primers to detect a specific SNP target. In some embodiments, the assay measures or detects the presence of an SNP by a change in fluorescence of the dyes associated with the probe. In such embodiments, probes hybridize to the target DNA between the two unlabeled primers and signal from the fluorescent dye on the 5’ end is quenched by the NFQ on its 3’ end by fluorescence resonance energy transfer (FRET). During PCR, Taq polymerase extends the unlabeled primers using the template as a guide and when the polymerase reaches the labeled probe, it cleaves the molecule separating the dye from the quencher. In some aspects, a qPCR instrument can detect fluorescence from the unquenched label. Exemplary reagents are commercially available SNP Assays, e.g., code C_25815666_10 for rs396991 (Applied Biosystems, Cat No. 4351379 for SNP genotyping of F158V in CD16). [0515] In some embodiments, subjects heterozygous or homozygous for the CD16 158V (F158V) polymorphism are identified. In some embodiments, subjects homozygous for the CD16 158V (F158V) polymorphism are identified. In some embodiments, NK cells or an NK cell subset are isolated or enriched from a biological sample from a subject identified as being heterozygous or homozygous for the CD16 158V polymorphism. In some embodiments, NK cells or an NK cell subset are isolated or enriched from a biological sample from a subject identified as being homozygous for the CD16 158V polymorphism.

[0516] In some embodiments, the method includes enriching NK cells from the biological sample, such as from a population PBMCs isolated or obtained from the subject. In some embodiments, the population of cells enriched for NK cells is enriched by isolation or selection based on one or more natural killer cell-specific markers. It is within the level of a skilled artisan to choose particular markers or combinations of surface markers. In some embodiments, the surface marker(s) is any one or more of the from the following surface antigens CDl la, CD3, CD7, CD14, CD16, CD19, CD25, CD27, CD56, CD57, CD161, CD226, NKB1, CD62L; CD244, NKG2D, NKp30, NKp44, NKp46, NKG2A, NKG2C, KIR2DL1 and/or KIR2DL3. In some embodiments, the surface marker(s) is any one or more of the from the following surface antigens CDl la, CD3, CD7, CD14, CD16, CD19, CD25, CD27, CD38, CD56, CD57, CD161, CD226, NKB1, CD62L; CD244, NKG2D, NKp30, NKp44, NKp46, NKG2A, NKG2C, SLAMF7 (CD319), KIR2DL1 and/or KIR2DL3. In particular embodiments, the one or more surface antigen includes CD3 and one or more of the following surface antigens CD16, CD56 or CD57. In some embodiments, the one or more surface antigen is CD3 and CD57. In some embodiments, the one or more surface antigen is CD3, CD56 and CD16. In other embodiments, the one or more surface antigen is CD3, CD56 and CD38. In further embodiments, the one or more surface antigen is CD3, CD56, NKG2A and CD161. In some embodiments, the one or more surface antigen is CD3, CD57, and NKG2C. In some embodiments, the one or more surface antigen is CD3, CD57, and NKG2A. In some embodiments, the one or more surface antigen is CD3, CD57, NKG2C, and NKG2A. In some embodiments, the one or more surface antigen is CD3 and CD56. In some embodiments, the one or more surface antigen is CD3, CD56, and NKG2C. In some embodiments, the one or more surface antigen is CD3, CD56, and NKG2A. In some embodiments, the one or more surface antigen is CD3, CD56, NKG2C, and NKG2A. Reagents, including fluorochrome-conjugated antibodies, for detecting such surface antigens are well known and available to a skilled artisan.

[0517] In some embodiments, the NK cell population is enriched, such as by isolation or selection, from a sample by the provided methods are cells that are positive for (marker-i- or marker^pos) or express high levels (marker^hlgh) of one or more particular markers, such as surface markers, or that are negative for or express relatively low levels (marker- or marker^neg) of one or more markers. Hence, 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 marker^neg herein 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 cases, such markers are those that are absent or expressed at relatively low levels on certain populations of NK cells but are present or expressed at relatively higher levels on certain other populations of lymphocytes (such as T cells). In some cases, such markers are those that are present or expressed at relatively higher levels on certain populations of NK cells but are absent or expressed at relatively low levels on certain other populations of lymphocytes (such as T cells or subsets thereof).

[0518] In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some embodiments, incubation is static (without mixing). In some embodiments, incubation is dynamic (with mixing).

[0519] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. The separation need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells. For example, in some aspects, a negative selection for CD3 enriches for a population of cells that are CD3^neg, but also can contain some residual or small percentage of other non-selected cells, which can, in some cases, include a small percentage of cells still being present in the enriched population that are CD3^pos. In some examples, a positive selection of one of the CD57^pos or CD16^pos population enriches for said population, either the CD57^pos or CD16^pos population, but also can contain some residual or small percentage of other non-selected cells, which can, in some cases, include the other of the CD57 or CD16 population still being present in the enriched population. [0520] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0521] In some aspects, the selection includes positive and/or negative selection steps based on expression of one or more of the surface antigens, such as in cells from a PBMC sample.

[0522] In some embodiments, the selection include positive selection for cells expressing NKG2C (NKG2C^pos) and/or negative selection for cells NKG2A (NKG2A^neg).

[0523] In some embodiments, the isolation includes positive selection for cells expressing CD56, cells expressing CD 16 or cells expressing CD57 and/or negative selection for cells expressing CD38 and/or negative selection for cells expressing non-NK cell markers, such as T cell markers, for example, negative selection for cells expressing CD3 (CD3^neg). For example, in some embodiments, the isolation includes positive selection for cells expressing CD56, cells expressing CD16 or cells expressing CD57 and/or negative selection for cells expressing non-NK cell markers, such as T cell markers, for example, negative selection for cells expressing CD3 (CD3^neg). In some embodiments, the isolation includes positive selection for cells expressing CD56, cells expressing CD16 or cells expressing CD57, and/or negative selection for cells expressing CD38 (CD38^neg), CD161 (CD161^neg), NKG2A (NKG2A^neg), and/or negative selection for cells expressing CD3 (CD3^neg). In some embodiments, the selection includes isolation of cells negative for CD3 (CD3^neg).

[0524] In some embodiments, the isolation includes negative selection for cells expressing CD3 (CD3^neg) and positive selection for cells expressing CD56 (CD56^pos). In some embodiments, the selection can further include negative selection for cells expressing CD38 (CD38^neg). In specific embodiments, the isolated or selected cells are CD3^negCD56^posCD38^neg.

[0525] In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD56 (CD56^pos), followed by negative selection for cells expressing NKG2A (NKG2A^neg) and CD161 (CD161^neg). In specific embodiments, the isolated or selected cells are CD3^negCD56^posNKG2A^neg CD161^neg.

[0526] In some embodiments, the selection includes negative selection for cells expressing CD3

(CD3^neg) and positive selection for cells expressing CD57 (CD57^pos). In specific embodiments, the isolated or selected cells are CD3^negCD57^pos. [0527] In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg) and positive for cells expressing CD16 (CD16^pos). In specific embodiments, the isolated or selected cells are CD3^negCD16^pos.

[0528] In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg) and positive selection for cells expressing CD57 (CD57^pos). In specific embodiments, the isolated or selected cells are CD3^negCD57^pos. For example, the NK cells may be enriched by depletion of CD3^pos cells (negative selection for CD3^pos cells) followed by CD57^pos cell selection, thereby isolating and enriching CD57^pos NK cells. The separation can be carried out by immunoaffinity-based methods, such as using MACS™ Microbeads. For example, CD3 microbeads can be used to deplete CD3^pos cells in a negative selection for CD3^neg cells. Subsequently, CD57 MicroBeads can be used for CD57 enrichment of CD3 cell-depleted PBMCs. The CD3^neg/CD57^pos enriched NK cells can then be used in expansion in the provided methods.

[0529] In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD57 (CD57^pos), and positive selection for cells expressing NKG2C (NKG2C^pos). In specific embodiments, the isolated or selected cells are CD3^negCD57^posNKG2C^pos. In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD57 (CD57^pos), and negative selection for cells expressing NKG2A (NKG2A^neg). In specific embodiments, the isolated or selected cells are CD3^negCD57^posNKG2A^neg. In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD57 (CD57^pos), positive selection for cells expressing NKG2C (NKG2C^pos), and negative selection for cells expressing NKG2A (NKG2A^neg). In specific embodiments, the isolated or selected cells are CD3^negCD57^posNKG2C^posNKG2A^neg.

[0530] In some of any of the provided embodiments, the selection can further include negative selection for cells expressing CD38 (CD38^neg). In specific embodiments, the isolated or selected cells are CD3^negCD57^posCD38^neg. In specific embodiments, the isolated or selected cells are CD3^negCD57^posCD38^negNKG2C^pos. In specific embodiments, the isolated or selected cells are CD3^negCD57^posCD38^negNKG2A^neg. In specific embodiments, the isolated or selected cells are CD3^negCD57^posCD38^negNKG2C^posNKG2A^neg.

[0531] In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg) and positive selection for cells expressing CD56 (CD56^pos). In specific embodiments, the isolated or selected cells are CD3^negCD56^pos. In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD56 (CD56^pos), and positive selection for cells expressing NKG2C (NKG2C^pos). In specific embodiments, the isolated or selected cells are CD3^negCD56^posNKG2C^pos. In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD56 (CD56^pos), and negative selection for cells expressing NKG2A (NKG2A^neg). In specific embodiments, the isolated or selected cells are CD3^negCD56^posNKG2A^neg. In some embodiments, the selection includes negative selection for cells expressing CD3 (CD3^neg), positive selection for cells expressing CD56 (CD56^pos), positive selection for cells expressing NKG2C (NKG2C^pos), and negative selection for cells expressing NKG2A (NKG2A^neg). In specific embodiments, the isolated or selected cells are CD3^negCD56^posNKG2C^posNKG2A^neg.

[0532] In some of any of the provided embodiments, the selection can further include negative selection for cells expressing CD38 (CD38^neg). In specific embodiments, the isolated or selected cells are CD3^negCD56^posCD38^neg. In specific embodiments, the isolated or selected cells are CD3^negCD56^posCD38^negNKG2C^pos. In specific embodiments, the isolated or selected cells are CD3^negCD56^posCD38^negNKG2A^neg. In specific embodiments, the isolated or selected cells are CD3^negCD56^posCD38^negNKG2C^posNKG2A^neg.

[0533] In some of any of the provided embodiments, the g-NK cells are cells having a g-NK surrogate surface marker profile. In some embodiments, the g-NK cell surrogate surface marker profile is CD16^pos/CD57^pos/CD7^dim/neg/CD161^neg. In some embodiments, the g-NK cell surrogate surface marker profile is NKG2A^neg/CD161^neg. In some of any such embodiments, the g-NK cell surrogate surface marker profile is CD38^neg. In some of any such embodiments, CD45^pos/CD3^neg/CD56^pos is used as a surrogate surface marker profile for NK cells. In some of any such embodiments, the g-NK cell surrogate surface marker profile further includes an NK cell surrogate surface marker profile. In some of any such embodiments, the g-NK cell surrogate surface marker profile further includes CD45^pos/CD3^neg/CD56^pos. In particular embodiments the g-NK cell surrogate surface marker profile includes CD45^pos/CD3^neg/CD56^pos/CD16^pos/CD57^pos/CD7^dim/neg/CD161^neg. In other particular embodiments, the g-NK cell surrogate surface marker profile includes CD45^pos/CD3^neg/CD56^pos/NKG2A^neg/CD161^neg. In other particular embodiments, the g-NK cell surrogate surface marker profile includes CD45^pos/CD3^neg/CD56^pos/CD38^neg.

[0534] In some embodiments, the methods of isolating, selecting and/or enriching for cells, such as by positive or negative selection based on the expression of a cell surface marker or markers, can include immunoaffinity-based selections. In some embodiments, the immunoaffinity-based selections include contacting a sample containing cells, such as PBMCs, with an antibody or binding partner that specifically binds to the cell surface marker or markers. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a sphere or bead, for example microbeads, nanobeads, including agarose, magnetic bead or paramagnetic beads, to allow for separation of cells for positive and/or negative selection. In some embodiments, the spheres or beads can be packed into a column to effect immunoaffinity chromatography, in which a sample containing cells, such as PBMCs, is contacted with the matrix of the column and subsequently eluted or released therefrom. [0535] The incubation generally is carried out under conditions whereby the antibodies or binding partners, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0536] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0537] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated and/or cultured; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0538] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0539] In some of any of such embodiments, the method comprises administering IL-12, IL-15, IL- 18, IL-2 and/or CCL5 to the subject prior to enriching, such as selecting and/or isolating, the NK cells or subset thereof.

[0540] In embodiments of the provided methods, the enriched NK cells are incubated or cultured in the presence of feeder cells, such as under conditions to support the proliferation and expansion of NK cell subsets, and in particular the g-NK cell subset.

[0541] In particular aspects, the feeder cells include cells that stimulate or promote expansion of NKG2C^pos and/or inhibit expansion of NKG2A^pos cells. In some embodiments, the feeder cells are cells that express or are transfected with HLA-E or a hybrid HLA-E containing the HLA-A2 signal sequence. For example, exemplary of such a hybrid is an AEH hybrid gene containing an MHC class I, such as HLA-A2, promoter and signal sequence and the HLA-E mature protein sequence, which, in some cases, can result in a mature protein identical to that encoded by the HLA-E gene but that can be stably expressed on the cell surface (see e.g., Lee et al. (1998) Journal of Immunology, 160:4951-4960). In some embodiments, the cell is an LCL 721.221, K562 cell or RMA-S cell that is transfected to express an MHC-E molecule stabilized in the presence of an MHC class I, such as HLA-A2, leader sequence. Cells lines that are engineered to express cell surface HLA-E stabilized in the presence of an MHC class I, such as HLA-A2, leader sequence peptide are known in the art (Lee et al. (1998) Journal of Immunology, 160:4951-4960; Zhongguo et al. (2005) 13:464-467; Garcia et al. (2002) Eur J. Immunol., 32:936-944). In some embodiments, 221. AEH cells, such as irradiated 221. AEH cells, can be used as feeder cells, or any other HLA-E -expressing cell line or irradiated HLA-E-expressing cell line that is otherwise HLA negative, such as K562. In some embodiments, the cell line can be transfected to express HLA-E. In some embodiments, K562 cells expressing membrane-bound IL-15 (K562-mbl5) or membrane-bound IL-21 (K562-mb21) can be used as feeder cells. Exemplary of such a cell line for use in the methods provided herein are 221 -AEH cells.

[0542] In embodiments, the HLA-expressing feeder cells are cryopreserved and thawed before use. In some embodiments, if the cells have been transfected to express HLA-E such as 221. AEH cells, the cells can be grown in the presence of appropriate nutrients, e.g., including serum or other appropriate serum replacement, and a selection agent prior to their use in the method. For example, in the case of 221.AEH cells, the cells can be cultured in cell culture media supplemented with Hygromycin B (e.g., 0.1% to 10%, such as at or about 1%) to maintain selective pressure on the cells to maintain the high level of plasmid HLA-E. The cells can be maintained at a density of 1 x 10⁵ cells/mL to 1 x 10⁶ cells/mL until use.

[0543] In particular embodiments, the HLA-E-expressing feeder cells, e.g., 221. AEH cells, added to the culture are non-dividing, such as by X-ray irradiation or gamma irradiation. The HLA-E-expressing feeder cells, e.g., 221. AEH, can be irradiated on the day of or just prior to their use in the provided methods. In some embodiments, the HLA-E-expressing feeder cells are irradiated with gamma rays in the range of about 1000 to 10000 rad, such as 1000-5000, rads to prevent cell division. In some embodiments, the HLA-E-expressing feeder cells are irradiated with gamma rays in the range of about 10 Gy to 100 Gy, such as 10-50 Gy to prevent cell division. In some embodiments, the cells are irradiated at 100 Gy. In other embodiments, irradiation is carried out by x-ray irradiation. In some embodiments, the HLA-E-expressing feeder cells are irradiated with x rays in the range of about 10 Gy to 100 Gy, such as 10-50 Gy to prevent cell division. In some embodiments, the A Rad-Sure™ blood irradiation indicator can be used to provide positive visual verification of irradiation. In aspects of the provided methods, the feeder cells are never removed; as a result of the irradiation the NK cells will be directly cytotoxic to the feeder cells and the feeder cells will die during the culture. [0544] In some embodiments, the enriched, selected and/or isolated NK cells are incubated or cultured in the presence of HLA-E-expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, at a ratio of feeder cells to enriched NK cells that is greater than or about 1:10 HLA-E feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, to enriched NK cells, such as from at or about 1:10 and at or about 10:1 of such feeder cells to enriched NK cells.

[0545] In some embodiments, the ratio of HLA-E-expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, is at a ratio of such feeder cells to enriched NK cells that is between at or about 1:10 and at or about 10:1, between at or about 1:10 and at or about 5:1, between at or about 1:10 and at or about 2.5:1, between at or about 1:10 and at or about 1:1, between at or about 1:10 and at or about 1:2.5, between at or about 1:10 and at or about 1:5, between at or about 1:5 and at or about 10:1, between at or about 1:5 and at or about 5:1, between at or about 1:5 and at or about 2.5:1, between at or about 1:5 and at or aboutkl, between at or about 1:5 and at or about 1:2.5, between at or about 1:2.5 and at or about 10:1, between at or about 1:2.5 and at or about 5:1, between at or about 1:2.5 and at or about 2.5:1, between at or about 1:2.5 and at or about 1:1, between at or about 1:1 and at or about 10:1, between at or about 1:1 and at or about 5:1, between at or about 1:1 and at or about 3:1, between at or about 1:1 and at or about 2.5:1, between at or about 2.5:1 and at or about 10:1, between at or about 2.5:1 and at or about 5:1 or between at or about 5:1 and at or about 10:1, each inclusive.

[0546] In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, is at a ratio of such feeder cells to enriched NK cells that is at or about 1.25:1, 1.5:1, 1.75:1, 2.0:1, 2.25:1, 2:5:1, 2.75:1, 3.0:1, 3.25:1, 3.5.:1, 3.75:1, 4.0:1, 4.25:1, 4.5:1, 4.75:1 or 5:1, or any value between any of the foregoing. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, is at a ratio of such feeder cells to enriched cells that is less than or less than about 5:1. In some embodiments, the ratio of HLA- expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, is at a ratio between at or about 1:1 and 2.5:1, inclusive. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221.AEH cells), such as an irradiated population thereof, is at a ratio of at or about 2.5:1. In some embodiments, the ratio of HLA-expressing feeder cells (e.g., 221. AEH cells), such as an irradiated population thereof, is at a ratio of at or about 2: 1.

[0547] In some cases, if the starting NK cell population has been cryopreserved prior to expansion, i.e., subject to freeze/thaw, a lower 221. AEH to NK-cell ratio can be employed than for methods using fresh NK cells. It is found here that a ratio of 1:1 221. AEH to freeze/thaw NK-cell resulted in comparable expansion in a culture containing a ratio of 2.5:1 221. AEH to fresh NK cells. In some aspects, the lower ratio ensures a higher number of NK cells in the culture to permit more cell-to-cell contact, which may play a role in promoting initial growth and expansion. In some embodiments, if initial enriched population of NK cells from a sample has been subject to freeze/thaw, a ratio of at or about 2:1 to 1:2 221.AEH to freeze/thaw NK-cells is used. In particular embodiments, the ratio is 1:1. It is understood that higher ratio, such as 2.5:1 221. AEH to freeze/thaw NK-cells can be used, but this may require a longer culture, e.g., at or about 21 days, to reach a desired threshold density or number.

[0548] In some embodiments, the NK cells are expanded by further adding to the culture nondividing peripheral blood mononuclear cells (PBMC). In some aspects, the non-dividing feeder cells can comprise X-ray-irradiated PBMC feeder cells. In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 1000 to 10000 rad, such as 1000-5000, rads to prevent cell division. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 10 Gy to 100 Gy, such as 10-50 Gy to prevent cell division. In some aspects, during at least a portion of the incubation, the irradiated feeder cells are present in the culture medium at the same time as the non-dividing (e.g., irradiated) HLA-E-expressing feeder cells. In some aspects, the non-dividing (e.g., irradiated) PBMC feeder cell, HLA-E-expressing feeder cells and enriched NK cells are added to the culture on the same day, such as on the day of the initiation of the incubation, e.g., at or about or near the same time.

[0549] In some embodiments, the incubation or culture is further carried out in the presence of irradiated PBMCs as feeder cells. In some embodiments, the irradiated PBMC feeder cells are autologous to, or from the same subject as, the enriched NK cells were isolated or selected. In particular embodiments, the PBMCs are obtained from the same biological sample, e.g., whole blood or leukapheresis or apheresis product, as used to enrich the NK cells. Once obtained, a portion of the PBMCs are reserved for irradiation prior to enrichment of NK cells as described above.

[0550] In some embodiments, irradiated PBMCs are present as feeder cells at a ratio of such feeder cells to enriched NK cells that is from at or about 1:10 to at or about 10:1, from at or about 1:10 to at or about 5:1, from at or about 1 : 10 to at or about 2.5:1, from at or about 1 : 10 to at or about 1:1, from at or about 1:10 to at or about 1:2.5, from at or about 1:10 to at or about 1:5, from at or about 1:5 to at or about 10:1, from at or about 1:5 to at or about 5:1, from at or about 1:5 to at or about 2.5:1, from at or about 1:5 to at or about 1:1, from at or about 1:5 to at or about 1:2.5, from at or about 1:2.5 to at or about 10:1, from at or about 1:2.5 to at or about 5:1, from at or about 1:2.5 to at or about 2.5:1, from at or about 1:2.5 to at or about 1:1, from at or about 1:1 to at or about 10:1, from at or about 1:1 to at or about 5:1, from at or about 1:1 to at or about 2.5:1, from at or about 2.5:1 to at or about 10:1, from at or about 2.5:1 to at or about 5:1 or from at or about 5:1 to at or about 10:1.

[0551] In some embodiments, the irradiated PBMCs are present as feeder cells at a ratio of such feeder cells to enriched NK cells that is between at or about 1:1 and at or about 5:1, such as at or about 1.25:1, 1.5:1, 1.75:1, 2.0:1, 2.25:1, 2:5:1, 2.75:1, 3.0:1, 3.25:1, 3.5.:1, 3.75:1, 4.0:1, 4.25:1, 4.5:1, 4.75:1 or 5:1, or any value between any of the foregoing. In some embodiments, the irradiated PBMCs are present at a ratio of such feeder cells to enriched cells that is or is about 5:1. [0552] In particular embodiments, during at least a portion of the incubation or culture one or more cells or cell types, such as T cells, of the irradiated PBMCs are activated and/or the incubation or culture is carried out in the presence of at least one stimulatory agent that is capable of stimulating the activation of one or more T cells of the PBMC feeder cells. In some embodiments, at least one stimulatory agent specifically binds to a member of a TCR complex. In some embodiments, the at least one stimulatory agent specifically binds to a CD3, optionally a CD3epsilon. In some aspects, the at least one stimulatory agent is an anti-CD3 antibody or antigen binding fragment. An exemplary anti-CD3 antibody includes mouse anti-human CD3 (OKT3).

[0553] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is present during at least a portion of the incubation that includes irradiated PBMC feeder cells. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is added to the culture or incubation at or about the same time as the irradiated PBMCs. For example, the anti-CD3 antibody or antigen-binding fragment is added at or about at the initiation of the incubation or culture. In particular aspects, the anti-CD3 antibody or antigen-binding fragment may be removed, or its concentration reduced, during the course of the culture or incubation, such as by exchanging or washing out the culture medium. In particular embodiments, after exchanging or washing, the methods do not include adding back or replenishing the culture media with the anti-CD3 antibody or antigen-binding fragment.

[0554] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is added, or is present during at least a portion of the culture or incubation, at a concentration that is between at or about 10 ng/mL and at or about 5 pg/mL, such as between at or about 10 ng/mL and at or about 2 pg/mL, between at or about 10 ng/mL and at or about 1 pg/mL, between at or about 10 ng/mL and at or about 500 ng/mL, between at or about 10 ng/mL and at or about 100 ng/mL, between at or about 10 ng/mL and at or about 50 ng/mL, between at or about 50 ng/mL and at or about 5 pg/mL, such as between at or about 50 ng/mL and at or about 2 pg/mL, between at or about 50 ng/mL and at or about 1 pg/mL, between at or about 50 ng/mL and at or about 500 ng/mL, between at or about 50 ng/mL and at or about 100 ng/mL, between at or about 100 ng/mL and at or about 5 pg/mL, between at or about 100 ng/mL and at or about 2 pg/mL, between at or about 100 ng/mL and at or about 1 pg/mL, between at or about 100 ng/mL and at or about 500 ng/mL, between at or about 500 ng/mL and at or about 5 pg/mL, between at or about 500 ng/mL and at or about 2 pg/mL, between at or about 500 ng/mL and at or about 1 pg/mL, between at or about 1 pg/mL and at or about 5 pg/mL, between at or about 1 pg/mL and at or about 2 pg/mL, or between at or about 2 pg/mL and at or about 5 pg/mL, each inclusive. In some embodiments, the concentration of the anti-CD3 antibody or antigen-binding fragment is at or about 10 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL, or any value between any of the foregoing. In some embodiments, the concentration of the anti-CD3 antibody or antigen-binding fragment is or is about 50 ng/mL. [0555] In some embodiments, the term “antibody” refers to immunoglobulin molecules and antigenbinding portions or fragments of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. The term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof, such as dAb, Fab, Fab’, F(ab’)2, Fv), single chain (scFv) or single domain antibody (sdAb). Typically, an “antigen-binding fragment” contains at least one CDR of an immunoglobulin heavy and/or light chain that binds to at least one epitope of the antigen of interest. In this regard, an antigen-binding fragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and variable light chain (VL) sequence from antibodies that bind the antigen, such as generally six CDRs for an antibody containing a VH and a VL (“CDR1,” “CDR2” and “CDR3” for each of a heavy and light chain), or three CDRs for an antibody containing a single variable domain.

[0556] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and singledomain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.

[0557] In some embodiments, the incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration that is at or about, or at least at or about, 0.05 x 10⁶ enriched NK cells/mL, at or about 0.1 x 10⁶ enriched NK cells/mL, at or about 0.2 x 10⁶ enriched NK cells/mL, at or about 0.5 x 10⁶ enriched NK cells/mL or at or about 1.0 x 10⁶ enriched NK cells/mL. In embodiments of the provided methods, the incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration that is between at or about 0.05 x 10⁶ enriched NK cells/mL and at or about 1.0 x 10⁶ enriched NK cells/mL, such as between at or about 0.05 x 10⁶ enriched NK cells/mL and at or about 0.75 x 10⁶, between at or about 0.05 x 10⁶ enriched NK cells/mL and at or about 0.5 x 10⁶, between at or about 0.05 x 10⁶ enriched NK cells/mL and at or about 0.20 x 10⁶ enriched NK cells/mL, between at or about 0.05 x 10⁶ enriched NK cells/mL and at or about 0.1 x 10⁶ enriched NK cells/mL, between at or about 0.1 x 10⁶ enriched NK cells/mL and at or about 1.0 x 10⁶ enriched NK cells/mL, between at or about 0.1 x 10⁶ enriched NK cells/mL and at or about 0.75 x 10⁶, between at or about 0.1 x 10⁶ enriched NK cells/mL and at or about 0.5 x 10⁶, between at or about 0.1 x 10⁶ enriched NK cells/mL and at or about 0.20 x 10⁶ enriched NK cells/mL, between at or about 0.20 x 10⁶ enriched NK cells/mL and at or about 1.0 x 10⁶ enriched NK cells/mL, between at or about 0.20 x 10⁶ enriched NK cells/mL and at or about 0.75 x 10⁶, between at or about 0.20 x 10⁶ enriched NK cells/mL and at or about 0.5 x 10⁶, between at or about 0.5 x 10⁶ enriched NK cells/mL and at or about 1.0 x 10⁶ enriched NK cells/mL, between at or about 0.5 x 10⁶ enriched NK cells/mL and at or about 0.75 x 10⁶, between at or about 0.75 x 10⁶ enriched NK cells/mL and at or about 1.0 x 10⁶ enriched NK cells/mL, each inclusive. In some embodiments, the incubation or culture is initiated in the presence of such enriched NK cells, such as selected and/or isolated NK cells, at a concentration that is at or about 0.2 x 10⁶ enriched NK cells/mL.

[0558] In some of any such embodiments, the amount of enriched NK cells, such as selected or isolated from PBMCs as described above, added or present at the initiation of the incubation or culture is at least or at least about 1 x 10⁵ cells, at least or at least about 2 x 10⁵ cells, at least or at least about 3 x 10⁵ cells, at least or at least about 4 x 10⁵ cells, at least or at least about 5 x 10⁵ cells, at least or at least about 6 x 10⁵ cells, at least or at least about 7 x 10⁵ cells, at least or at least about 8 x 10⁵ cells, at least or at least about 9 x 10⁵ cells, at least or at least about 1 x 10⁶ cells or more. In particular embodiments, the amount of enriched NK cells, such as selected or isolated from PBMCs as described above, is at least or about at least or is or is about 1 x 10⁶ cells.

[0559] In some embodiments, the population of enriched NK cells comprises at least at or about 2.0 x 10⁶ enriched NK cells, at least at or about 3.0 x 10⁶ enriched NK cells, at least at or about 4.0 x 10⁶ enriched NK cells, at least at or about 5.0 x 10⁶ enriched NK cells, at least at or about 6.0 x 10⁶ enriched NK cells, at least at or about 7.0 x 10⁶ enriched NK cells, at least at or about 8.0 x 10⁶ enriched NK cells, at least at or about 9.0 x 10⁶ enriched NK cells, at least at or about 1.0 x 10⁷ enriched NK cells, at least at or about 5.0 x 10⁷ enriched NK cells, at least at or about 1.0 x 10⁸ enriched NK cells, at least at or about 5.0 x 10⁸ enriched NK cells, or at least at or about 1.0 x 10⁹ enriched NK cells. In some embodiments, the population of enriched NK cells comprises at least at or about 2.0 x 10⁵ enriched NK cells. In some embodiments, the population of enriched NK cells comprises at least at or about 1.0 x 10⁶ enriched NK cells. In some embodiments, the population of enriched NK cells comprises at least at or about 1.0 x 10⁷ enriched NK cells.

[0560] In some embodiments, the population of enriched NK cells comprises between at or about 2.0 x 10⁵ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 5.0 x 10⁷ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 1.0 x 10⁷ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 5.0 x 10⁶ enriched NK cells, between at or about 2.0 x 10⁵ enriched NK cells and at or about 1.0 x 10⁶ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 5.0 x 10⁷ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁷ enriched NK cells, between at or about 1.0 x 10⁶ enriched NK cells and at or about 5.0 x 10⁶ enriched NK cells, between at or about 5.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 5.0 x 10⁶ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, between at or about 5.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells, between at or about 5.0 x 10⁶ enriched NK cells and at or about 5.0 x 10⁷ enriched NK cells, between at or about 5.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁷ enriched NK cells, between at or about 1.0 x 10⁷ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 1.0 x 10⁷ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, between at or about 1.0 x 10⁷ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells, between at or about 1.0 x 10⁷ enriched NK cells and at or about 5.0 x 10⁷ enriched NK cells, between at or about 5.0 x 10⁷ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 5.0 x 10⁷ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, between at or about 5.0 x 10⁷ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells, between at or about 1.0 x 10⁸ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells, between at or about 1.0 x 10⁸ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells, or between at or about 5.0 x 10⁸ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 2.0 x 10⁵ enriched NK cells and at or about 5.0 x 10⁷ enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10⁶ enriched NK cells and at or about 1.0 x 10⁸ enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10⁷ enriched NK cells and at or about 5.0 x 10⁸ enriched NK cells. In some embodiments, the population of enriched NK cells comprises between at or about 1.0 x 10⁷ enriched NK cells and at or about 1.0 x 10⁹ enriched NK cells.

[0561] In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 20% and at or about 90%, between at or about 20% and at or about 80%, between at or about 20% and at or about 70%, between at or about 20% and at or about 60%, between at or about 20% and at or about 50%, between at or about 20% and at or about 40%, between at or about 20% and at or about 30%, between at or about 30% and at or about 90%, between at or about 30% and at or about 80%, between at or about 30% and at or about 70%, between at or about 30% and at or about 60%, between at or about 30% and at or about 50%, between at or about 30% and at or about 40%, between at or about 40% and at or about 90%, between at or about 40% and at or about 80%, between at or about 40% and at or about 70%, between at or about 40% and at or about 60%, between at or about 40% and at or about 50%, between at or about 50% and at or about 90%, between at or about 50% and at or about 80%, between at or about 50% and at or about 70%, between at or about 50% and at or about 60%, between at or about 60% and at or about 90%, between at or about 60% and at or about 80%, between at or about 60% and at or about 70%, between at or about 70% and at or about 90%, between at or about 70% and at or about 80%, or between at or about 80% and at or about 90%. In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 20% and at or about 90%. In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 40% and at or about 90%. In some embodiments, the percentage of g-NK cells among the population of enriched NK cells is between at or about 60% and at or about 90%.

[0562] In some of these embodiments, the NK cells can be cultured with a growth factor. According to some embodiments, the at least one growth factor comprises a growth factor selected from the group consisting of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18 and IL-21. According to some embodiments, the at least one growth factor is IL-2 or IL-7 and IL-15. According to some embodiments, the at least one growth factor is IL-2, IL-21 or IL-7 and IL- 15. In some embodiments, the growth factor is a recombinant cytokine, such as a recombinant IL-2, recombinant IL-7, recombinant IL- 21 or recombinant IL- 15.

[0563] In some embodiments, the NK cells are cultured in the presence of one or more recombinant cytokines. In some embodiments, the one or more recombinant cytokines comprise any of SCF, GSK3i, FLT3, IL-2, IL-6, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof. In some embodiments, the one or more recombinant cytokines comprise any of IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-21. In some embodiments, the one or more recombinant cytokines further comprises IL-2, IL-7, IL-15, IL-12, IL-18, or IL-27, or combinations thereof. In some embodiments, at least one of the one or more recombinant cytokines is IL-2. In some embodiments, the one or more recombinant cytokines is at least IL-2 and IL-21. In some embodiments, the one or more recombinant cytokines are IL-21 and IL-2. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, and IL-15. In some embodiments, the one or more recombinant cytokines are IL-21, IL-12, IL-15, and IL-18. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, 11-12, IL-15, and IL-18. In some embodiments, the one or more recombinant cytokines are IL-21, IL-15, IL-18, and IL-27. In some embodiments, the one or more recombinant cytokines are IL-21, IL-2, IL-15, IL-18, and IL-27. In some embodiments, the one or more recombinant cytokines are IL-2 and IL- 15.

[0564] In particular embodiments, the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-2. In some embodiments, during at least a portion of the incubation, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, the recombinant IL-2 is present at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/mL and at or about 100 lU/mL, between at or about 100 lU/mL and at or about 500 lU/mL, between at or about 100 lU/mL and at or about 250 lU/mL or between at or about 250 lU/mL and at or about 500 lU/mL, each inclusive. In some embodiments, during at least a portion of the incubation, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, the concentration of the IL-2 is at or about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 125 lU/mL, 150 lU/mL, 200 lU/mL, or any value between any of the foregoing. In particular embodiments, the concentration of the recombinant IL-2 added at the initiation of the culturing and optionally one or more times during the culturing is or is about 100 lU/mL. In particular embodiments, the concentration of the recombinant IL-2 added at the initiation of the culturing and optionally one or more times during the culturing is or is about 500 lU/mL.

[0565] In particular embodiments, the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21. In some embodiments, during at least a portion of the incubation, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, the recombinant IL-21 is present at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/mL and at or about 100 lU/mL, between at or about 100 lU/mL and at or about 500 lU/mL, between at or about 100 lU/mL and at or about 250 lU/mL or between at or about 250 lU/mL and at or about 500 lU/mL, each inclusive. In some embodiments, during at least a portion of the incubation, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, the concentration of the IL-21 is at or about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 125 lU/mL, 150 lU/mL, 200 lU/mL, or any value between any of the foregoing. In particular embodiments, the concentration of the recombinant IL-21 added at the initiation of the culturing and optionally one or more times during the culturing, is or is about 100 lU/mL.

[0566] In particular embodiments, the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21. In particular embodiments, the concentration of recombinant IL-21 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL, between about 20 ng/mL and about 60 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 100 ng/mL, between about 30 ng/mL and about 90 ng/mL, between about 30 ng/mL and about 80 ng/mL, between about 30 ng/mL and about 70 ng/mL, between about 30 ng/mL and about 60 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, between about 40 ng/mL and about 100 ng/mL, between about 40 ng/mL and about 90 ng/mL, between about 40 ng/mL and about 80 ng/mL, between about 40 ng/mL and about 70 ng/mL, between about 40 ng/mL and about 60 ng/mL, between about 40 ng/mL and about 50 ng/mL, between about 50 ng/mL and about 100 ng/mL, between about 50 ng/mL and about 90 ng/mL, between about 50 ng/mL and about 80 ng/mL, between about 50 ng/mL and about 70 ng/mL, between about 50 ng/mL and about 60 ng/mL, between about 60 ng/mL and about 100 ng/mL, between about 60 ng/mL and about 90 ng/mL, between about 60 ng/mL and about 80 ng/mL, between about 60 ng/mL and about 70 ng/mL, between about 70 ng/mL and about 100 ng/mL, between about 70 ng/mL and about 90 ng/mL, between about 70 ng/mL and about 80 ng/mL, between about 80 ng/mL and about 100 ng/mL, between about 80 ng/mL and about 90 ng/mL, or between about 90 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 10 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is at or about 25 ng/mL.

[0567] In particular embodiments, the concentration of recombinant IL-15 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-15 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-15 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is at or about 10 ng/mL.

[0568] In particular embodiments, the methods include culture in the presence of IL-2, IL- 15 and IL-21. In embodiments of the provided methods, the concentration of recombinant cytokines, e.g., added to the culture at the initiation of the culturing and optionally one or more times during the culturing, is at between at or about 50 lU/mL and at or about 500 lU/mL IL-2, such as at or about 100 lU/mL or 500 lU/mL IL-2; between at or about 1 ng/mL and 50 ng/mL IL-15, such as at or about 10 ng/mL; and between at or about 10 ng/mL and at or about 100 ng/mL IL-21, such as at or about 25 ng/mL. In particular embodiments, 500 lU/mL of IL-2, 10 ng/mL of IL-15, and 25 ng/mL of IL-21 are added during at least a portion of the culturing, such as added at the initiation of the culturing and optionally one or more times during the culturing. In particular embodiments, 100 lU/mL of IL-2, 10 ng/mL of IL-15, and 25 ng/mL of IL-21 are added during at least a portion of the culturing, such as added at the initiation of the culturing and optionally one or more times during the culturing.

[0569] In some embodiments, the provided methods include incubation or culture of the enriched NK cells and feeder cells in the presence of recombinant IL-21 and the recombinant IL-21 is added as a complex with an anti-IL-21 antibody. In some embodiments, prior to the culturing, anti-IL-21 antibody is contacted with the recombinant IL-21, thereby forming an IL-21 /anti-IL-21 complex, and the IL- 21 /anti-IL-21 complex is added to the culture medium. In some embodiments, contacting the recombinant IL-21 and the anti-IL-21 antibody to form an IL-21 /anti-IL-21 complex is carried out under conditions that include temperature and time suitable for the formation of the complex. In some embodiments, the culturing is carried out at 37 °C + 2 for 30 minutes.

[0570] In some embodiments, anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/mL, between at or about 100 ng/mL and at or about 400 ng/mL, between at or about 100 ng/mL and at or about 300 ng/mL, between at or about 100 ng/mL and at or about 200 ng/mL, between at or about 200 ng/mL and at or about 500 ng/mL, between at or about 200 ng/mL and at or about 400 ng/mL, between at or about 200 ng/mL and at or about 300 ng/mL, between at or about 300 ng/mL and at or about 500 ng/mL, between at or about 300 ng/mL and at or about 400 ng/mL, or between at or about 400 ng/mL and at or about 500 ng/mL. In some embodiments, anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/mL. In some embodiments, anti-IL-21 antibody is added at a concentration of 250 ng/mL.

[0571] In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL, between about 20 ng/mL and about 60 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 100 ng/mL, between about 30 ng/mL and about 90 ng/mL, between about 30 ng/mL and about 80 ng/mL, between about 30 ng/mL and about 70 ng/mL, between about 30 ng/mL and about 60 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, between about 40 ng/mL and about 100 ng/mL, between about 40 ng/mL and about 90 ng/mL, between about 40 ng/mL and about 80 ng/mL, between about 40 ng/mL and about 70 ng/mL, between about 40 ng/mL and about 60 ng/mL, between about 40 ng/mL and about 50 ng/mL, between about 50 ng/mL and about 100 ng/mL, between about 50 ng/mL and about 90 ng/mL, between about 50 ng/mL and about 80 ng/mL, between about 50 ng/mL and about 70 ng/mL, between about 50 ng/mL and about 60 ng/mL, between about 60 ng/mL and about 100 ng/mL, between about 60 ng/mL and about 90 ng/mL, between about 60 ng/mL and about 80 ng/mL, between about 60 ng/mL and about 70 ng/mL, between about 70 ng/mL and about 100 ng/mL, between about 70 ng/mL and about 90 ng/mL, between about 70 ng/mL and about 80 ng/mL, between about 80 ng/mL and about 100 ng/mL, between about 80 ng/mL and about 90 ng/mL, or between about 90 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is at or about 25 ng/mL.

[0572] In particular embodiments, the concentration of recombinant IL-12 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-12 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-12 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is at or about 10 ng/mL.

[0573] In particular embodiments, the concentration of recombinant IL- 18 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-18 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL- 18 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is at or about 10 ng/mL.

[0574] In particular embodiments, the concentration of recombinant IL-27 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-27 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is between about 1 ng/mL and about 50 ng/mL. In particular embodiments, the concentration of recombinant IL-27 during at least a portion of the culturing, e.g., added at the initiation of the culturing and optionally one or more times during the culturing, is at or about 10 ng/mL.

[0575] In some embodiments, the methods include exchanging the culture medium, which, in some aspects includes washing the cells. For example, during at least a portion of the culture or incubation the culture medium can be exchanged or washed out intermittently, such as daily, every other day, every three days, or once a week. In particular embodiments, the culture medium is exchanged or washed out beginning within or within about 3 days to 7 days after initiation of the culture, such as at or about at day 3, day 4, day 5, day 6 or day 7. In particular embodiments, the culture medium is exchanged or washed out at or about beginning at day 5. For example, media is exchanged on day 5 and every 2-3 days afterwards.

[0576] Once the culture medium is removed or washed out, it is replenished. In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as any as described above. Hence, in some embodiments, the one or more growth factor or cytokine, such as recombinant IL-2, IL- 15 and/or IL-21, is added intermittently during the incubation or culture. In some such aspects, the one or more growth factor or cytokine, such as recombinant IL-2, IL-15 and/or IL-21, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the one or more growth factor or cytokine, such as recombinant IL-2, IL-15 and/or IL-21, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the one or more growth factor or cytokine, such as recombinant IL-2, IL-15 and/or IL-21. In some embodiments, the methods include adding the one or more growth factor or cytokine, e.g., recombinant IL-2, IL-15 and/or IL-21, at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation.

[0577] In particular embodiments, the culturing is carried out in the presence of at least one of IL-2, IL- 15 and IL-21 and the culture medium is replenished to include at least one of IL-2, IL- 15 and IL-21. In some embodiments, the culturing is carried out in the presence of IL-2 and IL-21 and the culture medium is replenished to include IL-2 and IL-21. In some embodiments, the culturing is carried out in the presence of IL-2 and IL- 15 and the culture medium is replenished to include IL-2 and IL- 15. In some embodiments, the culturing is carried out in the presence of IL- 15 and IL-21 and the culture medium is replenished to include IL- 15 and IL21. In some embodiments, the culturing is carried out in the presence of IL-2, IL- 15 and IL-21 and the culture medium is replenished to include IL-2, IL- 15 and IL-21. In some embodiments, one or more additional cytokines can be utilized in the expansion of the NK cells, including but not limited to recombinant IL-18, recombinant IL-7, and/or recombinant IL-12. [0578] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-2. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-2, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-2, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL-2, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL-2. In some embodiments, the methods include adding recombinant IL-2 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL-2 is added to the culture or incubation at a concentration of between at or about 1 lU/mL and at or about 500 lU/mL, such as between at or about 1 lU/mL and at or about 250 lU/mL, between at or about 1 lU/mL and at or about 100 lU/mL, between at or about 1 lU/mL and at or about 50 lU/mL, between at or about 50 lU/mL and at or about 500 lU/mL, between at or about 50 lU/mL and at or about 250 lU/mL, between at or about 50 lU/mL and at or about 100 lU/mL, between at or about 100 lU/mL and at or about 500 lU/mL, between at or about 100 lU/mL and at or about 250 lU/mL or between at or about 250 lU/mL and at or about 500 lU/mL, each inclusive. In some embodiments, the recombinant IL-2 is added to the culture or incubation at a concentration that is at or about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 125 lU/mL, 150 lU/mL, 200 lU/mL, or any value between any of the foregoing. In particular embodiments, the concentration of the recombinant IL-2 is or is about 100 lU/mL. In particular embodiments, the concentration of the recombinant IL-2 is or is about 500 lU/mL.

[0579] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-21. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-21, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-21, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL-21, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL-21. In some embodiments, the methods include adding recombinant IL-21 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL-21 is added to the culture or incubation at a concentration of between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL, between about 20 ng/mL and about 60 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 100 ng/mL, between about 30 ng/mL and about 90 ng/mL, between about 30 ng/mL and about 80 ng/mL, between about 30 ng/mL and about 70 ng/mL, between about 30 ng/mL and about 60 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, between about 40 ng/mL and about 100 ng/mL, between about 40 ng/mL and about 90 ng/mL, between about 40 ng/mL and about 80 ng/mL, between about 40 ng/mL and about 70 ng/mL, between about 40 ng/mL and about 60 ng/mL, between about 40 ng/mL and about 50 ng/mL, between about 50 ng/mL and about 100 ng/mL, between about 50 ng/mL and about 90 ng/mL, between about 50 ng/mL and about 80 ng/mL, between about 50 ng/mL and about 70 ng/mL, between about 50 ng/mL and about 60 ng/mL, between about 60 ng/mL and about 100 ng/mL, between about 60 ng/mL and about 90 ng/mL, between about 60 ng/mL and about 80 ng/mL, between about 60 ng/mL and about 70 ng/mL, between about 70 ng/mL and about 100 ng/mL, between about 70 ng/mL and about 90 ng/mL, between about 70 ng/mL and about 80 ng/mL, between about 80 ng/mL and about 100 ng/mL, between about 80 ng/mL and about 90 ng/mL, or between about 90 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the recombinant IL-21 is added to the culture or incubation at a concentration of between about 10 ng/mL and about 100 ng/mL, inclusive. The recombinant IL-21 is added to the culture or incubation at a concentration of at or about 25 ng/mL.

[0580] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-21, added as a complex with an antibody, such as an anti- IL-21 antibody. Hence, in some embodiments, the complex, such as an IL-21 /anti-IL-21 antibody complex, is added intermittently during the incubation or culture. In some such aspects, the complex, such as an IL-21 /anti-IL-21 antibody complex, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the complex, such as an IL-21 /anti-IL-21 antibody complex, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the complex, such as an IL-21/anti-IL-21 antibody complex. In some embodiments, the methods include adding the complex, such as an IL-21 /anti-IL-21 antibody complex, at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the anti-IL-21 antibody is contacted with the recombinant IL- 21, thereby forming an IL-21 /anti-IL-21 complex, and the IL-21 /anti-IL-21 complex is added to the culture medium. In any of such embodiments, contacting the recombinant IL-21 and the anti-IL-21 antibody to form an IL-21 /anti-IL-21 complex is carried out under conditions that include temperature and time suitable for the formation of the complex. In any of such embodiments, the culturing is carried out at 37 °C + 2 for 30 minutes. In any of such embodiments, anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/mL, between at or about 100 ng/mL and at or about 400 ng/mL, between at or about 100 ng/mL and at or about 300 ng/mL, between at or about 100 ng/mL and at or about 200 ng/mL, between at or about 200 ng/mL and at or about 500 ng/mL, between at or about 200 ng/mL and at or about 400 ng/mL, between at or about 200 ng/mL and at or about 300 ng/mL, between at or about 300 ng/mL and at or about 500 ng/mL, between at or about 300 ng/mL and at or about 400 ng/mL, or between at or about 400 ng/mL and at or about 500 ng/mL. In some embodiments, anti-IL-21 antibody is added at a concentration between at or about 100 ng/mL and at or about 500 ng/mL. In some embodiments, anti-IL-21 antibody is added at a concentration of 250 ng/mL. In any of such embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, between about 10 ng/mL and about 90 ng/mL, between about 10 ng/mL and about 80 ng/mL, between about 10 ng/mL and about 70 ng/mL, between about 10 ng/mL and about 60 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 100 ng/mL, between about 20 ng/mL and about 90 ng/mL, between about 20 ng/mL and about 80 ng/mL, between about 20 ng/mL and about 70 ng/mL, between about 20 ng/mL and about 60 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 100 ng/mL, between about 30 ng/mL and about 90 ng/mL, between about 30 ng/mL and about 80 ng/mL, between about 30 ng/mL and about 70 ng/mL, between about 30 ng/mL and about 60 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, between about 40 ng/mL and about 100 ng/mL, between about 40 ng/mL and about 90 ng/mL, between about 40 ng/mL and about 80 ng/mL, between about 40 ng/mL and about 70 ng/mL, between about 40 ng/mL and about 60 ng/mL, between about 40 ng/mL and about 50 ng/mL, between about 50 ng/mL and about 100 ng/mL, between about 50 ng/mL and about 90 ng/mL, between about 50 ng/mL and about 80 ng/mL, between about 50 ng/mL and about 70 ng/mL, between about 50 ng/mL and about 60 ng/mL, between about 60 ng/mL and about 100 ng/mL, between about 60 ng/mL and about 90 ng/mL, between about 60 ng/mL and about 80 ng/mL, between about 60 ng/mL and about 70 ng/mL, between about 70 ng/mL and about 100 ng/mL, between about 70 ng/mL and about 90 ng/mL, between about 70 ng/mL and about 80 ng/mL, between about 80 ng/mL and about 100 ng/mL, between about 80 ng/mL and about 90 ng/mL, or between about 90 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is between about 10 ng/mL and about 100 ng/mL, inclusive. In particular embodiments, the concentration of recombinant IL-21 used to form a complex with the anti-IL-21 antibody is at or about 25 ng/mL.

[0581] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-15. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-15, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-15, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL- 15, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL- 15. In some embodiments, the methods include adding recombinant IL- 15 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL- 15 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 15 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL-15 is added to the culture or incubation at a concentration of at or about 10 ng/mL. In particular embodiments, 500 lU/mL of IL-2, 10 ng/mL of IL- 15, and 25 ng/mL of IL-21 are added to the culture or incubation.

[0582] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-12. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-12, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-12, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL- 12, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL-12. In some embodiments, the methods include adding recombinant IL- 12 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL- 12 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 12 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL-12 is added to the culture or incubation at a concentration of at or about 10 ng/mL.

[0583] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-18. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-18, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-18, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL- 18, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL- 18. In some embodiments, the methods include adding recombinant IL- 18 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL- 18 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 18 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL- 18 is added to the culture or incubation at a concentration of at or about 10 ng/mL.

[0584] In some embodiments, the replenished culture medium includes the one or more growth factors or cytokines, such as recombinant IL-27. Hence, in some embodiments, the growth factor or cytokine, such as recombinant IL-27, is added intermittently during the incubation or culture. In some such aspects, the growth factor or cytokine, such as recombinant IL-27, is added at or about at the initiation of the culture or incubation, and then is added intermittently during the culture or incubation, such as each time the culture medium is exchanged or washed out. In some embodiments, the growth factor or cytokine, such as recombinant IL-27, is added to the culture or incubation beginning at day 0 (initiation of the incubation) and, at each exchange or wash out of the culture medium, it is further added to replenish the culture or incubation with the growth factor or cytokine, such as recombinant IL-27. In some embodiments, the methods include adding recombinant IL-27 at the initiation of the incubation (day 0), and every two or three days at each wash or exchange of the culture medium for the duration of the incubation, e.g., at or about at day 5, day 7, day 9, day 11, and day 14 of the culture or incubation. In any of such embodiments, the recombinant IL-27 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL, between about 1 ng/mL and about 40 ng/mL, between about 1 ng/mL and about 30 ng/mL, between about 1 ng/mL and about 20 ng/mL, between about 1 ng/mL and about 10 ng/mL, between about 1 ng/mL and about 5 ng/mL, between about 5 ng/mL and about 50 ng/mL, between about 5 ng/mL and about 40 ng/mL, between about 5 ng/mL and about 30 ng/mL, between about 5 ng/mL and about 20 ng/mL, between about 5 ng/mL and about 10 ng/mL, between about 10 ng/mL and about 50 ng/mL, between about 10 ng/mL and about 40 ng/mL, between about 10 ng/mL and about 30 ng/mL, between about 10 ng/mL and about 20 ng/mL, between about 20 ng/mL and about 50 ng/mL, between about 20 ng/mL and about 40 ng/mL, between about 20 ng/mL and about 30 ng/mL, between about 30 ng/mL and about 50 ng/mL, between about 30 ng/mL and about 40 ng/mL, or between about 40 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL-27 is added to the culture or incubation at a concentration of between about 1 ng/mL and about 50 ng/mL. In any of such embodiments, the recombinant IL-27 is added to the culture or incubation at a concentration of at or about 10 ng/mL.

[0585] In embodiments of the provided methods, culturing or incubating includes providing the chemical and physical conditions (e.g., temperature, gas) which are required or useful for NK cell maintenance. Examples of chemical conditions which may support NK cell proliferation or expansion include but are not limited to buffers, nutrients, serum, vitamins and antibiotics which are typically provided in the growth (i.e., culture) medium. In one embodiment, the NK culture medium includes MEMa comprising 10% FCS or CellGro SCGM (Cell Genix) comprising 5% Human Serum/LiforCell® FBS Replacement (Lifeblood Products). Other media suitable for use with the invention include, but are not limited to Glascow’s medium (Gibco Carlsbad Calif.), RPMI medium (Sigma-Aldrich, St Louis Mo.) or DMEM (Sigma- Aldrich, St Louis Mo.). It will be noted that many of the culture media contain nicotinamide as a vitamin supplement for example, MEMa (8.19 pM nicotinamide), RPMI (8.19 pM nicotinamide), DMEM (32.78 pM nicotinamide) and Glascow’s medium (16.39 pM nicotinamide).

[0586] In some embodiments, such as for applications in which cells are introduced (or reintroduced) into a human subject, culturing is carried out using serum-free formulations, such as AIM V™ serum free medium for lymphocyte culture, MARROWMAX™ bone marrow medium or serum-free stem cell growth medium (SCGM) (e.g., CellGenix® GMP SCGM). Such medium formulations and supplements are available from commercial sources. The cultures can be supplemented with amino acids, antibiotics, and/or with other growth factors cytokines as described to promote optimal viability, proliferation, functionality and/or and survival. In some embodiments, the serum-free media also may be supplemented with a low percentage of human serum, such as 0.5% to 10% human serum, such as at or about 5% human serum. In such embodiments, the human serum can be human serum from human AB plasma (human AB serum) or autologous serum.

[0587] In some embodiments, the culturing with feeder cells, and optionally cytokines (e.g., recombinant IL-2 or IL-21) is carried out under conditions that include temperature suitable for the growth or expansion of human NK cells, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. In some embodiments, the culturing is carried out at 37 °C + 2 in 5% CO2.

[0588] In embodiments of the provided methods, the culturing includes incubation that is carried out under GMP conditions. In some embodiments, the incubation is in a closed system, which in some aspects may be a closed automated system. In some embodiments, the culture media containing the one or more recombinant cytokines or growth factors is a serum-free media. In some embodiments, the incubation is carried out in a closed automated system and with serum-free media.

[0589] In some embodiments, the expansion of the NK cells is carried out in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g., G-Rex 10, G-Rex 10M, G-Rex 100 M/100M-CS or G-Rex 500 M/500M- CS). In some embodiments the culture vessel is a microplate, flask, bag or other culture vessel suitable for expansion of cells in a closed system. In some embodiments, expansion can be carried out in a bioreactor. In some embodiments, the expansion is carried out using a cell expansion system by transfer of the cells to gas permeable bags, such as in connection with a bioreactor (e.g., Xuri Cell Expansion System W25 (GE Healthcare)). In an embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g., gas permeable cell bag, with a volume that is about 50 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L, or any value between any of the foregoing. In some embodiments, the process is automated or semi-automated. In some aspects, the expansion culture is carried out under static conditions. In some embodiments, the expansion culture is carried out under rocking conditions. The medium can be added in bolus or can be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L/min, 0.05 L/min, 0.1 L/min, 0.2 L/min, 0.3 L/min, 0.4 L/min, 0.5 L/min, 1.0 L/min, 1.5 L/min, or 2.0 L/min or greater than 2.0 L/min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as with a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day.

[0590] In some aspects, cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.

[0591] The expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium. In some embodiments, any one or more of the steps of the method can be carried out in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, a closed system is used for carrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and/or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0592] In some of any of the provided embodiments, the culturing is carried out until a time at which the method achieves expansion of at least or at least about 2.50 x 10⁸ g-NK cells. In some of any of the provided embodiments, the culturing is carried out until a time at which the method achieves expansion of at least or at least about 5.0 x 10⁸ g-NK cells. In some of any of the provided embodiments, the culturing is carried out until the method achieves expansion of at least or at least about 1.0 x 10⁹ g- NK cells. In some of any of the provided embodiments, the culturing is carried out until a time at which the method achieves expansion of at least or at least about 5.0 x 10⁹ g-NK cells.

[0593] In some of any of the provided embodiments, the culturing is carried out for at or about or at least at or at least about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 day, 21 days, 22 days, 23 days, 24 days or 25 days. In some embodiments, the culturing is carried out for at or about or at least at or about 14 days. In some embodiments the culturing is carried out for at or about or at least at or about 21 days.

[0594] In some of any of the provided embodiments, the culturing or incubation in accord with any of the provided methods is carried out for at or about or at least at or at least about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 day, 21 days, 22 days, 23 days, 24 days or 25 days. In some embodiments, the culturing is carried out for at or about or at least at or about 14 days. In some embodiments, the culturing is carried out for at or about or at least at or about 21 days. In certain embodiments, a longer duration of culturing is typically necessary if the enriched NK cells at the initiation of the culturing have been thawed after having been previously frozen or cryopreserved. It is within the level of a skilled artisan to empirically determine the optimal number of days to culture the cells depending on factors such as the state of the cells at the initiation of the culture, the health or viability of the cells that the initiation of the culture or during the culturing and/or the desired number of threshold cells at the end of the culturing depending, for example, on the desired application of the cells, such as the dose of cells to be administered to a subject for therapeutic purposes.

[0595] At the end of the culturing, the cells are harvested. Collection or harvesting of the cells can be achieved by centrifugation of the cells from the culture vessel after the end of the culturing. For example, cells are harvested by centrifugation after approximately 14 days of culture. After harvesting of the cells, the cells are washed. A sample of the cells can be collected for functional or phenotypic testing. Any other cells not used for functional or phenotypic testing can be separately formulated. In some cases, the cells are formulated with a cryoprotectant for cryopreservation of cells.

[0596] In some embodiments, the provided methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, selection and/or enrichment. In some embodiments, the provided methods include steps for freezing, e.g., cryopreserving, the cells, either before or after incubation and/or culturing. In some embodiments, the method includes cryopreserving the cells in the presence of a cryoprotectant, thereby producing a cryopreserved composition. In some aspects, prior to the incubating and/or prior to administering to a subject, the method includes washing the cryopreserved composition under conditions to reduce or remove the cryoprotectant. Any of a variety of known freezing solutions and parameters in some aspects may be used. In some embodiments, the cells are frozen, e.g., cryofrozen or cryopreserved, in media and/or solution with a final concentration of or of about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9. 0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In particular embodiments, the cells are frozen, e.g., cryofrozen or cryopreserved, in media and/or solution with a final concentration of or of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% I, or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA. One example involves using PBS containing 20% DMSO and 8% human serum albumilHSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSInd HSA are 10% and 4%, respectively. The cells are generally then frozen to or to about -80° C. at a rate of or of about 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. In some embodiments, the cells are frozen in a serum-free cryopreservation medium comprising a cryoprotectant. In some embodiments, the cryoprotectant is DMSO. 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 cryopreservation medium is at or about 6% DMSO (v/v). In some embodiments, the cry opreservation 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 some embodiments, 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 or CS5). CryoStor™ CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). CryoStor™ CS5 is a cryopreservation medium containing 5% dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation media contains one or more additional excipients, such as plasmalyte A or human serum lumin (HSA). [0597] In some embodiments, the cells are cryopreserved at a density of 5 x 10⁶ to x 1 x 10⁸ cells/mL. For example, the cells are cryopreserved at a density of at or about 5 x 10⁶ cells/mL, at or about 10 x 10⁶ cells/mL, at or about 15 x 10⁶ cells/mL, at or about 20 x 10⁶ cells/mL, at or about 25 x 10⁶ cells/mL, at or about 30 x 10⁶ cells/mL, at or about 40 x 10⁶ cells/mL, at or about 50 x 10⁶ cells/mL, at or about 60 x 10⁶ cells/mL, at or about 70 x 10⁶ cells/mL, at or about 80 x 10⁶ cells/mL or at or about 90 x 10⁶ cells/mL, or any value between any of the foregoing. The cells can be cryopreserved in any volume as suitable for the cryopreservation vessel. In some embodiments, the cells are cryopreserved in a vial. The volume of the cryopreservation media may be between at or about 1 mL and at or about 50 mL, such as at or about 1 mL and 5 mL. In some embodiments, the cells are cryopreserved in a bag. The volume of the cry opreservation media may between at or about 10 mL and at or about 500 mL, such as between at or about 100 mL or at or about 200 mL. The harvested and expanded cells can be cryopreserved at low temperature environments, such as temperatures of -80°C to - 196°C. In some of any of the provided methods, the method produces an increased number of NKG2C^pos cells at the end of the culturing compared to at the initiation of the culturing. For example, the increase in NKG2C^pos cells at the end of culturing compared to at the initiation of the culturing can be greater than or greater than about 100-fold, greater than or greater than about 200-fold, greater than or greater than about 300-fold, greater than or greater than about 400-fold, greater than or greater than about 500-fold, greater than or greater than about 600-fold, greater than or greater than about 700-fold or greater than or greater than about 800-fold. 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. In some embodiments, the culturing or incubation in accord with any of the provided methods is carried out until a time at which the method achieves expansion of at least at or about 2.50 x 10⁸ NKG2C^pos cells, at least at or about 3.0 x 10⁸ NKG2C^pos cells, at least at or about 4.0 x 10⁸ NKG2C^pos cells, at least at or about 5.0 x 10⁸ NKG2C^pos cells, at least at or about 6.0 x 10⁸ NKG2C^pos cells, at least at or about 7.0 x 10⁸ NKG2C^pos cells, at least at or about 8.0 x 10⁸ NKG2C^pos cells, at least at or about 9.0 x 10⁸ NKG2C^pos cells, at least at or about 1.0 x 10⁹ NKG2C^pos cells, at least at or about 1.5 x 10⁹ NKG2C^pos cells, at least at or about 2.0 x 10⁹ NKG2C^pos cells, at least at or about 3.0 x 10⁹ NKG2C^pos cells, at least at or about 4.0 x 10⁹ NKG2C^pos cells, at least at or about 5.0 x 10⁹ NKG2C^pos cells, at least at or about 1.0 x 10¹⁰ NKG2C^pos cells, at least at or about 1.5 x 10¹⁰ NKG2C^pos cells, at least at or about 2.0 x IO¹⁰ NKG2C^pos cells, at least at or about 2.5 x IO¹⁰ NKG2C^pos cells or more.

[0598] In some of any of the provided methods, the method produces an increased number of NKG2A^neg cells at the end of the culturing compared to at the initiation of the culturing. For example, the increase in NKG2A^neg cells at the end of culturing compared to at the initiation of the culturing can be greater than or greater than about 100-fold, greater than or greater than about 200-fold, greater than or greater than about 300-fold, greater than or greater than about 400-fold, greater than or greater than about 500-fold, greater than or greater than about 600-fold, greater than or greater than about 700-fold or greater than or greater than about 800-fold. 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 3000-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 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. In some embodiments, the culturing or incubation in accord with any of the provided methods is carried out until a time at which the method achieves expansion of at least at or about 2.50 x 10⁸ NKG2A^neg cells, at least at or about 3.0 x 10⁸ NKG2A^neg cells, at least at or about 4.0 x 10⁸ NKG2A^neg cells, at least at or about 5.0 x 10⁸ NKG2A^neg cells, at least at or about 6.0 x 10⁸ NKG2A^neg cells, at least at or about 7.0 x 10⁸ NKG2A^neg cells, at least at or about 8.0 x 10⁸ NKG2A^neg cells, at least at or about 9.0 x 10⁸ NKG2A^neg cells, at least at or about 1.0 x 10⁹ NKG2A^neg cells, at least at or about 1.5 x 10⁹ NKG2A^neg cells, at least at or about 2.0 x 10⁹ NKG2A^neg cells, at least at or about 3.0 x 10⁹ NKG2A^neg cells, at least at or about 4.0 x 10⁹ NKG2A^neg cells, at least at or about 5.0 x 10⁹ NKG2A^neg cells, at least at or about 1.0 x 10¹⁰ NKG2A^neg cells, at least at or about 1.5 x 10¹⁰ NKG2A^neg cells, at least at or about 2.0 x 10¹⁰ NKG2A^neg cells, at least at or about 2.5 x 10¹⁰ NKG2A^neg cells or more.

[0599] In some of any of the provided methods, the method produces an increased number of NKG2C^posNKG2A^neg cells at the end of the culturing compared to at the initiation of the culturing. For example, the increase in NKG2C^posNKG2A^neg cells at the end of culturing compared to at the initiation of the culturing can be greater than or greater than about 100-fold, greater than or greater than about 200- fold, greater than or greater than about 300-fold, greater than or greater than about 400-fold, greater than or greater than about 500-fold, greater than or greater than about 600-fold, greater than or greater than about 700-fold or greater than or greater than about 800-fold. 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 lOOOO-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. In some embodiments, the culturing or incubation in accord with any of the provided methods is carried out until a time at which the method achieves expansion of at least at or about 2.50 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 3.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 4.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 5.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 6.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 7.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 8.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 9.0 x 10⁸ NKG2C^posNKG2A^neg cells, at least at or about 1.0 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 1.5 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 2.0 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 3.0 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 4.0 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 5.0 x 10⁹ NKG2C^posNKG2A^neg cells, at least at or about 1.0 x 10¹⁰ NKG2C^posNKG2A^neg cells, at least at or about 1.5 x 10¹⁰ NKG2C^posNKG2A^neg cells, at least at or about 2.0 x 10¹⁰ NKG2C^posNKG2A^neg cells, at least at or about 2.5 x 10¹⁰ NKG2C^posNKG2A^neg cells or more.

[0600] In some of any of the provided methods, the method produces an increased number of g-NK cells at the end of the culturing compared to at the initiation of the culturing. For example, the increase in g-NK cells at the end of culturing compared to at the initiation of the culturing can be greater than or greater than about 100-fold, greater than or greater than about 200-fold, greater than or greater than about 300-fold, greater than or greater than about 400-fold, greater than or greater than about 500-fold, greater than or greater than about 600-fold, greater than or greater than about 700-fold or greater than or greater than about 800-fold. 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 lOOOO-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. In some embodiments, the culturing or incubation in accord with any of the provided methods is carried out until a time at which the method achieves expansion of at least at or about 2.50 x 10⁸ g-NK cells, at least at or about 3.0 x 10⁸ g-NK cells, at least at or about 4.0 x 10⁸ g-NK cells, at least at or about 5.0 x 10⁸ g-NK cells, at least at or about 6.0 x 10⁸ g-NK cells, at least at or about 7.0 x 10⁸ g-NK cells, at least at or about 8.0 x 10⁸ g-NK cells, at least at or about 9.0 x 10⁸ g-NK cells, at least at or about 1.0 x 10⁹ g-NK cells, at least at or about 1.5 x 10⁹ g-NK cells, at least at or about 2.0 x 10⁹ g-NK cells, at least at or about 3.0 x 10⁹ g-NK cells, at least at or about 4.0 x 10⁹ g-NK cells, at least at or about 5.0 x 10⁹ g-NK cells or more, at least at or about 1.0 x 10¹⁰ g-NK cells or more, at least at or about 1.5 x 10¹⁰ g-NK cells or more, at least at or about 2.0 x 10¹⁰ g-NK cells or more, or at least at or about 2.5 x 10¹⁰ g-NK cells or more.

[0601] In some embodiments, the provided methods result in the preferential expansion of g-NK cells. In some aspects, g-NK cells are identified by the presence, absence or level of surface expression of one or more various marker that distinguishes NK cells from other lymphocytes or immune cells and that distinguishes g-NK cells from conventional NK cells. 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. 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. In some embodiments, fixation is achieved using formaldehyde (e.g., 0.01%) followed by disruption of membranes using a detergent (e.g., 0.1% to 1% detergent, for example at or about 0.5%), such as Triton, NP-50, Tween 20, Saponin, Digitonin or Leucoperm.

[0602] Antibodies and other binding entities can be used to detect expression levels of marker proteins to identify, detect, enrich and/or isolate the g“NK cells. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies and other forms of specific binding molecules.

[0603] 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.

[0604] 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.

[0605] 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.

[0606] In some embodiments, g-NK cells are cells having a phenotype of NK cells (e.g., CD45^pos, CD3^neg and/or CD56^pos) and express one or more markers that identify or that are associated with a g-NK cell subset.

[0607] In some embodiments, g-NK cells are identified as described in published Patent Appl. No. US2013/0295044 or Zhang et al. (2013) J. Immunol., 190:1402-1406.

[0608] In some embodiments, the g-NK cell subset of NK cells can be detected by observing whether FcRy is expressed by a population of NK cells or a subpopulation of NK cells. In some cases, g- NK cells are identified as cells that do not express FcRy. FcRy protein is an intracellular protein. Thus, in some aspects, the presence or absence of FcRy can be detected after treatment of cells, for example, by fixation and permeabilization, to allow intracellular proteins to be detected. In some embodiments, cells are further assessed for one or more surface markers (CD45, CD3 and/or CD56) prior to the intracellular detection, such as prior to fixation of cells. In some embodiments, g-NK cells are identified, detected, enriched and/or isolated as cells that are CD45^pos/CD3^neg/CD56^pos/ FcRy^neg.

[0609] In some embodiments, greater than at or about 50% of NK cells in the expanded population are FcRy^neg. In some embodiments, greater than at or about 60% of NK cells in the expanded population are FcRy^neg. In some embodiments, greater than at or about 70% of NK cells in the expanded population are FcRy^neg. In some embodiments, greater than at or about 80% of NK cells in the expanded population are FcRy^neg. In some embodiments, greater than at or about 90% of NK cells in the expanded population are FcRy^neg. In some embodiments, greater than at or about 95% of NK cells in the expanded population are FcRy^neg. For example, the methods herein generally result in a highly pure, e.g., 70-90%, g-NK cell product.

[0610] In some embodiments, it may be useful to detect expression of g-NK cells without employing intracellular staining, such as, for example, if cells of the sample are to be subjected to cell sorting or a functional assay. While treatments, e.g., fixation and permeabilization, to permit intracellular staining of FcRy can be used to confirm the identity of a substantially pure population of cells, in many cases cell-surface markers can be employed that can be detected without injuring the cells when identifying, detecting or isolating g“NK cells. Thus, in some embodiments, g-NK cells are identified using a surrogate marker profile that correlates with the lack of FcRy among a subset of NK cells. In some embodiments, a surrogate marker profile is of particular use when the presence or absence of an intracellular protein, such as FcRy, is difficult or not possible to assess depending on the particular application of the cells.

[0611] It is found herein that certain combinations of cell surface marker correlate with the g-NK cell phenotype, i.e., cells that lack or are deficient in intracellular expression of FcRy, thereby providing a surrogate marker profile to identify or detect g-NK cells in a manner that does not injure the cells. In some embodiments, a surrogate marker profile for g-NK cells provided herein is based on positive surface expression of one or more markers CD16 (CD16^pos), NKG2C (NKG2C^pos), or CD57 (CD57pos) and/or based on low or negative surface expression of one or more markers CD7 (CD7^dim/neg), CD161 (CD161^neg) and/or NKG2A (NKG2A^neg). In some embodiments, cells are further assessed for one or more surface markers of NK cells, such as CD45, CD3 and/or CD56. In some embodiments, g-NK cells can be identified, detected, enriched and/or isolated with the surrogate marker profile CD45^pos/CD3^neg/CD56^pos/CD16^pos/CD57^pos/CD7^dim/neg/CD161^neg. In some embodiments, g-NK cells are identified, detected, enriched and/or isolated with the surrogate marker profile CD45^pos/CD3^neg/CD56^pos/NKG2A^neg/CD161^neg. In some embodiments, g-NK cells that are NKG2C^pos and/or NKG2A^neg are identified, detected, enriched for, and/or isolated.

[0612] In some embodiments, greater than at or about 20% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 50% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 25% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 50% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 30% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 50% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 35% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 60% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 40% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 70% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 45% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 80% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 50% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 85% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 55% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 90% of NK cells in the expanded population are negative or low for NKG2A. In some embodiments, greater than at or about 60% of NK cells in the expanded population are positive for NKG2C and/or greater than at or about 95% of NK cells in the expanded population are negative or low for NKG2A.

[0613] In some embodiments, greater than at or about 70% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 70% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than at or about 75% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 75% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than at or about 80% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 80% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than at or about 85% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 85% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than at or about 90% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 90% of the g-NK cells in the expanded population are positive for granzyme B. In some embodiments, greater than at or about 95% of the g-NK cells in the expanded population are positive for perforin, and greater than at or about 95% of the g-NK cells in the expanded population are positive for granzyme B.

[0614] Cells expanded by the provided methods can be assessed for any number of functional or phenotypic activities, including but not limited to cytotoxic activity, degranulation, ability to produce or secrete cytokines, and expression of one or more intracellular or surface phenotypic markers. Methods to assess such activities are known and are exemplified herein and in working examples.

[0615] In some embodiments, antibody-dependent cell cytotoxicity (ADCC) cytotoxic activity against target cells can be used as a measure of functionality. For the ADCC cytotoxicity assays, cells from expansions can be co-cultured with appropriate targets cells in the presence or absence of an antibody specific to a target antigen on the target cells. For example, for anti-myeloma cytotoxicity any of a number of multiple myeloma (MM) target cells can be used (e.g., AM01, KMS11, KMS18, KMS34, LP1 or MM. IS) can be used and the assay performed with an anti-CD38 (e.g., Daratumumab) or anti- CD319 antibody (e.g., Elotuzumab). Cell killing can be determined by any number of methods. For example, cells can be stained with Propidium iodide (PI) and the number of NK-cells, live target cells, and dead target cells can be resolved, such as by flow cytometry.

[0616] In some embodiments, greater than at or about 10% of g-NK cells in the expanded population are capable of degranulation against tumor cells. Degranulation can be measured by assessing expression of CD107A. For example, in some embodiments, greater than at or about 20% of g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, greater than at or about 30% of g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, greater than at or about 40% of g-NK cells in the expanded population are capable of degranulation against tumor cells. In some embodiments, capacity for degranulation is measured in the absence of an antibody against the tumor cells.

[0617] In some embodiments, greater than at or about 10% of g-NK cells in the expanded population are capable of producing an effector cytokine, such as interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, greater than at or about 20% of g-NK cells in the expanded population are capable of producing an effector cytokine, e.g., interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, greater than at or about 30% of g-NK cells in the expanded population are capable of producing an effector cytokine, e.g., interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, greater than at or about 40% of g-NK cells in the expanded population are capable of producing an effector cytokine, e.g., interferon-gamma or TNF-alpha, against tumor cells. In some embodiments, capacity for producing interferon-gamma or TNF-alpha is measured in the absence of an antibody against the tumor cells.

[0618] Provided herein are methods for identifying or detecting g-NK cells in a sample containing a population of cells by employing a surrogate marker profile of g-NK cells. In some embodiments, the methods include contacting a sample of cells with a binding molecule, such as an antibody or antigenbinding fragment that is specific for one or more markers CD16, CD57, CD7, CD161, NKG2C, and/or NKG2A. In some embodiments, the methods further include contacting the sample of cells with a binding molecule, such as an antibody or antigen-binding fragment that is specific for CD45, CD3 and/or CD56. In some embodiments of the methods, the one or more binding molecules can be contacted with the sample simultaneously. In some embodiments of the methods, the one or more binding molecules can be contacted with the sample sequentially. In some embodiments, following the contact, the methods can include one or more washing under conditions to retain cells that have bound to the one or more binding molecule and/or to separate away unbound binding molecules from the sample.

[0619] In some embodiments, each of the one or more binding molecules, e.g., antibody, may be attached directly or indirectly to a label for detection of cells positive or negative for the marker. For example, the binding molecule, e.g., antibody, may be conjugated, coupled or linked to the label. Labels are well known by one of skill in the art. Labels contemplated herein include, but are not limited to, fluorescent dyes, fluorescent proteins, radioisotopes, chromophores, metal ions, gold particles (e.g., colloidal gold particles), silver particles, particles with strong light scattering properties, magnetic particles (e.g., magnetic bead particles such as Dynabeads® magnetic beads), polypeptides (e.g., FLAG™ tag, human influenza hemagglutinin (HA) tag, etc.), enzymes such as peroxidase (e.g., horseradish peroxidase) or a phosphatase (e.g., alkaline phosphatase), streptavidin, biotin, luminescent compounds (e.g., chemiluminescent substrates), oligonucleotides, members of a specific binding pair (e.g., a ligands and its receptor) and other labels well known in the art that are used for visualizing or detecting a binding molecule, e.g., an antibody, when directly or indirectly attached to said antibody. [0620] A number of well-known methods for assessing expression level of surface markers or proteins may be used, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of surface markers, such as by flow cytometry. In some embodiments, the label is a fluorophore and the methods for detection or identification of g-NK cells is by flow cytometry. In some embodiments, different labels are used for each of the different markers by multicolor flow cytometry.

[0621] In some embodiments, the methods include contacting a sample with a binding molecule specific to CD45, CD3, CD56, CD57, CD7 and CD161. In some such embodiments, g-NK cells are identified or detected as cells having the g-NK cell surrogate marker profile CD45^pos/CD3^neg/CD56^pos/CD 16^pos/CD57^pos/CD7^dim/neg/CD 161 ^neg.

[0622] In some embodiments, the methods include contacting a sample with a binding molecule specific to CD45, CD3, CD56, NKG2A and CD161. In some such embodiments, g-NK cells are identified or detected as cells having the g-NK cell surrogate marker profile CD45^pos/CD3^neg/CD56^pos/NKG2A^neg/CD161^neg.

[0623] In some embodiments, the provided methods also can include isolating or enriching g-NK, such as g-NK cells preferentially expanded in accord with any of the provided methods. In some such embodiments, a substantially pure population of g-NK cells can be obtained, such as a cell population containing greater than or greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more g-NK cells, such as determined using any of the described panel or combinations of markers. Antibodies and other binding molecules can be used to detect the presence or absence of expression levels of marker proteins, for use in isolating or enriching g“NK cells. In some embodiments, isolation or enrichment is carried out by fluorescence activated cell sorting (FACs). In examples of such methods, g- NK cells are identified or detected by flow cytometry using the methods as described above for staining cells for multiple cell surface markers and stained cells are carried in a fluidic stream for collection of cells that are positive or negative for markers associated with g-NK cells.

III. METHODS OF ASSESSING OR DETERMINING CLINICAL RESPONSE AND TREATING BASED ON CLINICAL RESPONSE

[0624] In some embodiments, the provided methods allow for subject selection, treatment, and/or clinical response assessment in subjects with a HLA-E cancer (e.g., subjects with multiple myeloma as described in Section I.B.3) to be treated in accord with any of the methods described in Section I following an initial treatment with a composition of g-NK cells (such as any composition described in Section I. A) based on biomarkers. In some embodiments, methods to assess, determine, and/or predict a likelihood a subject will respond to administration of a composition of g-NK cells include one or more steps for measuring, assessing, and/or determining the transcriptional expression profile of one or more RNA transcripts, e.g., a gene expression profile. In some embodiments, the upregulation or downregulation of the one or more genes as compared to a reference threshold value is predictive of, correlated with, and/or associated with response following administration of a composition of g-NK cells.

[0625] In some embodiments, the method comprises assessing the level of expression in one or more RNA transcripts or portions thereof in a biological sample from the subject. In some embodiments, the expression, upregulation, or down regulation of the one or more RNA transcripts is measured in a biological sample, e.g., a biological sample taken, collected, and/or obtained from the subject. In some embodiments, the biological sample is from bone marrow of the subject. In some embodiments, the biological sample is taken no later than one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks following the initial administration of a composition of g-NK cells to the subject. In some embodiments, the biological sample is taken no later than four weeks following the initial administration of a composition of g-NK cells to the subject.

[0626] In some embodiments, the one or more RNA transcript is a polynucleotide that is expressed by and/or encoded by the gene. In some embodiments, the RNA transcript is a messenger RNA (mRNA), a transfer RNA (tRNA), a ribosomal RNA, a small nuclear RNA, a small nucleolar RNA, an antisense RNA, long non-coding RNA, a microRNA, a small interfering RNA, and/or a short hairpin RNA. In particular embodiments, the RNA transcript is an mRNA.

[0627] In some embodiments, the amount or level of a polynucleotide in a sample may be assessed, measured, determined, and/or quantified by any suitable means known in the art. For example, in some embodiments, the amount or level of a polynucleotide gene product can be assessed, measured, determined, and/or quantified by polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, droplet digital PCR (including, e.g., NanoString nCounter® technology), real-time and quantitative PCR (qPCR) methods (including, e.g., TAQMAN®, molecular beacon, LIGHTUP™, SCORPION™, SIMPLEPROBES®; see, e.g., U.S. Pat. Nos.5,538,848; 5,925,517; 6,174,670; 6,329,144; 6,326,145 and 6,635,427); northern blotting; Southern blotting, e.g., of reverse transcription products and derivatives; array based methods, including blotted arrays, microarrays, or in situ-synthesized arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other methods known in the art, such as discussed in Shendure et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian, Euk. Cell 9(9): 1300-1310 (2010), including such specific platforms as HELICOS®, ROCHE® 454, ILLUMINA®/SOLEXA®, ABI SOLiD®, and POLONATOR® sequencing. In particular embodiments, the levels of nucleic acid gene products are measured by quantitative PCR (qPCR) methods, such qRT-PCR. In some embodiments, the qRT-PCR uses three nucleic acid sets for each gene, where the three nucleic acids comprise a primer pair together with a probe that binds between the regions of a target nucleic acid where the primers bind — known commercially as a TAQMAN® assay. [0628] In some embodiments, the expression of one or more RNA transcripts is predictive of or correlated with the likelihood of response following administration of the composition of g-NK cells to the subject.

[0629] In some embodiments, at least one or more RNA transcripts are negatively correlated to the likelihood of response following administration of the composition of g-NK cells. In some embodiments, the at least one RNA transcript 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. The inventors found that 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 are downregulated in subjects who respond to administration of g-NK cell compositions as a monotherapy compared to those who do not respond. Accordingly, in some embodiments, a level of expression of one or RNA transcripts 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. The inventors found that 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 that is downregulated in subjects who respond to administration of g-NK cell compositions as a monotherapy compared to those who do not respond.

[0630] In some embodiments, at least one or more RNA transcripts are positively correlated to the likelihood of response following administration of the composition of g-NK cells. In some embodiments, the at least one RNA transcript is transcribed from a gene selected from a group consisting of EGF, ITGB3, NID2, and PG4. The inventors found that EGF, ITGB3, NID2, and PG4 are upregulated in subjects who respond to administration of g-NK cell compositions as a monotherapy compared to those who do not respond.

[0631] In some embodiments, the method further comprises determining the likelihood of response of the subject to administration of the composition.

[0632] In some embodiments, determining the likelihood of response of the subject comprises: (i) calculating a fold-change, wherein the fold-change is a difference in the level of expression of the one or more RNA transcripts to a baseline level of expression, and (ii) comparing the fold-change to a threshold.

[0633] In some embodiments, the fold-change is calculated for one or more RNA transcripts that are negatively correlated to the likelihood of response (e.g., the RNA transcripts are transcribed from any one of genes 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. The inventors found that CD28, CEECE1, DEPTOR, DUSP2, DUSP5, FCGR2B, FCRE2, GPR160, HEA-DOB, ITGA6, EY9, MAGEA1, MAGEA12, MAGEC2, PDK1, PTCD2, SEAMF7, SMAD5, TNFRSF17 (BCMA), TNFSF8 (CD30 ligand), and WNT10A), and the threshold is a downregulation threshold. In some embodiments, the subject is determined to be responsive to the administration of the composition of g-NK cells when the fold-change is below or equal to the downregulation threshold. In some embodiments, the subject is determined to not be responsive to the administration of the g-NK cells when the fold-change is above the downregulation threshold.

[0634] In some embodiments, the downregulation threshold is between about -0.1 to -5 log2fold change. In some embodiments, the downregulation threshold is between about -0.2 to about -4 log2fold change. In some embodiments, the downregulation threshold is at about -0.1, about -0.2, about -0.3, about -0.4, about -0.5, about -0.6, about -0.7, about -0.8, about -0.9, about -1.0, about -1.1, about -1.2, about -1.3, about -1.4, about -1.5, about -1.6, about -1.7, about -1.8, about -1.9, or about -2.0 log2fold change. In some embodiments, the downregulation threshold is at or about -0.8 log2fold change. In some embodiments, the downregulation threshold is at or about -1 log2fold change. In some embodiments, the downregulation threshold is at or about -2 log2fold change.

[0635] In some embodiments, the fold-change is calculated for one or more RNA transcripts that are positively correlated to the likelihood of response (e.g., the RNA transcripts are transcribed from any one of genes EGF, ITGB3, NID2, and PG4), and the threshold is an upregulation threshold. In some embodiments, the subject is determined to be responsive to the administration of the composition of g- NK cells when the fold-change is above or equal to the upregulation threshold. In some embodiments, the subject is determined to not be responsive to the administration of the g-NK cells when the fold-change is below the upregulation threshold.

[0636] In some embodiments, the upregulation threshold is between about 0.1 to 5 log2fold change. In some embodiments, the upregulation threshold is between about 0.2 to about 4 log2fold change. In some embodiments, the upregulation threshold is at about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 log2fold change. In some embodiments, the upregulation threshold is at or about 0.8 log2fold change. In some embodiments, the upregulation threshold is at or about 1 log2fold change. In some embodiments, the upregulation threshold is at or about 2 log2fold change.

[0637] In some embodiments, the baseline level of expression is the level of expression of the one or more RNA transcripts in a reference subject. In some embodiments, the reference subject is a subject with untreated multiple myeloma. In some embodiments, the reference subject is the same subject that was administered the composition of g-NK cells at a time prior to the administration of the g-NK cells. [0638] In some embodiments, the reference subject is a subject that is not responsive to administration of a composition of g-NK cells. For example, in some embodiments, a reference subject has achieved a negative clinical outcome. In some embodiments, a reference subject has achieved less than a minor response (MR) based on IMWG, such as, e.g., progressive disease (PD) or stable disease (SD).

[0639] In some embodiments, the response is any positive clinical outcome. In some embodiments, the response is a minor response (MR) or better based on IMWG. In some embodiments, the response is a partial response (PR) or better based on IMWG. In some embodiments, the response is a very good partial response (VGPR) or better based on IMWG. In some embodiments, the response is a complete response (CR) based on IMWG. In some embodiments, the response is achieved at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year after the initial administration of the composition of g-NK cells.

[0640] In some embodiments, the method further comprises a method of adaptive treatment. In some embodiments, the method further comprises administering to the subject who is determined to not be responsive to the administration of the composition of g-NK cells an additional treatment. In some embodiments, the additional treatment is any method of treatment provided herein in Section I, including combination therapies as provided herein in Section I.D. In some embodiments, the additional treatment is administration of a dose of IL-2 to the subject. In some embodiments, the additional treatment is additional administration of a composition of g-NK cells to the subject or a higher dose of the composition of g-NK cells to the subject. In some embodiments, the additional treatment is administration of an antibody (e.g., a combination therapy). In some embodiments, the antibody is an anti-CD38 antibody, such as rituximab.

IV. ENGINEERED G-NK CELLS

[0641] In some embodiments, g-NK cells of the provided compositions are engineered to express a heterologous nucleic acid encoding an agent, such as a CAR or an immunomodulatory agent.

[0642] In some embodiments, the g-NK cells are engineered to express a CAR. In some embodiments, the CAR is a fusion protein generally including an ectodomain that comprises an antigen recognition region, a transmembrane domain, and an endo-domain. The ectodomain (i.e., the antigen recognition region or antigen binding domain) and the transmembrane domain may be linked by a flexible linker. The endo-domain may comprise an intracellular signaling domain that propagates the external cellular stimulus intracellularly. In some embodiments, the CAR comprises 1) an antigen binding domain; 2) a flexible linker; 3) a transmembrane region; and 4) and intracellular signaling domain. In some embodiments, the CAR binds to a target antigen and induces cytotoxicity upon antigen binding. [0643] In some embodiments, the engineered g-NK cells may further express one or more other additional heterologous protein agent. In some embodiments, the engineered g-NK cells also express an immunomodulator, 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.

[0644] Exemplary features of a CAR and immunomodulators are further described in the following subsections.

A. Chimeric A niigen Receptor

[0645] In provided embodiments, the g-NK cells are genetically engineered to express an antigen receptor(s) that binds to an antigen of interest. In certain embodiments, the antigen receptor is a chimeric antigen receptor (CAR). The antigen receptor can bind to a target antigen expressed on cells of the HLA- E expressing cancers, such as a target antigen expressed on autoreactive T and B cells. Thus, the engineered antigen receptor, e.g., CAR, is a recombinant antigen receptor that is intended to introduce a certain antigen specificity to the NK cell. In some embodiments, the antigen receptor, such as a CAR, is stably integrated into the g-NK cell. In other embodiments, the antigen receptor, e.g., CAR is transiently expressed by the g-NK cell. For instance, the g-NK cells comprise a CAR with a defined polypeptide sequence expressed from an exogenous polynucleotide that has been introduced into the immune effector cell, either transiently or integrated into the genome. In provided embodiments, the engineered NK cells provided herein that comprise an antigen receptor (e.g., CAR) may be used for immunotherapy to target and destroy cells associated with the HLA-E expressing cancer that express the target antigen recognized by the antigen receptor (e.g., CAR).

[0646] In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR). The CAR is typically encoded by a nucleic acid sequence (polynucleotide) that comprises a leader sequence, an extracellular targeting domain (also called ectodomain; e.g., antigen binding domain, such as an scFv), a transmembrane domain and one or more intracellular signaling domains. In some embodiments, a CAR is a fusion protein that includes an extracellular targeting domain (ectodomain) comprising an antigen recognition or antigen binding domain; a transmembrane domain; and an intracellular signaling domain. The ectodomain and transmembrane domains may be linked by a flexible linker (also called a spacer). In some embodiments, the antigen binding domain, such as a single-chain variable fragment (scFv) derived from a monoclonal antibody, recognizes a target antigen. In some embodiments, the antigen binding domain, e.g., an scFv, is linked or fused to the transmembrane domain via a spacer. In some embodiments, the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (IT AM). Activation of the CAR fusion protein results in cellular activation in response to recognition by the scFv (or other antigen binding domain) of its target. When a cell expresses such a CAR, it can recognize and kill target cells that express the target antigen. This property makes CAR- expressing cells particularly attractive agents for specific targeting of cellular activity to aberrant cells, including, but not limited to, autoreactive cells. Various CARs have been developed against target antigens, including B cell antigens, to mediate cytotoxic activity against target cells expressing the antigen and can be the engineered g-NK cells disclosed herein.

[0647] Any variety of chimeric antigen receptor can be expressed in the engineered NK cells, including those described in International PCT Application PCT/US2018/024650, PCT/IB2019/000141, PCT/IB 2019/000181, and/or PCT/US2020/020824, PCT/US2020/035752.

[0648] In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In some embodiments, the extracellular antigen-binding domain or targeting domain is derived from an antibody molecule, and comprises one or more complementarity determining regions (CDRs) from an antibody molecule that confer antigen specificity on the CAR. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab, which is optionally crosslinked. In certain embodiments, the extracellular binding doma’n is a F(ab')2- In certain embodiments, any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening scFv phage library with an antigen-Fc fusion protein.

[0649] In some embodiments, the scFv comprises the variable chain portion of an immunoglobulin light chain and an immunoglobulin heavy chain molecule separated by a flexible linker polypeptide. The order of the heavy and light chains is not limiting and can be reversed. The flexible polypeptide linker allows the heavy and light chains to associate with one another and reconstitute an immunoglobulin antigen binding domain. Suitably, the polypeptide linker comprises a length greater than or equal to 10 amino acids. Suitably, the polypeptide linker comprises a length greater than 10, 15, 20, or 25 amino acids. Suitably, the polypeptide linker comprises a length less than or equal to 30 amino acids. Suitably, the polypeptide linker comprises a length less than 15, 20, 25, or 30 amino acids. Suitably, the polypeptide linker comprises between 10 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 25 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 20 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 15 amino acids in length. Suitably, the polypeptide linker comprises between 15 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 20 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 25 and 30 amino acids in length. Suitably, the polypeptide linker comprises hydrophilic amino acids. Suitably, the polypeptide linker consists of hydrophilic amino acids. Suitably, the polypeptide linker comprises a G4S sequence (GGGGS). The G4S linker allows for flexibility and protease resistance of the linker. Suitably, the G4S linker is consecutively repeated in the polypeptide linker 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the flexible linker is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the flexible linker is a Whitlow linker, such as set forth in SEQ ID NO: 28. Suitably, the light chain variable region comprises three CDRs and the heavy chain variable region comprises three CDRs. Suitably, the CDRs for use in the antigen-binding targeting domain are derived from an antibody molecule of any species (e.g., human, mouse, rat, rabbit, goat, sheep) and the framework regions between the CDRs are humanized or comprise a sequence that is at least 85%, 90%, 95 or 99% identical to a human framework region.

[0650] In some embodiments, the antigen is an antigen of any cancer that expresses HLA-E.

Exemplary HLA-E expressing cancers include, but are not limited to, lymphomas (e.g., Non-Hodgkin’s Lymphoma), myeloma (e.g., multiple myeloma), and acute myeloid leukemia (AML). In some embodiments, the target antigen is a lymphoma antigen. In some embodiments, the target antigen is a B cell antigen. In some embodiments, the target antigen is a non-Hodgkin’s lymphoma (NHL) antigen. Exemplary lymphoma antigens, such as NHL antigens, include CD19, CD20, and CD22. In some embodiments, the antigen is CD19, CD20, or CD22. In some embodiments, the target antigen is a multiple myeloma (MM) antigen. In some embodiments, the target antigen is a plasma cell antigen. Exemplary multiple myeloma (MM) antigens include BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DL, and Kappa light chain. In some embodiments, the antigen is BCMA, CD19, SLAMF7, GPRC5D, CD138, CD38, CD70, NKG2DL, or Kappa light chain. In some embodiments, the target antigen is acute myeloid leukemia (AML). In some embodiments, the target antigen is a myeloid cell antigen. Exemplary AML antigens include CD123, CD33, and CLL-1. In some embodiments, the target antigen is CD 123, CD33, or CLL-1.

[0651] In certain embodiments, the antigen is a B cell antigen. In certain embodiments, the antigen is CD19, CD20 or CD22.

[0652] Binding of an extracellular antigen-binding domain (for example, an scFv or an analog thereof) of an antigen-targeted CAR can be confirmed by, for example, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein- antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0653] In some embodiments, the g-NK cell is engineered with a bispecific CAR or multiple different CARs, wherein their affinity is for two distinct ligands/antigens. Bispecific CAR-NKs can be used either for increasing the number of potential binding sites on cancer cells or, alternatively, for localizing cancer cells to other immune effector cells which express ligands specific to the NK-CAR. A bispecific CAR may bind to two separate cell markers, increasing the overall binding affinity of the NK cell for the target cell. In some embodiments, the bispecific CAR has specificity for any two of the following antigens: CD19, CD20, and CD22.

[0654] In some embodiments, the transmembrane domain of the CAR comprises hydrophobic amino acid residues and allows the CAR to be anchored into the cell membrane of the engineered NK cell. Suitably, the transmembrane domain comprises an amino acid sequence derived from a transmembrane protein. Suitably, the transmembrane domain comprises an amino acid sequence derived from the transmembrane domain of the alpha, beta, or zeta chain of the T-cell receptor, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, and CD 154. Suitably, the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD8. Suitably, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD8 alpha. In some embodiments, the CAR contains a transmembrane domain of CD8 alpha that has the sequence of amino acids set forth in SEQ ID NO:29 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:29. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:29. In some embodiments, the CAR contains a transmembrane domain of CD8 alpha that has the sequence of amino acids set forth in SEQ ID NO:30 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:30. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:30.

[0655] In some embodiments, suitably, the CAR comprises a transmembrane with an amino acid sequence derived from the transmembrane domain of CD28. Suitably, the CAR comprises a transmembrane domain with an amino acid sequence derived from the transmembrane domain of human CD28. In some embodiments, the CAR contains a hinge domain and a transmembrane domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:31 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:31. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:31. In some embodiments, the transmembrane domain of CD28 has the sequence of amino acids set forth in SEQ ID NO:32 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:32. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:32. In some embodiments, the CAR comprises a CD28 hinge domain and a CD28 transmembrane domain. In some embodiments, the CD28 hinge domain and transmembrane domain are set forth by the sequence of amino acids set forth in SEQ ID NO:33 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:33. In some embodiments, the CD28 hinge domain and transmembrane domain are set forth by the sequence of amino acids set forth in SEQ ID NO: 33.

[0656] In some embodiments, the CARs can also comprise a spacer region located between the antigen-binding targeting domain and the transmembrane domain. In some embodiments, the spacer region comprises hydrophilic amino acids and allows flexibility of the targeting domain with respect to the cell surface. Suitably, the spacer region comprises greater than 5, 10, 15, 20, 25, or 30 amino acids. Suitably, the spacer region comprises less than 10, 15, 20, 25, 30, or 35 amino acids. In some embodiments, the spacer region is a hinge region and includes a hinge sequence of CD8 or of an immunoglobulin molecule.

[0657] In some embodiments, the spacer region is or includes the CD8 hinge. In some embodiments the spacer is the hinge region of human CD8. In some embodiments, the CAR contains a CD8 hinge spacer sequence that has the sequence of amino acids set forth in SEQ ID NO:34 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:34. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:34. In some embodiments, the CAR contains a CD8 hinge spacer sequence that has the sequence of amino acids set forth in SEQ ID NO:35 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:35. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:35.

[0658] In some embodiments, the spacer region is or includes the CD28 hinge. In some embodiments the spacer is the hinge region of human CD28. In some embodiments, the CAR contains a CD28 hinge spacer sequence that has the sequence of amino acids set forth in SEQ ID NO:36 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:36. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:36.

[0659] In some embodiments, the spacer region includes all or a portion containing the hinge domain of an IgGl Fc or an IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer that has the sequence of amino acids set forth in SEQ ID NO:37 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:37. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:37. In some embodiments, the sequence of the spacer is the hinge portion of the IgGl Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has the sequence of amino acids set forth in SEQ ID NO: 38 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:38. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:38. In some embodiments, the IgG4 hinge spacer has the sequence of amino acids set forth in SEQ ID NO: 39 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:39. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:39.

[0660] In some embodiments, the intracellular signaling domain of the CAR increases the potency of the CAR and comprises an intracellular signaling domain derived from a protein involved in immune cell signal transduction. Suitably, the one or more intracellular signaling domains comprise an intracellular signaling domain derived from CD3 zeta CD28, OX-40, 4- IBB, DAP 10, DAP 12, 2B4 (CD244), or any combination thereof. Suitably, the one or more intracellular signaling domains comprise an intracellular signaling domain derived from any two of CD3 zeta CD28, OX-40, 4-1BB, DAP10, DAP 12, 2B4 (CD244), or any combination thereof.

[0661] In some embodiments, the endodomain of a CAR may include two more signaling domains. For instance, a CAR may include a primary intracellular signaling domain, such as a CD3zeta intracellular signaling domain, and an intracellular signaling domains from a costimulatory molecule to provide additional signal to the cells, such as to further enhance potency of the CAR-expressing immune cell. Thus, in some embodiments, the chimeric antigen receptor (CAR) comprises s: 1) an antigen binding domain; 2) a flexible linker; 3) a transmembrane region; and 4) an intracellular signaling region comprising a first primary intracellular signaling domain, such as a CD3 zeta intracellular signaling domain and second co-stimulatory intracellular signaling domain. In some embodiments, a costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3 costimulatory domains. In some embodiments, a costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), DAP10, DAP12, ICOS, and/or 2B4. In some embodiments, a co-stimulatory domain can be CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and/or B7-H3 costimulatory domains. In some embodiments, the costimulatory signaling domain is a signaling domain of CD28. In some embodiments, the costimulatory signaling domain is a signaling domain of 4- IBB.

[0662] In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:40 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:40. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:40. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:41 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:41. In some embodiments, the CAR contains an intracellular signaling domain that contains a signaling domain of CD3zeta that has the sequence of amino acids set forth in SEQ ID NO:41.

[0663] In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:42 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:42. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:42. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:43 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:43. In some embodiments, the CAR contains an intracellular signaling domain that contains the costimulatory signaling domain of CD28 that has the sequence of amino acids set forth in SEQ ID NO:43.

[0664] In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4- IBB that has the sequence of amino acids set forth in SEQ ID NO:44 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:44. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4- IBB that has the sequence of amino acids set forth in SEQ ID NO:44.

[0665] In some embodiments, an intracellular signaling domain can be a domain of CD3zeta, CD28 and/or 4-1BB. In some embodiments, an intracellular signaling domain contains a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:44 or a sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:44) and a CD3zeta signaling domain (e.g., SEQ ID NO:40 or 41 or a sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:40 or 41). In some embodiments, an intracellular signaling domain contains a CD28 costimulatory signaling domain (e.g., SEQ ID NO:42 or a sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:42) and a CD3zeta signaling domain (e.g., SEQ ID NO:40 or 41 or a sequence that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:40 or 41).

[0666] Suitably, the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and 4- IBB. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 40 and SEQ ID NO:44. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 41 and SEQ ID NO:44. [0667] In other embodiments, suitably the CAR comprises at least two intracellular signaling domains derived from CD3 zeta and CD28. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 40 and SEQ ID NO:42. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 40 and SEQ ID NO:43. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 41 and SEQ ID NO:42. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO: 41 and SEQ ID NO:43.

[0668] In some embodiments, the antigen receptor (e.g., CAR) is encoded by a polynucleotide that encodes a CAR with an NlE-terminal leader sequence. The leader sequence (also known as the signal peptide) allows the expressed CAR construct to enter the endoplasmic reticulum (ER) and target the cell surface. The leader sequence is cleaved in the ER and the mature cell surface CAR does not possess a leader sequence. In general, the leader sequence length will be in the range of 5 to 30 amino acids, and comprise a stretch of hydrophobic amino acids. Suitably, the leader sequence comprises greater than 5, 10, 15, 20, or 25 amino acids in length. Suitably, the leader sequence comprises less than 10, 15, 20, 25, or 30 amino acids in length. Suitably, the leader sequence comprises a sequence derived from any secretory protein. In some embodiments, the leader sequence can be any of the signal peptide sequences described herein. An exemplary CD8a signal peptide is set forth in SEQ ID NO:23. An exemplary GM- CSFRa signal peptide is set forth in SEQ ID NO:24. An exemplary IgK signal peptide is set forth in SEQ ID NO:25. An exemplary IgK signal peptide is set forth in SEQ ID NO: 26.

[0669] In some embodiments, the CAR is the CAR present in any of a variety of known engineered cell products. The CAR may include, but is not limited to a CAR engineered into cells of YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI®. In some embodiments, the CAR comprises a CAR of a commercial CAR cell therapy. Non-limiting examples of a CAR in commercial cell based therapies include the CAR engineered in cells of brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®).

[0670] In some embodiments, the g-NK cell is engineered with a CAR that binds to CD 19. Cluster of Differentiation 19 (CD 19) is an antigenic determinant detectable on leukemia precursor cells. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt/Swiss-Prot Accession No. P15391 and the nucleotide sequence encoding of the human CD19 can be found at Accession No. NM_001178098. CD19 is expressed on most B lineage cancers, including, e.g., acute lymphoblastic leukemia, chronic lymphocyte leukemia and non-Hodgkin’s lymphoma. It is also an early marker of B cell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157- 1165 (1997). The antigen-binding extracellular domain in the CAR polypeptide disclosed herein is specific to CD19 (e.g., human CD19). In some examples, the antigen-binding extracellular domain may comprise a scFv extracellular domain capable of binding to CD 19. In some embodiments, an anti-CD19 CAR may comprise an anti-CD19 single-chain variable fragment (scFv) specific for CD19, followed by a spacer and transmembrane domain that is fused to an intracellular co-signaling domain (e.g., a CD28 or 4-1BB) and a CD3zeta signaling domain.

[0671] In some embodiments, the extracellular binding domain of the CD 19 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:46 and the light chain variable region (VL) set forth in SEQ ID NO: 45. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:28. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:47. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:48. In some embodiments, the spacer is a CD8 hinge, such as set forth in SEQ ID NO: 34. In some embodiments, the spacer is an IgG4 hinge, such as set forth in SEQ ID NO: 38. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD19 and intracellular signaling and cytotoxic activity.

[0672] In some embodiments, the CAR comprises an anti-CD19 CAR of a commercial CAR cell therapy. Non-limiting examples of an anti-CD19 CAR in commercial cell based therapies include the anti-CD19 CAR engineered in cells of YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI®.

[0673] In some embodiments, the CAR is an anti-CD19 CAR that has the sequence of amino acids set forth in SEQ ID NO:49 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO: 49. In some embodiments, the CAR is the anti-CD19 CAR having the sequence of amino acids set forth in SEQ ID NO:49. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:49 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:49. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO: 49.

[0674] In some embodiments, the CAR is an anti-CD19 CAR that has the sequence of amino acids set forth in SEQ ID NO:50 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:50. In some embodiments, the CAR is the anti-CD19 CAR having the sequence of amino acids set forth in SEQ ID NO:50. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:50 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:50. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:50.

[0675] In some embodiments, the CAR is an anti-CD19 CAR that has the sequence of amino acids set forth in SEQ ID NO:51 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:51. In some embodiments, the CAR is the anti-CD19 CAR having the sequence of amino acids set forth in SEQ ID NO:51. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:51 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:51. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:51.

[0676] In some embodiments, the CAR is an anti-CD19 CAR that has the sequence of amino acids set forth in SEQ ID NO:52 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:52. In some embodiments, the CAR is the anti-CD19 CAR having the sequence of amino acids set forth in SEQ ID NO:52. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:52 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:52. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:52.

[0677] CD20 is a proven therapeutic target for hematologic malignancies, such as B-NHL, supported by approved and widely used monoclonal antibody therapy. Further, the universal presence of CD19, CD20, and CD22 antigens on malignant B-cells make them the perfect targets for cellular therapies. In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to CD20. In a particular embodiment, the CD20 CAR comprise a CAR directed to CD20, wherein the CAR directed to CD20 comprises a single chain Fv antibody or antibody fragment (scFv). In some embodiments, an anti-CD20 CAR may comprise an anti-CD20 single-chain variable fragment (scFv) specific for CD20, followed by a spacer and transmembrane domain that is fused to an intracellular cosignaling domain (e.g., a CD28 or 4-1BB) and a CD3zeta signaling domain. In some embodiments, the CAR contains an anti-CD20 scFv, followed by a IgG4-Fc spacer, a CD28 transmembrane domain, a 4- 1BB costimulatory domain and a CD3 zeta signaling domain. In some embodiments, the CAR is the Leul6 CAR as described in Rufener et al. Cancer Immunol. Res. 2016 4:509-519. See also, GenBank accession # KX055828). [0678] In some embodiments, the extracellular binding domain of the CD20 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:54 and the light chain variable region (VL) set forth in SEQ ID NO:53. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:28. In some embodiments, the anti-CD20 scFv is set forth in SEQ ID NO: 55. In some embodiments, the intracellular signaling domain contains a 4- 1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD20 and intracellular signaling and cytotoxic activity. In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, and IgG4 Fc spacer (e.g., SEQ ID NO: 37), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a CD28 costimulatory signaling domain (e.g., SEQ ID NO: 42), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the CD20 CAR has the sequence of amino acids set forth in SEQ ID NO:56 or a sequence that exhibits at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 56. In some embodiments, the CD20 CAR has the sequence set forth in SEQ ID NO: 56. In some embodiments, the CAR is encoded by a polynucleotide (e.g., mRNA) set forth in SEQ ID NO:57.

[0679] In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4- 1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 55, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD28 transmembrane domain (e.g., SEQ ID NO: 39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto.

[0680] In some embodiments, the extracellular binding domain of the CD20 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:58 and the light chain variable region (VL) set forth in SEQ ID NO:59. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:28. In some embodiments, the anti-CD20 scFv is set forth in SEQ ID NO: 60. In some embodiments, the intracellular signaling domain contains a 4- 1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto, and retain binding to CD20 and intracellular signaling and cytotoxic activity.

[0681] In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, and IgG4 Fc spacer (e.g., SEQ ID NO: 37), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a CD28 costimulatory signaling domain (e.g., SEQ ID NO: 42), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD8 transmembrane domain (e.g., SEQ ID NO:

30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD28 transmembrane domain (e.g., SEQ ID NO:

31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO: 60, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto.

[0682] In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to CD22. In a particular embodiment, the CD22 CAR comprise a CAR directed to CD22, wherein the CAR directed to CD20 comprises a single chain Fv antibody or antibody fragment (scFv). In some embodiments, the extracellular antigen binding domain of the CD22 CAR is derived from an antibody specific to CD22, such as m971, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies. In some embodiments, the extracellular binding domain of the CD22 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:61 and the light chain variable region (VL) set forth in SEQ ID NO:62. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:28. In some embodiments, the anti-CD22 scFv is set forth in SEQ ID NO: 63. In some embodiments, the extracellular binding domain of the CD22 CAR may comprise the heavy chain variable region (VH) set forth in SEQ ID NO:64 and the light chain variable region (VL) set forth in SEQ ID NO: 65. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker, such as set forth in SEQ ID NO: 27. In some embodiments, the linker separating the VH and VL in the scFv is the Whitlow linker set forth in SEQ ID NO:28. In some embodiments, the anti-CD22 scFv is set forth in SEQ ID NO: 66.

[0683] In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, and IgG4 Fc spacer (e.g., SEQ ID NO: 37), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a CD28 costimulatory signaling domain (e.g., SEQ ID NO: 42), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD28 transmembrane domain (e.g., SEQ ID NO:

31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 63, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto.

[0684] In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, and IgG4 Fc spacer (e.g., SEQ ID NO: 37), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a CD28 costimulatory signaling domain (e.g., SEQ ID NO: 42), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an CD8 hinge spacer (e.g., SEQ ID NO: 35), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD8 transmembrane domain (e.g., SEQ ID NO: 30), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an CD8 hinge spacer (e.g., SEQ ID NO: 71), a CD28 transmembrane domain (e.g., SEQ ID NO: 39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an CD28 hinge spacer (e.g., SEQ ID NO: 36), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, the anti-CD22 CAR contains the scFv set forth in SEQ ID NO: 66, an IgG4 hinge spacer (e.g., SEQ ID NO: 38 or 39), a CD28 transmembrane domain (e.g., SEQ ID NO: 31), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO: 44), and a CD3 zeta signaling domain (e.g., SEQ ID NO:40). In some embodiments, it is understood the CAR includes any sequences that exhibit some sequence variation to any of the above or described SEQ ID NOS, such as at least 85%, 90%, 95% or more sequence identity thereto

[0685] In some embodiments, an anti-CD22 CAR may comprise an anti-CD22 single-chain variable fragment (scFv) specific for CD22, followed by a spacer and transmembrane domain that is fused to an intracellular co-signaling domain (e.g., a CD28 or 4-1BB) and a CD3zeta signaling domain. In some embodiments, the CAR contains an anti-CD22 scFv, followed by a IgG4-Fc spacer, a CD28 transmembrane domain, a 4- IBB costimulatory domain and a CD3 zeta signaling domain.

B. /mmunomodu/ator

[0686] In provided embodiments, the g-NK cells are engineered to express a heterologous immunomodulatory, such as a cytokine or a functional portion thereof. In some embodiments, the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the g- NK cell. In other embodiments, the heterologous nucleic acid encoding the immunomodulator is transiently expressed. In some embodiments, the heterologous cytokine or functional portion thereof is secreted from the cell. In some embodiments, the heterologous cytokine or functional portion thereof is expressed as a membrane bound protein on the surface of the cell.

[0687] In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin or functional portion thereof is a partial or full peptide of one or more of IE-2, IE-4, IL-6, IL-7, IL-9, IL- 10, IL-11, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, Flt3-L, SCF, or IL-7. In some embodiments, the cytokine is IL-2 or a functional portion thereof. In some embodiments, the cytokine is IL- 12 or a functional portion thereof. In some embodiments the cytokine is IL- 15 or a functional portion thereof. In some embodiments, the cytokine is IL-21 or a functional portion thereof.

[0688] In some embodiments, the g-NK cell is engineered with a heterologous nucleic acid encoding IL- 15, such as a mammalian IL- 15. In some embodiments, the mammalian IL- 15 is a human IL-15. Human IL-15 amino acid sequences include, for example, Genbank Accession Nos: NR_751915.1, NP_000576.1, AAI00963.1, AAI00964.1, AAI00962.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, and CAA63913.1. In some embodiments, the engineered NK cell comprises a heterologous nucleotide sequence encoding IL- 15. In some embodiments, the IL- 15 nucleotide sequence is set forth in SEQ ID NO:67 or is a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:67. In some embodiments, the IL-15 is expressed by the cell in a mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, the IL-15 has the sequence of amino acids set forth in SEQ ID NO:68 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:68. In some embodiments, the amino acid sequence of IL-15 is or comprises SEQ ID NO:68.

[0689] In some embodiments, the g-NK cell is engineered with a heterologous nucleic acid encoding IL-2, such as a mammalian IL-2. In some embodiments, the IL-2 is a human IL-2. In some embodiments, the engineered NK cell comprises a heterologous nucleotide sequence encoding IL-2. In some embodiments, the IL-2 is expressed by the cell in a mature form lacking the signal peptide sequence and, in some cases, also lacking the propeptide sequence. In some embodiments, the IL-2 has the sequence of amino acids set forth in SEQ ID NO: 69 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:69. In some embodiments, the amino acid sequence of IL-2 is or comprises SEQ ID NO: 69.

[0690] In some embodiments, the g-NK cell is engineered with a heterologous nucleic acid encoding IL-21, such as a mammalian IL-21. In some embodiments, the IL-2 is a human IL-21. In some aspects, the IL-21 is a mammalian IL-21. In an embodiment, the IL-21 sequence is a human IL-21 sequence. Human IL-21 amino acid sequences include, for example, Genbank Accession Nos: AAU88182.1, EAX05226.1, CAI94500.1, CAJ47524.1, CAL81203.1, CAN87399.1, CAS03522.1, CAV33288.1, CBE74752.1, CB 170418.1, CBI85469.1, CBI85472.1, CBL93962.1, CCA63962.1,AAG29348.1, AAH66258.1, AAH66259.1, AAH66260.1, AAH66261.1, AAH66262.1, AAH69124.1, and ABG36529.1. In some embodiments, the IL-21 is expressed by the cell in a mature form lacking the signal peptide sequence and, in some cases, also lacking the propeptide sequence. In some embodiments, the IL-21 has the sequence of amino acids set forth in SEQ ID NO:70 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:70. In some embodiments, the IL-21 has the sequence of amino acids set forth in SEQ ID NO:71 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:71. In some embodiments, the amino acid sequence of IL-21 is or comprises SEQ ID NO:70.

[0691] The cytokine (e.g., IL-2, IL- 15, or IL-21) amino acid sequences may comprise any functional portion of mature cytokine, e.g., any functional portion of a mature, IL-2, mature, IL-15 or mature IL-21. The functional portion can be any portion comprising contiguous amino acids of the interleukin of which it is a part, provided that the functional portion specifically binds to the respective interleukin receptor. The term “functional portion” when used in reference to an interleukin refers to any part or fragment of the interleukin, which part or fragment retains the biological activity of the interleukin of which it is a part (the parent interleukin). Functional portions encompass, for example, those parts of an interleukin that retain the ability to specifically bind to the respective interleukin receptor, activate the downstream targets of the interleukin, and/or induce one or more of the differentiation, proliferation (or death) and activity of immune cells, e.g., NK cells, to a similar extent, the same extent, or to a higher extent, as the parent interleukin. The biological activity of the functional portion of the interleukin may be measured using assays known in the art. In reference to the parent interleukin, the functional portion can comprise, for instance, about 60%, about 70%, about 80%, about 90%, about 95%, or more, of the amino acid sequence of the parent mature interleukin.

[0692] Included in the scope of the cytokine or functional portion in accord with the provided embodiments are functional variants of the interleukins described herein. The term “functional variant” as used herein refers to an interleukin having substantial or significant sequence identity or similarity to a parent interleukin, which functional variant retains the biological activity of the interleukin of which it is a variant. Functional variants encompass, for example, those variants of the interleukin described herein (the parent interleukin) that retain the ability to specifically bind to the respective interleukin receptor, activate the downstream targets of the interleukin, and/or induce one or more of the differentiation, proliferation (or death) and activity of immune cells, e.g., NK cells, to a similar extent, the same extent, or to a higher extent, as the parent interleukin. Ln reference to the parent interleukin, the functional variant can, for instance, be at least about 80%, about 90%, about 95%, about 99% or more identical in amino acid sequence to the parent interleukin.

[0693] A functional variant can, for example, comprise the amino acid sequence of the parent interleukin with at least one conservative amino acid substitution. Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent interleukin with at least one nonconservative amino acid substitution. In some embodiments, the amino acid substitution, e.g., conservative or non-conservative amino acid substitution, does not interfere with or inhibit the biological activity of the functional variant as compared to the parental interleukin sequence. In some embodiments, the amino acid substitution, e.g., conservative or non-conservative amino acid substitution, may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent interleukin.

[0694] In some embodiments, the amino acid substitution(s) of the interleukin are conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, lie, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., lie, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0695] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21 or a functional portion of any of the foregoing) can be expressed by a g-NK cell as a secreted polypeptide in a variety of ways. For example, all or a functional portion of the cytokine can be expressed within the NK cell and secreted from the NK cell. In some embodiments, a secreted cytokine does not contain a transmembrane domain.

[0696] Although interleukins and other cytokines are generally secreted, they can also be membrane bound. In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21 or a functional portion of any of the foregoing) can be expressed by a g-NK cell as a membrane-bound cytokine in a variety of ways. In some embodiments, the cytokine or a functional portion thereof can be linked (e.g., conjugated or fused) directly or indirectly (e.g., ionic, non-ionic, covalent linkage) to the surface (e.g., at the surface, or within the membrane, of the NK cell) of the g-NK cell using any of a variety of linkers known in the art (Hermanson, G., Bioconjugate Techniques, Academic Press 1996). In some aspects, all or a functional portion of the cytokine is linked to all or a portion of a transmembrane protein. In one aspect, the NK cell expresses a fusion protein comprising all or a portion of the cytokine fused to all or a portion of a transmembrane protein. In some embodiments, the linker may be a peptide linker, such as a flexible linker. In some embodiments, the flexible linker comprises mainly glycine and serine residues. For example, the flexible linker may comprise one or more repeats of one or both of G4S and G3S (e.g., about 3 to about 15 or about 5 to about 12 repeats of G4S and G3S). In some embodiments, the linker is a cleavable linker, such as a furin cleavable sequence. Exemplary furin cleavage sequences are described in Duckert et al, Protein Engineering, Design & Selection, 17(1): 107- 112 (2004) and U.S. Patent 8,871,906, each of which is incorporated herein by reference.

[0697] Examples of transmembrane proteins include a receptor, a ligand, an immunoglobulin, a glycophorin or a combination thereof. Specific examples of transmembrane proteins include, but are not limited to, CD8a, CD4, CD3e, CD3y, CD35, CD3^, CD28, CD137, FceRIy, a T-cell receptor (TCR such as TCRa and/or TCRP), a nicotinic acetylcholine receptor, a GABA receptor, or a combination thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD or a combination thereof. Specific examples of glycophorin include glycophorin A, glycophorin D or a combination thereof.

[0698] In some embodiments, the heterologous cytokine is a membrane bound IL- 15 set forth in SEQ ID NO:72 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:72. [0699] In some embodiments, the IL- 15 is engineered into the cells with IL- 15 Receptor alpha (IL15RA). IL15RA specifically binds IL-15 with very high affinity, and is capable of binding IL-15 independent of other subunits. In some aspects, this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third cell. In some embodiments, the g-NK cells expresses a heterologous (e.g., exogenous) IL-15/IL-15Ra. In some embodiments, the g-NK cell is engineered with an IL-15/IL-15R fusion protein. In some embodiments, the g-NK cell is engineered with a single-chain IL-15/IL-15R fusion protein. In some embodiments, the IL-15/IL-15Ra is expressed as a membrane-bound IL-15. IL15Ra complex (e.g., Imamura et al., Blood, 2014 124(7): 108 and Hurton LV et al., PNAS, 2016). In some embodiments, the exogenous IL-15/IL-15Ra is secreted and is expressed as a soluble IL15Ra.IL15 complex (e.g., Mortier E et al., JBC 2006; Bessard A, Mol. Cancer Ther., 2009; and Desbois M, J. Immunol., 2016). In some embodiments, the provided engineered g-NK cells expresses a membrane-bound IL15/IL15Ra complex and a soluble (secreted) IL15Ra/IL15 complex. In some embodiments, the engineered g-NK cell expresses a membrane-bound from of IL15.IL15Ra complex with a cleavable linker.

[0700] In some embodiments, the heterologous cytokine is a membrane bound IL-21 set forth in SEQ ID NO:73 or a sequence that has at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:73.

V. KITS AND ARTICLES OF MANUFACTURE

[0701] Provided herein are articles of manufacture and kits comprising the provided compositions containing NK cells enriched for particular subsets, such as g-NK cells. In some embodiments, the compositions are produced by any of the provided methods. In some embodiments, the kit comprises any of the provided compositions and instructions for administering the composition as a monotherapy. In some embodiments, provided herein is a kit comprising any of the provided compositions and an additional agent. In some embodiments, the additional agent is an antibody. In some embodiments, the additional agent is a human, humanized, or chimeric antibody. In some of these embodiments, the additional agent is a full length antibody. Exemplary antibodies included any as described.

[0702] Kits can optionally include one or more components such as instructions for use, devices and additional reagents e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods. In some embodiments, the kits can further contain reagents for collection of samples, preparation and processing of samples, and/or reagents for quantitating the amount of one or more surface markers in a sample, such as, but not limited to, detection reagents, such as antibodies, buffers, substrates for enzymatic staining, chromagens or other materials, such as slides, containers, microtiter plates, and optionally, instructions for performing the methods. Those of skill in the art will recognize many other possible containers and plates and reagents that can be used in accord with the provided methods.

[0703] In some embodiments, the kits can be provided as articles of manufacture that include packing materials for the packaging of the cells, antibodies or reagents, or compositions thereof, or one or more other components. For example, the kits can contain containers, bottles, tubes, vial and any packaging material suitable for separating or organizing the components of the kit. The one or more containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the one or more containers hold a composition comprising cells or an antibody or other reagents for use in the methods. The article of manufacture or kit herein may comprise the cells, antibodies or reagents in separate containers or in the same container.

[0704] In some embodiments, the one or more containers holding the composition may be a singleuse vial or a multi-use vial, which, in some cases, may allow for repeat use of the composition. In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, therapeutic agents and/or package inserts with instructions for use.

[0705] In some embodiments, the kit can, optionally, include instructions. Instructions typically include a tangible expression describing the cell composition, reagents and/or antibodies and, optionally, other components included in the kit, and methods for using such. In some embodiments, the instructions indicate methods for using the cell compositions and antibodies for administration to a subject for treating a disease or condition, such as in accord with any of the provided embodiments. In some embodiments, the instructions are provided as a label or a package insert, which is on or associated with the container. In some embodiments, the instructions may indicate directions for reconstitution and/or use of the composition.

VI. EXEMPLARY EMBODIMENTS

[0706] Among the provided embodiments are:

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

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

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

6. The method of any one of embodiments 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 embodiments 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 embodiments 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 embodiments 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 of expressed by cells of the AML.

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

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

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

14. The method of embodiment 12 or embodiment 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 embodiment 12 or embodiment 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 embodiments 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 embodiments 1-16, wherein pathogenesis of the AML is associated with a viral infection.

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

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

20. The method of any one of embodiments 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 embodiments 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 embodiment 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:

(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:

(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 embodiments 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 of embodiments 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 embodiment 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 embodiments 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 embodiments 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 embodiments 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 embodiments 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 embodiments 1-36, wherein the NK cells are positive for NKG2C (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg).

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

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

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

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

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

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

45. The method of embodiment any one of embodiments 22-39 and 44, wherein the cancer is a Multiple Myeloma (MM). 46. The method of any one of embodiments 22-39, wherein the HLA-E expressing cancer is a myeloid cell marker expressing cancer.

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

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

49. The method of embodiment 47 or embodiment 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 embodiments 47-49, wherein the AML is a relapsed or refractory AML.

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

52. The method of embodiment 50 or embodiment 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 embodiment 50 or embodiment 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 embodiments 47-53, wherein the subject has received one or more prior treatment regimens for treating the AME selected from:

(i) At least 1 cycle of purine analogue containing intensive induction chemotherapy regimen, e.g.,

FEAG-Ida, CEIA or CEAG-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;

(iv) 2 cycles of venetoclax with HMA/LDAC +/- other agents; or

(v) 4 cycles of HMA alone.

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

56. The method of any one of embodiments 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 embodiment 55 or embodiment 56, wherein the antibody is directed against a lymphoma antigen.

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

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

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

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

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

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

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

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

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

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

71. The method of any one of embodiments 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 embodiments 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

77. The method of embodiment 75 or embodiment 76, wherein the lymphoma is NonHodgkin’s Lymphoma (NHL).

78. The method of any one of embodiments 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:

(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:

(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 embodiment 79 or embodiment 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 embodiments 65, 66, 68, 69, 79, and 80, wherein the anti- CD38 antibody is daratumumab or is isatuximab.

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

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

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

86. The method of any one of embodiments 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 embodiment 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 embodiments 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 embodiment 89, wherein the NK cells are positive for NKG2C (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg).

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

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

93. The method of any one of embodiments 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 embodiment 93, wherein the target antigen is a B cell antigen, plasma cell antigen, or myeloid cell antigen.

95. The method of any one of embodiments 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 embodiments 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 embodiments 1-96, wherein at least at or about 15% of the NK cells of the composition are positive for NKG2C (NKG2C^pos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2A^neg).

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

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

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

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

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

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

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

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

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

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

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

110. The method of any one of embodiments 89-99, wherein the disease or disorder associated with EBV is Multiple Myeloma (MM). 111. The method of any one of embodiments 30-56, 63, 64, 69, 71, 72, 93-101, and 110, wherein the antibody is an anti-CD38 antibody.

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

114. The method of embodiment 112 or embodiment 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 embodiments 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 embodiments 30-72, 75-88, and 93-115, wherein the antibody is administered intravenously.

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

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

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

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

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

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

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

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

128. The method of embodiment 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 embodiment 1-128, wherein at least at or about 20% of the cells in the composition of g-NK cells are FcRy-deficient (FcRy^neg) NK cells (g-NK).

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

131. The method of any one of embodiments 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 embodiment 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 embodiment 131 or embodiment 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 FcRy^pos; 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 FcRy^pos.

134. The method of any one of embodiments 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 embodiments 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 embodiments 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 embodiments 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 (anti-target antibody).

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

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

140. The method of any one of embodiments 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 embodiments 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 embodiments 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of cells that are NKG2C^pos cells cultured with irradiated HLA- E+ feeder cells, wherein the NKG2C^pos cells are enriched from a biological sample from a donor subject.

143. The method of any one of embodiments 1-139, wherein the composition of g-NK cells has been produced by ex vivo expansion of cells that are CD3^negNKG2C^pos cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3^negNKG2C^pos cells are enriched from a biological sample from a donor subject.

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

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

146. The method of any one of embodiments 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 158 V/F NK cell genotype.

147. The method of any one of embodiments 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 embodiments 140-147, wherein the irradiated feeder cells are deficient in HLA class I and HLA class II.

149. The method of any one of embodiments 140-148, wherein the irradiated feeder cells are 221. AEH cells.

150. The method of any one of embodiments 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 embodiment 150, wherein the recombinant cytokines are IL-21 and IL-2.

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

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

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

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

162. The method of embodiment 159 or embodiment 160, wherein the IL-2 is administered at a frequency of every other day (Q2W). 163. The method of any one of embodiments 159-162, wherein for each day of administration the IL-2 is administered once daily.

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

165. The method of any one of embodiments 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 embodiments 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 embodiment 165 or embodiment 166, wherein each 7-day cycle is the same.

168. The method of any one of embodiments 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 embodiments 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 embodiment 165 or embodiment 166, wherein each 7-day cycle is different.

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

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

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

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

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

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

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

185. The method of any one of embodiments 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 embodiment 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 embodiment 185 or embodiment 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 embodiments 110-187, wherein the lymphodepleting therapy comprises fludarabine and/or cyclophosphamide.

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

190. The method of any of embodiments 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/m² body surface area of the subject, optionally at or about 300 mg/m², daily, for 2-4 days.

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

192. The method of any of embodiments 185-191, wherein the lymphodepleting therapy comprises the administration of fludarabine at or about 30 mg/m² body surface area of the subject, daily, and cyclophosphamide at or about 400 mg/m² body 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 embodiments 1-192, wherein the method further comprises administration of a bispecific T cell targeting agent to the subject.

194. The method of embodiment 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:

(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 IE-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 embodiments 1-196, wherein the subject is a human subject.

VII. EXAMPLES

[0707] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Expression of NKG2C+/NKG2A- NK cells and Expansion of g-NK Cells

[0708] In this study, the preferential expansion of NKG2C+/NKG2A- NK cells was explored.

[0709] Human peripheral blood mononuclear cells (PBMC) were isolated by Histopaque® density centrifugation from whole blood from a CMV-positive human donor, or for comparison a CMV- seronegative donor, as per manufacturer’s instructions.

[0710] To enrich for NK cells deficient in FceRy (FcRy; g-NK cells), PBMCs from a CMV seropositive donor were harvested from huffy coat, washed, and assessed by flow cytometry for viable CD45^pos cells. NK cells were enriched by immunoaffinity-based magnetic bead separation using Miltenyi MACS™ Microbeads by CD3 depletion followed by positive selection for CD56 to enrich CD56^pos NK cells (CD3^negCD56^pos). The NK cells were co-cultured with gamma irradiated (100 Gy) 221. AEH feeder cells at a 1:1 221. AEH to NK cell ratio and expanded in the presence of IL-2 (500 lU/mL), IL- 15 (10 ng/mL), and IL-21 (25 ng/mL).

[0711] Specifically, the expansion method required >10% pre-expansion NKG2C+/NKG2A- NK- cells to preferentially expand g-NK cells. In a study of 10 expansions from CMV seropositive donors with high g-NK cells (>20% of total NK-cells), it was determined that all 8 donors with >10% preexpansion NKG2C+/NKG2A- NK-cells demonstrated a robust preferential expansion of g-NK cells that yielded a majority g-NK product (see Table El and FIG. 1). For every expansion, the percentage of NKG2C+/NKG2A- NK-cells increased from pre- to post-expansion (Table El).

Table El. A Comparison of Pre- and Post-expansion g-NK and NKG2C+/NKG2A- NK-Cells for 10 Expansions

[0712] FIG. 2A and 2B shows pre-expansion conventional NK cells and g-NK cells, respectively. Notably, FIG. 2B demonstrates that pre-expansion, g-NK cells are overwhelmingly NKG2C+ and NKG2A. Moreover, FIG. 3A demonstrates there is a strong correlation between post-expansion NKG2C+/NKG2A- NK and g-NK cells. Representative flow plots and histograms depicting postexpansion NKG2C+/NKG2A- NK and g-NK cells are shown in FIG. 3B.

[0713] The results demonstrate that the expansion of NK cells from CMV-seropositive donors with the 221. AEH feeder cell line preferentially expands NKG2C+/NKG2A- NK cells.

[0714] These results evidence the unique phenotype of a post-expansion g-NK cell, enriched in cells deficient for FcRy and indicate these cells also are enriched in cells that are NKG2C⁺ and NKG2A-. Without wising to be bound by theory, it is believed these cells will provide potent killing of HLA-E expressing targets via activating receptor NKG2C as seen in HLA-E expressing cancers such as AML.

Example 2: Administration of g-NK Cell Compositions as a Monotherapy or Combination Therapy for Treatment of NHL

[0715] Therapeutic g-NK cell compositions containing allogenic NK cells deficient in expression of

FcRy chain were administered to subjects with advanced non-Hodgkin’s lymphoma (NHL). As part of the study, subjects in different cohorts received different therapeutic g-NK cell composition regimens testing different g-NK cell composition dosing schedules/dosages, with or without the inclusion of IL-2 administration. Some cohorts also received a combination therapy with the anti-CD20 antibody rituximab.

[0716] The treated subjects were subjects with advanced NHL. Eligibility criteria included subjects with: (a) relapsed/refractory (R/R) NHL that was 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) have failed at least two lines of systemic chemotherapy and have the following additional criteria depending on type: (1) for DLBCL, HGBL, tFL, or PMBCL: failed a line of chemoimmunotherapy that includes an anti-CD20 monoclonal antibody plus anthracycline, (2) for FL or MZL: failed a line of chemoimmunotherapy that includes an anti-CD20 monoclonal antibody plus an alkylating agent, or (3) for MCL: failed a line of chemoimmunotherapy that includes an anti-CD20 monoclonal antibody plus an alkylating agent, as well as a Bruton’ s tyrosine kinase inhibitor; and (d) at least one measurable lesion according to the revised International Working Group Response Criteria for Malignant Lymphoma (Cheson, 2014). For (d), lesions that have been previously irradiated are considered measurable only if progression has been documented following completion of radiation therapy.

[0717] Different dosing regimens tested in subjects are set forth in Table E2. The regimens differed depending on g-NK cell composition dose (number of g-NK cells) and/or dosing schedules (e.g., a single dose schedule, a multiple dose Q2D (every 2 days) schedule, or a multiple dose QW (every week) dosing schedule), inclusion of IE-2 administration, and/or inclusion of the anti-CD20 antibody rituximab (e.g., a monotherapy or a combination therapy). For subjects receiving the g-NK cell composition as a monotherapy, IE-2 was not administered (N/A) or administered once a week for three weeks (e.g., 6 M IU QW x 3), or was administered as three times a week (e.g., 6 M IU Q2D x 3). Similarly, for subjects receiving combination therapy with rituximab in combination with g-NK cell compositions, IL-2 was either not administered (NA), administered once a week for three weeks (e.g., 6 M IU QW x 3), or was administered three times a week (e.g., 6 M IU Q2D x 3). The IL-2 may be omitted depending on the safety, tolerability, and pharmacokinetics. Dose level -1 (DL-1) is a de-escalating dose of g-NK cells that is pursued if dose-limiting toxicity (DLT) is observed at a higher dose level of g-NK cells, such as dose level 1 (DL1).

Table E2: Dosing Regimens for Subjects with NHL

DL-1 = dose level -1; DL1 = dose level 1; DL2 = dose level 2; DL3 = dose level 3

[0718] The g-NK cell compositions, prepared by expansion substantially as described in Example 1, were thawed from provided frozen liquid form prior to intravenous administration within 10 minutes (± 5 minutes) but not to exceed 30 minutes. Some subjects received g-NK cell composition as a single agent (i.e. without combination with an antibody), with or without IL-2 administered subcutaneously. For subjects receiving IL-2, IL-2 was administered 1 hour prior to infusion of the g-NK cell composition. For subjects receiving a combination therapy with rituximab, rituximab was provided in a single use vial in liquid form and was delivered intravenously. Administration of rituximab begins 7 days prior to the first infusion of g-NK cell composition. Pre -medication with steroids prior to the first administration of daratumumab is permitted at a dose <20 mg prednisone equivalent with approval by the Sponsor’s Medical Monitor. [0719] Prior to administration of therapeutic g-NK cell composition, subjects received administration of a lymphodepleting chemotherapy composed of fludarabine 30 mg/m², cyclophosphamide 400 mg/m², and mesna 300 mg/m², which was administered 3, 4, and 5 days prior to the first infusion of the g-NK cell composition.

[0720] A second cycle of treatment composed of repeat conditioning followed by up to 3 additional doses of g-NK cell composition can be given at the discretion of the investigator and medical monitor, assuming the subject meets the following criteria: (a) did not experience a dose-limiting toxicity (DLT) during the first cycle of treatment, (b) did not experience disease progression on study, (c) at least 30 days have elapsed since the last dose of g-NK cell composition, and (d) absolute neutrophil count is at least 1000 cells/uL.

[0721] Primary and secondary endpoints related to safety assessment and tolerability of g-NK cell compositions with or without IL-2 were assessed. For subjects receiving combination therapy with rituximab, similar primary and secondary endpoints related to safety assessment and tolerability were assessed. The primary and secondary endpoints include incidence of adverse events (AEs) or serious adverse events (SAEs) graded according to NCI CTCAE Version 5.0, incidence and nature of doselimiting toxicities (DLTs), changes from baseline in clinical safety laboratory values and vital signs, and incidence of potential immune -related toxicities. Additional primary and secondary endpoints included determining the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and maximum protocol dose (MPD) with and without IL-2 and with or without rituximab. Further endpoints related to efficacy and pharmacokinetics (PK) are also assessed, including the non-compartmental PK parameters of the therapeutic g-NK cell compositions (e.g., maximum drug concentration (C_max) and area under the concentration-time curve (AUC)) as well as objective response rate (ORR) according to the Lugano Classification criteria (Cheson, 2014).

[0722] Study results are assessed to determine efficacy (e.g., tumor response) according to the Lugano Classification (Cheson, 2014). Safety profiles, pharmacokinetics, and pharmacodynamics data were also assessed.

[0723] In an ongoing clinical trial, data available from one NHL subject who was administered g- NK cells at a dose of 5 x 10⁹ g-NK cells (henceforth referred to as dose level 1, or DL1) at a frequency of Q2D x 3 along with administration of IL-2 at 6 M IU at a frequency Q2D x 3 prior to a specific timepoint was assessed. Details regarding this subject is depicted in Table E3. The composition of g-NK cells was administered as a monotherapy (no antibody). This representative subject is a 77 y.o. male, with three prior regimens, which was inclusive of ADC (POLIVY®) with R-CHP, ADC (POLIVY®) with rituximab, and CD19 CAR-T cell (BREYANZI®) therapy.

Table E3. Representative NHL Subject Demographic and Clinical Summary

Auto SCT, autologous stem cell transplant; BCMA, B cell maturation antigen; CAR, chimeric antigen receptors; Dara, daratumumab; DL1, Dose level 1; Isa, isatuximab; Lymph, Lymphocyte count; Neut, Neutrophil count; PR, partial response; PD, progressive disease; Ritux, rituximab; TCE, T-cell engager; VGPR, very good partial response; WBC, White blood cell count; DL1 = dose level 1

[0724] The dosing schedule for this one NHL subject is detailed in Table E4.

Table E4. Dosing Schedule of NHL Subject

M= Million; IU = international units; DL1 = dose level 1; s.c. = subcutaneously

[0725] The initial efficacy of the g-NK monotherapy for the one NHL subject is detailed in Table

E5.

Table E5. Initial Efficacy of Representative NHL Subject

Response determined by: MM (IMWG, 2016), NHL (Lugano, 2014); VGPR =very good partial response;

PR=Partial response; MR= minor response

[0726] The radiologist’s impression of the patient included: (1) Partial treatment response evidence in decreased size in mesenteric nodal masses and masse predominantly along left pelvic musculature; (2) abdominal mass abut/encase bowel segments with no evidence of bowel obstruction, and (3) decreased retroperitoneal lymphadenopathy. FDG-PET images additionally demonstrated total reduction in tumor burden by 23.6%. Finally, FDG-PET images also demonstrated the central pericolic lymph node completely regressed. Overall, the one NHL patient had a significant reduction in tumor burden that did not reach criteria for a partial response.

[0727] Further, another subject with NHL was administered g-NK cells at a DL1 of 5 x 10⁹ g-NK cells at a frequency of Q2D x 3 along with administration of IL-2 at 6 M IU at a frequency Q2D x 3. The composition of g-NK cells was administered as a combination therapy with rituximab at a dose of 375 mg/m² once per week as described above for a total of six doses. This representative subject is a 64 y.o. male with Stage 3 DLBC. As shown in FIG. 4, a post-treatment biopsy of cecal lesion at baseline (top row) and on day 7 post-administration of the first dose of g-NK cell composition showed a rapid and deep depletion of CD19+ and CD20+ cells in tertiary tissue.

[0728] This result suggests that a combination therapy with g-NK cell compositions and an antibody can rapidly and deeply deplete targeted cell types, such as CD19 and CD20.

[0729] These results demonstrate tolerability and safety of a higher frequency dosing of every other day and with IL-2 and also a response in a heavily pre -treated subject. Furthermore, activity was observed with the g-NK cell therapy along with IL-2 in a subject with advanced NHL, whether in the absence of combination with ADCC mediating monoclonal antibodies or with an ADCC mediating monoclonal antibody rituximab.

Example 3: Administration of g-NK Cells as a Monotherapy or Combination Therapy in Treatment of Multiple Myeloma

[0730] Therapeutic g-NK cell compositions containing allogenic NK cells deficient in expression of FcRy chain were administered to subjects with advanced multiple myeloma (MM). Subjects in different cohorts received different therapeutic g-NK cell composition regimens testing different g-NK cell composition dosing schedules/dosages, with or without the inclusion of IL-2 administration. Some cohorts also receive a combination therapy with the anti-CD38 antibody daratumumab. Alternatively, the combination therapy uses the anti-CD38 antibody isatuximab.

[0731] Subjects with advanced MM were treated. Eligibility criteria included subjects with: (a) documented diagnosis of MM requiring systemic therapy, (b) relapsed or refractory (R/R) disease after >3 prior lines of therapy, (c) exposure to >1 proteasome inhibitors, > immunomodulatory agents, and >1 anti-CD38 monoclonal antibody, (d) a response (minimal response or better) to at least 1 prior treatment regimen, (e) diagnosis of MM that is 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. Induction with or without high-dose chemotherapy followed by autologous stem cell rescue and with or without maintenance therapy was considered a single prior line of therapy. Additionally, previous CAR T-cell and/or CD3 bispecific therapy was allowed.

[0732] Different dosing regimens are set forth in Table E6. The regimens differ depending on g- NK cell composition dose (number of g-NK cells) and/or dosing schedules (e.g., a single dose schedule, a multiple dose Q2D (every 2 days) schedule, or a multiple dose QW (every week) dosing schedule), inclusion of IL-2 administration, and/or inclusion of the anti-CD38 antibody, such as daratumumab or isatuximab (e.g., a combination therapy). [0733] In the regimens, for subjects receiving the g-NK cell composition as a monotherapy, IL-2 is not administered (NA) or is administered once a week for three weeks (e.g., 6 M IU QW x 3), or is administered as three times a week (e.g., 6 M IU Q2D x 3). Similarly, for subjects receiving combination therapy with daratumumab or isatuximab in combination with g-NK cell compositions, IL-2 is either not administered (N/A), administered once a week for three weeks (e.g., 6 M IU QW x 3), or is administered as three times a week (e.g., 6 M IU Q2D x 3). The IL-2 may be omitted depending on the safety, tolerability, and pharmacokinetics. Dose level -1 (DL-1) is a de-escalating dose of g-NK cells that is pursued if dose-limiting toxicity (DLT) is observed at a higher dose level of g-NK cells, such as dose level 1 (DL1).

Table E6: Dosing Regimens for Subjects with MM

DL-1 = dose level -1; DL1 = dose level 1; DL2 = dose level 2; DL3 = dose level 3; Dara = daratumumab; Isa = isatuximab

[0734] For dosing subjects according to certain regimens in Table E6, the g-NK cell compositions, prepared by expansion substantially as described in Example 1 , were thawed from provided frozen liquid form prior to intravenous administration within 10 minutes (± 5 minutes) but not to exceed 30 minutes. Some subjects received g-NK cell composition as a single agent, with or without IL-2 administered subcutaneously. IL-2 was administered 1 hour prior to infusion of the g-NK cell composition. For subjects receiving a combination therapy with daratumumab, daratumumab is provided in a single use vial in liquid form and is delivered through intravenous injection. Administration of daratumumab or isatuximab begins 7 days prior to the first infusion of g-NK cell composition. Pre-medication with steroids prior to the first administration of daratumumab is permitted at a dose <20 mg prednisone equivalent with approval by the Sponsor’s Medical Monitor.

[0735] For subjects in which a dosing regimen in Table E6 is given, prior to administration of therapeutic g-NK cell composition, subjects received lymphodepleting chemotherapy composed of fludarabine 30 mg/m², cyclophosphamide 400 mg/m², and mesna 300 mg/m², which was administered 3, 4, and 5 days prior to the first infusion of the g-NK cell composition.

[0736] A second cycle of treatment composed of repeat conditioning followed by up to 3 additional doses of g-NK cell composition can be given at the discretion of the investigator and medical monitor, assuming the subject meets the following criteria: (a) did not experience a dose-limiting toxicity (DET) during the first cycle of treatment, (b) did not experience disease progression on study, (c) at least 30 days have elapsed since the last dose of g-NK cell composition, and (d) absolute neutrophil count is at least 1000 cells/uE.

[0737] Primary and secondary endpoints related to safety assessment and tolerability of g-NK cell compositions, with or without IE-2, were assessed in subjects that received a monotherapy dosing regimen. Primary and second endpoints related to safety assessment and tolerability of g-NK cell compositions, with or without IL-2, and with an anti-CD38 antibody (e.g., combination therapy) are also assessed. Primary and second endpoints include incidence of adverse events (AEs) or serious adverse events (SAEs) graded according to NCI CTCAE Version 5.0, incidence and nature of dose-limiting toxicities (DLTs), changes from baseline in clinical safety laboratory values and vital signs, and incidence of potential immune -related toxicities. Additional primary and secondary endpoints include determining the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and maximum protocol dose (MPD) with and without IL-2 and with or without an anti-CD38 antibody, such as daratumumab or isatuximab. Further endpoints related to efficacy and pharmacokinetics (PK) are also assessed, including the non-compartmental PK parameters of the therapeutic g-NK cell compositions (e.g., maximum drug concentration (C_max) and area under the concentration-time curve (AUC)) as well as objective response rate (ORR), complete response (CR), stringent complete response (sCR), very good partial response (VGPR), partial response (PR), and minor response (MR) according to the IMWG Uniform Response Criteria for MM.

[0738] In an ongoing clinical trial, six subjects were enrolled and in the “S-DL-1” and “M-QW-DL- 1” cohorts as set forth in Table E6 and exposed to the corresponding single dose or multiple doses of g- NK cell compositions at the previously described schedule and dosing. All six subjects completed the 21 -day dose-limiting toxicity (DLT) assessment and none experienced a DLT. There were no fatal (Common Terminology Criteria for Adverse Events [CTCAE] toxicity, Grade 5), serious adverse reactions or serious AEs reported. Four subjects (66.7%) experienced a Grade 3 or Grade 4 treatment emergent adverse event (TEAE) due to blood count decreases (white blood cell, neutrophil, platelet or lymphocyte count decrease) or cytopenias (neutropenia or anemia). One subject reported a Grade 3 insomnia. Finally, 5 out of 6 subjects had myeloma with inherent bone marrow defects that synergized with the conditioning to exacerbate cytopenias.

[0739] Study results are assessed to determine efficacy (e.g., tumor response) according to the current version of the IMWG Uniform Response Criteria. Safety profiles, pharmacokinetics, and pharmacodynamics data are also assessed.

[0740] In the ongoing clinical trial, data available from nine subjects with MM that were administered g-NK cells as a monotherapy at a dose of 5 x 10⁹ g-NK cells (henceforth referred to as dose level 1, or DL1) at a frequency of either Q2D x 3 or QW x 3 with or without administration of IL-2 at 6 M IU at the same dosing schedule as the g-NK cells. Details regarding these nine subjects, including the best response by two different time points (TP1 and TP2) in the ongoing clinical trial, are depicted in Table E7. At time point 1, subjects were on the study for one to three months, whereas time point 2 depicts analysis of all subjects on the study at a time point about 5 months after time point 1.

Table E7. Representative MM Subject Demographic and Clinical Summary

Auto SCT, autologous stem cell transplant; BCMA, B cell maturation antigen; CAR, chimeric antigen receptors; Dara, daratumumab; DL1, Dose level 1; Isa, isatuximab; Lymph, Lymphocyte count; Neut, Neutrophil count; PR, partial response; PD, progressive disease; Ritux, rituximab; SD, stable disease; TCE, T-cell engager; VGPR, very good partial response; WBC, White blood cell count; NA, not available. [0741] The dosing schedules for eight of the MM subjects are detailed in Table E8.

Table E8. Dosing Schedules of MM Subjects

M= Million; IU = international units; DL1 = dose level 1; s.c. = subcutaneously

[0742] The initial efficacy of the g-NK monotherapy for the MM subjects at the first assessed time point (TP1) above is detailed in Table E9.

Table E9. Initial Efficacy of Representative MM Subjects

Response determined by: MM (IMWG, 2016); VGPR =very good partial response; PR=Partial response; MR= minor response

[0743] The therapy was well-tolerated in subjects and there have been no dose-limiting toxicities. The most common adverse events were cytopenias due to conditioning therapy. In particular, change in positron emission tomography (PET) standardized uptake units (SUVs) have been assessed for patients 03, 06, 07, and 09 at the time point 2 (TP2) in the ongoing clinical study. The mean change in SUV was determined by averaging the change in SUV of each evaluable lesion. Details regarding these four subjects are depicted in FIG. 5 and Table E10. For Table E10, the range of percent change in SUV from baseline is shown in parentheses. All post-treatment PET results were 2 months from baseline, except patient 03 that was 5 months.

Table E10. Change in PET SUVs of Patients with MM Following Treatment

MR = minimal response; PR = partial response; SD = stable disease; SUV = standardized uptake units

[0744] Moreover, in particular, for MM subject patient ID 04, the radiologist’s impression of the patient was that the 59 year old male with relapsed/refractory (R/R) MM who had 7 prior lines of therapy including: autologous stem cell transplant, BCMA CAR-T (ABECMA®), experienced a bone marrow plasma percentage decrease from 11% pre-treatment to 1% post-treatment (at day 28). FIG. 6A is a figure of patient ID 04’ s level of kappa light chains (mg/L) across time (days). Arrows indicate g-NK cell infusions with IL-2). Overall, patient ID 04 had a marked reduction in light chains, normalization of the marrow plasma cells, and a significant reduction in activity on PET scan.

[0745] Additionally, for MM subject patient ID 06, the 68 year old female with relapsed/refractory MM who had 5 prior lines of therapy, inclusive of RVD, Cytoxan followed by ASCT, daratumumab + dexamethasone, and Kyprolis, the radiologist’s impression of the patient was that there was evidence of treatment response with interval decrease in uptake within multiple myelomatous lesions involving hypermetabolic mediastinal lymph nodes and multiple osseous lesion in axial and appendicular skeleton and that there was stable focal hypermetabolic uptake in hepatic segment 2 with corresponding hypodensity. Overall, patient ID 06 had a significant reduction in disease activity by PET scan.

[0746] Further, MM subject patient ID 03, a patient who has R/R MM, showed impressive reduction of tumor by PET scan. First, pre-treatment, the bone marrow blast cells of patient ID 03 comprised 20- 25% monotypic plasma cells. These cells were considered “rare” (0.02% by flow cytometry) approximately four months after administration of the g-NK therapy. There was marked reduction and/or elimination of tumor lesions on PET at 4 months after g-NK cell therapy. Notably, FIG. 6B depicts FDG-PET images, with arrows pointing to FDG-avid lesions, at baseline (left) and at four months after treatment (right). FIG. 6B shows that at four months after g-NK cell therapy treatment, patient 03 demonstrated an improvement in multiple locations of the FDG-avid lesions identified at baseline. Patient ID 03 was progression free for 5 months (IWMG = SD) and then had CR on BCMA. The patient remained in CR at the time of last follow-up at around seven months.

[0747] Finally, patient ID 013, a 68 year old male patient diagnosed with R/R MM, also showed no disease progression following g-NK cell therapy treatment. Specifically, baseline PET imaging was negative for FDG-avid lesions. Patient ID 013 was progression free for 3 months after receiving g-NK cell therapy treatment and before receiving daratumumab (IWMG = SD). The patient showed no disease progression and remained progression free at the time of the last follow-up at 6 months. [0748] Finally, a summary of the maximum change in serum tumor biomarker (M-protein) is shown in FIG. 6C. As shown in FIG. 6C, the maximum change in nine different patients in serum tumor biomarker from baseline was determined based on either M-protein (serum and urine) or free-light chains (FLC) when M-protein was undetectable or unavailable. Asterisks indicate the patient did not progress on study but transitioned to other treatment. For patient 08, the expected ORR was 10-15%, VGPR was 5- 8%, and mPFS was 2-3 months. For patient 04, the expected ORR was 15-20%, VGPR was <10%, and median duration of response (mDOR) was 2-3 months. The results suggest that deep responses were achieved, even post-BCMA therapy with responses observed across all lines of therapy. The disease control rate was 100% and ORR was 67% with 6 of 9 patients responding.

[0749] The results demonstrated that g-NK cell therapy was well tolerated in both, every week x3 and every other day x3 regimens.

[0750] Furthermore, activity was observed with the g-NK cell therapy along with IL-2 in subjects with advanced MM, even in the absence of combination with ADCC mediating monoclonal antibodies. For example, g-NK cell therapy also induced favorable responses in highly pre-treated subjects with R/R MM including a 33% rate of VGPR. Highlights include: (1) quad-refractory post-BCMA subject with complex karyotype that achieved VGPR (-100% urine M protein) and was still alive around 11 months after receiving g-NK cell therapy, and (2) a penta-refractory, p53-deleted/3xmyc subject that achieved VGPR (-94% FLC). Results in these subjects exceeded expected outcomes, where the probability of VGPR+ was <10%, PFS was <3 months, and OS was around 3-5 months.

[0751] The responses in subjects administered g-NK cell compositions and an anti-CD38 antibody (e.g., a combination therapy), particularly isatuximab, are anticipated to be well tolerated and to enhance the response and/or increase the durability of response in treated subjects.

Example 4: Administration of g-NK Cells as a Monotherapy in Treatment of AML

[0752] Therapeutic g-NK cell compositions containing allogenic NK cells deficient in expression of FcRy chain are administered to subjects with acute myeloid leukemia (AML). As part of the study, subjects in different cohorts receive different therapeutic g-NK cell composition regimens testing different g-NK cell composition dosing schedules/dosages and/or inclusion of IL-2 administration.

[0753] Subjects with AML are treated. Eligibility criteria include subjects that have initial morphologic diagnosis of AML or “MDS/AML” per either World Health Organization, European Leukemianet, or International Consensus Classification criteria, and meet eligibility as either a subject with AML having measurable residual disease (MRD) or a subject with AML having low burden relapsed/refractory (R/R) disease.

[0754] Subjects with having low burden R/R disease must have the following: a. Patients will need to have <25% blasts in peripheral blood and BM. b. R/R disease defined by standard criteria as follows:

1. Relapsed: BM blasts >5%, reappearance of blasts in the blood, or development of extramedullary disease following achievement of CR/CRi/ morphologic leukemia-free state (MLFS).

2. Refractory: 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 <10,000. d. Appropriate prior therapy in order for patient to be deemed R/R includes any of the following:

• 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 or,

• At least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX- 351 with venetoclax or similar regimens or,

• At least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax or,

• 2 cycles of venetoclax with HMA/LDAC +/- other agents or,

• 4 cycles of HMA alone.

[0755] To qualify as a subject with AML having MRD, the following criteria are met: (a) be in compositive complete remission (cCR), which may include complete remission (CR), complete remission with partial hematologic recovery (CRh), or complete remission with incomplete hematologic recovery (CRi); (b) bone marrow must show MRD > 0.1% by multi-parameter flow cytometry difference from normal assay via central lab; (c) if they are in second or higher cCR for AML, have undergone at least one cycle of salvage therapy; (d) have MRD relapse after allogeneic stem cell transplantation (allo- SCR) or during consolidation or maintenance therapy; and I if they have AML in first remission cCR, have adverse risk AML per Dohner, 2022 criteria and have received at least: (1) 1 cycle of intensive induction and 1 cycle of consolidation chemotherapy with intermediate or high-dose cytarabine based regimen, (2) 4 cycles of venetoclax-based lower intensity regimen containing hypomethylating agent (HMA) or low dose cytarabine (LDAC), or (3) 4 cycles of HMA-based regimen.

[0756] To qualify as a subject with AML having low burden R/R disease, the following criteria are met: (a) need to have <25% blasts in peripheral blood and bone marrow; (b) have R/R disease defined by standard criteria as follows: (1) relapsed: bone marrow blasts >5%, reappearance of blasts in the blood, or development of extramedullary disease following achievement of CR/CRi/ morphologic leukemia-free state (MLFS), or (2) refractory: failure to achieve CR/CRi/MLFS following initial treatment, with evidence of persistent leukemia by blood and/or bone marrow evaluation with blasts >5%; (c) white blood cell (WBC) count <10,000, (d) having undergone an appropriate prior therapy, which includes the following:

(1) 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;

(2) at least 1 cycle of intensive induction chemotherapy with venetoclax, e.g., 7 + 3 or CPX- 351 with venetoclax or similar regimens;

(3) at least 2 cycles of intensive induction chemotherapy such as 7 + 3 or 5 + 2 or similar regimens without venetoclax;

(4) at least 2 cycles of venetoclax with HMA/LDAC +/- other agents; or

(5) 4 cycles of HMA alone;

(e) if a younger or fit subject in first relapse following intensive chemotherapy, the first remission (CR1) duration was <12 months; (f) must not have received more than 3 prior lines of therapies for active disease and 1 prior allo-SCT; and (g) if relapsing after allo-SCT, must have been >100 days since prior allo-SCT at the time of lymphodepletion, have recovered from all transplant-related toxicities and are off all immunosuppression, with no more than grade 1 chronic GVHD. Additionally, subjects with be eligible regardless of CR1 duration if they relapse with persistent or new TP53 mutations or are older or unfit subjects who relapse on HMA + venetoclax based maintenance regimen.

[0757] Additional eligibility criteria include having actionable mutations with available FDA- approved therapies, e.g., FLT3, IDH1/2 inhibitors may be enrolled after they have exhausted appropriate lines of FDA approved treatment options. Subjects with AML with antecedent hematological disorder (AHD), e.g., aplastic anemia, myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) or myeloproliferative disorder or neoplasm who have previously received a regimen appropriate for AML for the antecedent hematological disorder, as described above, and have progressed to AML, are eligible for the dose escalation phase only.

[0758] Different dosing regimens tested in subjects are set forth in Table Ell. The regimens differ depending on g-NK cell composition dosing schedules (e.g., a single dose schedule, a multiple dose Q2D (every 2 days) schedule, or a multiple dose QW (every week) dosing schedule) and inclusion of IL-2 administration.

Table Ell: Dosing Regimens for Subjects with AML

[0759] The IL-2 may be omitted depending on the safety, tolerability, and pharmacokinetics. Dose level -1 (DL-1) is a de-escalating dose of g-NK cells that is pursued if dose-limiting toxicity (DLT) is observed at a higher dose level of g-NK cells, such as dose level 1 (DL1).

[0760] The g-NK cell compositions, prepared by expansion substantially as described in Example 1, are thawed from provided frozen liquid form prior to intravenous administration within 10 minutes (± 5 minutes) but not to exceed 30 minutes. Some subjects receive g-NK cell composition as a single agent with or without IL-2 administered subcutaneously as per the approved prescribing information. IL-2 is administered 1 hour prior to infusion of the g-NK cell composition.

[0761] Prior to administration of therapeutic g-NK cell composition, subjects receive lymphodepleting chemotherapy composed of fludarabine 30 mg/m², cyclophosphamide 400 mg/m², and mesna 300 mg/m² 3, 4, and 5 days prior to the first infusion of the g-NK cell composition.

[0762] A second cycle of treatment composed of repeat conditioning followed by up to 3 additional doses of g-NK cell composition is given at the discretion of the study investigator and medical monitor, assuming the subject meets the following criteria: (a) did not experience a dose-limiting toxicity (DLT) during the first cycle of treatment, (b) did not experience disease progression on study, (c) at least 30 days have elapsed since the last dose of g-NK cell composition, and (d) absolute neutrophil count (ANC) is at least 1000 cells/uL and ANC and platelets have returned to baseline levels prior to receiving cycle 1.

[0763] Primary and secondary endpoints related to safety assessment and tolerability of g-NK cell compositions with or without IL-2 are assessed, including incidence of adverse events (AEs) or serious adverse events (SAEs) graded according to NCI CTCAE Version 5.0, incidence and nature of doselimiting toxicities (DLTs), changes from baseline in clinical safety laboratory values and vital signs, and incidence of potential immune -related toxicities. Additional primary and secondary endpoints include determining the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and maximum protocol dose (MPD) with and without IL-2. Further endpoints related to efficacy and pharmacokinetics (PK) are also assessed, including the non-compartmental PK parameters of the therapeutic g-NK cell compositions (e.g., maximum drug concentration (C_max) and area under the concentration-time curve (AUC)) as well as measurable residual disease (MRD) response (below limit of detection), objective response rate (ORR), and composite complete response (cCR).

[0764] Study results are assessed to determine efficacy (e.g., MRD status) using bone marrow or blood at a centralized testing lab using multi-parametric flow cytometry (MFC) at different time points throughout the study. Safety profiles, pharmacokinetics, and pharmacodynamics data are also assessed.

Example 5: Enrichment of g-NK Cells in the Tumor Microenvironment

[0765] In this study, the presence of g-NK cells in the tumor microenvironment was explored.

[0766] Single cell RNA sequencing data from the Obradovic dataset (Obradovic, A., et al (2021). Cell, 184(11): 2988-3005) was analyzed to evaluate measure g-NK cell frequency in the tumor microenvironment of patients with clear cell renal carcinoma or with HER2+ breast cancer. The single cell RNA sequencing data revealed the presence of g-NK cells in the tumor microenvironment.

[0767] For clear cell renal carcinoma, FIGS. 7A and 7B illustrates a specific g-NK cell cluster (cluster 2) from the Obradovic data set. FIG. 7A depicts the NK cell count categorized by tumor grade and indicates g-NK cells are enriched in the tumor microenvironment of patients with low grade clear cell renal carcinoma. FIG. 7B depicts the NK cell count by tumor stage, specifically stage 1 kidney cancer that is 4 centimeters or smaller in size and has not spread to other parts of the body (pTla) or stage 3 kidney cancer where the tumor extends beyond the kidney’s parenchyma and into the perinephric fat but is still contained within Gerota’ s fascia (pT3a) and indicates that g-NK cells are enriched in the tumor microenvironment of patients with early stage clear cell renal carcinoma. Conversely, as illustrated by FIGS. 7A and 7B, the number of g-NK cells is decreased in high grade and late- stage samples.

[0768] Using the same Obradovic dataset, a meta-analysis was additionally performed on the FinHER and CALGB40601 datasets of HER2+ breast cancer patients. Specifically, in the analysis of the CALGB40601 HER2+ breast cancer patient dataset, the pathologic complete response (pCR) rate of patients treated with trastuzumab, a monoclonal antibody that targets HER2 and is approved in HER2+ breast and gastric cancers, was determined. FIG. 8A shows 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. [0769] In the analysis of the FinHER HER2+ breast cancer patient dataset, there was a trend toward increased distant disease-free survival (DDES) in patients with a high proportion of g-NK cells when treated with trastuzumab and chemotherapy, as depicted by FIG. 8B.

[0770] In sum, the study demonstrates that g-NK cells can be identified in the tumor microenvironment of patients with various solid tumors. Specifically, g-NK cells have been identified in the tumor microenvironment of patients with clear cell renal carcinoma.

[0771] Moreover, the presence of high proportions of g-NK cells in the tumor and are correlated with an increased probability of DDES and pCR in HER2+ in trastuzumab-treated breast cancer patients. Collectively, these data support the capacity of g-NK to mediate single agent activity against solid tumors and to combine with therapeutic mAh to potentially rescue treatment non-small cell lung carcinoma (NSCLC) or refractory breast cancer patients.

Example 6: Faster Migration, Increased Synapse Frequency, Enhanced ADCC, and Enrichment of LFA-1 Expression

[0772] Experiments were conducted to evaluate antibody-dependent cellular cytotoxicity (ADCC), synapse frequency, and migration of g-NK cells. Additionally, using the same Obradovic dataset mentioned in Example 5, RNA profiles of g-NK cells (cluster 2) were analyzed.

[0773] Faster migration, quantified by effector displacement before synapse, was observed by g-NK cells, with or without daratumumab, compared to conventional NK cells (cNK cells), with or without daratumumab, as depicted by FIG. 9A. The median displacement rate of g-NK cells (with or without daratumumab) was approximately 1.2 pm/ min and FIG. 9A demonstrates the rate of g-NK cell motility was significantly higher compared to cNK cells (P-value generated using Fisher’s exact test). Interestingly, the rates at which g-NK cells found their targets (“tSeek” values are depicted in FIG. 9B) and formed a synapse (“tSynapse” values are depicted in FIG. 9C) were similar to those observed by cNK cells.

[0774] Even though the rates at which g-NK cells formed a synapse were similar to the rates at which cNK cells formed a synapse, it was observed that g-NK cells formed synapses at a higher frequency. FIGS. 10A-10C demonstrates the frequency of synapse formation between g-NK cells and target tumor cells was almost twice as high as compared to the frequency of synapse formation between cNK cells and target tumor cells. This higher synapse formation frequency demonstrated by g-NK cells is observed across different E:T (effectortarget ratios), at IE: IT as shown by FIG. 10A, at 1E:2T as shown by FIG. 10B, or at 1E:3T as shown by FIG. 10C. The increased rate of synapse formation was found to be the primary driver of enhanced g-NK ADCC. P-value generated using Fisher’ s exact test. [0775] Without being bound by theory, greater frequency of synapse formation led to overall greater single-target and serial killing by g-NK cells compared to cNK, as observed by FIG. 11A-11D. FIGS. 11A and 11C demonstrate daratumumab mediated for g-NK cells, as compared to cNK cells, a greater target killing of multiple myeloma target cells after synapse, at both a IE: IT ratio (FIG 11A) or a 1E:3T ratio (FIG. 11C). Additionally, FIG. 11C further demonstrates daratumumab significantly increased the percent of serial killing events in both g-NK and cNK cultures. FIG. 11B demonstrates that although daratumumab increased the killing and rate of killing for both g-NK cells and cNK cells, g-NK cells were much more responsive to daratumumab, especially at earlier time points, between 0 and 120 minutes, compared with cNK cells. FIG. 11D are representative images of cNK effector cells (top row) g-NK effector cells (bottom row) and alive or dead tumor cells.

[0776] Finally, analysis of RNA profiles of g-NK cells (cluster 2) are shown by FIG. 12A and 12B, along with other NK cell clusters. FIG. 12A depicts other gene markers that are positively expressed while FIG. 12B depicts gene markers that are negatively expressed. A green box in FIG. 12A denotes the CD2 expression among various NK cell clusters, highlighting the enriched expression of CD2, also known as Leukocyte Adhesion Protein- 1 (LFA-1) in cluster 2.

[0777] Moreover, a fraction of CD2 (LFA-1) expressing cells was analyzed within total NK (CD56+), conventional NK (FceRly-i-), and g-NK (FceRly-) populations of ex vivo expanded NK cells by flow cytometry. It was determined that the g-NK cell population expressed a significantly higher percentage of CD2 than the expression of either the total NK cell or the conventional NK cell population, as depicted by FIG. 12C (*p<0.05, **p<0.01, One-way ANOVA, Tukey post-hoc test for multiple comparisons).

Without being bound by theory, the higher expression of CD2 by g-NK cells likely contributed to enhanced motility of g-NK cells, and consequently, enhanced ADCC of g-NK cells. The results demonstrate that the enhanced ADCC of g-NK cells is partly due to their increased motility and ability to form synapses with target cells at higher frequencies.

Example 7: Kinetics of Cell Surface-Bound Antibody On g-NK cells

[0778] To assess the kinetics of binding of monoclonal antibody by CD16 expressed by g-NK cells, ex vivo expanded g-NK cells, generated substantially as described in Example 1, were incubated with 100 pg/mE of anti-CD19 monoclonal antibody (mAh). At regular intervals, up to nine hours (540 minutes), surface-bound anti-CD19 mAh was detected by flow cytometry using a fluorescently labeled recombinant CD 19 protein.

[0779] Results are shown in FIG. 13, which depicts the percentage of g-NK cell bound antibody (normalized to t = 0 minutes; at t = 0, the percentage was around 32%). FIG. 13 demonstrates that g-NK cells retain anti-CD19 mAh for up to 9 hours (540 minutes) in vitro. [0780] The results support that g-NK cells are able to retain bound antibodies on their cell surface, and thus facilitate transport of monoclonal antibodies including potential to increase tissue distribution. Without wishing to be bound by theory, these results support a model in which g-NK cells can support transport of mAbs across the blood-brain marrier by transporting the antibodies into lymph nodes and to the central nervous system (CNS) where they can effect cytolysis of autoreactive cells in CNS.

Example 8: Pharmacokinetics of IL-2 Administration

[0781] Experiments were conducted to evaluate how administration of cytokines, such as IL-2, affected g-NK cell compositions, including recovery and expansion of cryopreserved NK cells and pharmacokinetics of IL-2 following infusion in human subjects.

[0782] Post-thaw recovery and expansion of cryopreserved NK cells was assessed. G-NK cells were manufactured as described in Example 1 and then were cryopreserved. Freeze media for the cryopreserved cells was CS-10 (Biolife Solutions, Bothel, WA, USA). Cryopreserved cell products were thawed rapidly in a hot water bath prior and then were assessed for total cell number or viability immediately or were cultured post-thaw for up to 8 days in media with no cytokines or in media with IL- 2 at a concentration of 500 lU/mL or IL- 15 at a concentration of 10 ng/mL. Cytokines were added to the thawed NK cells on day 0, day 3, and day 6 post-thaw. At regular intervals for up to 8 days post-thaw, cell counts and viability were measured.

[0783] FIG. 14A depicts the total number of cells in the culture, and FIG. 14B depicts the percent viability of cells in the culture over time. When cultured with IL-2 or IL- 15, NK cells recovered viability over the first 48 hours post-thaw and then start to expand after 72 hours of cytokine exposure. In contrast, NK cells cultured without cytokines did not recover viability nor expand.

[0784] The results support that longer duration of IL-2, such as occurs when IL-2 is administered to subjects as g-NK cell support, can improve NK cell survival post-infusion.

[0785] To assess the pharmacokinetics of IL-2 administration during treatment of different cancers, such as NHL, MM, and AML, levels of IL-2 in serum were measured in different subjects receiving different dosing regimens of g-NK cells as previously described in Examples 2-4 (see Table E2, Table E6, and Table El l).

[0786] Briefly, subjects received g-NK cell composition doses either every 2 days (Q2D) or every week (QW) as a monotherapy or as a combination therapy with the anti-CD38 antibody daratumumab. Some subjects additionally were also administered IL-2 at a concentration of 6 M IU 1 hour prior to each infusion of the g-NK cell composition. To measure the pharmacokinetics of IL-2, serum samples were also taken from subjects on a regular basis and IL-2 levels measured.

[0787] FIG. 15A shows IL-2 serum concentrations in subjects receiving QW dosing of g-NK cell compositions, and FIG. 15B shows IL-2 serum concentrations in subjects receiving Q2D dosing of g-NK cell compositions. The results show that subjects that were administered IL-2 had consistently high levels at 1-hour post-infusion which then decreased to near-background levels of IL-2 within 48 hours, in contrast to subjects that were not administered IL-2. IL-2 levels were not measured in Subject D on day 10 post-g-NK cell administration.

[0788] FIG. 16A shows IL-2 serum concentrations in subjects on either a Q2D or QW dosing regimen that included administration of IL-2 when IL-2 levels are at their trough pre-dose (Day 0), Day 2 (pre-infusion), Day 4 (pre-infusion), and Day 7 post-first infusion of IL-2. FIG. 16B shows IL-2 serum concentrations in the same population of subjects when IL-2 levels at their peak after receiving their first, second, or third dose of IL-2. Peak IL-2 levels were measured following infusion of g-NK cell compositions.

[0789] The results show that subjects on Q2D dosing retained similar trough values on Days 2 and 4, while subjects on QW dosing exhibited lower trough values on Day 4 than Day 2.

[0790] Similar peak values were observed following the second and third doses of IL-2 for subjects on a Q2D or QW schedule, but IL-2 peak levels were higher in the first dose for patients on a Q2D schedule rather than a QW schedule. Additionally, the one subject receiving a combination therapy, as indicated by a “D” (noting administration with daratumumab) in FIG. 16B, had notably higher IL-2 peak levels at the second and third dose as compared to the other subjects, all of whom received g-NK cells as a monotherapy.

[0791] As administration of IL-2 may increase the persistence or survival of g-NK cells, these findings suggest that repeated dosing of IL-2, such as on a Q2D or QW schedule, may increase persistence of g-NK cells.

Example 9: Effect of g-NK Cells on Tumor Microenvironment Remodeling in Bone Marrow

[0792] The impact of the tumor microenvironment (TME) on patient outcomes following cell therapy including administration of g-NK cell compositions was determined by analyzing bone marrows from subjects treated with g-NK cell compositions.

[0793] Bone marrow samples from a subset of subjects with multiple myeloma (MM) who were administered g-NK cell compositions as described in Example 3 were analyzed using cytometry time-of- flight (CyTOF) using a 37 marker panel designed to identify immune populations. Some subjects included: a first subject (MM subject patient 04) who had achieved a partial response (PR) based on International Myeloma Working Group (IMWG) criteria in monoclonal protein and had improvements in the activity of lytic lesions on their PET scan; a second subject (MM subject patient 03) who had a minimal response (MR) based on IMWG criteria; and a third and fourth subjects (MM subject patient 02 and 01) who had stable disease (SD) based on IMWG criteria. Two of the three subjects (MM subject patient 01 and 03) with relatively stable monoclonal protein in the blood (MR or SD) had significant improvement in PET scans after NK-cell therapy. One of the subjects with SD (MM subject patient 01) had improvement in lytic lesions as measured by PET maintained SD for five months.

[0794] In particular, for MM subject patient ID 03, a patient who has R/R MM, showed impressive reduction of tumor by PET scan, as shown above in Example 3, with tumor microenvironment (TME) remodeling. Specifically, bone marrow cytometry by time of flight (CyTOF) showed TME remodeling at one month after initiation of g-NK cell therapy treatment. There was a massive influx of T cells as well as elimination of polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs). Analysis of T cells showed increased CD8 infiltration post treatment (post-tx), as demonstrated by FIGS. 17A-17B. Additionally, there was also a decrease in inflammatory markers (PD-1, CXCR3, and CD38), as demonstrated by FIG. 17C and elimination of PMN-MDSCs, as demonstrated by FIG. 17D.

[0795] Similarly, after g-NK cell therapy treatment, the induction of T cell infiltration with TME environment was also observed for another MM subject patient ID 13. Like patient ID 03, analysis of T cells for patient ID 13 showed an increase of CD8 infiltration post treatment (post-tx), as demonstrated by FIGS. 18A-18B. Patient ID 13 showed increase in bone marrow T cell infiltration specifically within the CD8 T cell subset at one month after initiation of g-NK cell therapy treatment.

[0796] Two subjects (MM subject patient 01 and 04) had post-treatment bone marrow samples showing decreased expression of the exhaustion biomarker PD1 by CD8+ T cells. The subjects who had improvement of their lesions on their PET scan experienced decreased in T cell expression of the inflammatory markers CXCR3 and CD38. The subject (MM subject patient 01) with SD and improvement in bone lesions that lasted five months saw a 5x increase in CD8+ T cells in his posttreatment biopsy with near complete clearance of myeloid-derived suppressor cells (MDSC) (<1%).

[0797] The data further suggested that pre-treatment condition of the bone marrow was predictive of anti-tumor activity. For example, of the four tested patients, the patient who achieved a PR had the highest percentage of CD8+ cells and lowest percentage of MDSC at baseline. The patient with SD and no improvement by PET scan had the highest percentage of MDSC, the lowest CD8+ T cell percentage, and the highest expression of PD-1 on CD8+ T cells on all the patients.

[0798] Collectively, the data suggests that administration of g-NK cell compositions can alter the TME and promote MM control by influx of non-exhausted T cells. The correlative CyTOF studies suggest anti-tumor mechanisms in addition to direct NK cell activity play a role in controlling tumors. Individual case studies show remodeling of the TME, including influx of non-exhausted CD8 T cells with resulting improved Teff to Treg ratios. These results support a combination therapy of the g-NK cells with a T cell targeting agent, such as a T cell engager (e.g. CD3 bispecific antibody also directed to a tumor target), for mediating anti-tumor activity and treatment of cancers. Furthermore, the results indicate the amount of MDSC in the bone marrow at time of treatment may be inversely correlated with anti-tumor activity. Example 10: Effect of g-NK Cells on Gene Regulation in Bone Marrow

[0799] The impact of g-NK cell therapy, as described in Example 3, on gene regulation was determined by analyzing bone marrows from subjects treated with g-NK cell compositions who responded or did not respond to treatment.

[0800] Bone marrow samples from a subset of subjects with multiple myeloma (MM) who were administered g-NK cell compositions as described in Example 3 were split into responder or nonresponders based on their response to treatment and analyzed for gene regulation. In particular, responders were defined as subjects who achieved greater or equal to a minor response (MR) by IMWG at day 28 post-administration of g-NK cell compositions. Results of this analysis are shown in FIGs. 19- 20 and Table E12.

Table E12: List of Select Genes that Are Regulated Between Baseline and Day 28 PostAdministration in Responders vs. Non-responders

DEPTOR, DEP domain-containing mTOR-interacting protein; WNT10A, Wnt Family Member 10A; CCL21, Chemokine (C-C motif) ligand 21; A2M, Alpha-2-macroglobulin; FAM124B, family with sequence similarity 1 4 member B; MSH6, mutS homolog 6; GNLY, granulysin; HLA-F, Human leukocyte antigen F [0801] FIG. 19 shows a volcano plot depicting differential gene expression in bone marrow of nonresponding and responding patients. Based on the volcano plot, the genes 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 were downregulated in responders compared to non-responders, while the genes EGF, ITGB3, NID2, and PG4 were upregulated in responders compared to non-responders. Functionally, the downregulated genes CD28, SEAMF7, and TNFRSF17 are associated with T cell co-stimulation; the downregulated genes HEA-DOB and CEECE1 are associated with antigen presentation, the downregulated genes SMAD5 and DUSP5 are associated with DNA damage response and repair, and the downregulated genes DEPTOR and PDK1 are associated with metabolic signaling.

[0802] FIG. 20 further shows the difference in gene expression in bone marrow samples between baseline and day 28 post-administration for non-responders (N) and responders (R) for select genes: DEPTOR, NDUFA4L2, WNT10A, CCL21, A2M, FAM124B, MSH6, GNLY, and HLA-F.

[0803] These results suggest that the g-NK cell therapy is an effective immunotherapy in responders. For example, downregulated genes included tumor-associated genes that align with effective immunotherapy, likely reflecting a reduction in tumor cells and/or modulation of the tumor microenvironment. Further, many of the downregulated genes are involved in immune evasion (e.g., MAGE family, Wnt signaling), so down-regulation of these genes suggests immunotherapy has successfully reactivated immune responses to target tumor cells. Further, downregulation of genes like CEECE1, WNT10A, and ITGA6 point to changes in the tumor microenvironment (TME) that reduce its support for tumor growth and survival. In addition, the results support that responders to g-NK cell therapy downregulate disease-associated genes (e.g., DEPTOR and WNT10A) and upregulate cytotoxic immune-associated genes (e.g., granulysin and HEA-F). Overall, the observed distinct regulation in genes associated with immune responses, pro-tumorigenic signaling, and reprogramming of the microenvironment in the bone marrow of responding individuals supports the efficacy of g-NK cell therapy.

[0804] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. SEQUENCES

Claims

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:

FLAG-Ida, CLIA or CLAG-M or similar regimens with or 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).

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

25. A method of treating an HLA-E expressing cancer, the method comprising:

(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:

28. A method of treating an HLA-E expressing cancer, the method comprising:

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:

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 (NKG2C^pos) and/or negative or low for NKG2A (NKG2A^neg).

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

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

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:

FLAG-Ida, CLIA or CLAG-M or similar regimens with or 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).

75. A method of treating lymphoma in a subject, the method comprising:

76. A method of treating lymphoma in a subject, the method comprising:

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.

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

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).

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

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

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

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 (NKG2C^pos) and at least about 70% of NK cells of the composition are negative or low for NKG2A (NKG2A^neg).

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 (FcRy^neg) 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 (FcRy^neg) NK cells (g-NK) or at least at or about 50% of the cells in the composition of g-NK cells are FcRy-deficient (FcRy^neg) 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 FcRy^pos; 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 FcRy^pos.

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 NKG2C^pos cells cultured with irradiated HLA-E+ feeder cells, wherein the NKG2C^pos cells 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 CD3^negNKG2C^pos cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3^negNKG2C^pos cells 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 IE

149. The method of any one of claims 140-148, wherein the irradiated feeder cells are

221. 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 10⁸ cells to at or about 50 x 10⁹ cells.

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 10⁸ cells.

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 10⁹ cells.

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 10⁹ cells.

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 10⁹ cells.

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/m² body 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/m² body 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.

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) administration of a dose of IL-2 to the subject,

(b) administration of a composition of g-NK cells to the subject, and/or

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